
Proceedings of the AACR

Volume 58 | April 2017

Part A: Abstracts 1-3597

TABLE OF CONTENTS

BIOINFORMATICS AND SYSTEMS BIOLOGY:

Bioinformatics and Analysis for Therapy and Immune System

Computational Cancer Biology

Functional and Genomic Analysis of Cancer

Computational Tools and Databases

CANCER CHEMISTRY:

Basic and Applied Nanotechnologies and Therapeutic Approaches

Proteomics and Mass Spectrometry 1

Novel Therapeutic Targets, Molecules, and Approaches for the Treatment of Cancer

Cheminformatics and Computational Strategies for Drug Discovery

Drug Delivery

Proteomics and Mass Spectrometry 2

CLINICAL RESEARCH:

Checkpoint Inhibitor and Prognostic Biomarkers

Childhood Cancer Clinical Translational Research

Early Detection

Molecular Diagnostics

Special Populations / Biostatistics in Clinical Trials

Cancer Survivorship and Quality of Life

Laboratory and Computational Tools to Enhance Clinical Decision Making

Adaptive Immunity to Cancer

Liquid Biopsies 1: Circulating Tumor Cells

Molecular Classification of Tumors

Predictive Biomarkers 1

Clinical Molecular Genetics and Laboratory Correlates

Liquid Biopsies 2: cfDNA

Outcomes in Patients and Survivors

Predictive Biomarkers 2

Clinical Biomarkers

Innate Immune Mechanisms in Cancer Treatment

Predictors and Drivers of Pediatric Tumors

ENDOCRINOLOGY:

Estrogen Receptor in Cancer Progression and Therapies

Prostate Cancer Biology and Therapy

EPIDEMIOLOGY:

Descriptive Epidemiology Including Trends in Incidence and Prognosis

Genetic Variation (Non-GWAS) and Cancer Risk, Prognosis, or Mechanisms

Genome-Wide Association Studies/Post-GWAS Studies

Biomarkers of Risk and Prognosis

Tobacco, Alcohol, and Other Risk Factors

Cancer Epidemiology and Prevention

EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

Alternative Small-Molecule Therapies

Antibodies, Bispecifics, and Antibody-Drug Conjugates

Antibody Technology

Gene Expression of Drug Resistance

Novel Agents

Targeting the PI3K Pathway

Tumor Microenvironment in Drug Development

Novel Mechanisms of Drug Resistance

Combination Strategies: Novel Agents and Standard Therapies

Combination Therapy 1

DNA Repair

New Targets 1

Novel Molecular Targets 1

Reversal of Drug Resistance

Targeting Growth Factor and Intracellular Signaling

Cell Death Targets

Combination Therapy 2

Drug Resistance: Other Topics

Growth Factor and Hormone Receptors as Therapeutic Targets

Mechanism of Drug Action

New Targets 2

Targeting p53, Apoptosis, and the Cell Cycle

Identification of Molecular Targets

IMMUNOLOGY:

Checkpoints 1

T-cell Immunity to Cancer: New Progress

Tumor Antigens and Adaptive Immunity

Novel Insights into Mechanisms of Response to Immunotherapy

Cytokines: The First Modern Immunotherapies

Tumor Microenvironment and Checkpoints

Tumor Models and Assays

Checkpoints 2: Small-Molecule Inhibitors

Immune Response to Hematopoietic Tumors: New Development in Tumor Immunology

Inflammation and Cancer

Adaptive Immunity in the Tumor Microenvironment

MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

Cell Cycle Checkpoint and Progression

Cell Growth Signaling Pathways 1

Cell Growth Signaling Pathways 2

Genomic Analyses across Cancer Types

Genomic Screening for Cancer Dependencies and Drug Response

Metabolic Pathways in Cancer

MicroRNA Regulation of Cancer Biology 1

Molecular Carcinogenesis and Chromosomal Alternations

Oncogenic Growth Factors and Signal Transducers

Chromatin Structure and Function

Oncogenes and Tumor Suppressors 1: Function and Therapeutic Vulnerabilities

Cell Growth Signaling Pathways 3

Cell Growth Signaling Pathways 4

Epigenetics 1

Genomic Instability and Cancer Therapy

Genomics in Inherited Susceptibility and Preneoplastic Conditions

MicroRNA Regulation of Cancer Biology 2

Mitochondria, Autophagy, and Cancer Metabolism

Targeting Aberrant Transcription in Cancer

Tumor Suppressors 1: Modeling, Metabolism, and Epigenetics

Apoptosis and Other Regulated Cell Death in Cancer

CDKs and CDK Inhibitors

Cell Growth Signaling Pathways 5

Epigenetics 2

Genome-Wide Analysis of Transcriptional Regulation

Genomic Landscape of Lymphoma, Leukemia, and Lung, Bladder, and Other Cancers

Homologous Recombination and DNA Double-Strand Break Repair

Metabolomics and Metabolic Regulation

MicroRNAs and Other Noncoding RNAs as Tumor Suppressors or Oncogenes 1

Nuclear Oncogenes and Tumor Suppressors

Breakthroughs in the Control, Function, and Drivers of Oncogenesis

High Throughput Genomics Dissecting Cancer Development and Vulnerabilities

The Riddle of the Noncoders: New Functional Roles for miRNAs, Long Noncoding RNAs, and mRNAs

PREVENTION RESEARCH:

Diet and Cancer

Biologic and Biochemical Mechanisms in Cancer Prevention

Molecular Targets and Cancer Prevention

TUMOR BIOLOGY:

Angiogenesis and Vascular Biology 1

Cell Culture and Animal Models of Cancer 1

Cellular Responses to Ionizing Radiation

Epithelial to Mesenchymal Transitions in Metastasis

Imaging Cancer Immunotherapy, Tumor Microenvironment, and Other Aspects of Tumor Biology

Migration and Invasion 1

Stem Cell Heterogeneity

Tumor Microenvironment 1

Animal Models of Human Oncogenesis

Niches and Routes of Metastasis

Angiogenesis and Vascular Biology 2

Cell Culture and Animal Models of Cancer 2

Imaging for Cancer Diagnosis and Image-Guided Therapy

Migration and Invasion 2

Normal and Neoplastic Stem Cells

Pediatric Cancer 1: Biomarkers, Preclinical Models, and New Targets

Regulation and Imaging of Tumor Metastasis

Tumor Microenvironment 2

Cell Culture and Animal Models of Cancer 3

Gene Expression in Metastatic Progression

Imaging Cancer Metabolism, Therapeutic Targets, and Treatment Response

Signaling in Cancer Stem Cells

Tumor Evolution and Heterogeneity 1

Tumor Microenvironment 3

Tumor Microenvironment 4

Early Stages of Metastatic Dissemination

Tumor and Stromal Cell Interactions

Advancing Clinical Trial Design in Regulatory Science and Policy

# 

# Sunday, April 2, 2017

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

### Alternative Small-Molecule Therapies

#1

Galectins in glioblastoma: opportunities for combined therapy.

David Cachia,1 Arindam Rano Chatterjee,1 William Alexander Vandergrift,1 Sunil J. Patel,1 Gabriel A. Rabinovich,2 Arabinda Das1. 1 _Medical University of South Carolina, Charleston, SC;_ 2 _Institute of Biology and Experimental Medicine, CONICET, Vuelta de Obligado, Argentina_.

Background: Despite aggressive treatment, including surgical resection, radiation and chemotherapy, over 90% of glioblastoma (GB) patients experience tumor recurrence. This may be due to high migratory potential, angiogenesis, molecular heterogeneity and a strong immunosuppressive environment. GB expresses high levels of carbohydrate-binding galectin proteins and histone deacetylase (HDAC) activity. The aim of this study was: 1) identify the differential expressions of galectin 1-15 protein levels in human GB patient samples as compared to normal tissue (obtained from Institutional Tissue Bank) and (2) modulate the galectin functions with and without a HDAC inhibitor (DATS: Dially Triisulfide and SAHA: Suberoylanilide Hydroxamic Acid ) or bevacizumab in in vitro GB models.

Methods: The effects of TMZ, ionizing radiation, or combined chemoradiation on galectin protein secretion and expression were assessed in human GB cells and human umbilical vein endothelial cells (HUVECs).

Results: We found increased galectin-1 protein expression in human GB tissue. We also observed that HUVEC co-culture with GB cells increased galectin-1 protein expression by 14 - 20% following bevacizumab, and conferred a bevacizumab protective benefit to GB cells. Our in vitro models promisingly demonstrated that 72 hr treatments with 25 µM of galectin 1 inhibitor + HDAC inhibitor induce antitumor activity in GB cells. Western blot and activities assay results also demonstrated that combination blockade of HDAC activity and galectin-1 augmented apoptosis in GB cells, which mechanistically involves activation of caspase-3 and inhibition of anti-apoptotic protein (survivin, p-Akt, and Mcl-1 expression).

Conclusion: Our in vitro culture results suggest possible benefit in combining a galectin inhibitor with an HDAC inhibitor in GB. Further studies in different animal models are warranted.

#2

Functional and mechanistic interrogation of BET bromodomain degraders for the treatment of metastatic castration resistant prostate cancer.

Steven Kregel, Rohit Malik, Irfan A. Asangani, Kari Wilder-Romans, Xia Jiang, Ingrid J. Apel, Gautham Ravi, Jean Tien, Xuhong Cao, Felix Y. Feng, Corey Speers, Shaomeng Wang, Arul M. Chinnaiyan. _University of Michigan, Ann Arbor, MI_.

Metastatic castration-resistant prostate cancer (mCRPC) is a lethal disease with about 30,000 estimated annual deaths in U.S, with a vast majority of CRPC driven by androgen receptor (AR) signaling. AR-signaling is critical for the development and progression of prostate cancer, and AR also the main therapeutic clinical target. AR-targeted therapies, such as AR-antagonists, provide substantial benefits in the treatment of mCRPC; however, majority of patients fail these therapies and succumb to the disease. Therefore, there is a clear and pressing need to develop new therapeutics against the AR axis in CRPC. One such novel strategy for targeting the AR-pathway and inhibiting the growth of CRPC has been the use of bromodomain and extraterminal (BET) protein inhibitors such a JQ1. However, a new class of molecules that target BET bromodomain proteins through their proteasomal degradation can improve efficacy and specificity of these inhibitors. Based on our findings, we hypothesized that pharmacologic BET degradation represents an important advance in the treatment of CRPC, and may provide a novel therapeutic strategy for advanced prostate cancer. These inhibitors affect AR-positive prostate cancer cells preferentially over AR-negative and benign prostate epithelial cells, and proteomic and genomic mechanistic studies confirm disruption of oncogenic AR and MYC signaling, both in vivo and in vitro. The overall goal is to develop very potent small molecule that leads to the proteasomal-degradation of BET proteins, with optimized in vivo properties, and to provide a compelling scientific rationale, including detailed mechanistic insight, to facilitate advancement of BET degraders as a novel potential therapeutic strategy for patients with mCRPC.

#3

Targeting epigenetic modifiers in diffuse large B-cell lymphoma.

Aarthi Goverdhan, Heng-Huan Lee, Mien-Chie Hung. _UT MD Anderson Cancer Center, Houston, TX_.

Diffuse Large B-Cell Lymphoma (DLBCL) is the most common type of Non-Hodgkin lymphoma, accounting for approximately 40% of all cases worldwide. In the last five years, researchers uncovered that DLBCL is characterized by widespread epigenetic deregulation. Lymphoma cells harbor loss-of-function mutations in transcriptional activators (KMT2D, EP300, CREBBP) and gain-of-function mutations in transcriptional repressors (EZH2). In our study, we aim to identify epigenetic vulnerabilities in lymphoma cells that can be targeted to deliver cytotoxic responses. We screened the therapeutic efficacy of a panel of lysine and arginine methyltransferase inhibitors in multiple lymphoma cell lines. Through these screens, we identified the Type I Protein Arginine Methyltransferase (PRMT) family as an attractive therapeutic target that induced cytotoxicity. Mechanistic dissection revealed that Type I PRMT inhibition caused apoptosis through deregulation of transcription factors and pro-survival genes. In addition, we uncovered novel crosstalk between PRMTs and other epigenetic modifiers that influenced cell fate in lymphoma. Our study demonstrates a critical role for Type I PRMTs in DLBCL, and adds a lucrative target to the growing list of promising epigenetics-based therapies.

#4

Inhibitory effects of aprotinin on survival and local invasion of human breast cancer cell lines.

Saeed Soleyman-Jahi, Kazem Zendehdel, Fatemeh Sadeghi, Ziba Afshari, Tahereh Barati, Samad Mohammadnezhad, Sevil Ghasemi, Saeed Amanpour. _Cancer Research Center, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran_.

Background: Aprotinin is a nonspecific serine protease inhibitor, which can inhibit kallikrein and many downstream extracellular matrix degrading enzymes. In this study, we assessed inhibitory effects of aprotinin on local invasion of human breast cancer cell lines.

Methods: We assessed effects of aprotinin on local invasion and survival of human breast cancer cell lines MDA-MB231 and SK-BR-3 in vitro. CHEMICON Cell Invasion Assay Kit was used to assess local invasion of tumoral cells. MTT assay was used to determine the anti-proliferative activity of aprotinin on different cancer cell lines. Mean optical densities of duplicate experiments were calculated for both assays. HDF-1 human fibroblast cell line was used as control normal cell. In addition, local invasion and survival of tumor cell lines treated with aprotinin were compared with untreated experiments.

Results: Cancer cell lines showed more invasion compared to HDF-1. Aprotinin significantly decreased the invasiveness of MDA-MB231 in concentration of 1 TIU/ml, 1.3 TIU/ml, and 1.7 TIU/ml in comparison with untreated group (ANOVA P <0.001). Treatment of SK-BR-3 with 1.3 TIU/ml aprotinin trended towards reduction of invasion optical density (P = 0.06). Treatment with different concentrations of aprotinin significantly decreased the surviving fraction and significantly inhibited the growth of all cell lines tested in this study (ANOVA P <0.001).

Conclusion: Aprotinin inhibited the survival and local invasion of human breast cancer cell lines MDA-MB231 and SK-BR-3. Although the growth inhibitory effect was significant in both cell lines, inhibition of local invasion was more pronounced in case of MDA-MB231. Future molecular studies could shed further lights on mechanisms underlying anti-neoplastic effects of aprotinin and its potential therapeutic effects.

#5

The effects of SIRT1 inhibitors nicotinamide and Ex-527 on lymphoma cells.

Manuela Guardi, Mariaelena Pistoni, Raffaele Frazzi. _Arcispedale S.Maria Nuova - IRCCS, Reggio Emilia, Italy_.

SIRT1 is a well-known lysine-deacetylase, having both histone and non-histone proteins as molecular targets. SIRT1 may function as a tumor promoter as well as a tumor suppressor in a context-dependent manner. We previously demonstrated that the treatment with resveratrol at concentrations able to induce apoptosis leads to SIRT1 down-regulation in lymphoma cells. Here we look for SIRT1 inhibition in order to investigate the effects on proliferation potential, apoptosis induction and targets modulation at a molecular level. The aim is to study whether SIRT1 inhibition affects cell cycle and proliferation capability of lymphoma cells. Experimental procedures. Diffuse large-B cell lymphoma derived cell line Toledo; human lymphoblastoid cell line GGB #7; SIRT1 inhibitors: nicotinamide (NAM) and Ex-527; cell cycle analysis; caspase-3 activity fluorimetric assay; genotoxicity assay gamma-H2AX; fluorescence-activated cell sorting intracellular staining for acetylated-H4K16 histone. New data. We found that the calculated IC50s for SIRT1 inhibition is 50mM for NAM and 343μM for Ex-527. The treatment with either NAM or Ex-527 leads to cell cycle arrest in both Toledo and GGB#7 cells, although in a different fashion. Namely, NAM causes an S-phase accumulation of Toledo while it leads GGB #7 to G0/G1 phase arrest with a concomitant decrease in S-phase already after 10mM. In both cell lines NAM 50mM induces apoptosis as demonstrated by caspase-3 activation and sub-G1 peak appearance in the cell cycle profiles. Ex-527 causes an accumulation in the S phase of Toledo cells and a decrease of G2/M in GGB#7 cells. No induction of apoptosis at the IC50 concentration was observed in both cell lines. Genotoxicity of the above mentioned treatments was investigated through the quantitation of gamma-H2AX histone released by treated cells. No genotoxic effects have been observed upon the treatments with these two inhibitors even at the concentration where NAM induces apoptosis. In order to correlate the observed changes with SIRT1 inhibition, we studied the acetylated H4K16 histone, a direct target of SIRT1 deacetylation. Our preliminary data indicate that NAM treatment increases the percentage of acetylated H4K16. Conclusions. Lymphoma and lymphoblastoid cells are affected by SIRT1 inhibition. NAM and Ex-527 cause cell cycle arrest and growth inhibition. Only NAM 50mM induces apoptosis without genotoxic effects. Overall, our data suggest the direct involvement of SIRT1 during the observed cell cycle arrest and growth inhibition mediated by these inhibitors.

#6

New chk1 inhibitor development & the feasibility of use of chk1 inhibitor with antimetabolite for NSCLC.

Injae Chung. _Duksung Women's University, Seoul, Republic of Korea_.

DNA damaging anticancer agents activate checkpoints that stop the cell cycle to prevent additional DNA replication & mitosis until damaged region has been repaired. One of the important regulatory proteins activated in DNA damage response pathway is Checkpoint kinase 1 (Chk1). Therefore, Chk1 inhibitors have emerged as promising new therapeutics for cancer. In order to find novel Chk1 inhibitor, we have conducted a structure based virtual screening. Several small molecules have shown 75 - 100 % inhibition of Chk1 in the binding affinity assay. Combined with the results of kinase assay with ATP & mechanism based cell assay, hit candidates has been led. Lung cancer is the leading cause of cancer related deaths in Korea. About 85 % of lung cancer is non-small cell lung carcinoma (NSCLC) worldwide. Chemotherapy for NSCLC is often regarded ineffective. To test the feasibility of Chk1 inhibitor to sensitize antimetabolite in NSCLC, growth inhibition induced by gemcitabine plus MK8776 was assessed in A549 & H1129 cells. These cells are insensitive to the MK8776 when treated as a single agent (IC50 >10 μM). In contrast, MK8776 reduced the concentration of gemcitabine required to inhibit H1299 or A540 cell growth by almost 3-30 fold or 6-15 fold, respectively, depending on the concentration used. These results would imply the potential clinical utility of Chk1 inhibitor as an adjuvant for antimetabolite such as gemcitabine for NSCLC. Further studies to evaluate the effectiveness of Chk1 inhibitor including new molecules would be warranted in additional preclinical and clinical settings. [The chemical library used in this study was kindly provided by Korea Chemical Bank & KIST. This study was supported by a grant (2015R1D1A1A01057014) from the National Research Foundation of Korea.]

#7

Ceftriaxone, an FDA-approved cephalosporin antibiotic, suppresses lung cancer growth by targeting Aurora B.

Xiang Li,1 Haitao Li,1 Shengqing Li,1 Feng Zhu,1 Dong Joon Kim,1 Hua Xie,1 Yan Li,1 Janos Nadas,1 Naomi Oi,1 Tatyana Zykova,1 Dong Hoon Yu,1 Mee-Hyun Lee,1 Myoung Ok Kim,1 Lei Wang,1 Wei Ya Ma,1 Ronald A. Lubet,2 Ann M. Bode,1 Ziming Dong,3 Zigang Dong1. 1 _University of Minnesota, Austin, MN;_ 2 _National Cancer Institute, Bethesda, MD;_ 3 _Zhengzhou University, Zhengzhou, China_.

Ceftriaxone, an FDA-approved third-generation cephalosporin antibiotic, has antimicrobial activity against both gram-positive and gram-negative organisms. Generally, ceftriaxone is used for a variety of infections such as community-acquired pneumonia, meningitis and gonorrhea. Its primary molecular targets are the penicillin-binding proteins. However, other activities of ceftriaxone remain unknown. Herein, we report for the first time that ceftriaxone has antitumor activity in vitro and in vivo. Kinase profiling results predicted that Aurora B might be a potential 'off' target of ceftriaxone. Pull-down assay data confirmed that ceftriaxone could bind with Aurora B in vitro and in A549 cells. Furthermore, ceftriaxone (500 µM) suppressed anchorage-independent cell growth by targeting Aurora B in A549, H520 and H1650 lung cancer cells. Importantly, in vivo xenograft animal model results showed that ceftriaxone effectively suppressed A549 and H520 lung tumor growth by inhibiting Aurora B. These data suggest the anticancer efficacy of ceftriaxone for the treatment of lung cancers through its inhibition of Aurora B.

#8

**Anandamide reduces cell migration, invasion, and induces apoptosis in an** in vitro **model of prostate cancer.**

Domenica Roberto,1 Laurence Klotz,2 Vasundara Venkateswaran1. 1 _Sunnybrook Research Institute, Toronto, Ontario, Canada;_ 2 _Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada_.

Introduction and Objective: Prostate cancer (PCa) is the most commonly diagnosed cancer in men and the second leading cause of cancer related death in Canada. A large body of evidence supports a possible role for cannabinoids in certain aspects of human health and disease, acting as palliative agents as well as inhibitors of cancer cell proliferation, migration, invasion, and the angiogenesis of tumours. Anandamide (AEA) is a highly potent endogenously produced compound that acts as an agonist of the cannabinoid receptors (CB1 and CB2) as well as the transient receptor potential vanilloid receptor 1. AEA is mainly metabolized by fatty acid amide hydrolase (FAAH) and overexpression of the enzyme in PCa tissues is correlated with greater invasivity, malignancy and increasing Gleason score. Majority of studies have focused on the effect of AEA treatment on cell proliferation, without much focus on prostate cancer biology. This study aims to determine the anti-cancer effect of AEA on PCa cells. We hypothesize that anandamide will reduce migration, invasion, and induce apoptosis in PCa cells.

Methodology: Human PCa cells (DU145, LNCaP, and PC3) were treated with AEA at concentrations ranging from 1μM to 40μM and growth of cells (using the MTS assay) was assessed at various times. Wound-healing assays were conducted to investigate the migratory potential of cells following 24hr exposure to treatment, and trans-well invasion assays were performed to explore the influence of AEA on cell invasion. Flow cytometry, using the FITC Annexin V dead cell apoptosis kit, was used to detect the proportion of apoptotic versus proliferating cells following anandamide treatment.

Results: There was a significant reduction in the proliferation of DU145 cells at concentrations of 20μM and 40μM AEA compared to controls (p=0.02, 0.005). LNCaP cells on the other hand required a higher concentration of AEA to achieve this effect (p=0.05). Cell migration and invasion studies on DU145 cells revealed a significant reduction in cell motility at 20μM and 40μM AEA (p=0.006, 0.0005) as well as a significant reduction in cell invasion at 20μM and 40μM ((p=0.0002, 0.0001). Flow cytometry results indicate a significant increase in the proportion of apoptotic cells compared to control (3 fold change) when treated with 20μM and 40μM in DU145 and LNCaP cells.

Conclusion: We have shown that AEA has greatest significant influence on cell migration, invasion, and apoptosis. Further analysis on the role of AEA in these pathways is underway exploring alterations in expression levels of key proteins implicated in cell migration and apoptosis.

Funding/ Conflicts of Interest: None

#9

Suppression of the invasion and growth of human head and neck squamous cell carcinomas via regulating STAT3 signaling and miR-21/β-catenin axis with HJC0152.

Yu Wang,1 Sinan Wang,2 Yansheng Wu,1 Jia Zhou,3 Qiang Shen,4 Xuan Zhou1. 1 _Tianjin Medical University Cancer Hospital, Tianjin, China;_ 2 _Tianjin Medical University General Hospital, Tianjin, China;_ 3 _University of Texas Medical Branch, Galveston, TX;_ 4 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Signal transducer and activator of transcription 3 (STAT3) is involved in the tumor growth and metastasis of human head and neck squamous cell carcinoma (HNSCC) and is therefore a target with therapeutic potential. In this study, we show that HJC0152, a recently developed anticancer agent and a STAT3 signaling inhibitor, exhibits promising antitumor effects against HNSCC both in vitro and in vivo via inactivating STAT3 and downstream microRNA-21/β-catenin axis. HJC0152 treatment efficiently suppressed HNSCC cell proliferation, arrested the cell cycle at the G0/G1 phase, induced apoptosis, and reduced cell invasion in both SCC25 and CAL27 cell lines. Moreover, HJC0152 inhibited nuclear translocation of phosphorylated STAT3 at Tyr705 and decreased VHL/β-catenin signaling activity via regulation of microRNA-21. Loss-of-function of VHL remarkably compromised the antitumor effect of HJC0152 in both cell lines. In our SCC25-derived orthotopic mouse models, HJC0152 treatment significantly abrogated STAT3/β-catenin expression in vivo, which leading to a global decrease of tumor growth and invasion. With its favorable aqueous solubility and oral bioavailability, HJC0152 holds the potential to be translated into the clinic as a promising therapeutic strategy for patients with HNSCC.

#10

A small molecule derivative effectively inhibits proliferation of pancreatic cancer cells by targeting Sp1 and survivin.

Myrna Hurtado,1 Umesh Sankpal,1 Aboubacar Kaba,2 Shahela Mahammad,1 Alvin Holder,3 Jamboor Vishwanatha,1 Riyaz Basha1. 1 _University of North Texas Health Science Center, Fort Worth, TX;_ 2 _Alabama State University, Montgomery, AL;_ 3 _Old Dominion University, Norfolk, VA_.

Pancreatic cancer is one of the most fatal malignancies due to its poor prognosis. With current standard treatment options, the overall 5 year survival rate is about 16% and these treatment plans often cause high toxicity. Therefore, there is an urgent need for identifying more effective and less toxic agents for the treatment of this malignancy. Tolfenamic acid (TA) is a generic drug used to treat migraine headaches but has been demonstrated to have anti-cancer activity in pre-clinical studies. It is known to downregulate the transcription factor Specificity protein 1 (Sp1). Sp1 regulates several genes involved in cell proliferation and apoptosis, including survivin, an inhibitor of apoptosis protein. Interestingly, a recent discovery proposed that copper(II) complex of TA can result in higher therapeutic response; however its efficacy was not tested in gastro-intestinal cancers. In this study, we assessed the therapeutic efficacy of a Cu(II)- containing complex of TA (Cu-TA) using human pancreatic cancer cell lines. MIA PaCa-2 and Panc-1 cells were treated with increasing concentrations of vehicle (DMSO), equimolar CuCl2 (negative control), TA or Cu-TA and the cell viability was measured at 24 and 48 h post-treatment using CellTiter-Glo kit. CuTA was further tested for its effect on Sp1 and survivin expression in MIA PACa-2 cells by Western blot (protein) and quantitative PCR (mRNA). The activation of apoptosis was determined by measuring the activity of effector caspases using the Caspase3/7-Glo kit and the apoptotic cell population through flow cytometric analysis using Annexin-V staining. Cell cycle arrest was assessed by flow cytometry with propidium iodide staining. While both TA and Cu-TA caused dose/time-dependent response to inhibit pancreatic cancer cell growth, Cu-TA showed higher efficacy when compared to TA. Cu-TA was highly effective in inhibiting Sp1 and survivin protein expression and showed similar trend for inducing apoptotic markers and causing cell cycle arrest in early phase (G0/G1). The results of qPCR demonstrated that the expression of survivin mRNA was significantly lower following both Cu-TA and TA treatment; however, the mRNA expression of Sp1 remained unchanged. This indicates that TA and Cu-TA could be working in similar mechanism by effecting Sp1 post-translationally, perhaps through proteasome-dependent degradation. These results demonstrate that Cu-TA is more effective than TA and potentially useful for pancreatic cancer treatment after clinical testing. Studies to precisely understand the underlying mechanisms of Cu-TA are currently under investigation through molecular profiling analysis.

#11

The oncolytic peptide LTX-315 enhances T cell clonality and induces synergy with chemotherapy.

Ketil André Camilio,1 Meng Yu Wang,2 Janne Nestvold,1 Gunhild Mælandsmo,2 Baldur Sveinbjørnsson,1 Øystein Rekdal1. 1 _Lytix Biopharma AS, Oslo, Norway;_ 2 _Oslo University Hospital, Oslo, Norway_.

LTX-315, a novel oncolytic peptide is effective against both drug-resistant and drug-sensitive cancer cells with lower toxicity towards normal cells. Intratumoral treatment with LTX-315 results in growth inhibition, complete regression and long lasting tumor-specific immune responses.

The oncolytic effect of LTX-315 involves perturbation of the plasma membrane and distortion of intracellular organelles including the mitochondria with subsequent release of Damage-Associated Molecular Pattern molecules (DAMPs) such as ATP, cytochrome c and HMGB1. LTX-315 effectively induces necrosis within the tumor followed by the release of tumor antigens as demonstrated by a greater increase in tumor infiltrating CD8+ T cells, expansion of T cell clonality, and number of clones within the tumor microenvironment.

LTX-315`s ability to modify the tumor microenvironment makes it ideal as a combination partner for other cancer therapies, including chemotherapy and immune checkpoint inhibitors.

In preclinical tumor models, combination of LTX-315 and chemotherapeutic agents such as cyclophosphamide and doxorubicin demonstrates significant synergy.

#12

Evaluation of metformin and clotam combination for medulloblastoma.

Umesh T. Sankpal,1 W Paul Bowman,1 Jeffrey C. Murray,2 Riyaz Basha1. 1 _Univ. of North Texas Health Science Ctr., Fort Worth, TX;_ 2 _Cook Children's Medical Center, Fort Worth, TX_.

Medulloblastoma (MB) is the most common malignant brain tumor in children. Standard treatment, which includes surgery, chemotherapy, and radiation, is successful for most patients, but survivors often suffer from significant long-term side effects affecting their neurocognitive and growth potential. Therefore, there is a critical need to understand the molecular processes that regulate MB growth and find less toxic therapies. Survivin (BIRC5) was identified as a protein belonging to the Inhibitor of Apoptosis Protein family that inhibits apoptosis by inhibiting caspase activation. Survivin expression is also associated with increased resistance of tumor cells to radiation and chemotherapy. In MB, several studies suggest that increased expression of survivin serves as a marker of tumor morphology and is associated with poor prognosis. Specificity protein 1 (Sp1) is one of the transcription factors that regulates the expression of survivin gene. Sp1 is also shown to be overexpressed in some cancer cells and is associated with poor prognosis. In this study, we are testing a strategy of targeting survivin in MB using anti-diabetic drug Metformin, which targets Sp1, and the NSAID Clotam (Tolfenamic acid, TA), which targets Sp1 and survivin. Dose response curves were established by treating MB cell lines DAOY and D283 with increasing concentrations of metformin or TA and measuring cell viability at 24 and 48h post-treatment using the CellTiter-Glo Cell Viability Assay. A time and dose dependent inhibition of cell proliferation was observed for both the drugs. Metformin dose response was then repeated in the presence of 25 or 50 µM TA. Our results indicate that TA significantly increased the growth inhibitory response of metformin. To further characterize this response, we determined the combination index (CI) using the Chou-Talalay method. We found that the CI values were between 0.85 to 0.61 for various combinations of metformin and TA, strongly suggesting a synergistic effect of the two drugs on cell proliferation. Combination of metformin and TA was also accompanied by a 3-4 fold increase in apoptotic cells at 48h post-treatment, as determined by AnnexinV staining of DAOY cells. Western blot analysis of DAOY cells treated with the two drugs also revealed a decrease in survivin and Bcl-2 protein levels, and an increase in cleaved-PARP. In conclusion, the anticancer activity of metformin in MB cells is enhanced in the presence of TA. Both drugs synergistically combine to inhibit MB cell proliferation and increase apoptosis, which in part may be the result of a decrease in survivin levels. This combination strategy may therefore represent a novel targeted therapy for MB. Studies are underway to determine whether this drug combination can enhance the response of chemotherapy in MB cells, since both TA and metformin are known to sensitize cancer cells to chemotherapy.

#13

Chromatin unfolding small molecules as a novel type of anticancer agents.

Katerina V. Gurova. _Roswell Park Cancer Institute, Buffalo, NY_.

Although targeted therapy was the major focus of anti-cancer drug development, recently revealed enormous heterogeneity of tumors planted a seed of doubt in it as a potential cure for cancer. Very few universal cancer targets are known and they lack cancer specificity. DNA is one of this sort, but activity of drugs targeting DNA was traditionally attributed to their ability to block DNA replication and cause DNA damage with a plethora of harmful long lasting effects in normal cells. We have found that reversible binding of DNA by non-DNA damaging small molecules, curaxins, has strong anticancer effect in different preclinical models without deleterious consequences associated with DNA damage (REF). In this study we explained the mechanism of activity of curaxin clinical lead, CBL0137, through alteration of chromatin organization in tumor cells. Intercalation of the curaxin carbazole moiety between base pairs and DNA major and minor groove binding with curaxin side chains cause an increase in inter base pair distance and untwisting of the double helix (~18o). Such alteration of DNA helical shape leads to decreased nucleosome stability and chromatin opening, creating sites for the high affinity binding of histone chaperone complex FACT. We named this phenomenon "chromatin trapping of FACT" or c-trapping. Consequences of C-trapping are much more deleterious for tumor, than for normal cells. First, c-trapping is an equivalent of functional inactivation of FACT. FACT is critical for oncogenic transformation and tumor, but not normal cell, viability and growth (REF). FACT is essential for the activity of several transcription factors, such as NF-kappaB, HSF1, MYC etc., with important roles in tumor cell proliferation and survival (REF). Further, deregulation of transcription caused by curaxin-dependent chromatin decondensation leads to upregulation of transcription from heterochromatin. This leads to the formation of double stranded RNAs from centromeric and pericentromeric repetitive elements, which mimics a viral infection and induces an IFN response, a powerful inhibitor of tumor cell growth. Additionally, c-trapping of FACT leads to casein kinase 2 mediated phosphorylation and activation of wild type p53 leading to p53-dependent death of cells. Thus non-DNA damaging DNA binding small molecules compromise cancer cell viability via alteration of nucleosome stability and chromatin organization. This is a novel mechanism of anti-cancer activity of small molecules with broad applicability to different types of cancer while lacking the harmful effects of DNA damaging chemotherapy.

#14

**Anticancer activities of terpenoid compounds isolated from the plant** centipeda minma **against nasopharyngeal carcinoma cells.**

Bei-bei Liu,1 Kui-yuan Zhu,1 Chi-on Chan,1 Ling-Hua Zhang,2 Daniel Kam-Wah Mok,1 Si-bao Chen1. 1 _State Key Laboratory of Chinese Medicine and Molecular Pharmacology, The Hong Kong Polytechnic University Shenzhen Research Institute, Shenzhen, China;_ 2 _NANOBIOTEC LLC, Whippany, NJ_.

Nasopharyngeal carcinoma (NPC) is one of common cancers throughout south-east Asia, southern China and other regions. Radiotherapy and chemotherapy are the standard treatments for NPC with severe adverse effects, multi-drug resistance, and insensitivity to advanced NPC. Complementary and novel treatments for NPC gain considerable interest around the world. The medicinal plant Centipeda minma is traditionally used in China for the treatment of various nasal diseases such as allergy, rhinitis, sinusitis and nasopharyngeal carcinoma. In the current study, we studied the anticancer potential of an ethanol extract (CME) and 3 pure sesquiterpenoids (A1, A2, A3) extracts from Centipeda minma against NPC cells in vitro and some underlying mechanisms of actions. The NPC cell lines CNE-1 and CNE-2 cells were treated with either CME or 3 pure compounds (A1, A2, A3). Proliferation of NPC cells was inhibited in a concentration- and time-dependent manner. For 48 h treatment, the IC50 values were 41.57 to 24.98 μg/mL for CME, and 2.3 to 7.5 μM for A1 to A3. While these extracts showed no appreciable effect on normal human LO2 hepatocytes, significant numbers of NPC cells underwent apoptotic morphological changes following treatments. Cytometric analysis demonstrated that treatments with CME and compounds results in NPC cell cycle arrest at the G2/M phase and eventually apoptosis in a concentration and time-dependent manner. Western blot analysis showed that these extracts could down-regulate Bcl-2 expression, up-regulate Bax expression and caspase-8, 9, 3 activities. In addition, the potential anti-NPC actions might be also associated with the marked inhibition of multiple signal transduction pathways including EGFR, VEGFR, HER2, FGFR, IGF-1R, PI3K/AKT/mTOR, MAP/ERK and Jan/Stat3 pathways. In summary, the Centipeda minma extract and terpenoids are potential apoptosis inducing agents to kill NPC cells and may be promising anti-cancer drug leads against nasopharyngeal carcinoma. The exact mechanism of actions remains further study.

#15

Suppression of adaptive responses to targeted therapies by transcriptional inhibition.

Maria Rusan,1 Kapsok Li,1 Yvonne Li,1 Camilla L. Christensen,1 Brian J. Abraham,2 Nicholas Kwiatkowski,1 Kevin Buczkowski,1 Bruno Bockorny,1 Ting Chen,1 Shuai Li,1 Haikuo Zhang,1 Hideko Terai,1 Tiffany Tavares,1 Tinghu Zhang,1 Tae Jung Kim,1 Michael Silkes,1 Tenny Mudianto,1 Li Tan,1 Takeshi Shimamura,3 Matthew Meyerson,1 Hideo Watanabe,4 Nathanael S. Gray,1 Richard A. Young,2 Kwok-Kin Wong,1 Peter S. Hammerman1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _MIT Whitehead Inst. for Biomed. Resea, Cambridge, MA;_ 3 _Loyola University Chicago, Maywood, IL;_ 4 _Icahn School of Medicine at Mount Sinai, New York, NY_.

Tumor cells become dependent on the expression of key genes that drive hallmark tumor traits, but these "addictions" also represent potential vulnerabilities for therapeutic intervention. Treating tumor cells with a small molecule inhibitor of the transcriptional kinase CDK7 selectively suppresses expression of key dependency genes in multiple cancers. Our previous work showed responses to the CDK7 inhibitor THZ1 were especially noteworthy in T cell leukemias, small cell lung cancers, and triple-negative breast cancers—tumor types where the prognoses are especially unfavorable. The genes whose expression is most sensitive to CDK7 inhibition and whose expression is essential for tumor cell survival are often associated with super-enhancers—clusters of constituent enhancers that are bound by unusually large amounts of transcription apparatus including CDK7 itself. This "Achilles' cluster" of super-enhancer-associated genes thus represents the set of addictions of tumor cells whose expression is important for tumor cell survival. Transcriptional inhibition synergizes with targeted therapies in models of multiple tumor types. Treating tumors with therapies that target kinase proteins such as EGFR, FGFR, and KRAS leads to resistance, which is concurrent with changes in the activities of enhancers and super-enhancers and gene expression programs of resistant cells. Treating cells with a transcriptional inhibitor in addition to targeted therapies prevents "rewiring" of the gene expression program and increases cell death in tumor models. Thus transcriptional inhibition represents a promising avenue in both monotherapy and combination settings where drug resistance is acquired.

#16

Evaluation of the efficacy of clotrimazole treatment in human melanoma cell lines that overexpress the multidrug resistance-associated protein (MRP) and the lung resistance-related protein (LRP).

Steven D. Blake, Shelby G. McKamey, Christopher M. Tweed, David W. Koh. _Ohio Northern University, Ada, OH_.

Drug resistance to chemotherapy, mediated in part by overexpression of P-glycoprotein (MDR-1), multidrug resistance-associated protein (MRP), or lung resistance-realted protein (LRP), continues to be a major problem in the treatment of melanoma. Because we previously demonstrated the ability of clotrimazole to enhance cytotoxicity in human metastatic melanoma cells, here we evaluated the ability of clotrimazole to treat drug-resistant human melanoma cells that overexpress MRP and/or LRP. We utilized human melanoma cell lines that overexpress either MDR or LRP, and human melanoma cell lines that overexpress both. Cell growth and cell death were analyzed by flow cytometry, proliferation assays, and immunoblotting after drug treatments and RNA interference. Clotrimazole caused significant decreases in proliferation and increases in cell death in both drug-sensitive melanoma lines and those overexpressing MRP and LRP. No extensive cell death was induced by clotrimazole treatment in noncancerous human skin cell lines. These results indicate that clotrimazole selectively induces cytotoxicity in both drug-sensitive and drug-resistant melanoma lines. As clotrimazole is known to inhibit various transient receptor potential (TRP) ion channels, we pretreated these melanoma lines with TRP inhibitors or RNAi. Inhibitors of the TRP melastatin-8 channel (TRPM8) or TRP vanilloid-1 (TRPV1) failed to produce comparable levels of cell death caused by clotrimazole. However, RNAi silencing of the TRPM2 cation channel caused significant levels of cell death in both drug-sensitive and drug-resistant melanoma lines. These results indicated that inhibition of TRPM2 channels may have a primary role in the ability of clotrimazole to treat drug-sensitive and drug-resistant melanoma lines.

In conclusion, this study demonstrated that clotrimazole selectively increases cell death in drug-resistant melanoma lines, with minimal deleterious effects in normal skin cells. Taken together, we conclude that clotrimazole has in vitro efficacy toward the treatment of drug-resistant human melanoma cells. Thus, these preliminary studies indicate that clotrimazole has the potential to successfully treat drug resistant melanoma in the future.

#17

Anti-metastatic platinum through glycan targeting in breast cancer.

Samantha J. Katner,1 Erica Peterson J. Peterson,1 Eriko Katsuta,2 Stephanie C. DeMasi,1 Jennifer Koblinski,1 Kazuaki Takabe,2 Nicholas P. Farrell1. 1 _Virginia Commonwealth University, Richmond, VA;_ 2 _Roswell Park Cancer Institute, Buffalo, NY_.

The high affinity of polynuclear platinum compounds (PPCs) for heparan sulfate (HS) is mediated through a "sulfate clamp" and PPC interactions can be conceptualized as "polyarginine" mimics. Strong HS-PPC binding protects the oligosaccharide against sulfate loss through metalloshielding.1 Metalloshielding will in principle affect HS interactions with relevant enzymes and proteins such as heparanase and growth factors, similar in concept to inhibition of DNA-protein binding through modification of DNA structure and conformation.2 PPCs inhibit cleavage of a model pentasaccharide by heparanase and further modulate bFGF binding to HS, and bFGF-induced migration and signaling in colon cancer cells.3 The end-point of functional modulation of HS interactions is inhibition of angiogenesis and metastasis.3 Following proof-of-principle of strong in vivo anti-metastatic activity of PPCs in clinically relevant breast cancer models,4 this contribution examines the underlying mechanism of platinums as anti-metastatic agents. We report on the cellular effects of PPC-HS metalloshielding on multiple growth factor signaling pathways (bFGF, HB-EGF) and specific syndecan (Sdc)-dependent signaling in breast cancer. Coupled with previously demonstrated DNA binding, PPCs represent intrinsically dual- function agents combining platinum cytotoxicity with anti-angiogenic effects derived from first principles through glycan targeting.

[1] Mangrum, J.B., Engelmann, B.J., Peterson, E.J., Ryan, J.J., Berners-Price, S.J., Farrell, N.P. Chemical Communications 2014, 50, 4056-4058.

[2] Chiodelli, P., Bugatti, A., Urbinati, C., Rusnati, M. Molecules 2015, 20, 6342-6388.

[3] Peterson, E.J., Daniel, A.G., Katner, S.J., Bohlmann, L., Chang,C., Bezos, A., Parish, C.R., von Itzstein, M., Berners-Price, S.J., Farrell, N.P. Chem. Sci. 2016, DOI: 10.1039/C6SC02515C.

[4] E. Katsuta, S. Demasi, S. Katner, H. Aoki, E. Peterson, E., N.P. Farrell and K. Takabe. (2016). Proc. AACR New Orleans, LA. Abstract #3064.

DC 1-5 April 2017

#18

**Vorinostat reexpressed estrogen receptor (ER) in triple negative breast cancer cell line subtypes and sensitized cells to tamoxifen and indole-3-carbinol** in vitro **.**

Beverly D. Lyn-Cook,1 Julie Getz,1 Beverly Word,1 Rhonda Moore,2 Gustav Miranda-Carboni3. 1 _FDA-NCTR, Jefferson, AR;_ 2 _FDA/Center for Tobacco Product, FDA, MD;_ 3 _University of Tennessee Health Science Center, Memphis, TN_.

Triple negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer. Although about 85% of breast cancers are estrogen positive, about 15% falls into this category.

This subtype of cancer lacks targeted therapies receptors, such as the estrogen receptor (ER), progesterone receptor (PR), and the human epidermal growth factor receptor-2 (HER2).

These patients are limited to cytotoxic chemotherapies with harsh side effects. In addition to occurring in younger women, other risk factors for TNBC include: being of African descent, BRCA1 mutation, a strong family history of breast cancer, lifestyle and environmental factors.

Although mutations are involved in the initiation of TNBC, research has revealed that individuals are controlled by factors other than DNA sequences such as epigenetic mechanisms. Environmental and lifestyle related factors, such as the lack of population-based screening, and lack of access to care-factors, the urban environment, food deserts, social stress, racism, diet, lack of exercise, alcohol intake, and tobacco use (i.e. cigarette smoking) have all been described as factors associated with breast cancer related disparities. This study investigated the role of epigenetic mechanisms in the re-expression of ER receptors in triple negative breast cancer (TNBC) cells by examining the effects of a FDA-approved epigenetic drug (vorinostat) and the dietary agent (indole-3-carbinol) on three subtypes of triple negative breast cancer. Basal-like 2 (HCC1806), mesenchymal stem cell-like (MSL) MDA-MB-231 and mesenchymal (BT-549) cell lines were treated with vorinostat for 6, 12, 24 and 48 hrs alone. The ER was expressed in HCC1806 (3-fold) and MDA-MB-231 (5-fold) at 6 hr. The ER was not expressed in BT-549 cells at any time point. MTS assay demonstrated a significant decreased in proliferation (60%) in MDA-MB-231 when treated with vorinostat (10, 20 or 30 µM) and 10 µM of tamoxifen. Furthermore, a significant decrease (40%) in proliferation was also detected in MDA-MB-231 cells treatmented with I3C (200 µM) and vorinostat (10, 20 or 30 µM). Our preliminary results show that three triple-negative cell lines representing three subtypes responded different to treatment with vorinostat in re-expressing the estrogen receptor. However, these results showed promising result for the use of this drug in sensitizing triple negative breast cancer cells to tamoxifen and a dietary agent, indole-3-carbinol. Further functional studies are currently underway.

### Antibodies, Bispecifics, and Antibody-Drug Conjugates

#20

Xentuzumab, a humanized IGF-1 and IGF-2 ligand neutralizing antibody, improves the antitumor efficacy of enzalutamide in preclinical models of prostate cancer.

Ulrike Weyer-Czernilofsky,1 Marco H. Hofmann,1 Paul J. Adam,1 Flavio Solca,1 Katrin Friedbichler,1 Norbert Kraut,1 Eva Corey,2 Thomas Bogenrieder1. 1 _Boehringer Ingelheim RCV GmbH & Co KG, Wien, Austria; _2 _University of Washington, Seattle, WA_.

Background: The proliferative and pro-survival signals driven by the insulin-like growth factor (IGF) ligands, IGF-1 and IGF-2, are transmitted through their binding to the IGF-1 receptor (IGF-1R). In addition, IGF-2 activates the insulin receptor variant A (IR-A) that is expressed during embryonic development as well as in many cancers. A large body of preclinical evidence suggests that IGF signaling plays a key role in cancer by driving therapy resistance, due to cross-talk with other signaling networks such as androgen receptor signaling. The aim of this study was to explore the potential of the IGF-1/-2 ligand blocking antibody, xentuzumab (BI 836845[1]), to enhance the anti-tumor activity of enzalutamide in prostate cancer cell lines and in a patient-derived prostate cancer xenograft model.

Methods: Effects of enzalutamide, xentuzumab and combinations thereof on in vitro proliferation, survival, cell cycle and signaling were evaluated using the prostate cancer cell lines VCaP, DuCaP, MDA PCa 2b, and LNCaP. The in vivo efficacy of enzalutamide, alone and in combination with xentuzumab was investigated using LuCaP 96CR, a patient-derived xenograft model of castration-resistant prostate cancer. Tumors were implanted s.c. into castrate SCID mice. When tumors exceeded 150mm3 animals were randomized into groups: 1) Control; 2) enzalutamide (50 mg/kg QD po), 3) xentuzumab (BI 836845[1], 200 mg/kg QW ip) in combination with enzalutamide.

Results: Cell viability was more effectively reduced by the combination of enzalutamide and xentuzumab than either drug alone in three of four cell lines expressing the IGF-1R and the androgen receptor (AR). In VCaP cells, prolonged inhibition of IGF pathway signaling and enhanced blockade of proliferation as well as induction of apoptosis was observed after combination treatment. In vivo, enzalutamide monotherapy did not show significant antitumor efficacy in the LuCaP 96CR model, however, combined treatment with xentuzumab significantly inhibited progression of LuCaP 96CR tumor growth (p<0.001 vs. enzalutamide alone). Reduced serum PSA levels were observed after enzalutamide and combination treatment. Enzalutamide plus xentuzumab inhibited tumor growth at tolerated doses and resulted in significant improvements in survival.

Conclusions: These studies demonstrated that addition of the IGF-1/-2 neutralizing antibody xentuzumab to enzalutamide results in improved anti-neoplastic activity in a subset of prostate cancer cell lines in vitro, and to re-sensitization to enzalutamide in a patient-derived xenograft model of CRPC.

Reference:

[1] Friedbichler K et al. (2014). Mol Cancer Ther 13(2):399-409.

#21

Regulation of proliferation and invasion through the inhibition of IGF signaling pathway on Epstein-Barr virus associated gastric cancer.

Inhye Jeong,1 Hoi Young Lee,2 Jae Kyung Roh,3 Tae Soo Kim,3 Suk Kyeong Lee,4 Woo-Ick Jang,5 Hyun Cheol Chung,1 Sun Young Rha6. 1 _Song-dang Institute for Cancer Research, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Republic of Korea;_ 2 _Department of Pharmacology, College of Medicine, Konyang University, Seoul, Republic of Korea;_ 3 _Song-dang Institute for Cancer Research, Yonsei University College of Medicine, Seoul, Republic of Korea;_ 4 _Research Institute of Immunobiology, Department of Medical Lifescience, College of Medicine, Catholic University of Korea, Seoul, Republic of Korea;_ 5 _Handok, Inc., Seoul, Republic of Korea;_ 6 _Song-dang Institute for Cancer Research, Brain Korea 21 PLUS Project for Medical Science, Division of Medical Oncology, Department of Internal Medicine, Yonsei Cancer Center, Yonsei University Health System, Seoul, Republic of Korea_.

Epstein-Barr virus associated gastric cancer (EBVaGC) is one subtype of four molecular classifications by TCGA group. Specifically, EBV related viral RNAs, microRNAs and proteins regulate cancer progression phenomena like aggressiveness, motility, invasion and metastasis. Nonetheless, understanding the mechanisms of viral growth dependency beyond malignant EBV transformation are still deficient. Finding the EBV dependent pathway with significant biological role and the clinically appropriated drug is needed for EBVaGC in this regard. We investigated new therapeutic target and its role of regulating in proliferation and invasion using EBVaGC cell line model. First, we determined proliferation both of AGS and EBV infected AGS (AGS-EBV) cell lines during 7 days by proliferation assay. For the test of BI836845 (IGF ligand neutralizing antibody, Boehringer Ingelheim, German) efficacy, CCK-8 assay was performed. mRNA expression levels of IGF-1R, IGF-1, IGFBP-3 and IGFBP-6 which is the key regulator of IGF-signaling were measured by RT-PCR. Also, protein expression levels of IGF associated and downstream factors were confirmed by Western blotting. Lastly, invasiveness of cells was detected by invasion assay. As a result, AGS-EBV showed the slower proliferation rate compared to AGS, but the sensitivity to BI836845 was significantly increased in a dose-dependent manner (p<0.001). Interestingly, baseline IGFBP-3 mRNA expression of was enhanced in AGS-EBV compared with AGS. In addition, BI836845 treated group showed up-regulated IGFBP-3 mRNA level in AGS and down-regulated IGFBP-3 mRNA level in AGS-EBV. When BI836845 treated to AGS-EBV, phospho-Akt level was highly inhibited. Moreover, invasiveness of AGS-EBV was elevated about 2 fold more than AGS. Treatment of BI836845 in AGS-EBV represented significant decrease the invasiveness (p=0.08). In conclusion, AGS-EBV seems to modulate their proliferation and invasion through the IGF signaling pathway. Inhibition of IGF signaling pathway would be the potential therapeutic strategy for EBV associated gastric cancer.

#22

MM-161, a first-in-class pan-FGFR antibody.

Tamara Dake, Greg Finn, Melissa Geddie, Neeraj Kohli, Maja Razlog, Lihui Xu, Violette Paragas, Haluk Yuzugullu, Sara Ghassemifar, Yasmin Hashambhoy-Ramsay, Charlotte McDonagh, Marco Muda, Birgit Schoeberl. _Merrimack Pharmaceuticals, Inc, Cambridge, MA_.

Aberrant signaling of the FGFR pathway has long been known to promote tumorigenesis and angiogenesis across multiple cancer indications. However, the development of an effective and well-tolerated FGFR targeted inhibitor has been hindered by the need to block the activation of multiple mitogenic receptors while avoiding significant toxicities associated with blocking endocrine FGF ligands.

Here we disclose for the first time a novel FGFR targeted antibody, MM-161, designed to block ligand-dependent signaling driven by all four FGF receptors, specifically the IIIc-isoforms. MM-161 is well tolerated in mice and cynomolgus monkeys with no significant weight loss observed in either species. Efficacy studies demonstrated that MM-161 monotherapy leads to significant tumor growth inhibition or tumor regression of xenografts of human lung, renal and endometrial cancer amongst others. Importantly, MM-161 has a dual mechanism of action by inhibiting both proliferation and angiogenesis. We will present data illustrating that inhibition of multiple FGFRs is desirable to achieve tumor regression. Furthermore, we will show combination studies with relevant standard of care therapies in models of lung and renal cancer.

Taken together, our preclinical data strongly supports the clinical evaluation of MM-161 in cancer patients.

#23

Antibody targeting of ADAM8 for treatment of triple-negative breast cancer.

Sonia G. Das, Stefania Pianetti, Gail E. Sonenshein, Nora D. Mineva. _Tufts Univ. School of Medicine, Boston, MA_.

Triple-Negative Breast Cancer (TNBC) accounts for 25% of breast cancer deaths. Current therapeutic options are restricted to chemotherapy (CT) and radiation, which fail to block disease progression. Recently, we identified the cell surface protein ADAM8 as a critical driver of TNBC tumor growth and of metastasis in half of all breast cancers (EMBO Mol. Med. 6:278, 2014). Elevated ADAM8 levels correlate with poor breast cancer patient outcome. One third of TNBC patient samples have high ADAM8 levels, while it is undetectable in normal breast tissue. ADAM8 Metalloproteinase (MP) and Disintegrin (DI) domains promote angiogenesis needed for tumor growth, and cancer cell adhesion to and invasion through the endothelium needed for dissemination, respectively. Using a prototype reagent in mice, we validated a unique antibody-based strategy that simultaneously targets ADAM8 MP/DI activities as an effective therapeutic intervention in TNBC.

Our group has now prepared a panel of pre-clinical, highly specific anti-human ADAM8 mouse monoclonal antibodies (ADPs) of either IgG1 or IgG2 subclass with dual MP/DI antagonist activity. The top 6 ADPs bind ADAM8 with low dissociation constants of 1.3 x 10-9 M to 7.23 x 10-8 M. The 4 most effective inhibitory ADPs in cell-based assays were screened in a single-dose efficacy study (10 mg/kg 3x/wk) against pre-existing MDA-MB-231 cell-derived tumors (50-75 mm3). ADP2 and ADP13 treatment resulted in significant tumor volume reduction, of 47% and 52%, respectively. Dose-response curves are in progress. ADP13 was also tested in a neo-adjuvant model for its ability to reduce metastases. Antibody treatment was initiated as above and continued for 6 weeks after tumors were resected at a volume of ~200 mm3. Metastases in dissected organs were examined using biophotonic imaging. ADP13 substantially decreased metastasis to multiple organs. Lastly, a neo-adjuvant survival experiment was performed, as above, except that following surgical resection of tumors mice were treated for 12 weeks. Recurrence at the primary site was assessed using palpation. Mice were sacrificed when recurrent tumors reached 900 mm3. ADP13 treatment resulted in significantly improved disease-free survival (P=0.05) and overall survival (P=0.03). More recently, we noted that ADAM8 regulates miRNAs associated with resistance to CT and showed that ADAM8 inhibition sensitizes TNBC cells to Cisplatin, Paclitaxel, and Doxorubicin. In vivo testing of a combinatorial anti-ADAM8 + CT approach is in progress and may present a promising strategy for early entry into the clinic.

Conclusions: A new anti-ADAM8 antibody therapeutic shows efficacy against TNBC tumor growth and dissemination, and improves survival in pre-clinical testing. We hypothesize that addition of an anti-ADAM8 antibody to current CT standards of care will enhance tumor killing, reduce metastasis, and increase survival of patients with ADAM8-expressing TNBC.

#24

HMBD001, a novel anti-ErbB3 antibody with a unique mechanism of action, effectively inhibits tumor growth in pre-clinical models of ErbB3+ solid tumors.

Jerome D. Boyd-Kirkup, Dipti Thakkar, Piers J. Ingram. _Hummingbird Bioscience, Singapore_.

Gastric cancer is the second highest cause of cancer mortality in Asia. Over-expression of ErbB3, a cell surface RTK, occurs in ~60% of mGC patients where it is significantly associated with poorer survival. Ligand binding to ErbB3 triggers heterodimerization and downstream signaling through Akt. Neutralizing antibodies that inhibit ligand binding show poor efficacy in clinical trials, likely due to high levels of ligand independent activation in many tumor types. Directly inhibiting heterodimerization could be more effective, however, current methods of antibody isolation offer limited control over the site of antibody binding and, therefore, mechanism of action, requiring extensive screening of antibodies and posing significant challenges to identifying suitable candidates for therapeutic development.

Hummingbird Bioscience has used its proprietary Rational Antibody Development Platform for the design and development of novel therapeutic antibodies against ErbB3. Computational sequence and structural analyses predicted highly specific surface epitopes, with strong antigenicity and good safety profiles, where antibody binding would inhibit dimerization. Subsequently, Hummingbird's directed evolution mouse immunization strategy efficiently isolated monoclonal antibodies that bound with low nM (Kd < 5nM) affinity to these epitopes on native folded ErbB3. Furthermore, these mAbs showed ELISA cross-reactivity to ErbB3 in model organisms (mouse, rat, monkey) but no binding to other EGFR family proteins.

Antibody HMBD001 was found to strongly inhibit the phosphorylation of ErbB3 and decrease downstream signaling through the Akt pathway (90% decrease in p-ErbB3 and 60% decrease in p-AKT observed at 24 hrs) resulting in the inhibition of tumor cell proliferation in models of ErbB3+ gastric cancer (over 90% inhibition of NCI-N87 proliferation after 5 days) and breast cancer (up to 85% inhibition of BT474 proliferation after 5 days). In mouse NCI-N87 gastric cancer CDX models, weekly doses of HMBD001 achieved greater than 90% inhibition of tumor growth (at 25 days) with no observable adverse toxicity.

The first in-human trial of HMBD001 is planned for 2018.

#25

Efect of nimotuzumab on malignant pleural mesothelioma cell lines.

Saé Muñiz-Hernández,1 Vanessa Izquierdo-Sánchez,1 Jorge A. Mendoza-Desión,1 Carolina González-Torres,2 Oscar Arrieta1. 1 _Instituto Nacional de Cancerología de México, Ciudad de México, Mexico;_ 2 _Instituto Nacional de Medicina Genómica, Ciudad de México, Mexico_.

Malignant Pleural Mesothelioma (MPM) is one of the most clinically aggressive malignancies, with a median survival time of less than 12 months. The monotherapy or combination of therapy with platinum-antifolate chemotherapy agents, may provide survival and life quality improvement in MPM patients. EGFR has been a target for new development drugs that could be used as oncologyc treatment. Nimotuzumab is a humanized antibody, design by Centro de Inmunología Molecular (Habana, Cuba), for treatment EGFR overexpress neoplasms. This antibody was approved for treatment of head and neck cancer and metastasic colorectal cancer. MPM exhibit an important expression of EGFR. In this study, we analyzed the efect of nimotuzumab on two cell lines derived of MPM and a xenograph model of one of them, in atimic nude mouse model. MSTO-211 and H2452 mesothelioma cell lines; MRC-5 normal lung cell line and A431 as EGFR positive cell line were used for all assays. We analyzed the viability of cell lines using violet cristal assays. Apoptosis were analyzed with Anexin-V-Fluos Staining kit accordin to manufacturer instructions. We analyzed the EGFR expression by WB assays. Finally, we generated a xenograft model with MSTO cells in nu/nu mice, and we treated a group with nimotuzumab or cisplatin-gemcitabine one doses/week. Nimotuzumab at any concentration (50, 100, 400 μg/mL) no modified the cellular viability, in both tumor and normal cells, the assays was followed from 24 h to 96 h after exposition. With respect at apoptosis, we no found significant diferences between control and exposured samples at 48 and 72 h after nimotuzumab treatment. In order to analyzed the EGFR expression in mesothelioma cell lines, we obtained protein total extract and then a WB was realized. The total protein from MSTO-211, H2452, A431 and MRC-5 cultures in normal conditions and exposed at nimotuzumab 100 μg/mL by 24h, were obtained; then a PAGE-SDS were realized. We observed that both mesotelioma derived cell lines showed similar EGFR expresion when it compared with A431 in normal culture conditions; while MRC-5 no showed EGFR expresion. When cellular cultures were exposed to nimotuzumab 100 μg/mL by 24h, H2452 and A431 cells showed similar EGFR expression while MSTO-211 showed high expression. Mice nu/nu (n=3) were subcutaneously inoculated in the dorsal right flank with MSTO-211 cells. Treatment was applied to animals when tumors were 50 mm3. First mice group received nimotuzumab, second group received cisplatin-gemcitabine; and third group no received treatmed. Nimotuzumab induced a reduction in tumor volumen and mice survival by 4 weeks more that those in the other groups. In vitro exposition of nimotuzumab in mesotelioma cell lines no affect their viability or induce apoptosis. According with the treatment scheme used in this work, nimotuzumab treatment induce a major survival and low toxicity in mice with mesothelioma pleural malignant xenograph.

#26

Panitumumab interacts with TAS-102 leading to combinational anti-cancer effects by blocking EGFR-mediated tumor response to trifluridine.

Kazuhide Nakamura,1 Yuji Baba,1 Toshiya Tamura,1 Yoshihiko Satoh,1 Masamitsu Gotou,1 Hiroshi Sawada,1 Shunsuke Ebara,1 Kazunori Shibuya,2 Jumpei Soeda2. 1 _Takeda Pharmaceutical Company Limited, Fujisawa, Japan;_ 2 _Takeda Pharmaceutical Company Limited, Tokyo, Japan_.

Panitumumab is a monoclonal antibody raised against the human epidermal growth factor receptor (EGFR). TAS-102 is a novel chemotherapeutic agent containing trifluridine (FTD) as the active cytotoxic component. Both panitumumab and TAS-102 have been approved for the treatment of metastatic colorectal cancer (mCRC). In this study, we show the mechanism underlying the anti-cancer effects of the panitumumab/TAS-102 combination in preclinical models. Co-treatment with panitumumab and FTD exerted additive and synergistic anti-proliferative effects in LIM1215 and SW48 colon cancer cells, respectively. Consistent with the in vitro effects, panitumumab/TAS-102 combination led to tumor regression in LIM1215 and COL-01-JCK colon cancer patient-derived xenograft models. In LIM1215 cells, FTD induced ERK/Akt/STAT3 phosphorylation and subsequent serine/threonine phosphorylation of EGFR, while it had no effects on EGFR tyrosine phosphorylation. Panitumumab and the tyrosine kinase inhibitor erlotinib reduced the basal level of EGFR tyrosine phosphorylation and reversed the FTD-induced ERK/Akt/STAT3 and EGFR serine/threonine phosphorylation. These results suggested that FTD together with the basal activity of the EGFR tyrosine kinase induced downstream pro-survival signaling through ERK/Akt/STAT3. Collectively, we propose that panitumumab interacts with FTD by targeting EGFR-mediated adaptive responses, thereby exerting anti-cancer effects in combination with TAS-102. These preclinical findings provide a compelling rationale to evaluate anti-EGFR antibodies combined with TAS-102 against mCRC.

#27

Combination of paclitaxel and Sym015, a mixture of two monoclonal antibodies directed at MET receptor, to increase anti-tumor effects in gastric cancer.

Hyun Jeong Kim,1 Sun Kyoung Kang,2 Ivan Horak,3 Michael Kragh,3 Woo Sun Kwon,2 Tae Soo Kim,2 Inhye Jeong,1 Sun Young Rha,4 Hyun Cheol Chung4. 1 _Song-Dang Institute for Cancer Research, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Republic of Korea;_ 2 _Song-Dang Institute for Cancer Research, Yonsei University College of Medicine, Seoul, Republic of Korea;_ 3 _Symphogen A/S, Ballerup, Denmark;_ 4 _Song-Dang Institute for Cancer Research, Brain Korea 21 PLUS Project for Medical Science, Division of Medical Oncology, Department of Internal Medicine, Yonsei Cancer Center, Yonsei University Health System, Seoul, Republic of Korea_.

MET is a proto-oncogene that encodes a protein known as hepatocyte growth factor receptor (HGFR), involved in proliferative, survival and invasive/metastatic abilities of cancer cells. MET has received considerable attention as a potential target for cancer therapy, including gastric cancer (GC). MET amplification is present in 4-5% of GC patients, and associated with poor outcomes and significantly shorter median survival. Although intensive efforts have been directed toward the development of HGF-MET axis inhibitors, there are some issues for clinical translation; 1) proper biomarker to select patients, 2) proper chemotherapy combination partner and 3) proper line of treatment. Therefore, exploring drug to inhibit HGF-MET axis is essential. Among them, Sym015 (Copenhagen, Demark), a mixture of two monoclonal antibodies directed at MET receptor, is one of the leading agents in the pipeline. In this study, we had 49 GC cell lines including 27 Korean cancer patients. Those cells were analyzed by whole exome sequencing (WES) and RNA sequencing to understand biological and molecular characteristics. Also, expression levels of MET and MET-associated molecules were determined by western blot and HGF expression was evaluated by ELISA. Sensitivity of Sym015 was screened in 49 GC cell lines and combination with paclitaxel was performed in 6 MET amplification cell lines by CCK-8 assay. Combination index (CI) and dose reduction index (DRI) were evaluated by CalcuSyn software. Among 49 GC cell lines, we confirmed 6 cell lines with MET amplification including 2 novel Korean cancer patient cell lines (YCC-31 and YCC-34) and 17 (34.7 %) cell lines were sensitive to Sym015 with 20~68 % inhibition rate at 100 nM. And our data showed that Sym015 was related to MET amplification, c-Met/p-Met overexpression and exon 14 deletion, as we expected. Also, the 6 MET amplified cell lines (SNU5, SNU520, MKN45, YCC-31, YCC-34 and Hs746T) were all sensitive to Sym015 and down-regulate expression of c-Met/p-Met. Using MET amplified GC cell lines, we evaluated the efficacy and potential mechanism of Sym015 in combination with paclitaxel, which is the standard agent for second line treatment of metastatic GC. CI values determined at the ED50 indicated that 3 of 6 cell lines (MKN45, SNU520 and YCC-31) had synergistic effects (CI values < 0.7). When used in combination to treat 4 cell lines (MKN45, SNU520, YCC-31 and Hs-746T), the dose at ED50 of Sym015 could be reduced by 2.2-13.6 fold, based on DRI analysis. Also, the DRI for paclitaxel indicated that dose reductions up to 4.7-7.1 fold (MKN45 and SNU520) could be obtained. Our results indicate that Sym015 was sensitive to MET amplified cell lines and the Sym015 combined with paclitaxel therapy had synergistic effects in MET amplified GC.

#28

**Cell penetrating proteins targeting Mcl-1 induce** in vitro **and** in vivo **on-target cancer cell killing of Mcl-1 dependent cell lines.**

Sabrina Deroo,1 Sophie Thiolloy,2 Johan Desmet,3 Franky Baatz,1 Karen Vandenbroucke,3 Eric Lorent,3 Paula Henderikx,2 Philippe Alard,3 Stefan Loverix,3 Ignace Lasters,3 Yvonne McGrath3. 1 _Complix Luxembourg S.A., Esch-sur-Alzette, Luxembourg;_ 2 _Complix N.V. Headquarters, Diepenbeek, Belgium;_ 3 _Complix N.V., Zwijnaarde (Ghent), Belgium_.

The pro-survival protein Myeloid Cell Leukaemia-1 (Mcl-1) plays an essential role in survival of numerous cancers. MCL-1 gene amplifications occur in a variety of human cancers and overexpression of the Mcl-1 protein is often associated with chemotherapeutic resistance and disease relapse. Complix has developed Cell Penetrating Alphabodies (CPAB), a novel and unique therapeutic class of proteins engineered to efficiently enter cells and inhibit proteins including Mcl-1.

High affinity Alphabodies (ABs) targeting Mcl-1 were engineered by a combination of rational design and phage library screening. In affinity assays, these ABs were shown to bind to Mcl-1 with picomolar affinities whilst binding to Bcl-XL and Bcl-2 was below the detection limit of the assay. In vitro, CPAB uptake was shown to occur rapidly with cytosolic levels reaching up to 1 μM within 2 hours of CPAB exposure. Uptake was associated with cell death of the Mcl-1 dependent multiple myeloma (MM) cell line NCI-H929 (IC50=0.5 µM) and killing was correlated with caspase-3/7 activation. Anti-Mcl-1 CPAB were also shown to disrupt Mcl-1-Bak and Mcl-1-Bim complexes in H929 cells and induced dose-dependent Bak activation. In a panel of MM cell lines, anti-Mcl-1 CPAB induced cell death with a median IC50 of 0.96 μM and cell killing was not restricted to a specific subset of MM cell lines (CCDN1, MAF, MMSET). Gene expression analysis revealed that the anti-Mcl-1 CPAB cell killing potency correlates with MCL-1 gene expression but correlates best with the MCL-1:BCL-2 gene expression ratio. The same gene expression correlation analysis of the Bcl-2 targeting agent Venetoclax revealed an inverse pattern to that achieved with the Mcl-1 specific CPAB.

In vivo, CPAB conferred with an albumin binding domain for extension of half-life, showed a serum half-life in mice of more than 1 hour and associated tumor concentrations of more than 1 µM. Immunohistochemistry and direct detection of fractionated tumor tissue confirmed the intracellular presence of the CPAB in the tumor cells. When given daily IV at 20 mg/kg, anti-Mcl-1 CPAB induced tumor growth inhibition of 50% versus control in two MM xenograft models (H929 and MOLP-8). Tumor growth remained significantly inhibited even two days after the last treatment in the MOLP-8 model and tumor growth inhibition was associated with increased staining of cleaved caspase-3 as compared to vehicle treated tumors.

In summary, anti-Mcl-1 CPAB efficiently kill Mcl-1 dependent cancer cell lines by on-target effects as demonstrated by (1) disruption of Mcl-1-Bak and Mcl-1-Bim complexes, (2) Bak activation and (3) correlation of potency with MCL-1:BCL-2 gene expression ratio. These CPAB induced a robust reduction in tumor growth in mouse models and represent a best-in-class cell penetrating protein therapeutics for tackling intracellular PPI critical to diseases with unmet medical need.

#29

Addition of Galunisertib to DC101 increased angiogenesis inhibition and tumor growth control in hepatocellular carcinoma (HCC).

Annemilai Tijeras-Raballand,1 Christian Hobeika,2 Elise Payen,3 Matthieu Martinet,1 Philippe Bonnin,3 Karim A. Benhadji,4 Clarisse Eveno,2 Marc Pocard,2 Sandrine Faivre,5 Eric Raymond,6 Armand de Gramont1. 1 _AFR Oncology, Paris, France;_ 2 _Inserm U965-Lariboisiere University Hospital, Paris, France;_ 3 _Inserm U965- Lariboisiere University Hospital, Paris, France;_ 4 _Eli Lilly Company, New Jersey, NJ;_ 5 _Beaujon University Hospital, Clichy, France;_ 6 _Saint-Joseph Hospital, Paris, France_.

Introduction: Galunisertib is a selective ATP-mimetic TGF-β receptor (TGFβR)-I inhibitor and DC101 is a rat antagonist antibody to mouse VEGFR-2 and is used to model VEGFR-2 blockade in murine tumor models. Ramucirumab and galunisertib being independently under clinical investigation in HCC patients, our study aimed at exploring the anti-tumoral potency of a combination of galunisertib and DC101 in an in vivo transgenic model of HCC.

Methods: Transgenic mice developing stage-defined HCC were treated for 8 weeks (W) from W8 to W16 with either vehicle, DC101 (40mg/kg, twice weekly, IP), galunisertib (100mg/kg, daily, oral gavage) or DC101 plus galunisertib. Tumor growth was evaluated by ultrasound (liver size) and by the number of macronodules at sacrifice. Angiogenesis was evaluated by doppler (blood flow in the coeliac trunk) and by CD31 staining.

Results: Liver size and the number of liver tumor macronodules were significantly lower in all treatment arms compared to placebo control at both the W12 intermediary sacrifice and W16 final sacrifice; the combination of galunisertib and DC101 showing increased tumor control at W16 (4,43±0,55 mean liver volume (in mm3) in the combination arm vs 5,22±0,86, 5,68±0,78, 7,09±1,54 in the DC101, galunisertib and placebo arms respectively). Angiogenesis assessed by measuring the mean blood flow in the coeliac trunk (TCm), decreased in all treatment arms compared to placebo. At W16, galunisetib potentiated the effect of DC101 with a TCm decrease of 66% compared to 59% and 10% in the DC101 and galunisertib, respectively. These results will be confirmed by the assessment of micronodules number on HPS section for tumor growth and CD31 staining for angiogenesis analysis (number of vessels and the vessel lumen area). The effects of the combination versus monotherapies will be evaluated on the immune landscape.

Conclusion: The combination of galunisertib and DC101 showed promising anti-tumor activities that were associated with decreased angiogenesis.

#30

S100A11 is a prominent therapeutic target in malignant pleural mesothelioma.

Hiroki Sato, Hiromasa Yamamoto, Kei Namba, Hidejiro Torigoe, Takahiro Yoshioka, Kazuhiko Shien, Junichi Soh, Shinichi Toyooka. _Okayama Univ. Hospital, Okayama City, Japan_.

[Background & Purpose] Malignant pleural mesothelioma (MPM) is an aggressive tumor arising from the mesothelial cells that show a serious malignancy in thoracic cavity with a median survival time of 9-12 months. The therapeutic approaches in clinical standard are limited to surgery and chemotherapy for early and advanced stages, respectively. The strategies unfortunately provide only palliation, thus further advanced approach is required. S100A11, a Ca2+-binding small protein with two EF-hands, is frequently upregulated in various human cancer tissues. It has been reported that S100A11 plays an important role in progression of many cancers derived from thyroid, ovarian and lung tissues, but its roles and signature in MPM are poorly understood. In this study, we investigated the impact of S100A11 in MPM cell lines and resected tumors.

[Material & Methods] We explored the expression of S100A11 in 7 MPM cell lines (HP-1, H28, MSTO-211H, H2052, H290, H2452, and YUMC44) and 2 normal mesothelial cell lines (Met-5A and LP-9). We analyzed the effect of S100A11 on cell proliferation, colony formation, migration, invasion and downstream signaling. The efficacy of anti-S100A11 antibody on cell proliferation and downstream signaling was also determined.

[Results] We found that S100A11 protein was consistently upregulated in 7 MPM cell lines at a significant level in comparison to 2 normal mesothelial cells. The same phenomenon was also confirmed in MPM tissue sections by immunohistochemistry, i.e., S100A11 was strongly stained at MPM cells, but not at surrounding normal lung cells. Specific knockdown of S100A11 by small interfering RNA turned the aggressive cells into the attenuated phenotypes for proliferation, invasion and migration. Those were notably observed in both 2 MPM cell lines (H2052 and H2452). Interestingly, we found that MPM cell lines but not normal cells actively secreted the S100A11 protein. To pursue this, we next tried to inhibit the function of the secreted S100A11. Administration of S100A11 neutralizing antibody significantly inhibited the proliferation of 4 MPM cell lines (H2052, H2452, H28, and H290). The antibody had no effect on the proliferation of only one MPM cell line, MSTO-211H, which showed no secretion of S100A11. Taken together, these results suggest that S100A11 secreted from MPM cells can be a prominent target for effective MPM therapy.

[Conclusion] Our results suggest that S100A11 is a possible therapeutic target in MPM.

#31

Preclinical development of a novel biparatopic HER2 antibody with activity in low to high HER2 expressing cancers.

Nina Weisser, Grant Wickman, Rupert Davies, Gerry Rowse. _Zymeworks, Vancouver, British Columbia, Canada_.

HER2-directed therapies have improved clinical outcomes for many patients with HER2 overexpressing breast and gastric cancer. Despite these successes, there remains a need to develop effective HER2-targeted therapies for these and other HER2-expressing tumors, particularly in the setting of recurrent or metastatic disease. One approach is to develop a single multifunctional antibody that has improved capacity and efficiency for binding HER2 compared with available HER2 inhibitors, can elicit ADCC, block ligand induced heterodimerization of HER2 with other HER family receptors, and down-regulate HER2 protein on the cell surface.

ZW25 is a novel humanized, IgG1-like bispecific antibody directed against two distinct epitopes on the HER2 receptor. ZW25 has low nM binding affinity (0.9-16 nM) to recombinant HER2 and to cultured cancer cells with a range of HER2 expression, and shows higher maximal binding (1.4-1.9x) than monospecific HER2 antibodies on all tumor cell lines tested. The unique structure of ZW25 favors crosslinking of multiple HER2 receptors which is thought to promote clustering, and improves HER2 internalization compared to monospecific HER2 antibodies. In vitro studies demonstrated that ZW25 alone caused significant inhibition in growth of human cancer cell lines with a wide range of HER2 expression (5-54% inhibition depending on the cell line), and inhibited ligand-mediated tumor cell proliferation. ZW25 elicited concentration-dependent ADCC at nM potency (0.05-64 nM) with maximal lysis up to 52% on TNBC cell lines expressing HER2 at a 0/1+ level. ZW25 also exhibited synergy and additivity with multiple chemotherapeutic agents including platins, taxanes, microtubule inhibitors, and DNA synthesis inhibitors in various HER2 expressing tumor cell lines.

In vivo studies demonstrated antitumor activity and/or improved survival against xenografts from a range of tumor types. In a HER2 3+ gastric cancer PDX model ZW25 induced significant tumor regressions, exhibiting 307% tumor growth inhibition relative to hIgG control and a 70% complete regression rate (trastuzumab exhibited 111% tumor growth control with no complete regressions). In a HER2 1+ breast cancer PDX model ZW25 significantly improved median survival (58 vs 28 days) and exhibited 141% tumor growth inhibition relative to hIgG control (trastuzumab exhibited 63% tumor growth inhibition, with median survival 39 days). In a low HER2-expressing NSCLC CDX model ZW25 improved median survival compared to hIgG control or cisplatin (> 66 vs 25 or 26 d respectively) and exhibited 159% tumor growth inhibition relative to hIgG control. In a GLP repeat dose toxicology study, ZW25 was well tolerated with no adverse effects at doses up to 150 mg/kg weekly IV for up to 8 weeks. Based on these findings, a first in human clinical trial of ZW25 has been initiated in patients with recurrent and/or metastatic HER2-expressing cancers.

#32

Preclinical evaluation of MCLA-158: A bispecific antibody targeting LGR5 and EGFR using patient-derived colon carcinoma organoids.

Rob Roovers,1 Bram Herpers,2 Mark James,3 Berina Eppink,4 Carme Cortina,3 David Maussang-Detaille,4 Ingrid Kolfschoten,4 Sylvia Boy,5 Marc van de Wetering,5 Wim De Lau,6 Robert Doornbos,4 Kuan Yan,2 Lucia Salinaro,2 Lex Bakker,4 john de Kruif,4 Hans Clevers,6 Robert Vries,5 Eduard Batlle,3 Leo Price,2 Mark Throsby1. 1 _Merus, Utrecht, Netherlands;_ 2 _OcellO BV, Leiden, Netherlands;_ 3 _IRB Barcelona, Barcelona, Spain;_ 4 _Merus NV, Utrecht, Netherlands;_ 5 _HUB, Utrecht, Netherlands;_ 6 _The Hubrecht Institute, Utrecht, Netherlands_.

Background. Colorectal cancer (CRC) is the third most common cancer and remains a large unmet need. Dysregulation of Wnt and receptor tyrosine kinase (RTK) signalling pathways are important oncogenic driving events in CRC. Due to this dysregulation, Wnt target genes are expressed at higher levels in CRC particularly in tumor initiating cells. We previously performed an unbiased screen of bispecific antibodies (bAbs) targeting Wnt and RTK targets that resulted in the selection of MCLA-158.

Methods. A cohort of 32 genetically and transcriptionally annotated patient-derived colorectal cancer and normal colon organoids were used to functionally characterize responses to antibodies based on morphological changes with high content 3D imaging. Binding affinity was measured by surface plasma resonance and cell based assays. The antibody binding epitopes were mapped by shotgun mutagenesis and FACS based screening. Ligand (R-spondin or EGF) blocking activity was measured in vitro by competition for ligand binding or functional inhibition of ligand dependent growth. In vivo activity was evaluated in xenograft models generated from organoids subcutaneously implanted into immunocompromised mice. Safety was evaluated via once weekly intravenous administration of MCLA-158 to cynomolgus monkeys for 4 weeks and monitoring for pathological changes.

Results. MCLA-158, an ADCC enhanced common light chain IgG1 bispecific antibody, binds in domain III of EGFR and in the N-Cap/1st LRR of LGR5, both ligand binding regions, however, only EGF binding was blocked by MCLA-158. MCLA-158 demonstrated inhibitory activity in 74% of tumor organoids independent of KRAS mutational status but was not active on organoids of the cohort harboring both KRAS and PIK3CA mutations. MCLA-158 was significantly more active on organoids derived from tumors than from normal tissue in contrast to cetuximab, which demonstrated equivalent activity on both (range 20-100 fold, n=4). In vivo activity was evaluated against tumor organoids with different KRAS mutation status shown to be sensitive to MCLA-158 in vitro. In all cases, MCLA-158 significantly inhibited the growth of the tumor compared to both control and cetuximab treatment. Inhibitors of both the Wnt and EGFR pathways have shown significant toxicity in humans. An initial evaluation of MCLA-158 toxicity in cynomolgus monkeys did not demonstrate any pathological finding after repeated dosing at 25mg/kg.

Conclusions. MCLA-158 demonstrates superior activity compared to reference antibodies in both in vitro and in vivo tumor organoid based assays regardless of KRAS status and was well tolerated in non-human primates. These preclinical data suggest MCLA-158 could benefit patients with metastatic CRC and warrant clinical evaluation.

#33

The binding mode of the bispecific anti-HER2xHER3 antibody MCLA-128 is responsible for its potent inhibition of HRG-driven tumorigenesis.

David Maussang-Detaille,1 Camilla de Nardis,2 Linda Hendriks,1 Carina Bartelink-Clements,1 Eric Rovers,1 Tristan Gallenne,1 Robert Doornbos,1 Lex Bakker,1 John de Kruif,1 Ton Logtenberg,1 Piet Gros,2 Cecile Geuijen,1 Mark Throsby1. 1 _Merus, Utrecht, Netherlands;_ 2 _Utrecht University, Utrecht, Netherlands_.

Introduction: MCLA-128 is as an ADCC-enhanced IgG1 bispecific antibody that targets the HER2:HER3 dimer and is currently being tested in Phase I/II clinical trials. MCLA-128 demonstrates an in vitro potency superior to other anti-HER2 and anti-HER3 antibodies in cells stimulated with high concentrations of heregulin (HRG) thereby overcoming one of the resistance mechanisms of current HER2 therapies. This study investigates the binding mode of MCLA-128 and proof of concept studies in HRG-driven tumor models. Methods: Alanine scanning shotgun mutagenesis was used to map the epitopes of MCLA-128 to HER2 and HER3. Fab fragments of MCLA-128 were crystallized with the soluble extracellular domains of HER2 and HER3. SAXS analysis on the HER2-HER3-MCLA-128 complex was performed to investigate the binding mode of the bispecific antibody in solution. Ligand-induced dimer specificity was investigated with PathHunter® heterodimerization assays. Bispecific anti-HER2xHER3 antibody and its parental anti-HER3 monoclonal antibody were labelled with 64Cu to compare their biodistribution profiles. The efficacy of MCLA-128 in HRG-driven systems was shown in vitro in MDA-MB-175 cells and in vivo in an orthotopic intracranial patient-derived xenograft (PDX) model originating from a breast cancer brain metastasis Results: The shotgun mutagenesis study identified that the bispecific antibody MCLA-128 binds amino acids T144, R166, R181 in HER2 domain I and R426 in HER3 domain III. Crystallographic studies confirmed the involvement of these critical residues and suggested that MCLA-128 locks the HER3 receptor in its ligand-unbound inactive confirmation. SAXS analysis suggests that the bispecific antibody MCLA-128 forms inter-dimer rather than intra-dimer interactions. In vitro, MCLA-128 specifically blocked HRG-induced signaling of HER2:HER3 but not HER2:HER4 heterodimers. Biodistribution of MCLA-128 in a xenograft model of breast cancer showed that the penetration of MCLA-128 in JIMT-1 HER2-amplified tumors is HER2-dependent despite the high affinity of the HER3 Fab arm for its receptor. MCLA-128 efficiently blocked tumor growth of the HRG-driven HER2 (1+) breast cancer cell line MDA-MB-175 in 3D in vitro. Treatment of orthotopically transplanted HER2-amplified breast cancer brain tumors in mice led to 100% survival with MCLA-128, in contrast to 38% and 0% survival in T-DM1 and vehicle treated mice respectively. Conclusion: MCLA-128 targets HER2-positive tumors via its HER2 arm and locks HER3 in an inactive confirmation. The potent anti-proliferative activity of MCLA-128 in vitro and in vivo supports the clinical development of this bispecific HER2xHER3 antibody in HRG-driven tumors.

#34

A MET x MET bispecific antibody that induces receptor degradation potently inhibits the growth of MET-addicted tumor xenografts.

John DaSilva. _Regeneron Pharmaceuticals, Tarrytown, NY_.

Aberrant regulation of MET receptor tyrosine kinase signaling is associated with development and progression of various human tumors. Elevated MET signaling during tumorigenesis results from MET amplification, mutation or up-regulation of the ligand, HGF. MET amplification is thought to be a key driver of oncogenesis in non-small cell lung cancers (NSCLC) and oesophagogastric malignancies. In addition, mutations resulting in exon 14 deletion of MET have been described as oncogenic drivers in a subset of NSCLC. Both preclinical and recent clinical results indicate that tumors harboring these genetic alterations respond to MET inhibitors, validating MET as a cancer driver.

We have developed a novel bispecific antibody that binds to two distinct epitopes on MET. The METxMET bispecific antibody blocks HGF binding and exhibits very low agonist activity (much weaker than either of its parental antibodies). Furthermore, the METxMET bispecific antibody effectively promotes MET degradation, thereby inhibiting ligand-independent signaling in MET-amplified tumor cells. The METxMET bispecific antibody exhibits strong anti-tumor efficacy in xenograft models harboring MET genetic alterations, either completely inhibiting tumor growth (EBC1 MET-amplified NSCLC) or promoting substantial tumor regression (SNU5 MET-amplified gastric cancer, Hs746T MET-amplified/MET exon 14 skip gastric cancer). Analysis of lysates from SNU5 tumors treated with the METxMET bispecific antibody confirms its ability to effectively promote MET degradation in vivo. Finally, the METxMET bispecific antibody strongly inhibits the growth of a glioblastoma xenograft model (U118) driven by autocrine HGF signaling. In summary, we have generated a novel METxMET bispecific antibody that potently inhibits the growth of MET-dependent tumor xenografts. Our data suggest that antibody-mediated MET degradation may be an effective clinical strategy in tumors harboring MET genetic alterations.

#35

The dual inhibition of Met and EGFR by ME22S, a novel Met/EGFR bispecific monoclonal antibody, suppresses the proliferation and invasion of laryngeal cancer.

Bok-Soon Lee,1 Dae Ho Kim,1 Yeon Soo Kim,2 Jae Won Chang,3 Bon Seok Koo,3 Ho Ryun Won,1 Haeng-Jun Kim,1 Hyun-Young Cha,1 Ji Min Lee,4 Kyung-Ah Kim,4 Chul-Ho Kim1. 1 _Ajou University, Suwon, Republic of Korea;_ 2 _Konyang University Hospital, Daejeon, Republic of Korea;_ 3 _Chungnam National University,, Daejeon, Republic of Korea;_ 4 _Samsung Advanced Institute of Technology (SAIT), Suwon, Republic of Korea_.

Background and purpose: It has been reported that the abnormal activation of receptor tyrosine kinases is associated with the development of many human carcinomas and the high activation of EGFR and Met mediates the tumorigenicity of laryngeal carcinoma. In this study, we have done the therapeutic efficacy of ME22S, a novel EGFR/Met bispecific antibody, in laryngeal carcinoma in vitro and in vivo was thoroughly evaluated.

Methods: The effects of ME22S on cell viability was assessed through MTT assays, then Western blotting and immunocytochemistry were used to determine the expression of EGFR and Met. Also, wound healing and invasion assays were performed to observe the inhibitory effects of ME22S.

Results: We found the ability of ME22S reducing the expression of both EGFR and Met and significantly inhibiting the cell migration, invasion, and proliferation of SNU899 and HN3 in vitro. Also the notably reduced levels of p-Met, p-ERK, and p-AKT were found when the cells were treated with only ME22S alone or with HGF together. Meanwhile, ME22S, interestingly enough, caused caspase-3-dependent apoptotic cell death when HN3 cells were treated with ME22S for 72h, decreased the HGF-induced Slug expression, and also inhibited the tumor growth of HN3 cells in a xenograft model in vivo.

Conclusion: Taken together, our findings suggest that the dual inhibition of EGFR and Met through ME22S largely suppresses the invasion and growth of laryngeal carcinoma both in vitro and in vivo, hence, can be a practical approach as a novel therapeutic strategy for the treatment of laryngeal carcinoma.

#36

**Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates (ADCs) demonstrate tumor specificity** in vitro **and** in vivo **antitumor efficacy.**

Jillian M. Prendergast,1 David A. Eavarone,1 Patricia E. Rao,1 Adam D. Curtis,2 Lindsay S. Shopland,2 Todd A. Hoffert,2 Jenna Stein,1 Jeff Behrens,1 Daniel T. Dransfield1. 1 _Siamab Therapeutics, Inc., Newton, MA;_ 2 _Eastern Maine Medical Center, Brewer, ME_.

Tumor-associated carbohydrate antigens (TACAs) historically have been challenging targets for antibody therapeutics. Sialyl-Tn (STn) is a cancer specific antigen that is expressed on the cell surface of carcinomas including ovarian, colon, prostate, and pancreatic tumors but is rarely present in normal tissue. STn expression has been linked to innate immune suppression, a chemoresistant phenotype, metastasis, and poor prognosis. Previous attempts to target this antigen in the clinic with synthetic glycan vaccines proved safe but lacked efficacy. We have developed highly selective humanized monoclonal antibodies and antibody drug conjugates (ADCs) targeting TACAs, such as STn. Remarkable sequence homology across all anti-STn mAbs was observed in both heavy and light chains, and hot spots for hypermutation were identified. These antibodies were selected using our glycan microarray that enriches for candidates whose binding is protein-independent, highly selective and demonstrates exceptional target affinity.

Lead humanized candidates demonstrated single digit nanomolar EC50s in ELISA/flow cytometric assays, STn selective cell internalization, and STn specific glycan binding on Siamab's proprietary glycan array. STn binding sites in common tumor lines (ovarian, gastric and breast) were determined per cell and subsequent cytotoxicity assays in these lines demonstrated in vitro efficacy. Tumor microarray experiments revealed membranous staining in cancerous tissues of various indications. Binding studies of anti-STn antibodies to primary human cancer samples by flow cytometry demonstrated that both tumor and Myeloid-Derived Suppressor Cells (MDSC, both myeloid and granulocytic) express STn. In an OVCAR3 xenograft model, 30 days after the last anti-STn ADC dose was given, groups treated (Q7Dx4) exhibited mean tumor volumes below the Day 1 pre-treatment mean tumor volumes (155mm3). Flow cytometric analysis of tumors from these mice demonstrated that anti-STn ADC treatment reduces STn expression on the primary tumor in a dose-dependent manner (Q7Dx4 vs. single dose) compared to the isotype-ADC control. Our data demonstrates that high-affinity, STn-selective mAbs show promise as therapies for solid tumors and could also target MDSCs to promote antitumor immune responses.

#37

Novel antibody-drug conjugates targeting ADAM9-expressing solid tumors demonstrate potent preclinical activity.

Stuart W. Hicks,1 Nicholas C. Yoder,1 Deryk Loo,2 Asli Muvaffak,1 Yinghui Zhou,1 Megan E. Fuller,1 Molly A. McShea,1 Marian Themeles,1 Katherine H. Mucciarone,1 Juniper A. Scribner,2 Bhaswati Barat,2 Thomas Sun,2 James Tamura,2 Francine Z. Chen,3 Kerry A. Donahue,1 Tom Chittenden1. 1 _Immunogen, Inc., Waltham, MA;_ 2 _MacroGenics, Inc., South San Francisco, CA;_ 3 _MacroGenics, Inc., Waltham, MA_.

ADAM9, also known as MDC9 or meltrin-γ, is a member of the ADAM (a disintegrin and metalloproteinase) family of proteases, which have been implicated in cytokine and growth factor shedding, and cell migration. Dysregulation of ADAM9 has been implicated in tumor progression and metastasis, as well as pathological neovascularization. ADAM9 overexpression has been shown to correlate with poor prognosis in prostate, renal, and pancreatic cancers. Using an immunization approach in which antibodies were raised to fetal progenitor and stem-like cancer cell lines followed by screening on tumor and normal tissues, we identified ADAM9 as a promising cell surface tumor target. FFPE-IHC expression analysis revealed that ADAM9 is overexpressed in multiple solid tumor indications relative to corresponding normal tissues. The overexpression of ADAM9 in tumors coupled with its restricted expression in normal tissues make ADAM9 an attractive target for antibody-drug conjugate (ADC) therapy.

Here, we describe two ADCs both of which are based on a high affinity anti-ADAM9 antibody to selectively target ADAM9-expressing tumors. The first ADC utilizes the maytansine-derived microtubule disruptor, DM4, linked via a hindered disulfide hydrophilic linker (sulfo-SPDB). The second ADC exploits an ultra-potent DNA alkylating payload, DGN549, which is conjugated to two engineered cysteines via a peptide linker. Both conjugates bound with similar subnanomolar affinity to ADAM9-expressing cells. In vitro cytotoxicity studies showed that anti-ADAM9 ADCs can kill a broad panel of ADAM9-positve tumor cell lines, including lung, pancreatic, renal, prostate, and colon tumor cell lines. In particular, the anti-ADAM9-DGN549 conjugate was extremely potent with IC50 values ranging from 0.1 to 65 pM and was at least 2 logs more active than a non-targeting conjugate. Surprisingly, efficient in vitro cytotoxicity was observed at ADAM9 expression levels as low as a few thousand cell surface receptors per cell.

Consistent with their in vitro activity, both anti-ADAM9 ADCs displayed compelling anti-tumor activity in xenograft models. In a CaLu3 non-small cell lung cancer xenograft model, anti-ADAM9-DM4 induced tumor growth delay at a single 1.25 mg Ab/kg dose. In the same model, a single intravenous dose of 0.25 mg Ab/kg of the anti-ADAM9-DGN549 produced durable complete remissions in 8/8 mice. A non-targeting DGN549 ADC was inactive even when dosed at 10 times that of the anti-ADAM9 ADC, demonstrating that targeted delivery of DGN549 through ADAM9 binding is required for activity.

These data demonstrate that anti-ADAM9 ADCs exhibit antitumor activity against a broad panel of ADAM9-positive malignancies and cause durable remissions in preclinical models at doses expected to be clinically achievable. Anti-ADAM9 ADCs represent a promising therapeutic strategy to target a wide range of ADAM9-expressing tumors.

#38

Target validation, antibody discovery and preclinical data supporting ADAM9 as an antibody-drug conjugate therapeutic target for solid tumors.

Juniper A. Scribner,1 Bhaswati Barat,2 Stuart W. Hicks,3 Nicholas C. Yoder,3 Thomas Son,1 Lusiana Widjaja,2 Gundo Diedrich,2 Sergey Gorlatov,2 Jeff Hooley,1 Ann Easton,1 Peter Lung,1 Anushka De Costa,1 Francine Chen,1 Michael Chiechi,1 Pam Li,1 Monica Licea,1 Timothy E. Hotaling,1 Michael Spliedt,2 Valentina Ciccarone,2 Nadia Gantt,2 James Tamura,2 Megan E. Fuller,3 Molly McShea,3 Scott Koenig,2 Syd Johnson,2 Paul A. Moore,2 Ezio Bonvini,2 Deryk Loo1. 1 _MacroGenics, Inc, South San Francisco, CA;_ 2 _MacroGenics, Inc, Rockville, MD;_ 3 _ImmunoGen, Inc., Waltham, MA_.

Introduction: A target-unbiased approach based on intact cell immunizations with fetal progenitor cells and cancer stem cells, followed by an immunohistochemistry (IHC) screen for cancer-specific candidates, led to the identification of anti-ADAM9 (a disintegrin and metalloproteinase) mAbs with highly differential tumor-versus-normal tissue binding. ADAM9 is a cell surface protein over-expressed in multiple tumors, with a possible role in promotion and progression of cancer through multiple mechanisms, including modulation of adhesion and migration as well as processing of tumorigenic and pro-angiogenic factors. In this preclinical study, we performed target/mAb validation and evaluated the therapeutic potential of anti-ADAM9 antibody-drug conjugates (ADCs) toward ADAM9-expressing solid cancers.

Methods: IHC was performed with anti-ADAM9 mAbs to confirm and extend available data of human normal and tumor tissue expression. Epitope mapping studies were conducted to define epitope-specificity. mAbs were also screened to identify those that efficiently internalized into tumor cells. In vitro cellular processing studies were performed to further evaluate the mAbs as ADC candidates. Selected mAbs were converted to ADCs via chemical conjugation to potent anti-microtubule (DM4) or DNA alkylating (DGN549) agents; in vitro cytotoxicity studies were conducted with tumor cell lines representing human cancer types that overexpress ADAM9. A lead mAb was then selected for humanization and affinity maturation to yield a development candidate.

Results: Anti-ADAM9 mAbs exhibited strong reactivity toward the tumor epithelium of solid cancers, including pancreatic, kidney, prostate, bladder, breast, colon, lung, and ovarian cancer, but limited reactivity toward normal tissues. Anti-ADAM9 mAbs were efficiently internalized and processed by tumor cell lines, including lines with only modest ADAM9 expression. Anti-ADAM9 ADCs exhibited specific, dose-dependent cytotoxicity toward ADAM9-positive cancer cell lines in vitro, with IC50 values in the sub-nanomolar range. Humanization and affinity maturation of the lead mAb yielded a development candidate that retains potent antitumor activity toward ADAM9-positive tumor cell lines and equivalent, high affinity binding to both human and cynomolgus monkey ADAM9.

Conclusion: ADAM9 is a cell surface antigen that is over-expressed on a wide range of solid cancers. Anti-ADAM9 mAbs that were strongly reactive with representative tumors exhibited high affinity for the antigen and were efficiently internalized and processed by ADAM9-bearing tumor cells. Anti-ADAM9 ADCs demonstrated dose-dependent cytotoxicity in vitro toward a panel of ADAM9-positive tumor cell lines. Our findings demonstrate that an ADC targeting ADAM9 may serve as a potential therapeutic for ADAM9-expressing solid tumors.

#39

Discovery of PEN-221, an SSTR2-targeting maytansinoid conjugate with potent activity in vitro and in vivo.

Brian H. White, Patrick Bazinet, Kerry Whalen, Michelle DuPont, James M. Quinn, Rossitza Alargova, Tsun Au Yeung, Adam Brockman, James Gifford, Haley Oller, Kristina Kriksciukaite, Charles-Andre Lemelin, Patrick Lim Soo, Benoit Moreau, Samantha Perino, Gitanjali Sharma, Rajesh Shinde, Beata Sweryda-Krawiec, Mary Simcox, Richard Wooster, Mark T. Bilodeau. _Tarveda Therapeutics, Watertown, MA_.

Here we describe the discovery and the structure of PEN-221, a somatostatin receptor 2 (SSTR2) targeting peptide conjugated to DM1. PEN-221 is the first clinical compound from Tarveda's Pentarin platform, which utilizes miniaturized drug conjugates that diffuse rapidly and deeply into solid tumors. Antibody drug conjugates (ADCs) have garnered a significant amount of attention in their ability to direct cytotoxic drugs to cancer cells; however, the efficacy of ADCs in solid tumors is limited by the slow diffusion of such large molecules through solid tumor tissue. Pentarins are designed to improve the efficacy of targeted therapies through effective tumor cell targeting and enhanced tumor penetration.

SSTR2, a GPCR overexpressed in multiple types of neuroendocrine tumors, including small cell lung cancers, internalizes rapidly upon agonist stimulation, making it an ideal vector for delivering cytotoxic payloads. Examination of a variety of SSTR2 targeting ligands, as well as several potential conjugation sites, led to the identification of the C-terminal side chain of [Tyr3]-octreotate amide as the best conjugation site for a lipophilic payload. The use of DM1 as a payload afforded superior receptor affinity and receptor internalization when compared to other similarly potent microtubule-targeting agents. In vitro studies show that PEN-221 has receptor-dependent cytotoxic effects, and preclinical studies demonstrate PEN-221 induces tumor regression in several SSTR2 expressing xenograft models.

#40

Development of a novel antibody-drug conjugate targeting endosialin/TEM-1: potent antitumor activity in sarcoma.

Gianluca Sala,1 Stefano Iacobelli,2 Emily Capone,2 Enza PIccolo,2 Jean-Fred Sauniere,2 Vanessa Vannucci Douet3. 1 _University of Chieti, Chieti, Italy;_ 2 _Mediapharma srl, Chieti, Italy;_ 3 _Mediapharma srl, Paris, France_.

The TEM-1/Endosialin/CD248 receptor is expressed in the cell surface of tumor-associated stroma cells, as well as in sarcoma and neuroblastoma cells. This receptor is emerging as an attractive molecule in diagnostics and therapeutics because of its expression across the stroma of many human tumors, the low to absent expression in normal tissues and accessibility from the vascular circulation. In this study, we present evidence of the preclinical efficacy of a novel Antibody-Drug Conjugate (ADC). It consists of a humanized TEM-1 monoclonal antibody (E.8-3) conjugated to a highly potent payload (TEM-1-ADC). In TEM-1 expressing cancer cell lines, this TEM-1-ADC demonstrated a powerful, specific and target-dependent killing activity. High expression levels of TEM-1 in cells correlated with efficient internalization, efficacy, and cytotoxic effects in vitro. Efficacy studies demonstrated that TEM-1-ADC treatment leads to a long lasting tumor growth inhibition of cell line-based models of human sarcoma. Taken together, our results demonstrated that TEM-1 is an attractive target in sarcoma and suggest that TEM-1-ADC has the potential to be developed into a biotherapeutic agent in these malignancies.

#41

An Antibody Drug Conjugate targeting HER-3 demonstrates promising antitumor efficacy in a wide range of human cancer.

Gianluca Sala,1 Manuela Iezzi,1 Alessia Lamolinara,1 Emily Capone,2 Stefano Iacobelli,2 Jean-Fred Sauniere,2 Sara Ponziani,3 Francesco Giansanti,3 Rodolfo Ippoliti3. 1 _University of Chieti, Chieti, Italy;_ 2 _Mediapharma srl, University of L'Aquila, Italy;_ 3 _University of L'Aquila, L'Aquila, Italy_.

The HER-3 receptor is emerging as an attractive molecule in therapeutics because of its overexpression across many human cancers and because of its role in in several compensatory processes that underlay emergence of resistance to certain cancer drugs. In this study, we present evidence of the preclinical efficacy of a novel Antibody-Drug Conjugate (ADC) targeting HER-3. It consists of a humanized HER-3 monoclonal antibody (mAb EV20), which recognizes the HER-3 extracellular domain, conjugated to different payloads (HER-3-ADC)s. In HER-3 expressing cancer cell lines, these HER-3-ADCs demonstrated a powerful, specific and target-dependent killing activity. High expression levels of HER-3 in tumor cells correlated with efficient internalization, efficacy, and cytotoxic effects in vitro. Efficacy studies demonstrated that HER-3-ADCs treatment leads to a long lasting tumor growth inhibition of cell line-based models of human head and neck, breast, pancreatic, prostatic, lung, stomach cancers and melanoma. Overall, these findings validate HER-3 as an attractive therapeutic target in multiple solid tumors and support further clinical development and application of HER-3 targeting ADCs.

#42

Preclinical development of a duocarmycin-based antibody-drug conjugate targeting B7-H3 for solid cancer.

Thomas Son,1 Juniper A. Scribner,1 Jeff Hooley,1 Michael Chiechi,1 Pam Li,1 Timothy E. Hotaling,1 Anushka De Costa,1 Yan Chen,1 Francine Chen,1 Bhaswati Barat,2 Valentina Ciccarone,2 Timur Gaynutdinov,2 James Tamura,2 Scott Koenig,2 Syd Johnson,2 Paul A. Moore,2 Ezio Bonvini,2 Deryk Loo1. 1 _MacroGenics, Inc., South San Francisco, CA;_ 2 _MacroGenics, Inc., Rockville, MD_.

Introduction: B7-H3, a member of the B7 family of immunomodulatory molecules, is overexpressed in a wide range of solid cancers. B7-H3 overexpression has been correlated with disease severity and poor outcome in several cancer types. Proof-of-concept studies targeting B7-H3 demonstrated that auristatin-based B7-H3 antibody-drug conjugates (ADCs) exhibited potent cytotoxicity in vitro and antitumor activity in vivo toward a range of B7-H3-expressing tumor cell lines. Based on these preliminary results, we undertook preclinical development of a B7-H3 ADC comprised of a humanized B7-H3 mAb conjugated to a potent DNA alkylating payload.

Methods: Chimeric B7-H3 mAbs were conjugated to vc-seco-DUocarmycin-hydroxyBenzamide Azaindole (DUBA) (ADC conjugated and provided by Synthon Biopharmecuticals B.V). In vitro and in vivo activity studies were conducted with tumor cell lines that overexpress B7-H3. Based on the potency analysis, together with the biophysical properties and immunohistochemistry (IHC) profiles of the candidates, a lead mAb was selected for preclinical development. The mAb was humanized via CDR grafting and conjugated to DUBA to yield the development candidate MGC018. In vitro and in vivo studies were then conducted with MGC018 to confirm and extend the results with the chimeric ADCs.

Results: Confirming our previous data and consistent with a growing body of literature, B7-H3 mAbs exhibited strong reactivity toward carcinoma cells and the vasculature of solid cancers. Chimeric B7-H3-DUBA ADCs demonstrated specific, dose-dependent cytotoxicity toward B7-H3-positive tumor cell lines in vitro and potent antitumor activity in vivo. The humanized ADC development candidate, MGC018, retained the favorable biophysical properties and the normal tissue-versus-tumor IHC profile of the parental mAb. MGC018 displayed cytotoxicity toward B7-H3-positive tumor cell lines in vitro, with IC50 values in the sub-nM range, and potent antitumor activity in vivo, resulting in tumor stasis and tumor regression in mice bearing B7-H3-positive human tumor xenografts, representing breast, lung and ovarian cancers.

Conclusion: MGC018, a preclinical candidate comprised of a humanized mAb targeting B7-H3 conjugated to the potent DNA alkylating payload DUBA via a cleavable peptide linker, exhibited a favorable preclinical profile, with strong reactivity toward tumor cells and tumor-associated vasculature, limited normal tissue reactivity, potent cytotoxicity in vitro and antitumor activity in vivo toward a range of B7-H3-expressing tumor cell lines representing several cancer types. Our findings support further preclinical development of MGC018 to evaluate its potential as an ADC therapeutic for B7-H3-expressing solid cancers.

#43

ASN004, a novel 5T4-targeted Dolaflexin ADC, achieves complete regressions and tumor-free survivors in a broad variety of solid tumor models.

Roger A. Smith, David J. Zammit, Sanjeeva P. Reddy. _Asana BioSciences, Lawrenceville, NJ_.

ASN004 is an Antibody Drug Conjugate (ADC) that targets the 5T4 oncofetal antigen (trophoblast glycoprotein). High expression of 5T4 is observed in a wide range of malignant tumors, while very limited expression is found in normal tissues. ASN004 incorporates a novel single-chain homo-dimer antibody, Fleximer® linker technology (Mersana Therapeutics), and several cytotoxic dolastatin (auristatin) analog warheads per ADC molecule with a drug/antibody ratio of ~15:1. ASN004 shows high affinity for the 5T4 antigen (Kd < 30 pM) and 5T4-expressing tumor cells; rapid cellular internalization; and potent, selective cytotoxicity. ASN004 provides complete tumor regressions and tumor-free survivors in multiple tumor xenograft models, derived from human tumor cell lines having both high and low 5T4 expression levels. Tumor-free survivors were achieved at doses that are well-tolerated, based on xenograft body weight measures and exploratory toxicology studies in pharmacologically-relevant non-human primates. As well, tumor-free survivors were achieved in xenograft tumor models following administration of a single dose of ASN004, as low as 1 mg/kg iv. The broad activity of ASN004 has been demonstrated by its superior efficacy in a head-to-head study against trastuzumab-DM1, in a low-5T4, high-HER2 expressing tumor xenograft model. Finally, tumor xenografts that initially responded to ASN004 and later showed tumor regrowth, had not developed resistance and responded well to subsequent treatment with ASN004. Overall, ASN004 is demonstrated to be an ADC with high therapeutic potential in multiple tumor types, encompassing a wide range of 5T4-expression levels. IND-enabling toxicology studies are ongoing, in preparation for advancement to clinical studies in patients with solid tumors.

#44

U3-1402, a novel HER3-targeting ADC, and a novel DNA topoisomerase I inhibitor inhibit the growth of non-small cell lung cancer with EGFR mutation.

Kimio Yonesaka,1 Koji Haratani,1 Kenji Hirotani,2 Kazuhiko Nakagawa1. 1 _Kindai University Faculty of Medicine, Japan;_ 2 _Daiichi Sankyo Co., Ltd., Japan_.

Background HER3, a member of the HER family, is overexpressed in non-small cell lung cancer (NSCLC), especially in those with EGFR mutation. Anti-HER3 antibody therapies, including patritumab, are effective, but limited in their efficacy, for patients with NSCLC. U3-1402 is a novel ADC composed of an anti-HER3 antibody (patritumab) and a novel potent topoisomerase I inhibitor DX-8951. U3-1402 achieved a high drug-to-antibody ratio (DAR: 7-8), because it is homogeneously conjugated with the payload. Here, we aimed to preclinically evaluate U3-1402's efficacy in NSCLC, especially in those with EGFR mutation.

Materials and methods An in vitro growth inhibition assay was used to evaluate the sensitivity of 14 NSCLC cell lines to U3-1402. Cells were treated with U3-1402 at different concentrations (0-10 μg/ml) over 7 days; 50% growth inhibitory concentration relative to control (IC50) was calculated. PC9, HCC827, HCC827GR5, Ma70, Ma70GR, 11-18, H1650, HCC4006, and H1975 cells had the EGFR mutation. HCC827GR and Ma70GR were EGFR-TKI resistant clones, with MET genomic amplification and an unknown resistance mechanism, established from HCC827 and Ma70 cells, respectively. HER3 mRNA expression levels were measured by quantitative reverse transcription-PCR (qRT-PCR) and the ratio was calculated against house-keeping genes in each cell line.

Results The in vitro growth inhibition assay indicated that HCC827GR5, Ma70GR, and 11-18 cells were sensitive to U3-1402 (IC50 values 1.0, 5.2, 3.2 μg/ml, respectively). However, other cells were relatively resistant to U3-1402, having IC50 values greater than 10 μg/ml. Notably, HCC827GR5 cells were more sensitive to U3-1402 than parental HCC827 cells were. Specifically, 1.0 μg/ml U3-1402 reduced the viable cell proportion to 50% of control in HCC827GR5 cells, but the effect was limited to 95% of control in HCC827 parental cells. Furthermore, EGFR-TKI erlotinib increased sensitivity to U3-1402 in HCC827GR5 cells. Specifically, 1.0 μM erlotinib reduced the viable cell proportion to 69% of control, and 1.0 μg/ml U3-1402 reduced it to 50% of control in HCC827GR5 cells, but both agents combined reduced it to 3% of control. In an effort to clarify the underlying mechanism by which EGFR-TKI resistant HCC827GR5 cells were more sensitive to U3-1402 than parental HCC827 cells were, we evaluated the HER3 mRNA expression in both cell lines. HCC827GR5 cells had significantly higher levels of HER3 mRNA than parental HCC827 cells did (1.85 vs 0.65, t-test; p = 0.003).

Conclusions U3-1402 preclinically exhibited its efficacy in NSCLC with EGFR mutation. Its efficacy was enhanced by EGFR-TKI combination. Sensitivity to U3-1402 might depend on HER3 expression levels. These results provide a rationale for U3-1402 alone or in combination with EGFR-TKI to be investigated in patients with NSCLC with EGFR mutation and aberrant HER3 expression.

#45

In vitro **and** in vivo **activity of a novel c-Met-targeting antibody-drug conjugate using a DNA-alkylating, indolinobenzodiazepine payload.**

Katharine C. Lai, Asli Muvaffak, Min Li, Marian Themeles, Surina Sikka, Kerry Donahue, Stuart W. Hicks, Angela Romanelli, Thomas Chittenden. _ImmunoGen, Inc., Waltham, MA_.

Purpose: c-Met dysregulation and/or overexpression are associated with tumor progression, metastasis and poor prognosis in numerous cancers. Despite strong pre-clinical evidence that blocking c-Met activity inhibits tumor cell growth and metastasis, targeted therapies have thus far failed to deliver an effective treatment option to the majority of patients. To address patients with both c-Met over-expressing and MET amplified tumors, we designed an antibody-drug conjugate (ADC) comprised of a humanized anti-c-Met monoclonal antibody linked to a highly potent indolinobenzodiazepine DNA-alkylating payload (DGN549) to enable activity against not only MET amplified but also c-Met over-expressing tumors.

Experimental Design: Panels of monoclonal antibodies (Abs) against c-Met were generated and screened for antagonistic and agonistic activity in the presence or absence of the c-Met ligand, HGF. Lead Abs were humanized and conjugated to DGN549 either through lysine (Drug-to-Ab ratio (DAR) = 2.5) or engineered cysteine residues (DAR 2.0). Abs were also conjugated via lysine residues to the potent anti-microtubule maytansine derivative, DM4, using a sulfo-SPDB linker (DAR 3.5). Binding and cytotoxicity of ADCs were tested in vitro on normal and cancer cell lines with varying c-Met levels. Expression of c-Met was evaluated in patient tumors and xenografts along with normal human tissues using the CONFIRM immunohistochemistry assay. In vivo efficacy of anti-c-Met-DGN549 and anti-c-Met-DM4 ADCs was tested in both MET amplified and c-Met over-expressed (but non-amplified) xenograft tumor models.

Results: A humanized anti-c-Met antibody, hucMet27, was identified which exhibits low c-Met agonist activity. Conjugates of hucMet27 were prepared with two different payloads, DGN549 and DM4, and in vitro and in vivo activity were determined. Both DGN549 and DM4 conjugates of hucMet27 bound with similar sub-nanomolar affinity to c-Met-expressing cells. hucMet27-DGN549 conjugates exhibited potent cytotoxicity against a large panel of c-Met expressing cell lines. By contrast, the potency of the hucMet27-DM4 conjugate was restricted mainly to cell lines harboring MET amplification, despite all cell lines demonstrating sensitivity to the unconjugated payload. When tested in mice bearing human xenograft tumors, both hucMet27-DGN549 and hucMet-DM4 conjugates were highly active in a MET amplified model, whereas hucMet27-DGN549 was more potent in inducing regressions in a model with c-Met over-expression without MET amplification.

Conclusion: hucMet27-DGN549 exhibits compelling c-Met targeted anti-cancer activity in vitro and in vivo, and represents a promising therapeutic strategy to deliver a potent cytotoxic agent to tumor cells bearing a wide range of c-Met expression.

#46

Preclinical activity of novel antibody-drug conjugates with pyrrole-based kinesin spindle protein inhibitors targeting different tumor antigens.

Anette Sommer,1 Sandra Berndt,1 Hans-Georg Lerchen,2 Beatrix Stelte-Ludwig,2 Sven Wittrock,2 Anne-Sophie Rebstock,2 Lisa Dietz,2 Christoph Mahlert,2 Simone Greven,2 Nils Griebenow,2 Yolanda Cancho-Grande,2 Rolf Jautelat,2 Heiner Apeler,2 Bertolt Kreft1. 1 _Bayer AG, Berlin, Germany;_ 2 _Bayer AG, Wuppertal, Germany_.

Antibody-drug conjugates (ADCs) are promising agents that are developed for targeted delivery of cytotoxic payloads to tumor cells. ADCs share a common design of antibody, linker, and cytotoxic payload. Despite significant efforts, the number of available payload classes with a differentiated mode-of-action that can successfully be employed to generate antibody-drug conjugates (ADCs) is still rather limited. So far, only ADCs with microtubule depolymerizing or DNA binding payloads have been approved. The identification of ADC payload classes with a novel mode-of-action will increase therapeutic options and potentially help to overcome resistance. Inhibitors of the kinesin spindle protein (KSP/Eg5/KIF11) have generated interest due to their high anti-tumor activity. However, the transfer of the potency of small molecule KSP inhibitors (KSPis) to highly efficient clinical regimens with a sufficient therapeutic window remains challenging. Through the conjugation of a novel pyrrole subclass of KSPis to antibodies targeting different cancer antigens, we generated a panel of ADCs and characterized them both in vitro and in vivo. ADCs targeting either EGFR or TWEAKR/Fn14 showed strong and specific internalization and displayed specific and potent anti-proliferative efficacy in vitro. In cytotoxicity assays, these ADCs exhibited sub-nanomolar potency in antigen-positive cancer cell lines (EGFR/TWEAKR-pos. NCI-H292; TWEAKR-pos. BxPC3, LoVo) and more than 100-fold selectivity versus non-targeted control-ADC containing the same linker and the same payload. Furthermore, selective anti-tumor efficacy of EGFR- and TWEAKR-KSPi-ADCs was demonstrated in vivo using both cancer cell line-derived models of NSCLC (NCI-H292), urothelial cell carcinoma (UCC) (KU-19-19), and renal cell carcinoma (A498), as well as in the TWEAKR-positive patient-derived xenograft UCC model BFX469. At doses of 5-10 mg/kg qw or bw potent anti-tumor efficacy with treated-to-control ratios (T/C) between 0.16 to 0.28 as well as complete regressions were observed. In summary, KSP inhibitors have been established as a promising new payload class allowing the generation of highly potent and selective ADCs for the treatment of solid tumors.

#47

SYD985, a novel duocarmycin-based HER2-targeting antibody-drug conjugate, shows antitumor activity in uterine and ovarian carcinosarcoma with HER2/neu expression.

Gulden Menderes, Elena Bonazzoli, Stefania Bellone, Jonathan Black, Gary Altwerger, Alice Masserdotti, Francesca Pettinella, Luca Zammataro, Natalia Buza, Pei Hui, Elena Ratner, Babak Litkouhi, Dan-Arin Silasi, Masoud Azodi, Peter Schwartz, Alessandro D. Santin. _Yale Univ. School of Medicine, New Haven, CT_.

Introduction: Carcinosarcoma (CS) is a rare and highly aggressive gynecologic malignancy. The amplification of HER2/neu has been reported to occur in 25%-56% of uterine and ovarian CSs with significant heterogeneity of the expression within epithelial and in-between epithelial and mesenchymal components. The reported high incidence makes HER2/neu an attractive target for new molecularly targeted therapies. We investigated the efficacy of SYD985, (Synthon-Biopharmaceuticals), a novel HER2-targeting antibody-drug conjugate (ADC) composed of a monoclonal antibody (mAb) similar to trastuzumab linked to a highly potent DNA-alkylating agent (i.e., duocarmycin) in uterine and ovarian carcinosarcoma. We also compared the anti-tumor activity of SYD985 to trastuzumab emtansine (T-DM1), a FDA-approved ADC, against multiple primary CS cell lines expressing different levels of HER2/neu in in vitro and in vivo experiments.

Methods: Eight primary CS cell lines were evaluated for HER2/neu surface expression by IHC and gene amplification using FISH assays. The cytotoxicity of SYD985 and T-DM1 was evaluated using these eight CS primary cell lines with differential HER2/neu expression (i.e., 0, 1+, and 3+). Antibody-dependent cellular cytotoxicity (ADCC), proliferation, viability and bystander killing experiments were performed using Cr51-release assays, propidium iodide-based and flow cytometry assays, respectively. SYD985 and T-DM1 in vivo activity was also studied in mouse xenograft models.

Results: Similar ADCC were induced by SYD985 and T-DM1 when effector cells were present against CS cell lines with different HER2/neu expression. In contrast, SYD985 was significantly more cytotoxic compared to T-DM1 in the absence of the effector cells. SYD985 is 7 to 54 fold more potent than T-DM1 in comparative experiments and unlike T-DM1, it is active against CS demonstrating moderate/low or heterogeneous HER2/neu expression. Specifically, in HER2/neu 0/1+ cell lines the mean IC50's were 0.060 µg/mL and 3.221 µg/mL for SYD985 vs T-DM1 (p<0.0001) and in HER2/neu 3+ cell lines 0.013 µg/mL and 0.096 µg/mL, (p<0.0001), respectively, (p<0.0001). Unlike T-DM1,

SYD985 induced efficient bystander killing of HER2/neu 0/1+ EOC cells admixed with HER2/neu 3+ cells. In vivo studies confirmed that SYD985 is more active than T-DM1 in CS and effective against HER2/neu 3+ xenografts.

Conclusions: We demonstrate for the first time that SYD985 is a novel ADC with remarkable activity against CS not only with strong (3+) but also with low (0/1+) HER2/neu expression . Clinical studies with SYD985 in patients harboring chemotherapy-resistant uterine and ovarian CS with low, moderate and high HER2 expression are warranted.

#48

Non-clinical pharmacokinetics of XMT-1522, a HER2 targeting auristatin-based antibody drug conjugate.

Alex Yurkovetskiy, Dmitry Gumerov, Elena Ter-Ovanesyan, Patrick Conlon, Michael Devit, Charlie Bu, Natalya Bodyak, Timothy Lowinger, Donald Bergstrom. _Mersana Therapeutics, Inc., Cambridge, MA_.

The ADC XMT-1522 consists of a novel human IgG1 anti-HER2 monoclonal antibody and a novel, auristatin-based cytotoxic payload (Auristatin F-hydroxypropylamide, AF-HPA). An average DAR of 12 AF-HPA molecules is achieved via a biodegradable polymer conjugation platform. The non-clinical DMPK properties of XMT-1522 have been characterized in vitro in plasma and microsomal stability studies, and in vivo in plasma and tissue disposition and excretion studies. Sample analysis for total AF-HPA drug payload and released (free) AF-HPA and its metabolites was performed by ESI+ LC/MS/MS; total antibody was determined by ELISA. The half-life for AF-HPA release in plasma was found to be greater than 120 hours in all species tested. Microsomal stability studies showed that AF-HPA was further converted to other metabolites including the carboxylic acid auristatin F (AF), as well as monomethyl auristatin F-HPA (MMAF-HPA) and MMAF. The pharmacokinetic profiles of XMT-1522 were evaluated in mouse, rat and cynomolgus monkey. The antibody of XMT-1522 is cross-reactive with monkey, but not rodent, HER2. In mouse and rat, XMT-1522 exposure was dose-proportional; exposure was slightly greater than dose-proportional in monkey consistent with saturation of target-mediated clearance. All species showed extended exposure to total AF-HPA drug payload, with measured clearance and volume of distribution similar for total AF-HPA and the antibody component of XMT-1522. Exposure to free AF-HPA and AF was less than 1/1000th the exposure of total AF-HPA. These data indicate the vast majority of AF-HPA in plasma is antibody-conjugated, indicating high stability of the ADC in systemic circulation. XMT-1522 tissue disposition was studied in NCI-N87 HER2-positive gastric cancer xenograft tumor bearing mice. After a single 3 mg/kg dose of XMT-1522, free AF-HPA and its metabolite AF were measurable in tumor tissue until the last time point measured (2 weeks). Total AF-HPA and free AF-HPA achieved peak tumor concentrations 48 hours after dosing. In contrast, AF achieved peak tumor concentration 7 days after dosing and showed only a slight decline in tumor concentration at 14 days, consistent with intracellular trapping of this poorly cell-permeable metabolite. Exposure to free AF-HPA or AF in other tissues was at least an order of magnitude lower than in tumor; in tissues with measurable free drug, AF was the predominant species. XMT-1522 excretion studies, conducted in rat, indicated that the AF-HPA payload was mainly excreted by the gastrointestinal route. In the first 96 hours after administration 33% of the AF-HPA dose was excreted in feces, compared to 3% excreted in urine. The major contributing metabolites both in feces and urine were conjugated AF-HPA, AF, and free AF-HPA. In conclusion, the plasma kinetics, tissue distribution and excretion profile of XMT-1522 are acceptable for clinical evaluation in cancer patients.

#49

Modulating insulin receptor splicing as a potential therapeutic approach for rhabdomyosarcoma.

Brianne Sanford,1 Chelsea Brown,1 Hemant Bid,1 Thomas Beebe,1 Daniel Comiskey,1 Frank Rigo,2 Peter Houghton,3 Dawn Chandler1. 1 _Nationwide Children's Hospital, Columbus, OH;_ 2 _Ionis Pharmaceuticals, CA;_ 3 _University of Texas Health Science Center, TX_.

The insulin receptor (IN-R) is subject to alternative splicing to produce two isoforms: the full-length IN-R B isoform and an isoform lacking exon 11 known as IN-R A which is the predominant isoform expressed in sarcoma cells. This short exon encodes for 12 amino acids but the resulting receptor has high affinity for a growth hormone called insulin-like growth factor 2 (IGF2), which exquisitely responds to autocrine and paracrine signaling. Increased IN-R A levels observed in rhabdomyosarcoma (RMS) coupled with increased expression of IGF2, programs the tumor cell for enhanced growth and angiogenesis, two important factors in tumorigenesis. Furthermore, IGF2 can maintain angiogenesis through IN-R A when the cognate receptor for IGF2, the IGF-1 receptor, is blocked. Interestingly we have shown that hypoxia increases alternative splicing to produce more IN-R A. This is an interesting finding since adaptation to the hypoxic environment is a hallmark of the neoplastic phenotype. Therefore, we hypothesize that expression of splicing factors is altered under hypoxic conditions which leads to increased alternative splicing and generation of IN-R A, allowing the cancer cells a growth advantage which contributes to metastasis beyond the micrometastatic phase.

To characterize sequence elements and splicing factors involved in the regulation of IN-R alternative splicing, we have developed a hypoxia-inducible splicing system, which recapitulates the splicing patterns observed in tumors. We have shown that sequence elements preceding exon 11 are critical to the increased alternative splicing we see under hypoxic conditions. As such, we have targeted these regions for antisense oligonucleotide (ASO) development to increase full-length splicing of IN-R, with the goal of restricting proliferative signaling by IGF2 activation of IN-R A. We performed an ASO walk to target regions important for exon inclusion or exclusion and have successfully targeted a region known to be a binding site for the splicing factor CUGBP1. RMS-derived cell lines almost exclusively express IN-R A but when treated with our lead ASO compound we see a dramatic decrease in alternative splicing resulting in increased levels of the full-length insulin receptor. We have also shown that RMS-derived cells treated with our splice modulating ASO exhibit reduced cell proliferation and migratory properties. With these promising data we postulate that modulation of IN-R splicing can be used in conjunction with already established anti-IGF-1 receptor therapies in the treatment of RMS. 

### Antibody Technology

#50

HTI-1511, a novel anti-EGFR-ADC, overcomes mutation resistance and demonstrates significant activity against multiple tumor types in preclinical studies.

Jesse D. Bahn,1 Feng Gao,1 Lei Huang,1 Barbara Blouw,1 Chunmei Zhao,1 Kelly Chen,1 Susan Zimmerman,1 Erin K. Wise,2 Maria L. Mancini,2 Matthew Bird,3 William McDowell,3 Curtis B. Thompson,1 Sanna Rosengren,1 Christopher D. Thanos1. 1 _Halozyme Therapeutics, San Diego, CA;_ 2 _Champions Oncology, Inc, Baltimore, MD;_ 3 _Abzena, Antitope Limited & PolyTherics Limited, Cambridge, United Kingdom_.

Multiple solid tumor types over-express epidermal growth factor receptor (EGFR). Antibodies that target the receptor are often accompanied by adverse skin reactions due to interaction with receptors expressed in normal tissue. Additionally, downstream mutations (KRAS, BRAF) within tumors can result in EGFR-independent activation and resistance to treatment. We have previously described HTI-1511, an antibody-drug conjugate in pre-clinical development that targets EGFR. HTI-1511 carries the potent cytotoxin MMAE and a novel bis-alkylating linker, connected to a monoclonal antibody engineered to have improved specificity for EGFR in the tumor microenvironment (Huang et. al. AACR National Meeting, 2016, New Orleans, LA). Here we screened a panel of over 70 tumor cell lines derived from various solid tumor malignancies for both EGFR expression by flow cytometry and sensitivity to cell growth inhibition by HTI-1511 in vitro. Cell lines derived from head and neck squamous cell carcinoma (SCC15, CAL27, FaDu, CAL33, SCC25 [IC50 0.52 nM - 3.1 nM]), non-small cell lung cancer (HCC827, NCI-H1666, PC-9, NCI-H1650 [IC50 0.04 nM - 6.2 nM]), and pancreatic carcinoma (BxPC-3, PANC-1, AsPC-1 [IC50 0.99 nM - 4.44 nM]) showed particular sensitivity to HTI-1511. In conjunction, HTI-1511 efficacy was assessed in vivo for tumor growth inhibition (TGI) in several human tumor xenograft models. Evaluations in the human xenografts A431 (epidermoid, 93% TGI at 3.0 mg/kg, p<0.05), BxPC3 (pancreatic, >100% TGI at 3.0 mg/kg, p<0.05), AsPC-1 (pancreatic, >100% TGI at 3.0 mg/kg, p<0.05), and FaDu (HNSCC, >100% TGI at 3.0 mg/kg, p<0.05) indicated dose dependent tumor regressions in all cases, and no observed toxicity when administered weekly at dose levels up to 3 mg/kg for up to eight weeks. HTI-1511 anti-tumor activity was also investigated in a group of patient derived xenograft (PDX) models. An initial study evaluated weekly administration of a single dose level of 2.5 mg/kg HTI-1511 in three different PDX models. TGI of 83% (p<0.05) was observed in a BRAFmut colorectal cancer model and 57% (p<0.05) in a wildtype colorectal cancer model, as well as 46% (not significant) TGI seen in a wild type renal cell carcinoma model. A separate study in a NSCLC (KRASmut) PDX model demonstrated a dose dependent response with greater than 100% TGI at 1.0 and 3.0 mg/kg (both p<0.05, and also p<0.05 to a non-cognate antibody ADC control). The non-cognate antibody control yielded 67.4% (not significant) TGI by itself compared to the vehicle control group. These results support further development of HTI-1511 as a possible treatment for EGFR overexpressing tumors, including those with downstream activating mutations in the KRAS/BRAF pathway.

#51

Characterization of the mechanism of action, pharmacodynamics and preclinical safety of ADCT-402, a pyrrolobenzodiazepine (PBD) dimer-containing antibody-drug conjugate (ADC) targeting CD19-expressing hematological malignancies.

Francesca Zammarchi,1 Simon Corbett,2 Karin Havenith,1 Narinder Janghra,2 Konstantinos Kiakos,2 Teresa Marafioti,2 David G. Williams,3 Simon Chivers,1 Phil W. Howard,3 John A. Hartley,2 Patrick H. van Berkel1. 1 _ADC Therapeutics, London, United Kingdom;_ 2 _University College London, London, United Kingdom;_ 3 _Spirogen/MedImmune, London, United Kingdom_.

ADCT-402, currently in Phase I clinical trials for B-cell hematological malignancies, is an ADC composed of a recombinant humanized IgG1 against human CD19, stochastically conjugated via a cleavable linker to a PBD dimer cytotoxin (DAR of 2.3). PBD dimers, DNA minor groove interstrand cross-linking agents, are gaining increasing attention and are currently being tested as the ADC warheads in several clinical trials. ADCT-402 has potent and targeted cytotoxicity against a panel of human lymphoma and leukemia cell lines in vitro. In vivo, ADCT-402 demonstrates dose-dependent antitumor activity against Burkitt's lymphoma xenograft models. Moreover, ADCT-402 is markedly superior to maytansinoid- and auristatin-based CD19-targeting ADCs in the Ramos xenograft model. In a rat toxicology study, a single dose of ADCT-402 at 2 mg/kg is well tolerated with a favorable PK profile and excellent stability in vivo. The current study aimed to further define the mechanism of action (MOA) of ADCT-402 and validate its pharmacology and preclinical safety for clinical development. CD19 is a clinically validated target with restricted normal tissue expression and a widespread expression in the majority of B-cell malignancies. Importantly, we show here the consistent expression of CD19 in matched samples (initial diagnosis and relapsed/refractory) from panels of lymphoma patients, indicating that relapsed/refractory patients are appropriate for treatment with ADCT-402. ADCT-402 was shown to be efficiently internalized by CD19+ cells in vitro. Moreover, in line with the PBD dimer MOA, following a 2 hour exposure to ADCT-402, DNA interstrand cross-links reached a peak between 8 - 12 hours and persisted for up to 36 hours post-treatment. In contrast, the peak of cross-link formation for the PBD dimer warhead alone was observed immediately after 2 hour incubation, while a non-targeted PBD-ADC did not yield any appreciable DNA cross-links. In SCID mice s.c. implanted with Ramos cells, a single dose of ADCT-402 was administered at 0.33 or 1 mg/kg. Twenty-four hours after treatment, excised tumors showed a dose proportional increase in intensity of staining by an anti-PBD payload antibody, as well as in DNA cross-linking and in γ-H2AX formation. In contrast, no DNA cross-linking was observed in matched lymphocyte samples. The toxicity of ADCT-402 was further evaluated in a repeat dose cynomolgus monkey study. ADCT-402 was clinically well tolerated with an acceptable off-target safety profile. The PK of the ADC was consistent with normal antibody clearance with a half-life of about 12 to 17 days. These data confirm the MOA of ADCT-402 and provide relevant pharmacodynamic assays and preclinical safety assessment to guide the clinical development of this promising ADC in B-cell malignancies.

#52

Mechanistic and benchmarking studies of ADCT-502, a pyrrolobenzodiazepine (PBD) dimer-containing antibody-drug conjugate (ADC) targeting HER2-expressing solid tumors.

Francesca Zammarchi,1 Halla W. Reinert,2 Narinder Janghra,2 Simon Corbett,2 Maria Mellinas-Gomez,3 Sajidah Chowdhury,3 Neha Arora,3 Peter Tyrer,3 Francois Bertelli,3 David G. Williams,3 Philip W. Howard,3 John A. Hartley,2 Patrick H. van Berkel1. 1 _ADC Therapeutics, London, United Kingdom;_ 2 _University College London, London, United Kingdom;_ 3 _Spirogen/MedImmune, London, United Kingdom_.

ADCT-502 is an ADC composed of an engineered version of humanized IgG1 trastuzumab, directed against human HER2, site-specifically conjugated to the highly cytotoxic PBD-based linker-drug tesirine (drug-antibody ratio of 1.7). In vitro, ADCT-502 has highly potent and targeted cytotoxicity against various solid cancer cell lines. In vivo, ADCT-502 demonstrates strong and durable antitumor activity in mouse xenografts with various levels of HER2, but is inactive in a HER2-negative xenograft. ADCT-502 is stable, well tolerated and has a favorable PK profile both in rat and cynomolgus monkey. The current study aimed to define further the mechanism of action of ADCT-502 and to benchmark its activity in xenograft models against ado-trastuzumab emtansine (T-DM1), the ADC currently approved for the treatment of HER2+ metastatic breast cancers. ADCT-502 bound and internalized efficiently in JIMT-1 cells (HER2+) and co-localized with lysosomes within 2 hours. PBD dimers bind in the DNA minor groove and exert cytotoxicity via the formation of DNA interstrand cross-links. Following a 2-hour exposure to ADCT-502, DNA interstrand cross-linking peaked between 12 and 24 hours, after which cross-links persisted at least 36 hours. In contrast, cross-link formation by an equimolar concentration of warhead alone, peaked immediately following drug exposure and a non-targeted ADC did not produce DNA crosslinks in these cells. Moreover, ADCT-502 showed indirect bystander killing activity in HER2-negative MDA-MB-468 cells incubated with conditioned medium from ADCT-502-treated HER2+ SK-BR-3 cells. In vivo, antitumor activity of ADCT-502 was compared to T-DM1 in both cell line- and patient-derived-xenograft (PDX) models. For example, in a HER2 1+, FISH- breast cancer PDX, ADCT-502 showed dose-dependent antitumor activity resulting in 1/8 and 8/8 TFS after a single dose at 0.1 and 0.2 mg/kg, respectively. Conversely, a single dose of T-DM1 at 30 mg/kg showed only marginal activity compared to the control. Similarly, in a HER2 1+, FISH- esophageal cancer PDX, while a single dose of ADCT-502 at 0.44 mg/kg resulted in strong and durable antitumor activity, single doses of T-DM1 at either 10 or 30 mg/kg showed no activity compared to the control. These data confirm that the mechanism of cell killing of ADCT-502 is via target-specific internalization and subsequent cross-linking of DNA. They also show superior in vivo antitumor activity of ADCT-502 compared to T-DM1 in various tumor xenografts, including those with low HER2 levels. Taken together, these results support the development of ADCT-502 not only in patients that have become resistant/refractory to T-DM1, but also in patients whose tumors express low levels of HER2, and are not eligible for treatment with T-DM1.

#53

Antibody-drug conjugates (ADCs) of peptide-linked Indolino-Benzodiazepine (IGN) DNA-alkylator provides improved anti-tumor activity over that of a crosslinker.

Michael L. Miller, Manami Shizuka, Jose F. Ponte, Leanne Lanieri, Dilrukshi Vitharana, Qifeng Qiu, Emily E. Reid, Katie E. Archer, Rui Wu, Erin K. Maloney, Olga Ab, Jan Pinkas, Ravi V. Chari. _ImmunoGen, Inc., Waltham, MA_.

We recently disclosed highly active antibody-drug conjugates (ADCs) that incorporated the novel DNA alkylating indolino-benzodiazepine (termed IGN) dimer, DGN549 (IGN-P1). The stereochemistry of the alanyl moiety of the protease-cleavable alanine-alanine linker used was shown to impact ADC catabolism, bystander killing activity, and in vivo efficacy (Shizuka, et al., AACR 2016 #2959). Building upon these results, here we describe preclinical results from a head-to-head comparison of ADCs of the mono-imine containing DGN549 with its corresponding DNA cross-linking diimine version, IGN-P1 diimine. IGN-P1 diimine and DGN549 were conjugated to a folate receptor-α (FRα)-binding antibody and an EpCAM-binding antibody. The resulting ADCs demonstrated similar high in vitro potency (IC50 ~3-100 pM) and specificity towards several cancer cell lines. Further in vitro studies revealed that the DNA alkylating anti-FRα-DGN549 ADC demonstrated superior bystander cell-killing activity compared to its DNA crosslinking counterpart, anti-FRα-IGN-P1 diimine. In vivo, this improved bystander killing ability translated into better in vivo activity for the DNA alkylating ADC. In an endometrial tumor xenograft model established with Ishikawa cells, the anti-FRα-DGN549 induced complete regressions at a single dose of 140 µg/kg Ab dose (equivalent to 5 µg/kg linked IGN). The cross-linking anti-FRα-IGN-P1 diimine had to be used at twice the dose to achieve the same level of anti-tumor activity. The in vivo tolerability in CD-1 mice also displayed differences in the two ADCs. We found that the ADC of the DNA crosslinker was at least two-fold less tolerated than the corresponding ADC of the DNA alkylator. These results indicate that a ~4 fold greater therapeutic index can be achieved when using a DNA alkylating mono-imine DGN549 ADC as compared to the DNA crosslinking IGN-P1 diimine ADC.

#54

The many faces of antibody in G protein-coupled receptor biology.

Lei Chen, Wing-Tai Cheung. _School of Biomedical Sciences, Faculty of Medicine, Chinese University of Hong Kong, Hong Kong_.

G protein-coupled receptors (GPCRs), the largest family of cell-surface receptor proteins mediating signal transmission, play a pivotal role in many physiological functions and are involved in multiple diseases. Although receptor activities of GPCRs are successfully modulated by many small molecules which represent 30% of all marketed drugs today, in fact only a few GPCR members are targeted and many more GPCRs of interest are intractable targets of small molecules, like orphan GPCRs and GPCRs with large binding sites. Besides, small molecule drugs rarely trigger GPCR-mediated apoptosis or induce direct cell killing, which is crucial for cancer therapies. Therefore, for these GPCRs, antibody-based drug would be a good alternative. Despite being eagerly sought for, the production of monoclonal antibody (mAb) targeting GPCR is hindered by the low expression of recombinant GPCR on cell surface, and relatively small exposed regions with glycosylation and conformational heterogeneity. Therefore those conventional methods that were well established for generation of mAbs targeting soluble proteins usually showed frustrating results in the case of GPCRs. To bypass the production and purification of recombinant GPCR proteins, we developed a novel approach of transplanting immunogenic conformational epitopes of GPCR into an antibody scaffold to make a water soluble surrogate antigen of GPCR, named as GPCR-antigenized antibody which can be easily and abundantly produced in bacteria for animal immunization. In coupled with phage display technology and cell panning strategy, which allow the retrieval of specific binders from a huge number of candidates, we have successfully isolated scFv antibodies for two human GPCRs, the MAS1 receptor and the chemokine receptor CXCR4. These antibodies showed good specificity in immunofluorescent staining, flow cytometry analysis, western protein immunoblot and immunoprecipitation assay. The results demonstrated this novel approach may offer a generic and effective method to generate specific monoclonal antibody targeting GPCR for diagnostic and therapeutic applications. [The project is partly funded by a CUHK direct grant (4054300)]

#55

Generation of half-life extended anti-CD33 BiTE® antibody constructs compatible with once-weekly dosing.

Tara L. Arvedson,1 Mercedesz Balazs,2 Pamela Bogner,3 Kurt Black,1 Kevin Graham,1 Anja Henn,3 Matthias Friedrich,3 Patrick Hoffmann,3 Roman Kischel,3 Peter Kufer,3 Ralf Lutterbuese,3 Markus Muenz,3 Tobias Raum,3 Benno Rattel,3 Karen Rex,1 Dan Rock,2 Oliver Thomas,3 Joachim Wahl,3 Andreas Wolf,3 Angela Coxon1. 1 _Amgen, Thousand Oaks, CA;_ 2 _Amgen, San Francisco, CA;_ 3 _Amgen, Munich, Germany_.

T cell engaging bispecific antibody constructs (BiTE®), such as blinatumomab which targets CD19-positive cells, have shown great promise for treating certain CD19-positive hematological malignancies. Blinatumomab comprises a single chain Fv (scFv) that binds CD19 and a scFv that targets the T cell CD3 protein. The molecular weight of this "canonical" BiTE® is ~ 55 kDa, making it susceptible to kidney-mediated clearance and resulting in a short serum half-life (~ 4 hours). To maintain effective serum concentrations, canonical BiTE® antibody constructs must be administered by continuous IV (cIV) infusion. While there are many advantages associated with cIV administration (e.g., safety and uniform PK profile), patient convenience could be enhanced if the BiTE® antibody construct were compatible with once-weekly administration. To achieve this, the serum half-life of the BiTE® antibody construct would need to be extended.

A canonical BiTE® targeting CD33 (AMG 330) is currently being evaluated in a phase I clinical trial. Like blinatumomab, AMG 330 is dosed cIV. To extend the serum half-life of AMG 330 and enable once-weekly dosing, several approaches were evaluated including fusion of AMG 330 to human albumin and Fc-containing moieties. Each of these half-life extended (HLE) constructs was evaluated in vitro, in mouse xenograft models and in non-human primates.

In vitro assays evaluated 1) binding to both human and cynomolgus CD33 and CD3 proteins, and 2) cytotoxicity using human and cynomolgus target and effector cells. In each of these assays the canonical and HLE BiTE® antibody constructs demonstrated similar activity: single-digit nM binding and single digit pM cytotoxicity. Canonical and HLE BiTE® antibody constructs were subsequently evaluated in an orthotopic mouse model in which MOLM13 cells were administered IV and activated human T cells were administered IP two days later. The Fc-based HLE BiTE® antibody constructs provided a similar survival advantage when administered Q4D or Q5D as the canonical BiTE® when administered QD. However, the albumin fusion--based HLE BiTE® was less efficacious when administered Q4D than the QD- administered canonical BiTE®. Lastly, the PK/PD relationship was evaluated for each of the constructs in non-human primates. The serum half-lives varied from 6 hours for the canonical BiTE® to 44-167 hours for the HLE BiTE® antibody constructs. Each of the HLE BiTE® antibody constructs showed on-target depletion of CD33-positive monocytes and neutrophils in the blood and depletion of CD33-positive cells in the bone marrow.

These data demonstrate that half-life extended BiTE® antibody constructs can be generated that retain comparable in vitro and in vivo activity as a canonical BiTE® and achieve a serum half-life compatible with once weekly dosing.

#56

Antibody-drug conjugates containing glucuronide-tubulysin payloads display activity in MDR+ and heterogeneous tumor models.

Patrick J. Burke, Joseph Z. Hamilton, Joshua H. Hunter, Julia H. Cochran, Thomas A. Pires, Christopher I. Leiske, Kim K. Emmerton, Peter D. Senter, Robert P. Lyon, Scott C. Jeffrey. _Seattle Genetics, Inc., Bothell, WA_.

While antibody-drug conjugates (ADCs) find increasing application in cancer treatment regimens, de novo or treatment-emergent resistance mechanisms could impair clinical benefit. Two resistance mechanisms that emerge under continuous ADC exposure in vitro include upregulation of transporters that confer multidrug resistance (MDR+) and loss of cognate antigen expression. New technologies that circumvent these resistance mechanisms may serve to extend the utility of next generation ADCs. Recently, we developed the quaternary ammonium linker system to expand the scope of conjugatable payloads to include tertiary amine-containing compounds and applied the linker to tubulysins, a highly potent class of microtubule disrupting agents that maintain activity in MDR+ cell lines. Quaternary ammonium-linked glucuronide-tubulysin drug-linkers were synthesized and evaluated as ADCs. The resulting conjugates were potent and immunologically specific across a panel of cancer cell lines, including those displaying the MDR phenotype. The ADCs also demonstrate potent bystander activity in a co-culture model containing a mixture of antigen-positive and -negative cell lines. Incorporation of a PEG12 side chain in the linker enabled loading at 8-drugs/Ab for increased in vivo potency while maintaining suitable ADC pharmacokinetic properties. In vivo, the glucuronide-tubulysin conjugates displayed activity in MDR+ xenograft models and bystander activity in an admixed Ag+/Ag- heterogeneous tumor model. Thus, the glucuronide-tubulysin drug-linkers represent a promising new payload for ADCs, combining conjugate potency in the presence of the MDR phenotype with robust activity in models of tumor antigen heterogeneity.

#57

Generation and characterization of novel anti-glycan monoclonal antibody against acute myeloid leukemia.

Chong Kok Teo,1 Wee Joo Chng,2 Andre Boon Hwa Choo1. 1 _Bioprocessing Technology Institute, Singapore, Singapore;_ 2 _National University Cancer Institute, Singapore, Singapore, Singapore_.

Hematological malignancies encompass a wide spectrum of cancers from hematopoietic and lymphoid tissues. Under this grouping, the various forms of leukemia, lymphoma and myeloma have been classified into more than 60 distinct disease types, each having particular clinical features and disease outcomes. Among these diseases, curative treatments with high long-term survival rate have been developed for some, e.g. Chronic Myeloid Leukemia, while such effective regimens are still unavailable for many others, e.g. Multiple Myeloma and Acute Myeloid Leukemia. In this study, a panel of antibodies was raised against SLAMF7, a cell surface marker that was associated to Multiple Myeloma. Among the antibodies, TAG-HC2 was surprisingly found to have preferential binding to SLAMF7 expressed on Acute Myeloid Leukemia (AML) cell lines instead. Characterization of TAG-HC2 revealed that the binding of TAG-HC2 to SLAMF7 requires the N-glycosylation of the antigen. Functionally, TAG-HC2 does not have non-specific binding to normal cell lines when tested via flow cytometry and immunohistochemistry. TAG-HC2, when complexed with saporin-conjugated secondary mAb, was able to internalize into and kill AML cell lines. These results suggest that TAG-HC2 has the potential to be developed into an Antibody Drug Conjugate against AML.

#58

Local intratumoral treatment with anti-mesothelin Immunotoxins overcomes resistance to anti-CTLA-4 therapy.

Yasmin Leshem,1 Xiu-fen Liu,1 Tapan Bera,1 Masaki Terabe,1 Jay A. Berzofsky,1 Birgit Bossenmaier,2 Gerhard Niederfellner,2 James O'Brien,1 Yoram Reiter,3 Ira Pastan1. 1 _NIH, Bethesda, MD;_ 2 _Roche Pharmaceutical Research & Early Development, Penzberg, Germany; _3 _Technion israel institute of technology, Haifa, Israel_.

Background: SS1P and RG7787 are immunotoxins composed of anti-mesothelin antibody fused to a fragment from pseudomonas exotoxin A. We previously observed delayed tumor responses in patients treated with SS1P leading us to hypothesis that immunotoxins can provoke anti-tumor immunity. We hypothesize that intra-tumoral injection of SS1P or RG7787 will produce immunologic death and convert living tumors to a source of antigens and boost the therapeutic effect of immune check point blockade.

Method: A BALB/c breast cancer cell line was transfected with human mesothelin (66C14-M) and grown in BALB/c mice transgenic for human mesothelin. SS1P or RG7787 was injected directly into established tumors (80-100 mm3) and anti-CTLA-4 administered IP.

Results: We observed total tumor regressions in 23 out of 38 mice (60%) treated with anti-CTLA-4 and either RG7787 or SS1P. No cure was achieved when the drugs were given as a monotherapy supporting the case for synergic anti-tumor activity. Pathologic evaluation of regressing tumors showed a massive inflammation of admixed eosinophils and mononuclear cells located in a collar surrounding the tumors. Tumor regression was associated with increased number of CD8+ cells and was blocked when CD8+ cells were depleted by an antibody. In addition, cured mice were resistant to re-implantation of 66C14 cells not expressing mesothelin indicating that a long-term anti-tumor immunity was formed. To determine if pathogen associated molecular patterns contributed to the response, we combined anti-CTLA-4 with an inactive RG7787 and observed a major reduction in anti-tumor activity (P<0.01) indicating that the effect is not mediated by recognition of a foreign bacterial protein.

Conclusions: We suggest that injecting immunotoxins into tumors is a useful approach to boost the activity of anti-CTLA-4. Based on the synergistic anti-tumor effect demonstrated in this study we believe that there is a strong rational to explore such combination therapies in patients.

#59

Engineering glypican-3 targeting immunotoxins for the treatment of liver cancer.

Bryan D. Fleming, Brittany Nixon, Ira Pastan, Mitchell Ho. _National Cancer Institute, Bethesda, MD_.

The expression of glypican-3 (GPC3) in hepatocellular carcinoma offers a target with high tissue specificity and cell signaling implications. A human single domain antibody discovered by phage display technology, HN3, was fused to domain II and domain III of Pseudomonas exotoxin A. This protein (HN3-PE38) showed a high level of cytotoxic activity with a IC50 of 0.4 nM on Hep3B cells, but a relatively low maximum tolerated dose of 0.8 mg/kg in mice. In order to produce an immunotoxin with reduced toxicity, a new version was constructed that removed domain II and seven B cell epitopes from the Pseudomonas toxin. This deimmunized immunotoxin (HN3-mPE24) was shown to have a similar IC50 of 0.2 nM on Hep3B cells. To determine if further deimmunization was possible, three new versions have been generated with T cell epitopes or a series of both B and T cell epitopes removed. These include the HN3-T20 immunotoxin which had six T cell epitopes removed, HN3-T19 with 4 B cell, 4 T cell and 2 shared epitopes, and HN3-M11 with 5 B cell, 4 T cell and 2 shared epitopes. A comparative analysis of these immunotoxins was made using in vitro cell proliferation assays using Hep3B. Both the HN3-T20 (IC50 = 0.6 nM) and HN3-T19 (IC50 = 0.8 nM) immunotoxins had similar activity to HN3-mPE24 (IC50 = 0.7 nM) in a side by side comparison. The HN3-M11 variant had poor cytotoxicity and was excluded from in vivo examination. A Hep3B subcutaneous xenograft model was generated in athymic nude mice and was followed by nine rounds of intravenous immunotoxin treatments. The mPE24, T20 and T19 immunotoxins all showed an increase in average survival rate 70 days (mPE24), 76 days (T20) and 72 days (T19) when compared to the 55 days for PE38 and the 41 days for PBS alone. Additionally, the T20 and the T19 showed a maximum tolerated dose that was similar to that of the mPE24 with dosages as high as 10 mg/kg being well tolerated. This data would suggest that the HN3-T19 immunotoxins has potential clinical applications because it represents the most deimmunized immunotoxin to date.

#60

Reducing toxicity of antibody-drug conjugates through modulation of pharmacokinetics.

Jessica Simmons, Francisco Zapata, Haley Neff-Laford, Joshua Hunter, Julia Cochran, Patrick Burke, Robert P. Lyon. _Seattle Genetics, Bothell, WA_.

Antibody-drug conjugates (ADCs) continue to emerge as effective therapeutics in a variety of oncology indications. Research on ADCs has revealed that the physicochemical properties of the drug-linker component can exert a significant impact on the disposition of the ADCs, particularly at higher levels of drug loading. We have recently reported (Nature Biotechnology 33, 733-735 (2015); Molecular Cancer Therapeutics, manuscript accepted) that these properties can be modulated through the judicious incorporation of small, discrete PEG chains of varying lengths into a monomethyl auristatin E (MMAE) drug-linker. Homogeneous DAR 8 ADCs prepared with these drug-linkers using native cysteine conjugation display a continuum of pharmacokinetic behaviors that mirror the length of the incorporated PEG chain. We selected four of these drug-linkers that span the range of observed pharmacokinetics and used them as model compounds to evaluate the impact of ADC clearance on the concentration profile of released MMAE in normal tissues and consequent toxicology in the Sprague-Dawley rat. Faster clearing ADCs (prepared with drug-linkers containing very short or no PEG modifier) produced higher tissue MMAE Cmax values at early post-dose time points relative to slower clearing ADCs that incorporate longer PEG chains. This finding indicates that MMAE concentrations in tissues are proportional to the rate at which the ADC is catabolized, a process which converts the conjugated payload into free drug. Faster clearing ADCs also exhibited diminished tolerability, with greater histologic depletion of bone marrow and more dramatic decreases and/or delayed recovery in select peripheral hematology parameters. These results provide a strategy for reducing the non-antigen-mediated toxicity of ADCs through modulation of pharmacokinetics.

#61

Zymelink drug conjugate platform: redefining the therapeutic window for ADCs.

Stuart Barnscher, John Babcook, Jamie Rich, Geoff Winters, Graham Garnett, Andrea Hernandez, Vincent Fung, Kevin Yin, Kevin Hamblett, Rupert Davies. _Zymeworks, Vancouver, British Columbia, Canada_.

Antibody drug conjugates (ADCs) combine the specificity of monoclonal antibodies with potent antineoplastic small molecules, and promise efficacy without the systemic toxicity of chemotherapy. Despite this tremendous potential, most clinical ADCs have failed to provide sufficient therapeutic benefit before the onset of off-target dose-limiting platform toxicities.

Here we report the development of proprietary protease cleavable N-acyl sulfonamide linked hemiasterlin and auristatin payloads, Zymelink, that allow the generation of an efficacious ADC platform with improved tolerability. Both Zymelink drug-linkers were conjugated via maleimides to endogenous cysteines. As Zymelink drug-linkers are more polar than maleimide valine citrulline monomethyl auristatin E drug-linker, the resulting ADCs can be produced with potential advantages in PK, efficacy and safety. The resulting ADCs exhibit potent in vitro cytotoxicity. A trastuzumab-based ADC prepared with Zymelink drug-linker and demonstrated at least equivalent efficacy compared to a trastuzumab-based ADC prepared with MMAE, promoting durable complete regressions in a patient-derived xenograft model.

Zymelink hemiasterlin and auristatin ADCs were tolerated at 5-6 fold higher doses compared to a MMAE ADC in cynomolgus monkeys. The maximum tolerated dose of the MMAE ADC was just 3 mg/kg based on severe neutropenia. Zymelink hemiasterlin ADC was tolerated at 15 mg/kg with no evidence of neutropenia or elevations in transaminases. Zymelink auristatin ADC was tolerated at 18 mg/kg based on increased levels of transaminases at 24 mg/kg. Moreover, Zymelink ADCs exhibited greater serum exposure at equivalent doses. These results suggest Zymelink ADCs have a greatly expanded therapeutic window compared to MMAE conjugates.

#62

CD19 - a potential target for Amanitin-based ADCs.

Torsten Hechler, Aniko Palfi, Christoph Müller, Christian Lutz, Andreas Pahl, Michael Kulke. _Heidelberg Pharma, Ladenburg, Germany_.

Background

ATACs (antibody-targeted Amanitin conjugates) comprise a new class of antibody-drug conjugates using amanitin as toxic payload. Amanitin binds to the eukaryotic RNA pol II and thereby inhibits the cellular transcription process at very low concentrations. In the current study, in vitro and in vivo data of new ATACs targeting CD19 (also known as B4, CVID3) are presented. CD19, a class I transmembrane glycoprotein with no significant homology to any known protein, is expressed in B cells and B-cell malignancies like in B-cell acute lymphocytic leukemia (B-ALL) and B cell chronic lymphocytic leukemia (B-CLL). Therefore it is an ideal target for Amanitin based ADCs.

Material and methods

Cell lines: Raji (Burkitt Lymphoma), Nalm-6 (B-ALL) and HL-60 (CD19-negative cell line).

Antibody: anti-CD19 Thiomab (licensed from the German Cancer Research Center; Heidelberg, Germany).

Synthesis of CD19-Thiomab-ATACs: Maleimide amanitin compounds were conjugated site specifically to engineered cysteine residues of the anti-CD19 Thiomab.

Cell proliferation assay: Quantitative determination of cytotoxicity was performed by CellTiter Glo 2.0 assay (Promega) or WST-1 assay (Roche).

Animal models: Mouse xenograft tumor models were performed. Tolerability was assessed in mice and non-human primates (NHP).

Results

CD19-Thiomab-ATACs showed in vitro cytotoxicity on CD19+ cell lines in picomolar range, whereas no cytotoxic activity on CD19- cells was observed.

In mouse xenograft models, CD19 showed dose-dependent tumor regression and complete remission after single dose i.v. of 2mg/kg and 4mg/kg. In a disseminating Raji xenograft model, median survival was increased from 6 days (vehicle control) to 105 days (CD19-Thiomab-30.2115 treated group at 6mg/kg, single dose). On day 154, 4 animals were still alive and in good overall condition.

Safety profiling in Cynomolgus monkey revealed a good tolerability after sequentially applied doses of 0.3, 1.0, 3.0 and 5.0 mg/kg. Hematology and clinical chemistry data show minor alterations: transient increase in liver enzymes (ALT and AST) in combination with a transient increase in LDH. The half-life of the ADC in serum is 7-11 days; the free toxin is detectable at serum levels close to the lower limit of quantification only (LLOQ = 1.2 nM).

Conclusions

In the current study, in vitro and in vivo data of Amanitin-ADCs targeting CD19 are presented. CD19 is expressed on cells of the B cell lineage, ranging from the pre-B cells until the terminal differentiation to plasma cells. It is expressed in most acute lymphoblastic leukemias (ALL), chronic lymphocytic leukemias (CLL) and B cell lymphomas (Kemeng et al. (2012); Exp Hematol Oncol 1: 36). The preclinical data show high cytotoxicity in picomolar range, very good efficacy in tumor xenograft models and tolerability in an exploratory tolerability study in NHP. The positive findings of these initial experiments encourage Heidelberg Pharma to further proceed with anti-CD19-Amanitin-based ADCs towards clinics.

#63

Targeting osteosarcoma with graphene oxide-associated anti-HER2 antibodies.

Xinjian Chen. _University of Utah, Salt Lake City, UT_.

Osteosarcoma (OS) is the most common primary cancer of the bone affecting children and adolescents. While over the last two decades the neoadjuvant chemotherapy has improved survival of the patients with resectable OS, the prognosis for unresectable or recurrent tumors remains poor due to the lack of effective treatment. Given that a majority osteosarcomas overexpress HER2, a phase II clinical trial of anti-HER2 antibody trastuzumab in conjunction with chemotherapy has been carried out to treat metastatic OS. However, no significant therapeutic benefit was observed. We recently reported that association of anti-CD20 antibody rituximab with a nanomaterial graphene oxide (GO) substantially enhances the anti-lymphoma activity of the antibody. Here we have studied antitumor activity of GO-associated trastuzumab (TRA). Similar to RTX, TRA could be stably associated with functionalized GO through non-covalent interactions, and GO-associated TRA (TRA/GO) showed markedly enhanced HER2 binding activity with capacity to aggregate (cap) HER2 on the target cells. Treating HER2+ OS as well as Ewing's sarcoma cell lines in culture with TRA/GO resulted in rapid sarcoma cell death within 12 hr, while free TRA or the cytotoxin-conjugated TRA (ado-trastuzumab emtansine) showed no significant cytotoxic effects. TRA/GO manifested no cytotoxicity to human lymphocytes, in contrast to chemotherapeutic drugs such as doxorubicin and oxaliplatin. We find that the ability to kill the sarcoma cells results from the unique capacity of TRA/GO to simultaneously induce oxidative stress as well as intense detrimental HER2 signaling, which leads to a complete disappearance of a major tyrosine-phosphorylated protein and caspase 8, and a partial loss of RIPK1 along with an increase in RIPK3 levels within 5 min, followed by necroptosis of the target cells. Intravenous administration of TRA/GO rapidly eradicated established xenograft human OS in the lung as well as at subcutaneous locations in NOD/ragko/gko immunodeficient mice in the absence of chemotherapy, resulting in indefinite survival of the animals. In contrast, free TRA treatment failed to do so. No appreciable side effects were observed of TRA/GO in vivo. These results therefore demonstrate a novel strategy to substantially enhance the therapeutic capacity of anti-cancer antibodies. Given the increasing variety of human malignancies that are found to overexpress Her2, including sarcomas, carcinomas and neurological malignancies, our findings should have broad therapeutic implications. As TRA/GO does not harm lymphocytes, TRA/GO-based therapy may constitute a unique opportunity to implement immunotherapy that is now known to play an important role in control tumor progression.

#64

**Meditope SnAP-body technology facilitates enhanced internalization and** in vivo **efficacy of antibody therapeutics.**

Karin Forster, Elisabeth Gardiner, Stephanie Hsieh. _Meditope Biosciences, San Diego, CA_.

Meditope Biosciences has developed a way to use SnAP technology to promote internalization of antibody receptor complexes through a novel construct called a SnAP-body. Meditope's SnAP platform functionalizes monoclonal antibodies to bind to specific meditope peptides; this property can be used to directly facilitate receptor crosslinking when meditope-enabled antibodies are bound to cell surface receptors. SnAP-bodies are self-crosslinking meditope-enabled antibodies which permit a meditope peptide to specifically interact with a meditope-enabled antibody at the cell surface upon antigen engagement. Antigen-antibody complex formation through the specific contact of the SnAP-bodies and their cognate epitopes on the target antigen can increase clustering or co-localization of a cell surface antigen on a cell, which can promote enhanced internalization of the SnAP-body. The increased internalization can be used to clear or reduce the concentration of a cell surface antigen from the surface of a target cell or tumor. Enhancing internalization can increase the efficacy of antibody-based therapeutics, and in the case of antibody-drug conjugates, which require receptor internalization to deliver cytotoxic payloads, can reduce the amount of drug necessary to achieve a therapeutic effect. In vitro studies of SnAP-body binding to cell surface antigens demonstrate accelerated internalization of the antibody. In vivo, this translates into enhanced efficacy. The ability to see equivalent efficacy in the absence of toxin suggests a unique role for SnAP-body constructs in cancer therapeutics, especially in cases where lack of efficacy for a specific target is correlated with poor receptor internalization.

#65

RESPECT (REsidue-SPEcific Conjugation Technology): A platform technology utilizing native cysteine and lysine residues for the generation of homogeneous antibody-drug conjugates.

Earl Albone, Jared Spidel, Xin Cheng, Young Chul Park, Sara Jacob, Arielle Verdi, Andrew Milinichik, Ben Vaessen, J. Bradford Kline, Luigi Grasso. _Morphotek, Inc, Exton, PA_.

The random conjugation of toxins, dyes, peptides, or other payloads to monoclonal antibodies often targets free thiol groups generated by partial reduction methods or lysine residues using succinimide- or isothiocyanate-based chemistry. There remains a need for conjugation technologies targeting specific amino acid residues as a way to produce a homogeneous antibody-drug conjugate (ADC) product with a defined drug-to-antibody ratio (DAR). Our REsidue-SPEcific Conjugation Technology (RESPECT) utilizes two methods by which payloads can be conjugated to specific residues in an antibody. Our cysteine-specific conjugation method exploits a unique intrachain disulfide bond in the light chain of rabbit antibodies between residues 80 and 171 of the variable and constant domains, respectively. Our humanization strategy allows retention of the cysteine at position 80 with a free thiol group that is both amenable for residue-specific conjugation and compatible with optimal antibody biophysical properties including antigen binding and structural stability. This platform has been optimized via antibody engineering strategy stems from in silico modeling, extensive mutagenesis, and crystallographic studies, which have allowed defining the contribution of neighboring residues to the retention of a reactive thiol group as well as the desired humanized antibody's properties. Our C-terminal lysine-specific linkage method employs the transglutaminase enzyme that catalyzes the formation of a stable isopeptide bond between the γ-carboxyamide group (acyl donor) of a glutamine and the ε-amino group (acyl acceptor) of a lysine. While we found no acyl acceptor sites in recombinant wild-type IgG, all antibodies investigated lacked the C-terminal Lys447 due to cleavage by carboxypeptidase B in the antibody production cell line. Blocking the cleavage of Lys447 by addition of a C-terminal amino acid resulted in transamidation of Lys447 by a variety of acyl donor substrates in the presence of any non-acidic, non-proline amino acid residue at position 448. Antibody-drug conjugates (ADCs) prepared using our RESPECT technology targeting the tumor associated-mesothelin protein produced uniform drug-to-antibody ratios (DAR) and were shown to be highly potent and specific in vitro and effective in vivo in reduction of tumor growth in a highly aggressive mesothelin-expressing xenograft tumor model.

#66

Anthracycline-based antibody drug conjugates with potent immune-stimulatory functions.

Roger R. Beerli. _NBE-Therapeutics AG, Basel, Switzerland_.

Antibody drug conjugates (ADCs) are highly potent and selective anti-tumor drugs, combining the specific targeting of monoclonal antibodies with the potency of small molecule toxic payloads. Here, we employed enzymatic, site-specific conjugation to generate homogenous ADCs based on a derivative of the highly potent anthracycline toxin PNU-159682 and a non-cleavable peptide linker, using the anti-HER-2 antibody trastuzumab (part of trastuzumab emtansine) and the anti-CD30 antibody cAC10 (part of brentuximab vedotin). Characterization of the resulting ADCs in vitro and in vivo showed that they were highly stable and exhibited potencies exceeding those of ADCs based on conventional tubulin-targeting payloads, such as trastuzumab emtansine and brentuximab vedotin. Anti-tumor activity in an immune-competent host involved activation of the immune system, as shown by evaluation of a trastuzumab-PNU ADC in a Kadcyla-resistant HER2-positive orthotopic breast cancer model. Depletion of CD8 T cells severely reduced the anti-tumor activity of the ADC, demonstrating an important role for T cells in driving tumor regression. Furthermore, when tumor free animals were re-challenged with the same tumor, tumor growth was entirely inhibited in the absence of any further ADC administration, indicating the development of an immunological memory. In summary, we present a novel ADC format endowed not only with highly potent cytotoxicity, but also effective immune-stimulatory functions.

#67

Characterization and preclinical development of STRO-001, a novel CD74-targeting antibody-drug conjugate (ADC) for the treatment of B-cell malignancies.

Cristina Abrahams, Xiaofan Li, Venita DeAlmeida, Millicent Embry, Abigail Yu, Stellanie Krim, Heidi Hoffmann, James Zawada, Maureen Bruhns, Shannon Matheny, Stuart Bussell, Toni Kline, Alice Yam, Ryan Stafford, Trevor Hallam, Mark Lupher, Arturo Molina. _Sutro Biopharma, South San Francisco, CA_.

CD74 is a type II transmembrane glycoprotein involved in the formation and transport of MHC class II protein. CD74 is highly expressed in many B-cell malignancies with limited expression in normal tissues (Stein R. et al., CCR 2007). STRO-001 is a novel CD74-targeting ADC containing an anti-CD74 aglycosylated human IgG1 antibody (SP7219) conjugated to a non-cleavable dibenzocyclooctyne (DBCO)-maytansinoid linker-warhead. SP7219 was discovered from a Fab ribosome display library based on Sutro's Xpress CFTM technology. Highly efficient site-specific conjugation enabled by our cell-free antibody production and click chemistry results in a well-defined homogeneous ADC drug product with a drug-antibody ratio (DAR) of 2. Conjugation sites were selected based on highest stability both in vitro and in vivo, thereby limiting loss of drug moiety from STRO-001 in circulation. Due to its limited cell permeability, the major catabolite released by STRO-001 has 1000X lower cell killing activity on CD74 positive and negative cells compared to the reference cytotoxic maytansine. In vitro cytotoxicity assays show potent activity of STRO-001 in a diverse panel of B-cell tumor lines including 4 multiple myeloma (MM), 9 germinal center B-cell (GCB) diffuse large B-cell lymphoma (DLBCL), 3 activated B-cell (ABC) DLBCL, and 3 mantle cell lymphoma (MCL) cell lines with IC50 ranging from 0.17-20 nM. CD74 cell surface expression is required for STRO-001 cytotoxic activity but expression level, as measured by antibody-binding capacity, does not correlate with in vitro potency (R2=0.4640). STRO-001 inhibits the formation of visceral tumors (p<0.004) and prevents growth of CD138+ plasma cells in bone marrow (BM) after 4 weekly doses of 3 mg/kg in the ARP-1 disseminated MM xenograft model. STRO-001 dosed at 3 mg/kg weekly x 3 also eradicates malignant BM plasma cells (p<0.0001) and prolongs survival in the MM.1S disseminated model (100% animals alive at >90 days). STRO-001 exhibits dose-dependent tumor growth inhibition in SU-DHL-6 xenografts starting at 2.5 mg/kg weekly x 3 doses. The combination of bendamustine/rituximab (BR) + STRO-001 further improves tumor suppression in SU-DHL-6 xenografts compared to vehicle (p = 0.002) or BR alone (p = 0.02). Preliminary studies with a MCL xenograft model, Jeko-1, demonstrate potent anti-tumor activity compared to vehicle (p<0.0001) starting at a single STRO-001 dose of 3 mg/kg, with ongoing tumor stasis up to 21 days after treatment. STRO-001 reduces normal B-cells in cynomologous monkeys, providing pharmacodynamic evidence of B-cell targeting. Based on these encouraging observations, STRO-001 is advancing to IND-enabling studies for the treatment of CD74-expressing B-cell malignancies.

#68

Methotrexate prevents primary immune responses against recombinant immunotoxin in murine models.

Emily M. King, Ronit Mazor, Ira Pastan. _National Cancer Institute, NIH, Bethesda, MD_.

Recombinant immunotoxins (rITs) are composed of a tumor antigen-targeting antibody fragment fused to a portion of Pseudomonas exotoxin A. rITs have been effective in clinical trials for patients with hematologic malignancies. The CD22 targeting rIT Moxetumomab Pasudotox has achieved overall response rates of 86% and complete remission rates of 46% in patients with relapsed-refractory Hairy Cell Leukemia. However, the therapeutic efficacy of rITs against solid tumors is limited by their immunogenicity in immune-competent patients. In clinical trials to treat mesothelioma patients with SS1P, a rIT targeting mesothelin, 90% of patients developed neutralizing antibodies against SS1P after one cycle of treatment. When immunosuppressive chemotherapy and SS1P were combined, more cycles of rIT could be given and several patients with advanced chemo-refractory mesothelioma had striking tumor regressions. This implicates high therapeutic potential for rITs against solid tumors once immunogenicity is surmounted.

Methotrexate (MTX) is a folate antagonist which interferes with purine biosynthesis, and is used to treat osteosarcomas and other cancers. MTX also interferes with T cell responses and is used to treat autoimmune diseases. Based on its immunosuppressive properties, Joly et al. demonstrated that low-dose MTX prevented the formation of ADAs against recombinant human alglucosidase alfa in mice in an antigen-specific manner. We hypothesized that MTX would similarly prevent the formation of ADAs against rITs in an antigen-specific manner. To test our hypotheses, mice were treated with the mesothelin-targeting rIT RG7787 with or without MTX given 0, 24, and 48 hours after RG7787 treatment. Serum was collected and anti-RG7787 ADAs were measured by direct ELISA. We found that six doses of RG7787 combined with low dose MTX (1 mg/kg) inhibited the formation of ADAs against RG7787. This inhibition was sustained through six challenges with RG7787 without additional MTX. Further, we found that immunization with RG7787 plus MTX induced RG7787-specific tolerance, and had no effect on the ADA response against a second protein, ovalbumin.

We conclude that combination of MTX and RG7787 is effective at preventing primary immune responses in a durable, antigen-specific manner. We propose to combine this agent in immune-competent cancer patients receiving rIT therapy to prevent rIT immunogenicity.

#69

Anti-podocalyxin cancer-specific monoclonal antibody: preclinical study.

Shinji Yamada. _Tohoku University, Sendai, Japan_.

Background: Podocalyxin, a CD34-related sialomucin, is expressed in many tumors including colorectal cancers, breast cancers, mesothelial tumors, and brain tumors. Overexpression of podocalyxin has been reported to be an independent predictor of progression, metastasis, and poor outcome. However, PODXL is known to be highly expressed in normal cells including many epithelial cells and endothelial cells; therefore, podocalyxin could not be a target of antibody therapy. Although many monoclonal antibodies (mAbs) against podocalyxin have been established, they bind to both cancer and normal cells. We recently established a novel technology for developing cancer-specific mAbs (CasMabs), which could target only cancer cells although those membrane proteins are highly expressed in both cancer and normal cells.

Methods: We first produced a podocalyxin-expressing glioblastoma cell line. We purified human podocalyxin using PA-tag, and immunized mice with those proteins. CasMabs were screened using flow cytometry against podocalyxin-expressing cancer cells and podocalyxin-expressing normal cells. The cancer specificity was confirmed using immunohistochemistry against breast cancer tissues. Furthermore, a human-mouse chimeric anti-podocalyxin mAb was produced. Antibody-dependent cellular cytotoxicity (ADCC) was investigated in vitro using glioblastoma cells as target cells and human NK cells as effector cells. The in vivo efficacy was evaluated using xenograft models of podocalyxin-expressing cell lines. Furthermore, we investigated the toxicity of chPcMab-6 using cynomolgus monkey.

Results: A cancer-specific anti-podocalyxin mAb (clone: PcMab-6; mouse IgG1, kappa) was established. PcMab-6 reacted with podocalyxin-expressing many cancer cell lines including brain tumors, malignant mesotheliomas, colon cancers, and pancreatic cancers whereas it did not bind to vascular endothelial cells in flow cytometry. Furthermore, PcMab-6 reacted only with podocalyxin-expressing cancer cells, not with VECs in breast cancer tissues using immunohistochemistry. Furthermore, we produced chPcMab-6, a human-mouse chimeric mAb of PcMab-6, which also reacts with only podocalyxin-expressing cancer cells. ChPcMab-6 possesses ADCC activity in vitro and antitumor effect in vivo. Single injection of chPcMab-6 induced no clinical abnormality in male cynomolgus monkey (2 mg/kg and 20 mg/kg) . In addition, abnormal values were not observed in hematologic test, urine analysis, and measurement of serum inflammatory cytokines. Tissue disorders were not found in histology and relative organ weight on postmortem examination.

Conclusion: ChPcMab-6, a cancer-specific human-mouse chimeric anti-podocalyxin mAb could be useful for targeting podocalyxin in cancer, although podocalyxin is highly expressed in many normal cells.

#70

Elucidating the roles of antibody pharmacokinetics and maleimide stability in the toxicology of antibody-drug conjugates.

Haley Neff-LaFord, Franciso Zapata, Wendi Schultz, Cindy Balasubramanian, Paul Pittmen, Shawna Hengel, Russell Sanderson, Nagendra Chemuturi, Jocelyn Setter, Robert P. Lyon. _Seattle Genetics, Bothell, WA_.

Antibody-drug conjugates (ADCs) continue to emerge as effective therapeutics in a variety of oncology indications, with two agents currently approved and many more in late-stage clinical trials. These ADCs employ drug-linkers that were developed many years ago, and are now recognized to have properties that may adversely impact the activity and toxicology of the ADCs prepared with them. Two such properties that are now well appreciated are the reversibility of maleimide-based drug conjugation, and the impact of drug conjugation on the pharmacokinetics of the ADC. We recently reported advances in drug-linker design that independently address both of these properties, resulting in the irreversible conjugation of drugs which have minimal impact on antibody pharmacokinetics, even at high levels of drug loading (Nature Biotechnology 32, 1059-1062 (2014), Nature Biotechnology 33, 733-735 (2015)). We have now prepared drug-linkers of monomethylauristatin E (MMAE) that orthogonally employ these features to enable a systematic evaluation of the relative contributions of maleimide instability and accelerated plasma clearance on the in vivo behavior of MMAE ADCs. Biodistribution studies with these molecules have revealed that the concentration of released MMAE in normal tissues is greatly impacted by the rate of ADC clearance (fast clearance results in greater Cmax of free drug), while stabilization of the maleimide has a relatively small effect. These differences in observed free drug concentrations were paralleled in tolerability studies, with ADC clearance rates exerting a greater impact on hematology parameters than maleimide stability. Collectively, these results suggest that ADC pharmacokinetics dominate the biodistribution and toxicology profiles for a given drug payload, with conjugate stability playing a relatively minor role.

#71

**Bystander activity and** in vivo **efficacy of a folate receptor α (FRα)-targeting antibody-drug conjugate with a novel peptide linker.**

Qifeng Qiu, Rui Wu, Leanne Lanieri, Erin Maloney, Anna Skaletskaya, Shan Jin, Lintao Wang, Olga Ab, Joe Ponte, Yulius Setiady, Wayne Widdison, Thomas Keating, Ravi Chari, Richard Gregory, Erica Hong. _ImmunoGen, Inc, Waltham, MA_.

Folate receptor α (FRα) is an antigen that is overexpressed on the cell surface of solid tumors including ovarian cancer. The differential expression on cancer cells makes FRα an attractive target for antibody-drug conjugates (ADCs), and an ADC targeting FRα, Mirvetuximab soravtansine, has demonstrated promising activity and safety profiles in the clinic. Here, we employed a new linker (NL) to enhance the bystander activity of ADCs, which is the ability of ADCs to generate cell-permeable catabolites that can diffuse into and kill proximal cancer cells with little or no target expression. With the goal of improving the potency of anti-FRα ADC in solid tumors with heterogeneous FRα expression, we constructed the M9346A-NL-DM. M9346A-NL-DM is a conjugate of the tubulin-disrupting maytansinoid (DM), linked via a novel linker to M9346A, a humanized antibody that binds FRα with high affinity. After cellular uptake and catabolism of the ADC, the new linker undergoes lysosomal cleavage followed by self-immolation to generate free DM that can readily penetrate neighboring cancer cells. Correspondingly, M9346A-NL-DM showed enhanced bystander cytotoxic activity against proximal antigen-negative cells in vitro. In the xenograft tumor models in vivo, M9346A-NL-DM demonstrated enhanced efficacy against tumors with heterogeneous expression of FRα. Interestingly, M9346A-NL-DM also showed improved antitumor activity against a tumor model with homogeneous expression of FRα, possibly due to better tumor penetration of the cell-permeable catabolite. In summary, M9346A-NL-DM is a novel ADC with enhanced bystander activity and antitumor activity that can target tumors with heterogeneous expression of FRα.

#72

Induction of immune tolerance to recombinant immunotoxin LMB-100 using synthetic vaccine particles encapsulating rapamycin.

Ronit Mazor,1 Emily King,1 Takashi Kei Kishimoto,2 Ira Pastan1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Selacta Bioscince Inc., Boston, MA_.

Recombinant Immunotoxins (RITs) are genetically engineered proteins designed for cancer therapy. LMB-100 is a second generation RIT that is composed of a humanized Fab targeting mesothelin and a de-immunized fragment of Pseudomonas Exotoxin A. Due to the bacterial origin of the toxin, LMB-100 is immunogenic, although less immunogenic than first generation immunotoxins. Almost all patients treated with LMB-100 made anti-drug antibodies (ADAs) after two or more cycles of treatment that neutralized the RIT and greatly lowered efficacy. Kishimoto et al. demonstrated that Synthetic Vaccine Particles containing Rapamycin (SVP-R) inhibited the formation of ADAs, when administered with a foreign protein such as KLH or Pegsiticase. The SVP-R are taken up by macrophages and dendritic cells and increase the number of regulatory T cells in treated mice. Here we evaluated the efficacy of combination therapy of LMB-100 and SVP-R to eradicate mesothelin expressing tumors while preventing ADA formation. To evaluate if SVP-R can prevent ADAs against LMB-100, we immunized immune-competent mice with a combination of SVP-R and RIT and measured ADAs titers by ELISA and by a functional neutralization assay. We found that the treatment reduced ADA titers by more than 99%. To determine if the mice were tolerized to LMB-100, we treated the mice with two doses of SVP-R and six doses of LMB-100 to induce tolerance; and followed by nine challenges of LMB-100 given over 6 weeks. We found that the ADAs were reduced by 98%, indicating development of immunological tolerance to LMB-100. To show that the tolerance is transferable, we tolerized the mice with six doses of SVP-R and LMB-100 and adoptively transferred their splenocytes to naïve recipient mice. The recipient mice were challenged with six doses of LMB-100 and ADA titers were measured. We found that splenocytes from tolerized mice induced a 66% decrease in ADA formation in the recipient mice indicating that the tolerance was mediated by cells of the immune system. To demonstrate that immune suppression is useful in treating tumors, we implanted mouse breast cancer cells expressing human mesothelin into Balb/c mice with a normal immune system and treated them with LMB-100. LMB-100 produced tumor regressions when given before ADAs developed, but was inactive in mice with pre-existing ADAs. However, when mice with preexisting ADAs were treated with LMB-100 and SVP-R, anti-tumor activity was restored and ADAs suppressed (P≤0.0001). SVP-R are being evaluated in humans to prevent ADA to Pegsiticase, an enzyme for refractory gout treatment. Our data indicates that combining SVP-R with LMB-100 should be useful in treating cancer by allowing more treatment cycles and better efficacy. This approach can be used to increase the efficacy of other immunogenic agents such as CAR-T cells, antibody drug conjugates and viral gene therapy vectors

#73

Role of macrophages in the antitumor activity of an anti-transferrin receptor 1 antibody ch128.1 in a xenograft model of multiple myeloma.

Lai Sum Leoh, Yoon Kyung Kim, Pierre V. Candelaria, Otoniel Martínez-Maza, Tracy R. Daniels-Wells, Manuel L. Penichet. _UCLA, Los Angeles, CA_.

The transferrin receptor 1 (TfR1), also known as CD71, is a membrane glycoprotein involved in cellular iron uptake and regulation of cell growth. The high level of TfR1 expression on malignant cells and its key role in cancer cell pathology make this receptor an attractive target for antibody cancer therapy. We previously developed a mouse/human chimeric IgG3 specific for human TfR1 (ch128.1). This antibody exhibits direct cytotoxicity against certain human malignant B cells in vitro through TfR1 degradation and iron deprivation. Importantly, ch128.1 shows remarkable anti-tumor activity in xenograft models of disseminated multiple myeloma (MM) in immunosuppressed mice (SCID-Beige). Interestingly, this anti-tumor protection was observed even against MM cells (KMS-11 cells) that show no sensitivity to this antibody in vitro, suggesting the in vivo contributions of antibody effector functions. This possibility was supported by the lack of anti-tumor protection observed using a ch128.1 Fc mutant with impaired binding to FcγRs and to the complement component C1q. To examine host effector functions involved in ch128.1-mediated protection in our mouse model bearing KMS-11 tumors, depletion studies of complement and macrophages were performed. Complement depletion using cobra venom factor (CVF) did not affect protection, suggesting that complement-mediated cytotoxicity (CDC) is not a relevant mechanism of action. Notably, we now report that macrophage depletion using clodronate liposomes (clodrolip) significantly reduced protection, suggesting that these effector cells play a relevant role in the anti-tumor activity. Consistent with this result, we also report that ch128.1 is capable of eliciting antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) against KMS-11 cells in the presence of the murine macrophage cell line J774.2 or murine bone marrow-derived macrophages. To examine the role of iron deprivation in ch128.1-mediated tumor death in vivo, animals treated with ch128.1 were systemically supplemented with iron in a preliminary study. However, no difference in survival was observed, suggesting that iron deprivation is not a contributor to the effects of ch128.1 in our in vivo model or the iron dose tested was not optimal. Our results suggest that macrophages play a key role in ch128.1-mediated anti-tumor protection in our model and that ch128.1 can be an effective therapy of incurable human B-cell malignancies such as MM.

#74

Discovery of new therapeutic monoclonal antibodies to challenging GPCRs, ion channels and transporters.

Lewis J. Stafford, Ross Chambers, Sharon H. Willis, Moniquetta Hall, Brad Screnci, Manu Mabila, David Tucker, Trevor Barnes, Rachel Fong, Andrew Ettenger, Jennifer Pfaff, Chidananda Sulli, Nicholas Molino, Andrew Hudacek, Benjamin J. Doranz, Joseph Rucker. _Integral Molecular, Philadelphia, PA_.

The objective of this work was to evaluate the ability to generate panels of monoclonal antibodies against a set of highly challenging targets including GPCRs (CB1, C5AR, CXCR5 and CGRPR), transporters (GLUT4), and ion channels (P2X3). Integral membrane proteins are important drug targets and monoclonal antibodies (MAbs) directed against them are highly sought for therapeutic purposes. However, the complex structure of multispan membrane protein targets makes the discovery of these MAbs especially challenging. To address this need, Integral Molecular has developed the MPS Discovery Engine® to enable the isolation, characterization, and engineering of monoclonal antibodies for GPCRs, ion channels, and transporters. MPS utilizes a collection of technologies to address each of the barriers to monoclonal antibody development against the native extracellular epitopes of multispan membrane proteins. These include, antigen engineering to attain high levels of surface expression, DNA and Lipoparticle immunization to present native epitopes to the immune system, diverse immunization host species to deal with highly conserved proteins, Lipoparticles (high concentration native membrane proteins) to enable phage display and microfludic B-cell isolation, and shotgun mutagenesis (comprehensive Alanine scanning) for epitope mapping. Using the MPS Discovery Engine® we were able to successfully generate large panels of antibodies to the targets that were able to bind to the native extracellular epitopes on cells by flow cytometry. A subset of the antibodies had antagonist activity. With this technology we have the ability to target intact, conformation specific, and functional antibodies to complex membrane proteins.

#75

Comparison of site-specific and lysine-linked indolino-benzodiazepine antibody-drug conjugates (ADCs).

Chen Bai,1 Nicholas C. Yoder,1 Alan Wilhelm,1 Sharlene Adams,1 Kathleen Whiteman,2 Jenny Lee,1 Katie O'Callaghan,2 Erin Maloney,1 Manami Shizuka,1 Yelena Kovtun,1 Thomas A. Keating1. 1 _ImmunoGen, Inc., Waltham, MA;_ 2 _Current Address: Unum Therapeutics Inc., Cambridge, MA_.

ADCs are a promising modality for cancer therapy enabled by chemical conjugation of potent cytotoxic compounds to monoclonal antibodies. While many ADCs in clinical evaluation employ heterogeneous conjugation chemistries where the payload is linked through lysine or endogenous cysteine residues, there has recently been considerable interest in site-specific conjugation. ADCs prepared using site-specific methods are believed to have a wider therapeutic index compared to heterogeneous ADCs. We have previously shown that site-specific ADCs incorporating the maytansinoid-based tubulin inhibitor DM1 were less efficacious in vivo when compared to analogous lysine-linked conjugates (Yoder et al AACR 2015 Abstract #545). More recently, we presented results from the evaluation of 2.5-3.0 drug-to-antibody ratio (DAR) heterogeneous lysine-linked and 2 DAR site-specific ADCs using antibodies with engineered cysteines at position 442 in the heavy chains (known as CYSMABTM). These ADCs used the peptide-linked indolino-benzodiazepine DNA alkylator DGN549 (also known as IGN-P1) as the effector. Unlike the DM1 case, site-specific DGN549 ADCs were at least as active in vivo when compared to lysine-linked ADCs (Yoder et al AACR 2016 Abstract #2960). We have made further pharmacological comparisons between CYSMAB and lysine-linked DGN549 ADCs at matched payload doses using two different antibodies targeting distinct cell surface receptors. In the case of mAb1, CYSMAB and lysine-linked ADCs were comparably active. For mAb2, the CYSMAB ADC was more active than the lysine-linked ADC in some models and similarly active in others. The mAb1 CYSMAB ADC exhibited a significantly higher maximum tolerated dose (MTD) compared to the lysine-linked ADC. In contrast, the MTDs of the mAb2 ADCs were similar. However, the mAb2 CYSMAB conjugate was better tolerated in terms of median lethal dose. The mechanism for the improved tolerability of the mAb1 CYSMAB conjugate is not apparent. In an effort to understand whether it is a consequence of conjugation chemistry or DAR, we compared the tolerability of the mAb1 CYSMAB conjugate to that of a ~4 DAR site-specific analog and found that both factors contribute. To determine if our observations can be rationalized in terms of in vivo disposition, we compared the pharmacokinetics of mAb1 CYSMAB and lysine-linked ADCs at matched antibody doses. Intriguingly, the CYSMAB ADC showed slightly greater exposure. These results, along with our previous work on site-specific DM1 ADCs, indicate that in some cases site-specific conjugation can lead to improved efficacy and tolerability. However, generalizations cannot be made across different combinations of antibody, linker, and payload. We conclude that the advantages and disadvantages of site-specific conjugation should be carefully considered for every candidate ADC.

#76

**Synthetic lethal targeting of** BRCA **mutant tumors with antibody linked pyrrolobenzodiazepine dimers.**

Haihong Zhong, Ravinder Tammali, Cui Chen, Christine Fazenbaker, Kennedy Maureen, Noel Monks, Jay Harper, Ronald Herbst, Dave Tice. _Medimmune, Gaithersburg, MD_.

Pyrrolbenzodiazepine dimers (PBDs) are amongst the most potent DNA alkylating agents, with activity against a broad spectrum of tumors. PBDs form cross-links within the minor groove of DNA causing double strand breaks (DSB). DNA repair genes such as BRCA1 and BRCA2 play important roles in homologous recombination repair (HRR) of DSB. Cells defective in BRCA1 or BRCA2 are known to be sensitive to DNA interstrand crosslinks. Accordingly, it is possible that PBD-based ADCs will have enhanced killing of cells (synthetic lethality) in which HR processes are defective by inactivation of BRCA1 or BRCA2 genes in breast, ovarian and other cancers. To determine anti-tumor activity of PBD dimers, we have used MEDI0641, PBD-dimer conjugated to anti-5T4 antibody, against BRCA wild type and mutant xenograft tumor models. MEDI0641 was >3-fold more potent in BRCA1 or BRCA2 mutant models than in wild-type xenografts. Similar observations were seen in 25 patient-derived xenograft (PDX) models (19 breast and 6 ovarian) bearing mutations in BRCA1 or BRCA2 (blinded to 5T4 expression) treated with MEDI0641. Out of a total of 25 PDX models, 17 models had tumor regression with a single administration of MEDI0641 at 0.3 mg/kg (response rate = 68%), and 14 models showed response to 0.1 mg/kg of MEDI0641 (response rate = 56%). In BRCA wild-type PDX models, a higher dose of 1 mg/kg was required to achieve full anti-tumor efficacy. Retrospective analysis of 5T4 expression in PDX tumors demonstrated no correlation between efficacy and target expression in BRCA mutant PDX models. To further delineate the role of BRCA1/2 mutations in determining sensitivity to PBD, we used siRNA knock-down of both BRCA1 and BRCA2 in the DNA repair wild type HeLa cells. Knockdown of BRCA genes sensitized Hela cells to PBD payload and MEDI0641 in vitro. Anti-tumor activity of MEDI0641 was further examined in isogenic BRCA2 knockout xenograft models. Genetic deletion of BRCA2 markedly increased anti-tumor activity of MEDI0641. In conclusion, PBD based ADCs may have improved therapeutic window in cancer patients with somatic BRCA mutations.

#77

Preclinical evaluation of HDP-101, an anti-BCMA antibody-drug conjugate.

Torsten Hechler, Aniko Palfi, Christoph Müller, Christian Lutz, Andreas Pahl, Michael Kulke. _Heidelberg Pharma GmbH, Ladenburg, Germany_.

Background:

ATACs (antibody-targeted Amanitin conjugates) comprise a new class of antibody-drug conjugates using amanitin as toxic payload. Amanitin binds to the eukaryotic RNA pol II and thereby inhibits the cellular transcription process at very low concentrations. In the current study, in vitro and in vivo data of new ATACs targeting BCMA (B Cell Maturation Antigen, also known as CD269) are presented. BCMA is selectively expressed on malignant plasma cells like in multiple myeloma (MM) and hence considered an ideal target for Amanitin based ADCs.

Material and methods:

MM cell lines: NCI-H929, MM.1S Luc (stable luciferase transfected) and CCRF-CEM (BCMA negative).

Antibody: anti-BCMA Thiomab (Max Delbrück Centrum, Berlin; derivatization and production at Heidelberg Pharma).

Synthesis of HDP-101: Maleimide amanitin compounds were conjugated to substituted cysteine residues of the anti-BCMA Thiomab.

Cell proliferation assay: Quantitative determination of cytotoxicity was performed by CellTiter Glo 2.0 assay (Promega) or WST.1 assay (Roche).

Animal models: Subcutaneous and metastatic mouse xenograft tumor models with MM cell lines NCI-H929 and MM.1S Luc were performed in single-dose and multiple-dosing experiments. Tolerability was assessed in mice and nonhuman primates (NHP).

Results:

HDP-101 showed in vitro cytotoxicity on BCMA+ cell lines in picomolar range, whereas no cytotoxic activity on BCMA- cells was observed.

In mouse xenograft models, HDP-101 caused dose-dependent tumor regression and complete remission after a single i.v. dose of 2.0 mg/kg and 4.0 mg/kg in s.c. xenografts and after single i.v. doses from 0.1 mg/kg to 2.0 mg/kg in disseminating xenografts.

Safety profiling in Cynomolgus monkeys revealed a good tolerability and therapeutic index after sequentially applied doses of 0.3, 1.0, and multiple dose application of 4 x 3.0 mg/kg. Hematology and clinical chemistry parameters were unaffected except liver enzymes and LDH: A mild to moderate and transient increase was observed. The half-life of the ADC in serum was 7-9 days; the free toxin was detectable at levels close to the lower limit of quantification only (LLOQ = 1.2nM).

Conclusions:

Targeted cytotoxic drug delivery to BCMA positive MM cell lines was achieved by using HDP-101, an anti-BCMA-ATAC. The mode of action of the payload Amanitin led to an efficient anti-tumor potential in vitro and in vivo with good tolerability in NHP studies. Using ADCs in the therapy of multiple myeloma is a promising approach, especially by using a cytotoxic agent whose mode of action differs from other commonly used toxins, like ATACs. First-in-human trial is expected to start in 2018.

#78

Highly efficient antibody drug conjugation with a novel photoactivation strategy.

Elisabeth Gardiner, Karin Forster, Robert McKenzie. _Meditope Biosciences, San Diego, CA_.

Meditope SnAP (site-specific novel antibody platform) technology is a novel way to target molecules to antibodies in a site specific, non-destructive fashion. A "meditope" is a proprietary peptide that can bind a meditope-enabled antibody in a unique site and in such a way that antigen binding is not compromised. This specific peptide antibody interaction can be readily employed for site-specific covalent conjugation of cytotoxic payloads to any meditope-enabled antibody. To facilitate covalent conjugation, the meditope peptide has been rationally designed with a photoactivatable side chain and the generation of the antibody-drug conjugate (ADC) occurs in a highly efficient manner following the specific binding of the meditope to the enabled antibody. To test the hypothesis that photoactivation would be an efficacious and homogenous way to make an ADC, meditope-enabled ADCs were designed and tested in vitro and in vivo. Toxin conjugation was tested, as was efficiency of covalent interaction following short-term exposure of the antibody peptide conjugates to a nondestructive wavelength of 340 nM. Photoactivation proved to be a highly efficient and scalable way to make an ADC. Payload stability was confirmed in vivo with multiple toxins and in vivo potency of two unique ADCs were tested in an EGFR positive gastric cancer xenograft as well as a HER2 positive ovarian cancer xenograft. The meditope-enabled photogenerated anti-HER2 ADC was benchmarked against conventionally conjugated T-DM1 and showed equivalent efficacy, but with the site-directed conjugated meditope ADC using less toxin. Meditope-directed photoactivated-controlled conjugation can be readily extended to any meditope-enabled antibody, resulting in a novel way to develop ADCs. This methodology, with its high efficiency, speed and homogeneity has evident advantages for the development of antibody-drug conjugates.

#79

Trastuzumab and C6.5 diabody armed with deBouganin overcome drug resistance to ADCs comprised of anti-microtubule agents.

Shilpa Chooniedass,1 Rachelle L. Dillon,1 Arjune Premsukh,1 Gregory P. Adams,2 Glen C. MacDonald,3 Jeannick Cizeau1. 1 _Viventia Bio Inc., Winnipeg, Manitoba, Canada;_ 2 _Eleven Biotherapeutics Inc., Philadelphia, PA;_ 3 _Eleven Biotherapeutics Inc, Winnipeg, Manitoba, Canada_.

DeBouganin (deB) is a de-immunized form of bouganin, a Ribosome Inactivating Protein (RIP) that when internalized blocks protein synthesis thereby leading to cell death. When conjugated to trastuzumab (T-deB) or genetically attached to the C6.5 diabody, deBouganin was more potent than DM1 and unaffected by mechanisms of resistance to which DM1 is susceptible. To further highlight the differentiating mechanism of action of deBouganin, HCC1419 and BT-474 tumor cells that survived T-DM1 or trastuzumab-MMAE (T-MMAE) treatment in vitro were re-exposed to T-DM1, T-MMAE, or treated with T-deB or an anti-HER2 C6.5 diabody-deBouganin fusion protein. C6.5 diabody-deBouganin and T-deB were potent against HCC1419 and BT-474 cells surviving T-DM1 or T-MMAE treatment. However, the surviving cell populations were resistant to T-DM1, T-MMAE, DM1, MMAE and taxol treatment. In addition, cross-resistance was seen against trastuzumab-duocarmycin which contains a payload with a cell cycle independent mechanism of action. The contribution of multi-drug resistance, Bcl-2 family members and other survival pathways accounting for the resistant phenotype will be discussed. Overall, the data suggest that treatment with chemotherapeutics or ADCs comprised of small molecule compounds such as anti-microtubule agents, can lead to the outgrowth of tumor cells resistant to similar agents. In contrast, antibodies and antibody fragments armed with deBouganin can overcome these mechanisms of resistance and may therefore represent a more effective treatment option.

### Gene Expression of Drug Resistance

#80

Pooled shRNA screening using mouse xenografts of human hepatocellular carcinoma cells identifies CDK5 as a potential mechanism mediating sorafenib resistance.

Tzu-Hsuan Lin,1 Wen-Chi Feng,1 Yu-Yun Shao,1 Li-Chun Lu,1 Ya-Jhen Chen,2 Ann-Lii Cheng,1 Chih-Hung Hsu1. 1 _National Taiwan University College of Medicine, Taipei, Taiwan;_ 2 _National Taiwan University Hospital, Taipei, Taiwan_.

Background: Sorafenib is the only approved systemic agent for hepatocellular carcinoma (HCC). However, the efficacy of sorafenib is modest for HCC with low objective response rate and short time to tumor progression, indicating that the inherent and acquired resistance underlies the limited success of sorafenib in HCC. We conducted a pooled shRNA screening in mouse xenografts of human HCC cells treated with sorafenib, and intended to identify genes that are involved in the therapeutic efficacy of sorafenib.

Materials and Methods: We introduced a pool of 3920 shRNAs targeting 1960 genes, including various kinases, transcriptional factors, phosphatase and cytokine, into Huh7 HCC cells by retrovirus infection. The Huh7 cells after infection with pooled shRNAs were implanted subcutaneously in immunocompromised mice, and were treated with vehicle or sorafenib at the dose of 30mg/kg/day for 4 weeks. Genomic DNAs were extracted from HCC xenografts, PCR-amplified of the shRNA regions, and deep-sequenced of the PCR products to quantify the abundance of each shRNA. Candidate genes were identified from those shRNAs with statistically different frequencies between vehicle-treated and sorafenib-treated xenografts, and were further validated by their impacts on sorafenib-sensitivity in HCC cells.

Results: By calculating the abundance frequencies of each shRNA of sorafenib- treated xenografts versus that of vehicle-treated xenografts, we found that 3 candidate sorafenib- resistance genes because their shRNA abundance was significantly decreased in sorafenib-treated xenografts. Among them, only the cyclin dependent kinase 5 (CDK5) was further investigated because its expression was confirmed to be increased in sorafenib- treated xenografts. In PLC5 and a sorafenib-resistant subline of Huh7 cells (Huh7-SR), downregulation of CDK5 by RNA interference or roscovitine, a CDK5 inhibitor, enhanced the sensitivity to sorafenib- induced growth-suppression. On the other hand, overexpression of CDK5 increased the resistance to sorafenib in Huh7 cells. Mechanistically, sorafenib combined with shRNA-mediated CDK5 downregulation augmented the induction of apoptosis, and resulted in an increased downregulation of multiple anti-apoptosis molecules such as XIAP and survivin in PLC5 and Huh7-SR cells. In 8 paired human HCC tissues, we found 3 of them have unequivocally increased expression of CDK5 in HCC cells of post-sorafenib HCC tissues compared with those of pre-treatment tissues.

Conclusions: Our results suggest that increased CDK5 expression may mediate resistance to sorafenib in HCC in vivo, and targeting CDK5 is warranted further studies in the treatment of HCC. (This study was supported by grants MOST 103-2314-B-002 -181 -MY2, MOST 105-2314-B-002-194-, MOST 103-2314-B-002 -090 - & MOST 104-2314-B-002 -073).

#81

E2F4/p107 complex regulates chemotherapy resistance in human colorectal cancer stem cells.

Junichi Matsubara,1 Yong F. Li,2 Piero Dalerba,3 Debashis Sahoo,4 Taichi Isobe,2 Russ B. Altman,2 Michael F. Clarke2. 1 _Kyoto University, Kyoto, Japan;_ 2 _Stanford University, Stanford, CA;_ 3 _Columbia University, New York, NY;_ 4 _University of California San Diego, San Diego, CA_.

Most cancer patients experience disease progression despite one or multiple lines of treatment with cytotoxic chemotherapy. Evidence from human trials and in vivo experiments on patient-derived xenograft (PDX) tumor lines indicates that cancer stem cells (CSCs) can survive chemotherapy treatment. However, the mechanism of chemotherapy resistance in CSCs is not fully elucidated. To identify new mechanisms of the drug-resistance in colorectal cancer, we used gene-expression microarrays to examine the gene expression profile of CSCs and non-CSCs in four human PDX models of colorectal cancer, both before-and-after treatment with cytotoxic chemotherapy. The gene-expression data were analyzed using Independent Component Analysis (ICA) followed by Transcription Factor Target Genes (TFTG) enrichment analysis, which identified the transcription factor E2F4 as a candidate regulator of chemotherapy resistance in colorectal CSCs. E2F4 was predominantly expressed in the CSC-enriched cell population (CD44+/CD166+ human colorectal cancer cells). E2F4 knock-down sensitizes cancer cells to chemotherapy in three different PDX tumors as well as HT29 cells. Co-immunoprecipitation experiments showed that E2F4 forms a complex with p107 and DP-1, which also contributed to the chemotherapy resistance. Simultaneous knock-down of both E2F4 and p107 enhanced chemotherapy sensitivity more than E2F4 knock-down alone. In normal intestinal crypt cells E2F4 was dispensable for chemotherapy resistance. ChIP-seq and RNA-seq experiments revealed that LDHA is a key downstream target of E2F4. LDHA knock-down sensitizes cancer cells to chemotherapy. High LDHA expression in tumor tissues was an independent poor prognostic factor in colorectal cancer patients (n=466), as well as in non-small cell lung cancer (n=1,926) and breast cancer (n=3,554). Thus, the E2F4/p107-LDHA pathway represents a novel therapeutic target to overcome chemotherapy resistance in CSCs.

#82

**MiR-206-mediated c-MET suppression modulates BCRP/ABCG2 levels in** NRF2 **-silenced cancer cells.**

Bo-hyun Choi, In-geun Ryoo, Donghyeok Kim, Sujin Lee, Mi-Kyoung Kwak. _The Catholic University of Korea, Bucheon, Gyeonggi-do, Republic of Korea_.

Breast cancer resistance protein (BCRP/ABCG2), a xenobiotic efflux transporter, is responsible for anticancer resistance in tumors. NF-E2-related factor 2 (NRF2) is a critical transcription factor in cellular defense system by regulating the expression of antioxidant and detoxifying enzymes; however, its overexpression is often associated with tumor resistance to chemotherapy. In the present study, we demonstrate that NRF2 modulation affects the expression of hepatocyte growth factor receptor (HGFR/c-MET) and consequently suppresses BCRP/ABCG2 activity in cancer cells. Interfering RNA-mediated stable inhibition of NRF2 in both ovarian carcinoma SKOV3 and renal carcinoma A498 cell lines reduced the expression of c-MET and this was accompanied by BCRP/ABCG2 down-regulation. The treatment of cells with pharmacological or genetic inhibitor of c-MET decreased BCRP/ABCG2 level and subsequently increased intracellular accumulation of doxorubicin and Hoechst 33342, indicating a link between c-MET and BCRP/ABCG2 activity. As a potential molecular mechanism of altered c-MET expression, miR-206 was identified as a predictive regulator of c-MET. As experimental evidence, transient and stable expressions of miR-206 in SKOV3 and A498 cells repressed c-MET and BCRP/ABCG2 levels. In addition, NRF2-knockdown cancer cells expressed higher levels of miR-206 compared to the control cells, and the treatment of NRF2 knockdown cells with the miR-206 inhibitor could restore c-MET and BCRP/ABCG levels. Collectively, our results showed that the NRF2 silencing-mediated miR-206 regulation could suppress BCRP/ABCG2 levels through c-MET modulation, which is providing an additional evidence of chemosensitization of tumor cells by NRF2 inhibition.

#83

Irinotecan resistance in type 2 interleukin-1 receptor overexpressed colorectal cancer cells is overcome by inhibitor of MEK.

Ai-Chung Mar,1 Chun-Ho Chu,2 Te-Chang Lee1. 1 _Academia Sinica - Inst. of Biomedical Sci., Taipei, Taiwan;_ 2 _Department of Surgery, Taipei, Taiwan_.

We have previously demonstrated that the expression of interleukin-1 receptor type II (IL1R2) is closely associated with the advanced staging and distant metastasis in patients with CRC. We also found that enhanced expression of IL1R2 played certain roles to resist the targeted therapeutics, regorafenib, in CRC cells. Whether IL1R2 expression is associated with the resistance to chemotherapeutics is unclear. Irinotecan, a water-soluble and semisynthetic derivative of camptothecin, is one of widely used first- and second-line chemotherapeutics for treatment of patients with metastatic colorectal cancer (CRC). Herein, we first demonstrated that enhanced expression of IL1R2 resulted in increased resistance of CRC cells to irinotecan using IL1R2 ectopically expressed HCT116 and DLD-1 cells, which were IL1R2 low expressing CRC cells, and IL1R2 silenced HT29 cells, which were IL1R2 high expressing CRC cells. Since we have shown that IL1R2 may activate MEK/ERK signaling by inhibition of PP2A, we further revealed that a specific inhibitor of MEK, CI-1040, could overcome the irinotecan resistance in IL1R2-overexpressing DLD-1 and HT29 cells. Our result showed that the combination of irinotecan and CI-1040 synergistically suppressed the cell growth in high IL1R2 expressing CRC cells but less significant in low IL1R2 expressing cells. We also confirmed that treatment of IL1R2-overexpressing CRC cells with CI-1040 significantly suppressed the pERK, a downstream target of MEK. These results support that IL1R2 mediates through activation of MEK/ERK signaling to cause irinotecan resistance in human CRC cells. The investigation how IL1R2 induces irinotecan resistance is ongoing. Accordingly, we may suggest that combined treatment of irinotecan and MEK-ERK inhibitor is a potential therapeutic strategy against IL1R2 overexpression related irinotecan resistant CRC.

#84

CCN1/Cyr61 regulation of gemcitabine-resistant phenotype in pancreatic cancer: involvement of CTGF and dCK.

Vijayalaxmi G. Gupta, Gargi Maity, Inamul Haque, Sushanta K. Banerjee, Snigdha Banerjee. _Kansas City VA Medical Center and University of Kansas Medical Center, Kansas City, KS_.

With an estimated half a million new cases and similar mortality rates for 2016, pancreatic ductal adenocarcinoma (PDAC) remains a life-threatening and challenging disease to diagnose and treat. As per American Cancer Society (ACS), varying efficacy in different patients has led to an increase in the mortality rate of PDAC. Gemcitabine (GEM) remains the drug of choice either alone or in combination, but is unsuccessful in reducing or curing PDAC in most patients. The limited efficacies of these drugs are due to the acquisition of chemo-resistant characteristics of PDAC. Although several molecular and physiological factors have been shown to correlate with the GEM-resistance, defined molecular mechanism(s) of GEM-resistance remains a mystery. Previous studies have shown that CCN1, which is overexpressed in PDAC and known to associate with PDAC progression, is critical for drug resistance. Here, we found that while the pancreatic cancer cell lines (i.e., Panc-1 and AsPC-1) in which CCN1 is overexpressed are typically GEM-resistant, the knocking down of CCN1 makes them sensitive to GEM. Mechanistic studies revealed that CCN1 regulates two important genes that are directly involved in regulation GEM-sensitivity in PDAC. These include cancer cell-secreted connective tissue growth factor (CTGF), a regulator of desmoplasia, and Deoxycytidine kinase (dCK), an enzyme that enhances gemcitabine sensitivity and efficacy in cancer cells. The deletion of CCN1 in PDAC cells blocks CTGF expression and under co-cultured conditions prevent the growth of alpha-smooth muscle actin (α-SMA)-positive stellate cells, which are required for desmoplastic growth in pancreatic cancers. CCN1 ablation upregulates dCK expression in PDAC cell lines as compared to CCN1 expressed PDAC cells. These two events enhance the anti-proliferative effect of GEM and can be rescued by CTGF-treatment or blocking dCK. In conclusion, CCN1 promotes GEM-resistance in PDAC cell through the regulation of CTGF and dCK and the mechanistic insights provided by these studies may help in designing future therapeutic strategies to combat PDAC.

#85

Establishment of cabazitaxel-resistant prostate cancer cell lines.

Atsushi Mizokami,1 Kazuaki Machioka,1 Kouji Izumi,1 Maolake Aerken,1 Ariunbold Natsagdorji,1 Yoshifumi Kadono,1 Yuta Takezawa,1 Hiroaki Iwamoto,1 Evan T. Keller2. 1 _Kanazawa University, Kanazawa, Japan;_ 2 _University of Michigan, Ann Arbor, MI_.

Background: The final treatment for castration-resistant prostate cancer (CRPC) is generally cabazitaxel treatment. However, once CRPC becomes resistant to cabazitaxel, the patients are obliged to best supportive care. Therefore, the elucidation of the mechanism of the cabazitaxel-resistance and the conquest are important themes to improve the prognosis of the patients. We already established paclitaxel/docetaxel-resistant prostate cancer, PC-3-TxR and DU145-TxR cells, and characterized those cell lines previously. Then we further tried to establish cabazitaxel-resistant prostate cancer cell lines, and characterized those cell lines.

Materials and Methods: We established two cabazitaxel-resistant cell lines, PC-3-TxR/CxR and DU145-TxR/CxR by increasing concentration of cabazitaxel from 1 nM to 30 nM gradually when PC-3-TxR and DU145-TxR cells were passaged. We confirmed the IC50 of docetaxel and cabazitaxel of these cells. Next we purified total RNA and performed cDNA microarray (Agilent and compared gene expression profiles among these cells.

Results and Conclusion: IC50 of PC-3-TxR and PC-3-TxR/CxR for cabazitaxel was 1.3 nM and 15.4 nM, respectively. IC50 of DU145-TxR and DU145-TxR/CxR was 7.0 nM and 30. 8 nM, respectively. Comparison of cDNA microarray between PC-3-TxR and PC-3-TxR/CxR cells or between DU145-TxR and DU145-TxR/CxR cells revealed that 4,470 genes in PC-3-TxR/CxR, and 1,345 genes in DU145-TxR/CxR cells were upregulated more than 3-fold and 4,683 genes PC-3-TxR/CxR and 1,537 genes were downregulated less than 0.3-fold, respectively. Especially, although expression of ABCB1 (MDR) gene was upregulated in DU145-TxR by 500-fold compared with DU145 cells, it was not upregulated in not upregulated in DU145-TxR/CxR cells any more. In contrast, expression of ABCB1 gene was upregulated in PC-3-TxR by 20-fold compared with PC-3 cells and it was further upregulated in PC-3-TxR/CxR by 40-fold compared with PC-3-TxR. In conclusion, various genes were regulated in cabazitaxel-resistant PCa cells and different mechanisms might be involved in this resistance in different cell lines.

#86

Time course analysis of gene expression and epigenetic interactions in acquired cetuximab resistance in head and neck squamous cell carcinoma.

Luciane T. Kagohara,1 Genevieve Stein-O'Brien,1 Sijia Li,1 Manjusha Thakar,1 Ruchira Ranaweera,2 Michael Considine,1 Joseph A. Califano,3 Christine H. Chung,2 Daria Gaykalova,1 Elana J. Fertig1. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _Moffitt Cancer Center, Tampa, FL;_ 3 _University of California San Diego, San Diego, CA_.

The current study performs time course genomics and epigenomics profiling to determine the complex dynamics of interactions between gene expression and epigenetic changes that alter cellular signaling and drive acquired therapeutic resistance. Targeted therapeutic agents block the activity of specific molecules critical for tumor progression. Cetuximab is a monoclonal antibody against EGFR currently used in the treatment of head and neck squamous cell carcinoma (HNSCC) and metastatic colorectal cancers. Similar to many targeted therapeutics, cetuximab improves survival for a brief period of two years, after which most patients acquire resistance resulting in tumor progression or recurrence. The mechanisms that alter cellular signaling pathways and cause resistance are poorly understood. We hypothesize that the molecular alterations that precede the resistance phenotype are more likely to be responsible for the resistance than the widespread alterations observed once the resistance is established. To test this hypothesis, we develop a novel time course experimental and bioinformatics model of acquired resistance. Specifically, this protocol enables weekly profiling of RNA, DNA, and proliferation as cetuximab resistance developed in vitro. We apply the CoGAPS bioinformatics algorithm to perform integrated analysis of gene expression and DNA methylation. This analysis distinguishes signatures of sustained activation of cellular pathways in response to treatment from signatures of clonal expansion associated with acquired cetuximab resistance. Despite identifying massive changes in gene expression driven by therapeutic response, only the expression changes associated with the acquired resistance, and not response to treatment, have corresponding epigenetic signatures. The genomic signature associated with acquired resistance is enriched for gene targets of the AP-2 family of transcription factors. In a previous study, we found that these gene targets were overexpressed after short-term treatment in cetuximab sensitive cell lines and HNSCC tumors. Our data suggest that this family of transcription factors serves as a feedback mechanism to maintain homeostasis to growth factors receptor signaling. As a result, AP-2 transcription factors serve as a natural mechanism to cause subsequent therapeutic resistance without addition of genetic alterations. Further investigation is needed to determine the role of AP-2 transcription factors in acquired cetuximab resistance.

#87

Treatment schedules influence acquisition of Doxorubicin resistance through epigenetic mechanism in breast cancer cells.

Logeswari Ponnusamy, Prathap Kumar S. Mahalingaiah, Kamaleshwar P. Singh. _The Institute of Environmental & Human Health, Texas Tech University, Lubbock, TX_.

Breast cancer is the leading cause of cancer related mortality in women. Chemotherapy resistance is a major limitation in clinical treatment of breast cancer. Mounting evidence implicates the epigenetic aberrations in acquired chemoresistance. However, the influence of treatment schedules and associated epigenetic changes on acquisition of doxorubicin resistance in breast cancer is not clear. Therefore, objective of this study was to evaluate the impact of treatment schedules on acquisition of doxorubicin resistance and the molecular mechanistic basis of this process in breast cancer cells. To address this question, two different treatment strategies viz, continuous exposure and intermittent exposure were used to generate doxorubicin resistant cells lines from ER-positive MCF-7 and triple negative MDA-MB-231 breast cancer cell lines. The level of resistance against doxorubicin and the temporal sequence of molecular changes associated with the resistance phenotype were evaluated at 4 months and 18 months' following exposure to doxorubicin. Results revealed that intermittent exposure to doxorubicin resulted in significantly higher level of resistance as compared to continuous exposure. ER-positive MCF-7 cells developed relatively earlier and high level of resistance when compared to MDA-MB-231 cells. Additionally, the resistance to doxorubicin-induced cytotoxicity was associated with transient acquisition of EMT and CSC-like phenotype in breast cancer cells around 4 months following doxorubicin. Gene and protein expression analysis further revealed temporal changes in the expression of epigenetic regulatory genes and histone modifications respectively. The temporal changes in expression of genes were positively correlated with doxorubicin exposure time during resistance development. In summary, the result of this study for the first time suggests that the treatment schedules influence the acquisition of doxorubicin resistance potentially through epigenetic mechanisms. The findings of this study will be helpful in chemotherapy of breast cancer to achieve the best clinical result

#88

The role of RBPMS in cisplatin resistant ovarian cancer.

Perla M. Báez-Vega,1 Fatma Valiyeva,1 Ginette Santiago,2 Pablo Vivas-Mejia1. 1 _University of Puerto Rico Comprehensive Cancer Center, San Juan, PR;_ 2 _University of Puerto Rico Medical Sciences Campus, San Juan, PR_.

Epithelial ovarian cancer is the fifth leading cause of cancer-associated deaths among women in the United States. High-grade serous ovarian cancer (HGSOC) is the most common type of epithelial ovarian cancer and accounts for 70-80% of ovarian cancer patient deaths. RNA-Binding Protein With Multiple Splicing (RBPMS) is a member of a family of proteins that bind to the nascent RNA transcripts and regulate their processing, including the pre-mRNA splicing and the transport, localization, and stability of the RNA molecules. However, little is known about the biological function of RBPMS family in ovarian cancer. We hypothesized that decreasing the expression of RBPMS in high-grade serous ovarian cancer increases ovarian cancer progression and platinum-based drug resistance. Quantitative real-time PCR (qPCR) and Western blot (WB) analysis were conducted to determine the basal mRNA and protein expression of RBPMS in ovarian cancer cells. The invasion ability of the ovarian cancer cells after targeting RBPMS with silencing RNA (siRNA) was determined by invasion assays. The proliferation rate of the ovarian cancer cells overexpressing RBPMS was determine by colony formation assay. RBPMS is highly expressed in A2780 cells when compared to the cisplatin resistance ovarian cancer cells and HGSOC cancer cells. Targeting RBPMS increased the invasion ability of the A2780 cells. Cisplatin resistant, A2780CP20 cells overexpressing RBPMS showed a decreased in cell proliferation when compared to the A2780CP20 empty vector clones. We described here for the first time the mRNA and protein expression profile of RBPMS in HGSOC and epithelial ovarian cancer cells. These studies will help to better understand the role of RBPMS in ovarian cancer and may support the use of RBPMS as an adjuvant treatment to overcome the platinum-based chemotherapy resistance characteristic of the high-grade serous ovarian cancer.

#89

Targeting Rho/MRTF regulated gene transcription in drug-resistant melanoma.

Sean A. Misek,1 Scott D. Larsen,2 Kathleen A. Gallo,1 Richard R. Neubig1. 1 _Michigan State Univ., East Lansing, MI;_ 2 _University of Michigan, Ann Arbor, MI_.

Much of the recent focus of melanoma targeted therapy has been on the ERK pathway, which is aberrantly activated in approximately 90% of melanoma tumors (over half of which express BRAFV600E). Current targeted therapies such as vemurafenib (BRAFV600E inhibitor), or a combination therapy using dabrafenib (BRAFV600E inhibitor) and low dose trametinib (MEK inhibitor) shows profound initial effects in a majority of BRAFV600E expressing tumors. However, these responses are often short-lived and resistances typically develops within months. Resistance to these targeted therapies can arise from multiple mechanisms, including activation of pro-survival signaling pathways parallel to the ERK pathway. The goal of this work is to identify pharmacologically targetable resistance mechanisms so that effective combination therapies can be developed.

Despite the clear role of the RhoA subfamily of Rho GTPases (RhoA/B/C) as melanoma oncogenes, their role in drug resistance is not well understood. It is challenging to develop small molecule inhibitors which directly target the activity of small Rho GTPases, so an alternative approach is to inhibit downstream pathways. Through modulation of the actin cytoskeleton Rho can induce gene transcription through multiple transcriptional co-activators including Myocardin-Related Transcription Factor (MRTF) and Yes-Associated Protein 1 (YAP).

My bioinformatics analysis demonstrates that MRTF-A gene expression is correlated with poor overall survival in a large cohort of cutaneous melanoma patients. Furthermore, expression of a set of 216 MRTF target genes is enriched in dabrafenib/trametinib resistant cutaneous melanoma tumors compared to matched pre-treatment tumors, suggesting that MRTF activation may be involved in drug resistance. Based upon these results I hypothesized that small Rho GTPases may promote resistance to MAPK pathway targeted therapies through activation of MRTF/YAP.

To test this hypothesis, I generated vemurafenib resistant melanoma cells through chronic exposure to vemurafenib. This vemurafenib-resistant cell population is enriched for actin stress fiber positive cells, and these cells have increased Myosin Light Chain 2 (MLC2) phosphorylation, suggesting that there is increased Rho activation. Furthermore, these drug resistant cells are more sensitive to pharmacological inhibition of MRTF activity. These preliminary data suggest that vemurafenib resistant melanoma cells may be re-wired to depend on the Rho-induced gene transcription for their survival, and that a combination therapy simultaneously targeting these two pathways may be an effective treatment strategy for BRAF inhibitor-resistant melanomas.

#90

STING colocalizes with gamma-H2AX upon treatment of breast cancer cells with genotoxics: A new role in DNA repair.

Laura Cheredame,1 Julie Gaston,1 Vanessa Yvonnet,1 Olivier Deas,1 Marie-Françe Poupon,1 Jean-Gabriel Judde,1 Vincent Goffin,2 Stefano Cairo1. 1 _XenTech, Evry, France;_ 2 _Inserm U1151, Institut Necker Enfants Malades (INEM), University of Paris Descartes, Faculty of Medicine, Paris, France_.

One of the current tumor immunotherapy strategies involves the use of STING agonist, a well-known inducer of interferon (IFN) signaling in the immune system, to promote tumor-rejection. However, recent evidence also indicates that constitutive activation of IFN signaling in the tumor may lead to a bad outcome. For instance, increased expression of IFN- stimulated genes (ISGs) at time of surgery was associated with early breast cancer recurrence, and an IFN-related DNA damage resistance signature (IRDS) was identified as a predictive marker of recurrence after radiotherapy. Thus, IFNs secreted into the tumor microenvironment may have complex opposite effects on tumor behavior and response to treatment.

Using patient-derived xenograft (PDX) models, we previously showed that the IFN/STAT1 pathway was activated within breast cancer cells in response to chemotherapy and that this pathway may be involved in treatment resistance and recurrence. The aim of this study was to elucidate the mechanisms by which IFN-signaling is triggered in breast cancer cells following chemotherapy and how its activation leads to tumor survival and recurrence. To this aim, the breast cancer cell line MCF7 was treated in vitro with mafosfamide and the activity of different IFN pathway effectors was monitored using western blot, immunofluorescence and cell fractionation techniques.

We found that in breast cancer cells, similarly to what is observed in immune cells, type I IFN expression is triggered in a STING-dependent manner. STING silencing abrogated chemotherapy-induced type I IFN production and signaling, while potentiating genotoxic treatment efficacy by promoting cell death and delaying cell colony regrowth. Surprisingly, while STING is described as an endoplasmic reticulum resident protein that relocalizes to perinuclear vesicles upon activation in immune cells, we showed that STING is constitutively present in the nucleus of breast cancer cells and, under activation, forms nuclear clusters that co-localize with gH2AX at DNA breaks.

This study provides the first demonstration of STING activation in breast cancer cells and describes a potential new role for the nuclear form of STING in DNA damage response. Our data suggest that genotoxic stress-induced STING activation is a cell-intrinsic mechanism of breast cancer cell survival and regrowth. Whether STING pro-survival role is IFN-dependent (through the activation of given ISGs), IFN-independent (through its involvement in the DNA damage response) or both is currently under investigation.

#91

**Molecular insight into drug resistance in acute myeloid leukemia: An** in-silico **and** in-vitro **approach.**

Urja Desai, Sheefa Mirza, Sonia Parikh, Rakesh Rawal. _The Gujarat Cancer & Research Institute, Ahmedabad,Gujarat,India, India_.

INTRODUCTION:

Drug resistance of leukemic stem cells is a surmountable obstacle to effective chemotherapy in acute myeloid leukemia (AML). P-glycoprotein (Pgp) and FLT3 undoubtedly contribute to worse prognosis and mechanisms of these proteins lead to shorter survival and chemotherapy resistance. Due to heterogeneity of stem cells in AML, outcome of patients with a normal karyotype is highly challenging and changes in drug resistance genes likely to be identified which will contribute to a better understanding of the disease biology, eventually leading to the development of alternative therapeutic approaches regarding drug resistance in AML.

METHODOLOGY:

Blood and Bone-marrow samples were collected from newly diagnosed adult AML patients with normal karyotype. Gene expression analysis of MDR genes like Pgp, MRP1, BCRP and LRP was done by qRT-PCR in FLT3-ITD+/- and CD34+/- subgroups. Higher expression of Pgp amongst all MDR genes instigated to undertake in-silico analysis of Pgp with natural compound library using YASARA. MTT assay was used to find out IC50 value of natural compounds shortlisted from in-silico analysis and used alone or with cytarabine on THP-1 cell line and in primary AML stem cells. Pgp inhibitory activity of natural compounds was assessed by fluorimetric MDR assay.

RESULTS:

A significant upregulation of Pgp expression was observed in FLT3-ITD+, CD34+ group after cytarabine exposure during their induction (7+3) therapy. Docking analysis revealed curcumin, hesperidin and silymarin having better binding affinity with Pgp as compared to its known inhibitor Verapamil. Furthermore, these selected compounds were used in-vitro, and results of MTT assay suggested 6.1µM, 40.73µM, 63.09µM and 54.95µM IC50 values of cytarabine, curcumin, hesperidin and silymarin respectively in THP-1 cell line. Combination index (CI) analysis revealed that curcumin (25µM), hesperidin (30µM) and silymarin (35µM) having synergistic effect with cytarabine and decreased IC50 value of cytarabine (1µM). In addition to this, Silymarin at 250nM and 500nM concentration showed equivalent inhibition of Pgp as compared to control Verapamil.

CONCLUSION:

A combination of in-silico and in-vitro screening revealed Curcumin, Hesperidin and Silibinin can be used as MDR modulators as well as chemosensitizer to reduce the cytotoxicity profile and drug resistance in AML.

#92

Acquisition of temozolomide resistance: Identification of a new drug tolerant stage in glioblastoma cells.

Marion Rabé,1 Hicham Janati,2 Solenne Dumont,1 Christelle Thibault-Carpentier,3 Jean Clairambault,2 François M. Vallette,1 Catherine Gratas1. 1 _Université de Nantes, UMR INSERM892/CNRS6299, Nantes, France;_ 2 _Inria-Paris-Rocquencourt, Le Chesnay, France;_ 3 _IGBMC, Illkirch-Graffenstaden, France_.

Glioblastoma multiforme (GBM) are the most aggressive and common brain tumors in adults. Despite surgery and combined radio-chemotherapy with temozolomide (TMZ), tumor reccurence always occurs. The median survival time for patients diagnosed with GBM is about 14 months with less than 5% survival at 5 years. Today the main marker of TMZ resistance is the methylation status of MGMT promoter. Patients with a methylated promoter usually have a better response to treatment than patients with an unmethylated promoter. Indeed MGMT is an enzyme involved in DNA repair mechanisms that abrogates TMZ effects. However, in clinical trials targeting the MGMT enzyme, median survival of patients was not improved. It is thus essential to decipher the mechanisms involved in the acquisition of TMZ resistance to identify new therapeutic targets. To achieve this goal TMZ resistant cells were generated by continuous treatment of the U251 human glioblastoma cell line. These cells are sensitive to TMZ and do not express MGMT. We performed transcriptomic analysis by RNA-Seq on U251 treated with TMZ (50µM) for different time and selected differentially expressed genes. We also evaluated target genes expression in single cells by RT-qPCR using C1-HD-Biomark technology (Fluidigm). Transcriptome profiling allowed to identify a transient phase with TMZ tolerant cells before acquisition of complete resistance. These cells are characterized by a modified morphology and a no proliferative state. In this population we identified a subset of genes with the same transient overexpression. Similar results were observed in other glioblastoma cell lines and under other stress conditions. In contrast, stable expression of MGMT appeared later with the emergence of the TMZ resistant population. Interestingly single cell qPCR showed that MGMT expression in the resistant cells could not be explained by clonal selection of MGMT positive cells. Drug screening on the TMZ tolerant cells revealed a potent killing activity of histone deacetylating agents (HDAC inhibitors). In conclusion, we have shown that glioblastoma cells become resistant after a transient state of TMZ tolerance. Identification of this singular population highlights new molecular targets and a new therapeutic window. Targeting these tolerant cells could avoid emergence of resistance and tumor recurrence, thereby patients survival could be improved.

#93

Transcriptional control of glucocorticoid responses in leukemia.

Lai N. Chan, Zhengshan Chen, Gang Xiao, Jae Woong Lee, Kadriye Nehir Cosgun, Huimin Geng, Valeria Cazzaniga, Hilde Schjerven, Ross A. Dickins, Markus Muschen. _UCSF, San Francisco, CA_.

Glucocorticoids (GCs) are central to all major therapy regimens for pre-B cell-derived acute lymphoblastic leukemia (ALL), but have no activity in myeloid leukemia. Such divergent responses represent an empirically established clinical standard; however, neither the mechanism by which GCs induce cell death nor the biological basis for the distinct responses in B-cell and myeloid leukemias is clear. Studying patient-derived samples revealed that NR3C1 (glucocorticoid receptor) levels were 6- to 20-fold higher in pre-B ALL compared to chronic myeloid leukemia (CML). High levels of Nr3c1 were reduced upon B- to myeloid-lineage conversion, suggesting that regulation of NR3C1 expression and GC responsiveness depend on a B-cell transcriptional program. B-cell transcription factors (e.g. PAX5, IKZF1) are critical for B-cell development, yet they are genetically lesioned in more than 80% of pre-B ALL cases. Despite such high frequency, the significance of these inactivating lesions remains elusive. Combining ChIP-seq and RNA-seq analyses, we identified a novel B-cell transcriptional program for activation of NR3C1 and its transcriptional target TXNIP (a negative regulator of glucose uptake). Reconstitution of PAX5 or IKZF1 expression in haploinsufficient patient-derived pre-B ALL cells increased NR3C1 and TXNIP levels. Conversely, expression of dominant negative mutant of PAX5 or IKZF1 abolished NR3C1 expression. Loss of Nr3c1 or Txnip in murine BCR-ABL1-driven pre-B ALL cells resulted in survival advantage in competitive growth assays. Importantly, loss of Nr3c1 or Txnip significantly elevated glucose uptake, lactate production and cellular ATP levels. These findings suggest that GCs induce cell death by exacerbating glucose and energy depletion. Notably, reconstitution of PAX5 or IKZF1 rendered haploinsufficient patient-derived pre-B ALL cells more sensitive to dexamethasone (dex) treatment. In contrast, dominant-negative PAX5 or IKZF1 largely de-sensitized pre-B ALL cells expressing wildtype PAX5 or IKZF1. These findings suggest that B-cell transcription factors set the threshold for GC responsiveness in pre-B ALL. Since relapsed ALL cells often acquire GC resistance, drug-combinations may be useful to prevent GC-resistance. As expected, loss of Nr3c1 abrogated responses to GCs. Interestingly, loss of Txnip also largely rescued GC-induced cell death in pre-B ALL cells. On this basis, we tested drug interactions between GCs and TXNIP agonists, 3-O-methylglucose (3-OMG) and D-allose. Treating patient-derived GC-refractory pre-B ALL cells with 3-OMG or D-allose strongly synergized with GC-treatment. Collectively, our findings provide a mechanistic explanation for the empiric finding that GCs are effective in the treatment of B-cell but not myeloid malignancies, and identify TXNIP as a novel therapeutic target in pre-B ALL.

#94

Association of xCT overexpression with RTKI resistance and metastases in clear cell renal cell carcinoma.

Sreenivasulu Chintala, Remi Adelaiye-Ogala, Ashley Orillion, Sreevani Arisa, May Elbanna, Nur P. Damayanti, Roberto Pili. _Indiana University-Purdue University, Indianapolis, IN_.

Background: Cystine/glutamate exchanger xCT is a catalytic component of system xc- involved in transport of 'conditionally indispensable' amino acid cystine. Cystine transport is a rate limiting step for the synthesis of glutathione, a major intracellular redox regulator. Recently, we and other groups reported the overexpression of xCT and its association with drug resistance in several human cancers including bladder, glioma, breast, and colon. There are no studies to show the xCT expression in clear cell renal cell carcinoma (ccRCC) and its association with tyrosine kinase inhibitors resistance and metastasis. In the current study we have evaluated xCT expression in human ccRCC tumors arranged in tissue microarray (TMA) and determined its role in receptor tyrosine kinase inhibitor (RTKI) resistance and metastases using the patient derived tumor xenografts (PDX) models and TKI resistance ccRCC cells.

Methods: Human Renal cell carcinoma tumor nephrectomy specimens arranged in tissue microarray (TMA) were used to determine xCT expression by immunohistochemistry. Patient derived tumor xenografts (PDX) of primary, metastasis, and sunitinib resistance were used to determine the role of xCT in RCC. To understand the molecular alterations associated with RTKI resistance, we have generated sunitinib resistance 786 OR ccRCC cells and performed RNAseq analysis. To determine the xCT inhibition effect on ccRCC tumor metastases, sulfasalazine, an inhibitor of xCT was used to treat metastatic ccRCC tumor xenografts transplanted in SCID mice.

Results: Immunohistochemical evaluation of xCT in RCC TMA revealed that 70 % (19 out of 27) of the tumors express different levels of xCT. Association with tumor response to RTKI will be presented. RTKI less responsive PDX RP-R-02 was developed using the dose escalation treatment strategy and was found to have an upregulation of xCT when the tumors become less responsive to sunitinib. RNAseq analysis revealed differential gene expression of various pathway genes including lysosome biogenesis and function such as SLC7A11, HPS4, HPS5, CTSB, IFI30, PPT1, SCPEP1, TPP1, ATP6AP1L, MCOLN1, PRKAG2, and VPS18 in sunitinib resistant 786 OR cells compared to parental cells supports the role of lysosomes function in RTKI drug resistance. Furthermore, xCT inhibitor sulfasalazine treatment significantly decreased the metastasis lung nodules of RP-R-02LM in SCID mice demonstrated the xCT role in RCC tumor metastasis.

Conclusions: We found preliminary evidence that overexpression of xCT may be associated with RTKI resistance in ccRCC. These results suggest that targeting the xCT in ccRCC may reverse the resistance and enhance the efficacy of RTKI. Additional studies using larger numbers of ccRCC tumors are required to identify xCT as a potential predictive biomarker for response/resistance to RTKI in ccRCC patients.

#95

Targeting BRD4 overcomes cetuximab resistance in HNSCC.

Toni Brand, Yan Zeng, Brandon Leonard, Rachel O' Keefe, Hua Li, Daniel Johnson, Jennifer Grandis, Neil E. Bhola. _University of California San Francisco, San Francisco, CA_.

Background: Nearly 600,000 people are diagnosed with head and neck cancer worldwide and 60% succumb to the disease within 5 years. The epidermal growth factor receptor (EGFR) is a major driver of HNSCC and in 2006 the EGFR monoclonal antibody cetuximab was FDA-approved for HNSCC treatment. However, cetuximab has not conferred significant long-term benefit compared to chemoradiation in patients with poorly differentiated recurrent-metastatic HNSCC. We and others have demonstrated that cetuximab treatment activates alternative receptor tyrosine kinases (RTKs) including Fibroblast Growth Factor (FGF) receptors, Met and Axl in HNSCC. To circumvent administration of multiple RTK inhibitors in combination with cetuximab, we sought to identify a common molecular target that regulates the expression of these RTKs. Bromodomain-containing protein-4 (BRD4) has been shown to regulate the transcription of RTKs in breast cancer models.

Hypothesis: We hypothesized that targeting BRD4 will overcome cetuximab resistance by depleting the expression of alternative RTKs in HNSCC. We further hypothesize that genetic and pharmacological targeting of BRD4 will synergize with cetuximab.

Results: Using a phospho-RTK array, 72-hour cetuximab treatment increased or sustained phosphorylated levels of EGFR, HER2, HER3, MET, and AXL in several HNSCC cell lines. Treatment with the BRD4 inhibitor JQ1 abrogated both phosphorylated and total RTK abundance in the presence of cetuximab. Cetuximab and JQ1 robustly decreased phosphorylated Src and induced the senescence marker p21. Phenotypically, cetuximab and JQ1 significantly decreased survival and increased apoptosis in 6 HNSCC cell line models, while the normal oral keratinocyte cell line NOKsi had approximately 10-fold higher IC50s for the BRD4 inhibitors, JQ1 and I-BET762, relative to HNSCC cell lines. Importantly, two HNSCC models of acquired cetuximab resistance exhibited robust sensitivity to pharmacological (JQ1, IBET-762) and genetic (RNAi) BRD4 targeting strategies. Moreover, exogneous overexpression of different RTKs (HER3, ALK, and ROR1) resulted in cetuximab resistance that was reversed upon BRD4 targeting (RNAi and JQ1). Combination of cetuximab and JQ1 in co-culture experiments with T cells decreased the CD4+/CD25+ Treg population and PD-L1 expression on HNSCC cell lines. Further, preliminary findings indicate that JQ1 treatment prevents outgrowth of cetuximab-treated HNSCC patient-derived xenograft models.

Conclusion: Our findings indicate that targeting BRD4 decreases the activation and expression of multiple RTKs that mediate resistance to the FDA-approved EGFR inhibitor cetuximab. Furthermore, BRD4 abrogates expression of immunosuppressive markers, making it a promising tumor intrinsic and extrinsic therapeutic strategy for HNSCC.

#96

Didhydropyrimdine dehydrogenase (DPYD) provides resistance to 5-flurouracil in mutant p53 (mtp53) expressing colorectal cancer cells.

Prashanth Ravishankar Gokare,1 Niklas Finnberg,1 David Dicker,1 Maureen Murphy2. 1 _Fox Chase Cancer Ctr., Philadelphia, PA;_ 2 _Wistar Institute, Philadelphia, PA_.

5-Flurouracil (5-FU) is a mainstay of colorectal cancer therapy. Even though much is known about the 5-FU mechanism of action, very little is known about the genetic determinants which provide sensitivity and resistance to 5-FU. Here we provide first insights into p53, a major predictor of 5-FU response, modulating nucleotide catabolism specifically through the control of the rate limiting enzyme dihydropyrimidine dehydrogenase (DPYD). Colorectal cancer cells expressing mtp53 specifically show higher expression of DPYD as compared to the wildtype p53 (wtp53) carrying cells. Higher expression of DPYD in mtp53 cells specifically provides increased resistance to 5-FU as indicated by cellular proliferation and long term clonogenic assays. Furthermore, knockdown of DPYD in mtp53 cells preferentially resensitizes them to 5-FU as opposed to cells expressing Wtp53 by decreasing the viability and increasing apoptosis observed in these cells. Pharmacological inhibition of DPYD with gimeracil, clinical inhibitor of 5-FU phenocopies the effects seen by siRNA/shRNA mediated knockdown of DPYD. Finally, invivo studies with tumor xenograft from mtp53 carrying cells shown increase resistance to 5-FU therapy which is abrogated by stable knockdown of DPYD expression or in combination gimeracil. Preliminary analysis of TCGA patients treated with 5-FU indicate differential expression of DPYD based on specific Tp53 mutation status. Taken together our study provides first insights into crosstalk between nucleotide catabolism and mtp53 under conditions of DNA damage and metabolic stress having major implications on the therapeutic response to 5-FU the in the clinic.

#97

RNA sequencing of bladder cancer patient-derived xenograft models identifies genes associated with chemoresistance.

Kelly A. Martin,1 Nicholas R. Hum,1 Aimy Sebastian,1 Deepa K. Murugesh,1 Chong-Xian Pan,2 Ai-Hong Ma,2 Ralph de Vere White,2 Gaby Loots1. 1 _Lawrence Livermore National Laboratory, Livermore, CA;_ 2 _UC Davis Comprehensive Cancer Center, Sacramento, CA_.

Background: Bladder cancer is among the top ten most common cancers, with about ~380,000 new cases and ~150,000 deaths per year worldwide. Platinum-based combination chemotherapy is commonly used to treat advanced bladder cancer. It has been shown that only ~50% of the patients with advanced bladder cancer respond to platinum-based therapy.

Methods: We have utilized a patient-derived bladder cancer xenograft (PDX) platform to characterize the molecular mechanisms that contribute to resistance of gemcitabine-cisplatin combination therapy in advanced bladder cancer. We have also identified key regulatory pathways in our PDX models that can be targeted to treat chemotherapy resistant bladder cancer using RNAseq analysis. Transcriptome profiling of P0 (passage 0) bladder cancer xenograft tumors from 4 PDX lines (2 gemcitabine-cisplatin resistant lines and 2 drug sensitive lines) was performed by RNA-Seq analysis, before and after a 21-day cisplatin/gemcitabine drug treatment regimen.

Results: The RNA-seq data has indicated significant differences between the transcriptional profiles of drug-sensitive and drug-resistant tumors. PDXs retained morphology and shared 92-97% of genetic alterations of parental cancer cells. We identified 333 genes >2 fold up or down regulated in the drug resistant tumors compared to the drug sensitive tumors. Significantly up-regulated genes in drug resistant tumors analyzed include metabolic enzymes ALDH2, ALDH3A1, ALDH4A1 and ALDH7A1, transporter proteins ABCA1, SLC1A4, SLC2A5, SLC30A1, SLC39A6, SLC7A5 and SLC9A3, Notch ligand JAG2, Growth hormone receptor GHR and transmembrane protein GPNMB. Consistent with the change of cell surface proteins such as GHR and GPNMB, the MAPK and the PI3K-AKT pathways were upregulated when PDXs became resistant to cisplatin treatment. Additional changes in gene expression based on RNA-seq data before and after drug treatment were also found.

Conclusion: Chemoresistance to gemcitabine and cisplatin is associated with altered expression of several cell surface proteins and upregulation of the downstream signaling pathways. Targeting these cell surface proteins can possibly be harnessed to overcome chemoresistance. GPNMB is a type I transmembrane protein that has previously been shown to be up-regulated in many metastatic cancers including breast cancer. Potentially targeting GPNMB with glembatumumab vedotin, an antibody-drug-conjugate has shown promising results in treating several cancers including breast cancer and osteosarcoma. Further studies will elucidate whether targeting GPNMB is an effective strategy for the treatment of chemotherapy resistant bladder cancer.

This study received funding by a developmental grant from the UCDCC. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). IM number: LLNL-688318

#98

TET suppresses protein translation, differentially effects polysome loading of cell cycle regulatory proteins and inhibits cell growth and proliferation of prostate cancer cells.

Praveen K. Jaiswal, Sweaty Koul, Qin Dong, Hari Koul. _LSU Health Sciences Center - Shreveport, Shreveport, LA_.

Introduction: Translational control is a critical component in development and progression of cancer and could be explored to develop antineoplastic therapies. Results from our laboratory have shown that TET (a derivative of Tetrandrine) inhibits cell growth and promotes apoptosis of prostate cancer (PCa) cells. However, the underlying molecular mechanisms are not clearly understood. We observed that TET inhibited 4EBP phosphorylation, suggesting that it might inhibit translational initiation. In the present study we evaluated the effects of TET on Translation in general and cell cycle related proteins in particular.

Methods: Androgen dependent PCa cells LNCaP and Enz resistant CRPCa cells 22Rv1 were maintained in supplemented RPMI as recommended. Where indicated cells were treated with TET (0 or 20µM) for various time points (2h, 8h, 18h and 24h). At the end of the experimental periods cells were pulsed with cycloheximide for 10 minutes in RPMI media. Cytoplasmic RNA was collected by lysing cells in polysome lysis buffer. Cell lysates were centrifuged and the resulting cytosolic supernatant was separated by density (a 10% to 50% sucrose) gradient and centrifugation. Fractions were collected using a TELEDYNE ISCO Density Gradient Fractionation System with continuous monitoring of untranslated mRNAs. Polysome (P)/Monosome (M) ratio were measured. The total RNA in each fraction was extracted using TRIzol LS (Invitrogen) and qRT PCR was done for p21, p27, Cyclin D1 and c-Myc gene for each fraction. Cell viability was measured by MTT assay at various time points.

Results: TET treatment resulted in decreased protein synthesis. TET treatment resulted in decrease of RNA associated with Polysomes. There were a significant shift in P/M ratio from 0.82 (control) to 0.48 (24 h. TET) [TET 2hrs (0.75), 8h (0.69) and 18hrs (0.52)] in LNCaP and P/M ratio form 0.91 (control) to 0.32 (8 h TET) [TET 2h (0.78) and 4h (0.53)] in 22Rv1 cells. The decrease of the P/M ratio suggests that TET inhibits translation initiation in prostate cancer cells independent of the androgen dependence. While there was generalized decrease in translation and specific decrease in mRNA for Cyclin D1, c-Myc associated with polysomes following TET treatment. In quite contrast to these findings, we observed an increase in mRNA for p21 and p27 in polysomal fractions of TET treated cells as compare to control at 24 h in LNCaP cells. These data suggest differential effects of TET treatment on polysome loading of cell cycle regulatory proteins. Moreover, TET inhibited growth and proliferation of LNCaP and 22Rv1 cells in dose as well as in time dependent fashion.

Conclusions: TET targets translational machinery and differentially effects translation of cell cycle regulatory genes by effecting their polysome loading and effects cell growth and proliferation of androgen dependent and castrate resistant PCa cells.

#99

Identify potential kinetochore protein inhibitors to overcome cisplatin resistance.

Chen Yin-Ju, Jeng-Fong Chiou. _Taipei Medical University, Taipei, Taiwan_.

Cisplain is the most commonly used chemotherapeutic drug for cancer, while the resistance causes treatment failure. Cisplatin resistance may involve in many molecular changes so identify aberrations in cisplatin-resistant (CR) genes and find new therapeutic strategies could provide valuable information to reduce therapeutic resistance. The aim of this study is to search CR genes and identify potential novel drugs to overcome cisplatin resistance. To identify aberrations in CR genes, three CR genetic signatures were collected and analyzed differential expression genes from lung, ovarian and oral cancer cells. After integration of CR signatures, the kinetochore associated proteins, including NUF2, SPC25, PC24, DSN1, SKA2, KNTC1, MIS18A and SKA1 were up-regulated in various CR cells. Silencing of kinetochore associated proteins increased cisplatin sensitivity. For identification of kinetochore associated protein inhibitors to overcome cisplatin resistance, CR signatures were queried to the Connectivity map (Cmap) database to search for potential drugs which may reverse cisplatin resistance. 3 compounds exhibited cytotoxicity effect on the cisplatin-resistant cells and reduced kinetochore associated proteins expression level. Taking together, this study demonstrates kinetochore associated proteins participate in cisplatin resistance and identify potential inhibitors to overcome cisplatin resistance.

#100

Pancreatic cancer cells rely on the NADPH producing enzyme, IDH1, for adaptive survival against acute metabolic stress.

Ali Vaziri-Gohar, Mahsa Zarei, Jonathan R. Brody, Jordan M. Winter. _Thomas Jefferson University, Philadelphia, PA_.

We recently demonstrated that pancreatic cancer cells adapt to low nutrient conditions and chemotherapeutic stress through an adaptive response where HuR (ELAVL1) protects cells from oxidative damage induced by metabolic stress. RNA sequencing data and a series of protein-RNA interaction assays proved that HuR stabilizes transcript levels of the NADPH producing enzyme, isocitrate dehydrogenase 1 (IDH1). HuR-knockout cells had near-complete loss of IDH1 expression (manuscript under review). In light of the fact that IDH1-null mice are particularly sensitive to oxidative damage, we hypothesize that this enzyme plays a critical role in PDA survival of acute stress. We examined the expression levels of all eight well-characterized NADPH-generating enzymes in pancreatic cancer cells in vitro, and demonstrate that only IDH1 and phosphogluconate dehydrogenase (PGD) are upregulated by >2-fold after incubation in low glucose (5 mM) for 48 hours. IDH1-knockout MiaPaCa2 cells were generated through CRISPR gene editing, such that mRNA expression was detected at <5% of control levels. ROS levels detected by DCFDA measurements were increased by 55% in IDH1-knockout cells compared to isogenic controls under normal culture conditions, with even higher levels under low glucose and chemotherapy stress for 48 hours. Additionally, IDH1-knockout cells were more sensitive to chemotherapy under both high and low glucose conditions, compared to IDH1-proficient control cells. Taken together these results suggest that IDH1 expression is important for acute antioxidant defense in pancreatic cancer cells in the face of metabolic stress, and represents a potential therapeutic target.

#101

Adaptive feedback reactivates MAPK signaling in KRAS-mutant cancers with inhibition of MEK, but not ERK.

Leanne G. Ahronian, Sandra Misale, Jason T. Godfrey, Koki Nishimura, Lifeng Chen, Jeffrey A. Engelman, Ryan B. Corcoran. _Massachusetts General Hospital Cancer Center, Charlestown, MA_.

Activating mutations in the KRAS oncogene occur in about 40% of colorectal cancers (CRCs) and over 90% of pancreatic ductal adenocarcinomas (PDACs). Since development of small molecules capable of inhibiting KRAS directly has proven difficult, alternative strategies have instead focused on inhibiting downstream effector pathways, such as the MAPK pathway. However, inhibition of the MAPK pathway alone with MEK inhibitors, such as selumetinib and trametinib, produces only cytostatic effects and is insufficient to kill KRAS-mutant cancer cells.

We hypothesized that inhibition of an additional kinase during MEK inhibitor treatment could improve response. We performed a kinase-targeting shRNA screen to find kinases whose knockdown would cooperate with trametinib in KRAS-mutant CRC and PDAC cell lines. The kinases found in this screen represent potential therapeutic targets to inhibit in combination with MEK.

Interestingly, despite using a very high concentration of trametinib in the screen to enrich for hits outside of the MAPK pathway, the most highly ranked kinases in the screen were members of the MAPK pathway, including ARAF, BRAF, CRAF, and MEK1. This suggests that even at high concentration, trametinib produces suboptimal MAPK inhibition. Indeed, we found that while MEK inhibitors produce robust inhibition of MAPK signaling initially, pathway reactivation was observed by 48-96 hours despite regular replenishment of drug. This feedback reactivation was accompanied by marked increases in active CRAF and phosphorylated MEK. In fact, experimental approaches that artificially increased upstream signaling flux through the MAPK pathway led to a >10-fold reduction in the ability of MEK inhibitors to inhibit the MAPK pathway.

Remarkably, despite triggering the same degree of adaptive upstream MAPK signaling as seen with MEK inhibitor, we found that ERK inhibitors were able to maintain MAPK pathway suppression. Importantly, these differences in MAPK pathway suppression amount to differences in cell viability. Over four weeks, ERK inhibitor treatment reduces the outgrowth of KRAS-mutant cell lines compared to those treated with MEK inhibitors. Additionally, as these inhibitors are not used as monotherapies, replacement of trametinib with an ERK inhibitor in therapeutically relevant combination treatments improved cell responses over four weeks.

Despite the feedback reactivation of the MAPK pathway, we find that ERK inhibitors are less sensitive to this signaling than MEK inhibitors, and can effectively maintain suppression of MAPK signaling. The findings of our screen demonstrate that MAPK pathway targeting is key to successful treatment of KRAS-mutant cancers, and that ERK inhibition provides greater opportunity for inactivating MAPK. Further exploration into the mechanisms of pathway feedback will be necessary to developing valuable clinical combinations for KRAS-mutant cancers.

#102

Overcoming MET inhibitor resistance in GBM therapy.

Nichola A. Cruickshanks,1 Ying Zhang,1 Sarah Hatef,1 Julia Wulfkuhle,2 Isela Gallagher,2 Alexander Koeppel,1 David Schiff,1 See Phan,3 Stephen Turner,1 Emanuel Petricoin,2 Roger Abounader1. 1 _University of Virginia, Charlottesville, VA;_ 2 _George Mason University, Manassas, VA;_ 3 _Genentech, Menlo Park, CA_.

Glioblastoma (GBM) is the most common and most lethal primary malignant brain tumor. The receptor tyrosine kinase MET is frequently upregulated or over activated in GBM. Clinically applicable MET inhibitors have been developed and tested in the lab and in clinical trials. However, resistance to single modality anti-MET drugs frequently occurs, rendering these agents ineffective. This study aimed to determine the mechanisms of MET inhibitor resistance in GBM and to use the acquired information to develop novel therapeutic approaches to overcome resistance. We investigated two clinically applicable MET inhibitors: PF-02341066, an ATP-competitive small molecule inhibitor of MET, and MetMab, a monovalent monoclonal antibody that binds to the extracellular domain of the MET receptor. We generated PF-02341066 and MetMab resistant GBM cell lines and primary cells by subjecting them to increasing concentrations of drug over a period of time. We utilized RNA sequencing (RNA-seq) and reverse phase protein arrays (RPPA) in addition to death and proliferation assays to identify the pathways altered in the resistant GBM cells compared to wild type GBM cells. We discovered many critical proteins that were altered in the resistant cells lines compared to wild type cells. These included FAK, COX-2, p-FGFR1, Vimentin, mTOR and p-STAT3. There was substantial but not complete overlap between the molecules that were altered in cells resistant to the small molecule as compared to molecules that were changed in cells resistant to the antibody. The protein changes in resistant GBM cells were verified by western blotting. Notably, we discovered that both COX-2 and p-FGFR1 were upregulated in GBM resistant cells and thus investigated whether inhibition of these targets could restore MET inhibitor sensitivity. Celecoxib, an FDA-approved drug, acts to inhibit COX-2 and Debio-1347 acts to inhibit the FGFR family, with a higher affinity for FGFR1. Combining Celecoxib or Debio-1347 with PF-02341066 or MetMab led to increased cell death in resistant cells compared to either agent alone. In addition, the drug combination decreased resistance to cell proliferation inhibition compared to either agent alone, indicating restored sensitivity to both MET inhibitors. We are currently undertaking in vivo experiments to establish whether the combination of celecoxib and MET inhibitors can restore sensitivity to MET inhibition in resistant cells as effectively in vivo as seen with in vitro experiments. These data indicate that MET inhibitor resistance can be overcome by targeting the resulting upregulated pathways using FDA-approved drugs and that multi-drug combinations may revert resistance during treatment.

#103

Blockage of Cdc7/CDK9 signaling sensitizes triple negative breast cancer to EGFR-targeted therapy.

Ronan P. McLaughlin,1 Jichao He,1 Vera van der Noord,1 Jevin Redel,1 Marcel Smid,2 Lambert Dorssers,2 John Martens,2 John Foekens,2 Yinghui Zhang,1 Bob van de Water1. 1 _Leiden University, Leiden, Netherlands;_ 2 _Erasmus University Medical Center, Rotterdam, Netherlands_.

The treatment of Triple-Negative Breast Cancer (TNBC) still represents a profound clinical challenge, the disease being disproportionately responsible for breast cancer-associated deaths. As a consequence, the identification of targeted agents, which synergize with current therapeutic options, is paramount. Resistance to EGFR-tyrosine kinase inhibitors (TKIs), despite frequent EGFR overexpression and reliance on downstream signalling pathways in TNBC, remains endemic. Recent data has also suggested that kinase-inhibition of EGFR in itself is insufficient, given the kinase-independent functions of the protein in transcriptional regulation and DNA-repair may permit prolonged EGFR-mediated signalling in the presence of inhibitors. We performed a kinase inhibitor drug screen (378 kinase inhibitors targeting ~40 different cancer-related kinases) to identify synergistic activity with EGFR inhibition in TNBC. We demonstrate that the dual cdc7/CDK9 inhibitor PHA-767491 synergizes with multiple EGFR-TKIs (Erlotinib, Gefitinib and Lapatinib) in vitro to overcome resistance to EGFR-targeted therapy in various TNBC cell lines. Combined inhibition of EGFR and cdc7/CDK9 resulted in reduced cell proliferation, accompanied by induction of apoptosis and G2-M cell cycle-arrest. Combination therapy inhibited crucial components of the DNA-replicative machinery and also proteins involved in CDK9-mediated transcriptional elongation. Moreover, higher expression of cdc7 and POLR2A was found to be significantly correlated with poorer survival rates in breast cancer patients. Additionally, this synergistic combination effectively inhibited the growth of cells isolated from patient-derived TNBC xenografts under ex vivo culture conditions, highlighting the utility of targeting common transcriptional nodes in cancers addicted to growth factor-mediated signalling pathways.

#104

miRNAs in the 14q32 cluster are involved in lapatinib resistance.

Juliette Chupin,1 Ai Nagano,1 Victoria Haley,2 Catherine Lenihan,1 Francesca Cavicchioli,1 Karen O'Leary,1 Natasha Sahgal,1 Cristiana Lo Nigro,3 Alice Shia,1 Claude Chelala,1 Peter Schmid1. 1 _Barts Cancer Institute, London, United Kingdom;_ 2 _Open University, London, United Kingdom;_ 3 _Ospedale Cuneo, Italy_.

HER2 is over-expressed in around 20-25% of breast cancers. Due to increased cell growth and differentiation signaling resulting from HER2 over-expression, this sub-type of the disease is associated with aggressive tumor progression and poor prognosis. HER2 targeted therapy has improved patient prognosis, however, only a subgroup of patients derives the optimal benefit, while other patients have refractory disease or develop resistance. miRNAs are small non coding RNAs involved in the RNAi machinery. They are often aberrantly expressed in tumors and miRNA profiles have been seen to evolve over the course of treatment implicating them in therapeutic resistance.

Methods: miRNA array completed on the Human TaqMan miRNA card. Gene expression profiling performed by Illumina Human HT12 v4.0 array. Differential gene expression used LIMMA1 empirical Bayes statistics module. Adjusted p-values calculated by Benjamini and Hochberg procedure. In silico target prediction used miRWalk. Lapatinib resistant cells generated by culturing cells in increasing doses of lapatinib up to 2µM. IC50 obtained from drug cytotoxicity measured by MTT analyzed by nonlinear regression.

Results: A panel of miRNAs differentially expressed in paired lapatinib sensitive/resistant BT-474 HER2 positive breast cancer cells were identified by miRNA array. Three miRNAs, located in the 14q32 region, were selected for further investigation of their role in resistance to HER2-targeted therapy: miR-127-3p, miR-409-3p, and miR-495-3p. RT-qPCR also confirmed upregulation of these miRNAs in HCC1954 and SKBR-3 HER2 positive cells with acquired resistance to lapatinib, linking increased miRNA expression with acquired resistance. To understand the mechanism behind the miRNA expression in resistant cells we explored epigenetic changes between the sensitive and resistant lines. Global methylation reversal revealed upregulation of all miRNAs in the sensitive cells with a further increase upon addition of an HDAC inhibitor. De-methylation had less effect in resistant cells, indicating loss of methylation as a key component in miRNA upregulation in the process of lapatinib resistance. Using differential gene expression analysis in paired lapatinib-sensitive/resistant BT-474 cells combined with in silico analysis we identified putative targets of these miRNAs. BASP1, a c-MYC transcriptional inhibitor, was down-regulated in the BT-474/L resistant cells. Knock-down altered sensitivity to lapatinib, with an 8 fold shift in IC50 towards resistance, indicating reduced expression of the protein could be involved in the resistance machinery.

Conclusions: miR-127, miR-409, and miR-495, clustered to the 14q32 region are subject to epigenetic control, which is altered in lapatinib resistant cells. Resulting upregulation of the miRNAs appears to post-transcriptionally regulate genes, such as BASP1, involved in an acquired resistance mechanism to lapatinib in HER2 positive breast cancer cell lines. 

### Novel Agents

#105

Depletion of extracellular cystine and cysteine by a mutagenized human enzyme causes ROS mediated cytotoxicity in pancreatic cancer cells .

Sabin Kshattry, Achinto Saha, Shira Cramer, John DiGiovanni, Stefano Tiziani, Nathalie Munoz, George Georgiou, Everett Stone. _University of Texas at Austin, Austin, TX_.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal 5-year survival rate at 7%. The oncogene KRAS that is mutationally activated in over 90% of PDACs, has been shown to be involved in regulating redox homeostasis. Hence, perturbation of oxidative balance might provide a therapeutic window to effectively treat pancreatic cancer. One strategy to achieve this is to target the tripeptide glutathione (GSH) – a major intracellular antioxidant. Cysteine (Cys), which has the functional moiety of GSH, can either be synthesized de novo or imported, predominantly as cystine (CSSC) that is then reduced intracellularly to Cys. In cancer, intracellular Cys synthesis has to be supplemented with extracellular import in order to fulfil the excessive metabolic demand of proliferation, which includes maintenance of oxidative balance through GSH synthesis. We hypothesize that this increased requirement for Cys/CSSC import in tumor cells will make them selectively sensitive to prolonged depletion of these amino acids in the serum by a genetically engineered human enzyme called Cyst(e)inase. In addition, we believe that combining Cyst(e)inase with other redox balance perturbing agents will produce a synergistic therapeutic effect.

In our study, Cyst(e)inase treatment of cultured pancreatic cancer cell lines (Panc1, MIA-PaCa2, BxPC3) decreased intracellular Cys and GSH, and inhibited cell growth. Sensitivity to Cyst(e)inase was correlated with ROS accumulation (Panc1>MIA-PaCa2>BxPC3). Panc1 cells exhibited G2-arrest and apoptotic cell death following 24 hours of treatment whereas BxPC3 cells underwent only a G1-arrest and no cell death even after 72 hours of treatment. Further mechanistic investigation showed activation of AMP kinase and other stress related kinases (p38, ERK and JNK), and DNA damage signaling (ATM) only in the more sensitive cell lines. Inhibition of the mTORC1-p70S6K-S6 ribosomal protein signaling pathway was observed in all 3 cell lines. Cyst(e)inase displayed synergistic cytotoxicity when combined with buthionine sulfoximine (GSH synthesis inhibitor), auranofin (thioredoxin reductase inhibitor), sulfasalazine (inhibitor of cystine import) and the natural compound curcumin known to increase intracellular ROS, indicating that concurrently inhibiting alternative cellular antioxidant pathways or directly increasing intracellular ROS might improve the anti-tumor efficacy of Cyst(e)inase. The effect of biweekly intraperitoneal Cyst(e)inase treatment on growth of pancreatic cancer cell xenografts in nude mice will also be reported. Collectively, the current data suggest that depletion of extracellular Cys/CSSC using Cyst(e)inase may have utility either as a monotherapy or a combination therapy for pancreatic cancer.

#106

Traditional medicinal plants effect on five cancer cell lines.

Elbert L. Myles. _Tennessee State Univ., Nashville, TN_.

Cancer refers to as a group of numerous diseases that can originate in various parts of the body. From the beginning of human history natural compounds have been used for medical purposes. Many of the most frequently used natural compounds have their origin from plants. Plants are very diverse and responsible for the synthesis of a number different secondary compounds. Scientist and non-scientist have taken advantage of these secondary compounds treatment of a variety of diseases. In the current study, we are investigating the viability of specific cancer cell lines after exposure to methanolic extracts from each of the following plants. The plants are Costus, Fenugreek, and Rhamnus. The cell lines used in this investigation are lung cancer (A549), colon cancer, (SW620 and Colo320), breast cancer (BT549), and cervical cancer (HeLa). The determination of cell viability found inhibition. The three test compounds were methanolic extracts from the plants Costus, Fenugreek, and Rhamnus. All showed different levels of viability and was dependent on the specific cell line.

#107

Mechanism of anticancer activity of BPS-001 (lyophilized leech saliva extract).

Amr Ammar,1 Emma Guns,1 Omer Kucuk,2 Abdualrahman Abdualkader,3 Mohamed Alaama,3 A.B.M. Helal Uddin,3 Abbas Ghawi,3 Mohamed Hassona1. 1 _Univ. of British Columbia, Vancouver, British Columbia, Canada;_ 2 _Emory University, Atlanta, GA;_ 3 _International Islamic University Malaysia, Kuantan, Malaysia_.

Recent studies, investigating the composition and therapeutic potential of leech saliva have identified many peptides and proteins with multiple therapeutic properties including anti-thrombotic, antimicrobial and anti-metastatic. In vitro anti-cancer effects were shown in breast, prostate and lung cells lines. In vivo anticancer activity of the extract was shown in multiple breast and prostate cancer xenografts studied by our lab. In our previous studies, BPS-001 was non-toxic and demonstrated various activities including pro-apoptotic properties, inhibition of cellular adhesion, angiogenesis and downregulation of AR expression. To investigate the affected pathways, mechanistic studies were carried out using in vivo and in vitro treated samples. Protein expression in samples was determined by Western blotting of in vitro and in vivo samples. AR expression was measured using an eGFP reporter assay in LNCaP cells, with PSA also measured in the media using cobas e 411 analyzer. BPS-001 inhibited Proteinase Activated Receptor 1 (PAR-1) which is responsible for hormone like cellular signals in different cancer cells including those of the breast and prostate. Moreover, in LNCaP cells AR activation and PSA expression were significantly inhibited after treatment with BPS-001. Inhibition of PI3K and PLCG2 were also observed. It has been found that BPS-001 modulates a plethora of pathways which makes it a very promising candidate for treatment of cancer with an important advantage of low toxicity and potential resistance.

#108

In vitro **and** in vivo **characterization of novel scorpion venom-based peptides for the treatment of colon cancer.**

Bin Li, Hang Fai KWOK. _University of Macau, Macau, China_.

In the last decade, many of the venom-based bioactive peptides demonstrated with a broad and diverse spectrum of pharmacological activities, which help to enlarging the current drug-screening library for searching of new specific biomarkers and novel prototype drugs for diagnosis of diseases such as cancer. Moreover, it paved a new insight to overcome the current drug discovery problems, which including drug resistance problem, side effect and so on.

Here we report the discovery of a panel of novel venom-based peptides, which can significantly inhibit the growth of human colon cancer cells. Through our in-house developed high throughput screening techniques and bioinformatics analysis, we successfully isolated eight novel peptides from scorpion venoms with anticancer activities. We performed a series of anti-proliferative assays to demonstrate that these peptides could inhibit the growth of a broad spectrum of tumor cells, especially in human colon cancer cell line; while almost have no effect on normal human epithelial cells. Haemolysis assay was also performed to show our peptides do not cause any harm on normal human red blood cells, which point to the direction of bringing those peptides into the preclinic trial studies. Before the in vivo studies, we are currently investigating the anticancer mechanisms and involved signal pathways of these peptides. LDH assay and real-time live cell analysis were performed, and from those data it indicates that the peptides might actually work by inhibiting the cancer cell growth instead of disrupt their cell membrane directly. Flow cytometry and western bolt results also showed that the peptides have a significant G1 phase cell cycle arrest effect through inhibiting cyclin dependent kinases CDK4 and up-regulating the expression of cell cycle protein regulator Cyclin D3, p27, p21. Moreover, from our current data, it demonstrated that our peptides wouldn't induce any apoptosis during the cell cycle process. With the solid anticancer mechanism/pathway data, the potential anticancer peptides to target in vivo malignant tumor will be evaluated in cancer xenograft animal model to demonstrate the growth inhibitor activity of our lead peptides.

In summary, our study demonstrate that our peptides can inhibit tumor cell growth via cell-cycle arrest, and almost have no cytotoxicity on human epithelial cells, which could lead our lead peptides to further clinical investigation for the new strategy of peptide mono-/combination therapy.

#109

Effects of trifluoperazine analogue on A549 human lung cancer cells.

Joo Yeon Jeong, Jinhyun Ryu, Nal Ae Yoon, Seokmin Kang, Hyemin Seong, Ahmad Fudhaili, Juyeong Park, Jimin Sim, Jiyeon Lee, Soohyun Hwang, Sang Soo Kang. _Gyeongsang National University, Jinju, Republic of Korea_.

Although there have been great advancement in technology, molecular diagnosis, therapeutics, lung cancer is still the leading cause of cancer related mortality all over the world. Recently, some antipsychotic drugs have been shown to possess the anticancer activity. Thus the present study was designed to evaluate the anticancer effects of trifluoperazine (TFP), a commonly used antipsychotic drug, and its synthetic analogs on human lung cancer cell lines. To this end, effects of TFP and its selected analog on A549 cells were investigated in vitro as well as in vivo experiments. Synthetic TFP analogs were evaluated by the viability of A549 cells following drug treatment and compared to TFP. KCFC51, a selected TFP analog, significantly inhibited the proliferation of A549 cells. Further experiment showed that TFP and KCFC51 had activities to inhibit the migration, and anchorage dependent/independent colony formation of A549 cells. Western blot analysis revealed that TFP and KCFC51 affect the protein expression levels related to cell apoptosis and cell cycle. KCFC51 showed stronger anticancer effects in all the experiment than TFP. Flow cytometric analysis showed that KCFC51 induced sub-G1 population and reduced cell population in S and G2/M phase. In addition, the two in vivo experimental models, KCFC51 also showed powerful anticancer effect in skin xenograft tumor growth and orthotropic lung cancer development than TFP. Thus present study demonstrates that a synthetic TFP analog has anti-lung cancer activity and provides a potential therapeutic candidate for lung cancer.

#110

Quantitative high throughput screening as a tool to identify novel therapies in bladder cancer.

Reema Railkar, Thomas Sanford, L. Spencer Krane, Piyush K. Agarwal. _NCI-NIH, Bethesda, MD_.

Introduction: Bladder cancer (CaB) is the 4th most common cancer among men in the US. It is the most expensive malignancies to treat from diagnosis to death. No new pharmacological agents have been approved for treatment of bladder cancer since the approval of BCG in 1990. Thus, there is an urgent need for development of new treatment therapies. Quantitative high throughput screening (qHTS) of representative cancer lines with oncology drugs may identify new treatments or re-purpose already existing therapies for different disease. We utilized this technique to identify new therapies in two primary bladder lines (T24 and UMUC3) and their metastatic lines (T24T, SLT3 and FL3 of T24 and LUL-2 for UMUC3).

Methods: We screened 7 bladder cancer cell lines against 1,912 oncology drugs using a 48 hour cell proliferation assay with an ATP−based readout (CellTiterGlo) to determine activity and potency of compounds in a dose response manner. One of the candidate drugs inhibitory in all cell lines tested is flavopiridol, a pan CDK inhibitor. We further characterized the mechanism of action and in vivo effects of flavopiridol using various cell based assays and mouse xenograft studies.

Results: The initial screen identified 95 compounds active in 7 cell lines. The top 50 compounds were further analyzed for molecular size of >200 g/mol and TPSA<90. This identified mitomycin C and 8 novel compounds. Further testing revealed Flavopiridol to be most consistent with qHTS data having IC50 of 100-300nM in all the cell lines tested. Flavopiridol induces G2/M arrest; however, very little apoptosis was seen suggesting cytostatic rather than cytotoxic mechanism of flavopiridol action. Flavopiridol showed dose dependent inhibition of migration, invasion and colony formation in CaB cell lines tested. Xenograft studies in rapidly growing UMUC-3 cells showed slowing of tumor growth but not complete reduction indicating cytostatic mechanism of flavopiridol. However, in slow growing cells, 5637, 5/8 treated mice showed complete tumor reduction.

Conclusions: qHTS can identify novel compounds. Flavopiridol seems to be a very effective inhibitor both in vitro and in vivo. Physical properties of Flavopiridol are most suited for intravesical use which may lead to it being an effective inhibitor of CaB in the bladder at higher doses without any/few systemic toxicities. Studies are underway to elucidate the use of flavopiridol as a single intravesical agent.

#111

Targeting STAT3 and telomerase for the treatment of colorectal cancer.

Seyung Chung,1 Quincy Okobi,2 Debbie Adekoya,3 Jaydutt Vadgama1. 1 _Charles Drew University, Los Angeles, CA;_ 2 _UC Santa Cruz, Los Angeles, CA;_ 3 _University of Dublin, Ireland_.

There is an increasing evidence of pro-inflammatory cytokines involvement in cancer development. Here, we found that two cytokines, IL-6 and TNF-α, induced colorectal cancer cells more active and invasive. Combined treatments of IL-6 and TNF-α phosphorylated transcription factors STAT3 in a synergistic manner. STAT3 and NF-ҡB physically interacted upon cytokine stimulation. Similarly, STAT1 hetero-dimerized with STAT3 and the binding affinity was enhanced with cytokine treatments. STAT3 bound the promoter region of human telomerase reverse transcriptase (hTERT), and IL-6 and TNF-α stimulation further enhanced this STAT3 binding affinity. Withaferin A, an anti-inflammatory steroidal lactone, inhibited the IL-6 and TNF-α induced cancer cell invasiveness and decreased colonosphere formation. Notably, withaferin A inhibited STAT3 phosphorylation and abolished the STAT3 and NF-ҡB interactions. STAT3 binding to hTERT promoter was inhibited and telomerase activity was decreased with the withaferin A treatments. Taken together, pro-inflammatory cytokines induced-cancer cell invasiveness is mediated by STAT3 regulated mechanism in colorectal cancer cells. Our study suggests the novel natural compound therapy for the metastatic colorectal cancer in clinical settings.

#112

Identifying drugs that target ovarian cancer tumor initiating cells.

Michelle K. Ozaki, Carrie D. House, Christina M. Annunziata. _National Cancer Institute, Bethesda, MD_.

Ovarian cancer is the most lethal gynecological cancer, with overall five-year survival for women with advanced disease being at only 25%. The current standard of care is treatment with a platinum-based chemotherapy drug. Unfortunately, a subpopulation of tumor cells often persists, contributing to platinum-based chemotherapy resistance and disease recurrence. In this study we investigated drugs to target these putative tumor-initiating cells (TICs), and explored the pathways of these drugs in both adherent proliferative cell culture conditions and non-adherent TIC-enriched cultures. We conducted a drug screen under both growth conditions, in order to identify compounds that are able to inhibit growth of TICs. Of the drugs identified, we focused further efforts on those involved with inhibiting the NF-kappaB pathway because prior studies have linked NF-kappaB activity with drug resistance and poor survival. Cell viability assays done with the four drugs, bardoxolone methyl, salinomycin, disulfiram, and elesclomol, show these drugs inhibit the growth of ovarian cancer cell lines both as TICs and in corresponding adherent cultures. Disulfiram showed preferential killing of TICs in some of the cell lines tested. All drugs showed some evidence for inhibiting NF-kappaB on Western blot, and ability to decrease CD133/ALDH double-positive TICs on flow cytometry. Carboplatin is known to kill proliferative ovarian cancer cells, and increase the relative percentage of CD133/ALDH positive TICs. Our ongoing studies will address combination of each drug with carboplatin, with the hypothesis that these drugs will increase in vivo efficacy by suppressing TICs, and thereby decrease recurrence of platinum-resistant ovarian cancer.

#113

Evaluating the anticancer activity of two flexible heteroarotinoid analogs on breast cancer.

Emily Ginn,1 Hongye Zou,1 Maryam M. Fallatah,1 Shengquan Liu,2 Maggie C. Louie1. 1 _Dominican University, San Rafael, CA;_ 2 _Touro University, Vallejo, CA_.

Flexible heteroarotiniods (flex-hets) are compounds derived from retinoic acid and recent studies have indicated that these compounds exhibit anti-cancer activity. Amongst the flexible heteroarotinoids, SHetA2 has been to shown block the growth of cervical, head and neck, kidney, lung, and ovarian cancers, and most recently, prostate and breast cancers. However, due to SHetA2's limitations— a high degree of hydrophobicity, non-selectivity, and potential liver toxicity— a second generation of analogs was developed. Results from our recent study suggest that one of the second-generation analogs, 1-(1-(naphthalen-1-yl)ethyl)-3-(4-nitrophenyl)thiourea (SL-1-09) exhibits anti-cancer activities against both ERα+ and ER- breast cancer cells at micromolar concentrations. Since SL-1-09 is a racemic mixture, the R (SL-1-29) and S (SL-1-30) enantiomers were purified and investigated for their anti-cancer properties. Our results suggest that SL-1-30 has greater growth inhibitory effects on T47D, MCF7, MDA-MB-453, MDA-MB-468 cells in comparison to SL1-29, suggesting that the activity observed in SL-1-09 is likely associated with the S enantiomer. Consistent with this data, breast cancer cells treated with SL-1-09 and SL-1-30 express lower levels of proteins that regulate the cell cycle (i.e, cyclin A, cyclin B, cyclin D1, cyclin E and cdk2). These results demonstrate that SL-1-30 and SL-1-09 inhibit breast cancer cell growth, potentially by blocking cell cycle progression, however further studies are necessary for determining the mechanisms of action.

#114

TG02 induces cell cycle arrest in glioblastoma.

Yu-Ting Su, Robert Chen, Hallie Lappin, Herui Wang, Dragan Maric, Orieta Celiku, Aiguo Li, Mark R. Gilbert, Jing Wu. _NIH, Bethesda, MD_.

Dysregulated cell cycle contributes to the limitless replicative potential of cancer cells. The cyclin-dependent kinases (CDKs) and cyclins, the CDK-regulatory proteins are the key cell cycle machinery and can be targets for cancer therapy. TG02, a pyrimidine-based multi-kinase inhibitor has an anti-glioma effect which was demonstrated by our previous studies. An in vitro kinase spectrum assay of TG02 demonstrated inhibitory effects in several CDKs at the nanomolar level. To further investigate the molecular mechanisms of TG02-induced cell cycle regulation in glioma, a cell cycle analysis was performed by flow cytometry using the Click-It Edu Flow Cytometric Assay Kit in GSC923 and U251 cell lines, a human stem-like cell and patient derived cell line, respectively. Protein expression of CDKs and cyclins was tested by Western blotting. RNAseq analysis using Next Generation Sequencing was performed (only GSC923) and followed by a supervised hierarchical clustering analysis of cell cycle pathways that were derived from Qiangen's Ingenuity Pathway Analysis (IPA). Our results showed that TG02 treatment results in G2/M-phase arrest in glioma cell lines. The protein expression of CDK1, CDK2, cyclin A2 and cyclinB1 are downregulated in both TG02 treated cell lines. Additionally, RNAseq analysis revealed downregulation of cyclins A2, B1 and B2 and significant irregulation in mRNA expression of G2/M checkpoint and spindle formation proteins, indicating G2 checkpoint and mitosis progression are also affected by TG02. Taken together, we demonstrated that TG02 induced cell cycle G2/M arrest through downregulating CDK and cyclin expression signaling in the preclinical models of glioblastoma. These findings strongly support the further investigation of TG02 as a potential therapy for malignant gliomas. <!--EndFragment-->

#115

Flubendazole targets cancer stem-like properties and the HER2/Akt signaling pathway in HER2-positive breast cancer with trastuzumab resistance.

Daeil Sung, Youngkwan Cho, Eunhye Oh, Tae-Min Cho, Yoon-Jae Kim, Ji Young Kim, Jae hong Seo. _Korea University, Seoul, Republic of Korea_.

Background and Propose: HER2-positive breast cancers are associated with an aggressive phenotype and trastuzumab resistance leading to poor clinical outcomes. Flubendazole (FLU) is a potent anthelmintic agent that inhibits microtubule polymerization, and also exhibits anticancer activity in several cancer types. The objective of the present study was to investigate the effect of FLU on apoptosis, HER2/Akt signaling pathway, cancer stem cell (CSC)-like properties and trastuzumab-resistance in HER2-positive breast cancer cells. Methods: FLU-induced antitumor effects were characterized by MTS assay, Sub-G1 and G2/M-phase arrest population, Annexin-V analysis, ALDH1 activity assay, Western blotting and immunofluorescence. For in vivo study, trastuzumab-resistant JIMT1 cells were injected into the mammary fat pads of BALB/c nude mice. We then determined the tumor growth and CSC-like properties. Results and conclusion: FLU significantly reduced cell viability and induced apoptosis with accompanied by activation of caspase-3, -7, and -8, as well as PARP cleavage in HER2-positive cell lines (MDA-MB-453, SKBR3 and BT474). FLU-induced apoptosis is associated with significant decreases in expression levels of HER2, p-HER2 (Tyr1221/1222), HER3, and p-HER3 (Tyr1289). FLU treatment also resulted in G2/M-phase arrest of the cell cycle, as evidenced by a marked downregulation of phospho-Histone H3 expression. These results were closely related to the decline of CSC-like properties, coinciding with the suppression of ALDH1 activity. These effects were also observed in trastuzumab-resistant JIMT-1 cells, and significant reductions in tumor growth and ALDH1A1 downregulation were observed in xenograft models together with downregulation of HER2 expression in vivo. Our findings suggest that FLU may be potentially effective for the treatment of HER2-positive breast cancer with trastuzumab resistance.

#116

Erufosine, a third-generation alkylphosphocholine with cytotoxic and cytostatic effects in breast and colorectal cancer cells.

Asim Pervaiz,1 Saqib Mahmood,1 Martin R. Berger2. 1 _Univ. of Health Sciences, Lahore, Pakistan;_ 2 _German Cancer Research Center (DKFZ), Heidelberg, Germany_.

Introduction Breast and colorectal cancers are highly malignant diseases with low 5-year survival rate (<21%) in advanced stages, which highlights the need of finding new therapeutic options. Considering this, we are interested in exploring the anticancer potential of erufosine, a membrane targeting synthetic lipid analogue belonging to a class of antineoplastic agents known as alkylphosphocholines (APCs). Properties such as intravenous administration and significantly reduced hemolytic activity as compared to previous APC generations, and efficient cytotoxic effects against malignant cells make it an attractive anticancer compound.

Materials and Methods In the present experiments, we studied the effects of erufosine against breast (MDA-MB-231 and MCF-7) and colorectal (SW480 and SW620) cancer cell lines. Following exposure to erufosine (0.78-100µM) for 24, 48 and 72h, cytotoxic effects were measured by MTT dye reduction assay and inhibitory concentrations (IC) were identified by GraphPad Prism software 6. Erufosine mediated cytostatic effects were highlighted by means of propidium iodide (PI) based labeling of the DNA followed by flow cytometry analysis. Furthermore, the cells were exposed to increasing concentrations of erufosine (IC25, 50, 75) and alterations in cell cycle relevant genes (CCNA1, CCND1, CCNE1, CCNE2, CDK1, CDK4) were investigated by qRT-PCR methodology.

Results Erufosine was highly active against the selected cell lines (IC50 < 10µM, 24h) and induced significant arrest in the G2/M phase of the cell cycle. The cytostatic effects were more pronounced in the two metastatic cell lines of breast (MDA-MB-231) and colorectal (SW620) cancers. At molecular levels, erufosine induced expression of CCNA1 (≥ 2fold) in SW480, SW620, and MCF-7 cells, while the expression was inhibited in MDA-MB-231 cells at high concentrations (IC50, IC75). There were no significant alterations (≤ 2fold) in the expression levels of CCND1, CCNE1 and CDK4 in the four cell lines. CCNE2 was significantly downregulated in breast cancer cell lines only. Noteworthy, alterations in CDK1 were associated with cell lines from primary cancers, where the expression was upregulated in SW480 cells and downregulated in MCF-7 cells.

Conclusion Antineoplastic effects of erufosine contribute to decreased proliferation of breast and colorectal cancer cell lines. This synthetic lipid induces arrest in G2/M phase and alterations in cell cycle relevant genes in the selected panel of breast and colorectal cancer cells. Characteristics like in vivo stability, minimal hemolytic activity, no toxicity to bone marrow and antineoplastic effects (cytotoxic/cytostatic) against cancer cells make it an attractive therapeutic compound. Further understanding at molecular levels supported by in vivo experimentation will pave the way to validate its therapeutic potential against cancers.

#117

Anti-proliferative and cytotoxic effects of verteporfin in endometrial cancer cells.

Radhika P. Gogoi,1 Jessica M. Castañeda-Gill,2 Raghu P. Metpally,1 Sarath B. Krishnamurthy,1 Jamboor K. Vishwanatha,2 David J. Carey,1 John D. Nash,1 Venkata Ramesh Dasari1. 1 _Geisinger Health System, Danville, PA;_ 2 _University of North Texas Health Science Center, Fort Worth, TX_.

Endometrial cancer (EMCA) is the most prevalent gynecologic cancer in women in the United States. The relatively poor prognosis and limited therapeutic options in advanced endometrial cancer underscores the urgency in developing new and more effective treatment modalities. On the basis of clinical and histopathological features, EMCA is classified into type I and type II disease groups. Type 1 is estrogen potentiated, estrogen receptor (ER) and progesterone receptor (PR) positive, and generally carries a favorable prognosis. Type 2 is ER/PR negative and carries a much poorer prognosis. We investigated the therapeutic efficacy and mechanism of action of Verteporfin (VP), a benzoporphyrin derivative in EMCA. Treatment with VP resulted in a decrease in cell viability, invasion and an increase in cytotoxicity of EMCA cells. Similarly, VP treatment increased apoptosis in organoids, developed from patient-derived xenografts. Since the half-life of verteporfin is very short, we evaluated the longer duration efficacy of VP, encapsulated in mPEG-PLGA polymers. Our results demonstrated that VP nanoparticles (NP) decreased cell viability in both Type 1 (HEC-1-B) and Type 2 (ARK-1) EMCA cell lines over a period of 4 days. We did not observe any lethal effects of blank NP to the EMCA cells. In order to determine the efficacy of VP in changing cellular transcriptome, we performed RNASeq of EMCA cells after treatment with VP. RNASeq data analysis was carried out using latest version of Kallisto and Sleuth software. Based on the global gene expression whole transcriptome analysis, we observed that HEC-1-A VP treated cells show 225 differentially expressed transcripts (out of 204 genes); whereas HEC-1-B VP treated cells show 12315 differentially expressed transcripts (out of 7870 genes). There were 114 differentially expressed transcripts (out of 105 genes) common to both the cell lines. We observed that ATM, ATR, BRCA2, CDK4, ERBB2, JAK2, NF1, NOTCH and TET2 are some of the differentially expressed genes after VP treatment. To test the efficacy of VP under in vivo conditions, we developed a subcutaneous mouse model of EMCA using HEC-1-B-GFP cells. IP administration of VP in this model achieved 21% tumor regression. To more accurately simulate the results in vivo in a clinically relevant model, we have developed an orthotopic tumor model with IV injection of VP. Taken together, our results suggest that VP is a promising chemotherapeutic agent for the treatment of endometrial cancer.

#118

Targeting cancer with a novel BET bromodomain inhibitor ODM-207.

Anu-Maarit Moilanen,1 Mari Björkman,1 Reetta Riikonen,1 Chandrasekhar Abbineni,2 Mahaboobi Jaleel,2 Sivapriya Marappan,2 Tarja Ikonen,3 Girish Daginakatte,2 Aravind A B,2 Elina Mattila,1 Juha Rantala,4 Susanta Samajdar,2 Murali Ramachandra,2 Pekka Kallio1. 1 _Orion Corporation Orion Pharma, Turku, Finland;_ 2 _Aurigene Discovery Technologies Limited, Bangalore, India;_ 3 _Orion Corporation Orion Pharma, Finland;_ 4 _Misvik Biology, Turku, Finland_.

Background: Bromodomain and extra-terminal (BET) family proteins are dual bromodomain-containing epigenetic readers that bind to acetylated-lysine residues at gene promoter and enhancer elements in histones and recruit protein complexes to promote transcriptional elongation. Recent evidence demonstrates that BET bromodomain inhibition leads to anti-proliferative activity in pre-clinical models of many hematological malignancies and solid tumors. Selective inhibition of BET bromodomains by small molecule inhibitors has emerged as a promising therapeutic strategy for the treatment of cancer. In this study, we evaluated the antitumor activity of ODM-207, a novel, potent and highly selective BET bromodomain inhibitor.

Methods and Results: ODM-207 is a potent and selective BET inhibitor that is structurally unrelated to the benzodiazepine like inhibitors such as JQ1, I-BET762, and OTX015. We tested the preclinical activity of ODM-207 across multiple tumor types in a 4-day growth inhibition in vitro assay. ODM-207 potently inhibits cell viability of a wide range of hematological and solid tumor cell lines. ODM-207 also shows potent antiproliferative effects in patient-derived cancer cells representing various tumor types. In VCaP prostate cancer cell lines, ODM-207 induced apoptosis consistent with increased expression of pro-apoptotic regulators, whereas potent antiproliferative effects associated with cell cycle arrest and cellular senescence were seen in e.g. LNCaP prostate cancer cell line. To gain insight on mechanisms of acquired BET inhibitor resistance, we generated a cell line with resistance to BET-inhibition by culturing LNCaP prostate cancer cells with increasing concentrations of OTX015, a potent and selective BET-inhibitor. Interestingly, ODM-207 is also able to inhibit proliferation and downregulate Myc levels in cells that have acquired resistance to BET-inhibitors OTX015 and I-BET762. In xenograft models, oral treatment with ODM-207 significantly inhibits tumor growth at a dose which is well tolerated.

Conclusions: In summary, these studies demonstrate that ODM-207 is a potent inhibitor of BET proteins in models of hematologic malignancies and solid tumors in vitro and in vivo and support its clinical development for the treatment of cancer.

#119

Vitamin K2 targets castration-resistant prostate cancer VCaP cells by reactive oxygen species mediated apoptotic cell death.

Subramanyam Dasari,1 Maarten C. Bosland,2 Andre Kajdacsy- Balla,2 Gnanasekar Munirathinam1. 1 _University of Illinois, College of Medicine at Rockford, Rockford, IL;_ 2 _University of Illinois at Chicago, Chicago, IL_.

Prostate Cancer (PCa) is the second most common cancer in western countries especially in US population, in which castration-resistant prostate cancer (CRPC) is the major cause for patient mortality. Current treatment options available for CRPC are not efficient and have undesirable side effects. Hence there is an urgent need to develop non-toxic and effective treatment strategies for CRPC. Vitamin K2 (VK2), a natural menaquinone has several medicinal values including anti-cancer activity and anti-osteoporosis effect. The aim of this study is to evaluate the therapeutic effects of Vitamin K2 (VK2) and its anti-cancer mechanism against CRPC. In this study, we have used VCaP cell line (ATCC) which is established from a patient with hormone refractory prostate cancer. VCaP cells were treated with various concentrations of VK2 to evaluate its effects on cell viability by MTT assay, anchorage independent growth by soft agar assay, cellular senescence by beta-galactosidase staining assay and cancer cell migration by wound healing assay. We have also assessed the VK2-induced production of intracellular reactive oxygen species (ROS) using DCF (2′,7′-dichlorofluorescein) probe based fluorescence assay. VK2 induced apoptosis was detected by Annexin-V FITC and TUNEL assays. Western blot analysis was utilized to uncover the anti-proliferative and anti-metastatic mechanisms of VK2 against CRPC. Our results showed that VK2 significantly inhibits the proliferation of VCaP cells in a dose dependent manner at 48 hrs treatment in vitro. MTT data also showed that anti-proliferative effects of VK2 were significantly abrogated in the presence of anti-oxidant N-acetyl cysteine (NAC) and caspase inhibitor Z-VAD-FMK suggesting that ROS and caspase activation as the underlying anti-cancer mechanisms of VK2 in CRPC cells. In addition, VK2 reduced the migration potential of VCaP cells in wound healing assay and inhibited anchorage independent growth of these cells when compared to untreated cells. Annexin-V and TUNEL assays confirmed that VK2 induces apoptosis in VCaP cells. Our results also suggested that the VK2 has the ability to enforce growth arrest in CRPC cells by activating cellular senescence. Western blot analysis revealed that VK2 downregulated the expression of BiP, survivin, MMP-2, and PCNA while activating PARP-1, p21 and DNA damage response marker, phospho-H2AX in VCaP cells. Furthermore, VCaP cells treated with VK2 resulted in the activation of Caspase-3 and-7 apoptotic mediators. These results correlated with translocation of Bax and Cytochrome C to cytoplasm following VK2 treatment in VCaP cells as determined by confocal immunofluorescence analysis. In conclusion our study suggests that VK2 might be an effective anti-proliferative and anti-metastatic agent for CRPC by specifically targeting key anti-apoptotic, cell cycle progression and metastasis promoting signaling molecules.

#120

A study of in vitro and in vivo effects of a novel peptide and rhenium compounds on prostate cancer.

Hirendra N. Banerjee,1 Sasha Hodge,1 William Kahan,1 Santosh Mandal,2 David Weber,3 Rena Lapidus,4 Fazlul Sarkar,5 Somiranjan Ghosh6. 1 _Elizabeth City State University, Elizabeth City, NC;_ 2 _Morgan State University, Baltimore, MD;_ 3 _University of Maryland Medical School, Baltimore, MD;_ 4 _Greenbaum Cancer Center, University of Maryland, Baltimore, MD;_ 5 _Wayne State University Koromanos Cancer Center, Detroit, MI;_ 6 _Howard University, Washington, DC_.

Emerging evidence suggests that acquisition of the epithelial-to-mesenchymal transition (EMT), a process that resembles the genesis of cancer stem-like cells, contributes to tumor aggressiveness and is mediated by deregulated expression of microRNAs (miRNAs), such as miR-200 and let-7 family. Loss of miR-200 expression results in the over-expression of Lin28B, which is prevalent in human Prostate Cancer( PCa) . Lin28B is also known to block the processing of another miRNA (pre-let-7 and pri-let-7), resulting in decreased mature let-7, thereby leads to increased Suz12 and EZH2 expression, which are important components of the polycomb repressive complex 2 (PRC2). Thus, over- expression of Lin28B and loss of miR-200 and let-7 appear to be responsible for PCa aggressiveness. A group of novel rhenium compounds have shown promising anti-cancer properties in various cancer cell lines tested in our laboratories. Our current investigations show that re-expression of miR-200b, miR-200c, and let-7 could be achieved by treating cells with our newly developed rhenium compounds by down-regulating the expression of Lin28B and EZH2. Based on our preliminary results, we hypothesize that over-expression of Lin28B leads to the acquisition of invasive and metastatic characteristics in PCa cells (EMT-phenotype cells) via down-regulation of miR-200b and miR-200c, resulting in increased expression of Suz12, ZEB1, and ZEB2. We also hypothesize that over-expression of Lin28B represses the maturation of let-7 family, leading to increased expression of EZH2, and these processes can be attenuated by treatment of cells with novel rhenium compounds in vitro. Our experiments were done on prostate cancer cell lines derived from both Caucasian(CA) and African American (AA)patients and also biopsy samples obtained from both CA and AA patients at Henry Ford Hospital and Karmanos Cancer Center of Wayne State University, Detroit, MI. We confirmed using gene expression studies by micro array and Real-Time PCR and then data analysis by Ingenuity software systemthe validity of our hypothesis and continued our research by testing one of these rhenium compounds-RPR1 on prostate cancer model of nude mice at Greenbaum Cancer Center(GCC) of University of Maryland at Baltimore. In vitro studies by Real -Time PCR, Flow Cytometry, Western Blotting ,Smart-Flare technology, cell death and proliferation assays along with inhibition of spheroid forming assays showed efficacy of these rhenium compounds as anti cancer agents. In vivo studies also showed decrease of tumor volume and mass ,no toxicity, thus efficacy of these novel drugs. ACKNOWLEDGEMENT: Supported by NIH- 3R01CA164318-03S1 and NIH-T-34-GM100831, a NSF-VESTEM award and NSF-LSAMP award .

#121

Identification and characterization of calcium channel blockers as inhibitors of YAP/TAZ and glioblastoma cells.

Wei Li, Zhijun Liu, Jennifer Xavier, Hong-Gang Wang. _Penn State Univ. College of Medicine, Hershey, PA_.

Glioblastomas (GBM) are the most aggressive primary brain tumors. Despite major advances in neuroimaging and neurosurgical techniques over the past decades, the neurosurgical management of GBM patients remains challenging. Median survival of the patients is about 14.6 months. In order to radically alter the clinical course of these tumors, it is important to develop targeted therapies based on identified oncogenic mutations and signaling pathways that drive their development and sustain their maintenance. In a screen for small molecules, which can inhibit the growth of human glioblastoma cells, we identified a set of calcium channel blockers (CCB). We found that these CCB induce cell death through an apoptosis-independent process. To understand the mechanisms of the non-apoptotic cell death induced by these CCB, we dissected cellular signaling which is responsible for the cell death induction. We found that these CCB are able to inhibit YAP and TAZ, two oncogenic effectors of the Hippo tumor suppressor pathway. Importantly, GBM cells expressing the YAP active mutant or overexpressing YAP are less sensitive to these CCB. These results suggested that inhibition of YAP/TAZ is a mechanism by which CCB suppress tumor cell growth. The Hippo pathway is an essential signaling network in regulating tissue homeostasis. In this pathway, the transcription co-activators YAP and TAZ promote a gene expression program favoring cell proliferation, survival and self-renewal. In normal brain, the nuclear accumulation of YAP is largely limited to regions harboring neural progenitor cells. However, increased nuclear accumulation of YAP is found in various brain tumors, especially those more aggressive subsets of gliomas. In addition, high expression of YAP is associated with reduced mean survival in astrocytoma patients. Our results identified a novel approach to target the Hippo pathway for GBM treatment. We will present our work in further characterizing these regulations and therapeutic potentials of these calcium channel blockers in the conference.

#122

**Preclinical update on targeting** KRAS **wild-type colorectal cancer with an EGFR-targeted monoclonal tri-body mixture, MM-151.**

Shawn P. Carey, Hongfang Wang, Erika Handly, Brittany Ahlstedt, Daniel Gaddy, Jeffrey D. Kearns, Greg Finn, Birgit Schoeberl, Rachel Nering. _Merrimack Pharmaceuticals, Cambridge, MA_.

Within KRAS wild-type colorectal cancer (KRAS-wt CRC), standard of care treatment includes EGFR-directed monoclonal antibodies (mAb) in combination with an irinotecan-based chemotherapy regimen. While such combinations have clinically established synergy, the underlying mechanisms of synergy remain elusive, and resistance invariably develops against either the EGFR-targeted therapy or irinotecan, leaving patients with limited treatment options. MM-151 consists of three anti-EGFR IgG1 mAbs that bind simultaneously to non-overlapping epitopes on the EGFR extracellular domain (ECD). Preclinical and clinical studies have demonstrated that oligoclonal targeting of EGFR with MM-151 results in blockade of the EGFR pathway via potent inhibition of ligand-driven signal amplification and receptor downregulation. Critically, these activities are uniquely maintained in the presence of high-affinity EGFR ligands as well as mutations in the ECD of EGFR, both of which drive resistance to cetuximab and panitumumab. Here, we tested the preclinical hypothesis that comprehensive inhibition of the EGFR pathway by MM-151 leads to enhanced antitumor activity when combined with chemotherapy in models of KRAS-wt CRC. In vitro studies to investigate the activity of MM-151 in combination with SN-38, the active metabolite of irinotecan, showed that MM-151 potentiates SN-38-induced cell death in KRAS-wt CRC models. Treatment with MM-151 potently suppresses EGFR pathway activation caused by SN-38, and the combination enhances stress and pro-apoptotic signaling, leading to increased apoptosis. Notably, these synergistic activities are maintained in the presence of two key mechanisms of resistance to cetuximab: high-affinity ligands and EGFR ECD mutations. Dose scheduling studies demonstrated that sustained treatment with MM-151 following transient SN-38 exposure - a hallmark of small molecule therapy - partially overcomes the sub-optimal activity of chemotherapy alone. Finally, data from in vivo xenograft and PDX studies strongly supports the addition of MM-151 to irinotecan-based chemotherapy regimens for KRAS-wt CRC. In summary, we present data supporting the hypothesis that comprehensive antagonism of the EGFR pathway via oligoclonal targeting of EGFR with MM-151 leads to antitumor activity when combined with chemotherapy in preclinical KRAS-wt CRC models. The ability of MM-151 and SN-38 to overcome both de novo and acquired mechanisms of resistance within CRC models supports further clinical evaluation of this combination in metastatic CRC patients.

#123

A novel J-series prostamide mediates D-series prostamide-induced apoptosis in skin cancer: receptor-independent signaling.

Eman Soliman,1 Daniel Ladin,2 Hussam Albassam,2 Ahmed E. Elhassanny,2 Andrew Morris,3 Allison Danell,3 Rukiyah Van Dross4. 1 _Faculty of Pharmacy, Zagazig University, Zagazig, Egypt;_ 2 _Brody School of Medicine, East Carolina University, Greenville, NC;_ 3 _Department of Chemistry, East Carolina University, Greenville, NC;_ 4 _Brody School of Medicine and Center for Health Disparities East Carolina University, Greenville, NC_.

Non-melanoma skin cancer (NMSC) is the most common cancer in the United States. The absence of selective toxicity is a major problem that limits the utility of chemotherapeutic and radiation therapy for NMSCs. Our previous data showed that the endocannabinoid, anandamide, selectively induced apoptosis in non-melanoma skin cancer (NMSC) cells which overexpress cyclooxygenase-2. The cytotoxic effect of anandamide was mediated by the production of ethanolamide-conjugated D- and J-series prostaglandins (PGs), also known as prostamide D2 (PMD2) and prostamide J2 (PMJ2), respectively. The aim of the current study was to determine if tumorigenic keratinocytes are more sensitive to PMD2 than non-tumorigenic cells and to examine whether the cytotoxicity of PMD2 is mediated by its downstream metabolite PMJ2. To determine if PMD2 demonstrates preferential toxicity, tumorigenic (JWF2) and non-tumorigenic (HaCaT) keratinocytes were utilized. A significant reduction in cell viability was observed in JWF2 but not in HaCaT cells treated with PMD2. In tumorigenic keratinocytes, PMD2 induced apoptotic cell death, oxidative stress and increased the expression of apoptotic ER stress protein, C/EBP homologous protein-10 (CHOP10). In addition, use of the antioxidant, trolox, and ER stress inhibitor, salubrinal, inhibited the cytotoxic effect of PMD2. Furthermore, the use of prostaglandin D receptor (DP1 and DP2) antagonists did not inhibit PMD2-induced apoptosis indicating that the activity was mediated by a receptor-independent pathway. Similar effects were observed in keratinocytes treated with the structurally-related arachidonic acid metabolite, prostaglandin D2 (PGD2). Interestingly, PMD2 increased the production of J-series prostaglandins in both tumorigenic and non-tumorigenic keratinocytes. LC-ESI-MS/MS analysis detected ethanolamide- conjugated J series PG (15-deoxy, Δ12,14 prostamide J2, 15d-PMJ2) in PMD2-treated cell culture media. Since selective inhibitors of the J-series prostaglandins are not available, 15d-PMJ2 was synthesized to examine its direct activity. Exogenous 15d-PMJ2 mimicked the activity of PMD2 demonstrating preferential cytotoxicity towards tumorigenic compared to non-tumorigenic keratinocytes. In addition, 15d-PMJ2 induced oxidative stress, ER stress and apoptosis in tumorigenic keratinocytes. These findings suggest that the cytotoxicity of PMD2 is mediated by 15dPMJ2. Since PMD2 and its metabolite 15d-PMJ2 are preferentially toxic towards tumorigenic cells, PMD2 or 15d-PMJ2 may be an ideal topical treatment for NMSC that will elicit minimal toxicity in healthy surrounding skin cells.

#124

Imipridone ONC201 promotes intra-tumoral accumulation of CD3+/NK+ cells that contribute to its anti-tumor efficacy.

Jessica Wagner,1 C. Leah Kline,1 Lanlan Zhou,1 Andrew Zloza,2 Charles Chesson,2 Jenna Newman,2 Howard Kaufman,2 Joseph Bertino,2 Mark Stein,2 Wafik El-Deiry1. 1 _Fox Chase Cancer Center, Philadelphia, PA;_ 2 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ_.

ONC201, a first-in-class oral anti-tumor agent, upregulates the pro-apoptotic immune cytokine TRAIL and activates the integrated stress response leading to upregulation of death receptor 5 in bulk tumor and cancer stem cells. We previously demonstrated that ONC201 exerts a dose- and schedule-dependent effect on tumor progression in vivo while suppressing Akt/ERK signaling in tumors in a dose/frequency-dependent manner (Wagner et al., AACR, 2016). We also provided evidence that ONC201 exhibits a potent anti-metastatic effect (Wagner et al., AACR, 2016). We observe accumulation and activation of TRAIL-secreting NK+ cells within ONC201-treated tumors in C57/BL6, Balb/c, and athymic nude tumor-bearing mice. Importantly, ONC201 exerts in vivo anti-tumor efficacy on tumor cell lines that are ONC201-resistant in vitro, including acquired stable resistance. Using the NK-depleting antibody GM1, we demonstrate that the activation and TRAIL secretion of NK cells by ONC201 significantly contributes to in vivo anti-tumor efficacy, including TRAIL/ONC201-resistant tumors. We are currently investigating how ONC201 recruits NK cells to the tumor by examining NK-recruiting chemokine factors within the tumor site. We have also demonstrated upregulation of CD3+ T cells by ONC201 in syngeneic mice. Finally, we observed an increase in activated TRAIL-secreting NK cells in the peripheral blood of patients upon ONC201 administration in the clinic. Our results demonstrate novel and potentially significant increases in cytotoxic NK cell recruitment to tumors. The results offer a unique pathway of immune stimulation for cancer therapy that may be combined with immune checkpoint or targeted cancer therapy strategies. We are currently investigating the role of NK cells and CD3+ cells in ONC201's ability to inhibit metastasis by using a metastatic model that involves surgically removing the primary tumor and allowing metastases to grow in vivo before treatment. These findings indicate that ONC201 possess immunomodulatory activity and provide a rationale for combining ONC201 with PD-1/PDL-1 inhibitors, a combination we are currently testing in syngeneic immunocompetent mouse models.

#125

Differential effects of inhibitors of epigenetic modifiers on IDH mutant cell lines.

Alişan Kayabolen, Tugba Bagci Onder. _Koç University, İstanbul, Turkey_.

Our aim is to find effective drug treatments for IDH mutant glioma cells and investigate their mechanism of actions by analysing genetic and epigenetic alterations upon drug treatments.

Drug screen was performed on IDH mutant and wild type cell lines by using a library of epigenetic modifier inhibitors. Hits for IDH mutant cells were validated with individual treatments. Combination treatments of these inhibitors were also performed to see synergistic effects. Then, IDH wild type glioma cells and BJ fibroblasts were transduced with mutant IDH1 overexpression plasmid, and checked if they were sensitized to these drugs or not.

Based on drug screen performed on IDH mutant and wild type glioma cell lines, we found that IDH mutant cell lines, MGG119 and MGG152, were highly sensitive to 5-azacytidine, Chaetocin and GSK-J4. For IDH1 mutant MGG152 cell line, IC50 value for GSK-J4 is 5.2 ± 1.3 µM, for 5-azacytidine is 2.2 ± 0.3 µM, and for Chaetocin is 10.2 ± 1.0 nM at 48h. On the other hand, these drugs did not affect viability of IDH wild type glioma cell lines and fibroblasts around these concentrations. Moreover, combination of drugs, especially combination of Chaetocin and 5-azacytidine had a highly synergistic effect on IDH mutant cells while it was still ineffective on IDH wild type cells. After transduced with mutant IDH overexpression plasmid, fibroblasts became more sensitive to these drugs. On the other hand, although their growth rate was reduced, IDH wild type glioma cells were not significantly sensitized to these drugs upon mutant IDH overexpression.

IDH mutation mainly inhibits DNA and histone demethylation, and leads to hypermethylation phenotype. Therefore, it is an expected result that IDH mutant cell lines are sensitive to 5-azacytidine which is an inhibitor of DNA methyltransferases, and Chaetocin which is an inhibitor of H3K9 methyltransferases. However, it was interesting to observe that GSK-J4 which is an inhibitor of H3K27 demethylases is also effective on IDH mutant cells. This may be a result of dependency on low number of active genes since IDH mutation causes downregulation of many genes by methylation. GSK-J4 may downregulate these genes by increasing H3K27 methylation. On the other hand, these effects might also be results of induced cell stress upon drug treatment. To answer this question, now we are trying to knock-down target genes of these drugs by shRNA, and obtaining compatible results. We are also planning to perform RNA-seq analysis to see genes with altered expressions upon drug treatments.

To conclude, unlike IDH wild type glioma cells or healthy fibroblasts, IDH mutant glioma cells were found to be sensitive to few epigenetic drugs in low concentrations. This was confirmed by sensitization of fibroblasts to these drugs upon mutant IDH1 overexpression. In addition, combination of these drugs had a highly synergistic effect on mutant cells which may give opportunity to use drugs in very low concentrations efficiently.

#126

A chemogenomic approach reveals the action of splicing modulators at the branch point adenosine binding pocket defined by the PHF5A/SF3b complex.

Teng Teng, Jennifer Tsai, Xiaoling Puyang, Michael Seiler, Shouyong Peng, Daniel Aird, Silvia Buonamici, Benjamin Caleb, Betty Chan, Laura Corson, Jacob Feala, Peter Fekkes, Craig Karr, Manav Korpal, Yoshiharu Mizui, Eunice Park, James Palacino, Peter Smith, Vanitha Subramanian, Jeremy Wu, Lihua Yu, Agustin Chicas, Markus Warmuth, Nicholas Larsen, Ping Zhu. _H3 Biomedicine Inc., Cambridge, MA_.

Dysregulation of RNA splicing can cause various forms of cancer and neuromuscular disorders. Thus, developing compounds with splicing-modulating activity represents a promising therapeutic approach for these diseases. Natural products such as pladienolide, herboxidiene, and spliceostatin have been identified as potent splicing modulators that bind SF3B1, a member of the SF3b subcomplex that assembles into the U2 snRNP. Using integrated chemogenomic, structural and biochemical approaches, we show that PHF5A, another core component of the SF3b complex, is also targeted by these modulators. Whole exome sequencing of E7107 (pladienolide analogue) and herboxidiene resistant clones identified common mutations in either PHF5A-Y36, SF3B1-K1071, SF3B1-R1074, or SF3B1-V1078, which confers resistance to these modulators as assessed by splicing modulation and cell growth inhibition, suggesting a common site of interaction for these splicing modulators. We determine the crystal structure of human PHF5A and find that Y36 is located on the surface in a region of high sequence conservation. Analysis of the cryo-EM spliceosome Bact complex from yeast shows that these mutations cluster in a well-defined pocket surrounding the branch point adenosine suggesting a possible competitive mode of action for these modulators. Whole-transcriptome RNA-seq analysis reveals that PHF5A Y36C alters the profile of splicing modulators from inducing intron-retention events to exon-skipping events. Furthermore, the differential in GC content between adjacent introns and exons correlates with the relative intron strength, making some splicing events more susceptible to modulation. Collectively, we propose that PHF5A-SF3B1 is a central node for binding to these small-molecule splicing modulators offering novel approaches to modulate specific splicing events.

#127

Efficacy of novel IRAK4 inhibitor CA4948 in AML and MDS.

Gaurav S. Choudhary,1 Tushar D. Bhagat,1 Maria Elena S. Samson,2 Shanisha Gordon,1 Dagny Von Ahrens,1 Kith Pradhan,1 Aditi Shastri,1 Andrea Pellagatti,3 Jacqueline Boutlwood,3 Robert N. Booher,2 Ulrich Steidl,1 Amit Verma1. 1 _Albert Einstein College of Medicine, Bronx, NY;_ 2 _Curis, Inc., MA;_ 3 _University of Oxford, United Kingdom_.

Myelodysplastic syndrome (MDS) & Acute Myeloid Leukemia (AML) are hematologic malignancies that arise from a population of aberrant hematopoietic stem cells (HSCs). Overactivated innate immune signaling pathways such as IRAK1, TRAF6, IL1RAP, S100A9 and IL8 have been demonstrated in MDS/AML and play important roles in propagation of disease. IRAK4 (interleukin-1 receptor-associated kinase 4), is a protein kinase involved in signaling innate immune responses and forms a critical signaling complex with IRAK1. To determine its role in disease pathobiology, we analyzed transcriptomic data from CD34+ stem and progenitor cells from 183 MDS patients and found significantly increased expression of IRAK4 in MDS samples belonging to the high risk RAEB category (Refractory anemia with excess of blasts, N=80, P Value <0.05 when compared to healthy controls). Furthermore, increased IRAK4 expression was predictive of significantly adverse prognosis (P value < 0.05, median survival of 2.6 years compared to 5.2 years for group with lower IRAK4). Clinical correlations revealed that MDS patients with higher IRAK4 expression in stem/progenitor cells had significantly higher transfusion dependence and had higher leukemic blast counts (Mean Blast Count 9.3% vs 3.9%, P<0.05), further demonstrating IRAK4 to be an adverse prognostic marker in MDS. IRAK4 was also overexpressed in highly purified FACS sorted disease initiating stem cell populations (Long Term-HSC, CD34+/CD38-/CD90+/Lin –ve) from AML patients with complex cytogenetics when compared to healthy controls.

To functionally determine the role of IRAK4 in MDS/AML pathogenesis, we utilized CA-4948, a potent, oral, small-molecule inhibitor of IRAK4, to assess the effect of inhibiting IRAK4 catalytic activity. In vitro, CA-4948 blocked downstream NF-κB pathway signaling, including secretion of proinflammatory cytokines, in Toll-like receptor stimulated THP1 leukemic cells. CA-4948 was tested in clonogenic assays from primary MDS and AML samples. MDS and AML are associated with block in differentiation that leads to cytopenias that are the cause of morbidity in these patients. Treatment with CA-4948 led to increased erythroid and myeloid differentiation in a majority of samples. Furthermore, drug treatment led to decreased viability of MDS/AML stem cells (CD34+/CD38-/Lin-ve) In vivo studies using a THP1 leukemia xenograft model in NSG mice demonstrated that CA-4948 was well tolerated and led to significantly decreased disease burden after 6 weeks of treatment.

In conclusion, we demonstrate that IRAK4 is upregulated in stem and progenitor cells in MDS and AML and is an adverse prognostic marker. Importantly, a novel, specific, inhibitor of IRAK4 shows preclinical in vitro and in vivo efficacy in MDS and AML models.

#128

The next-generation CDK2/9 inhibitor CYC065 elicits marked antineoplastic effects in lung cancer by engaging antimetastatic pathways.

Masanori Kawakami, Jason Roszik, Lin Zheng, Jonathan Kurie, Lisa Maria Mustachio, Xi Liu, Ethan Dmitrovsky. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

We previously reported CDK2 antagonism with the first generation CDK2/9/7 inhibitor seliciclib (CYC202; Cyclacel) triggered anaphase catastrophe. This occurred when genomically unstable cancer cells with supernumerary centrosomes (a hallmark of cancer) fail to cluster excessive centrosomes at mitosis. This causes multipolar cell division and apoptotic death. Anaphase catastrophe is conferred after CDK2 antagonism of aneuploid cancer cells, sparing bipolar normal cells with two centrosomes. CYC065 (Cyclacel) is a next-generation CDK2/9 inhibitor that is undergoing clinical trial. Here, we explored CYC065 activity against lung cancer cells, some with known high metastatic potential. CYC065 substantially inhibited growth, triggered apoptosis, and induced anaphase catastrophe in murine (ED1, LKR13, and 393P) and human (Hop62, A549, and H1299) lung cancer cells. In marked contrast, these effects were largely unseen in bipolar immortalized pulmonary epithelial (murine C10 and human BEAS-2B) cells. We sought to explore whether CYC065 antineoplastic effects engaged antimetastatic pathways. In vitro migration and invasion assays were performed. CYC065 markedly inhibited migration and invasion of lung cancer cells (murine: 344SQ and KC2; human: A549 and H1299). Reverse Phase Protein Arrays (RPPAs) interrogated nearly 300 growth-regulatory proteins in murine (344SQ and KC2) and human (A549 and H1299) lung cancer cells over time (6, 12, 24, and 48 hours) after CYC065 or vehicle treatments. These lung cancer cells have high metastatic as well migration or invasive potentials. When highlighted proteins were clustered after CYC065 treatment, some species were clustered as significantly up-regulated or down-regulated in all cells over studied time points. Not surprisingly, up-regulated proteins included those involved in DNA damage or apoptosis. In addition to known CDK targets like phosphorylated retinoblastoma protein, novel proteins were markedly down-regulated, including mTOR pathway and integrin pathway proteins such as FAK phosphorylation and Src phosphorylation. Affected pathways were identified using Ingenuity Pathway Analysis (IPA). IPA revealed up-regulation of pathways that engaged ATM signaling, G2/M DNA damage checkpoint regulation, or apoptosis signaling. Down-regulated pathways affected mTOR signaling, cyclins, cell cycle regulation, or integrin pathways. Mouse studies will be presented that examine in vivo effects of CYC065 in reducing metastases. Taken together, the next-generation CDK2/9 inhibitor, CYC065, elicits marked antineoplastic effects by antagonizing migration and invasion of lung cancer cells. Comprehensive RPPA and IPA studies found that distinct pathways trigger these effects.

#129

Assessing the mechanism and therapeutic potential of modulators of the human mediator complex-associated protein kinases CDK8 and CDK19.

Paul A. Clarke,1 Maria-Jesus Ortiz-Ruiz,2 Robert Te Poele,1 Olajumoke Adeniji-Popoola,1 Gary Box,1 Christina Esdar,2 Kenneth Ewan,3 Sharon Gowan,1 Alexis De Haven Brandon,1 Phllip Hewitt,2 Wolfgang Kaufmann,2 Aurelie Mallinger,1 Florence Raynaud,1 Felix Rohdich,2 Kai Schiemann,2 Stephanie Simon,2 Richard Schneider,2 Melanie Valenti,1 Julian Blagg,1 Trevor Dale,3 Suzanne Eccles,1 Paul Workman,1 Dirk Wienke Dirk Wienke2. 1 _Institute of Cancer Research, London, United Kingdom;_ 2 _Merck KGaA, Darmstadt, Germany;_ 3 _Cardiff University, Cardiff, United Kingdom_.

Mediator-associated protein kinases CDK8 and CDK19 are context-dependent drivers or suppressors of tumorigenesis. Their inhibition is predicted to have pleiotropic effects, but it is unclear whether this will impact on the clinical utility of CDK8/19 inhibitors. We identified two structurally differentiated chemical series, suitable for exploring their function. In addition to tools that fulfil the criteria set out for chemical probes, the lead compounds from each series, CCT251921 and MSC2530818, had optimal pharmacological and pharmaceutical properties making them suitable for preclinical studies. Having potent, highly selective, orally bioavailable exemplar compounds from these series in hand, we were well positioned to investigate the therapeutic potential of dual CDK8/19 inhibition. The compounds exhibited modest anti-tumor activity in colorectal cancer cell line xenograft models with modulation of p-STAT1SER727, a target engagement biomarker, and altered gene expression profiles, including super-enhancer regulated gene expression, consistent with the inhibition of CDK8/19. In PDX-derived cell cultures we observed inhibition of soft-agar growth in cells derived from different tumor types. However, we only detected significant antitumour activity in 1 of 6 colorectal PDX models tested in vivo, and one example of sensitization to standard of care chemotherapy, despite showing inhibition of p-STAT1SER727. Acute myeloid leukemia cells were the most sensitive cancer type in the PDX panel with therapeutic potency seen in systemic and sub-cutaneous models. Significantly, the compounds impacted on stem cell biology. In a bone progenitor model we saw dose-responsive activation and inhibition of markers of bone matrix and bone deposition that was distinct from WNT blockade. Treatment of a diverse collection of normal cell co-culture models detected a unique response profile consistent with stimulation of an immune/inflammatory response. In vivo treatment of a genetically engineered mouse model expressing oncogenic beta-catenin shifted cells within hyperplastic intestinal crypts from a stem cell to a transit amplifying phenotype. Finally, in pre-clinical tolerability studies we observed a similar, widespread adverse safety profile at therapeutically relevant exposures for both CCT251921 and MSC2530818. At the concentrations tested we detected >80% inhibition of p-STAT1SER727 and increased IL-12 plasma levels. Since the observed pathological effects were generated with two potent, highly selective, but structurally distinct compounds, we conclude that the adverse consequences of treatment are the direct result of inhibition of CDK8 and/or CDK19. The serious and complex nature of the toxicity observed indicates that the clinical development of either series of CDK8/19 modulators, or other chemotypes with similar profiles, will be extremely challenging.

#130

Discovery and characterization of small molecules targeting the DNA-binding ETS domain of ERG in prostate cancer.

Miriam S. Butler,1 Mani Roshan-Moniri,1 Michael Hsing,1 Desmond Lau,2 Ari Kim,1 Paul Yen,1 Marta Mroczek,1 Mannan Nouri,1 Scott Lien,1 Peter Axerio-Cilies,1 Kush Dalal,1 Clement Yau,1 Fariba Ghaidi,1 Yubin Guo,1 Takeshi Yamazaki,1 Sam Lawn,1 Martin Gleave,1 Cheryl Y. Gregory-Evans,1 Lawrence P. McIntosh,1 Paul S. Rennie,1 Artem Cherkasov,1 Michael E. Cox1. 1 _Vancouver Prostate Centre, Vancouver, British Columbia, Canada;_ 2 _University of British Columbia, Vancouver, British Columbia, Canada_.

Genomic alterations involving translocations of the ETS-related gene ERG occur in approximately half of prostate cancer cases. These alterations result in aberrant, androgen-regulated production of ERG protein variants that directly contribute to disease development and progression. This study describes the discovery and characterization of a new class of small molecule ERG antagonists identified through rational in silico methods. These antagonists are designed to sterically block DNA binding by the ETS domain of ERG and thereby disrupt transcriptional activity. We confirmed the direct binding of a lead compound, VPC-18005, with the ERG-ETS domain using biophysical approaches. We then demonstrated VPC-18005 reduced migration and invasion rates of ERG expressing prostate cancer cells, and reduced metastasis in a zebrafish xenograft model. These results demonstrate proof-of-principal that small molecule targeting of the ERG ETS domain can suppress transcriptional activity and reverse transformed characteristics of prostate cancers aberrantly expressing ERG. Clinical advancement of the developed small molecule inhibitors may provide new therapeutic agents for use as alternatives to, or in combination with, current therapies for men with ERG-expressing metastatic castration-resistant prostate cancer.

#131

**Chemotherapeutic effects of naturally occurring** colossolactones **against solid tumor cells in-vitro.**

Mohammed A. Baghdadi,1 Fahad A. Al-Abbasi,2 Ali M. El-Halawany,3 Ahmed M. Al-Abd4. 1 _King Faisal Specialist Hospital & Research Centre, Jeddah, Saudi Arabia; _2 _King Abdulaziz University, Jeddah, Saudi Arabia;_ 3 _Faculty of Pharmacy, Cairo University, Cairo, Egypt;_ 4 _National Research Centre of Egypt, Giza, Egypt_.

Colossolactones were isolated previously from the Vietnamese mushroom ganoderma colossum, such triterpenoids. The different colossolactone compounds (schisanalactonoe a, colossolactone I, ergosterol, colossolactone III, colossolactone II, colossolactone IV, colossolactone E) characterized by the presence of a six-membered lactone ring (ring D) with or without a seven-membered lactone rings (ring A) were isolated. In the current study, using DPPH free radical scavenging assay, all colossolactone compounds showed weak antioxidant activity with EC50's of 99.46, 220.32, 219.65, 100.81, 238.54, 116.57 and 131.94 µM, respectively. Isolated compounds were examined for their potential anticancer activity against breast (MCF-7), cervix (HeLa), colorectal (HCT-116) and liver (HepG2) cancer cells. Cytotoxicity of the isolated compounds was assessed in the aforementioned cell lines using SRB assay after 72 h of exposure and fitted using Emax model. Amongst colossolactones, schisanalactonoe-A and colossolactone-IV showed the best cytotoxic profile in all cell lines under investigation with IC50's ranged from 4.9 to 64.2 µM; and R-fraction less than 47.5%. Further mechanistic studies for the detailed mechanism of cytotoxicity of schisanalactonoe-A and colossolactone-IV are currently carried out.

#132

African polyherbal formulation alleviates benzene-induced leukemia in Wistar rats.

Olufemi E. Akanni,1 Ayodeji Faremi,2 Aminat O. Agboola,1 Adekemi R. Akanni,3 Oluseyi E. Bamisaye4. 1 _Ladoke Akintola University of Technology, Department of Medical Laboratory Science, College of Health Sciences, Osogbo, Nigeria;_ 2 _Ladoke Akintola University of Technology, Department of Chemical Pathology; College of Health Sciences, Osogbo, Nigeria;_ 3 _Ladoke Akintola University of Technology, Department of Medical Microbiology and Parasitology, College of Health Sciences, Osogbo, Nigeria;_ 4 _Afe Babalola University, Department of Medical Laboratory Science, College of Medicine and Health Sciences, Ado Ekiti, Nigeria_.

Background: Chemotherapy and radiotherapy are effective cancer treatment options but they are accompanied by serious side effects. Therefore, more effective therapies are sorely needed. This study investigated the chemotherapeutic effects of a polyherbal formulation on benzene induced leukemia in Wistar rats. The polyherbal formulation is composed of fruits such as Orange, Lime, Lemon, Pineapple, Grape and Vegetables like; Pumpkin leave, Garden egg in honey medium.

Method: Leukemia was induced by injecting 0.2 ml of benzene solution intravenously through the tail at 48 h intervals for four weeks. Leukemia developed in 92% of rats within 2 to 4 weeks after the last benzene injection and further observed for leukemia development in appropriate rat groups. Leukemia burden was assessed using indicator parameters such as total leukocyte, red blood cell count, hematocrit, and hemoglobin concentration. Immunophenotyping enumeration of CD 19 expression also corroborated the leukemia assessment by providing specific cellular marker for differentiation of the developed leukemia type. Exactly 0.48 ml of the polyherbal formulation was administered orally by gavage using oral cannular once daily post leukemia induction for four weeks.

Results: Leukemia induction reflected in significant reduction in hematocrit, hemoglobin concentration, red cell count and a marked and a significant increase in leukocyte count and CD 19 expression over the control (p < 0.05). There is significant difference between the leukemia group treated with polyherbal formulation and the leukemia positive control group untreated.

Conclusion: This study reveals profound chemotherapeutic activity of the polyherbal formulation against experimentally developed leukemia by reversing the induced leukemia in the positive control rat group, hence the ability of the polyherbal formulation in alleviating the cancer symptoms.

Table1.Hematological parameters in polyherbal formulation treated leukemia bearing Wistar rats. | |

|

---|---|---|---

Estimated parameters | Negative control rats

(Mean±SD) | Leukemia Positive rats. (Mean±SD) | Polyherbal formulation treated leukemic rats (Mean±SD)

CD 19 (X 109/L) | 47.59±1.39 | 89.41±1.72 | 62.32 + 1.42*

Total Leukocyte count (X 109/L) | 6.84 + 0.30 | 11.08 + 0.31 | 7.45 + 0.65*

Red blood cells(X 1012/L) | 6.30 + 0.38 | 3.5 + 0.38 | 5.10 + 0.03*

Hematocrit (L/L) | 0.43 + 0.17 | 0.26. + 0.20 | 0.37 + 0.08*

Hemoglobin (g/dl) | 13.62 + 0.69 | 6.63 + 0.96 | 11.80 + 2.96*

Platelets (X 109/L) | 554.80 + 92.23 | 389.50 + 146.10 | 425.10 + 60.8*

*Significance at p<0.05

#133

Aloe-emodin as a potential lead targets lung cancer stem cells.

Pei-Jung Lee,1 Wan-Jiun Chen,2 Chao-Chi Ho,3 Pan-Chyr Yang,3 Huei-Wen Chen4. 1 _Graduate Institute of Oncology, National Taiwan University Medical College, Taipei, Taiwan;_ 2 _Institute of Statistical Science, Academia Sinica, Taipei, Taiwan, Taipei, Taiwan;_ 3 _Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University Medical College, Taipei, Taiwan;_ 4 _Graduate Institute of Toxicology, National Taiwan University Medical College, Taipei, Taiwan_.

Cancer stem cells (CSCs) have been proposed to be responsible for tumor initiating, drug resistance, metastasis, and recurrence. Many novel therapeutic strategies have been designed to target and eliminate CSCs. According to our previous study, we have established a model of CSCs and cancer associated fibroblasts (CAFs) co-culture system for anti-CSCs drug screening. Here, we report one of the potential hits screened via this platform and the anti-CSCs activity was further investigated both in vitro and in vivo. Human lung CSCs and CAFs were primary cultured from patient with lung adenocarcinoma according to our previous study. Image-based high content screening system was used to analyze different parameters after drug treatment. Tumorogenicity and self-renew ability are examined by sphere forming ability. Aldehyde dehydrogenase (ALDH) activity was used to analyze stem cell population by flow cytometry. The expression level of stemness-related genes, Nanog, Oct3/4 and Sox2 were validated by real-time reverse transcriptase Q-PCR. The efficacy of the lead on tumor growth was examined by the xenograft model. Lung cancer stemness markers of the xenograft tumor tissues were also evaluated by immunohistochemistry. Using the image-based high content screening system to screen over one thousands of compounds, we have identified aloe-emodin (AE) shows higher potency targeting on lung cancer stem cells (under the concentration of 1 μM) and high specificity targeting on the cancer cell lines (IC50 < 20 μM); compared to normal human bronchial epithelium cells and human normal fibroblast represented by IC50 (26.77 μM v.s. 39.13 μM). The level of stemness markers, Nanog, Sox2 and Oct3/4 were significantly down regulated after AE treatment compared to cisplatin treatment. AE could suppress tumor initiating abilities and self-renew capacities represented by the ability to grow as tumors spheres in CL152 ALDH+ cells. Besides, AE could inhibit ALDH population in CL152 cells (40% reduced). Also, the AE can inhibit the cisplatin-induced ALDH population as well. Furthermore, we found that AE combine with cisplatin could inhibit tumor growth as comparing to cisplatin treatment in subcutaneous models in NOD/SCID mice, whereas, AE can inhibit the level of Nanog in mice tumor tissues. According to these results, AE is a potential lead targeting on lung cancer stem cells. To discover the pharmacological mechanism of AE on cancer stem cells will be helpful to develop new strategy for lung cancer therapy.

### Targeting the PI3K Pathway

#134

A dual mTOR-PI3K inhibitor DCB-HDG2-57 with Hedgehog signaling pathway antagonist activity.

Ying-Shuan Lee, Mann-Yan Kuo, Chia Wei Liu, Yu-Wen Tseng, Win Yin Wei, Shian-Yi Chiou, Her Sheng Lin, Y.-Y. Lu, Chu-Bin Liao, Shao-Zheng Pen. _Development Center for Biotechnology, New Taipei City, Taiwan_.

The Hedgehog (Hh) signaling pathway is a critical regulator of embryonic patterning, and aberrant Hh pathway activation has been implicated in a diverse spectrum of cancers. Therefore, components of the Shh pathway (such as Shh, SMO, and GLI1/2) are viable therapeutic targets for anti-tumor strategy. Smo antagonists such as GDC-0449 and NVP-LDE225 have received FDA approval for treating basal cell carcinoma. However, acquired resistance has emerged as a challenge to targeted therapeutics and may limit their anti-cancer efficacy. Studies have linked the hyperactive phosphatidylinositol-3-kinase (PI3K)/AKT/mTOR signaling pathway in a variety of human cancers and drug resistance condition. The synergistic role of PI3K/AKT/mTOR in Hh signaling in embryonic development and Hh-dependent tumors has been reported. Both the PI3K/Akt/mTOR and Hh pathway also play important role in maintaining the stem cell-like properties of cancer stem cell (CSCs). We report here the development of a potent dual mTOR-PI3K inhibitor designated DCB-HDG2-57 with Hh signaling pathway antagonist activity. In vitro biochemical assays showed that DCB-HDG2-57 inhibited recombinant pI3Kα and mTOR kinase with IC50 of 27.2 and 133 nM, respectively. DCB-HDG2-57 is also highly active in cellular assays, as evidenced by inhibition of phosphorylation of cellular downstream targets of PI3K-mTOR kinases ribosomal protein S6 kinase (S6K1, Ser240/24) in the colon (LS-180) and pancreatic cancer (MiaPaCa2 and PANC1) cell lines. Interestingly, DCB-HDG2-57 demonstrated Hh signaling pathway antagonist activity in a 293 cell-based Gli-luciferase inhibition assay upon agonist treatment, and retains inhibition activity against the Smo wild-type and D473H mutant co-transfection with IC50 of 261.8 and 327.7 nM, respectively. In addition, DCB-HDG2-57 can significantly decrease the cancer stem cell liked side population of PANC1 pancreatic cell, and inhibited the migration of E3LZ10.7 pancreatic cancer cell. DCB-HDG2-57 demonstrated in vivo tumor growth inhibition activity in LS-180 colon cancer xenograft model and reduced the Gli-1 RNA abundance in the tumor. Taken together, our data demonstrate that combined pharmacological blockade of mTOR-PI3K and Hh pathway of DCB-HDG2-57 can provide a therapeutic strategy for targeting ligand-dependent Hh cancer.

#135

**Targeted therapy of hormone receptor-positive breast cancer harboring** PIK3CA **and** AKT1 **genetic aberrations.**

Maryam Shariati, Kurt W. Evans, Stephen M. Scott, Huiqin Chen, Funda Meric-Bernstam. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Introduction: Molecular alterations in the PI3K/Akt/mTOR pathway is implicated in the pathogenesis of ER positive breast cancer with high frequency and therefore is the major focus of drug development. Activating mutations of this signaling pathway occur in more than 70% of breast tumors. The genetic alterations affecting major components of PI3K/Akt/mTOR include genes encoding the PI3K catalytic subunits p110α (PIK3CA) and the PI3K effector AKT1. Despite the efficacy of the available small molecule inhibitors for targeting different components of PI3K pathway, the contribution of PIK3CA and AKT1 genetic alterations to targeted therapy is not well understood. We speculated that ER positive breast tumors with PIK3CA and AKT1 alterations are more sensitive to PI3K and Akt inhibitors due to differential downstream pathway effectors.

Methods: We used a panel of isogenic MCF7 cell lines with oncogenic mutations of PIK3CA and AKT1 established through somatic cell gene targeting. A series of pharmacological compounds currently approved for breast cancer therapy or in clinical trials in targeting PI3K/Akt/mTOR pathway were evaluated using viability, clonogenic and cell cycle analysis assays. We performed functional proteomic profiling using reverse phase proteomic analysis (RPPA) to investigate differential protein expression in response to PI3K pathway inhibition in PIK3CA and AKT1 mutants MCF7 cell lines. In vivo experiment was done to evaluate the efficacy of the inhibitors in suppressing tumor growth.

Results: We demonstrated that PIK3CA (E545K) and AKT1 (E17K) sensitized cells to the inhibitory effects of BYL719 (p110α catalytic subunit inhibitor) and AZD5363 (pan-AKT kinase inhibitor) on survival, cell cycle progression and colony formation ability. The presence of PIK3CA and AKT1 mutations conferred growth advantage and exhibited increased proliferation in vitro and in vivo. These alterations dramatically increased Akt phosphorylation and induced activation of PI3K downstream effectors. RPPA analysis revealed several proteins differentially expressed in PIK3CA and AKT1 mutant cells compared with wild type (p-value < 0.05).

Conclusion: PIK3CA and AKT1 mutations showed distinct effects on sensitivity to targeted pathway inhibitors in an isogenic luminal breast cancer model. The presence of these genetic alterations in estrogen receptor (ER) positive breast cancer provided proliferative advantage and enhanced their sensitivity to targeted PI3K inhibition. These results will contribute to identify single agent and combination therapies targeting PI3K pathway with maximal activity at tolerated dose and selection for individuals most likely to be responsive based on their genomic alterations.

#136

Phospho-S6 levels correlate with response to Copanlisib (BAY 80-6946) in multiple myeloma.

Sarah Larson, Mao Yu Peng, Monica Mead, Andrae Vandross, Dylan Conklin, Erika Von Euw, Dennis J. Slamon. _UCLA, Santa Monia, CA_.

Background: Copanlisib (BAY 80-6946) is a reversible, pan-class I PI3K inhibitor with preferential activity for the alpha isoform, which may be of particular importance in multiple myeloma. Here we demonstrated the in vitro efficacy of copanlisib in a panel of 20 multiple myeloma cell lines. Baseline levels of phospho-S6 (P-S6) correlated with sensitivity to copanlisib, resulting in a potential biomarker of response. In addition, the change of P-S6 post-treatment could be used as a pharmacodynamic biomarker for copanlisib treatment.

Methods: We screened a panel of 20 multiple myeloma cell lines and selected 3 sensitive: NCI-H929, MM.1S, L-363, and 3 resistant: AMO-1, JJN3, COLO-677 for further analysis. We performed apoptosis and cell senescence assays following 72 hours of 50nM and 100nM copanlisib exposure. Cell cycle analysis and induction of apoptosis were performed by FACS after propidium iodide or PI/ANX-V FITC staining, respectively. Cellular senescence was determined by measuring β-galactosidase activity in cells treated for 96 hours. Reverse phase protein array (RPPA) was performed at baseline and post treatment for proteomic analysis with confirmatory western blots. Flow cytometry was also performed to monitor the post-treatment P-S6 level changes.

Results: Copanlisib treatment induced apoptosis in the sensitive cell lines (50-80% AN-V+ cells) but not in resistant cell lines (1-5% AN-V+ cells). An increased cell cycle arrest in G1 was also observed in the sensitive cell lines but not in the resistant lines. The cell senescence assays confirmed apoptosis rather than cell senescence as the mechanism of inhibition of proliferation. RPPA analysis demonstrated lower baseline p-S6 (S235/236, S240/244) protein levels in sensitive compared to resistant cell lines and this was confirmed with western blot analysis. Treatment with copanlisib resulted in a greater decrease in p-S6 in the sensitive cell lines NCI-H929 and L363 (53-83%, 73-93% respectively) than in the resistant cell lines COLO-677 and JJN3 (5-27%, and 38-67%, respectively), which was validated by western blot and phospho-flow. We also showed by RPPA and WB that copanlisib down-regulates pro-survival and proliferation molecules including p-S6K1, p-S6 and p-4EBP1, and upregulates pro-apoptotic PDCD4 in all cell lines, but to a greater extent in sensitive cell lines. Finally, pharmacodynamic p-S6 response remained at different post-treatment time points.

Discussion: A differential response to copanlisib is seen in the myeloma cell line panel. A subgroup of multiple myeloma cell lines demonstrated median IC50 values in the low nanomolar range (5-100nM), and responses correlated with low baseline P-S6. This p-S6 stratified response was only observed with PI3K-alpha inhibitors, but not with inhibitors targeting other PI3K isoforms or pan-PI3K inhibitors. Further studies may allow development of a new patient screening method or companion diagnostic.

#137

**FT-1518, a new generation selective and potent mTORC1 and mTORC2 inhibitor: an** in vitro **and** in vivo **profile.**

Alain C. Mita,1 Monica M. Mita,1 Anthony D. William,2 Khalid Pasha,3 Chandra Siddamadappa,2 Kevin Zikaras,2 Felix T. Garzon2. 1 _Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA;_ 2 _FTG BIO, Hackensack, NJ;_ 3 _Vipragen Biosciences Pvt. Ltd., Mysore, India_.

Mammalian target of rapamycin (mTOR) is a clinically validated target in the treatment of cancer. mTOR forms two distinct multiprotein complexes, mTORC1 and mTORC2 which regulate cell growth, metabolism, proliferation, and survival. Rapamycin analogues target only the mTORC1 complex but do not affect the mTORC2 complex, which is an important driver for cancer cell growth and survival. The new generation of "Selective" mTOR inhibitors, blocking both mTORC1 and mTORC2 signaling might increase the efficacy and safety while expanding the therapeutic potential of these anticancer agents. Herein we describe FT-1518, a low nanomolar potent, kinase and PI3K sub family selective mTOR inhibitor. FT-1518 not only exhibited high oral bioavailability in preclinical species but has demonstrated excellent microsomal stability with no inhibitory activity towards undesired CYPs. FT-1518 showed high sustained tumor exposure and target Inhibition in a single oral dose xenograft model. FT-1518 depicted very good growth inhibitory activity across a large panel of hematologic and solid tumor cell lines with most activities falling into low nanomolar range. mTOR kinase inhibition in cells, by FT-1518, resulted in more potent inhibition of the mTOR pathway biomarkers (mTORC 1 & 2 biomarkers [pAkt(S473) and pS6(S240/244) or p70 S6K), no inhibition of PI3K biomarker [pAkt(T308)], and improved anti-proliferative activity as compared with rapamycin. FT-1518 exhibited dose-dependent and higher tumor growth inhibition (TGI) in multiple solid tumor xenografts compared with rapalogs and is poised to enter the clinic with a favorable toxicology profile.

#138

FOXO proteins mediate adaptive resistance to PI3K inhibition in mucinous colorectal cancers.

Murali R. Kuracha, Peter Thomas, Brian W. Loggie, Venkatesh Govindarajan. _Creighton University, Omaha, NE_.

Purpose: Mucinous colon adenocarcinomas (MCAs) comprise a substantial fraction (10-15%) of sporadic colorectal cancers (CRCs). MCAs show a distinct range of genetic modifications compared to nonmucinous CRCs and are more prone to peritoneal dissemination and distant metastasis. These aggressive cancers show a poorer response to chemotherapy and are often difficult to treat. A higher mutation rate in effectors of the KRAS-RAF-MEK (80% vs 42%) and PI3K-AKT-mTOR (60% vs 22%) pathways in MCAs over nonmucinous CRCs suggested potential targets for therapy. Our previous results showed that, MCAs, though initially sensitive to PI3K inhibition (PI3Ki), later develop adaptive resistance. The purpose of this study was to test whether FOXO transcription factors mediate this adaptive resistance to PI3Ki single agent treatment.

Methods: PI3K was inactivated in MCA cell lines, LS174T (KRAS G12D, PI3K H1047R) and RW7213 (KRAS G12C, PI3K WT), by treatment with GDC0941 (Pictilisib), a small molecule inhibitor of class I PI3K. Alterations in phosphorylation of effectors in the PI3K-AKT or MEK-ERK pathways were determined using western blots. FOXO nuclear localization was assessed by immunocytochemistry, confocal microscopy and nuclear fractionation studies. Expression of FOXO proteins (FOXO1, FOXO3 and FOXO4) was reduced by short inhibitory RNAs (siRNAs). FOXO1 was also inhibited in MCA cells using AS1842856, a small molecule inhibitor.

Results: Immunohistochemical analysis and nuclear fractionation studies showed significant nuclear enrichment of FOXO1 and FOXO3 in MCA cell lines in response to PI3K inhibitor treatment. Upon siRNA-mediated knockdown of FOXOs, rebound ERK phosphorylation seen in response to PI3Ki treatment was substantially reduced. Concomitant with this decrease was a reduction in expression of receptor tyrosine kinases (RTKs) IR, IGFR, HER2 and HER3. Combinatorial treatment of PI3K and FOXO1 inhibitors resulted in a more pronounced loss of cell viability compared to single agent treatment.

Conclusions: Our results suggest that resistance to PI3K single agent inhibition seen in MCA cells is mediated, at least in part, by nuclear translocation of FOXO proteins, particularly FOXO1. Our results support a model where PI3K inhibition results in increased FOXO nuclear localization which in turn, leads to transcriptional upregulation and activation of RTKs which then lead to rebound activation of the MEK-ERK pathway and increased survival and resistance. A role for FOXO proteins in mediating PI3Ki resistance in KRAS-mutant MCA cells has not been described before. Our results suggest that PI3Ki single agent therapy is unlikely to be successful in KRAS-mutant MCAs due to FOXO-mediated resistance thus providing a rationale for combination treatment strategies.

#139

M2698, a novel dual inhibitor of p70S6K and Akt: preclinical efficacy in gastric cancer.

Shota Fukuoka,1 Takashi Kojima,1 Yoshikatsu Koga,2 Mayumi Yamauchi,1 Masahiro Yasunaga,2 Yasuhiro Matsumura,2 Toshihiko Doi,1 Takayuki Yoshino,1 Toshio Kuronita,3 Anderson Clark,4 Brian Elenbaas,4 Atsuhi Ohtsu1. 1 _Division of Gastrointestinal Oncology, National Cancer Center Hospital East, Kashiwa, Japan;_ 2 _Division of Developmental Therapeutics, Research Center for Innovative Oncology, National Cancer Center Hospital East, Kashiwa, Japan;_ 3 _Merck Serono Co Limited, Tokyo, Japan;_ 4 _EMD Serono Research & Development Institute, Inc, Billerica, MA_.

M2698 is a selective, ATP-competitive dual inhibitor of p70S6K and Akt1/3 that is being evaluated in a phase I clinical trial in cancer patients. The dual nature of M2698 may result in improved clinical efficacy by blocking the increased AKT activity in a compensatory feedback loop induced by PI3K/AKT/mTOR (PAM) pathway inhibition. In previous preclinical studies, M2698 was shown to have potent anti-proliferative activity in vitro, and inhibit tumor growth in some xenograft models in vivo. The current study examined the effects of M2698 on cell proliferation in a panel of 13 gastric cancer cell lines, as gastric cancers often harbor mutations in PAM pathway genes that deregulate this signalling pathway. Cells were treated with M2698 at a range of concentrations and proliferation was evaluated at least twice using the WST-8 cell proliferation assay kit (Dojindo Molecular Technologies, Inc. Japan). Two cell lines, HGC-27 and IM95m, were particularly sensitive to M2698 (50% growth inhibition concentration [GI50] 84 and 160 nM, respectively). The GI50 values for all other cell lines were >10-fold higher. Although HGC-27 and IM95m each carry a PIK3CA hotspot point mutation, the presence of an activating PI3K pathway mutation was not the sole determinant of sensitivity, as a third PIK3CA mutant cell line, MKN1, was far less sensitive to M2698 (GI50 9.0 uM). Western blot analysis of pharmacodynamic biomarkers showed that M2698 (1 uM) blocked the PAM pathway in both sensitive and resistant cell lines, inhibiting phospho[p]‐S6 and p‐PRAS40, despite increased p‐Akt. Some biomarkers from the PAM pathway and other signaling pathways appeared to be associated with sensitivity to M2698, but these candidate biomarkers need to be validated in a larger panel of cell lines. Treatment of HGC-27-tumor-bearing mice with M2698 (10, 20, 30 mg/kg/day) for 14 days resulted in significant tumor growth inhibition (80.2-98.6%) at Day 29 compared to treatment with vehicle (p<0.01). In conclusion, we have shown that gastric cancer cell lines have a range of sensitivities to M2698 and the sensitivity cannot be explained solely by genetic mutations in the PAM pathway. M2698 provides potent PAM pathway inhibition in both sensitive and resistant gastric cancer cell lines by blocking both p70S6K and Akt. M2698 significantly inhibits tumor growth in the HGC-27 xenograft model in vivo.

#140

Discovery and biological evaluation of PQR530, a highly potent dual pan-PI3K/mTORC1/2 inhibitor.

Denise Rageot,1 Florent Beaufils,2 Anna Melone,1 Alexander M. Sele,1 Thomas Bohnacker,1 Marc Lang,2 Jürgen Mestan,2 Petra Hillmann,2 Paul Hebeisen,2 Doriano Fabbro,2 Matthias P. Wymann1. 1 _University of Basel, Basel, Switzerland;_ 2 _PIQUR Therapeutics AG, Basel, Switzerland_.

The PI3K/AKT/mTOR signaling pathway plays a fundamental role in cell proliferation, growth and survival and aberrant activation of this signaling pathway has been shown to drive the progression of malignant tumors.[1] Drugs targeting the pathway at multiple points, such as dual PI3K/mTOR inhibitors appear to have the broadest activity profile to address cancer therapeutic strategies and are currently being explored in numerous clinical studies. Recently, we presented PQR309, a novel, brain-penetrant pan-PI3K/mTOR inhibitor, which entered phase II clinical trials in 2016.[2] Here, we report the lead optimization of PQR530, a potent and brain-penetrant follow-up compound as pan-PI3K/mTORC1/2 inhibitor.

The development of a follow-up compound concentrated on the improvement of both, the potency and the selectivity for all targeted kinases, namely the class IA PI3K isoforms as well as mTOR. We present a detailed ligand-based structure-activity relationship study which was obtained by systematic modifications of the hinge region as well as the affinity binding substituents. This study led to the identification of PQR530, a dual pan-PI3K/mTORC1/2 inhibitor showing excellent activities in cellular assays as well as in PI3Kα and mTOR enzymatic binding assays.

In A2058 melanoma cells PQR530 inhibited protein kinase B (PKB, pSer473) and ribosomal protein S6 (pS6, pSer235/236) phosphorylation with IC50 values of 0.07 µM. PQR530 showed excellent selectivity over a wide panel of kinases, as well as excellent selectivity versus unrelated receptor enzymes and ion channels. Moreover, PQR530 displayed potency in a panel of 44 cancer cell lines (NTRC OncolinesTM) to prevent cancer cell growth (mean value for GI50 of 426 nM). Oral application of PQR530 to mice resulted in a dose-proportional PK and demonstrated good oral bioavailability and excellent brain penetration.[3]

An optimized, robust synthetic route allowed rapid access to multi-gram quantities of PQR530 for pre-clinical development in only 4 steps. In conclusion, PQR530 inhibits all PI3K isoforms and the mammalian target of rapamycin (mTOR) complexes C1/2 potently and selectively, and shows anti-tumor effects in vitro and in vivo.

[1] M. P. Wymann, M. Zvelebil, M. Laffargue (2003). Phosphoinositide 3-kinase signalling – which way to target? Trends Pharmacol Sci.; 24, 366-376.

[2] V. Cmiljanovic et. al. "PQR309: Structure-Based Design, Synthesis and Biological Evaluation of a Novel, Selective, Dual Pan-PI3K/mTOR Inhibitor" presented at AACR Annual Meeting 2015, April 18-22, Philadelphia, Pennsylvania, USA.

[3] P. Hillmann et al. "Pharmacological Characterization of the Selective, Orally Bioavailable, Potent Dual PI3K/mTORC1/2 Inhibitor PQR530" abstract submitted for AACR Annual Meeting 2017, April 1-5, Washington, D. C., USA.

#141

CD44 positive and sorafenib resistant hepatocellular carcinomas respond to the ATP-competitive mTOR inhibitor INK128.

Mohamed Badawi,1 Jihye Kim,1 Dhruvitkumar Sutaria,1 Tasneem Motiwala,2 Ryan Reyes,2 Nissar Wani,2 Samson Jacob,2 Mitch Phelps,1 Thomas Schmittgen3. 1 _College of Pharmacy, The Ohio State University, Columbus, OH;_ 2 _College of Medicine, The Ohio State University, Columbus, OH;_ 3 _College of Pharmacy, University of Florida, Gainesville, FL_.

The PI3K/AKT/mTOR pathway is activated in about 50% of patients with hepatocellular carcinoma (HCC). Allosteric mTOR inhibitors, also known as rapalogs, fail to show any significant clinical utility. In an effort to identify new pathways and compounds to treat advanced HCC, we considered the ATP-competitive mTOR inhibitor INK128. ATP-competitive mTOR inhibitors attenuate both mTORC1 and mTORC2. We evaluated INK128 in sorafenib sensitive and insensitive HCC cell lines, CD44low and CD44high HCC and those cell lines with acquired sorafenib resistance. CD44 was significantly increased in Huh7 cells made resistant to sorafenib. Forced expression of CD44 enhanced cellular proliferation and migration, and rendered the cells more sensitive to the anti-proliferative effects of INK128. INK128 suppressed CD44 expression by blocking phosphorylation of eukaryotic translation initiation factor 4EBP1 in HCC cells while allosteric mTOR inhibitors do not. Moreover, INK128 exhibited potent anti-proliferative and anti-migration effects on the mesenchymal-like HCC cells, CD44high and sorafenib resistant HCC cells compared to the allosteric mTOR inhibitor everolimus. Combination studies of INK128 and sorafenib showed an additive to synergistic anti-proliferative effect in CD44high HCC cells. Our findings suggest that ATP-competitive mTOR inhibitors are effective in treating advanced HCC patients who express CD44, and are insensitive or resistant to sorafenib. It also highlights the potential use of CD44 as a biomarker of response in these patients.

#142

Exploiting replicative vulnerabilities to counter incomplete responses to PI3K/Akt/mTOR inhibition.

Sameer Chopra,1 Mario Niepel,2 Anne Jenney,2 Marc Hafner,2 Peter Sorger2. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Harvard Medical School, Boston, MA_.

While a broad range of cancers harbor mutations that dysregulate PI3K-Akt-mTOR signaling, most small molecule drugs targeting this pathway have failed to demonstrate efficacy for the treatment of solid tumors. In this study, we investigated the mechanisms that account for drug efficacy and failure in PI3K-pathway dysregulated triple negative breast cancer (TNBC) cell lines using a diverse collection of 25 PI3K-Akt-mTOR inhibitors. Drug response phenotypes and changes in signaling were quantified using time-lapse imaging and quantitative single cell immunofluorescence microscopy, respectively. We identified GSK2126458 and Torin2 as having superior potency and efficacy in PI3K-pathway dysregulated TNBC. Unlike other PI3K pathway inhibitors, whose ineffectiveness arises from insufficient induction of apoptosis and variable inhibition of cell cycle progression at G1/S, GSK2126458 and Torin2 each rapidly induce caspase 3/7 activity and durably inhibit the proliferation of surviving cells. While the effectiveness of GSK2126458 in vitro appears to arise from near-complete suppression of PI3K-Akt-mTOR signaling, this approach has already proved unachievable in clinical trials. An alternative therapeutic strategy was identified by characterizing the mechanism of action of Torin2, a tool compound that inhibits both mTOR kinase and the DNA damage response kinases ATR, ATM, and DNA-PK. Unlike all other PI3K-Akt-mTOR inhibitors studied, Torin2 counters incomplete drug block at G1/S by concomitant induction of intolerable replication stress in S phase cells. The unique cell cycle pharmacology of Torin2 is recreated by combining inhibitors of mTOR and ATR/Chk1 kinases presently undergoing evaluation in clinical trials. In the context of combination therapy, where cytotoxicity arises from targeting S phase vulnerabilities rather than from PI3K-Akt-mTOR inhibition in G1, submaximal doses of mTOR kinase inhibitors are sufficient and confer benefit by preventing the outgrowth of cells that survive fractional killing from ATR/Chk1 inhibition. These findings suggest a novel strategy for mitigating the failure of precision monotherapy and have implications for the treatment of tumors where genetic lesions in the PI3K pathway co-occur with replicative vulnerabilities.

#143

Preclinical studies on potential therapeutic combination partners for the potent and selective PI3Kδ inhibitor INCB050465 in DLBCL.

Matthew C. Stubbs, Robert Collins, Leslie Hall, Alla Volgina, Holly Koblish, Sang Hyun Lee, Timothy Burn, Phillip C. Liu, Jin Lu, Eddy Yue, Yun-Long Li, Andrew P. Combs, Wenqing Yao, Gregory Hollis, Reid Huber, Bruce Ruggeri, Peggy Scherle. _Incyte Corp., Wilmington, DE_.

The delta isoform of PI3K (PI3Kδ) plays an essential role in B-cell development and function by mediating the signaling of key receptors on B cells. Increased malignant B cell proliferation and survival has also been associated with aberrant activation of PI3Kδ, making selective inhibition of this isoform an attractive therapeutic approach for the treatment of B cell malignancies. INCB050465 is a potent inhibitor of PI3Kδ, with a >20,000 fold selectivity over other PI3K isoforms. Emerging clinical data indicate that INCB050465 monotherapy is well tolerated and results in promising clinical responses in patients with various lymphoma histologies, including those with DLBCL. We therefore sought to explore rational combination strategies for INCB050465 using mouse xenograft models of ABC-subtype (HBL-1), GCB-subtype (Pfeiffer), and GCB/double-hit (WILL-2) human DLBCL, evaluating standard of care agents such as bendamustine and rituximab, as well as with targeted agents. PIM inhibition is a logical addition to PI3Kδ inhibition as a therapeutic approach as both kinases play a critical role in the AKT signaling pathway, having overlapping substrates. Likewise BET inhibition is a rational addition to PI3Kδ inhibition in "double-hit" DLBCL due to de-regulation of MYC transcriptional activity. In vivo studies performed in the Pfeiffer xenograft model demonstrate that INCB050465 combined with the pan-PIM inhibitor INCB053914 yielded complete tumor regressions. This profound decrease in tumor cell survival was due in part to the significant reduction in pBAD levels resulting from dual PIM and PI3Kδ inhibition. Despite modest single agent activity in vivo, the combination of INCB050465 with BET inhibitors, INCB054329 or INCB057643, resulted in significant anti-tumor efficacy in all of the DLBCL models studied, and caused a marked repression in tumor MYC expression. To study the transcriptional effects of combining PI3Kδ and BET inhibitors in this lymphoma model, WILL-2 xenograft tumors from mice treated with single dose INCB050465, INCB054329, the combination, or vehicle control were analyzed by RNAseq. INCB050465 enhanced the ability of INCB054329 to repress a MYC-driven transcriptional program, and the combination also regulated multiple developmental and inflammatory pathways. Together, these data support the clinical evaluation of the PI3Kδ inhibitor INCB050465 as part of a combination regimen with PIM or BET inhibitors for the treatment of DLBCL.

#144

Src leads to a novel mechanism of resistance to PI3K inhibitors through regulation of PI3K/p85 activation.

Gui Chul Kim, Hae Yun Nam, Hyang Ju Lee, Min Kyung Kim, Geun Hee Lee, Myung Woul Han, Seong Who KIM, Sang Yoon Kim. _Univ. of Ulsan, Seoul, Republic of Korea_.

Activation of the PI3K pathway is commonly observed and is correlated with tumor development, progression, poor prognosis, and resistance to cancer therapies, such as radiotherapy, in most cancers. As a central node of this pathway, PI3K is an attractive target for PI3K-addicted cancer therapy and PI3K inhibitors may thus restore sensitivity to other treatments when administered as part of combination regimens. Here, we found that PI3K/p85 was expressed predominantly in the radioresistant head and neck cancer cell line (HN31 cell line). And then, we investigated whether PI3K modulation was crucial for the development of novel treatment strategies for radioresistant cancer cell line. Interestingly, we found that head and neck cancer cell lines with PI3K/p85 activation showed the resistance to PI3K inhibitors and the resistance mechanism was associated with Src activation which is a member of a superfamily of membrane-associated nonreceptor protein tyrosine kinases. Src inhibitor improves the efficacy of PI3K inhibitor treatment through suppression of Src and PI3K/p85 activation in HN31 cell line. Collectively, our study highlights the role of p85 and Src activation in the resistance for PI3K inhibition and the potential clinical application of combination regimens of Src and PI3K inhibitors in head and neck cancers. This is the first investigation to analyze the role of Src in resistance to the PI3K inhibitors of head and neck cancer. As a consequence, a greater understanding of resistance mechanisms through our results will enable the rational design of combination regimens and sequential treatment algorithms to improve clinical outcomes.

#145

PIK3CB inhibitors selectively block the survival of glioblastoma cells.

Lamvy Le,1 Zhi Sheng2. 1 _Virginia Tech Carilion Research Institute, Roanoke, VA;_ 2 _Virginia Polytechnic Inst. & State Univ., Roanoke, VA_.

Objective: To explore the divergent role of PI3K isoforms in glioblastoma

Background: Glioblastoma multiforme is the most malignant brain tumor in adults. Despite aggressive treatments, the median survival and five-year overall survival of glioblastoma patients remains low (14.6 months and 4.7%, respectively). Nearly 90% of patients experience recurrence within two years, leaving patients with few treatment options. Recently, PI3K pan inhibitors have been used to treat recurrent glioblastoma and achieved modest effect in the clinic. PI3K has four catalytic isoforms (PIK3CA, B, D, and G); hence, it is possible that these isoforms may have different roles in glioblastoma and need to be selectively targeted. This idea is further supported by our recent results, in which we have shown that elevated expression of PIK3CB, but not other PI3K isoforms, significantly correlated with higher rate, risk, and poor prognosis of recurrent glioblastomas. Hence, we hypothesize that PIK3CB-selective inhibitors are more effective for glioblastoma.

Design/Methods: We used a panel of glioblastoma cells with different levels of PIK3CB, treated them with PI3K-isoform-selective inhibitors, and compared them to pan PI3K inhibitors. We measured cell viability using the MTS cell viability assay. Normal human astrocytes were used as controls to determine toxicity.

Results: We found that PIK3CB inhibitors TGX-221 and GSK2636771 significantly blocked the proliferation of PIK3CB-high U87MG and SF295 cells, while it had no effect on the viability of PIK3CB-low A172 and LN229 cells. In contrast, other PI3K inhibitors and pan inhibitors either non-selectively blocked or had no effect on these cells. Importantly, these compounds, but not PIK3CB inhibitors, blocked the growth of astrocytes.

Conclusions: Selective blockade of PIK3CB, but not other PI3K isoforms, is an effective therapy for glioblastoma with a low toxicity to normal tissues.

#146

The PI3K inhibitor, taselisib, has a unique mechanism of action that leads to enhanced potency in PIK3CA mutant models.

Kyung W. Song, Kyle A. Edgar, Donald S. Kirkpatrick, Lilian Phu, Stephen Schmidt, Michelle Nannini, Rebecca Hong, Eric Cheng, Lisa Crocker, Amy Young, Deepak Sampath, Lori Friedman. _Genentech, South Sanfrancisco, CA_.

Activating mutations in PIK3CA are commonly found in a wide variety of human cancers, and the dysregulation of the phosphoinositide-3 kinase (PI3K) signaling pathway has been implicated in tumor cell growth and survival. Taselisib (GDC-0032), a novel, oral, selective inhibitor of p110alpha, sparing inhibition of p110beta, is more potent in cancer cells bearing PIK3CA mutants than those with wildtype PIK3CA. Preclinical studies demonstrate that taselisib induces more apoptotic cell death in PIK3CA mutant cancer cells than other PI3K inhibitors.

We have discovered that taselisib has a dual mechanism of action, both blocking PI3K signaling, and inducing a decrease in p110a protein levels. Mass spec analysis reveals that taselisib treatment leads to the specific depletion of mutant p110alpha without significant change in wildtype p110alpha protein levels. This drug-induced p110a protein depletion is rescued by E1 inhibitors and by proteasome inhibitors. Other clinical PI3K inhibitors, including p110alpha selective and pan-PI3K inhibitors, are unable to induce the depletion of mutant p110 alpha protein. Furthermore, we have discovered that taselisib more effectively maintains pathway suppression in PIK3CA mutant cells at 24 hrs in response to feedback. In comparison to other clinical-stage PI3K inhibitors administered at a maximum tolerated dose, taselisib has superior efficacy with increased tumor regressions in PIK3CA mutant xenograft models. In summary, these preclinical studies indicate that PI3K inhibitors, which have the ability to trigger degradation of mutant p110a protein, can more effectively suppress the signaling pathway, which may result in greater anti-tumor activity and improved therapeutic index in PIK3CA mutant tumors.

#147

Metformin suppresses triple-negative breast cancer stem cells by targeting KLF5 for degradation.

Peiguo Shi, Zhongmei Zhou, Rong Liu, Ceshi Chen. _Kunming Inst. of Zoology, Kunming, China_.

Out of the breast cancer subtypes, triple-negative breast cancer (TNBC) has the poorest prognosis without effective targeted therapies. A stem cell transcription factor KLF5 is over-expressed in basal type TNBC and promoting cell proliferation, survival and stemness. Previously, we demonstrated that Mifepristone suppresses basal TNBC stem cells by down-regulating KLF5 expression through inducing the expression of miR-153. In this study, Metformin, a first-line drug for type 2 diabetes mellitus, was demonstrated to target breast cancer stem cells selectively. However, the efficiency and the mechanism of action of metformin in TNBC are unclear. We demonstrated that metformin decreased the percentage of TNBC stem cells partially through the downregulation of the expression of KLF5 and its downstream target genes, such as Nanog and FGF-BP1, in TNBC cell lines. Metformin induced GSK3β-mediated KLF5 protein phosphorylation and degradation through the inhibition of PKA activity in TNBC cells. Consistently, PKA activators increased the expression levels of KLF5. We observed a positive correlation between p-CREB and KLF5 protein levels in human TNBC samples. These findings suggest that metformin suppresses TNBC stem cells partially through the PKA-GSK3β-KLF5 signaling pathway.

#148

A dual and selective small molecule inhibitor of EGFR and PI3 kinase shows promising preclinical activity against KRAS and BRAF mutant colorectal tumors.

Joel D. Maust, Elizabeth K. Ziemke, Christy L. Frankowski-McGregor, Jun Beom Ku, Rachel Mumby, Karin M. Hardiman, Christopher E. Whitehead, Judith S. Sebolt-Leopold. _Univ. of Michigan, Ann Arbor, MI_.

Agents targeting epidermal growth factor receptor (EGFR) have met with limited success in the clinical management of colorectal cancer (CRC). Mutations in KRAS, BRAF, and PIK3CA are important drivers of resistance to EGFR-targeted therapy. Conversely, EGFR-mediated feedback mechanisms serve to mediate resistance to MEK inhibitor-based treatment of CRC by reactivating MAP kinase signaling. Our central hypothesis is that a dual small molecule inhibitor that potently and selectively targets only EGFR and PI3KA, when combined with a MEK inhibitor, will be highly efficacious against subpopulations of BRAF mutant or KRAS mutant colorectal cancers that are dependent upon these kinase molecules to drive tumor progression. Employing a computational modeling approach, we exploited the known binding modes of structurally related ATP binding site inhibitors of EGFR and PI3K to design small molecules that simultaneously inhibit both kinases in a selective manner. To the best of our knowledge, the lead compound MTX-211, whose binding mode is flipped in PI3K compared to EGFR, represents a first in class selective inhibitor of these two critical oncogenic kinases. MTX-211 exhibits a favorable pharmaceutical and selectivity profile, possessing sub- to low nanomolar potency against both targets, >70% oral bioavailability, strong pharmacodynamic modulation of both EGFR and PI3K signaling, and strong in vivo single agent efficacy against multiple BRAFmt and KRASmt colorectal cancer models, as evidenced by T/C values of 29 to 36% after 10-14 days of oral dosing of 50 mg/kg. A significantly higher degree of in vivo activity is seen when MTX-211 is co-administered with the MEK inhibitor trametinib (>400% increase in survival compared to single agent arms), lending support for this polypharmacology approach over triple drug combination strategies. Based on its promising therapeutic profile, MTX-211 is the focus of an ongoing mouse trial of a large panel of patient-derived xenograft BRAFmt and KRASmt CRC models to inform the design of future human clinical trials.

#149

Co-targeting mTORC and EGFR signaling as a potential therapeutic strategy in HNSCC.

Adam D. Swick, Prashanth J. Prabakaran, Amal Javaid, Margot Miller, Michael Fisher, Emmanuel Sampene, Irene M. Ong, Kwangok Nickel, Randall J. Kimple. _Univ. of Wisconsin-Madison, Madison, WI_.

Background - Head and neck squamous cell carcinomas (HNSCCs) have high rates of mutation and other alterations along the PI3K/AKT/mTORC signaling axis. This has led to interest in the use of therapeutics targeting this pathway, however identifying reliable predictive biomarkers to guide patient selection remains challenging. Despite excellent preclinical data, the use of these compounds as monotherapy has been underwhelming in initial clinical trials. The EGFR monoclonal antibody cetuximab remains the only approved targeted agent for HNSCC and with reasonable toxicity profiles, has potential use in combination therapy.

Methods - Both catalytic mTORC (AZD8055) and PI3K/mTORC(NVP-BEZ-235) inhibitors were tested +/- cetuximab in several in vitro and in vivo pre-clinical models. A panel of HNSCC cell lines and patient derived xenografts (PDX) were evaluated for PI3K/AKT/mTORC pathway mutation by sequencing and potential protein biomarker by immunoblot and IHC. Cell lines were assayed for sensitivity to all three agents by growth inhibition and clonogenic survival assay. DNA replication(BrdU uptake) and apoptosis (Capase 3/7 activity) were investigated to assess the mechanism of inhibition. The specificity of the molecular targeted effects was confirmed by siRNA knockdown. Five unique PDX models that presented PIK3CA mutation or intrinsic cetuximab resistance were treated with a combination of cetuximab and the dual mTORC inhibitor AZD8055 in a nude mouse model.

Results - Assessment of the panel of HNSCC cell lines by mutational hotspot sequencing did not reveal any obvious sensitizing mutations, whereas putative protein biomarkers (e.g. PIK3CA, pAKT) were elevated in some cell lines. All cell lines showed modest response to both PI3K/mTORC and dual mTORC inhibition. The addition of cetuximab to either agent produced modest additive effect. Mechanistic studies revealed that growth inhibition rather than death induction was the major anti-cancer effect. SiRNA knockdown showed similar molecular signaling and functional effects to drug inhibition. Using the PDX models, in vivo single agent mTORC inhibition inhibited growth of a PIK3CA mutant cancer, but had no effect on any PIK3CAWT or a second PIK3CA mutant model. In all models the combination therapy showed greater growth delay than monotherapy.

Conclusions -The uniform ability of PI3K/mTORC and mTORC inhibition to suppress the growth of HNSCC cells highlights the role of this signaling pathway to drive the proliferation. In vivo, despite some PDX models meeting likely selection criteria, the single agent therapy was largely ineffective. Conversely the combination treatment produced growth delay and suggests the potential for adding a catalytic mTORC inhibitor to cetuximab therapy for HNSCC patients. Overall, these results add to a growing body of evidence suggesting that attempts to match genetic alternation or other biomarker to the optimal therapy in HNSCC remains complex and challenging.

#150

Identification of determinants of sensitivity to AKT inhibition using breast cancer (BC) patient-derived tumor xenografts (PDX).

Albert Gris-Oliver,1 Mafalda Oliveira,1 Marta Guzman,1 Olga Rodríguez,1 Judit Grueso,1 Maurizio Scaltriti,2 William J. Howat,3 J Carl Barrett,4 Javier Cortés,5 José Baselga,2 Gaia Schiavon,3 Barry R. Davies,3 Cristina Saura,1 Violeta Serra1. 1 _Vall d'Hebron Institute of Oncology, Barcelona, Spain;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 3 _AstraZeneca, Cambridge, United Kingdom;_ 4 _AstraZeneca, Waltham, MA;_ 5 _Ramón y Cajal University Hospital, Madrid, Spain_.

The antitumor activity of AKT inhibitors is being investigated for the treatment of BC with activation of the PI3K/AKT pathway. Putative predictive biomarkers that are being tested in ongoing trials are PTEN loss/mutation (mut) (around 40% of triple negative BC) and AKT1 mutation (4-5% ER+/HER2- BC). The BEECH trial is evaluating the activity of the pan-AKT1/2/3 inhibitor (AKTi) AZD5363 in combination with the antimicrotubule agent paclitaxel in HER2-negative (HER2-) metastatic BC. We aimed to identify response biomarkers to the single agent AKTi and its degree of synergy in combination with paclitaxel, using HER2- patient-derived tumor xenografts (PDX).

Fifteen PDX have been established from BC patients receiving standard treatment at our Hospital, as well as from those participating in the BEECH trial, when sample was available, by implanting their tumor biopsies at baseline, on-treatment and at progression. The antitumor activity of AZD5363 as single agent and in combination with paclitaxel has been measured in these PDX. Genotyping and protein levels have been analyzed by exome sequencing, capture-based sequencing, Western blot and immunohistochemistry (IHC).

AZD5363 monotherapy results in disease stabilization or tumor regression in 3 out of 15 PDX (2/9 ER-/HER2- and 1/6 ER+/HER2-). We found a positive association between high levels of pAKT S473 by Western blot and antitumor response. Interestingly, the two ER-/HER2- responding PDX harbor a PTEN frameshift mutation or gene loss concomitant with an activating mutation within the PI3K pathway (PIK3CA or PIK3R1). The ER+/HER2- PDX responder harbors an AKT1-p.E17K mutation. AZD5363 plus paclitaxel, at a clinically relevant dose, results in disease stabilization in 2/11 PDX that progress to either single agent (1/7 ER-/HER2- and 1/4 ER+/HER2-); the latter PDX being derived from the baseline tumor biopsy of a BEECH trial patient who benefited from this combination for one year.

Altogether these results suggest that double-altering events in the PI3K pathway, including PTEN loss/mut and a second PI3K-pathway alteration in ER-/HER2- tumors, or AKT1-p.E17K in ER+/HER2- tumors, and consequent high pAkt S473 levels could be associated with intrinsic sensitivity to AKT inhibition. The predictive value of these alterations as determinants of response to AKTi deserves further investigation.

#151

Inhibition of AKT-1 for the treatment of human non-small cell lung cancer (NSCLC) in-vitro.

Paige M. Chorner, Roger A. Moorehead. _University of Guelph, Guelph, Ontario, Canada_.

AKT is a serine-threonine kinase implicated in tumorigenesis as a central regulator of cellular growth, proliferation, survival, metabolism, and migration. Activated AKT is overexpressed in 50-70% of NSCLC tumors and has exhibited an association with poor prognosis as well as chemotherapeutic resistance to platinum-based therapy. Accordingly, AKT inhibitors such as MK-2206 are currently undergoing clinical investigation for the treatment of human NSCLC however, these agents broadly target all three (1-3) AKT isoforms. Recent evidence suggests opposing roles of the AKT isoforms in tumorigenesis where loss of AKT-1 inhibits while the loss of AKT-2 enhances lung tumor development in transgenic mouse models. Based on these findings, we hypothesized that preferential inhibition of AKT-1 would warrant a more effective therapeutic strategy for NSCLC compared to the current clinical approach of broad AKT inhibition. WST-1 cell viability assays have revealed that a selective AKT-1 inhibitor A-674563 is a more potent regulator of survival in 6 NSCLC cell lines compared to a pan-AKT inhibitor MK-2206. Furthermore, off-target CDK2 inhibition likely contributes to the observed benefits of the AKT-1 inhibitor as the reduction in cell viability largely parallels the effects of a CDK2 inhibitor PHA-848125. In addition, the cell lines with higher endogenous CDK2 and p-CDK2 expression are more sensitive to the AKT-1 inhibitor relative to the pan-AKT inhibitor MK-2206. Basal protein and RNA levels in each of the cell lines have also shown that high AKT-3 expression may confer resistance to the pan-AKT inhibitor MK-2206. Thus, AKT-3 expression has the potential to serve as a predictive marker for patient response to AKT-1 versus broad AKT-inhibition. Cell cycle analysis demonstrated that the AKT-1 inhibitor decreases the proportion of cells in the Go/G1 phase and increases the proportion of cells in the S-phase, indicating a possible S-phase cell cycle arrest. These differences are also more significant in the cell lines with augmented sensitivity to the AKT-1 inhibitor. Therefore, altered cell cycle progression could be the major driver of the therapeutic benefits of the AKT-1 inhibitor. Overall, our findings suggest that AKT-1 inhibition is significantly more effective at reducing NSCLC cell viability in-vitro compared to pan-AKT inhibition. Furthermore, cell lines with higher CDK2 and AKT-3 expression have marginally increased sensitivity to the AKT-1 inhibitor A-674563 compared to the pan-AKT inhibitor MK-2206. Future research will focus on understanding the mechanism of action of the AKT-1 inhibitor through combined western blot, AKT antibody array, flow cytometry, and confocal microscopy data. Additionally, we will investigate the toxicity of these inhibitors on normal human small airway epithelial cells (HSAECs) to ensure preferential activity against malignant over somatic cells.

#152

Dual mTORC1/2 inhibition sensitizes testicular cancer cell lines toward cisplatin treatment.

Fernanda Ximena Rosas Plaza, Gerda de Vries, Albert J. Suurmeijer, Jourik A. Gietema, Marcel A.T.M. van Vugt, Steven de Jong. _UMCG, University of Groningen, Groningen, Netherlands_.

Testicular cancer (TC) patients with metastatic disease and poor prognosis have a 50% 5-year survival. It has been reported that resistant TC cell lines show hyperactivation of the PI3K/Akt/mTOR pathway. In this study we investigated the potential benefit from PI3K, Akt and mTOR inhibition in combination with cisplatin in TC.

Our panel of embryonal carcinoma cell lines include the cisplatin-sensitive cells: Tera and 833KE and the cisplatin-resistant cells: Scha, TeraCP and NCCIT. Western blotting showed that the resistant TC cell line TeraCP expresses higher levels of p-Akt compared to the sensitive cell line Tera. To evaluate sensitization towards cisplatin, TC cell lines were treated with cisplatin and/or PI3K inhibitor GDC0941, Akt inhibitor MK2206, mTORC1 inhibitor everolimus and mTORC1/2 inhibitors AZD8055 and AZD2014 for 24 hours and DilC5/PI staining was performed to estimate apoptosis with flow cytometry. All TC cell lines were strongly sensitized by mTORC1/2 inhibition by increasing cisplatin induced apoptosis to 60-80% when cisplatin alone induced only 15-30%. TC cells were also sensitized by mTORC1 and PI3K inhibition but to a lesser extent. Akt inhibition did not sensitize Scha or Tera cells to cisplatin, while TeraCP was marginally sensitized. We then evaluated clonogenic capacity in cells pretreated with AZD8055 for 24 hours and seeded in the presence of different concentrations of cisplatin for 6 days. Clonogenic capacity was reduced in Tera and TeraCP after mTORC1/2 inhibition with AZD8055 compared with control cells treated with cisplatin only. Western blot done with Scha, Tera and TeraCP lysates treated for 24 hours with AZD8055, everolimus, GDC0941 and MK2206 showed that only mTOR inhibition was able to block S6 phosphorylation. In addition, we performed immunohistochemistry of p-S6 and Ki-67 in paraffin embedded tissue from TC patients. IHC showed that patient derived xenografts showed high expression of these markers compared to non-cancerous tissue, meaning that the mTOR pathway is very active in this tumor type.

These data indicate that TC relies on the PI3K/Akt/mTOR pathway for survival and mTORC1/2 inhibition showed the stronger sensitizing effect towards cisplatin treatment. We consider adding mTORC1C/2 inhibition to cisplatin based treatment a potential therapeutic option for chemoresistant TC patients that warrants further in vivo investigations.

Supported by CONACYT grant 381543 and Dutch Cancer Society grant RUG 2014-6691

#153

Tricyclic fused pyrimidinopyrrolo-oxazines reveal conformational preferences of morpholine for PI3K hinge region binding.

Alexander M. Sele,1 Denise Rageot,1 Florent Beaufils,1 Anna Melone,1 Thomas Bohnacker,1 Eileen Jackson,1 Jean-Baptiste Langlois,1 Paul Hebeisen,2 Doriano Fabbro,2 Matthias P. Wymann1. 1 _University of Basel, Basel, Switzerland;_ 2 _PIQUR Therapeutics AG, Basel, Switzerland_.

Class I phosphoinositide 3-kinases (PI3Ks) are lipid kinases, produce PtdIns(3,4,5)P3 and trigger intracellular signaling pathways that are vital to cell growth, proliferation, survival and migration. Constitutive activation of PI3K is frequently observed in many tumor types, which defines PI3K as a valuable drug target in oncology.1

Numerous PI3K inhibitors in clinical development contain a morpholine moiety that mediates hinge region binding in the ATP pocket of PI3K by a hydrogen bond with the active site valine backbone nitrogen (Val851 in PI3Kα)2. We present here novel pyrimidinopyrrolo-oxazines related to the clinically advanced, pyridinylmorpholine and triazinylmorpholine derived pan-PI3K/mTOR inhibitors BKM120 and PQR309. The novel fused tricyclic core of these compounds contains two morpholine moieties of which one is conformationally restricted by the introduction of a methylene bridge that links the pyrimidine core with one of the two morpholine moieties. This modification leads to the generation of two regioisomers, each existing as a set of enantiomers.

We investigated the influence of this conformational restriction on PI3K inhibitory activity and analyzed the distinct selectivity profiles and potencies of the respective stereo- and regio-isomers. The design and preparation of specific compounds in combination with biological assays (phosphorylation of PKB and S6, binding affinity to p110α), structure-activity relationship (SAR) and molecular modelling studies allowed us to understand the binding mode of these compounds and acquire valuable information that potentially lead to the development of derivatives with a distinct selectivity profile (e.g. PI3K versus mTOR).

A selection of compounds demonstrated inhibition of protein kinase B (pSer473) and ribosomal protein S6 (pSer235/236) phosphorylation with IC50 values in the nanomolar range and high inhibitory potency of all PI3K isoforms (Ki(p110α) > 40 nM). Single p.o. administration of our lead compound to SD rats resulted in good oral bioavailability as well as excellent brain penetration. Furthermore, mechanism of action-based increases in glucose levels and insulin levels have been observed.

In conclusion, we present here the development, optimization, preparation and biological evaluation of a novel class of potent, orally available and brain-penetrant pan-PI3K inhibitors that represent an innovative extension to known pyrimidinomorpholine derived PI3K inhibitors. Moreover, our results add to the understanding of how introducing specific structural and conformational modifications can lead to the development of optimized, selective PI3K and mTOR inhibitors.

[1] Thorpe, L. M.; Yuzugullu, H.; Zhao, J. J. Nat. Rev. Cancer 2015, 15, 7-24.

[2] Andrs, M.; Korabecny, J.; Jun, D.; Hodny, Z.; Bartek, J.; Kuca, K. J. Med. Chem. 2015, 58, 41-71.

#154

The phosphatidylinositol-3-kinase (PI3K) inhibitor (i) copanlisib is active in preclinical models of B-cell lymphomas as single agent and in combination with conventional and targeted agents including venetoclax and palbociclib.

Eugenio Gaudio,1 Ivo Kwee,1 Filippo Spriano,1 Chiara Tarantelli,1 Andrea Rinaldi,1 Thibaud Jourdan,2 Melanie Berthold,2 Alberto Arriibas,1 Anastasion Stathis,1 Davide Rossi,1 Ningshu Liu,2 Martin Lange,2 Oliver Politz,2 Emanuele Zucca,1 Francesco Bertoni1. 1 _Institute of Oncology Research - IOR, Bellinzona, Switzerland;_ 2 _Bayer AG, Berlin, Germany_.

Introduction

Copanlisib (BAY 80 6946) is a highly selective pan class I PI3K-i with predominant inhibitory activity against PI3Kδ and PI3Kα, in clinical development as single agent and in combination for lymphoma patients. To address single agent antitumor activity in different lymphomas and to understand the molecular basis of resistance mechanisms for rational combination, we performed a screening of copanlisib as single agent and in combination with 15 other anticancer agents in 17 cell lines derived from mantle cell lymphoma (MCL), marginal zone lymphoma (MZL) and chronic lymphocytic leukemia (CLL).

Methods

MCL (Jeko1, Rec1, JVM2, Granta519, Maver1, Mino1, SP-49, SP-53, UPN1, Z138), MZL (Karpas1718, VL51, SSK41, ESKOL, HAIR-M, HC-1) and CLL (MEC1) cell lines were exposed to increasing doses of copanlisib alone and in combination with other compounds using the fixed ratio set-up. Tested compounds included approved and experimental inhibitors of key regulatory pathways. Synergy was assessed via Chou-Talalay combination index (CI). Gene expression profiling (GEP) was done using Illumina Human HT12Expression BeadChips and GSEA (FDR<0.25).

Results

Copanlisib showed antitumor activity in most cell lines (median IC50=22nM; 95%C.I.: 15-98). The other most active drugs were bortezomib (5nM; 5-7), romidepsin (34nM; 2-94), roniciclib (23nM; 18-29), panobinostat (161nM; 11-1263), MI2 (490nM; 224-1000). The remaining had median IC50s >500nM.

Copanlisib-containing combinations often gave synergy/additive effects: copanlisib with venetoclax was beneficial in 16/17; with MI2 in 15; with palbociclib or ibrutinib in 14; with BAY 1125976 or panobinostat in 13; with lenalidomide or BAY 1238097 in 12; with rituximab in 11; with romidepsin in 10; with roniciclib in 9; with bortezomib in 8; with BAY 1143572 in 7; with bendamustine in 6; with ruxolitinib in 2. Combinations with venetoclax and with palbociclib were the most promising, achieving CI values <0.5 in 7 and 6 cell lines, respectively. GEP before treatment identified genesets associated with different sensitivity to these 2 combinations. High expression of genes involved in IFN signaling, oxidative phosphorylation, fatty acid metabolism, apoptosis, PI3K/AKT/mTOR and IL6/JAK/STAT signaling and low expression of cell cycle genes were associated with synergism to copanlisib/venetoclax. Largely the opposite was observed for the palbociclib combination, more active with high expression of E2F/MYC targets and cell cycle genes and low expression of genes involved in IFN PI3K/AKT/mTOR and IL6/JAK/STAT signaling.

Conclusion

Copanlisib was active in MCL, MZL and CLL models. Combinations with BCL2-i venetoclax and CDK4/CDK6-i palbociclib were the most synergistic. Specific GEP features might predict lymphomas that could benefit from these regimens.

#155

**Addition of RP6530, a dual PI3Kδ/γ inhibitor, accentuates Romidepsin activity in NHL cells** in vitro **.**

Srikant Viswanadha,1 Satyanarayana Eleswarapu,1 Seeta Nyayapathy,1 Swaroop Vakkalanka2. 1 _Incozen Therapeutics Pvt. Ltd., Shameerpet Hyderabad, India;_ 2 _Rhizen Pharmaceuticals SA, La Chaux-de-Fonds, Switzerland_.

Background: Relapsed or refractory Non-Hodgkin's Lymphoma (NHL) represents a significant unmet medical need. Novel treatment options currently being explored include Romidepsin, a histone deacetylase inhibitor, which has demonstrated significant preclinical and clinical activity. Because the PI3K pathway is often activated in NHL, combining Romidepsin with a PI3K δ/γ inhibitor may be a viable alternative to chemotherapy. RP6530 is a novel, potent, and selective PI3K δ/γ inhibitor that demonstrated high potency against PI3Kδ (IC50=25 nM) and γ (IC50=33 nM) enzymes with selectivity over α (>300-fold) and β (>100-fold) isoforms. Dose-escalation trials to evaluate the safety and efficacy of RP6530 in patients with hematological malignancies are currently underway at several sites in US and Europe. The objective of this study was to evaluate the effect of a combination of Romidepsin and RP6530 in NHL cells.

Methods: Combination of Romidepsin and RPRP6530 was evaluated in the following NHL cell lines: OCI-LY-1, OCI-LY-10, RAJI, DAUDI, TOLEDO, JEKO, MAVER, and SU-DHL-1. Synergism or additivity between Romidepsin and RP6530 was determined using different concentrations of the compounds in a 5 x 5 grid and calculated based on the BLISS score. Effect on apoptosis and cell cycle were estimated by flow cytometry. Phosphorylated AKT and PARP were determined by Western Blotting.

Results: Synergism or additivity between Romidepsin and RP6530 for anti-proliferative activity was noticed in all the cell lines tested with effect being most pronounced in OCI-LY-1, OCI-LY-10, JEKO, and SU-DHL-1. Incubating cells with 3-5 nM Romidepsin and 3 or 5 μM RP6530 resulted in a 2 to 5-fold increase in the total apoptotic population. Cell cycle analysis indicated that the combination of Romidepsin (1-5 nM) and RP6530 (1-5 μM) caused a G0/G1 arrest (2 to 3-fold) with a corresponding increase in percent of apoptotic cells. While Romidepsin had minimal effect of pAKT, treatment with RP6530 reduced expression by 40-90% across cell lines. Further, Romidepsin + RP6530 enhanced expression of cleaved PARP (8 to 60-fold) compared to either agent alone

Conclusions: Addition of RP6530, a potent and selective dual PI3Kδ/γ inhibitor, potentiated Romidepsin activity in NHL cells. Findings provide a rationale for use of the combination in future clinical trials involving naïve or relapsed NHL patients thereby providing a safer alternative to currently available therapy.

#156

Preclinical characterization of GDC-0077, a specific PI3K alpha inhibitor in early clinical development.

Kyle Edgar, Emily Hanan, Steven Staben, Stephen Schmidt, Rebecca Hong, Kyung Song, Amy Young, Patricia Hamilton, Alfonso Arrazate, Cecile de la Cruz, Marcia Belvin, Michelle Nannini, Lori S. Friedman, Deepak Sampath. _Genentech, South San Francisco, CA_.

The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/ mammalian target of rapamycin (mTOR) signaling pathway is a major regulator of tumor cell growth, proliferation and survival. Dysregulation of the PI3K/Akt/mTOR signaling pathway through multiple mechanisms has been described in solid tumor malignancies, including activating and transforming "hotspot" mutations as well as amplification of PIK3CA that encodes the p110 alpha subunit of PI3K. Hotspot mutations of PIK3CA mutation are frequently observed in breast cancer with a prevalence of approximately 30%. GDC-0077 is a potent inhibitor of PI3K alpha (IC50 = 0.038 + 0.003 nM) and exerts its activity by binding to the ATP binding site of PI3K, thereby inhibiting the phosphorylation of PIP2 to PIP3. Biochemically, GDC-0077 is more than 300-fold selective over other Class I PI3K isoforms such as beta, delta, and gamma and more than 2000 fold more selective over PI3K class II and III family members. Importantly, GDC-0077 is more selective for mutant versus wild-type PI3K alpha in cell based assays. Compared to the PI3K inhibitor, taselisib, the improved biochemical selectivity of GDC-0077 against PI3K delta is demonstrated in human CD69+ B-cells, which are primarily dependent on PI3K delta for proliferation and survival and were more sensitive to taselisib than GDC-0077. Mechanism of action studies indicate that GDC-0077 induces depletion of mutant PI3K alpha protein resulting in reduction of PI3K pathway biomarkers such as pAkt and pPRAS40, inhibition of cell proliferation and increased apoptosis in human PIK3CA mutant breast cancer cell lines to a greater extent when compared to PIK3CA wild-type cells. In vivo, daily oral treatment with GDC-0077 in cell-culture-derived and patient derived PIK3CA mutant breast cancer xenograft models, resulted in tumor regressions, induction of apoptosis and a reduction of pAkt, pPRAS40, and pS6RP in a dose-dependent fashion. In vivo efficacy in a PIK3CA-mutant human breast cancer xenograft model was also improved when GDC-0077 was combined with standard-of-care therapies for hormone-receptor positive (HR+) breast cancer such as anti-estrogens (fulvestrant) or CDK4/6 inhibitor (palbociclib). Collectively, the preclinical data provide rationale for evaluating GDC-0077, a PI3K alpha mutant selective inhibitor, as a single agent and in combination with standard-of-care endocrine and targeted therapies that may provide additional benefit to patients that harbor PIK3CA mutations.

#157

Targeting distinct nodes of the PI3K/AKT/mTOR cascade in prostate cancer cells: impact on cell proliferation, apoptosis and pathway signaling.

Rute B. Marques, Ashraf Aghai, Wendy Stam, Wytske M. van Weerden. _Erasmus MC, Rotterdam, Netherlands_.

Inactivation of the PTEN gene is a common event in PCa, leading to activation of the PI3K/AKT/mTOR pathway and promoting PCa progression. Inhibitors targeting this pathway are currently being developed as anticancer agents. While recent clinical trials with mTORC1 inhibitors have shown limited single-agent efficacy in castration-resistant prostate cancer (CRPC), it is still not known whether targeting alternative nodes of the PI3K/AKT/mTOR cascade may improve response. The current study aimed to characterize the response of PCa cell lines to multiple inhibitors targeting the PI3K, AKT and mTOR nodes of the cascade and identify possible mechanisms of resistance.

The impact of the different PI3K/AKT/mTOR inhibitors on cell proliferation and apoptosis was measured by WST-1 and ApoTox-Glo assays, respectively, in a broad panel of established PCa cell lines and primary cell cultures generated from patient-derived xenografts. PTEN-negative PCa cell lines showed strong response to PI3Kβ/δ, AKT and mTOR inhibition, whereas PTEN-positive cells were resistant to PI3Kβ/δ and showed variable sensitivity to mTOR inhibitors. MTORC1 inhibitor showed low IC50 but reached plateau at about 70-80% growth inhibition. In contrast, mTORC1/2 inhibitor could reach complete growth inhibition at ~1μM concentration, in sensitive cell lines. Furthermore, treatment with PI3K or AKT inhibitor in combination with androgen depletion induced apoptosis in various PTEN-negative models, which is consistent with previously reported synergistic effect of PI3K/AKT and androgen receptor co-targeting. The effect of the inhibitors on phosphorylation of PI3K downstream targets (AKT, PRAS40, GSK3, S6K1 and 4EBP1) was analyzed by phospho-specific western blotting. Perturbation of different nodes of the PI3K/AKT/mTOR cascade resulted in differential phosphorylation of downstream targets, but there was no evident correlation with in vitro growth inhibition.

In conclusion, the current study validates PI3Kβ and AKT as alternative targets in PTEN-negative PCa, whereas PTEN-positive models showed preferential yet variable responses towards mTOR inhibitors.

#158

ASN003, a highly selective inhibitor of B-Raf and PI3 kinases, shows strong antitumor activity in B-Raf inhibitor resistant patient-derived xenograft models.

Scott K. Thompson,1 Roger A. Smith,1 Niranjan Rao,1 Michael J. Wick,2 Sanjeeva P. Reddy1. 1 _Asana BioSciences, Lawrenceville, NJ;_ 2 _South texas Accelerated Research Therapeutics, San Antonio, TX_.

The RAS-RAF-MEK and PI3K-AKT-mTOR pathways are two major signaling pathways involved in human cancer. Components of these two pathways are frequently mutated in a wide variety of solid tumors. Concurrent double mutations in the two pathways are also observed quite often in a broad range of tumor types. Additionally, inhibition of one of these pathways often leads to the upregulation of the other pathway and development of resistance. In preclinical models, combined inhibition of both pathways has been shown to impart greater efficacy as compared to inhibition of either pathway alone. ASN003 is a novel, highly selective, small-molecule inhibitor of both RAS-RAF and PI3K pathways, discovered using a rational design approach. ASN003 shows potent inhibitory activity against B-RAF and PI3K kinases (low nM IC50). Within the PI3K family, ASN003 has high selectivity for inhibition of PI3Kα and PI3Kδ over PI3Kβ. In a panel of 292 kinases, ASN003 showed high selectivity for inhibiting B-RAF and PI3 kinases, and associated mutant kinases. In cell-based mechanistic studies, ASN003 inhibited phosphorylation of ERK, AKT and S6, and showed strong antiproliferative activity (IC50 = 60-300 nM) in cell lines with B-RAF and PI3K pathway mutations as well as in vemurafenib-resistant cell lines. In pharmacodynamic studies in multiple tumor models, ASN003 showed strong inhibition of the phosphorylation of downstream targets of B-RAF and PI3K, confirming appropriate target engagement. In in vivo efficacy studies, ASN003 showed strong tumor growth inhibition or regression in multiple tumor xenograft models, including A375 (B-Raf V600E mutation), RKO (B-Raf V600E and PIK3CA mutations), and A2058 (B-Raf V600E mutation and PTEN loss). We now report that ASN003 also showed strong tumor growth inhibition (>80%) in a patient-derived xenograft (PDX) model established from a relapsed patient with progressive B-Raf mutant melanoma who showed initial response to vemurafenib. Sequencing analysis showed that the vemurafenib resistant tumor acquired a concurrent PIK3CA mutation. Dual targeting of the B-RAF and PI3K pathways with ASN003 has the potential to treat and/or prevent the acquired resistance to selective B-RAF inhibitors, and may also treat a broader patient population and provide greater efficacy and survival benefit than selective B-RAF inhibitors or selective PI3K pathway inhibitors alone. ASN003 is currently in Phase I clinical development in patients with advanced solid tumors, including tumors with B-Raf V600 mutation, PI3 kinase pathway alterations or PTEN loss.

#159

Pharmacological characterization of the selective, orally bioavailable, potent dual PI3K/mTORC1/2 inhibitor PQR530.

Petra Hillmann,1 Denise Rageot,2 Florent Beaufils,1 Anna Melone,2 Alexander Sele,2 Robert A. Ettlin,1 Jürgen Mestan,1 Vladimir Cmiljanovic,1 Marc Lang,1 Elisabeth Singer,3 Carolin Walter,3 Hoa HP Nguyen,3 Paul Hebeisen,1 Matthias P. Wymann,2 Doriano Fabbro1. 1 _PIQUR Therapeutics, Basel, Switzerland;_ 2 _University of Basel, Basel, Switzerland;_ 3 _University of Tübingen, Tübingen, Germany_.

Introduction: The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays a fundamental role in many cellular processes like growth, survival, proliferation, differentiation and motility. In cancers several mutations have been identified that lead to constitutive activation of PI3K. PQR530 is a novel, ATP site directed inhibitor of all PI3K isoforms and the mammalian target of rapamycin (mTOR) complexes C1/2 that is currently in pre-clinical development. PQR530 potently binds to its targets, inhibits cell proliferation and shows excellent selectivity versus related and unrelated kinases [1].

Results: PQR530 inhibits PI3K signaling in stimulated MCF7 cells as detected by PathScan analysis. Excellent tolerability has been found for PQR530 during GLP toxicological testing in rats and dogs. Increase in insulin and blood glucose, a treatable class effect of PI3K inhibitors, has been observed after PQR530 administration to mice. Investigation of mutagenicity and hERG binding resulted in a clean profile. PQR530 exhibited dose-proportional pharmacokinetics (PK) in male C57BL/6J mice. A maximum concentration (Cmax) in plasma and brain was reached after 30 minutes (7.8 μg/ml and 112.6 μg/ml, respectively) indicating that efficacious concentrations were reached in both tissues. The calculated half-life (t1/2) for plasma and brain was approximately 5 hours. PQR530 potently inhibited PI3K signaling in vivo for several hours after administration of a single oral dose of 50 mg/kg. Tumor growth was significantly decreased in SUDHL-6 lymphoma, RIVA lymphoma and OVCAR-3 ovarian cancer mouse xenografts using daily, oral administration.

Conclusion: PQR530 is a potent, ATP competitive pan-PI3K and mTORC1/2 inhibitor. The physico-chemical properties of PQR530 result in good oral bioavailability and excellent brain penetration. PQR530 is well tolerated and efficiently inhibits tumor growth in xenograft models. Preclinical data allow for further development of the compound.

[1] Rageot D, et al., Discovery and biological evaluation of PQR530, a highly potent dual pan-PI3K/mTORC1/2 inhibitor, abstract submitted for AACR Annual Meeting 2017, April 1-5, Washington, D. C., USA.

#160

High target binding affinity with long lasting cellular target engagement and high dose intermittent schedule of PI3K inhibitor copanlisib contribute to the potent anti-tumor activity and good safety profile.

Amaury E. Fernández-Montalván,1 Victoria Georgi,1 James Vasta,1 Sarah Glaeske,1 Vera Puetter,1 Matthew B. Robers,2 Ursula Moenning,1 Andrea Sturz,1 Julien Lefranc,1 Karl Ziegelbauer,1 Michael Brands,1 Christian Stegmann,1 William J. Scott,1 Ningshu Liu1. 1 _Bayer AG, Berlin, Germany;_ 2 _Promega Inc, Berlin, Germany_.

Introduction: Several generations of PI3K inhibitors have been tested in clinic. However, thus far, clinical activity has been moderate. Different from other oral PI3K inhibitors dosed continuously, copanlisib (BAY 80-6946) is an intravenous PI3K inhibitor given intermittently to patients. Copanlisib dosed once weekly demonstrated clinical benefit with an improved safety profile, and therefore challenges the concept of default continuous dosing of PI3K inhibitors. However, it is still unclear if this concept can be generalized and whether 'micropharmacokinetic parameters' also contributed to the potent anti-tumor profile of copanlisib. Here, we report the characterization of binding kinetics for copanlisib, as well as the functional consequence in vivo.

Methods: A set of PI3K inhibitors were characterized in 1) a kinetic probe competition assay (kPCA); 2) a cellular nanoBRET target engagement assay; 3) a cellular washout study with the assessment on pathway engagement; and 4) in vivo pharmacokinetics analysis.

Results: Copanlisib showed nearly diffusion-controlled on- and relatively slow off-rates with kon = 3.45E+7 [M-1*s-1] and koff = 1.67E-3 [s-1] to PI3Kα. Consequently, it exhibited very high affinity to PI3Kα ( Ki ePCA = 9.31E-11[M] and KD kPCA = 4.77E-11 [M]). In a cellular nanoBRET target engagement assay, the apparent half-life (t1/2) of ca. 2 hours greatly surpassed the 6.9 min measured using kPCA. The high affinity to PI3Kα also translated into potent cellular pathway engagement demonstrated by inhibition of p-AKT and p-PRAS40 in the PIK3CAmut KPL4 cell line. In a cellular washout study, p-AKT and p-PRAS40 were assessed till 168 h after incubation with copanlisib for 1 h followed by a washout step. A dose- and time-dependent pathway engagement was observed even at 72 h post washout. This result indicated that in cells, copanlisib engages PI3Kα for an extremely long time, likely due to rebinding effects facilitated by the fast equilibration kinetics of the compound and its micropharmacokinetic properties. Interestingly, in vivo, BAY 80-6946 levels were approximately 100-fold higher in the tumor than in plasma at 48 hours and drug clearance from the tumor occurred more slowly than from plasma. This high and prolonged tumor exposure might be explained, at least in part, by the high expression of PI3Kα and long lasting target occupancy of copanlisib in tumors.

Conclusion: Copanlisib demonstrated high affinity to PI3Kα with protracted target engagement at cellular and in vivo levels. This 'micropharmacokinetic feature' not only supports intermittent dosing but likely also explains the high exposure in tumors vs plasma, potent anti-tumor activity and good safety profiles.

#161

Medulloblastoma therapy targeting Hedgehog and PI3K-mTOR signaling pathways in combination with chemotherapy.

Nagendra K. Chaturvedi, Don W. Coulter, Timothy R. McGuire, Matthew J. Kling, Sutapa Ray, Shantaram S. Joshi, John G. Sharp. _University of Nebraska Medical Center, Omaha, NE_.

Medulloblastoma (MB) is the most frequent malignant brain tumor in children. MB patients with high-risk disease have poorly understood biology and few targeted therapies available. Preclinical studies and molecular profiling of MB have revealed that the aberrant activation and interaction of the SHH and PI3K/AKT/mTOR signaling networks are frequently associated with poor prognosis MB cases. Emerging evidence also demonstrate the key role of activated PI3K/AKT/mTOR pathway components in SHH-driven MB therapy-resistance, thus combined targeting of the SHH and PI3K/AKT/mTOR pathways may be a viable therapeutic strategy to treat high-risk patients. Therefore, we investigated the combined efficacy of SHH inhibitor vismodegib and PI3K-mTOR dual inhibitor BEZ235 or their combination, individually with the chemotherapeutic drug cisplatin against high-risk MB. Using four MB cell lines, including non-MYC and MYC amplified cell lines, and a xenograft mouse model, the in vitro and in vivo efficacies of the proposed therapies on cell growth/survival along with associated molecular mechanism(s) were investigated. Our results showed that both inhibitors as single agents significantly decreased MB cell growth and induced apoptosis by targeting the key molecules of the associated pathways in vitro. BEZ235 as single agent showed a greater anti-MB efficacy compared to vismodegib. Combined treatment of vismodegib and BEZ235 together or with cisplatin significantly decreased MB cell growth/survival and anchorage-independent growth in a dose-dependent fashion compared to single agent activity. Corresponding changes in the expression of the targeted molecules following therapy were observed. Results from the combined approach suggested that the inhibitors not only suppressed MB cell growth/survival when combined, but also significantly enhanced the cytotoxic effects of cisplatin. Of these combinations, BEZ235 exhibited a significantly greater efficacy in enhancing the cisplatin-mediated MB cytotoxicity. In vitro studies also demonstrated that the MYC amplified MB cell lines showed a higher sensitivity to these combined therapies compared to relatively non-MYC amplified cell lines. Therefore, as a next logical step, we tested the efficacy of above combined approaches against MYC-amplified MB in vivo using NSG mice. Our in vivo results showed that the combination of vismodegib and BEZ235 or their combinations individually with cisplatin significantly delayed tumor growth and increased survival of xenograft mice compared to single agent activity. These combination not only significantly reduced tumor growth and increased survival of the mice but also significantly enhanced anti-MB efficacy by targeting SHH and mTOR pathways in MB in vivo. Thus, our studies lay a foundation for translating these combined therapeutic strategies to the clinical setting to determine their efficacies in high-risk MB patients. 

### Tumor Microenvironment in Drug Development

#162

A prodrug of green tea polyphenol (-)-epigallocatechin-3-gallate served as a potentially novel angiogenesis inhibitor in endometrioid endometrial cancer.

Jianzhang Wang,1 Chi Wai Man,1 Xueying Yang,1 Joseph Kwong,1 Q. Ping Dou,2 Tak Hang Chan,3 Chi Chiu Wang1. 1 _The Chinese University of Hong Kong, Hong Kong, Hong Kong;_ 2 _Wayne State University, Detroit, MI;_ 3 _The Hong Kong Polytechnic University, Hong Kong, Hong Kong_.

Background and aim: Anti-angiogenesis is a promising treatment in endometrioid endometrial cancer (EEC) while the present anti-angiogenic drugs still have limitations such as drug tolerance, toxicity profile and substantial costs. (-)-Epigallocatechin-3-gallate (EGCG), the major polyphenol of green tea, is associated with anti-cancer benefits, but it is unstable and has poor bioavailability. These limitations can be solved through peracetate ester of EGCG as a prodrug (ProEGCG), while little is known about its anti-angiogenesis effect in cancer. The objective is to study the effect and underling mechanism of ProEGCG on EEC angiogenesis.

Methods and Results: Tumor xenografts were established by subcutaneous injection of a human EEC cell line (RL95-2) into nude mice, and ProEGCG or control was orally administered every day. 35 days later, mice treated with ProEGCG showed significant reduction in lesion size and MVD without major toxic effects compared to control. To find out how ProEGCG inhibits tumor progress and angiogenesis, microarray analysis of excised lesions was conducted and indicated that HIF-1α, VEGFA and CXCR4 were downregulated by ProEGCG. Compared to control, immunohistochemistry showed that ProEGCG reduced expression of HIF-1α and VEGFA in RL95-2 cells and decreased CXCR4 expression in tumor-associated macrophages (TAMs) and endothelial cells (ECs). To further study the role of ProEGCG on EEC cells, qRT-PCR and Western blotting proved that hypoxia (1% O2) upregulated the level of HIF-1α and VEGFA in EEC cell lines, which was reversed by ProEGCG through inhibiting hypoxia-induced ROS formation. It is known that TAMs play an important role in tumor angiogenesis, and double immunofluorescence of F4/80 and CD206 antibodies in xenografts was applied and found that TAMs infiltration was reduced in ProEGCG group with decreased VEGFA in TAMs. Moreover, proliferation and migration of ECs are critical for tumor angiogenesis. To study the effect of ProEGCG on ECs, tube formation and invasion assays were conducted. The addition of ProEGCG on ECs showed less capillary tube-like structure formation and less invaded ECs by inhibition of CXCR4 expression on ECs. However, it was still unclear if CXCL12, the ligand of CXCR4, was affected. Immunostaining for CXCL12 in tumor lesions indicated that ProEGCG decreased CXCL12 expression in cancer and stromal cells, which was further confirmed in primary human endometrial stromal cells by addition of ProEGCG.

Conclusion: Our study was the first to reveal the mechanism of anti-angiogenesis of ProEGCG, which decreased HIF1α/VEGFA expression in tumor cells, inhibited recruitment and differentiation of TAMs and tube formation of endothelial cells via CXCL12/CXCR4 reduction. These findings provided that ProEGCG would be an effective, safe and economic anti-angiogenic drug for EEC.

#163

Evaluation of ETS2101 and its combination with anti-mCTLA4 monoclonal antibody on CD4 and CD8 TILs in a subcutaneous colorectal cancer model of CT-26.

Lorcan Sherry,1 Mark Anderson,1 John Waller,1 Adam Sardar,2 Victoria Flores,2 Daniel Hynes2. 1 _OracleBio, Biocity Scotland, United Kingdom;_ 2 _e-Therapeutics, Long Hanborough, United Kingdom_.

ETS2101, (HU-211, Dexanabinol) is a synthetic cannabinoid with limited affinity to cannabinoid receptors 1 and 2 (CB1 and CB2), reducing its psychotropic potential in comparison to other molecules in the cannabinoid class. ETS2101 has been found to have effects on immune cell function in vitro. The presence of Tumor Infiltrating Lymphocytes (TILs) has been previously shown to correlate with better patient outcomes during various antitumor therapies in a multitude of cancers. This study utilized immunohistochemistry (IHC) combined with digital image analysis to quantify CD4+ and CD8+ TILs within CT-26 tumors treated with ETS2101 in combination with a checkpoint inhibitor, anti-mCTLA4.

A CT-26 colon carcinoma tumor line was implanted s.c. into immune-competent mice. Ten days post implantation groups were treated with ETS2101 administered at 200 mg/kg QD in combination with anti-mCTLA-4 administered as 10 mg/kg i.p. BiW. Thirty seven days post treatment, tumors were collected and IHC was performed for CD4+ and CD8+ TILs on FFPE tumor sections (n=12-15 per group). Whole slide images were generated per IHC section and customized algorithms developed within the Indica Labs HALO platform to classify viable tumor present across each section and to quantify CD4+ or CD8+ stain area within the viable tumor region of interest (ROI).

Anti-mCTLA4 monotherapy resulted in regression of 44% tumors (7% CR) and anti-mCTLA4 + ETS2101 combination therapy resulted in regression of 74% tumors (36% CR). There was a significant increase in CD8+ % staining of viable tumor observed following anti-mCTLA4 monotherapy treatment versus control (2.16% v. 1.16%, P<0.0027) and anti-mCTLA4 + ETS2101 combination therapy (2.18% v. 1.10%, P<0.0019) compared to control. A similar trend was observed for CD4+ % staining of viable tumor with a significant increase observed following anti-mCTLA4 monotherapy treatment versus control (1.82% v. 0.49%, P<0.0083) and anti-mCTLA4 + ETS2101 combination therapy (1.93% v. 0.41%, P<0.0028) compared to control. Although, there was no significant difference between anti-mCTLA4 monotherapy treatment and anti-mCTLA4 + ETS2101 combination therapy for either marker alone, following treatment with the combination a statistically significant relationship between best overall response and the ratio of CD8+:CD8+ TILs was observed (P=0.0186).

In summary, there was an observed difference in the number of mice exhibiting a partial/complete regression in tumor volume for the combination arm 74% (36% CR) versus the antibody monotherapy arm 44% (7% CR). A statistically significant relationship (p=0.0186) was observed between the best overall tumor response and the ratio of CD8+:CD4+ TILs. IHC combined with image analysis can be utilized to evaluate therapeutic response on TILs within the context of their tumor microenvironment.

#164

Enhanced antitumor efficacy by recruiting macrophages as effector cells via bispecific antibodies mediated by CD89.

Bingyu Li, Lijun Xu, Kun Xie, Fei Tao, Renhao Li, Hua Gu, Jianmin Fang. _Tongji University, Shanghai, China_.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is one of key mechanisms through which antibody therapeutics can lyse tumor target cells. ADCC is usually mediated by IgG through recruiting effector cells, mostly NK cells, via IgG Fc receptor. In order to enhance specific tumor lysis by ADCC, a common strategy is to design a bispecific antibody that binds a tumor cell surface antigen and T cell marker CD3, thus to recruit T cells as effector cells. An example of this type of design is the FDA-approved bispecific antibody blinatumomab. We are interested in designing novel bispecific strategies to achieve improved ADCC effect. Since tumors are often infiltrated by macrophages and neutrophils, it would be advantageous to turn these types of cells into effector cells for ADCC. We have designed several bispecific strategies that can target a tumor associated antigen (TAA) and CD89 (FcαRI). The aim is to lyse tumors through connecting tumor cells to CD89-expressing immune effector cells, such as macrophages and neutrophils. The CD89 bispecific antibodies were produced by HEK293 cells and showed binding activities to both CD89 and TAA. The CD89 bispecific antibodies can recruit CD89-expressing immune cells and induced enhanced tumor cells-killing in human whole blood ADCC assays. Since mouse does not express human CD89 homologue, we generated a transgenic mouse strain with specific expression of CD89 on macrophages and monocytes. In this transgenic mouse model, the CD89 bispecific antibodies showed significant anti-tumor activities, demonstrating that the bispecific antibodies can redirect macrophages, including M2 macrophages, to mediate additional effector function in tumor microenvironment.

#165

**A novel** in vitro **microenvironment modeling platform for HCC therapeutics and biomarker development.**

Mohd Feroz Mohd Omar, Benny Tang, Sheng Chun Chang, Sarah Hong Hui Low, Gim Hwa Tan, Richie Soong, Bhaskar Bhattacharya. _National University of Singapore, Singapore, Singapore_.

Background: Hepatocellular carcinoma (HCC) is characterized by hypoxia, hypoglycemia and lactic acidosis within the microenvironment due a highly glycolytic phenotype. However, routine culture conditions of HCC cells employ supra-physiological glucose, pH-buffered media, and normoxia. The aim of the study was to simulate the microenvironment features of HCC in vitro by culturing HCC cells in their native (NAT) conditions (hypoglycemia, hypoxia and lactic acidosis) and examine phenotypic, transcriptomic and pharmacological differences compared to cells cultured in standard (ST) culture conditions.

Methods: Six HCC cell lines were cultured in NAT or ST conditions. Pharmacological response to cytotoxic drugs and targeted agents were evaluated using the MTS assay. Phenotypic differences were examined using standard techniques. Transcriptomic analysis was carried out using the Illumina HumanHT-12 expression kit.

Results: HCC cells cultured in NAT conditions have higher doubling time than cells cultured in ST conditions. However, no remarkable differences in the cell cycle profiles were observed when cultured in either NAT or ST conditions. Protein analysis revealed an increase in phosphorylation of AKT and decrease in the levels of AMPK in all cells cultured in NAT but not in ST conditions. Increase in the protein levels of GLUT1, HK2, LDHA, and a decrease in PDHA was observed only in cells cultured in NAT conditions. Furthermore, the HCC cells in NAT conditions exhibited lower levels of reactive oxygen species and ATP consistent with the elevated glycolysis and inefficient oxidative phosphorylation phenotype of HCC. Thirty-one genes were found to be aberrantly expressed by gene expression analysis, most notably NDRG1, a hypoxia-associated gene was upregulated in NAT cells, validated by qPCR. Upregulated NDRG1 was maintained even up reversal of the NAT condition. The panel of thirty-one genes were found to be associated with poor prognosis exclusively in HCC based on data available via TCGA supporting the validity of NAT culture condition. Differences in the IC50 of doxorubicin, sorafenib, PI3K, c-MET and HDAC inhibitors but not AKT, MEK, mTOR, Wnt inhibitors were observed in cells cultured in NAT compared with ST conditions suggesting microenvironment modelling can influence pharmacological response of certain class of compounds. Additional genomic and metabolomics analysis are currently being undertaken to characterize the HCC cells in NAT conditions.

Conclusion: In conclusion, HCC cells when cultured in NAT condition exhibit more of the tumor characteristics than cells in ST condition and this can be utilized as an informative platform for better understanding of disease biology and pharmacology.

#166

Recombinant protein MBP-NAP restricts tumor progression by triggering T-cell immunity in mouse metastatic melanoma model.

Ting Wang,1 Cong Ding,1 Zhenyu Ji,2 Xin Liu,1 Mingxuan Du,1 Qiaozhen Kang1. 1 _Zhengzhou University, Zhengzhou, China;_ 2 _Henan Academy of Medical and Pharmaceutical Sciences, Zhengzhou, China_.

The pro-inflammatory and immunomodulatory properties of Helicobacter pylori neutrophil activating protein (Hp-NAP) not only make it play an important role in disease pathogenesis, but also make it a potential candidate for applications, including vaccine and drug development. Our previous work demonstrated that the recombinant Hp-NAP fused with the maltose-binding protein of Escherichia coli (rMBP-NAP) exert an important role in regulating the differentiation of Th1 cells. As a potential TLR2 ligand, it was reported to possess the ability to induce systemic antitumor immunity in murine hepatoma H22 and sarcoma S180 tumor models. To further understand the antitumor and immunomodulatory effect of rMBP-NAP, we elucidate the effect and mechanism of rMBP-NAP at the local immune response modulation in established mouse B16-F10 melanoma pulmonary metastasis model. Our results demonstrated that metastatic lung tumor growth was significantly arrested after rMBP-NAP treatment, along with marked reduction in the number of lung nodules and significant increase in survival. Flow cytometry immunophenotyping and Quantitative RT-PCR analyses demonstrated that rMBP-NAP could induce both local and systemic immune responses, which associated with higher influx of CD3+CD4+T cells, CD3+CD8+T cells and higher secretion of interferon (IFN)-γ and interleukin (IL)-27 cytokines. The intraperitoneal administration of rMBP-NAP in mice promoted infiltration of lymphocyte in the lungs and reduced the production of several proinflammatory cytokines, such as IL-6 and transforming growth factor beta (TGF) -β, indicating an anti-inflammatory effect in local area. By comparing with control mice, mRNA expression of chemokines also revealed that rMBP-NAP treatment substantially decreased the expression of CCL2 and CCL20 in tumor tissues, which are central to recruit myeloid-derived suppressor cell (MDSC) and regulatory T cells (Treg) to tumor microenviroment and form immunosuppression. Moreover, we assess the expression of vascular marker CD34 in pulmonary section using immunohistochemical method, the results showed the vascularization was apparently inhibited in rMBP-NAP treated tumor bearing mouse. In conclusion, rMBP-NAP could induce the activation of effective Th1/Tc1 cells and the production of relevant inflammatory cytokines and chemokines, which are responsible for enhencing T cell immunity and reversing immunosuppression against metastatic cancer progression. Our findings strongly indicate that rMBP-NAP treatment might be a novel therapeutic approach against metastatic melanoma, and rMBP-NAP might be a potential template for the development of agents that could be used as immunomodulatory adjuvant in the therapy against melanoma.

#167

Ibrutinib regulates tumor microenvironment and enhances response to everolimus in renal cell carcinoma mouse models.

Jun Chen, Chun-Te Chen, Jing Liu, Jeff Hsu, Taisei Kinoshita, Betty Y. Chang. _Pharmacyclics, LLC, an AbbVie Company, Sunnyvale, CA_.

Introduction: Ibrutinib (ibr), a first-in-class, once-daily, oral inhibitor of Bruton's tyrosine kinase (BTK), is indicated for the treatment of patients with CLL/SLL, MCL and WM. Ibr also inhibits EGFR/HER2 and has demonstrated efficacy against EGFR+ NSCLC and HER2+ breast cancer in vitro and in xenograft models (Gao 2014; Chen 2016). Further, ibr modulated host immunity and enhanced anti-PD-L1 activity in solid tumor models otherwise insensitive to BTK or HER kinase inhibition (Sagiv-Barfi 2015), suggesting that ibr may be active in renal cell carcinoma (RCC) via multiple mechanisms. Here we determined the impact of ibr alone and in combination with everolimus (eve) on tumor growth and the tumor microenvironment in syngeneic and xenograft RCC mouse models.

Methods: Cell proliferation was determined with CellTiter-Glo using a 3-day treatment regimen. Subcutaneous implantation of 786-0 and Renca cells established xenograft and syngeneic models in nude and immune-competent mice. Treatment started when tumor volume reached 150 mm3 (786-0) or 55 mm3 (Renca) and was measured twice weekly. Immunophenotying of tumor infiltrates was determined by flow cytometry. For Treg differentiation, magnetically-sorted CD4+CD25- mouse splenic T cells were stimulated with anti-CD3/CD28 antibodies and TGF-β1 for 5 days.

Results: Ibr significantly inhibited the growth of syngeneic Renca tumors (TGI%=32.4 at Day 19, p≤0.05) in Balb/c mice and enhanced the efficacy of eve, although Renca cells were not sensitive to ibr in vitro. The combination significantly reduced the numbers of splenic and tumor Tregs in addition to reducing PD-1 expression on tumor CD8+ T cells. Consistently, in vitro Treg differentiation assays revealed that, while the combination of ibr and eve significantly reduced Treg differentiation, either drug alone showed little effect. In 786-0 xenograft model, ibr alone slowed tumor growth in a subgroup of mice and significantly enhanced the effect of eve (p≤0.05 or 0.01). To identify whether BTK or EGFR/HER2 is the target, CGI-1746, a BTK inhibitor, and lapatinib, an EGFR/HER2 inhibitor, were tested. Surprisingly, both CGI-1746 and lapatinib were active in inhibiting tumor growth (TGI%=26.7 or 37.6 respectively, p≤0.05 or 0.01), and slightly potentiated the efficacy of eve. However, consistent with ibr results, neither CGI-1746 nor lapatinib showed any effect on the cell growth of 786-0 cells.

Conclusion: This study suggests that ibr has antitumor activity against RCC alone and when combined with eve in animal models. This effect may be mediated by modulation of the tumor microenvironment, such as inhibiting Treg differentiation and suppressing PD-1 expression on CD8+ T cells, and both BTK and EGFR/HER2 are involved. Further investigation is needed to clarify the mechanism of action, but the results here provide a rationale for ibr as a novel agent for RCC in combination with mTOR inhibitors.

#168

**Loss of function mutation in** TIMP2 **gene accelerates tumorigenesis and mortality in murine model of lung cancer through EGFR signaling.**

Sarvesh Kumar, Sandra Jensen, David Peeney, Ananda Chowdhury, Beiyang Wei, William G. Stetler-Stevenson. _NCI/NIH, Bethesda, MD_.

The aim of this study is to determine the effect of the tissue inhibitor of metalloproteinase-2 (TIMP-2) on lung tumorigenesis and investigate the underlying molecular mechanisms using an orthotopic mouse model with a loss of function mutation in the Timp2 gene (T2M). T2M and wildtype (WT) control mice were given 1x106 Lewis lung carcinoma cells transfected with luciferase (LL/2-Luc-M38, Caliper) in 50μL PBS via intratracheal installation. IVIS imaging revealed a higher tumor burden in T2M mice compared to WT littermates, suggesting that loss of function of the Timp2 gene enhances tumor growth (p<0.05). We also conducted a Kaplan-Meier analysis to determine the effect of this mutation on mortality following cancer development. We found that LL/2 tumor-induced mortality was significantly higher in T2M mice compared to WT controls (p = 0.01). Histologic analysis and H&E staining of lung tissue sections revealed a significant increase in the number of tumor nodules of T2M mice compared to WT controls (p <0.01). CD31 staining showed a significant increase in microvessel density (MVD) in T2M mice compared to WT controls (p<0.01). Given that VEGF is a primary driver of tumor neovascularization, we determined mRNA levels of VEGF expression in healthy and tumor bearing mice by qRTPCR analysis. Interestingly, basal VEGF expression levels were increased in lungs of both non-tumor bearing and tumor bearing T2M mice compared to WT controls (p<0.01). VEGF is the direct downstream target of HIF-2α, a transcription factor implicated in tumor hypoxia and expression correlates with decreased overall survival in non-small cell lung cancer (NSCLC) patients. Similarly, an examination of TIMP-2 and HIF-2α mRNA expression in a small cohort of patient samples revealed decreased TIMP-2 and increased HIF2α in NSCLC tumors compared to surrounding non-tumor lung tissue. Hypoxia induces EGFR signaling through HIF-2α. Thus, we determined the effect of mutated T2M on the EGFR signaling pathway. We found increased levels of EGFR phosphorylation as well as downstream ERK and Akt activation in tumor bearing and non-tumor bearing T2M lungs compared to WT controls. In conclusion, these findings offer new avenues for TIMP-2 research in regulating hypoxic mediators within the tumor microenvironment through EGFR signaling, suggesting TIMP-2 as a novel bio-therapeutic for lung cancer therapy.

* Corresponding author This work is supported by center for cancer research, intramural research program, NCI/NIH research grants ZIA BC011204 & ZIA SC 009179 to WGSS

#170

An innovative immunotherapeutic strategy for ovarian cancer: Glycomimetic peptides.

Katherine F. Roby,1 Laura L. Eggink,2 J. Kenneth Hoober2. 1 _Univ. of Kansas Medical Ctr., Kansas City, KS;_ 2 _Susavion Biosciences, Inc., Tempe, AZ_.

Successful treatment strategies for women with ovarian cancer remain elusive. We hypothesize that novel means of activating anti-cancer immune activity will be an important component of a multifaceted approach to successful treatment. The present set of studies tests the hypothesis that novel peptide mimetics of C-type lectin receptor ligands, sv4L and sv6D, enhance anti-cancer immune activity and limit the progression of ovarian cancer in a mouse model. We further test the hypothesis that sv6D will function in synergy with additional immune modulators and conventional cytotoxic therapy. C-Type lectin receptors were targeted that are specific for N-acetylgalactosamine (GalNAc). Both svL4 and sv6D bind GalNAc-specific C-type lectin receptors including CLEC10A/CD301 with a KD in the low nanomolar range. CLEC10A is a transmembrane, endocytic receptor expressed on dermal dendritic cells, macrophages and immature dendritic cells. Further, studies with the B16 mouse melanoma model and spontaneous tumors (histiocytic sarcoma and mammary gland tumor) in dogs showed that treatment with svL4 correlated with reduced tumor-associated Treg cells.

In the present studies subcutaneous injection of svL4 or sv6D every other day over 5 days stimulated a several-fold proliferation of immune cells in the peritoneal cavity of healthy mice. These results indicated that svL4 and/or sv6D might exhibit significant activity on peritoneal tumors. Efficacy of svL4 and sv6D each as a single agent and as a combination therapy with paclitaxel or anti-PD-1 was tested in C57BL6 female mice bearing ovarian ID8 intraperitoneal tumors. As a single agent, 0.1 nmole/g doses of svL4 or sv6D had a significant effect on suppressing ascites formation, a measure of tumor progression, and overall survival.

Drug combination studies revealed a positive therapeutic interaction with sv6D and the cytotoxic paclitaxel. As single agents, sv6D and paclitaxel each had a significant effect on extending survival (median survival 140.5 and 150 days, respectively, vs. 122 days with no treatment). Survival was extended further with combination treatment when sv6D was administered to mice previously treated with paclitaxel (median survival 169 days). Also, a positive interaction was observed with sv6D and the check-point inhibitor anti-PD-1. Administration of sv6D following anti-PD-1 treatment resulted in a significant survival advantage compared to treatment with either agent alone.

These data demonstrate 1) sv4L and sv6D mobilize immune cells in the peritoneal cavity, 2) svL4 or sv6D as single agents slow progression of ovarian cancer and enhance survival in a mouse model of ovarian cancer, 3) sv6D in combination with paclitaxel or anti-PD-1 extends survival past that of either agent alone. Taken together these date demonstrate the potential for this novel approach of harnessing lectin receptors as a means toward effective cancer treatment.

#171

Targeting the tumor microenvironment: inhibition of carbonic anhydrase 9 impedes brain tumor initiating cell chemoresistance and delays glioblastoma growth in vivo.

Nathaniel H. Boyd,1 Kiera Walker,1 Paul C. McDonald,2 Mark O. Bevensee,1 Yancey G. Gillespie,1 Burt Nabors,1 Shoukat Dedhar,2 Anita B. Hjelmeland1. 1 _University of Alabama at Birmingham, Birmingham, AL;_ 2 _BC Cancer Research Centre, Vancouver, British Columbia, Canada_.

Brain tumor initiating cells (BTICs) are resistant to chemo- and radiotherapy, providing a reservoir for tumor recurrence and a desirable target for glioma treatments. Standard of care for glioblastoma (GBM; grade IV astrocytoma) includes the chemotherapeutic agent temozolomide, which prolongs life expectancy by months and is not curative. Prior studies suggested the efficacy of chemotherapies including temozolomide was increased by reducing expression of carbonic anhydrase 9 (CA9). CA9 is a hypoxia responsive gene elevated in tumors that is important for regulating intracellular pH and contributing to the acidic extracellular microenvironment. After confirming basal and hypoxia-induced expression of CA9 in GBM BTICs, we targeted CA9 activity with the small molecule inhibitor SLC-0111 alone or in combination with temozolomide. In multiple GBM BTIC lines, SLC-0111 reduced cell growth in vitro and showed additional benefit when used concurrently with temozolomide. Importantly, SLC-0111 inhibited the enrichment of BTICs after temozolomide treatment as determined via BTIC marker expression and neurosphere formation capacity. These data suggested the potential of SLC-0111 as a chemosensitizer, which we next evaluated in preclinical studies using a subcutaneous recurrent GBM model. GBMs treated with SLC-0111 in combination with temozolomide significantly regressed and the resulting in vivo growth delay was greater than that of temozolomide or SLC-0111 alone. Together, our data suggest that SLC-0111 can sensitize GBM BTICs to the chemotherapy temozolomide and significantly delay disease progression.

## CANCER CHEMISTRY:

### Basic and Applied Nanotechnologies and Therapeutic Approaches

#172

Oxidative stress and hepatotoxicity induced by graphene oxide in Sprague-Dawley Rats.

Anita K. Patlolla, Paul B. Tchounwou. _Jackson State Univ., Jackson, MS_.

Graphene oxide (GO) has been extensively explored as a promising nanomaterial for applications in biology because of its unique properties. Therefore, systematic investigation of GO toxicity is essential to determine its fate in the environment and potential adverse health effect. The aim of this study was to investigate the effect of graphene oxide on the induction of reactive oxygen species (ROS), the activity of certain liver enzymes (Alanine ALT, Aspartate AST, alkaline phosphatases ALP), and concentration of lipid hydroperoxide (LHP) in serum and histopathological evaluation of liver tissue in Sprague-Dawley rats. Four groups of five male rats were orally administered GOs, once a day for five days, with doses of 0, 10, 20 and 40 mg/Kg GO. A control group was also made of five rats. Blood and liver were collected 24 h after the last treatment following standard protocols. GO's exposure increased the induction of ROS, the activities of the liver enzymes (ALT, AST, ALP), concentration of lipid hydroperoxide (LHP) and morphological alterations of the liver tissue in exposed groups compared to control. The highest two doses, 20 and 40 mg/kg, showed statistically significant (p < 0.05) increases in the induction of ROS, activities of ALT, ALP , LHP concentration, and morphological alterations of liver tissue compared to control. However, AST activity showed no effect. Taken together, the results of this study demonstrate that GO is hepatotoxic, and its toxicity may be mediated through oxidative stress.

#173

Synthesis of substituted tetrahydroisoquinoline derivatives as anticancer agents.

Kinfe Ken Redda, Madhavi Gangapuram, Suresh Eyunni. _Florida A &M University, Tallahassee, FL_.

Breast cancer is the second leading cause of cancer-related deaths in women today and is the most common cancer among women, excluding non-melanoma skin cancers. Over 246,000 women will be diagnosed with breast cancer and 40,450 are expected to die of it in 2016. Estrogen receptors ERα, ERβ and their associated steroid hormones play vital role in breast cancer development and progression. Estrogen binds and activates the estrogen receptors in certain breast cancer cells. Endocrine therapy which aims to block ER action (antagonism) on breast tumor cells and thereby stopping the proliferation of cancer cells is a very useful strategy in treating breast cancer. Tamoxifen is the first selective estrogen receptor modulator (SERM) and is the leading drug used in treating breast cancer. Although Tamoxifen has shown great benefit in treating breast cancer, its agonistic effect on the uterus is said to be associated with an increased risk of developing endometrial cancer. We are interested in designing a single chemical entity which acts at specific multiple biomolecular targets. This strategy may exert favorable advantages in improving efficacy at desired multiple targets and lower incidence of side effects. Thus, alternative chemical entities, preferably non-steroidal molecules which act as estrogen receptor modulators and microtubule disruptors are sought. The tetrahydroisoquinoline (THIQ) core structure is an important pharmacophore in natural products and small molecules which act as drug molecules. The steroidomimetic tetrahydroisoquinoline moieties were reported to be selective SERMs and microtubule disruptors. Based on these considerations and in continuation with our previous research, herein we report in vitro cytotoxic and in silico docking studies of substituted THIQs. Novel substituted THIQ derivatives were designed and synthesized. N-amination of substituted isoquinolines by the aminating agent, O-mesytelene sulfnylhydroxylamine led to the formation of ylides. The ylides were reduced using sodium borohydride to yield the desired substituted tetrahydroisoquinolines in moderate to good yields. These compounds were evaluated for their cytotoxic effects using MCF-7, MDA-MB-231 and Ishikawa cells using the CellTiter-Glo luminescent cell viability assay. Among all the compounds screened, 4-ethyl-N-(8-hydroxy-3,4-dihydroisoquinolin-2(1H)-yl)benzamide showed IC50 values of 0.61, 1.36 and 0.09 µg/ml on MCF-7, MDA-MB-231 and Ishikawa cells respectively.

This research was supported by the National Center for Research Resources and the National Institute of Minority Health and Health Disparities of the National Institutes of Health through Grant Number 8 G12MD007582-28.

#174

Preclinical activity of new liposomal formulation of doxorubicin (TLD-1).

Massimo Broggini,1 Federica Guffanti,1 Roberta Affatato,1 Lavinia Morosi,1 Patrick Buschor,2 Christoph Matthieu,2 Florian Weiss,2 Andreas Konig,2 Peter Halbherr,2 Stefan Halbherr2. 1 _Mario Negri Inst. for Pharmacol. Research, Milan, Italy;_ 2 _InnoMedica, Bern, Switzerland_.

Background: InnoMedica is a young company with focus on clinical translation of nanomedicine. The company employs its own manufacturing technology that gives rise to a novel liposome platform. The platform allows for specific shaping of nanosurfaces. This leads to a myriad of different particle designs with highly distinct biological functions. In a first approach, the lead-formulation TLD-1 (targeted liposomal doxorubicin) was developed in order to ameliorate chemotherapeutic outcome for patients, taking into account the patterns of biodistribution in the entire organism as well as nanoparticle-cell interactions and subcellular localisation.

Methods: Pharmacologic properties of TLD-1 were investigated in vitro and in vivo. Human ovarian cancer cells A2780 were cultured in both 2D and 3D settings and antineoplastic activity of different formulations of doxorubicin was measured by quantification of cell viability. The same formulations were also compared in three animal models of cancer including murine breast cancer 4T1, human breast cancer MDA-MB231, and A2780. MDA-MB231 and A2780 cells were grown in vitro and injected subcutaneously (sc) in immunodeficient mice (Athymic Nude-Foxn1nu). 4T1 cells were injected either sc or in the mammary fat pad of immunocompetent mice (BALB/c). The different drug products were injected intravenously twice weekly for a total of three weeks. Activity was determined with caliper measurements of tumor diameters every three days. Animal body weight was recorded as index of toxicity. Doxorubicin was determined in plasma and tissues by HPLC-MS.

Results: In 2D and 3D cell culture assays, TLD-1 showed a marked increase in cytotoxicity compared to Caelyx and was close to free drug. In all three animal models, TLD-1 showed activity superior to that of free doxorubicin given at the same dose and compared well to Caelyx both in terms of activity and toxicity.

Conclusions: A couple of nanoparticulate features of TLD-1 liposomes are believed to attribute to the difference in antitumor activity. Cytotoxicity data from cell culture experiments indicate that TLD-1 liposomes are readily taken up by cancer cells and release their drug load into the cytoplasm. Commercial liposomes as in Caelyx seem to largely remain outside of cancer cells. In sum, TLD-1 is a novel and highly active antineoplastic nanodrug that has the potential to outperform free drug as well as commercial liposomal formulations of doxorubicin.

#175

Combination of plasmonic photothermal therapy with surgery applied to naturally occurring mammary tumors in canines and felines: clinical outcomes and molecular studies.

Moustafa R. Ali,1 Haithem A. Farghali,2 Hala R. Ali,2 Ahmed H. Osman,2 Yousef A. Soliman,2 Yue Wu,1 Ibrahim M. Ibrahim,2 Salah A. Selim,2 Dong M. Shin,3 Mostafa A. El-Sayed1. 1 _Georgia Inst. of Technology, Atlanta, GA;_ 2 _Veterinary Medicine, Cairo University, Cairo, Egypt;_ 3 _Emory University, Atlanta, GA_.

Plasmonic Photothermal Therapy (PPTT) is a cancer therapy where gold nanorods (AuNRs) are injected at the tumor site and near-infrared light (safe to bio-system) is applied to generate localized heat causing cancer cell death. PPTT is a potentially good alternative to replace traditional surgery for localized tumors. However, for large tumors (volume ≥10 cm3), PPTT could be ineffective due to an uneven distribution of injected AuNRs causing possible inhomogeneity of heat. Surgery is frequently recommended in those cases. However, it carries a high risk of cancer recurrence. For effective treatment of large tumors, we combined both PPTT and surgical resection and applied it to naturally occurring tumors in mammary glands of dogs and cats, which could realistically represent their human equivalents at the molecular level. For the experimental design, we divided the animals into three different groups. 20 cases (7 cats and 13 dogs) were all diagnosed with adenocarcinoma; the animals were monitored for 1-2 years after treatments. Group (I): three cases were solely treated by mastectomy (control group); all of them died within a few weeks. Group (II): five cases were treated with mastectomy first. Then, each tumor wound was divided into two halves, and only one half was exposed to PPTT. After treatment, two cases in this group rendered complete remission. In the other three cases, the half wound that was not exposed to PPTT had tumor recurrence causing animal death within one year. Group (III): 12 cases were treated with surgery followed by PPTT treatment. This regime showed complete remission without any recurrence for eight cases. However, four cases died 4-12 months after therapy for reasons such as pneumonia (no tumor found, based on X ray). Histopathology results showed a decrease of cancer grades before (variant grades from 1-4) and after two weeks of treatment via PPTT and surgery (grade 0). Blood tests (conducted 1 year after therapy) showed no obvious change in liver and kidney functions in groups II and III. In addition, X-ray diffraction showed no metastasis 1- 2 years after treatment. We have performed quantitative, real time-PCR analysis two weeks before and after treatment to study the expression levels of several important genes. The genes that are responsible for repairing cancer cells such as BRCA1, BRCA2, and CD163-IL-10 were significantly diminished two weeks after treatment (group III). Furthermore, tumor microenvironment cells such as tumor-associated macrophages (TAMs) were greatly altered after treatment. TAM 1, which retards tumor growth, augmented, and TAM 2, which promotes tumorigenesis, was diminished, which explains the animals' increased survival rate. In conclusion, our study demonstrates the feasibility of applying PPTT after surgery for large tumors in dogs and cats.

#176

Early development of GMC1, a novel molecule targeting FKBP52 for the treatment of hormone-refractory prostate cancer.

Huan Xie, Oscar Ekpenyong. _Texas Southern University, Houston, TX_.

Purpose: GMC1 directly inhibits FKBP52, effectively blocking androgen receptor dependent gene expression and androgen-stimulated proliferation. This make it an attractive option for the treatment of hormone-dependent and hormone-independent prostate cancer. This study investigated an analytical method for GMC1 quantification, pre-formulation characteristics of GMC1, and developed intravenous formulations for the evaluation of GMC1 in animal models.

Method: An LC/MS/MS method for the quantification of GMC1 in solution, plasma and urine was developed, validated and applied to the determination of the stability, log P, plasma protein binding and solubility of GMC1 in various solvents. Liposomal formulations and co-solvent systems with various ratios of high capacity vehicles were formulated and the optimal formulation applied, at 2 mg/kg single IV bolus dose, to the pharmacokinetic study of GMC1 in a rat model.

Result: The intra- and inter-day accuracy (%RE) and precision (%CV) of the LC/MS/MS method ranged from 1.6 – 11.7 % and 1.4 – 8.8 %, respectively. GMC1 is stable in solid and solution state, moderately lipophilic (log P = 1.38 ± 0.05), poorly water soluble (0.4 ± 0.01 mg/mL), and highly plasma protein bound (>71%). The optimal formulation consisting of PEG 300 and Labrasol ® (1:1, v/v) allowed us to achieve a GMC1 concentration of 10 mg/mL, and tolerated an aqueous environment. GMC1 has a tri-exponential disposition with a Cmax of 7.6 ± 1.97 mg/L, clearance of 0.53 L/kg/hr, α-distribution, β-phase and terminal elimination half-lives of 0.1 ± 0.04 hr, 1.2 ± 0.34 hr, and 19.7 ± 5.09 hr respectively.

Conclusion: The LC/MS/MS method, formulations and pharmacokinetic study can be applied to the pre-clinical and clinical development of GMC1.

Grant Support: This work was performed under funding from NIH/NIGMS grant (5SC3GM102018) and NIH/NIMHD/RCMI grant (5G12MD007605).

#177

Thymidine quinoxaline conjugates as a novel selective and effective photosensitizer for anticancer photodynamic therapy.

Qibing Zhou, Zhiwei Wang, Rong Yang, Ting Qian, Dejun Zhang. _Huazhong Univ. of Science & Technology, Wuhan, China_.

Thymidine quinoxaline conjugates are a new class anticancer agent that exhibits selective anticancer activity by targeting the abnormally high level of thymidine kinase of the thymidine salvage pathway in cancer cells. In addition, the conjugated quinoxaline moiety could also act as a photosensitizer to generate reactive oxygen species that would significantly enhance the anticancer activity. Therefore, a variety of structural derivatives were synthesized and investigated for the potential of combined anticancer activity. We found that the thymidine moiety was critical for the accumulation of compounds in cancer cells and thus their activity. On the other hand, the substitutions and additional conjugation of quinoxaline moiety had significant impact on the effectiveness to generate reactive oxygen species for enhanced potency. Among all the compounds studied, dT-QP exhibited a potent activity against liver cancer cells with IC50 at 20 nM under photo-irradiation at 400 nm wavelength for 20 min. This potent activity of dT-QP was 12 times more effective than that of the clinically used photosensitizer protoporphyrin IX (IC50 at 250 nM) under the same condition and 5 times more potent than anticancer drug doxorubicin (IC50 at 100 nM). In addition, thymidine quinoxaline conjugates were found to form aggregates with an average size of 200 nm. Further optimization of the aggregating condition led to a highly stable suspension of 90 nm size nanoparticles containing a tumor-specific peptide that could be used for in vivo study. Therefore, thymidine quinoxaline analogs could be a selective anticancer agent and effective photosensitizer for potential photodynamic therapy.

#178

Peptide nanofibers: targeted therapies for glioblastoma multiforme.

Diana M. Leite,1 Rong Zhu,2 Eugen Barbu,1 Peter Hinterdorfer,2 Geoffrey J. Pilkington,1 Aikaterini Lalatsa1. 1 _University of Portsmouth, Portsmouth, United Kingdom;_ 2 _Johannes Kepler University, University of Linz, Linz, Austria_.

Glioblastoma multiforme (GBM) is a malignant brain tumor with poor prognosis due to tumor heterogeneity, poor drug blood-brain barrier (BBB) permeability and targeting. GBM biopsies indicated the overexpression of G-protein coupled receptors (GPCRs) that when activated by neuropeptide agonists result in an antiproliferative effect. However, translation of these neuropeptides into novel therapies for GBM is frustrated by their short half-life (<5 minutes) and inability to cross the BBB. Here, we present a novel strategy based on the lipidization of peptide agonists which results in peptide amphiphiles able to self-assemble into nanofibers that can entrap brain impermeable drugs, possess an enhanced stability to enzymatic degradation, permeate across an all human in vitro BBB model and are able to target GBM cells resulting in a significant antiproliferative effect. Peptide amphiphiles were synthesized using solid-phase peptide synthesis and characterised using pyrene, thioflavin T, circular dichroism and transmission electron microscopy experiments. Nanofibers were loaded with brain impermeable cytotoxic drugs (e.g. paclitaxel) and their stability was studied in biological media (plasma, brain, liver and GBM cell lysates). BBB permeation was studied in an all human in vitro Transwell model. The antiproliferative effect of the nanofibers was evaluated on U-87 MG cells. Targeting of the nanofibers to the GPCR was evaluated using single-molecule force spectroscopy (SMFS). Peptide amphiphiles self-assemble into stable nanofibers at concentrations above 189 μM and are able to solubilize high amounts of paclitaxel (>1.8 mg) ensuring that the resulting nanomedicine can be clinically translatable. Both unloaded- and paclitaxel loaded-nanofibers showed superior stability compared to the parent neuropeptide in the presence of plasma, brain, liver and cell homogenates (>6-fold). Nanofibers elicited a strong antiproliferative effect (IC50: 5.06 μM) resulting in a cell cycle arrest at G2/M in a GPCR positive GBM cell line (U-87 MG). Loading paclitaxel (1nM) within the nanofibers resulted in a synergistic effect evidenced by a decrease in cell survival by 32%. Nanofibers counteracted the forskolin-induced increase of intracellular cAMP levels indicating that the GPCR is linked to the GαI protein known to mediate the antiproliferative effect on GBM. Confocal studies confirmed the internalization of the peptide while SMFS studies supported specific binding of the nanofibers to GPCR on U-87MG cell surface with equivalent binding probability to peptide agonists and increased residence time. Texas Red labelled nanofibers permeate across an in vitro BBB model enabling the permeation of paclitaxel (Papp: 8.45 x10-6 cm/s). Thus, described peptide nanofibers are a novel targeted nanomedicine for GBM therapy able to be clinically translated. In vivo proof of concept studies and pharmacokinetics are under way.

#179

Plasma membrane lipid therapy: disruption of oncogenic Ras spatiotemporal organization by membrane-targeted dietary bioactives (MTDB).

Natividad R. Fuentes,1 Rola Barhoumi,1 Mohamed Mlih,2 Jason Karpac,2 Paul Hardin,1 Trevor Steele,1 Spencer Behmer,1 Ian Prior,3 Robert S. Chapkin1. 1 _Texas A &M University, College Station, TX; _2 _Texas A &M Health Sciences Center, College Station, TX; _3 _University of Liverpool, Liverpool, United Kingdom_.

Approximately 30 to 50% of colorectal cancers contain KRas mutations, which confer resistance to standard therapy and have therefore been termed "undruggable." Since no curative treatments for KRas driven colon cancer are available, there is a critical need to develop toxicologically innocuous KRas therapeutic approaches that are free of safety problems intrinsic to drugs administered over long periods of time. High fidelity signaling of Ras is dependent on its spatial organization into defined nanoclusters on the plasma membrane. This is noteworthy, because select nonsteroidal anti-inflammatory drugs, through direct modulation of the biophysical properties of the plasma membrane, alter oncogenic Ras nanoclustering and attenuate signal transduction. These findings suggest that Ras nanoclusters represent a novel target for future interventions. Consistent with this rationale, we hypothesize that select amphiphilic membrane targeted dietary bioactives (MTDB's), e.g., polyunsaturated fatty acids (n-3 PUFA) docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19) and eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17), [1] modulate the rigidity of the plasma membrane, [2] modify Ras nanocluster formation, [3] disrupt oncogenic Ras driven signaling (pERK), and [4] suppress phenotype (hyper-proliferation) in vitro and in vivo.Our studies utilized a variety of complementary models including mouse colonic crypts, Drosophila midguts, and isogenic human and mouse colonic cell lines. Using a membrane order sensitive dye, Di-4-ANEPPDHQ, we initially characterized the rigidity of the plasma membrane in mouse colonic crypts and young adult mouse colonocytes (YAMC) cells. Quantification of Ras nanocluster formation was determined by fluorescence lifetime imaging microscopy (FLIM) combined with fluorescence resonance energy transfer (FRET) of fluorescently tagged Ras membrane targeting domains. In addition, oncogenic Ras driven signaling (pERK) and phenotype (hyper-proliferation) was determined by targeting expression of RasV12 or KRasG12D to Drosophila midgut stem cells and mouse colon, respectively. In vitro and in vivo incorporation of n-3 PUFA consistently reduced plasma membrane rigidity and tH-Ras clustering, while increasing tK-Ras clustering and the mixing of tH and tK proteins. These changes in membrane spatiotemporal organization were associated with a reduction in oncogenic RasV12 driven ERK phosphorylation and intestinal stem cell proliferation.These novel findings demonstrate a unique role for MTDBs in the modulation of Ras nanoscale spatial organization and signaling. Establishing a role for MTDBs in Ras-dependent oncogenesis would have a major translational impact because these bioactives are safe, well tolerated, relatively inexpensive, and provide additional health benefits, such as reduction in mortality. This work was support by NIH grant R35CA197707.

#180

PhAc-ALGP-Dox is a new tumor selective peptide prodrug of doxorubicin that shows improved efficacy and systemic tolerance in triple negative breast cancer models.

Andrea Casazza,1 Massimiliano Mazzone,2 Peter Pokreisz1. 1 _CoBioRes NV, Leuven, Belgium;_ 2 _VIB Vesalius Research Center, Leuven, Belgium_.

Use of traditional chemotherapeutic drugs is restricted by severe side effects and lack of tumor specificity of these cytotoxic agents. Less toxic prodrug that can be selectively activated in tumor tissue have been explored in attempts to improve the therapeutic index. Some approaches to the development of tumor activating prodrugs take advantage of inherent properties of the tumor, for example, selective enzyme expression, hypoxia, or low extracellular pH in the vicinity of the tumor. CoBioRes makes use of capped, tetrapeptidic prodrug of existing chemotherapeutics. Doxorubicin (Dox) was the first chemotherapeutic that was linked to the tetrapeptide sequence and tested experimentally (PhAc-ALGP-Dox). This approach makes the prodrug impermeable to cell membranes of both normal and tumor cells. Furthermore, it remains stable in blood by the use of the capping group on the amino-terminal group of the peptide that prevents aspecific activation by circulating esopeptidases. The prodrug as such is thus not active. The peptide sequences have been developed to be sensitive to the hydrolytic action of a selected group of peptidases that are released within the tumor microenvironment. These peptidases, which accumulate in the tumors, also play an important role in cancer cell invasion and metastasis. What makes this approach unique is that it is not targeting a single enzyme, but requires, for its selectivity, a two-step activation based on enzymes with an increasing selectivity. In vivo activation starts when the tetrapeptide prodrug is exposed to endonucleases (CD10 and THOP1) leading to the formation of intermediates that are still inactive and poorly permeable through cell membranes. This leads to a tissue distribution that is determined by the expression of the endonucleases. Since these are known to be overexpressed in several tumors, and only present at lower level in some normal tissues, the prodrug will be preferentially located and activated in the vicinity of tumors. The second step of the activation is the cleavage at the prolyl by dipeptidases FAP and DPPIV, releasing the free doxorubicin. The simultaneous expression of all the enzymes involved in the prodrug activation was confirmed both in human triple negative breast cancer biopsies and in experimental triple negative breast cancer models. In vitro analysis of PhAc-ALGP-Dox reveals higher tumor cells tropism and higher cytotoxicity in cancer cells than in normal cells. In vivo PhAc-ALGP-Dox gives promising preliminary results in terms of systemic tolerance even at high concentration, and in vivo efficacy in xenograft mouse models compared to free doxorubicin. Taken together, these results provide a strong rationale for further investigation aimed at unleashing the potential clinical value of this compound.

#181

Discovery of novel targeted therapeutics for metastatic breast cancer.

Cristina Del Valle,1 Eliud Hernández,1 Cornelis P. Vlaar,1 Luis A. Cubano,2 Suranganie Dharmawardhane,1 Linette Castillo-Pichardo2. 1 _University of Puerto Rico, Medical Sciences Campus, San Juan, PR;_ 2 _Universidad Central del Caribe, Guaynabo, PR_.

Metastatic disease is the primary cause of breast cancer mortality, due to the lack of effective therapy. The Rho GTPase Rac is integral for the promotion of cancer cell migration/invasion, proliferation, and survival. Since metastatic breast cancers often overexpress or exhibit high Rac activity, inhibition of Rac is a viable strategy against metastatic cancer. Recently, we characterized EHop-016, a small molecule that inhibits Rac activity of metastatic breast cancer cells with an IC50 of 1 µM. EHop-016 is 10-100 times more active than previously available Rac inhibitors, and is the first compound shown to inhibit the activation of Rac by the oncogenic GEF Vav. EHop-016 inhibits the activity of the Rac downstream effector p21 activated kinase (PAK), lamellipodia extension, and cell migration of metastatic breast cancer cells. We also reported that EHop-016 at ≥ 25 mg/kg Body Weight (BW) significantly reduced tumor growth, metastasis, and angiogenesis in a mouse model. However, our recent pharmacokinetic study of EHop-016 in a mouse model demonstrated that the bioavailability of Ehop-016 needs to be improved for further pharmacological development. Therefore our hypothesis is that improvement of the EHop-016 structure will provide probes with increased potency against Rac and, therefore, increased bioavailability. Herein we have tested several Ehop-016 derivatives for their effects on breast cancer cell viability and Rac activation. Using MTT assays we found that the Ehop-016 derivatives, HV-107 and HV-118, significantly inhibit the viability of metastatic breast cancer cell lines MDA-MB-231 and MDA-MB-435. The effects of HV-107 and HV-118 on the inhibition of Rac activation were tested by ELISA-based Rac activity assays and pulldown assays. Results show that at 250nM, HV-107 inhibits Rac activation by 55%, whereas HV-118 has a similar effect at 100nM in MDA-MB-231 and MDA-MB-435 cells. Taken together, our findings suggest HV-107 and HV-118 as promising Ehop-016 derivatives with potential as anti-metastatic agents, which should be further characterized.

This study was supported by awards from the Susan Komen for the Cure, NIH/NIMHHD U54MD008149, and the Puerto Rico Science and Technology Trust to SD; NIH/NCRR R25GM061838 to UPR MSC; NIH/NIMHHD RCMI 8G12MD007583RCMI, Title V PPOHA 031M10505 and Title V Cooperative P031S130068 from U.S. Department of Education to UCC; and PRINBRE (NIH/NIGMS P20GM103475-13) Pilot Project to LCP.

#182

A new anticancer agent derived from decenoic acid for the treatment of colon cancer.

Masumi Suzui,1 Saeko Ando,1 Harutoshi Matsumoto,1 Katsumi Fukamachi,1 Mitsuru Futakuchi,1 Naoki Yoshimi2. 1 _Department of Molecular Toxicology, Nagoya City University Graduate School of Medical Sciences and Medical School, Nagoya, Japan;_ 2 _Department of Pathology and Oncology, Graduate School of Medicine and Faculty of Medicine, University of the Ryukyus, Okinawa, Japan_.

The final goal of the current study is to create a promising lead compound that targets human colon carcinoma. After discovering in vitro antiproliferative activity of a naturally occurring compound, 10-hydroxy-2-decenoic acid, we designed and synthesized over 150 derivatives. The initial screening resulted in the discovery of three primary lead compounds, we then proceeded with modifications in side chain and eventually obtained a second-generation lead compound, named PPI (patented in 2014), whose IC50 was 0.3 μM. We focused on the transcription factor STAT3 that is able to drive carcinogenesis, apoptosis, and angiogenesis-related genes. In silico docking simulation exhibited that PPI can bind to SH2 domain of STAT3, suggesting that this drug blocks dimerization of STAT3, thereby PPI inactivates function of STAT3. PPI inhibited transcriptional activity of STAT3 when SW480, SW837, and HT29 human colon carcinoma cells were treated with 3.7 to 5 μM PPI for 24h. Flowcytometry analysis indicated that when HT29 cells were treated with 1 μM PPI, the percentage of cells in G1 increased by 5% and this was associated with a concomitant decrease of cells in the S and G2-M phases of the cell cycle. The results indicate that PPI causes carcinoma cells to arrest in the G1 phase. When HT29 cells were treated with 10 μM PPI, subG1 fraction and DNA fragmentation were seen, indicating induction of apoptosis. We then performed western blot analysis to determine whether treatment of carcinoma cells with PPI alters cellular levels of the G1 cell cycle control protein cyclin D1 and apoptosis/angiogenesis-related molecules. When HT29 cells were treated with PPI for 24 to 48h, there was a marked decrease in the levels of expression of the Bcl-2, Bcl-xL, and VEGF proteins and a marked increase in the levels of expression of the cleaved caspase 3, 7, 8, 9, and PARP proteins. PPI also inhibited expression levels and nuclear tanslocation of pSTAT3. To see if PPI exerts preventive effect on tumor promotion, F344 rats received sc injections of a carcinogen azoxymethane and then treated with soybean oil or PPI. There was a dose dependent decrease in multiplicity of aberrant crypt foci (ACF) in the colon. PPI inhibited the occurrence of larger ACF consisting of more than 4 aberrant crypts, indicating inhibitory effect of PPI on tumor promotion. In a mouse xenograft model, PPI decreased the size and number of blood vessels in the tumor. All mice survived without causing significant body weight loss during experiment. No histopathological abnormality was found in any of organ site. To confirm the effects of PPI on angiogenesis, we performed chorioallantoic membrane assays and found a dose-dependent decrease in average number of blood vessels. Inhibition of STAT3 by PPI may affect the function of molecules that are related to apoptosis, angiogenesis, and cell cycle progression and eventually contributes to PPI-induced growth inhibition.

#183

Brassinolide, a plant steroid hormone, reverses drug resistance in human small-cell lung carcinoma cells.

David Sadava, Susan E. Kane. _City of Hope, Duarte, CA_.

Small-cell lung carcinoma (SCLC) has a dismal prognosis in part because of multidrug resistance (MDR). Epibrassinolide (EB) is a steroid hormone present in higher plants, where it has numerous physiological effects and acts via a LRR-RLK membrane receptor and GSK3/SHAGGY pathway, resulting in stabilization of a transcription factor. The parallels to the Wnt signaling pathway, which is activated in SCLC and results in increased β-catenin, prompted investigations of the effects of EB on SCLC cells, particularly showing MDR. SCLC cells were NCI-H69 and a derived line, VPA17, showing resistance to etoposide (9-fold), doxorubicin (20-fold) and vincristine (10-fold). EB was cytotoxic to both cell lines (IC50 = 2 uM), indicating a lack of cross-resistance. EB was pro-apoptotic after 24 h as measured by ELISA of BUdR-labeled DNA fragments. Apoptosis was also indicated by an increase in caspase-3 specific activity (to 2.5 enzyme units/mg protein vs. 0.01 for untreated controls). Matrigel assays showed that EB reduced the SCLC cell invasion phenotype by 80% in a time-dependent fashion. Pre-incubation of VPA17 cells in 1 µM EB for 96 h reduced the IC50 for etoposide (6.0 uM to 1.8 µM) and doxorubicin (0.37 µM to 0.09 µM). Synergism between EB and chemotherapy drugs was determined by exposure of VPA17 cells to 1:1 ratios at the appropriate IC50 values of EB: etoposide or EB: doxorubicin. After 4 days, the combination index (CI) was determined using CalcuSyn software. EB and etoposide showed modest synergism (CI = 0.80 at ED50 and CI = 0.90 at ED95); EB and doxorubicin also showed synergism (CI = 0.65 at ED50 and CI = 0.90 at ED95). The Wnt - β-catenin signaling pathway was investigated as a possible explanation for the pharmacological effects of EB. In the presence of EB, SCLC cells showed a time- and dose dependent reduction of β-catenin (maximum 80% reduction). Cell fractionation studies showed that the reduction was primarily in nuclear β-catenin. Transcription analyses of SCLC cells showed EB led to significant reduction in expression of β-catenin-dependent genes that are anti-apoptotic (e.g., c-Jun, survivin), cell division-related (e.g., CCND1 cyclin, sox9), and metastasis-related (e.g., MMP7, uPAR). Down-regulation of the β-catenin promoter was confirmed by transfection of SCLC cells with a construct containing the promoter coupled to a luciferase reporter (reduced by 85%). WIKI4, a known inhibitor of Wnt signaling, was cytotoxic to SCLC cells (IC50 = 0.02 uM). Synergism between EB and WIKI4 was determined by exposure of VPA17 cells to 1:1 ratios at the appropriate IC50 values of EB: WIKI4, as above. EB and WIKI4 showed antagonism (CI = 1.09 at ED50 and CI = 1.35 at ED95), suggesting that EB and WIKI4 act on the same pathway. Taken together, these data indicate that EB, a natural product with widespread occurrence in plants, is pharmacologically active in both drug-sensitive and drug-resistant SCLC cells and acts through the Wnt signaling pathway.

#184

Effects of the magnetic-organic compound Fe(salen) on anaplastic thyroid cancer cells.

Shuzo Tamura,1 Takashi Oshima,1 Mariko Kamiya,1 Shinya Amano,1 Toru Aoyama,1 Hirotaka Nakayama,1 Nobuyasu Suganuma,2 Tsutomu Sato,1 Hiroshi Harada,1 Manabu Shiozawa,2 Takaki Yoshikawa,2 Yohei Miyagi,2 Yasushi Rino,1 Munetaka Masuda1. 1 _Yokohama city university, Yokohama city, Japan;_ 2 _Kanagawa cancer center, Yokohama city, Japan_.

Introduction Although anaplastic thyroid cancer (ATC) is a rare disease, it is a cancer with one of the worst prognoses. Even in patients who receive multidisciplinary treatment, the 1-year survival rate is less than 30%. Because about 50% of patients with ATC die of airway obstruction, control of local progression is very important in the management of ATC. Novel treatments for ATC, especially those that can control local disease, are expected to emerge. Recently, several studies have reported that the magnetic organic compound μ-oxo N,N-bis(salicylidene)ethylenediamine iron (Fe(salen)) is effective against malignant melanoma and lingual cancer. Fe(Salen) itself is magnetic and might be effective as a local treatment by externally applying a magnetic field. Fe(salen) thus has the potential to become a novel treatment for ATCs. In this study, we evaluated uselessness of Fe(salen) as an anticancer drug for ATC.

Methods The in vivo anticancer effect of Fe(salen) against ATC cell lines (KTA1, KTA2, TTA2, TTA3, 8305c, 8505c) was evaluated by XTT assay. In addition, the effectiveness of combined therapy with paclitaxel, which is currently a standard chemotherapy for ATCs, was evaluated by the Chou and Talalay method. Colony formation assay was performed to evaluate differences in effects depending on the order of drug administration. In addition, to clarify the mechanism of Fe(salen) on cancer cells, changes in the cell cycle after Fe(salen) treatment were examined by flow cytometry.

Results Fe(salen) showed anticancer activity with an IC50 value of less than 100μM against KTA1, KTA2, TTA2, TTA3, 8305c, and 8505c cells. In particular, the IC50 values were less than 10 μM against TTA3 (8.82±4.58 μM) and 8305c (7.06±4.11 μM) cells. Combination therapy with paclitaxel had a synergistic effect, with a combination index of less than 1. No significant difference was detected in anticancer effect depending on the order of drug administration. G2/M cell cycle arrest was observed in 8305c cells on flow cytometry after Fe(salen) treatment.

Conclusion Our results showed that Fe(salen) has an anticancer effect on ATC cells and that Fe(salen) and paclitaxel have synergistic activity. We will evaluate the in vivo effectiveness of Fe(salen) treatment in an orthotopic mouse model, focusing on local control achieved by applying a magnetic field.

#185

Development of a novel non-diuretic brain-penetrating ethacrynic acid analog and demonstration of its potent efficacy in orthotopic glioblastoma (GBM) models.

Surendra R. Punganuru, Hanumantha Rao Madala, Kalkunte S. Srivenugopal. _Texas Tech Univ. Health Sciences Ctr., Amarillo, TX_.

The incidence of pediatric and adult brain tumors has continued to rise and temozolomide (TMZ) has remained the sole choice for chemotherapy of these cancers. A number of factors including the inefficient drug entry through the BBB, intratumoral heterogeneity, overexpression of MGMT repair protein and bone marrow toxicity due to alkylating regimens have impeded successful therapy of these cancers. There is, therefore, an urgent need to design new, more effective brain-penetrating drugs that can effectively eliminate brain tumors. We are highly interested in exploiting the elevated oxidative stress present in gliomas and have synthesized a hydrophobic, non-diuretic analog of ethacrynic acid (EA) called KSS72 [1-(2,3-dichloro-4-methoxyphenyl)-2-methylenebutan-1-one] by removing the carboxylate side chain. KSS72 was selectively cytotoxic to cancer cells including gliomas. Compared to EA which does not cross the BBB, Pharmacokinetics following intravenous or oral administrations in mice showed its excellent penetrance through the blood brain-barrier, KSS72 accumulating at levels equivalent to TMZ (25% of plasma levels) in the brain In vitro assays measuring protein carbonyl content, GSH content, ROS generation, GSTpi enzyme activity and others showed that KSS72 triggers a redox imbalance by inhibiting GSTpi and by lowering the antioxidant (GSH) and reducing equivalent (NADPH) levels, leading to a significant elevation of ROS levels. We also showed the upregulation of endoplasmic reticulum (ER) stress-responsive proteins, activation of MAPK, autophagy and apoptotic pathways by KSS72 in several cell lines. KSS72-induced autophagy was a post event of redox perturbation that displayed a wide array of characteristic features including double membranous vacuoles with entrapped organelles, acidic vesicular organelles, and increased expression of LC3-II and beclin-1. In view of its excellent brain penetrance and multiple cytocidal events mediated by ROS and autophagy, we tested the antitumor efficacy of KSS72 in human GBM cell lines and intracranial GBM xenografts. SF-188 human GBM cells expressing firefly luciferase were injected into the brain just left of bregma for the development of orthotopic GBM in nude mice. These mice were given 25 mg/kg/day of KSS72 intraperitoneally for 2 weeks. The animals when imaged by IVIS after luciferin injections showed a complete lack of intracranial tumors in all KSS72 administered animals. H&E staining of mouse brain sections confirmed the total elimination of GBM by KSS72. KSS72 did not exert any toxicity on host tissues and serum ALT and AST levels were not altered. In summary, we conclude that KSS72 acting through multiple pathways of oxidative stress is a hugely promising non-toxic anti-glioma drug with potential to enter clinical trials (supported by CPRIT grants RP130266 and RP170207 to KSS).

#186

FDA approved drug Bazedoxifene as a novel inhibitor of IL 6 and IL 11/GP130 signaling for osteosarcoma therapy.

Xiaojuan Wu,1 Hui Xiao,2 Chenglong Li,3 Jiayuh Lin4. 1 _Tongji Hospital, Huazhong University of Science and Technology, Wuhan, China;_ 2 _Center for Childhood Cancer and Blood Diseases, the Research Institute at Nationwide Children's Hospital, College of Medicine, The Ohio State University, Department of Pediatrics, OH;_ 3 _College of Pharmacy, University of Florida, Gainesville, FL;_ 4 _University of Maryland School of Medicine, Baltimore, MD_.

IL-6 and L-11 signaling pathways play an important role in cancer cell survival and progression, including osteosarcoma. IL-6/IL-11 binds to IL-6/IL-11 Rα to form a binary complex, then recruits GP130 to form the IL-6/IL-6 Rα/GP130 or IL-11/IL-11 Rα/GP130 heterotrimer and triggers a signaling cascade downstream. One of the major downstream effectors of IL-6 is STAT3. STAT3 is persistently activated in many human osteosarcoma specimens and cell lines and STAT3 is required for osteosarcoma cells survival. So IL-6 and IL-11 present a viable novel target for osteosarcoma therapy. To date, however, no small molecules that target both IL-6/GP130 and IL-11/GP130 signaling pathways are available for cancer therapy. To overcome this critical problem, we have utilized a novel drug discovery approach combining Multiple Ligand Simultaneous Docking and drug repositioning to target GP130. Drug repositioning refers to reuse fragments from the existing FDA-approved drugs for new applications and could potentially reposition the existing drugs as novel, off-target inhibitors of the GP130 as their new target. Using this novel method, we have identified a FDA-approved drug Bazedoxifene with a novel function to inhibit IL-6 and GP130 proteinprotein interactions. Furthmore, because IL-11 binds to the same domain of GP130 as IL-6, Bazedoxifene should also inhibit IL-11/GP130 signaling. Accordingly, Bazedoxifene can indeed inhibit the induction of P-STAT3 by both IL-6 and IL-11. Bazedoxifene appeared specific to IL-6 and IL-11 as phosphorylation of STAT1 and STAT3 by IFN-γ and LIF respectively were not affected by the compound. Bazedoxifene inhibited P-STAT3 and induced apoptosis in human osteosarcoma cell lines expressing IL-6 and IL-11. In addition, Bazedoxifene can inhibit colony formation after the drug treatments in cancer cells.IL-6 but not IFN-γ could rescue the Bazedoxifene-mediated inhibition of cell viability in osteosarcoma cells. These results further support that IL-6/GP130 signaling pathway is one of the main targets of Bazedoxifene-mediated inhibition in osteosarcoma cells. To determine the in vivo activity of Bazedoxifene, we further tested the efficacy of Bazedoxifene in tumor xenografts generated from the SJSA osteosarcoma cells that show persistent IL-6/STAT3 activation. Bazedoxifene via oral gavage inhibited P-STAT3 and the growth of SJSA tumor xenografts. These data demonstrated that Bazedoxifene is potent in suppressing tumor growth and is orally bioavailable in inhibiting P-STAT3. It further indicates that Bazedoxifene is a promising IL-6/GP130-targeting drug, which likely to have in vivo anti-tumor activity in osteosarcoma. In summary, Bazedoxifene already approved for safety by the FDA as a novel inhibitor of IL-6/GP130 and IL-11/GP130 signaling should provide an easier path to clinical trials and have a potential to improve the outcome of osteosarcoma.

#187

Efficient delivery of Bcl2 siRNA by DNA nanoparticles to inhibit cellular growth and cancer progression.

Mohammad Aminur Rahman,1 Pengfei Wang,2 Dongsheng Wang,1 Selwyn J. Hurwitz,2 Zhengjia Chen,2 Zhuo G. Chen,1 Yonggang Ke,2 Dong M. Shin1. 1 _Emory Univ. Winship Cancer Inst., Atlanta, GA;_ 2 _Emory University, Atlanta, GA_.

Background: Short interfering RNA (siRNA) has emerged as a promising molecular therapeutic tool for targeted cancer treatment. However, systemically administered siRNA has demonstrated only limited success, due to limited delivery to cancer cells. Therefore, the lack of a robust and versatile siRNA delivery system is a critical issue in translating this therapeutic tool for cancer treatment. Recent developments in DNA nanotechnology have made programmable DNA nanoparticles (DNPs) a potent drug delivery platform. This study focuses on the development of a novel DNP-based siRNA delivery system to knockdown Bcl2 gene, as a targeted cancer therapeutic. Methods: Structural DNA technology was applied to design a library of DNPs with different sizes and shapes. Flow cytometry, confocal imaging, and electron microscopy were utilized to study the cellular internalization of DNPs. The efficacy of Bcl2 knockdown by DNP-siBcl2 and the resulting influence on cell growth and progression were assessed in cancer cells (in vitro) and in mice bearing corresponding tumor xenografts (in vivo). Results: DNPs of varying sizes (10-120 nm) and shapes (polyhedral or rod) were constructed using DNA origami techniques and successfully verified by agarose gel electrophoresis, atomic force microscopy, and transmission electron microscopy. The cell internalization capabilities of three DNPs including a tetrahedron (TET, diameter: 10 nm), a small rod (SR, 5x5x30 nm), and a large rod (BR, 10x10x120 nm) were examined. BR internalized with relatively higher efficiency and rate compared to TET and SR, and also demonstrated the most efficient knockdown of Bcl2. BR-siBcl2 demonstrated significant cell growth inhibition of DMS53 and H146 small cell lung cancer (SCLC) cell lines after 48hrs of treatment. A pilot study with BR-siBcl2 (1.25 mg/kg, iv) in mice bearing DMS53 tumor xenograft (n=3) slowed tumor growth compared with buffer control and naked siBcl2. Significant differences were observed in tumor volume by pairwise comparison between the two groups: Buffer vs DNP-BR-siBcl2 (p=0.007), and siBcl2 vs DNP-BR-siBcl2 (0.028), respectively. No toxicity was observed in lung, liver, kidney, heart, brain, or spleen. Conclusions: Our novel DNP formulations demonstrated substantial cellular internalization of siBcl2. Targeting Bcl2 and its downstream signaling intermediaries reduced cellular growth. We validated the strategy of silencing of Bcl2 using DNPs in order to inhibit cancer progression in vivo. We believe that the DNPs and methodologies developed in this project will be applicable to knockdown of Bcl2 and other gene targets and may be applicable to future anti-cancer therapy. (This work is supported by NIH grant R21EB022828-01).

#188

Preventive effect of aerosolized bexarotene in three major subtypes of lung cancer: adenocarcinoma, squamous cell carcinoma and small cell lung cancer in mice.

Qi Zhang,1 Jing Pan,1 Marker S. Miller,2 Ronald A. Lubet,2 Yian Wang,1 Ming You1. 1 _Medical College of Wisconsin, Milwaukee, WI;_ 2 _National Cancer Institute, Rockville, MD_.

Lung cancer is the leading cause of cancer-related deaths in the United States. The 5 year survival rate for lung cancer patients has remained a dismal 15% for 3 decades. Thus, the development of chemopreventive agents that could prevent lung cancer could potentially reduce the incidence and mortality of pulmonary neoplasms. Bexarotene has exhibited inhibitory effects in preclinical in vivo models of mammary and lung tumorigenesis, has been approved for clinical use in the treatment of cutaneous T-cell lymphoma, and has shown efficacy in phase I/II trials of non-small cell lung cancer (NSCLC). Preclinical studies have demonstrated that it is highly effective in the prevention of all three major subtypes of lung cancer in mouse models: adenocarcinoma (AD), squamous cell carcinoma (SCC), and small cell lung cancer (SCLC). The major side effects of bexarotene when administered orally to rodents or human patients have been hypertriglyceridemia and hypercholesterolemia. Previous studies in a mouse model of lung AD have demonstrated that aerosol delivery of bexarotene through nasal inhalation exhibited potent chemopreventive activity similar to that observed following oral administration (59 to74% reductions in lung tumor multiplicity). The significant decreases in tumor multiplicity and tumor load were achieved without hypertriglycerides that accompany oral bexarotene administration. In this study, aerosolized delivery of 10-30 mg/ml bexarotene showed a significant chemopreventive effect in all three major subtypes. In the N-nitroso-tris-chloroethylurea (NTCU) induced mouse SCC model, 1 week after the first dose of NTCU a dose dependent decrease in tumor formation was observed, with the highest dose causing 75% (p<0.001) and 42% (p<0.01) decreases in SCC tumor burden and the percentage of SCC tumors, respectively. Tumor load was decreased by 73% in A/Jp53 mouse AD model and by 41% in A/Jp53Rb mouse SCLC model. Aerosol delivery of bexarotene had no effect on animal body weight and other signs of toxicity, and no effect on triglyceride and cholesterol level. Aerosolized bexarotene formulation was effective against all 3 major lung cancer cell types and would be a major advance achieving significant reductions in preventing lung cancer incidence in persons at high risk of lung cancer e.g. former or present smokers.

#189

**Development of peptidomimetic inhibitors of the** ERG **gene fusion product in prostate cancer.**

Xiaoju Wang. _The University of Michigan, Ann Arbor, MI_.

Transcription factors play a key role in the development of diverse cancers, and therapeutically targeting them has remained a challenge. In prostate cancer, the gene encoding the transcription factor ERG is recurrently rearranged and plays a critical role in prostate oncogenesis. Here, we identified a series of peptides that interact specifically with the DNA binding domain of ERG. ERG inhibitory peptides and derived peptidomimetics (EIPs) bound with high affinity and specificity leading to proteolytic degradation of ERG protein. The EIPs attenuated ERG-mediated transcription, chromatin recruitment, protein-protein interactions, cell invasion, and tumor growth. Thus, peptidomimetic targeting of transcription factor fusion products may provide a promising therapeutic strategy for prostate cancer as well as other cancers.

#190

**Identification** **of** **compound isolated from** Beilschmiedia tsangii **as a liver cancer specific NRF2 inhibitor.**

Yi Siao Chen, Chih Chung Lai, Yi Ping Kuo, Hsun Shuo Chang, Ih Sheng Chen, Chia-Hung Yen. _Kaohsiung Medical Univ., Kaohsiung City, Taiwan_.

Drug resistance is the main cause of cancer recurrence and a major obstacle to the success of anticancer therapy. NRF2, a pivotal transcription factor regulates antioxidant response and detoxification, has been shown to participate in the development of cancer drug resistance. Functional suppression of NRF2 rendered cancer cell more susceptible to anticancer treatments. Beilschmiedia tsangii Merr. (Lauraceae) is a medium-sized evergreen tree. It has been reported that Beilschmiedia extract showed a strong antioxidant activity. In continuation of our bioactivity studies on this species, we tested the effects of 23 compounds isolated from the B. tsangii on NRF2 activity. We identified rel-(7R,8R,7′R,8′R)-3,4,3′,4′-dimethylene-dioxy-5,5′-dimethoxy-7,7′-epoxylignan (BT04) significantly inhibited NRF2 activity in liver cancer cell (Huh7) with an IC50 value of 17 μM, but not in keratinocyte (HacaT cell). By contrast, luteolin, a known NRF2 inhibitor, suppressed NRF2 activity in both Huh7 cell and HacaT cell. Moreover, the mRNA level of NRF2 target genes, NQO1 and HO1, were significantly decreased in Huh7 upon BT04 treatment, while those NRF2 target genes remained unchanged in BT04-treated HacaT cell. A moderate cytotoxic effect of BT04 on Huh7 cell was also observed. Accordingly, our result suggested that BT04 can specifically inhibit NRF2 activity in liver cancer, which in turn indicated that BT04 could be a potential adjuvant to improve chemoresistance.

#191

Double blockade of interacting CK2 and EGFR pathways by tumor-targeting nanobioconjugates increases therapeutic efficacy against glioblastoma multiforme.

Julia Y. Ljubimova. _Cedars-Sinai Medical Center, Los Angeles, CA_.

Introduction: Glioblastoma multiforme (GBM) remains the deadliest brain tumor in adults, and is notorious for drug and radiation resistance. To inhibit GBMs more effectively, polymalic acid-based blood-brain barrier crossing nanobioconjugates were synthesized that are delivered to the cytoplasm of cancer cells and specifically inhibit the master regulator serine/threonine protein kinase CK2 and the wild-type/mutated epidermal growth factor receptor (EGFR/EGFRvIII), which are overexpressed in gliomas according to The Cancer Genome Atlas (TCGA) GBM database.

Methods and Results: The used nanobioconjugates are novel nanotherapeutics where all moieties are covalently connected to poly(β-L-malic acid) (PMLA). Our biodegradable and non-toxic nanodrugs bind to the receptors enriched on tumor vasculature and cross the BBB by transcytosis. They specifically bind to cancer cells and after internalization exit to the tumor cell cytoplasm using pH-sensitive endosomal disruption unit. Two xenogeneic mouse models bearing intracranial human GBMs from cell lines LN229 and U87MG that expressed both CK2 and EGFR were used. The knockdown of CK2α and EGFR/EGFRvIII suppressed their downstream prosurvival signaling. Treatment also markedly reduced the expression of programmed death-ligand 1 (PD-L1), a negative regulator of cytotoxic lymphocytes. Downregulation of CK2 and EGFR also caused suppression of heat shock protein 90 (Hsp90) co-chaperone Cdc37, which may inhibit the activity of key cellular kinases. Inhibition of either target was associated with downregulation of the other target as well, which may underlie efficacy of the dual nanobioconjugate that is directed against both CK2 and EGFR. Importantly, the single nanodrugs, and especially the dual nanodrug, markedly suppressed the expression of cancer stem cell markers c-Myc, CD133, and nestin, which could contribute to the efficacy of these nanodrugs. In both tumor models, the dually targeting nanobioconjugate significantly increased (up to 2-fold) animal survival compared with the control group.

Conclusion: The versatile nanobioconjugates developed in this study, with the ability of anti-cancer drug delivery across biobarriers and inhibition of key tumor regulators, offer a promising nanotherapeutic approach to treat GBMs and to potentially prevent drug resistance and retard the brain tumor recurrence.

Support: NIH grants U01 CA151815, R01 CA136841, R01 CA188743, R01 CA209921, R01 EY013431

#192

Targeting stearoyl CoA desaturase 1 (SCD1) in hepatobilliary carcinoma.

John Alton Copland,1 Laura A. Marlow,1 Ilah Bok,1 James L. Miller,1 Matsuda Akiko,1 Yan W. Asmann,1 Vivekananda Sarangi,2 Steven R. Alberts,2 Kabir Mody,1 Lewis R. Roberts,2 Mark J. Truty,2 Tushar C. Patel1. 1 _Mayo Clinic Florida, Jacksonville, FL;_ 2 _Mayo Clinic, Rochester, MN_.

Background: The paucity of effective therapeutic agents for hepatocellular cancer (HCC) underscores the critical need for more effective therapeutic strategies. Recent studies indicate lipid biosynthesis and desaturation is required for HCC survival. Targeting these may prove beneficial because such changes contribute to therapeutic resistance. Stearoyl CoA desaturase (SCD1), a key mediator of fatty acid (FA) biosynthesis and rate-limiting in conversion of saturated fatty acids (SFAs) to mono-unsaturated fatty acids (MUFAs), is upregulated in HCC and many other cancers. As such, we therapeutically targeted a novel lipogenic tumor survival mechanism mediated by SCD1 as a means to combat the chemoresistance associated with HCC. In so doing, we evaluated a novel lead SCD1 inhibitor in HCC.

Methods: Paraffin embedded patient HCC tissues were examined for SCD1 expression. Using combined computational and synthetic chemistry approaches, we synthesized four novel specific SCD1 inhibitors with SSI-4 being the lead SCD1 inhibitor. HCC cell lines were examined using proliferation assays for response to SSI-4. IC50 concentrations for blocking SCD1 enzyme activity was determined. Blood half-life and bioavailability of single dose SSI-4 was determined. Mechanisms of action of SCD1 were examined that included Endoplasmic reticulum (ER) stress. In vivo, antitumor activity was determined using HCC patient derived xenograft (PDX) mouse models.

Results: We identified elevated SCD1 mRNA and protein in HCCs tissues. SSI-4 dose-dependently inhibits cell proliferation in HCC cell lines with specificity demonstrated by oleic acid (MUFA) co-culture. Single dose oral gavage SSI-4 demonstrated a half-life of ~4 hours and excellent oral bioavailability. SSI-4 was well tolerated with long-term daily dosing. SSI-4 treatment of HCC cells and tumors led to endoplasmic reticulum (ER) stress followed by apoptotic cell death. Single agent SSI-4 demonstrated antitumor activity in HCC PDX mouse models with suppression of ER stress regulated proteins.

Conclusions: Targeting a novel lipid metabolic pathway in HCC may provide effective therapy for aggressive HCC.

#193

Inhibitors of MPS1: Discovery of CCT289346, a highly potent, selective and orally available preclinical candidate.

Hannah L. Woodward, Paolo Innocenti, Kwai-Ming J. Cheung, Sébastien Naud, Angela Hayes, Alan T. Henley, Amir Faisal, Grace Mak, Gary Box, Isaac M. Westwood, Michael Carter, Melanie Valenti, Alexis De Haven Brandon, Lisa O'Fee, Harry Saville, Rosemary Burke, Rob van Montfort, Florence Raynaud, Suzanne A. Eccles, Spiros Linardopoulos, Julian Blagg, Swen Hoelder. _Institute of Cancer Research, Sutton, United Kingdom_.

MPS1 (also known as TTK), is a dual-specificity protein kinase and one of the main components of the spindle assembly checkpoint. Cancer cells heavily rely on MPS1 to cope with aneuploidy resulting from aberrant numbers of chromosomes and MPS1 has been found to be upregulated in a large number of tumor types. Extensive work by us and other groups has shown that MPS1 inhibitors are effective against a variety of cancers, particularly when used in combination with other drugs, for example, tubulin-targeting agents. We recently reported the structure-based design and discovery of a series of pyrido[3,4-d]pyrimidines inhibitors of MPS1 (1). Advanced compounds showed very potent inhibition of MPS1 in biochemical and cellular assays. However, these compounds suffered from high lipophilicity and pronounced metabolism in human liver microsomes preventing progression into preclinical development. Here we report the optimisation of this series ultimately yielding CCT289346, our preclinical candidate. CCT289346 shows excellent potency, kinase selectivity, and ADME properties including stability in human liver microsomes. The compound has been produced on a kilogram scale and is currently undergoing preclinical development. We will discuss our design approach and hypotheses leading to the discovery of CCT289346 and disclose in vivo efficacy data. References 1. Innocenti P et al. Rapid Discovery of Pyrido[3,4-d]pyrimidine Inhibitors of Monopolar Spindle Kinase 1 (MPS1) Using a Structure-Based Hybridization Approach. Journal of Medicinal Chemistry. 2016; 59(8):3671-88.

#194

Effective targeting of MYC expression with a novel nucleic acid binding (G4-quadruplex) small molecule coupled with HDAC inhibition synergizes to limit myeloma growth.

Snehal M. Gaikwad,1 David R. Calabrese,2 Elena C. Leon,1 John K. Simmons,1 Shuling Zhang,1 Aleksandra Michalowski,1 Sayeh Gorjifard,1 Zaw Phyo,1 Daniel Connors,1 John S. Schneekloth,2 Beverly A. Mock1. 1 _CCR, NCI, NIH, Bethesda, MD;_ 2 _CCR, NCI, NIH, Frederick, MD_.

MYC is deregulated in many malignancies and its aberrant expression is associated with tumorigenesis and tumor maintenance. We employed a therapeutic strategy to target both MYC transcription, and protein stability. Previously, we developed a small molecule microarray and screened it for MYC G4-binding compounds. The stabilization of G-quadraplex (G4) structures by nucleic acid binding small molecules in the NHE III region of the MYC promoter slows MYC transcription. We identified a benzofuran containing small molecule that could selectively and reversibly stabilize MYC-G4 DNA and reduce MYC expression. This compound demonstrated a G4-dependent mechanism of action showing toxicity in multiple myeloma cell lines containing a G4 in their MYC promoter whereas minimal effects were seen in a Burkitt's lymphoma line lacking the MYC G4 sequence. Gene expression analysis validated that this compound selectively inhibited MYC and other MYC effectors, but did little to limit the expression of other G4 containing genes. The synthesis of more than 15 analogs identified a new benzofuran-containing analog with enhanced inhibitory activity in a diverse panel of human cancer cell lines (NCI-60), including a number of myeloma cell lines. EDGE-seq data confirmed that the new analog is similar to the original compound in having higher affinity for the G4 in MYC compared to the G4 in BCL2, KRAS, VEGFA and HIF1A. In addition, the analog was synergistic with entinostat, an HDAC inhibitor that can affect MYC protein stability, in limiting myeloma cells, but not PBMCs. The more potent growth inhibitory activity of the new analog allowed us to generate sufficient quantities to evaluate its pharmacokinetics and toxicity in mice. The compound was well tolerated in a dose-escalation toxicity study; no adverse effects were observed and preliminary pharmacokinetic studies showed promising serum bioavailability and exposure properties when administered either intravenously, intraperitoneally or orally. In an assessment of short term in vivo activity, MYC protein expression was inhibited in multiple myeloma xenografts. Longer term studies to evaluate the anti-tumor activity of the compound are currently in progress. Thus, our data provide evidence that small molecule stabilization of the MYC G4 can drive transcriptional silencing of oncogenic MYC both in vitro and in vivo. Furthermore, synergistic tumor cell killing was achieved when the MYC G4 inhibitor was coupled with HDAC inhibition.

#195

Evaluation of the cell surface binding of phycocyanin and associated mechanisms causing cell death in prostate cancer cells.

Paramjot Kaur,1 Sivanesan Dhandayuthapani,1 Shona Joseph,2 Syed Hussain,2 Miroslav Gantar,3 Appu Rathinavelu1. 1 _Rumbaugh Goodwin Institute for Cancer Research, Nova Southeastern University, FL;_ 2 _Halmos College of Natural Sciences and Oceanography, Nova Southeastern University, Fort Lauderdale, FL;_ 3 _Florida International University, Miami, FL_.

Plant and microbial metabolites are constantly explored to identify novel therapeutics with promising potential for treating diseases such as cancer. Marine-derived compounds are also tapped frequently for their efficacy in treating cancers and are used both in mono and combination therapies. The potential of cyanobacteria (blue-green algae) as the source of anticancer agents has been explored for many decades and, as a result of continuing efforts, several compounds have emerged as templates for the development of new anticancer drugs from these microorganisms. The C-phycocyanin (C-PC) tested in our experiments was derived from the cyanobacteria Limnothrix sp., strain 37-2-1, which is found abundantly in Florida's Everglades. In our previous studies, this fluorescent compound was found to potentiate the cytotoxic effects of Taxol and Topotecan. In LNCaP prostate cancer cells, C-PC was able to induce apoptosis by itself through activation of the apoptotic pathway and completing DNA fragmentation. It was originally anticipated that C-PC might penetrate the cell membrane to induce mitochondrial damage and trigger the apoptotic process through activations of caspases. In order to verify this possibility, experiments were conducted to determine the uptake of C-PC using LNCaP cells. When we incubated the cells with C-PC at the concentrations of 250 and 500 µg/mL for 12 hrs and assessed the cellular uptake by capturing the fluorescence signals at the wavelength of 605 nm, using a fluorescence microscope, the signals were observed only with the periphery of the cells. Furthermore, with subsequent washes using PBS, the fluorescence signal that was seen to be associated with the plasma membrane was reduced gradually and was removed completely after the 3rd wash without any signs of penetration into the cytoplasm. The binding of C-PC to the plasma membrane was very transient and quickly dissociated at room temperature. However, after incubating the cells with the same concentrations of C-PC, there was up-regulation of proapoptotic markers such as p53 and p21 and as a result, the cells showed a significant reduction (>65%) in viability. So far, our results suggest that the cytotoxicity towards LNCaP cells might have been triggered by the binding of C-PC to the cell membrane receptors such as FasR, TRAIL-R, TNF-R, which may be linked to the mediators of extrinsic and intrinsic apoptotic signals. Hence, our findings are significant for explaining some of the apoptotic events triggered by C-PC. However, additional studies are required to identify the actual receptors involved in triggering the pathway. (The support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida is gratefully acknowledged).

#196

Identification of small molecule inhibitors of the notch transcriptional activation complex.

Luisana Astudillo, Anthony Capobianco. _Univ. of Miami Miller School of Medicine, Miami, FL_.

Deregulation of the Notch pathway underlies many aspects of cancer physiology depending on cell type and context. In many human cancers, aberrant Notch activity has been demonstrated to play a role in the initiation and maintenance of the neoplastic phenotype. Notch also plays a central role in cancer stem cells, which may underlie a role in metastasis and resistance to therapy. The important and diverse role played by Notch in cancer makes it an exceedingly attractive target for anti-neoplastic therapeutics. However, the full range of potential targets in the pathway have been under-explored. Through computer-aided drug design we explored potential ligand binding sites and screened for compounds that could disrupt the assembly of the Notch transcriptional activation complex. An in vitro assay that quantitatively measures the assembly of the Notch transcriptional complex on DNA was used for screening. We have recently reported the identification and characterization of a small molecule inhibitor of the Notch transcriptional activation complex, termed Inhibitor of Mastermind Recruitment-1 (IMR-1). Herein we report the characterization of small molecule inhibitors of Notch derived from IMR-1 that were identified through a combination of structure-activity relationship studies and molecular docking simulations. We demonstrate that these compounds inhibit the growth of Notch dependent cell lines and decrease Notch target gene transcription.

#197

**Anti-cancer activity of extract from the Jamaican round-leaf yellow yam (RLY** 2 **) (Dioscorea cayenensis).**

Sasha-Gay A. Wright,1 Wesley G. Gray,2 Helen Asemota1. 1 _University of the West Indies, Mona Campus, Kingston, Jamaica;_ 2 _Southern University and A &M College, Baton Rouge, LA_.

The Jamaican Round-Leaf yellow yams (RLY2), Dioscorea cayenensis, are edible tubers, bulbils or rhizomes that are of considerable economic value and possess several health benefits. These RLY2 are a rich source of anti-oxidants, vitamins and phytochemicals. In addition to its nutritional value, the Jamaican populace regards RLY2 as an herbal medicine useful in treatment of diabetes mellitus, hypertension and certain kind of cancers. Chemical analysis of RLY2 revealed the presence of a myriad of bioactive ingredients that are likely to be lost due to the method of preparation for consumption. Thus, we hypothesized that mild extraction of RLY2 will preserved the bioactive analytes that give RLY2 its anti-cancer property. To test this hypothesis, different ethanolic extracts of RLY2 were prepared, and their bioactivity demonstrated in two models of prostate cancer. The present study demonstrates dosage and time-dependence inhibition in both the androgen sensitive LNCaP and androgen insensitive DU-145 prostate cancer cell lines. We observed that DU-145 was three-times more sensitive to RLY2 ethanol extract than LNCaP. Growth of LNCaP for 3-days with varying concentration of RLY2 ethanol extract resulted in an IC50 of 750 ppm (95% C.I. 726-791ppm), whereas DU-145 resulted in an IC 50 of 250 ppm (95% C.I. 236-281ppm). The maximum growth inhibition by RLY2 extract occurs within twenty-four hours for both LNCaP and DU-145 with no significant changes after 5-days. To determine the mechanism of toxicity by RLY2, we induced both LNCaP and DU-145 with RLY2 for 0-24h and prepared cells for flow cytometer analysis. The effect of RLY2 on cell cycle and degree of apoptosis will be discussed. Our data indicated that the ethanol extract of RLY2 was more sensitive toward androgen-insensitive prostate cancer. Thus, this study suggests that RLY2 contain bioactive chemical that may be used in grade II prostate adenocarcinoma represented by the DU-145 phenotype.

#198

Low molecular weight pyrrolobenzodiazepine (PBD) monomers have potent cytotoxicity in haematological tumor cells.

David B. Corcoran,1 Thomas Lewis,2 Amrit Varsha,1 Chris Pepper,2 David E. Thurston,1 Khondaker Miraz Rahman1. 1 _King's College London, London, United Kingdom;_ 2 _University of Cardiff, Cardiff, United Kingdom_.

The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) have long been of interest as potential chemotherapeutic agents due to their ability to form covalent adducts within the minor groove of the DNA helix. The most effective synthetic modifications to PBD cores have involved the conjugation of two DNA-interactive moieties via their C8/C8'-positions to create PBD dimers capable of cross-linking duplex DNA which improves cytotoxicity. Research to date has focussed on adding substituents to the C8-position of the PBD core to improve DNA-interaction and cytotoxicity. All of these approaches tend to increase the molecular weight of the compounds, although this has not prevented their successful development to the clinic, either as stand-alone agents (e.g., SJG-136) or as a component of Antibody-Drug Conjugates (ADCs) (e.g., SGN-CD33A). During a structure-activity relationship (SAR) study, we embarked on a "molecular pruning" exercise to sequentially reduce the length and bulk of the C8-substituent of a PBD monomer expecting the cytotoxicity to reduce with a decrease in length/bulk, and to establish the minimal pharmacophore. Surprisingly, we found that reducing the length of the C8-substituent maintained cytotoxicity and in some cases enhanced it. A 20-member library of short C8-substituted PBD monomers has been synthesized featuring C8-substituents of various chemical composition and length, with some containing aniline substituents. Cytotoxicity evaluation in several tumour cell types (e.g., primary CLL, JJN-3 and MDA-MB-231) was carried out, and low nanomolar to high picomolar IC50 values were obtained for several library members including DC-1-194 (IC50 = 4.2nM in CLL, and 0.79nM in MDA MB 231), DC-1-255 (IC50 = 9.5nM in CLL, and 1.1nM in MDA MB 231), DC-1-253 (IC50 = 8.4nM in CLL, and 10nM in JJN-3) and DC-1-275 (IC50 = 7.6nM in CLL, and 9.6nM in JJN-3). Remarkably, many of these more-active compounds had very short C8-substituents. HPLC and FRET based DNA binding studies were also carried out, in order to confirm the capacity of these molecules to form adducts with DNA. The observations reported here are significant, as they add to an evolving understanding of the SAR of PBD monomer structures. The results presented here may also have significance for the future development of PBD-based therapeutic agents where there may be advantages to working with lower molecular weight molecules.

#199

Silibinin suppresses bladder cancer through down-regulation of actin cytoskeleton and PI3K/Akt signaling pathways.

Mitsuho Imai-Sumida,1 Takeshi Chiyomaru,2 Shahana Majid,1 Priyanka Kulkarni,1 Pritha Dasgupta,1 Sharanjot Saini,1 Taku Kato,1 Shigekatsu Maekawa,1 Yutaka Hashimoto,1 Marisa Shiina,1 Guoren Deng,1 Varahram Shahryari,1 Hannah Nip,1 Rajvir Dahiya,1 Yuichiro Tanaka,1 Soichiro Yamamura1. 1 _UCSF VA Medical Ctr., San Francisco, CA;_ 2 _National Hospital Organization Kagoshima Medical Center, Kagoshima, Japan_.

Silibinin is the major active constituent of silymarin, an extract of milk thistle seeds. Silibinin has been shown to have significant anti-cancer effects in a variety of malignancies. However, the molecular mechanisms of silibinin action in bladder cancer have not been studied extensively. In the present study, we found that silibinin (10 μM) significantly suppressed proliferation and invasion of T24 and UM-UC-3 human bladder cancer cells. In this study, we investigated the molecular mechanisms underlying these effects of silibinin. Our results showed that silibinin down-regulates actin cytoskeleton-related Ras-Rac-PAK1 pathways and the phosphatidylinositide 3-kinase (PI3K)/Akt signaling pathway in these bladder cancer cell lines. These pathways were found to promote cellular transformation and cancer development and have crosstalk through Ras cascades. We investigated the role of silibinin on Ras, and found that silibinin suppresses levels of trimethylated histone H3 lysine 4 and acethylated H3 at the K-RAS promoter. We also found that silibinin downregulates long non-coding RNAs: HOTAIR and ZFAS1, which are known to play roles as oncogenic lncRNAs in various cancers. This study shows that silibinin exerts anti-cancer effects through down-regulation of the actin cytoskeleton and PI3K/Akt pathways and thus suppresses the development and progression of bladder cancer.

#200

Long lasting inhibition of TGF-β receptor Type 1 inhibitor (TGF-β R1) for treatment of pancreatic cancer.

Cristian Rodriguez-Aguayo,1 Emine Bayraktar,1 Haifa Shen,2 Gabriel Lopez-Berestein1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _The Methodist Hospital Research Institue, Houston, TX_.

Pancreatic cancer stroma is a high component of total tumor volume and activators of fibro-genesis such as Transforming growth factor-β (TGF-β) are abundantly secreted by pancreatic stellate cells (PSCs) to the tumor microenvironment. Aberrant TGF-β signaling pathways are associated with many human diseases, including bone diseases, immune-suppression, fibrosis, cancer progression and metastasis. However, the precise mechanism of this phenomenon in pancreatic cancer remains unclear. In this study we hypothesized that inhibition of TGF-β Type I receptor kinase (TGF-β R1) in pancreatic cancer and stellate cells regulates proliferation and fibrosis through the regulation of TGF-β R1 signaling pathway in PC cells, resulting in decreased migration, and decreased proliferation. We analyzed a TGF-β R1 data set of PC patients from the Cancer Genome Atlas. Furthermore, the analysis of a panel of pancreatic cancer cell lines showed that TGFR-Β1 at mRNA and protein levels were higher in PC cells when compared with human pancreatic duct epithelial (HPDE) normal cells. In vitro cell viability, growth, cell-cycle progression, migration, invasion and apoptosis, as well as in vivo therapeutic effectiveness in murine xenograft models, were also assessed following the inhibition of TGF-β R1 in pancreatic cancer and stellate cells. The experimental validation identifies TGFR-β1 associated with PC survival. Significant inhibition of cell growth, invasion, migration, angiogenesis and fibro-genesis. Furthermore, treatment with LY-DMPC:DMPG-MSV inhibited growth of CAPAN-II tumors. These findings identify TGFR-β1 as a potential therapeutic target in pancreatic cancers expressing high levels of this oncogenic protein.

#201

Targeting colorectal cancer lung metastasis microenvironment with PI3K inhibitors and chemotherapy.

Piotr Rychahou, Younsoo Bae, Derek A. Reichel, Yekaterina Zaytseva, Eun Y. Lee, Heidi L. Weiss, B. Mark Evers. _University of Kentucky, Lexington, KY_.

Introduction: Colorectal cancer (CRC) is the second leading cause of cancer deaths in the US; systemic metastasis to the lungs occurs in approximately 10-20% of patients with CRC. The toxicity of currently available anticancer drugs and the inefficiency of chemotherapeutic treatments, especially for advanced stages of the CRC metastatic disease, have severely limited the treatment options in patients with CRC lung metastasis. Here, we evaluated nanocarrier that accumulates selectively in normal lung stroma and lung metastasis tumor microenvironment. The purpose of our study was to: (i) demonstrate lung-selective delivery of nanoparticles into the tumor microenvironment and surrounding normal parenchyma as a targeted therapy for CRC lung metastasis, and (ii) evaluate effect of lung selective delivery of PX-866 and SN-38 loaded nanoparticles on CRC lung metastasis growth.

Methods: (1) HT29 LungM3 cell line was derived from the human CRC line HT29 following multiple rounds of in vivo selection for lung metastasis in immunodeficient mice. (2) Polymeric nanoparticles were constructed and loaded with fluorescent dye (Alexa 547), pan-PI3K inhibitor PX-866 or SN-38. Fluorescent nanoparticles were administered intravenously into mice at 1, 4, 8, 24h timepoints. (3) Nanoparticle drug delivery was quantified in lung, liver and kidney tissue samples by HPLC. (4) CT26 and HT29 LungM3 cell lines were injected intravenously to establish lung metastasis. Mice were then treated with PX-866 and SN-38 loaded nanoparticles every 3 days intravenously for 31 days.

Results: Lung selective accumulation of Alexa-546 fluorescently-labeled nanoparticles was confirmed by confocal imaging of frozen tissue sections from lung, liver, kidney and spleen. Selective PI3K inhibition in lung tissue was confirmed by western blot analysis of protein extracts from lung, liver, kidney and spleen for pAkt (S473) expression. HPLC analysis showed predominant PX-866 drug delivery to lungs and minimal drug delivery to kidney and liver tissues. Treatment with PX-866 loaded nanoparticles demonstrated a marked suppression of lung metastasis growth; CRC lung metastases were not detected after treatment with SN-38 loaded nanoparticles. Notably, administration of PX-866 and SN-38 nanoparticles had no deleterious effect on normal lung, kidney or liver parenchyma.

Conclusions: We demonstrate selective and efficient delivery of drug-loaded nanoparticles to lungs with minimal accumulation in kidneys, liver and immune system, suggesting that lung selective drug delivery is a viable treatment strategy for CRC lung metastasis. Moreover, treatment of mice with CRC lung metastases with either PX-866 or SN-38 loaded nanoparticles resulted in a marked suppression of metastatic growth. Polymeric nanocarriers have a potential to revolutionize drug delivery and to significantly improve current approaches to the treatment of patients with CRC lung metastasis.

### Proteomics and Mass Spectrometry 1

#202

Comprehensive spatial mapping by MALDI imaging mass spectrometry of the N-glycan tissue glycome and database generation for eighteen tumor types.

Richard R. Drake, Kacey Talbot, Kim Norris-Caneda, Evelyn Bruner, Peggi M. Angel. _Medical Univ. of South Carolina, Charleston, SC_.

Glycoproteins account for approximately 80% of the proteins located at the cell surface and in the extracellular environment, and serve as binding ligands for cell adhesion, extracellular matrix molecules, signaling receptors, immune cells, lectins and pathogens. Alterations and changes in cell surface glycosylation during tumorigenesis are well documented, including the major N-linked glycan class of glycosylated proteins. We have recently developed a MALDI imaging mass spectrometry (MALDI-IMS) method to spatially profile N-linked glycans in frozen and formalin-fixed paraffin-embedded (FFPE) tissue sections and tissue microarrays (TMAs). In order to facilitate method development and maximize glycome determinations for many cancer types, we have generated two custom TMAs comprised of multiple tissue core pairs of non-tumor and tumor regions representing eighteen different tumor types (pancreatic, colon, prostate, breast, lung, melanoma, sarcoma, head/neck, kidney, liver, glioma, ovarian, thyroid, uterine, cervical, testicular, gastric, bladder). Tissues are incubated with a molecular coating of peptide N-glycosidase, and released N-glycans are detected directly using MALDI-FTICR imaging, linked directly with tissue histopathology. The method is able to simultaneously identify and distinguish 50 or more components of the N-glycome on a single slide. The approach allows comparison of the similarities and differences in N-glycome compositions across each tumor tissue type, as well determine the relative abundance of each individual glycan across all tumor types. For example, an increase in high mannose glycans is common to many tumor types. From the TMA data, this glycan class is consistently elevated for colon, prostate, lung, breast and bladder cancers, while the other tumor types are variable. Differences in branching complexity, bisecting N-acetylglucs0amine, and the degree of fucosylation and sialylation are also readily evaluable. Additionally, data from each of the TMA core pairs is being compared systematically to the N-glycans detected in the source FFPE blocks. The goal is to generate an extensible reference database of expected N-glycans for each tumor tissue type. This will also provide a framework to expand to other classes of glycans, and complementary glycan data generated by other methods on the same TMA cores.

#203

Automated, high throughput mass spectrometry imaging of serial sections for investigation of tumor heterogeneity in three dimensions.

Emrys A. Jones,1 Fiona Henderson,2 David Coope,2 Joanna Denbigh,2 Steven Pringle,1 Adam McMahon,2 Kaye Williams2. 1 _Waters, Wilmslow, United Kingdom;_ 2 _University of Manchester, Manchester, United Kingdom_.

For the purpose of investigating the potential of automation and high throughput mass spectrometry imaging, we have combined a desorption electrospray ionisation (DESI) MS system with a robotic slide loader. The analysis of tissue sections on multiple slides could allow, for example, large patient cohorts to be analysed with almost no input from the operator. Here we present another use of such a system, the mapping and visualisation of tissues in 3D from serial sections.

All experiments were carried out on a Waters Xevo G2-XS Q-ToF equipped with a Prosolia (Indianapolis) 2D DESI stage. The automated slide loader and modified plate holder were provided by Prior Scientific (Cambridge). As an ambient ionisation technique, the DESI sampling procedure occurs outside of the vacuum of the instrument and is therefore suited to accommodate slide handling devices. A modified top plate was created that, once in place, allows the Prior PL200 microscope slide loader to load slides from the holding cassettes to the stage and back. A snapshot of the slide being taken and object definition algorithms is used to identify the location of the tissue(s) on the slide. These co-ordinates are then written to the experiment file and the acquisition commences. This process will continue until all slides are analysed.

Recent technological advances have led to the time taken for a single MS tissue image being reduced from the region of hours to minutes. This then opens up the possibility of automatic sample loading and high throughput usage. Fifteen sections taken at 120µm steps through a human glioma were analysed automatically by the DESI system. Data was collected in both positive and negative ion mode from the same tissue to increase the molecular coverage. Sections taken from between those use for the imaging were used for H&E staining and CA-9, Ki67 and SCD immunohistochemistry staining. Once all sections were analysed, recently developed software allowed for the reconstruction of the sections into a three dimensional representation of the chemically distinct regions within the whole tissue volume. This process which involves peak picking, object alignment and rotation as well as a non negative matrix factorisation step is all done automatically. When these regions identified from the 3D mass spectrometry mapping are compared to the CA-9 and Ki67 IHC stains the regions of hypoxia and proliferation clearly correlate indicating that mass spectrometry imaging could be a useful tool for the determination of hypoxia within excised tissue. Further to these identified regions was the discovery of molecular signals that were only present on the boundary between the hypoxic and normoxic regions; the identities and roles of which are currently under investigation.

Faster and automated mass spectrometry imaging allows whole tissues to be chemically mapped in three dimensions in a few hours providing insights into tumour heterogeneity.

#204

In vitro **metabolomics of mesothelioma: Challenges and outcomes.**

Sabrina Lagniau,1 Kevin Lamote,1 Lore Vandermeersch,1 Herman Van Langenhove,1 Jan P. van Meerbeeck,2 Karim Y. Vermaelen1. 1 _Ghent University, Ghent, Belgium;_ 2 _Antwerp University Hospital, Edegem, Belgium_.

Introduction

Early detection of malignant pleural mesothelioma (MPM) is likely to improve outcome in affected patients. From our previous studies (MesoBreath 1 & 2) we developed a screening tool in which volatile organic compounds (VOCs) in breath allowed discrimination of MPM patients from at risk controls. However it is not yet clear which VOCs arise from the neoplastic cells themselves or from the host response. Identifying the cancer cell-specific compounds should increase the specificity of our breathomic signature model. Therefore, we set up an in vitro experimental setup model for VOC analysis from the headspace of mesothelioma cell lines.

Methods

Standard polystyrene culture flasks (NunclonTM Delta Surface, Thermo Scientific) were mounted with an adapted cap accommodating teflon inlet and outlet tubes. The inlet tube provides environmental air while an external pump (Gilian GilAir Plus, Sensidyne) draws headspace air from the outlet tube over an adsorption column (Tenax GR, Markes International, UK). The adsorption columns were further processed on a GC-MS platform (GC: Focus GC, Thermo Finnigan, Milan, Italy; DSQII Single Quadrupole MS, Thermo Finnigan, Austin, TX, USA). VOC profiles were processed using supervised clustering algorithms and principal component analysis.

Results

Using this setup, we have investigated the impact of flow and total sampled volume on the concentration and range of VOCs detected. For a constant flow, VOCs were detected in higher concentrations with increasing sample volume. For a constant volume, a lower sampling flow yielded a higher concentration of detected VOCs. Most of the VOCs obtained were alkanes and ketones. We will show data illustrating the differential VOC profiles of container plastic, cell culture medium and growing mesothelioma cancer cells, as well as the differential VOC output of epithelioid vs sarcomatoid mesothelioma cell lines.

Conclusions

We have succesfully developed an in vitro experimental setup allowing collection and analysis of VOCs emanating from mesothelioma cancer cell line cultures. This platform will allow us to investigate how VOC profiles are impacted in different experimental conditions (e.g. hypoxia, nutrient deprivation, cytostatics, co-culture with leukocytes). The data collected will help us to pinpoint the cancer cell-derived VOCs from patient exhaled breath, and increase the robustness of our mesothelioma breathomic signature.

#205

Underlying mechanisms of genome-proteome discordance in squamous cell lung cancer.

Paul A. Stewart, Robbert J. Slebos, Eric A. Welsh, Ling Cen, Yonghong Zhang, Zhihua Chen, Chia-Ho Cheng, Fredrik Pettersson, Anders Berglund, Guolin Zhang, Bin Fang, Victoria Izumi, Sean Yoder, Katherine Fellows, Ann Chen, Jamie K. Teer, Steven A. Eschrich, John M. Koomen, Eric B. Haura. _Moffitt Cancer Center, Tampa, FL_.

Introduction: Genomic analyses have yielded a tremendous amount of data on the genetic changes in lung cancers, but translating these experiments into actionable information benefitting lung squamous cell carcinoma (SQLC) patients has proven more difficult. Studies by the NCI Clinical Proteomic Tumor Analysis Consortium (CPTAC), our group, and others have demonstrated that gene and protein expression show only moderate correlation, demonstrating limitations in explaining phenotypic changes from genomics alone. These findings indicate a clear need for integrative proteogenomics to better understand tumor biology, especially in a complex disease like SQLC.

Experimental: We have assembled a comprehensive proteogenomic dataset including DNA copy number (Affymetrix CytoScan HD Assay), targeted exome sequencing (Agilent Comprehensive Cancer Panel), RNA-sequencing (Illumina NextSeq), and shotgun proteomics (Q Exactive LC-MS/MS) on 116 surgically resected SQLC tumor samples with extensive clinical and follow up data.

Results: We have identified 6584 high confidence proteins from preliminary proteomic analysis. After quality control filtering, we utilized 5562 gene-protein pairs for further analysis. Clustering of patient RNA expression in this patient cohort has been unable to fully reproduce the molecular classification previously published for SQLC. Furthermore, proteomic results indicate yet another potential classification strategy selecting patient subgroups that differ at protein level. We observed a 0.29 median Spearman's correlation of 5562 gene-protein pairs. There were 2781 highly correlated gene-protein pairs (greater than median) and 2781 poorly correlated gene-protein pairs (less than median) including 773 anti-correlated gene-protein pairs (less than 0). We hypothesized that poorly correlated gene-protein pairs could be functionally related in a pathway-dependent manner. Enrichment analysis of poorly correlated proteins identified pathways related to mRNA processing, growth factor signaling (EGFR, FGFR), and nonsense-mediated decay (NMD). Interestingly, there were 9 frequently mutated SQLC genes in the low correlation gene-protein pairs but only 3 in the highly correlated pairs. We found three distinct patient subgroups by clustering poorly correlated proteins. Analysis of these subgroups showed differentially expressed pathways related to mRNA processing, ubiquitination, and NMD.

Conclusion: Differential modulation of the proteome outside of genomic regulation may suggest important regulatory mechanisms in cancer and give new insights into treating SQLC. Analysis of poorly correlated gene-protein pairs suggests certain pathways are dysregulated in cancer, and ongoing DNA analysis and future analyses involving miRNAs, RNA-binding proteins, and the ubiquitin proteome system will help elucidate our preliminary findings.

#206

Proteogenomic classifications and outcome in squamous cell carcinoma of the lung.

Robbert J.C. Slebos, Paul A. Stewart, Eric A. Welsh, Ling Cen, Yonghong Zhang, Zhihua Chen, Chia-Ho Cheng, Fredrik Pettersson, Anders E. Berglund, Guolin Zhang, Bin Fang, Victoria Izumi, Sean J. Yoder, Katherine M. Fellows, Y Ann Chen, Jamie Teer, Steven Eschrich, John Koomen, Eric Haura. _Moffitt Cancer Center, Tampa, FL_.

Introduction: Genomic analyses have yielded a tremendous amount of knowledge on the genetic changes in lung cancers, but translating this information into actionable data benefitting patients has proven difficult. The integration of proteomic analyses with genomics and gene expression profiling allows a more detailed description of the biological processes, thus improving our understanding of cancer phenotypes. These insights can potentially be used for better classification and help to guide patient selection for targeted therapies.

Experimental: We analyzed 116 surgically resected squamous cell lung carcinoma samples for copy-number alterations, gene expression profiling, targeted exome-sequencing and global proteomic profiling. The cohort consisted of mostly early stage tumors (83% Stage I or II) with complete follow-up (median 58 months). Copy number status was analyzed using the Affymetrix CytoScan array, DNA mutation status was assessed using a customized version of the Agilent Comprehensive Cancer Panel for targeted sequencing, and gene expression profiling was carried out by RNA-sequencing. Proteomic analysis was performed using TMT labeling, 12- fraction bRPLC separation and LC-MS/MS analysis with a Thermo Q-Exactive mass spectrometer. Database searches were performed using multiple search engines against RefSeq version 78, and summarized using IDPicker 3.

Results: The non-redundant protein inventory consisted of more than 6,000 protein groups with a protein FDR <5%. Tumors were classified according to well established gene expression criteria into 4 classes: Classical, Basal, Primitive and Secretory. At the protein level, the Classical subtype was associated with xenobiotic and energy metabolism; the Basal subtype with defense responses and extracellular matrix changes; the Primitive subtype with nucleic acid metabolism; and the Secretory subtype with p38 signaling. These findings confirm and expand on previous mRNA expression studies of squamous cell lung carcinoma. Proteomics-based classification identified two sub-categories within the Classical subtype, which were characterized by inflammatory and stress response signaling. Within this group, patients with high expression of inflammation-associated proteins had better cancer-specific survival than those with low expression (p=0.04, Log-rank test). Targeted exome sequencing of 154 cancer-associated genes revealed frequent mutations in TP53, CDKN2A, NFE2L2, and other genes. Proteomic expression of genes located in amplified chromosomal regions was used to identify driver genes in squamous cell lung carcinoma.

Conclusion: Our results provide new biological insights from the addition of protein measurements to genomic datasets that have the potential to improve classification. The data suggest that proteins involved in immune responses are important for the biological behavior and outcome of the Classical subtype in squamous cell lung carcinoma.

#207

Reliable identification of mutations in bottom-up proteomics.

Miroslav Hruska, Lakshman Varanasi, Jiri Voller, Petr Dzubak, Marian Hajduch. _Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital, Olomouc, Czech Republic_.

Knowledge of missense mutations is of significant biological importance as it provides valuable insights into alterations of phenotype. While identification of reference proteins is routinely performed in proteomics, identification of mutations remains still fairly uncommon. Well-established methods for reference peptides are generally insufficient for altered ones; e.g., utilizing mutant-augmented database search usually results in inadmissible proportion of false positives. This undesirable situation is, however, rather natural consequence of peptide fragmentation, its computational modelling and evaluation of their correspondence. Often, there are many peptides having the same or similar agreement with acquired spectrum, thereby preventing agreement-based decision. These situations happen frequently in identification of mutant peptides (e.g., homologous peptides with PTMs, semi-specific peptides), therefore their reliable identification requires additional treatment.

To deeper understand properties of identification, we have formally studied generalized version of the identification problem and derived its optimal solution under particular assumptions. In proteomics, the selection of optimal solution can be always found in finite time; moreover, the strategy is straightforward to implement, enables parallelization and provides guarantees over claimed interpretations. The solution utilizes almost exclusively spectral data of product ions without additional LC/MS information. In practice, however, it is beneficial to employ precursor isotopic distribution analysis for correction of non-monoisotopic selection of precursor as that would otherwise systematically result in artefacts.

The behaviour of proposed method was validated in a variety of scenarios that could be broadly categorized as direct (known spectral content) and indirect (partial knowledge of sample content). For direct validation, synthetic combinatorial peptide library of 400 peptides (all coded amino acids in 12. and 13. codon of KRAS peptide) was measured and interpreted; the sensitivity and specificity obtained was 0.81 and 0.99 respectively. Indirect validation was, at first, performed on in-house samples with RNA-Seq data available (HCT116), giving 97 missense RNA-Seq supported mutations. Similarly, the analysis was done on proteome data of NCI60 cell lines, resulting on average in 23 SNVs with maximum of 56 SNVs (HCC-2998). Identified alterations correspond, in general, to highly expressed genes. In summary, the approach was shown to reliably identify missense mutations in a range of cancer cell lines; its wide applicability enhances interpretation of standard bottom-up proteomics data.

#208

Proteomic measurements of protein abundance and phosphorylation identify novel kinase-substrate relationships in ovarian cancer.

Jason E. McDermott,1 Tao Liu,1 Samuel Payne,1 Vladislav Petyuk,1 Hui Zhang,2 Zhen Zhang,2 Daniel Chan,2 Richard Smith,1 Karin Rodland,1 Clinical Proteomic Tumor Analysis Consortium. 1 _Pacific Northwest National Laboratory, Richland, WA;_ 2 _Johns Hopkins Medical Insitutions, Baltimore, MD_.

As part of the Clinical Proteomic Tumor Analysis Consortium (CPTAC), we have recently published the first large-scale proteomic and phosphoproteomic analysis of high-grade serous ovarian tumors. We observed that phosphorylation status was an excellent indicator of pathway activity and could discriminate between patient survival times. This dataset covers tumor samples from 69 patients with deep phosphoproteomic depth (>20,000 phosphopeptides confidently identified). Our continuing analysis of this dataset, reported here, has revealed that the correlation between kinase protein abundance and abundance of phosphorylated target peptides is very low, indicating that kinase abundance is not a good proxy for phosphorylation status overall. However, highly correlated kinase-substrate pairs are significantly more likely to be true relationships (from existing knowledge), demonstrating that this method could be used to predict novel kinase targets in some cases. Using this approach we predicted novel kinase-target relationships and constructed a kinase activity network of ovarian cancer. To better analyze cancer-relevant pathway activity we developed a novel approach that characterizes correlation, differential abundance, and statistical interactions between components to analyze multiple omics types in the context of signaling and functional pathways. We used this approach, called the Layered Enrichment Analysis of Pathways (LEAP), to identify active pathways in molecular subtypes of ovarian cancer, contrasting observations in patients stratified for short versus long overall survival. This analysis resulted in a pre-treatment protein-based signature that is significantly predictive of overall survival. Our results show that integration of multiple omics types has the ability to inform our understanding of novel kinase-substrate interactions, and potentially identify novel interactions associated with patient survival.

#209

Proteomic analysis of therapeutic biomarkers in decalcified bone metastases.

Fabiola Cecchi,1 Shankar Sellappan,1 Sarit Schwartz,1 Chao Gong,2 Marlene Darfler,1 Kerry Scott,1 Steven W. Mamus,3 Mike Emmert-Buck,4 Todd Hembrough1. 1 _NantOmics, Ellicott City, MD;_ 2 _NantOmics, Rockville, MD;_ 3 _Cancer Center of Sarasota-Manatee, Sarasota, FL;_ 4 _NIH, Bethesda, MD_.

Introduction: Targeted therapies successful in treating primary tumors are often ineffective against corresponding metastatic lesions. Tumors and their bone metastases are biologically distinct, as reflected by discordant biomarker status. Decalcification of bone destroys protein and can preclude molecular analysis. A profiling method that does not depend on tissue antigenicity or nucleic acid preservation could increase the diagnostic value of decalcified bone samples. We assessed the effects of decalcification on proteomic analysis of tumor tissue. We also quantified 27 therapeutically-relevant proteins in decalcified bone metastases of cancer patients using mass spectrometry-based proteomic analysis.

Methods: To examine the effects of decalcification on protein quantification, we used 3 non-bone tissue specimens from lung adenocarcinoma, lung squamous cell carcinoma, and colon medullary carcinoma. Non-bone tumor tissue was expected to perform similarly in proteomic analyses to bone tissue, which was not available. Tissue specimens were processed without decalcification and with hydrochloric acid-based Decal-Stat™ decalcification solution for 1, 3, 12, and 24 hours prior to paraffin embedding, tissue sectioning, and mass spectrometric analysis. Proteomic analysis was also performed on 26 previously decalcified biopsies of metastatic bone lesions from patients with cancers of the lung (n=7), breast (n=7), stomach (n=3) and 8 other indications. Archived tumor tissue was microdissected and solubilized to tryptic peptides. Target proteins in each liquefied tumor sample were quantitated in triplicate with a multiplexed, selected reaction monitoring mass spectrometry assay.

Results: In the non-bone tumors, non-decalcified & decalcified samples yielded similar quantities of total protein (range: 19.2 - 24.1 µg) and of all 20 protein biomarkers detected. The 26 bone metastases expressed 20 of the 27 protein targets tested. Of the 7 bone samples from lung cancer patients, 5 expressed EGFR protein and 5 expressed hENT1 protein (a marker of response to gemcitabine). A metastatic bone tumor from a breast cancer patient expressed hENT1 (129 amol/µg) and overexpressed HER2 (5750 amol/µg; this exceeds a level previously correlated with increased survival in trastuzumab-treated patients). Bone lesions from prostate & gynecologic cancers overexpressed AR, hENT1, EGFR and TOPO1 proteins.

Conclusions: A commonly used decalcifying solution had no discernable effects on proteomic quantification of biomarker proteins in archived tumor samples. Targeted proteomics can quantify an entire panel of therapeutically-relevant proteins from a single decalcified bone biopsy specimen. Proteomic analysis of bone metastases upon diagnosis of metastasis or at relapse could inform treatment decisions, particularly in patients who have disease progression only in bone lesions or whose bone biomarkers are discordant from those of the primary tumor.

#210

Development of scores reflective of biological processes underlying human disease states from mass spectrometry of serum.

Carlos Oliveira, Julia Grigorieva, Krista D. Meyer, Joanna Roder, Heinrich Roder. _Biodesix, Inc., Boulder, CO_.

A method was developed that allows the evaluation of complex biological processes from mass spectrometry of human serum samples. Applying gene set enrichment analysis ideas to matched protein expression data from a panel of 1129 proteins and deep MALDI® mass spectral data from a set of 49 human serum samples with lung cancer (n=45) or non-cancer (n=4), subsets of mass spectral features associated with selected biological functions were identified. Biological functions included acute response, complement system, and wound healing and the protein members of each function were assigned using the intersection of gene ontologies and the protein panel. Using mass spectral data collected from an independent NSCLC sample cohort (n=85) from patients treated with targeted therapy, principal component analysis was used to derive scoring functions (combinations of the feature subsets) for each biological class. These scoring functions were validated in an additional NSCLC sample set (n=123) treated with chemotherapy. The scoring functions can be applied to mass spectra obtained from any human serum sample to generate scores associated with individual biological processes. We have developed scoring functions for several biological processes. Acute response score was associated with outcome in Cox proportional hazard analysis in several independent patient cohorts across multiple therapies and indications, including lung cancer, as mentioned, and ovarian cancer (n=165) treated with platinum doublets following surgery. Choice of a single cutoff allowed stratification of patients into groups with significantly better or worse outcome. In a cohort of nivolumab treated NSCLC patients (n=67) with available longitudinal samples, outcome was also found to depend on changes in scores during therapy. Interestingly, while the distributions of acute phase scores were quite similar across multiple tumor types, scores for other biological functions, such as wound healing, varied considerably. This may reflect differences in relative importance of individual biological functions between tumor types. A scoring system based on biological functional categories has a wide range of uses that could be tested and applied in a clinical setting. Currently, tests that measure a single biomarker for monitoring a particular disease have questionable utility and limit the usefulness to the clinician and patient treatment decision making. A tool that measures the activity of a complex biological process could be useful in deciding when an intervention is needed (screening), or for monitoring the effects during therapy.

#211

Protein painting identifies PD-1: PDL-1 therapeutic targets at protein-protein interfaces.

Alessandra Luchini, Luisa Paris, Virginia Espina, Kelsey Mitchell, Angela Dailing, Lance A. Liotta. _George Mason Univ., Manassas, VA_.

The next generation of molecular cancer therapeutics will target pivotal protein-protein interaction interfaces participating in immune cell receptor signaling, oncogenes, and suppressor genes. We have created a wholly novel, technology "protein painting" for the rapid direct sequencing of hidden native protein-protein interaction hot spots. Our technology, employs previously unexplored small molecule (12 Å) aryl hydrocarbon dyes or "paints" to cut out, and MS sequence, only the hidden unmodified contact interfaces between two or more interacting native proteins.

Novel Technology: Paint chemistries have extremely high affinities (rapid on-rates, and very slow off-rates that are ten to 100 times higher than most protein-protein interactions). When mixed with a native pre-formed protein complex for only 5 minutes at physiologic pH and salinity, the paints non-covalently coat all external sites on the protein without altering the 3D conformation of the complex, but cannot gain access to the solvent inaccessible hidden protein-protein interaction domains. Each paint molecule spans less than 3 amino acids, and has high affinity for protease cleavage consensus sites. Following painting, the proteins are dissociated. This leaves the paint molecules coating surfaces not participating in the interface. Following dissociation, the proteins are linearized, digested with trypsin, and sequenced by standard MS. The paint molecules remain non-covalently bound after the proteins are denatured. Trypsin will not cleave the regions of the protein that are "painted". Following proteolysis peptides emerging from MS will be generated exclusively from the unmodified opposing points where the proteins were in intimate contact.

Results: Protein Painting identified hot spot domains between PD-L1:PD-1, including two surface interface regions that are separated in the linear sequence but adjacent in the 3D structure. We created novel cyclized multivalent inhibitors that block both sides of the PD-L1:PD-1 interface and markedly suppress cell-cell coupling and abolished downstream signaling through this complex in cultured tumor cell immune cell interactions. A very high correlation (p&lt0.0003) was found for known contact points predicted by crystal structure, with a 97% specificity for true positive hot spots: 95% agreement with Robetta prediction software for known complexes. Protein painting outperforms (425%) hydrogen deuterium exchange and cross linking for number of positive hits and % true positive hits.

Conclusions: Protein painting is a new tool to identify highly specific drug targets located within protein interaction interfaces, yielding inhibitors that abolish protein signaling relevant to cancer immunotherapy.

#212

Identification by mass spectrometry of unique phosphoproteins subsequent to signaling through c-ErbB2.

C Sidhanth, Manoj Garg, P Manasa, S Krishna Priya, S Bindhya, S Sneha, R.P. Nagare, S Shirley, M Kanchan, Trivadi S. Ganesan. _Cancer Institute (WIA), Chennai, India_.

The receptor tyrosine kinase c-ErbB2 is amplified in breast and ovarian cancer. The linear pathways through which signals by c-ErbB2 are transduced is well known. However, second generation questions that address spatial aspects of signaling remain. To address this, we have undertaken a mass spectrometry approach to identify phosphoproteins. We have used two tyrosine kinase inhibitors, Lapatinib and CP724714, that inhibit phosphorylation of c-ErbB2 to identify phosphoproteins. SKOV-3, an ovarian cancer cell line that endogenously overexpresses c-ErbB2 was grown in culture without serum for 72 hrs. Cells were then stimulated in the presence or absence of inhibitor with EGF (100ng/ml) as a ligand for 60 mins. Subsequently, cells were lysed and evaluated by western blotting with anti-phosphotyrosine antibody (4G10). Following stimulation of cells with EGF, maximal phosphorylation of c-ErbB2 was observed at 60 minutes. Lapatinib (10μM) and CP724714 (15μM) completely inhibited phosphorylation of c-ErbB2, which was confirmed by immunoprecipitation. This was further confirmed by the inhibition of downstream effectors (Erk1/2, Akt). Lapatinib (10 μM) also completely inhibited phosphorylation of EGFR while CP724714 (15μM) only inhibited partially. Cellular lysates were prepared from quiescent cells (grown without serum), after stimulation with EGF in the presence or absence of inhibitors. Purified phosphoproteins from all three samples following digestion with trypsin were subjected to mass spectrometry (Nano LC ESI MS/MS). We identified totally 62 phosphoproteins. Twenty seven phosphoproteins were observed in all the 3 samples while 17 phosphoproteins were identified both in the EGF stimulated and lapatinib treated samples. Eighteen unique phosphoproteins were observed only in the EGF stimulated sample suggesting that they are specific to signaling by c-ErbB2. The novel phosphoproteins included the proteins that partcipate in carbohydrate metabolism,cytoskeleton, cell migration and proliferation. We have evaluated two phosphoproteins, LASP-1 and Aldose reductase that has not been previously described following phosphorylation of c-ErbB2. LASP-1 is an oncogene and is located as the same arm 17q21 as c-ErbB2. It was not expressed in the normal ovary or fallopian tube. However, it was over-expressed in 17% of tumours (n=85) from patients with ovarian cancer. c-ErbB2 was not expressed in tumours that expressed LASP1. Aldose reductase is a cytosolic NADPH dependent oxidoreductase that catalyzes the reduction of glucose to sorbitol, the first step in polyol pathway of glucose metabolism. The activity of aldose reductase in reducing NADPH as a substrate was significantly higher in lysates from EGF stimulated as compared to the starved cells. Identification of phosphoproteins by using mass spectrometry is promising in identifying novel substrates and pathways following phosphorylation of c-ErbB2.

#213

Integrated proteogenomic analysis of laser capture microdissected breast tumors.

Viswanadham Sridhara,1 Tao Liu,2 Marina A. Gritsenko,2 Lori A. Sturtz,1 Albert J. Kovatich,3 Vladislav A. Petyuk,2 Brenda Deyarmin,1 Jason E. McDermott,2 Anil K. Shukla,2 Ronald J. Moore,2 Matthew E. Monroe,2 Bobbie-Jo M. Webb-Robertson,2 Jeffrey A. Hooke,3 Leigh Fantacone-Campbell,3 Praveen Kumar Raj Kumar,1 Leonid Kvecher,1 Jianfang Liu,1 Jennifer Kane,1 Jennifer Melley,1 Stella Somiari,1 Joji Iida,1 Stephen C. Benz,4 Justin Golovato,4 Shahrooz Rabizadeh,5 Patrick Soon-Shiong,5 Richard D. Smith,2 Richard J. Mural,1 Craig D. Shriver,6 Hai Hu,1 Karin D. Rodland2. 1 _Chan Soon-Shiong Institute of Molecular Medicine at Windber, Windber, PA;_ 2 _Pacific Northwest National Laboratory, Richland, WA;_ 3 _CBCP, Henry Jackson Foundation, Rockville, MD;_ 4 _NantOmics, LLC, Culver City, CA;_ 5 _NantWorks, LLC, Culver City, CA;_ 6 _Murtha Cancer Center, Walter Reed National Military Medical Center, Bethesda, MD_.

Introduction: Molecular characteristics of breast tumors play an important role in determining patients' survival outcome. Here, we report preliminary findings of proteogenomic profiling of 50 breast tumors using RNA-Seq and mass-spectrometry (MS) based proteomic technologies. An additional 60 tumors are being analyzed, including WGS for all samples. We are also collecting patient survival outcome data.

Methods: Cases used in this study were drawn from the Clinical Breast Care Project, where patients were consented using an IRB-approved protocol. A total of 50 breast tumors were selected and processed by laser capture microdissection (LCM). This cohort includes 36 Caucasian Americans (CA) and 8 African Americans (AA), and the age of the patients is 57 ± 13 years. Protein and RNA were extracted using the Illustra triplePrep kit, which isolates DNA from the same cells as well. Quantitative global proteomics and phosphoproteomics analyses were performed using isobaric TMT 6-plex labeling with the "universal reference" strategy and IMAC enrichment of phosphopeptides. Mass spectrometry data were acquired using a Q-Exactive instrument and analyzed using Proteome Discoverer with Byonic node. Phosphopeptide abundance was normalized to abundance measurements of the parent protein for all of the phosphorylation analyses. Phosphoproteomic data was also searched for the presence of O-GlcNAc modifications. RNA-Seq analyses were done on Illumina HiSeq and the data were analyzed using GSNAP.

Results: There were 19 Luminal A, 7 Luminal B, 8 HER2-enriched, and 16 basal-like subtypes based on the PAM50 algorithm. In the global proteomics data, we were able to quantitate >8600 proteins. Unsupervised clustering on the highly varying proteins across the samples resulted in two primary clusters, with one being luminal-enriched. The other cluster contains a basal-like tumor sub-cluster and a sub-cluster of mixed subtypes. Differential protein expression analyses between the two primary clusters confirmed known markers (e.g., overexpression of KRT8/KRT18 in luminal-enriched cluster). The luminal-enriched cluster is primarily CA with post-menopausal status.

A similar search of the phosphoproteomic data yielded quantitation of >12500 phosphopeptides. Unsupervised clustering of the phosphoproteins resulted in four primary groups, with one being basal-enriched and another being luminal-enriched. We also observed >50 overexpressed phosphopeptides. While some of these phosphosites have been previously reported (e.g., on RANBP2), other phosphosites appeared to be novel (e.g., on IRF2BP2).

Conclusion: Analysis of LCM breast tumors using proteogenomic technologies resulted in basal- and luminal-enriched clusters, thus enabling us to study protein and phosphopeptide markers across multiple platforms.

The views expressed in this article are those of the author and do not reflect the official policy of the Department of Defense, or U.S. Government.

#214

ARID1A and ARID1B dependent proteomics.

Yutaka Shoji,1 Kumiko Kato-Shoji,1 Kelly A. Conrads,2 Rusheeswar Challa,1 Brian L. Hood,2 Nicholas W. Bateman,2 Thomas P. Conrads,2 John I. Risinger1. 1 _Michigan State Univ., Grand Rapids, MI;_ 2 _Women's Health Integrated Research Center at Inova Health System, Annandale, VA_.

Protein sub-units of the SWI/SNF nucleosome and chromatin remodeling complexes are frequently mutated in cancer. The ARID1A and ARID1B DNA interacting component are among these mutated proteins. Alterations in ARID1A have been reported in breast, colon, lung, kidney, pancreatic, bladder, cervical, ovarian and uterine cancers whereas ARID1B mutation is less common. Additional data suggests ARID1A mutant cancers are dependent on functional ARID1B and that targeting ARID1B may be a therapeutic strategy. However, ARID1A and 1B are often co-inactivated in aggressive un/dedifferentiated carcinomas of the ovary and endometrium suggesting tumor suppressive functions might exist not only in ARID1A but also in 1B. In this study, we examined the effects of restoring ARID1A or 1B in an undifferentiated endometrial adenocarcinoma cell line harboring inactivating mutations in both genes. ACI-98 cells derived from a stage IV undifferentiated endometrial cancer were found to lack expression of ARID1A and 1B protein. We subsequently identified two individual truncating mutations in ARID1A and 1B by genomic DNA and cDNA sequencing indicating no wild-type message was expressed. Restoration of ARID1A in ACI-98 cells using a Tet-on system revealed remarkable growth inhibition, however, ARID1B restoration showed only a moderate cell growth inhibition with cells taking on a flattened phenotype. Clones of ARID1A/1B restored cells were treated w/wo doxycycline (Dox) for 48h a time point prior to cell death or morphologic changes and protein changes catalogued using LC MS/MS. We identified 771 and 1168 differentially expressed proteins (z-score <0.05) from ARID1A or 1B restored clones compared to dox free control and 138 proteins were common in these two groups (up-regulated: 57, down-regulated: 37, reverse regulation between ARID1A and 1B: 44). We performed pathway analysis of the ARID1A and 1B proteome by Advaita Pathway Guide software and this analysis identified 10 significantly impacted pathways in ARID1A and 17 pathways in ARID1B. However, there was no common pathway between the two. Wnt signaling pathway (KEGG: 04310) was among ARID1A distinguished pathways with 9 of 46 proteins being differentially expressed. Among these NKD1 a negative regulator of the Wnt-β-catenin-Tcf signaling pathway was increased in ARID1A restored cells more than 10 fold. We confirmed the differential expression of NKD1 using qRT-PCR following ARID1A expression. The p53 signaling pathway (KEGG: 04916) was significantly (11 of 34 proteins) affected following ARID1B complementation. We confirmed the differential expression of FAS was up-regulated 6 fold in proteomics and 3.4 fold in qPCR. These results suggest that restoration of ARID1A or 1B both effect cell proliferation to varying degree. The lack of overlap between the proteomes of ARID1A/1B restored cells indicates distinct pathways underlying these induced phenotypes and different function of SWI/SNFARID1A or SWI/SNFARID1B.

#215

Characterization by mass spectrometry of protein kinase C substrates differentially phosphorylated in LNCaP cells in response to phorbol ester and bryostatin 1 treatment.

Noemi Kedei,1 Sudipto Das,2 Thorkell Andresson,2 Peter M. Blumberg1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Frederick National Laboratory for Cancer Research, Frederick, MD_.

Bryostatin 1 (bryo 1) is a natural product of therapeutic interest for cancer and Alzheimer disease. Its unique behavior as a protein kinase C activator that paradoxically antagonizes many but not all phorbol ester responses has led to intense interest in its mechanism of action. Recently, using microarray analysis in two different cellular systems (LNCaP prostate cancer and U937 leukemia cell lines) where the typical phorbol ester PMA and bryo 1 have different biology, we have shown that a core mechanism contributing to the unique biology of bryo 1 is transiency of action resulting in a variable extent of decreased or missing late responses. We have excluded that there is a class of genes whose transcription is uniquely regulated at early times by bryo 1. In continuing to explore the mechanisms underlying the variable transiency of the responses by bryo 1 we have evaluated the substrates phosphorylated after treatment with PMA and bryo 1. Since PMA and bryo 1 induce differential subcellular localization of PKCs, they should result in differential access to substrates and consequent differences in the pattern of substrate phosphorylation. Whole cell lysates of LNCaP cells treated for 30 min with fully effective doses (100 nM) of PMA and bryo 1 and with DMSO as vehicle control were subjected to mass spectroscopic analysis. The comprehensive analysis identified several thousand phosphopeptides after drug treatment including the expected phosphopeptides for ERK2 (S185, Y187) and PKCdelta (S299, S302, S304). Many of peptides were similarly phosphorylated in response to both drugs (e.g. S641/S646 of Fam129B, S310 of actin-related protein 2/3 complex subunit 1B, S876 of RhoGEF and pleckstrin domain-containing protein 2, or multiple previously unknown sites for PKD1 (S239, S247), while a limited number were differentially phosphorylated. PMA specific phosphorylations included PKCdelta at Y313, mTOR at T2471, MAP4 at S624 and/or T627, E3 ubiquitin-protein ligases ZNRF2 at S82 and HUWE1 at S3818. Bryo 1 specific phosphorylations included BCL2-like 13 at S303, TOM1L2 at S424, and MAP2K2 in the T17, T25, S26 region. Selected specific phosphorylations are being validated using phostag gels, a method that enables the separation of phosphorylated proteins from their non-phosphorylated counterparts on SDS-gels. The identification of PKC substrates that are differentially phosphorylated by bryo 1 should both facilitate screening of other ligands capturing the biological behavior of bryo 1 as well as further illuminate the specifics of the pathways downstream of PKC activation by these differently acting ligands.

#216

Tracking expression, post-translational modifications and interactions of EGF signalling proteins in A431 cells with antibody microarrays.

Lambert Yue, Steven Pelech. _University of British Columbia, Vancouver, British Columbia, Canada_.

Elucidation of epidermal growth factor (EGF) signalling pathways in cancer cells has helped to define the mechanisms for their neoplastic transformation and potential drug targets for therapeutic intervention. Antibody microarrays are promising tools to evaluate alterations in the levels and phosphorylation status of hundreds of proteins of interest with only microgram amounts of crude cell and tissue lysate protein. However, interpretations of the results from traditional antibody microarray approaches have been hampered by the problems associated with sample preparation and protein detection, even when reliable antibodies are deployed in these arrays. The Kinex™ KAM-900P antibody microarray permitted semi-quantitative measurements of the expressions, post-translational modifications and interactions of proteins with 100 µg or less of lysate proteins. These microarrays utilize approximately 878 different pan- and phosphosite-specific antibodies for tracking protein kinases, phosphatases and other low abundance regulatory proteins. Multiple detection protocols were developed with the KAM-900P slides to enable high depth profiling of protein levels, phosphorylation and protein-protein interactions in A431 cells in response to EGF treatment. One method (KAM) involved the capture of in vitro biotin-labeled proteins, followed by their detection with a secondary dye-labeled anti-biotin antibody. False positive signals from associated proteins in complexes with the targets were reduced by chemical cleavage with NTCB prior to their capture on the array, and this also produced more uniformity of the dye signals for protein targets despite vast differences in their sizes. Transient changes in protein phosphorylation in EGF treated cells that were typically lost when processed by conventional methods were better preserved by chemical cleavage right at time of sample homogenization. Biotin-labeling and subsequent detection of the protein on the array with a dye-labeled secondary antibody further reduced non-specific background signals, allowed for a greater dynamic range of detection, and enhanced discrimination of subtle changes. In conjunction with other detection protocols, such as the usage of dye-labeled reporter antibodies for generic protein-tyrosine phosphorylation in sandwich antibody microarrays (SAM format) or generic protein phosphorylation with nanoparticles such as pAMIGO (PAM format), it is also feasible to monitor changes in general post-translational modifications of target proteins or their specific association with other adapter, scaffolding and chaperone proteins of interest for which antibody probes are available.

#217

Quantitative analysis of two cancer signaling pathways using multiplex immunoprecipitation and targeted mass spectrometry.

Bhavin Patel, Leigh Foster, Gregory Potts, Abid Haseeb, Alex Behling, John Rogers. _Thermo Fisher Scientific, Rockford, IL_.

Background: The AKT/mTOR and RAS/ERK pathways represent key mechanisms for cells to regulate cell survival, proliferation, and motility. In addition to their independent signaling cascades, which provide offsetting mechanisms, these two pathways extensively engage in cross-talk in order to both positively and negatively regulate each other. The quantitation of protein expression and modifications of pathway targets are critical for characterization of disease, monitoring cancer progression and determining treatment response. Some major bottlenecks in the accurate quantitation of these proteins are the lack of rigorously validated reagents and a reliance on semi-quantitative results from available immunoassays. Mass spectrometry (MS) is increasingly becoming the detection method of choice for proteins and their post-translational modifications (PTMs). Immunoprecipitation coupled with MS (IP-MS) enables assessment of antibody specificity and identification of low-abundant targets. Multiplexed IP coupled with targeted MS (mIP-tMS) can quantitate multiple proteins of interest, PTMs and interacting partners in a single MS run. The objective of this study was to determine the efficacy of mIP-tMS for quantitation of the AKT/mTOR and RAS/ERK pathway proteins and to compare these results versus Western blotting (WB).

Methods: Serum starved and LY294002 treated HCT116 and A549 cells were stimulated with IGF-1. Antibodies to targets in the AKT/mTOR and RAS/ERK pathways were selected for verification of antibody specificity by IP-MS. mIP-tMS assays were developed and validated for absolute quantitation of targets in these pathways and benchmarked with WB across two unstimulated, IGF-1 stimulated and LY294002 treated cell lysates as well as several tissue lysates.

Results: Previously, we showed that an optimized IP-MS workflow for Protein A/G and Streptavidin magnetic beads increases target protein yield with low non-specific background. In this study, we validated antibodies to several AKT/mTOR and RAS/ERK pathway targets using the optimized IP-MS workflow. mIP-tMS assays allowed absolute quantitation for multiple total and phosphorylated targets from both pathways in low to sub-nanogram concentrations across two unstimulated, IGF-1 stimulated and LY294002 treated cell lysates. The benchmarking of mIP-tMS assays showed low correlation for quantitation of total and phosphorylated targets relative to WB. This lower correlation may be due to differences in the specificity of antibodies used for each assay technique.

Conclusion: Overall, the mIP-tMS assays can be used for effective identification and quantitation of AKT/mTOR and RAS/ERK pathway proteins in multiple cancer cell lines and tissue samples. Major advantages of this assay are high confidence in target identity coupled with simultaneous absolute quantitation of multiple targets and their PTMs from cancer signaling pathways.

#218

Functional proteomic analysis of TβRI immunocomplex in TGF-β signaling.

Liuya Tang,1 Zhaojing Meng,2 Mary Heller,1 Ming Zhou,2 Ying Zhang1. 1 _NCI-CCR, Bethesda, MD;_ 2 _Frederick National Laboratory for Cancer Research, Frederick, MD_.

Transforming growth factor-beta (TGF-β) regulates a wide array of biological responses ranging from proliferation to apoptosis, and alterations in its signaling pathway are associated with a variety of human diseases, including cancer. During the activation of TGF-β signaling, TGF-β receptor I (TβRI) plays an important role in transducing signals to downstream effectors. To identify TβRI interacting proteins which are responsible for Smad-dependent pathways as well as Smad-independent pathways, we employed a large-scale quantitative proteomic analysis of TβRI immunocomplex. AML12 cells were labeled with L-Lysine and L-Arginine (K0R0, light) or L-Lysine-13C6-15N2 and L-Arginine-U-13C6-15N4 (K8R10, heavy). The cells cultured in K8R10 medium (H: Heavy) were transfected with Flag-TβRI, while the cells cultured in K0R0 medium (L: Light) were transfected with Flag vector. The cell lysates were combined at equal protein amount, and TβRI immunocomplex were purified using FLAG M2 agarose. The resulting peptides from eluted TβRI immunocomplex were subjected to mass spectrometry quantitation. If a protein whose ratio of H/L was not smaller than 1.3, it was categorized as a constitutively interacting protein of TβRI. A total of 687 proteins were quantitated and 325 proteins were defined as constitutively interacting proteins. Pathway enrichment analysis found these proteins are involved in a number of signaling pathways, which indicates the cross-talks between TGF-β signaling and other signaling. Future work will include validation of interaction between TβRI and candidate proteins, and understand functional significance of candidate proteins in TGF-β signaling or signaling cross-talks by a series of molecular biological approaches.

#219

Identification of the mechanistic target of rapamycin complex 2-associated interactome involved in brain cancer cell motility by affinity purification-mass spectrometry.

Naphat Chantaravisoot,1 Piriya Wongkongkathep,1 Fuyuhiko Tamanoi,2 Trairak Pisitkun1. 1 _Chulalongkorn University, Bangkok, Thailand;_ 2 _UCLA, Los Angeles, CA_.

The Mechanistic Target of Rapamycin Complex 2 (mTORC2), a multiprotein complex with serine-threonine kinase activity, drives several cellular processes of normal cells to promote proper metabolic activity, survival, proliferation, differentiation and movement. On the other hand, mTORC2 has been implicated as a critical player in tumorigenesis, stimulation of cell growth, cancer metabolic reprogramming, and development of highly migratory and invasive capabilities of cancers. Therefore, characterization of mTORC2 is necessary not only for better understandings of known mTORC2-mediated functions, but also for the discovery of new roles and more interacting partners of mTORC2 signaling network in cancers, particularly ones with hyperactivated mTORC2 such as glioblastoma. However, until now mTORC2 is still partially characterized. Previously, mTORC2 was reported to be associated with Filamin A and Myosin-9 which have been known to play important roles in cellular activities requiring locomotion, including cancer migration and invasion. The complex participates in regulating glioblastoma migration and invasion, and Filamin A acts as a physiological downstream target of mTORC2. Currently, proteomics has emerged as a powerful tool to identify, quantify, and examine a large number of proteins. Therefore, in order to extensively unravel mTORC2 signaling pathway, the use of proteomics is necessary.

We characterized mTORC2 by proteomic analysis based on affinity purification coupled to mass spectrometry (AP-MS) using antibodies against RICTOR, a specific component of mTORC2. The protein-protein interactions between RICTOR and other associated proteins were assessed. In addition to canonical components of mTORC2, the study has revealed more insights into a number of mTORC2 interacting partners which can be categorized and related to the main functions previously identified such as actin cytoskeleton reorganization, fatty acid metabolism, protein translation. Interestingly, we also found other types of cytoskeletal proteins, nucleotide metabolism, less-characterized and low abundance proteins associated with mTORC2 such as Hornerin and Desmoplakin. In addition, we have carried out the identification of condition-specific interactions for a dynamic view of mTORC2 interactome and found changes in the mTORC2-associated proteins from different conditions. Furthermore, proteomic approaches can also be relevant to investigation and prediction of mTORC2 upstream regulators, downstream targets, and complex localization. Ultimately, this project will uncover some unknown molecular mechanisms, and result in a significant impact on the field of signal transduction machinery in cancers.

#220

Proteomic identification of therapeutics targets for Enzalutamide resistance in Castration Resistant Prostate Cancer.

Lauriane VELOT,1 Dominique Levesque,2 François-Michel Boisvert,2 Nicolas Bisson,1 Frédéric Pouliot1. 1 _Centre de recherche sur le cancer, Centre de recherche du CHU de Quebec - Université Laval, Québec, Quebec, Canada;_ 2 _Faculté de Médecine et des sciences de la santé - Université Sherbrooke, Sherbrooke, Quebec, Canada_.

Prostate cancer (PC) is the most frequently diagnosed cancer in Canadian men and is the 3rd cause of cancer mortality. The Androgen Receptor (AR) is activated by androgens (e.g. testosterone), which leads to prostatic cell proliferation. The primary treatment for advanced PC is androgen-deprivation therapy, which is achieved via surgical or chemical castration. Nevertheless, most PC will become castration-resistant (CRPC). Although new anti-androgens (e.g. Enzalutamide) were developed to improve patients' survival, their efficacy is still very limited and most CRPC patients will die from the disease within a few years. We postulate that by gaining insights into AR signalling networks in the context of CRPC, we could better direct patients towards the best-suited therapy and propose new therapeutic targets.

To this aim, we took advantage of an innovative proteomics approach, namely proximity-labeling (BioID), to characterize global AR signalling networks in hormone-dependant LAPC4 cells. We identified 45 AR-associated proteins in non-stimulated cells, 35 of which were not previously reported. Upon androgenic stimulation, the AR signalling network increased to 320 proteins, including 278 (253 were novel) that were restricted to androgen-stimulated cells. Enzalutamide treatment resulted in a loss of 259 proteins from the network when compared to stimulated cells. As expected, this reproduced quite faithfully the status of non-stimulated LAPC4 cells. Interestingly, we identified 4 proteins in the AR network specifically after Enzalutamide treatment. These are interesting targets that may be relevant for the acquisition or the prediction of Enzalutamide resistance. Hence, they could become in time alternative therapeutic targets for CRPC treatment, to be used when Enzalutamide fails.

#221

Integrated functional proteomics of MET/VEGFR inhibitors reveals complex mechanism of action of foretinib in NSCLC.

Natalia J. Sumi, Bin Fang, Lily L. Rix, Muhammad Ayaz, Fumi Kinose, Eric A. Welsh, Steven A. Eschrich, Harshani R. Lawrence, John M. Koomen, Eric B. Haura, Uwe Rix. _Moffitt Cancer Center, Tampa, FL_.

BACKGROUND: Foretinib (FORE) and cabozantinib (CABO) are two MET/VEGFR inhibitors with similar chemical structures. CABO is FDA-approved for medullary thyroid and renal cancer; in addition, it is in clinical trials for treatment of non-small cell lung cancer (NSCLC). Through an unbiased viability screen we have observed potent cellular activity of FORE, but not CABO, in several NSCLC cell lines. We have previously shown that most NSCLC cell lines are insensitive to MET or VEGFR inhibition, suggesting off-target activity of FORE in these cells. The aim of this project is to identify the mechanism of action of FORE in NSCLC and design an optimized combination therapy.

METHODS: Cellular viability assays were done using CellTiter-Glo, cell cycle analysis by flow cytometry. Western blotting was performed to evaluate the induction of apoptosis through PARP1 and caspase cleavage, as well changes in signaling. We synthesized FORE and CABO analogues and performed differential quantitative chemical and phosphoproteomics to determine the target kinase profile and pathway effects in NSCLC cells. Changes in gene expression upon drug treatment were measured by RNA-seq. RNAi in combination with pharmacological inhibitors was performed to interrogate targets and pathways.

RESULTS: FORE showed greater potency in NSCLC cell lines than CABO with regard to inhibition of viability and induction of apoptosis. FORE decreased phosphorylation of AKT and ERK. Chemical and phosphoproteomics revealed several kinases, such as MEK and MAP4K5, to bind preferentially by FORE over CABO that differentially affect the adherens junction and MAPK signaling pathways. Target validation showed differential inhibition of MEK1/2 and MAP4K5. Cellular validation with RNAi in combination with pharmacological inhibitors suggested that MEK1/2, MAP4K5 and IGF1R are involved in the mechanism of action of foretinib in NSCLC cells. RNA-seq pathway analysis furthermore suggested regulation of chromatin organization and Wnt pathway signaling by foretinib.

CONCLUSION: Our results suggest that the difference in the efficacy between FORE and CABO is related to polypharmacology of FORE, which simultaneously targets IGF1R, MEK1/2 and MAP4K5. This difference results in divergence in signaling pathway inhibition and induces distinct effects in NSCLC. The establishment of FORE targets and signaling pathways can lead to optimized combination therapy for NSCLC and identification of new actionable kinases in lung cancer cells.

#222

Protein network mapping of bladder cancer: tumor compartment and microenvironment analysis.

Kimberley A. Hodge,1 Andrew Hau,2 Mariaelena Pierobon,1 Donna Hansel,2 Emanuel Petricoin1. 1 _George Mason University, Manassas, VA;_ 2 _University of California: San Diego, San Diego, CA_.

Background: Interactions between tumor and stroma are of intense interest in the field of oncology and provide new opportunities to understand the driving mechanisms behind tumor progression, metastases and responsiveness to therapy. Laser capture microdissection (LCM) coupled with reverse phase protein microarrays (RPPA) are ideal technologies for isolating different cellular compartments from heterogeneous tissues and for exploring protein signaling networks.

Material and Methods: We explored protein signaling networks of epithelium and matched surrounding stroma of 16 bladder cancers (BC) including papillary (n=8) and invasive (n=8) tumors. Tumor epithelium and matched surrounding stroma were isolated with LCM for each case. Signaling network analysis of dissected material was performed using RPPA, allowing for the measurement of 111 proteins and phosphoproteins. Wilcoxon Rank Sum Test was used to compare the signaling network of papillary and invasive lesions. Spearman Rho correlation analysis (correlation coefficient > 0.90) was used to explore signaling networks of each cellular compartment across histotypes.

Results: Mean comparison of tumor epithelium revealed increased PD-L1 and PDGFRβ expression in invasive tumors compared to papillary lesions (p= 0.015 and p=0.028 respectively). Correlation analysis of the epithelium compartments collected from papillary and invasive cancers revealed different signatures with invasive cases showing a phenotype of proliferation and survival in contrast to papillary tumors which were characterized by wound healing and metabolic network activation. Analysis of stroma showed increased expression of MMP9 in invasive compared to papillary stroma (p= 0.05). While stroma surrounding both invasive and papillary tumors showed high correlation between members of the PI3K/AKT pathway, only invasive BC presented with interconnections between immune response proteins. Finally, receptor tyrosine kinases (RTKs) in the invasive epithelium appeared to be highly correlated with RTKs in the stroma as well as their downstream targets suggesting cross-talk between the two compartments.

Conclusions: LCM-RPPA workflow is a unique tool for exploring tumor and surrounding microenvironment interactions in BC. Signaling networks of epithelial cells and surrounding stroma of invasive BC has unique characteristics compared to papillary tumors including increased expression of immune checkpoint PD-L1 and PDGFRβ in the epithelium, two potential therapeutic targets for this group of patients, along with increased interaction between PI3K/AKT pathway and proteins involved in immune modulation. If validated in a larger cohort of patients, these findings may lead to better understanding of the molecular mechanisms driving invasive tumors of the bladder especially in the context of tumor-stroma interactions.

#223

Targeting critical signaling nodes using multiplexed antibody based phosphopeptide enrichment with iMRM validation.

Charles L. Farnsworth, Yiying Zhu, Matthew Stokes. _Cell Signaling Technology, Danvers, MA_.

Introduction: A challenge for biomedical researchers is to develop assays to analyze complex systems that interrogate whole cellular signaling networks. Here we show the results of employing antibody based immunoaffinity enrichment of phosphopeptides derived from critical signaling nodes to quantitate changes in phosphorylation upon kinase inhibitor treatment. Human cancer cell lines, including gastric and breast adenocarcinomas were treated with kinase inhibitors and cellular extracts were digested with trypsin and peptides were purified. A pool of phosphorylation dependent site-specific antibodies was employed to enrich for known sites of phosphorylation. Following antibody based enrichment these peptides were identified and quantified using liquid chromatography/tandem mass spectrometry (LC-MS/MS). Phosphopeptides were identified using SEQUEST, and differences in abundance were determined by employing label-free MS1 based quantification using Progenesis from Non-Linear Dynamics. We also show validation data for a number of the antibodies employed using immuno-multiple reaction monitoring, or iMRM. Employing a QExactive mass Spectrometer (Thermo Scientific) we developed calibration curves for a number of the targeted tryptic phosphopeptides using combinations of heavy and light peptides with endogenous cancer cell line peptides as a background matrix.

Experimental Results: Cell lines were treated with DMSO or inhibitors to which the cell lines should be sensitive/insensitive. Following peptide purification, a pool of antibodies were multiplexed and used to enrich for phosphopeptides derived from signaling proteins across a number of signaling pathways. The results show significant changes in abundance for many proteins/sites, including activation loop tyrosine residues of tyrosine kinases, changes in sites on proteins in the AKT/PI3K pathway, sites in the MAPK pathway, and other critical regulators of cellular signaling such as sites on PKC family members. Targeted LC-MS/MS methods using iMRM were used to confirm these results and show the promise of using such targeted strategies for quantifying changes in phosphorylation consistently across many samples.

Conclusion:The strategy of multiplexing antibodies for immuno-enrichment allows for simultaneous detection and quantification of hundreds to thousands of known phosphorylation sites that have been shown to be important nodes of regulation. Targeted LC-MS/MS methods such as iMRM will allow researchers to confidently profile changes in both total levels as well as phosphorylation site occupancy for critical signaling proteins.

#224

Critical variables for automated protein immunodetection using western blot.

Alexander Margulis,1 David P. Chimento,2 Russ Yukhananov1. 1 _Precision Biosystems, Mansfield, MA;_ 2 _Rockland Immunochemicals, Inc., Limerick, PA_.

Reliable immunodetection of proteins is a critical step in the discovery of novel biomarkers. It is often performed manually. The immunodetection protocol consists of multiple steps including blocking of nonspecific binding sites, incubation using primary and secondary antibodies and extensive washing between steps that easily introduce bias and errors. The quality of results depends on multiple subjective and objective factors such as the qualification and technical skills of the personnel performing the assay and the accuracy of temporal and temperature control, especially during the immunodetection step. BlotCycler™, automated western blot processor, use fluidic control system that allows to eliminate the variability associated with immunodetection and to achieve higher sensitivity by optimized washing procedure. We used BlotCycler to analyze critical variables for immunodetection including temperature, incubation time and reagent mixing. Relatively small changes in duration and temperature of incubation significantly affect not only the intensity of signal but also the specificity of antibody interaction with antigen. Automated immunodetection at 4ºC significantly improve the specificity of detection and sensitivity that allows reproducible detection of low expressing proteins. Automated processing using BlotCycler allows optimization and control of critical variables leading to improved reproducibility and specificity of immunodetection and should be routinely used for biomarker candidate discovery and confirmation, reliable protein quantification, primary antibody specificity testing, and optimization of primary and secondary antibodies concentrations.

#225

Chemoproteomic evaluation of target engagement in clinical samples.

Tyzoon K. Nomanbhoy. _ActivX Biosciences, Inc., La Jolla, CA_.

One of the challenges during the clinical development of kinase inhibitors is understanding whether or not a compound is actually binding the kinase of interest, in the appropriate tissue, during the course of compound treatment. While compound concentration in blood can be determined from pharmacodynamics, there are very few approaches that directly measure whether or not the compound is binding the target kinase. Herein, we apply a chemoproteomics platform (KiNativ®) using a desthiobiotin ATP acyl phosphate probe (ATP probe), to monitor target engagement in clinical samples. As a proof of concept, we demonstrate that compound can be added to whole blood, after which peripheral blood mononuclear cells (PBMCs) are isolated, lysed, labeled with the ATP probe, and analyzed by mass spectrometry to determine whether or not compound treatment blocks the probe-labeling of the target. We successfully applied this approach in the analysis of both the reversible JNK inhibitor CC-930, as well as the covalent BTK inhibitor Ibrutinib (Imbruvica®). We then extended the study to monitor the inhibition of BTK by Ibrutinib in PBMCs isolated from patients undergoing drug treatment. Finally, we demonstrate that in addition to profiling kinases in PBMCs isolated from whole blood, this method can also be used to profile kinases in solid tumors. Thus, the chemoproteomics approach described here could be applied as a general method to monitor target engagement for inhibitors developed against a variety of different kinases in clinical samples.

#226

Verification of prostate cancer genomics biomarker candidates at protein level using PRISM-SRM.

Hui Wang,1 Yuqian Gao,1 Athena Schepmoes,1 Gyorgy Petrovics,2 Jennifer Cullen,2 Thomas Fillmore,1 Tujin Shi,1 Wei-Jun Qian,1 Richard Smith,1 Brandi Weaver,3 Robin Leach,3 Ian Thompson,3 Sudhir Srivastava,4 Jacob Kagan,4 Albert Dobi,2 Karin Rodland,1 Shiv Srivastava,2 Tao Liu1. 1 _Pacific Northwest National Laboratory, Richland, WA;_ 2 _Uniformed Services University of the Health Sciences, Bethesda, MD;_ 3 _University of Texas Health Science Center at San Antonio, San Antonio, TX;_ 4 _National Cancer Institute, Bethesda, MD_.

Mass spectrometry (MS) based targeted proteomics such as selected reaction monitoring (SRM) provides an antibody-independent strategy for sensitive, specific and multiplexed verification of genomics biomarker candidates at the protein level. In order to identify a panel of proteins with the potential to discriminate between aggressive and indolent forms of prostate cancer and predict prostate cancer progression, we have selected 52 protein candidates from existing prostate cancer genomics data sets and validated cancer drivers, and performed quantitative proteomics analysis in tumor and control tissue samples using the highly sensitive PRISM (high-pressure, high-resolution separations coupled with intelligent selection and multiplexing)-SRM approach. PRISM-SRM assays have been developed for the 52 prostate cancer biomarker candidates including: prostate cancer prognosis associated genes, prostate cancer associated genes that were up-regulated in transcriptomics studies, and other cancer-related genes (including the ERG or ETV1 isoforms). Two sets of tissue samples were analyzed using PRISM-SRM with heavy isotope-labeled synthetic peptides as internal standards: 1) 10 high Gleason-score (7-9) primary prostate tumors and 10 benign prostatic hyperplasia (BPH) tissues (OCT-embedded specimens); and 2) 10 primary tumors from patients showing metastatic progression, 10 primary tumors from patients who showed biochemical recurrence (BCR), and 10 primary tumors from patients with no BCR or metastatic progression after more than ten years of follow-up after radical prostatectomy (FFPE whole mount prostate specimens). Overall, PRISM-SRM analyses of all the patient tissue samples enabled the detection of 48 out of 52 biomarker candidates, suggesting extremely low level of expression of the remaining 6 genes (HXC6, OSTP, TWST1, and ERG8); in comparison regular LC-SRM can only detect 21 of these candidates at the protein level. In the 10 high Gleason-score tumors and 10 BPH controls, 13 proteins were found differentially abundant with P<0.05. In the 10X10X10 FFPE sample analysis, there were three proteins discriminating between "metastatic progression" and "no progression" tumors, one protein discriminated between BCR and "no progression" tumors, and four proteins discriminated between metastatic progression and BCR tumors (P<0.05). These promising biomarker candidates will be further evaluated, individually and in panels, in independent, larger cohort for their potential prognostic applications. In summary, PRISM-SRM provides a highly sensitive method for quantification and rapid screening of multiple potential biomarker candidates at the protein level. This approach holds great potential for rapidly translating genomics-based discovery candidates into protein-based biomarkers.

#227

Mass spectrometry-based proteomics analysis of the non-small cell lung cancer secretome.

Emmanuel K. Cudjoe, Tareq Saleh, David A. Gewirtz, Adam M. Hawkridge. _Virginia Commonwealth University, Richmond, VA_.

Non-small cell lung cancer (NSCLC) is the more common subtype (~80%) of lung cancer, a leading cause of cancer death worldwide. NSCLC has one of the lowest 5-year relative survival rates due to a combination of late stage diagnosis and treatment relapse. Autophagy, a stress response mechanism in which cancer cells recycle organelles, and proteins to generate the necessary nutrients and metabolic intermediates for survival, is thought to be one of the principal mechanisms of treatment resistance and relapse in cancer. Due to its relatively refractory nature to chemo/radiotherapy, our long-term goal is to identify molecular signatures and pathways of autophagy that can be targeted to improve the sensitivity of NSCLC to radiotherapy. H1299 +/-p53 cells were cultured to ~85% confluency, the media exchanged to serum-free DMEM, and treated with 6Gy of ionizing radiation. After 12 hours, secreted proteomes were collected, processed and analyzed by reverse-phase nanoLC-MS/MS (Q-Exactive, ThermoFisher). A total of 364 secreted proteins were identified, of which 163 were quantified in at least 2 replicates of one condition. 29 unique proteins were identified to be present in all samples except H1299 +p53 cells which were not treated with ionizing radiation. These proteins included HMGA1, LSM8, CAST, CAPZB, CHGB, GSS, and HSPA9 and were associated principally with protein secretion and chaperone activities. Functional enrichment analysis using FunRich revealed that the secretome was enriched for exosomal, lysosomal, cytoplasmic, and cytoskeletal proteins (>2 fold, p-value < 0.0001). Using ANOVA, 24 proteins were found to be significantly differentially secreted between IR treated and untreated +/-p53 cells. Amongst these proteins are GPI, TXNRD1, FAM3C, DSTN, CLEC11A, HSPA9, and ALDOA. Of the significant genes, only GPI and TXNRD1 showed an interaction effect i.e. the effect of p53 status on the expression of GPI and TXNRD1 in the secretome depends on IR treatment status. In the pairwise comparison between –p53 and +p53 cells treated with IR, proteins over-secreted in –p53 cells by ≥ 2 fold include LSM8, CANX, CHGB and SCG2 whereas GNB2L1 was over-expressed by >2 fold in +p53 cell secretomes. Calnexin, an ER chaperone protein that is associated with senescence, has been identified in previous studies as a sero-marker of lung cancer but not as a marker for distinguishing between the autophagic responses of NSCLC to IR. The novel functional and potential diagnostic value of these differentially secreted proteins as a function of p53 expression, irradiation and functional autophagy status in the context of NSCLC will be discussed.

#228

Identification of a novel biomarker to predict castration-resistant prostate cancer (CRPC) by the proteome analysis.

Hiroji Uemura,1 Noriaki Arakawa,2 Yusuke Itoh,3 Takashi Kawahara,1 Yasuhide Miyoshi,1 Masahiro Yao,3 Shouhei Akiba,4 Norihisa Ohtake5. 1 _Yokohama City University Medical Center, Yokohama, Japan;_ 2 _Yokohama City University, Yokohama, Japan;_ 3 _Yokohama City University Graduate School of Medicine, Yokohama, Japan;_ 4 _Tosoh, Tokyo, Japan;_ 5 _Tosoh, Yokohama, Japan_.

Prostate specific antigen (PSA) is useful for screening prostate cancer, while it has no reliable correlation with pathological malignancy of prostate cancer and is not a predictor for castration resistant prostate cancer (CRPC). In order to explore novel biomarkers to predict the development of CRPC, we performed proteomics from secreted proteins from human prostate cancer cells and identified the candidates of biomarker for CRPC.

Materials and Methods: The proteins secreted from 6 prostate cancers in culture medium were analyzed and compared with 8 other cancer cells including renal and urothelial cancers using LTQ Orbitrap mass spectrometer. With the focus on high tissue specificity, the candidate biomarker proteins were then identified through analysis of gene expressions in proteins common to human prostate cancers by real time qPCR. Next, a system to measure the identified mouse monoclonal antibodies against the focused proteins was established. Finally, serum levels of these proteins from 33 patients with benign prostate hyperplasia (BPH), 31 with untreated prostate cancer (PCa) and 35 with CRPC, were measured.

Results: The proteome analysis identified 12 candidates of secreted cell membrane proteins as new biomarkers. The proteome analysis indicated that not only matured GDF15, but pro-peptide as well as fragments (GDPP) are released from prostate cancer cells. Patients' serum was analyzed for matured and pro-peptide GDF15 using ELISA and immunoprecipitation-MRM mass spectrometry. The results showed that the serum level of GDDP-1, one of the processing forms of GDPP, was significantly higher in CRPC than those in BPH and untreated PCa (P<0.01). ROC analysis also showed that the AUC of GDPP-1(0.86) was higher than that of matured GDF15 (0.76). When the cutoff value of GDPP-1 was set at 4.0 ng/mL, there was a significant difference of overall survival (OS) in CRPC patients between those with more than 4.0 ng/mL compared to less than 4.0 ng/mL of GDPP-1, whereas there was no significant difference of OS measurable by PSA in CRPC patients. These data suggest that GDPP-1 may be a novel biomarker for CRPC.

Conclusion: GDPP-1 shows potential as a novel biomarker for CRPC.

#229

Establishing reference intervals of human urine proteome for monitoring physiological and pathological changes.

Wenchuan Leng,1 Xiaotian Ni,2 Changqing Sun,3 Anna Malovannaya,4 Yi Wang,4 Jun Qin1. 1 _National Center for Protein Sciences (The PHOENIX Center, Beijing), Beijing, China;_ 2 _East China Normal University, Shanghai, China;_ 3 _Tianjin Baodi Hospital, Tianjin, China;_ 4 _Baylor College of Medicine, Houston, TX_.

Urine as a true non-invasive sampling source holds great potential for biomarker discovery. But the lack of method for profiling urine proteome in high throughput and systematic evaluation of variations in urine proteomes based on large number of population have been being the major obstacles for successfully finding new biomarkers from urine. Due to low throughput, only limited number of urine samples can be measured in discovery phase of biomarker studies, which make it hard to determine whether proteins differentially expressed between groups represent actual differences between control and disease states or just physiological variations among individuals. So, candidate biomarkers often fail in validation phase. Here, we report a streamlined workflow with capacity of measuring 8 urine proteomes per day per MS at the coverage of more than 1500 proteins. With this workflow, we systematically evaluated variations in 497 human urine proteomes from 167 apparently healthy donors, allowing us to evaluate day to day and inter-personal variation in human urine proteome. Then personal and pan-human reference intervals (RIs) of urine proteome were established based on this large-scale dataset. We demonstrated that RIs can be used to monitor physiological changes by detecting transient outlier proteins, such as trans-continental travel and common flu. And it was also found that if the underlying cause is physiological variation, outlier proteins will fall back to the normal range in the follow up measurement, as exemplified in the intercontinental travel case. Persistent outlier proteins may be indicative of non-physiological conditions. These results indicate that periodical measurements of a person's urine proteome could establish a personal health archive that would be valuable for detecting future health issues. Furthermore, we proposed a complete novel strategy dependent on RIs-based algorithm for biomarker discovery and validation to screen for diseases, which were exemplified by analyzing 154 urine proteomes from patients with 7 types of cancers. The algorithm can distinguish normal people from cancer patients with specificity of 0.95 and sensitivity of 0.85. This study paves a way to use urine proteome for health monitoring and disease screening.

#230

Proteomic approaches in the discovery of novel drug targets or potential biomarkers in breast cancer.

Ioanna-Maria Orfanou, Theodoros Karampelas, George Mermelekas, Konstantinos Vougas, Constantin Tamvakopoulos. _Biomedical Research Foundation Academy of Athens, Athens, Greece_.

The aim of this study is the detection, identification and quantification of known and new candidate cell membrane receptors that are overexpressed in Breast Cancer (BC) cells. In order to achieve this goal we have developed State-of-the-Art Proteomic approaches. These novel membrane target receptors can be used either as biomarkers or for the design of targeted drugs against aggressive disease subtypes with poor prognosis and therapeutic outcomes, such as Triple Negative BC (TNBC) and HER2 overexpressing BC.

For our study, four well characterized BC epithelial cell lines were selected: HCC-1954 and SKBR3 (HER2 overexpressing), MDA-MB-231 (TNBC) and MCF-10A (benign control). We employed a combination of subcellular fractionation and membrane enrichment protocols and combined those with quantitative, tandem Mass-Spectrometry (MS) based Proteomics. The discovery phase of our approach included: the GeLC-MS/MS technique, where protein fractions were first separated by 1D-gel electrophoresis followed by In-Gel enzymatic digestion prior to identification by a high resolution Orbitrap mass analyser. A complementary approach for known proteins associated with BC that cannot be readily detected in the discovery phase, such as GPCR receptors, was based on nano LC-MRM analysis. Proof of Concept experiments, with the use of a Triple Quadrupole (QqQ) instrument, were based on the detection of standard peptides derived from the protein of interest after following an in solution tryptic digestion protocol.

Using GeLC-MS/MS approach we identified the well-known BC involved receptors, EGFR and HER2, as well as other potential protein targets in concordance with the literature (e.g. TFR1, EPHA2, GPCR5A). We further confirmed these results by Western blot analysis. Certain GPCRs expected to be present in the cell lines tested were not detected in the studies used on the GeLC-MS/MS analysis, thus we proceeded to the more sensitive and quantitative nanoLC-MRM approach. As a paradigm target we chose the GnRH receptor, an established target for personalized therapy known to be expressed in BC. The quantitative nanoLC-MRM approach revealed GnRHR significant expression in the MDA-MB-231 BC line and in the WPE-NB26-3 Prostate cancer cell line (used as an overexpressing GNRHR positive control).

Our results indicate that the strategy of combining and applying various Proteomic approaches into selected cell lines for the identification of new protein targets will add more information to the genetic and histological classification of the tumor in order to achieve our goal, which is a holistic view of a patient's molecular profiling that can ultimately lead to treatment in the most effective way. 

## PREVENTION RESEARCH:

### Diet and Cancer

#231

The polyphenolic plant lignan secoisolariciresinol diglycoside reduces mammary tumor growth, possibly via inhibition of local inflammatory signaling.

Claire G. Lineberger,1 Laura W. Bowers,1 Nikki A. Ford,2 Emily L. Rossi,1 Bruce K. Kimler,3 Carol J. Fabian,3 Stephen D. Hursting1. 1 _The University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 2 _The University of North Carolina at Chapel Hill, Austin, TX;_ 3 _The University of Kansas Medical Center, Kansas City, KS_.

Background: Secoisolariciresinol diglycoside (SDG) is a polyphenolic plant lignan found in flax and sesame seeds as well as legumes, whole grains, fruits and vegetables. It is metabolized by the gut bacteria into two major enterolignans: enterolactone (ENL) and enterodiol (END). These enterolignans have been associated with reduced breast cancer risk and progression in population studies as well as decreased tumor growth in preclinical models of breast cancer.

Methods: The impact of SDG supplementation on tumor growth in a mouse model of basal-like breast cancer was examined. C57BL/6 mice were fed a control diet (10% kcal from fat) or control diet with SDG supplementation (100 mg/kg food) for eight weeks, then both groups were orthotopically injected with E0771 mammary tumor cells. An inflammatory signaling qPCR array (Qiagen) was performed on mammary tissue distal to tumor. Tumors were stained by immunohistochemistry (IHC) for Ki67 to measure proliferation levels and phospho-p65 to determine inflammatory signaling pathway activation. Tumors and mammary tissue were also stained for F4/80 to quantify macrophage infiltration. Serum level of hormones, adipokines, and cytokines were measured via luminex assay (Bio-Rad).

Results: SDG supplementation significantly decreased tumor weight (p<0.05). SDG did not affect body weight or body fat percentage but did significantly decrease expression of F4/80, CRP, and other pro-inflammatory markers in the mammary tissue. IHC staining revealed no difference in tumor proliferation; however, SDG supplementation did reduce inflammatory signaling in the tumors, indicated by a significant decrease in phospho-p65 staining. However, serum cytokine levels were not significantly different between the groups. Tumors are currently being stained by IHC for cleaved caspase 3 to measure levels of apoptosis. In addition, cell culture experiments will be conducted to define the impact of ENL treatment on protumorigenic cross-talk between tumor cells, adipocytes, and macrophages. Specifically, the effects of conditioned media from adipocyte/macrophage co-cultures (with +/- ENL treatment) on mouse E0771 and human MDA-MB-231 tumor cell proliferation, migration and invasion will be examined.

Conclusions: SDG supplementation reduced mammary tumor growth in association with SDG's effects on local, but not systemic, inflammatory signaling.

#232

Modification of dietary sugar on the chemotherapeutic potential in breast cancer.

Yan Jiang, Patrea R. Rhea, Lorenzo Cohen, Peiying Yang. _UT MD Anderson Cancer Ctr., Houston, TX_.

Background: The estimated cost of breast cancer (BCa) care in the U.S. is over 16 billion dollars yearly, more than any other cancer. Thus, identifying contributors to BCa development and barriers in BCa treatment is a continuing focus of BCa research. The per capita consumption of sugar in Americans has surged to 70 lb per year, and an increase in the consumption of added sugars is identified as a pivotal contributor to worldwide epidemics of non-communicable diseases including cancer. However, whether dietary sugar affects the efficacy of the chemotherapy in BCa is questionable. In current study, we investigated the impact of glucose or sucrose on the chemotherapeutic effect of chemo-agents, especially doxorubicin, in the growth of BCa cells or tumors.

Methods: BCa cell (human MDA-MB-231, SUM159, T47D, BT474, MDA-MB-468 and MCF-7) proliferation was assessed in 3-D culture system. BALB/C nu/nu mice were used to investigate tumor growth with/without doxorubicin treatment upon feeding sucrose enriched diet. Apoptotic gene array and microarray analysis were used to determine gene expression profile.

Results: Among six human BCa cells tested, glucose treatment (30 mM) promoted the growth of MDA-MB-231 and SUM159 cells after 10 days by 2.5 to 3 fold, respectively. In contrast, T47D, BT474, MDA-MB-468 and MCF-7 cells did not respond to glucose stimulation. Glucose treatment abrogated doxorubicin-induced cell apoptosis in triple negative BCa cells including MDA-MB-231 and SUM159 cells, and 4T1 (mouse) cells. Additionally, the apoptotic effect of chemotherapeutic agents, such as epirubicin, 5-FU and cyclophosphamide in MDA-MB-231 cells were also blocked by glucose treatment. Furthermore, gene expression analysis demonstrated that glucose significantly altered apoptotic genes in doxorubicin treated MDA-MB-231 cells. Compared to doxorubicin alone, glucose/doxorubicin co-treatment decreased a number of pro-apoptotic gene expression of including PEA15, BAX, and BcL2L11; while increased anti-apoptotic gene NFκB family. Finally, in mice bearing MDA-MB-231 cell derived tumors, doxorubicin (4 mg/kg body weight weekly for 4 weeks) inhibited the growth primary tumors by 50% compared to that in control mice. In the same study, for the mice fed with sucrose diet, treatment of doxorubicin only reduced the tumor growth by less than 20% compared to control diet fed mice without doxorubicin treatment, suggesting that dietary sugar might negatively impact the chemotherapeutic agent in BCa.

Conclusion: The results of our study indicated that glucose (sucrose) altered BCa responses to doxorubicin in vitro and in vivo. Therefore, dietary sugar may potentially interfere with the efficacy of chemotherapeutic agents in BCa patients which warrants further investigation. This study was support by a generous donation of Leighton and Lynda Steward, The William A. and Madeline Welder Smith Foundation and the University of Texas Startup Fund.

#233

The impact of omega-3 fatty acid supplementation on obesity-induced inflammatory signaling within the breast tumor microenvironment.

Duan K. Quach,1 Brittany Harlow,1 Laura Winikka,1 Andrew Brenner,2 Murali Beeram,3 Stefano Tiziani,1 Lucy Lengfelder,1 Gloria Galvan,1 Christopher Jolly,1 Linda deGraffenried1. 1 _The University of Texas at Austin, Austin, TX;_ 2 _UT Health San Antonio MD Anderson, San Antonio, TX;_ 3 _The START Center for Cancer Care, San Antonio, TX_.

The Impact of Omega-3 Fatty Acid Supplementation on Obesity-induced Inflammatory Signaling within in the Breast Tumor Microenvironnment. Obesity is associated with a worsened prognosis in breast cancer. This is in part due to the role of prostaglandin E2 (PGE2) in the obesity-inflammation-aromatase axis. Omega-3 polyunsaturated fatty acids (PUFAs) have demonstrated anti-cancer effects through multiple pathways, including suppression of the pro-inflammatory COX2-PGE2 pathway. In order to determine if supplementation with omega-3 fatty acids can effectively suppress PGE2 production in obese postmenopausal women, we conducted a 30 day non-interventional study with correlative biomarker endpoints. Forty (40) postmenopausal women were provided oral daily supplements of 1500mg of docosahexaoic acid (DHA) and 2500mg eicosapentanoic acid (EPA). Serum samples were collected prior to and on day 29 of taking the supplement and analyzed for PGE2 levels. Fifty-five percent (55%) of the subjects demonstrated a significant suppression of PGE2 levels. To test if response could be based upon the ratio of omega-6 to omega-3 fatty acids on inflammatory signaling within the breast, pre-clinical studies were performed on different cell types found in the tumor microenvironment. Macrophages, breast cancer epithelial cells and pre-adipocytes were exposed to omega-6 and omega-3 fatty acids at ratios of 46:1, 20:1, 10:1 and 1.3:1 for 24 hours. While breast cancer epithelial cells demonstrated limited response to PUFA concentrations, the macrophage and adipocyte cells produced high levels of PGE2 when exposed to higher ratios of omega-6 fatty acids, which was effectively suppressed in a dose-dependent manner with increasing levels of omega-3 fatty acids. These data suggest that obese breast cancer patients may have a particular benefit to omega-3 fatty acid supplementation. Ongoing studies will assess how PUFA-modulated changes in inflammatory signaling from different cells within the microenvironment impact breast cancer cell proliferation, therapeutic resistance, and migration as measures of breast cancer progression. These mechanistic studies, in combination with our on-going NCI-funded prospective clinical study in newly diagnosed breast cancer patients, will significantly contribute to understanding how ratios of omega-6 to omega-3 fatty acids can modulate inflammatory signaling within the tumor microenvironment, and if this can be used to improve therapeutic response in the obese breast cancer population.

#234

Association between fecal microbiome, diet, and colon adenomas and hyperplastic polyps.

Yang Yu,1 Joshua Millstein,1 Amie E. Hwang,2 Bing Ma,3 Guoqin Yu,4 Laura H. Buchanan,5 Michael Humphreys,3 Ann S. Hamilton,5 John Zadnick,6 Myles G. Cockburn,7 James Buxbaum,2 Heinz-Josef Lenz,5 Thomas M. Mack,5 Jacques Ravel,3 Wendy Cozen5. 1 _Keck School of Medicine of USC, University of Southern California, Los Angeles, CA;_ 2 _University of Southern California Keck School of Medicine, Los Angeles, CA;_ 3 _University of Maryland, Baltimore, MD;_ 4 _National Cancer Institute, National Institutes of Health, Bethesda, MD;_ 5 _USC/Norris Comprehensive Cancer Ctr., Los Angeles, CA;_ 6 _University of Southern California, Los Angeles, CA;_ 7 _University of Colorado Cancer Center, Denver, CO_.

Background: Colorectal cancer arises from adenomatous and serrated colon polyps that are identifiable with colonoscopy. Generally, a healthy gut is characterized by higher fecal microbiota. We conducted a study to determine whether fecal microbiota composition and structure are associated with colon adenomas and hyperplastic polyps in a set of identical (MZ, monozygotic) twins.

Methods: We enrolled 83 individual twins from the California Twin Program. Of these, 56 (representing 38 twin pairs) had documented past colonoscopy and no gastrointestinal diseases, cancer or recent antibiotic use. Body mass index (BMI) and food frequency information was collected 15 years before stool collection and again at the time of stool collection. The V4 region of the 16S rRNA gene was sequenced using the HiSeq 2500 system. Alpha diversity measures including number of OTU (operational taxonomic units) and Shannon index were calculated for each sample. Mixed ANOVA models with a random effect accounting for twin pair status, were used to examine the association of alpha diversity with polyp status.

Results: Alpha diversity measured by Shannon index and number of unique OTUs was inversely associated with obesity and high beef consumption and positively associated with yogurt consumption. Surprisingly, we found that fecal microbiota alpha diversity was higher in subjects with adenomas and hyperplastic polyps compared to subjects with no polyps. When restricted to subjects with colonoscopy in the 3 years prior to stool collection, differences were stronger and statistically significant (mean OTUs for subjects with adenoma, hyperplastic polyps and no polyps = 908, 850, and 801, respectively; Padenoma vs. no = 0.017, Phyperplastic vs no = 0.007; Plinear trend= 0.018). In order to determine whether diet changes may have played a role, we examined dietary change from food frequency questionnaires collected 15 years apart. We found that subjects with no polyps increased yogurt consumption by 50% over the 15 year period, whereas subjects diagnosed with adenomas and hyperplastic polyps increased their yogurt consumption more (140% and 130%, respectively).

Conclusion: Participants diagnosed with adenomas and hyperplastic polyps had higher fecal microbiota alpha diversity compared to those without polyps. The timing of measurement of fecal microbiota years after colonoscopy obscures the causal relationship between adenoma and fecal microbiome. One possibility is that subjects may have increased their yogurt consumption after a polyp diagnosis, resulting in increased fecal alpha diversity, compared to subjects who were not diagnosed with polyps. Alternatively, polyp removal may alter microbial diversity. A third possibility is that polyp susceptibility is associated with an outgrowth of deleterious bacteria, still present after polyp removal, reflected as increased alpha diversity. Twin comparisons will be presented.

#235

**Probiotics ameliorate** Porphyromonas gingivalis **-promoted pancreatic cancer progression in oncogenic** Kras **transgenic mice.**

Ming-Shiou Jan,1 Wan-Ting Chen,1 Yu-Jen Chen,1 Chia-Wei Lin,1 Wen-Wei Chang,1 Chung-Hung Tsai,1 Jia-Shiou Peng,2 Li-Jin Hsu3. 1 _Chung Shan Medical University, Taichung, Taiwan;_ 2 _Chung-Jen Junior Collerge of Nursing, Health Science and Management, Chiayi, Taiwan;_ 3 _National Cheng Kung University, Tainan, Taiwan_.

Periodontal disease has been shown to play important roles in the pathogenesis of many chronic diseases. Recent epidemiological studies and microbiome analysis have suggested that periodontal disease may be associated with the progression of pancreatic cancer. Our recent study has demonstrated that oral-smear of a periodontitis pathogen Porphyromonas gingivalis (P. gingivalis) significantly induced pancreatic cancer deterioration in the KrasG12D/-Pdx-1-Cre transgenic mouse model. Higher expression levels of epithelial-mesenchymal transition (EMT) markers, such as N-Cadherin, αSMA, SNAIL-1, Vimentin and ZEB1, were observed in P. gingivalis-treated KrasG12D/-Pdx-1-Cre mice than the untreated controls. In an attempt to develop a prophylactic treatment for pancreatic cancer, the mice were fed with a combination of two probiotics, Lactobacillus reuteri and Lactobacillus paracasei, for four consecutive weeks after oral-smear of P. gingivalis. These probiotic strains have been demonstrated to have the efficacy of either anti-inflammation or enhancement of cell-mediated immunity, respectively. Our results demonstrated that oral administration of the probiotics significantly suppressed oncogenic Kras-induced pancreatic cancer development and reversed P. gingivalis-stimulated cancer deterioration in mice. Significantly reduced expression of the aforementioned EMT markers in pancreatic cancer tissues was observed in KrasG12D/-Pdx-1-Cre transgenic mice treated with the probiotics. Our results suggest that the microbiota profile is very important for pancreatic cancer development. The manipulation of microbiota for enhancing antitumor immunity may be an emerging strategy for cancer prevention.

#236

Stearoyl-CoA desaturase-1, a novel target of omega-3 fatty acids for reducing breast cancer risk in obese postmenopausal women.

Andrea Manni, John P. Richie, Susann E. Schetter, Ana Calcagnotto, Neil Trushin, Cesar Aliaga, Karam El-Bayoumy. _Penn State Univ. Hershey Medical Ctr., Hershey, PA_.

Preclinical and epidemiologic data suggest that a unique feature of lipogenesis in cancer cells which has received limited attention is the accumulation of monounsaturated fatty acids (MUFA) which are largely derived from saturated fatty acids (SFA) by the action of stearoyl-Co-A desaturase (SCD-1). Activation of SCD-1, a delta-9 fatty acid desaturase, is considered to be an important factor in the development of obesity and several types of cancer including breast cancer. However, no data are available on how changes in SCD-1 activity induced by potential chemopreventive agents relate to established biomarkers of breast cancer risk. To address this issue, we measured the activity of SCD-1, expressed as the ratios of palmitoleic acid (C16:ln7) to palmitic acid (C16:0) (SCD-16) and oleic acid (C18:ln9) to steric acid (C18:0) (SCD-18) in plasma samples of postmenopausal women enrolled in our recently published clinical trial (Sandhu N, et al Cancer Prev Res 9:275, 2016) designed to test the individual and combined effect of the antiestrogen Raloxifene and the omega-3 preparation Lovaza on breast density, a validated biomarker of breast cancer risk. We observed that daily administration of Lovaza (1,860 mg eicosapentaenoic [EPA] + 1,500 mg docosahexaenoic [DHA]) significantly reduced SCD-1 activity, an effect which was sustained for the two-year duration of the trial. Raloxifene, on the other hand, did not significantly alter SCD-1 activity in our subjects. SCD-1 activity was positively correlated with BMI (for SCD-16, r=0.45, p<01; for SCD-18, r=0.23, p<0.01) and paralleled changes in BMI in the same direction over the two years of the study. These findings support the role of this enzyme in the development of obesity. Importantly, decreasing levels of SCD-1 were found to be associated with a progressive reduction in breast density in obese women (BMI≥30) (for SCD-16; r=0.47, p<0.01; for SCD-18; r=0.36, p<0.05). No correlation between breast density and SCD-1 was observed in non-obese subjects (r=0.02 for SCD-16 and 0.04 for SCD-18). Our results suggest that BMI-related factors play an important role in the reduction of breast density by omega-3 fatty acids. They also indicate that SCD-1 may be a useful biomarker in future clinical trials testing the benefit of nutritional interventions in reducing obesity associated breast cancer risk.

#237

Increased NSCLC tumorigenesis in mice fed a high fat diet is associated with increased plasma IGF-1 levels and PD-1 expression in CD4+ tumor-infiltrating lymphocytes.

Regan M. Memmott,1 Krista Pearman,2 Joell Gills,3 Tony Tullot,2 Valerie Wong,2 Benjamin Singer,4 Kristin Lastwika,5 Franco D'Alessio,6 Phillip Dennis,7 Jeffrey William Norris2. 1 _University of Arizona Phoenix, Phoenix, AZ;_ 2 _Midwestern Univ. - Glendale Campus, Glendale, AZ;_ 3 _Johns Hopkins Medical Institute, Baltimore, MD;_ 4 _Northwestern University, Evanston, IL;_ 5 _Fred Hutchinson Cancer Research Institute, Seattle, WA;_ 6 _Johns Hopkins University - School of Medicine, Baltimore, MD;_ 7 _Astra-Zeneca, MD_.

Lung cancer is the leading cause of cancer-related mortality worldwide, and 85% of lung cancer cases are associated with tobacco use. Activating mutations in K-ras have been identified in ~25% of tobacco-associated lung adenocarcinomas. Using mouse models of K-ras-driven lung tumorigenesis, we previously demonstrated that deletion of the IGF-1 gene or reduction of systemic IGF-1 levels using the antidiabetic drug metformin markedly reduced tumor burden. Preclinical and clinical studies suggest that diet composition is the best predictor of IGF-1 levels. Therefore, we hypothesized that diets high in fat or carbohydrate would promote lung tumorigenesis by increasing systemic IGF-1 levels. To assess the effect of diet on systemic IGF-1 levels, 9 week old C57Bl/6J and A/J mice were fed standard cereal, high-carbohydrate, or high-fat (HFD) diets for 12 weeks. Compared to cereal-fed control mice, plasma IGF-1 and insulin levels were increased in both strains of mice fed a HFD, but not in mice fed a diet high in carbohydrate. This was not due to obesity, as only the C57Bl/6J mice fed a HFD had an increase in body weight. We then investigated the effect of HFD on lung tumorigenesis using two mouse models. In the first, C57Bl/6LA2 mice, which harbor a mutation in K-ras, were fed either cereal diet or HFD for 10 weeks following weaning. Lung tumor burden in the mice fed HFD was increased 2.7-fold compared to littermates fed cereal diet. In the second model, the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1 butanone (NNK) was given by intraperitoneal injection to A/J mice beginning at 6 weeks of age. This carcinogen causes lung tumor development by inducing K-ras mutations. After 3 weekly injections of NNK, the mice were randomized to cereal diet or HFD for ten weeks. Mice fed HFD had a 60% increase in lung tumor burden. In both models, there was no relationship between the weight of the mice and lung tumor burden. Immunohistochemical analysis of the proliferation marker Ki-67 showed no significant difference in expression between tumors from mice fed a HFD or a cereal diet. Therefore, we evaluated tumor-infiltrating lymphocytes (TIL) from mice on both diets. Interestingly, both immunohistochemical analysis and flow cytometry demonstrated a 50% reduction in the number of TIL in mice fed HFD compared to mice fed a cereal diet. Additionally, HFD was associated with a 2-fold increase in PD-1+ CD4+ TIL, and the PD-1 staining intensity in these lymphocytes was significantly greater than in mice fed a cereal diet. These results may suggest that a high fat diet increases lung tumorigenesis by increasing systemic IGF-1 levels and by creating an immune-permissive tumor microenvironment.

#238

Evidence of early colorectal cancer risk and prevention pathways in the fecal microbiome of colonoscopy patients: associations with diet and circulating adipocytokines.

Carrie R. Daniel,1 Kristi L. Hoffman,1 G S. Raju,1 Samir M. Hanash,1 Diane S. Hutchinson,2 Nadim J. Ajami,2 Richard G. Fowler,1 Gladys J. Browman,1 Akhil Sood,1 Paul Scheet,1 Xifeng Wu,1 Joseph F. Petrosino2. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Baylor College of Medicine, Houston, TX_.

Accumulating evidence suggests that the gut microbiome's role in early colorectal cancer etiology extends beyond the pro-carcinogenic activities of specific pathogens and is largely influenced by the wider microbial community of commensal bacteria. To identify early microbiome-related pathways and potential dietary intervention targets, we conducted an epidemiologic study among cancer-free colonoscopy patients at known and varied risk of colorectal neoplasia. Sporadic patients undergoing colonoscopy screening provided consent and fasting blood. Polyps, if found, were removed at colonoscopy and ~1 month later (prior studies show the effect of the colonoscopy prep dissipates within this time period), eligible patients provided a stool sample via mail kit (n=47). Patients completed comprehensive dietary assessments and clinicopathologic factors, including screening history, were abstracted from electronic medical records. We characterized the fecal microbiome via whole genome shotgun sequencing (Illumina HiSeq) and circulating blood adipocytokines via Luminex multiplex assays (Millipore). Majority of the patients were female and recently diagnosed with precancerous polyps, primarily tubular and sessile serrated adenomas. Polyp patients were more likely to be obese and to not consume alcohol, but otherwise similar by age, diet composition, and other risk factors to patients with normal colonoscopy findings. We observed no effects of time interval between colonoscopy and fecal sample collection, or of recent polyp diagnosis/removal, on microbial alpha or beta diversity. Correlation networks between species revealed that Faecalibacterium prausnitzii inversely correlated with E. coli, the most abundant species in our sample, and co-occurred with other short chain fatty acid-metabolizing bacteria, including Anaerostipes hadrus and Roseburia hominis, known butyrate-producers. Among recurrent patients (n=14), Bacteroides fragilis co-occurred with Bilophila wadsworthia (r=0.6; P=0.02). Higher intake of fiber and/or overall diet quality, as defined by the Healthy Eating Index, was associated with several bacteria largely linked to butyrate production (e.g., Bifidobacterium animalis, F. prausnitzii, Roseburia intestinalis, Coprococcus eutactus, Eubacterium eligens). These same dietary factors were inversely correlated with Blautia hydrogenotrophica, an acetogen, and other bacteria implicated in inflammation and colorectal cancer (e.g., B. fragilis). Biologically plausible associations between microbial (functional gene content) pathways important to cancer risk (e.g., one-carbon, lysine, carbohydrate and fatty acid metabolism) with dietary factors and circulating adipocytokines involved in immunity, inflammation, and glucose metabolism support the functionality of these diet-microbe relationships.

#239

Associations of polyunsaturated fat, bile acids, and gut microbiota on colorectal adenoma risk.

Tengteng Wang, Nicole M. Brown, Amber N. McCoy, Robert S. Sandler, Temitope O. Keku. _University of North Carolina at Chapel Hill, ChapeL Hill, NC_.

Background: Intestinal microbiota, and their metabolites are increasingly recognized as important players in the genesis of colorectal adenomas and cancer. Epidemiological studies have also shown that polyunsaturated fat intake is associated with decreased risk of colorectal adenomas. This study examined the association between adherent gut microbiota and mucosal bile acids, and total polyunsaturated fat intake in relation to colorectal adenomas in a case-control study.

Methods: Participants were 217 adenoma cases and 218 adenoma-free controls who underwent screening colonoscopies and completed food frequency questionnaires. The mucosal adherent microbiota was characterized by bacterial 16S rRNA sequencing. Global metabolomics of colonic mucosal tissue was conducted by gas chromatography and mass spectrometry on a subset of samples. Genus level microbiota and bile acids were compared between cases and controls using t-test with correction for multiple comparison. Logistic regression and Pearson correlations were used to estimate adjusted odds ratios (ORs) and 95% confidence intervals (CIs), and correlation coefficients for the association among microbiota abundance, bile acids concentration, and total polyunsaturated fat intake.

Results: Adenoma subjects had a significantly higher bacterial abundance of Sphingomonas, Marinomonas and Ralstonia, but lower abundance of Adlercreutzia compared to controls. The concentration of eleven bile acids (Chenodeoxycholate, Cholate, Deoxycholate, Glycochenodeoxycholate, Glycocholenate sulfate, 7-ketodeoxycholate; 12-dehydrocholate, Taurochenodeoxycholate, Taurocholate, Taurodeoxycholate, Taurolithocholate 3-sulfate, and Tauroursodeoxycholate) were significantly elevated in cases compared to controls. Subjects with increased abundance of Sphingomonas and Ralstonia were significantly more likely to have adenomas (OR 2.17, 95% CI 1.31-3.57; OR 1.19, 95% CI 1.02-1.38) respectively. Subjects with increased abundance of Adlercreutzia were less likely to have adenomas (OR 0.44, 95% CI 0.21-0.93). The multivariable adjusted OR for those in the high relative to the low level of total polyunsaturated fat intake was 0.46 (95% CI 0.27-0.80). The correlations between bacterial diversity and eleven bile acids also significantly differed by case control status.

Conclusion: The interactions of the gut microbiota, bile acids and consumption of polyunsaturated fat are associated with colorectal adenoma risk.

#240

Dietary patterns and risk of colorectal neoplasia in Puerto Rican Hispanics: A case control study.

Julyann Perez-Mayoral,1 Sachelly Julian,2 Marievelisse Soto-Salgado,2 Michael J. Gonzalez,2 Marcia Cruz-Correa1. 1 _University of Puerto Rico Comprehensive Cancer Center, San Juan, PR;_ 2 _University of Puerto Rico Medical Sciences Campus, San Juan, PR_.

Colorectal cancer (CRC) is the leading cause of cancer death in Puerto Rico and second cause of death in the US. It is well accepted that modifiable lifestyle habits such as diet, contribute to CRC development. Diet could reduce up to 70% of CRC cases. Dietary patterns vary according to race/ethnicity. Puerto Rican Hispanics (PRH) have been shown to have a distinct diet compared to US non-Hispanic Whites (NHW). According to the BRFSS, only 17.7% of PRH and 23.4% of NHW consume fruit and vegetables five times per day. The objective of this study was to describe the association of specific food groups consumption with the risk of colorectal neoplasia (CRN: polyps and/or CRC) in PRH. The validated Colon Cancer Family Registry food frequency questionnaire was administered to healthy individuals (controls) and individuals with CRN (cases) recruited through the Puerto Rico Colorectal Cancer Registry (PURIFICAR). Models adjusted by age, gender, educational attainment and family history of CRC were fitted to estimate the odds ratios (OR) with 95% confidence intervals (CI) through a polytomous logistic regression models comparing cases and controls. A total of 577 subjects were included in the study (controls=254; polyps=50; and CRC=273). The frequency of consumption of fruits, low fat dairy products, eggs, pasta/rice, non-fried chicken, and seafood were significantly different between controls and CRN cases (p<0.05). Adjusted models showed that a higher consumption of fruits (OR≥once per day=0.29) and full-fat dairy products (OR≥once per day=0.22) were associated with decreased risk of polyps (p<0.05). A frequency of consumption of 2-4 times per week, when compared to ≤ 1 per week, of full-fat dairy products (OR2-4 times per week=0.51), seafood (OR2-4 times per week=0.49), and red meats (OR2-4 times per week=0.56) was associated with a decreased risk of CRC (p<0.05). Our results showed that a higher consumption of fruits and full-fat dairy products significantly reduce the risk of polyps in PRH. In addition, consumption of seafood, full-fat dairy, and red meats up to 4 times per week may reduce the risk of CRC in PRH. Educational interventions for cancer prevention in PRH should incorporate nutritional information tailor to our community.

#241

Vimentin offers a potential target for the prevention of ovarian tumor progression and recurrence.

Aparna Yellapa,1 Janice M. Bahr,2 Sameer Sharma,1 Pincas Bitterman,1 Sanjib Basu,1 Animesh Barua1. 1 _Rush University Medical Center, Chicago, IL;_ 2 _University of Illinois at Urbana-Champaign, Urbana-Champaign, IL_.

Background: Aggressive rates of progression and frequent recurrence are the main causes of high rates of death due to ovarian cancer (OVCA). Vimentin, an intermediate filament protein, expressed by ovarian mesenchymal cells may be involved in OVCA metastasis and recurrence. Ashwagandha (Withania somnifera, ASH, a natural product) has shown to improve tumor-associated stress against several malignancies.

Objectives: The goals of this study were to examine whether (1) OVCA progression is associated with vimentin expression; and whether (2) OVCA progression and vimentin expression can be prevented by dietary supplementation of Ashwagandha (ASH) in laying hen model of spontaneous OVCA.

Methods: Exploratory study: Clinical specimen from normal ovaries (n=10), benign tumors (n=5) and malignant ovarian epithelial cancers at early (n=7) and late stages (n=16) were collected from patients and examined for vimentin expression. Prospective study: 4-year old laying hens with normal ovaries or ovaries with early stage OVCA were supplemented with 2% dietary ASH root powder or basal diet (24 hens in each group containing 18 healthy, 6 with early stage OVCA). Hens were provided with feed and water ad libitum for 120 days and euthanized thereafter. Tumor stages were recorded; serum, normal or cancer ovaries were collected. Expression of vimentin both in clinical and preclinical treated or untreated specimens was examined. Statistical differences in the intensity of vimentin expression among different pathological and ASH supplemented groups were determined using ANOVA and t-tests.

Results: Vimentin was localized in the nucleus and cytoplasm of normal ovarian, benign and malignant cells. Compared with normal and benign tumors, the intensity of vimentin expression was significantly (P<0.0001) lower in early stage OVCA and increased remarkably in late stage OVCA. Similar to OVCA patients, intensity of vimentin expression was lower in hens with early OVCA than normal hens, and increased significantly (P<0.0001) in hens with late stage OVCA. As compared with untreated hens, tumor incidence and progression as well as vimentin expression decreased significantly in hens treated with 2% dietary ASH.

Conclusions: Results of the present study suggest that vimentin expression increased during tumor metastasis. ASH treatment reduced OVCA metastasis by inhibiting vimentin expression. Therefore, vimentin may represent a potential target for ASH to prevent OVCA progression and metastasis. Support: Support: R21CA187309-02.

#242

The anti-tumor effect and the impact on gut microbiota of gypenosides Rd and Rb3 in ApcMin/+ mice.

W.L. Wendy Hsiao,1 Guoxin Huang,1 Imran Khan,1 Lei Chen,2 Xiaoang Li1. 1 _State Key Lab of Quality Research in Chinese Medicine, Macau Univ of Sci. & Tech, Macau, China; _2 _Rutgers University, Piscataway, NJ_.

Gut commensal microbiota (GM) is closely associated with health and diseases, including cancer. In our previous studies, we have demonstrated that Gynostemma pentaphyllum saponins (GpS) exert anticancer effects in both athymic and ApcMin/+ mice. Our recent study also showed that GpS exhibited prebiotic effect by up regulating the beneficial bacteria and improving the epithelial microenvironment of the gut. Gypenoside IV (Rb3) and gypenoside VII (Rd) are the two constituents of GpS. The Rd exhibits stronger anticancer effect than Rb3 in an in vitro cell model system established in our lab. Our aim is to confirm the antitumor effect of these two compounds, as well as their impact on GM in the ApcMin/+ mouse model. In the study, single daily dose of Rb3 or Rd at 20mg/kg, and solvent control were orally fed to the mice for 8 weeks, started at 6 weeks of age before the appearance of spontaneous intestinal polyps. The fecal microbiome was analyzed by enterobacterial repetitive intergenic consensus (ERIC)-PCR and 16S rRNA gene pyrosequencing technique. We found that Rd was more effective than Rb3 in reducing polyp formation, and improving the intestinal lining by up-regulating anti-inflammatory cytokines IL-4 and IL-10. Rd, not Rb3, might facilitate intestinal tissue repair by enhancing M2 marker while suppressing M1 markers expression. Moreover, Rd treatment greatly enhanced the ratio of Bacteroidetes vs Firmicutes, while profoundly reduced the abundance of cancer-cachexia-associated bacteria, such as Parabacteroides goldsteinii (22.25%), Dysgonomonas wimpennyi (48.54%) and Blautia coccoides (36.77%). These findings suggested that the level of antitumor effect of the gypenoside might be correlated with its ability to modulate the gut microbiota from dysbiosis state to symbiosis.

This study was supported by Macau Science and Technology Development Fund 015/2014/A1.

#243

Diet and exercise-induced weight maintenance, alone and in combination with a whole tumor cell vaccine, delays mammary tumor growth and reduces MDSC accumulation.

Yitong Xu, William J. Turbitt, Andrea M. Mastro, Connie J. Rogers. _Pennsylvania State University, University Park, PA_.

Obesity and physical inactivity increase breast cancer risk, while the prevention of weight gain by diet and exercise can be protective. Numerous biological mechanism(s) have been proposed to explain the beneficial effects of weight maintenance; however few studies have examined the immune response to energy balance. We have previously shown that diet and exercise-induced weight maintenance (WM) achieved via a 10% restriction in calories and access to voluntary running wheels in combination with a whole tumor cell vaccine (VAX) significantly reduced mammary tumor growth and metastases in the 4T1 mammary tumor model. This WM-induced reduction in tumor growth occurred concurrently with an elevation in tumor specific IFN-γ production and a reduction in the number of myeloid-derived suppressor cells (MDSCs). However, because tumor size is positively correlated with immune suppression, the goal of the current study was to investigate if mice in the WM+VAX intervention had enhanced anti-tumor immune responses and/or fewer MDSCs controlling for tumor size. Female BALB/c mice were randomized into weight gain (WG) and WM groups (n=8/group) and had access to voluntary running wheels or standard cages, respectively. WG mice were fed ad libitum while WM mice were energy-restricted by 10% to maintain a stable body weight. After 8 weeks on the intervention, all mice were orthotopically injected with 5x104 4T1.2 cells into the fourth mammary fat pad and continued on their intervention. Once injected, both WG and WM mice were further randomized into vaccination (VAX) and vehicle control (VEH) groups (n=4/gr) and administered 1x106 irradiated 4T1.2 cells (VAX) or HBSS (VEH) at day 7 post-tumor implantation. Primary tumor growth was quantified, and mice were sacrificed when tumor volume reached 0.1-0.2 cm3. WM mice weighed significantly less than WG mice over the course of the study (p<0.001). Mice in both WM+VEH and WM+VAX groups took a significantly longer number of days post tumor implantation to reach a tumor size of 0.1-0.2 cm3 (26.3 ± 3.3 d, p=0.003; 23.8 ± 0.5 d, p=0.039, respectively) compared to the WG+VEH group (18.3 ± 2.5 d). Despite the fact that mice were sacrificed when the tumor volumes were standardized at 0.1-0.2 cm3, the combination of WM+VAX resulted in a significant reduction of splenic MDSC accumulation (p=0.049) and enhanced CD4+ T cell proliferation ex vivo. These results suggest that diet and exercise-induced WM was highly effective in delaying primary mammary tumor growth. The combination of WM and an allogenic whole tumor cell vaccine reduced pro-tumorigenic MDSC accumulation and enhanced effector T cell function. Furthermore, these data suggest that diet and exercise may be changing the tumor microenvironment at an early stage of tumor growth favoring tumor clearance. This work is supported by R21 CA209144; T32AI074551.

#244

Eicosapentaenoic acid reduces effects of some adipocyte derived factors on breast cancer cell inflammation and glucose metabolism.

Arwa Aljawadi,1 Sara Alhaj,1 Suranganie Dharamawardhane,2 Shane Scoggin,1 Lauren Gollahon,1 Preethi Gunaratne,3 Naima Moustaid-Moussa1. 1 _Texas Tech Univ., Lubbock, TX;_ 2 _University of Puerto Rico Medical Sciences Campus, San Juan, PR, Puerto Rico;_ 3 _University of Houston, Houston, TX_.

Breast cancer is still the leading cause of death in women among all cancer types. Obesity, a chronic low grade inflammatory disease, is a key contributor to the progression of breast cancer especially in women, post-menopause. Given the current understanding of cancer-related inflammation and associated microvesicles in restructuring the microenvironment, we hypothesized that adipocyte-derived cytokines and exosomes negatively impact breast cancer progression. By contrast, eicosapentaenoic acid (EPA), a dietary omega 3 polyunsaturated fatty acid and a well-established anti-inflammatory compound will reduce adipocyte-secreted pro-inflammatory factors, thereby reducing breast cancer progression. To test these hypotheses, we investigated the effects of conditioned media or exosomes from 3T3-L1 adipocytes or human mesenchymal stem cells (HMSC), treated +/- 100uM EPA on MCF7 and MDA-MB231 breast cancer cells for 24 – 72 h. Following treatment, changes in breast cancer cell gene expression were measured using RT-qPCR and glycolytic rate was measured using XFe24 Seahorse extracellular flux analyze. We observed that conditioned medium from HMSC significantly increased the mRNA expression levels of oncogenic genes such as signal transducer and activator of transcription 3 (STAT3), baculoviral IAP repeat-containing protein3 (BIRC3) known as cIAP2, and the lipogenic fatty acids synthase (FASN). In contrast, conditioned media from EPA-treated human adipocytes reduced the expression levels of these genes. Similarly, exosomes isolated from EPA-treated adipocytes showed a significant reduction in mRNA expression levels of STAT3 and cIAP2 in both cancer cell lines. Furthermore, glycolysis was significantly reduced in MCF7 but not MDA-MB231 cells incubated with 3T3-L1 adipocyte-conditioned medium pretreated with EPA for 24 hrs. Taken together, our data suggest that adipocytes play a significant role in promoting breast cancer progression by providing a microenvironment that increases survival and inflammation. EPA is a promising anti-inflammatory nutrient that may help reduce breast cancer cell inflammation and survival, possibly by modulating adipocyte-derived cytokines and exosomes in obesity and thus warrants further investigations.

#245

Dietary long-chain omega-3 fatty acids reduce adipose inflammation in mammary tissue of mice fed moderate fat-isocaloric diets.

Saraswoti Khadge,1 Geoffrey M. Thiele,1 John Graham Sharp,1 Lynell W. Klassen,1 Timothy R. McGuire,1 Michael J. Duryee,1 Holly C. Britton,1 Alicia J. Dafferner,1 Jordan Beck,2 Paul Black,2 Concetta C. DiRusso,2 James E. Talmadge1. 1 _Univ. of Nebraska Medical Ctr., Omaha, NE;_ 2 _Univ. of Nebraska,-Lincoln, Lincoln, NE_.

Increased adipose tissue Inflammation and breast density; including ductal epithelial hyperplasia have been associated with increased risks for breast cancer. Omega 6 (ω6) and omega 3 (ω3) fatty acids (FAs); serve as substrates for pro-inflammatory and inflammation resolving mediators respectively, emphasizing the potential regulatory role for dietary intake of these FAs in inflammation. Western diets have a ω6:ω3 FA ratio of >15:1 with low levels of long-chain (LC)-ω3FA. White adipose tissue inflammatory foci, characterized by crown-like structures (CLS) consisting of dead adipocytes and adjacent macrophages in breast tissue have been related to breast cancer risk in overweight and obese women presumably by the obesity-inflammation-aromatase axis. However, a role of dietary ω6:ω3 FA in adipose inflammation, independent of obesity is not clear. Herein, we examined effects of dietary ω6:ω3 ratio on the mammary tissue microenvironment and adipose inflammation using a moderate fat, iso-caloric diets, and pair-fed model. The LieberDeCarli diet containing 21:1 ratio of ω6:ω3 FA was used as a ω6 diet, whereas encapsulated fish oil containing a 3:1 ratio of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid was used to decrease ω6:ω3 ratio to 0.7:1 in the ω3 diet. Both iso-caloric diets contained 35.5% of calories derived from fat and were pair-fed to maintain iso-intake. Female BALB/c mice were established on the ω6 and ω3 diets for 10 weeks and weight gain and diet consumption monitored. There were no differences in the volume of diet consumed and weight gain between dietary groups. At autopsy, mammary fat pads (MFP) were collected and analyzed for fatty acid composition, histopathology, epithelial proliferation and macrophage infiltration. Arachidonic acid (AA) levels in the MFPs were not different between the groups but EPA and DHA were absent in the MFPs from the ω6 diet fed mice. Whereas, (2.41+/- 0.5) mole% of EPA and (1.52+/-0.29) mole% of DHA were detected in MFP of ω3 diet fed mice. The MFP of ω6 diet fed mice had significantly increased areas of unilocular adipocytes relative to adipocytes of the ω3 group. Similarly, ω6 diet fed mice had increased connective tissue in the ductal stroma, significantly higher numbers of proliferating cells in the ductal epithelium, as well as in adipose tissue area of MFP. In addition, ω6 diet fed mice had a significant increase in the numbers of CLS in mammary adipose tissue. In summary, our studies demonstrated that despite the comparable levels of AA in MFP in both of groups, the presence of LC-ω3 FA (EPA and DHA) was able to reduce inflammation in the MFP of ω3 diet fed mice, thus regulating the MFP microenvironments by reducing macrophage infiltration and ductal epithelial proliferation in an obesity-independent manner.

#246

Assessing microbial dysbiosis of electronic cigarettes and cigarette smokers using oral and lung microbiome.

Kevin Ying,1 Min-Ae Song,1 Daniel Y. Weng,1 Quentin Nickerson,1 David Frankhouser,1 Pearlly S. Yan,1 Ralf Bundschuh,1 Mark D. Wewers,1 Ewy Mathé,1 Jo L. Freudenheim,2 Peter G. Shields1. 1 _The Ohio State University, Columbus, OH;_ 2 _University at Buffalo, Buffalo, NY_.

The link between smoking tobacco and changes in the oral microbiome in response to tobacco smoking are well established. It is not known if there are changes in response to electronic cigarettes (e-cig). These changes in the microbiome are associated with increased numbers of disease causing pathogens. Currently there are no published studies that have investigated the relationship of smoking tobacco on both the oral and lung microbiome. There is insufficient evidence showing whether changes in oral cavity and lung microbiome are also seen in e-cig users. We will study the oral cavity and lung of non-smokers, smokers and e-cig users to examine concordance between oral cavity and the lungs as well as comparing the three groups, examining the microbiomes and expression of inflammatory markers. We hypothesize that microbial dysbiosis and expression of inflammatory cytokines will differ for smokers and non-smokers; and that e-cig users will have microbial dysbiosis similar to cigarette smokers. A cross-sectional study is being conducted on three groups, 1) never-smokers, 2) cigarette smokers, and 3) e-cig users. For each study participant, saliva and bronchoalveolar lavage (BAL) are being collected to measure microbiome. RNA is extracted from saliva and BAL samples for total transcriptome analysis using RNA-seq. This analysis will detect human and bacterial reads thereby allowing observations of bacterial communities as well as human inflammatory cytokine response to bacterial presence. 85% to 98% of BAL sample reads aligned to the human genome compared to less than 50% from saliva samples. The alignment results allow us to deduce that the majority of reads from BAL samples are human and that the majority of the reads in saliva samples are bacterial.

Preliminary results show detection of human RNA expression and of bacterial reads are present in both saliva and BAL samples. More samples are being processed and the comparison of BAL and saliva samples between the three groups will be discussed.

#247

Nutrient stress via folic acid modulation causes systemic and cancer-specific metabolic reprogramming and differential effects on primary and metastatic mammary tumor growth in lean and obese mice.

Ciara H. O'Flanagan,1 Xuewen Chen,1 Zahra Ashkavand,2 Sergey A. Krupenko,2 Stephen D. Hursting1. 1 _Univ. of North Carolina at Chapel Hill, Chapel Hill, NC;_ 2 _Nutrition Research Institute, Kannapolis, NC_.

Many foods are currently fortified with folic acid (FA), a synthetic folate (Vitamin B9). Folate deficiency causes many human health defects, most notably anemia and neural tube defects. The effects of excess supplementation on human health have to date been understudied. The rise in obesity in the last three decades further complicates this issue, with the combined effects of excess or insufficient folic acid intake and an obese phenotype being unknown. Obesity is associated with a number of cancers, including triple negative breast cancer (TNBC). TNBC comprises 16% of all breast cancers, is highly aggressive and more likely to recur and metastasize than other breast cancers. Unlike other subtypes, TNBC does not respond to hormone-targeted therapies and treatment options are limited to cytotoxic chemotherapy. Here, we examined the effects of FA supplementation and deficiency on tumor growth, metastasis and metabolism in obesity-responsive models of primary (M-Wnt) and metastatic (metM-Wnt; MDA-MB-231) TNBC. FA supplementation and deficiency significantly enhanced primary tumor growth and invasiveness in lean mice, while no difference in tumor size was detected in obese groups. FA supplementation reduced while deficiency increased survival and reduced lung tumor metastasis incidence in lean, but not obese mice. Liver and tumor metabolomic profiling revealed that modulation of dietary FA caused systemic and tumor-specific metabolic reprogramming, altering pathways involved in fatty acid, purine, amino acid, glutathione and energy metabolism. Short term in vitro FA withdrawal resulted in reduced proliferation, migration and invasion and energy production in all cell lines, as well as significant changes in gene expression profile, particularly of many metabolic pathways. In contrast, chronic in vitro FA depletion resulted in heightened oxidative stress, autophagy and apoptosis in metastatic TNBC cells, with nonmetastatic TNBC cells being able to adapt to and withstand the nutrient stress via the pentose phosphate pathway and glutathione redox signaling. Taken together, these results suggest that modulation of dietary folic acid in lean (but not obese) individuals causes systemic and tumor-specific metabolic reprogramming, which may confer a growth advantage in nonmetastatic cells and from which metastatic TNBC cells cannot recover. Moreover, obesity and FA excess cause similar metabolic and procancer effects and in combination, are not synergistic.

#248

Eicosapentaenoic acid attenuates obesity-related hepatocellular carcinogenesis.

Akane Inoue-Yamauchi, Hiroko Itagaki, Hideaki Oda. _Tokyo Women's Medical University, Tokyo, Japan_.

The population of obesity caused by excess energy intake and insufficient physical activity is increasing worldwide. Excess accumulation of body fat is a risk of health, indeed obesity is associated with the pathogenesis of various disease including type II diabetes, cardiovascular disease and cancer. Among various kinds of cancer, the risk of hepatocellular carcinoma (HCC) is strongly influenced by obesity. Studies using diet-induced obesity mouse models show that obesity enhances tumor development inducing chronic inflammation in liver. In fact, obesity failed to promote HCC formation in the absence of inflammatory cytokines IL-6 and TNF-α. Eicosapentaenoic acid (EPA), an omega-3 polysaturated fatty acid, has been shown its anti-inflammatory properties and could be therapeutically useful to ameliorate inflammatory diseases. However, little is known about the effects of EPA on liver cancer which is linked to obesity-related inflammation. Therefore, we tested the effects of EPA on obesity-induced inflammatory response and following tumor development. We used the hepatic procarcinogen dietylnitrosamine (DEN) to induce HCC formation in mice. Since high-fat diet (HFD) can induce obesity and significantly enhance HCC formation in DEN-injected mice, this model is useful to assess tumor promotion on obesity. Using this model, we examined therapeutic potential of EPA in obesity-related cancer. As a result, we found that tumor growth in mice fed HFD supplemented with EPA was reduced compared to HFD-fed mice, indicating that EPA attenuates obesity-related hepatocarcinogenesis. Interestingly, EPA had no obvious effects on obesity-induced inflammation, instead, inhibited tumor growth by suppression of the protumorigenic transcription factor Stat3 signaling. These data suggest translational implications for the treatment of obesity-related HCC.

#249

Dietary supplementation with methylseleninic acid reduces male mammary tumorigenesis in MMTV-PyMT mice.

Sneha Sundaram. _Grand Forks Human Nutrition Research Center, ARS, USDA, Grand Forks, ND_.

Male breast cancer makes up approximately 1% of all breast cancers and less than 1% of all cancers in men in the United States. However, it is an aggressive disease with poor prognosis and the incidence of male breast cancer is on the rise. The present study investigated the effects of dietary supplementation of selenium on male breast carcinogenesis in MMTV-PyMT mice (FVB/N background). Three-week-old male mice were weaned onto the AIN93G diet with or without supplementation of selenium in the form of methylseleninic acid (2.5 mg Se/kg). Mice were euthanized 10 weeks after the first palpable mammary tumor was detected. There were no differences in food intake and body weight between the groups. The median latency (the age at which the first palpable tumor was detected) was 14.1 and 13.9 weeks and the incidence of palpable mammary tumor was 71% and 70% for the control and the selenium group, respectively. Selenium supplementation compared to the control diet reduced mammary tumor progression by 258% and tumor weight by 84%, respectively. Tumor progression was defined as the change in tumor volume from detection of the first palpable mammary tumor to termination 10 weeks later. Incidence of lung metastasis was 74% and 50% in control and selenium group, respectively. In mice bearing lung metastases, selenium supplementation significantly reduced the number of metastases by 83% compared to the control group. In conclusion, dietary supplementation with selenium in the form of methylseleninic acid reduces male breast carcinogenesis and its metastasis in mice. It indicates that selenium may be useful in male breast cancer prevention.

#250

Methionine restriction increases macrophage tumoricidal activity and significantly inhibits prostate cancer growth.

Ashley R. Orillion,1 Sreenivasulu Chintala,1 Remi Adelaiye-Ogala,1 Li Shen,2 Nur Damayanti,1 May Elbanna,1 Sreevani Arisa,1 Bennett Elzey,3 Chinghai Kao,1 Luigi Fontana,4 Roberto Pili1. 1 _Indiana Univ.-Purdue Univ. Indianapolis, Indianapolis, IN;_ 2 _Roswell Park Cancer Institute, Buffalo, NY;_ 3 _Purdue University, Lafayette, IN;_ 4 _Washington University, St. Louis, MO_.

Background: Our previous work showed a significant reduction of tumor growth, macrophage infiltration, circulating IGF-1, and mTOR activation with low protein diet in a patient derived xenograft model of prostate cancer. The evolutionarily conserved, nutrient sensing, mTOR pathway plays a central role in both development of advanced stage prostate cancer and immune responsiveness. This study presents novel data on the impact of dietary protein modification on the function of the host immune system in response to prostate cancer and immunotherapy.

Methods: Our In vitro studies utilized bone marrow or tumor derived (RP-B6 Myc) macrophages. In vivo studies utilized the recently characterized RP-B6 Myc model. Mice were fed ad libitum control or methionine restricted diets for four weeks prior to S.C. implantation with ~1mm2 tumor pieces. Treatment of survivin peptide vaccine (1mg/ml S.C. 1 X week) and anti-PD-1 (20mg/kg I.P. 2 X week) began at ~50mm2 tumor size. Tumor volumes were blindly recorded 2 X week. End point analyses include: tumor weight, flow cytometric analysis, proteomic profiler analyses, and microbiome analyses from each diet and treatment group.

Results: We show here that while methionine restriction (MR) has little impact on our RP-B6 Myc prostate cancer cell line, it does yield a significant alteration in both the polarization and function of M1 and M2 macrophages. In the in vitro MR conditions, we observed significantly enhanced polarization of M1 macrophages and reduced polarization of M2 macrophages. Functional analysis revealed increased tumoricidal activity of both M1, 'antitumor', and M2, 'pro-tumor,' macrophages suggesting a flip in M2 function from tumor-promoting to tumoricidal. Further analysis of the released cytokines in MR media conditions yielded significant increase of antitumor cytokines & chemokines, such as IL-12, IL-27, CXCL9, CXCL10, CXCL11, CCL2, CCL4, and TNF-alpha, a double-edged sword which in our system correlates with decreased cancer cell viability upon co-culture with amino acid restricted M1 and M2 macrophages. Our preliminary results show dietary MR yielding a significant inhibition of prostate cancer growth in the RP-B6-Myc model and our ongoing study with survivin peptide vaccine and anti-PD-1 immunotherapies will be available to present in the conference.

Conclusions: Our data suggest that restricting methionine is sufficient to alter both the polarization and tumoricidal function of macrophages. Our preliminarily results show that restricted dietary methionine is able to inhibit prostate cancer growth, modify the host immune response and better inhibit prostate cancer growth alone or in combination with immunotherapy. These results provide a strong basis to consider diet restriction as a means to limit cancer growth targeting tumor immune system and potentially to enhance cancer patient responses to immunotherapy.

#252

Resveratrol and pterostibene selectively chemosensitize Burkitt's lymphoma cells to 5-Fluorouracil.

Connor J. Peck, Michelle H. Townsend, Kim L. O'Neill. _Brigham Young University, Provo, UT_.

In the current study, we assess the chemosensitization activity of the naturally occurring polyphenols resveratrol (RES) and pterostilbene (PTER) to 5-Fluorouracil (5-FU) in Burkitt's B-cell lymphoma. RES is a stilbenoid compound found in a variety of fruits, plants, and nuts. In addition to antioxidant and anti-aging properties, RES has been shown to exhibit significant anti-tumor activity against a variety of malignancies. Since the compound is typically protective of normal cells, its anti-cancer activity makes it a promising candidate as both a chemopreventative nutritional supplement and a chemotherapeutic agent. PTER, an analog of RES, has likewise shown anti-cancer activity. Its therapeutic potential, however, is much less understood. Recent studies have investigated the use of these and other natural compounds in adjuvant chemotherapy strategies, many of which continue to produce underwhelming rates of response. Here, we investigate the chemosensitization capacity of both RES and PTER to 5-FU, a commonly used drug in chemotherapy, in healthy lymphocytes and in Burkitt's B-cell lymphoma (Raji) cells. MTS assays are used to measure the viability of malignant and normal lymphocytes after 5-FU treatment with or without co-incubation of RES, PTER, or RES/PTER combinations. We find that Raji co-incubation with physiologically relevant concentrations (<10uM) of either RES or PTER significantly increases 5-FU toxicity in Raji cells, with data suggesting PTER to be slightly more effective than RES. Interestingly, RES/PTER combinatorial treatments further increase chemosensitization, an important finding considering the typically low bioavailability of each individual compound in vivo. Together, these data support the potential use of RES as a chemosensitizing adjuvant and provide new evidence that its analog, PTER, also possesses significant chemosensitization capacity. Furthermore, our studies suggest that low physiological concentrations of PTER and RES may be still be sufficient to induce significant anti-cancer effects.

#253

Mammary tumorigenesis causes bone loss and dietary selenium supplementation does not affect such bone loss in male MMTV-PyMT mice.

Lin Yan. _USDA-ARS, Grand Forks, ND_.

Cancer progression is accompanied by wasting that eventually results in cachexia characterized by significant weight loss and multi-organ functional failures. Limited clinical trials indicate that bone is adversely affected by cancer-associated wasting. To determine the effects of breast cancer on skeletal health, we performed micro-computed tomographic analysis of femurs and vertebrae collected from a recently completed study showing that dietary supplementation with selenium (methylseleninic acid, 2.5 mg Se/kg) reduced male mammary tumorigenesis in MMTV-PyMT mice. Compared to age-matched non-tumor-bearing mice (MMTV-PyMT negative), the presence of mammary tumors significantly reduced bone volume fraction, trabecular thickness and bone mineral density and increased the structure model index (an indicator of the plate- and rod-like geometry of trabecular structure) in femoral trabecular bone. Mammary tumor development did not affect vertebral trabeculae nor femoral and vertebral cortical bone, except it significantly reduced cortical bone thickness of vertebrae. There were no differences in aforementioned measurements between groups with or without selenium supplementation. In conclusion, mammary tumorigenesis causes bone loss and dietary selenium supplementation at 2.5 mg Se/kg, which is antitumorigenic, does not affect mammary tumor-associated bone loss in this male MMTV-PyMT breast cancer model.

#254

**The potential of** Lactobacillus **probiotic treatments in colorectal cancer (CRC).**

Imen Kahouli,1 Meenakshi Malhotra,2 Susan Westfall,1 Moulay Alaoui-Jamali,1 Satya Prakash1. 1 _McGill University, Montreal, Quebec, Canada;_ 2 _Standford University, Palo Alto, CA_.

Colorectal cancer (CRC) is the third leading cause of death worldwide. It is known to be a type of cancer that is preventable by changes in diet and lifestyle. Mounting evidence are supporting the role of gut microbiome in the etiology of CRC and emphasize on the potential of probiotics as biotherapeutics in the prevention and management of CRC. Lactobacillus probiotic bacteria were suggested to balance pathogenic and oncogenic microbial community in the colon and produce anti-tumorigenic and anti-inflammatory effects in healthy individuals at risk and CRC patients. There is a need, however, for studies that focus on identifying potent probiotic strains with activity against CRC and inhibit cancer growth. This report discusses and present findings about the formulation of novel probiotic Lactobacillus biotherapeutic for CRC based on anti-CRC proliferative effect, immune modulation and metabolic activity, in vitro and in vivo, using a genetically-induced animal CRC model. Results and metabolomic analysis demonstrated the potential acrion of probiotic treatment to change host and gut microbiome co-metabolic profiles, produce local and systemic anti-inflammatory effects, inhibit cancer-causing events, and improve overall gut health.

#255

Dietary amphiphilic polyphenols modulate the biophysical properties of plasma membrane organization and membrane-dependent macropinocytosis.

Michael L. Salinas, Natividad R. Fuentes, Rola Barhoumi, Robert S. Chapkin. _Texas A &M University, College Station, TX_.

Ras-driven cancers exhibit a distinct high metabolic demand necessary for biosynthetic growth/proliferation, in some cases resulting in over stimulation of macropinocytosis, a process intended for nutrient acquisition. A hallmark of Ras-driven macropinocytosis is plasma membrane ruffling resulting in invaginations and the formation of macropinosomes, extracellular nutrient-packed vesicles distinct from other endocytic processes due to their size, independence of vesicle-coating proteins, and are the result of rapid polymerization and branching of cytoskeletal actin filaments. These cytoskeletal manipulations are driven by spatial/temporal regulators downstream of Ras along with lipid rafts and their components. Drugs and other extrinsic factors, e.g., polyunsaturated fatty acids, have been shown to biophysically modulate these lipid domains. We hypothesize that unique dietary polyphenolic amphiphilic molecules [1] modulate plasma membrane fluidity, [2] disrupt macropinocytosis, and [3] suppress macropinocytosis-dependent proliferation.

Young adult mouse colonocyte (YAMC) cells, expressing an HRas mutation, were treated with 1, 10, and 100 µM (+)-catechin or procyanidin B2 for 30 min. For macropinocytosis assessment, cells were stimulated with 25 ng/mL epidermal growth factor (EGF) for 5 min following a 30-minute pretreatment. Membrane order was measured in whole cells and giant plasma membrane vesicles (GPMV) utilizing a lipid-packing detection dye, Di-4-ANEPPDHQ, and assayed using a combination of fluorescence image-based flow cytometry (Amnis FlowSight) and confocal microscopy. In addition, macropinocytosis was determined by uptake of fluorescently labeled 70 kDa dextran (FITC-dextran). The uptake was visualized by fluorescence image-based flow cytometry. In the GPMV model, (+)-catechin increased membrane fluidity, and procyanidin B2 decreased membrane fluidity in a dose-dependent manner. In contrast, in the whole cell model, interaction of (+)-catechin and procyanidin B2 with the plasma membrane resulted in an increase in membrane fluidity in a dose-dependent manner.

Interestingly, plasma membrane fluidification in the whole cell model was associated with a significant 27-67% inhibition of macropinocytosis. These observations indicate that dietary amphiphilic molecules (DAMs) modulate plasma membrane organization, which is linked to macropinocytosis, an essential fuel-obtaining process. Establishing a role for DAMs in membrane-dependent oncogenesis is noteworthy because these molecules are innocuous and found in high abundance in a variety of fruits, vegetables, and walnuts.

This work was support by NIH grant R35CA197707.

#256

Delta tocopherol inhibits urothelial tumorigenesis in the UPII mutant Ha-ras transgenic mouse model and induces apoptosis via activation of the ATF4/CHOP-DR5 pathway.

Christopher A. Blair,1 Maggie Wu,1 Tim Huynh,1 Hanze Hu,1 Arman Walia,1 Chung S. Yang,2 Xiaolin Zi1. 1 _UC Irvine, Irvine, CA;_ 2 _Rutgers University, Piscataway, NJ_.

Epidemiological studies have reported that Vitamin E intake was inversely related to the risk of multiple cancers including human urinary bladder cancer. Tocopherols (T) are the major forms of vitamin E in the U.S. diet, exist as α-T, β-T, γ-T and δ-T. Compared to α-T, the anti-cancer effect of other Ts and their mechanisms of action remain largely unknown. We have shown that δ-T is the most effective one among the Ts in reducing the viabilities of bladder cancer cell lines with IC50s of under 10 μM, whereas there is no or minimal effect of α-T at a concentration of up to 50 μM on the viability of bladder cancer cell lines. δ-T treatment of bladder cancer cell lines, RT4 and UMUC-3, resulted in apoptosis via marked induction of death receptor-5 (DR5) expression and then activation of caspase 3, 8, and 9 leading to PARP cleavage and apoptotic morphology. siRNA knockdown of DR5 expression significantly attenuated the apoptotic effects of δ-T in bladder cancer cells. Consistent with the above results, δ-T treatment significantly upregulated the expression of ER stress sensors PERK and IRE1α, as well as downstream components GRP78, ATF4, and CHOP. These results suggested that δ-T can induce endoplasmic reticulum stress and the Unfolded Protein Response, triggering its apoptotic effect. Furthermore, homozygous male UPII mutant Ha-ras transgenic mice that mimic the development and progression of human urothelial cell carcinoma (UCC) with H-ras activation were fed with vehicle control diet (n=20) or diet supplemented with δ-T (0.2% in diet) for 150 days (n=18). The δ-T diet significantly decreased the mean bladder weights (serves as a surrogate of tumor weight) of survived Ha-ras mutant mice by 56% (control vs δ-T, 0.259 ± 0.015 gram vs. 0.114 ± 0.012 gram, P<0.0001) with no significant difference in overall body mass and no detectable indication of toxicity. Our results suggest that δ-T is a potent anti-cancer agent and deserves further investigation in preventing the recurrence and progression of non-muscle-invasive UCC.

#256A

Validation of methyl donors between two food measurements in a colorectal cancer study.

Lufei Young, S. Pamela K. Shiao. _Augusta University, Augusta, GA_.

Purpose: The purpose of this study was to validate methyl donor intakes between 30-day food frequency questionnaire (FFQ) and 24-hour food record (FR) in a colorectal cancer study.

Background: Dietary methyl donors play pivotal roles in cancer prevention by promoting adequate metabolism and DNA methylation. The FFQ and 24-hour FR are the most commonly used methods to assess adequate nutrient intake, including methyl donors and other vital micronutrients that are essential to promote metabolism and methylation. While FFQ provides best estimates in dietary intakes, a 24-hour food record is less time consuming for subject burden.

Methods: A total of 134 participants' dietary data were collected using FFQ and 24-hour FR by using US Department of Agriculture computerized programs. Bland and Altman methodology was used to validate the bias and agreement between these two measurements.

Results: Overall, the 24-hour measurements presented underestimated values across all nutrients in comparison to FFQ measurements. Using a 10% criterion on the differences between the two measurements, the percentage bias for 24-hour FR compared to 30-day FFQ was acceptable for most B vitamins, including B1 (-6.9 ±0.15), B2 (-8.66± 0.12), B3 (-9.25± 0.12), B6 (-5.02, ± 0.14), and B9 (folate: -7.90 ±0.24). However, the bias was greater for protein based methyl donors, including B12 (-17.23± 0.14), choline (-13.38± 0.11), Glycine (-17.53± 0.10), and Methionine (-18.88± 0.09). The correlations between the two measurements on all methyl donors ranged from 0.92 to 0.99. The precisions for all measurements based on the standard error (SE) were less than and within 0.03% (0.007 -0.021%). And, the agreements between the two measurements were less than and within 10% for Glycine and methionine (8.96 - 9.58%), based on the standard deviation (SD); however, greater than 10% for other protein based methyl donors including choline and other B vitamins (10.5-24.11%).

Conclusion: Proper dietary intakes of methyl donors are vital for cancer prevention. These findings point out the importance of further validation and use of more accurate food measurement to monitor dietary intake, and the interpretation of nutrient reports with caution.

## EPIDEMIOLOGY:

### Descriptive Epidemiology Including Trends in Incidence and Prognosis

#257

Estrogen receptor positive and negative breast cancer secular trends in Ireland (2004-2013).

Maeve Mullooly,1 Jeanne Murphy,1 Gretchen L. Gierach,1 Brittny Davis,1 Paul Walsh,2 Sandra Deady,2 Thomas I. Barron,3 Mark E. Sherman,4 Philip S. Rosenberg,1 William F. Anderson1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _National Cancer Registry Ireland, Ireland;_ 3 _Johns Hopkins Bloomberg School of Public Health, Baltimore, MD;_ 4 _Mayo Clinic, Jacksonville, FL_.

Background: Estrogen receptor (ER) status in breast cancer is strongly associated with etiologic factors, prognosis and treatment response. Data suggest that population-based incidence rates of ER-positive (ER+) tumors are increasing and ER-negative (ER-) cancers have fallen over time in the United States (US), Denmark and Scotland. Whether these trends are consistent in other populations with different etiologic exposures and across ER±/HER2± subtypes is unknown. We, therefore, analyzed breast cancer secular trends in Ireland by ER expression; and for the first time, by joint expression of ER±/HER2±, a well-established clinical marker of uncertain etiological significance.

Methods: This study included invasive breast cancers (n=24,845; 2004-2013) among women aged 20-84 years within the population-based National Cancer Registry of Ireland (97% coverage). The general population at risk (n=10,401,986) was obtained from the Irish Central Statistics Office. Using a validated approach to account for missing ER/HER2-status, we assessed ER± and ER±/HER2± secular trends with age-standardized incidence rates (ASR) and estimated annual percentage change (EAPC) with 95% confidence intervals (95%CI).

Results: Over the 10-year follow-up period, 19,264 ER+ and 4,161 ER- breast cancers were diagnosed (1,420 were ER-unknown). ASRs increased significantly for ER+ (EAPC: 2.20%/year (95%CI: 0.97, 3.45%/year)) and decreased significantly for ER- cancers (EAPC: -3.43%/year (95%CI: -5.05, -1.78%/year)). The ER-specific trends were qualitatively similar among three age groups (<50, 50-64 and ≥65 years). With further stratification by HER2± expression, ER+/HER2- cancers rose (EAPC: 2.87%/year (95%CI: 1.33, 4.44%/year)), ER+/HER2+ cancers were statistically flat, while ER-/HER± cancers significantly declined.

Conclusion: Secular trends for ER± cancers in Ireland were similar to those previously observed in the US and Denmark. Further, trends for ER/HER2 followed those for ER, although rates were constant for ER+/HER+ tumors. The divergence of cancer incidence patterns by ER among independent populations over time could reflect comparable changes in risk factor exposures with dual or opposite effects for risk of ER+ and ER- cancers (e.g., obesity, age at first birth/parity). Additionally, although HER2 is a well-established clinical predictive and prognostic marker for breast cancer outcome, it did not appear to substantively impact breast cancer incidence trends.

#258

The changes of esophageal malignant tumors by histopathology over 40 years (1975-2014) at a single institute in northern China.

Xue Ke Zhao,1 Hai Jun Yang,2 Zong Min Fan,1 Ji Lin Li,3 Li Min He,4 Yun Zhou,5 Liu Qin Yang,6 Ying Fa Zhou,7 Sa Tang,6 Yang Yang,1 Lei Ma,4 Wei Li Han,8 Li Dong Wang1. 1 _The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China;_ 2 _Anyang Tumor Hospital, China;_ 3 _Linzhou Esophageal Cancer Hospital, China;_ 4 _The First People's Hospital of Nanyang, China;_ 5 _Henan Provincial People's Hospital, Zhengzhou, China;_ 6 _Central Hospital of Xinxiang, China;_ 7 _The Second Affiliated Hospital of Zhengzhou University, Zhengzhou, China;_ 8 _Xijing Hospital of the Fourth Military Medical University, China_.

It has been well recognized that esophageal squamous cell carcinoma (ESCC) is the major histopathological type of esophageal cancer in China. However, it is not even clear how many histopathological types of esophageal malignant tumor (EMT) exist and if the histopathological patterns have changed over the past decades in China. The present study was thus undertaken to characterize the histopathological patterns over 40 years at a single institute in northern China, the high incidence area for esophageal cancer. All the patient information was from esophageal cancer database in Henan Key Laboratory for Esophageal Cancer Research of the First Affiliated Hospital, Zhengzhou University in Henan, China (1973-2015). A total of 66,216 patients with MET and detailed histopathological records were enrolled in this study, including 39,873 males with a mean age of 60±6 and 26,343 females with a mean age of 60±6 from 1975-2014. All the patients were classified into four period groups based on the diagnosed time, group I (1975-1984, 4,331/6.5 %), group II (1985-1994, 14,577/22%), group III (1995-2004, 20,781/31.4%) and group IV (2005-2014, 26,527/40.1%). The results demonstrated 12 different histopathological types of EMT. The most common type was ESCC (64,800, 97.4%), followed by esophageal adenocarcinoma (EAC, 1268, 1.9%), esophageal small cell carcinoma (201, 0.3%), esophageal adenosquamous carcinomas (EASC, 119, 0.18 %), esophageal spindle cell carcinoma (76, 0.11%), esophageal undifferentiated carcinoma (37, 0.06%), esophageal malignant melanoma (5, 0.008%), esophageal stromal tumor (3, 0.005%), esophageal clear cell carcinoma (2, 0.003%), esophageal malignant schwannoma (1, 0.002%), esophageal lymphatic tumor (1, 0.002%) and esophageal smooth muscle cell sarcoma (1, 0.002%). Time trending analysis showed that, over the past 40 years, ESCC was relatively stable (98.9%, 98.0%, 97.0%, 97.1%, respectively). However, EAC (0.97%, 1.66%, 2.30%, 1.87%, respectively), small cell carcinoma (0.02%, 0.17%, 0.28%, 0.43%, respectively) and spindle cell carcinoma (0.02%, 0.01%, 0.10%, 0.22%, respectively) increased apparently. The present study indicates that ESCC remains the major histopathological type for Chinese esophageal cancer. Interestingly, esophageal adenocarcinoma, small cell carcinoma and spindle cell carcinoma seem to increasing over the past decades. The improved molecular diagnostic biomarkers and the changes of lifestyle including the diet patterns resulted from recent economic development in China, may contribute to the changes of esophageal histopathological types. [Supported by Joint Funds of the National Nature Science Foundation of Chian (U1301227), National Nature Science Foundation of China (81472323), and Correspondence to: Li Dong Wang, Email: ldwang2007@126.com]

#259

A case series of vitreoretinal lymphoma in New York City with exposure to Chernobyl nuclear disaster decades earlier.

Sanford Kempin,1 Paul Finger,2 Robert Gale,3 John Rescigno,1 Jeffrey Rubin,2 Walter Choi,1 Rebecca Fisher,4 Alexander Aizman,2 Ilona Genis,5 Roxana Moslehi* (Corresponding Author)6. 1 _Mount Sinai / Beth Israel Comprehensive Cancer Center, New York City, NY;_ 2 _New York Eye and Ear Infirmary of Mount Sinai, New York City, NY;_ 3 _Imperial College London, London, NY;_ 4 _Mount Sinai / St. Luke's Roosevelt Hospital, New York City, NY;_ 5 _Maimonides Medical Center, Brooklyn, NY;_ 6 _University at Albany, State University of New York, Albany, NY_.

Background: Vitreoretinal lymphoma (VRL) is an unusual presentation of primary central nervous system lymphoma and a subcategory of intraocular lymphoma (IOL). The etiology and epidemiology of this rare malignancy is unknown.

Methods: We diagnosed 10 subjects with VRL in our clinical practices in New York City (NYC) during 2010-2013. Upon systematic collection of demographic and exposure history, 6 patients reported residence in regions proximal to the Chernobyl nuclear disaster (4 Ukraine, 1 Poland, 1 Moldova). We studied clinical variables including presentation, diagnostic parameters, therapies and survival of this case series. To provide context, we examined population-based incidence data from the Surveillance, Epidemiology and End Results (SEER) program to determine age-adjusted incidence rates of IOL (there is no unique code for VRL) per 100,000 person-years in the United States (US) during 1992-2013. New York State (NYS) rates are not reported to SEER and were examined separately using NYS Cancer Registry (NYSCR). Incidence trends using 10-year intervals (1992-2002 and 2003-2013) were also analyzed.

Results: All six subjects (5 females) were diagnosed with diffuse large B-cell lymphoma with bilateral presentation. Diagnosis in all cases was made by immunohistochemical analysis on tumor specimen obtained by vitreous fine needle aspiration, vitreous biopsy or stereotactic brain biopsy. Median age at diagnosis was 76 years (range, 62-84 years). Median interval from Chernobyl accident (1986) to diagnosis was 26 years (range, 24-27 years). We identified no other common exposure or environmental risk factor. There were 68 cases of primary IOL during 1992-2013 in the 13 SEER areas. IOL rates per 100,000 person-years for both sexes were highest among Asian/Pacific Islanders (0.010 for males and 0.014 for females) followed by whites (0.009 for males and 0.008 for females) and (based on small numbers) by blacks. Analysis of incidence trends showed an increase in rates among Asian/Pacific Islander females from 0.005 in 1992-2002 to 0.018 in 2003-2013 and among males from 0.006 to 0.013. Rates among whites increased for both genders. Analysis of NYSCR data revealed 44 cases of primary IOL. IOL rates per 100,000 person-years for both genders were highest among whites (0.010 for males and 0.012 for females) followed by Asian/Pacific Islanders (0.007 for males and females). Analysis of trends revealed increase in rates for both genders and racial groups.

Conclusions: Our analyses highlight the rarity of VRL and reveal an increase in incidence, particularly among Asian/Pacific Islanders where IOL rates tripled within two decades. Based on incidence rates, 8 cases of IOL (with a subset being VRL) are expected in 4 years in NYS. Our observation of 10 cases of VRL in 4 years in our practices in NYC is unanticipated. The sole common environmental factor we identified was residence in regions proximal to the Chernobyl nuclear disaster.

#260

Incidence rates and outcomes of fallopian tube carcinomas: Data from the North American Association of Central Cancer Registries.

Mark E. Sherman,1 Sally B. Coburn,2 Hannah Yang,2 William Anderson,2 Philip Rosenberg,2 Gretchen Gierach,2 Nicolas Wentzensen,2 Kathy Cronin,2 Britton Trabert2. 1 _Mayo Clinic, Jacksonville, FL;_ 2 _National Cancer Institute, Bethesda, MD_.

Background: Serous tubal intraepithelial carcinoma (STIC) is hypothesized to give rise to many cancers that have been historically classified as ovarian primaries. Although STIC is gaining status as a diagnostic entity, similar appearing lesions would likely have been reported as fallopian tubal carcinoma in-situ (FT-CIS) in the past. Accordingly to characterize patterns of reporting and behavior for tubal cancers, we analyzed population-based data for incidence rate trends, co-occurrence with cancer at other sites and survival of FT-CIS and invasive fallopian tube carcinoma (FT-Inv).

Methods: We analyzed data for FT-CIS and FT-Inv included within 33 registries using the Cancer Incidence in North America Deluxe Analytical Files provided by the North American Association of Central Cancer Registries (1995-2012). Primary site of invasive carcinomas were defined according to the International Classification of Diseases for Oncology (3rd edition) topography codes (ovarian (C56.9), fallopian tube (C57.0), and primary peritoneal cancers (C48.1-.2, 48.8)). We excluded cancers with non-epithelial histology. Total counts, incidence rates per 1 million adjusted to the 2000 standard US population and age-standardized stage-specific survival were computed. Temporal incidence rate patterns were analyzed by joinpoint regression with estimates of annual percentage change (APC).

Results: Cases in which the first cancer diagnosis was FT-CIS, included 98 FT-CIS alone; 27 FT-CIS followed by another cancer diagnosis within 1 year and 172 cases in which FT-CIS was diagnosed concurrently with another cancer as compared with 5513 invasive tubal carcinomas over the same period. The incidence rate of FT-CIS was stable from 1995-2002, then significantly increased from 2002-2012 [APC (95% CI) = 16.2% (10.9-21.7)]. Rates of early stage high-grade serous FT-Inv increased significantly from 2002-2012 [10.4% (6.1-14.9)] and rates of late stage high-grade serous FT-Inv rose sharply from 2002-2012 [20.0% (17.5-22.6)]. Five-year age-standardized survival for women with FT-CIS only was 75.6% overall, reflecting 89.1% survival for women less than 50 years and 70.9% for women 50 years or older; survival for high-grade early stage serous FT-Inv was 77.8%.

Conclusions: Diagnoses of FT-CIS and FT-Inv have increased dramatically, likely reflecting changes in diagnostic pathology practice. Developing standardized reporting for FT-CIS (and in the future, STIC) when present with concurrent invasive cancer are needed, given that this occurs often. Based on limited data, 5-year survival for FT-CIS and early stage FT-Inv are similar, but future studies to evaluate data for STIC will be required, which will likely be achievable only through national registries that are large enough to capture sufficient cases.

#261

Pancreatic cancer incidence trends and recent patterns overall and by histologic type among US men and women by racial/ethnic group: evidence from the Surveillance, Epidemiology, and End Results (SEER) program.

Vanessa L. Gordon-Dseagu,1 Susan Devesa,1 Mike Goggins,2 Rachael Stolzenberg-Solomon1. 1 _NCI, Rockville, MD;_ 2 _Johns Hopkins University, MD_.

Introduction: Pancreatic cancer incidence has been rising. We examined incidence patterns by sex, race, age, and histologic subtype.

Methods: We used data from the Surveillance, Epidemiology and End Results (SEER) registries (9, 13 and 18) to calculate counts, age-adjusted rates (2000 US Standard Population), annual percent changes (APCs) and incidence rate ratios (IRRs) for all pancreatic cancers and main histologic subgroups.

Results: Pancreatic cancer incidence rates declined among males between 1974 and 1991, and increased in the 1992-2013 period among white Hispanics and non-Hispanics (APC= 0.73 and 0.84, respectively). Among females, incidence rates also rose significantly during 1992-2013 among white non-Hispanics, white Hispanics, and Asians (APC= 0.81, 0.56, and 1.23, respectively). In contrast, rates among black males and females changed little. Within age groups, rates significantly increased among males who were white non-Hispanic age ≥55 years and white Hispanics in the 55-74 age group. Among black males, rates declined in the 45-54 age group at a statistically significant pace. Pancreatic cancer incidence rates for females rose among white non-Hispanics in all age groups <85 years (range: 0.47-4.01), among Asians of ages 55-84, and white Hispanics in the 55-64 age-group.

Most pancreatic cancers were specified as adenocarcinoma, not otherwise specified (NOS) or ductal adenocarcinoma. Rates increased among all racial/ethnic groups for adenocarcinoma, NOS and ductal adenocarcinoma while those for cystic mucinous ductal adenocarcinoma and poorly specified type decreased. The incidence rates of non-secretory pancreatic endocrine cancer rose >6% per year among white non-Hispanics and Asian/Pacific Islanders.

Overall incidence rates were significantly higher among males than females (M/F IRR=1.28). The IRR was >1.00 at all ages >35, but rates among females were significantly higher at younger ages (IRR <0.81). The M/F IRRs were elevated for acinar cell adenocarcinoma (2.85), non-secretory endocrine cancers (1.47) and ductal cystic or mucinous adenocarcinomas (1.11), while for solid pseudopapillary adenocarcinoma there was a significant female excess (0.22).

Conclusion: Pancreatic cancer incidence rates vary within demographic groups and histologic subtypes.

#262

Analysis of demographics, survival and patterns of care of pediatric glioblastoma using National Cancer Database.

Jigisha Thakkar, Meng Liu, Emily Van Meter Dressler, John L. Villano. _University of Kentucky, Lexington, KY_.

Background

We analyzed the largest clinical database in the United States, the National Cancer Database (NCDB), comprising over 70% of cases diagnosed and/or treated at commission on Cancer approved institutions. We analyzed current hospital-based epidemiologic frequency, survival and patterns of care of pediatric glioblastoma (GBM).

Methods

Cases included patients 0-19 years between 1998-2011. Inclusion criteria for histology codes included for GBM 9440, 9441 and 9442 (9440 (GBM), 9441(Giant Cell) and 9442 (Sarcomatas component) and tumor sites (C70.0-C72.9, C75.1-C75.3) related to the brain: brain stem, cerebellum, cerebrum, ventricle and brain not otherwise specified (NOS). Kaplan-Meier survival estimates were calculated for each demographic criteria and treatment plan and Cox proportional hazards models were employed to assess the risk of mortality. Demographic variables analyzed were age, gender, race, Hispanic origin, income, education, region and insurance status. Comparisons were made among histologic subtypes and primary site.

Results

1173 patients with GBM from age 0 to 19 years were identified from 1998 to 2011 in the United States, of which 21.5%, 24.5% and 54.0% were in age group 0-5, 6-10 and 11-19 years old. The largest group (48%) received advanced care including a combination of radiation (XRT), chemotherapy and surgery. On the other hand, the no treatment group was the smallest comprising of 4% cases. 14% received surgery only, 8% received a combination of XRT and surgery and 9% received a combination of XRT and chemotherapy.

Patients that received combination of XRT, chemotherapy and surgery had significantly improved survival when compared to those without any treatment (p = 0.0048). Patients that received surgery or chemotherapy had significantly improved survival as compared to patients that did not receive the above treatments (p = 0.0027).

Conclusion

We report an extensive demographic and survival analysis of pediatric GBM. Observed difference likely reflect biology across age, gender, race and origin groups. Our analysis demonstrates concern in the delivery of optimal care to a large percentage of pediatric patients. These treatments include surgery and chemotherapy that have demonstrated survival benefits. Adverse effects of chemotherapy and XRT can limit the appropriate administration of treatment.

#263

Epidemiology of primary lung cancer and its trend northeast of Iran during 1985-2012.

Maryam Salehi,1 Pardis Shojaee,1 Soodabeh Shahidsales,1 Seyed Amir Aledavood,1 Mehdi Seilaniantoosi,1 Golboo Goshayeshi,1 Fahimeh Khoshroo2. 1 _Mashhad University of Medical Sciences, Mashhad, Islamic Republic of Iran;_ 2 _Razavi Hospital, Mashhad, Islamic Republic of Iran_.

Introduction: In this study we investigated epidemiology and trends of primary lung cancers in northeast of Iran during 1985-2012.

Methods: In this cross-sectional study records of primary lung cancers from 1985 to 2012 in Mashhad, northeast of Iran, were investigated. Data were obtained from the referral oncologic hospital and private radiation oncology clinic. To study trends, the duration of study was categorized into three periods: 1985-1995, 1995-2005 and 2005-2012. Statistically significant level was considered <0.05.

Results: Of 939 cases with mean age of 60.57 (±12.31) years, 660(70.3%) were male. Male to female ratio was 2.36. Mean age was 61.47±12.01 years for males and 58.45±12.75 for females (p=0.001). Mean age at diagnosis and rate of smokers was unchanged in three intervals (table 1). | |  | |

---|---|---|---|---

Time period | 1985-1995 | 1995-2005 | 2005-20012 | P value

Age(years) | 60.67±11.74 | 60.57±12.24 | 60.49±12.94 | 0.98

Gender:

Male

female | 167(78.4)

46(21.6) | 330(68.6)

151(31.4) | 163(66.5)

82(33.5 | 0.01

Male to female ratio | 3.63 | 2.18 | 1.99

|

Smoking history:

Yes

no | 69(76.7)

21(23.3) | 211(67.8)

100(32.2) | 100(65)

54(35) | 0.15

Smokers:

Male

Female | 59(85.5)

10(14.5) | 159(75.4)

52(24.6) | 74(74)

26(26) | 0.16

Male-to-female ratio of smokers | 5.9 | 3.06 | 2.84

|

Squamous cell carcinoma with relative frequency of 44.2% was the most frequent pathologic subtype in total population followed by adenocarcinoma (18.2%), Small Cell Lung cancer (16.8%), large Cell carcinoma (1.3%) and other subtypes (19.5%). Relative frequency of pathologic subtypes had significant changes in three intervals (p <0.001). Relative frequency of adenocarcinoma and large cell carcinoma were increasing while it was decreasing for SCC and SCLCs during three recent decades. There was statistically significant difference in mean age at diagnosis (p<0.001), rate of smokers (p<0.001) and male to female ratio (p=0.01) between four leading pathologic subtypes. Mean age at diagnosis (55.92±12.90 years), male to female ratio (1.69), and rate of smokers (46.8%) were lowest in adenocarcinomas.

Conclusion: Similar to universal picture, adenocarcinoma showed increasing rates during recent three decades with higher prevalence among younger aged patients, women and nonsmokers. These trends are indicative of changes in exposures and smoking habits and reveal the need for regional studies in this context.

#264

Time trending for the ratio of rural and urban patients with esophageal squamous cell carcinoma in a single institute in northern China over the past 30 years.

Shou Jia Hu,1 Hai Jun Yang,2 Neng Chao Wang,2 Xiu Min Li,3 Shuang Lv,4 Qi De Bao,5 Wen Bin Yue,6 Hui Meng,1 Dan Feng Du,1 Xue Min Li,7 Fu You Zhou,2 Jian Li,8 Li Dong Wang1. 1 _Henan Key Laboratory for Esophageal Cancer Research, the First Affiliated Hospital of Zhengzhou University, Zhengzhou City, China;_ 2 _Anyang Tumor Hospital, Anyang, China;_ 3 _Cancer Research Center, Xinxiang Medical University, Xinxiang City, China;_ 4 _School of Basic Medical Sciences, Xinxiang Medical University, Xinxiang City, China;_ 5 _Anyang District Hospital, Anyang, China;_ 6 _Puyang City Oil Field General Hospital, Puyang, China;_ 7 _Cixian Hospital, Cixian, Handan, China;_ 8 _Henan Provincial People's Hospital, Zhengzhou City, China_.

Esophageal squamous cell carcinoma (ESCC) has been well characterized as a major histological type in China and occurred predominantly in the mountain rural region with poor economics. Nutritional deficiencies have been recognized as major risk for esophageal carcinogenesis in these rural regions. In the past decades, the rural economic status in China has improved apparently. However, the time trending for ESCC in rural regions has not been characterized. The present study was thus undertaken to determine the time trending for the ratio of rural and urban patients with ESCC in a single institute in northern China over the past 30 years (1985-2014). All the 66,515 ESCC patients in this study were derived from the esophageal and gastric cardia carcinoma databases of 500 thousands patients (1973-2015) established by Henan Key Laboratory for Esophageal Cancer Research of the First Affiliated Hospital of Zhengzhou University in Henan, China. Of the ESCC patients, 50,313 patients had been recorded with detailed address. Based on the diagnosed time, these patients were divided into three groups with periods of 1985-1994, 1995-2004 and 2005-2014. The rural and urban regions were recognized based on the government administrative division of China. The rural region in China refers to the farmer village with hundreds to thousands population. Chi-Square test was used to analyze the different distributions of ESCC patients with a p-value of less than 0.05 as significant. The results demonstrated that more than 84% of the ESCC patients occurred in rural regions with an overall ratio of 5:1 for rural and urban regions (p < 0.0001). Moreover, over the past 30 years with three periods, the ratios for rural and urban regions were 5.4:1, 5.5:1 and 5.4:1, respectively. The present results also demonstrated that over the past 30 years with three periods, ESCC occurred predominantly in males with ratios for male and female as 1.5:1, 1.4:1 and 1.5:1, respectively in rural regions and 1.6:1, 1.6:1 and 1.8:1, respectively in urban regions. Interestingly, the mean age both in male and female in urban regions was slightly older than that in rural regions (urban vs. rural: 60.7±9.86 vs. 59.8±9.38 in male and 62.2±9.57 vs. 60.6±9.47 in female, respectively). The present study demonstrated that ESCC remains the major healthy burden in rural regions in China. Considering the relative low socioeconomic status in China, the prevention and control of ESCC should be enhanced in rural regions in terms of more control programs, medical and education resources. [Supported by National High-Tech Research and Development Program of China (SQ2015AA0202183), Project for Characteristic and Advantage Discipline of Henan Province (20151208)]

#265

Global transition in cardia and non-cardia gastric cancer incidence, 1998-2007.

Aesun Shin,1 Dalis Seungeun Kim,2 Hyungtaek Woo1. 1 _Seoul National Univ. College of Medicine, Seoul, Republic of Korea;_ 2 _USF Morsani College of Medicine, Seoul, FL_.

Gastric cardia and non-cardia cancer differs in their descriptive patterns and risk factors. The objective of this study was to determine the global trends in incidence of cardiac and non-cardiac gastric cancer. Ninety two high quality registries with the proportion of Not Otherwise Specified (NOS) gastric cancer (ICD-10 code C16.8-9) below 50% and the total number of gastric cancer cases above 300 were selected from 225 registries in Cancer Incidence of Five Continent (CI5) volume 9 and 262 registries from CI5 volume 10. The NOS cases were re-distributed to cardia and non-cardia according to the registry-specific proportion. Age-standardized incidence rates (ASRs) per 100,000 population and incidence rate ratios (IRR) from 1998-2002 to 2003-2007 were calculated. Non-cardia cancer incidence was significantly decreased in 5 out of 10 Asian registries, 17 out of 18 European registries, 27 out of 56 North American registries, and all 5 Oceanian registries in men. Only two registries in Asia showed a significant increase in non-cardia cancer incidence. In contrast, cardia cancer significantly increased in one Asian registry, two European registries, and two North American registries. Significant decrease in cardia cancer was observed in 3 Asian registries, 6 European registries, 11 North American registries, and 3 Oceanian registries in men. In women, six registries (1 Asian, 2 European, and 3 North American) showed significant increase in non-cardia cancer, whereas registries 25 (3 Asian, 13 European, 8 North American, and 1 Oceanian) showed significant decrease in non-cardia cancer incidence. Cardia cancer in women increased in 16 registries (2 Asian and 14 North American) and decreased in 9 registries (1 Asian, 5 European, and 3 North American). In conclusion, non-cardia cancer incidence decreased in most registries, whereas decrease in cardia cancer is more prominent in men but not in women.

#266

Trends in age distribution of breast cancer, in north east of Iran: Report of six thousand patients over 30 years.

Maryam Salehi, Soodabeh Shahidsales, Mehdi Seilaniantousi, Gholamhosein Noferesti, Mahta Salehi, Amirhossein mohammadian bajgiran. _Mashhad University of Medical Sciences, Mashhad, Islamic Republic of Iran_.

Aim: This study was aimed to evaluate the age at the time of diagnosis of breast cancer and it's probably changes in distribution among patients during 30 years.

Methods: This cross- sectional study was conducted in Mashhad, Iran from 1981 until 2011. The data of patients were obtained from case records. The duration of study was categorized into three periods: 1981-1990, 1991-2000 and 2001-2011. SPSS software was used for analyses. The difference was considered significant if P<0.05.

Result: From 6274 breast cancer cases, 174 patients were male (2.5%) and the others 6100 were female (97.5%).Mean age at the time of diagnosis was 49.07 ± 12.07 years (male: 56.48 ± 14.14, female: 48.8 ±12.01). There was significant difference in age by sex (p <0.001). Table 1 presents the mean age of studied patients in each 10 years of study. ANOVA test showed that there was not statistical significant difference (P value= 0.1). | |  | |

|

---|---|---|---|---|---

Decades | Frequency | Mean age | SD | Youngest | Oldest

1981-1990 | 467 | 47.97 | 12.043 | 18 | 88

2000-1991 | 1771 | 49.03 | 12.338 | 20 | 99

2001-2011 | 3949 | 49.22 | 11.954 | 20 | 98

The most histologic type of breast cancer was ductal carcinoma 65.6% others percent in order were: NOS 19.6%, lobular 3.7%, medullary 2.8%, adenocarcinoma 2.8%, tubular and papillary 0.2% and 5.1% of other types. Relative frequency of histologic types had significant differences among 3 intervals (p <0.001), the frequency of adenocarcinoma was decreasing but ductal carcinoma was increasing during 3 decades. Mean age of ductal carcinoma, lobular, medullary and adenocarcinoma was 48.9±11.8, 50.6±12.4, 46.4±11.4, 50.2±11.9, respectively. Mean age had significant differences among these main histologic types (p =0.002).98% of patients were urban and 2% were rural. Mean age of these 2 groups has statistical significant difference (P = 0.001) and is 10 years older in rural patients. Frequency of people who live in urban area was increased significantly (p<00.1) during these 30 years compare with rural area.

Conclusion: breast cancer occurs in Iranian women at least one decade younger than women in developed countries we found no change in mean age of diagnosis of breast cancer during these 30 years from 1981 to 2011.

.

#267

GeoSurveillance of invasive cervical cancer incidence by counties in the state of Maryland.

Sally Peprah, Frank Curriero, Amber D'Souza. _Johns Hopkins University, Baltimore, MD_.

Background: Invasive cervical cancer (ICC) is a highly preventable cancer whose rates have dramatically declined over the past few decades. In spite of the progress made, new ICC cases are recorded annually in Maryland and in other parts of the United Sates. We assessed if ICC incidence varied significantly 2005-2012 and across counties within the state of Maryland.

Methods: Using a discrete Poisson model we evaluated significant space-time clusters of both high and low ICC incidence rates over a retrospective 10-year period. All analyses were conducted at the county level, using 2-year aggregates of time. We adjusted for age, percent minority, cervical cancer screening rates, median income, obesity as well as smoking rates within each county in our final cluster detection

analysis.

Results: ICC was 13.1 per 100,000 female population and did vary by county and over the 10-year period assessed. We identified five significant space-time clusters of ICC incidence, three of which were clusters of higher than expected rates. The most likely cluster identified was a cluster of low ICC incidence [aRR=0.08, p-value<0.01] observed for the period 2009-2012 and comprised six high-income suburban counties. The other cluster of low rate [aRR=0.41, p-value<0.01], which was a secondary cluster, was observed for the same time period, 2009-2012 in three high to moderate-income suburban counties. There were two clusters of high ICC incidence, observed for recent time periods. One cluster [aRR=1.74, p-value<0.01], which was made up of a single moderate to low-income urban county was identified for the period 2009-2012. The other cluster of elevated rates [aRR=2.47, p-value<0.01] was identified in a suburban moderate-income county for the period 2011-2012. Additionally, for the period 2005-2008 we identified a historical cluster of elevated incidence [aRR=2.53, p-value<0.01].

Conclusion: Overtime some counties in Maryland have experienced significantly lower rates of disease than expected. However there are more recent clusters of higher than expected rates of disease, which are not explained by the county level variables adjusted for. These clusters of high rates need to be further examined and addressed.

#268

Tracking HPV vaccination of young boys: is parental recall as dependable as physician records.

Nosayaba Osazuwa-Peters,1 Betelihem B. Tobo,2 Meera Muthukrishnan,2 Daphne Lew,2 Betty Chen,3 Eric Adjei Boakye2. 1 _St. Louis Univ. Cancer Center, Saint Louis, MO;_ 2 _St. Louis Univ., Saint Louis, MO;_ 3 _St. Louis Univ. School of Medicine, Saint Louis, MO_.

Introduction: HPV vaccine is administered to young boys and girls aged 11-12 years in multiple doses rather than single dose. The interval between doses is critical as well as completion of the dose series. Moreover, the administration of the HPV vaccine in 2 doses is a recent change from the 3 doses previously recommended for children aged 11-12 years. Therefore, it is critical that HPV vaccination uptake records are accurately kept as interventions are developed to increase HPV vaccine uptake. Previous studies on the validity of parental reported HPV vaccine uptake have focused only on adolescent girls. This is because the HPV vaccine recommendation for boys came 5 years after that for girls. Currently, vaccination rates among boys lag behind those for girls across the United States. This study compared the validity of parent- vs. provider-reported HPV vaccine initiation and completion rates among a national sample of adolescent boys in the United States.

Methods: We analyzed the 2014 National Immunization Survey-Teen data for adolescent boys only (n = 9,493), and obtained information on the records of HPV vaccine uptake [initiation (≥1 dose) and completion (≥ 3 doses)] for both parents (parental recall) and provider reports (electronic medical records). We compared the validity of parent- versus provider-reported HPV vaccination by computing validity measures [sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and kappa].

Results: Rates reported by provider and parental recall were similar for HPV vaccine initiation (41.8% vs. 42.7%, respectively), and completion (19.6% vs. 21.9%, respectively). Compared to provider report, parent-reported HPV vaccine initiation had a sensitivity of 83.0%, a specificity of 88.8%, a PPV of 84.1%, a NPV of 87.9%, and a kappa coefficient of 0.72. Compared to provider report, parent-reported HPV vaccine completion had a sensitivity of 68.6%, a specificity of 92.9%, a PPV of 72.9%, a NPV of 91.4%, and a kappa coefficient of 0.63.

Conclusions: Both parent- and provider-reported HPV vaccine initiation and completion rates were comparable; however, parental recall of HPV vaccine completion among boys was less sensitive. Ongoing surveillance of HPV vaccination rates among boys is important as more studies are designed to improve HPV vaccine uptake and understand parental HPV vaccine barriers. In this regard, parental recall may be used as an effective alternative in assessing HPV vaccine uptake in the United States.

#269

The descriptive epidemiology of gynecologic cancers: an international comparison of incidence, survival and mortality.

Zoubida Zaidi, Mokhtar Hamdi Cherif. _University Hospital of Setif, Setif, Algeria_.

Introduction: Gynecologic Cancers are the most common cancers in women, in 2012, Cervical Cancer (CC) is ranking as the fourth with an estimated 528.000 new cases and Ovarian Cancer (OC) is the seventh most common for females with nearly 239.000 new cases worldwide.

Aim: this communication presents the latest international descriptive epidemiological data for invasive GC, including incidence, survival and mortality in the worldwide.

Methods: -The incidence and mortality statistics presented for GC worldwide were taken from the International Agency for Research on Cancer IARC: * the Cancer Incidence in five Continents Vol X and * GLOBOCAN database, 2012.

-The data of cancer survival are taken from: * Cancer survival in five continents, a worldwide population-based study (CONCORD) version 2, 1995-2009. Estimated five-year net survival, adjusted for background mortality by single year of age, sex, calendar year in each country.

Results: CC is the most common cancer among women in 45 countries mainly in sub-Saharan Africa, part of Asia and some countries in Central and South America, the lowest incidence rates in western Europe, North America, Australia and the eastern Mediterranean. Almost 55% of all new cases of OC occurred in countries very high level of human development mainly northern Europe and America and Oceanic, Africa presents the lowest incidence.

-Data for CC are available for 602 225 women, CC survival was 50% or higher in most countries, except for Libya (Benghazi, 39%) and India (Karunagappally, 46%). During 2005-09, age-standardised 5-year net survival was 70% or higher in Iceland, Mauritius, Norway, South Korea, and Taiwan, for Qatar is also above 70% (based on only 16 cases and is not age-standardised).

-Data for ovarian cancer are available for 779 302 women, during 2005-09, age-standardised 5-year net survival was 40% or higher in Ecuador, the USA, nine countries in Asia, and eight countries in Europe. Survival in other countries was mostly in the range 30-40%, except for Libya (22%).

Conclusions: The evolution of cancers in women shows a consistent and very striking pattern during the epidemiological transition with rapid declines in the incidence of cervical cancer. Comparison of population-based cancer survival CONCORD study 2 showed very wide variations in survival from gynecologic cancer worldwide.

#270

Trends in colorectal cancer survival in Arab World, 1990-2009.

Zoubida Zaidi, Mokhtar Hamdi Cherif. _University Hospital of Setif, Setif, Algeria_.

Introduction: Cancer survival is a key measure of the effectiveness of health-care systems. Globally, colon and rectum cancer ranked third for cancer incidence and fourth for cancer death in 2013. For developed countries it ranked second for incidence and mortality, and in developing countries it ranked fourth for both incidence and mortality. An increasing trend in incidence is reported from various registries of Arab world, Kuwait and Saudi Arabia present the highest incidences worldwide.

Objective: To describe the trends of the survival of colorectal cancer patients diagnosed in Arab countries.

Material and Methods: This report is a summary of the two survival figures of CONCORD study 1 (1990-1994) and CONCORD study 2 (1995-2009). Individual colon and rectum tumour records were submitted by 06 population-based cancer registries in Arab countries (Jordan, Saoudi Arabia, Qatar, Algeria, Lybia and Tunisia) for 9050 patients (15-99 years) diagnosed during 1990-2009 and followed up to 31 December 2009 . Estimated five-year net survival, adjusted for background mortality by single year of age, sex, calendar year in each country.

Results : For patients diagnosed during the period 2005-2009, the age-standardized five-years net survivals were respectively higher 68,2% for colon cancer and 77,8% for rectum cancer in Qatar and the lowest rate for rectal cancer 21% in Jordan, between 1995-1999 and 2005-2009, Survival increased in Algeria, but this trend is less reliable

Conclusions: Comparison of population-based cancer survival CONCORD study showed very wide variations in survival from colorectal cancer in Arab world. Cancer survival research is being used to formulate cancer control and the need to implement effective strategies of primary prevention.

#271

Clinicopathological and molecular study of breast cancer in Algerian women from the Aures region: Data from the anti-cancer center of Batna.

Farid Cherbal,1 Chiraz Mehemmai,2 Khadidja Gassi,1 Abdelmoumene Guedioura,3 Yosr Hamdi,2 Wassila Benbrahim,4 Rabah Bakour1. 1 _Unit of Genetics, LMCB, Faculty of Biological Sciences, USTHB, Algiers, Algeria;_ 2 _Laboratoire de Génomique Biomédicale et Oncogénétique (LR11 IPT 05) Institut Pasteur de Tunis, Tunis, Tunisia;_ 3 _LOBEM, Faculty of Biological Sciences, USTHB, Algiers, Algeria;_ 4 _Anti-cancer center of Batna, Batna, Algeria_.

Background:Breast cancer is currently the leading cause of cancer morbidity and mortality among Algerian women. The aim of this study was to analyze the different proportions and distribution of breast cancer molecular subtypes and to determine their associations with some clinical and pathological characteristics in women from the Aures region. We also screened for the prevalence of BRCA1 germline mutations in HBOC patients.

Materials and Methods: Clinical and tumor characteristics data of 1144 breast cancer patients were collected from cancer registry of anticancer center of Batna. Breast cancers were diagnosed between 2011 and 2015. Breast cancer subtypes definitions were as follow: Luminal A (ER+ and/or PR+,HER2-), Luminal B (ER+ and/or PR+,HER2+),TNBC (ER-, PR-,HER2-) and HER2+ (ER-, PR-, HER2+). We also compared patients with TNBC and those with other breast cancer subtypes. Differences between the two groups with regard to clinicopathologic parameters: age at diagnosis, menopausal status, histological type, histological grade, breast feeding and oral contraception were examined using Chi square test. BRCA1 was screened by PCR direct sequencing in 22 HBOC patients including all exons where a mutation was previously found in Algerian population (exons 3, 5, 11).

Results: The mean age at diagnosis cancer was 48.5 years. Proportions of luminal A, luminal B, TNBC and HER2+ breast cancer subtypes were 47.29%, 21.94%, 21.24% and 9.52%, respectively. We noted a significant difference in the distribution of age at diagnosis among the four cancer subtypes (P = 0.04). Invasive Ductal Carcinoma was the most common histological type in all breast cancer subtypes. Tumors with histological grade 2 and 3 were more common in patients for the four breast cancer subtypes. Our data showed significant differences between premenopausal and postmenopausal status (P=0.00) and tumor histological grade (P<0.03) between TNBC and non TNBC patients. The proportion of premenopausal status was higher in TNBC and non TNBC. Among tumor with histological grade III, TNBC accounted for 33.33% for all TNBC patients and non TNBC accounted for 30.23% for all non TNBC patients. The three BRCA1 recurrent mutations c.83_84delTG, c.181T>G and c.798_799delTT have not been detected in our present study.

Conclusion: We report here for the first time, the distribution of molecular subtypes of breast cancer and some clinical and pathological characteristics in a cohort of female breast cancer patients from the Aures region. Interestingly, the proportion of TNBC subtype in women found in our study is similar to African-American breast cancer patients and higher than reported in white women in Europe and America. Further studies are needed to determine the outcome of the different breast cancer subtypes in the Aures region. Large studies of the screening of BRCA1 and BRCA2 genes are on going for the implementation of the genetic testing.

#272

Circadian disruption and cancer risk in the United States.

Neil E. Caporaso,1 Fangyi Gu,1 Shangda Xu,1 Susan S. Devesa,1 Fanni Zhang,2 Elizabeth B. Klerman,3 Barry Graubard1. 1 _NCI-DCEG, Rockville, MD;_ 2 _IMS Corporation, Rockville, MD;_ 3 _Harvard Medical School, Boston, MA_.

Circadian disruption is a probable human carcinogen. Dawn and dusk times occur latter in western compared to eastern regions of each time zone, producing discrepancies between individuals' biological clock and social time which may lead to circadian disruption and increased risk of cancer. We examined associations between longitude distance from the time zone meridian and age-standardized county-specific cancer incidence rates for total and 23 specific cancers by gender. Four million whites were diagnosed with cancer during 2000-2012 in the Surveillance, Epidemiology and End Results Program in residents of 607 counties in 11 continental US states. The models were adjusted for latitude, poverty, smoking, and state as well as rural/urban location, obesity, and Hispanic composition.

The total cancer incidence rates for counties located in the western regions of time zones were significantly elevated for both men and women after Bonferroni correction. The rate ratio (RR) per five degrees of longitude toward the west (corresponding to 20 minutes delay of sunrise) was 1.03 (95% confidence intervals, CI, 1.02-1.04, p=2.7×10-6) for men and 1.04 (95% CI, 1.03-1.05, p=3.8 ×10-10) for women. Chronic lymphocytic leukemia rates for counties in the western portions of time zones were significantly elevated for both men (RR=1.13, 95% CI=1.08-1.20, p=3.1 x10-6) and women (RR=1.12, 95%CI=1.05-1.19, p=3.7 x10-4). Among men, western location within a time zone was associated with elevated risk of prostate cancer (RR=1.04, 95% CI=1.02-1.07, p=4.8 x 10-4). Among women, western location within a time zone was associated with an increase in breast cancer (RR=1.04, 95% CI=1.02-1.06, p=6.5 x 10-5) and corpus uteri cancer (RR=1.10, 95% CI=1.07-1.14, p=2.4 x 10-9 among others.

The increased cancer risk with western residency within a time zone is in accord with the circadian disruption hypothesis. Our findings suggest that circadian disruption is not a rare phenomenon affecting only shift workers or international travelers but may be widespread in the general population which has broader implications for public health than generally appreciated.

#273

Risk of primary central nervous system lymphomas in solid organ transplant recipients.

Parag Mahale, Meredith S. Shiels, Eric A. Engels. _National Cancer Institute, Rockville, MD_.

Background: The risk of primary central nervous system lymphoma (PCNSL) is greatly increased in HIV-infected people. Case series have described PCNSL in immunosuppressed solid organ transplant (SOT) recipients (SOTRs). Herein, we examine the incidence and risk factors for PCNSL in SOTRs.

Methods: We used data from the Transplant Cancer Match Study, which links the US transplant registry with 17 cancer registries (1987 - 2014). PCNSL risk relative to the general population was estimated as a standardized incidence ratio (SIR = observed/expected cases). Poisson regression was used to estimate adjusted incidence rate ratios (aIRR) of PCNSL across subgroups of SOTRs. Logistic regression was used for case-case comparisons of PCNSL with other non-Hodgkin lymphomas (NHL).

Results: We included 288,637 SOTs. There were 173 PCNSL cases (SIR 57.7; 95%CI 49.4-66.9) and 2,583 other NHLs (SIR 7.3; 95%CI 7.0-7.6). Most PCNSL were diffuse large B-cell lymphomas (n=118; 68.2%). Compared to kidney SOTRs, PCNSL risk was lower in liver SOTRs (aIRR 0.5; 95%CI 0.3-0.9), not different in heart and/or lung SOTRs (aIRR 0.9; 95%CI 0.6-1.5) and higher in other/multiple SOTRs (aIRR 2.4; 95%CI 1.5-3.8). Asians/Pacific Islanders had higher PCNSL risk than non-Hispanic whites (aIRR 2.0; 95%CI 1.2-3.3). People who received induction therapy with alemtuzumab (aIRR 2.8; 95%CI 1.5-5.5) or polyclonal antibodies (aIRR 1.9; 95%CI 1.3-2.8) had higher PCNSL risk. SOTRs who were seronegative for Epstein-Barr virus (EBV) at transplant had higher risk (aIRR 2.0; 95%CI 1.1-3.5) than seropositive SOTRs. PCNSL risk was high in the first 1.5 years after SOT (0.5-1 year, aIRR 2.6; 1-1.5 years, aIRR 2.3; vs. 0-0.5 year) and progressively decreased over time (ptrend<0.0001). Risk did not differ according to the age at SOT, sex, or maintenance immunosuppressive regimen. Compared to other NHL, PCNSL cases were more likely to be middle aged (18-64 years) at transplant (p=0.009), Asians/Pacific Islanders (p=0.02), or have received induction therapy with polyclonal antibodies (p=0.002), and less likely to be liver or heart and/or lung SOTRs (p=0.02). EBV serostatus did not differ between PCNSL and other NHL (p=0.11). Conclusions: PCNSL risk is very elevated among SOTRs. Because EBV-seronegative SOTRs are at risk of primary infection after SOT, these results highlight the important contribution of EBV to PCNSL. Risk is highest within 1.5 years after transplant, in people who receive multiple non-thoracic organs, and is associated with induction therapy with alemtuzumab or polyclonal antibodies. Case-case differences with other NHLs suggest unique etiologic factors leading to PCNSL.

#274

Early-onset colorectal cancer is distinct from traditional colorectal cancer.

Doron Betel, Heather Yeo, Rhonda Yantiss, Xi E. Zheng, Jonathan S. Abelson, Manish A. Shah. _Weill Cornell Medicine, New York, NY_.

Background & Objective: Colorectal cancer (CRC) is the fourth most commonly diagnosed cancer worldwide, responsible for over 700,000 deaths annually. Although CRC rates have declined 3.1%/year annually over the past decade, annual incidence rates of CRC among patients between 20-49 years old have increased by more than 1.4% per year over the same time period and these early-onset CRCs (E-CRC) now comprise 10-18% of newly diagnosed cases.

Our hypothesis is that E-CRC represents a distinct subtype of CRC with unique drivers. To examine this, we analyzed national data sources such as the SEER database, the Behavioral Risk Factor Surveillance Survey (BRFSS), and the Cancer Genome Atlas (TCGA) to characterize the epidemiology and genetics differences between early onset CRC (E-CRC) and typical CRC (T-CRC).

Methods: The SEER database was analyzed to characterize the clinical and pathological differences between E-CRC patients (20-49 years) and T-CRC group (> 50 years). We compared rates of age-adjusted CRC with known lifestyle risk factors using population data from the BRFSS. Based on differences in clinical presentation and anatomical location we hypothesized that there are underlying molecular differences between E-CRCs and T-CRC that may explain these differences. To investigate this we analyzed the TCGA CRC dataset for genomic markers that can distinguish between E-CRC and T-CRC tumors.

Results: E-CRC incidence has risen at an annual rate of 1.4% per year from 2000-2011, whereas T-CRC incidence has declined by 3.1% per year among patients 50 years or older during the same period.

Relative to T-CRC, E-CRC rates are significantly more prevalent in the Black population than the White or Hispanic populations.

Anatomical location of E-CRC is significantly increased towards the distal colon when compared to T-CRC locations suggesting a distinct etiology.

Analysis of risk factors confirmed that U.S counties with high rates of diabetes, obesity and smoking were significantly correlated with higher T-CRC rates. However, no such correlations were observed for E-CRC rates.

Analysis of the limited number of E-CRC in the TCGA cohort identified a number of known oncogenes that are suggestive of distinct E-CRC tumorigenesis.

Conclusions: Our results suggest that E-CRC appears to be distinct from T-CRC. Young patients with CRC represent a distinct patient group with unique epidemiology and mechanisms of disease development. The increase in left sided colon and rectal cancers is consistent with earlier reports and although the underlying reasons are unknown, they are suggestive of differences in tumor genesis and mutational characteristics. To follow up on this hypothesis we are performing additional genetic and epigenetic characterization of E-CRC and matching T-CRC samples collected at Weill Cornell.

#275

The prevalence of endometrial cancer in women with postmenopausal bleeding: a systematic review and meta-analysis.

Megan A. Clarke,1 Beverly J. Long,2 Arena Del Mar Morillo,1 Jamie N. Bakkum-Gamez,2 Nicolas Wentzensen1. 1 _National Cancer Institute, Rockville, MD;_ 2 _Mayo Clinic, Rochester, MN_.

Background: Endometrial cancer is highly curable when detected early, but clinical symptoms are often missed. Most cancers arise in women with abnormal postmenopausal bleeding (PMB). However, PMB is not specific for endometrial cancers. In previous reports, the prevalence of endometrial cancer in women with PMB was reported to range from 3-25%. Because of the importance for guiding clinical management and endometrial cancer risk prediction, we conducted a systematic review and meta-analysis to obtain precise prevalence estimates of endometrial cancer in women with PMB.

Methods: A systematic review and meta-analysis summarized all peer-reviewed studies reporting endometrial cancer prevalence in women with PMB, published before Sept. 1, 2016. We calculated a pooled prevalence estimate and 95% confidence interval (CI) using a random-effects model, and quantified heterogeneity across studies using the I2 statistic. Subgroup meta-analyses were performed to investigate sources of variability according to selection criteria for endometrial thickness (ET) and hormone replacement therapy (HRT). We also stratified by study location to determine whether prevalence estimates varied by country or region if ≤5 studies included per country.

Results: 105 studies were included, contributing data for 37,000 women with PMB and 3,028 with cancer from 34 countries. Overall, the pooled prevalence of endometrial cancer in women with PMB was 11% (95% CI 10-12%), with I2 =94.3%. Among studies that selected women with a minimum ET (≥ 4mm; n=14), the pooled prevalence of endometrial cancer was 17% (95% CI 14-20%) compared with studies that did not select for ET (10%, 95% CI 9-11%). In studies that excluded women using HRT (n=34), the pooled prevalence of endometrial cancer was 15% (95% CI 12-17%) compared with studies that did not exclude women using HRT (9%, 95% CI 8-10%). Stratification by study location revealed variation in prevalence estimates. For example, in the United States (n=9) and the United Kingdom (n=16), prevalence of endometrial cancer was 4% (95% CI 2-7%) and 5% (95% CI 4-6%), respectively, whereas in Italy (n=14), the prevalence was 11% (95% 8-14%). Regional differences were also observed, ranging from 8% (95% CI 3-14%) in South Central Asia (n=5) to 18% (95% CI 12-23%) in Southern Europe (n=11).

Conclusions: The overall prevalence of endometrial cancer among unselected women with PMB is approximately 10% with substantial heterogeneity across studies. Marked geographic differences in the prevalence endometrial cancer were observed. Factors related to selection for ET and/or HRT use may partially contribute to this variability. Risk prediction models depend on precise prevalence estimates for accurate prediction and utility in clinical management. More research is needed to test the applicability of endometrial cancer risk prediction models in high-risk subgroups and in study populations from diverse geographic regions.

#276

Retrospective analysis of epidemiological variants of malignancies in Sokoto, Northwestern Nigeria.

Saddiku M. Sahabi, Kabiru Abdullahi. _Usmanu Danfodiyo University Teaching Hospital, Sokoto, Nigeria_.

Background: The burden of cancer in Nigeria is increasing. Information on incidence, prevalence, pattern, and high risk factors are essential for evaluation and cancer control. This report is a follow up from earlier previous report published in 2005 on cancers seen in Usmanu Danfodiyo University Teaching Hospital (UDUTH) Sokoto.

Objectives: The aim of this study is to determine epidemiological variants of all malignancies seen from 1 January 2006 to 31 December 2015.

Materials and Methods: This is a retrospective review of all malignancies seen at the Usmanu Danfodiyo University Teaching Hospital in Sokoto during the period 2006 to 2016. There was no restriction on gender or age groups. Mean, frequencies, percentages, and independent sample t-test were performed using SPSS version 21.0 for Windows. Significant level set at p < 0.05.

Results: A total of 3933 cancer patients were seen during the ten-year period. There were more female malignancies (n=2260, 57.5%) compared to male malignancies (n=1673, 42.5%). The overall mean age (years) was 44.28, median age was 45.0, modal age was 50, and SD was 18.69; the age range was 1 to 99 years. Males (mean age 45.77, median age in male 50.0, modal age 60, and SD 21.03) were significantly older than females (mean age 43.18, median age 45.0, modal age 50, and SD 16.67). The adult malignancies account for 90.9% (n=3575) and childhood malignancies account for 9.1% (n=358). Overall, the ten most common malignancies were: breast 763 (19.4%), cervix 312 (7.9%), prostate 267 (6.8%), lymph node 248 (6.3%), skin 225 (5.7%), bladder 191 (4.9%), rectum 114 (2.9%), ovary 110 (2.8%), eye 97 (2.5%), soft tissue 95 (2.4%). In males, the ten most frequent malignancies were: prostate 267 (16.00%), bladder 160 (9.60%), skin 138 (8.20%), nasophraynx 87 (5.2%), lymph node 85 (5.10%), rectum 61 (3.60%), eye 55 (3.30%), salivary gland 38 (2.3%), larynx 37 (2.20%), and anal 37 (2.20%). In females, the ten most common malignancies were breast 731 (731%), cervix 302 (13.40%), lymph node 163 (7.20%), ovary 110 (4.90%), skin 87 (3.80%) endometrial 61 (2.70%), rectum 53 (2.30%), nasophraynx 45 (2.0%), soft tissue 43 (1.90%), and eye 42 (1.90%).

Conclusion: Our study concludes that malignancies of the breast, cervix, and prostate are the three most common in our environment. These malignancies tend to present at younger age (less than 50 years of age) compared to other developing countries. Therefore, public education and well developed screening programs are urgently needed to overcome this burden.

#277

A retrospective analysis of pediatric and adolescent oncology patients and congenital anomalies.

Jeannette R. Wong-Siegel, Kimberly Johnson, Katie Gettinger, Todd Druley. _Washington University in St. Louis, St. Louis, MO_.

Introduction: Congenital anomalies (CAs) are a leading cause of infant death and contribute to long-term disability and repeated hospitalizations. Many epidemiology studies have suggested that children with CAs have an increased cancer risk compared to those without CAs. This retrospective case series further investigates associations between CAs and cancer in a large academic center's pediatric and adolescent oncology patient population.

Methods: Electronic medical records of 1,435 oncology patients diagnosed from birth to 23 years of age at St. Louis Children's Hospital from January 1, 2004 - December 31, 2014 were reviewed. CA information was extracted and verified with ICD-9 codes when available. Patients followed for <1 year at SLCH (n=193), and those diagnosed with a chromosomal anomaly or known cancer predisposition syndrome (n=113) were excluded from the analysis. Bivariate analyses compared demographic and other characteristics between patients with and without a CA with significant differences determined by chi-square tests. We calculated age-adjusted standardized rate ratios (SRRs) to evaluate whether the observed number of CAs among specific cancer types (benign, bone, CNS, epithelial, leukemia, lymphoma, germ cell, and soft tissue tumors) varied from the expected number using the CA rates from the entire cohort as the reference. Differences in survival time distributions in cancer cases with CAs versus those without CAs were evaluated with the log-rank test.

Results: Of 1,129 SLCH pediatric cancer patients, 156 (14%) patients were identified with a CA. Overall there was an increased proportion of patients with a CNS tumor who also had a CA compared to those without a CA (p=0.005). Neurological anomalies were specifically found to be in excess in CNS tumor cases versus the overall population of pediatric cancer patients (SRR= 1.42 95% CI 1.02-1.92 p=0.038). There were no significant differences by age at primary tumor diagnosis, but patients with a CNS tumor and CA were diagnosed an average of 1.5 years earlier (7.7 vs. 9.2 years, p=0.075) compared to those without a CA. The rate of CAs did not vary significantly by sex, but a significant excess of males with a neurological anomaly was observed among all patients diagnosed <5 years of age (M/F ratio=2.55 95% CI 1.15-5.56 p=0.02). Finally, survival between those with and without a CA was not significantly different (p=0.24).

Conclusions: This study provides additional insight into the association between specific types of CAs and cancer development. Our results suggest children with neurological anomalies are more likely to develop CNS cancers and may be more likely to develop cancer at an earlier age, particularly in males. Our study supports the need for additional longitudinal surveillance and research that may improve outcomes as well as translational research to investigate any associated developmental mechanisms that may underlie tumor predisposition.

#278

The Sister Study: Morbid conditions at enrollment in a breast cancer cohort.

M. Elizabeth Hodgson,1 Whitney D. Arroyave,1 Sandra Deming-Halverson,1 Aimee A. D'Aloisio,1 Dale P. Sandler2. 1 _Social and Scientific Systems, Inc., Durham, NC;_ 2 _Epidemiology Branch, National Institute of Environmental Health Sciences, Research Triangle Park, NC_.

The Sister Study is a prospective cohort of 50,884 unaffected sisters of women diagnosed with breast cancer; given that participants were at increased risk of developing breast cancer, we wanted to investigate the prevalence of other conditions at enrollment. We compared demographic, lifestyle, and selected self-reported morbid conditions between Sister Study participants and women in NHANES, a representative sample of the US population, using age-adjusted prevalence, overall and by race/ethnicity. Demographically, women in the Sister Study are more likely than women in NHANES to be non-Hispanic white, well-educated and to have had higher household incomes. They were also somewhat more likely to have been post-menopausal at enrollment and have ever used hormonal birth control or hormone replacement therapy. The Sister Study has a lower prevalence of current smokers and a higher prevalence of alcohol consumption than among women in NHANES. BMI was lower in the Sister Study. Sister Study participants generally had a lower age-adjusted prevalence of self-reported chronic morbid conditions at baseline than did women in NHANES. For cardio- and cerebrovascular conditions, for example, heart attack and stroke were lower in the Sister Study (0.9% vs. 2.4%; 0.6% vs. 3.1%, respectively) as were self-reported hypertension and high cholesterol (22.8% vs. 34.5%; 28.6% vs. 40.7%). This pattern was similar across all race/ethnicity groups with the exception of angina, which was similar to NHANES overall (2.1% vs. 2.2%). Prevalence of diabetes was lower in the Sister Study (4.9% vs. 9.5%) as were cancers other than breast or non-melanoma skin (4.4% vs. 8.1%) and rheumatoid arthritis (3.3% vs. 6.2%). Again, this was similar across race/ethnicity groups. A few conditions had a higher prevalence in the Sister Study: uterine fibroids, endometriosis, osteoporosis, and allergies (22.0% vs. 13.7%; 12.2% vs. 7.7%; 17.0% vs. 8.6%; and 54.1% vs. 44.2%). Thyroid disease (excluding cancer), asthma, Crohn's disease, and ulcerative colitis were similar in the Sister Study and NHANES (19.4% vs. 18.5%; 14.6% vs. 15.0%; 0.5% vs. 0.4%; and 1.4% vs. 1.3%). Two or more morbid conditions were reported by just over one third of Sister Study participants. After restriction to college graduates, differences between the Sister study and NHANES were no longer apparent. The few exceptions were differences that would be expected, given Sister Study participants' ability to participate in a long-term study, healthy behaviors and access to care: lower prevalence of hypertension and high cholesterol; higher prevalence of self-reported fibroids, osteoporosis, and allergies. Although Sister Study participants do differ in a few respects from the NHANES population, the cohort is a unique and well characterized resource, well-suited for research on the incidence of number of chronic conditions, in addition to breast cancer.

#279

Clinicopathologic features and management of bronchopulmonary and pleural malignancies in Ile Ife, Nigeria.

Olanisun O. Adewole. _Obafemi Awolowo University/Teaching Hospital, Ile Ife, Nigeria_.

CLINICOPATHOLOGIC FEATURES AND MANAGEMENT OF BRONCHOPULMONARY AND PLEURAL MALIGNANCIES IN NIGERIA

Adewole O.O, G.E Erhabor.

Department of Medicine, Obafemi Awolowo University Teaching Hospitals Complex, Ile Ife, Nigeria.

Introduction. Lung cancer is the most common cause of cancer related death worldwide with increasing incidence in developing countries, Nigeria inclusive. The aim of this study is to evaluate the clinical patterns, diagnostic procedures and management of bronchopulmonary cancer (BPC) and pleural cancer (PLC) in Ile Ife, Nigeria.

Method. All cases of bronchopulmonary and pleural cancer seen over an eighteen year period (1990-2015) at OAUTHC, Ile Ife, Nigeria, a tertiary center were reviewed. Cases were identified from the admission and discharge files on the wards and were retrieved fro the main medical record library. All retrieved cases were analyzed and presented, using a descriptive statistics.

Results. Thirty seven cases of BPC and PLC were identified of which twenty two case files were fully retrievable, and these are presented. There were twenty one cases of BPC and two PLC. The male to female ratio of BPC was 2:1 while it is 1:1 for PLC. The mean age of patients was 51 years (range 48-85yrs) and 45(30-66yrs) for BPC and PLC respectively. Cough and weight loss were the most common symptoms among al the patients.45% had chest pain while 15% had hemoptysis. Two patients had progressive dyspnea. Smoking and indoor/outdoor pollution were identified as the risk factors in 20% each of the patients with BPC .10% of patients with BPC had a past history of TB about 10 years prior to the presentation. One of the cases of PLC had some exposure to asbestos in his work as a carpenter.

Three patients with BPC presented with features of SVCO.

Pleural effusion was the main chest pathology, it was found in 65% of the BPC cases and the entire PLC. The right hemithorax were mainly involved. Diagnosis were made by pleural fluid cytology, pleural biopsies, FNAC, LN biopsies, bronchoscopy in 41%, 10%, 10%,15% and 5% of BPC respectively. Other diagnostic procedures done in PLC included open thoracotomy.

Adenocarcinoma was the commonest histologic type of BPC, while it was mesothelioma for PLC. Few of the patients with Adenocarcinoma had history of smoking.

30% 0f the BPC were secondaries from breast, cervix, bone and skin. Death rate was 50% within two weeks of diagnosis, three patients discharge against medical advice.

The remaining were referred to another center.

Conclusion. Primary BPC are the commonest in this review except in females. With increasing industrialization, atmospheric pollution, cigarette smoking and poverty in most developing countries, the incidence and problems of BPC and PLC would be expected to rise. More studies to identify risk factors and training for physicians in diagnostic procedures and treatment of BPC are urgently needed in resource limited countries.

#280

The maximum tumor length and lymph node metastasis in esophageal squamous cell carcinomas.

Shuang Lv,1 Shuang Lv,2 Fu You Zhou,3 Hai Jun Yang,3 Bei Li,2 Li Na Liu,2 Yan Jie Xia,2 Yan Ru Qin,2 Rui Tai Fan,2 Fan Kai Xiao,2 Li Dong Wang2. 1 _Xinxiang Medical University, Xinxiang, China;_ 2 _The First Affiliated Hospital of Zhengzhou University, China;_ 3 _Anyang Tumor Hospital, China_.

The lymph node metastasis (LNM) is one of the crucial risk factors for esophageal squamous cell carcinomas (ESCC) prognosis. However, it is difficult to precisely determine the LNM status before surgery. The maximum tumor length (MTL) may reflect the ESCC growth speed and could be calculated accurately either with computed tomography or ultrasound endoscopy before esophagectomy. Therefore the present study was designed to determine if the MTL could reflect the LNM status for ESCC. A total of 22,660 ESCC patients was enrolled in this study from the ESCC database (1973-2015) in Henan Key Laboratory for Esophageal Cancer Research of the First Affiliated Hospital, Zhengzhou University. Of these patients, there were 13,793 males with an mean age of 60±8, and 8,867 females with an mean age of 61±8. All the cases had been performed radical esophagectomy. The MTL and LNM records were retrieved from the medical records in hospitals. Based on the 6th version of UICC criteria, the LNM was classified as N0 and N1. The MTL was classified as 5 groups: <2cm, ≥2 and <4cm, ≥4 and <6cm , ≥6 and <8cm , ≥8cm. The SPSS21.0 software and Spearman rank correlation were used to determine the correlation of MTL and LNM. The results demonstrated that the average and median of MTL were 4.1±1.8 cm and 4.0 cm (range: 0.1-18 cm) in these ESCC patients. The positive lymph node metastasis rates in each MTL groups were 19.1%, 34.1%, 45.8%, 51.5%, and 54.1%, respectively. Spearman rank correlation analysis indicated that MTL was strongly correlated with LNM (r=0.997, P<0.001), suggesting that MTL could reflect the LNM status in ESCC and that MTL could be used as one of clinical staging parameters for ESCC. [Supported by Major Science and Technology Projects of Henan Province (161100311300). National Key Research and Development Program: Precision Medicine (2016).]

#281

Site specific risk factors for colorectal cancer.

Samir Gupta,1 Ranier Bustamante,1 Ashley Earles,1 Maria E. Martinez,2 Karen Messer,2 Christina D. Williams,3 Andrew J. Gawron,4 Tonya Kaltenbach,5 Lin Liu2. 1 _VA San Diego Healthcare System, San Diego, CA;_ 2 _University of California, San Diego, La Jolla, CA;_ 3 _Durham VA Medical Center, Durham, NC;_ 4 _VA Salt Lake City Health Care System, Salt Lake City, UT;_ 5 _San Francisco VA Health Care System, San Francisco, CA_.

Background: Most studies of colorectal cancer (CRC) risk factors pool cases across anatomical sites, though tumor characteristics and embryologic origins differ. Our aim was to conduct a site-stratified (proximal, distal, rectal) case control study of candidate risk factors for CRC.

Methods: Our study base was US Veterans with >1 colonoscopy 1999-2011. CRC cases at baseline colonoscopy were identified with the Veterans Affairs Central Cancer Registry. Controls had normal colonoscopy without biopsy, no history of CRC, and no CRC within 3 years of index colonoscopy. Age, sex, race/ethnicity, body mass index (BMI), diabetes, and smoking were considered candidate risk factors. Primary outcome was CRC, stratified by site. Site-specific odds ratios (OR) and 95% confidence intervals (CI) for each risk factor were computed with unadjusted and adjusted multinomial logistic regression models.

Results: We identified 21,739 CRC cases and 616,323 normal colonoscopy controls. For cases combined vs. controls, median age was 68 vs. 61 years, 98% vs. 95% were male, median BMI was 27.8 vs. 28.8 kg/m2, 28% vs. 24% had diabetes, and 25% vs. 29% were non-smokers; race/ethnicity was similar. Presence and strength of associations differed by risk factor and CRC site (Table). Smoking was more closely associated with rectal (OR=1.84) than proximal (OR=1.59) or distal cancer (OR=1.50). Diabetes was more closely associated with proximal than distal or rectal cancer (OR=1.28, 1.17 and 1.12, respectively). Blacks had reduced odds for rectal (OR=0.89), but increased odds for distal (OR=1.27) and particularly proximal cancer (OR=1.60) when compared to non-Hispanic whites. For males vs. females, odds of rectal was higher than distal or proximal cancer (OR=2.64, 1.95 and 1.32, respectively).

Conclusions: Presence and strength of association of cancer risk factors may differ by CRC site. Site should be a key consideration in future studies of CRC risk.

Site specific risk factors for 21,739 CRC cases compared to 616,323 normal colonoscopy controls

---

|  | Anatomic Site of CRC*

|

|

Proximal (n=7,686) | Distal (n=7,036) | Rectal (n=7,017)

Candidate Risk Factor | OR (CI) | OR (CI) | OR (CI)

Age, years† | 1.100 (1.096-1.103) | 1.065 (1.062-1.069)‡ | 1.055 (1.051- 1.059)‡§

Gender, male vs. female | 1.318 (1.080-1.608) | 1.950 (1.538-2.471)‡ | 2.636 (2.006-3.463)‡

Race/ethnicity | |

|

Black vs. non-Hispanic white | 1.596 (1.480-1.720) | 1.270 (1.167-1.381)‡ | 0.886 (0.808-0.971)‡§

Hispanic vs. non-Hispanic white | 1.291 (1.123-1.485) | 1.531 (1.337-1.753) | 1.219 (1.053-1.411)§

Asian vs. non-Hispanic white | 0.872 (0.634-1.201) | 0.917 (0.662-1.272) | 0.556 (0.371-0.832)

BMI, kg/m2† | 0.994 (0.988-1.000) | 1.005 (0.999-1.012)‡ | 0.963 (0.957-0.970)‡§

Diabetes, yes vs. no | 1.278 (1.196-1.366) | 1.170 (1.089-1.257) | 1.119 (1.038-1.207)‡

Smoking | |

|

Current vs. never | 1.594 (1.476-1.722) | 1.500 (1.383-1.627) | 1.837 (1.695-1.992)‡§

Former vs. never | 0.997 (0.926-1.073) | 1.082 (1.001-1.171) | 1.085 (0.998-1.119)

* Multinomial analyses adjusted for other risk factors, as well as year of colonoscopy, 1999-2011

† by one unit increment

‡ p<0.05 when compared to proximal cancer

§ p<0.05 when compared to distal cancer

CRC, colorectal cancer; BMI, body mass index.

#283

Establishing the predominant type of leukemia among patients attending the Uganda Cancer Institute.

Gerald Nsubuga. _Uganda Cancer Institute, Kampala, Uganda_.

Introduction: Leukemia is a cancer of white blood cells. It is characterized by an abnormal accumulation of WBC in the bone marrow which leads to bone marrow failure and is manifested in form of anemia and thrombocytopenia. This arises from neoplastic transformation usually of a single cell. There are four common types of leukemia: 1) Acute lymphoblastic leukemia (ALL); 2) Acute myeloblastic leukemia (AML); 3) Chronic lymphocytic leukemia (CLL); and Chronic myeloid/myclocytic leukemia (CML).

Methods: The study was designed to find out the predominant type of leukemia. 80 leukemia cases which comprised of all bone marrow aspirates and peripheral blood slide results with specific age, sex and ward of the patients was analyzed in the hematology laboratory.

Results: The study was revealed that leukemia occurred in following order of types of predominant that is AML 15 (18.75%), CLL 12(15%) and least type was CML 11(13.75%). The result also showed that the most affected age group by all age. Brackets of (01-20) years. From the study a male preponderance (M: F) of 4:3 was found in cases of ALL. The study emphasizes that special staining techniques be adopted in diagnosing and typing of leukemia.

Conclusions: From the results presented, ALL is the predominant type of leukemia affecting children and young adults. AML was found to affect children, young adults, and adults. CLL and CML were found to occur at the same incidence, the elderly (61-70 and 71-80 years). Other types were rare in these age group similarly CLL and CML were rare in children and young adults.

#284

The trending of lymph node dissection for esophageal squamous cell carcinoma over 40 years in China.

Pei Nan Chen,1 Fu You Zhou,2 Hong Mei Chen,1 Xiao Shan Feng,3 Lei Sun,4 Ling Yuan,5 Ping He,6 Ying Pan,7 Tao Jiang,3 Min Jie Wu,8 Wen Jun Gao,9 Jin Chang Wei,10 Li Dong Wang1. 1 _The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China;_ 2 _Anyang Tumor Hospital, China;_ 3 _The First Affiliated Hospital of Henan University of Science and Technology, China;_ 4 _People's Hospital of Qixian, China;_ 5 _Cancer Hospital of Henan Province, Zhengzhou, China;_ 6 _The Second People's Hospital of Xinxiang, China;_ 7 _The Third Affiliated Hospital of Xinxiang Medical University, China;_ 8 _The Fifth Affiliated Hospital of Zhengzhou University, Zhengzhou, China;_ 9 _People's Hospital of Linzhou, China;_ 10 _The Hospital of Linzhou Esophageal Cancer, China_.

The accumulated evidences have indicated the importance of lymph node dissection (LND) in improving survival for esophageal squamous cell carcinoma (ESCC). However, it is largely unclear in terms of standard lymph node dissection, e.g., how many is suitable and which node station is more important, etc. The present study was undertaken to summarize the trending of lymph node dissection for ESCC over 40 years at a single institute in northern China. A total of 27,260 ESCC patients were enrolled in this study from 1974 to 2016, including 16,565 males with a mean age of 59.72±7.82 and 10695 females with a mean age of 60.3±8.51. All the patients were from the ESCC database in Henan Key Laboratory for Esophageal Cancer Research of the First Affiliated Hospital, Zhengzhou University (1973-2016). All the patients had pathological LND records, including the number dissected. The patients were classified into four period groups based on the diagnosed time, group I (1974 to 1986, 367/1.3%), group II (1987 to 1996, 3182/11.7%), group III (1997 to 2006, 10, 494/38.5%) and group IV (2007 to 2016, 13,217/48.5%). The SPSS21.0 software was used to evaluate the trending changes of LND at different periods. The results showed that the total number of LND/the mean number by each patient from group I to group IV was 3,000/8.43, 22,001/7.07, 87,740/8.36, and 159,340/12.06, respectively (p<0.0001). Further analysis indicated that the total number of LND/the mean number by each patient from group I to group IV in males was 1,634/7.93, 13,428/6.99, 53,143/8.53 and 100,073/12.19, respectively and 1,366/8.48, 8,573/6.80, 34,597/8.11, 59,267/11.83, respectively in females. The present results indicate an apparent increasing in number of LND from 1987 to 2016 for esophageal cancer surgical treatment in China. Further analysis is needed to correlate the LND and metastasis and survival. [National High-Tech Research and Development Program of China (SQ2015AA0202183), Major Science and Technology Projects of Henan Province (161100311300).]

#285

Standardized prediction estimates for cancerous and noncancerous gynecological lesions: a preliminary study in an oil city of Niger Delta region.

Felix M. Onyije,1 Anthony A. Ngokere,2 Aloysius E. Ligha,1 Osaro O. Mgbere,3 Godwin O. Avwioro4. 1 _Niger Delta University, Wilberorce Island, Amassoma, Nigeria;_ 2 _Nnamdi Azikiwe University, Awka, Nigeria;_ 3 _Institute of Community Health, University of Houston, Texas Medical Center, Houston, TX;_ 4 _Delta State University, Abraka, Delta State, Nigeria_.

The objective of this pilot study was to determine the epidemiology of gynecologic cancer and non-cancerous lesions and to develop standardized prediction estimates for the diseases in thirty-five years time. The impact of cancerous and non-cancerous lesions on women's health cannot be underestimated, especially where there is constant emission of hydrocarbons and contaminated water level through oil spillage. Data used for this retrospective study comprised of 679 records and covering the period 2010-2014 were retrieved from the archive of Braithwaite Memorial Specialist Hospital (BMSH) Histopathology laboratory in Port Harcourt, Nigeria. Total of 32 (4.6%) cancerous and 665 (95.4%) non-cancerous lesions were diagnosed with the distribution pattern differing significantly (p<0.001) by year of diagnosis, developmental stage and age category. Leiomyoma recorded the highest 5-year standardized prevalence rate of 0.508, and without intervention, it is estimated that the number of cases diagnosed will rise from 235 in 2015 to 1883 by the year 2050. This was followed by ovarian cyst with a prevalence rate of 0.124 and projected increase from 57 in 2015 to 461 by the year 2050. Similarly, the product of conception is also predicted to increase from 34 to 277 by the year 2050. The 700% increase prediction of cancerous and non-cancerous lesions by 2050 calls for urgent attention for both governmental and private agencies to fund awareness campaigns and screening for women, especially for those residing in oil and gas producing areas where oil and gas pollution are common. This may help reduce morbidity and mortality associated with these conditions.

#286

Forecast of the incidence and etiology of liver cancer in Taiwan, Japan, United States, and United Kingdom: toward harmonization of East and West.

Emily Han-Chung H. Hsiue,1 Wei-Cheng Lo,2 Hsien-Ho Lin,2 Chih-Hung Hsu,3 Ann-Lii Cheng3. 1 _Johns Hopkins School of Medicine, Baltimore, MD;_ 2 _Institute of Epidemiology and Preventive Medicine, College of Public Health, National Taiwan University, Taipei, Taiwan;_ 3 _National Taiwan University Hospital, Taipei, Taiwan_.

Background The global epidemiology of primary liver cancer (PLC), primarily hepatocellular carcinoma (HCC), has evolved significantly in the past decades because of improved control of viral hepatitis and the growing prevalence of metabolic factors. We conducted a quantitative forecast of the incidence and etiology of PLC. Methods Incidence rates of PLC were projected in two endemic Asian countries (Taiwan and Japan) and two Western countries (the United States and United Kingdom) with rigorous cancer registry. The annual incidence of PLC (ICD-10 C22.0) between 1980 and 2013 were obtained from the Taiwan Cancer Registry Database, the Japan National Cancer Center, the US SEER 9 database, Cancer Research UK, and the European Network of Cancer Registry. Incidence rates were projected to year 2035 by an age-period-cohort model with a power link function. The population attributable fractions (PAF) of chronic hepatitis B (HBV), chronic hepatitis C (HCV), alcohol consumption, obesity, and diabetes mellitus (DM) for HCC in Taiwan were projected to 2045. The prevalence of each risk factor was obtained from national surveys and projected inferentially by using a log-linear model or simple linear assumption. The relative risk of each factor was derived from pooled literature data. Results From 2013 to 2035, incidence rates of PLC in Taiwan are estimated to decrease by more than 50% in both men (49.3 to 24.2/100,000) and women (19.7 to 9.2/100,000). In Japan, incidence rates have plateaued in the 1990s, and are expected to continue to decline to 17.0/100,000 for men and 6.3/100,000 for women. Conversely, rates in the UK are projected to increase by more than 30% in both men (13.5 to 18.2/100,000) and women (6.0 to 8.1/100,000). A more modest increase is expected in the US, from 10.2 to 12.5/100,000 for men and 3.3 to 3.5/100,000 for women. In Taiwan, the PAF of HBV, the predominant risk factor of HCC, is predicted to decrease from 54.3% in 2015 to 34.4% in 2045. By 2045, the PAFs of obesity and DM are estimated to reach 11.9% and 15.4%, respectively, the latter similar to that of HCV (15.0%). Conclusion PLC incidence rates are forecast to follow a trend of convergence in four representative countries. Metabolic factors are expected to play increasingly important etiologic role in HCC even in regions currently endemic for viral hepatitis. 

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

### Cell Cycle Checkpoint and Progression

#287

BAY 1816032, a novel BUB1 kinase inhibitor with potent antitumor activity.

Gerhard Siemeister,1 Anne Mengel,1 Wilhelm Bone,1 Jens Schröder,1 Sabine Zitzmann-Kolbe,1 Hans Briem,1 Amaury E. Fernández-Montalván,1 Simon Holton,1 Ursula Mönning,1 Oliver von Ahsen,1 Sandra Johanssen,1 Arwed Cleve,1 Marion Hitchcock,1 Kirstin Meyer,1 Franz von Nussbaum,2 Michael Brands,1 Dominik Mumberg,1 Karl Ziegelbauer1. 1 _Bayer Pharma AG, Berlin, Germany;_ 2 _Bayer S.A.S, Lyon, France_.

The spindle assembly checkpoint represents a highly conserved surveillance mechanism which safeguards correct chromosome segregation by delaying anaphase onset until all chromosomes are properly bi-oriented on the spindle apparatus. Non-catalytic functions of the mitotic kinase BUB1 (budding uninhibited by benzimidazoles 1) were reported to be essential for spindle assembly checkpoint activation. In contrast, the catalytic function of BUB1 plays a minor role in spindle assembly checkpoint activation but is required for chromosome arm resolution and positioning of the chromosomal passenger complex for resolution of spindle attachment errors. Here, we disclose for the first time the structure and functional characterization of a novel, first-in-class Bub1 kinase inhibitor. Medicinal chemistry efforts resulted in BAY 1816032 featuring high potency, long target residence time and good oral bioavailablity. It inhibits BUB1 enzymatic activity with an IC50 of 7 nanomol/L, shows slow dissociation kinetics resulting in a long target residence time of 87 min, and an excellent selectivity on a panel of 395 kinases. Mechanistically BAY 1816032 abrogated nocodazole-induced Thr-120 phosphorylation of the major BUB1 target protein histone H2A in HeLa cells with an IC50 of 29 nanomol/L, induced lagging chromosomes and mitotic delay. Persistent lagging chromosomes and missegregation were observed upon combination with low concentrations of paclitaxel. Single agent BAY 1816032 inhibited proliferation of various tumor cell lines with a median IC50 of 1.4 micromol/L and demonstrated synergy or additivity with paclitaxel or docetaxel in almost all cell lines evaluated (minimal combination index 0.3). In tumor xenograft studies BAY 1816032 only marginally inhibited tumor growth as single agent upon oral administration, however, upon combination with paclitaxel or docetaxel a strong and statistically significant reduction of tumor size as compared to the respective monotherapy was observed. Intratumoral levels of phospho-Thr120 H2A were found to be strongly reduced, and no hints on drug-drug interactions were found. In line with the good tolerability in xenograft studies, no relevant findings from non-GLP 2 weeks toxicological studies in rat and dog were reported. Our findings validate the innovative concept of interference with mitotic checkpoints and justify clinical proof of concept studies evaluating BUB1 inhibitor BAY 1816032 in combination with taxanes in order to enhance their efficacy and potentially overcome resistance.

#288

Loss of a branched actin checkpoint in cancer cells.

Nicolas Molinie, Svetlana Rubtsova, Alexis Gautreau. _CNRS / Ecole Polytechnique, Palaiseau, France_.

Cell proliferation depends on multiple signals, such as growth factors, adhesion to the substratum and to neighboring cells. Cancer cells proliferate independently of these signals. The same stimuli also induce actin polymerization. Here we investigate whether the actin cytoskeleton plays a role in transmitting the proliferation signal. We found that inactivation of the Arp2/3 complex by either a chemical inhibitor or by siRNAs blocks S phase entry in untransformed human breast cell lines and in primary breast epithelial cells. In contrast, breast cancer cell lines did not respond to Arp2/3 inhibition. Inactivating the tumor suppressor p21WAF1/CIP1, or Rb, converts sensitive cells into unresponsive cells further highlighting that this response to Arp2/3 inactivation is at the heart of the transformation process. The depletion of the WAVE complex, the Arp2/3 activator that generates branched actin in lamellipodia, but not of other Arp2/3 activators, blocks cell cycle progression. Conversely, activation of WAVE by active Rac, or depletion of Arpin, which antagonizes WAVE, potentiates cell cycle progression. Together, these results show that branched actin networks generated in lamellipodia are sensed and convey an essential signal for S Phase entry. The signal that branched actin delivers integrates stimulation from soluble growth factor with mechanotransduction from cell adhesions to the substratum and to neighboring cells. This signaling pathway qualifies as a checkpoint, which is required in normal cells, but lost in transformed cells, that monitors the integrity of branched actin networks and instructs the cell as a response that the required conditions to proliferate are fulfilled.

#289

The key role of Mitostatin in the maintenance of genome stability.

Angela Lauriola,1 Andrea Caporali,2 Sabine Mai,3 Domenico D'Arca1. 1 _University of Modena and Reggio Emilia, Modena, Italy;_ 2 _University/BHF Centre For Cardiovascular Science, The Queen's Medical Research Institute, Edinburgh, United Kingdom;_ 3 _Manitoba Institute of Cell Biology, CancerCare Manitoba, University of Manitoba, Winnipeg, Manitoba, Canada_.

Genomic instability is a characteristic of most cancers and it refers to an increased tendency of alterations in the genome during the life cycle of cells. The fidelity of DNA replication is highly ensured by different checkpoints; the activation of spindle checkpoints prevents cells from premature entry into mitosis, avoiding incorrect chromosome segregation and aneuploidy, a typical feature of many cancers. Mitostatin, a novel protein, endowed with tumor suppressor activity, has been reported to bind centrosomal proteins Odf2 and ninein, and its depletion causes an alteration of the anchorage of microtubules to the centrosome. Since functional defects of centrosomes are associated to mitotic failure, Mitostatin may have a key role in guarding the fidelity of mitosis in cells.Here we show that the depletion of Mitostatin in cancer cells, synchronized by aphidicolin (G1/S) block and released into nocodazole-containing medium, leads to mitotic slippage and adaptation to the spindle checkpoint in the presence of a spindle inhibitor. Concomitantly, Mitostatin depletion promotes the early degradation of Mad2 and cyclin B1. Since the activated spindle checkpoint delays cell exit from mitosis by preventing cyclin B1 proteolysis, the cyclin B1 early degradation leads to mitotic checkpoint escape and resulting chromosome instability. In particular, we observed a premature sister-chromatid separation, chromosome bridges and mis-segregation in anaphase that are consistent with defective activation of the spindle checkpoint.In this study, we report for the first time that the depletion of Mitostatin induces an increase of numerical and structural chromosomal aberrations compared to control cells. These aberrations include aneuploidy (P=.0005), the formation of triradials (P=.0061) and broken chromosomes (P=.0066). Moreover, 3D nuclear telomere analysis using TeloView shows decreases in telomeric signals (P=.0061), in the total number of aggregates (P=.0027), and in total intensity (P=.018) and single intensity (P=.019) in cells depleted of Mitostatin. These findings suggest that telomere dysfunction is increased in the absence of Mitostatin; the decrease in telomere intensity is associated with ongoing proliferation (mitotic slippage), and the maintenance of telomeric aggregates is indicative of ongoing genomic instability. Taken together, these observations suggest that Mitostatin plays a critical role in guarding the fidelity of mitosis, enabling the optimal activation of the spindle checkpoint. Thus, low levels of Mitostatin found in certain human tumors may contribute to cellular transformation by promoting genomic instability.

#290

A novel function of HGF in the activation of Chk1 phosphorylation in colon cancer cells.

Na Song, Xiaofang Che, Xiujuan Qu, Lu Xu, Kezuo Hou, Yunpeng Liu. _The First Hospital of China Medical University, Shenyang, China_.

ATR/Chk1 pathway plays an essential role in modulation of DNA damage response (DDR) and homologous recombination (HR). Particularly, Chk1 phosphorylation is involved in cancer prognosis and therapeutic resistance. Although some receptor tyrosine kinases (RTKs) participate in the regulation of Chk1 phosphorylation, the effect of hepatocyte growth factor (HGF) on Chk1 phosphorylation is unknown. In the present study, Western blotting and subcellular fractionation were performed to identify the expression and the location of Chk1 phosphorylation in the presence of HGF. With small interfering RNA transfections, co-immunoprecipitations were performed to detect the interaction between TopBP1 and ATR complex. Our results demonstrated that HGF moderately activated Chk1 phosphorylation in colon cancer cells via upregulation of TopBP1, RAD51, and complex formation of TopBP1 and ATR. Furthermore, AKT activation promoted by HGF served as an important intermediator linking HGF/MET signaling and Chk1 phosphorylation. Depletion of AKT activity attenuated expression of basal line p-Chk1 and induced Chk1 activation due to HGF stimulation. Meantime, activation of AKT directly regulated expression of TopBP1 and RAD51. Suppression of AKT restored HGF-induced upregulation of TopBP1, RAD51, and enhancement of TopBP1/ATR complex as well. Our studies showed that HGF was implicated in regulating Chk1 phosphorylation and further demonstrated that AKT activity was responsible for HGF-induced Chk1 phosphorylation, which might potentially result in management of prognosis and therapeutic sensitivity in cancer therapy.

#291

Sequential treatment with the Wee1 inhibitor, AZD1775, enhances the effect of trabectedin in the L-sarcomas.

Elgilda Musi, Grazia Ambrosini, Gary K. Schwartz. _Columbia University Medical Center, New York, NY_.

Soft tissue sarcomas are heterogeneous mesenchymal neoplasms and account for 1% of all cancers in adults. Over 50 sarcomas subtypes have been classified and many have very limited treatment options. Chemotherapies such as doxorubicin, dacarbazine and gemcitabine have had very low response rates. Two of the most common subtypes of sarcomas are liposarcoma and leiomyosarcoma, also referred to as the L-sarcomas. Trabectedin, a marine derived compound from the sea sponge, Ecteinascidia turbinate, has recently been approved for the treatment of L-sarcomas which has shown a modest effect over common therapies. As a DNA damaging agent, we hypothesized that trabectedin would induce a G2 cell cycle arrest and subsequent inhibition of Wee1 would abrogate this cell cycle checkpoint and induce apoptosis. We therefore examined the effects of trabectedin followed by AZD1775, a Wee1 inhibitor, in a panel of 6 sarcoma cell lines including liposarcoma (LS141, DDLS) and leiomyosarcoma (SK-LMS, SK-UT1, SK-UT1b). IC50s concentrations of trabectedin were determined to be between 0.5 to 2nM, while IC50s for AZD1775 were in the range of 100-200 nM. In vitro results have shown an enhanced inhibition of cell viability with combination treatment in all cell lines at concentrations of 1nM trabectedin for 24hrs followed by 100nM AZD1775 for 48hrs. We also examined biochemical effects with sequential drug treatment (24hrs trabectedin followed by 24hrs AZD1775) which presented an increase of cyclin A1 and cyclin B1 indicative of G2 cell cycle arrest along with increase of phospho-H2AX, indicative of DNA damage by trabectedin, while subsequent treatment with the Wee1 inhibitor showed decrease of phosphorylation of CDC2 (Tyr15). Flow cytometric analysis indicated G2/M cell cycle arrest with trabectedin and abrogation of G2/M with AZD1775. These observations correlated with enhanced apoptosis as measured by biochemical PARP cleavage and DNA content (sub-G1 population). Taken together these results provide a foundation for the development of the sequential treatment of trabectedin followed by the Wee1 inhibitor, AZD775, in the L-sarcomas.

#292

Synthesis and characterization of the novel benzylindazole-based BUB1 kinase inhibitor BAY 1816032 with potent anti-tumor activity.

Marion Hitchcock,1 Anne Mengel,2 Carl Nising2. 1 _Bayer AG, Cambridge, MA;_ 2 _Bayer AG, Berlin, Germany_.

BUB1 (budding uninhibited by benzimidazoles 1) is a serine/threonine protein kinase. The protein is bound to kinetochores and plays a key role in the establishment of the mitotic spindle checkpoint and chromosome congression prior to anaphase.

Inhibition of BUB1 kinase represents a novel approach for cancer treatment: whereas cell cycle arrest is the predominant mode of action of a number of antimitotic cancer drugs (e.g. taxanes and vinca alkaloids), BUB1 inhibition results in aneuploidy and cell death by driving cells through mitosis irrespective of misattached chromosomes.

Here, we disclose for the first time the structure and functional characterization of a novel, first-in-class Bub1 kinase inhibitor. Medicinal chemistry efforts resulted in BAY 1816032 featuring high potency, long target residence time and good oral bioavailability. BAY 1816032 is highly selective for BUB1 displaying single digit nanomolar biochemical potency and double-digit nanomolar cellular potency (H2A induced HeLa-cells). Synergistic effects can be observed when BUB1 inhibitor BAY 1816032 is combined with low doses of paclitaxel affecting chromosome segregation and cell proliferation. X-ray data of benzylindoles allowed a better understanding of the binding mode. Further data on structure-activity relationships including pharmacokinetic characterization, drug metabolism and the synthesis of BAY 1816032 and analogues will be presented.

These results validate the innovative concept of interference with mitotic checkpoints and justify clinical proof of concept studies evaluating BUB1 inhibitor BAY 1816032 in combination with taxanes in order to enhance their efficacy and to potentially overcome resistance.

#293

Screening the druggable genome for synthetic lethal interactions with the CHK1 inhibitor PNT737.

Rebecca Rogers,1 Mike I. Walton,1 Paul Clarke,1 Ian Collins,1 Michelle D. Garrett,2 Paul Workman1. 1 _The Institute of Cancer Research, London, United Kingdom;_ 2 _University of Kent, Kent, United Kingdom_.

Check point kinase 1 (CHK1) is a key regulator of the cell cycle, DNA damage repair and DNA replication. CHK1 inhibition sensitises cancer cells to genotoxic agents and recent studies have indicated that CHK1 inhibitors could be used as single agents to treat cancers with high levels of replication stress. We have recently described the discovery of a highly selective and orally bioavailable CHK1 inhibitor, PNT737, that not only has potent antitumour activity in combination with standard-of-care genotoxic agents but also as a single agent in defined tumour types. Here we sought to identify gene products whose loss would be synthetically lethal with CHK1 inhibition, with the aim of identifying patient populations likely to be sensitive to single agent CHK1 inhibition or to novel combinations utilising CHK1 inhibitors. To do this, we performed a large siRNA screen of the druggable genome (~6500 genes) in A549 (NSCLC) and SW620 (colon cancer) cell lines, with and without PNT737 treatment, and determined effects on cell viability by SRB. POLA1, POLE and POLE2 (B-family DNA polymerases) were identified as significant hits causing synthetic lethality with PNT737 in both cancer cell lines. Treatment with additional siRNA sequences subsequently validated these genes in both the original two cell lines and extra NSCLC and colon cancer cell lines. Interestingly, a number of biomarkers for replication stress, pRPA2 and pCHK1, were increased in cells treated with POLA1, POLE and POLE2 siRNA in combination with PNT737, in comparison to cells treated with the siRNA or drug alone. Further studies conducted with PNT737 and the B-family DNA polymerase inhibitor aphidicolin showed that these agents had a synergistic effect on inhibiting cell viability on 8 out of 9 NSCLC and colon cancer cell lines tested. In addition, immunofluorescence analysis revealed that there was an increase in the level of γH2AX, a marker of DNA damage, in 4 out of 5 cell lines that exhibited synergy when treated with a combination of aphidicolin and PNT737, as compared to cells treated with either agent alone. Our data indicate that the combination of a reduction in POLA1, POLE or POLE2 activity (by siRNA transfection or aphidicolin treatment) and CHK1 activity (PNT737 treatment) increases replication stress and DNA damage in NSCLC and colon cancer cells. Encouragingly, our data support the case for the use of the clinically relevant combination of PNT737 and gemcitabine, as gemcitabine is metabolised it is incorporated into DNA, inhibiting the B-family DNA polymerases. Furthermore, it will now be important to establish if subsets of colon and endometrial cancers with mutations in their POLE proofreading domain are sensitive to CHK1 inhibitors.

#294

Override the doxorubicin-induced G2/M checkpoint using cell-cycle checkpoint inhibitors on acute lymphoblastic leukemia.

Andrea Ghelli Luserna di Rorà,1 Ilaria Iacobucci,2 Enrica Imbrogno,1 Anna Ferrari,1 Valentina Robustelli,1 Cristina Papayannidis,1 Maria Chiara Abbenante,1 Antonella Padella,3 Giovanni Marconi,1 Sandro Grilli,1 Giovanni Martinelli1. 1 _University of Bologna, Bologna, Italy;_ 2 _St. Jude Children's Research Hospital, Memphis, TN;_ 3 _University of Bologna, Montecassiano, Italy_.

The topoisomerase 2 inhibitor, doxorubicin, has been showed by different groups to induce cell cycle arrest in various kind of tumor cells. Specifically doxorubicin-treated cells activate the G2/M cell cycle checkpoint as a consequence of the induction of DNA damages. During the last years many studies have been showed the efficacy of different cell cycle checkpoint inhibitors in single agent or in combination with various DNA damaging agents. These studies showed that the inhibition of key proteins of the cell cycle, like Chk1 and Wee1, deeply sensitize tumor cells to the treatment with genotoxic agent. On these bases, the aim of the study was to evaluate the efficacy of a selective Chk1/Chk2 inhibitor and a Wee1 inhibitor in combination with doxorubicin for the treatment of acute lymphoblastic leukemia. Firstly we evaluate the effect of doxorubicin treatment on a panel of human B and T ALL cell lines in term of reduction of the cell viability, modification of cell cycle profile and activation of the DNA damage response. For this reason the cells were treated with doxorubicin (0.25, 0.5 and 1uM) for 24 and 48 hours and the reduction of the cell viability was quantified using WST-1 reagents. In all the cell lines treated the cytotoxic effect of doxorubicin was time and dose dependent. Then the induction of the apoptosis (Pi/Annexin V) and the effect on cell cycle profile (Pi staining) was evaluated in all the cell lines. In line with the literature the treatment with doxorubicin arrested the cells in G2/M phase. Then the effect of the combinations between doxorubicin and the two checkpoint kinase inhibitor was assessed in all the cell lines. Different cell lines were treated with doxorubicin (5, 10, 25 and 50 nM for the more sensitive cell lines; 50, 100, 250 and 500 nM for the less sensitive cell lines) in combination with the Chk1/Chk2 inhibitor (2, 5 and 10 nM) for 24 and 48 hours. The combination showed a additively effect in term of reduction of the cell viability and induction of apoptosis. Different cell lines were pre-treated for 18 hours with doxorubicin and then with Chk1/Chk2 inhibitor for different time points. Interestingly the inhibition of both Chk1/Chk2 proteins removed the G2/M arrest induced by the pre-treatment with doxorubicin, progressively reducing the number of cells in G2/M phase, increasing the percentage of cells in sub-G1 phase. Similar results were seen combining a Wee1 inhibitor with doxorubicin on several ALL cell lines. In our opinion the combination between the cell cycle checkpoint inhibitors and doxorubicin could be a promising strategy for the treatment of B/T-ALL. Supported by ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project.

#295

CASC-578, a novel Chk1 inhibitor, is active as a single agent in solid tumors and displays synergistic anti-tumor activity in combination with Wee1 inhibition.

Alex Vo,1 Janelle Taylor,1 Robert Rosler,1 Julia Piasecki,1 Dina Leviten,1 Teresa Sierra,1 Ashley Dozier,1 Kevin Klucher,2 Bob Boyle,3 Rich Boyce,3 Scott Peterson1. 1 _Cascadian Therapeutics, Seattle, WA;_ 2 _none, WA;_ 3 _Sentinel Oncology, Cambridge, United Kingdom_.

Background: Checkpoint kinase 1 (Chk1) is a serine/threonine protein kinase that regulates cell division in response to genotoxic stress by arresting cell cycle progression in the S & G2 phases. Pharmacological inhibition of Chk1 is proposed to target tumor cells with increased DNA replication stress, resulting in the uncoupling of DNA replication checkpoint function and the induction of DNA damage and cell death. These properties make Chk1 inhibition a novel therapeutic approach as a single agent in cancers with high replication stress that is driven by oncogenic signaling and loss of parallel DNA damage response pathway function.

Methods and Results: This report highlights the activity of the orally bioavailable, selective small molecule Chk1 inhibitor, CASC-578, in solid tumor derived cell lines. CASC-578 is a sub-nanomolar enzymatic inhibitor of Chk1 with limited off-target activity against a panel of protein kinases. When evaluated in a large cell line panel in vitro, CASC-578 demonstrated a broad potency range as a single agent in solid tumor derived cells lines, with IC50s ranging from 30 nM to greater than 50 μM. Several solid tumor types demonstrated enriched sensitivity to CASC-578 in vitro, including gastric, non-small cell lung and ovarian cancers. Treatment of sensitive cell lines with CASC-578 resulted in the induction of DNA damage, as measured by phosphorylated histone H2AX, and the induction of cell death. CASC-578 was active as a single agent in SK-MES-1 and NCI-H727 NSCLC tumor xenograft models in vivo with minimal effects on body weight in treated mice. In addition to the potent single agent activity of CASC-578, combination with the Wee1 inhibitor AZD-1775 was highly synergistic in vitro in multiple solid tumor cell lines and the combination was more efficacious than either agent alone in NSCLC tumor xenograft models. These data support the advancement of CASC-578 into clinical development as a potential therapeutic agent for the treatment of solid tumor diseases. Experiments are ongoing to identify biomarkers associated with sensitivity to CASC-578 as a single agent in solid tumor cell lines to prospectively identify tumor genotypes that are more responsive to the drug.

#296

**Interplay of human MGMT DNA repair protein with PCNA / p21** cip1 **and MGMT's novel role as an S-phase checkpoint.**

AGM Mostofa, Kalkunte S. Srivenugopal. _Texas Tech University Health Sciences Center, Amarillo, TX_.

O6-Methylguanine-DNA methyltransferase (MGMT) is a critical antimutagenic DNA repair protein that protects the genome and an established target for improving the efficacy of alkylating agents. In contrast with the stoichiometric repair reaction performed by a single small MW protein, we show for the first time that MGMT in human glioblastoma cells specifically associates with PCNA, p21cip1, and undergoes selective degradation at mid-S-phase along with replication-licensing components to maintain genomic integrity. First, we identified a PCNA-Interacting Protein (PIP box) motif between amino acids 61-70, QCTAWLNAYF in the MGMT protein. PCNA encircles the DNA and functions as a sliding clamp by interacting with DNA metabolic proteins having a PIP-box to make the replication processive. In p53-null H1299 lung cancer cells engineered to express the p21cip1, either by Tet-off conditional or lentiviral stable transfections, a reciprocal immunoprecipitation/western blot analyses using antibodies to PCNA or MGMT confirmed the specific association of MGMT and PCNA proteins. Expression of the CDK inhibitor p21 disrupted the interaction between PCNA and MGMT in cells, indicating its regulatory role in DNA repair during cell cycle blockade. Confocal immunofluorescence imaging in glioblastoma cells and isogenic HCT116 cells with and without p21cip1 expression, showed a co-localization of MGMT and PCNA proteins in glioma cells; when cells were subjected to alkylation DNA damage, the co-localization pattern was punctate and more prominent, suggesting that PCNA functions to recruit the repair protein to the damage sites. To probe the cell-cycle dependent regulation of MGMT, we used synchronized human GBM cells at the G1/S boundary using double thymidine-block or single thymidine-mimosine blocks. Cells released into the S-phase showed that the MGMT disassociates from PCNA during the late S-phase and undergoes specific degradation before re-accumulating in G2/M. The replication-licensing protein Cdt1 and p21cip1 were also observed to undergo co-degradation in S-phase, which has been established as a key step in marking the replication origin sites. MGMT inhibition, either by O6-benzylguanine or specific shRNAs greatly impeded the progression of cells into the S-phase in synchronized cells. DNA synthesis measured by 3H-thymidine or BrdU incorporation was also curtailed significantly by MGMT inhibition. Furthermore, enforced expression of MGMT in two GBM cell lines led to a moderate endo-reduplication of the genome. Collectively, we show new non-repair functions for MGMT, its requirement for cell cycle progression and timed elimination to maintain genomic stability. On the clinical front, the observations provide a clear biochemical rationale for combining MGMT inhibitors (apart from the alkylators) with antimetabolites [supported by CPRIT grants RP130266 & RP170207 to KSS].

#297

The novel orally available sub-nanomolar potent and selective checkpoint kinase 1 inhibitor CASC-578 is highly active in mantle cell lymphoma as a single agent and in combination with Wee1 inhibition.

Robert Rosler,1 Janelle Taylor,1 Dina Leviten,1 Teresa Sierra,1 Ashley Dozier,1 Kevin Klucher,2 Rich Boyce,3 Bob Boyle,3 Alex Vo,1 Scott Peterson1. 1 _Cascadian Therapeutics, Seattle, WA;_ 2 _none, CA;_ 3 _Sentinel Oncology, Cambridge, United Kingdom_.

Background: Checkpoint kinase 1 (Chk1) is a serine/threonine protein kinase that regulates cell division by arresting progression in the S & G2 phases of the cell cycle in response to genotoxic stress. Pharmacological inhibition of Chk1 uncouples the completion of DNA replication from G2/M phase transition in tumor cells that have impaired DNA damage response networks, resulting in DNA damage induction, mitotic catastrophe and cell death. These properties make Chk1 inhibition an attractive therapeutic approach as a single agent, or to enhance the efficacy of other cancer drugs that target DNA damage response pathways.

Methods and Results: This report highlights the activity of the orally bioavailable, selective, small molecule Chk1 inhibitor, CASC-578, in multiple mantle cell lymphoma (MCL) models in vitro and in tumor mouse xenograft studies. MCL have the common genetic biomarker t(11;14)(q13;q32), a chromosomal translocation leading to the constitutive expression of the oncogene CCND1 which encodes cyclin D1, a cell cycle-regulating protein. This genotypic characteristic has been observed in the clear majority of patients with MCL in the clinic and may provide a rationale for sensitivity to Chk1 inhibition. Consistent with this idea, CASC-578 demonstrated remarkable potency as a single agent in blocking the proliferation of MCL cell lines in vitro, with an average IC50 of 61 nM. Treatment with CASC-575 as a single agent resulted in the induction of DNA damage, as measured by phosphorylated histone H2AX and activation of apoptosis, as measured by cleaved caspase. In a JEKO-1 xenograft tumor study, CASC-578 as a single agent completely regressed subcutaneously implanted tumors in the majority of mice after oral drug administration using 20 mg/kg daily or when dosed on an intermittent schedule at 25 mg/kg, with minimal effects on body weight. In addition to the observed single agent activity of CASC-578, the combination of CASC-578, and the Wee1 inhibitor AZD-1775, was highly synergistic in MCL cell lines including JEKO-1, REC-1, Z-138 and MAVER-1 in vitro. CASC-578 was also highly potent as a single agent, and in combination with AZD-1775, in blocking the proliferation of a variety of leukemia derived cell lines in vitro. Taken together, these data support the advancement of CASC-578 into clinical development as a potential therapeutic agent for the treatment of MCL, as well as other hematological malignancies.

#298

WEE1 inhibitor activity correlates to AXL/mTOR expression and exhibits synergy with temozolomide (TMZ) in small cell lung cancer (SCLC).

Triparna Sen, Pan Tong, Lixia Diao, You Hong-Fan, C. Allison Stewart, John V. Heymach, Jing Wang, Lauren A. Byers. _UT MD Anderson Cancer Center, Houston, TX_.

Background: WEE1 inhibitors (WEEi) are a novel class of small molecule inhibitors that are now under early clinical trials for small cell lung cancer (SCLC). WEE1 is a protein kinase that plays a key role in regulating the G2 checkpoint in response to DNA damage. Moreover, because WEE1 inhibition induces a HR deficient state, dual targeting of WEE1 and PARP (another promising target previously identified in our group) may induce synthetic lethality. In the present study we evaluate the efficacy of AZD1775 alone and with the PARP inhibitor, olaparib; investigate the mechanisms of primary resistance to AZD1775 in SCLC models; and assess the combinatorial efficacy of AZD1775 treatment with the chemotherapy drug, temozolomide (TMZ), that has shown promise in patients with recurrent SCLC and has been included in the National Comprehensive Cancer Network guidelines for standard treatment for SCLC.

Results: Combination of AZD1775 with olaparib revealed an additive effect in vitro in 90% of SCLC cell lines. AZD1775 combined with TMZ synergistically decreased viability, increased DNA damage and apoptosis in SCLC cell lines (n=10) irrespective of MGMT status or initial response to AZD1775 alone. However, unlike the sensitive cells, SCLC cell lines showing primary resistance to AZD1775 had an intact DNA repair mechanism (after DNA damage), which may contribute to the resistance mechanism. Proteomic analysis revealed AXL (receptor tyrosine kinase) and phospho-S6K (S240/244) as markers of AZD1775 resistance and treatment with AXL inhibitor, TP0903 (40nM), resensitized the cells to AZD1775. Pre and post-AZD1775 treated samples revealed sustained activation of mTOR pathway in AZD1775 primary resistant lines. We further demonstrated that pre-treatment of the cells with the mTOR inhibitor everolimus sensitized SCLC cells to AZD1775 by causing downregulation of AKT/mTOR pathway.

Conclusion: WEE1 inhibitors are currently in clinical trials for SCLC patients. However, as with any targeted therapy, drug resistance is an important barrier to clinical benefit which could be addressed with therapeutic combinations. We show the efficacy of single agent AZD1775 and in combination with olaparib. WEE1 inhibitor, AZD1775 synergizes with TMZ irrespective of MGMT status in all tested in vitro models which warrants further clinical investigation. We also show that the activity of the WEE1 inhibitors might be limited in cancer cells overexpressing of AXL and activated mTOR pathway and that AXL and mTOR inhibition re-sensitized the cells to AZD1775. Our work supports further exploration of the combination of PARP and WEE1 in SCLC and also the possibility of AXL/mTOR inhibition as a mechanism to overcome WEE1 inhibition resistance in SCLC. SCLC is a disease with very limited therapeutic options and no targeted agents with proven benefit; thus the results from this study have clear translational benefit.

#299

RNA editing enzyme ADAR1 accelerates normal hematopoiesis cell cycle by regulation microRNA biogenesis.

Qingfei Jiang, Maria Anna Zipeto, Nathan Delos Santos, Sheldon Morris, Catriona Jamieson. _University of California, San Diego, La Jolla, CA_.

Compelling murine studies demonstrate that adenosine-to-inosine (A-to-I) RNA editing mediated by adenosine deaminase associated with RNA1 (ADAR1) is vital for both fetal and adult hematopoiesis. While genetic ablation of ADAR1 leads to murine embryonic lethality due to severe defects in erythropoiesis, conditional deletion in the hematopoietic system impairs maintenance indicative of cell type and context specific roles for ADAR1 in cell fate specification and self-renewal. By regulating mRNA and microRNA (miRNA) stability, ADAR1 exhibit wide-ranging effects on embryonic development and stem cell regulation. We have previously shown that inflammation-responsive ADAR1 plays important roles in both stem cell differentiation and self-renewal in CML (chronic myeloid leukemia) disease progression. Here, we describe a novel function of ADAR1 in cell cycle regulation of normal hematopoietic stem cell and progenitors (HSPC) by regulation of miRNA biogenesis.

Our results demonstrated that ADAR1 induces G0 to G1 phase transition in normal cord blood HSPCs, as demonstrated by elevated expression of Ki67, reduced DiR signal, and enhanced in vivo cord blood engraftment. Cell cycle qRT-qPCR microarray of 84 cell cycle transcripts and whole transcriptome RNA-sequencing analysis of KEGG cell cycle pathway indicate that several cell cycle genes are differentially expression upon overexpression of ADAR1 WT or an A-to-I editing deficient ADAR1 mutant (ADAR1E912A). We previously demonstrated that impaired biogenesis of let-7 miRNAs by ADAR1 WT induces enhanced self-renewal in cord blood CD34\+ HSPCs. To determine the miRNA targets of ADAR1-mediated RNA editing, we performed miRNome miScript PCR array of 1008 miRNA candidates in cord blood CD34\+ HSPCs overexpressing ADAR1 WT or ADAR1E912A. Total of 263 miRNAs were differentially expressed (142 upregulated and 121 downregulated) by comparing ADAR1 WT to the backbone control. Interestingly, ADAR1E912A mutant also exhibit A-to-I editing independent regulation of miRNAs (307 upregulated and 59 downregulated). We found that the expression of miR-26a-5p, a miRNA frequently downregulated in leukemia, is inhibited by ADAR1-mediated RNA editing. ADAR1 directly binds and edits the DROSHA cleavage site of primary miR-26a transcript, thereby prevent miR26a-5p maturation. Moreover, lentiviral expression of mature miR26-5p reverses the effect of ADAR1 WT, including enhanced CDKN1A expression, inhibition of cord blood proliferation in vivo, as well as reduced HSC self-renewal as measured by colony-formation assay.

Our finding suggests carefully regulated A-to-I editing by ADAR1 is essential for the maintenance of proper cell proliferation in HSC. For future study, it will be interesting to investigate if the elevated expression of ADAR1 in CML BC LSC contributes to false regulation of cell cycle that leads to the expansion of malignant leukemia stem cells.

#300

**Taeumjowi-tang (TJ001) induces G** 2 **/M cell cycle arrest but not apoptosis in p53-mutant prostate cancer via up-regulation of p21** WAF/CIP1 **.**

Soo-Yeon Kang, Hyo In Kim, Se Hyang Hong, Jin Mo Ku, Kangwook Lee, Myeong-Sun Kim, Yu-jeong Choi, Chunhoo Cheon, Youme Ko, Ching Wen Huang, Yui Sasaki, Sohyeon Kang, Hye-Sook Seo, Tai Young Kim, Ji Hye Kim, Yong Cheol Shin, Seong-Gyu Ko. _Kyung Hee University, Seoul, Republic of Korea_.

Taeumjowi-tang (TJ001) is a traditional Korean medicine that usually prescribed for Tae-um person to regulate stomach-related symptoms including headache, indigestion, and jaundice. Other studies on anti-obesity effect of TJ001 have also been researched, but have never been reported as a cure for cancer. In the present study, we investigated the molecular mechanism that TJ001 induces G2/M cell cycle arrest in DU145 (p53-mutant) prostate cancer cells. The missense mutation in human p53 gene (TP53) confers oncological effect to tumor suppressor p53-mutant protein. In prostate cancer, this gain-of-function of p53-mutant is associated with androgen-independence, increased angiogenesis, and metastasis. Our in vitro studies showed that a water extract of TJ001 induced G2/M cell cycle arrest via p53-mutant status and p21WAF/CIP1 up-regulation. Conventinally, p21WAF/CIP1 is induced by mediating p53 regulation, but, in experiments, handling the p53-dependent and -independent pathway. Although serine15-phosphorylated p53 and p53 proteins remained unchanged, p21WAF/CIP1 expression is induced and cyclin B1/Cdc2 complex is inactivated by a decrease of cyclin B1. In addition, in p53-independent pathway, the level of Cdc25C expression decreased and Serine-216 phosphorylated Cdc25C increased. Generally, concomitant with G2/M cell cycle arrest come the apoptosis cell death, but in the study, prolonged G2/M cell cycle arrest developed not apoptosis but cell senescence. Therefore, our data suggest that TJ001 is helpful for p53-mutant prostate cancer treatment.

#301

Prognostic value of metastatic tumoral caveolin-1 expression in patients with resected gastric cancer.

Jihyung Hong,1 Der Sheng Sun,2 Soon Auck Hong,3 Hye Sung Won,2 Han Hong Lee,4 Okran Kim,5 Yoon Ho Ko6. 1 _Incheon St. Mary's Hospital, The Catholic University of Korea, Incheon, Republic of Korea;_ 2 _Division of Oncology, Department of Internal Medicine, The Catholic University of Korea, Seoul, Republic of Korea;_ 3 _Department of Pathology, Soonchunhyang University Cheonan Hosptial, Cheonan, Republic of Korea;_ 4 _Department of General Surgery, The Catholic University of Korea, Seoul, Republic of Korea;_ 5 _Cancer Research Institute, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea;_ 6 _Division of Oncology, Department of Internal Medicine, The Catholic University of Korea, Seoul, Republic of Korea_.

Background : Caveolin-1 (Cav-1), as the main component of caveolae, has complex roles in tumourigenesis and clinical outcomes in a variety of human malignancies. We investigated Cav-1 in primary and metastatic tumor of gastric cancer (GC) and its association with clinical outcomes.

Method : All specimens evaluated were obtained from 145 patients with GC who had undergone curative gastrectomy. The primary outcomes measured were the expression levels of Cav-1 by immunohistochemistry and its association with clinicopathologicalparameters and patient survival.

Results : Among the 145 patients, the frequency of high expression of stromal Cav-1 within primary tumor and tumoral Cav-1 protein in metastatic lymph node were 22.5% (18/145) and 16.6% (15/91). In the multivariate analysis tumoral Cav-1 protein in metastatic lymph node showed prognostic significance for relapse-free survival (RFS, HR, 3.934; 95% CI, 1.882-8.224; P&lt;0.001) and cancer-specific survival outcome (CSS, HR, 3.985; 95% CI, 1.620 - 8.623; P = 0.002). In addition, it remained as a strong indicator of poor prognosis for RFS (HR, 3.137; 95% CI, 1.445 - 6.810; P = 0.004) and CSS (HR, 2.509; 95% CI, 1.079 - 5.838; P = 0.033).

Conclusions : High expression of tumoral Cav-1 protein in metastatic lymph node shows a strong negative association with the clinical outcomes in patients with curatively resected GC, suggesting that this protein have potential uses as novel prognostic markers.

#302

A novel epigenetic pathway in medulloblastoma.

Nagi Ayad, Clara Penas. _Univ. of Miami Miller School of Medicine, Miami, FL_.

Medulloblastoma is the most common malignant pediatric brain tumor with variable prognosis due to its clinical and genomic heterogeneity. Despite recent treatment advances, approximately 40% of children experience tumour recurrence, and 30% will die from this disease. Therefore there is a need to develop novel therapies for patients. We recently reported that Casein kinase 1δ (CK1δ) may be an attractive therapeutic target for medulloblastoma. CK1δ is a serine/threonine kinase that controls cell cycle progression, signal transduction and neurogenesis. We found high levels of CK1δ protein in mouse models of medulloblastoma and human medulloblastoma samples. Furthermore, CK1δ inhibition dramatically reduced medulloblastoma tumor progression. We demonstrate here that CK1δ phosphorylates the epigenetic reader bromodomain-containing protein 4 (BRD4). BRD4 has been identified as a therapeutic target in several cancers, including medulloblastoma. We demonstrate that CK1δ phosphorylates BRD4 and that CK1δ is required for BRD4 binding to the Gli1 promoter in vitro and in vivo. Furthermore, combined CK1δ/BRD4 inhibition is a novel means of reducing medulloblastoma growth downstream of SUFU and SMO. These studies define a novel therapeutic means of inhibiting SMO inhibitor resistant medulloblastoma.

#303

Oncogenic activity of amplified miniature chromosome maintenance 8 in human malignancies.

Jian-Hua Luo, Dong-Mei He, Baoguo Ren, Yan Yu. _Univ. of Pittsburgh Medical Ctr., Pittsburgh, PA_.

Miniature chromosome maintenance (MCM) proteins play critical roles in DNA replication licensing, initiation and elongation. MCM8, one of the MCM proteins playing critical role in DNA repairing and recombination, was found to have over-expression and increased DNA copy number in a variety of human malignancies. The gain of MCM8 is associated with aggressive clinical features of several human cancers. Increased expression of MCM8 in prostate cancer is associated with cancer recurrence. Forced expression of MCM8 in RWPE1 cells, the immortalized but non-transformed prostate epithelial cell line, exhibited fast cell growth and transformation, while knocked down of MCM8 in PC3, DU145 and LNCaP cells induced cell growth arrest, and decreased tumor volumes and mortality of severe combined immunodeficiency mice xenografted with PC3 and DU145 cells. MCM8 bound cyclin D1 and activated Rb protein phosphorylation by cyclin-dependent kinase 4 in vitro and in vivo. The cyclin D1/MCM8 interaction is required for Rb phosphorylation and for S phase entry in cancer cells. As a result, our study showed that copy number increase and overexpression of MCM8 may play critical roles in human cancer development.

#304

CLIC1 membrane insertion is a pivotal regulator of glioblastoma stem cell G1-S transition by promoting an increase of chloride permeability.

Ivan Verduci,1 Valentina Carlini,1 Federica M. Raciti,1 Matteo Conti,1 Federica Barbieri,2 Tullio Florio,2 Michele Mazzanti1. 1 _University of Milano, Milano, Italy;_ 2 _University of Genova, Genova, Italy_.

Glioblastoma (GBM) is the most aggressive and lethal brain tumor and, despite aggressive surgery and adjuvant radiotherapy and/or chemotherapy, the prognosis remains invariantly poor. As for most of solid and hematological malignancies, it was demonstrated that the bulk of tumor cells in GBM is generated by a rare fraction of self-renewing, multipotent cancer stem cells (CSCs) and the persistence of CSCs within the tumor mass is considered the main determinant of GBM development, progression, recurrence and radio- or chemoresistance. Thus, one of the main goals of current research is to identify specific biological mechanisms or intracellular pathways of CSCs whose pharmacological targeting might affect their survival and proliferation. In particular, little is known about the possibility that the molecular mechanisms underlying cell-cycle control in GBM CSCs are endowed with specific and unique features as compared with normal cells. Our study is based on the observation that GBM cells express higher levels of chloride intracellular channel 1 (CLIC1) as compared to nonmalignant brain cells and that in CSCs CLIC1 is mainly localized in the membrane forming an active channel. Conversely, in physiological conditions CLIC1 is mainly a cytoplasmic protein only transiently translocating to the membrane. We recently showed that the different level of activity of CLIC1 in CSCs and normal mesenchymal stem cells confers CLIC1-targeting drugs (for example the biguanide metformin) selective cytotoxicity toward tumor cells. Here we report, that in response to stress conditions, CLIC1 increases the probability to modify its structure going from a cytoplasmic hydrophilic form to a transmembrane conformation. Once in the membrane, CLIC1 acts as a chloride permeability, participating, together with NADPH oxidase, to the generation of a chronic state of oxidative stress that favor the transition between G1 and S phase. The peculiarity of CLIC1 exposure on the external face of the GMB CSC plasma membrane support the idea that this protein could represent a main determinant of the cell cycle progression in this tumor cell subpopulation and thus an accessible and relevant pharmacological target to eradicate CSCs in GBM.

#305

TET blocks cell cycle progression in pancreatic cancer cells at G1-S boundary by decreasing cyclin D1 and increasing CDK inhibitors (CKIs), p21 (WAF1, Cip1) and p27 (Kip1).

Karnika Singh, Qin Dong, Sweaty Koul, Hari K. Koul. _LSUHSC, Shreveport, LA_.

Introduction: Pancreatic cancer (PaCa) is the fourth leading cause of cancer related deaths in the United States with an overall five-year survival rate of less than five percent. The current standard treatment/s for PaCa are largely ineffective thus there is an urgent need for development of therapeutic agents for the treatment of PaCa. Owing to its mutational landscape, which involves mutated KRAS and p53 genes these cancer cells have a deregulated G1 checkpoint of cell cycle control and therefore confer high rate of proliferation and therapeutic resistance. Recently we observed that Tetrandrine derivative (TET), a bis-benzylisoquinoline alkaloid halts cell cycle progression at G0/G1 boundary in vitro and in vivo. In the present study we evaluated potential mechanism of TET induced cell cycle arrest in two pancreatic cancer cells with either wild type KRAS or mutant KRAS, and mutant p53 by evaluating critical cell cycle regulatory proteins involved in G0/G1 to S transition.

Methods: Pancreatic Cancer cell lines: BxPC3 (Pancreatic Ductal Adeno-Carcinoma) and MiaPaCa2 (pancreatic carcinoma) were used in the study. Cells were treated with various concentrations of TET for different time points. Cell proliferation was evaluated using the MTT survival assay. Cell cycle was analyzed following PI staining via Flow cytometry. Changes in protein expression were analyzed by Western Blot analysis in cells. Changes in mRNA were evaluated by real time PCR.

Results: Treatment of pancreatic cancer cells with TET resulted in decreased levels cyclin D1 and phospho-Rb, two critical proteins required for G0/G1 to S transition. Pretreatment of the cells with proteasomal inhibitor MG132 was able to increase the levels of cyclin D1 in control cells but was unable to prevent its loss upon TET treatment. Our results also show that levels of CKIs, p21WAF1/Cip1 and p27Kip1 were increased at the protein level. Gene expression analysis revealed that cyclin D1 mRNA was decreased and p21WAF1/Cip1 mRNA was increased while there was no significant change in p27 mRNA levels. The cycloheximide chase revealed that TET stabilizes p27Kip1 and to some extent p21WAF1/Cip1 as well, suggesting that TET increased p27Kip1 and also p21WAF1/Cip1 levels in part by increasing protein stability. These data suggest a complex mechanism by which TET regulates expression of critical cell cycle regulatory molecules at both transcriptional, translational and post-translational level. Such a mechanism would provide for cell cycle arrest in pancreatic cancer cells following TET treatment.

Conclusion: These results show that TET halts cell cycle at G0/G1 boundary in PaCa cells by modulating the cell cycle regulatory proteins and stabilizing the CKIs by altering their protein stability, suggesting that TET can not only halt deregulated cell cycle but also overcome the therapeutic resistance in pancreatic cancer cells.

#306

Antitumor effect of Wee1 inhibitor in gastric cancer cells.

Seongyeong Kim,1 Ahrum Min,1 So Hyeon Kim,1 Dong Hyeon Ha,1 Hyemin Jang,1 Yu JIn Kim,1 Hee Jun Kim,2 Kyung-Hun Lee,1 Tae-Yong Kim,1 Do-Youn Oh,1 Yung-Jue Bang,1 Seock-Ah Im1. 1 _SNU Cancer research Institute, Seoul, Republic of Korea;_ 2 _Chung-Ang University College of Medicine, Seoul, Republic of Korea_.

Background: A cell cycle checkpoint is a key step to watch any damages and, it is needed to maintain genomic stability. Thus, to jeopardize cell cycle checkpoint is an attractive strategy for cancer treatment. Wee1 is a G2 cell cycle regulator that phosphorylate cdc2 on tyrosine 15 for inhibiting its activity. The activation of Wee1 can delay G2 phase progression and cells can have a chance to repair DNA damages. Therefore, to inhibit wee1 can accelerate DNA damage accumulation in the cell. AZD1775 is a first-in-class wee1 inhibitor. So far, previous studies showed the antitumor effect of AZD1775 as a mono-therapeutic agent or combination treatment with other chemo-agents. However, the activity of wee1 in gastric cancer (GC) cells is not yet fully understood. Thus, we would like to study whether AZD1775 has an antitumor effect against GC or not.

Materials and methods: We determined the antitumor effects of AZD1775 on GC cells using a cytotoxicity assay, cell cycle analysis, immunofluorescence assay and western blotting by AZD1775 treatment.

Results: AZD1775 effectively inhibit GC cell proliferation. The TP53 gene status or p-Wee1 expression levels were not associated with AZD1775 sensitivity. AZD1775 effectively down-regulated p-Wee1 and p-cdc2 expressions in SNU-601, a sensitive cell line. However, these effects were not significant in KATO-III cells, which is less sensitive to AZD1775. Cell cycle analysis revealed that the cells had different responses by AZD1775 treatment. SNU-601 cells were enriched in S phase when we decide cell cycle only by its DNA contents. However, KATO-III cells were arrested in G2/M phase. The sub-G1 population was dramatically increased only in SNU-601 cells. A population of BrdU-positive and p-HH3 positive cells was significantly increased in the SNU-601 cells which indicate that premature mitosis was increased. However, no differences were observed in KATO-III cells. Moreover, due to premature mitotic entry, multi-nucleated cells were observed only in SNU-601 cells.

Conclusion: AZD1775 effectively inhibited the growth of GC cell lines. The inhibition of cdc2 by AZD1775 was observed only in a sensitive cell line. Moreover, early mitotic entry was induced by AZD1775, and it led the induction of mitotic catastrophe. This result indicates that AZD1775 have an antitumor effect on GC and the possibility of adoption to the clinical trial design.

#307

TRIB3 regulates cell cycle progression and programmed cell death in non-small cell lung cancer.

Abeer Almiman, Daotai Nie, Jamila Adom. _Southern Illinois Univ. School of Medicine, Springfield, IL_.

Introduction: The pseudokinase Tribbles pseudokinase 3 (TRIB3) is known as a regulator in cellular responses to a variety of stresses such as glucose insufficiency and (ER) stress. TRIB3 has been described in some studies as a tumor suppressor due to its effect on inactivating PI3K/Akt pathway, but other studies suggest TRIB3 as a stimulator of tumor progression. In this study, we aimed to define the functions of TRIB3 in in non-small cell lung cancer.

Methods: TRIB3 expression was altered using a lentiviral vector to overexpress TRIB3 in non-small cell lung cancer. A CRISPR-CAS9 construct with guiding sequence matching to TRIB3 gene was used to knock out its expression. Cell proliferation was evaluated using MTS and trypan blue assays. Boyden chamber assay was used to assess the cell migration while cell cycle phases were determined using flow cytometry. Heatmap analysis was performed to assess the changes in the expressions of genes in cell cycle progression and apoptosis.

Results: TRIB3 overexpression led to reduced cell proliferation and migration. Moreover, it contributed to the increased of cell cycle arrest at G0/G1 phase. QPCR analyses of cell cycle related genes showed an upregulation of CDK inhibitors in (NCI-H358) TRIB3 overexpression cells, while depletion of TRIB3 led to the down-regulation of CDK inhibitors. TRIB3 overexpression led to downregulation of LC3B and other autophagy markers while increasing the expression of apoptotic genes.

Conclusion: Increased TRIB3 expression in non-small cell lung cancer cells inhibited proliferation by blocking cell cycle progression through the upregulation of CDK inhibitors, led to activation of cell death through apoptosis. Our study reveals a significant role of TRIB3 in regulating cell cycle progression, apoptosis and autophagy.

#308

The PP2A-B56 phosphatase opposes cyclin E autocatalytic degradation via site-specific dephosphorylation.

Ryan J. Davis, Jherek Swanger, Bruce E. Clurman. _Fred Hutchinson Cancer Research Ctr., Seattle, WA_.

Cyclin E, in conjunction with its catalytic partner cyclin-dependent kinase 2 (CDK2), regulates cell cycle progression as cells exit quiescence and enter S-phase. Multiple mechanisms control cyclin E-CDK2 activity during the cell cycle, including phosphorylation-dependent cyclin E ubiquitylation by the SCFFbw7 ubiquitin ligase. Serine 384 (S384) is the critical cyclin E phosphorylation site that stimulates Fbw7 binding and subsequent cyclin E degradation. Because S384 is autophosphorylated by CDK2, cyclin E therefore instigates its own degradation in an autocatalytic manner. This presents a paradox as to how cyclin E-CDK2 is able to phosphorylate its numerous substrates prior to cyclin E autophosphorylation-catalyzed degradation. Here we find that the PP2A-B56 phosphatase specifically dephosphorylates cyclin E at S384, thereby uncoupling cyclin E degradation from cyclin E-CDK2 activity. Furthermore, the rate of S384 dephosphorylation is high in interphase and low in mitosis, allowing PP2A-B56 to oppose autocatalytic cyclin E degradation and maintain cyclin E-CDK2 activity at the G1/S transition.

#309

Effect of histone deacetylase (HDAC) inhibitor on gene expression in MDM2 transfected prostate cancer cells.

Thiagarajan Venkatesan,1 Ali Alaseem,2 Khalid Alhazzani,2 Thanigaivelan Kanagasabai,1 Appu Rathinavelu1. 1 _Rumbaugh Goodwin Institute for Cancer Research, Nova Southeastern University, Fort Lauderdale, FL;_ 2 _College of Pharmacy, Nova Southeastern University, Fort Lauderdale, FL_.

Deacetylation of histone gives a tag for epigenetic repression and plays an important role in transcriptional regulation, cell cycle progression, and developmental events. HDACs catalyse the removal of the acetyl moiety from the lysine residues of proteins including the core nucleosomal histones. Through removal of critical acetyl groups from histones, HDACs can create a chromatin conformation that can prevent the transcription of genes that encode for proteins involved in cell cycle regulation. Thus together with histone acetyltransferases (HATs), HDACs regulate the level of acetylation and alter multitude of cellular functions and their characteristics. Several alterations of HDAC and HAT levels and activities have been found to be enacted by translocation, amplification, overexpression, or mutation of the relevant genes in a variety of cancers. In many cancer cell lines, overexpression or activation of the HDAC enzymes result in histone hypo-acetylation and consequent promotion of pro-cancerous mechanisms. Therefore, HDAC inhibitors represent a potential new class of antitumor agents with cytotoxic activity and the ability to regulate gene expression in tumor cells. In this study we evaluated the effects of Vorinostat (suberoylanilide hydroxamic acid), which is a potent inhibitor of HDAC activity, on cell cycle regulation in MDM2 (mouse double minute 2 homolog) overexpressing cells. MDM2 amplification or overexpression is found in many tumors that eventually lead to the inactivation of the cell cycle control and loss of pro-apoptotic functions through both p53 dependent and independent mechanisms. The PCR array, qRT-PCR, and western blot analysis of MDM2 overexpressing prostate cancer cells (LNCaP-MST), after treating with Nutlin-3 (20 µm) and 17-AAG (10 µm), was able to trigger p21 expression and down-regulation of BIRC5 (Baculoviral IAP Repeat Containing 5). Similarly, when we treated the MDM2 transfected LNCaP-MST cells with vorinostat (7.5 µm for 24 hrs), some of the above mentioned changes, similar to Nutlin-3 treatment, were observed. As a result of HDAC inhibition the mRNA levels of p21, p53 and TIMP-1 were significantly elevated, while the levels of BIRC5 was significantly down-regulated. Thus, treatment of MDM2 overexpressing cell lines with HDAC inhibitor resulted in activation of p21 and consequent decrease in cell proliferation due to resumption of cell cycle arrest. Our results with LNCaP-MST cells offer convincing evidence to suggest that the inhibition of HDAC can control cell proliferative signals in MDM2 overexpressing prostate cancer cells. (The generous support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida is gratefully acknowledged).

#310

Enhanced stability of D-type cyclins correlates with glioblastoma resistance to ionizing radiation.

Fadila Guessous, Mouadh Benamar, Hui Zhong, Tarek Abbas. _Univ. of Virginia, Charlottesville, VA_.

Transition from G1 to S phase of the cell cycle is promoted by D-type cyclins (D1, D2 and D3), regulatory subunits critical for the activation of cyclin dependent kinases 4 and 6 (CDK4/6), which phosphorylate and inactivate the retinoblastoma protein (pRb) and activate the transcription factor E2F1 to promote the expression of genes essential for DNA synthesis. Deregulated expression of D-type cyclins is frequently observed in human malignancies and correlates with increased proliferation. Cyclin Ds overexpression is also associated with radioresistance, leading to radiotherapy failure and disease recurrence. Cyclin D1 undergoes ubiquitin-dependent degradation following the exposure of cancer cells to ionizing radiation (IR), and this has been shown to be critical for IR-induced G1 growth arrest and sensitivity of cancer cells to IR. While the role of cyclin D1 overexpression in drug resistance is already well documented, less is known about the role of cyclins D2 and D3 in radio-resistance in human cancer cells, particularly in brain neoplasms. In the present study, we investigated the effect of IR on different D-type cyclins (D1, D2, D3) in human glioblastoma cell lines as well as in tumor-derived mouse oligodendrocytes progenitor cells (OPCs). Here, we show that exposure of human astrocytes to increasing levels of IR led to a rapid proteasomal degradation of all three D-type cyclins. On the other hand, IR failed to induce significant cyclin Ds degradation in a number of human glioblastoma cells (U87, A172, U251, Snb19). Analysis of cell cycle profile of all glioblastoma cells tested and exposed to IR showed prominent cell cycle arrest in G1 starting at 24 hours following exposure. Furthermore, we show that the steady-state levels of D-type cyclin is higher in OPCs compared to mouse NIH3T3 cells and fail to undergo proteasomal degradation following IR exposure. Collectively, these results suggest that the degradation of D-type cyclins is not critical for IR-induced G1 cell cycle arrest in GMB and may underlie their resistance to IR. Understanding the role of cyclin Ds in promoting radioresistance may lead to a useful pharmacological strategy to enhance radiotherapy outcome and help implement a more effective treatment modality.

#311

**Development of AZD2811, an aurora kinase B inhibitor, incorporated into an Accurin** TM **nanoparticle for use in haematological and solid cancers.**

Susan Ashton,1 Nicholas Floch,2 Paula Taylor,1 Colin Howes,2 Doug Ferguson,3 Matthew Ling,4 Maureen Hattersley,3 Shenghua Wen,3 Kim Maratea,3 Adina Hughes,4 Sean Redmond,3 Wolfram Brugger,2 Simon Smith,2 Alexander MacDonald,2 Keith Parry,5 Howard Burris,6 Young-Ho Song,7 Jim Nolan,7 Elizabeth Pease,2 Simon T. Barry2. 1 _AstraZeneca, Macclesfield Cheshire, United Kingdom;_ 2 _AstraZeneca, Cambridge, United Kingdom;_ 3 _AstraZeneca, Gatehouse Park Boston, MA;_ 4 _AstraZeneca, Macclesfield, United Kingdom;_ 5 _Sarah Cannon Research Institute, London, United Kingdom;_ 6 _Sarah Cannon Research Institute, Nashville, TN;_ 7 _Pfizer, New York, NY_.

A nanoparticle formulation of AZD2811, a selective aurora kinase B inhibitor, is currently under clinical development for the treatment of both haematological and solid tumour disease. AZD2811 is the active derivative of the prodrug Barasertib (AZD1152) which gave promising clinical activity in elderly AML patients delivered as a 7-day infusion (Kantarjian et al, Cancer, 119, 2611-2619, 2013). To address the limitations associated with the clinical utility of Barasertib and other cell cycle inhibitors in the clinic, AZD2811 has been incorporated into an AccurinTM nanoparticle using a pamoic acid ion pairing approach to optimise drug release rate (Song et al, Journal of Controlled Release, 229, 106-119, 2016), improve the drug exposure to tumour and reduce the duration of administration. A proof of principle formulation of AZD2811 as an AccurinTM nanoparticle established the principle that anti-tumour activity and improved therapeutic index could be achieved (Ashton et al, Science Translational Medicine, 325, 1-10, 2016). The clinical nanoparticle formulation of AZD2811 has been optimised for drug loading and release rate. In pre-clinical models, the clinical formulation can be used flexibly to optimise drug delivery for use in both haematological disease such as AML, or in solid tumour settings. Anti-tumour activity in solid tumours can be achieved at doses where bone marrow toxicity is reduced compared to Barasertib. In sensitive xenograft and PDX solid tumour models greater than 90% tumour regression is observed after a total dose of 50mg/kg with no tumour progression for greater than 40 days. In contrast, for AML, increasing the dose intensity by 2-4 fold leads to neutropenia and to complete tumour regression in a range of AML xenograft models for greater than 60 days. These data establish the concept that drug delivery using nanoparticles is able to resolve therapeutic index challenges, and is able to do so across different disease types. AZD2811 is currently in Phase 1 clinical trial (D6130C0000). The current pre-clinical and clinical data with this novel approach to inhibition of the cell cycle will be discussed.

#312

Anti-proliferative effects of cinnamon extract in colon cancer.

Patrick P. Carriere, Hina Mir, Neeraj Kapur, Clarence E. Clark, Shailesh Singh. _Morehouse School of Medicine, Atlanta, GA_.

Colon cancer is the third most lethal cancer worldwide, and is estimated to cause 49,190 cancer-related deaths in the U.S. in 2016 alone. Current treatments for colon cancer are limited in their capacity to target tumor cells employing adaptive mechanisms necessary for survival and proliferation, often resulting in poor prognosis. Therefore, it is critical to develop novel target-specific therapeutic approaches with minimal cytotoxicity to normal cells, in order to mitigate potential side effects. Proanthocyanidins represent a subclass of flavonoids, which have been widely investigated for their chemopreventive and therapeutic potential. Cinnamtannin B-1 (CTB-1) is a proanthocyanidin shown to have anti-cancer effects in several cancers, but its anti-cancer potential in colon cancer has not been tested. Hence, the focus of this study was to determine the anticancer effects of CTB-1 in colon cancer. Our results show that CTB-1 treatment significantly decreases the cell viability and proliferation of DLD-1 and COLO 201 human colon cancer cells in a time and dose-dependent manner. In addition, CTB-1 treatment modulated the cell cycle progression of colon cancer cells via G2/M arrest, coupled with a reduction of cells in the S phase. The expression and/or phosphorylation of key molecules involved in apoptosis and cell cycle progression were also modulated in response to CTB-1 treatment. Collectively, these findings, for the first time, shed light on the anti-proliferative effects of CTB-1 in colon cancer, and further demonstrate its potential as a novel therapeutic/preventive agent.

#313

Tbata induces G2/M cell cycle arrest and sensitizes osteosarcoma cells to etoposide in a p53-independent manner.

Milos D. Miljkovic, Francis A. Flomerfelt, Ronald E. Gress. _National Cancer Institute, Bethesda, MD_.

Thymus, brain, and testes-associated (Tbata) is a negative control cell-cycle gene highly expressed in murine thymic epithelial cells (TECs). Tbata protein binds to Uba3, inhibiting formation of Nedd8 E1 and subsequent target degradation via neddylation of several cell cycle control proteins needed for G2/M transition, which may be a major mechanism of TEC growth arrest during thymic involution. Etoposide is a cytotoxic drug which targets the enzyme toposimerase II — increased 2-3 fold during the G2 phase. To further characterize effect of Tbata on the cell cycle, we modified p53 wild-type U2OS cells derived from human osteosarcoma to express a Tbata/mCherry fusion protein when mifepristone is added to culture media. Tbata-expressing cells identified on flow cytometry did exhibit growth arrest, with quantitative assessment of DNA content in U2OS cells by flow cytometry establishing that 32% of Tbata/mCherry-expressing cells were in the G2 phase when exposed to mifepristone for 24 hours, compared to 11% of mCherry-expressing cells. Gene expression studies were consistent with these results. Cells expressing Tbata/mCherry were also more sensitive to etoposide at 0.5, 1, and 5 times the IC50 dose. To test whether p53 function was required, we further modified the cells to overexpress a dominant negative p53 mutant along with Tbata/mCherry. Similar G2 arrest and increased sensitivity to etoposide were observed, indicating that the effects of Tbata did not require normal p53 function. Potential Tbata analogues or mimetics may therefore be used as an adjunct to G2-targeted chemotherapy.

#314

Evaluation of effective drug combinations of sonidegib and ribociclib for treatment of Sonic hedgehog medulloblastoma using mathematical models of the cell cycle and Hedgehog pathways.

Zack Jones, Jessica Roberts, Haley Houke, Martine Roussel, Clinton Stewart, Carl Panetta. _St. Jude Children's Research Hospital, Memphis, TN_.

Sonic hedgehog (SHH) tumors account for roughly 25% of all pediatric medulloblastoma. Aberrant signaling of the SHH pathway, typically via Patched-1 (PTCH) mutation, results in cell proliferation and subsequent tumor formation. Smoothened (SMO) inhibitors such as sonidegib have been used clinically to combat tumor progression and have shown initial promise. However, many tumors acquire resistance to therapy. Combination therapy with an additional agent targeting another node in pathways affecting cell-cycle progression such as ribociclib, a cyclin D-dependent kinase (CDK) 4/6 inhibitor, may improve therapy and reduce tumor burden by delaying or preventing the development of resistance. Identification and evaluation of effective anti-cancer drug combinations, along with their schedule, sequence, and dosage are a major challenge due to the large number of possible combinations in addition to the cost and time involved in experimentally testing each possible permutation. Mathematical models can aid in the discovery of optimal drug combinations. A mathematical model of the SHH and Cyclin D / CDK / Rb pathways was designed to evaluate effective sequences and schedules of sonidegib and ribociclib for the treatment of SHH medulloblastoma. The model was parameterized using in vitro data from studies treating the NIH3T3 cell line with sonidegib and ribociclib singly or in combination. Flow cytometry analysis was used to quantify response to treatment by obtaining cell-cycle phase distributions for each drug as a single agent or in combination. Uncertainty and sensitivity analysis of the model parameters were evaluated via Latin Hypercube Sampling and Partial Rank Correlation Coefficient analysis to interpret the effects of amplification or deletion of nodes in the pathway, evaluate potential targetable nodes, and identify sensitive parameters that need robust quantification. Sensitivity analysis indicated that the formation of Cyclin D/CDK4/6 complexes and the dephosphorylation of Rb were significant nodes that should be included in the model. Uncertainty analysis indicated that the majority of the variability lies in the distribution of cells in G0/G1 and S phase. Simulations of single agent treatment predict a similar response between sonidegib and ribociclib, with co-treatment indicative of an additive effect. Optimal control theory methods are being applied to this model to help determine effective sequence, schedule, and dose of sonidegib and ribociclib to improve efficacy and reduce drug resistance in the treatment of SHH medulloblastoma.

### Cell Growth Signaling Pathways 1

#315

S100B regulates IL-6 signaling via the p90 ribosomal S6 kinase (RSK) in malignant melanoma.

Milad J. Alasady,1 Kaylin A. Adipietro,1 Adam D. Pierce,1 Paul T. Wilder,1 Kira G. Hartman,1 David J. Weber,1 Nissim Hay2. 1 _University of Maryland, Baltimore, Baltimore, MD;_ 2 _University of Illinois, Chicago, Chicago, IL_.

S100B is a tumor marker that is highly elevated during the progression of malignant melanoma. However, the function of S100B in the progression of melanoma is not well understood. Here we propose a regulatory mechanism that defines interplay between S100B, Interleukin-6 (IL-6), STAT3, and CREB. First, we show that S100B inhibits IL-6 mRNA and protein levels, and that silencing S100B in melanoma cell lines induces the expression and secretion of IL-6. Consistent with this result, STAT3 phosphorylation and its activation occur upon S100B ablation and IL-6 production. Our data suggest that S100B exerts its effect on the IL-6 pathway via p90 ribosomal S6 kinase (RSK), leading to activation of transcription factor CREB. Elevated S100B in melanoma cells were shown to bind directly to RSK, sterically block its phosphorylation (at Thr573) and sequester RSK in the cytoplasm (Hartman et al., 2014). Silencing of S100B enables RSK nuclear translocation, which in turn elevates CREB phosphorylation and its transcriptional activity in the nucleus to induce IL-6 expression, which is consistent with these newer observations. Because IL-6 can inhibit the proliferation of melanocytes and early stage melanoma cells, we propose that the suppression of IL-6 by elevated S100B may be a mechanism for cancer cells to circumvent the inhibitory effect of IL-6 at early stages of melanoma. Additionally, the suppression of IL-6 by elevated S100B may also curb local immune response(s), which otherwise would be elevated by the secreted IL-6.

#316

Identification of SGK1 as a potential therapeutic target in castrate resistance prostate cancer.

Massar I. Alsamraae, Urzsula McClurg, Craig N. Robson, Stuart McCracken. _Northern Institute For Cancer Research, Newcastle upon Tyne, United Kingdom_.

Introduction: Prostate cancer (PC) is the most common male cancer in the UK, with approximately 1 in 8 men developing the disease within their lifetime (Prostate Cancer UK). The androgen receptor (AR) has a crucial role in the proliferation and progression of prostate cancer. Patients respond to anti-androgen therapy in the early stage of the disease, however many will develop resistance, entering a "castrate-resistant" disease state (CRPC), carrying a very poor prognosis, posing a major clinical challenge (1). The development of second generation anti-androgen therapies, such as Enzalutamide and ARN-509, have shown promise in the treatment of CRPC patients, but response rates of just 50% and the development of resistance to these drugs have limited their success in the clinic (2,3). This study aims to interrogate the global gene expression consequences of anti-androgen resistance in an LNCaP prostate cancer cell line model, resistant to Enzalutamide. In our gene microarray, SGK1 demonstrates high expression in Enzalutamide resistant cells. Our subsequent experiments suggest SGK1 may serve as a biomarker of resistance or perhaps an exploitable target in CRPC.

Methods: A gene microarray was used to determine the gene profile of parental LNCaP cells, sensitive to anti-androgen drugs, versus in-house generated LNCaP-Enzalutamide Resistant cells. QPCR to determine the mRNA level of SGK1 +/- Dihydrotestosterone (DHT), +/-Dexamethasone. Western blot used to detect the protein level of the SGK1. IncuCyte used to determine the proliferation. Wound Healing Assay to detect the direction of the cells toward the edges

Results: These preliminary data demonstrate an increase in expression of SGK1 in the LNCaP-Enz-R cell line model, compared with parental LNCaP cell, at both the protein and mRNA levels. We have demonstrated that stimulating AR by DHT also increases SGK1 expression at both the protein and mRNA levels, in both cell lines. In addition, it is shown that activation of the glucocorticoid receptor (GR) by stimulation with Dexamethasone increases SGK1 expression at the mRNA level only in LNCaP cells, however high SGK1 expression is observed at the mRNA AND protein level in the LNCaP-Enz-R cell line. Inhibition of SGK1 using small molecular inhibitors significantly decreases proliferation and migration of the LNCaP-Enz-R cell line, whereas no significant difference is seen in the parental, androgen sensitive LNCaP cell line.

Conclusion: Increased expression of SGK1 in Enzalutamide, Casodex and ARN509 resistant cell line models was observed, compared with parental LNCaP cells. AR regulates SGK1 in both parental LNCaP and LNCaP-Enz-R cell line models. GR regulates SGK1 in the LNCaP-Enz-R cell line alone. SGK1 has a vital role in the proliferation and migration of the LNCaP-Enz-R cell line model. SGK1 could represent either a future biomarker of Enzalutamide-resistance, or a potential therapeutic target in advanced disease.

#317

Combination of a novel ERK1/2 inhibitor (LY3214996) with CDK4 and CDK6 inhibitor (abemaciclib) enhances antitumor efficacy in KRAS mutant non-small cell lung cancer (NSCLC).

Wenjuan Wu, Shripad V. Bhagwat, Constance King, Susan Pratt, Xueqian Gong, Julie Stewart, Bonita Jones, Robert Flack, Richard Beckman, Beverly Falcon, Jason Manro, William T. McMillen, Ramon V. Tiu, Sheng-Bin Peng, Christoph Reinhard, Sajan Joseph, Sean Buchanan. _Eli Lilly and Company, Indianapolis, IN_.

ERK1/2, a key downstream effector of RAS mutations, is involved in the signaling network which drives cell proliferation, survival, metastasis and cancer resistance to drug treatment (including MEK and BRAF inhibitors). Lung cancer is a leading cause of cancer death worldwide. KRAS mutation present in up to 30% of NSCLC patients is associated with a poor prognosis and represents an unmet medical need. In KRAS mutant NSCLC, enhanced ERK activation cooperates with dysregulation of the cell cycle checkpoint (e.g., cyclin D, CDK4 and CDK6 complex), and contributes to tumor progression; thus, the simultaneous inhibition of ERK and the CDK4/6 pathway is hypothesized to augment tumor growth inhibition. LY3214996, a novel and highly selective small molecule inhibitor of ERK1 and ERK2, is currently in phase I clinical trial and has been shown to inhibit cell proliferation in RAS or BRAF mutant tumor cells in vitro and xenograft tumor growth in vivo. Abemaciclib, a CDK4 and CDK6-selective inhibitor is currently in phase III studies for ER positive breast cancer and KRAS mutant NSCLC. In this study we explore the potential efficacy of combined inhibition of ERK1/2 and CDK4 and CDK6 in KRAS mutant NSCLC. The combination of LY3214996 and abemaciclib synergistically inhibited cell proliferation in 85% of KRAS mutant cells in an unbiased NSCLC panel. Combination treatment with LY3214996 and abemaciclib significantly decreased levels of phospho- p90RSK, phospho-Rb, phospho-S6 and Ki67; and synergistically inhibited cell proliferation and survival in KRAS mutant NSCLC cell lines including NCI-H2122 (G-12C), A549 (G-12S) and NCI-H441 (G-12V). Subsequent in vivo studies showed that the combination treatment with LY3214996 and abemaciclib was well tolerated and led to more robust tumor growth inhibition or regression in all KRAS mutant NSCLC xenograft models (H2122, A549 and H441) compared with either single agent treatment (p≤0.002). Furthermore, in xenograft tumors the combination of LY3214996 and abemaciclib resulted in more significant reduction of phospho-p90RSK, phospho-Rb, phospho-S6 and Ki67 in H2122 tumors compared with either single agent. Overall, the combined inhibition of ERK1/2 and CDK4 and CDK6 was tolerated and enhanced antitumor efficacy in several KRAS mutant NSCLC preclinical models. These data support the feasibility of combining ERK inhibitor LY3214996 with CDK4 and CDK6 inhibitor abemaciclib as a promising strategy for the treatment of KRAS mutant NSCLC patients, and provides the rationale for the combination study in the on-going phase I LY3214996 clinic trial (NCT02857270).

#318

Identification of FERM domain-containing protein 5 (FRMD5) as a novel target of β-catenin/TCF7L2 complex.

Yoichi Furukawa, Chi Zhu, Tomoyuki Ohsugi, Yumi Terakado, Rei Noguchi, Tsuneo Ikenoue, Kiyoshi Yamaguchi. _Univ. of Tokyo, Tokyo, Japan_.

Deregulated Wnt signaling is one of the earliest steps in colorectal tumorigenesis. The impairment results most frequently from APC mutations, which leads to the accumulation of β-catenin and subsequent activation of TCF7L2. Although previous studies have identified a number of target genes of the activated β-catenin/TCF7L2 transcriptional complex such as cMYC and CCND1, the comprehensive effect of the activation on tumorigenesis remains to be elucidated. To understand the precise molecular mechanisms underlying colorectal cancer, we searched for novel genes regulated by the complex in colorectal tumors. We performed expression profile analysis of HCT116 and SW480 colon cancer cells treated with β-catenin siRNAs, and combined these data with public microarray data of LS174 cells treated with a dominant-negative form of TCF7L2. As a result, we identified a total of 134 genes that were regulated by both β-catenin and TCF7L2. Subsequent ChIP-sequence with TCF7L2 antibody selected 11 genes from the 134 genes as candidates of direct target genes. In this study, we focused on FERM domain-containing protein 5 (FRMD5) among the 11 candidates. Quantitative PCR confirmed that its expression was reduced by β-catenin siRNA in HCT116 and SW480 cells. ChIP-quantitative PCR analysis with ant-TCF7L2 antibody also corroborated the interaction of TCF7L2 with a region (hg19; chr15:44,449,680-44,450,487) within intron 1 of FRMD5. Reporter assay with plasmids containing this region revealed that the reporter activity was down-regulated by the knockdown of β-catenin. These data suggested that FRMD5 is a direct target of β-catenin/TCF7L2 complex, and that the region is involved in the transcriptional activation through an interaction with the complex. Consistently, its expression was elevated in colorectal tumors compared to normal colonic mucosa in public microarray data. To uncover the role of FRMD5 in colorectal carcinogenesis, we analyzed expression profile of HCT116 cells treated with FRMD5 siRNA, and combined the data with profile with β-catenin siRNA. Gene set analysis with 53 commonly up-regulated and 36 commonly down-regulated genes by both FRMD5 and β-catenin disclosed that the gene sets of DNA replication, cell cycle, and extracellular matrix (ECM) were altered by FRMD5. These data may be useful for the future studies of colorectal carcinogenesis.

#319

Cholesterol inhibition reduces Hh mediated chondrosarcoma.

Qingxia Wei,1 Eyal Ramu,2 Mushriq AL-Jazrawe,1 Raymond Poon,1 Jay Wunder,2 Benjamin Alman3. 1 _The Hospital for Sick Children, Toronto, Ontario, Canada;_ 2 _Mount Sinai Hospital, Toronto, Ontario, Canada;_ 3 _Duke University, Durham, NC_.

Constitutive activation of Hh signaling is a common occurrence in chondrosarcoma(CSA). Gene profiling analysis showed that Gli transcription regulates genes that govern cholesterol homeostasis, which alters cholesterol accumulation in chondrocytes; a higher level of Gli-mediated transcription results in accumulation of intracellular cholesterol. Here we determined if targeting cholesterol-processing genes downstream of Hh signalling could be used as a novel treatment approach. With institutional review board approval, human CSA samples were obtained fresh from surgery. For in vitro studies, CSA explants of 2mmx2mm x2 mm cubic in size establihed as organ cultures. For in vivo studies, one million CSA cells were subcutanously injected into NSG mice. Cells from five CSAs were treated both in vitro and in vivo with a hedgehog inhibitor, Cur61414, a cholesterol inhibitor, Lovastatin, or both. In vitro, CSA explants were treated for 48 hrs at concentration of 20 μM of each drug. In vivo, mice were treated with 4.5mg/kg/day of Cur61414, Lovastatin, or both by intraperitoneal injection for 4 wks. At the end of treatment, the explants or xenografts were harvested and processed for further analysis. RT-PCR was used to meausre the expression of Hh and cholesterol target genes. Tumor size was meausred from the xenografts. Blockade of Hh signaling significantly decreased Gli1 gene expression by 30%, increased 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) expression for more than 300% indicating decreased intracellular cholesterol. Treatment with the cholesterol inhibitor Lovastatin increased expression of HMGCR for more than 500%. The combination of Hh and cholesterol blockage resulted in increased expression of HMGCR for more than 3400%. Analysis of chondrosarcoma xenografts in vivo showed a significnat decrease in tumor size with Lovastatin (32% decline), 3 folds reducation of Brdu(+) cells, and 2.4 fold increase of Caspase-3 + cells, treatment with Cur 61414 reduced the tumor growth by 5% with no significant reduction of Brdu(+) cells but 2.6 fold increase of Caspase-3 (+) cells . The combination treatment of lovastatin and Cur61414 on xenografts resulted in a significnat decrease in tumor size (32% decline), 3 fold reduction of Brdu (+) cells, but no significant changes of Caspase-3 (+) cells . These data suggest that cholesterol functions downstream of Hh signaling pathway in CSA. The more effective reduction in tumor growth with cholesterol inhibition compared to Hh blockade suggests cholesterol blockade is an effective therapeutic approach.

#320

Potential of targeting of MYC-driven cancers via CIP2A without detrimental physiological effects.

Xi Qiao. _Turku University, Turku, Finland_.

Transcription factor MYC is a driver of many human cancers due to its widespread effects on gene expression. An understanding of the mechanisms determining MYC's transcriptional and proliferation-promoting activities in vivo could facilitate approaches for MYC targeting. However, post-translational mechanisms that control MYC function in vivo are poorly understood. Here, we demonstrate that MYC phosphorylation at serine 62 enhances MYC accumulation on Lamin A/C-associated nuclear structures and that the protein phosphatase 2A (PP2A) inhibitor protein CIP2A is required for this process. CIP2A is also critical for serum-induced MYC phosphorylation and for MYC-elicited proliferation induction in vitro. Complementary transgenic approaches and an intestinal regeneration model further demonstrated the in vivo importance of CIP2A and serine 62 phosphorylation for MYC activity upon DNA damage-induced proliferation. However, targeting of CIP2A did not influence the normal function of intestinal crypt cells or general well-being of the mice. Meanwhile, in breast cancer cells, CIP2A depletion was shown to inhibit the MYC serine 62 phosphorylation, MYC-mediated gene expression, and anchorage-independent growth. Furthermore, CIP2A supports MDA-MB-231 xenograft growth in nude mice. Therefore, results of these studies collectively suggest for a novel opportunity to target MYC's function in MYC- driven cancers via CIP2A, and without detrimental physiological effects.

#321

MUC1 regulates TGFβ function in pancreatic cancer.

Priyanka Grover,1 Monica D. Nye,1 Mahboubeh Yazdanifar,1 Mohammad Ahmad,1 Ru Zhou,1 Lopamudra Das Roy,1 Kajal Grover,2 Shu-ta Wu,1 Sritama Nath,1 Pinku Mukherjee1. 1 _Univ. of North Carolina - Charlotte, Charlotte, NC;_ 2 _Univ. of North Carolina - Chapel Hill, NC_.

In 2016, Pancreatic Cancer (PC) has moved to 3rd leading cause of cancer-related deaths in the USA with 94% dying within 5 years of diagnosis. 90% of PC is Pancreatic Ductal Adenocarcinoma (PDA), of which 80% of PDA overexpress tumor associated Mucin-1 (tMUC1), a membrane bound glycoprotein that is hypoglycosylated. Overexpression of tMUC1 is associated with metastasis and poor prognosis. However the mechanism remains obscure.

Transforming growth factor-β (TGF-β) plays a role in poor prognosis. TGF-β is a cytokine with dual functionality. Within normal cells and early carcinogenesis, TGF-β functions as a tumor suppressor and induces apoptosis. This effect is mediated by activation of the canonical Smad pathway via engagement of TGF-β Receptor 1 (TGF-βRI). However during later stages of cancer, TGF-β becomes a tumor promoter and stimulates epithelial to mesenchymal transition, migration, and invasion thus enhancing metastasis. This TGF-β effect is mediated by the noncanonical Erk1/2 pathway.

We have recently shown correlation between tMUC1 and TGF-β within an exogenous tMUC1 model of PDA. Therefore, it is hypothesized that the mechanism between the two newly connected pathways exists in PDA tumors with high endogenous tMUC1 levels. We propose that the tyrosine kinases present in the cytoplasmic tail of tMUC1 are intermediary between the two pathways, thus leading to enhanced metastasis.

First the secretion levels of TGF-β1 were assessed in a panel of 13 PDA cell lines with variable expression levels of tMUC1 by ELISA. The effects of TGF-β1 to induce apoptosis versus invasiveness on a variety of tMUC1 high and tMUC1 low PDA cell lines were determined. The effects of tMUC1 knockdown were evaluated in tMUC1 high cell lines via siRNA. We studied the role of tyrosine kinases and other protein complexes in mediating the interactions between tMUC1 and TGF-β via proteomics analysis. Finally, we evaluated the anti-tumor efficacy of neutralizing TGF-β1 in vivo in high and low tMUC1-expressing PDA tumors.

In tMUC1 high PDA cells, TGF-β1 is secreted less when compared to tMUC1 low PDA cells, allowing for the more resistant cells to diminish TGF-β mediated effects endogenously. It is shown that with TGF-β1 treatment, tMUC1 high cells are more invasive, resist apoptosis, and activate Erk pathway, while tMUC1 low cells activate SMAD pathway. However, tMUC1 knockdown via siRNA does not affect TGF-β receptor levels. When studying the tyrosine kinases, c-Src becomes more phosphorylated in the presence of TGF-β1 in tMUC1 high cells, while in tMUC1 low cells c-Src becomes more deactivated in the presence of TGF-β1. Finally, within the mouse model, tMUC1-high PDA tumors respond to the neutralization of TGF-β1 by decreasing tumor size, while having no statistical effect on tMUC1-low PDA tumors.

tMUC1 expression is influential in TGF-β function in an endogenous model. Neutralizing TGF-β in tMUC1 high expressing tumors can be considered as a possible treatment. This has high clinical significance for patients with PDA.

#322

Distinct pools of ShcA coupled tyrosine kinase signaling influences breast tumor heterogeneity and therapeutic responsiveness.

Jacqueline R. Ha,1 Ryuhjin Ahn,1 Young Kyuen Im,1 Valerie Sabourin,1 Harvey W. Smith,2 Ivan Topisirovic,1 Tony Pawson,3 Peter Siegel,2 William J. Muller,2 Josie Ursini-Siegel1. 1 _Lady Davis Institute for Medical Research, McGill University, Montreal, Quebec, Canada;_ 2 _Goodman Cancer Research Centre, McGill University, Montreal, Quebec, Canada;_ 3 _Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada_.

Phospho-tyrosine (pTyr) signaling networks are frequently activated in breast cancers (BrCa) and are considered to be major oncogenic drivers of tumor progression. Therapeutic interventions, such as the tyrosine kinase (TK) inhibitor Trastuzumab, focus on targeting TK activity and downstream effectors. Although successful for early stage tumors, a subset of patients experience relapse due to intrinsic or acquired resistance. This includes activation of alternative receptor tyrosine kinase (RTK) and/or cytoplasmic TKs, many of which require recruitment of the adaptor protein, ShcA. ShcA is a key convergence point downstream of RTKs and serves to integrate multiple signal transduction pathways dysregulated in BrCa. Specifically, ShcA contains two pTyr binding motifs including an amino-terminal PTB domain and a carboxy-terminal SH2 domain which facilitate its interactions with TKs including ErbB2 and Src Family Kinases (SFK), respectively. The CH-1 domain houses three tyrosine phosphorylation sites at residues 239/240 and 317 which transduce Ras-dependent and independent signals. Using a well characterized transgenic mouse model of BrCa where ShcA can no longer engage the transforming oncogene through its PTB domain, we demonstrate that loss of PTB-driven ShcA (ShcA-PTBMut) signaling delays mammary tumor onset. However, once formed, the growth and angiogenic potential of these tumors is significantly increased relative to control mice. Increased growth potential of ShcA-PTBMut tumors is associated with the hyper-activation of the c-Src tyrosine kinase. Deletion of c-Src in ShcA-PTBMut breast tumor cells significantly delays tumor onset but is dispensable for the growth of tumors that retain an intact ShcA PTB domain. These data suggest that the ShcA PTB domain can recruit negative regulators that limit the activation of downstream tumorigenic signaling networks. Interestingly, tumors expressing ShcA-PTBMut debilitated in SH2 driven pTyr interactions (SH2Mut), are significantly delayed in tumor onset relative to ShcA-PTBMut controls. Paradoxically, deletion of c-Src in the context of ShcA-PTBMut-SH2Mut further accelerates tumor growth which is attributed to increased levels of Fyn and Lyn. These observations support the high dependence of intracellular ShcA pools on other SFK family members to retain tumorigenic potential when adapting to low levels and/or activity of c-Src. We demonstrate that uncoupling of PTB-driven ShcA signaling from upstream RTKs can potentiate ShcA signaling from intracellular pools to hyper-activate SFKs. This data is clinically relevant as c-Src is frequently hyper-activated in Trastuzumab-resistant BrCa. This is the first study to identify a tumor suppressive role of the ShcA PTB domain and to characterize an intrinsic ShcA SH2 domain-SFK dependent resistance mechanism downstream of activated RTKs in mammary tumorigenesis.

#323

A rapid flow cytometry method for analyzing NF-κB activation and translocation in whole blood.

George C. Brittain, Sergei Gulnik. _Beckman Coulter, Inc., Miami, FL_.

Background: The end point of most cell-signaling cascades is the translocation of protein complexes into the nucleus to modify transcriptional activity. For this reason, the analysis of cytoplasmic vs. nuclear localization provides important insights into cell activation. However, assessing nuclear translocation in complex cell mixtures, such as whole blood, is a difficult task. Leukocytes are a rare event in blood, with many subpopulations, requiring the cells to first be concentrated, sorted, rested and often expanded prior to further molecular analyses. This process can take weeks, and questions regarding physiological relevance arise from even the first preparatory steps. Here, we present a buffer-based system to lyse RBCs and differentially stain leukocytes in order to enable the quantitative analysis of cytoplasmic vs. nuclear localization in endogenous cells by flow cytometry. We demonstrate the use of this system by analyzing the activation of NF-κB signaling in whole-blood monocytes following lipopolysaccharide (LPS) stimulation.

Methods: The main components of the Whole Blood Nuclear Localization Kit are two buffers: Buffer 1 permeabilizes the cytoplasmic membrane, leaving the nuclear membrane intact, while Buffer 2 permeabilizes both the cytoplasmic and nuclear membranes. Briefly, blood was treated for different times with LPS, fixed for 10 min, split into 2 fractions, and then one was lysed for 30 min with Buffer 1 and the other with Buffer 2. Each fraction was washed, stained and acquired on a CytoFLEX-S flow cytometer (Beckman Coulter, Brea, CA). The cytoplasmic and nuclear signals were calculated using the compensated results from the two tubes. Blood was collected onsite daily from healthy, consenting human adults.

Results: Following LPS stimulation, nuclear p50 and RelA increased maximally in monocytes by 10 min. Phospho-RelA S536 was maximal by 5 min mostly in the cytoplasm, while phospho-RelA S529 was maximal by 10 min in the nucleus. IκBα degraded maximally by 10 min. Additional kinase signaling will be presented, as well as controls to demonstrate the partitioning efficiency.

Conclusions: This system is very fast and user friendly, and can help to improve our understanding of normal and aberrant signaling in blood, while also saving researchers time and resources compared to conventional procedures, e.g., purifying leukocytes and performing Western blots. It provides clear delineation of the nucleus with minimal perinuclear staining, and can even be used to improve the quantitation of nuclear signals from 2D microscopic images. Finally, this system can enable research where blood volumes are limited, such as with rare and precious samples and small-animal studies. ***This kit and the CytoFLEX-S are for Research Use Only. Beckman Coulter and the Beckman Coulter product and service marks mentioned herein are trademarks or registered trademarks of Beckman Coulter, Inc. in the United States and other countries.***

#324

Investigating the dependence of canine bladder transitional cell carcinoma on activated mutant BRAF.

Kathryn Cronise, Belen Hernandez, Daniel L. Gustafson, Dawn L. Duval. _Colorado State Univ., Fort Collins, CO_.

Transitional cell carcinoma (TCC) accounts for approximately 2% of diagnosed malignancies in canines. The majority of canine TCCs are invasive, intermediate to high grade tumors sharing similarities with human muscle invasive TCCs in risk factors, histology, sites of metastasis, and therapeutic response to single agents. Whole exome sequencing of canine TCC tumors was performed to identify somatic mutations in known cancer driver and repressor genes that could potentially contribute to canine TCC pathogenesis. A valine to glutamic acid mutation in BRAF homologous to the activating V600E mutation identified in human melanoma, colorectal and thyroid cancers was identified in 70% of sequenced tumors. Sensitivity to the BRAFV600E inhibitor Vemurafenib was tested in three BRAF mutant canine TCC cell lines (Bliley, Tyler1 and Tyler2) and two BRAF wild type canine TCC cell lines (Angus1 and Kinsey). All five canine TCC cell lines exhibited IC50s greater than 10μM, with BRAF mutant cell lines being slightly more sensitive. These sensitivity ranges are similar to those of some mutant human colorectal cancer cell lines, indicating that additional mechanisms may contribute to Vemurafenib resistance. Western blot analysis was performed to measure relative abundance of pERK, a downstream target of BRAF, in canine TCCs in response to serum starvation. All TCC cell lines showed sustained pERK expression in the absence of serum, indicating constitutive activation of the MAPK pathway. The five canine TCC lines were treated with 15μM Vemurafenib for 6 and 24 hours and their lysates were analyzed for pERK protein expression. pERK abundance was decreased in only the BRAF mutant cell lines after 6 hours of treatment. However, this decrease was less pronounced after 24 hours, suggesting that resistance mechanisms are bypassing BRAF to activate ERK. Sequence analysis of an additional panel of formalin-fixed paraffin embedded canine TCCs also revealed a mutation in RanBP2 in 31% of samples. Strikingly, the RanBP2 mutation appeared to be mutually exclusive to BRAF V to E mutant tumors with only two of the analyzed samples carrying both mutations. Significant tumor heterogeneity was implicated due to low level mutant amplification in these samples. It has been reported that loss of RanBP2 is synthetic lethal in BRAF V600E mutant colorectal cancer. Since RanBP2 forms complexes with CRM1 at the nuclear pore complex for nuclear export and at the kinetochore during mitosis, canine TCC cell lines were treated with KPT-185, a CRM1 inhibitor. BRAF mutant TCC cell lines had IC50 values ranging from 45nM to 65nM and were approximately ten-fold more sensitive than wild type cell lines. Overall, this data indicates that the pathogenesis of canine TCC likely depends on driving factors in addition to constitutive BRAF signaling, but Vemurafenib resistant BRAF mutant tumors can be targeted through inhibition of the nucleopore complex.

#325

Pediatric relapsed acute lymphoblastic leukemia patients display enrichment of the PI3K/mTOR pathway and respond to the dual PI3K/mTOR inhibitor PKI-587.

Mohiuddin Gazi, Sausan A. Moharram, Alissa Marhäll, Kinjal Shah, Julhash U. Kazi. _Lund University, Lund, Sweden_.

Although significant improvements have been observed in the treatment of acute lymphoblastic leukemia, there is a substantial subset of high-risk T-ALL patients with relatively poor prognosis. T-cell acute lymphoblastic leukemia (T-All) is a hematopoietic malignancy affecting lymphoblast of T-cell lineage which cause lots of hematopoietic cancer related deaths every year all over the world. The current overall cure rates of newly diagnosed childhood ALL are more than 80% in Europe, but approximately 20% of patients relapse due to treatment failure in childhood ALL. Like other leukemia types, alterations of PI3K/mTOR pathway are predominant in T-ALL which is also responsible for treatment failure and relapse. In this study, two different gene expression data sets of relapsed patients also displayed enrichment of the PI3K/mTOR pathway. Out of 88 different inhibitors targeting multiple components of this pathway, we observed that PKI-587 was the most selective to the T-ALL cell line CCRF-CEM. Thus, we characterized the activity of the novel dual PI3K/mTOR pathway inhibitor PKI-587 using CCRF-CEM and Molt3 cells. We observed that PKI-587 blocked proliferation, colony formation and induced apoptosis in the T-ALL cell lines and selectively abrogated PI3K/mTOR without affecting the MAPK signaling. In vivo PI3K/mTOR inhibition delayed tumor progression, and reduced tumor load in a NSG/SCID xenograft mouse model. Since no deceptive body weight decrease was noticed, our conclusion is that this dose is effective and well tolerated. The beneficial effects of PKI-587 on T-ALL cells that has been observed in this study both in vitro and in vivo warrant further investigation.

#326

Baseline IL-17 receptor signaling is essential for controlling aberrant JNK-dependent cellular proliferation via maintenance of endogenous level of ubiquitin-editing enzyme A20.

Chi Yan, Yang Lei, Anna L. Greenshields, David W. Hoskin, Tong-Jun Lin, Jun Wang. _Dalhousie University, Halifax, Nova Scotia, Canada_.

The molecular mechanisms underlying aberrant activation of NF-κB and JNK in cancer remain incompletely understood. Here, we demonstrate that baseline IL-17 receptor (IL-17R) signaling is essential for controlling aberrant NF-κB and JNK activation, and restraining JNK-dependent homeostatic cellular proliferation. Using a shRNA knockdown approach, we demonstrated in B16 melanoma and 4T1 breast carcinoma murine cell lines that IL-17RA silencing markedly enhanced tumor cell growth in vitro and in vivo. Through mapping IL-17R signaling pathways, we further demonstrated that baseline IL-17A/IL-17R signaling actively restrained JNK phosphorylation in vitro and in vivo via the maintenance of basal expression of the ubiquitin-editing enzyme A20, a negative regulator of NF-κB and JNK. Remarkably, IL-17RA reconstitution evidently restored the A20 level, and suppressed cell proliferation and JNK activity in tumor cells. The reconstitution of A20 in IL-17RA knockdown subclones was able to restore the normal rate of cellular proliferation and associated JNK/c-Jun activity. Finally, meta-analysis of human cancer microarray and RNA-Seq datasets confirmed significant co-expression of IL-17RA and A20. Furthermore, alterations (mutation, upregulation or downregulation) of IL-17RA level in melanoma, ER+ breast cancer and colorectal cancer patients were associated with poorer overall survival compared to the respective patients with normal baseline IL-17RA expression. Together, our data demonstrates a previously unrecognized molecular mechanism underlying aberrant activation of NF-κB and JNK in cancer cells. This work highlights the unique biological role of proinflammatory IL-17R signaling in the maintenance of A20 to regulate the pathogenesis of human cancer, which draws caution on the utility of IL-17A neutralizing antibody in cancer therapy.

CY is a PhD student supported by the graduate student scholarship from the IWK Health Centre and received a trainee award from the Beatrice Hunter Cancer Research Institute with funds provided by the Cancer Research Training Program as part of The Terry Fox Foundation Strategic Health Research Training Program in Cancer Research in CIHR.

#327

EpCAM/EpEX regulate tumor progression through EGFR signaling in colon cancer cells.

Kang-Hao Liang, Jun-Kai Lai, I-I Kuan, Hsien-Cheng Tso, Han-Chung Wu. _Academia Sinica - Inst. of Cell. & Organismic Bio., Taipei, Taiwan_.

Epithelial cell adhesion molecule (EpCAM) is highly expressed in advanced epithelial cancers and tumor-initiated cells (TICs), but its roles in cancer progression remain to be elucidated. Here, we showed that the extracellular domain of EpCAM (EpEX) could bind to EGFR through EGF-like domain I, and subsequently activated its downstream molecules, ERK1/2 and Akt. EGFR inhibitor and knockdown of EGFR by shRNA ablated EpEX-induced ERK1/2 phosphorylation. Regulated intramembrane proteolysis (RIP) of EpCAM was induced similarly by EpEX and EGF through EGFR-dependent activation of ERK pathway. MEK inhibitor, U0126, could abolish ADAM17 and PS2 phosphorylation induced by EpEX. EpAb2-6, an anti-EpEX neutralizing monoclonal antibody, inhibits EpEX-activated EGFR-PI3K-AKT pathway in detached colon cancer cells. Moreover, intracellular domain of EpCAM (EpICD), the product of RIP-induced cleavage of EpCAM, is necessary for nuclear accumulation of β-catenin, and their target gene expressions in vitro and in mouse xenograft models. We also found that an increase of nuclear EpICD observed in CRCs predicted metastasis and poor prognosis in CRC patients. Finally, in animal model studies, EpAb2-6 therapy exhibited an enhanced antitumor effect and markedly extended the survival time of mice with human colorectal cancer in metastatic and orthotopic models. These results demonstrate that EpEX works as a growth factor in activating EGFR-mediated signaling, and as a potential target for treatment of colon cancer. This research was supported by grants from Academia Sinica and Ministry of Science and Technology [MOST 104-0210-01-09-02,MOST 105-0210-01-13-01], and the National Science Council (NSC103-2321-B-001-064), Taiwan (to H-C Wu).

#328

PARP inhibitors suppress growth of EGFR-mutant cancers by targeting nuclear PKM2.

Nan Li, Steven H. Lin. _UT MD Anderson Cancer Ctr., Houston, TX_.

Poly(ADP-robose) polymerase-1 (PARP1) or ADP-ribosyltransferase diphtheria toxin-like 1 (ARTD1) is the most abundant and the best understood member of the 17 PARP family proteins. PARP1 binds to both single strand breaks (SSBs) and double-strand breaks (DSBs) and participates in the recognition, excision and repair of DNA damage. The most extensively studied role of PARP1 is its involvement in base excision repair (BER) and PARP inhibition induced-trapping of PARP1 during BER. Moreover, suppression of PARP1/2 leads to synthetic lethality in BRCA1/2-defecient tumors, indicating that PARP1-dependent BER and BRCA-dependent homologous repair pathway have overlapping and redundant functions in DNA repair. Recent studies have also pointed to a broader utility of PARP inhibitors beyond hereditary BRCA-deficient cancers. Pyruvate kinase isoform M2 (PKM2) is a glycolysis enzyme that converts phosphoenolpyruvate (PEP) into pyruvate. Up-regulation of PKM2 has been shown recently to be an important feature of tumorigenesis. In tumor cells, PKM2 forms a dimmer that is catalytically inactive as a glycolysis enzyme, but provides advantage for tumor progression due to Warburg effect. In this report, we demonstrated that poly-ADP-ribose (PAR) is required for nuclear retention and nuclear function of PKM2. PKM2 translocates into nucleus and binds to PAR upon EGF stimulation. PARP inhibition, or the PKM2-C/A mutant, which abolishes the PKM2/PAR interaction, suppresses the nuclear function of PKM2. In addition, PARP inhibition also diminishes the nuclear PKM2-dependent glycolysis and tumor growth. Moreover, we showed that nuclear localization of PKM2 correlates with PAR expression in EGFR-mutant human lung cancer tissues. Interestingly, PARP inhibition leads to growth suppression of some EGFR-mutant lung cancer cells, which are resistant to EGFR inhibitor. These data together support an unexpected function of PARP inhibition in tumor suppression and indicate that nuclear PKM2 may serve as a promising biomarker for the further development of PARP inhibitor-based therapies.

#329

Selinexor or KPT-8602 mediated XPO1 inhibition synergizes with dexamethasone to repress convergent pathways in the mTORC1 signaling network and drive cell death in multiple myeloma.

Christian Argueta, Trinayan Kashyap, Boris Klebanov, Hua Chang, Sharon Friedlander, Erkan Baloglu, Yosef Landesman, Margaret Lee, Humphrey Gardner, Sharon Shacham, William Senapedis. _Karyopharm Therapeutics Inc., Newton, MA_.

Background: Dexamethasone (DEX), a synthetic glucocorticoid (GC), is administered to nearly all multiple myeloma (MM) patients as a single agent and in combination with other chemotherapies or targeted agents. DEX and other GCs bind to glucocorticoid receptors (GR) in the cytoplasm, induce nuclear translocation and regulate GR-dependent gene expression networks in the nucleus. Selective Inhibitor of Nuclear Export (SINE) compounds (selinexor and KPT-8602) exhibit potent anti-tumor activity in MM especially when combined with DEX. SINE compounds enhance nuclear localization of tumor suppressor proteins (TSPs) through inhibition of the nuclear export protein, XPO1. We discovered that inhibition of the mechanistic Target of Rapamycin Complex 1 (mTORC1) pathway is a primary driver of the combination effect. Here we further elucidate the molecular mechanism of action of the SINE-DEX synergy in MM.

Methods: GR+ MM.1S and GRnull MM.1R MM cell lines were treated with SINE compounds and/or DEX for 24 hours. Whole cell lysates were subjected to SDS-PAGE and western blot analysis. Gene expression and GR transcriptional activity was analyzed using qPCR and ELISA, respectively. Cell viability was examined using the Celltiter-Fluor assay.

Results: We found that MM cell lines treated with SINE compounds (selinexor or KPT-8602) have increased basal GR protein levels. Consistent with these results, the SINE-DEX combination shows enhanced GR transcriptional activity. Several GR-DEX target genes are known to inhibit the GTPase, Ras homolog enriched in brain (RHEB), which is required for mTORC1 activation. We discovered that the SINE-DEX combination not only reduces RHEB protein but also induces the RHEB inhibitory pathways containing REDD1 and the KLF15-BCAT2 axis. Although SINE compound-mediated inhibition of mTORC1 (i.e. reduced phosphorylation of S6K1 and 4E-BP1) is GR independent, SINE-DEX inhibition is more robust in GR+ MM.1S cell line when compared to the GRnull MM.1R cells. The combination resulted in the selinexor IC50 in MM.1S cells shifting from 40 nM to 11 nM in the presence of low dose DEX. As expected, DEX did not modulate the SINE compounds IC50s in MM.1R cells.

Conclusion: We show that SINE compound inhibition of MM cell viability is enhanced with DEX. Our results indicate that this combinatorial effect is due to convergent suppression of mTORC1 signaling by GR targets. The findings provide mechanism of action data around the SINE-DEX combination in MM with suggestive biomarkers (REDD1, KLF15, BCAT2 and GR) that may predict best response to the combination. Therefore, these data may translate directly to the current clinical development of SINE compounds.

#330

SGK1 activation is essential for PI3K-dependent tumor development.

Arturo Orlacchio, Antonio Di Cristofano. _Albert Einstein College of Medicine, Bronx, NY_.

The PI3K signaling cascade is frequently activated in human cancer, with AKT being commonly considered its major transforming conduit.

Conditional deletion of Pten in the mouse thyroid epithelium induces constitutive activation of the PI3K pathway, which causes thyroid hyperplasia at birth that progresses to invasive and metastatic follicular carcinoma. Concomitant loss of p53 or activation of KRAS dramatically accelerates the development of aggressive and lethal tumors.

The PDK1 kinase plays a key role in the PI3K signaling cascade, directly activating, in a PI3K-dependent manner, not only AKT but also a set of additional AGC kinases, including, S6K, PKC, and SGK.

Using in vivo, ex vivo, and in vitro genetic and pharmacological approaches, we now show that AKT activation is not sufficient to transform thyroid epithelial cells. Concomitant activation of additional PDK1-dependent pathways is absolutely required to develop neoplastic lesions in vivo, and to induce cell proliferation ex vivo, in mouse models based on Pten loss, alone and in combination with additional genetic alterations.

In particular, we show that one member of the SGK family, SGK1, is an essential mediator of the transformation process downstream of PI3K.

Genetic and pharmacological SGK1 inhibition strongly reduces cell proliferation in Pten-/-and PIK3CA-mutant cell lines.

Moreover we show that concomitant SGK inhibition significantly increases the efficacy of inhibitors targeting AKT or PI3K.

Taken together, our data identify an essential and druggable signaling cascade that critically cooperates with AKT activation to transform thyroid epithelial cells.

#331

SIRPB1 promotes prostate cancer cell proliferation & migration.

Qiong Song,1 Siyuan Qin,1 Chunlin Zou,1 Wenchu Wang,1 Lihui Wang,1 Haibo Tong,1 William J. Catalona,2 Jian Zhang,1 Yi Lu,1 Zhou Wang3. 1 _Guangxi Medical University, Nanning, China;_ 2 _Northwestern University Feinberg School of Medicine, Chicago, IL;_ 3 _University of Pittsburgh School of Medicine, pittsburgh, PA_.

Signal-regulatory-protein beta 1(SIRPB1) is a member of the signal-regulatory-protein (SIRP) family that belongs to the immunoglobulin superfamily and is capable of modulating receptor tyrosine kinase-coupled signaling. The copy-number variations (CNV) at the SIRPB1 locus were associated with aggressiveness of prostate cancer in patients. To test if SIRPB1 could affect prostate cancer development and progression, we investigated the potential role and mechanisms of SIRPB1 action in prostate cancer cell lines in vitro and in xenograft tumors. Knockdown of SIRPRB1 by RNA interference resulted in significant suppression of cell colony formation, cell mobility, cell migration, and invasion. The knockdown also induced cell cycle arrest during the G0/G1 phase and a remarkable enhancement of apoptosis in PC3 prostate cancer cells. In contrast, overexpression of SIPRB1 significantly induced cell migration, invasion, colony formation and cell cycle in C4-2 prostate cancer cells. Furthermore, overexpression of SIRPB1 in C4-2 cell model enhanced its tumor take rate in nude mice. Also, SIRPB1 mRNA expression was increased in up to 39% of the prostate cancer specimens based on in silico analysis of several public databases. These results suggest that SIRPB1 is a potential oncogene in the prostate and could be used as a biomarker to identify patients at risk of developing aggressive prostate cancer.

#332

Loss of primary cilia promotes LPA-driven proliferation in glioblastoma.

Yuriy Loskutov. _West Virginia University, Morgantown, WV_.

Glioblastoma is one of the most common brain cancers with a median survival of 13 months. Therapeutic approaches for this type of cancer are very limited, creating an urgent need to develop new strategies for the treatment. Recently several groups reported that glioblastoma have a drastic decrease in primary cilia, suggesting that loss of cilia might be important for glioblastoma biology. The primary cilium is a ubiquitous microtubule based organelle presented on most of human cells. It plays significant role in embryonic development and tissue homeostasis, serving as a hub for multiple signaling cascades including RTK, Sonic Hedgehog, Wnt and Hippo. Here we report that loss of primary cilia in immortalized astrocytes stimulate cell proliferation and drastically change ERK1/2 activation in response to serum. These changes depend on lysophosphotidic acid (LPA), a water-soluble lipid metabolite, implicated in chemotactic and proliferative signaling. The receptor to LPA type 1 tends to accumulate in the primary cilia, indicating its potential role in inhibition/restriction of LPA-dependent mitogenic signaling via dynamic changes in primary cilium. Loss of primary cilia can lead to LPA receptors 1 redistribution to the plasma membrane and switch cellular response for the LPA, allowing it to act as mitogen. Inhibition of LPA receptors with a small molecule inhibitor, Ki16425, is significantly decreasing the growth of glioblastoma PDXs. Overall, our findings clearly indicate that loss of primary cilia is sufficient to severely change mitogen-driven signal transduction and point towards the new therapeutic target for glioblastoma.

#333

Identification of a natural product small molecule inhibitor against Wnt/β-catenin signalling pathway.

Mohd Saleem Dar,1 Aehtesham Hussain,2 Paramjeet Singh,1 Qazi Parvaiz Hassan,2 Satdarshan Paul Monga,3 Mohd Jamal Dar*1. 1 _Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Jammu, J &K, India; _2 _Microbial Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Srinagar, J &K, India; _3 _Department of Pathology, University of Pittsburgh, School of Medicine S-417 BST, Pittsburgh, PA, PA_.

Wnt/β-catenin signalling pathway plays an important role in many processes like cell proliferation, differentiation, regeneration, carcinogenesis and regulates stem cell pluripotency. β-Catenin, the central component of Wnt/β-catenin signalling pathway has a vast number of binding partners and thus plays different roles in the cells. Mutations of β-catenin are seen in many cancers, including liver cancer, colorectal cancer, lung carcinoma, ovarian cancer and malignant breast tumours. Till date there is not even a single small molecule in clinical use that can block the β-catenin activity or the activities of this signalling pathway. In this study, we established many assays for screening small molecule inhibitors of this signalling pathway. Initially, we created a library of β-catenin deletion and point mutants and carried out a comprehensive comparative analysis of these mutants by luciferase reporter assays, GFP-fluorescence, immunoblotting and protein-protein interactions. We then screened many known and unknown natural and synthetic molecules against the wnt/β-catenin pathway and got 2 hits: C-18 and 055A, bioactive metabolites from actinomycetes. The IC50 (cell toxicity) of these molecules along with salinomycin, a known ionophore and inhibitor of β-catenin, were determined in different human cancer cells by MTT assay. The IC50 of 055A, C-18 and salinomycin in HepG2 cells was 7.5µM, 9.7µM and 25µM respectively. Interestingly, C-18 was ineffective against non-cancerous cell line (HEK-293 cells) even upto 60µM. This shows that C-18 is a very promising hit because it is active against cancer cells and spares non-cancerous cells. The sub lethal doses of these molecules were then tested for β-catenin mediated transcription by luciferase reporter assay in HepG2 cells which harbours a β-catenin deletion responsible for enhanced β-catenin mediated transcriptional activity. Our results show that C-18 and 055A significantly block the β-catenin activity in a dose dependent manner. The inhibition of wnt/β-catenin pathway by C-18 was observed at lower nanomolar concentrations in comparison to 055A and salinomycin which shows activity in micromolar concentrations. The β-catenin target gene expression analysis showed marked decrease in cyclinD1 by immunoblot analysis upon treatment of HepG2 cells with C-18. Interestingly, C-18 treated HepG2 cells failed to show any apoptosis as confirmed by Parp-1 and caspase-3 immunoblotting indicating that C-18 can be targeted to block the activity of wnt/β-catenin pathway, and could act as a lead molecule. Therefore, it warrants further experimentation and validation which are underway to draw the further conclusions.

#334

PTEN-L regulates epithelial growth and macrophage function.

Andrew L. Wolfe,1 Benjamin D. Hopkins,2 Sebastián A. Riquelme,3 Kipyegon Kitur,3 Sait Ozturk,2 Kyeongah Kang,2 Romain Remark,2 Adeeb Rahman,2 Chyuan-Sheng Lin,3 Miriam Merad,2 Matthias Szabolcs,3 Shu-Hsia Chen,2 Alice Prince,3 Ramon Parsons2. 1 _University of California San Francisco, San Francisco, CA;_ 2 _Mount Sinai School of Medicine, New York, NY;_ 3 _Columbia University, New York, NY_.

PTEN is among the most frequently mutated and deleted tumor suppressor genes in many malignancies, including breast cancer. An alternatively translated long form of PTEN, termed PTEN-L, has divergent functionality from PTEN, although its function at the organism level has not been studied. Here, we report a knockout mouse with specific ablation of PTEN-L expression but intact expression of PTEN. These mice display mammary ductal hyperplasia characterized by increased luminal growth and increased numbers of macrophages in the surrounding stroma. Macrophages are particularly affected by PTEN-L loss, with significant changes to their secretomes and functional deficiencies in clearing bacterial infections, consistent with a shift toward an M2-like polarization. Overall, these findings demonstrate that PTEN-L has unique functions in regulating mammary epithelial growth and macrophage functionality that are independent of canonical PTEN.

#335

In situ **protein complexes as a measure of AXL signaling and inhibitor response in cell line models of lung cancer.**

Anurima Majumder,1 Yun Bai,1 Fumi Kinose,1 Erin D. Lew,2 Yumi Yokoyama,2 Danielle Murphy,2 Gary Li,1 Eric B. Haura1. 1 _Moffitt Cancer Center, Tampa, FL;_ 2 _Ignyta, Inc, San Diego, CA_.

AXL kinase overexpression is associated with metastasis and poor prognosis in various cancers. Recent studies show that epithelial-mesenchymal transition (EMT) and associated activation of AXL is one of the mechanisms by which tumors develop resistance to targeted therapies, such as EGFR tyrosine kinase inhibitor (TKI) in lung cancer. These studies have made AXL an attractive drug target. However, the lack of genetic alterations in AXL and recent evidence suggesting that ligand secretion into the tumor microenvironment plays a key role in signaling and resistance in cancer cells, underscore the need for assays to visualize active AXL signaling associated complexes. In this study, we aimed to develop a Proximity Ligation Assay (PLA) to effectively measure AXL activation in tumor tissues in situ, and to use the assay to evaluate the pharmacodynamic effect of a novel AXL TKI, RXDX-106, in lung cancer models. To this end, several lung cancer cell lines were analyzed for total and phosphorylated AXL (pAXL) expression as well as effects of AXL TKI on downstream signaling. Co-immunoprecipitation studies were also conducted to identify adaptor proteins that form active signaling complexes with AXL kinase, and PLAs were developed to detect these complexes in situ.

H1299 and Calu1 cells have the highest pAXL among the cells screened. RXDX-106 potently inhibits pAXL and downstream pAKT, but does not affect pERK or cell viability, in these cell lines. We also observe an increase in total AXL in response to the RXDX-106. Co-immunoprecipitation shows RXDX-106-dependent reduction in AXL-PI3KR1 (Phosphoinositide-3-Kinase, Regulatory Subunit 1) interaction that is consistent with the drug-induced reduction in pAKT by western blot. Based on this observation, we developed PLA to detect active AXL:PI3KR1 and AXL:pY100 signaling complexes in these cells. H1299 and Calu1 cells have high basal AXL:PI3KR1 and AXL:pY100 PLA foci that are abrogated by RXDX-106, in both fresh fixed and formalin-fixed paraffin-embedded cells. As expected, the HCC827 cells, which lack ligand independent pAXL, do not show significant labeling by either PLA.

Overall, we demonstrate that RXDX-106 is a potent inhibitor of AXL kinase and its downstream PI3K/AKT signaling pathway. We also show that activated AXL signaling complexes can be annotated specifically by PLA. The data provides justification for the extension of our PLA as a tool to measure drug-targetable signaling complexes to identify new models of activated AXL and enrich for activated AXL in patient tissues.

#336

PIK3CB/p110B is a survival factor in glioblastoma.

Kevin J. Pridham, Sujuan Guo, Zhi Sheng. _Virginia Polytechnic Institute and State University, Blacksburg, VA_.

Glioblastoma multiforme (GBM) is lethal even after surgical removal of the tumor, radiation, and chemotherapy. Residual tumor cells form an intractable tumor in nearly all patients within two years. Recurrent GBM is incurable due to resistance to current therapies. Inhibitors of PI3K (phosphatidylinositol-4,5-biphosphate 3-kinase)-a signaling pathway that causally contributes to tumor formation/recurrence-have been used to treat recurrent GBMs and achieved modest clinical effect. This is perhaps attributed to non-selective inhibition of PI3K isoforms, which yields intolerable toxicity. Class IA PI3K isoforms include three catalytic subunits (PIK3CA, B, or D that encodes p110α, β, or δ) and three regulatory subunits (PIK3R1-3 that encodes p85 isoforms). Our recent work indicates that PIK3CB levels positively correlate with the chances/risk of GBM recurrence, while being inversely associated with patient prognosis. This suggests that PIK3CB/p110β is important for GBM cell survival. To test this hypothesis, we first measured the expression of PI3K isoforms in a panel of 9 GBM cell lines. We found that U87MG, SF295, and U251 expressed much higher levels of p110β, coinciding with the levels of phosphorylated AKT. We then knocked down PIK3CA, B, and D in human U87MG cells and found that only depletion of PIK3CB/p110β resulted in an inactivation of downstream AKT. Moreover, knockdown of PIK3CB/p110β, but not other isoforms, induced substantial growth inhibition in U87MG, SF295 and U251 cells. This is congruent with the result that inhibition of PIK3CB/p110β activated apoptosis in U87MG cells. We also report that treatment of p110βhigh GBM cells with isoform specific inhibitors selectively represses the viability of these cells. Finally, we report that ectopic expression of p110β, but not p110α or δ, partially rescued U87MG cells from growth inhibition induced by TGX-221, a p110β-selective inhibitor. Collectively, our results demonstrate that PIK3CB/p110β is an important selective survival factor in GBM, underscoring the divergent roles of PI3K isoforms in GBM disease progression/recurrence and future therapeutic intervention.

#337

Sonic Hedgehog ligand drives tumor progression and chromosomal instability in a mouse model of small cell lung cancer.

Anette Szczepny,1 Samuel Rogers,2 Samantha Jayasekara,1 Kwon Park,3 Rachael McCloy,2 Julien Sage,4 Craig Peacock,5 Jason Cain,1 Andrew Burgess,2 David Neil Watkins2. 1 _Hudson Institute of Medical Research, Victoria, Australia;_ 2 _Garvan Institute of Medical Research, Darlinghurst, Australia;_ 3 _University of Virginia, Charlottesville, VA;_ 4 _Stanford Cancer Institute, Stanford, CA;_ 5 _Cleveland Clinic, Cleveland, OH_.

Activation of the Hedgehog (Hh) signaling pathway is well documented in many cancers including Small Cell Lung Cancer (SCLC). Whilst it has been shown that Smoothened, the central Hh pathway mediator, is required for the initiation and progression of SCLC in a mouse model, it is unclear what drives activation of this pathway in these tumors.

To address this question, we used a well-characterized conditional genetic mouse model of SCLC in which inhalation of recombinant adenovirus expressing Cre can trigger recombination at loxP sites in the airway epithelium. When the virus is administered to mice double homozygous for the conditional p53 and Rb knockout alleles (p53lox/lox;Rblox/lox), mice develop multiple tumors over 9 months.

In order to manipulate Shh expression in this model, we crossed these animals with either gain-of-function or loss-of-function Shh alleles to observe the effects on SCLC initiation and progression in vivo. Aged cohorts of AdenoCre-infected p53lox/lox;Rblox/lox;ShhTg mice developed more frequent and significantly larger tumors compared to p53lox/lox;Rblox/lox littermate controls, with tumors exhibiting a highly malignant and proliferative phenotype. Conversely, deletion of Shh resulted in no significant change in tumor number but a dramatic reduction in tumor size in p53lox/lox;Rblox/lox;Shhlox/lox mice compared to littermate controls.

Interestingly, Shh ligand overexpression also induced marked chromosomal instability and the Smoothened-independent nuclear translocation and activity of Cyclin B1. In turn, overexpression of Cyclin B1 induced chromosomal instability in cells lacking both p53 and Rb. These results suggest that Shh ligand drives progression of SCLC by activating both the canonical and non-canonical arms of the Hh pathway and that Shh may be a potentially useful therapeutic target.

#338

Unexpected activity of multiple targeted cancer drugs in regulating ciliary dynamics.

Anna Kiseleva,1 Vladislav Korobeynikov,2 Anna Nikonova,1 Alexander Deneka,1 Margret Einarson,1 Emmanuelle Nicolas,1 Petr Makhov,1 Vladimir Kolenko,1 Jeffrey Peterson,1 Zeng-jie Yang,1 Erica Golemis1. 1 _Fox Chase Cancer Center, Philadelphia, PA;_ 2 _Columbia University, New York, NY_.

The primary cilium is an antenna-like structure protruding from the cell surface, which provides a platform for receptors for signaling systems including PDGF-alpha, Hedgehog, Wnt, and others that influence cell differentiation and proliferation decisions. Under normal physiological growth conditions, the cilium forms after mitosis and in quiescent cells, extending from a basal body centered at a centriole, with timed resorption in G0 or early G1. Cancer cells have altered ciliary dynamics, with some (medulloblastomas and basal cell carcinomas) often dependent on cilia, and other tumor types downregulating them. In prior work, we defined Aurora-A, an oncogenic kinase typically thought of as a mitotic regulator, as transiently activated at the basal body, and absolutely required for resorption of cilia at the G0/G1 boundary, and we showed the targeted Aurora-A inhibitor entirely blocked ciliary resorption. In subsequent work, we found that a second drug, the EGFR inhibitor erlotinib, also affected ciliary resorption, positioning erlotinib and alisertib to influence cilia-dependent signaling. In this study, we broadly assessed the activity of a panel of targeted preclinical and clinical agents of known specificity for action in controlling ciliary dynamics. For this, we developed stable hTERT-RPE1 cell line models with integrated Arl13b-GFP reporters to visualize cilia, and performed a mid-throughput screen of 180 drugs for activity in 1) independently inducing ciliary resorption in quiescent G0 cells, or 2) blocking ciliary resorption in cells induced to cycle by serum treatment. With a ~5% hit rate overall, we identified 9 compounds inducing, and 7 compounds blocking resorption. This included some agents commonly used in the clinic, such as sunitinib, which had never previously been linked to control of the ciliary protrusion/resorption cycle. Mechanistic analysis confirmed these compounds controlled activation of Aurora-A at the basal body; in vivo testing demonstrated sunitinib resulted in loss of cilia not only in vitro, but also in normal kidney, kidney tumors, and in renal cysts associated with polycystic kidney disease (PKD), a disease dependent on defective signaling from intact cilia. Further, sunitinib was successful in controlling PKD-dependent cyst development, as predicted by current models for this ciliopathy. These data for the first time suggest new mechanisms for activity of sunitinib and other drugs, in which they can influence activity of lateral signaling pathways such as Wnt and PDGFa by regulating the availability of receptors on a ciliary signaling platform.

#339

Identification of a novel isoform of WNT/planar cell polarity VANGL2 in breast cancer.

Jean-Paul Borg, Alexandra Walton, Eric Bailly, Sylvie Marchetto, Stéphane Audebert. _CRCM Inserm-Institut Paoli-Calmettes, Marseille, France_.

The WNT/PCP pathway is an evolutionarily conserved developmental process which is essential in embryogenesis and development of polarized structures in metazoans. Its importance in human diseases is best demonstrated in neural tube closure defects and cancer. WNT/PCP signaling involves a set of evolutionarily conserved WNT/PCP genes encoding WNT ligands, transmembrane (vangl2, frizzled, fat, dachsous) and cytoplasmic (scribble, prickle, dishevelled, diego) molecules triggering a genetically well-defined non-canonical WNT-JNK pathway. Recent work has linked defects of this pathway to breast cancer aggressiveness. We have shown that the non-canonical WNT/PCP transmembrane receptor VANGL2 is overexpressed in poor prognosis basal breast cancers and implicated in tumor growth. We also found that VANGL2 binds through its C-terminal sequence to Scribble and p62/SQSTM1 which are involved in cancer cell migration and growth. A recent in silico study has suggested the existence of an N-terminally extended VANGL2 isoform (VANGL2-Long), owing to the occurrence of an alternative non-AUG translation initiation site, upstream of the conventional start site. Translation initiation from this alternative site is expected to add an N-terminal extension of 48 amino acids. Accordingly, by analyzing VANGL2 immunoprecipitates by mass spectrometry, we identified a peptide mapping upstream of the first methionine supporting the existence of a longer isoform of VANGL2. While missing in Drosophila, this N-terminal extension is found in all vertebrate VANGL2 sequences that have been examined, suggesting a function for VANGL2-Long. We provide further experimental evidence in favor of the VANGL2-Long isoform, using polyclonal antibodies that have been raised against the predicted VANGL2 N-terminal extension. Using these tools, we characterized VANGL2-Long and showed that it dimerizes with VANGL2 and VANGL1, a close homologue, at the endogenous level in living cells. Our data depict a complex organization of VANGL1/VANGL2 molecules at the plasma membrane and improve the mechanistic understanding of VANGL2 in normal and cancer cells.

#340

SRMS regulates normal and oncogenic KIT signaling.

Sachin Raj M Nagaraj, Julhash U. Kazi, Lars Rönnstrand. _Lund University, Lund, Sweden_.

SRMS (Src-related tyrosine kinase lacking C-terminal regulatory tyrosine and N-terminal myristoylation sites) belongs to a family of non-receptor tyrosine kinases, which harbours a Src homology 3 and a Src homology 2, as well as a protein kinase domain. SRMS was first identified in a screen for the genes that regulate the growth and differentiation of neuroepithelial cells. SRMS, however, is an understudied member of this family. The present study was undertaken in order to explore the role of SRMS in signaling downstream of KIT. The receptor tyrosine kinase KIT, also known as the stem cell factor receptor, plays a key role in several developmental processes and have been implicated in many human cancers such as gastrointestinal stromal tumors, acute myeloid leukemia and testicular carcinoma. To understand the role of SRMS in KIT signaling, we generated Ba/F3 cell lines overexpressing KIT and SRMS. We observed that SRMS regulates normal and oncogenic KIT signaling differentially with respect to cell proliferation and apoptosis. SRMS association triggers KIT ubiquitination which in turn downregulates the receptor. Further, expression of SRMS downregulates wild-type KIT-mediated phosphorylation of AKT, ERK1/2, p38. Taken together the data demonstrates that SRMS regulates both normal KIT and an oncogenic mutant of KIT, and have differential impact on receptor downstream signaling.

#341

Investigating a novel GPCR ITIM motif in regulating Shp2 binding, signaling, and migration.

Ryan D. Paulukinas, Lili T. Belcastro, Catherine C. Moore. _Philadelphia College of Pharmacy, University of the Sciences, Philadelphia, PA_.

Aberrant expression of CXCR4, a chemokine G protein-coupled receptor (GPCR), promotes a migratory and invasive phenotype during breast cancer metastasis, however the mechanism is unknown. Here we investigate the role of CXCR4 tyrosine phosphorylation within a novel ITIM motif. Following activation, CXCR4 is normally phosphorylated on Ser/Thr residues, which promotes receptor desensitization and internalization. We found previously that Ser/Thr phosphorylation of CXCR4 is delayed upon SDF gradient sensing, and correlates with sustained signaling to SHP2, a protein that drives hyperproliferation and invasion of breast cancer. Since SHP2 is known to be recruited to tyrosine phosphorylated ITIM motifs (immmunoreceptor tyrosine-based inhibitory consensus motifs), we investigated if CXCR4 contains an ITIM motif, and if tyrosine phosphorylation within this motif regulates SHP2 binding, signaling, and migration. Specifically, we assessed 1) if CXCR4 is tyrosine phosphorylated as measured by recombinant phosphatase assay, phospho-Ab development, and tyrosine mutagenesis (YF); 2) if tyrosine mutagenesis alters CXCR4 membrane localization, internalization, and SDF gradient sensing, by ELISA; 3) if tyrosine mutagenesis disrupts CXCR4 interaction with SHP2, by co-IP; 4) if tyrosine mutagenesis alters CXCR4 sustained signaling to SHP2, by Western; and 5) if tyrosine mutation (YF) within the novel ITIM motif in CXCR4 alters migration of metastatic breast cancer cells, by transwell motility assays. Our results demonstrate that CXCR4 phosphorylation is sensitive to recombinant tyrosine phosphatase treatment and tyrosine mutagenesis, suggesting that CXCR4 is indeed tyrosine phosphorylated within the ITIM motif. We determined that while tyrosine phosphorylation within the ITIM motif is not required for CXCR4 membrane localization, internalization, or SDF gradient sensing, it does regulate CXCR4 binding and signaling to SHP2. Investigating the role in CXCR4 mediated migration is on going. To date, our data support a working model that tyrosine phosphorylation of CXCR4 within an ITIM motif is critical for binding to SHP2 and transducing sustained signaling to SHP2. These data have implications on aggressive breast cancers with dysregulated CXCR4 and SHP2. These studies were supported by NIH grant GM-097718, PA Department of Health grant SAP4100057688, and the Milton Lev Memorial Faculty Research Fund.

#342

WHSC1L1-mediated EGFR mono-methylation enhances the cytoplasmic and nuclear oncogenic activity of EGFR in head and neck cancer.

Vassiliki Saloura,1 Theodore Vougiouklakis,1 Makda Zewde,1 Xiaolan Deng,1 Kazuma Kiyotani,1 Jae-Hyun Park,1 Yo Matsuo,2 Mark Lingen,1 Naoshi Dohmae,3 Takehiro Suzuki,3 Ryuji Hamamoto,1 Yusuke Nakamura1. 1 _Univ. of Chicago, Chicago, IL;_ 2 _Oncotherapy Science, Japan;_ 3 _RIKEN, Japan_.

While multiple post-translational modifications have been reported to regulate the function of epidermal growth factor receptor (EGFR), the effect of protein methylation on its function has not been well characterized. In this study, we show that WHSC1L1 mono-methylates lysine 721 in the tyrosine kinase domain of EGFR, and that this methylation leads to enhanced activation of its downstream ERK cascade without EGF stimulation. We also show that EGFR K721 mono-methylation not only affects the function of cytoplasmic EGFR, but also that of nuclear EGFR. WHSC1L1-mediated methylation of EGFR in the nucleus enhanced its interaction with PCNA in squamous cell carcinoma of the head and neck (SCCHN) cells and resulted in enhanced DNA synthesis and cell cycle progression. Overall, our study demonstrates the multifaceted oncogenic function of the protein lysine methyltransferase WHSC1L1 in SCCHN, which is mediated through direct non-histone methylation of the EGFR protein with effects both in its cytoplasmic and nuclear functions.

#343

Proteomic and functional studies identify DCAF7 as major partner of DYRK1A.

Varsha Anathapadmanabhan,1 Selene Swanson,2 Siddharth Saini,1 Vijay Menon,1 Larisa Litovchick1. 1 _Virginia Commonwealth University, Richmond, VA;_ 2 _Stowers Institute for Biomedical Research, Kansas City, MO_.

Human DYRK1A protein kinase is encoded by dosage-dependent gene since an extra copy contributes to Down syndrome (DS) pathogenesis while loss of one allele causes severe developmental defects. Current knowledge of DYRK1A's role in phosphorylation of proteins involved in cell cycle control, transcription and tumor suppression does not fully explain dosage-dependent function of this important kinase. Using MudPIT proteomic analysis, we identified DYRK1A interacting proteins in human T98G cell line. Four independent DYRK1A-HA pull-down samples were analyzed to identify 50 proteins that were specifically detected in 3 out of 4 replicates. This analysis identified WD-repeat protein DCAF7 as major partner of DYRK1A that was most highly enriched among DYRK1A-binding proteins. Furthermore, glycerol gradient ultracentrifugation showed almost exact co-fractionation of DCAF7 and DYRK1A. DCAF7 has been shown to bind several protein kinases including DYRK1A, DYRK1B, HIPK2 and MAP3K1 suggesting that it serves as a scaffold protein in signal transduction. However, the function of DCAF7 is not established yet. In order to understand the functional relationship between DCAF7 and DYRK1A, we undertook MudPIT proteomic analysis of DCAF7 binding proteins in T98G cells and compared the data with the DYRK1A interactome dataset. Analysis of three independent DCAF7-HA pull-down samples identified 32 proteins specifically detected in all three replicates including DCAF7, DYRK1A and five proteins earlier detected in DYRK1A MudPIT analysis. Given the proposed role of DCAF7 as a scaffold protein, we tested whether DCAF7 mediates its interaction with some of its interacting proteins. Interestingly, we found that RNF169, a recently characterized RING-domain ubiquitin ligase involved in the DNA double-strand break (DSB) repair, binds to N-terminus of DYRK1A independently of DCAF7. Furthermore, our data show that DYRK1A is required for DCAF7-RNF169 interaction because this interaction was abolished in human U-2 OS cells where DYRK1A gene was disrupted using CRISPR-Cas9 editing (DYRK1A-KO cells). Interestingly, stable overexpression of DCAF7 in DYRK1A-KO U-2 OS cells rescued some of the phenotypes observed in these cells, suggesting that DCAF7 could be an effector downstream of DYRK1A. Our findings revealed an unexpected scaffolding role of DYRK1A that is required for mediating the interaction between DNA repair protein RNF169 and the WD-repeat protein DCAF7. While further studies are needed to understand the exact role of the RNF169-DCAF7 interaction in DNA repair, the novel role of DYRK1A as a scaffold protein could explain some aspects of the dosage-dependent function of this protein kinase. In addition, we report here an initial functional characterization of the DCAF7-DYRK1A interaction in human cells and discuss novel functional interactions of DCAF7 that could be independent of DYRK1A.

#344

Chondroitin sulfatases regulate Wnt signaling through effects on Shp2, phospho-Erk1,2, c-Myc, and histone methylation of DKK3 in prostate cancer and prostate stem cells.

Joanne Kramer Tobacman, Sumit Bhattacharyya, Leo Feferman. _Univ. of Illinois at Chicago, Chicago, IL_.

The chondroitin sulfatases N-acetylgalactosamine 4-sulfatase (arylsulfatase B; ARSB) and N-acetylgalactosamine 6-sulfatase (galactose 6-sulfatase, GALNS) remove the 4-sulfate and 6-sulfate groups of chondroitin sulfates. ARSB acts to remove 4-sulfate groups of chondroitin 4-sulfate (C4S) or dermatan sulfate, and GALNS acts to remove 6-sulfate groups of chondroitin 6-sulfate (C6S) or chondroitin 4,6-disulfate, as well as keratan sulfate. In human prostate cancer tissues, ARSB activity was markedly reduced and GALNS activity increased. By immunohistochemistry of malignant prostate tissue captured by laser microdissection and by assay of human prostate stromal and epithelial cell lines, ARSB was predominant in stromal cells of the extracellular matrix, whereas GALNS predominated in prostate epithelial cells. When ARSB was reduced by silencing in human prostate stem cells or GALNS was increased by overexpression in the stem cells, SHP2 binding to chondroitin 4-sulfate increased, leading to inhibition of phosphatase action and sustained phosphorylation of Erk1,2. Erk activation enabled increase in c-Myc binding to nuclear DNA and increased expression of DNMT 1a, 3a, and 3b. DNMT activation increased following ARSB silencing or GALNS overexpression, and was blocked by ERK inhibition. Increased DNMT activity led to the increased promoter methylation of DKK3, and the subsequent inhibition of nuclear β-catenin nuclear translocation and Wnt signaling. Decline in Wnt signaling was manifested by reduced TCF-LEF nuclear binding, and decline in the mRNA expression of c-Myc and GATA. Since c-Myc activation can lead to increased expression of cell cycle regulators, the changes in chondroitin sulfation mediated by sulfatases can lead to profound effects in cell proliferation. Hence, the modification of sulfatase activity, manifested as increased chondroitin 4-sulfate, provides the platform for an extensive extracellular-intracellular signaling network that can regulate phosphorylation, DNA promoter methylation, and proliferation, and affect development and malignant transformation. 

### Cell Growth Signaling Pathways 2

#345

PTEN tumor suppressive effects in glioblastoma are partly mediated by MET downregulation via a p53-miR-34a axis.

Fadilla Guessous,1 Nichola A. Cruickshanks,1 Ying Zhang,1 David Schiff,1 Jann Sarkaria,2 Roger Abounader1. 1 _University of Virginia, Charlottesville, VA;_ 2 _Mayo Clinic, Charlottesville, VA_.

Glioblastoma (GBM) is the most common and most lethal primary malignant brain tumor. Two common alterations in GBM are loss of the tumor suppressor PTEN and upregulation of the oncogenic receptor tyrosine kinase MET. We uncovered a new connection between PTEN and MET expressions and functions in GBM. We found that PTEN expression in GBM cell lines (U87, U373, A172, LNZ-308) and primary cells (GBM8) inhibits MET expression. We uncovered the molecular basis and functional significance of this connection. Based on our previous work and published literature, PTEN stabilizes p53, p53 induces miR-34a expression, and miR-34a inhibits MET expression. We therefore hypothesized that PTEN inhibits MET by regulating p53 and miR-34a and that the PTEN tumor suppressive effects are partly mediated by MET downregulation. To verify the hypothesis, we first showed that PTEN restoration leads to miR-34a upregulation in GBM cells. We then showed that this latter effect is p53-dependent as p53 knockdown with a specific shRNA partly reversed PTEN induction of miR-34a. We also showed that PTEN-induced downregulation of MET expression can be rescued by inhibition of miR-34a with an anti-miR3a. We therefore established a new molecular cascade that links PTEN, p53, miR-34a and MET in GBM. To determine the functional relevance of this cascade, we assessed the effects of PTEN on GBM cell death and invasion also in the settings of shRNA-mediated knockdown of p53 and anti-miR-mediated inhibition of miR-34a. PTEN strongly induced GBM cell death (measured by Annexin V/7AAD flow cytometry) and inhibited transwell invasion though a collagen IV matrix. The PTEN effects on cell death and invasion were partly reduced by p53 knock-down and miR-34a inhibition. This demonstrates that the tumor suppressive effects of PTEN in GBM are partly mediated by the PTEN/p53/miR-34a/MET cascade. In ongoing experiments, we are investigating the expressions of PTEN, MET, p53 and miR-34a in human GBM tissues to determine if there are potential correlations that are consistent with our above findings. Altogether, this study establishes a new molecular and functional link between PTEN and MET that involves p53 and miR-34a in GBM.

#346

Fatty acid synthase is a risk factor for bleomycin-induced lung fibrosis in mice.

Mi-Yeon Jung. _Mayo Clinic, Rochester, MN_.

Pulmonary fibrosis is a significant complication in cancer patients treated with radiation or various chemotherapeutic agents. One such reagent, bleomycin, is used to treat a variety of cancers including, but not limited to, lymphoma, ovarian cancer, and melanoma. Its efficacy, however, is impacted by the incidence of significant pulmonary fibrosis, which often becomes dose limiting. In studies investigating the role(s) of metabolism in the profibrotic actions of transforming growth factor beta, we previously determined that increased fatty acid synthase (FASN) expression was observed in the fibrotic areas of lungs from idiopathic pulmonary fibrosis (IPF) patients compared to healthy controls. In that FASN levels are significantly increased in numerous human tumors including breast, prostate, colorectal, and ovarian, we wished to integrate these findings by examining whether inhibiting FASN activity could reduce pulmonary fibrosis. To that end, the cerulenin-derived FASN inhibitor C75 was tested in a murine treatment model of bleomycin-induced lung fibrosis. Following addition of C75, there was significant diminution in a number of profibrotic targets including collagen 1α1, fibronectin, connective tissue growth factor, and total lung collagen as assessed by hydroxyproline content. Not only was there a C75 dose-dependent decrease in the aforementioned targets, but lung function as assessed by peripheral blood oxygenation (on room air) was also stabilized/improved. Thus, FASN inhibition might reflect a possible target to maintain normal lung physiology in the context of cancer chemotherapy.

This work was supported by Public Health Service grants GM-055816 and GM-054200 from the National Institutes of General Medical Sciences.

#347

The role of Pin1 in chemosensitivity of BRCA1-deficient breast cancers.

Catherine Knowlson, Paul Mullan, Niamh Buckley. _Queen's University Belfast, Belfast, United Kingdom_.

BRCA1-deficient breast cancers are largely triple negative (ERα, PR and HER2 negative) and express markers representing the basal-like subgroup. Triple receptor negativity means that there are no targeted therapies available for this subtype, which consequently has the poorest overall survival rates. Given that BRCA1 dysfunction or 'BRCAness' is often observed in triple negative breast cancers (TNBCs), it is likely that loss of BRCA1 also plays a key role in their pathogenesis. Therefore to improve TNBC treatment responses, investigation into the underlying biology is required. It has been reported that levels of the prolyl isomerase Pin1 are upregulated in the absence of functional BRCA1, and that levels correlate with tumor grade. Pin1 alters the conformation of target proteins to affect their regulation and function. Many of these target proteins are implicated in key oncogenic signalling pathways, including BRCA1-associated pathways, such as cell cycle and growth regulation, DNA damage response, transcriptional regulation and NFκB signalling. We have observed that Pin1 levels are indeed upregulated in BRCA1-deficent cell lines compared to matched BRCA1 reconstituted cells, and that Pin1 plays a role in their growth rate and responsiveness to chemotherapy. BRCA1-deficient cells are sensitive to DNA damaging agents due to loss of DNA repair functions, notably homologous recombination (HR) repair of double strand breaks. We have identified a correlation between Pin1 levels and sensitivity to PARP inhibitors, which are toxic to HR deficient cells due to synthetic lethality. Ongoing work will establish whether Pin1 levels may be utilized as a biomarker of BRCAness and PARP inhibitor sensitivity. In contrast, during treatment of breast cancer cells with microtubule damaging agents, the presence of BRCA1 is required to promote apoptosis through MEKK3 and JNK, therefore BRCA1 deficient cells are less sensitive. Knockdown of Pin1 in BRCA1-deficient cells sensitizes them to the taxane Paclitaxel, due to repression of the anti-apoptotic protein Mcl-1, while Pin1 knockdown in BRCA1 proficient cells has no effect on sensitivity as Mcl-1 levels are repressed through the MEKK3 pathway. We have also identified that several Src family kinases (SFKs) are upregulated by Pin1 in a BRCA1-deficient background, and may play a role in taxane sensitivity. Pre-treatment with the Src/SFK inhibitor Dasatinib increases cytotoxicity of Paclitaxel, suggesting that combination treatment of Dasatinib and Paclitaxel may be an effective treatment option for BRCA1-deficient breast cancers. In conclusion, we have found that Pin1 may be an important oncogenic mediator downstream of dysfunctional BRCA1 facilitating the viability of TNBCs. We propose that Pin1 could serve as a predictive marker of PARP and Src inhibitor treatments, allowing clinicians to make better informed treatment decisions for this heterogeneous and aggressive subtype of breast cancer.

#348

Comparison of the response of the NCI60 NSCLC panel with the response of patient-derived NSCLC lines to approved and investigational agents.

Beverly A. Teicher,1 David Evans,2 Thomas Silvers,2 Michael Selby,2 Rene Delosh,2 Julie Laudeman,2 Chad Ogle,2 Russell Reinhart,2 Joel Morris,3 Gurmeet Kaur,1 James Doroshow1. 1 _National Cancer Inst., Bethesda, MD;_ 2 _Leidos Biomedical Research, Inc., Frederick, MD;_ 3 _National Cancer Inst., Rockville, MD_.

An 800 compound screen with the NCI60 cell lines and 5 patient-derived NSCLC lines (PD NSCLC), was conducted at 9 concentrations and included the FDA approved oncology agents and an investigational agents library. The screen was conducted using 384-well monolayer cultures, an exposure time of 72 hrs and compound concentrations from 1 nM to 10 uM. CellTiter-Glo was used to measure viability as an endpoint. The NCI60 NSCLC panel consists of 9 cell lines: A549, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H322M, NCI-H460 and NCI-H522. The response of these lines was compared with the response of 5 PD NSCLC lines and 60 SCLC lines. While both sets of NSCLC lines had similar responses to the majority of compounds, there were some marked differences. Differences in response of the NCI60 NSCLC lines and the PD NSCLC lines were notable upon exposure to tubulin fragmenters, KSP/EG5 inhibitors, GAR transformylase inhibitors, Polo-like kinase-1 inhibitors (PLK1), MEK inhibitors, and IAP inhibitors. The NCI60 NSCLC lines were more sensitive to the tubulin fragmenters than were the PD NSCLC and the SCLC lines. Verubulin had a mean GI50 in NCI60 NSCLC of 0.0037 uM and 0.0042 uM in SCLC versus 0.047uM in the PD NSCLC lines. The NCI60 NSCLC lines were more sensitive to the KSP inhibitor, ARRY-520 (mean GI50 0.0049 uM) than the SCLC lines (0.028 uM) and the PD NSCLC lines (0.26 uM). The NCI60 NSCLC lines (mean GI50 0.019 uM) and the SCLC lines (mean GI50 0.04 uM) were more sensitive to PLK1 inhibitors such as BI-2536 than the PD NSCLC lines (0.13 uM). However, the NCI60 NSCLC lines (mean GI50 0.8 uM) and the SCLC lines (mean GI50 0.134 uM) were less sensitive than the PD NSCLC lines (mean GI50 0.025uM) to GAR transformylase inhibitors such as pelitrexol. The NCI60 NSCLC lines (mean GI50 6.3 uM) and SCLC lines (mean GI50 10 uM) were also less responsive to IAP inhibitors, than were PD NSCLC lines (mean GI50 1.6 uM). A heterogeneous response to the MEK inhibitors such as cobimetinib (GDC-0973) was observed with the SCLC lines (mean GI50 8.9 uM) versus 1.15 uM in the NCI60 NSCLC lines and 0.56 uM in the PD NSCLC lines. PD NSCLC lines exhibit some interesting differences in response from established lung cancer lines upon in vitro exposure to anticancer agents and thus add to our knowledge and understanding of NSCLC and help inform discovery efforts and/or clinical development of therapeutics in this disease. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261200800001E.

#349

Eribulin induces cortical localization of E-cadherin, p120-catenin and beta-catenin in breast cancer cells.

Alison D. Clark, Nicholas F. Dybdal-Hargreaves, Susan L. Mooberry. _UTHSCSA, San Antonio, TX_.

Eribulin provides a survival advantage for breast cancer patients, particularly those with triple-negative and HER2-negative subtypes.1,2 Mechanistically, eribulin was shown to reverse epithelial-to-mesenchymal transition (EMT) in breast cancer models.3 E-cadherin is a central regulator responsible for maintenance of the epithelial phenotype and loss of cortical E-cadherin and its downstream signaling is an early and significant event in EMT. Short term treatment of HCC1937 breast cancer cells with eribulin initiated cortical E-cadherin localization in manner different from other microtubule targeting agents. Cortical E-cadherin plays essential roles in cell adhesion and in regulating the Wnt pathway, whose aberrant activity is implicated in EMT. At the adherens junctions p120-catenin and β-catenin bind to E-cadherin, an interaction facilitated by microtubules. Cortical sequestration of p120- and β-catenin at the adherens junctions stabilizes these complexes and inhibits their ability to stimulate signaling pathways that contribute to EMT. We tested whether eribulin, which binds to the plus ends of microtubules, promotes EMT reversal by influencing p120- and β-catenin localization and activity. Our results show that eribulin induces cortical localization of p120-catenin and β-catenin with E-cadherin in HCC1937 cells, consistent with the formation of normal adherens junctions. Eribulin also stimulated the phosphorylation of Ser33/37 and Thr41 on β-catenin which promotes proteasomal degradation, which would be expected to further inhibit oncogenic signaling. Collectively, our results suggest that eribulin contributes to reversal of EMT through modulation of downstream signaling initiated by cortical localization of p120- and β-catenin at the adherens junctions. Funding for this work was provided by Eisai Inc.

1. Cortes J., et al., Lancet 377:914-923, 2011

2. Twelves, C., et al., Breast Cancer Res Treat 148:553-561, 2014

3. Yoshida, T., et al., Br J of Cancer 110: 1497-1505, 2014

#350

Pregnancy at early age is associated with a reduction of progesterone-responsive cells and epithelial Wnt signaling in human breast tissue.

Robert Mechera,1 Simone Münst,1 Silvio Däster,1 Salvatore Piscuoglio,1 Charlotte Ng,1 Fabienne Meier-Abt,2 Walter P. Weber,1 Savas D. Soysal1. 1 _University Hospital Basel, Basel, Switzerland;_ 2 _University Hospital Zuerich, Zuerich, Switzerland_.

Background: Pregnancy at early age is the most significant modifiable factor which consistently decreases lifetime breast cancer risk. However, the underlying mechanisms haven't been conclusively identified. Studies in mice suggest a reduction in progesterone-receptor (PR) sensitive epithelial cells as well as a downregulation of the Wnt signaling pathway as being one of the main mechanisms for the protective effect of early pregnancy. The aim of our study was to validate these findings in humans.

Methods: We collected benign breast tissue of 123 women who had been stratified according to age at first pregnancy and the occurrence of subsequent breast cancer, and performed immunohistochemistry for PR, Wnt4 and the Wnt-target Versican.

Results: The number of PR positive epithelial cells was significantly lower in the group of women with early pregnancy and no subsequent breast cancer compared to the group of nulliparous women with subsequent invasive breast cancer (p=0.017). In women with early pregnancy, expression of Versican and Wnt4 was significantly lower compared to nulliparous women (p=0.0064 and p=0.0156 respectively), and Versican expression was also significant lower compared to women with late pregnancy (p<0.0001).

Discussion: Our results confirm prior observations in mice and suggest a role of downregulation of epithelial Wnt signaling in the protective effect of early pregnancy in humans. This results in a decreased proliferation of stem/progenitor cells; therefore, the Wnt signaling pathway may represent a potential target for breast cancer prevention in humans.

#351

mTOR-ERK co-targeting strategies for head and neck cancer therapy.

Zhiyong Wang,1 Esteban Delgado,2 Kosuke Yamaguchi,1 Ramiro Iglesias-Bartolomé,3 Panomwat Amornphimoltham,1 Alfredo Molinolo,1 Qianming Chen,4 J.Silvio Gutkind1. 1 _Moores Cancer Center, University of California, San Diego, La Jolla, CA;_ 2 _San Diego State University, San Diego, CA;_ 3 _National Cancer Institute, NIH, Bethesda, MD;_ 4 _West China School of Stomatology, Sichuan University, Chengdu, China_.

Despite therapeutic advances in the past decade, head and neck squamous cell carcinoma (HNSCC) continues to be a major public health concern. Although recent studies of human cancer genomes identified remarkable complexity of genomic alterations in HNSCC, surprisingly, most aberrations have fallen within key driver signaling pathways. Among them, PI3K/AKT/mTOR pathway is most frequently activated in over 80% of the HNSCC patients. We showed that the majority of HNSCC lesions exhibit high levels of phosphorylated ribosomal S6 protein (pS6), key downstream target of the AKT-mTOR pathway, and that inhibition of mTOR by the use of rapamycin causes a rapid decrease in the level of pS6 and the apoptotic death of HNSCC tumor xenografts, thereby causing tumor regression. Our initial studies identified the AKT-mTOR pathway as a potential therapeutic target for HNSCC and provided strong rational to initiate multiple clinical trials. Indeed, inhibitors of PI3K/AKT/mTOR block mTOR activity and exert a beneficial response in HNSCC patients. However, therapeutic resistance was evidenced in several trials, which might be due to the activation of adaptive survival signaling in tumor cells. To explore the therapeutic option that can overcome the resistance, we performed a synthetic lethality screen in HNSCC cell lines using shRNA libraries. We found that multiple molecules involved in ERK pathway were highly represented. Furthermore, treating HNSCC cell lines harboring activated mTOR with small molecule inhibitors that target PI3K/AKT/mTOR pathway can sequentially induce ERK activation, which is consistent with the findings observed in numerous clinical trials. We demonstrated that co-targeting mTOR and ERK using trametinib, a MEK1/2 inhibitor or cetuximab, an EGFR monoclonal antibody, exhibited the synergistic effect by sensitizing HNSCC to priming with the mTOR inhibitor. Specifically, mTOR-ERK co-targeting prevented the growth of HNSCC tumor xenografts by decreasing cell proliferation, reducing lymphangiogenesis and activating apoptosis. In addition, we have recently used a phosphoproteomics approach to identify the molecular mechanisms of ERK feedback activation caused by PI3K/AKT/mTOR inhibition. Overall, our promising findings from these preclinical studies suggest that the use of mTOR-ERK co-targeting strategies may provide the novel alternative approach to achieve durable responses (cure) in HNSCC cancer patients.

#352

The role of p62 (SQTSM1) in the transforming growth factor β signaling pathway.

Evelyn Ng, Adrian Gunaratne, John Di Guglielmo. _Western University, London, Ontario, Canada_.

Transforming growth factor beta (TGFβ) is a cytokine that regulates many cellular processes, including cellular adhesion, proliferation and apoptosis. Its canonical downstream effectors include Smad2/3 proteins, which are phosphorylated and then translocate to the nucleus to alter gene transcriptional programs and promote processes such as epithelial-to-mesenchymal transition (EMT). Previous studies in our lab have shown that atypical Protein Kinase C (aPKC) isoforms associate with TGFβ receptors and modulate receptor trafficking and signal transduction. An aPKC-associated protein, p62 (SQSTM1) has been implicated in TGFβ-dependent EMT, however the mechanisms remain unclear.Here, we investigate the localization of p62 and its potential roles in modulating TGFβ signaling via knockdown and overexpression studies. Using antibody feeding and immunofluorescence microscopy, we support previous findings showing that p62 localizes to late endosomes. In addition, using a co-immunoprecipitation approach, we observed that p62 may associate with TGFβ type II receptor. To explore the functional role of p62 in TGFβ signaling, we conducted protein silencing using siRNA. We observed TGFβ-independent decreases in E-cadherin expression. However these changes were independent of the phosphorylation status or nuclear translocation of Smad2.P62 also plays an important role in autophagy by targeting proteins for degradation. Recently, prolonged TGFβ stimulation has been shown to induce cellular autophagy. Therefore, it is in our interest to characterize the relationship between TGFβ-dependent EMT and autophagy, and to determine whether p62 has a regulatory role between these two processes.

#353

ROR1 inhibits ASK1-mediated pro-apoptotic signaling in lung adenocarcinoma.

Lisa Ida, Tomoya Yamaguchi, Taisuke Kajino, Kiyoshi Yanagisawa, Yukako Shimada, Motoshi Suzuki, Takashi Takahashi. _Division of Molecular Carcinogenesis, Center for Neurological Diseases and Cancer; Nagoya University, Nagoya, Japan_.

We previously reported that the receptor tyrosine kinase-like orphan receptor 1 (ROR1) is transcriptionally activated by TTF-1/NKX2-1 lineage-survival oncogene in lung adenocarcinoma and maintains a favorable balance between pro-survival PI3K-AKT and pro-apoptotic ASK1-p38 signaling. Although in-depth mechanistic insight into how ROR1 sustains EGFR-mediated PI3K-AKT signaling in both kinase-dependent and -independent manners has been obtained, it remains elusive how ROR1 inhibits pro-apoptotic signaling. In the present study, we investigated the underlying mechanism of ROR1-mediated inhibition of the ASK1-p38MAPK signaling pathway. Co-treatment with siASK1 resulted in partial but significant alleviation of siROR1-mediated growth inhibition in the lung adenocarcinoma cell lines PC-9 and NCI-H1975, which readily express both ROR1 and ASK1. Significant decreases in auto-phosphorylation of ASK1 at threonine 845 and p38 phosphorylation at threonine 180 and tyrosine 182 were observed in ROR1-transfected MSTO-211H cells in both steady state and oxidative stress-elicited conditions, suggesting ROR1-mediated negative regulation of the ASK1-p38 axis. ROR1 was shown to interact physically with ASK1 through the C-terminal serine/threonine-rich domain of ROR1. An in vitro kinase assay using cellular ASK1 prepared from ASK1-transfected 293T cells as well as recombinant MBP-tagged kinase-deficient MKK6 revealed that co-incubation of ASK1 with recombinant GST-tagged ROR1 clearly diminished ASK1 auto-phosphorylation and MKK6 phosphorylation. We further examined whether the kinase activity of ROR1 is required for inhibition of ASK1 activity using MSTO-211H cells stably transfected with either a wild-type or a kinase-dead mutant of ROR1. In contrast to wild-type ROR1, kinase-dead ROR1 failed to significantly repress hydrogen peroxide-induced ASK1 phosphorylation as well as consequential p38 phosphorylation and increase in sub-G1 cells. Since we previously found that ROR1 activates SRC, we also examined whether SRC is involved in the ROR1-sustained ASK1 inhibition. Introduction of constitutively active SRC significantly reduced siROR1-induced ASK1activity in PC-9 cells, while co-transfection of SRC and ASK1 resulted in tyrosine phosphorylation of ASK1, which was accompanied with diminished ASK1 phosphorylation at threonine 845. Finally, the interaction between SRC and ASK1 was found to be enhanced by the presence of ROR1 by an in vitro pull-down assay. Taken together, the present findings support our notion that ROR1 sustains lung adenocarcinoma survival, at least in part, through direct physical interaction with both ASK1 and SRC, which consequently results in repression of the pro-apoptotic ASK1-p38 axis in a ROR1 kinase activity-dependent manner.

#354

Inhibition of Smad3 signaling with a TAT fusion peptide prevented TGF-β-induced fibronectin expression and cell migration in malignant gliomas.

Jeonghan Kang. _Mayo Clinic, Rochester, MN_.

Malignant primary brain tumors represent one of the most common causes of cancer death in the world. Grade IV gliomas (glioblastoma multiform, GBM) are routinely resistant to current chemoradiationchemo radiation therapy with a median progression-free and overall survival for glioblastoma following current chemoradiation of ~7 and 15 months, respectively. While TGF-β functions as a growth inhibitor for most normal cell types, many tumors (including GBMs) have lost this inhibitory response and TGF-β now functions to promote tumor progression and/or dissemination. As aberrant TGF-β signaling is often considered a hallmark of high-grade gliomas, we examined whether inhibition of specific components would impact the malignant phenotype of glioblastoma. The primary mediators of TGF-β action are the Smad proteins (R-Smads), Smad2 and Smad3. Following receptor activation and R-Smad phosphorylation, they translocate to the nucleus to modulate gene expression. Since we previously determined that a cell penetrating peptide conjugate of the HIV TAT protein fused to a 30 amino acid sequence from sorting nexin 9 (SNX9) prevented Smad3, but not Smad2, nuclear import and target gene expression in models of organ fibrosis, we further investigated whether it might similarly impact aspects of GBM proliferation. To that end, twenty-one patient-derived xenografts (PDXs) were examined for their responsiveness to TGF-β. Of those, TGF-β stimulated migration and fibronectin expression in seven (33%), which was prevented by inhibiting Smad3 signaling with the TAT-SNX9 peptide but not a control peptide unable to block Smad3 action. These findings indicate that the TGB-b/Smad3 axis is a potentially important mediator of glioma cell invasion in a significant minority of GBM models. specifically targeting Smad3 might be effective in a subset of gliomas.

This work was supported by Public Health Service grants GM-055816 and GM-054200 from the National Institutes of General Medical Sciences and P50 CA-108961 Developmental Pilot Award from the National Cancer Institute.

#355

TET2-loss modifies androgen signaling in prostate cancer.

Michael L. Nickerson,1 Sudipto Das,2 Kate Im,1 Sevilay Turan,1 Sonja Berndt,1 Hongchuan Li,1 Hong Lou,1 Seth Brodie,1 Kalpit Shah,1 Jean-Noel Billaud,3 Tongwu Zhang,1 Joseph Boland,1 Stephen Anderson,1 Meredith Yeager,1 Michael Dean,1 Thorkell Andresson2. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Leidos Biomedical, MD;_ 3 _Ingenuity, CA_.

Molecular markers for metastatic prostate cancer (PCa) can be identified by sequencing metastatic tumor genomes. We recently identified cancer gene mutations in a patient with metastatic disease using next generation sequencing (NGS) of DNA from 5 metastatic tumors and blood. We characterized a somatic non-conservative substitution in the methylcytosine dioxygenase ten-eleven translocation 2 (TET2) in all metastatic tumors but not the blood or primary adenocarcinoma. Analysis of metastatic tumors from additional patients revealed frequent somatic loss and sequence alteration of TET2, which was previously observed to be altered in 5-15% of myeloid, kidney and colon cancer. Genome-wide association studies previously identified non-coding risk variants associated with PCa and melanoma, and rare germline missense variants are observed in African-Americans with PCa. We sought to further investigate the role of TET2 in PCa. We performed fine-mapping of PCa risk across the TET2 locus using genotypes from the PEGASUS case-control cohort and identify six new risk variants in introns 1 and 2. Electrophoretic mobility shift assays show two risk SNPs are bound by the transcription factor octamer-binding protein 1 (Oct1). Full length TET2 (2002 aa) is expressed in normal prostate and cancer tissue and is significantly reduced in a subset of the Cancer Genome Atlas (TCGA) PCa tumors that are associated with metastatic disease and reduced disease-free survival. TET2-loss drives a cancer phenotype as siRNA-mediated knockdown (KD) significantly increases LNCaP and DU145 cell proliferation, and LNCaP transwell migration and wound healing. Affinity chromatography, mass spectrometry and co-immunoprecipitation confirm that endogenous TET2 binds the androgen receptor (AR) in LNCaP cell extracts. TET2 KD alters the expression of a subset of androgen-responsive genes including increased prostate-specific antigen (KLK3/PSA) expression, and published data indicate TET-catalyzed hydroxymethylcytosine (hmC) and TET2 binding sites proximal to KLK3. Analysis of TCGA PCa tumor RNA-seq reveals TET2 expression is co-regulated with the expression of genes encoding functions metabolizing 2-oxoglutarate and succinate including the lysine demethylase KDM6A, BRCA1-associated BAP1, and citric acid cycle enzymes IDH1-2, SDHA-B and FH. Co-expression is conserved across all 31 TCGA cancers examined. Examination of genomic locations associated with TET2-binding and hmC, and gene expression changes during androgen signaling indicate a putative role for TET2 as an energy sensor that modifies androgen-AR signaling. Decreased TET2 mRNA expression in TCGA PCa tumors that are associated with reduced patient survival indicates TET2 expression may be an informative biomarker of PCa disease progression.

#356

DARPP-32, a bridge between pro-inflammatory NF-kB and STAT3 signaling in gastric cancer.

Shoumin Zhu, Mohammed Soutto, Zheng Chen, Wael El-Rifai. _Surgery, Nashville, TN_.

Background: Gastric cancer is one of the most frequently diagnosed malignancies with poor prognosis in the world. We have previously shown that Dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32) is a novel cancer gene, which is overexpressed in 2/3 of gastric cancers patients; and associated with increased cancer cell survival, drug resistance, and invasion. However, the mechanisms of how DARPP-32 promotes gastric carcinogenesis remain unclear.

Methods: DARPP-32 promoter activity and stat3 activity were measured by luciferase. The mRNA and protein levels were evaluated by quantitative real-time PCR and Western blot analysis. The association between DARPP-32 and STAT3 was evaluated by immunofluorescence. SOCS3 degradation was analyzed by Ubiquitination Assay. The interaction between DARPP-32 and IGF1R was evaluated by co-immunoprecipitation assays.

Results: Promoter analysis of DARPP-32 indicated the presence of NF-κB transcription factor putative binding sites in the DARPP-32 promoter region. We cloned the DARPP-32 promoter into the luciferase reporter (pGL3-Luc). TNF-α treatment induced DARPP-32 reporter activity (P < 0.01). Using deletion constructs of DARPP-32 promoter and ChIP assay, we demonstrated that the sequence (-996 to -1008 bp), containing putative NF-κB binding sites, is the most active region. We confirmed that activation of NF-kB by TNF-α and H.pylori infection leads to induction of DARPP-32 mRNA and protein level. Interestingly, we found that DARPP-32 induction leads to increased phosphorylation of STAT3 and activation of STAT3 luciferase reporter (P<0.01). We detected a decrease in the protein level of SOC3, a negative regulator of STAT3. DARPP-32 shortened the SOCS3 protein half-life by mediating an increase in phosphorylation and ubiquitination of the SOCS3 protein following treatment with MG132 and IL6. Furthermore, we detected an increase in SRC and IGF1R phosphorylation in DARPP-32-overexpressing cells. SRC inhibitor (Dasatinib) and SRC-siRNA blocked DARPP-32-induced activation of STAT3. We further examined our hypothesis that DARPP-32 could interact with IGF1R and enhance IGF1R autophosphorylation. Using co-immunoprecipitation, we found that DARPP-32 and IGF1R co-exist in the same protein complex.

Conclusion: We demonstrated, for the first time, the role of NF-κB in DARPP-32 transcriptional regulation. The in vitro studies indicate that DARPP-32 plays a role in activation of STAT3 signaling through inhibition of SOCS3 and activation of IGF1R and SRC. DARPP-32 interacted with IGF1R, which increasing the phosphorylation of IGF1R and SRC, and induced SOCS3 phosphorylation and degradation. This cascade of events leads to STAT3 activation in gastric cancer cells. We propose DARPP-32 as an important signaling bridge between NF-kB and STAT3 in response to pro-inflammatory signaling in gastric carcinogenesis.

#357

NKX2-1 regulates SMAD and non-SMAD pathways in thyroid stem cells.

Manabu Iwadate, Yoshinori Takizawa, Shioko Kimura. _National Cancer Institute, MD_.

NKX2-1 plays a critical role in thyroid differentiation. Recently, we established Side Population cells-derived Thyroid cell line (SPTL) from mouse thyroid Side Population cells. In SPTL cells, NKX2-1, PAX8 and E-cadherin were not expressed when examined by western blotting. Immunocytochemistry revealed that only 1% of SPTL cells were NKX2-1 positive. When GFP-SPTL cells were directly injected into mouse thyroid, a few GFP-SPTL cells with weak expression of NKX2-1 were found in part of thyroid follicle, suggesting that SPTL cells have potential of thyroid stem cells. Comprehensive gene expression analysis indicated that TGF-beta signaling pathway is critical for SPTL cells. TGF-beta signaling and NKX2-1 appear to play an important role in thyroid stem cells and thyroid differentiation. Here we show that NKX2-1 regulates both SMAD and non-SMAD pathways in SPTL cells. To examine the role of NKX2-1 in SPTL cells, we produced SPTL cells having doxycycline-inducible Nkx2-1 expression (named SPTL(Nkx2-1on)). In SPTL(Nkx2-1on) cells, doxycycline induced NKX2-1 as expected, while PAX8 and E-cadherin expression was not. Upon overexpression of NKX2-1 in SPTL(Nkx2-1on) cells, TGF-beta-induced phospho-SMAD3 expression was inhibited. Additionally, NKX2-1 enhanced the expression of phospho-AKT. In GFP-myrAKT(constitutively active AKT)-overexpressing SPTL cells, AKT was found localized in their cytoplasm. TGF-beta did not induce phospho-SMAD3 in these cells. The data indicate that NKX2-1 inhibits SMAD pathway through AKT activation. Ourwork demonstrates that TGF-beta signaling is controlled by NKX2-1 in thyroid stem cells.

#358

LTB and LTBR mediates alternative Nf-kB activation through NIk and RELB/NF-kB2 to promote cell migration of HNSCC.

Rita Das, Jamie Coupar, Anthony saleh, Paul E. Clavijo, Zhong Chen, Carter VanWaes. _NIH-NIDCD, Bethesda, MD_.

Nuclear factor-κB (NF-κB) includes a family of NF-κB/REL transcription factors that regulate key inflammatory and cancer genes, and which are aberrantly activated in head and neck squamous cell carcinomas (HNSCC). Lymphotoxin beta/Lymphotoxin beta receptor (LTβ/LTβR) can mediate activation of the "alternative" NF-κB pathway that includes NF-κB-inducing kinase (NIK), Inhibitor-κB kinase α (IKKα), and transcription factors RELB/NF-κB2. While LTβ is expressed in lymph nodes where HNSCC spread, the role of this pathway in pathogenesis of HNSCC is not well studied. Analysis of The Cancer Genome Atlas (TCGA) data reveals that LTβR is amplified or overexpressed in larynx or oral cavity HNSCC, while LTα/β, NIK, (MAP3K14) and RELB are overexpressed in oropharynx and tonsil cancers. Similarly, we observed overexpression of LTβR, NIK, and RELB proteins in subsets of HNSCC cell lines. In UM-SCC46, LTβ enhanced nuclear translocation of RELB/NF-κB2 (p52) subunit proteins, while LTβR siRNA knockdown decreased the expression of its target kinase NIK and RELB/NF-κB2 (p52). NIK knockdown also decreased RELB/NF-κB2 (p52) protein expression, whereas LTβ treatment stabilized NIK, and RELB/NF-κB2 (p52). Functional LTβ-mediated NF-κB activation was examined using a stably transfected NF-κB reporter cell line for β-lactamase/Fret activity. Knockdown of LTβR and NIK decreased NF-κB activity, whereas treatment with LTβ partially restored it. Treatment with a NIK inhibitor (1, 3[2H, 4H]-Iso-Quinoline Dione) reduced the protein expression of NIK, IKKα, and RELB/NF-κB2 (p52) in the cytoplasm and/or in the nucleus, and blocked LTβ induced translocation of RELB to nucleus. NIK and RELB knockdown by siRNA or using NIK inhibitor slowed HNSCC migration. Knockdown of NIK inhibited the LTβ-induced expression of MET, and SERPINE1 genes involved in migration, and knockdown of MET by siRNA inhibited the migration of HNSCC cell lines. Our findings show that LTβ/LTβ receptor promotes activation of the alternative NIK-IKKa-RELB/NF-κB2 pathway, to enhance MET-mediated cell migration in HNSCC. This may help explain why HNSCC exhibit migration and metastasis to local lymph nodes, where LTβ is expressed. The constitutive and LTβ induced activation of NIK and downstream NF-κB alternate pathway proteins, RELB and NF-κB2/p52 could be therapeutic targets in HNSCC.

(Supported by NIDCD intramural projects ZIA-DC-000016, 74)

#359

C1GALT1 activates multiple RTKs and promotes malignant behaviors in pancreatic adenocarcinoma cells.

Ting-Chun Kuo,1 Hsueh-Fen Juan,2 Yu-Wen Tien,1 Min-Chuan Huang2. 1 _National Taiwan University Hospital, Taipei, Taiwan;_ 2 _National Taiwan University, Taipei, Taiwan_.

Pancreatic adenocarcinoma (PDAC), stands up to the 4th in all cancer mortalities and remains the most in progress in America. Curative resection remains the most and only potential strategy but only 15~20% of patients are candidates. Even though, many of them have dismal prognosis. Aberrant glycosylation illustrates an attribution of cancer, especially fot promoting malignant behaviors in adenocarcinomas. Altered expression of glycans, such as GalNAcα1-O-S/T (Tn antigen) and Galβ1,3-GalNAc-S/T (T antigen), is hallmark of cancers. Core 1 β1,3-galactosyltransferase (C1GALT1) catalyzes the transfer of Gal to Tn antigen to form T antigen. C1GALT1 is reported to contribute to clinicopathological features and malignant phenotypes in various cancers. However, the expression and function of C1GALT1 in PDAC remain unrevealed. Our data showed that C1GALT1 was overexpressed in PDAC tissues and correlated with poor prognosis. C1GALT1 knockdown inhibited viability, migration, and invasion analyzed by MTT, transwell migration, and Matrigel invasion assays, respectively, in PDAC cells, associated with increased Tn antigen but decreased T antigen expression. Conversely, C1GALT1 overexpression promoted these malignant behaviors. C1GALT1 knockdown inhibited AKT and ERK activities in PDAC cells. Consistently, phospho-receptor tyrosine kinase (p-RTK) array showed decreased phosphorylation of several RTKs including EGFR, IGF1R, and FLT3. Additionally, flow cytometry of C1GALT1 knockdown cells showed enhanced apoptosis, which was associated with decreased catalase, Bcl-x, and death receptor DR5 revealed by apoptosis array. We also found that C1GALT1 knockdown arrested G1 progression in PDAC cells. Furthermore, gene set enrichment analysis of cDNA microarray indicated that C1GALT1 regulated cell cycle, cytoskeleton, and chromatin structure in PDAC cells. These findings indicate that higher C1GALT1 expression predicts worse survival of PDAC and enhances malignant characters in PDAC cells, highlighting a fundamental role of C1GALT1 in PDAC development.

#360

Leveraging a novel ITIM motif in GPCRs for targeted antibody design.

Lili T. Belcastro,1 Anastasia Jancina,2 Christina Adams,2 Ryan D. Paulukinas,2 Catherine C. Moore1. 1 _Cancer Biology Program, The Wistar Institute and University of the Sciences, Philadelphia, PA;_ 2 _Philadelphia College of Pharmacy, University of the Sciences, Philadelphia, PA_.

CXCR4, a chemokine GPCR, is essential for migration of neuronal, hematopoietic, and breast cancer cells during metastasis whereby CXCR4 dysregulation promotes migration and invasion. Following SDF stimulation, CXCR4 is phosphorylated on Ser/Thr residues which initiates adaptor recruitment, receptor desensitization, and trafficking to endocytic sites. Here we show that stimulation with gradient SDF, delays receptor phosphorylation and trafficking, leading to sustained signaling to a novel CXCR4-SHP2-ERK pathway. SHP2 is a tyrosine phosphatase implicated in HER2(+) and triple-negative breast cancers, whereby it transduces mitogenic and migratory signals driving hyperproliferation and invasion. SHP2 is recruited to tyrosine phosphorylated ITIM motifs (immmunoreceptor tyrosine-based inhibitory consensus motifs), a hallmark found in inhibitory immune receptors with little evidence in GPCRs. Here we identify an ITIM motif in CXCR4 that regulates both SHP2 binding and signaling. Specifically, we assessed if gradient SDF stimulation of CXCR4 1) delays receptor phosphorylation and trafficking, 2) sustains signaling to SHP2-ERK, 3) induces SHP2-dependent migration; and if CXCR4 Tyr mutation within the ITIM motif 4) maintains SDF gradient sensing ability, and 5) disrupts interaction with and signaling to SHP2. Our data demonstrate that gradient SDF delays receptor Ser/Thr phosphorylation and internalization thereby sustaining signaling to SHP2-ERK and driving SHP2-dependent migration. Furthermore, the ITIM mutant maintains SDF gradient sensing ability, but disrupts interaction with and signaling to SHP2. Our data support a working model that CXCR4 contains a functional ITIM motif which we are currently leveraging for targeted antibody design for use in migration studies of aggressive breast cancer cells with dysregulated CXCR4. These studies were supported by NIH grant GM-097718, PA Department of Health grant SAP4100057688, and the Milton Lev Memorial Faculty Research Fund.

#361

Loss of cholangiocyte primary cilia induces LKB1 downregulation and defective AMPK signaling.

Adrian P. Mansini, Kristen M. Thelen, Sergio A. Gradilone. _University of Minnesota, Austin, MN_.

Cholangiocarcinoma (CCA) is a malignancy arising from cholangiocytes, the epithelial cells lining the biliary tree. CCA is an uncommon, but devastating cancer that is increasing in incidence. Over the past 3 decades, 5-years survival rates have remained at 10%. Although surgical resection and liver transplantation are potentially curative therapies, most patients are diagnosed at late stages and are not eligible for these options. Therefore, it is imperative to identify novel targets leading to new therapeutic strategies for this devastating disease.

Cholangiocytes express primary cilia that function as chemo, mechano, and osmosensors controlling several molecular pathways. We showed cilia are absent in CCA cells, and experimental deciliation of normal cholangiocytes induced a malignant like phenotype, with significant invasion and proliferation, suggesting the loss of cilia could be associated with CCA development. LKB1 is a tumor suppressor described to be expressed in primary cilia in MDCK cells, and is involved in AMPK activation through a ciliary dependent mechanism. AMPK functions as metabolic and stress check points. Interestingly, patients with intrahepatic CCA and low expression of LKB1 have poor prognosis. Therefore, we hypothesized that primary cilia function as tumor suppressor organelles through a LKB1-AMPK-p53 pathway. To test this hypothesis, first LKB1 subcellular localization was evaluated by confocal microscopy. We show for the first time that LKB1 is enriched in cholangiocyte primary cilia in a normal human cholangiocyte cell line (NHC). However, in experimental deciliated NHC and the CCA cell line KMCH, LKB1 is found dispersed in the cell cytoplasm. Next, we analyzed cellular expression levels of LKB1, AMPK (T172), p53 (s15), p53 and p21 by western blot. We found experimentally deciliated cholangiocytes and CCA cells exhibit defective AMPK signaling characterized by lower levels of LKB1 (-70% and -85%), AMPK (T172) (-72% and -71%), p53 (-37% and -83%), p53 (S15) (-38% and -100%) and p21 (-59% and -87%) relative to NHC. These molecular characteristics correlated with increased cell proliferation in deciliated cells (28%) and CCA cells (27%). Finally, we attempted to rescue the phenotype by using the AMPK activator, ampkinone. This treatment induced 20% inhibition of proliferation on experimental deciliated cholangiocytes and 18% in CCA cells, while not affecting NHC cells and the inhibition was associated with phosphorylation of AMPK and p21 up-regulation. In summary, our data suggest cholangiocyte primary cilia may normally function as tumor suppressors via a mechanism involving LKB1 and AMPK. The loss of cilia in CCA impairs LKB1-AMPK-p53-p21 signaling inducing a proliferative phenotype that may be rescued by specific activation of AMPK, warranting further studies to assess its use as a potential therapeutic approach.

#362

Repression of Smad3 by Stat3 and c-Ski/SnoN induces gefitinib resistance in lung adenocarcinoma.

Yojiro Makino,1 Jeong-Hwan Yoon,2 Eunjin Bae,3 Mitsuyasu Kato,4 Keiji Miyazawa,5 Tatsuo Ohira,1 Norihiko Ikeda,1 Masahiko Kuroda,3 Mizuko Mamura3. 1 _Tokyo Medical University Hospital, Tokyo, Japan;_ 2 _Kyungpook National University Hospital, Daegu, Republic of Korea;_ 3 _Tokyo Medical University, Tokyo, Japan;_ 4 _University of Tsukuba, Ibaraki, Japan;_ 5 _University of Yamanashi, Yamanashi, Japan_.

Cancer-associated inflammation develops resistance to the epidermal growth-factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in non-small cell lung cancers (NSCLCs) harboring oncogenic EGFR mutations. Stat3-mediated Interleukin (IL)-6 signaling and Smad-mediated transforming growth factor-β (TGF-β) signaling play crucial regulatory roles in cancer-associated inflammation. Here we show that Stat3 represses Smad3 in cooperation with c-Ski and SnoN, whereby renders gefitinib-sensitive HCC827 cells resistant. IL-6 signaling via phosphorylated Stat3 induced gefitinib resistance. By contrast, TGF-β upregulated gefitinib sensitivity. We found that IL-6 signaling via phosphorylated Stat3 repressed, whereas TGF-β upregulated the expression of Smad3 in HCC827 cells. Promoter analyses showed that Stat3 synergized with c-Ski/SnoN to repress Smad-induced transcription of the Smad3 gene. Smad3 induced apoptosis by upregulating pro-apoptotic genes such as Caspase 3 and downregulating anti-apoptotic genes such as Bcl2. Our results suggest that preventing IL-6/Stat3-induced loss of Smad3 can be a therapeutic strategy to prevent gefitinib resistance in NSCLCs with gefitinib-sensitive EGFR mutation.

#363

STAT5 inhibition induces apoptosis in peripheral T cell lymphoma.

Haley M. Simpson, Aki Furusawa, Kavitha Sadashivaiah, Arnob Banerjee. _University of Maryland Baltimore, Baltimore, MD_.

Peripheral T cell lymphoma (PTCL) is a heterogeneous malignancy with extremely poor prognosis. Five-year overall survival is <40% and can be as low as 12% for specific subtypes. There is, therefore, an urgent need to identify and develop novel therapeutic targets and approaches. A number of recent sequencing studies, including a whole exome sequencing analysis of primary PTCL patients conducted by our laboratory, have revealed recurrent oncogenic driver mutations in the common gamma chain/JAK/STAT signaling pathway, resulting in the aberrant activation of the transcriptional regulator STAT5. STAT5 has additionally been found to be upregulated or contain activating mutations in 30-40% of PTCL cases of specific subtypes. These findings highlight STAT5 as an oncogenic driver of PTCL and suggest a role for it in novel therapeutic approaches. Based on these data, we aimed to evaluate STAT5 mechanistically as a therapeutic target in PTCL.

The drug, pimozide, which is an FDA approved neuroleptic agent, was recently identified by drug screen to be a STAT5 inhibitor. We assessed the efficacy of pimozide in PTCL using resazurin dye assay, Fluorescence Activated Cell Sorting (FACS), and immunoblot analysis. Our data demonstrate a concentration dependent reduction in STAT5 activity and the number of viable cells in PTCL cell lines after culture with pimozide. This is shown to be due to an increase in cell death by apoptosis. To verify that pimozide's efficacy is due to its inhibition of STAT5 activity, we transduced PTCL cell lines with anti-STAT5 shRNA to knock down STAT5 and assessed PTCL cell survival and proliferation. We show that STAT5 knockdown results in a three-fold reduction in PTCL cell viability, which occurs due to an increase in apoptosis. Furthermore, our data supports a TRAIL dependent mechanism for induction of apoptosis shown by an upregulation of TRAIL by PTCL cells after STAT5 knockdown or culture with pimozide. Mitochondrial membrane potential is also disrupted. These findings have potential clinical implications, as we further demonstrate that pimozide inhibits STAT5 phosphorylation, reduces cell viability, and induces apoptosis in primary PTCL patient samples ex vivo.

This research supports further exploration of STAT5 as a therapeutic target in PTCL and the development of STAT5 inhibition in the treatment of PTCL. To facilitate the development of non-chemotherapeutic dependent therapeutic approaches for PTCL, we are now assessing STAT5 inhibition in combination with small molecule inducers of apoptosis including TRAIL pathway and PARP inhibitors. PARP inhibitors have shown efficacy in malignancies with DNA damage response pathway mutations, which have been identified in up to half of cases of certain subtypes of PTCL. We also aim to further investigate the use of JAK inhibitors, kinases responsible for STAT5 activation, in combination in PTCL, as several have demonstrated clinical efficacy in other hematologic malignancies.

#364

Mass cytometry of human glioblastoma characterizes more than 99 percent of cells and reveals intratumoral cell subsets defined by contrasting signaling network profiles.

Nalin Leelatian,1 Justine Sinnaeve,1 Bret C. Mobley,2 Akshitkumar M. Mistry,2 Daniel Liu,1 Kyle D. Weaver,2 Reid C. Thompson,2 Lola B. Chambless,2 Rebecca A. Ihrie,1 Jonathan M. Irish1. 1 _Vanderbilt University, Nashville, TN;_ 2 _Vanderbilt University School of Medicine, Nashville, TN_.

Background: Glioblastoma (GBM) remains largely incurable despite intense study of resected tissue. Prior studies have revealed GBM cell subsets (Patel et al., Science 2014) and have implicated subset emergence as a potential mechanism of poor outcome in other cancer types. Signaling in rare cells or a mix of cell subsets may enable therapy resistance and recurrence of GBM. For example, STAT3 RNA expression has been previously shown to correlate with poor outcome in GBM (Jahani-Asl et al., Nat Neurosci 2016 and TCGA). The complexity of GBM, combined with the interconnectedness between cancer and host cells in the microenvironment, means that a single cell biology approach is needed to comprehensively characterize patient biopsy cells and determine how protein expression, signaling, and functional capabilities impact treatment response.

Methods: We developed a novel mass cytometry approach to characterize human GBM that identified ~90-95% of tumor cells (Leelatian & Doxie et al., Cytometry B 2016). Here, we applied this approach using a newly created 35-antibody mass cytometry panel focused on basal phospho-protein signaling. The published panel of 16 identity proteins included SOX2, CD44, Nestin, PDGFRα, S100B, and NCAM. This panel was augmented to measure 10 additional proteins and 9 phospho-proteins including p-STAT3, p-EGFR, and p-NFκB. Signaling measurements were chosen to match prior single cell studies of signaling networks that stratified clinical outcomes in blood cancers (Irish et al., Cell 2004; PNAS 2010, Levine et al., Cell 2015). Between 10,000 and 250,000 viable cells were characterized for each tumor (N = 7). Tumors were collected with informed consent and in accord with the Declaration of Helsinki.

Results: This new 35-antibody mass cytometry panel positively identified >99% of GBM cells. Subsets of GBM cells displayed protein expression that matched previously observed transcriptional molecular subclasses (Verhaak et al., Cancer Cell 2010 and TCGA). Strikingly, this panel revealed novel GBM cell subsets defined by contrasting basal signaling profiles. An inverse correlation was observed between baseline STAT3 phosphorylation and the abundance of CD45+ leukocytes. Additionally, similar signaling patterns were seen in cells that expressed proteins associated with distinct functions, such as proliferation and migration.

Conclusions: The correlation between low STAT3 signaling and high immune cell abundance provides evidence for the idea that an intimate relationship exists between immune cells and GBM tumor growth and survival. Moreover, single cell analysis may reveal biomarkers of treatment response and allow prediction of clinical outcomes. The abnormal signaling mechanisms observed here in some GBM cell subsets should be studied further as potential targets for novel cancer-selective combination therapies.

#365

Cross-talk between BRAF and Hippo/YAP1 signaling in melanoma.

Mohan Kumar Durai Raj,1 Jonathan Nguyen,1 Namrata Bora-singhal,1 Jane Messina,1 Geoffrey Gibney,2 Srikumar Chellappan1. 1 _H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL;_ 2 _MedStar Georgetown University Hospital, Lombardi Comprehensive Cancer Center, Washington, DC_.

Hippo/YAP1 signaling pathway is a tumor suppressive pathway that controls the organ size by modulating the cell growth, proliferation and apoptosis and is conserved from Drosophila to mammals. In mammals, the Hippo tumor suppressor pathway consists of cascade of kinases in which MST1/2 phosphorylates and activates LATS1/2. The latter phosphorylates the oncogenic transcriptional coactivators YAP1 and TAZ, leading to their cytoplasmic retention by 14-3-3 proteins and/or degradation. Inactivation of MST and LATS kinases allows YAP1 and/ or TAZ nuclear translocation and subsequent activation of their target genes.

Deregulation of Hippo pathway can induce tumors in model organisms and occurs in wide range of human cancers including melanoma. Merlin, a key component of this pathway which inhibits YAP1, is mutated/deleted in 8% of melanoma. Majority of uveal melanomas are driven by Gq/11 mutations that trigger YAP1 nuclear translocation, promoting tumor growth. High levels of YAP1 in BRAFV600E mutant tumors confer resistance to RAF- and MEK- targeted therapy in patients.

Our results show that YAP1 level is elevated in melanoma and the YAP1 inhibitor verteporfin alone or in combination with B-RAF inhibitor PLX4720 reduces the viability, invasion and anchorage-independent growth of B-RAF V600E mutant SK-MEL-28 and SK-MEL-5 cells. In addition, verteporfin treatment also reduced the viability of PLX4720 resistant 1205 cells. Western blot analysis of verteporfin and PLX4720 treated SK-MEL-28 and SK-MEL-5 cells displayed reduced levels of YAP1, B-RAF, pERK, MEK and pMEK.

We also report a novel physical interaction between YAP1 and B-RAF; this could be detected using double immunofluorescence and immunoprecipitation-western blotting techniques in both B-RAF V600E mutant and N-Ras mutant melanoma cells. Proximity ligation assays on tissue microarray showed that YAP1-B-RAF interaction is elevated in metastatic melanoma compared to normal skin. These novel findings highlight the crosstalk between B-RAF and Hippo/YAP1 signaling which might have a

potential role in melanoma development and progression. Further, experiments are in progress to elucidate the functional significance of YAP1-B-RAF interaction in melanoma.

#366

Disruption of TCF7L1 mediated transcriptional repression promotes pancreatic tumorigenesis.

Kathleen M. Kershaw, Bridgette T. Ho, Missael I. Vasquez, Anna R. Lay, David W. Dawson. _UCLA, Los Angeles, CA_.

While phenotypic studies demonstrate Wnt/β-catenin signaling is critical for the initiation and progression of pancreatic adenocarcinoma (PDA), the downstream transcriptional effectors tied to its effects are largely unknown. Previous work from our group has identified differential patterns of LEF/TCF expression linked to variations in Wnt activation and function in PDA, including reduced TCF7L1 (aka TCF3) message and protein levels in pancreatic tumors with elevated Wnt pathway activity. Consistent with this observation, we found TCF7L1 protein levels rapidly diminished in PDA lines after Wnt pathway activation mediated by either Wnt3A ligand or GSK3β inhibitor. RNAi-mediated knockdown of TCF7L1 in PDA lines phenocopied most of the pro-tumorigenic effects seen with Wnt3A ligand treatment. Because TCF7L1 commonly represses transcription and is rapidly downregulated in PDA upon Wnt activation, we explored its transcriptional derepression as a potentially important mechanism through which Wnt signaling promotes pancreatic tumorigenesis. Candidate genes linked to TCF7L1-mediated transcriptional repression in PDA were identified by RNA-sequencing of MiaPaCa-2 and PANC1 lines following RNAi-mediated knockdown of TCF7L1. A total of 196 genes upregulated at least 1.5-fold in both cell lines were identified, representing potential direct targets of TCF7L1-mediated transcriptional repression. Gene ontology analysis among these 196 upregulated genes revealed enrichment of categories including small GTPase-mediated signal transduction, RAS protein signal transduction, regulation of axon extension, and regulation of synaptic plasticity. Among top upregulated genes was prostaglandin E synthase (PTGES), an enzyme catalyzing the isomerization of prostaglandin H2 to prostaglandin E2 (PGE2) as the final step in PGE2 synthesis from arachidonic acid. Notably, PGE2 stimulates pancreatic cancer proliferation, invasion, angiogenesis and metastasis. Validation experiments in TCF7L1-expressing PDA lines confirmed PTGES message and protein levels rapidly increased in response to TCF7L1 depletion and were associated with a corresponding increase in PGE2 production as measured by ELISA. PGE2 increased Wnt reporter activity in PDA cell lines, a potentially important feedforward mechanism whereby Wnt signaling may be reinforced upon its activation. In conclusion, TCF7L1 represses target genes with known roles in PDA, while reduction in TCF7L1 through Wnt pathway activation or alternative means is apparently tied to a program of transcriptional derepression linked to the promotion of pancreatic tumorigenesis.

#367

**Trop-2 activates a dormant Na** + **/K** + **-ATPase/PKCα/CD9/ezrin signaling axis to override the basal growth program of cancer cells.**

Marco Trerotola,1 Valeria Relli,1 Romina Tripaldi,1 Andrea Sacchetti,2 Kristina Havas,3 Pasquale Simeone,1 Emanuela Guerra,1 Annalaura Aloisi,4 Rossana La Sorda,1 Rossano Lattanzio,1 Daniele Vergara,5 Isabelle Fournier,6 Michel Salzet,6 Mauro Piantelli,1 Saverio Alberti1. 1 _"G. D'Annunzio" University of Chieti-Pescara, Chieti, Italy;_ 2 _Josephine Nefkens Institute, Erasmus MC, Rotterdam, Netherlands;_ 3 _IFOM the FIRC Institute of Molecular Oncology, Milan, Italy;_ 4 _San Raffaele Scientific Institute, Milan, Italy;_ 5 _University of Salento, Lecce, Italy;_ 6 _Université de Lille 1, Cité Scientifique, Villeneuve D'Ascq Cedex, France_.

Trop-2 is overexpressed in most human cancers, suggesting selective pressure for a key, conserved function. Here we show that Trop-2 stimulates cancer cell growth through the activation of a constitutively expressed, yet otherwise dormant, growth-control module. We discovered that crosslinking of membrane Trop-2 with specific Abs leads to a cytoplasmic Ca2+ raise through interaction with the Na+/K+-ATPase α1 subunit and mobilization of the intracellular stores. This triggers a feed-forward loop through Trop-2-dependent activation and membrane recruitment of PKCα;, which in turn phosphorylates the Trop-2 cytoplasmic tail at two target sites, activating the molecule to stimulate its downstream signaling targets Akt and ERK. Our findings indicate that the Trop-2-triggered cell growth operates through binding and extensive crosstalk with CD9, CD81, CD82 and CD151 through PKCα. Detailed analysis of CD9 and CD81 indicates that they bind to the HIKE region of the Trop-2 intracellular tail. Correspondingly, we found that the HIKE region of Trop-2 mediates its anchoring to the β-actin cytoskeleton through direct interaction with the ERM protein ezrin. Consistently, the Trop-2-dependent dynamic remodeling of the cell cytoskeleton is discovered to occur through activation of myosin II and binding of annexins A1/A11, α-actinin and gelsolin. Systematic drug screening, gene expression silencing and site-directed mutagenesis revealed that cytoskeleton disassembly, HIKE deletion and CD9 inhibition revert the growth of Trop-2-expressing cancer cells to that of their Trop-2-null counterparts. On the other hand, these inhibitors have no effects on basal cell growth. This indicates that Trop-2-centered protein interactions and activations are an essential step for the Trop-2-dependent cancer growth. Tight co-expression of the key components of the Trop-2 growth-stimulatory complex is found in a large breast cancer case series, thus indicating strong clinical relevance. Hence, Trop-2 triggers a universal, but otherwise dormant, layer of cancer growth, that overrides basal cell growth regulatory mechanisms and sensitizes tumors to targeted anticancer therapies.

#368

Shoc2 scaffold protein role in Ras-MAPK signaling and cell survival in pancreatic adenocarcinoma cells.

Camilla R. Borges, Pamela V. Andrade, Mirella Baroni, Vanessa S. Silveira. _Ribeirao Preto Medical School - University of Sao Paulo, Ribeirao Preto, Brazil_.

Background: The highly malignant potential and aggressiveness of pancreatic adenocarcinoma cells is mainly promoted by oncogenic KRAS activation, which triggers innumerous signaling pathways and controls key processes for tumor progression such as cell proliferation, cell survival and metabolic reprograming. Interestingly, recent studies suggest that the major adjustments in metabolic reprogramming, mediated by KRAS activity, are regulated by MAP kinase activities. As SHOC2 scaffold protein plays an important role linking RAS to downstream signal transducers in the RAS/ERK MAP kinase signaling cascade, this study aimed to evaluate the mechanisms underlying RAS/ERK activation mediated by SHOC2 and the outcome in pancreatic adenocarcinoma cells proliferation and survival.

Experimental design: RNAseq data from Pancreatic Adenocarcinoma (data generated by the TCGA Research Network: http://cancergenome.nih.gov/) was analyzed by using the cBioPortal (Sci. Signal. 6, pl1 (2013)). Gene expression analysis and protein expression was performed by quantitative RT-PCR and western blot respectively in pancreatic cell lines and normal pancreatic tissue. To address the role of SHOC2 in human pancreatic carcinoma cell lines (AsPC-1, PANC-1 and MIA PaCa-2) shRNA lentiviral constructs containing shRNA for SHOC2 or empty vector were used. Puromycin selection was performed to generate stable transformed lineages. Gene knockdown was confirmed by qRT-PCR and western blot. Doubling time and cell proliferation assay was performed in a 4-day MTT assay. Colony-forming assay (CFA) was tested in triplicate and t-test was used to analyze the data (alpha value was set at 5%).

Results: Gene expression analysis by RNA-seq revealed that SHOC2 is highly expressed (a 10-fold log scale upregulation) in pancreatic adenocarcinoma samples. SHOC2 was also upregulated in pancreatic carcinoma cell lines in comparison to normal pancreatic tissue in both mRNA and protein analysis. After SHOC2 gene knockdown cell lines showed proliferation rates reduction and clearly increased their doubling time. In survival analysis both PANC-1 and MIA PaCa-2 cell lines showed a significant decrease in self-renewal capability (p = 0.003 and p = 0.002 respectively) in shRNA#SHOC2 transduced cells compared to controls (empty vector) suggesting that SHOC2 may play a role in pancreatic carcinoma cells survival. To further address if this outcome was ERK-mediated, we investigate ERK activation after SHOC2 knockdown. Unexpectedly, we did not observe any modulation on ERK phosphorylation, suggesting that SHOC2 function in pancreatic carcinoma cell survival may occur through other molecules rather than only ERK-mediated. SHOC2 knockdown had no major effects in ERK activation and cell growth in AsPC-1 cells.

Conclusion: In pancreatic carcinoma cells we suggest that SHOC2 can play an important role in oncogenic signaling and lead to tumor cell survival.

#369

Therapeutic targeting of the Wnt antagonist DKK1 with a humanized monoclonal antibody in oncology indications.

Michael H. Kagey,1 Yinyuan Wu,2 Xinjun Zhang,2 Cynthia A. Sirad,1 Shane E. Mulligan,1 Xi He,2 Christopher K. Mirabelli1. 1 _Leap Therapeutics, Cambridge, MA;_ 2 _Boston Children's Hospital, Harvard Medical School, Boston, MA_.

Wnt signaling is a fundamental pathway that is dysregulated in oncology. The Wnt antagonist DKK1 is expressed in a variety of tumor types which frequently correlates with a poor prognosis, including overall survival. DKK1 has known oncogenic activity by stimulating proliferation, metastasis, and angiogenesis, and recently been implicated in contributing to an immunosuppressive tumor microenvironment. The neutralization of DKK1 is hypothesized to have efficacy from both a direct antitumor effect and through an immune stimulated response. Here we describe the characterization of DKN-01, a humanized monoclonal therapeutic antibody to DKK1. DKN-01 binds DKK1 with high affinity and selectivity, disrupts the interaction of DKK1 with the LRP6 co-receptor, and neutralizes DKK1 activity in a cell based assay. In vivo, DKN-01 has efficacy both as a monotherapy and in combination with chemotherapies in a non-small cell lung (NSCLC) cancer A549 xenograft model. Results suggest that DKN-01 has an antiangiogenic effect and may stimulate a NK cell mediated antitumor response. Clinically, DKN-01 is being evaluated in relapsed/refractory esophageal cancer patients in combination with paclitaxel, and preliminary results demonstrate promising activity. Archival patient tumor samples are currently being analyzed genetically and by IHC for DKK1 and β-catenin staining for biomarker identification. Taken together, our results suggest that DKN-01, a novel therapeutic, has clinical efficacy by disrupting Wnt signaling, which results in a direct anti-tumor effect and stimulates a pro-inflammatory tumor response.

#370

T-PLL cells resemble memory-type T-cells with aberrant effector functions implicating a leukemogenic cooperation of TCL1A and TCR signaling.

Alexandra Schrader,1 Kathrin Warner,2 Sebastian Oberbeck,1 Giuliano Crispatzu,1 Petra Mayer,1 Sabine Pützer,1 Hans Diebner,3 Stephan Stilgenbauer,4 Georg Hopfinger,5 Jan Dürig,6 Torsten Haferlach,7 Mark Lanasa,8 Ingo Roeder,3 Michael Hallek,1 Dimitrios Mougiakakos,9 Michael von Bergwelt-Baildon,1 Monika Brüggemann,10 Sebastian Newrzela,2 Hinrich Abken,1 Marco Herling1. 1 _Department I of Internal Medicine, Center for Integrated Oncology (CIO) Köln-Bonn, University of Cologne (UoC), Germany;_ 2 _Senckenberg Institute of Pathology, Goethe-University, Frankfurt/M., Germany;_ 3 _Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Institute for Medical Informatics and Biometry, Dresden, Germany;_ 4 _Department III of Internal Medicine, University Hospital, Ulm, Germany;_ 5 _Department of Internal Medicine I, Bone Marrow Transplantation Unit, Medical University of Vienna, Vienna, Austria;_ 6 _Clinic for Hematology, University Hospital Essen, Essen, Germany;_ 7 _MLL Munich Leukemia Laboratory, Munich, Germany;_ 8 _Duke University Medical Center, Durham, NC;_ 9 _Department of Medicine 5, Haematology and Oncology, University Hospital Erlangen, Germany;_ 10 _Medical Department II of Hematology and Oncology, University Hospitals of Schleswig Holstein, Campus Kiel, Germany_.

The pathogenesis of the rare and aggressive T-cell prolymphocytic leukemia (T-PLL) is poorly understood, which particularly applies to a mechanistic concept around its hallmark oncogene TCL1A. Existing data implicate TCL1A as a catalytic enhancer of the oncogenic kinase AKT, a central node in a T-cell's antigen receptor (TCR) signaling cascade, which mediates proliferation and differentiation. The levels and role of TCR activation in T-PLL's pathogenesis are not known.

To first clarify which physiological T-cell subset T-PLL cells most resemble, we performed comprehensive global gene expression profiling and immunophenotyping of primary T-PLL (n=79) in comparison to healthy-donor derived T-cell populations. Principle component analyses and gene signature alignments revealed a high similarity of T-PLL cells to (central) memory T-lymphocytes over naïve T-cells. Surface markers revealed a spectrum of memory-type differentiation (n=69/79; 87%) with predominant central-memory stages (n=35/79; 44%). The usually TCR and/or CD28-coreceptor positive T-PLL cells revealed no restrictions to genetic or surface TCR-clonotypes. The abnormally high basal activation levels (surface CD25, CD38, CD69) correlated in their degree with inferior clinical outcomes (med. survival 20.8 vs 58.3 mo.; p=0.0012). In parallel, T-PLL cells lost expression of negative-regulatory TCR-co-receptors (e.g. CTLA-4, LAG3). Fittingly, TCR engagement of primary T-PLL cells revealed a trend to hyperactive intracellular responses and interleukin(IL)-2 release alongside a prominent Th1-cytokine program. T-PLL cells also showed a robust resistance to stimulation-induced cell death and agonistic CD95 ligation. TCR-derived signals (phospho-kinase induction, IL-2 release) were enhanced in vitro by the modulated presence of TCL1A with kinetics indicative of a sensitizer relationship, mainly in the CD3 axis as opposed to the CD28 branch. A mouse model with TCL1A-initiated protracted development of T-PLL (Lckpr-TCL1Atg) revealed congruent findings with the aberrant T-cell phenotype of human T-PLL. TCL1A expressing T-cells of this model, that were further equipped with monoclonal epitope-defined TCRs against ovalbumine or a chimeric-antigen-receptor (CAR) against carcinoembryonic antigen, gained a pre-leukemic growth advantage in scenarios of pulsed or continuous low-level receptor stimulation.

Overall, we establish that T-PLL cells resemble antigen-experienced memory T-cells. Retention of functional effector responses to TCR stimulation and loss of restricting activation regulators underlie a highly activated phenotype and a marked resistance to death-inducing signals. TCL1A proactively enhances TCR responses and we postulate that this leukemogenic cooperation drives accumulation of memory-type cells that utilize amplified, hence permissive, low-level cognate antigen input.

#371

Developing small molecule therapeutics to target AKT signaling in non-small cell lung cancer.

Balaji Chandrasekaran,1 Deeksha Pal,1 Venkatesh Kolluru,1 Srinivasa R. Ramisetti,2 Arun K. Sharma,2 Murali Ankem,1 Chendil Damodaran1. 1 _University of Louisville, Louisville, KY;_ 2 _Pennsylvania State University, Hershey, PA_.

Lung cancer remains a leading public health problem which is evidenced by its increasing death rate. Platinum-based chemotherapy is the first-line of treatment for patients in advanced stages of non-small cell lung cancer (NSCLC), however the success rates are not quite impressive. Hence, developing individualized treatment strategies for metastatic lung cancer gains momentum, such as Tyrosine kinase inhibitors erlotinib and gefitinib, or ALK inhibitors ceritinib and alectinib are commonly used in the clinic. Recent reports suggest that pAKT(ser473) is highly expressed in NSCLC and higher nuclear expression of pAKT correlated with poor prognosis and an independent prognostic marker for survival. Our lab is interested to develop novel small molecules which specifically inhibit AKT signaling in NSCLC. Structure-activity relationship (SAR) studies in our laboratory have recently ideintifed one such compound, AKS-407, that effectively inhibited cell growth at nanomolar concentration in NSCLC cell lines (A549 and H460; 250nM). Molecular studies revealed AKS-407 inhibited AKT signaling by down regulating pAKT(ser473) expression and downstream events including NFκB activation, BCl-2 expression in both the cell lines. As signaling through AKT regulates epithelial-mesenchymal transition (EMT) in NSCLC, we determined the effect of AKS-407 on EMT phenotype on NSCLC cells. Treatment of AKS-407 inhibited the mesenchymal markers includes snail, MMP9, N-cadherin, β-catenin and vimentin expression that resulted in blocking invasion and migration of A549 and H-460 cells. These results suggest AKS-407 to be a promising small molecule targeting AKT signaling pathway which remains an important target for the development of effective treatment of metastatic NSCLC. Validating in-vivo efficacy of this potential drug candidate would further support our overall goal of the study.

#372

Optimizing vertical MAPK pathway inhibition for RAS mutant non-small cell lung cancer.

Jens Köhler,1 Cloud P. Paweletz,2 Yanan Kuang,2 Prafulla Gokhale,2 Margaret K. Wilkens,2 Hong Tiv,2 Atsuko Ogino,1 Jihyun Choi,1 Paul T. Kirschmeier,2 Pasi A. Jänne1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Belfer Center for Applied Cancer Science, Boston, MA_.

Introduction: Non-small cell lung tumors (NSCLC) with mutations in genes encoding for Ras proteins (H-, N-, K-Ras) exhibit activation of the MAPK signaling pathway. Mek inhibitors, however, have limited efficacy in patients affected by this genotype-defined tumor subtype and major resistance derives from insufficient suppression or reactivation of the extracellular signal-regulated kinase (Erk) as a result of dynamic kinome reprogramming. Erk inhibitors may overcome limitations of Mek inhibition due to the bottleneck function of Erk proteins in submitting mitogenic and anti-apoptotic signals. Combinations of both drug classes ("vertical pathway inhibition") may further increase treatment efficacy but inevitably potentiate toxicity hence requiring alternative dosing schedules.

Methods: We investigated the efficacy of vertical MAPK inhibition with selumetinib (Mek inhibitor) and SCH772984 (Erk inhibitor) in commercially available and patient-derived RAS mutant NSCLC models. A quantitative RT-PCR based 12-gene signature was used as a surrogate to assess Erk-dependent transcriptional output. Pulsatile drug regimens were investigated in xenograft models.

Results: In a genetically simple, patient-derived NRAS-mutant lung cancer cell line (DFCI168), we observed strongest in vitro suppression of Erk-dependent transcriptional output and delay of feedback reactivation by vertical MAPK pathway inhibition with a selumetinib/SCH772984 combination compared to either drug alone. Deeper MAPK pathway suppression in our model cell line was accompanied by higher anti-proliferative activity in a panel of RAS mutant cell lines in vitro (n=5 patient-derived, n=19 commercially available cell lines) and in vivo (n=2). The degree of transcriptional output suppression of cell lines (n=8) with different sensitivities towards Mek/Erk inhibition did not differ significantly at 24 hrs in vitro. Gastrointestinal toxicities (i.e. peritonitis-like symptoms) occurred when continuously administered SCH772984 was intermittently combined with selumetinib (4 days ON, 3 days OFF) but resolved when both drugs were given intermittently (4 days ON, 3 days OFF). None of the drug treatments, however, could cure the mice.

Conclusion: We have demonstrated that deeper and prolonged transcriptional output suppression by vertical MAPK pathway inhibition translates into superior efficacy in RAS mutant lung cancer models compared to single agent treatment. Other factors (e.g. signaling pathways) are likely to influence the overall outcome of vertical MAPK pathway inhibition. Toxicities observed in PDX models overlap with those reported from genetically engineered mouse models (GEMM) with loss of Mek1/2 or Erk1/2 and can be mitigated by pulsatile dosing schedules.

#373

Wnt signaling induces anti-apoptotic effect in colorectal cancer cells through the suppression of IFITs.

Tomoyuki Ohsugi, Kiyoshi Yamaguchi, Chi Zhu, Tsuneo Ikenoue, Yoichi Furukawa. _Division of Clinical Genome Research The Institute of Medical Science The University of Tokyo, Tokyo, Japan_.

Impaired Wnt signaling pathway plays a crucial role in the development of colorectal cancer through the activation of β-catenin/TCF complex. Although genes up-regulated by Wnt/β-catenin signaling has been well studied, the down-regulated genes are poorly understood. To clarify the comprehensive changes regulated by the signaling in colorectal cancer cells, we explored a global gene expression of CRC cells transfected with β-catenin siRNAs or dominant negative form of TCF7L2 (dnTCF7L2). Consequently, a set of genes that were negatively regulated by β-catenin/TCF were identified. Among the genes, three members of interferon-induced proteins with tetratricopeptide repeats (IFIT) family (IFIT1, IFIT2, and IFIT3) expression were significantly increased by the inhibition of β-catenin/TCF. Comparison of gene expression data from normal colonic mucosa and the tumor tissues showed that the expression of IFIT1 and IFIT2 in the tumors was significantly lower than that in normal tissues. To elucidate the mechanism of IFITs expression regulated by β-catenin/TCF, we performed a reporter assay using plasmid containing 1.2-kb of 5'-flanking region of the IFIT2 gene. As a result, the reporter activity was significantly enhanced by either transduction of β-catenin or dnTCF7L2, suggesting that blockage of β-catenin/TCF stimulated IFITs through the promoter. In addition, we found that overexpression of IFIT2 increased apoptosis and decreased cell proliferation in SW480 and HCT116 cells. These results imply that Wnt signaling may promote anti-apoptotic effect in cancer cells through the suppression of IFIT2. Our findings suggest that analysis of down-regulated genes in response to activated Wnt/β-catenin signaling provides a better understanding of human colorectal carcinogenesis.

#374

KMT2A promotes tumor growth by activating hTERT and CBP signaling and predicts poor prognosis in human melanoma.

Changlin Zhang,1 Ranran Tang,2 Kefang Zhang,3 Wenlin Huang,1 Wuguo Deng1. 1 _Sun Yat-sen University Cancer Center; State Key Laboratory of Oncology in South China; Collaborative Innovation Center for Cancer Medicine, Guangzhou, China;_ 2 _Dalian Medical University, Dalain, China;_ 3 _Global Life Care Federation, Hong Kong, China_.

Melanoma is an aggressive type of cutaneous malignancy. Although the inhibitors targeting BRAF and/or MEK pathways provide a therapeutic option for non-resectable melanoma driven by BRAF mutation, melanoma, especially metastatic melanoma, has still become one of the most threatening malignancies. Thus, identifying the exact molecular mechanisms involved in melanoma growth and discovering the novel targets for melanoma therapy is urgently needed. In this study, we screened a siRNA library targeting 6024 human genes in human melanoma cells and identified KMT2A as a potential therapeutic target for melanoma. KMT2A was highly expressed in melanoma cell lines and tumor tissues of melanoma patients. Knockdown of KMT2A by siRNA or shRNA significantly inhibited cell viability and colony formation, whereas exogenous expression of KMT2A effectively promoted cell growth in various melanoma cell lines. Further mechanism studies showed that the KMT2A-mediated regulation of melanoma growth was through targeting the hTERT and CBP signaling. Knockdown of KMT2A significantly inhibited hTERT promoter activity and protein expression and attenuated telomerase activity. Overexpression of hTERT rescued the KMT2A knockdown-mediated melanoma cell growth. By contrast, exogenous expression of KMT2A activated hTERT transcription and expression and increased telomerase activity in melanoma cells. Moreover, we found that KMT2A promoted hTERT expression and melanoma cell growth by cooperating with the transcriptional co-activator CBP, which interacted with and acetylated KMT2A. Inhibition of CBP by siRNA or a CBP-specific inhibitor suppressed the acetylation of KMT2A, abrogated the binding of KMT2A on the hTERT promoter and down-regulated hTERT expression, thereby inhibiting the growth of melanoma cells. Conversely, overexpression of CBP increased KMT2A acetylation and the binding to hTERT promoter, resulting in the promotion of hTERT expression and cell growth. The in vivo studies also showed that KMT2A promoted melanoma growth by activating the hTERT signaling in a xenograft tumor mouse model. Furthermore, the analyses for the clinical samples demonstrated that KMT2A expression was positively correlated with hTERT in tumor tissues of melanoma patients, and the high expression of both KMT2A and hTERT was associated with the worse clinical TNM staging and poor prognosis in melanoma patients. Taken together, our results indicate that KMT2A promotes melanoma growth by activating hTERT and CBP signaling in human melanoma. Our study therefore provides new insights into understanding the regulatory mechanism of melanoma growth and suggests that the KMT2A/CBP/hTERT signaling may be a potential therapeutic target for human melanoma.

Funding support: This work was supported by the funds from the National Natural Science Foundation of China (81071687, 81272195), the State "973 Program" of China (2014CB542005).

### Genomic Analyses across Cancer Types

#375

Oncologist use and perception of large panel next generation tumor sequencing.

Alison M. Schram, Dalicia Reales, Jesse Galle, Roy Cambria, Robert Durany, Darren Feldman, Eric Sherman, Jonathan Rosenberg, Gabriella D'Andrea, Shrujal Baxi, Yelena Janjigian, William Tap, Maura Dickler, José Baselga, Barry Taylor, Debyani Chakravarty, Jianjiong Gao, Nikolaus D. Schultz, David B. Solit, Michael F. Berger, David M. Hyman. _Memorial Sloan Kettering Cancer Center, New York, NY_.

At Memorial Sloan Kettering, we have used a clinically validated custom hybridization capture-based NGS assay (MSK-IMPACT) to sequence the tumors of more than 10,000 patients. We sought to determine physician perception and use of these results, including whether they changed management and the reasoning behind this decision-making. All physicians who ordered MSK-IMPACT testing for patients where it was not considered routine were asked to complete a questionnaire quarterly (Table). Physician determination of genomic "actionability" was compared to OncoKB, a curated knowledge base of somatic variants (OncoKB.org).

Responses were received from 146 of 258 physicians emailed (57%) regarding 1932 of 9147 patients (21%). Physician respondents comprised a diversity of cancer specialties including medical oncology (67%), pediatric oncology (8%), surgery (6%), radiation oncology (5%), and neuro-oncology (5%). A total of 49 cancer types were represented. Physicians indicated that sequencing altered management in 331 (20%) of profiled patients in need of a treatment change. Among those in whom treatment was reportedly not altered, physicians indicated the presence of at least one actionable alteration in 55% (805/1474) of cases. However, only 45% of these cases harbored a genomic variant annotated as actionable by OncoKB. Among patients in whom physicians deemed the report non-actionable, 12% had OncoKB annotated actionable variants. Across the cases annotated as potentially actionable by OncoKB, physicians identified an actionable alteration in 81% of cases. At the time of data analysis, 297 (15%) patients had been enrolled in at least one clinical trial of targeted therapy at MSKCC including 224 (12%) patients on genomically-matched trials, 76% of whom participated after IMPACT profiling. As the clinical adoption of NGS panels expands, continued education of physicians as well as maintained knowledge bases for annotation will be necessary to expand the utility of this approach and the opportunity for precision medicine.

TABLE

|

---|---

Questions | Responses, N (%)

DID alter treatment, as follows:

|

1. Patient enrolled to a therapeutic protocol at MSKCC | 265 (14)

2. Patient enrolled to a therapeutic protocol at another institution | 15 (1)

3. Patient treated with off-label use of an FDA approved therapy | 43 (2)

DID NOT alter treatment, as follows:

|

4. Actionable mutation(s) identified, but no therapeutic protocol was available | 175 (9)

5. Actionable mutation(s) identified, but patient declined participation in, or was ineligible for, available therapeutic protocol | 115 (6)

6. Actionable mutation(s) identified, but patient deteriorated, progressed, or died before results could be used | 176 (9)

7. Actionable mutation(s) identified and therapeutic study available, but patient has not recurred/progressed since MSK-IMPACT result | 339 (18)

8. No actionable mutation identified | 669 (35)

Other | 135 (7)

TOTAL | 1932

#376

Biospecimen and data resources for cancer research from NCI's BPV program.

Ping Guan, Helen M. Moore. _National Cancer Institute, Bethesda, MD_.

The Biospecimen Preanalytical Variables (BPV) Program was initiated by the National Cancer Institute's Biorepositories and Biospecimen Research Branch to evaluate the impact of preanalytical factors on the molecular integrity of biospecimens. Selected preanalytical factors including cold ischemic time (delay to formalin fixation (DTF)), time in formalin (TIF), freezing methods, and storage temperatures and durations were examined for their potential effects on molecular profiles from surgical resection tissues and matched blood from four cancer types (kidney, ovary, colon and lung). The BPV program has collected tumor specimens from 364 cancer patients. Each specimen was annotated with 300+ data elements that cover steps in the collection, handling, and processing procedures, pathology review, and clinical information. NCI conducted multiple studies using these specimens to evaluate the preanalytical impacts on different analytical platforms including gene expression profiling, copy number variation, proteomics and metabolomics profiling. The program invites interested organizations to work with NCI through collaboration to further evaluate preanalytical effects on molecular analyses (https://techtransfer.cancer.gov/availabletechnologies/e-000-2013). The remaining specimens are available to support relevant research focusing on biospecimen science and/or clinical biomarker assay development (https://specimens.cancer.gov/search/). The IT infrastructure that was developed to support BPV biospecimen collection and management has been further developed into open source products (https://github.com/NCIP/CDR and https://github.com/NCIP/CDR-Lite). The controlled vocabulary that records the terms and definitions used in describing the overall biospecimen collection efforts has been refined and published at publicly accessible CDE repositories: NCI's caDSR (https://cdebrowser.nci.nih.gov/CDEBrowser/ ) and NIH's CDE portal (https://cde.nlm.nih.gov/cde/search ). An ongoing collaboration with an academic ontology consortium will map the CDEs to existing biobanking ontology frameworks and make them publicly available. The BPV program has generated a wide range of ~omics data. We are preparing a BPV data compendium to be submitted to dbGaP at NCBI. These data will be used as the experimental evidence to develop evidence-based best practices for fit-for-purpose collection, processing, and storage of biospecimens for cancer research.

#377

International Cancer Genome Consortium (ICGC).

Jennifer L. Jennings,1 Lincoln D. Stein,1 Fabien Calvo2. 1 _Ontario Institute for Cancer Research, Toronto, Ontario, Canada;_ 2 _Inst. Gustave Roussy, Villejuif, France_.

The International Cancer Genome Consortium (ICGC) was established to bring together researchers from around the globe to comprehensively analyze the genomic, transcriptomic, and epigenomic changes in 50 different tumor types or subtypes that are of clinical and societal importance across the globe (International network of cancer genome projects. Nature 464, 993-998 (15 April 2010)). As of November 2016, the ICGC has received commitments from researchers and funding organizations in Asia, Australia, Europe, North America and South America for 103 project teams in 17 jurisdictions to study more than 25,000 tumor genomes. Processed data is available via the Data Coordination Centre (http://dcc.icgc.org) based at the Ontario Institute for Cancer Research and is updated semi-annually. The August 2016 release (Version 22) in total comprises data from more than 16,000 cancer donors spanning 70 projects and 21 tumor sites. The Pan-Cancer Analysis of Whole Genomes (PCAWG) project of the ICGC and The Cancer Genome Atlas (TCGA) is coordinating analysis of more than 2,600 cancer genomes, with the extensive use of cloud computing. Because of the very large size of the pan-cancer dataset, with 5,000 whole genome sequences, PCAWG is using a distributed compute cloud environment (generated by computing centres in the USA, Europe and Asia) that meets the project's technical requirements and the bioethical framework of ICGC and its member projects. Each genome is being characterized through a suite of standardized algorithms, including alignment to the reference genome, uniform quality assessment, and the calling of multiple classes of somatic mutations. Scientists participating in the research projects of PCAWG are addressing a series of fundamental questions about cancer biology and evolution based on these data. The first phase of ICGC, which is slated for completion in 2018, has focused on developing extensive catalogs of tumor genomic information. The proposed second phase, ICGCmed, will link genomics to clinical information and health, including lifestyle, patient history, response to therapies, and underlying causes of disease, for a broad spectrum of cancers, including preneoplastic lesions, early cancers and metastases. The goal will be to accelerate the movement of genomic information into the clinic to guide prevention, early detection, diagnosis, and prognosis, and provide the information needed to match a patient's disease to the most effective combinations of therapy. The ICGC develops policies and quality control criteria to help harmonize the work of member projects located in different jurisdictions. Data produced by ICGC projects are made rapidly and freely available to qualified researchers around the world via the data cloud and through the ICGC Data Coordination Center at (http://dcc.icgc.org). More information can be found on www.icgc.org.

#378

The Cancer Genome Collaboratory.

Christina K. Yung,1 George L. Mihaiescu,1 Bob Tiernay,1 Junjun Zhang,1 Francois Gerthoffert,1 Andy Yang,1 Jared Baker,1 Guillaume Bourque,2 Paul C. Boutros,1 Bartha M. Knoppers,2 BF Francis Ouellette,1 Cenk Sahinalp,3 Sohrab P. Shah,4 Vincent Ferretti,1 Lincoln D. Stein1. 1 _Ontario Institute for Cancer Research, Toronto, Ontario, Canada;_ 2 _McGill University, Montreal, Quebec, Canada;_ 3 _Simon Fraser University, Vancouver, British Columbia, Canada;_ 4 _BC Cancer Agency, Vancouver, British Columbia, Canada_.

The Cancer Genome Collaboratory is an academic compute cloud designed to enable computational research on the world's largest and most comprehensive cancer genome dataset, the International Cancer Genome Consortium (ICGC). The ICGC is on target to categorize the genomes of 25,000 tumors by 2018. A subproject of ICGC, the PanCancer Analysis of Whole Genomes (PCAWG) alone has generated over 800TB of harmonized sequence alignments, variants and interpreted data from over 2,800 cancer patients. A dataset of this size requires months to download and significant resources to store and process. By making the ICGC data available in cloud compute form in the Collaboratory, researchers can bring their analysis methods to the cloud, yielding benefits from the high availability, scalability and economy offered by cloud services, avoiding a large investment in static compute resources and essentially eliminating the time needed to download the data.

To facilitate the computational analysis on the ICGC data, the Collaboratory has developed software solutions that are optimized for typical cancer genomics workloads, including well tested and accurate genome aligners and somatic variant calling pipelines. We have developed a simple to use, but fast and secure, data transfer tool that imports genomic data from cloud object storage into the user's compute instances. Because a growing number of cancer datasets have restrictions on their storage locations, it is important to have software solutions that are interoperable across multiple cloud environments. We have successfully demonstrated interoperability across The Cancer Genome Atlas (TCGA) dataset hosted at University of Chicago's Bionimbus Protected Data Cloud, the ICGC dataset hosted at the Collaboratory, and ICGC datasets stored in the Amazon Web Services (AWS) S3 storage. Lastly, we have developed a non-intrusive user authorization system that allows the Collaboratory to authenticate against the ICGC Data Access Compliance Office (DACO) when researchers require access to controlled tier data. We anticipate that our software solutions will be implemented on additional commercial and academic clouds.

The Collaboratory is actively growing, with a target hardware infrastructure of over 3000 CPU cores and 15 petabytes of raw storage. As of November 2016, the Collaboratory holds information on 2,000 ICGC PCAWG donors (500TB total). We anticipate expanding the Collaboratory to host the entire ICGC dataset of 25,000 donors (approximately 5PB) and to extend its data management and analysis facilities across multiple clouds. During the current closed beta phase, the Collaboratory has been successfully utilized by multiple research groups, most notably PCAWG project researchers who analyzed thousands of genomes at scale over a few weeks' time. The Collaboratory will open to the public during the second quarter of 2017. We invite cancer researchers to learn more about our cloud resources at cancercollaboratory.org, and apply for access to the Collaboratory.

#379

Genomic profiling of the tumors harboring activating epidermal growth factor receptor mutations.

Jingrui Jiang, Meaghan Russell, Cheryl Eifert, Angeliki Pantazi, Ruobai Sun, Stephen Lyle, Joerg Heyer, Alexei Protopopov. _KEW Inc, Cambridge, MA_.

Introduction: EGFR is a kinase of the HER/ERBB family and an oncogenic driver, especially for NSCLC, HNSCC and CRC. EGFR targeted therapies have been successfully used for treating cancer patients harboring activating EGFR mutations. However a main challenge is acquired drug resistance due to mutations or alternative signaling. While developing next generation drugs is a promising strategy, targeting other alternatives is also an attractive therapeutic option. The purpose of our study is to investigate the mutational landscape of the tumors harbored activating EGFR mutations and to pinpoint potential strategies for overcoming acquired drug resistance and for combinational therapies.

Methods: CancerPlex, a NGS large panel test, which includes targeted, full-gene sequencing of over 400 genes, was applied to analyze a cohort of 2294 patient FFPE samples across majority of solid tumor types.

Results: 1, Among 293 (out of 2294, 12.8%) samples harbored EGFR mutations, 86 (86/293, 29.4%) samples harbored actionable EGFR mutations (namely AE group), such as exon 19 deletion and L858R, which includes 84 lung cancer (79 NSCLC), 1 glioblastoma, and 1 urothelial carcinoma. 2, The genes/variants in the AE group are then subjected to further functional analysis and mapped to several pathways/ biological functions. Our results show that the mutational landscape of activating mutant EGFRs covers most main signaling pathways and biological processes with several features: a), Receptors at different classes, including PKHD1, ROS1, and ERBB2, and genes involving in genome and epigenome stabilities, including ARID1A,, KMT2D, and XPC, have the highest mutation rate in the context of activating EGFR mutations. b), PI3K/AKT/mTOR and Ras-Raf-MEK are two main pathways transducing ligand activated EGFR signals. Our results show that the mutant variants in the PI3K-AKT-mTOR pathway are almost 50% more than those in Ras-Raf-MEK pathway, suggesting that activating mutant EGFRs might preferentially rely on effectors in the PI3K pathway, such as PIK3CA and RICTOR, to transduce oncogenic signals. Of note, no KRAS mutation is found in the AE group. c), As expected, mutations are also found in the TP53/apoptosis and Rb/cell cycle pathways with higher variant number in the TP53/apoptosis axis. d) Strikingly, the number of mutant variants in the Wnt/beta-catenin pathway tops that in the PI3K and TP53 pathways. Several recent publications demonstrated that the Wnt/beta-catenin pathway is abnormally activated in NSCLC and may be a main mechanism of the drug resistance. Our finding that the genes in the Wnt/beta-catenin pathway, including APC, AXIN2, RNF43, and BCL9, are mutated to a great extent in the context of activating EGFR mutations supports the notion that targeting the Wnt/beta-catenin pathway, in combination with EGFR targeted therapies, is a promising therapeutic strategy for the treatment and overcoming drug resistance of EGFR-driven tumors.

#380

Systematic identification of novel functional tumor-specific mutations in receptor tyrosine kinases based on their pan-cancer mutational profiles in Japanese patients with cancer.

Masakuni Serizawa,1 Takeshi Nagashima,2 Yuji Shimoda,2 Shumpei Ohnami,3 Sumiko Ohnami,3 Keiichi Ohshima,4 Tohru Mochizuki,4 Takashi Nakajima,5 Kenichi Urakami,3 Masatoshi Kusuhara,6 Ken Yamaguchi7. 1 _Drug Discovery and Development Division, Shizuoka Cancer Center Research Institute, Shizuoka, Japan;_ 2 _SRL, Inc., Tokyo, Japan;_ 3 _Cancer Diagnostics Division, Shizuoka Cancer Center Research Institute, Shizuoka, Japan;_ 4 _Medical Genetics Division, Shizuoka Cancer Center Research Institute, Shizuoka, Japan;_ 5 _Division of Pathology, Shizuoka Cancer Center, Shizuoka, Japan;_ 6 _Region Resources Division, Shizuoka Cancer Center Research Institute, Shizuoka, Japan;_ 7 _Shizuoka Cancer Center, Shizuoka, Japan_.

Background: Recent accumulation of large amounts of cancer genome information has revealed the presence of tumor-specific mutations with unknown functions; characterization of these mutations should facilitate the identification of novel therapeutic targets. Some tumor-specific mutations in receptor tyrosine kinases (RTKs) act as oncogenic drivers in multiple cancer types. Here, we aimed to identify novel, functional, tumor-specific, single nucleotide variants (SNVs) in cancer-related RTKs based on pan-cancer mutational profiles in Japanese patients with cancer.

Methods: Surgically resected tissue and corresponding peripheral blood samples of 1,685 patients enrolled in Project HOPE (High-tech Omics-based Patient Evaluation), an ongoing prospective molecular profiling study at the Shizuoka Cancer Center, collected between January 2014 and December 2015, were subjected to whole-exome sequencing with an ion torrent proton platform. Newly detected tumor-specific SNVs that could represent functional mutations between the transmembrane and kinase domains of 35 cancer-related RTKs were subjected to serum response element (SRE)-reporter assay to assess effects on extracellular signal-regulated kinase (ERK) signaling relevant to cancer cell survival.

Results: We identified 201,422 nonsynonymous tumor-specific SNVs in 1,685 patients. Of these SNVs, 1,049 were observed in our focused 35 cancer-related RTKs. Approximately 45% of patients with lung squamous cell carcinoma, which was the most frequent cancer type, followed by lung adenocarcinoma (39%), harbored tumor-specific SNVs in RTKs. The relative frequencies of tumor-specific SNVs in RTKs in other tumor types were 32%, 31%, 29%, 28%, and 19% in liver cancer, colorectal cancer, gastric cancer, head and neck cancer, and breast cancer, respectively. After filtering out of SNVs registered in multiple cancer-related databases, such as COSMIC, dbSNP, and DoCM, to select putative novel tumor-specific SNVs, 22 SNVs were selected as novel tumor-specific SNVs with high potential as functional mutations through an additional selection process based on amino acid substitution patterns and multiple alignment of amino acid residues coupled with information regarding functional mutations. Among these SNVs, 13 had already been assessed using an SRE reporter assay. We identified two SNVs in NTRK1 and IGF1R that were involved in activation of the ERK pathway, indicating that these SNVs may be activating mutations. Moreover, inactivating mutations were also identified. Nine SNVs in FLT3, NTRK1, EPHA5, ERBB4, FGFR3, and KDR significantly reduced ERK activity.

Conclusions: A systematic evaluation of functionally unknown tumor-specific SNVs detected in cancer genome sequencing is necessary to expand the range of molecularly targeted cancer therapeutics.

#381

Genomic alterations in mucins in cancer.

Ryan J. King, Fang Yu, Pankaj K. Singh. _University of Nebraska Medical Center, Omaha, NE_.

Mucins promote the growth, aggressiveness, and invasiveness of various cancer types. Changes in the expression or localization of mucins can significantly alter patient survival. Due to their predominance in multiple cancers, biomarkers and therapeutic agents have been developed to improve patient care. However, an extensive investigation across multiple cancer subtypes examining the genomic status of the cohort of mucins has not been performed and could yield significant leads into new roles of mucins in different cancers. Utilizing The Cancer Genome Atlas, the cancers of 11 mucin expressing tissues were examined for the genomic alterations in mutations, mRNA expression levels, copy numbers, methylation status, and the impact on patient survival due to these genomic features. It was discovered that different mucins across multiple tissue origins and cancer subtypes might have a different rate, pattern, and survival impact. MUC1 was not mutated in the majority of cancers; however, we did observe a frequent T112P mutation in pancreatic tumors that was also present in other cancers. MUC4 was frequently mutated at H4205 and MUC4 mutations were seen to have survival differences. Multiple significant differences were discovered in mRNA expression, which varied distinctly between cohorts. One such pattern was observed with de novo expression of MUC21 in colorectal cancers, in which mRNA expression was suddenly induced in cancer and increased in a stage dependent manner. Copy number alterations were seen for only a few cancer subtypes and mucins, including MUC4 in lung squamous cell carcinomas. Global promoter demethylation was generally observed across the cancers. MUC15 in renal cancers was seen to be one of the most demethylated mucins, in which mRNA was seen to be substantially decreased or expunged. This extensive study investigated the status of multiple mucins in multiple cancer settings and highlights a few noticeable mucins that are suggested to be further studied for roles in biomarkers, survival, and etiology for cancer progression.

#382

Genome-wide location analysis of DNA adducts in vivo.

Brian Woo, Christopher Fang, Paolo Abada, Stephen B. Howell, Olivier Harismendy. _UC San Diego, La Jolla, CA_.

DNA adducts are the biochemical consequences of exposure to UV light, carcinogens or DNA-reactive drugs. They play a central role in malignant transformation and the selective killing of cancer cells by chemotherapeutic agents. The functional consequences of DNA adducts is likely to depend on their exact location in the genome with respect to histones and sites where histones and DNA are modified. However, tools that permit precise genome-wide mapping of adduct locations have poor resolution or are too specific to the type of DNA-adduct. Here we present the development of a molecular assay (Ad-Seq) capable of identifying the genomic location of damaged DNA bases in whole cells. Using naked DNA treated with cisplatin (cDDP) or ultra-violet (UV) light as the damaging agents, the strategy relies on differential exonuclease digestion to enrich genome-wide libraries for fragments containing DNA adducts, followed by high-throughput sequencing. Consistent with the expected adduct chemistry, the 5' end of the reads were enriched in TT, TC or CT di-nucleotides in the UV treated DNA (>4x) and in AG or GG in the cDDP treated DNA (1.5x), suggesting a positive predictive value of 76% and 44% and UV (TT) and cDDP (GG) adduct detection, respectively. Applied in vivo to IMR90 cells, Ad-Seq revealed that the fraction of AG/GG loci was higher in DNA from the cDDP-treated cells at all coverage depths, reaching 1.5x enrichment for the highest depth quartile. This indicates that the method is selective and that even low covered loci show enrichment of AG/GG sequences. The enrichment in purines was limited to 3 nucleotides downstream of the read start site, confirming the high resolution of the exonuclease digestion. A total of 11 x 103 AG/GG loci were identified in replicate in cDDP-treated samples and were absent from untreated controls. With about 60 x103 expected adducts per genome, the current assay therefore captured up to 18% of cDDP adducts. The most covered adduct sites were significantly enriched in the quiescent chromatin domains (depleted of histone modifications, p=6.7x10-31), and depleted in the transcription start sites (p=4.9x10-12), as defined by the ENCODE consortium. This may be reflective of the differences in DNA accessibility or the mechanisms of repair in these distinct regions of the genome. The analysis was performed using the PipeDuct computational package, which was developed to align the sequencing reads and provide general statistics and quality control information of Ad-Seq data. Ad-Seq is therefore a novel, unbiased method to map DNA-adducts and study in vivo the genome-wide specificity of their formation and repair.

#383

Survey of spliceosome gene mutations and associated splicing defects across 33 cancer types.

Michael Seiler, Shouyong Peng, Anant Agrawal, Jennifer Tsai, James Palacino, Silvia Buonamici, Lihua Yu. _H3 Biomedicine, Inc, Cambridge, MA_.

Hotspot mutations in the core spliceosome genes SF3B1, U2AF1, SRSF2 have been reported at high frequency in hematological malignancies and with lower occurrence in solid tumors. The TCGA Exome-seq and RNAseq data from 33 tumor types, the majority solid tumors, provides a unique opportunity to define the landscape of spliceosome mutations and associated splicing alterations in human malignancies.

To this end, we compiled a list of 404 genes known to be involved in splicing. We performed 2 different mutational analyses; for the first analysis each tumor type was evaluated independently using MutSig2CV and 68 significantly mutated genes (q ≤ 0.1) were identified. The second analysis was performed by systematically looking for genes across all tumor types with loss of function (LoF) or hotspot in-frame mutations, which identified an additional 12 genes. Among the 80 genes, EEF1A1, HNRNPCL1, PCBP1, PHF5A and ZC3H4 carry hotspot mutations in addition to the previously reported spliceosome genes SF1, SF3B1, SRSF2 and U2AF1. Interestingly, we observed that known leukemia mutation/deletion near P95 in SRSF2 were also present in uveal melanoma. Furthermore, the 2 hotspot mutations in U2AF1 identified in leukemia and lung adenocarcinoma (LUAD) were detected in an additional 9 tumor types. We identified new SF3B1 hotspot mutations p.L833F, p.E862K, p.E902K/G, and p.R957Q located in the heat domains (HD) 9-12 in AML, bladder (BLCA), and endometrial cancers. Unexpectedly, alternative exon usage was the most common splicing event observed in p.E902K mutant BLCA samples. This observation differs from the reported alternative 3' splice site usage induced by SF3B1 hotspot mutations located in the HDs 4-8.

The majority of mutated spliceosome genes (52/80) contained LoF mutations across multiple tumor types. In particular, RBM10 showed the highest mutation frequency in LUAD (6.5%) and BLCA (3.8%), and FUBP1 in low grade glioma (8.0%). Differential splicing analysis comparing RBM10 LoF mutant and wild type LUAD identified exon inclusion and intron skipping as major splicing alterations, consistent with data showing RBM10 knock-down induces alternative exon usage within specific genes. Additionally, we identified cassette exon usage as the major splicing alteration in FUBP1 mutant versus wild type glioma samples. FUBP1 has been reported to bind and repress inclusion of AT rich exons, and confirming this finding we observed higher AT content in exons included in FUBP1 LoF mutant samples when compared to unaffected following exons. These findings suggest that LoF mutations in spliceosome genes impact splicing regulation and may play a critical role in cancer.

In conclusion, the landscape of hotspot and LoF mutations in multiple spliceosome genes and associated splicing alterations highlight the increasing importance of the splicing machinery in tumorigenesis in solid tumors beyond hematological malignancies.

#384

The implications of splicing variant of AIMP2 lacking exon 2 among various cancer types: An analysis of the ICGC/TCGA database and clinical validation.

Dong Chan Kim,1 Ryul Kim,2 Daeyoon Kim,1 Hyojin Song,1 Dong-Yeop Shin,2 Inho Kim,2 Kwang-Sung Ahn,3 Nam Hoon Kwon,4 Sunghoon Kim,4 Sung-Soo Yoon,2 Youngil Koh2. 1 _Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea;_ 2 _Internal Medicine, Seoul National University Hospital, Seoul, Republic of Korea;_ 3 _Functional Genome Institute, PDXen Biosystem Inc., Seoul, Republic of Korea;_ 4 _Medicinal Bioconvergence Research Center, Seoul National University, Suwon, Republic of Korea_.

Aminoacyl-tRNA synthetase interacting multifunctional proteins (AIMP) is the multiple tRNA synthetase complex protein called the multi-tRNA complex (MRC). In cancer, the splicing variant of AIMP2 derives a several signaling cascades, which are crucial for cancer proliferation. Detecting an exon-2 depleted splicing variant (AIMP2-DX2) is an issue of growing importance in cancer therapy. This study suggests the evidence for interrelation between the AIMP2-DX2 and cancer development. We analyzed AIMP2 and AIMP2-DX2 gene expression and their ratio on 7 commercial cancer cell lines and Multiple myeloma patient derived 536MM cell line by RT-PCR and targeted RNA sequencing. Extended this profile, the distribution of AIMP2-DX2/AIMP2 ratio and AIMP2-related major cancer pathways were analyzed using the samples in the ICGC/TCGA database. Over 23 cancer types, 753 samples were used in WTS analysis. In the DEG set analysis, 10 pre-defined major cancer pathways were analyzed among 16 cancer types. Some cancer types, especially acute myeloid leukemia (AML) showed most significant association with AIMP2-DX2 in terms of cancer signaling pathways. We focused on clinical implications of AIMP2-DX2/AIMP2 ratio in the ICGC/TCGA database. 19 AML samples were used, Overall survival (OS) showed that patients with AIMP2-DX2/AIMP2 ratio higher than Q1 shows poor OS and Most of the genes including MEK1/2, ERK, MNK1/2 in this pathway had positive association with AIMP2-DX2/AIMP2 ratio. In colon carcinoma and hepatocellular carcinoma, OS curves had a tendency in a similar way to AML. For the clinical validation of the prognostic value of AIMP2-DX2, 51 AML patients were included in this analysis. The correlation between AIMP2-DX2 expression and survival outcomes was investigated in clinical validation cohort of AML. The AIMP2-DX2-positive group had significantly inferior OS rate and had worse RFS compare to AIMP2-DX2-negative group. Our sequential data shows that the AIMP2-DX2/AIMP2 expression and their ratio can possibly be an indicator to measure malignancy of various cancer types.

#385

Network-driven discovery of cancer drivers and pathways using 2,500 whole cancer genomes.

Jüri Reimand. _Ontario Institute for Cancer Research, Toronto, Ontario, Canada_.

Cancer driver genes exhibit unexpectedly high mutation rates in large cancer genomic datasets. We hypothesize that driver mutations specifically alter molecular interaction networks by disrupting "active sites" - interaction interfaces in proteins and DNA. We present ActiveDriverWGS, a novel computational method to discover cancer drivers in whole-genome sequencing (WGS) data. ActiveDriverWGS finds genome regions that are significantly enriched in somatic single nucleotide variants (SNVs) and indels and ascertains whether these associate to known active sites. Analysis of active sites allows us to predict the mechanisms of mutation on three layers of the central dogma: regulatory DNA with transcription factor (TF) binding sites (TFBS), mRNA with microRNA binding sites in untranslated regions (UTRs), and post-translational modification (PTM) sites in proteins. To discover cancer driver genes and pathways, we analysed the WGS dataset of >2,500 samples from the International Cancer Genome Consortium (ICGC) Pan-Cancer Analysis Working Group (PCAWG). We found 61 protein-coding candidates with 34 known drivers (P=10-40), validating the high accuracy of our method. 40 genes have significant mutations of PTM sites, suggesting that rewiring of PTM signalling networks is a common oncogenic mechanism. For example, the BRAF V600E SNV flanks two phosphorylation sites and one ubiquitination site (FDR P=10-44), a novel interpretation and potential avenue for precision therapies targeting the kinase and ubiquitin network of BRAF. In the non-coding genome, we detected known lncRNAs (NEAT1, MALAT1), promoters (TERT, WDR74) and novel candidates with mutation enrichment. For example, an enhancer on chr6 has a mutation hotspot in 33 patients (FDR P=10-19), with 20 SNVs affecting binding motifs of cancer-associated TFs FOXO3, SOX2, HMGA2 (FDR P=10-10). Thus our method discovers non-coding drivers and their candidate mechanisms in a single analysis. Our ActiveDriverPW method extends coding and non-coding mutations to biological pathways. We found >600 mutation-enriched pathways in the PCAWG pan-cancer dataset. Of these ~200 are also significant when only non-coding mutations are analysed, showing that the non-coding genome includes previously unstudied mutations in pathways. The DNA double-strand break response pathway (FDR p=10-10) includes non-coding SNVs in ~20 histones and chromatin modifiers, such as the demethylase KDM4B with 46 SNVs in its promoter and enhancers. ActiveDriverPW maps mutations of the long tail that affect genes in hallmark cancer processes yet remain undiscovered in gene-focused analyses. Our methods accurately capture known drivers in the ICGC-PCAWG dataset and suggest specific mechanistic details. Our benchmarks also emphasize the robust performance of our methods. ActiveDriverWGS and ActiveDriverPW are valuable additions to the toolbox for cancer genome analysis.

#386

OnkoInsight: an end-to-end cancer informatics pipeline to generate insights from large sequencing datasets.

Li Tai Fang, Marghoob Mohiyuddin, Yao Fu, Lijing Yao, Narges Bani Asadi, Hugo Y. Lam. _Roche Sequencing Solutions, Belmont, CA_.

OnkoInsight is a pipeline designed to detect cancer driver genes from large sequencing datasets. It includes the somatic mutation detection module SomaticSeq, and the novel driver gene detection module GSMuta. SomaticSeq leverages an ensemble approach and machine learning to accurately detect somatic mutations. In Stage 5 of the ICGC-TCGA DREAM Somatic Mutation Challenge, SomaticSeq v1 placed #1 and #2 in the INDEL and SNV sub-challenges. In the current project, we used the improved SomaticSeq v2.2.2, which now extracts most features directly from the BAM files instead of SAMtools, and has the added function of handling multiple variant calls at the same position. We incorporated MuTect, Indelocator, VarScan2, SomaticSniper, VarDict, MuSE, and LoFreq. GSMuta detects regions, genes, and pathways that are enriched for somatic mutations. It identifies cancer drivers and distinguishes between oncogenes and tumor-suppressing genes.

As a demonstration of the capability and scalability of OnkoInsight, we deployed the tools as docker images, developed the pipeline using common workflow language, and analyzed over 1,000 TCGA lung cancer patients with tumor-normal whole exome sequencing data on Cancer Genomics Cloud. The project involved 569 adenocarcinoma (LUAD) and 490 squamous cell carcinoma (LUSC) samples.

On average, SomaticSeq detected over 700 somatic mutations per sample. The predicted mutation rate was consistent with the expected mutation rates of LUAD and LUSC. Once we obtained the high-confidence somatic mutations from SomaticSeq, we used GSMuta to detect driver genes in LUAD and LUSC separately. We detected 97 and 50 driver genes for LUAD and LUSC, respectively. To assess the quality of GSMuta's driver gene prediction, we compared the results with known lung cancer driver genes. Indeed, GSMuta reproduced 16 out of the 18 LUAD driver genes reported by TCGA's landmark study such as EGFR, KRAS, and BRAF. It reported 9 out of 10 LUSC driver genes reported by TCGA such as PTEN. LUAD and LUSC shared nine predicted driver genes, and the pathway disruption was homogenous across the two subtypes. It also detected some potential new driver genes.

The project was completed in less than a week for over 1,000 pairs of exomes on the cloud at a cost of less than ten dollars per pair. This demonstrated that our OnkoInsight pipeline is highly scalable and can be deployed to reliably analyze population-sized cancer data sets in a reasonable time frame. (L.T.F., M.M., and Y.F. had equal contribution)

#387

Targeted exome sequences of cancer-related genes in human cancers using amplicon sequencing.

Yasushi Sasaki, Takafumi Nakagaki, Miyuki Tamura, Hisayo Fukushima, Hiroshi Ikeda, Ryota Koyama, Masashi Idogawa, Takashi Tokino. _Sapporo Medical University, Sapporo, Japan_.

Objectives: Next-generation sequencing technologies have revolutionized cancer genomics research by providing a comprehensive method of detecting somatic cancer genome alterations. Platforms for genomic DNA alterations are more common in clinical practice and include whole genome/exome sequencing analysis. These tests are still very expensive, although the costs are coming down substantially. Here, we aimed to determine the efficacy and advantages of targeted exome sequencing of known cancer-related genes in human cancers using amplicon sequencing.

Methods: DNA was extracted from 61 human cancer specimens and their corresponding non-cancerous tissues, including oral squamous cell carcinomas (OSCCs) and multiple myelomas (MMs). Forty nanograms of DNA were used for multiplex PCR amplification with an Ion Ampliseq Comprehensive Cancer Panel that offers targeted coverage of all exons in 409 tumor suppressor genes and oncogenes frequently cited and frequently mutated in human cancers. This platform was designed to be amplification based capture with 15,992 regions (1.6 megabases in total size). Purified DNA libraries were sequenced with 6-8 samples on Ion Proton P1 chip. Sequence reads of tumor and normal samples were aligned to the hg19 genome, and generated BAM files were used to detect somatic mutations (point mutation, insertion and deletion) and copy number variations.

Results: Each sample underwent on mean 8.4 million sequencing reads after quality filtering. The mean base coverage depth was 530, and >95% of targeted bases were represented by at least 20 reads. The number of non-synonymous somatic mutations in 47 patients with OSCC ranged from 1 to 21 with a mean of 7.5 (6.4/Mb). The most frequent mutations in OSCC were in TP53 (63.8%), NOTCH1 (25.5%), CDKN2A (19.2%), TAF1L (17.0%), SYNE1 (14.9%) and PIK3CA (8.5%). We also detected a mean of 6.1 (range 3-11) non-synonymous mutations per MM patient. Somatic mutations were found in known MM-associated genes, including TP53 and NRAS. Pathway assessment has shown that somatic aberrations within MM genomes are mainly involved in several important pathways, including cell cycle regulation, RTK-MAPK-PI3K and NF-kB. We found several genetic alterations that may have been associated with the poor prognosis and poor response to chemotherapy of MM patients.

Conclusions: This study demonstrates the utility of using a semiconductor-based sequencing to efficiently identify somatic genetic alterations in human cancers. The targeted next-generation sequencing using low amounts of FFPE DNA is a valuable tool for rapid (5 days) and high-throughput genetic testing in research and clinical settings.

#388

A benchmark study for identifying cancer drivers in the non-coding part of the genome.

Damien Drubay,1 Daniel Gautheret,2 Stefan Michiels1. 1 _INSERM, Gustave Roussy, Villejuif, France;_ 2 _Université Paris-Sud, CNRS, CEA, Gif-sur-Yvette, France_.

Purpose: Prioritizing potential deleterious variants is an essential task to guide research and validation of new pathological variants in the immensity of the genome. Many tools have been introduced to detect new variants in the coding part of the genome. Detailed knowledge of coding sequences led to efficient statistical models for cancer driver discovery. The challenge is greater for the non-coding part of the genome due to its large size (>98% of the genome) which contains many non-functional or unknown features. Several deleteriousness scores have been proposed in the last decade, but no large-scale comparison has been realized to date to assess their ability to identify cancer drivers.

Material and method: We compared the leading scoring systems (CADD, FATHMM-MKL, Funseq2 and GWAVA) and some recent competitors (DANN, SNP and SOM scores) for their ability to discriminate assumed pathologic variants in the non-coding genome (as identified by 928 ClinVar variants / 44,158 recurrent COSMIC mutations) from assumed non-pathologic variants (100,000 randomly sampled 1000 Genomes project variants with minor allele frequency > 1%). To define the pathogenic variants using COSMIC as reference, we varied the threshold for number of COSMIC recurrences from 2 to 10. We compared the sensibility, specificity and precision of the scoring systems using the area under the curve (AUC) of receiver operating characteristic (ROC) and precision-recall (PR) curves.

Results: Most scores had good sensibility and specificity for the detection of the ClinVar variants (AUCROC>0.90). As far as precision for ClinVar variants was concerned, the top performing methods were CADD (AUCPR=0.84), DANN (AUCPR=0.83) and, to a lesser extent, FATHMM-MKL (AUCPR=0.75).

When using a threshold of 3 recurrences to define true pathogenicity of COSMIC variants, the AUCROC ranged from 0.52 (DANN) to 0.80 (GWAVA) but precision was low with AUCPR ranging from 0.05 (DANN, SOMmelanoma) to 0.18 (GWAVA). Increasing the pathogenicity threshold to 10 recurrences increased AUCROC values (ranging from 0.50 (SOMmelanoma) to 0.89 (GWAVA)) but decreased precision values (AUCPR ranging from 0 to 0.02).

Discussion: This large scale benchmark study distinguished CADD as the best tool to detect variants with features similar to those of ClinVar, which are mainly located in protein coding regions. However, based on the results using COSMIC, GWAVA outperformed CADD for variants in other regions, including lincRNAs, pseudogenes and other parts of the genome "dark matter", for which there is increased interest. This should nevertheless be balanced by the potential presence of non-pathologic variants in the COSMIC database due to sequencing errors and limitation of the recurrence criteria to define pathologic status in the instable fragile genome regions. The development of a gold standard as consistent as ClinVar for these regions will be necessary to confirm our tool ranking.

#389

Integrating diverse transcriptomic alterations to identify cancer-relevant genes.

Natalie R. Davidson,1 PanCancer Analysis of Whole Genomes 3 (PCAWG-3) for ICGC, Alvis Brazma,2 Angela N. Brooks,3 Claudia Calabrese,2 Nuno A. Fonseca,2 Jonathan Goke,4 Yao He,5 Xueda Hu,5 Andre Kahles,1 Kjong-Van Lehmann,1 Fenglin Liu,5 Gunnar Rätsch,1 Siliang Li,6 Roland F. Schwarz,7 Mingyu Yang,5 Zemin Zhang,5 Fan Zhang,5 Liangtao Zheng5. 1 _ETH Zurich, Zurich, Switzerland;_ 2 _European Molecular Biology Laboratory - European Bioinformatics Institute, Hinxton, United Kingdom;_ 3 _University of California, Santa Cruz, Santa Cruz, CA;_ 4 _Genome Institute of Singapore, Singapore, Singapore;_ 5 _Peking-Tsinghua Center for Life Sciences, Beijing, China;_ 6 _BGI-Shenzhen, Shenzhen, China;_ 7 _Berlin Institute for Medical Systems Biology,, Berlin, Germany_.

Introduction:

We present a novel method to identify cancer driver genes that jointly examines any number of diverse transcriptomic alterations with the goal to uncover highly recurrent and heterogeneous patterns in 1190 samples across 26 cancer types as part of the PanCancer Analysis of Whole Genomes (PCAWG) of the International Cancer Genome Consortium (ICGC).

Motivation:

Previous pan-cancer genomic studies have focused on the analysis of somatic mutations as the driver of phenotypic changes. Here, we propose a method to integrate a wide variety of RNA and DNA changes to redefine the concept of driver events and account for the transcriptome's role in tumorigenesis. PTK2 provides a motivating example, since it has many RNA alterations that correlate with patient survival, such as overexpression, exon-skips, and alternative promoter usage.

In our analysis, we integrate an unprecedented amount of various alterations including gene fusions, RNA editing, alternative splicing, expression outliers, alternative promoters, allele specific expression, and somatic mutations. This enables us to also identify mutually exclusive (MutE) and co-occurring (CoO) patterns between different types of alterations within a gene.

Methods:

Our method has 3 main strengths: flexibility to handle any number or type of alteration, sensitivity to different frequencies of alterations so rare events are not lost in the recurrence analysis, and diversity of ranking such that genes with multiple alterations are prioritized. Our method is summarized in two steps:

1) Identify genes that are both recurrently and heterogeneously altered across many samples by calculating a rank-based score for each gene.

2) Identify MutE and CoO patterns between alteration types for the genes identified in the previous step.

To ensure that alterations were comparable, we applied a thresholding model to binarize all alterations for gene-sample pairs, allowing us to account for the properties of the different modalities involved.

Step 1 of our method calculates a score for each gene that takes into account: 1) the number of alterations to a gene across all samples, 2) the rarity of each alteration, and 3) how many types of alterations are observed per gene. The score is then used to rank the genes and top genes are considered for MutE and CoO analyses.

Results:

Our top 100 ranked genes were highly enriched for cancer census genes (adjusted p-value: 2.06e-9), indicating that we identify cancer relevant genes. Our top five ranked cancer census genes were IGF2, ERBB2, RARA, CREBBP, and ARID1A; all of which had at least 4 of 7 possible alterations, showing our scoring method prioritizes genes with diverse alterations. We also found that alternative promoter usage and alternative splicing were highly co-occurring alterations, with PTK2 having the highest co-occurrence between them. In summary, we propose a new method to analyze various RNA disruptions and show it can yield new insights beyond genomic variation.

#390

Identification and characterization of survival-associated genomic features across tumor types.

Joan Smith,1 Ann Lin,2 Chris Giuliano,2 Jason M. Sheltzer2. 1 _Google, New York, NY;_ 2 _Cold Spring Harbor Laboratory, Cold Spring Harbor, NY_.

The basic biology that differentiates a primary cell from a transformed cell has been elucidated over the past several decades. Mutations in a limited number of oncogenes and tumor suppressors free the cell from growth-inhibitory checkpoints and allow proliferation in the absence of external stimuli, leading to tumor formation. Yet, primary tumors themselves are rarely deadly: greater than 90% of cancer mortality results from metastasis. What drives metastasis, and, more broadly, what distinguishes a lethal cancer from one with a favorable prognosis, is poorly understood.

To address this question, we have performed a comprehensive meta-analysis on genomic data from primary tumors that are linked to patients' clinical outcomes. Utiliizing data from >20,000 patients, we have identified protein-coding genes, lncRNAs, miRNAs, methylation sites, and CNVs in primary tumors that are significantly associated with patient prognosis across cancer types. Multivariate analysis reveals a hierarchy of survival determinants: the strongest mortality-associated factors are enriched for components of the mitotic cell cycle, while secondary clusters of genes are involved in extracellular matrix remodeling, cell motility, and angiogenesis. Survival-associated genomic features are indicative of immune infiltration into primary tumors and oxidative phosphorylation activity.

In vitro and in vivo analyses reveal that mortality-associated genes rarely promote the direct transformation of primary cells. Instead, CRISPR mutagenesis reveals that these genes largely represent tumor dependencies, and in their absence cancer cells fail to proliferate. Through this analysis, we have identified new genetic dependencies common across cancer cells, including C16ORF59 and C5ORF46. In total, our results represent the largest assessment of genomic features linked to cancer prognosis completed to date, and offer several lines of insight into the biological differences between fatal and benign cancers.

#391

A comparative genomics approach to understanding the control of cell context dependent P53 binding.

Varsha Sundaresan,1 Ying Li,2 Benedetto DiCiaccio,1 Victor T. Lin,1 Lei Zhou1. 1 _University of Florida, Gainesville, FL;_ 2 _Macau University of Science and Technology, Macau, China_.

Cancer is considered to be a genetic disease characterized by sequential accumulation of mutations. Recent studies have provided evidence that epigenetic changes and regulatory sequence mutations could also dysregulate oncogenes and tumor suppressors. As a transcription factor, P53 is activated in response to oncogenic stress and exerts distinct anti-proliferative functions based on the stressor and cell type. Though a number of ChIP-Seq studies have identified thousands of P53 binding sites in mammalian genomes, the functionality of these binding sites remains to be established. In addition, we know little about what controls stress and cell context specific binding profile of P53.

Traditionally, mutations in the coding regions of p53 have been extensively studied to gain insights on its role in cancer and to identify strategies to restore the functions of p53 in cancer cells. We hypothesize that mutations in or epigenetic silencing of functionally important P53 binding sites play an important role in tumorigenesis as well. Since functional regulatory regions tend to be more evolutionarily conserved, in this project we propose using a comparative genomics approach to identify functional P53 binding sites and determine if these regions are involved in tumorigenesis.

In order to study how P53 binding following DNA damage differs between cell types, we aim to perform comparison between P53 ChIP-Seq data generated in our lab from Drosophila embryos at different developmental stages and a cell line (Kc167) as well as published datasets from mammalian stem cells and differentiated cells. Differential expression analysis using RNA-Seq exhibited that at an early stem cell-like stage there is P53-dependent induction of pro-apoptotic genes in response to DNA damage but not in the differentiated stages. We are seeking to establish functionally significant P53 binding sites by combining ChIP-Seq data and gene expression data obtained using RNA-Seq. The importance of these binding sites will be verified by CRISPR-Cas9 -mediated genome editing. We will also perform comparison studies of DNA damage-induced P53 binding in human, mouse and Drosophila to identify analogous patterns.

Knowledge gained from this study will help us to understand the role of non-coding regulatory regions in tumorigenesis, and predict patient response to apoptosis inducing therapeutic agents. It may also lead to novel strategies to restore cellular sensitivity to chemotherapy or radiation.

#392

Application of somatic mutation-based expression profiles for high-throughput phenotyping in cancer.

Frederick S. Varn, Yue Wang, Chao Cheng. _Geisel School of Medicine at Dartmouth College, Hanover, NH_.

Somatic mutations have been heavily implicated in initiating and driving cancer growth and metastasis. These alterations serve as upstream events that have broad transcriptional effects by altering protein structure and function and interfering with signaling pathways. Here, we apply an iterative approach that utilizes patient gene expression profiles to define mutation-associated transcriptomic signatures. We use these signatures to quantify the level at which a given somatic mutation affects its gene's downstream activities. Using TP53 in breast cancer as an example, we show that while TP53 mutations are the primary predictor of altered p53 activity, events such as copy number alterations and methylation changes may represent alternative routes to high p53 disruption. Conversely, we identify a class of TP53 mutations that have minimal downstream transcriptomic effects. By expanding this approach to other genes and tumor types, we show that mutation signatures derived from different tissue types exhibit varying levels of conservation, suggesting a degree of tissue-specificity in the downstream effects of somatic mutations. To provide functional context to our results, we examine the associations between different mutation signatures and various cancer-related features, including patient survival, immune infiltration, and pathway activity. Together, our results indicate the utility of using patient gene expression profiles to study mutation function. Going forward, these results may be useful in functionally annotating variants of unknown significance and broadening the potential of precision medicine-based therapeutic approaches.

#393

Predicting DNA accessibility in the pan-cancer tumor genome using RNA-Seq, WGS, and deep learning.

Kamil Wnuk,1 Jeremi Sudol,1 Shahrooz Rabizadeh,1 Patrick Soon-Shiong,2 Christopher Szeto,3 Charles Vaske3. 1 _NantOmics, Culver City, CA;_ 2 _NantWorks, Culver City, CA;_ 3 _NantOmics, Santa Cruz, CA_.

DNA accessibility, chromatin regulation, and genome methylation are key drivers of cancer transcription. However, there is much left to be understood about the functional implications of sequence-level data to the regulation of gene expression, especially when it comes to the noncoding genome. Recently [Kelley, D., Snoek, J., and Rinn, J., Genome Res. 2016] trained neural networks to effectively predict DNA accessibility in multiple cell types. These models make it possible to explore the impact of mutations on the predicted accessibility and thus directly link one aspect of the gene regulation puzzle all the way down to the sequence level. We present a model with improved performance on the original dataset of 164 ENCODE and Roadmap Epigenomics Consortium sample types, and then extend the method to provide predictions on any sample with RNA-Seq data without need of DNase-seq for the sample.

We first demonstrate that with several model and algorithmic changes we improve performance across 164 cell types from a mean AUC of 0.895 to a mean AUC of 0.910.

Unfortunately current accessibility models require DNase-seq for each new cell type. Models for detecting transcription factor binding sites, which rely on ChIP-seq for training data, also share this issue. In order to generalize sequence-based predictive models to apply to unseen cell types without requiring re-training we investigate using RNA-Seq as a proxy signature of cell type. The model aims to capture the interdependence of gene expression levels that characterize a cell with the regulatory logic in which sequence-level signatures are combined to determine accessibility without restriction to cell type. We explore the model's performance when applied to held-out cell types in the ENCODE and Roadmap Epigenomics Consortium data as well as data from the TCGA Pan-Cancer initiative. We look for the impact of non-coding changes in whole-genome sequencing data in TCGA samples, and report on predicted differences in DNA accessibility across cancer subtypes.

#394

Saturation mutagenesis of KRAS reveals the functional landscape of missense variants.

Eejung Kim,1 Seav Huong Ly,1 Nicole S. Persky,1 Belinda Wang,1 Xiaoping Yang,1 Federica Piccioni,1 Katherine Labella,1 Mihir Doshi,1 Robert E. Lintner,1 Cong Zhu,1 Scott Steelman,1 David E. Root,1 Cory M. Johannessen,1 Alex B. Burgin,1 Laura E. MacConaill,2 William C. Hahn,1 Andrew J. Aguirre1. 1 _Broad Institute, MA;_ 2 _Dana-Farber Cancer Institute, MA_.

KRAS is the most commonly mutated oncogenes and is a major driver of tumor initiation and progression. Understanding the functional consequences of cancer-associated KRAS variants may have important clinical implications. For example, KRAS mutation status defines those that are likely to respond to EGFR-directed therapy in KRAS-mutant metastatic colorectal cancer. A compendium of all possible oncogenic KRAS alleles would serve as a roadmap for future therapeutic strategies directed at KRAS itself or downstream signaling effectors. Comprehensive mutagenesis of KRAS may also elucidate structure-function relationships that reveal novel biochemical properties that may be exploited for therapeutic gain. We performed saturation mutagenesis of both a wild-type (WT) and a G12D mutant form of KRAS cDNA and generated lentiviral expression libraries of 3,553 and 3,534 single amino acid substitution mutants of each backbone. We utilized these WT and G12D mutagenesis libraries for functional genetic screening to identify gain- and loss-of-function missense variants that alter critical oncogenic properties of KRAS. First, we sought to comprehensively identify all possible oncogenic missense mutations in KRAS that mediate oncogenic transformation. We stably transduced the WT library into immortalized human epithelial cells and evaluated growth in low attachment (GILA), an assay that is highly correlated with in vivo tumor formation. We identified all previously known hotspot oncogenic alleles of KRAS as well as many functionally relevant alleles that are also discovered at lower frequency in human tumors. Moreover, we also discovered a group of transforming KRAS variants that have not been well described in human tumors, thus revealing potentially novel activating mechanisms for oncogenic KRAS. In parallel, we utilized the G12D mutagenesis library to perform second-site suppressor screening to identify loss-of-function single amino acid changes that abrogate the transforming ability of oncogenic KRAS. We performed positive-selection screening in primary cell lines for variants that enable bypass of oncogene-induced senescence. Additionally, we conducted a negative-selection screen with the G12D library in a KRAS-dependent cancer cell line with inducible suppression of endogenous KRAS, thus identifying all possible second-site mutations that abolish KRAS-driven signaling necessary for maintenance of cellular proliferation and viability. Structure-function analysis of these data may reveal novel patterns of amino-acid changes that result in inactivation of oncogenic KRAS. In summary, this comprehensive dictionary of gain- and loss-of-function KRAS mutants will facilitate understanding of clinically important mutations and also yield novel insights into structure-function relationships that may improve our understanding of the KRAS oncogene.

#395

Activation mechanisms of cancer associated MEK1 mutants.

Yijun Gao,1 Matthew T. Chang,1 Daniel McKay,2 Rona D. Yaeger,1 Merna Torres,1 Keven Muniz,1 Drosten Matthias,3 Omar I. Abdel-Wahab,1 Mariano Barbacid,3 Giordano Caponigro,2 Darrin Stuart,2 David Solit,1 Barry S. Taylor,1 Zhan Yao,1 Neal Rosen1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Novartis Institutes for BioMedical Research, Cambridge, MA, USA., MA;_ 3 _Centro Nacional de Investigaciones Oncológicas, Madrid, Spain., Spain_.

Activating BRAF mutants drive human tumors by dysregulating ERK signaling despite ERK-dependent feedback suppression of RAS. These RAF mutants become RAS independent, by either of two mechanisms, and they are thus unaffected by feedback inhibition of upstream signaling. Recently, it has become clear that mutation activation of MEK1 or MEK2 occur at appreciable frequency in human tumors, but the mechanism of activation of these mutants and whether they remain dependent on upstream activation of RAS or RAF signaling remains unknown. Here we characterized the mechanism of activation of 18 recurrent MEK1 mutants identified in human cancer. Based on these data, they fall into three classes. The kinase activity of first class remains dependent on RAF mediated phosphorylation of S218 and S222; the second class of MEK mutants has basal RAF-independent activity, but can be further stimulated by RAF and the third class no longer requires phosphorylation of S218 and S222 and signals in a RAF independent manner. These features determined their sensitivity to ERK-dependent feedback regulation and the ability to drive ERK signaling output in cells. The more RAF-independent activity the mutants acquired, the better they could activate downstream ERK pathway, leading to increased transforming activities in MEF cells in the absence of RAF. This is consistent with their genetic association with RAS, RAF and NF1 mutations in human tumors. The Class 3 RAF-independent MEK1 mutants tend to be mutually exclusive with those mutants, while coexistence with RAS/RAF/NF1 mutations is frequently observed in tumors with Class 1 or 2 MEK1 mutants. Moreover, functional class correlated with sensitivities of ERK signaling driven by these mutants to MEK inhibitors that function by different mechanisms. Unlike RAF dependent or regulated MEK1 mutants, the ERK signaling driven by RAF independent MEK1 mutants is insensitive to an allosteric MEK inhibitor that functions by preventing RAF mediated MEK phosphorylation. However, signaling driven by all classes of MEK1 mutants is sensitive to an ATP competitive MEK1 inhibitor which targets MEK1 kinase activity.

#396

Detecting copy number variations using WES datasets in patient derived xenografts.

Jia Xue,1 Wubin Qian,1 Sheng Guo,1 Jie Cai,1 Henry Qixiang Li2. 1 _Crown Bioscience, Inc., Santa Clara, CA;_ 2 _Crown Bioscience, Inc., Beijing, China_.

Amplification or deletion of oncogenes and tumor suppressors can be oncogenic, which may serve as drug targets and biomarkers for disease prognosis and drug response. Traditionally, array-based assays, e.g. Affymetrix SNP6.0® and lately OncoScan®, have been used to detect DNA copy number variations (CNVs). These assays have limitations including insufficient probe availability for some genes, cross hybridization, imprecise measurement of fluorescent signal, less suitability of tumor samples, and high cost. Whole exome sequencing (WES) is now widely used to profile tumor samples, and provides a fast and efficient determination for point mutations, insertions and deletions at the DNA level. Recently, WES is being used to profile CNVs with some success, but also suffers from many drawbacks, such as the requirement of paired normal tissues, the need of a large batch of samples, the inadequacy of detecting chromosomal-level CNVs, the incapability to detect CNVs in low coverage genomic regions. We have developed a CNV detection pipeline on both genomic (segment) level and gene level from WES data using the concept of off-target and on-target reads1, and evaluated it in a set of 155 patient-derived xenografts (PDXs), with head-to-head comparison to Affymetrix SNP6.0® and OncoScan® on 5 models with 2 passages from each. PDX is a well-accepted experimental model mimicking original patient in histo- & molecular pathology2. Reads derived from mouse contaminants were removed from WES datasets to avoid mouse signal interference, which cannot be done in array-based techniques. RT-PCR was used to experimentally validate CNVs for selected genes. We found that the average off-target rate for our PDX models is approximately 15%, and off-target reads were uniformly distributed across genome. The comparison with array-based technologies indicates that 1) WES has the highest resolution (20kb) while OncoScan® is the second (50-100 kb), followed by SNP6.0® (100-200kb), 2) the OncoScan® CNV calls are very similar to our WES methods at the genome level, yet 3) WES gives a much higher accuracy on CNV inference for genes flanking the ~900 cancer related genes with enhanced probe densities on OncoScan®, suggesting that our WES method has the highest accuracy. RT-PCR results confirmed the observations. In summary, our WES-based approach gives a better solution in CNV detection in PDX models.

References

1. Kuilman T, Velds A, Kemper K, Ranzani M, Bombardelli L, Hoogstraat M, Nevedomskaya E, Xu G, de Ruiter J, Lolkema MP, Ylstra B, Jonkers J, et al. CopywriteR: DNA copy number detection from off-target sequence data. Genome biology 2015;16:49.

2. Guo S, Qian W, Cai J, Zhang L, Wery JP, Li QX. Molecular Pathology of Patient Tumors, Patient-Derived Xenografts, and Cancer Cell Lines. Cancer research 2016;76:4619-26.

#397

Whole genome copy number variation analysis using a SNP-focused targeted sequencing panel for tumor analysis.

Jiashi Wang,1 Kristina Giorda,1 Zhongwu Lai,2 Daniel Stetson,2 Mirna Jarosz1. 1 _Integrated DNA Technologies, Redwood City, CA;_ 2 _Astrazeneca, Boston, MA_.

Accurate genome-wide copy number variation (CNV) analysis is critical for disease and cancer research. Current approaches for CNV analysis include fluorescence in situ hybridization (FISH), array comparative genomic hybridization (array CGH), and SNP arrays. Unfortunately, these methods are not sensitive enough for real world cancer samples because of tumor ploidy, purity and heterogeneity. NGS-based targeted sequencing is increasingly being used for CNV analysis due to throughput, coverage, cost, and sample input requirements. For CNV analysis, detection power is improved by combining both read depth and SNP allele frequency analysis, particularly for copy-neutral events such as loss of heterozygosity. A custom xGen Lockdown CNV backbone panel was developed for broad, uniform genome coverage and to enrich for population-based SNPs. We demonstrate use of the panel as an addition to the xGen Exome Research Panel and a custom cancer focused panel. Downstream analysis incorporates both read depth and observed minor allele frequencies to determine CNVs with enhanced sensitivity. To increase the resolution for large-scale alterations of chromosome 7, a hot-spot for disease-associated CNVs, probe density was increased 6 fold. A known standard, NA12878, was used to validate the panel's ability to detect heterozygous SNPs with high confidence. In addition, mixtures of cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE) were tested with varying levels of background copy-neutral genomic DNA. The sensitivity and specificity of the panel to detect CNV and LOH events with was assessed using deep exome and Affymetrix SNP array data. The ability to detect copy number alterations with high resolution and accuracy would be a valuable resource for disease and cancer research.

#398

Using off-target data from comprehensive genomic profiling to characterize the genomic architecture of copy number alterations in tumor sequencing data.

Caitlin F. Connelly, Zachary R. Chalmers, Philip J. Stephens, Garrett M. Frampton. _Foundation Medicine, Inc., Cambridge, MA_.

Background

Copy number alterations are common driver mutations in cancer. Identifying genes which show altered copy number and knowing the breakpoints of copy number alterations gives insight into their functionality as well as the mechanisms responsible for the alterations. While copy number alterations have been well-studied in common tumor types, there are still many rare tumor types which have not been characterized. Targeted genomic profiling using capture methods results in sequencing a subset of genes to extremely high coverage (>500x) and sequencing off-target regions spanning the rest of the genome to low (0.5x – 1x) coverage. We used this data to identify the approximate breakpoints of alterations that extend beyond targeted regions and to identify novel, recurrent copy number alterations in clinical tumor samples.

Methods

We used standard methods to identify copy number alterations in the off-target regions. Briefly, we calculated coverage in 10 kb windows across the genome, excluding the targeted regions which are sequenced at high coverage, corrected for GC-bias, calculated the log ratio of coverage for the tumor compared to an unrelated normal sample, and then used a segmentation algorithm to segment the genome into copy number regions and identify regions with significantly different copy number than the surrounding regions.

We sequenced cell line DNA with known copy number variants and used this data to assess the accuracy and precision of these methods. Copy number alterations were filtered against known common copy number variant regions, and we identified copy number alterations overlapping known genes to identify potentially functional alterations.

Results

We applied these methods to tumor samples that underwent comprehensive genomic profiling as part of clinical care. Our cohort consisted of a large and diverse set of "real world" unbiased clinical specimens and contained many rare and unusual tumors not included in larger sequencing efforts. Using this data, we characterized the landscape of copy number variants genome-wide in rare tumor types which have not previously been characterized and identified unique copy number alterations in these tumor types. Further, we characterized the breakpoints of copy number alterations across the cohort.

Conclusions

We will present novel copy number alterations in rare tumor types and the recurrent breakpoints for common alterations across multiple tumor types. These findings will provide insight into the mechanisms of cancer progression.

#399

CPTAC phase II final report.

Mathangi Thiagarajan. _Leidos Biomedical Research Inc., Rockville, MD_.

National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium (CPTAC) is a comprehensive and coordinated effort to accelerate the understanding of the molecular basis of cancer through the application of robust, quantitative, proteomic technologies and workflows. The primary goal for this program is to systematically identify proteins that derive from alterations in cancer genomes and related biological processes, integrate the genomic and proteomic data from analysis of common cancer biospecimen and provide this data with accompanying assays and protocols to the community. The phase II effort managed by the Leidos Biomedical Research Inc. had a goal to procure and qualify a minimum of 100 cases from 3 cancer types (Breast, Ovarian and Colon). Tissues and analytes from the 100 cases were sent to proteomic and genomic characterization centers for analysis. The proteomic data were made available through the Data Coordinating Center (DCC) and the genomic data were made available through NCI's Genomic Data Commons (GDC). The success from the phase II effort has set precedence for a much larger phase III effort that will focus on additional cancer types and cases. Preliminary analysis results confirm the need for the combination of the genomic and proteomic approaches to produce a more comprehensive inventory of the detectable proteins in a tumor and advance our understanding of cancer biology.

#400

NCI's CPTAC Phase III, proteogenomic analysis of additonal cancer types.

Linda I. Hannick. _Frederick National Lab for Cancer Research, Rockville, MD_.

The Clinical Proteomics Tumor Analysis Consortium (CPTAC) is a National Cancer Institute initiative that seeks to uncover the molecular basis of cancer using a proteogenomic approach to study prospective cancer specimens. Leidos Biomed provides an infrastructure for supporting the collection of high quality biospecimens and data, in addition to project and subcontract management for the program. CPTAC applies the understanding of the molecular basis of cancer to identify biomarker candidates. Phase II of CPTAC, completed in 2016, collected over 500 cases from breast, colon and ovarian patients. In early 2016, CPTAC Phase III began to collect and analyze 200 cases of each of ten additional cancers. The goal is to collect 200 qualified cases of five to six tumor types over a five-year period, although the program is currently accruing from ten. The study entails collection and pathology evaluation of biospecimens, high-quality clinical data and images from clinical sites around the world. A biorepository evaluates and processes the biospecimens, sending nucleic acids to a sequencing center and tissues to proteomics groups. Data are combined and analyzed by translational centers. Genomic data are made available to the research community through the NCI Genomic Data Commons. Proteomic data are made available through the Data Coordinating Center. We report here on progress in collection of tissues and clinical data, and use of biorepository, proteomics and genomics and analysis centers. 

### Genomic Screening for Cancer Dependencies and Drug Response

#401

Designing for success: the right CRISPR design strategies for the right experiment.

Leigh Brody, Neil Humphryes-Kirilov, Victor Dillard, Riley Doyle. _Desktop Genetics, London, United Kingdom_.

CRISPR design is a complex multivariate problem. The weight of each variable differs depending on the experiment under consideration. Current methods of designing CRISPR experiments are based on a single-variable, resulting in poor experimental outcomes. From the numerous experiments enabled by the DESKGEN platform, we can demonstrate key design improvements to maximise experimental outcome.

Our current scoring methods were tested and validated using an sgRNA library of over 3,500 sgRNAs in essential genes with a variety of predicted sgRNA activities and specificities. The predicted 'good' sgRNAs achieved significantly higher read depletion for essential genes, with fewer failing sgRNAs, compared to sgRNAs that didn't meet the scoring criteria. Our scoring algorithm incorporates multiple published observations for choosing effective sgRNAs, which differs from other CRISPR tools that tend to focus on one factor such as sgRNA sequence or specificity. We were unable to find correlations for some currently accepted sgRNA selection criteria, such as position within a gene, which demonstrates the need to constantly challenge and improve sgRNA design rules.

#402

Biomarker-based pooled CRISPR-Cas9 screening: Platform development and validation.

Tim M. Scales, Benedict C. Cross. _Horizon Discovery, Cambridge, United Kingdom_.

Functional genomic screening with CRISPR-Cas9 has provided a powerful and precise new way to interrogate the phenotypic consequences of gene loss in high-throughput, unbiased analyses. Rapid development of pooled lentivirus and deep-sequencing-led approaches have allowed us and others to exploit this technology in target ID, target validation, drug MOA analysis and patient stratification. Adaptation of the Cas9 nuclease by domain fusion and catalytic inactivation has further provided a novel way to both up and down-regulate gene expression using targeted, high-throughput CRISPRi and CRISPRa tools. To date, the vast majority of screens have been conducted using sgRNA abundance in surviving cell populations as an NGS-based proxy for proliferation, for example for in vitro resistance analysis to a cytotoxic treatment regimen. However, where cellular pathophysiology is uncoupled from cell health, the enormous promise of pooled CRISPR-Cas9 screening has been largely untapped. We now report on the development of a FACS-based platform that can be used to monitor biomarker-based cellular response in combination with high-throughput genetic screening. The endogenous interaction between CD28 and CD80 was used as a proof-of-concept paradigm and the performance of our platform was measured in screen-simulation conditions. We find that FACS-linked NGS was able to robustly identify the CD28 deleted cells from a complex population using a FITC-labelled CD80-Fc. Pseudo-blinding of the data yielded unambiguous identification of the expected target guides, with robust screening metrics, validating a new platform for myriad discovery programmes.

#403

CRISPR/Cas9 genome-wide sgRNA libraries for loss-of-function and gain-of-function genetic screens.

Donato Tedesco, Paul Diehl, Mikhail Makhanov, Sylvain Baron, Alex Chenchik. _Cellecta, Inc., Mountain View, CA_.

Genome-wide loss-of-function screening is a fundamental method to identify genes responsible for driving biological processes. Complex pooled lentiviral-based libraries expressing large numbers of genetic disruptors, such as shRNAs (RNAi) or sgRNAs (CRISPR), make large-scale cell screening practical. While RNAi-based approaches have proven to be an effective strategy for these screens, recent work has showed CRISPR-based technologies offer not only an effective alternative, but distinct advantage. We will present unpublished data from genetic screens in human cancer cell lines using CRISPR-Knockout (KO), CRISPRi and CRISPRa technologies, as well as RNAi. Although loss-of-function shRNA and sgRNA pooled library screens are similar in concept, the gene loss of function is achieved by different mechanisms (mRNA degradation with RNAi, full gene disruption with CRISPR-KO, transcriptional inhibition with CRISPRi), so some divergences are expected and indeed observed when comparing results obtained using one method versus the other. Furthermore, contrary to RNAi, CRISPR technology can be modified to activate gene expression (CRISPRa), thus enabling the use of genome-wide gain-of-function screening in gene function studies.

#404

A bi-clustering based computational approach to identify molecular markers for Neoadjuvant chemotherapy outcome prediction in complement to cancer subtype classifications.

Chi Zhang,1 Sen Liang2. 1 _Indiana University, School of Medicine, Indianapolis, IN;_ 2 _Jilin University, Chang Chun, China_.

Molecular stratification models have been well established for most cancer types to classify tumors into sub-types by using genome level molecular characteristics. Tumors of certain sub-type have distinct response to specific therapies. Hence a reliable molecular classification model can be applied to determine treatment strategy. Recent neoadjuvant chemotherapy, immunotherapies and gene targeted drugs and their possible combinations raise a demand for small subsets of novel molecular markers for outcome prediction in addition to the complete molecular profile based classification. However, it is unknown what genes have predictive power for a certain therapeutic strategy while the highly crossed clinical features increase the difficult to determine sample groups for marker identification that form a bi-clustering problem. In addition, predictive maker may be varied among different cancer sub-types. In this work, we developed a bi-clustering based computational framework to identify transcriptomic markers for predicting the outcome of neoadjuvant chemotherapy and adjuvant chemotherapy. Our computational approach identifies functional related gene expression modules show significant expression patterns in a subset of samples that are highly associated with one or several disease sub-types and with high predictive power for the outcome of certain. By applying the analysis to 8 breast carcinoma, 10 colorectal carcinoma and 3 acute myeloid leukemia data sets in public domain, we have identified sets of gene markers with high predictive power for chemotherapy outcome complementary to the existing molecular classifications, suggesting the bi-clustering based approach can improve the whole genome level molecular classification based outcome prediction.

#405

A novel panel of 325 biomarkers is part of a large interconnected network representing multiple cell signaling pathways and allowing development of predictive tests for oncology drugs.

Edward C. Goodwin, Said Attiya, Marcia Fournier. _BioArray Genetics, Inc., Farmington, CT_.

Background: We developed an algorithm based on the gene expression of tumor biopsies to identify the best combination of biomarkers to reliably predict a patient's response to relevant cancer treatments. This algorithm is derived from 325 genes whose expression showed significant changes during differentiation of non-malignant human mammary epithelial cells cultured in laminin-rich extracellular matrix. Of these 325 genes, 251 are novel and not present in 9 other cancer based gene expression panels such as FoundationOne or PAM50. These differentiated cells formed multicellular structures with defined lumens and tight junctions and with specific localizations of cadherin and integrins. In contrast, cells from breast cancers displayed a general loss of structure. Previous work showed that different sets of these 325 biomarkers accurately predicted overall breast cancer patients' survival or response to neoadjuvant therapy in multiple independent studies.

Objective: Predict cell-signaling pathways, drug associations, and disease associations for the 325 biomarkers (BA325) in contrast to other cancer gene panels.

Methods and Results: The Qiagen Ingenuity program was used to identify pathways and disease states containing significant overlap with BA325. Multiple cell signaling pathways including cell proliferation, migration, invasion, and metabolism were found in BA325, while most other cancer biomarker panels were highly concentrated in cell proliferation. Examples of significant pathway associations relevant for oncology drug discovery and targeted treatments include Cell Cycle Control of Chromosomal Replication (p=8.1E-14), Polo-like-kinase and HSP90 complex (p=6.3E-07), G2/M DNA Damage Checkpoint (p=6.4E-07), Integrin Signaling (p=3E-05), Integrin Linked Kinase Signaling (p=4.51E-05), BRCA1 DNA Damage Response (p=1.75E-04), Estrogen Mediated S-phase entry (p=4.24E-04), Regulation of Actin Based Motility by RHO (p=4.55E-04), Adherins Junction Remodeling (p=5.6E-04), Actin Cytoskeleton Signaling (p=7.25E-04), Mismatch Repair (p=1.6E-03), RAN Signaling (p=1.64E-03), Protein Ubiquitination (p=1.78E-03) and Cholesterol Biosynthesis (p=4.8E-03). This wide coverage of cellular signaling pathways provides an advantage in developing predictive tests for treatments targeting highly complex cell signaling networks. Surprisingly, even though the BA325 panel was obtained during non-malignant differentiation, the most significant disease state associated with these genes was mammary tumor, demonstrating the validity of this approach to probe breast cancer biology.

Conclusion: This analysis demonstrates that the BA325 panel is useful both in understanding non-malignant mammary epithelial differentiation and breast cancer tumors.

#406

CRISPR pooled screening of hundreds of cancer cell lines identifies differential dependencies on epigenetic pathways and synthetic lethal relationships.

Alexandra R. Grassian, Darren Harvey, Julian Fowler, Allison E. Drew, Igor Feldman, Richard Chesworth, Robert Copeland, Jesse J. Smith, Scott Ribich. _Epizyme, Inc., Cambridge, MA_.

Target identification is a critical step in drug discovery, but the process has many challenges including non-specific reagents, limited ability to test numerous models, and incomplete target inhibition. Pooled screening with CRISPR/Cas9 permits the quick and accurate examination of proliferation effects across many genes and many cell lines. To determine the specific dependencies of cell lines on epigenetic pathways, we designed a CRISPR/Cas9 library to target 640 epigenetic genes and screened more than 200 cell lines covering a variety of oncology indications, including breast, lung, and renal cell carcinoma (RCC). We find that CRISPR pooled screening is a highly effective approach for target identification and provides robust, highly reproducible data as long as a sufficient number of small guide RNAs are used. We identify known pan-essential genes, including in the transcription (CDK9), translation (EIF4A1 and EIF4A3) and splicing (SRSF2) machinery. We additionally identify many novel pan-essential genes across a variety of epigenetic pathways, including histone acetylases and deacetylases, chromatin remodeling factors, helicases and others. We also investigated epigenetic synthetic lethal interactions that have been previously reported. For example, it has been reported that the SWI/SNF family displays paralog synthetic lethality for SMARCA2 in the context of SMARCA4 mutations, and for ARID1B in the context of ARID1A mutations. While we do see that some of the same trends hold, the synthetic lethal relationship appears to be more complex than previously realized, including the need to examine mRNA levels in addition to mutation type. Most importantly, we identify more than 100 epigenetic genes which show selective sensitivity, i.e. where knockout shows an anti-proliferative effect in only a subset of the cell lines. These are the most promising targets for further drug discovery programs. We have used additionally CRISPR/Cas9-domain based screening to identify the functionally relevant sites for many of these genes. Furthermore, we can overlay gene expression and mutation data to identify novel synthetic lethal relationships. One gene that displays selective sensitivity is EGLN1, the prolyl hydroxylase for the hypoxia-inducible factor, HIF1α. We find that EGLN1 is required for proliferation only in RCC cell lines which retain wild-type VHL, another component of the hypoxia response pathway, which is frequently lost in RCC. As such, EGLN1 loss is synthetically lethal in the presence of wild-type VHL in RCC cells. Thus this approach not only identifies an enzymatic drug target but also a patient stratification method. Other novel synthetic lethal interactions have also been identified. Our data demonstrates that CRISPR pooled screening is a powerful technique for identification of epigenetic synthetic lethal interactions.

#407

Whole-genome pooled CRISPR/Cas library screens to identify new hepatocellular carcinoma drivers and drug targets in combination with sorafenib.

Takahiro Kodama,1 Justin Y. Newberg,2 Nancy A. Jenkins,2 Tetsuo Takehara,1 Neal G. Copeland2. 1 _Osaka University Graduate School of Medicine, Suita, Japan;_ 2 _Houston Methodist Research Institute, Houston, TX_.

Backgrounds and Aims: Hepatocellular carcinoma (HCC) is a deadly cancer and its mortality rate ranks third among all cancer types worldwide. To comprehensively understand its genetic drivers, over 500 HCC genomes have been sequenced but they reveal the existence of thousands of infrequently mutated genes. This intra- and inter-tumor heterogeneity, combined with large number of passenger mutations, has made it difficult to identify the true drivers of HCC. In addition, sorafenib is the currently only FDA-approved molecular target drug in HCC but its anti-tumor effect is very limited. To overcome these problems, we performed two forward genetic screens using recently innovated whole-genome pooled CRISPR/Cas library. Methods: Two human liver cancer cell lines, Huh-7 and SNU-398, were lentivirally transduced with Geckov2 library and transduced cells were selected with puromycin. First, transduced cells were orthotopically injected into the liver of the Nude mice and monitored for tumor formation. To identify potential tumor suppressor genes (TSGs) in the liver, gRNAs enriched in the orthotopic liver tumors were determined by sequencing of gDNA in these tumors. Next, transduced cells were treated with vehicle or sorafenib in vitro for 14 days. To identify potential targets which inhibition show synergistic anti-tumor effect with sorafenib, we determined gRNAs depleted in cells with sorafenib treatment compared to cells with vehicle treatment. Results: In the first in vivo screening, we identified 62 genes targeted by 2 or more highly enriched gRNAs in Huh-7 cells-derived tumors and 47 genes in SNU-398 cells-derived tumors. Twenty-nine genes were common including 4 miRNAs. TCGA analysis revealed that HCC patients with mutation or copy number alteration in any of these genes showed worse overall survival and disease free survival compared to HCC patients without them. In addition, gene expression levels of 3 candidate TSGs, BPHL, CTSZ and PLCG2 were significantly negatively correlated with poor survival of HCC patients. In the second in vitro screening, CYP7B1, HS3ST5, NLRC4, and Mir-1237 were identified as potential drug targets in combination with sorafenib in HCC. Conclusion: Whole-genome pooled CRISPR/Cas library screens discovered potential new hepatocellular carcinoma drivers and drug targets in combination with sorafenib.

#408

Investigating the biology of atypical teratoid/rhabdoid tumors by whole genome CRISPR/Cas9 screening.

Matthew P. Selby, Martina A. Finetti, Matthew Bashton, Ruth E. Cranston, Yura Grabovska, Alicia Del-Carpio-Pons, Amanda Smith, Simon Bailey, Steven C. Clifford, Daniel Williamson. _Newcastle University, Newcastle upon Tyne, United Kingdom_.

Atypical Teratoid/Rhabdoid Tumors are rare, highly aggressive, paediatric tumours with a dismal prognosis. Rhabdoid Tumours (RT) have one key genetic defect (biallelic inactivation of SMARCB1 in > 90% of patients) which, when re-expressed in RT cells, causes differentiation and growth arrest. We used whole genome CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 screening; a technique by which mutations are created systematically in each gene in the genome - to identify novel SMARCB1-dependent therapeutic targets and synthetic lethal relationships in RT.

A panel of RT cell lines were transduced with a GeCKO (Genome Scale CRISPR Knock-Out) library which contains 122,441 guide constructs targeting 19,050 genes and 1,864 miRNAs; each cell contained a single CRISPR insert. This panel was further transduced with a SMARCB1 expression lentivirus or an empty vector control, cells were collected at 0, 7 and14 days post infection (300x library coverage, 3.7 x 107 cells per time point).

CRISPR inserts were sequenced, counted, normalized and analyzed to identify those whose abundance was significantly altered, representing candidate gene targets which promote or inhibit RT cell growth/viability in a SMARCB1-dependent manner. CRISPR hits were further cross-validated with primary patient and functional model genomic data (RNA-sequencing/450k DNA methylation array) to identify several high confidence deleterious events with strong SMARCB1 dependency.

Hundreds of significantly enriched (both positively and negatively) genes were identified which alter when SMARCB1 is present or absent including MYC family targets, cell cycle targets, SHH pathway and the Aurora kinase family. TGFβ was identified as a top upstream SMARCB1/methylation dependent regulator and when SMARCB1 re-expression data was overlaid onto the TGFβ canonical pathway showed a convincing enrichment in pathway regulation. Pathway members SMAD 2/3 showed a 'synthetic lethal' phenotype and represent a functional SMARCB1-dependent relationship which is also observed in SMARCB1 re-expression and methylation model data and is characteristic of primary RT. This phenotype was validated functionally by expression of a dominant negative TGFBR2.

Combining RNA-sequencing and 450K DNA methylation microarray data from primary tumours and experimental models, coupled with whole-genome functional screening, provides a genome-wide view of key RT tumorigenic genes/pathways and their SMARCB1 dependence. These data suggest synthetic lethal phenotypes and enable prioritisation of true SMARCB1-dependent events and therapeutic targets, in this rare yet lethal paediatric tumour.

#409

Genome engineering to generate models of chromosome arm-level aneuploidies in lung carcinoma.

Alison M. Taylor, Gavin Ha, Juliann Shih, Xiaoyang Zhang, Joshua M. Francis, Matthew Meyerson. _Dana-Farber Cancer Institute/Broad Institute, Boston, MA_.

Almost 90% of tumors are aneuploid and have arm- or whole- chromosome level copy number changes. Arm-level copy number alterations cluster by tumor type, suggesting that specific arm-level changes are influenced by cell type. Systematic methods of generating copy number changes on a particular chromosome have not been tested, leaving the effects of specific aneuploidies in cancer unclear. The most frequent genomic alteration in lung squamous cell carcinomas (SCCs) is loss of chromosome 3p. This alteration occurs in 90% of lung SCCs, and covers the entire length of the chromosome arm. Over two-thirds of 3p genes show significantly decreased expression. However, the phenotypic effects of 3p loss in tumorigenesis are not known.

We used the CRISPR-Cas9 system to generate double-strand breaks (DSBs) and produce partial aneuploidies of arm-level loss. We have targeted guide RNAs adjacent to centromeric satellite-repeat sequences on chromosome arm 3p. At this location, we induced homologous recombination with a selection cassette and artificial telomere. We have successfully isolated almost 90 clones of immortalized lung epithelial cells with deletion of the 3p arm, with 8 validated by whole genome sequencing. Consistent with patient data, expression of 3p genes is also decreased upon deletion. Phenotypic characterization revealed that cells with chromosome 3p deletion proliferate more slowly than their siblings. 3p deleted cells show increased G1 arrest, but do not undergo increased apoptosis or cell death. We are currently testing 3p loss in combination with other frequent SCC alterations, such as SOX2 and TP63 gain. We have also identified expression changes in trans, outside of chromosome 3p. These studies provide a robust model that will address a gap in our understanding of aneuploidy in cancer by using targeted endonuclease technology to create models of partial aneuploidies. Future work will include investigation of how different chromosomal changes contribute to cancer formation, which will have implications on our understanding of tumorigenesis.

#410

Identifying ovarian cancer specific targeted drugs using high-throughput drug sensitivity profiles of primary cancer cells.

Astrid Murumägi,1 Akira Hirasawa,2 Suleiman Khan,1 Daniela Ungureanu,3 Mariliina Arjama,1 Teijo Pellinen,1 Samuli Eldfors,1 Riitta Koivisto-Korander,4 Arto Leminen,4 Ralf Bützow,4 Tero Aittokallio,1 Olli Kallioniemi1. 1 _Helsinki Univ., Inst. for Molecular Medicine, Helsinki, Finland;_ 2 _Keio University School of Medicine, Tokyo, Japan;_ 3 _Tampere Univ., Inst. of Biosciences and Medical Technology, Tampere, Finland;_ 4 _Helsinki University Hospital, Helsinki, Finland_.

Ovarian cancer (OvCa) is the sixth most common cancer in women and leading cause of death from gynecologic diseases. Poor prognosis in OvCa is due to late diagnosis and acquired resistance to the commonly used platinum-based regimens. A significant setback for OvCa treatment is the lack of reliable biomarkers and effective targeted therapies. In order to discover novel therapeutic opportunities with approved and emerging drugs for OvCa, we have established primary cultures using ascites or tumor tissue samples from chemo-refractory ovarian cancer patients for ex vivo Drug Sensitivity and Resistance Testing (DSRT) and genomic profiling. In addition, we have performed DSRT with 31 established OvCa cell lines. Comparison of the drug sensitivity profiles of ten primary cancer cell cultures and 31 OvCa cell lines revealed previously unanticipated cancer selective drug vulnerabilities. Several drug groups were identified suggesting that the sensitive samples were addicted to the corresponding signaling networks. For example, in a 53-year old chemorefractory low grade serous OvCa patient, genomic and transcriptomic analyses revealed a fusion gene of NRG-1, a target that was recently reported to involve the NRG1/ERBB3 activation loop in OvCa. We found high expression of ERBB2 and ERBB3 by RNA-seq as well as high levels of phospho-ERBB3, phospho-ERBB2 and phospho-EGFR by immunohistochemistry. In agreement with the molecular mechanism, DSRT analysis identified significant sensitivity of primary cancer cells to EGFR inhibitors, such as erlotinib and to dual EGFR and Her2 inhibitor afatinib. The patient has received combination therapy of gemcitabine and erlotinib during nine months, followed by afatinib monotheraphy that has lead to complete remission after six months treatment. We have detected activated NRG1/ERBB3 activation loop also in several additional ovarian cancer patient cases, highlighting the importance of this signaling pathway in ovarian cancer pathogenesis. Our study reveals that a subset of serous ovarian cancer patients with activating NRG1/ERBB3 signaling loop have clinical benefits from repurposing dual EGFR/Her2 inhibitor afatinib, providing a basis for the clinical use of EGFR and dual EGFR/Her2 tyrosine kinase inhibitors in clinical treatment. In conclusion, DSRT technology together with molecular profiling provides a powerful strategy to identify tumor driver signals and select clinically actionable inhibitors. Hence, this type of systems medicine approach can significantly improve the power of mainly genomics-oriented personalized medicine approaches.

#411

In vivo **pooled shRNA library identifies KPNB1 as a new drug target for epithelial ovarian cancer.**

Michiko Kodama,1 Takahiro Kodama,1 Kosuke Yoshihara,2 Kae Hashimoto,1 Seiji Mabuchi,1 Kenjiro Sawada,1 Tadashi Kimura,1 Neal Copeland,3 Nancy Jenkins3. 1 _Osaka University Graduate School of Medicine, Suita, Japan;_ 2 _Nigata University Graduate School of Medical and Dental Sciences, Japan;_ 3 _Houston Methodist Research Institute, Houston, TX_.

Objective

Epithelial ovarian cancer (EOC) is a most lethal cancer in gynecology, of which cure rate is 30%. To seek its new therapeutic targets, we performed in vivo loss of function screen.

Method

Human EOC cell line, SKOV3, was transduced with pooled druggable shRNA library containing 42450 shRNAs targeting 7490 genes. Transduced cells were intraperitoneally injected into 12 female nude mice and monitored for PC tumor formation. Genomic DNA of 12 biggest PC tumors were sequenced to identify shRNAs depleted in these tumors.

Results

Ten potential drug targets were identified, including 2 known oncogenes, ERBB2 and RAF1. Second highest ranked gene, KPNB1, is known as a nuclear transporter. KPNB1 inhibition significantly decreased and overexpression increased in vitro cell proliferation in multiple EOC cell lines. KPNB1 inhibition caused multi-phase cell cycle delay at both of G1/S and G2/M transition via elevation of p21 and p27 and induced apoptosis. KPNB1 inhibition significantly decreased in vivo tumor formation through the same mechanism as in vitro, because more cleaved PARP and less Ki67 positive cells were found in KPNB1 knockdown tumors via immunochemical staining. We found a positive correlation between KPNB1 mRNA levels and poor survival of EOC patients, suggesting the oncogenic role of KPNB1 in human EOC. Comprehensive mass spectrometric study identified that KPNB1 positively regulated several members of anaphase promoting complex/cyclosome. Lastly, we found that ivermectin, a well-known anti-parasitic drug, exerted an anti-proliferative effect on EOC cell lines via KPNB1 inhibition, and its combination with paclitaxel synergistically suppressed cell proliferation in vitro and inhibited tumor formation in vivo.

Conclusion

In vivo pooled shRNA library screen identified KPNB1 as a new drug target for EOC. Ivermectin can be a new therapeutics for EOC especially combined with paclitaxel.

#412

Genetic variation in platinum-sensitive and platinum-resistant high-grade serous epithelial ovarian cancer.

Tara Castellano, Leslie H. Clark, Naim Rashid, Victoria Bae-Jump. _University of North Carolina at Chapel Hill, Chapel Hill, NC_.

Background: We examined the association of platinum resistance with genetic mutations in high grade serous ovarian cancer (HSOC) patients undergoing high-throughput genomic tumor sequencing.

Methods: Snap-frozen and fresh frozen paraffin embedded tissue samples were collected from HSOC patients enrolled on UNCseq (NCT01457196). UNCSeq is an institutional protocol which uses next generation sequencing to detect genetic mutations in a wide array of malignancies. Illumina libraries were prepared separately from tumor and a matched normal sample from each patient. Relevant targets were enriched by a custom designed Agilent SureSelect hybrid capture enrichment library using standard protocols. Samples were sequenced on Illumina HiSeq machines in a variety of formats. Mutations with a quality score <100 were filtered from the data set, and only mutations rated to have a moderate to high impact were retained. Medical record review determined platinum sensitivity or platinum resistance. Tumors were defined as platinum sensitive or resistant if patients were noted to have > or < 6 months of disease free interval following completion of induction therapy, respectively.

Results: Overall 39 HSOC cases met inclusion criteria; 32 tumors met criteria for platinum sensitive and 7 platinum resistant. 308 mutations were noted in at least one individual across all patients. The top observed mutations in platinum sensitive HSOC were TP53 (41%, n= 13), GucylA2 (19%, n=6), MLL2 (19%, n=6) and MTOR (16%, n=5). The top observed mutations in platinum resistant tumors were TP53 (71%, n=5), TET1 (43%, n=3), NF1 (43%, n=3) and MLL3 (43%, n=3). There was a trend toward more p53 mutations in resistant tumors (71% versus 41%, p=0.21) There was no difference in the total number of mutations per tumor in platinum sensitive and resistant patients, (3 vs. 4, p=0.516).

Conclusions: We did not detect a difference in the number of genetic mutations in HSOC according to platinum sensitivity. Platinum resistant tumors had a trend toward higher frequency of TP53 mutations than platinum sensitive HSOCs. Furthermore, we identified 3 frequently mutated genes in platinum resistant HSOC: TET1, an epigenetic regulator, NF1, a tumor suppressor gene and MLL3, a histone modifier gene. Ongoing tumor sequencing of HSOCs on UNCseq will help to confirm these results.

#413

Neo-epitope detection and immune infiltrate analysis of colorectal cancer samples.

Fang Yin Lo,1 Nitin Mandloi,2 Timothy Yeatman,3 Kiran Paul,2 Ashwini Patil,2 Steven Anderson,4 Ravi Gupta,2 Anup Madan1. 1 _Covance Genomics Laboratory, Redmond, WA;_ 2 _MedGenome Inc, Foster City, CA;_ 3 _Gibbs Cancer Center, Spartanburg, SC;_ 4 _Covance Genomics Laboratory, Durham, SC_.

Colorectal cancer (CRC) is the third most common type of cancer in the United States. Targeted therapies that use monoclonal antibodies (mAbs) to EGFR have been shown to benefit some CRC patients. Until recently, KRAS has been the only predictive biomarker for anti-EGFR therapy for metastatic CRC. However, 40% to 60% of patients with wild-type KRAS do not respond to anti-EGFR therapy. Previously we have stratified 55 CRC samples by applying a RAS gene signature score which measures MEK pathway functional output independent of tumor genotype. We showed that samples that have RAS activating mutations such as KRAS and BRAF have significant higher RAS scores (p<0.001). Here, we further investigate the potential immune reactivity in these CRC samples, and thereby the potential benefit of immunotherapy, by evaluating the tumor neo-epitope burden, and the quality of immune cell infiltration based on exome-seq and RNA-seq analysis. In the 53 samples that were sequenced, 779 unique non-synonymous mutations were detected by exome-seq. These 779 mutations spanned across 263 genes. The majority of these mutations are not shared between samples (~ 5% of the mutations were shared by more than 2 samples). Several driver gene mutations were identified in this study, including KRAS, TP53, PIK3CA, APC and HER2. HLA prediction based on Exome-Seq and RNA-Seq data shows that ~86.7% of the alleles predicted to be present in 53 samples were concordant between the two RNA-seq datasets. The predicted alleles based on exome-seq and RNA-seq results have 67-69% concordance. Prediction of neo-epitopes show that HLA-binding neo-epitopes are more frequent than TCR-binding ones, and that most neo-epitopes are private and not shared between samples. A more in-depth analysis of the tumor microenvironment was performed using the RNA-seq data. The epithelial, stromal and immune content of the tumors was comparable to tumors from TCGA CRC data. Next, the immune cell compartment was further stratified into 7 different immune cell types using signatures specific to CD8 andCD4 T, T-regulatory, NK, B-cells, Macrophages and Myeloid derived suppressor cells (MDSC). The immune make up of colorectal cancer is dominated by macrophages and MDSCs. Interestingly, both granulocytic G- and monocytic M-MDSCs are present together, supporting the idea that MDSCs confer an immune suppressive microenvironment in this cancer. Significantly, high MDSC infiltrated tumors showed upregulated expression of pro-tumorigenic insulin-like growth factor pathway genes. Additionally, tumors with lower burden of MDSC showed signature of complement activation suggesting innate cell-mediated anti-tumorigenic mechanisms of tumor control in CRC. These analyses provide potential biomarkers to stratify CRC patients based on their immune reactivity and predict response to cancer immunotherapy drugs.

#414

**Identifying selective vulnerabilities in colorectal cancer molecular subtypes using** in vivo **functional genomic screens.**

Akira Inoue, Bahar Salimian Rizi, Alessandro Carugo, Sahil Seth, Christopher Bristow, Giannicola Genovese, Andrea Viale, David G. Menter, Scott Kopetz, Giulio F. Draetta. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Colorectal cancer (CRC) is a leading cause of cancer-related morbidity and mortality with significantly heterogeneous features and drug responses. Recently, the international Colorectal Cancer Subtyping Consortium identified four robust consensus molecular subtypes of CRC (CMS1-4) using large-scale gene expression data. These findings may enable us to identify molecularly homogenous subsets of CRC patients and accelerate effective drug development strategies. To identify potential therapeutic targets and novel selective vulnerabilities in CRC molecular subtypes, we developed an in vivo loss-of-function genomic screen using CRC patient-derived xenografts (PDXs) for each molecular subtype. Our PDX-derived CRC models underwent comprehensive integrated molecular characterization of mRNA profiles, DNA mutations, and histochemical profiles upon confirmed serial retransplantation to determine whether characteristics of the subtypes are recapitulated in vivo. Because the original CMS classification algorithm was trained and validated using Affymetrix data, profiling the PDX-derived cell lines using this technology provided the most robust analysis of the CMS subtypes. In vivo pooled short hairpin RNA (shRNA) screens rely on specific elimination of individual shRNAs in a cell population and require that the infected tumor cell population is adequately endowed with engraftment capacity when implanted into recipient mice. Therefore, we determined the transduction efficiency of the PDX models, the frequency of tumor-initiating cells, and the maximum library complexity allowed by each model. To identify targets that represent selective vulnerabilities in specific CRC molecular subtypes, we screened each model in vivo with an shRNA library targeting about 200 genes specifically belonging to U.S. Food and Drug Administration-approved targeted therapies (FDAome; 10 shRNAs/gene ). We leveraged redundant shRNA activity analysis to evaluate "hits" (or top-scoring genes) emerging from our screening. We further applied ranking-based analytics in combination with integromic approaches (use of computational packages to unravel relationships between -omics) to inform on selective CMS specific top-scoring genes. One of the benefits of using an FDAome library is the direct correspondence of target genes with clinically available drugs. We therefore tested these drugs for validation in fully annotated PDXs. These efforts, in association with systematic profiling of the CMS subtypes at the patient level through adaptation of NanoString technology, may enable us to stratify CRC patients who will benefit from selective U.S. Food and Drug Administration-approved drugs and to rapidly design successful preclinical and clinical trials in CRC patients.

#415

In vivo **shRNA library screening to identify novel targets for head and neck squamous cell carcinoma.**

Curtis R. Pickering,1 Tongxin Xie,1 Manish Kumar,1 Ramya L. Parimi,1 Liang P. Yang,1 Jiping Wang,1 Sahil Seth,1 Christopher A. Bristow,1 Alessandro Carugo,1 Frederick S. Robinson,1 Giulio F. Draetta,1 Heath Skinner,1 Mitchell J. Frederick,2 Jeffrey N. Myers1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Baylor College of Medicine, Houston, TX_.

Head and neck squamous cell carcinoma (HNSCC) is a diverse group of tumors from the upper aerodigestive tract that are treated primarily with surgery, chemotherapy, and/or radiation. Recent genomic studies have characterized the genomic alterations in HNSCC but they failed to identify novel oncogenic drivers for therapeutic targeting. In order to identify novel targets we have utilized an in vivo shRNA library screening platform in a panel of genomically characterized HNSCC cell lines. The screens were performed alone or in combination with platinum-based chemotherapy or radiation, and the screening libraries included known druggable targets, DNA repair genes, and HNSCC candidate driver genes. Initial results indicate that this in vivo screening is able to identify targets that may not be found through traditional in vitro screening approaches and related to tumor-stromal interactions and metabolism. These genes emphasize the importance of including the tumor microenvironment in studies to understand driver genes and identify novel therapeutic interventions.

#416

Identification of therapeutic combinations in glioblastoma using personalized gene expression networks.

Vasileios Stathias, Michele Forlin, Bryce Allen, Stephan Schürer, Nagi G. Ayad. _University of Miami, Miami, FL_.

The goal of our study was to identify patient-specific gene expression networks from Glioblastoma Patient-Derived Xenografts (PDXs) and determine novel therapeutic compound combinations using those networks.

Glioblastoma is the most common malignant primary adult brain tumor with a standard of care consisting of maximal surgical resection followed by radiotherapy and adjuvant temozolomide (TMZ) chemotherapy. However, despite medical advances in the field, recurrence is almost universal, suggesting the need for more personalized and targeted therapeutic approaches.

For this, we obtained, transcriptional data from Glioblastoma PDXs and used them to identify their respective differentially expressed genes. Patient-specific gene expression networks were then created and their biological relevance was supplemented by integrating them with TCGA Glioblastoma transcriptional data. In order to identify compound combinations specific for those networks, we used the extensive chemical perturbation signatures from the Library of Network-based Cellular Signatures (LINCS). From the large number of L1000 transcriptional data we extracted gene expression signatures that were indicative of specific LINCS compounds. We then compared those signatures to the patient-specific networks in order to prioritize compound combinations that were inducing discordant transcriptional changes in distinct sub-networks of the PDXs transcriptome. The most orthogonal compound combinations were then chosen and used in in-vitro cell viability assays of Glioblastoma PDXs in order to evaluate their effectiveness. The above process can be used to prioritize compound combinations with potential therapeutic effect in a patient-specific manner.

#417

**Defining molecular mechanisms of resistance to glioblastoma immunity using a novel CRISPR/Cas9** in vivo **loss-of-function screening platform.**

Martha R. Neagu,1 Robert T. Manguso,1 Hans Pope,2 Maria C. Speranza,2 Gordon J. Freeman,2 John Doench,3 Arlene H. Sharpe,1 William Nicholas Haining2. 1 _Harvard Medical School, Boston, MA;_ 2 _Dana Farber Cancer Institute, Boston, MA;_ 3 _Broad Institute of Harvard and MIT, Cambridge, MA_.

OVERVIEW: Despite the impact of cancer immunotherapy, expanding its clinical utility requires a rational method for identifying combination therapies and resistance mechanisms. This is pertinent to glioblastoma (GBM), where initial trials show uncertain response rates. Functional genomic screens used to identify new therapies and escape mechanisms were generally conducted in vitro, where interaction with the immune system is absent. Here we present the development of a high-throughput, in vivo, loss-of-function screening platform for GBM immune-escape mechanisms.

METHODS: CT2A- and GL261-astrocytoma cells bearing Cas9-endonuclease were engineered to express a library of barcoded single guide RNAs (sgRNAs). These cells form tumors when implanted intracranially in immunocompetent mice. Tumor-bearing mice were treated with vaccination or PD-1 checkpoint blockade. Dropout of sgRNAs targeting putative immune evasion molecules or enrichment of sgRNAs mimicking resistance mechanisms were detected using next-generation sequencing at the time of tumor implantation and harvest post-immunotherapy.

RESULTS: We first developed an in vivo, pooled, loss-of-function genetic screen using Cas9/CRISPR genome editing in mouse transplantable tumors subjected to titratable, selective immune-pressure. Screening 2,400 genes expressed by melanoma cells for those that synergize with or cause resistance to checkpoint blockade recovered known immune-evasion molecules PD-L1 and CD47. Novel immunotherapy targets validated individually, identifying essential pathways of immune-evasion. We then sought to recapitulate this approach in the CNS, and showed that 500-1000 genes can be functionally screened under graded immune-pressure. In optimized immune-competent CT2A and GL261 GBM models, tumors derived from cancer stem cell CD133hi-rich neurospheres were sensitive to immunotherapy and more aggressive and infiltrative than tumors derived from adherent tumor cells. A neurosphere based immune-competent model can be scaled up to a whole-genome screen due to a shorter experimental time requirement and improved engraftment allowing for functional screening of >1000 genes. We curated a GBM-specific library based on differential in vitro and in vivo gene expression profiles of CT2A and GL261 cells exposed to graded immune pressure. We are now screening this GBM-specific library in our optimized in vivo pooled loss-of-function genetic screen using immune-pressure titration to identify novel immunotherapy targets in GBM.

CONCLUSIONS: This assay provides the first high-throughput method for systematically identifying resistance mechanisms and new candidate targets for immunotherapy in CNS tumors. Our optimized model could be scaled up to whole-genome loss of function screens, serving as an important tool for identification of next-generation and combination immunotherapies.

#418

Adaptive resistance to chemotherapy in triple-negative breast cancer revealed by single cell DNA and RNA sequencing.

Charissa Kim,1 Ruli Gao,1 Emi Sei,1 Rachel Brandt,1 Nicola Crosetto,2 Theodoros Foukakis,2 Nicholas Navin1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Karolinska Institutet, Stockholm, Sweden_.

Triple-negative breast cancer (TNBC) is an aggressive subtype that displays extensive intratumor heterogeneity and frequently (46%) develops resistance to neoadjuvant chemotherapy (NAC). Currently, the genomic basis of chemoresistance remains poorly understood. An important question is whether resistance to chemotherapy is driven by the selection of rare pre-existing subclones with genomic mutations and transcriptional programs that confer resistance to chemotherapy (adaptive resistance) or by the spontaneous induction of new mutations and expression changes that confer a resistant phenotype (acquired resistance). To investigate this question we applied single cell DNA and RNA sequencing methods and deep-exome sequencing to longitudinal time-point samples collected from a cohort of 20 TNBC patients. Deep-exome sequencing of the cohort at three time points revealed a random death model, wherein multiple clones were targeted, as opposed to the selection of specific somatic mutations. In contrast, single cell copy number profiling of ~800 cells from 8 patients identified an adaptive resistance model, wherein minor subclones from the pre-treatment tumors were selected and expanded in response to NAC. Similarly, single cell RNA sequencing of ~8000 cells from 8 patients identified subclones with chemoresistant phenotypes that were selected in response to NAC, resulting in the expansion of the resistant tumor mass. These data suggest that chemoresistance evolves through the selection of copy number changes and expression changes in signaling pathways associated with chemoresistance, rather than point mutations. This adaptive resistance model has important translational implications in clinical diagnostics, by suggesting that resistant clones can be detected in TNBC patients prior to the administration of chemotherapy.

#419

Integrative analysis of multi-omics tumor profiles identifies pathways associated with resistance to anti-HER2 therapy in early stage breast cancer.

Salendra Singh,1 Hannah Gilmore,1 Maysa Abu-Khalaf,2 George Somlo,3 William Sikov,4 Lyndsay Harris,5 Vinay Varadan1. 1 _Case Western Reserve Univ., Cleveland, OH;_ 2 _Sidney Kimmel Medical College at Thomas Jefferson University, Philadelphia, PA;_ 3 _City of Hope National Med Center, Duarte, CA;_ 4 _Warren Alpert Medical School of Brown University, Providence, RI;_ 5 _National Cancer Institute, Rockville, MD_.

Background: HER2 positive breast cancers are heterogeneous at both the clinical and molecular levels, with the HER2-enriched subtype exhibiting increased levels of immune infiltration signatures and the highest rate of pathologic complete response (pCR), while the HER2-Basal subtype is resistant to anti-HER2 therapy (Varadan et al, CCR 2016). Here we aim to characterize the molecular underpinnings of response and their interaction with increased immune infiltration across these HER2-subtypes using integrative analyses of genomic and transcriptomic data from two multicenter trials (DFCI 03-311 and BrUOG 211B).

Methods: Fresh tumor core biopsies were taken at baseline and a 2-week time point after a single dose of trastuzumab. 80 HER2+ early breast cancer (EBC) patients were enrolled in the 03-311 trial, and 60 patients in the 211B trial. Biopsy samples were profiled for gene expression (Microrrays:03-311; RNAseq:211B), somatic mutations (Whole-exome sequencing: 211B; Targeted Sequencing: 03-311) and somatic copy-number aberrations (SNP-arrays: 03-311; Whole-exome Sequencing: 211B). Subtyping was performed using gene expression data and tumors were classified into HER2-Enriched, HER2-Luminal and HER2-Basal subtypes. Integrative analysis of gene expression and copy-number data to infer signaling network activities per sample, was performed using the recently developed InFlo framework (Dimitrova et al, Oncogene 2016).

Results: HER2-Basal tumors exhibited lower average copy number for HER2 and were less likely to have high-level amplifications of co-amplicons (e.g. 11q13, 20q13). In the 211B and 03-311 trials, respectively, 62% and 63% of somatic mutations persisted after one dose of therapy, while 21% and 19% of mutations were undetectable after one dose of therapy. Tumors harboring amplifications in the 8p11 (FGFR1) genomic locus exhibited higher indices immune signatures associated with macrophages (P=0.0073) and T-cells (P = 0.0493) in 211B, but this association did not achieve significance in the 03-311 trial. Integrative InFlo-based analysis of tumor gene expression and copy-number profiles after one dose of trastuzumab in the 211B trial revealed significantly higher activity of signaling pathways associated with CD4+ T-cells in the responders (P=0.008), while higher activity of mTOR pathway was observed in non-responders (P=0.0014).

Conclusions: Changes in mutational profiles over time may either be related to therapy-induced alterations of clonal architecture or the consequence of intra-tumor heterogeneity, thus warranting further exploration. Integrative analysis of gene expression and copy-number profiles reveal signaling pathways associated with response and resistance, enabling the discovery of biomarkers of response to anti-HER2 therapy.

#420

Preclinical evaluation of the SF3B1 inhibitor E7107 in triple negative breast cancer.

Praveen Sridhar, Stefanie Chan, Ying-Jie Lock, Fabio Petrocca. _Boston University, Boston, MA_.

Triple-negative breast cancers (TNBC) are the most aggressive subtype of breast cancer and account for roughly 15% of human breast cancers. No effective targeted therapies are currently available against these tumors, at least in part due to their genetic and epigenetic heterogeneity. Yet, TNBCs are commonly enriched for cells arrested in a progenitor-like state. We therefore performed a genome-wide siRNA lethality screen to identify selective TNBC dependencies associated with this particular phenotype in vitro. These studies led to the identification of several spliceosome genes, including the core splicing factor SF3B1, as selectively and recurrently essential for progenitor-like (or basal-A) TNBC cell lines. On this basis, we examined the antitumor effect of E7107, a clinically tested SF3B1 inhibitor, on multiple TNBC cell lines. In vitro, treatment with E7107 for 24-48 hr selectively killed basal-A cell lines relative to differentiated luminal cell lines, decreasing viability by ≈40% or more in 5 of 6 basal-A lines tested. E7107-treated cells appeared to die of apoptosis based on immunoblotting for multiple apoptotic markers. In mice, E7107 (5 mg/kg i.v. for 4 consecutive days) suppressed the growth of preformed tumors by ≈80% and ≈50% in 2 different cell-line-derived xenograft models and was remarkably well-tolerated at this dose. Of note, tumor response in vivo closely mirrored cell sensitivity in vitro, suggesting that in vitro analysis provides a fair estimate of TNBC response to E7107 in mice. Amongst 2 additional patient-derived xenograft (PDX) models tested, E7107 inhibited tumor growth by ≈50% in one model. Thus, E7107 was therapeutically active against 3 of 4 xenograft models tested. Preliminary mechanistic studies in vitro and in vivo pointed to MCL1 inactivation as a major mechanism of action of E7107 in top responders. Together, these data suggest that the spliceosome is a druggable target in TNBC, and that E7107 or similar SF3B1 inhibitors could be effective in a TNBC subset that needs to be further defined.

#421

**Comprehensive genomic analysis of metastatic breast cancers reveals** ESR1 **fusions as a recurrent mechanism of endocrine therapy resistance.**

Ryan J. Hartmaier,1 Nolan Priedigkeit,2 Laurie Gay,1 Michael E. Goldberg,1 James Suh,1 Siraj Ali,1 Jeffery Ross,1 Michaela Tsai,3 Barbara Haley,4 Julio Peguero,5 Rena D. Callahan,6 Irina Sachelarie,7 John Cho,8 Amir Bahreini,2 Shannon L. Puhalla,2 Steffi Oesterreich,2 Aju Mathew,2 Peter C. Lucas,2 Nancy E. Davidson,2 Adam M. Brufsky,2 Philip J. Stephens,1 Juliann Chmielecki,1 Adrian V. Lee2. 1 _Foundation Medicine, Cambridge, MA;_ 2 _University of Pittsburgh Cancer Institute, Pittsburgh, PA;_ 3 _Minnesota Oncology, Minneapolis, MN;_ 4 _UT Southwestern Medical Center, Dallas, TX;_ 5 _Memorial Hermann Hospital, Houston, TX;_ 6 _UCLA Medical Center, Santa Monica, CA;_ 7 _Carlisle Regional Medical Center, Carlisle, PA;_ 8 _New Bern Cancer Care, New Bern, NC_.

Metastatic breast cancer is often intractable due to its inherent ability to overcome current therapies. Genomic alterations are frequently responsible for therapeutic resistance. To better understand genomic mechanisms of acquired resistance in breast cancer we undertook a detailed characterization of single nucleotide variation (SNV) and structural variation (SV) in paired primary-metastasis metachronous tumors from 6 breast cancer patients (median time to recurrence 7.3 years). In ER-positive recurrent tumors treated with endocrine therapies, we identified multiple metastatic-acquired variants in ESR1 including a novel constitutively active, ligand-independent ESR1-DAB2 gene fusion. Importantly, this fusion resulted from a breakpoint in intron 4, retaining the DNA-binding domain but eliminating the ligand-binding domain (LBD), concordant to a similar fusion reported previously in a xenograft model. Hybrid capture based genomic profiling from >7,800 breast cancers identified similar exon/intron 4 fusions in 5 tumors with direct paired-read evidence. Using a novel copy number shift detection strategy, 58 additional tumors showed indirect evidence of a rearrangement at exon 4 based on a novel copy number shift detection strategy. ESR1 fusion and copy number shift positive tumors are strongly enriched in metastatic disease (78%; p<10-4) supporting their expected involvement in endocrine therapy resistance. Clinical follow up was available for 7 patients. 6/7 tumors were clinically ER-positive and received extensive endocrine therapy with progressive disease. Together, these data indicate that ESR1 fusions involving exon/intron 4 are a recurrent, albeit rare, mechanism of endocrine therapy resistance in breast cancer. The absence of the LBD implies these fusions will not respond to other ERα targeted therapies. Additional studies are needed to identify appropriate treatment options to overcome this mechanism of resistance.

#422

Lymph node metastasis evolution drives immune evasion and targeted therapy resistance in gastro-esophageal adenocarcinomas (GEAs).

Matthew N. Davies,1 Louise J. Barber,1 Georgia Spain,1 Filipa Lopes,1 Katharina von Loga,1 Beatrice Griffiths,1 Andrew Woolston,1 Donat Alpar,1 Marta Gomez,1 Kamil A. Lipinski,1 Kerry Fenwick,1 Zakaria Eltahir,2 Stefano Lise,1 Emese I. Agoston,3 Laszlo Harsanyi,3 Richard Marais,4 Andrew Wotherspoon,2 A Szasz,3 Caroline Springer,1 Marco Gerlinger1. 1 _The Institute of Cancer Research, London, United Kingdom;_ 2 _The Royal Marsden Hospital, London, United Kingdom;_ 3 _Semmelweis University, Budapest, Hungary;_ 4 _Cancer Research UK Manchester Institute, Manchester, United Kingdom_.

GEAs are aggressive tumors in which several targeted therapy trials have failed. We assessed intratumor heterogeneity (ITH) and its impact on progression and therapy failure by applying an 81-gene NGS panel and SNP array copy number aberration (CNA) analysis to multiple primary tumor (T) regions and lymph node (LN) metastases from 9 GEAs.

Analysis of 39 samples found ITH in all cases. 8 chromosomally instable (CIN) GEAs predominantly evolved through CNAs, with 17-76% of the genome affected by heterogeneous CNAs. A microsatellite instable GEA showed parallel evolution and diversified exclusively through point mutations (58% ITH). This demonstrates ongoing genomic instability rather than punctuated evolution and that specific instability mechanisms impact evolutionary trajectories.

LN metastases contributed more to ITH (p<0.01) than any anatomic location within T. Further, subclonal aberrations that activate the Mitogen Activated Protein Kinase-pathway (MAPK-pw), including ERBB3, ERK2, KRAS and NRAS amplifications (amp) and NRAS mutations, were detected in LN metastases from 4/8 CIN GEAs. Subclonal MAPK-activating amp were enriched in LN (p=0.019) compared to T regions that only exhibited a single subclonal MET amp. Convergent evolution of LN subclones across several GEAs suggests that selection pressures differ systematically between LN and T ecosystems.

To assess the phenotypes established by MAPK-activating amp evolution, we analyzed 135 published primary CIN subtype GEAs. Cytolytic activity (CYT), estimating tumor immune recognition from RNA expression data, correlated with the mutation load in GEAs with EGFR, ERBB2 or MET amp (p=0.04). In contrast, CYT did not correlate with mutation load in GEAs with KRAS or ERBB3 amp (p=0.22, NRAS/ERK2: insufficient data), indicating that these specific alterations, that also recurrently evolved in LN, may enable immune evasion. Downregulation of TAP and Class I MHC genes (p<0.05) in KRAS or ERBB3 amp GEAs suggested impaired antigen processing and presentation as the mechanisms driving T cell immune evasion.

Moreover, ITH of MAPK-activating amp is likely to confer resistance to upstream tyrosine kinase inhibition. We used GEA cell lines with various MAPK-activating amp (ERBB2, MET, NRAS) to investigate downstream MAPK-pw inhibition as a novel strategy to broadly target heterogeneous subclones. Growth control was incomplete with ERK- and MEK-inhibitors but the panRAF/SRC inhibitor CCT196969 was effective in all lines, suggesting that it can effectively intercept subclonal heterogeneity in GEAs.

In conclusion, we identified ITH with parallel and convergent evolution in 9/9 metastatic GEAs. Distinct selection pressures in LN foster the evolution of subclonal MAPK-activating amp that decrease immunogenicity and drive evolutionary pre-adaptation to future targeted drugs that can be intercepted by panRAF/SRC inhibitors.

#423

Detection of genetic alterations to predict the chemotherapeutic responses in unresectable pancreatic ductal adenocarcinomas.

Joo Kyung Park, Kwang Hyuck Lee, Donghyun Park, Woong-Yang Park, Dae-Soon Son, Jong Kyun Lee, Kyu Taek Lee. _Samsung Medical Center, Seoul, Republic of Korea_.

Background & Aims: Next-generation sequencing (NGS) that enables the analyses of massively parallel sequences of DNA can advance the understanding of the underlying molecular pathophysiologies of cancer. Such recent genomic analyses have revealed a complex mutational landscape for PDACs. The aims of this study were to investigate the genomic profile to predict the chemotherapy response in unresectable PDACs.

Methods: The total of 80 pathologically confirmed PDACs were enrolled and genomic DNA was extracted and quality control metrics of DNA analytes were measured. The specimens that passed a quality control test underwent targeted deep sequencing using a customized cancer panel (CancerSCAN) enriched in the exons of 83 genes.

Results: Clinical prognostic factors associated with survival in PDAC were gender, tumor mass size, stage and chemotherapy response rate. (P=0.078, 0.009, 0.052 and <0.001, respectively). Multivariate Cox proportional-hazards analysis revealed chemotherapy response rate (P < 0.001, hazard ratio (HR) = 1.908, 95% CI, 1.281 to 2.840) is an independent prognostic factor. There were 56 (71%) and 9 (11%) study patients who underwent gemcitabine based chemotherapy and FOLFIRINOX respectively. Response rate of study patients were as follows; CR+PR 14 (20%), SD (27%) and PD 28 (41%). There were only 9 (16%) and 4 (50%) patients who had CR+PR response gemcitabine based chemotherapy and FOLFIRINOX respectively.

Conclusion: Targeted sequencing using EUS-FNA specimens in PDAC showed excellent compatibilities to analyze genomic profiles of PDACs. Furthermore, novel genes associated with survival, metastasis and chemotherapy response in PDAC were identified.

#424

Landscape of somatic mutations in drug-resistant acute myeloid leukemia.

Samuli Eldfors,1 Mika Kontro,2 Yevhen Akimov,1 Olli Kallioniemi,1 Kimmo Porkka,2 Caroline Heckman1. 1 _Inst. for Molec. Medicine Finland (FIMM), Helsinki, Finland;_ 2 _Helsinki University Central Hospital Cancer Center, Helsinki, Finland_.

Introduction: Most patients with acute myeloid leukemia (AML) initially respond to cytarabine-anthracycline induction chemotherapy. However, in many patients, the disease recurs in a lethal drug-resistant form. Somatic mutations underlying the pathogenesis of AML have been extensively characterized by sequencing of newly diagnosed AMLs. However, the mutations driving therapy resistance and disease progression at relapse have not been well characterized. In this study, we have exome sequenced a cohort of relapsed and refractory AMLs and compared the landscape of somatic mutations at relapse to diagnosis phase AMLs to identify mutations that contribute to therapy resistance and disease progression.

Materials and Methods: We performed exome sequencing of diagnosis phase AMLs (n=70) and relapsed or primary refractory AMLs (n=54). Patients with AML M3 subtype were excluded from the study. Paired diagnosis and relapse samples were available from 27 patients. A skin biopsy was used as the germline control. Nine patients had received an allogeneic hematopoietic stem cell transplant before relapse. Somatic mutations were called using varscan2 and copy number aberrations using copyCat. Since the identification of large insertions from next-generation sequencing data remains challenging using existing algorithms, FLT3 internal tandem duplications (FLT3-ITDs) were identified using a novel custom algorithm optimized for FLT3-ITD detection. Population variants were filtered out to remove donor-derived germline variants in chimeric post-transplant relapse samples.

Results: Comparison of somatic mutation frequencies in diagnosis and relapse and refractory samples showed that on average relapsed tumors have a higher number of driver mutations than tumors at diagnosis. WT1, TP53, CBL, IDH1 and PTPN11 were mutated at a higher frequency in relapsed samples than at diagnosis, with 13 %, 11 %, 11 %, 9 % and 9 % of relapsed or refractory samples and 4 %, 6 %, 3 %, 4 % and 7 % of diagnosis mutated respectively. Analysis of paired diagnosis-relapse samples showed that in patients with WT1, CBL or PTPN11 mutation at diagnosis the second allele is frequently mutated or lost due to uniparental disomy occurring at relapse.

Conclusions: On average relapsed AMLs have a higher number of driver mutations than diagnosis phase AMLs indicating that acquisition of additional driver mutations contributes to relapse. AMLs frequently acquire additional mutations in the same genes and pathways that already harbored mutations at diagnosis.

#425

SMRT® Sequencing of full-length androgen receptor isoforms in prostate cancer reveals previously hidden drug resistant variants.

Manish Kohli,1 Yeung Ho,2 David W. Hillman,1 Jamie L. Van Etten,2 Christine Henzler,2 Rendong Yang,2 Yingming Li,2 Elizabeth Tseng,3 Ting Hon,3 Tyson A. Clark,3 Liguo Wang,1 Kevin Silverstein,2 Liewei Wang,1 Scott M. Dehm2. 1 _Mayo Clinic, Rochester, MN;_ 2 _University of Minnesota, Minneapolis, MN;_ 3 _Pacific Biosciences, Menlo Park, CA_.

Prostate cancer is the most frequently diagnosed male cancer. For prostate cancer that has progressed to an advanced or metastatic stage, androgen deprivation therapy (ADT) is the standard of care. ADT inhibits activity of the androgen receptor (AR), a master regulator transcription factor in normal and cancerous prostate cells. The major limitation of ADT is the development of castration-resistant prostate cancer (CRPC), which is almost invariably due to transcriptional re-activation of the AR. One mechanism of AR transcriptional re-activation is expression of AR-V7, a truncated, constitutively active AR variant (AR-V) arising from alternative AR pre-mRNA splicing. Noteworthy, AR-V7 is being developed as a predictive biomarker of primary resistance to androgen receptor (AR)-targeted therapies in CRPC. Multiple additional AR-V species are expressed in clinical CRPC, but the extent to which these may be co-expressed with AR-V7 or predict resistance is not known. Here we utilized long read sequencing to identify and quantify AR isoforms expressed in CRPC. To unambiguously characterize all AR isoforms, we prepared Iso-Seq™ libraries via 3' rapid amplification of cDNA ends (RACE) with RNA isolated from prostate cancer cell lines and xenograft tissues using a forward primer anchored in AR exon 1. 3'RACE reactions were subjected to single molecule, real-time (SMRT®) long-read sequencing with a Pacific Biosciences RSII System. Our work identified AR-V9 as a truncated isoform that is frequently co-expressed with AR-V7 in CRPC. Mechanistically, our work re-annotated AR-V7 and AR-V9 mRNAs, showing these two species shared a common 3' terminal exon containing separate splice acceptor sites. Taking into account this new information, novel siRNAs and antibodies which could distinguish between AR-V7 and AR-V9 were designed, validated and used to measure the relative expression of these two AR isoforms in CRPC cells with a view to determining the potential of AR-V9 as a predictive biomarker of primary resistance to AR-targeted therapies.

#426

Targeted genome profiling in patients with advanced hepatocellular carcinoma treated with sorafenib.

Wonseok Kang, Kyung Kim, Joon Hyeok Lee, Ho Yeong Lim, Woong-Yang Park, Jeeyun Lee, Yong Han Paik. _Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea_.

Background/Objectives: Sorafenib is the only approved targeted agent as first-line treatment for advanced hepatocellular carcinoma (HCC). Unfortunately, many HCC patients are initially not responsive to sorafenib. Due to anatomic limitations and underlying liver dysfunction, genomic studies of HCC patients have not been actively pursued yet compared to other solid tumors; especially, in sorafenib-treated subset. In this study, we have retrospectively identified advanced HCC patients who had archival tumor tissues available for targeted genomic sequencing and who had been treated with sorafenib.

Methods: We performed targeted genomic profiling for 381 cancer-related genes from 42 HCC (32 hepatitis B virus-related, 1 alcohol-related, 7 unknown) patients treated with sorafenib at Samsung Medical Center, Seoul, Korea from July 2008 to October 2013. We excluded 2 patients who were lost to follow up for evaluation of treatment response to sorafenib.

Results: Of the 40 patients, there were 5 confirmed PRs, 7 SDs, and 28 PDs. In 40 patients, there were 6 CDKN2B amplifications, 4 NTRK1 amplifications, 2 MET amplifications, 2 FGF19 amplifications, 2 CCND1 amplifications, 1 EGFR amplification, 1 FGF23 amplification and 1 CCNE1 amplification. In cell cycle-related genes, those patients with aberrations were less likely to respond to sorafenib. Of note, two patients whose tumor had CCND1 amplifications had de novo resistance to sorafenib.

Conclusions: Our study demonstrates inter-patient heterogeneity in advanced HCC patients and the role of cell cycle-associated genes in resistance mechanism to sorafenib is being investigated.

#427

A path towards determining tumor mutation burden and identifying neoantigens using next-generation sequencing (NGS).

Alex So, Shannon Kaplan, Nai-yu Wang, Shile Zhang, Aaron Wise, Kristina Kruglyak, Karen Gutekunst. _Illumina, San Diego, CA_.

Introduction: The recent clinical demonstrations that cancer immunotherapy is effective for some patients, but not others has led to widespread interest in identifying cancer genetic factors that can predict positive response. Importantly, it has been shown by various studies that tumor mutation burden (TMB) and predicted neoantigen load correlate with patient response to checkpoint inhibitors. This project aims to develop a clinical next-generation sequencing (NGS) assay that utilizes TMB and neoantigen information to guide cancer immunotherapy selection.

Experimental Procedures: Whole exome sequencing (WES) and whole transcriptome sequencing (WTS) libraries were generated using Illumina's TruSeq® Exome and RNA Access library preparation kits, respectively. The samples were pair-end sequenced (2x75bp) using HiSeq® 2000 and 2500 instruments.

Data Summary: Here, we demonstrate a workflow using tumor/normal WES and tumor-only WTS to determine expressed TMB. Using the WES data, we were also able to accurately identify human leukocyte antigen (HLA) major histocompatibility complex (MHC) Class I genes at four-digit resolution. We then identified putative neoantigens, defined as peptides with mutated amino acids, that were expressed in the tumor samples and predicted to bind to the respective HLA sequences. In addition, as a path towards developing a clinical assay for TMB and neoantigen determination, we tested the ability of reagents developed for Illumina's Trusight® Tumor 170 panel to be used for an exome panel. With workflow modifications, we achieved comparable quality of sequencing metrics as compared to the TruSeq® Exome kit.

Conclusion: Our data demonstrate a path towards developing a clinical assay that can be used to assess TMB and neoantigen candidates.

#428

Supporting neoantigen identification for personalized cancer vaccines through analytical validation of an augmented content enhanced (ACE) exome.

Ravi K. Alla, Jennifer Yen, Sean M. Boyle, Richard Chen. _Personalis, Inc., Menlo Park, CA_.

The identification of neoantigens has become a critical step in the development of neoantigen-based personalized cancer vaccines and other immunotherapy applications. Since neoantigens can be generated from tumor specific mutations in any expressed gene, the first step in identification of neoantigens typically involves deep exome and transcriptome sequencing on the tumor and exome sequencing of the matched normal. As personalized vaccines enter clinical trials with the potential for clinical use, there is a growing need for strong analytical validation of these platforms.

To address this we have developed our ACE Exome (~200X) and Transcriptome platforms for neoantigen identification which utilitize an augmented exome approach designed to increase sensitivity for neoantigens in low complexity, traditionally hard to sequencing regions. To enable this platform for neoantigen based personalized cancer vaccines, we have performed a validation of both our ACE Exome (tumor and normal) and ACE transcriptome platforms for detecting DNA-based SNVs and Indels, as well as for RNA based small variant and fusion calls. These are variant types are especially important for neoantigen identification. In this abstract we describe the ACE Exome validation.

We used 11 cancer cell lines and their matched normals to assess analytical sensitivity and limits of detection (LOD) for small variant (SNV and Indel) detection using our ACE exome and Tumor Normal bioinformatics pipeline. We identified a gold set of variants, 875 SNVs and 19 Indels that were previously validated in these 11 cell lines (COSMIC, CCLE and Sanger Sequencing confirmed variants). These gold set variants were used to calculate our analytical sensitivity (percent of gold variants detected across the 11 cell line pairs using our assay). To determine our LOD, we chose 3 of the 11 cancer cell lines and created 6 dilutions (5%, 10%, 20%, 30%, 50% and 80% tumor purity) with their matched normal. We then determined Positive Predictive Agreement (PPA, percent of pure cell line variants detected in a diluted samples) and False Discovery Rate (FDR, percent of erroneously detected variants in the diluted sample that were not detected in the pure cell lines) metrics for variants across different minor allelic frequencies (MAF) in the diluted samples.

The ACE "Tumor Normal" Exome assay had a high sensitivity of 98% for SNVs and 95% for Indels. The assay also showed robust PPA (sensitivity) of 97% and FDR (specificity) of 2% for SNVs with MAF &gt;= 10% and PPA of 87% and FDR of 3% for Indels with MAF &gt;= 10%.

We demonstrate that the ACE "Tumor Normal" Exome assay is highly accurate for identification of SNVs and Indels in cancer exomes. With high analytical sensitivity, PPA and low FDR we believe this assay provides augmented ability to detect cancer driver and potential neoantigen generating mutations across various tumor types.

#429

Integrated genomic DNA and RNA profiling to predict cancer immunotherapy response.

Pan Du, Xiaohong Wang, Zhixin Zhao, Huiquang Wang, Shidong Jia. _Predicine Inc, Hayward, CA_.

Immunotherapy response varies widely, making it difficult for physicians to know whether immunotherapy will be effective for a given patient. Recent studies have reported that patients with high PD-L1 gene expression are likely to respond to checkpoint blocking drugs, but there are still many patients whose tumor test for the PD-L1 protein are negative and can respond to the drugs. In addition to the potential link between mismatch repair (MMR) gene mutations and clinical response to anti-PD1 immunotherapy drug, recent findings show that tumor mutation burden and microsatellite instable (MSI) are good indicators of the cancer immunotherapy responses. Using Predicine's proprietary Gene RADAR (RNA and DNA single molecular digital Readings) technology, here we report the development of PrediSeq-CI (Cancer Immunotherapy) panel for comprehensive genomic profiling of DNA and RNA that are associated with cancer immunotherapy response. The panel has been tested using both tissue biopsy and plasma samples. The development of PrediSeq-CI test has potential to enable precision medicine in cancer immunotherapy. Finally, we also developed a liquid biopsy droplet digital PCR test to measure PD-L1 expression in blood.

### Metabolic Pathways in Cancer

#430

Combination of CB-839 and everolimus is effective in inhibiting growth of endocrine resistant breast cancer in vivo.

Yassi Fallah,1 Diane M. Demas,1 Susan Demo,2 Ayesha N. Shajahan-Haq1. 1 _Georgetown Lombardi Comp. Cancer Ctr., Washington, DC;_ 2 _Calithera Biosciences, South San Francisco, CA_.

About 70% of all breast cancers are estrogen receptor alpha positive (ER+). Anti-hormone therapy such as antiestrogens are often used to treat ER+ breast cancer but breast cancer cells can develop resistance to these drugs (endocrine resistance). Unfortunately, ~50% percent of all antiestrogen treated tumors eventually develop endocrine resistance, and therefore, there is an urgent need to find ways to treat this incurable disease. We have shown that endocrine resistant breast cancer cells show an increased dependence on the amino acid glutamine and this process is regulated by MYC activation via the unfolded protein response (UPR). Metabolites of glutamine such as glutamate and proline are significantly elevated in endocrine resistant cells. Cellular changes in glutamine are sensed by the mammalian target of rapamycin (mTOR) complex, mTORC1, which is known to be deregulated in endocrine resistant breast cancer. In this study, we used human antiestrogen (Fulvestrant/Faslodex or Tamoxifen) sensitive and resistant ER+ breast cancer cells and xenografts to test the efficacy of CB-839, an anti-glutaminase, and everolimus (Afinitor), a mTORC1 inhibitor. Combination of CB-839 and everolimus, but not each drug alone, inhibited growth of antiestrogen resistant tumors compared to vehicle alone at 4 weeks post-treatment. The combination treatment did not significantly inhibit growth of antiestrogen sensitive tumors compared with its respective vehicle alone treatment. Thus, a combination strategy that targets glutamine dependence and increased mTOR activation may be a novel strategy in treating endocrine resistant breast cancer.

#431

IDH1-mutated gliomas rely on anaplerosis of glutamate and lactate whereas IDH1 wild-type gliomas rely on glycolysis and acetate anaplerosis.

Mohammed Khurshed,1 Krissie Lenting,2 Remco J. Molenaar,1 William P. Leenders,2 Cornelis J. van Noorden1. 1 _Academic Medical Center, Amsterdam, Netherlands;_ 2 _Radboudumc, Amsterdam, Netherlands_.

Hotspot mutations in isocitrate dehydrogenase 1 (IDH1MT) initiate low grade glioma (LGG) and secondary glioblastoma and induce neomorphic activity that converts α-ketoglutarate (α-KG) to the oncometabolite D-2-hydroxyglutarate (D-2-HG). This causes metabolic rewiring that is not fully understood and in vitro studies have shown that IDH1MT cancer cells rely on glutaminolysis, providing cells with α-KG via activities of glutaminase (GLS) and glutamate dehydrogenase (GLUD). Here, we first show by in silico analysis of 269 IDH1 wild-type (IDH1WT) and 408 IDH1MT gliomas, obtained from the The Cancer Genome Atlas (TCGA) database, that IDH1WT gliomas have high expression levels of genes encoding for enzymes that are involved in glycolysis and acetate anaplerosis. On the other hand, the tricarboxylic acid (TCA) cycle, rather than glycolytic lactate production, is the predominant metabolic pathway in IDH1MT gliomas and is driven by lactate and glutamate anaplerosis to facilitate production of α-KG, and ultimately D-2-HG. IDH1WT\- and IDH1MT-related differences in expression were found in both LGG and glioblastoma. Furthermore, via in situ enzymatic activity mapping, we show in human gliomas and in xenocraft models that GLUD activity is increased and GLS activity is decreased in IDH1MT glioma, indicating that IDH1MT gliomas depend on glutamatolysis, rather than glutaminolysis. We show that transcript levels in our xenocraft models are in good agreement with our in silico analysis of the TCGA database. Finally, we confirmed the glutamate dependency of IDH1MT gliomas by MRS-flux analysis, whereas IDH1WT gliomas show high lactate production. Taken together, we show that IDH1WT gliomas have a typical Warburg phenotype and rely on acetate anaplerosis whereas IDH1MT gliomas are glutamate and lactate dependent. This metabolic rewiring in IDH1MT glioma, enables targeting of glutaminolysis rather than direct inhibition of IDH1MT for therapy. It diminishes the supply of glutamate-derived α-KG and directly inhibits the production of D-2-HG and simultaneously worsen the redox status of the glioma cells by inhibiting NAD(P)H production by GLUD. A candidate drug to inhibit GLUD is epigallocatechin-3-gallate gallate, a component of green tea that is currently receiving high interest as anti-cancer agent.

#432

xCT inhibition disrupts redox homeostasis in CD44v-expressing tumor cells showing glutaminolytic metabolism in head and neck squamous cell carcinoma.

Shogo Okazaki,1 Kenji Tsuchihashi,2 Oltea Sampetrean,1 Hideyuki Saya,1 Osamu Nagano1. 1 _Institute for Advanced Medical Research, School of Medicine, Keio University, Tokyo, Japan;_ 2 _Kyushu University Graduate School of Medical Sciences, Fukuoka City, Japan_.

Metabolic changes favoring aerobic glycolysis and glutaminolysis may influence the sensitivity of tumor cells to anticancer drugs. We previously showed that sulfasalazine, an inhibitor of xCT-dependent cystine transport, selectively induces oxidative damage in undifferentiated tumor cells that express CD44 variant (CD44v) at a high level, without affecting CD44v-negative differentiated tumor cells, in head and neck squamous cell carcinoma (HNSCC). However, whether metabolic changes contribute to the xCT dependency of CD44v-expressing HNSCC cells has remained unknown. Here we show that CD44v-expressing HNSCC cells manifest a highly glutaminolytic phenotype and constitutively generate reactive oxygen species through glutamine metabolism pathway, and that xCT plays a key role in maintaining redox homeostasis in these cells. Inhibition of xCT suppressed the consumption of glutamine without affecting that of glucose in these cells, indicating that CD44v-expressing HNSCC cells also rely on xCT for maintenance of glutaminolysis. Furthermore, administration of sulfasalazine, but not that of the anticancer drug cisplatin, selectively depleted CD44v-expressing undifferentiated tumor cells positive for the glutamine transporter ASCT2 in human HNSCC tumors formed in nude mice. Targeting of xCT is thus a potential therapeutic approach for ASCT2-expressing HNSCC tumors in which cellular metabolism is shifted toward enhanced glutaminolysis.

#433

Glycogen accumulation in aggressive breast cancers during hypoxic exposure.

Megan Altemus, Joel Yates, ZhiFen Wu, LiWei Bao, Sofia Merajver. _University of Michigan, Ann Arbor, MI_.

Isolation of specific metabolic alterations in aggressive triple negative and inflammatory breast cancers represents a compelling avenue for the development of treatments for these diseases. The ability of cancer to utilize diverse metabolic pathways to modulate increased survival and proliferation is well established. Indeed, we have previously described a series of metabolic adaptations in the triple negative inflammatory breast cancer cell line SUM149 and demonstrated a role for the small GTPase RhoC in the metabolic phenotype of the cell line.

In this work we seek to describe a survival mechanism for cancer cells that are subjected to hypoxic environments. Hypoxic survival or growth is often necessitated at the center of a growing tumor mass or if the tumor is growing in the lymphatic system, as is often the case for inflammatory breast cancers. Here we investigate the regulation of glycogen levels in breast cancer and normal-like cell lines in atmospheric and 1% oxygen environments. Reserves of glucose are typically stored in liver and muscle cells as the polysaccharide glycogen. Elevated levels of glycogen have previously been observed in various cancers, and recent studies have implicated the importance of glycogen metabolism in promoting cancer cell survival. We hypothesize that aggressive breast cancers utilize modifications of the glycogen synthesis and degradation pathways to provide nutritional flexibility as they proliferate and invade into diverse environments. MCF10A, SUM149, and SUM190 cell lines demonstrate a greater than 10-fold increase in glycogen accumulation when subjected to hypoxic growth conditions. MDA-MB-231 cells also increase glycogen storage, though at levels on the order of 2-3 times higher in hypoxia when compared to normoxic growth. Conditioned media for all cell lines in normoxic and hypoxic conditions were analyzed for glucose and lactate levels to determine changes in metabolite consumption.

The data presented indicate an unexpected accumulation of glycogen in response to hypoxic conditions. Studies to determine the mechanisms through which these changes occur and why they are important for continued survival and growth of the cancer cells are ongoing and will be presented.

#434

Characterizing the role of serine metabolism in pediatric sarcomas.

Sameer Issaq,1 Ria Kidner,1 Jason Rohde,2 Matthew Boxer,2 Lee Helman1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _National Institutes of Health, Rockville, MD_.

Sarcomas represent a diverse group of malignancies with unique molecular and pathological characteristics. In order to improve sarcoma treatment, a better understanding of the alterations associated with specific sarcoma subtypes is critically important. Renewed interest in the altered metabolic properties of cancer cells has led to an exploration of targeting metabolic dependencies as a novel therapeutic strategy. Metabolism of the amino acid serine is frequently altered in cancer, supporting a number of critical biological processes, including protein, lipid, and nucleotide synthesis, and redox balance. The first, rate-limiting step in the serine synthesis pathway is catalyzed by the enzyme 3-phosphoglycerate dehydrogenase (PHGDH), which is overexpressed in several cancers. Previous work has shown that PHGDH loss or inhibition is selectively toxic to cancer cells with high PHGDH expression or increased flux through the serine synthesis pathway. In this study, we have characterized the dependency of pediatric sarcomas on serine metabolism by examining expression of PHGDH in Ewing sarcoma and rhabdomyosarcoma cell lines, and evaluating the effects of PHGDH inhibition and serine deprivation on cellular proliferation and bioenergetic properties. We show that PHGDH is highly expressed in pediatric sarcoma cell lines, and that PHGDH knockdown resulted in decreased proliferation, especially under conditions of serine limitation. Moreover, pharmacological inhibition of PHGDH resulted in a dose-dependent decrease in proliferation and mitochondrial bioenergetic function. Furthermore, individual sarcoma cell lines were differentially sensitive to serine deprivation, indicating that some sarcoma cells may depend on extracellular serine in addition to de novo serine synthesis. Our findings suggest that the dependency of pediatric sarcomas on serine metabolism should be further investigated in order to identify vulnerabilities that could be targeted for potential therapeutic benefit.

#435

Regulation of macropinocytosis-dependent cell survival in pancreatic cancer cells.

Sung Eun Kim, Man-Tzu Wang, Frank McCormick. _UCSF, San Francisco, CA_.

Cancer cells utilize multiple scavenging mechanisms to support growth in nutrient-poor, hypoxic environments. Signaling pathways to accommodate these mechanisms may not be required for oncogenesis per se but are crucial for cancer cell survival. These processes, known as non-oncogene addiction, can be conferred by specific oncogenes that reprogram metabolism or by the tumor microenvironment. Understanding how these pathways are regulated can provide novel opportunities for therapeutic intervention.

Studies showed that KRAS-mutant cancer cells, including pancreatic ductal adenocarcinoma (PDAC), upregulate macropinocytosis, a caveolin- and clathrin-independent endocytic process, to import extracellular protein to support growth upon nutrient depletion. Similarly, high levels of macropinocytic uptake are observed in PDAC murine models and also in human PDAC specimens. This process serves as a scavenging mechanism for cancer cells to survive and proliferate in nutrient-deprived tumor microenvironments. Thus, uncovering the key players of this pathway and the mechanisms underlying their regulation will be important to find potential therapeutic targets.

Here, we used the Informer set of small molecules to screen regulators of survival in culture conditions supplemented with either glutamine or extracellular protein. The Informer set drug library consists of small molecules including FDA-approved drugs and clinical candidates that have high selectivity for their targets and regulate many cellular pathways. We find that survival of cells grown in conditions supplemented with glutamine or exogenous protein is differentially regulated by distinct drug subsets. This suggests that different pathways of nutrient uptake, processing, and utilization are used in cells depending on the nutrient source. Further examination to determine the mechanism of pathway inhibition and applicability may unravel potential uses for therapeutics.

#436

Folate cycle represents a new metabolic vulnerability for hepatocellular carcinoma treatment.

Derek Lee, Iris Ming-Jing Xu, David Kung-Chun Chiu, Robin Kit-Ho Lai, Chun-Ming Wong, Irene Oi-Lin Ng, Carmen Chak-Lui Wong. _The University of Hong Kong, Hong Kong_.

Hepatocellular carcinoma (HCC), primary liver cancer, ranks the third most lethal cancer worldwide due to late symptom presentation and lack of promising curative therapy. Metabolic reprogramming has been recognized as a major and new hallmark of cancer in recent years. Better understanding of its underpinning molecular mechanisms favoring cancer growth will be crucial for the development of effective HCC therapeutics. The folate cycle fuels metabolic processes and the production of metabolites essential to cell growth and tumorigenesis maintenance. Through the shuttling of a single carbon unit by a folate derivative through the tetrahydrofolate (THF) backbone in the cytoplasmic and mitochondrial compartments, metabolites like NADPH - a major cellular antioxidant for redox balance, s-adenosyl methionine (SAM) - precursor of DNA and histone methylation, and pyrimidine and purine - the building blocks of DNA are produced. We found folate to be indispensable for HCC cell growth. Furthermore, methylene-THF dehydrogenase 1-like (MTHFD1L), a key enzyme facilitating the folate cycle from the mitochondria, was found to be significantly up-regulated in HCC with association to poorer clinical features for patients. Genetic inhibition of MTHFD1L by knockdown and knockout by shRNA and CRISPR-Cas9 systems, respectively, blocked NADPH production. Rapid elevation in oxidative stress induced DNA damage and cell cycle delay; ultimately inhibiting HCC proliferation. Binding of the transcription factor NRF2, a potent protector of oxidative stress, and MTHFD1L was confirmed by ChIP assay. NRF2 over-expression using the CRISPR-activating system in HCC cells further highlighted the dependent relationship between NRF2 and MTHFD1L. Metabolomics analysis showed that MTHFD1L knockdown caused a disruption to the folate cycle and accumulation of serine. Surprisingly, MTHFD1L knockdown did not reduce the levels of SAM and nucleotides. Knockdown of MTHFD1L in HCC cells significantly inhibited primary liver tumor growth and lung metastasis in orthotopic liver implantation model. Therapeutically, the administration of methotrexate, an anti-folate agent, sensitized HCC cells towards Sorafenib treatment both in vitro and vivo. The folate cycle represents a metabolic vulnerability and attractive therapeutic target for HCC. Inhibition of MTHFD1L disrupts the folate cycle and sensitizes HCC cells towards its convention treatment agent, Sorafenib in various HCC mouse models. Our investigation unravels a metabolic vulnerability in cancer which contributes to better understanding and is beneficial for the development of precise inhibitors specifically targeting associated pathways.

#437

Stromal support of pancreatic tumor metabolism.

Costas A. Lyssiotis. _University of Michigan, Ann Arbor, MI_.

Pancreatic Ductal Adenocarcinoma (PDAC) is an aggressive disease characterized by a prominent desmoplastic stromal reaction and deregulated metabolism. The role of stroma in PDAC biology is complex and has been shown to play critical roles that may differ depending on the biological context. The intense stromal reaction also impacts the vasculature, leading to a highly hypoxic and nutrient poor environment. As such, these tumors must adapt how nutrients are captured and utilized to support their metabolic needs. In this talk, I will describe how stromal-associated pancreatic stellate cells (PSCs) are critical for PDAC metabolism through the secretion of non-essential amino acids (NEAA). Specifically, we uncovered an undescribed role for alanine, which outcompetes glucose and glutamine-derived carbon in PDAC to fuel the tricarboxylic acid (TCA) cycle, and thus NEAA and lipid biosynthesis. This shift in fuel sources decreases the dependence on glucose and serum-derived nutrients, which are limiting in the pancreatic tumor microenvironment. Moreover, we demonstrated that PSC alanine secretion is dependent on PSC autophagy, a process stimulated by the cancer cells. Thus, our results demonstrate a novel metabolic crosstalk between PSCs and cancer cells, with PSC-derived alanine as an alternative carbon source, and highlight a previously unappreciated metabolic network within pancreatic tumors where diverse fuel sources are utilized to promote growth in an austere tumor microenvironment.

#438

Cholesterol biosynthesis is a critical metabolic dependency in pancreatic cancer.

Linara Gabitova, Alena Klochkova, Diana Restifo, Aleksandra Mazitova, Edna Cukierman, Tiffiney Hartman, Igor Astsaturov. _Fox Chase Cancer Center, Philadelphia, PA_.

Pancreatic cancer is rapidly rising to become the 2nd leading cause of cancer deaths by 2020 in the USA. The rise in pancreatic cancer incidence is paralleled by the epidemic of obesity, type 2 diabetes, and associated increase in blood cholesterol, although mechanistic explanations for this are currently unknown. We and others have demonstrated that cancers with activated EGFR and KRAS signaling have increased demands for cholesterol and are vulnerable to interference with cholesterol uptake or endogenous cholesterol biosynthesis. We recently identified a metabolic step in the distal cholesterol pathway, mediated by SC4MOL and NSDHL enzymes, as a critical regulator of cell growth in the keratinocyte model of KRAS-induced carcinogenesis via the LXR agonistic effect of accumulating C4-methylsterols. Based on these preliminary data, we set out to test if pancreatic cancer initiation and progression depend on accelerated cholesterol biosynthesis in a genetic mouse model containing a conditional cholesterol pathway enzyme deficiency (conditional knockout of Nsdhlf/f) in the context of pancreatic cancer development in KPC mice (LSL-KrasG12D;Tp53f/f;Pdx1-Cre).

Results: Conditional inactivation of NSDHL alone in pancreatic tissue during normal development produced no apparent phenotype. As expected, the NSDHL-sufficient KPC mice as well as heterozygous Nsdhlf/+ littermates did not survive beyond 8 weeks of age due to rapid development of progressive pancreatic tumors showing complete displacement of pancreatic tissue with adenocarcinoma and high-grade PanIN lesions. Contrastingly, the survival of pancreatic conditional NSDHL-null mice was significantly extended beyond the median survival of 50 days in NSDHL-sufficient age-matched controls. Furthermore, the progression of pancreatic lesions from ADM to PanIN3 was remarkably delayed on NSDHL-null background, with only a proportion of animals developing adenocarcinoma. Evaluation of pancreatic tissues revealed a dramatic reduction of tumor-induced desmoplasia at all stages of pancreatic cancer development. Our ongoing studies will address the role of cholesterol metabolism in pancreatic cancer progression via regulation of tumor-specific production of stroma-recruiting growth factors.

Conclusions: Our studies demonstrated for the first time essential genetic evidence for metabolic dependency of pancreatic cancer on cholesterol metabolism. We identified NSDHL as a critical target in the endogenous pathway of cholesterol biosynthesis, and determined that blockade of NSDHL has dramatic consequences on the reciprocal signaling between the KRAS-transformed pancreatic cancer cells and the stroma.

#439

Metabolic flux analysis reveals targets to sensitize chemoresistance in acute myeloid leukemia induced by mesenchymal stromal cell-derived exosomes.

Abhinav Achreja,1 Hongyun Zhao,1 Ziwen Zhu,1 Jonathan Gerszberg,1 Marina Y. Konopleva,2 Michael Andreeff,2 Deepak Nagrath1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

The tumor microenvironment has a pleiotropic role in supporting cancer cell growth, metastasis and drug resistance. Exosomes from mesenchymal stromal cells (MSC) were found to regulate metabolism of acute myeloid leukemia (AML) cells that led to a chemoresistant phenotype. Exosomes carry a host of proteins, nucleotides and metabolites that can induce metabolic reprogramming recipient cells via direct supply of metabolite cargo or through signaling. We utilized 13C tracer techniques and two metabolic flux analysis techniques to reveal the mechanism of metabolic reprogramming induced by MSC-derived exosomes. First, 13C metabolic flux analysis was used to quantify intracellular fluxes of central carbon, amino acids and fatty acid metabolism in AML cells cultured with and without exosomes in media with 13C substrates. Second, we employed a novel technique, exosome-mediated metabolic flux analysis (Exo-MFA), to estimate direct support provided by metabolite supply from exosome cargo by introducing 13C-labeled exosomes. The combined analysis dissected the metabolite supply from exosome cargo from the overall reprogramming of metabolic pathways due to exosomes. Our results revealed key metabolic pathways that could be targeted to inhibit exosome-induced reprogramming to reverse the chemoresistant phenotype.

#440

The oncogenic activity of a pro-oxidant intracellular milieu is associated with redox dependent activation of NF-kB.

Michelle Y. Yee, Stephen J. Chong, Shazib Pervaiz. _National Univ. of Singapore, Singapore, Singapore_.

Evidence over the years has highlighted that intracellular redox status plays a critical role in determining cell fate. To date, we have shown that a pro-oxidant milieu, specifically a mild elevation of intracellular superoxide, promotes cancer cell survival and chemoresistance. To that end, we demonstrated in vitro that elevated superoxide, upon silencing of superoxide dismutase (SOD1), resulted in sustained phosphorylation of Bcl-2 at serine 70, thereby stabilizing its anti-apoptotic activity. These findings were corroborated by the abundance of Serine 70 Bcl2-phosphorylation and low SOD1 expression in lysates of patients with aggressive lymphomas and/or disease with poor prognosis. In addition to the anti-apoptotic protein Bcl-2, the pro-inflammatory transcription factor nuclear factor kappa B (NF-κB)- mediated signaling was also shown to be modulated by intracellular redox milieu. As constitutive activation of NF-κB has been reported in various human tumors, which is often associated with malignant progression and chemotherapeutic resistance, here we set out to investigate whether a pro-oxidant environment, specifically the upregulation of intracellular superoxide, was associated with the induction of NF-κB transcriptional activity. Indeed, we demonstrate that elevated intracellular superoxide via the pharmacological inhibition and genetic silencing of SOD1 results in enhanced activation of canonical NF-κB signalling. This is accompanied by increased p65 nuclear localization and transcription of NF-κB regulated genes, which potentially confer survival advantages to tumour cells upon chemotherapeutic treatments. Considering the association between NF-kB activation, chronic inflammation and the process of carcinogenesis, these preliminary data provide a novel mechanism of activation of this master transcriptional factor with potential therapeutic implications.

#441

GOT1 regulates anaplerotic glutamine metabolism under chronic acidosis stress in pancreatic cancer.

Jaime Abrego, Venugopal Gunda, Enza Vernucci, Surendra Shukla, Ryan J. King, Aneesha Dasgupta, Nina Chaika, Pankaj Kumar Singh. _UNMC Eppley Inst. for Cancer Research, Omaha, NE_.

Epithelial-cell derived tumors exhibit the Warburg effect that is characterized by an increased rate of glycolysis and lactate release, as well as, reduced oxidative metabolism. It is known that these metabolic alterations of cancer cells result in a tumor microenvironment with a lower pH than that of the plasma. However, little is known regarding the physiology and metabolism of cancer cells enduring chronic acidosis. Here, we cultured pancreatic cancer cells in chronic acidosis, i.e. pH 6.9 to 7.0, and observed a shift from glycolytic metabolism to oxidative metabolism that also results in reduced cell growth and increased intracellular ROS levels. We identified that this is due to an increase in glutamine uptake and increased expression of the transaminase enzyme GOT1 that enhances glutamine metabolism. Survival in low pH is reduced upon depletion of GOT1 due to further increased intracellular ROS levels. Thus, GOT1 plays an important role in energy metabolism and ROS balance in chronic acidosis stress. Our studies suggest the therapeutic potential of targeting anaplerotic glutamine metabolism in pancreatic cancer.

#442

Small molecule inhibitors of SHMT1/2 validate serine metabolism as a target in the treatment of c-Myc positive solid tumors.

Tomasz Rzymski, Anna Wrobel, Karolina Pyziak, Agnieszka Sroka, Marta Sowinska, Agnieszka Dreas, Marcin Krol, Pawel Guzik, Agnieszka Adamus, Agnieszka Przybylowicz, Katarzyna Hamara, Magdalena Sieprawska-Lupa, Artur Biela, Krzysztof Brzozka. _Selvita S.A., Krakow, Poland_.

Many oncogenes modulate metabolic pathways and altered metabolism is one of the hallmarks of cancer. In order to sustain proliferation, cell growth and adopt to a very specific tumor microenvironment, cancer cells have to undergo metabolic reprogramming. Increased uptake of glucose, consumed in anaerobic manner, allows to maintain essential bioenergetic and biosynthetic pathways. Various reports indicated that many cancers cells are crucially dependent on serine, which could be either imported or synthesized by the serine synthesis pathway (SSP) branched from glycolysis. Serine can be converted to glycine by Serine Hydroxymethyltransferases isoforms 1 and 2 (SHMT1/2), which provide also carbon for the folate cycle. There is a growing interest in targeting SSP and SHMT1/2 have been proposed as druggable targets for the treatment of various cancers. One of the major challenges is validation of these concepts by high quality pharmacological and genetic tools, particularly in the context of high tumor heterogeneity, artificial tissue culture conditions and many branching points of tumor metabolism, which eventually result in acquired resistance. We have identified and characterized a series of sub micromolar dual SHMT1/2 inhibitors. Affinity of these compounds to protein targets has been confirmed in biochemical and binding assays and further corroborated by X-ray crystallography studies. In order to confirm efficacy of these compounds in cancer cells, both sensitive and resistant cells to the inhibition of SSP have been identified by using conditioned serine and glycine depleted media. Moreover functional roles of both paralogs: SHMT1, which fuels cytoplasmic folate cycle and SHMT2, which is responsible for the mitochondrial branch, were further confirmed by gene knockdown studies. Cell lines resistant to depletion of serine in cell culture media were characterized by elevated levels of proteins involved in the synthesis of serine, namely PHGDH, PSAT1, SHMT2, c-Myc amplification and increased 13C flux from glucose to serine and glycine. Metabolic flux analysis further indicated that treatment with presented SHMT1/2 inhibitors effectively blocked the production of glycine from glucose and serine in cancer cells. Viability studies confirmed anti-cancer efficacy of SHMT1/2 inhibitors at concentrations consistent with metabolic flux studies in the same cells. Moreover rescue experiments with media supplemented with glycine and formate, which is a crucial intermediate between mitochondrial and cytoplasmic branches of folate cycle, were sufficient to reduce activity of SHMT1/2 inhibitors. Finally synergistic studies with antifolates provided an insight how efficacy of SHMT1/2 could be exploited therapeutically also in rational combinations with approved drugs.

#443

Lymphatic endothelium protects breast cancer cells from death by inducing metabolic adaptations.

Mirela Berisa,1 Simona Podgrabinska,1 Brandon Nicolay,2 Raul Mostoslavsky,2 Jerry Chipuk,1 Mihaela Skobe1. 1 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 2 _Massachusetts General Hospital Cancer Center, Boston, MA_.

The lymphatic vasculature is an important pathway for breast cancer dissemination, yet it is not understood whether and how the lymphatic vessel microenvironment influences cancer metastasis. We demonstrate that lymphatic endothelial cells (LECs) promote survival of triple-negative breast cancer cells (TNBCs) under stress by improving mitochondrial function and inducing metabolic shift to promote cellular energy production. LECs protected TNBCs from death in vitro induced by the loss of attachment and nutrient deprivation. Cell death was preceded with a sharp increase in reactive oxygen species (ROS), strong up-regulation of Nrf2-mediated oxidative stress response genes and a rapid decline of mitochondrial activity. LECs lowered ROS levels, decreased mitochondrial superoxide formation and enhanced mitochondrial activity in TNBCs. RNAseq transcriptome analysis identified key regulator of mitochondrial metabolism and cellular bioenergetics, peroxisome proliferator-activated receptor gamma coactivator (PPARGC1A/PGC-1α, to be specifically up-regulated in breast cancer cells by LEC-derived factors. Inhibition studies demonstrated that the TNBC survival was dependent on pentose phosphate pathway (PPP) activity. Notably, LECs induced a metabolic shift from glycolysis to fatty-acid oxidation (FAO) and oxidative phosphorylation to maintain ATP and sustain cell viability. These data demonstrate that lymphatic endothelium promotes survival of breast cancer cells by regulating energy production and maintaining redox homeostasis. Our findings suggest that lymphatic endothelium may facilitate metastasis by promoting survival of breast cancer cells within the lymphatic vasculature.

#444

Monocarboxylate transporter 1 inhibition with AZD3965 increases cancer cell dependence on bioenergetic metabolism predicating combination therapy with mitochondrial inhibitors.

Mounia Beloueche-Babari,1 Teresa Casals Galobart,1 Slawomir Wantuch,1 Paul D. Smith,2 Martin O. Leach1. 1 _Institute of Cancer Research, London, Sutton, United Kingdom;_ 2 _AstraZeneca, Cambridge, United Kingdom_.

Monocarboxylate transporters (MCTs) are key mediators of lactate transport that have emerged as promising targets for anti-cancer therapy. The MCT1 inhibitor AZD3965 (AstraZeneca) has shown promising activity in various pre-clinical models and is currently in phase I/II clinical testing. Understanding the impact of this drug on tumour cell metabolism may unravel dependencies that could be exploited for combination therapy. Here we investigate changes in glucose metabolism induced by AZD3965 treatment using 13C NMR isotopomer analysis, and examine their significance for cell survival using mitochondrial metabolism inhibitors.

Exposure of Raji human lymphoma cells to AZD3965 in media supplemented with [1-13C]glucose led to a marked reduction in glucose uptake and lactate production in the cellular growth media alongside a build-up in intracellular [3- 13C]lactate and [1-13C]glucose levels, indicative of blockade of lactate excretion and inhibition of overall glycolytic activity. These effects were concomitant with increased [4-13C]glutamate levels, consistent with re-routing of pyruvate towards mitochondrial metabolism and enhanced flux through oxidative pyruvate dehydrogenase.

Further, AZD3965 treatment was paralleled with a significant increase in levels of steady state Krebs cycle-related metabolites (including succinate, fumarate, acetate and NAD+/NADH) and cellular ATP, as revealed by bioluminescent and 1H NMR analyses, indicating improved mitochondrial metabolism and cellular re-energization.

Co-administration of the mitochondrial complex I inhibitor metformin or the mitochondrial pyruvate carrier inhibitor UK5099 markedly potentiated the anti-proliferative effects of AZD3965 and led to significantly increased cell death, indicating that the observed upregulation in mitochondrial metabolism was necessary to maintain cell survival under MCT1 inhibitor-induced metabolic stress. Similar effects were observed with a second human lymphoma cell line, Hut78.

Our findings show that MCT1 inhibition leads to a shift in cellular metabolism towards mitochondrial bioenergetic metabolism, creating a metabolic vulnerability that could be exploited for combinatorial therapy to increase the efficacy of AZD3965.

#445

Efficient tumorigenesis after genetic dissociation of glycolysis and the TCA cycle.

Laura E. Jackson,1 Sucheta Kulkarni,1 Huabo Wang,1 Jie Lu,1 Sivakama Bharathi,1 Radha Uppala,1 Mulchand S. Patel,2 Eric S. Goetzman,1 Edward V. Prochownik1. 1 _Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA;_ 2 _State University of New York at Buffalo, Buffalo, NY_.

Virtually all tumors undergo some form of metabolic re-programming to match anabolic needs with energy supply. The best-known form of this is the Warburg effect by which tumors become more reliant on glycolysis over mitochondrial oxidative phosphorylation, even when their oxygen supply is abundant. Nonetheless, mitochondria retain some residual activity in order to provide both ATP and critical metabolic building blocks and other substrates for processes such as lipid biosynthesis and protein acetylation. The pyruvate dehydrogenase (PDH) complex (PDC) is the main enzymatic link between glycolysis and the TCA cycle and irreversibly converts the end-stage glycolytic product pyruvate into the critical TCA substrate, acetyl-coenzyme A (AcCoA). In a mouse model of the pediatric liver cancer hepatoblastoma (HB), we recently showed PDH to be highly up-regulated, despite reduced mitochondrial function. We postulated that this might represent a response to pyruvate depletion due to upstream shunting of glycolytic intermediates into anabolic pathways. To test this idea, we examined in vivo tumorigenesis in mice bearing a hepatocyte-specific conditional knockout of the pdha1 subunit (KO mice). The survival of these mice was marginally longer than the wild-type (WT) controls, although tumor sizes at time of death were ~20% smaller. KO mice expressed virtually no PDH (and hence no PDC activity) and had high blood lactate levels. This suggested that they succumbed with smaller tumors as a result of severe lactic acidosis that could not be corrected by hyperventilation due to respiratory compromise by the large HBs that restricted lung volumes. This was supported by studies performed in a modified form of this model in which only tumors were PDC deficient whereas the surrounding liver parenchyma was not. These mice now survived longer than did their WT counterparts and their tumors reached comparable sizes. They also had lower lactate levels than the pan-KO mice. Oxygen consumption rates of WT and KO tumors were similar yet were lower than those of normal livers. Rates of fatty acid β-oxidation by WT and KO tumors were also lower than those of liver, indicating that both types of tumors preferentially divert fatty acids into membrane synthesis rather than toward supplying AcCoA. Taken together, these findings demonstrate the remarkable capacity for nearly normal HB pathogenesis despite the severing of glycolysis from the TCA cycle.

#446

Differential effects of folate depletion on metabolic reprogramming and oxidative stress in nonmetastatic and metastatic claudin-low breast cancer cells.

Xuewen Chen, Ciara H. O'Flanagan, Stephen D. Hursting. _University of North Carolina, Chapel Hill, NC_.

Folate coenzymes play an important role in biosynthesis and methylation reactions. Aberrant folate metabolism has been implicated in the development of several cancer types, though mechanisms underlying folate metabolism and cancer development remain unclear. We previously showed that a folate-restricted diet can exert differential effects on metastatic versus nonmetastatic murine claudin-low breast cancer cells in vivo. Specifically, folate restriction increased growth and invasion of orthotopically transplanted M-Wnt (nonmetastatic) tumor cells, but decreased growth and lung metastases of transplanted metM-Wnt cells, a metastatic subclone of M-Wnt cells. The current study set out to explore the underlying mechanism. To examine the effect of long-term folate depletion (LFD) on M-Wnt and metM-Wnt cell metabolism, oxidative stress and autophagic flux in vitro, the two cell lines were grown in standard and folate-depleted media for 14 days. metM-Wnt cells showed higher oxidative stress, as measured by ROS staining and Nrf2 expression, and phosphorylation of the key nutrient sensor, AMPK, compared to M-Wnt cells when grown in standard growth medium. LFD M-Wnt cells showed an increased dependence on glycolysis compared to those cultured in standard medium. Both M-Wnt and metM-Wnt cells displayed a high autophagy level in LFD, measured by LC3B cleavage, and AMPK phosphorylation. However, LFD metM-Wnt cells showed low viability, increased apoptosis and loss of redox defense, as measured by cleaved-caspase 3 and Nrf2 expressions. These results suggest that non-metastatic M-Wnt cells undergo metabolic reprogramming, including a shift from oxidative phosphorylation to glycolysis that may fuel cell growth and proliferation. Further, an elevated autophagic flux may mitigate nutrient stress induced by folate depletion, which allows them to withstand LFD and which may contribute to a more invasive primary tumor in response to folate restriction. In contrast, metM-Wnt cells are unable to undergo this metabolic adaptation, and display increased oxidative stress and cell death in response to LFD, preventing the development of metastatic lesions in vitro. This study highlights different responses of primary and metastatic breast cancer cells to folate depletion. The results provide additional rationale for targeting folate metabolism as a potential strategy for treating metastatic breast cancer.

#447

ZAG promotes cachexia-associated white adipose tissue browning and energy wastage.

Sawsan Elattar, Satyanarayana Ande. _Augusta University, Augusta, GA_.

Cancer cachexia is a complex condition of tissue wasting that affects up to 80% of cancer patients. To date, there is no effective treatment for cachexia due to the complex nature of this condition. Energy wasting in cachexia is caused by excessive lipid and protein turnover in the body, browning of white adipose tissue (WAT), and futile metabolic cycling such as glucose recycling between the liver and tumor. Therefore, identifying and inhibiting factors that promote WAT browning, and glucose and lipid recycling can reduce energy wasting and ameliorate cachexia in cancer patients. Zinc-α2-glycoprotein (ZAG), a lipid mobilizing factor secreted by multiple cancers, has been shown to promote lipolysis and inhibit lipogenesis in WAT. However, whether ZAG plays any role beyond lipolysis and participates in other energy wasting mechanisms of cachexia such as glucose and lipid recycling, and WAT browning has not been explored. Our initial studies indicate that while ZAG is highly expressed in the heart, kidney, and liver of wild-type mice, the basal expression of ZAG is very low in WAT at both the mRNA and protein levels. To investigate the metabolic functions of ZAG in vivo, ZAG-expressing 293 cells were implanted subcutaneously in athymic nude mice, and analyzed them after 3 and 6 weeks. We detected a 3-fold increase in the circulating plasma levels of ZAG. Importantly, the increase in ZAG levels was associated with a decrease in body weight gain without any differences in food consumption and physical activity. Morphological examination of eFAT pads of ZAG-cell implanted mice showed smaller size compared to controls. In addition, histological examination of eFAT showed cell shrinkage and almost complete depletion of fat stores, suggesting enhanced lipolysis in the WAT. Notably, ZAG induced the expression of beige, mitochondrial, and thermogenesis genes at mRNA level in the eFAT. Moreover, the expression of brown adipocyte-specific thermogenic genes such as Ebf2, Prdm16, PGC1α, UCP1, SRC1 and IRF4 were strongly induced at the protein level in eWAT of ZAG-cell implanted mice suggesting that ZAG causes WAT browning. Furthermore, metabolic analysis revealed increased O2 consumption and heat production in ZAG-cell implanted mice compared to controls indicating increased total body energy expenditure. ZAG-cell implanted mice also displayed reduced respiratory exchange ratio indicating increased consumption of lipids. Overall, our findings suggest that ZAG functions beyond lipolysis and causes significant energy wasting by causing WAT browning and promoting glucose and lipid catabolism in the beige cells. These findings suggest that ZAG plays a predominant role in the development of cachexia, and thus, may represent a novel therapeutic target to block energy wasting and delay or prevent cachexia in cancer patients.

#448

Role of fructose in prostate cancer.

Daniela Carreño,1 Nestor Corro,1 Marcia Arredondo,1 Carmen Navarro,1 Verónica Torres,1 Viviana Montecinos,1 Paula Sotomayor,2 Francisco Nualart,3 Julio Cesar Cárdenas,4 Alejandro S. Godoy1. 1 _Pontificia Universidad Católica de Chile, Santiago, Chile;_ 2 _Universidad Andres Bello, Santiago, Chile;_ 3 _Universidad de Concepción, Concepción, Chile;_ 4 _Universidad de Chile, Santiago, Chile_.

The elevated level of glucose uptake and metabolism in cancers is the basis for the clinical localization of primary cancers and sites of metastasis by positron emission tomography (PET scanning), based on the enhanced cellular uptake of 2-deoxy-2-[18F]-fluoro-D-glucose (FDG). In prostate cancer (CaP), however, FDG-PET imaging has shown limited clinical applicability. This striking difference suggests that CaP cells utilize hexoses other than glucose, such as fructose, as the principal energy source. The purpose of this study was to determine whether or not fructose is a/the principal source of energy for CaP cells. mRNA and protein expression for the glucose transporter Glut-1 and fructose transporters Glut-2, Glut-5, Glut-7, Glut-9 and Glut-11 was analyzed in benign (PWR-1E, RWPE-1) and malignant (LNCaP, vCaP, LNCaP-C4-2, DU-145, and PC-3) human prostate cell lines using qRT-PCR and western blot, respectively. In addition, Glut(s) protein expression was analyzed on a tissue microarray containing 200 formalin-fixed paraffin-embedded benign and malignant human prostate tissues using immunohistochemistry. Fructose and glucose uptake was measured in vitro in benign and malignant human prostate cell lines using radiolabelled D-[U-14C]-fructose or 2-[1,2-3H]-deoxy-D-[3H]-glucose, respectively. Lastly, the effect of fructose or glucose on the levels of ATP, mitochondrial metabolism, and expression of the enzymes hexokinase-2 (HK2), type-C fructokinase (KHK-C), pyruvate kinase M2 (PKM2) and type-A lactate dehydrogenase (LDH-A) was analyzed in benign and malignant human prostate cell lines using chemiluminescence, seahorse, and qRT-PCR analyses, respectively. Our results indicated that expression of the fructose transporters, Glut-5 and Glut-9, was increased in CaP cell lines and in human CaP tissues compared to benign cell lines and benign prostate tissues, respectively. Glut-1 expression, however, did not differ between benign and malignant human prostate cells. Transport assays demonstrated that CaP cell lines have a higher capacity to transport fructose compared to benign cell lines. However, glucose uptake was not altered between benign and malignant human prostate cell lines. ATP levels in CaP cells were similar in the presence of fructose or glucose. Fructose, but not glucose, significantly altered mRNA expression of HK2, KHK-C, PKM2, and LDH-A in malignant human prostate cells. Taken together, our results suggest that fructose may represent an alternative energy source and may reprogram hexose metabolism in CaP cells.

#449

Reduced argininosuccinate synthetase expression in refractory sarcomas: impacts on therapeutic potential and drug resistance.

Eisuke Kobayashi,1 Youngji Kim,1 Daisuke Kubota,2 Yoshiyuki Suehara,1 Akira Kawai,1 Shigehisa Kitano1. 1 _National Cancer Center Hospital, Tokyo, Japan;_ 2 _Juntendo University, Tokyo, Japan_.

Introduction: Treating drug-resistant sarcomas remain a major challenge. The present study aimed to identify a novel therapy for drug-resistant sarcomas based on a metabolic errors involving argininosuccinate synthetase1 (ASS1).

Methods: We assessed the expression of ASS1 and P-glycoprotein (P-gp) in osteosarcoma (KHOS), doxorubicin (Dox)-resistant osteosarcoma (KHOSR2), epithelioid sarcomas (ES-X and VAESBJ), alveolar soft part sarcoma (ASPS-KY), and each clinical specimen. Each cell was cultured in arginine-containing and arginine-free media. Cell growth was assessed using an XTT assay and flow cytometry. We analyzed the induction of autophagy in arginine-free medium. Moreover, we assessed the expression of P-gp in after suppressing ASS1 in Dox-sensitive cells (MCF-7, KHOS) and in after transfecting the ASS1 into Dox-resistant cells (ES-X, VAESBJ, ASPS-KY and KHOSR2).

Results: The expression of ASS1 was reduced in Dox-resistant sarcoma cells. Immunohistochemistry (IHC) and real-time PCR showed that there was interestingly an inverse relationship between the expression of ASS1 and the expression of P-gp. The inhibition of cellular proliferation with G1-arrest was shown to lead to autophagy with arginine deprivation. In addition, the combination of autophagy inhibitor plus arginine deprivation was more effective than arginine deprivation alone. In cells in which the expression of ASS1 was suppressed, the expression of P-gp was upregulated in comparison to negative controls.

Discussion: These results indicate that the reduced expression of ASS1 expression in Dox-resistant sarcomas may contribute to drug resistance. ASS1 deficiency is a potential target for novel drug therapies. The combination of arginine deprivation therapy and an autophagy inhibitor may have anti-tumor effects in refractory sarcomas.

Significance: As the induction of autophagy by the deprivation arginine may have a pro-survival role in patients with ASS1-deficient sarcomas, the combination of arginine deprivation therapy with autophagy modulators might potentiate anti-tumor effects in patients with drug-resistant sarcomas.

#450

Aldo-keto reductase family 1 member b1 links glucose metabolism to epithelial-to-mesenchymal transition.

Annemarie Schwab,1 Aarif Siddiqui,1 Maria Eleni Vazakidou,1 Francesca Napoli,1 Martin Boettcher,2 Bianca Menchicchi,3 Ida Rapa,4 Maximilian Waldner,3 Dimitrios Mougiakakos,2 Marco Volante,4 Florian Greten,5 Thomas Brabletz,6 Paolo Ceppi1. 1 _IZKF Junior Group 1, University Hospital Erlangen, Erlangen, Germany;_ 2 _Department of Internal Medicine 5, Hematology and Oncology, University Hospital Erlangen, Erlangen, Germany;_ 3 _Department of Medicine 1, University Hospital Erlangen, Erlangen, Germany;_ 4 _Pathology Unit, San Luigi Hospital, University of Turin, Turin, Italy;_ 5 _Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany;_ 6 _Experimental Medicine I, FAU Erlangen-Nürnberg, Erlangen, Germany_.

Introduction: We performed a bioinformatic analysis to identify metabolic genes connected with the process of epithelial-to-mesenchymal-transition (EMT) and the results indicated a possible role for aldo-keto reductase family 1 member B1 (AKR1B1). AKR1B1 is a member of the polyol pathway responsible for catalyzing the reduction of numerous aldehydes, such as glucose. Based on the importance of EMT during carcinogenesis and metastatic progression and on the relevance of enhanced glycolytic rate in cancer cells, we investigated a direct role for AKR1B1 during EMT and tumor progression.

Experimental Procedure: The bioinformatic analysis was performed on datasets from the NCI60 panel of cancer cell lines. Cancer cells from lung, breast and ovarian origin have been investigated in vitro: changes in EMT markers were monitored by western blotting as well as immunofluorescence, growth assays were performed using the IncuCyte® ZOOM, migration rate was tested by wound-healing assays. Changes in stem-like properties were determined by western blotting, FACS and sphere-formation assays. Immunohistochemistry (IHC) was performed on FFPE specimens from lung cancer patients. Additionally, IHC was performed on samples from a mouse model of AOM-induced colon tumorigenesis in mice with an intestinal epithelial cell-specific p53 deletion (which were shown to undergo EMT) and in the wildtype counterparts.

Results: AKR1B1 gene and protein expression was found significantly higher (7-fold) in mesenchymal-like cells. ShRNA-mediated knockdown of AKR1B1 lead to mesenchymal-to-epithelial transition in vitro and suppressed EMT induced by TGF-β or by high glucose levels. Besides reduced migration, AKR1B1-deficient cells displayed decreased proliferation rate and colony-formation ability. Moreover, AKR1B1 knockdown or its inhibition with specific drugs diminished cancer stem cells. The phenotypes observed with AKR1B1 knockdown could be obtained by targeting sorbitol dehydrogenase (SORD), the second and last enzyme of the polyol pathway. Suppression of each enzyme resulted in an impaired glycolytic and oxidative metabolism and adding fructose, the end-product of the polyol pathway, rescued the expression of EMT markers. IHC on samples from the AOM-induced colon cancer model indicated a higher AKR1B1 expression in invasive tumors from p53ΔIEC mice as compared to both p53-deficient non-invasive or wildtype tumors. Finally, AKR1B1 staining of cancer tissues from a cohort of lung cancer patients confirmed a significant correlation with EMT and a negative prognostic value.

Conclusion: In summary, we describe a glucose-related pathway with a previously unknown role in regulating cancer plasticity and EMT, which links altered glucose metabolism to growth and migratory ability, with several potential implications. Targeting polyol pathway enzymes could be a potentially effective therapeutic strategy to arrest cancer progression.

#451

Germ free mice accelerate cachexia-associated cancer.

Rodrigo Xavier das Neves,1 Soumen Roy,1 Amiran Dzutsev,1 April Huang,1 Loretta Smith,1 Simone Difilippantonio,1 Hawes Misty,1 Marília Seelaender,2 Giorgio Trinchieri1. 1 _NCI, Bethesda, MD;_ 2 _ICB, Sao Paulo, Brazil_.

The syndrome of cancer cachexia is currently defined as a state of ill health, malnutrition and physical wasting with marked white adipose tissue (WAT) and skeletal muscle mass wasting, representing the clinical consequence of a chronic and systemic inflammatory response. Over the last decade, WAT has been recognized as an important endocrine organ, and earning a lot of attention during cancer cachexia development. We investigated the role of microbiota along the cachexia associated cancer. We performed experiments with conventional and Germ Free mice (GF) (n=6 in each group) of 8-10 weeks old C57B/6, which were subcutaneously injected with LLC cells [4x106 cells in 0.2 mL; Tumor-bearing, (TB) or PBS control (C)]. We performed Immunohistochemistry, RT- qPCR, and Western Blot. We observed that GF Tumor-bearing mice have increased several symptoms of the cachexia compared to conventional TB mice. The WAT mass was decreased 50% in GF Tumor-bearing mice compared to all groups, which indicates a pathway related to lipolysis, as we found increased level of phosphorylated enzymes in GF Tumor-bearing mice. We also observed that GF Tumor bearing mice decreased skeletal muscle mass and gene expression that are related with atrophy were increased in GF Tumor bearing mice. Our data suggested that homeostasis of microbiota may impair the development of the cachexia syndrome.

#452

Activation of Akt pathway and autophagy promotes resistance to FASN inhibition in colorectal cancer patient-derived xenograft models.

Yekaterina Y. Zaytseva,1 Piotr G. Rychahou,1 Anh-Thu Le,1 Robert M. Flight,1 Timothy L. Scott,1 Jennifer W. Harris,1 Sally Hodges,1 Brent J. Hallahan,1 Dana L. Napier,1 Jinpeng Liu,1 Chi Wang,1 Manjula Sunkara,1 Andrew Morris,1 Ji Tae Kim,1 Sivakumaran Theru Arumugam,1 Andrew Lane,1 Teresa W. Fan,1 Hunter Moseley,1 Tianyan Gao,1 Eun Y. Lee,1 Heidi L. Weiss,1 Timothy S. Heuer,2 George Kemble,2 B Mark Evers1. 1 _University of Kentucky, Lexington, KY;_ 2 _3-V Biosciences, Menlo Park, CA_.

Fatty Acid Synthase (FASN), a key enzyme of de novo lipogenesis, is upregulated in many cancers including colorectal cancer (CRC); increased FASN expression is associated with poor prognosis. Potent FASN inhibitors developed by 3-V Biosciences demonstrate anti-tumor activity in vitro and in vivo and a favorable tolerability profile in a Phase I clinical trial in solid tumor patients. However, CRC characteristics associated with responsiveness to FASN inhibition are not fully understood. The purpose of our study was: (i) to determine the effect of FASN inhibition on tumor growth in CRC patient-derived xenografts (PDXs); (ii) to identify potential biomarkers associated with CRC responsiveness to FASN inhibition; and (iii) to explore new combination strategies with FASN inhibitors. METHODS. Tumor growth was assessed in 9 PDXs established in NSG mice using freshly resected specimens. Once the xenografts grew to ~100 mm3, mice were randomized into two groups (n=5) to receive either vehicle or TVB-3664 or four groups (n=10) for TVB-3664 treatment in combination with either MK2206 or Chloroquine (CQ). Tumor volume and animal weights were measured weekly. Western blot analysis and immunohistochemistry staining were used to identify FASN-mediated changes in signaling pathways. Changes in metabolites and lipids were analyzed by nuclear magnetic resonance and mass spectrometry in plasma and tumor tissues. Next Generation Sequencing was used to assess the mutation profile of 198 oncogenes in patient tumors and PDXs. RESULTS. PDXs showed a wide range of sensitivity to FASN inhibition: TVB-3664 treatment attained significant response (reduced tumor volume) in 3 PDXs, significant response followed by developed resistance in one PDX, and no response in 5 PDXs. Activation of Akt and AMPK pathways was associated with resistance to FASN inhibition and combination of TVB-3664 with either MK2206 or CQ led to a significant reduction in tumor volume as compared to either drug alone. Moreover, TVB-3664 treatment significantly decreased the total palmitate level in plasma and the levels of triglycerides, diglycerides, phosphatidylserines, phosphatidylethanolamines, and phosphatidylcholines in tumor tissues. Furthermore, a significant decrease in the levels of AXP-1, AXP-2 and myo-Inositol-2 was observed in tumors responsive to FASN inhibition. CONCLUSIONS. Our studies demonstrate that TVB-3664 shows anti-tumor activity in CRC. Importantly, our results suggest that activation of Akt and autophagy are major mechanisms of resistance to FASN inhibition and demonstrate that combine inhibition of these pathways and FASN may be a new therapeutic approach in CRC. Ongoing studies of correlation between mutation and metabolic profiles of tumors and tumor response to FASN inhibition aim to identify a subset of CRC patients that are likely to respond to FASN-targeted therapy.

#453

Orlistat as a novel targeted therapy for obesity-driven endometrial cancer.

Stephanie Sullivan,1 Arthur-Quan Tran,2 Weiya Wysham,2 Paola Gehrig,2 Chunxiao Zhou,2 Victoria Bae-Jump2. 1 _University of North Carolina, Chapel Hill, OH;_ 2 _University of North Carolina, Chapel Hill, NC_.

Introduction: Obesity is associated with increased risk for and worse outcomes from endometrial cancer (EC). Orlistat is a weight loss medication that has been shown to be a potent inhibitor of fatty acid synthase (FAS), and FAS is known to be upregulated in ECs. Thus, we sought to investigate the anti-tumorigenic potential of orlistat in EC cell lines and a genetically engineered mouse model of endometrioid EC (LKB1fl/flp53fl/fl mouse model).

Methods: Two endometrial cell lines, ECC-1 and KLE, were used. Cell proliferation was assessed by MTT assay after exposure to orlistat. Western immunoblotting was performed to evaluate the effect of orlistat on downstream targets of fatty acid metabolism. LKB1fl/flp53fl/fl mice were fed a control low fat diet (LFD; 10% calories from fat, lean group) versus a high fat diet (HFD; 60% calories derived from fat, obese group) to mimic diet-induced obesity, starting at 3 wks of age. AdCre was injected at 6 wks of age to induce invasive EC. Mice were treated with placebo or orlistat (200 mg/kg/day, IM) following tumor onset for 4 weeks (N=10 mice/group). Immunohistochemistry was performed to assess for the effect of orlistat on expression of Ki-67 (marker of cell proliferation), vascular endothelial growth factor (VEGF) and phosphorylated (p)-acetyl-CoA carboxylase (ACC).

Results: Orlistat inhibited growth in a dose-dependent manner for both endometrial cell lines with a mean IC50 for ECC-1 of 88.7 uM and 145.4 uM for KLE (p<0.05). In addition, orlistat decreased expression of FAS, ACC and carnitine palmitolytransferase 1A, consistent with an inhibitory effect on fatty acid metabolism. In both the obese and lean LKB1fl/flp53fl/fl mice, treatment with orlistat resulted in decreased tumor weight/size as well as Ki-67 expression (p<0.05), but the difference was more pronounced in obese mice. Orlistat decreased tumor weight/size by 66% in the obese mice and 23% in the lean mice (p<0.05). Immunohistochemistry revealed that p-ACC and VEGF expression was higher in the obese versus lean mice at baseline (p<0.05). Orlistat reduced VEGF in the endometrial tumors of both the obese and lean mice (p<0.05) but only decreased expression of p-ACC in the tumors of obese mice (55%, p<0.05).

Conclusions: Orlistat inhibited EC cell proliferation and endometrial tumor growth in an endometrioid EC mouse model, with increased efficacy in obese versus lean mice. Therefore, orlistat may be worthy of drug repurposing as an anti-tumorigenic agent in EC, with the potential added benefit of weight loss in this obesity-linked disease.

#455

Phosphoenolpyruvate carboxykinase coordinates metabolic flexibility to control tumor growth.

Emily Montal, Ashley Ropell, Geoffrey Girnun. _Stony Brook University, Stony Brook, NY_.

Colorectal cancer (CRC) is the third most common cancer (excluding skin cancer) and the third leading cause of cancer death in the United States. While rates are declining among individuals over 50 years of age due to increased screening, the incidence of CRC in the US population under 50 years of age is increasing. Therefore, better treatment approaches are urgently needed. Studies are emerging that highlight an important role for the TCA cycle in regulating cancer cell proliferation. The TCA cycle integrates glucose, amino acid and lipid metabolism depending on cellular needs. In addition, biosynthetic pathways crucial to tumor growth require the TCA cycle for the processing of glucose and glutamine derived carbons. Phosphoenolpyruvate carboxykinase (PEPCK) is well known for its role in gluconeogenesis. However, PEPCK plays an important role in mediating TCA cycle flux. We recently showed that PEPCK promotes colon cancer growth in vitro and in vivo, independent of its role in gluconeogenesis. This growth advantage was mediated in part through regulating metabolic flux of glucose and glutamine to biosynthetic precursors, such as lipids and nucleic acids. These effects of PEPCK on glucose metabolism and cell proliferation are in part mediated via activation of mTORC1. Currently we are investigating the mechanism(s) responsible for how PEPCK regulates cell signaling via metabolic flux. We are also seeking to identify ways in which we can utilize PEPCK as a therapeutic target.

#456

Functional analysis of DEC1 and DEC2 in cancer metabolism.

Fuyuki Sato, Yasueru Muragaki. _Wakayama Medical University, Wakayama city, Japan_.

Basic helix-loop-helix (bHLH) transcription factor DEC1 (bHLHE40/Stra13/Sharp2) and DEC2 (bHLHE41/Sharp1) are clock genes that show a circadian rhythm in various tissues. However, the function of DEC1 and DEC2 in metabolism has not been poorly understood. AMP-activated protein kinase (AMPK) activity plays important roles in the metabolic process and circadian rhythm. However, how circadian rhythm of phosphorylation of AMPK (pAMPK) is regulated has been yet understood. The aim of this study is to investigate whether there is a direct correlation between DEC1 and DEC2 expression and AMPK activity in normal and cancer cells. There was an inverse relationship between DEC1 expression and AMPK activity. DEC1 protein and pAMPK showed a circadian rhythm in the mouse liver with different peak levels. Knocking down DEC1 expression increased pAMPK, whereas overexpression of DEC1 decreased it. DEC1 bound to the E-box of the LKB1 promoter, decreased LKB1 activity and total protein levels. In addition, pAMPK immunoreactivity was strongly detected in the liver and lung of DEC2 knockout mice compared with that of wild-type mice. Interestingly, medium change induced the circadian rhythm of DEC2, but not DEC1 in cancer cells. We conclude that DEC1 and DEC2 negatively regulates AMPK activity via LKB1, and play important roles in cancer cell metabolism.

#457

**Satisfying the fatty acid demand of prostate cancer: Outlier overexpression of genes involved in** de novo **synthesis vs fatty acid uptake define two different and potentially synergistic prostate cancer phenotypes.**

Sandra M. Gaston,1 James Kearns,1 George W. Adams,2 Soroush Rais-Bahrami,3 Jeffrey W. Nix,3 Peter N. Kolettis,3 James E. Bryant,3 Mark S. DeGuenther,2 Denise Oelschlager,3 Dennis Otali,3 William E. Grizzle3. 1 _Tufts Medical Center, Boston, MA;_ 2 _Urology Centers of Alabama, Birmingham, AL;_ 3 _The University of Alabama at Birmingham, Birmingham, AL_.

Malignant transformation increases cellular demand for fatty acids (FA). Many cancers, including prostate cancer (PCa), show increased expression of fatty acid synthase (FASN), the enzyme that catalyzes the de novo synthesis of the FA palmitate. De novo FA synthesis is energetically expensive; in most normal cells FASN expression is low and de novo synthesis suppressed in favor of utilization of exogenous FA. In contrast, in cancer cells, de novo FA synthesis is an important source of precursors needed to support tumor growth. Much of the research effort on FA in cancer has focused on FASN and the other genes involved in de novo FA synthesis. However a number of observations suggest that alternate mechanisms for FA acquisition are also important, most notably studies showing that cancer cells can be rescued from the pro-apoptotic effect of FASN inhibition by exogenous palmitate. Using a biopsy-based approach designed to identify high risk prostate cancer phenotypes that are common in African American (AA) men, we identified a PCa subtype with outlier (> 10 fold) mRNA overexpression of fatty acid binding protein 5 (FABP5). FABP5 facilitates the utilization of palmitate and other FAs from outside the cell, and a FABP5 overexpression phenotype may confer a selective advantage when dietary FAs are abundant or when FASN is targeted as a therapy. The FABP5 outlier overexpression phenotype was observed in 7% Gleason sum (GS) 7-10 PCas. We also observed a second molecular subtype with outlier FASN overexpression in 9% of GS 7-10 PCas. With few exceptions these two PCa subtypes showed an "either-or" pattern with top quartile overexpression of FABP5 or FASN but not both (Spearman r = 0.385; P < 0.0001). Interestingly, in low grade (GS 6) PCas, median FABP5 mRNA overexpression is higher in AA than in European American (EA) men (P < 0.01) with GS6 PCas from AA men showing median FABP5 expression levels as high as that observed in GS 7-10 PCas. IHC confirmed variable expression of both FABP5 and FASN, including "patchwork" PCas with high Gleason pattern (GP) areas overexpressing FABP5 next to low GP areas overexpressing FASN. The net result may be a selective advantage for the high grade cancer if FABP5 allows it to exploit the FA being synthesized by the adjacent low grade focus. In a subset of PCas, FABP5 IHC showed robust nuclear staining consistent with its proposed role in regulating fatty acid mediated gene expression; the ratio of FABP5 nuclear/cytoplasmic staining was higher in PCa from AA than from EA patients (P < 0.05). Identification of a PCa subtype with high levels of FABP5 overexpression suggests a previously unrecognized mechanism by which some PCas can increase FA supply without de novo synthesis. Such a PCa subtype might be particularly sensitive to dietary interventions and relatively insensitive to FASN inhibitors.

#458

Choline metabolism in cancer cells modifies PD-L1 expression.

Balaji Krishnamachary, Marie-France Penet, Yelena Mironchik, Zaver M. Bhujwalla. _Johns Hopkins Univ., Baltimore, MD_.

Increased expression of choline kinase-α (Chk-α), the enzyme that converts choline to phosphocholine has been observed in most cancer cells including breast (1). This increase of choline kinase results in increased production of phosphocholine that is used in the synthesis of membrane phosphatidylcholine. We investigated the relationship between Chk-α and the programmed death ligand-1 (PD-L1) expression in triple negative MDA-MB-231 human breast cancer cells. Expression of PD-L1 in response to the secretion of interferon gamma (IFNγ) by cytotoxic T cells and its binding to the immune checkpoint PD-1 on T cells results in cancer cells escaping destruction by the immune system (2). Quantitative real-time PCR analysis (q-RT-PCR) did not reveal any change in PD-L1 mRNA expression between MDA-MB-231 expressing an empty vector control plasmid (231-EV) and MDA-MB-23 stably expressing shRNA against Chk (231-Chk). However, there was a difference in the mean intensity fluorescence (MIF) between 231-EV (325 ±11.5, n=2) and 231-Chk (527±18.5, n=2) in the FACS analysis. Upon induction with IFNγ (10ng/ml) for 24h, there was 1.23 fold increase in PD-L1 mRNA in 231-Chk compared to 231-EV. Interestingly, the MIP for cell surface expression of PD-L1 was 600±21 (n=2) for 231-EV compared to 872±16 (n=2) for 231-Chk cells. Values represent mean + SE. These data suggest that the cell surface increase of PD-L1 in 231-Chk cells could be due to translocation. Increased PD-L1 expression creates an opportunity to deliver PD-L1 targeted nanoparticles carrying therapeutic cargo. Since most conventional treatments result in a decrease of Chk-α and phosphocholine, our data suggest that these treatments may also result in cancer cells escaping immune surveillance through expression of PD-L1.

Acknowledgements: This work was supported by NIH R01CA136576 and P50 CA103175.

Reference: 1) Glunde K et al, Cancer Res 65, 11034 (2005)., 2) Zou W, Chen L, Nat Rev Immunol 8(6), 467-77 (2008).

#459

Targeting MUC1 mediated nucleotide metabolism sensitizes pancreatic tumors to radiation therapy.

VENUGOPAL GUNDA, Joshua Souchek, Jaime Abrego, Gennifer Goode, Enza Vernucci, Surendra K. Shukla, Aneesha DasGupta, Nina V. Chaika, Ryan King, Fang Yu, Tadayoshi Bessho, Chi Lin, Wang Shou, Li Sicong, Pankaj K. Singh. _UNMC, Omaha, NE_.

Pancreatic cancer has the lowest survival rate of six perecent among all the cancers in the US and is projected to be the second leading cause of cancer related deaths in a decade. Radiation therapy provides only marginal increases in the survival rate in pancreatic cancer, due to poor responsiveness of pancreatic tumors. Clinical trials indicate a significant response to radiation only in twenty percent of primary pancreatic tumors. While multiple factors cause resistance to radiation therapy, the biological mechanisms mediating such innate resistance are currently being explored. Overexpression of MUC1 facilitates chemo and radiation resistance in pancreatic cancer. MUC1 overepxression also promotes pancreatic tumor growth through metabolic upregulation. We investigated the in vivo role of MUC1 mediated metabolic alterations in radiation response of pancreatic tumors. Our findings indicate that MUC1 expressing pancreatic tumors survive better upon radiation treatment. Metabolomic analysis through liquid chromatography coupled tandem mass spectrometry approach revealed that MUC1 expressing pancreatic tumors exhibit higher glycolytic and nucleotide metabolites upon irradiation. MUC1 expressing tumors also possess relatively higher PPP and nucleotide metabolites. Glycolytic inhibition using bromopyruvate revealed that MUC1 induced radiation resistance could be abrogated through inhibition of glucose carbon flux into nucleotide metabolism by BrPA. Treatment with BrPA effectively reduced glycolysis, pentose phosphate pathway and nucleotide levels in irradiated MUC1 expressing cells. Furthermore, a combination of BrPA and radiation reduced tumor growth in MUC1 expressing tumors. Metabolomic analysis showed a decrease in the glycolysis, PPP and nucleotide metabolites in MUC1 expressing tumors upon combination treatment with radiation and BrPA. Hence, our findings demonstrate that glycolytic inhibition could be used to effectively target MUC1 mediated radiation-resistance in pancreatic tumors. 

### MicroRNA Regulation of Cancer Biology 1

#460

A central microRNA checkpoint for the inflammatory stem cell niche.

Xiling Shen. _Duke Univ., Durham, NC_.

The microRNA miR-34a is a well-known tumor suppressor in various types of cancer. Previous studies largely focused on its role in suppressing oncogenic pathways such as Bcl2 and Myc. This study will demonstrate a previously unknown function of miR-34a—a central protector of tissue integrity in the inflammatory colon stem cell niche by modulating both proinflammatory response and stem cell behavior. Epigenetic silencing of miR-34a by a long non-coding RNA (lncRNA), Lnc34a, subverts this checkpoint to promote cancer stem cell proliferation.

In DSS- and bacteria-induced colitis models, miR-34a (1) suppresses differentiation of CD4+ T cells into Th17 cells by directly targeting multiple interleukin receptors, (2) blocks recruitment of Th17 cells to the inflammatory site by directly targeting chemokine CCL22 from epithelial cells, (3) attenuates the effect of IL17 on stem cells by directly targeting IL17 receptors in stem cells, and (4) switches on asymmetric division to curb stem cell proliferation.

We further show that a novel lncRNA, Lnc34a, is upregulated by Notch signaling during colorectal cancer (CRC) progression and epigenetically silences miR-34a promoter by recruiting Dnmt3 and HDAC1. This process removes the miR-34a checkpoint of the inflammatory niche and contributes to proliferation of stem-like cancer cells.

Our finding of miR-34a as an inflammatory checkpoint suggests potential therapeutic strategies to reinforce or restore such miR-34a functions in colitis patients to prevent CRC and in CRC patients to prevent proliferation of cancer stem cells. Furthermore, MRX34, a miR-34a mimic, is the first microRNA mimics to reach phase II clinical trial for cancer therapy, which is ongoing. However, delivery efficiency into the tumor is always a challenge for microRNA mimics. Our discovery of Lnc34a, which recruits epigenetic regulator to silence miR-34a, provides a promising therapeutic target, because it is much easier to develop small molecule inhibitors to disrupt lncRNA structure or block its functional interaction with protein than to restore microRNA expression.

#461

Dichotomous roles of Dicer1 in rhabdomyosarcoma and angiosarcoma.

Jason A. Hanna, Matthew R. Garcia, Catherine J. Drummond, Jonathon C. Go, David Finkelstein, Jerold E. Rehg, Mark E. Hatley. _St. Jude Children's Research Hospital, Memphis, TN_.

Pediatric and adult angiosarcomas are rare and highly aggressive soft tissue sarcomas with an extremely poor prognosis. Due to the rarity of this disease especially in children, the molecular drivers and optimized treatment strategies for patients are lacking, highlighting the need for genetic and in vivo animal models. MicroRNAs are a class of small RNAs that regulate gene expression and are often dysregulated in cancers including sarcomas. DICER1 is required for microRNA biogenesis and germline DICER1 mutations result in a cancer predisposition syndrome associated with increased risk of benign and malignant tumors including rhabdomyosarcoma (RMS), a pediatric soft tissue sarcoma resembling developmentally arrested skeletal muscle. Here we show Dicer1 expression is required for tumorigenesis in a mouse model of RMS driven by activation of oncogenic Smoothened by Cre recombinase expressed from the adipose protein 2 (aP2) promoter (aP2-Cre). Unexpectedly, in studying the role of Dicer1 in RMS, we found that Dicer1 deletion with aP2-Cre leads to aggressive angiosarcoma. Angiosarcoma development was independent of the Smoothened oncogene and other genetically engineered oncogenes or tumor suppressor loss, providing the first in vivo mouse model of biallelic Dicer1 loss alone driving tumorigenesis. Angiosarcomas in aP2-Cre;Dicer1Flox/- mice histologically and genetically resemble human angiosarcoma and were enriched for microRNA-23 target genes including the oncogene Ccnd1 as well as Adam19, Plau, and Wsb1 that promote invasiveness and metastasis. The aP2-Cre;Dicer1Flox/- model provides a simple in vivo animal model to study angiosarcoma for novel therapeutics and the molecular mechanisms of cancer initiation, progression, and metastasis. In addition, our results demonstrate DICER1 and microRNAs play major and opposing roles in sarcomagenesis.

#462

The role of miR-24 as a race-related genetic factor in prostate cancer.

Yutaka Hashimoto,1 Marisa Shiina,1 Taku Kato,1 Soichiro Yamamura,1 Yuichiro Tanaka,1 Shahana Majid,1 Sharanjot Saini,1 Varahram Shahryari,1 Priyanka Kulkarni,1 Pritha Dasgupta,1 Yozo Mitsui,1 Mitsuho Sumida,1 Guoren Deng,1 Laura Tabatabai,1 Deepak Kumar,2 Rajvir Dahiya1. 1 _University of California at San Francisco & VA Medical Center, San Francisco, CA; _2 _University of the District of Columbia, Washington, DC_.

The incidence of prostate cancer (PCa) among African-Americans (AfA) is significantly higher than Caucasian-Americans (CaA) but the genetic basis for this disparity is not known. To address this problem, we analyzed miRNA expression in AfA (n=81) and CaA (n=51) PCa patients. Here, we found that miR-24 is differentially expressed in AfA and CaA PCa patients and attempt to clarify its role in AfA patients. Also, the public sequencing data of the miR-24 promoter confirmed that it was highly methylated and down-regulated in PCa patients. Utilizing a VAMCSF and NDRI patient cohorts, we discovered that miR-24 expression was linked to a racial difference between AfA/CaA PCa patients. Interestingly, miR-24 was restored after treatment of PCa cells with 5Aza-CdR in an AfA cell line (MDA-PCa-2b), while restoration of miR-24 was not observed in CaA cells, DU-145. Ectopic expression of miR-24 showed decreased growth and induced apoptosis, though the effect was less in the CaA cell line compared to the AfA cell line. Finally, we found unique changes in biological pathways and processes associated with miR-24 transfected AfA cells by quantitative PCR-based gene expression array. Evaluation of the altered pathways showed that AR, PDPK1, IGF1, and IGFBP5 were markedly decreased in the AfA derived cell line compared with CaA cells, and there was a reciprocal regulatory relationship of miR-24/target expression in prostate cancer patients. These results demonstrate that miR-24 may be a central regulator of key events that contribute to race-related tumorigenesis and has potential to be a therapeutic agent for PCa treatment.

#463

MicroRNA-7 suppresses RB1 expression leading to chromosomal instability in leukemia cells harboring c-Kit mutation.

Keiji Kurata,1 Shinichiro Kawamoto,1 Ryota Masutani,2 Kimikazu Yakushijin,1 Katsuya Yamamoto,1 Hiroshi Matsuoka,1 Takayuki Takubo,3 Hironobu Minami1. 1 _Kobe University Hospital, Kobe City, Japan;_ 2 _Osaka Medical College Hospital, Osaka, Japan;_ 3 _Japanese Red Cross Osaka Blood Center, Osaka, Japan_.

C-Kit mutation D816V is a well-known indicator of poor prognosis in acute myeloid leukemia (AML) harboring t(8;21) chromosomal translocations. However, the mechanism the D816V mutation promotes therapeutic resistance is still under investigation. C-Kit V814 mutation is a murine counterpart of the human D816V. We utilized a murine IL-3 dependent cell line Ba/F3 with or without the c-Kit V814 mutation to investigate its downstream signaling pathway and the effect on the expression of bcl-2 family members and critical granulocytic transcription factors. The c-Kit V814 and wild type (WT) c-Kit were retrovirally transduced into Ba/F3 cells. Unexpectedly, Gata1 mRNA was significantly downregulated to one-tenth in V814+Ba/F3 cells (p < 0.01), contrary to the previous reports that WT c-Kit signaling induces Gata1 expression. When V814+Ba/F3 cells were treated with several c-Kit signaling pathway inhibitors, mRNA and protein expression levels of Gata1 recovered to normal levels only with MEK 1/2 inhibitor (PD325901) treatment. We then hypothesized that miRNAs might control Gata1 transcription. We used an array to identify differences in miRNA expression between Ba/F3 cells and V814+Ba/F3 cells, and between V814+Ba/F3 cells treated with or without PD325901. Only miR-7a-5p and miR-706 were significantly upregulated in V814+Ba/F3 cells compared with Ba/F3 without the V814 mutation, and downregulated (> 2-fold) in cells treated with PD325901 compared with controls. We focused our further analysis on miR-7 as it is highly conserved in vertebrates. The differences in miR-7 expression were confirmed using qRT-PCR (10.5-fold change, p < 0.01). In silico analysis implicated Rb1 as a candidate miR-7 target that may regulate Gata1 transcription. Luciferase reporter constructs containing murine and human Rb1 miR-7 target sequence exhibited 0.51 and 0.49 times lower luciferase activity than the controls, respectively (p < 0.01). To further determine the biological significance of the c-Kit mutation that would transform the cells to more aggressive phenotype, we focused on DNA and chromosomal instability caused by RB1 deterioration. We exposed WT c-Kit Ba/F3 and V814+Ba/F3 cells to 4 Gy irradiation and then used immunohistochemistry to analyze the frequency of γH2AX foci in the cells. The percentage of cells with more than 10 γH2AX foci was significantly higher in V814+Ba/F3 cells than WT Ba/F3 cells (27% vs. 14%, p < 0.05), Here, we clearly show that a single mutation of c-Kit is sufficient to regulate miR-7 expression leading to RB1 translational suppression. Since c-Kit mutation requires only a single step to inhibit RB1, this mechanism is more likely to facilitate oncogenesis than bi-allelic RB1 chromosomal and/or gene alterations in leukemia cells. Taken together, we identified miR-7 as a suppressor of RB1 in V814 mutation-positive cells, which might reflect the responsibility of D816V to refractory feature in AML.

#464

Novel role of xpo1 in regulating MicroRNAs related to pancreatic ductal adenocarcinoma invasion and metastasis.

Asfar S. Azmi,1 Yiwei Li,1 Irfana Muqbil,1 Amro Aboukameel,1 William Senapedis,2 Erkan Baloglu,2 Yosef Landesman,2 Michael Kauffman,2 Sharon Shacham,2 Philip A. Philip,1 Ramzi M. Mohammad1. 1 _Wayne State Univ., Detroit, MI;_ 2 _Karyopharm Therapeutics, Newton, MA_.

Objective: There are no known reports on the role of exportin 1 (XPO1; also known as chromosome maintenance region 1 [CRM1]) in microRNA biology. In this study, we for the first time demonstrate that interfering with XPO1 machinery can influence miRNA signaling leading to suppression of pancreatic ductal adenocarcinoma (PDAC) proliferation, invasion and metastasis.

Methods: miRNA arrays (LCSciences, Houston, TX) were performed on total RNA samples from PDAC cell lines (HPAC, MiaPaCa-2, AsPc-1 and L3.6pl) and normal human pancreatic ductal epithelial (HPDE) cells. PDAC cells were treated with XPO1 inhibitor (Selinexor) or transfected with control siRNA, XPO1 siRNA (Santa Cruz), miR-control or miR-145 mimic (Applied biosystems) all at a final concentration of 20 nM using DharmaFact Transfection Reagent (Dharmacon, Lafayette. CO). The total RNA and total protein from treated or transfected cells were subjected to real-time PCR or immunoblot analysis in order to measure expression level of miR-145, let-7d, miR-34c, miR-320, miR-205, and miR-145 target or downstream genes including EGFR, MMP1, MT-MMP, c-Myc, Sox-2, and PAK4. The impact of XPO1 inhibitor Selinexor on PDAC growth, proliferation, invasion and migrations was also evaluated using MTT and scratch assay.

Results: In this study, we show that PDAC cells have significantly reduced expression of miR-145 when compared to normal pancreatic duct epithelial cells. Similarly, forced expression of miR-145 in PDAC cells inhibited cell proliferation and migration. Conversely, we demonstrate that RNAi of XPO1 by siRNA knockdown or chemical inhibition of XPO1 by selective inhibitor of nuclear export compound (Selinexor) restores miR-145 expression in PDAC cells ultimately leading to inhibition of cell proliferation and migration. In addition, we show that the inhibition of cell proliferation and migration by Selinexor is mediated through the down-regulation of known miR-145 signals including EGFR, MMP1, MT-MMP, c-Myc, PAK4 and Sox-2. Selinexor also induced the expression of two important tumor suppressive miRNAs, miR-34c and let-7d, leading to the up-regulation of p21WAF1. We also observed the down-regulation of oncomir mir-205.

Conclusions: These results are the first to show that targeted inhibition of the nuclear exporter protein XPO1 by RNAi or Selinexor could restore tumor suppressive miRs in PDAC.

#465

Downregulation of miR-193a and its correlation with clinical and pathological behavior of colorectal cancer.

Afraa M. Mamoori,1 Riajul Wahab,1 Farhadul Islam,1 Katherine Lee,1 Robert A. Smith,2 Vinod Gopalan,1 Alfred Ky Lam1. 1 _Cancer Molecular Pathology, School of Medicine, Menzies Health Institute Queensland, Gold Coast, Australia;_ 2 _Genomics Research Centre, Institute for Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Australia_.

Aim: Deregulation of miR-193a expression was reported to have significant roles in cancer development and progression. This study aimed to investigate the expression pattern and clinicopathological implication of miR-193a-3p in patients with colorectal cancer. Also, the bio-physiological effects of miR-193a in colon cancer cells were examined.

Methodology: Fresh frozen tissues from 70 matched colorectal cancers (adenocarcinomas) and adjacent noncancer tissue were prospectively collected with no selection bias. Expression level of miR193a-3p was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemical staining was used to detect the expression level of K-ras protein as a predicted downstream target for miR-193a. Restoration of the miR-193a level in the cell lines was achieved by permanent transfection. Multiple functional and immunological assays were performed to analyze miR-193's cellular implications and target affinity in colon cancer cell lines.

Results: Downregulation of miR-193a-3p was noted in the majority of the colorectal cancer tissues when compared to nontumor colorectal tissues (70%, n= 49/70). In addition, high prevalence of miR-193a downregulation was noted in early stage colon cancer tissues and cell lines (SW480) when compared to advanced staged colorectal carcinomas. Immunohistochemical analysis revealed inverse correlation between miR-193a and K-ras expression in which high K-ras protein expression was reported in 28/49 cases with miR-193a downregulation. Analogues findings were also observed in cell lines using Western blot (P value< 0.05). Overexpression of miR-193a in colon cancer cell lines resulted in reduced cell proliferation, increased apoptosis and a less accumulation of cells in the G0-G1 phase cell cycle.

Conclusion: The current study confirms the downregulation of miR-193a-3P in colorectal cancer and its tumour suppressor effects in vitro plays a key role in the modulation of K-ras regulated molecular signalling pathways in colon cancers

#466

Role of miR-15b-3p in mitoquinone induced autophagy of breast cancer cells.

Francesca Mascia, Kaytee Pokrzywinski, Ashutosh Rao. _FDA/CDER, Silver Spring, MD_.

Mitoquinone, a redox-active mitochondrially targeted ubiquinone, activates autophagy more potently in breast cancer cells (MDA-MB-231) than in normal breast cells (MCF-12A). Mitoquinone induces the production of reactive oxygen species (ROS) and a concomitant activation of the Nrf2-dependent antioxidant response. MicroRNA are endogenous small double stranded RNA that bind to the 3' UTR of several target mRNAs and function through translational repression or mRNA degradation. The goal of this study was to evaluate microRNA as a link between oxidative stress and autophagy signaling. We performed RNA sequencing analysis of the microRNAs modulated by mitoquinone treatment of MDA-MB-231 cells. miRNAs were identified using Illumina RNA sequencing. DEseq analysis revealed 97 miRNAs that were differentially expressed at an α < 0.05. Within this set, 15 miRNAs were down-regulated by stimulation with mitoquinone making them attractive as potential autophagy inhibitors. We characterized the role of a miR-15b-3p by transfecting the cells with miR-mimic and anti-miR before inducing an autophagy response. Western blot analysis showed a reduction in the expression of the autophagy marker LC3, the autophagosome chaperon p62 and the transcription factor Nrf2 in presence of the miR-15b-3p mimic. Conversely, miR-15b-3p mimic pre-treatment increased the expression of the cell cycle inhibitor p27 both at the basal level and after mitoquinone stimulation for 48 hrs. The administration of the anti-miR-15b-3p did not change the expression level of these proteins suggesting that miR-15b-3p may work with other miRs toward regulating autophagy. Combining the action of miR-15b-3p mimic with redox active chemotherapeutic drugs could represent a novel strategy for breast cancer therapy by reducing survival signaling through autophagy.

#467

Analysis of human hypoxia related miRNAs in MDM2 transfected prostate cancer cells.

Saad Ebrahim Alobid,1 Thiagarajan Venkatesan,2 Ali Alaseem,1 Khalid Alhazzani,1 Appu Rathinavelu1. 1 _Nova Southeastern Univ. College of Pharmacy, Davie, FL;_ 2 _Rumbaugh Goodwin Institute for Cancer Research, Nova Southeastern University, Davie, FL_.

Recent studies have shown the crucial role of miRNAs in regulating cancer initiation, progression and metastatic process via influencing gene expression patterns. However, the distinct mechanism by which miRNA regulates tumorigenesis remains as an area requiring detailed research investigations. In our earlier studies, we have demonstrated a positive correlation between MDM2 expression and hypoxia inducible factor -1alpha (HIF-1α) levels in both normoxic and hypoxic conditions using prostate cancer cell lines. Subsequently, we postulated that the upregulation of HIF-1α is most likely regulated by MDM2 in cancers cells that are positive for this gene expression. Hence, to confirm our hypothesis, we utilized miScript miRNA PCR Array analysis (Qiagen, USA) and compared the gene expression profile of the hypoxia signaling pathway in LNCaP (prostate cancer cells) and LNCaP-MST

(MDM2 transfected prostate cancer cells) under normoxic conditions. This study was expected to outline how MDM2 impacts the differential expression of miRNAs and leading to enhancement of tumor angiogenesis via increase in HIF-1α or other genes of the hypoxia signaling pathway. Our preliminary data indicate that hsamiR99a5p, hsamiR125b5p, hsamiR1385p, hsamiR199b5p and hsalet7c5p are significantly upregulated in LNCaP-MST cells compared to LNCaP. In addition, some of the miRNAs such as hsalet7d5p, hsalet7e5p, hsalet7f5p, hsalet7g5p and hsalet7i5p are significantly downregulated in the same cells. Among the upregulated miRNAs, hsamiR125b5p emerged as an important gene that is known to downregulate the protein levels of hypoxia inducible factor 1alpha inhibitor (HIF1AN or FIH), which can normally inhibit the transcriptional activity of HIF-1α in both normoxic and hypoxic conditions. Therefore, overexpression of hsamiR125b5p seems to be associated with the increase in HIF-1α level. From the aforementioned findings, it is confirmed that the MDM2 overexpression is impacting miRNA levels such as hsamiR125b5p that are involved in hypoxia related signaling pathway. Thus, our results offer a convincing evidence that MDM2 can promote angiogenesis and increase metastatic potential of cancers through altering the miRNA expression levels. (The financial support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida is gratefully acknowledged).

#468

Evaluation of microRNA inhibition in medulloblastoma.

Alexander Vavra,1 Sydney Stoops,2 Jonathan White2. 1 _Kansas City Univeristy, Kansas City, MO;_ 2 _MRIGlobal, Kansas City, MO_.

Medulloblastoma is the most common pediatric malignant brain tumor. The current treatment plan consists of a combination of surgery, radiation, and chemotherapy which results in an 80% five year survival rate. However, the survival rate does not take into account the developmental delays and learning disabilities inherited as a result of the aggressive treatment plan on a developing brain. As such the development of new treatment options is needed. While many protein targets are currently being explored for therapeutic intervention, the role of non-coding RNAs is of increasing interest. Specifically microRNA profiling in medulloblastoma has been performed by others to highlight expression profiles which can be mapped to the disease state and also to potentially identify associated protein targets. Moreover, it is particularly intriguing that microRNAs themselves may be directly targeted as therapeutic points of intervention. With this in mind, our lab is currently investigating methods for directly targeting microRNAs in medulloblastoma and we have recently validated several upregulated miRNAs for further investigation. Specifically, we evaluated the expression profile eighteen selected miRNAs in DAOY and D341 cell lines. Our results indicate that four miRNAs (miR-196a, miR-183, miR-96, and miR-182) are statistically overexpressed in both cell lines. Results of these findings and our initial efforts to target these selected miRNAs will be described.

#469

Microrna signature as a potential biomarker for predicting survival in colon cancer.

Havjin Jacob, Luka Stanisavljevic, Kristian Eeg Storli, Olav Dahl, Mette Pernille Myklebust. _Clinical Department of Science, Bergen, Norway_.

Background: Colon cancer is one of the most common cancers with increasing incidence and high mortality worldwide. Prognosis and choice of treatment is largely based on the tumor stage at presentation. Thus, finding novel biomarkers for predicting survival is highly desirable. Lately, several studies have been looking at microRNAs (miRNAs) in several cancers, including colon cancer. MicroRNAs are conserved, non-coding RNA molecules that play an important role in the regulation of post-transcriptional gene expression.

Material and Methods: In the present study, we have profiled miRNA in one hundred and seventy two TNM stage I-IV colon cancer patients and 10 corresponding normal colon tissue samples. Total RNA was extracted from freshly frozen tissues, and the expression of miRNA profile were assessed using Pick and Mix focus panels from Exiqon containing 84 miRNAs that have been linked to cancer.

Results: The results were visualized in a heatmap (Qlucore omics Software) and more than 20 miRNAs were found to be differentially expressed in tumors compared to the normal colon. Further, twelve miRNAs were found to discriminate between relapse and no-relapse patients in TNM- stage II and III, and four of these miRNAs (miR-23a, miR-25, miR-30d and miR-31) were found to be statistically significant in binary logistic regression with relapse as outcome variable. In univariate analysis, low expression of the four-miRNA signature was associated with better 3-year disease-free survival (DFS), 88 % versus 63% in low versus high signature, respectively (P=0.001). Moreover, the signature was a predictor of poor relapse-free survival in multivariate analyses (P=0.001; HR 31; 95% CI: 3.8-248.9). Another regression analyses method (LASSO) identified a 16-miRNA signature, and the four miRNAs found earlier were among them. The 16-miRNA signature was associated with better survival (P<0.001).

Conclusion: The present study has identified a four-miRNA signature predicting relapse in colon cancer stage II and III patients.

#470

**Epstein-Barr virus-encoded miR-BART5 modulates PIAS3-pSTAT3 and p21** waf1 **in gastric carcinoma cells.**

Dong Ha Kim,1 Chan Jin Yoon,1 Jin-Seoub Kim,1 Sunyoung Park,2 Euno Choi,2 Jun Hee Woo,3 Mee Soo Chang2. 1 _Asan Institute for Life Sciences, University of Ulsan College of Medicine, Seoul, Republic of Korea;_ 2 _Seoul National University College of Medicine Boramae Hospital, Seoul, Republic of Korea;_ 3 _Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea_.

The ubiquitous Epstein-Barr virus (EBV) is associated with a subset of gastric carcinomas. MicroRNAs (miRs) are 22±2 nucleotide non-coding RNAs, and regulate various functions in cells. EBV-encoded miRs in gastric carcinoma cells have been identified, but the targets and roles of EBV-encoded miRs remain elusive. In the present study, we investigated cellular target of miR-BART5, and its possibility as an oncomiR. In naturally EBV-infected gastric carcinoma cells and EBV-positive gastric carcinoma tissues, the comprehensive EBV-miR profile revealed the expression of 22 EBV miRs (each having -3p and -5p) composed of miR-BART cluster 1 and cluster 2, without miR-BHRFs. Using bioinformatics analysis, we focused on miR-BART5 which shared seed sequence homology with hsa-miR-18a and miR-18b having oncomiR function, and protein inhibitor of activated STAT3 (PIAS3) mRNA. The western blot results confirmed that PIAS3 protein expression was reduced in miR-BART5-expressing gastric carcinoma cells (EBV-negative gastric carcinoma cells transfected with miR-BART5), compared with miR-control mock cells. Also PIAS3 expression was significantly lower in naturally EBV-infected gastric carcinoma cells than in EBV-negative gastric carcinoma or EBV-infected lymphoma cells. Moreover, there was a statistically significant inverse correlation between the expression of miR-BART5 and PIAS3. The luciferase reporter activity in cells diminished after co-transfection of pEZX-MT06 vector-PIAS3 3′UTR and miR-BART5, compared with co-transfection of empty vector and miR-control or co-transfection of empty vector and miR-BART5. When we tested twenty proteins as downstream candidates of miR-BART5, pSTAT3 increased and p21waf1 decreased in miR-BART5-expressing gastric carcinoma cells, compared with mock cells, and nuclear translocation of pSTAT3 was observed in miR-BART5-expressing gastric carcinoma cells. In a reverse context that naturally EBV-infected gastric carcinoma cells were transfected with anti-miR-BART5, protein levels were changed likewise; PIAS3 increased, pSTAT3 decreased and p21waf1 increased. However, there were no statistically significant differences in cellular biologic properties such as proliferation, apoptosis, invasion and migration between miR-BART5-expressing gastric carcinoma cells and mock cells, or between naturally EBV-infected gastric carcinoma cells transfected with anti-miR-BART5 and mock cells. Taken together, miR-BART5 not only targets directly PIAS3 and then modulates PIAS3-pSTAT3 axis, but also decreases p21waf1 protein, although it fails to alter cellular proliferation in gastric carcinoma cells. This suggests that a single viral microRNA is not sufficient to implement oncogenic function, despite directly targeting cellular protein involved in oncogenesis. Instead, a whole cluster like miR-BART cluster 1 or cluster 2 may be advocate to control cellular biologic property.

#471

HER2 regulates PARP-1 expression by suppressing the let-7a microRNA in HER2+ breast cancer.

Eddy Shih-Hsin Yang, Monicka Wielgos, Rajani Rajbhandari, Shi Wei, Susan Nozell. _Univ. of Alabama at Birmingham, Birmingham, AL_.

Background:

HER2+ breast cancers are sensitive to PARP inhibition and express elevated levels of the PARP-1 protein. However, the mode of regulation of PARP-1 expression by HER2 is not well understood. MicroRNAs are small non-coding RNAs that function in RNA silencing and post-transcriptional regulation of gene expression. In this study, we investigate whether PARP1 expression in human breast cancer cells is regulated by HER2 regulation of microRNAs.

Methods:

Human HER2+ breast cancer cell lines BT-474 and SKBR3 were used in this study. MDA-MB-231 (non-HER2 expressing breast cancer cells) stably transfected with a HER2 wild-type plasmid (231 HER2) or the vector control (231 NEO) were also utilized. To identify candidate microRNAs regulated by HER2 overexpression, we performed the nCounter miRNA Expression Assay. MicroRNA and mRNA expression were validated via qRT-PCR analysis in breast cancer cell lines or patient primary tumors. Cells were also transfected with a HER2 siRNA, a let-7a mimic, and an inhibitor of let-7a as well as their respective controls. Western blot analysis and firefly luciferase assays were used to determine whether the 3'UTR of PARP1 was being directly targeted by the let-7a microRNA.

Results:

HER2 did not regulate PARP-1 at the mRNA level but increased PARP-1 protein in HER2+ breast cancer cells. Specifically, ectopic HER2 overexpression correlated with increased PARP-1 protein levels in the 231 HER2 cell line. Conversely, silencing HER2

reduced PARP-1 protein levels in the BT-474 and SKBR3 cell lines. NanoString nCounter analysis revealed that the HER2+ breast cancer cell lines expressed low levels of the let-7a microRNA. Further, let-7a expression was upregulated after HER2 knockdown in the two native HER2+ breast cancer cell lines. The let-7a mimic also reduced both PARP1 protein expression and luciferase activity in the 231 HER2 and BT-474 cell lines, whereas the let-7a inhibitor reversed these effects in the 231 NEO cell line. Importantly, human HER2+ breast tumors expressed higher levels of PARP-1 and lower levels of let-7a; whereas, the HER2- breast tumors expressed lower levels of PARP-1 and higher levels of let-7a. Further, overexpression of the let-7a mimic reduced cell proliferation in HER2+ breast cancer cells.

Conclusions:

These results suggest that let-7a regulates PARP-1 expression in HER2+ breast tumors. Let-7a may also be a potential therapeutic target and predictive biomarker of PARPi sensitivity in HER2+ breast cancer patients.

#472

Role of miRNAome deregulation in the pathogenesis of non-alcoholic steatohepatitis (NASH)-derived hepatocellular carcinoma.

Juliana F. Ortega,1 Aline DeConti,1 Kostiantyn Dreval,1 Fernando S. Moreno,2 Frederick A. Beland,1 Igor P. Pogribny1. 1 _NCTR/FDA, Jefferson, AR;_ 2 _University of Sao Paulo, Sao Paulo, Brazil_.

Recent epidemiological studies have attributed the growing incidence of liver cancer, including hepatocellular carcinoma (HCC), in the United States to NASH, an advanced form of non-alcoholic fatty liver disease. Elucidating the molecular pathways that lead to the development of NASH-derived HCC is critical not only for identifying early diagnostic biomarkers of the disease, but also for treatment and prevention. We have investigated the role of microRNAs (miRNAs) in the development of NASH-derived HCC by using a Stelic Animal Model (STAM) of liver carcinogenesis. Using Liver miFinder PCR arrays, we examined the miRNA profiles in the livers of STAM mice and identified 25 and 35 miRNAs that were differentially expressed at a NASH-fibrotic stage (12 weeks) and in full-fledged HCC (20 weeks), respectively. Among these differentially expressed miRNAs, 18 miRNAs were in common. Multi-algorithm target prediction analysis of the common differentially expressed miRNAs revealed the involvement of these miRNAs in the deregulation of major molecular processes associated with the development of HCC, including epithelial-mesenchymal transition, HCC stem cell activation, and the induction of the β-catenin/Wnt and Hippo signaling pathways. These findings were confirmed by a marked up-regulation of Yes-associated protein 1 (YAP1), the main effector of the Hippo-pathway, the activation of epithelial-mesenchymal transition, and an increased-expression of hepatic progenitor cell markers. In addition to the common differentially expressed miRNAs, 10 miRNAs, including 5 members of the polycistronic oncomir miR-17-92 cluster, were found to be over-expressed in only HCC. These results indicate the fundamental role of miRNAs in the development of NASH-associated HCC. This is evidenced by the early occurrence of miRNA alterations at the preneoplastic stage of liver carcinogenesis, the persistence of these changes in HCC, and the accumulation of additional miRNA alterations in HCC, and by the existence of a mechanistic link between miRNA alterations and deregulation key cancer-related pathways.

#473

PRKRA/PACT expression promotes chemoresistance in mucinous ovarian cancer.

Takeshi Hisamatsu,1 Michael McGuire,1 Sherry Y. Wu,1 Rajesha Rupaimoole,1 Sunila Pradeep,1 Kyunghee Noh,1 Justyna Filant,1 Jean M. Hansen,1 Yasmin Lyons,1 Kshipra M. Gharpure,1 Archana S. Nagaraja,1 Lingegowda S. Mangala,1 Takashi Mitamura,1 Cristian Rodriguez-Aguayo,1 Geoffrey A. Bartholomeusz,1 Cristina Ivan,1 Ju-Seong Lee,1 Koji Matsuo,2 Michael Frumovitz,1 Kwong K. Wong,1 Gabriel Lopez-Berestein,1 Anil K. Sood1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _University of Southern California, Los Angeles, CA_.

Purpose: To investigate the mechanisms of chemotherapy resistance and developing strategies to enhance therapeutic responses in mucinous ovarian cancer (MOC).

Experimental design: We carried out a kinome-based siRNA screen using human MOC to identify novel targets to enhance the efficacy of chemotherapy in MOC cell lines. In vitro and in vivo validation studies were carried out using MOC models. We specifically interrogated the role of PRKRA in MOC based on our screen results.

Results: Among the 939 genes in the screen, we focused on PRKRA/PACT because it was one of the top 5 target genes that exhibited the greatest extent of synthetic lethality in the target gene-siRNA plus oxaliplatin group relative to the target gene-siRNA group. The combination of oxaliplatin plus siPRKRA treatment resulted in significantly reduced cell viability compared with oxaliplatin plus control siRNA in RMUG-L-ip1 or RMUG-S-ip1 MOC cells (p<0.001), while knockdown of PRKRA did not result in a significant change in cell viability compared with the control. We also observed a 2.1-fold increase in cell apoptosis in vitro after treatment with oxaliplatin plus siPRKRA in both MOC cells (p<0.05). Using orthotopic mouse models of MOC, we observed an 88% reduction (p<0.01) in tumor weight and 75% reduction in the number of tumor nodules (p<0.01) in the siPRKRA plus oxaliplatin group compared with the control siRNA plus oxaliplatin group. PRKRA expression in human MOC was significantly higher relative to high-grade serous ovarian tumors as evaluated by immunohistochemistry. Furthermore, we found that the interaction between PACT and Dicer can regulate maturation of microRNA. In particular, mature-miR-515-3p was found to be inhibited by the interaction between PACT and Dicer. Expression of miR-515-3p promotes chemosensitivity in MOC by targeting the mRNA of the anti-apoptotic gene AXL.

Conclusion: The PRKRA/PACT axis represents an important therapeutic opportunity in MOC for enhancing oxaliplatin efficacy.

#474

**Estrogen-responsive miRNAs as modulators of E** 2 **-induced apoptosis in AI-resistant breast cancer.**

Reiner Hoppe,1 Ping Fan,2 Stefan Winter,1 Florian Büttner,3 V. Craig Jordan,2 Hiltrud B. Brauch3. 1 _Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, and University of Tuebingen, Tuebingen, Germany;_ 2 _MD Anderson Cancer Center, University of Texas, Houston, TX;_ 3 _Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, and University of Tuebingen, Tuebingen, German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany_.

Long-term estrogen deprivation with tamoxifen or aromatase inhibitors (AI) is the basic principle of endocrine treatment of ER-positive breast cancer. Acquired resistance is however a major obstacle in treatment success. Based on clinical observations and in vitro as well as animal in vivo experiments it has been suggested that tumor cell clones evolve over time and become vulnerable to E2-induced apoptosis, thereby potentially providing a promising second line treatment option. In vitro, this vulnerability is mimicked in the AI-resistant breast cancer models MCF-7:5C and MCF-7:2A, which during long-term E2-deprivation reconfigure their survival signaling including endoplasmic reticulum, oxidative and inflammatory stress related pathways. E2-stimulation of these cells initiates an unfolded protein response which in turn triggers apoptosis through the intrinsic and subsequently extrinsic pathway (Jordan VC 2015). Recently, we identified miRNA profiles matching the biology of AI resistance and vulnerability to E2-induced apoptosis (Hoppe R et al. 2016). Here we investigate the modulatory role of miRNAs in E2-induced apoptosis through the identification of their global expression changes in E2-stimulated 5C and 2A models compared to MCF-7:WS8 reference. Each cell line was treated with 10-9 M E2 or vehicle over a 72h time course (6, 12, 24, 72 h). miRNA profiles were generated using Affymetrix GeneChip miRNA2.0 arrays. At each time point relative miRNA expression changes (E2/control) were evaluated resulting in a total of 72 (5C), 104 (2A) and 94 (WS8) differentially expressed miRNAs (FC > 1.5 or < 1/1.5, Pt-test < 0.05). Differential expression analyses between consecutive time points revealed 16 (5C), 43 (2A) and 27 (WS8) miRNAs (maximum absolute FC difference > 1.5, PF-test < 0.05). Differential area under the curve (dAUC) analyses at 6-72, 6-24, and 24-72 h mined the overall, early, and late-responding miRNAs of the different phenotypes (Ppermutation test < 0.05). Representative early down-regulated miRNA candidates potentially modulating E2-induced apoptosis in 5C cells are miR-543 and miR-432 of the DLK1-DIO3 locus on Chr. 14q32.31. Late responding up-regulated miRNA candidates are miR-150* and miR-149* the low expression of which in tumor tissues registered in The Cancer Genome Atlas (miRNA-Seq v. 3.1.17.0) was associated with worse outcome in all PAM50 breast cancer subtypes and Lum B, respectively (HR = 2.1, 95% CI: 1.4-3.2; P = 0.00017; HR = 6.8, 95% CI: 1.5-3.0; P = 0.0036). We are currently in the process to subject respective miRNA sets to functional enrichment analyses (KEGG, GO) and to correlate E2-responsive miRNA candidates with respective transcriptome signatures. In summary, we will present miRNAs matching to the biological processes inherent to E2-induced apoptosis. These may serve as potential targets for the amplification of the apoptotic trigger upon E2-treatment.

#475

Role of microRNA-34a in regulating oncoprotein STMN1.

Balabhadrapatruni V.S.K. Chakravarthi,1 Rohit Mehra,2 Rui Wang,2 Darshan Shimoga Chandrashekar,1 Sai Akshaya Hodigere Balasubramanya,1 Irfan A. Asangani,3 Robert J. Lonigro,2 Arul M. Chinnaiyan,2 Sooryanarayana Varambally1. 1 _University of Alabama at Birmingham, Birmingham, AL;_ 2 _University of Michigan, Ann Arbor, MI;_ 3 _University of Pennsylvania, Philadelphia, PA_.

MicroRNAs, as negative regulators of gene expression, play a major role in cellular homeostasis. Downregulation of miRs is common in cancer which results in upregulation of multiple oncogenes in cancer. Here we show that stathmin (STMN1), which is over-expressed and oncogenic in wide variety of cancers, is regulated by microRNA-34 in prostate cancer. Earlier studies suggest that stathmin is involved in vital cellular processes such as cell proliferation, motility and metastasis. In the present study, we show elevated expression of stathmin in metastatic prostate cancer. Functionally, knockdown of stathmin in prostate cancer cells resulted in reduced cell proliferation and invasion in vitro, and tumor growth and metastasis in vivo. We also show that tumor suppressor microRNA miR-34a targets stathmin and mir-34 is regulated by transcriptional corepressor CtBP1 (C-Terminal Binding Protein 1). Further, Microarray analyses using stathmin modulated prostate cancer cell line RNA revealed multiple molecular alterations which may play a critical role in STMN1-mediated oncogenic activity. Thus, our study suggests that CtBP1-regulated miR-34a modulates stathmin expression in prostate cancer. This study therefore demonstrates functional role of CtBP1-miR-34a-Stathmin axis in prostate cancer progression.

#476

MicroRNA mediated regulation of ATF5 contributes to homeostasis and benign to malignant transformation in breast cancer cells.

Kari Ann Gaither, Bhanupriya Madarampalli, David X. Liu. _Washington State University, Spokane, WA_.

The transcription factor ATF5 modulates survival, proliferation, differentiation, and homeostasis. In unstressed conditions, ATF5 has a short half-life and is rapidly degraded due to post-translational modifications. Conversely, ATF5 is upregulated in cells under cellular stress. Furthermore, we have found that ATF5 is elevated and is a survival factor in transformed C6 glioma and MCF7 breast cancer compared to non-transformed cells. Regulation of ATF5 expression is not fully understood. We hypothesized that microRNA (miRNA) play a role in regulating the expression of ATF5 at the 3' UTR and sought to better understand the role of ATF5 in the transformation to a malignant cell phenotype. To date, no studies have examined the regulation of ATF5 by miRNA. MiRNAs are endogenous small non-coding RNAs 20-25 nucleotides in length that contribute to regulation of gene expression at the translational level. We used in silico modeling programs to identify miRNAs predicted to bind to the 3' UTR of ATF5. We then aimed to identify the presence of specific miRNA and their ability to downregulate ATF5 during cellular stress and other physiological conditions in vitro. Luciferase reporter assays and immunoprecipitations of ATF5 3' UTR mRNA and cell lysate were performed and microRNA quantity analyzed via qPCR analysis. Subsequently, transfections of precursor microRNA were carried out in human MCF10A breast epithelial, MCF7 mammary epithelial, and breast adenocarcinoma MDA-MB-231 cell lines, and expression levels of ATF5 were measured under varying physiological conditions via Western Blot analysis. In vitro migration and invasion assays were performed and transformation was studied via MCF10A mammary epithelial cells with inducible SRC. We demonstrate that miRNA are bound to the 3' UTR of ATF5 and that miRNAs 433-3p and 520b help to regulate the expression of ATF5 under varying stress conditions and at steady state in MCF10A, MCF7, and MDA-MB-231 cells. Additionally, preliminary data indicate that ATF5 is upregulated during transformation of MCF10A cells with inducible SRC and is overexpressed in MDA-MB-231 cells as compared to MCF7 and MCF10A cell lines, and suggest that ATF5 may enhance migration and invasion. Further studies are necessary to elucidate the role of ATF5 in transformation, migration, and invasion. Better understanding of the regulation of ATF5 could have implications in a broad range of human malignancies.

#477

Down-regulated miR-23a contributes to invasion and metastasis of cutaneous melanoma by promoting autophagy.

Weinan Guo, Huina Wang, Yuqi Yang, Sen Guo, Weigang Zhang, Tao Zhao, Lin Liu, Zhe Jian, Ling Liu, Gang Wang, Tianwen Gao, Qiong Shi, Chunying Li. _Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi'an, China_.

Background: The occurrence of invasion and metastasis is the major cause of mortality in melanoma. Recent studies suggest that dysregulated miRNAs play critical roles in this procedure, but the underlying mechanism remains elusive. Here, we show that down-regulated miR-23a can promote invasion-metastasis cascade through autophagy in melanoma.Methods: The role of miR-23a in prognosis was assessed in a cohort of melanoma patients (n = 192) with Kaplan-Meier analysis. The effects of miR-23a overexpression were investigated using assays of invasion, migration and in a xenograft model (n = 10 mice per group). Autophagy-related target of miR-23a was confirmed by bioinformatics analysis, luciferase assays and immunoblotting. Molecular studies were performed to determine the downstream cellular and molecular mechanisms. All statistical tests were two-sided.Results: Serum miR-23a level was significantly down-regulated in melanoma patients (P< .001) and was highly correlated with poor clinical outcomes (P = .027, log-rank test). In addition, miR-23a level was remarkably decreased in metastatic melanoma tissues and cell lines (P<.05, P<.01). Moreover, miR-23a overexpression prevented the invasion and migration in vitro and lung and liver metastasis in vivo (both P< .05), by targeting ATG12 and inhibiting autophagy. Mechanically, miR-23a-ATG12 axis attenuated invasion and migration through autophagy-mediated AMPK-RhoA pathway. Finally, the down-regulation of miR-23a in metastatic melanoma was caused by RUNX2 in a transcriptional repression-manner. Conclusion: MiR-23a can act as a crucial epigenetic repressor of melanoma invasion and metastasis in an autophagy-dependent way, which indicates that miR-23a-mediated autophagy inhibition can be exploited to restrain invasion-metastasis cascade in melanoma treatment.

#478

Regulation of cross talk between AR and wnt/beta-catenin pathways in prostate cancer.

Alexis Plaga, Girish C. Shukla. _Cleveland State University, Cleveland, OH_.

Prostate Cancer (PCa) is the second most commonly diagnosed cancer in the United States with 180,890 males diagnosed and is predicted to cause approximately 26,000 deaths in 2016. The current treatment of androgen deprivation therapy (ADT) initially depletes circulating androgens; however, intratumoral androgens rescue androgen receptor (AR) signaling and promotes the development of castration resistant prostate cancer (CRPC). The dysregulation of the Wnt/β-catenin signaling pathway has been implicated in the development of many cancers including PCa. The disruption of this signaling leads to the stabilization of β-catenin which upregulates many genes involved in tumorigenesis. Additionally, β-catenin acts as AR cofactor. Our previous studies show that AR is a direct target of miR-644a. We hypothesized that targeting the expression of both the β-catenin pathway and AR by conventional drugs and/or tumor suppressor miR-644a would have synergistic therapeutic benefits. In this

study, we are investigating miR-644a mediated posttranscriptional downregulation of GSK3-β and β-catenin in the wnt/β-catenin signaling pathway. The effect of miR-644a in combination with inhibitors (β-catenin or GSK3-β) was also assessed using proliferation assays and a significant downregulation was observed. We will further study the posttranscriptional effects of tumor suppressor miR-644a on the wnt/β-catenin pathway and also the processing and regulation of miR-644a in PCa.

#479

Modeling miRNA induced silencing in breast cancer with PARADIGM.

Andrew J. Sedgewick,1 Panayiotis V. Benos,2 Shahrooz Rabizadeh,3 Patrick Soon-Shiong,3 Charles J. Vaske1. 1 _Nantomics LLC, Santa Cruz, CA;_ 2 _University of Pittsburgh, Pittsburgh, PA;_ 3 _Nantworks, Culver City, CA_.

Introduction: MicroRNAs play an important role in regulation of gene expression and are known biomarkers for breast cancer as well as other malignancies. PARADIGM is a pathway based algorithm that allows for integration of multiple genomic data types with a curated pathway database to make pathway activity predictions. We added a model of gene silencing due to miRNA to the PARADIGM algorithm in order to study miRNA expression in a pathway context.

Results: We curated a set of 7751 miRNA-mRNA interactions from the intersection of 3 target prediction algorithms. These interactions involved 66 miRNA and 2814 mRNA transcripts. We ran this model on global DNA copy number, RNAseq and miRNAseq data from 697 patients in the TCGA breast cancer cohort, and studied changes in the interactions between miRNAs and their targets between different tumor subtypes. The median activity of the RNA-induced silencing complex (RISC) predicted by our model is significantly higher in Basal tumors than other subtypes. In addition, RISC activity is significantly associated with overall survival of patients with Luminal A tumors. The miRNA-target pairs with the largest correlation changes between Basal and Luminal A subtypes were enriched for putative oncogenes and oncomirs. The mRNA targets are involved in a number of important signaling pathways including PI3K-AKT, JAK-STAT, and Ras. Many of these highly differential links involved the miR-16 family of miRNAs which are known tumor suppressors. miR-16 shows significantly lower activity in basal tumors than other subtypes.

Conclusions: By looking at changes in miRNA-target links between tumor subtypes, our algorithm was able to identify both miRNAs and target genes involved in pathways relevant to breast cancer. Our predictions of overall RNA-induced silencing activity show prognostic value in both determining subtype and predicting overall survival within subtypes.

#480

Novel miRNA regulation in an early progression model of PDA.

Nina J. Chu, Todd D. Armstrong, Elizabeth M. Jaffee. _The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD_.

The success of immunotherapy is dependent on infiltration and function of T cells within the tumor microenvironment. However, for many cancers, inflammatory and stromal cells provide formidable barriers to T cell access. Emerging data suggests that the cellular barriers to T cell access and function are regulated by both genetic and epigenetic factors. Identification of these regulators should provide new targets for enhancing immunotherapy.

The overall goal of this research is to construct a comprehensive profile of miRNA expression within the inflammatory and stromal cells that develop in the earliest pre-malignant pancreatic intraepithelial neoplasias (PanINs) in the KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) mouse model, a spontaneous model of pancreatic ductal adenocarcinomas (PDA) tumorigenesis. Specifially, we aim to investigate the functional roles of key differentially expressed miRNAs in propagating the tumor microenvironment via modulating the signaling between transformed ductal epithelial cells and the recruited cancer associated fibroblasts (CAFs) that comprise the majority of the desmoplastic stroma that characterizes PDA. Although numerous studies have described molecular alterations that are involved in pancreatic cancer development and progression, little is known about the miRNA regulatory profile and the associated inflammatory changes within the tumor microenvironment in the earliest PanIN lesions.

We conducted miRNA microarray analysis to determine the levels of 750 unique miRNAs in the pancreata of KPC mice ranging from 4 to 12 weeks of age (pre-PanIN1 to PDA). miRNA was isolated from normal pancreatic ducts, PanIN grades 1 through 3, and PDA via laser capture microdissection. miRNA expression was quantified by Taqman miRNA OpenArrays and confirmed by qPCR analysis. Out of the 750 rodent miRNAs, 4 miRNAs (miR-21, miR-16, miR-19b, and miR-224) were significantly upregulated throughout PDA development. miR-21 and miR-224 are of particular interest for their regulation of targets in cancer promoting inflammatory pathways and epithelial-mesenchymal transition (EMT).

To investigate the roles that miR-21 and miR-224 play in the developing microenvironment, primary KPC pancreatic ductal epithelial and fibroblast cell lines were established via fluorescence activated cell sorting (FACS) in order to perform in vitro miRNA knock-in and knock-out studies. Additionally, miRNA fluorescence in situ hybridization (miR-FISH) was performed to examine the spatial expression of miR-21 throughout progression of the early microenvironment. miR-21 is expressed at low levels in wildtype pancreata, but is highly expressed particularly in ductal epithelial cells of late stage KPC pancreata. Additional studies are underway to determine the functional role of this miRNA in PDA development and progression.

#481

Diminished microRNA-29b results in overexpression of BRD4 and BRD4-regulated oncogenes in cutaneous T-cell lymphoma.

Rebecca Kohnken,1 Jing Wen,1 Bethany Mundy-Bosse,1 Max Yano,1 Leah Grinshpun,1 Kathleen McConnell,1 Ashleigh Keiter,1 Alex Hartlage,1 James Bradner,2 Michael Caligiuri,1 Pierluigi Porcu,1 Anjali Mishra1. 1 _Comprehensive Cancer Center, The Ohio State University, Columbus, OH;_ 2 _Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA_.

Cutaneous T-cell lymphoma (CTCL) is a CD4+ T-cell malignancy that affects the skin but may disseminate systemically, and there are no long-term effective therapeutic strategies. Here, we characterized the therapeutic efficacy of manipulating the tumor suppressor microRNA-29b (miR-29b) and its target bromodomain-containing protein 4 (BRD4) in CTCL pathogenesis. Using primary CD4+ T-cells we determined that miR-29b expression is significantly decreased in patients compared to healthy controls (0.007±0.002, n=9 vs 1.008±0.052, n=6, p<0.0001). We utilized miR-29b-/- mice and bortezomib, a proteasome inhibitor known to increase miR-29b levels, to confirm the inverse relationship between miR-29b and BRD4 (Mishra, A. et al. Cancer Cell, 2012). Diminished miR-29b level resulted in increased BRD4 protein expression (1.87±0.29, p=0.014), while increase in miR-29b, as in bortezomib-treated CTCL cell lines, results in nearly undetectable BRD4 protein. We also observed increased genome-wide occupancy of BRD4 at regulatory regions in CTCL patients, an effect that is reversed with BRD4 inhibitor, JQ1, to similar levels as observed in healthy control CD4+ T-cells. Specifically, oncogene loci of NOTCH1 and RBPJ demonstrated enhanced BRD4 binding in CTCL patients, with resultant increases in mRNA in patient vs normal donors (4.16±0.98, p=0.024; 3.02±0.54, p=0.012). Further, in vivo treatment of interleukin-15 (IL-15) transgenic mice (Mishra, A. et al. Cancer Discovery, 2016) with JQ1 prevented CTCL development and decreased lesion severity (3.37±0.49, n=8 vs 6.0±0.45, n=5, p=0.004). Skin from JQ1-treated mice demonstrated decreased protein expression of BRD4, NOTCH1, and RBPJ, consistent with our findings in patients and CTCL cell lines. Bortezomib treatment of CTCL mice yielded similar results to JQ1 in vivo, while miR-29b levels were elevated in treated mice vs control animals. Since IL-15 signals through its receptor complex, we evaluated and observed increased BRD4 binding at IL-15 receptor αβγ gene loci in CTCL patients, which was reversed with both JQ1 and bortezomib treatment. Thus, we conclude that diminished miR-29b results in increased expression and subsequent binding of BRD4 at regulatory regions of known oncogenes in CTCL cells including NOTCH1, RBPJ, and the IL-15 receptor complex. We confirm the potential therapeutic utility of targeting this pathway in vivo, by direct displacement of BRD4 by JQ1, or by rescue of miR-29b expression by bortezomib. We therefore describe a novel targetable oncogenic pathway featuring IL-15, miR-29b, and BRD4 in CTCL.

#482

miRNAs involved in LY6K and estrogen receptor-α contribute to tamoxifen susceptibility in breast cancer.

Yesol Kim, Dasom Son, Kyung Hyun Yoo, Jong Hoon Park. _Sookmyung Women's University, Seoul, Republic of Korea_.

Estrogen receptor-alpha is a clinically important therapeutic target for breast cancer. However, tumors that lose ERα are less responsive to anti-estrogens such as tamoxifen. MicroRNAs are small RNAs that regulate expression of their target gene and dysregulations of miRNA has been identified in many diseases including human cancer. However, only a few miRNAs associated with tamoxifen resistance has been reported. In this study, we found that lymphocyte antigen 6 complex, which is a member of the Ly-6/μPAR superfamily and related to breast cancer progression and metastasis, is inversely correlated with ERα expression. We, for the first time, found miRNAs involved in the regulatory molecular mechanism between ERα and LY6K and related to tamoxifen susceptibility in breast cancer. miR-192-5p, induced by LY6K, downregulates ERα directly and induced tamoxifen resistance in ERα-positive breast cancer cells. In addition, re-expression of ERα in ERα-negative breast cancer cells increased miR-500a-3p expression and directly inhibits LY6K expression. Ectopic expression of miR-500a-3p sensitized ERα-negative cells to tamoxifen by increasing apoptosis. Finally, we observed an inverse correlation between LY6K and ERα in primary breast cancer samples. We found that patients with recurrence showed high expression of miR-192-5p after tamoxifen treatments. In addition, expression of miR-500a-3p was significantly correlated to survival outcome. As miRNAs involved in the regulatory mechanism between LY6K and ERα can affect tamoxifen resistance, downregulating miR-192-5p or re-expressing miR-500a-3p could be a potential therapeutic approach for treating tamoxifen resistant patients.

#483

Differential expression of miR-34b and androgen receptor pathway regulate prostate cancer aggressiveness between African Americans and Caucasians.

Marisa Shiina, Yutaka Hashimoto, Taku Kato, Soichiro Yamamura, Yuichiro Tanaka, Shahana Majid, Sharanjot Saini, Shahryari Varahram, Priyanka Kulkarni, Prita Dasgupta, Mitsuho Sumida, Guoren Deng, Rajvir Dahiya. _UCSF/VA Medical Center, San Francisco, CA_.

African-Americans are diagnosed with more aggressive prostate cancers and have worse survival than Caucasians, however a comprehensive understanding of this health disparity remains unclear. To clarify the mechanisms leading to this disparity, we analyzed the potential involvement of miR-34b expression in African-Americans and Caucasians. We found that miR-34b expression is lower in human prostate cancer tissues from African-Americans compared to Caucasians. DNA hypermethylation of the miR-34b-3p promoter region showed significantly higher methylation in prostate cancer compared to normal tissues. We found that AR and ETV1 genes are differentially expressed in MDA-PCa-2b and DU-145 cells after overexpression of miR-34b. Direct interaction of miR-34b with the 3' untranslated region of AR and ETV1 was validated by luciferase reporter assay. We found that miR-34b downregulation in African-Americans is inversely correlated with high AR levels that lead to increased cell proliferation. Overexpression of miR-34b in cell lines showed higher inhibition of cell proliferation, apoptosis and G1 arrest in African-American cells (MDA-PCa-2b) compared to the Caucasian cell line (DU-145). Taken together, our results show that differential expression of miR-34b and AR are associated with prostate cancer aggressiveness in African-Americans.

#484

Epigenetic regulation of hsa-miR-3663 in colon cancer.

Laia Pique, Humberto Jorge Ferrerira, Manel Esteller. _Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Spain_.

Colon Cancer is the third most common cancer in the world and a major cause of morbidity and mortality. Molecular mechanisms of colon carcinoma have been deeply studied and current evidences indicate that microRNAs play a pivotal role in its tumorigenesis and progression. MicroRNAs (miRNAs) are small 19 to 22 nucleotides of RNA classified as non-coding RNAs that negatively regulate gene expression at the post-translational level controlling numerous biological processes including development, cell proliferation, apoptosis, differentiation and cell migration. Aberrant expression of miRNAs due to epigenetic alterations has been associated with carcinogenesis. Aimed to identify epigenetically regulated miRNAs involved in colon cancer, we analyzed DNA methylation profiles available in The Cancer Genome Atlas. One of the main hits identified was hsa-miR-3663, hypermethylated in 26% of colon cancer patients (n=286, p< 0.001) in comparison with complete lack of methylation in normal tissues (n=38), suggesting its potential function as a putative tumor suppressor. In order to study the role of DNA methylation controlling hsa-miR-3663, HumanMethylation450K methylation profiles were generated for a panel of colon cancer cell lines (n=10), including the HCT116-DNA methyltransferase knock-out model (DKO, double knockout of DNMT1 and DNMT3B). We found a significant correlation between methylation status of hsa-miR-3663 and expression in colon cancer cell lines. Furthermore, epigenetic regulation of hsa-miR-3663 was confirmed in HCT116 methylated cell line by restored expression upon treatment with DNA demethylating agent 5-aza-2′-deoxycytidine and in the HCT116-DKO model. In order to elucidate the function of hsa-miR-3663, we stably expressed hsa-miR-3663 in a panel of five methylated colorectal cancer cell lines (HCT116, RKO, DLD1, SW48 and SW480). Our initial functional assays revealed that hsa-miR-3663 is not involved in cell proliferation or migration. We are now performing additional assays to further study hsa-miR-3663 and its potential role in colon cancer.

#485

Epigentic silencing of miR-142-3p promotes metastasis by targeting ZEB2 in NPC.

Yingqin Li, Na Liu, Jun Ma. _Sun Yat-sen University Cancer Center, Guangzhou, China_.

Metastasis is the major cause of cancer-related death in nasopharyngeal carcinoma (NPC). However, the molecular mechanisms governing metastasis remain poorly understood in NPC. Here, we demonstrated that miR-142-3p was downregulated and significantly suppressed NPC cell migration, invasion in vitro and in vivo. We identified zinc finger E-box binding homeobox 2 (ZEB2) as a direct target of miR-142-3p, and its overexpression rescued the inhibitory effects of miR-142-3p in NPC. Importantly, miR-142-3p downregulation was associated with DNA methylation within the miR-142 locus. Enhancer of zeste homologue 2 (EZH2) suppressed miR-142 methylation and miR-142-3p expression. Systemic reversal of miR-142 silencing suppressed lung metastasis of NPC cells. Furthermore, miR-142 hypermethylation was associated with poor metastasis-free survival. This study demonstrates a suppressive role of miR-142-3p in metastasis and uncovers an epigenetic mechanism for miR-142-3p silencing in NPC, suggesting a potential prognostic factor and therapeutic target to combat metastasis in NPC.

#486

Production of an unbiased, highly reproducible small RNA library for NGS using a novel circularization technology.

Marta Gonzalez-Plasky, Amanda McNulty, Konstantinos Charizanis, Jing Ning, Edward Jan, Matthew Carroll, Emmanuel Kamberov, Karl Hecker. _Rubicon Genomics, Inc., Ann Arbor, MI_.

MicroRNA sequencing (miRNA-Seq) is a useful tool for aiding researchers in the examination of miRNA expression patterns, the characterization of novel miRNAs, and for uncovering miRNA-disease associations. Since miRNAs are also unusually well-preserved in a range of samples (e.g. urine, FFPE tissue, plasma), profiling their expression is becoming a powerful diagnostic tool. However, current methods for sequencing miRNA require large amounts of total RNA, are not very reproducible and, more importantly, have considerable systematic bias resulting in loss of many prospective biomarkers. This bias severely affects the trustworthiness of results as libraries are not a true representation of the biological state of the sample.

We have recently developed a technology that efficiently captures miRNA species with extremely low bias. Through a circularization approach, Illumina P5 and P7 adapters are ligated to miRNA, resulting in sequenceable libraries in under 5 hours in a single tube. These libraries are then purified and size-selected prior to sequencing.

Analysis of read distributions of libraries prepared from an equimolar mixture of 963 miRNAs demonstrated the technology is able to efficiently capture all species with high reproducibility between replicates and input amounts (correlation coefficient of ≥0.97). Furthermore, 80-86% of these captured miRNA read numbers fall within a +/- 2-fold variation of the expected read number demonstrating unbiased representation. In contrast, frequency distribution analyses for other technologies reveal that 47-56% of miRNAs are greatly under-represented (i.e. less than 2X fewer reads than expected), 29-38% of miRNAs are represented within a +/- 2-fold variation of the expected read number, and around 15% are over-represented by more than 2X.

These findings highlight the fact that the current technical state of library preparation for miRNA sequencing fails to accurately represent miRNA expression patterns. This in turn leads researchers to perform more costly validation experiments than necessary. Our product is designed and developed for more accurately reflecting the true biological state of a sample. This will provide researchers with a tool that can aid in advancing miRNA sequencing in translational research.

### Molecular Carcinogenesis and Chromosomal Alternations

#487

A comprehensive target enrichment panel for fusion detection.

Ashesh Saraiya,1 Brandon Young,2 Tobias Meißner,2 Brian L. Jones,2 Stephanie C. Huelga,1 Doug A. Amorese1. 1 _NuGEN, San Carlos, CA;_ 2 _Avera Cancer Institute for Precision Oncology Genomics, San Diego, CA_.

Growing interest in cancer classification and progression has accelerated the rate of novel gene fusions discovery with increasing recognition of their roles as biomarkers. RNA-Seq is an attractive method for expressed fusion discovery and detection because of its ability to provide unbiased fusion sequencing information. The ability to detect low expressing fusion transcripts, however, require high sequencing depth and represents a significant financial barrier and identification of clinically relevant fusion sequences from a large data set can be a bioinformatics challenge.

To address these challenges we have tested the Ovation® Fusion Panel Target Enrichment System V2, a targeted RNA sequencing method using the Single Primer Enrichment Technology (SPET), with a number of control and clinical samples. Initial studies were performed using a comprehensive target enrichment panel targeting 502 genes with three samples from Horizon DX containing known fusions. Target enriched libraries were constructed with 10 ng and 100 ng inputs and the data was analyzed using the NuFuseD pipeline (available as a point and click BaseSpace application or downloadable linux package) which has been optimized for fusion analysis from this data. Expected fusions were identified at both input levels, even when down sampled to 500K reads, with fewer fusion calls compared to other publically available fusion detection software (Chimerascan and SOAPFuse), suggesting a lower false positive rate. NuFuseD fusion calls are provided with a P-value to help prioritize the identified fusions for subsequent validation. Additionally, NuFuseD detected novel fusions in the control samples demonstrating the advantage of a comprehensive panel compared to more restricted panels. We further validated the target panel using control RNA (UHR and Human Brain) and fresh or FFPE cell lines (NCI-H2228, HCC1937) to further demonstrate our ability to identify known fusions.

Finally, the system was evaluated at an external site using patient FFPE samples. These samples (N=8) were from a set of breast, liver and ovarian cancers, containing a unique fusion in 4 of the samples based on DNA based sequencing. Only 1 of the 4 expected fusions were identified using whole transcriptome data (100 million reads) while 3 of the 4 fusions were detected with this assay (10 million reads) demonstrating its ability to generate targeted RNA sequencing libraries with increased sensitivity of gene fusion detection and reduced sequencing costs compared to standard RNA-Seq methods.

#488

E2F2-mediated copy number changes drive metastasis and therapeutic response of HER2-positive tumors through Col1a1, CHAD, and AKT-dependent mechanisms.

Jonathan P. Rennhack, Kelian Sun, Jordan Honeysett, Eran Andrechek. _Michigan State University, East Lansing, MI_.

The E2F family of transcription factors is classically known to regulate G1 to S-phase transition in cell cycle but has emerging roles in HER2+ breast cancer. A loss of E2F1 or E2F2, in a HER2 mouse model, MMTV-Neu, leads to a decrease in tumor metastasis1. It is not known what mechanistic roles specific E2Fs are playing in this process. To investigate this, we leveraged and bioinformatic principles, including genomics and transcriptomics with traditional laboratory science and high throughput drug screening projects. This experimental approach immediately revealed that loss of E2F2 is significantly associated with a more unstable tumor including an increase in copy number alterations, single nucleotide variants, and translocations. Further analysis revealed a conserved copy number alteration in both mouse and humans. Specifically we noted the amplification of 17q21.33 in 25% of HER2+ patients. The analogous region chromosome 11D was lost in 30% of the less metastatic MMTV-Neu E2F2 knockout mice indicating a role of the region in tumor metastasis. Transcriptomic data revealed that two genes, Collagen Type I, alpha 1 (Col1a1) and Chondroadherin (CHAD), were potential genes of significance in the amplification event. CRISPR mediated knockout studies were conducted to determine each gene's effect on tumor cell migration and metastasis in mouse and human derived cell lines. Wound healing assays and tail vein injection have shown the Col1a1 and CHAD KO cell lines have a delay in cell migration (P<.01) and reduced ability to colonize the lung (P<.05) respectively. Oncogenic signaling data shows that the 17q21.33 amplification event has higher AKT and E2F2 signaling than HER2 positive tumors without the event. It was hypothesized that the tumors would be dependent on the signaling and perturbation of the network might be an effective therapy for patients with the 17q21.33 amplification event. To investigate this we identified deferentially lethal siRNAs and compounds between HER2+ tumors with and without the 17q21.33 event in the Achilles, CCLE, and PDX datasets. A String-DB analysis showed many of the deferentially lethal genes and compounds centered around AKT. A decrease in AKT signaling through siRNA or chemical compound results in the death of the cell in the 17q21.33 amplified samples but not the HER2+ samples without the event. This study reveals that patients' with a 17q21.33 amplification event have more metastatic tumors mediated through Col1a1 and CHAD and may be responsive to AKT targeting therapy.

References1. Andrechek ER: HER2/Neu tumorigenesis and metastasis is regulated by E2F activator transcription factors. Oncogene 2013.

#489

Mismatch repair defects and endocrine therapy resistance in estrogen receptor positive breast cancer.

Svasti Haricharan,1 Jacob Schmelz,1 Cheryl Schmidt,1 Purba Singh,1 Kimberly R. Holloway,1 Meenakshi Anurag,1 Shunqiang Li,2 Shyam M. Kavuri,1 Shixia Huang,1 Dean P. Edwards,1 Vera Suman,3 Kelly Hunt,4 John A. Olson,5 Jeremy Hoog,2 Cynthia X. Ma,2 Matthew N. Bainbridge,1 Matthew J. Ellis1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Washington University, St. Louis, MO;_ 3 _Mayo Clinic, Rochester, MB;_ 4 _UT MD Anderson Cancer Center, Houston, TX;_ 5 _University of Maryland, School of Medicine, Baltimore, MD_.

Estrogen receptor positive (ER+) breast cancer is treated with endocrine therapy but intrinsic resistance occurs in ~1/3 of patients and acquired resistance in ~1/5 of the remainder. While many resistance mechanisms have been explored, therapeutic strategies to overcome resistance in the clinical setting have seen mixed outcomes, and appear most effective in the acquired resistance setting. Understanding mechanisms of resistance and finding therapeutic strategies to target them, therefore, remain important challenges facing breast cancer researchers. In this study we systematically examine the role of DNA damage repair defects in inducing endocrine therapy resistance, a relatively understudied question of recent interest. We use in silico analysis of clinical datasets, in vitro experiments evaluating endocrine therapy resistance in response to DDR dysregulation in multiple breast cancer celllines, and in vivo validation using cellline xenograft and patient-derived xenograft models. We also use gene expression microarrays and RPPA data from cell lines, patient-derived xenografts and primary ER+ breast tumors to uncover therapeutic options that are validated in vitro and in vivo and corroborated by clinical trial data. The results of this study uncover an intriguing link between mismatch repair (MMR) deficiency, specifically of the MutL complex (MLH1/3, PMS1/2), and poor prognosis in ER+ disease. We find a direct role for MutL loss in endocrine therapy resistance in vitro and in vivo by knocking down multiple MutL genes using CRISPR and stable shRNA approaches validated using standard rescue experiments. We identify the underlying mechanism: MutL deficiency in ER+ breast cancer abrogates Chk2-mediated feedback inhibition of CDK4/6 that appears necessary for endocrine therapy responsiveness. Consequently, pharmacological targeting of CDK4/6 in vitro and in vivo significantly inhibits growth of endocrine therapy resistant MutL-deficient ER\+ breast cancer cells. These results are corroborated by data from a neoadjuvant clinical trial demonstrating that cell cycle regulation of MutL-mutant tumors tends to be estrogen-independent but sensitive to CDK4/6 inhibitors. The results of this study provide important biological and clinically relevant insights. 1) MMR deficiency is unexpectedly causal to intrinsic endocrine therapy resistance 2) This causal effect appears to be mediated by abrogation of cell cycle checkpoint activation in response to endocrine therapy 3) MMR deficiency in a subset of ER+ tumors explains why CDK4/6 inhibition is effective against some de novo endocrine therapy resistant tumors. While there are currently no biomarkers to guide the use of CDK4/6 inhibitors for ER+ breast cancer, markers of MMR dysregulation could identify patients in whom CDK4/6 inhibition should be used to prevent disease recurrence.

#490

Identification of oncogenic gene fusions in primary colon cancers.

Robert R. Coebergh van den Braak,1 Wigard P. Kloosterman,2 Mark Pieterse,2 Markus van Roosmalen,2 Anieta S. Sieuwerts,1 Christina Stangl,2 Ronne Brunekreef,2 Zarina S. Lalmahomed,1 Salo Ooft,3 Anne van Galen,1 Marcel Smid,1 Armel Lefebvre,2 Fried J. Zwartkruis,2 John W. Martens,1 John A. Foekens,1 Katharina Biermann,1 Marco J. Koudijs,2 Jan N. IJzermans,1 Emile E. Voest1. 1 _Erasmus University Medical Center, Rotterdam, Netherlands;_ 2 _University Medical Center Utrecht, Utrecht, Netherlands;_ 3 _Netherlands Cancer Institute, Amsterdam, Netherlands_.

Introduction: Fusion genes (such as ALK-EML) are potentially attractive targets for anti-cancer treatment. In the era of personalized medicine exploring the identification, incidence, and functionality of fusion genes may contribute to effective treatment approaches.

Methods: We performed a comprehensive and unbiased screening for gene fusions in a clinically well-defined prospectively collected cohort of 278 primary stage I to III colon cancers. Illumina RNA sequencing was performed using RNA from the fresh frozen samples. The STAR fusion gene detection pipeline and GATK RNA-seq variant calling were used to identify fusion genes and detect somatic genetic variations. Gene fusions were considered relevant when recurrent, when resulting in divergent expression (outlier analysis) or when functional relevance was predicted (i.e. kinase fusions).

Results: 2.5% of all samples contained a relevant gene fusion. Kinase fusions were most prevalent with a frequency of 1.8%. Three BRAF-fusions were identified, two known (TRIM24-BRAF & AGAP3-BRAF) and one novel fusion (DLG1-BRAF). Co-expression with ERK1 in HEK293 cells resulted in enhanced EKR1 phosphorylation. The outlier analysis revealed four unique fusions (EML4-NTRK3, RRBP1-RET, USPX9-ERAS & EIF3E-RSPO2). The EML4-NTRK3 fusion led to increased expression of the tyrosine kinase encoding domain exons, which is retained in the fusion transcript. Co-expression with ERK1 in HEK293 cells resulted in enhanced EKR1 phosphorylation. The tumor with the RRBP1-RET fusion was pan negative for known driver mutations, as reported in literature. Mate-pair sequencing of the USPX9-ERAS fusion gene region revealed that the fusion gene was caused at the genomic level by a highly local chromothripsis event on chromosome X spanning solely the region covered by USP9X and ERAS, leading to high ERAS expression. ERAS is a constitutionally active RAS protein and normally only expressed in embryonic stem cells. Analysis of phosphorylated AKT after expression of the USP9X-ERAS fusion gene in NIH-3T3 A14 cells showed activation of AKT signaling. The occurrence of one R-spondin fusion in our set (0.4%) is low compared to the previously described 9.5%, which may be explained by difference in incidence of KRAS- or BRAF-mutations in the two cohorts, since all R-spondin fusions occurred in a tumor with a KRAS- or BRAF-mutation. All fusion genes were expressed in cell lines resulting in the activation of AKT signaling. Finally, oncogenic fusions were only found in lymph node negative tumors and were not observed in tumors with classical driver mutations in BRAF and KRAS except for the R-spondin fusion.

Conclusions: We found oncogenic gene fusions including novel fusions with a frequency of 2.5%, including an USP9X-ERAS fusion with strong oncogenic activity in vitro. Although recurrent fusion genes are rare events in colorectal cancer they may represent functional drivers and provide potential novel leads for personalized therapeutic strategies.

#491

**Elucidating** de novo **PATRR-mediated t(3;8) balanced translocation and clear cell renal cell carcinoma.**

Marilena Melas,1 Kevin J. McDonnell,1 Christopher K. Edlund,1 Sarah J. Tash,2 Duveen Y. Sturgeon,1 Chenxu Qu,1 Charalampos Lazaris,3 Peter J. Gruber,4 Thomas W. Glover,5 Beverly S. Emanuel,2 Stephen B. Gruber1. 1 _USC Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA;_ 2 _Division of Human Genetics, Children's Hospital of Philadelphia, Philadelphia, PA;_ 3 _Laura & Isaac Perlmutter Cancer Center and Helen L. & Martin S. Kimmel Center for Stem Cell Biology School of Medicine, New York, NY; _4 _Division of Cardiothoracic Surgery, Department of Surgery, University of Iowa Carver College of Medicine, Iowa City, IA;_ 5 _Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI_.

Palindromic AT-Rich Repeat (PATRR) - mediated translocations entail exchange across chromosomes at sites enriched in palindromic repeats of the nucleotides adenine (A) and thymine (T). Their precise embryologic origin and associated pathobiology with clear cell renal cell carcinoma (ccRCC) remain incompletely described. In the present study we document an individual with familial non-VHL ccRCC (7 primary renal tumors) who harbors a germline de novo PATRR mediated balanced translocation involving chromosomes 3 and 8 [t(3;8)] validated by spectral karyotyping (SKY).

Using translocation specific PCR and DNA sequencing we determined the chromosome 3 breakpoint to be located in an AT-rich palindromic sequence in the third intron of the FHIT gene (chr3p14.2) and the chromosome 8 breakpoint in the first intron of the TRC8 gene (chr8q24.1). Genotyping analysis, using a high density custom exomechip array by Illumina, revealed a loss of the entire aberrant chromosome 8 carrying the 3p segment [der(8)] in all renal tumors tested. We also determined that the [46, XY, t(3;8), (p14.2, q24.1)] translocation was paternally derived by performing a genotypic assessment of the regions that differ between the parental alleles and then establishing which haplotypes are associated with the translocation. The somatic mutational landscape was assessed by Whole Exome sequencing of the renal tumors and the proband's germline DNA. No germline or somatic deleterious mutations were detected in VHL gene suggesting that the ccRCC phenotype is not associated with Von Hippel-Lindau disease.

Furthermore, we measured the transcriptomic profiles of the renal tumors and matched normal tissues employing barcoded probe hybridization technology (NanoString) and RNASeq to fully characterize differential gene expression and define gene ontology networks that are dysregulated. Most significantly, no difference in expression of the VHL gene was detected between tumors and normal counterparts. Aberrant regulation was detected in members of WNT signaling pathway, TGF-beta and MAPK pathways, in addition to NOTCH signaling pathway genes and members of the VEGF family. In summary, in this study we employed advanced genomic and transcriptomic techniques, to delineate the embryologic origin and deleterious biological consequences of the PATRR-mediated t(3;8) balanced translocation associated with clear cell renal cell carcinoma.

#492

Chronic inflammation contributes to neoplastic progression in prostate tissue: a needle biopsy study.

Shardul C. Soni, Michael Glover, Qinghu Ren, Gregory T. MacLennan, Pingfu Fu, Sanjay Gupta. _Case Western Reserve University, Cleveland, OH_.

The relationship between inflammation and prostate cancer has not been established, although chronic inflammation has frequently been identified in prostate biopsies, radical prostatectomy and benign prostatic hyperplasia specimens. In the peripheral zone of the prostate, sometimes adjacent to foci of high-grade PIN and cancer, certain morphologic changes are often identified, which may represent active and terminal phases of chronic inflammation. These changes are designated as proliferative inflammatory atrophy (PIA) and post atrophic hyperplasia (PAH), and their morphology is well documented. In our previous studies, we have identified chronic inflammation as a putative contributor to neoplastic progression in prostate epithelial cells, and hypothesized that its adverse effects were related to an increase in Bcl2, a survival protein involved in cell survival and carcinogenesis. We hypothesize that changes in the stromal microenvironment, characterized by infiltration of immune cells, with generation of reactive oxygen species, can induce oxidative stress in the surrounding proliferating epithelium and cause permanent genomic alterations. In the present study, we focused on several key proteins involved in the inflammatory process, COX2 and iNOS; cell survival, Bcl2 and GSTPπ; and evaluated immunohistochemical expression of alpha-methylacyl coenzyme A racemase (AMACR) and basal cell-specific markers 34βE12 and/or p63 to evaluate possible neoplastic alterations in epithelial cells in an inflammatory environment. We evaluated 16 prostate core needle biopsy specimens that exhibited the presence of chronic inflammation as well as PIA and PAH lesions along with immunohistochemical staining for various markers was performed in each set of biopsies. The integrity of the basal layer was maintained in the area of chronic inflammation with high expression of p63 in 72% of these cells. Approximately 68% of luminal cells expressed high to moderate levels of iNOS and COX-2, whereas 55% of these cells express modest levels of GSTP1 and Bcl2. Interestingly, prostatic glands near the areas of chronic inflammation in the PIA lesions exhibit high AMACR expression in luminal cells and weak to patchy p63 expression in basal cells, which was associated with increased expression of the inflammatory markers COX2 and iNOS, as well as loss in pro-survival signal GSTP1 and Bcl2 in the adjacent luminal cells. These neoplastic alterations were observed in 6/16 (38%) of the needle biopsy specimens. Our findings suggest that basal cells undergo alterations in a setting of chronic inflammation. This is important because basal cells are considered to be progenitor cells capable of differentiating into secretory luminal cells, but under the influence of chronic inflammation, they may instead transform into the neoplastic cells that characterize high grade prostatic intraepithelial neoplasia and prostatic adenocarcinoma.

#493

**Pathologic findings at RRSO in germline** BRCA **mutation carriers with breast cancer: significance of bilateral RRSO at the optimal age in germline** BRCA **mutation carriers.**

YoungJae Lee, YongMan Kim, ShinWha Lee. _Asan Medical Center, Seoul, Republic of Korea_.

Objective: Most BRCA1/2 carriers do not undergo risk-reducing salpingo-oophorectomy (RRSO) by the recommended age. This study aimed to find the incidence of precursor lesions and cancer after RRSO.

Methods: We retrospectively reviewed breast cancer patients identified as BRCA mutation carriers who underwent RRSO at Asan Medical Center, Seoul, Korea, from 2010 to 2014. From 2013, all cases were examined according to the SEE/FIM protocol and underwent immunohistochemically (IHC) staining. RRSO was performed in 63 patients, 27 in 2010-2012 and 36 in 2013-2014.

Results: The median age at RRSO was 46.5 years (32-73 years). Occult invasive cancer was detected in 8 patients, of ovarian origin in 5 and of tubal origin in 3. All occult invasive cancer cases with metastasis were detected in patients older than 40 years. Of the 36 patients from the 2013-2014 cohort, 7 showed p53 overexpression, 1 showed Ki-67 overexpression, 2 showed serous tubal intraepithelial carcinoma (STIC), and 3 showed occult cancer. The detection rate of precursor lesions or cancer was 36.1% (13/36). In the analysis according to age, precursor lesions were more common in BRCA1 mutation carriers younger than 40 years old (66.7% vs 20.0%). In BRCA2 mutation carriers, precursor lesions were only detected in those older than 40 years of age, indicating the possible faster occurrence of precursor lesions in BRCA1 mutation carriers.

Conclusions: Many patients still tend to delay RRSO until after they are 40 years old. Our findings support the significance of RRSO before the age of 40 in germline BRCA mutation carriers.

#494

**Frequent** NRG1 **genomic rearrangements in invasive mucinous adenocarcinoma from caucasian patients.**

Domenico Trombetta,1 Giulio Rossi,2 Angelo Sparaneo,1 Federico P. Fabrizio,1 Maria C. Manzorra,1 Evaristo Maiello,1 Vito M. Fazio,1 Paolo Graziano,1 Lucia A. Muscarella1. 1 _IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy;_ 2 _Azienda USL Valle d'Aosta Hospital Parini, Aosta, Italy_.

Invasive Mucinous Adenocarcinoma (IMA) accounts for 2-5% of lung adenocarcinomas and it is associated with an unfavorable clinical course, mainly due to lack of effective treatments. Current knowledge of the molecular alterations involved in IMAs is limited. Recently, the NRG1 genomic rearrangement was identified as a subtype-specific molecular feature of Asiatic IMA cohorts since it acts as a strong inductor of the aberrant tyrosine kinase activity of ErbB2/ErbB3 heterodimers through PI3K–AKT and MAPK cellular cascades.

In light of these premises we explored the occurrence and frequency of NRG1 fusions in a cohort of 90 Formalin Fixed Paraffin Embedded (FFPE) lung adenocarcinoma from Caucasian patients (35 non-IMAs and 55 IMAs) by performing FISH analysis to study the NRG1 genomic region (β-III isoform, chr 8p12). In total, 16 out of 55 (29%) IMAs showed NRG1 rearrangements, whereas in non-mucinous lung adenocarcinoma group it was found with a frequency of 3% (1/35). The functional effect of the genomic rearrangement was confirmed by RT-PCR and sequencing in three cases with available RNA, where NRG1-CD74 fusion transcripts were identified. An aberrant expression of pErbB3 was also observed in these three NRG1 rearranged cases by performing immunohistochemistry analysis, thus confirming the ErbB3 cascade activation.

Our results strongly confirm NRG1 rearrangements as potentially treatable oncogenic driver alterations associated with a definite lung adenocarcinoma subtype in Caucasian population and support a clear molecular rationale to novel therapeutic opportunity for these aggressive tumors.

#495

Somatic copy number variants detection using the NEBNext Direct target enrichment method.

Kruti M. Patel,1 Sarah K. Bowman,1 Noa Henig,1 Amy B. Emerman,1 Andrew Barry,2 Charles Elfe,1 Scott Adams,1 Salvatore Russello,2 Ted Davis,2 Cynthia L. Hendrickson1. 1 _Directed Genomics, Ipswich, MA;_ 2 _New England Biolabs, Ipswich, MA_.

Variations in copy number are the most common type of DNA structural variations. Many of these alterations are associated with disease or indicate disease susceptibility. Detection of copy number variants (CNVs) for clinical applications requires efficient, fast and cost effective methods.

Here we describe the use of the NEBNext Direct® hybridization-based target enrichment method to identify somatic CNVs with high sensitivity. This approach begins with target enrichment followed by enzymatic digestion of off-target sequences and ligation of adapters that contain unique molecular identifiers (UMIs). The UMIs enhance the identification of duplicate reads and further increase the final number of on-target reads used for variant detection.

We used a cell line known to contain a deletion in the CDKN2A gene and spiked DNA isolated from the cell line into a HapMap DNA sample that contains full diploid copies of CDKN2A. We were able to detect the gene deletion as a somatic CNV in a dose dependent manner with high sensitivity. Thus, we demonstrate that the NEBNext Direct approach is an efficient technique to detect somatic CNVs of high and low frequencies.

#496

Ubiquitin specific peptidases 37 promotes constitutive replication fork movement by stabilizing Chk1 via its deubiquitination.

Mayank Singh,1 Amy C. Burrows,2 Andrew Dickson,2 Komal Komal,1 Debjani Pal,1 Matthew K. Summers1. 1 _Ohio State University, Columbus, OH;_ 2 _Cleveland Clinic, Cleveland, OH_.

Protein deubiquitination controls many intracellular processes, including cell cycle progression, transcriptional activation, and signal transduction. Ubiquitin specific peptidases (USPs) remove ubiqiuitin tags from target proteins to control both protein fate and function. USP37 (Ubiquitin specific peptidase 37) has been implicated in cancer and we have previously shown that antagonizes the tumor suppressor APCCDH1and promotes S phase entry. However, the role of USP37 during S-phase was unknown. Here, we report that in cells experiencing replication stress USP37 overexpression confers survival advantage while its depletion enhances sensitivity. USP37 overexpressing cells were able to resolve different DNA damage markers much more effectively then the control cells or cells in which USP37 was depleted. Mechanistically, our data indicate that USP37 binds and stabilizes the active form of CHK1 and deubiquitinates Chk1 to increase its stability and promote the checkpoint response. USP37 overexpression results in constitutive replication fork movement and long tract DNA synthesis while USP37 depleted cells were unable to carry out long tract DNA synthesis. Notably, expression of low level of Chk1 in USP37 depleted cells rescues cell survival and DNA damage response. Overall our data suggest that inhibition of USP37 may represent a novel mechanism to modulate Chk1 activity.

#497

Higher nucleolar index of nucleolin as an indicator of aberrant cellular DNA damage response (DDR).

Iqra Nadeem,1 Amna Aslam,2 Jingyuan Wang,1 Anna Kozlova,1 Danielle Gordon,1 Rumsha Javed,2 Ruchama Steinberg,1 Rachele Dolce Rameau,3 Xinyin Jiang,1 Anjana D. Saxena3. 1 _CUNY, Brooklyn College, New York, NY;_ 2 _Midwood High School, Brooklyn, New York, NY;_ 3 _CUNY, Brooklyn College and Graduate Center, New York, NY_.

Nucleoli provide a survival advantage for various tumor types, exemplified in prostate neoplasia, leukemia, lung, and breast carcinomas. Unregulated expression and increased nucleolar localization of many stress-responsive factors often correlate with hyper-proliferative status. Therefore, nucleolar changes in cancer cells are the first cytological indicators that can serve as an index for diagnostic and prognostic purposes. In this study, we examine an abundant nucleolar stress factor, nucleolin (NCL) for its sub-nuclear localization under normal and DNA damage conditions. NCL is a multifunctional RNA-binding phosphoprotein that plays critical role/s in many cellular processes including regulation of gene expression during normal cell cycle as well as in the cellular DNA damage response (DDR). NCL-mediated DDR involves changes in its phosphorylation status, sub-nuclear translocation and a multi-level regulation of p53-checkpoint signaling. However, the role/s of p53 status and the signaling pathways triggered during NCL-mediated stress response remain elusive, and forms the focus of this study. Taking advantage of two human ductal breast epithelial tumor cell lines with different genetic backgrounds (wild type, wt-p53 and mutant, mut-p53 L194F), we demonstrate that NCL is predominantly nucleolar in unstressed conditions irrespective of the p53 status. Upon genotoxic stress, however, wt-p53 is required for NCL to mobilize from nucleoli to the nucleoplasmic region. We observed a significant increase in nucleoplasmic index for NCL that is wt-p53 dependent and specific to the type of DNA damage. In contrast, in the presence of mut-p53, NCL failed to translocate even under various DNA damage conditions, resulting in a higher nucleolar index. Interestingly, another nucleolar factor, nucleophosmin (NPM) remained unaltered upon stress conditions even in the presence of wt-p53. This study corroborates the previous research that suggests NCL translocation requires wt-p53 and provides new insights into the role of p53-mutations (e.g. the zinc finger mutation in the central DNA binding region-L194F) in NCL-mediated DDR. Further, we also assessed the role of NCL and p53 (wt vs. mut) in regulating stress signaling (ATM/ATR and p38-kinase) pathways and gene expression in apoptosis. Together, our study elaborates a novel approach that correlates NCL sub-nuclear localization, downstream signaling pathways and gene expression during the cellular DDR.

#498

Attenuation of polycyclic aromatic hydrocarbon (PAH)-mediated pulmonary carcinogenesis in mice by omega-3-fatty acids.

Bhagavatula Moorthy,1 Weiwu Jiang,1 Lihua Wang,1 Sudha R. Kondraganti,1 Guodong Zhou2. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Texas A &M University, Houston, TX_.

3-Methylcholanthrene (MC) and benzo[a]pyrene (BP) are potent polycyclic aromatic hydrocarbons (PAHs) carcinogens. PAHs are present in cigarette smoke, diesel exhausts, and charcoal broiled meats, etc. Cytochrome P450 (CYP) 1A/1B1 enzymes play key roles in the activation of PAHs to carcinogenic metabolites, which initiate carcinogenesis by binding covalently to DNA, and the adducts, if not repaired, could lead to tumorigenesis. In this study we tested the hypothesis that pre-treatment of mice with omega-3-fatty acids, i.e. [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) will lead to attenuation of PAH-mediated pulmonary DNA adduct formation and pulmonary tumorigenesis. Twelve week old male and female A/J mice received EPA (60 mg/kg) and DHA (40 mg/kg) from day 1 to day 24. Control mice were treated with vehicle corn oil. On day 3, mice were treated with BP (40 µmol/kg) or 3-methylcholanthrene (MC, 40 µmol/kg) by i.p. In the short-term experiment (DNA adduct studies), 3-5 mice from each group were terminated at day 10 (7 days after BP or MC treatment). EPA/DHA significantly suppressed formation of BP-DNA and MC-DNA adducts in lung and liver of both male and female mice. In the long-term experiment (tumor studies), 8 mice for each group continued to receive EPA/DHA twice per week till end of experiment (16 weeks). Pulmonary tumor incidence and multiplicity was significantly suppressed in mice given EPA+DHA, compared to those given the PAHs, BP or MC alone. These studies suggest that the omega 3-fatty acids could be developed as novel candidates for the prevention of PAH-mediated lung cancers in humans.

#499

**IgH enhancers hs3b/hs4 are dispensable for c-** Myc **deregulation in mouse plasmacytomas with T(12;15) translocations.**

Alexander L. Kovalchuk, Herbert C. Morse. _NIH-NIAID, Rockville, MD_.

c-Myc deregulating t(12;15) chromosomal translocation is the hallmark cytogenetic abnormality of murine plasmacytomas (PCTs). In the great majority of PCTs, the immunoglobulin heavy chain (IgH) locus is broken between Eμ enhancer and the 3' regulatory region (3'RR), making the latter the major candidate for orchestrating c-Myc deregulation. To elucidate the role of 3'RR in tumorigenesis, we induced PCTs in mice deficient for the major 3'RR enhancer elements, namely hs3b and hs4 (3'KO). Unexpectedly, and contrary to the previous observations made in a mouse lymphoma model, Hs3b/Hs4 3'KO mice did develop t(12;15)-positive PCTs, although with a lower incidence than wild-type controls. In heterozygous mice, there was no allelic bias in targeting IgH for the t(12;15). Molecular analysis of IgH/Myc junctions revealed dominance of Sμ region breakpoints compared to the prevalence of hits into Sγ or Sα in the controls. Analysis of c-Myc expression as well as Ig secretion in 3'KO PCT cell lines revealed no significant differences from the controls. Our current findings highlight the complexity of the 3'RR and the potential of its components to compensate for each other in the context of differentiation from B-lymphocyte to plasma cell. We next extended our observations to include a PCT model where Cre-mediated deletion can remove all four 3'IgH enhancers: hs3a, hs1,2, hs3b and hs4. In PCT cell lines where c-Myc is translocated to an IgH BAC transgene with a floxed 3'IgH enhancer region, induction of retrovirally-expressed Cre-recombinase causes gradual Myc downregulation and complete cell death within 96 hours. The later observation confirms the requirement of the full 3'RR for c-Myc deregulation by T(12;15).

#500

Impairment of DNA double strand break repair in human primary alveolar type II cells in emphysema.

Beata Kosmider, Liudmila Vlasenko, Nathaniel Marchetti, Sudhir Bolla, Chenna Mandapati, Nathaniel Xander, Gerard Criner, Karim Bahmed. _Temple University, Philadelphia, PA_.

Rationale Emphysema is caused by the destruction of alveolar wall septa. The major risk factor for this disease is cigarette smoke and effective therapies are very limited. Alveolar type II (ATII) cells are in the gas exchange portion of the lung. They make and secrete pulmonary surfactant, and proliferate to restore the epithelium after damage to the more sensitive alveolar type I cells.

Methods Control ATII cells were isolated from deidentified control non-smoker and smoker organ donors whose lungs were not suitable for transplantation and donated for medical research. Furthermore, as a unique approach, we have developed a new method on how to isolate ATII cells from excess tissue from lung transplants obtained from patients with emphysema using magnetic microbeads. We determined DNA damage, DNA repair, oxidative stress, injury, and inflammation in human primary ATII cells isolated from these individuals in comparison with controls.

Results Our data indicates high oxidative stress in human ATII cells induced by cigarette smoke extract in vitro as measured by 4-HNE staining by immunocytofluorescence. We also observed DNA double strand breaks, high DNA damage, and low DNA repair in these cells. Moreover, we found greater proinflammatory response as determined by IL-8 and IL-6 levels by ELISA. Furthermore, ATII cells obtained from patients with emphysema had greater injury compared to cells obtained from control non-smokers and smokers.

Conclusion Our results indicate oxidative ATII cell damage induced by cigarette smoke and in emphysema. Observed DNA damage/repair imbalance may contribute to cell death in this disease. The study of ATII cell injury may improve our knowledge on this disease pathogenesis and may lead to novel therapeutic strategies to slow the progression of emphysema.

#501

Tyrosyl-DNA phosphodiesterase I cellular function dependent on its N-terminal residues.

Selma M. Cuya, Robert C.A.M. Van Waardenburg. _Univ. of Alabama at Birmingham, Birmingham, AL_.

Tyrosyl-DNA phosphodiesterase I (Tdp1) is a highly conserved eukaryotic DNA repair enzyme that catalyzes the resolution of 3' and 5' phospho-DNA adducts. Tdp1 has been implicated in the repair of DNA topoisomerase I (Top1)-DNA covalent complexes reversibly stabilized by FDA approved camptothecins (CPTs) derivatives topotecan and irinotecan. Tdp1 activity relies on two catalytic histidines that function as a nucleophile and an acid-base residue. A mutation of the acid-base His to Arg (H493R) in human Tdp1 is associated with the rare recessive ataxia SCAN1. We defined alternative substitution of either catalytic histidine that induce cytotoxicity, reduce catalytic activity and enhances the requisite Tdp1-DNA covalent adduct lifetime in the cell. The phenotypes of the catalytic mutants provide excellent tools to study Tdp1 cellular function. Biochemical studies revealed that Tdp1 catalysis in vitro is independent of the N-terminal domain. Among Tdp1 proteins, the N-terminal domain is poorly conserved in sequence and size (79aa for yeast and 148aa for human Tdp1). Conversely, the N-terminal domain regulates the in vitro activity of these Tdp1 mutants. Additionally, we investigated the role of the N-terminal domain for Tdp1 activity in the yeast and human cell models. Expression of N-terminal truncated proteins showed similar cellular distribution as the full-length proteins. However, these N-terminal truncated Tdp1 mutants did not display the toxicity that was observed with the full-length Tdp1 mutant proteins. Our data suggests that the N-terminal domain is required to resolve protein-DNA covalent complexes, such as Top1. Indeed, preliminary results suggest that this domain is also critical to process Top2-DNA covalent complexes, but only in the presences of etoposide. We are currently investigating other protein-DNA adducts that are resolved by Tdp1. These results suggests that the N-terminal domain is a critical determinate of Tdp1 cellular function. However, further studies are necessary to ensure that these constructs are properly distributed and retain their catalytic activity. Additionally, the N-terminal domain of human Tdp1 is post-translational modified, while our preliminary results suggest that this domain is important for protein-protein interaction and Tdp1 recruitment to its substrates. Understanding Tdp1 substrate and protein-interactions are important in the development of Tdp1 as therapeutic target. This work is in part supported by the ADDA, UAB Cancer Comprehensive Center (P30CA013148), ACS-IRG-60-001-53, DOD OCRP (W81XWH-15-1-0198).

#502

Electronic cigarette aerosols increase cellular reactive oxygen species and induce significant oxidative DNA damage.

Vengatesh Ganapathy,1 Jimmy Manyanga,1 Dehra McGuire,1 Daniel Brobst,1 Theodore Wagener,1 David Rubenstein,1 Ilangovan Ramachandran,2 Lurdes Queimado1. 1 _Univ. of Oklahoma Health Sciences Ctr., Oklahoma City, OK;_ 2 _University of Madras, Chennai, India_.

Background: E-cigarettes (ECs), also known as electronic nicotine delivery systems (ENDS) are battery-operated devices that deliver nicotine through inhaled aerosols. The health risks associated with EC use are unclear, but ECs have been promoted as a safer alternative to tobacco smoking and a smoking cessation aid. Currently, more than 13% of American high school students and 10% adults report using ECs. EC aerosols contain unique constituents (e.g. silicate beads, tin and flavorants), as well as other toxicants also present in tobacco smoke, including carcinogens and reactive oxygen species (ROS). The potential of EC aerosols to induce DNA damage has not been fully characterized.

Aims: (1) To examine the effects of EC aerosol exposure in the levels of cellular reactive oxygen species (ROS); and (2) To evaluate the potential of EC aerosols to cause oxidative DNA damage.

Methods: EC aerosols and mainstream smoke extracts were generated from distinct brands of ECs containing diverse nicotine concentrations and a reference combustible cigarette, in controlled conditions using a modified smoking apparatus. Human normal epithelial and oral cancer cell lines were exposed up to 2 weeks to diverse doses of EC aerosol extracts equivalent to 1 to 100 EC puffs. Levels of ROS were evaluated using the 2',7' -dichlorofluorescin diacetate (DCFDA) cellular ROS detection assay kit (Abcam). Overall DNA damage was quantified using two distinct assays q-PADDA and Comet assay. 8-Oxoguanine, one of the most common DNA lesions resulting from ROS, was quantified using a commercially available ELISA kit. Data were analyzed by Student's t-test.

Results: Exposure to EC aerosols caused a significant increase in ROS levels. Exposure to EC extracts induce significant DNA damage measurable by q-PADDA and Comet assay. Overall, EC aerosol extracts induced significantly less DNA damage than mainstream smoke extracts. Yet, a significant increase in 8-oxoguanine lesions was observed after acute and long-term exposure to EC extracts compared to the control.

Conclusion: Both short- and long-term exposure to EC aerosols can increase cellular ROS levels and cause significant DNA damage. Moreover, EC aerosols cause oxidative DNA lesions which are highly mutagenic, and can lead to tumor initiation and progression. Our study emphasizes the urgent need to further investigate the potential short- and long-term health effects of EC aerosols.

Grant support: This work was supported by the Oklahoma Tobacco Research Center (LQ). Dr. Queimado holds a Presbyterian Health Foundation Endowed Chair in Otorhinolaryngology.

#503

Enhanced repair of bulky DNA adducts induced by a tobacco carcinogen and UV light in human oral epithelial cells by black raspberry extract.

Joseph B. Guttenplan,1 Kun-Ming Chen,2 Yuan-Wan Sun,2 Ross Teicher,3 Wieslawa Kosinska,3 Krishne Gowda,2 Shantu Amin,2 Gary D. Stoner,4 Karam El-Bayoumy2. 1 _New York University Dental and Medical Schools, New York, NY;_ 2 _Penn State College of Medicine, Hershey, PA;_ 3 _New York University Dental School, New York, NY;_ 4 _Medical College of Wisconsin,, Milwaukee, WI_.

We were the first to report that an anthocyanin-enriched black raspberry extract (BE) inhibited mutagenesis, and reduced levels of DNA adducts induced by metabolites of the tobacco carcinogen dibenzo(a,l)pyrene (DBP), in a rat oral fibroblast cell line, by enhancing removal of DNA adducts (Guttenplan, et al., Cancer Prev Res; 9(8) August 2016). Here we extend these findings to the repair of DBP-induced DNA damage in a human oral leukoplakia cell line (MSK leuk1), and a reduction in the toxicity (assayed by dead cell numbers and cell survival) of DBP diolepoxide (DBPDE) in these cells. In addition, we examined the effect of BE on DNA damage and toxicity induced by UV light. Treatment of the MSK cells with DBP, and 2 of its metabolites, DBP-dihydrodiol and DBPDE led to a major adenine adduct, (-)-anti-trans-DB[a,l]PDE-dA (DBPDE-dA). The order of potency was DBPDE>DBP>DBP-dihydrodiol with one µM DBP, producing adduct levels of about 20/10E6 dA. BE in the range of 75-150 µg/ml significantly inhibited adduct formation when cells were pretreated with BE before addition of DBP. However, when cells were first treated with DBP and one day later BE was added, adduct levels were reduced by about 50% two days after treatment with DBP, indicating that the BE was enhancing DNA repair. This conclusion results from the fact that BE was not present during the metabolic activation of DBP to DBPDE, and hence couldn't modulate the activation steps. We also tested whether BE, added 4 hr after treatment with the short-lived DBPDE, inhibited toxicity to MSK cells. Initial toxicity was measured by counting the numbers of dead cells in the medium 24 hr after addition of DBPDE; and the relative levels of surviving cells were determined using an MTT cell viability assay. It was found that 100ug/ml BE reduced toxicity induced by 25 - 200 nM DBPDE by about 50%, with a concomitant increase in survival in the BE-treated cells. As DBP produces bulky DNA adducts we also investigated the effects of BE on toxicity and levels of DNA adducts produced by UV light. Cells were irradiated for 30 - 120 seconds with 254 nm light and 15 minutes later, treated with 50 - 150 µg/ml BE. Toxicity was measured, as above, and a logarithmic decrease in cell death was observed with increasing concentration of BE. Similar to results with DBPDE, BE increased cell survival. The effects of BE on relative levels of the UV-light-induced cyclopyrimidine adducts were also determined, using an ELISA assay. BE at levels of 150 - 300 µg/ml reduced adduct levels by 30 - 60%. These results indicate that BE may provide chemopreventive effects on initiation of carcinogenesis by environmental agents that produce bulky DNA adducts - by enhancing DNA repair, likely via the nucleotide-excision repair pathway. Supported by NIH grant #CA173465.

#504

Quantification of nucleic acid quality in postmortem tissues from a cancer research autopsy program.

Jun Fan,1 Raya Khanin,1 Hitomi Sakamoto,1 Yi Zhong,1 Chelsea Michael,1 Derwin Pena,1 Breanna Javier,1 Laura Wood,2 Christine Iacobuzio-Donahue1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Johns Hopkins Hopital, New York, NY_.

The last decade has seen a marked rise in the use of cancer tissues obtained from research autopsies. Such resources have been invaluable for studying cancer evolution or the mechanisms of therapeutic resistance to targeted therapies. Degradation of biomolecules is a potential challenge to usage of cancer tissues obtained in the post-mortem setting and remains incompletely studied. We analysed the nucleic acid quality in 371 different frozen tissue samples collected from 80 patients who underwent a research autopsy, including eight normal tissue types, primary and metastatic tumors. Our results indicate that RNA integrity number (RIN) of normal tissues decline with the elongation of post-mortem interval (PMI) in a tissue-type specific manner. Unlike normal tissues, the RNA quality of cancer tissues is highly variable with respect to post-mortem interval. The kinetics of DNA damage also has tissue type-specific features. Moreover, while DNA degradation is an indicator of low RNA quality, the converse is not true. Finally, we show that despite RIN values as low as 5.0, robust data can be obtained by RNA sequencing that reliably discriminates expression signatures. Based on this promising pilot data, we are currently identifying samples for multiregion RNA sequencing of pancreatic cancers with divergent morphologies within the primary and/or metastatic sites. Thus far we have screened 554 RNA samples from 10 autopsy cases. These samples represent 198 geographically distinct regions of tumor with duplicate or triplicate RNA extractions. The number of samples per patient ranges from 17 to 86 with an average of 55. Our data shows that high quality RNA can be acquired from cases with long PMI (>48h). While extensive screening of samples is required to identify those with optimal quality, the downstream data produced is of high quality and can support novel hypotheses or validations of experimental data.

#505

**Somatic and inherited riboSnitches in** TPT1 **and** LCP1 **mRNA secondary structures.**

Lela L. Lackey,1 Aaztli Coria,1 Chanin Tolson,1 Evonne McArthur,2 Alain Laederach1. 1 _University of NC, Chapel Hill, Chapel Hill, NC;_ 2 _Vanderbilt University, Nashville, TN_.

RiboSnitches are single nucleotide variants that change the secondary structures of RNA. These structural changes can have a regulatory or functional impact on the RNA. Many inherited single nucleotide variants function as riboSNitches. We wanted to investigate the potential of somatic mutations to act as riboSNitches. We determined the secondary structure of two cancer-associated mRNAs, TPT1 and LCP1, using the chemical probing SHAPE-Map technique. Both of these mRNAs are highly structured and have similar folds in vivo and in vitro. We analyzed the structural impact of all the synonymous somatic mutations identified in the COSMIC cancer sequencing database for both TPT1 and LCP1 mRNAs. In TPT1 we also tested several inherited variants. We found that both somatic and inherited variants can be riboSNitches, at approximately the same ratio. Our results are in accord with previous work indicating that ~15% of SNVs are capable of acting as riboSNitches. Although the impact of non-coding and synonymous somatic mutations are difficult to measure, it is becoming clear that these mutations can contribute to cancer progression and phenotype. The majority of our identified riboSNitches are detrimental according to FATHMM-MKL functional impact prediction. Our experiments indicate that a substantial fraction of somatic mutations can cause a measurable change to RNA structure, potentially changing the regulatory or functional behavior of that mRNA.

#506

The role of Nek2 in hepatocellular carcinoma.

Chan Shing Ho Vincent. _The University of Hong Kong, Hong Kong, Hong Kong_.

Cancer is a disease of aging. Among different types of cancer, liver cancer is one of the most prevalent cancers over the world and particularly affects people in some East Asia countries such as Korea, Japan and China. The NIMA-related kinase 2 (Nek2), which regulates centrosome cohesion, is a potential player in carcinogenesis. Our evidence showed Nek2 mRNA and protein levels are overexpressed in liver cancer cells and promotes HCC cell growth. Since Nek2 is pertinent to centrosome, we speculated that it might take part in centrosome regulation. To study the effect of Nek2 in chromosomal instability, centrosome splitting and cell cycle, a stable isoform Nek2A overexpression inducible system was generated in different Hepatocellular Carcinoma cell lines for investigations through immunofluorescent staining and cell-cytometry. For the study of centrosome, we observed that higher proportion of cells with centrosome splitting occurred in the Nek2A overexpressing cells in both HepG2 and SMMC-7721 inducible cell lines. Moreover, cell cycle analysis showed an increased content of DNA when Nek2A is overexpressed, suggesting that Nek2A induces more aneuploidy cells. To conclude, overexpression of Nek2 exacerbates chromosomal instability. Dysregulation of centrosome may be the key for understanding of regulatory mechanism of chromosomal instability by Nek2.

#507

A signature of stress-induced mutagenesis in cancer.

Kimberly J. Bussey, Luis Cisneros. _NantOmics, LLC, Tempe, AZ_.

The action of the AID/APOBEC family of cytosine deaminases contributes to mutational clustering but fails to explain 50% of the clusters observed in cancer genomes. Stress-induced mutagenesis in bacteria occurs when double-strand breaks (DSB) happen in DNA in the context of additional cellular stress sufficient to initiate the SOS response. This results in mutational clustering driven by DinB where mutational abundance decays as a function of the distance from the DSB, but remains above background rates of mutagenesis up to 1 MB away. In humans, the orthologous genes to DinB have become specialized for translesion synthesis (TLS). The dysregulation of cell cycle and DNA repair that characterizes most tumors would logically increase the need for TLS in cancer. Therefore, we hypothesized that stress-induced mutagenesis in cancer would result in peaked clusters of SNVs driven by TLS.

We used data from 764 cases with somatic mutation and structural variant calls from WGS from the ICGC database, release 19, and private de novo mutations derived from 130 trios in the 1000 Genomes project with WGS. SNV context was determined based both on the reference and the mutant allele, and called when only one mechanism (TLS, APOBEC, or AID) could be assigned, thereby underestimating the actual number of TLS events. We observed that as the total number of SNVs increases so does the number of clusters, the proportion of SNVs in clusters, and the average size of clusters in both de novo private mutations and somatic mutations in cancer. In normal, a median of 15.2% of the SNVs occurring in clusters occur in a sequence context indicative of TLS, compared to 0.53% and 1.6% for APOBEC and AID, respectively. In contrast, in cancer, TLS accounts for a median of 30.6% of SNVs in clusters. APOBEC and AID account for 2.3% and 2.6%, respectively. We devised a measure of cluster shape based on empirical cumulative distributions, termed the Stress-Introduced Heterogeneity (SItH) score. It ranges from -1 to 1 and quantifies how sharply peaked clusters are with more positive numbers indicative of a faster decay in mutational load over distance relative to the putative DSB. In normal, SItH scores range from 0.457 to 0.578 with a median of 0.504. In cancer, SItH scores ranged from 0.17 to 0.999 with a median of 0.46 and vary significantly by organ site (ANOVA, F=44.96, p<2.2x10-16). We assessed the contribution of TLS, APOBEC, and AID to SItH score using an additive, linear model of SItH scores derived solely from SNVs in that specific context and including organ site as a variable. We found that in both normal and cancer that TLS was the only context that contributed significantly to SITH score. Therefore, we conclude that the clustering seen in cancer is being driven primarily by TLS and these clusters have a shape characteristic of a process of stress-induced mutagenesis.

#508

DNA repair status in a patient derived ovarian cancer xenobank.

Federica Guffanti,1 Maddalena Fratelli,1 Monica Ganzinelli,1 Francesca Ricci,1 Roberta Affatato,1 Maria Rosa Cappelletti,2 Daniele Generali,2 Francesca Bizzaro,1 Massimo Broggini,1 Raffaella Giavazzi,1 Giovanna Damia1. 1 _IRCCS - Institute for Pharmacological Research Mario Negri, Milan, Italy;_ 2 _Azienda Socio-Sanitaria Territoriale di Cremona, Cremona, Italy_.

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy with a 5-year relative survival rate of 45%. The high mortality rate is in part due to the development of platinum chemoresistance occurring in more than 70% of patients after the first-line therapy. DNA repair capacity has been reported to be a key determinant for the cellular response to platinum agents. Since half of the high grade serous EOCs lacks Homologous Recombination repair, we aimed to profile the DNA repair status in a panel of well characterized 42 ovarian patient derived xenografts (PDXs) recently established in our laboratory and to correlate it with the in vivo response to a platinum based therapy. We evaluated by real time PCR (ABI-7900, Applied Biosystems) the mRNA levels of genes with a key role in Base Excision Repair (OGG1, POLB and PARP1), Homologous Recombination (BRCA1, PALB2, TP53BP1 and RAD51), Nucleotide Excision Repair (ERCC1, XPA, XPF, XPD and XPG), Fanconi Anemia pathway (FANCA, FANCC, FANCD2 and FANCF), Translesion Repair (POLEta), Mismatch Repair (MLH1), Microhomology End Joining (POLQ), Non Homologous End Joining (XRCC4, XRCC5, XRCC6 and XRCC7) and CDK12, a kinase regulating the transcription of some DNA repair genes. The methylation status of BRCA1, ERCC1, MLH1, XPA, XPG and FANCF was investigated by standard techniques. Our results show that the DNA repair genes considered were variably expressed in all the 42 PDXs analyzed, with no specific histotype-specific cluster of expression. The expression of PALB2, FANCC, FANCD2, OGG1, POLQ and RAD51 was found to correlate with the expression of at least six other genes. In high grade serous/endometrioid PDXs, the CDK12 mRNA expression levels positively correlated with the expression of TP53BP1, PALB2, XPF and POLB. BRCA1 was found to be hypermethylated in 51% of the xenografts. TP53 mutated PDXs showed statistically significant higher levels of POLQ, FANCD2, RAD51, and POLB genes. The expression of CDK12 [p=0.017], PALB2 [p=0.019] and XPF [p= 0.016] was negatively associated with the in vivo response to DDP, with resistant PDXs showing higher mRNA levels than responsive ones. We looked for association with overall survival in the TCGA data set and we found that high levels of CDK12 were associated with a worse overall survival in patients with a residual tumor after surgery minus than 2cm. These data suggest that some DNA repair genes can have a role in EOC patients' response to DDP therapy. Particularly, CDK12 was significantly able to predict worse survival in patients undergoing optimal debulking surgery. Our xenobank will be a valid instrument to set up functional DNA repair assays, as suggested by preliminary data on primary cultures.

#509

**Genomic profiling of acute lymphoblastic leukemia in ataxia telangiectasia patients reveals tight link between** ATM **mutations and chromothripsis.**

Manasi Ratnaparkhe,1 Mario Hlevnjak,1 Thorsten Kolb,1 Anna Jauch,2 Kendra Maass,1 Frauke Devens,1 Agata Rode,1 Volker Hovestadt,1 Andrey Korshunov,1 Agata Pastorczak,3 Wojciech Mlynarski,3 Stephanie Sungalee,4 Jan Korbel,4 Jessica Hoell,5 Ute Fischer,5 Till Milde,1 Christof Kramm,6 Michaela Nathrath,7 Krystyna Chrzanowska,8 Eugen Tausch,9 Masatoshi Takagi,10 Takashi Taga,11 Shlomi Constantini,12 Jan Loeffen,13 Jules Meijerink,14 Stefan Zielen,15 Gudrun Goehring,16 Brigitte Schlegelberger,16 Eberhard Maass,17 Reiner Siebert,18 Joachim Kunz,2 Andreas Kulozik,2 Barbara Worst,1 David Jones,1 Stefan Pfister,1 Marc Zapatka,1 Peter Lichter,1 Aurelie Ernst1. 1 _German Cancer Research Center, Heidelberg, Germany;_ 2 _University Heidelberg, Heidelberg, Germany;_ 3 _Medical University of Lodz, Lodz, Poland;_ 4 _EMBL, Heidelberg, Germany;_ 5 _Heinrich Heine University, Duesseldorf, Germany;_ 6 _University of Halle, Halle, Germany;_ 7 _Technical University Munich, Munich, Germany;_ 8 _The Children's Memorial Health Institute, Warsaw, Poland;_ 9 _University of Ulm, Ulm, Germany;_ 10 _Graduate School of Medical and Dental Sciences, Tokyo, Japan;_ 11 _Shiga University of Medical Science, Japan;_ 12 _Tel-Aviv Medical Center, Israel;_ 13 _Sophia Children's Hospital, Rotterdam, Netherlands;_ 14 _Princess Máxima Center for Pediatric Oncology, Netherlands;_ 15 _Goethe-University, Frankfurt, Germany;_ 16 _Hannover Medical School, Germany;_ 17 _Children's Hospital, Klinikum Stuttgart, Germany;_ 18 _University Hospital Schleswig-Holstein Campus Kiel/Christian-Albrechts University Kiel, Germany_.

Recent developments in sequencing technologies lead to the discovery of a novel form of genome instability, termed chromothripsis. This catastrophic genomic event, involved in cancer formation, is characterized by tens to hundreds of locally clustered rearrangements on one chromosome, acquired simultaneously. We hypothesized that leukemias developing in individuals with Ataxia Telangiectasia, who are born with two mutated copies of the ATM gene, essential guardian of genome stability, would show a higher prevalence for chromothripsis due to the defect in DNA double-strand break repair. Using whole-genome sequencing, fluorescence in situ hybridization and RNA sequencing, we characterized the genomic landscape of Acute Lymphoblastic Leukemia (ALL) in patients with Ataxia Telangiectasia. We detected a high frequency of chromothriptic events in these tumors, specifically on acrocentric chromosomes, as compared to tumors from individuals with other types of DNA repair syndromes (27 cases in total, of which 10 with Ataxia Telangiectasia). Our data show that the genomic landscape of Ataxia Telangiectasia ALL is clearly distinct from that of sporadic ALL. Mechanistically, short telomeres and compromised DNA damage response in cells of Ataxia Telangiectasia patients are linked with frequent chromotripsis. Additionally, we show that ATM loss is associated with increased chromothripsis prevalence in further tumor entities.

#510

Topoisomerase IIβ silencing increases R loops at specific genomic loci associated with an increase of γH2AX and cell cycle progression delay in human cancer cells.

Maria Delcuratolo, Jessica Marinello, Giovanni Capranico. _University of Bologna, Bologna, Italy_.

DNA topoisomerases (Top) are important players in maintaining the genome stability of cells by removing negative and positive DNA supercoils during transcription, replication and other DNA transactions. Recent evidence demonstrated that Top 1 silencing and poisoning by camptothecin affect the formation of R-loops, which are RNA/DNA hybrid structures involved in genome instability and are favored by negative supercoils of the DNA. The findings overall demonstrate that Top 1 reduces R loop levels during transcription whereas it favors R loop formation at early origins of DNA replication. Here, we address the question of whether DNA topoisomerase IIβ can also contribute to steady-state levels of R loop structures in the genome of U2OS cancer cells. We have then investigated the effects of Top2β silencing and Top2 poisons, doxorubicin and etoposide, on R loops and DNA damage in human U2OS cancer cells. Similarly to published data with camptothecin (Marinello et al., Nucleic Acids Research, 2013), Top2 poisons increase cellular R loops by IF after short treatment times and reduce them after 1 hour of treatment. The bi-phasic effect of poisons is mainly dependent on transcription. Moreover, we have determined the changes of R-loop levels by IF after Top2β silencing and the findings demonstrate that the enzyme can strongly modulate the formation of R loops as a full Top2β depletion increases nuclear R-loop levels. In addition, Top2β depletion leads to a slight increase of phosphorylation of H2AX histone along with cell cycle delay and eventually cell death. Thus, Top2β depletion can trigger genomic DNA breakage through alterations of R-loops. In order to establish which are the genomic regions of altered R loop levels, we have mapped Top2β-dependent R loop alterations by the DRIP method showing that specific genomic sites are affected by Top2β. Bioinformatic analyses of R loop maps in U2OS cells will be presented and discussed at the meeting. Altogether the findings demonstrate a critical role of Top2β in governing R loop structures in human cancer cells indicating that DNA torsional tension is a main driving factor of R loop formation and hence genome instability in cancer cells.

#511

Chromosome instability mechanisms in breast cancer among African Americans and Caucasians.

Jose Thaiparambil, Oula Mansour, Susan Haley, Randa EL-Zein. _Houston Methodist Research Institute, Houston, TX_.

Significant disparities exist in breast cancer among African Americans (AAs) women compared to Caucasians. Even though AAs are less likely to undergo regular mammograms due to access barriers, it is unlikely that differences in screen based detection accounts entirely for outcome disparities. While the current guidelines recommend women begin mammography at age 40 or 50, AA women may be at increased risk for delayed diagnosis since >10% of breast cancer cases in AAs are diagnosed in women younger <40 years compared with 5% of Caucasians. This highlights the importance of defining optimal ethnically appropriate age of screening especially given the disproportionate mortality among younger black women patients. Therefore, an alternative approach using biomarkers should be considered in order to develop ethnically appropriate measures for detecting the disease in the earliest stages. Genomic instability has long been recognized as a major driver of carcinogenesis occurring early on and increasing in complexity with disease progression. However, to date little is known about the underlying mechanisms associated with such instability among the different ethnic groups. In this study we hypothesized that alterations in telomere length and dysfunctional centrosome apparatus are underlying mechanisms associated with ethnic-based genomic instability. We investigated alterations in centrosome apparatus, misaligned chromosomes and spindle angle orientation in AA [HCC 70] and Caucasian [HCC1395] cell lines using confocal microscopy. Our results demonstrated significant differences in structural (30%) and numerical alterations (60%) in centrosome apparatus between AA and Caucasian cell lines (p<0.001). We also noticed a significant increase of misaligned chromosomes (35%) and (68 %) in the metaphase of Caucasians and AA's respectively (p<0.002). In addition, a significant difference in spindle orientation was observed among AAs and Caucasians suggesting that mitotic phase abnormalities may be, in part, responsible for the underlying genetic instabilities associated with breast cancer. Using ingenuity pathway analysis, we compared gene expressions in both AAs and Caucasian cell lines and observed a significant change in telomerase signaling expression between the cell lines, suggesting a role for telomere lengths in the generation of genetic instability. Together, identifying the specific mechanisms that drive tumorigenesis may provide opportunities for future targeted prevention efforts among high risk cohorts. (This work is supported in part by CA189240)

#512

Knockdown of the spindle assembly checkpoint gene Bub1b results in increased cell death and cell cycle impairment in breast cancer.

Dilara Koyuncu, Erik T. Goka, Philip C. Miller, Marc E. Lippman. _University of Miami, Miami, FL_.

Genomic instability is an enabling hallmark of cancer that provides cancer cells a replicative advantage. Accumulation of genomic aberrations can also compromise the genomic integrity and put cells under mitotic stress. Maintaining balance between the instability that gives cancer cells a replicative advantage and the instability that could lead them to mitotic catastrophe is crucial for survival of cancer cells. Mitotic catastrophe can be caused by rapid progression through mitosis before crucial checkpoints are met, resulting in cell death. To overcome this challenge, cancer cells may acquire overexpression of spindle assembly checkpoint (SAC) genes, which can prevent mitotic catastrophe that would occur if they undergo mitosis prematurely. Bub1b is an important part of SAC and inhibits the onset of anaphase until all chromosomes are aligned at the metaphase plate. Our analysis of clinical datasets show Bub1b expression is elevated in breast cancer, compared to normal breast, and is exemplified by a pattern of increasing overexpression in more aggressive variants, such as triple negative breast cancer (TNBC). Bub1b overexpression also correlates with decreased overall survival in patients. Expression analysis of breast cancer cell lines corroborates this clinical data. We hypothesize that the requirement for Bub1b expression indicates a vulnerability of rapidly proliferating breast cancers cells, and the inhibition of Bub1b will result in cell death through mitotic catastrophe.

Using RNA interference with siRNAs we knocked down Bub1b expression in a variety of breast cancer cells. This resulted in significant decrease in cellular viability and clonogenicity in soft agar. Furthermore, analyses of apoptosis using Annexin V and PI costaining showed that Bub1b knockdown results in significantly increased apoptosis and cell death, especially in the TNBC cell line MDA-MB-468. However, the viability and apoptosis levels of the normal breast epithelial cell line, MCF12A, were not affected. Bub1b knockdown resulted in silencing of SAC activity, as measured by Cyclin B levels, and also significantly impaired cell cycle progression in breast cancer cell lines. Additionally, knockdown of Bub1b in breast cancer cell lines demonstrated an additive effect when combined with DNA-damaging agents.

Our data support that Bub1b is a critical player in breast cancer cell survival, by enabling them to overcome mitotic stress. We demonstrate that impairment of Bub1b has detrimental effects on cell cycle and progression of mitosis. Further investigation of the role of Bub1b in promoting successful chromosome alignment and proliferation of breast cancer cells with genomic instability could provide a novel pathway on how cancer overcomes the mitotic stress caused by genomic instability. These studies may also lead to novel therapeutic strategies that combine SAC inhibitors with standard genotoxic treatments.

#513

The link between polyploidy and replication stress in melanoma.

Anna E. Vilgelm,1 C. Andrew Johnson,1 Kiran Malikayil,2 Dayanidhi Raman,3 David Flaherty,4 Brian Higgins,5 Ann Richmond6. 1 _Vanderbilt University, Nashville, TN;_ 2 _Meharry Medical College, Nashville, TN;_ 3 _University of Toledo, Toledo, OH;_ 4 _Vanderbilt University Medical Center, Nashville, TN;_ 5 _Roche Innovation Center, New York, NY;_ 6 _Tennessee Valley Healthcare System, Nashville, TN_.

Genomic instability is a hallmark of cancer implicated in tumor evolution and resistance to therapy. However, the molecular mechanisms that underlie genomic instability are still poorly understood. One common feature of cancer cells is an elevated genomic content (polyploidy) which is also associated with the acquisition of therapy resistance. Here we demonstrate a link between genomic instability and polyploidization in melanoma cells. We show that polyploidy induced by treatment with small molecule inhibitors of various mitotic kinases, such as AURKA, AURKB and PLK1, leads to DNA damage in cells with elevated (>4n) DNA content. This DNA damage results from replication stress exacerbated by limited activation of p53 in malignant melanocytes. Pharmacological induction of p53 in polyploid cells using an MDM2 antagonist reduced DNA damage by blocking re-replication of the polyploid genome as a result of p53-mediated p21 activation and transactivation of DNA repair genes. Notably we found that p21 blockade using siRNA knockdown or genetic knockout shifted polypoid cell response to p53 activation from cytostatic to cytotoxic. As a result, p21-deficient cells exhibited enhanced sensitivity to mitotic blockade combined with p53 induction. Furthermore, TCGA dataset analysis showed poor progression free survival in melanoma patients with high p21 protein expression. These data argue for administering mitotic inhibitors and MDM2 antagonists, which are currently in clinical development, in conjunction with agents that target p21. In summary, our data here reveal that polyploidization can be a mechanism for induction of DNA damage and genomic instability associated with drug resistance in cancer and suggest a novel strategy for targeting these pathways to improve melanoma therapy.

#514

Genetic load in cancer cell populations.

Yuezheng Zhang,1 Xu Shen,2 Yawei Li,2 Tianqi Zhu,2 Yong Tao,2 Tao Li,2 Di Wang,1 Xueying Li,3 Qin Ma,2 Xuemei Lu,2 Hurng-Yi Wang,4 Chung-I Wu2. 1 _University of Washington, Seattle, WA;_ 2 _Beijing Institute of Genomics, Beijing, China;_ 3 _Peking University, Beijing, China;_ 4 _Institute of Clinical Medicine, National Taiwan University, Tai Pei, Taiwan_.

Populations accumulate deleterious mutations that have yet to be removed by natural selection. This mutated portion of the population that does not contribute to its fitness is called the genetic load and is proportional to the deleterious mutation rate. Given the genomic instability of cancer cells, the genetic load could be substantial if the instability indeed leads to loss of cell fitness. We hypothesize that because aneuploidy and copy number variations (CNVs) are the most common forms of genome instability, their fitness consequences may determine the genetic load in cancer cells. To test this hypothesis, we randomly selected single cells from a HeLa cell line and measured the cell growth rate via daily cell counting and an MTT cell proliferation assay. We then measured the genetic changes and resulting growth rate variation. We observed rapid generation of heterogeneity in the growth rate within the population. The growth rate of different progeny cells was stable indicating that it is genetically determined. Next, using whole genome sequencing of recently descended clones of a single HeLa cell, we found that cells with fewer newly acquired CNVs have higher proliferation rates vice versa. This indicated that CNV accumulation decreased cell fitness systematically. We next estimated the rate of CNV mutation by measuring the growth rate of two daughter cells from single proliferation and found that there is approximately 1 deleterious mutation in every 4 cell divisions. We speculated that due to a high, deleterious CNV mutation rate, tumor cells inevitably accumulate deleterious CNVs and a large percentage of tumor cells are genetically defective. Accordingly, we observed that the average growth rate of tumor cell populations decreased in the short term since defective cells accumulated in the population and the variation of cellular growth rates within the population increased. By modeling the process of mutation accumulation and measuring cell growth rate, we estimated that the deleterious mutation rate in HeLa cells is about 0.26-0.31 per cell division, and that HeLa cells reduce roughly 5%-6% of fitness for every cell division. The observations of a high proliferation rate and high genetic load in this representative tumor cell line indicates a "high risk, high reward" evolution strategy for tumor cells and suggests that increasing the level of genomic instability may cause the meltdown of tumor cell population by forcing cells to accelerate the cell cycle. 

### Oncogenic Growth Factors and Signal Transducers

#515

Alterations in phosphatidylinositol 3-phosphate (PI3P) pathway and cAMP pathway confirm poor prognosis and reduced overall survival (OS) in a series of 209 acute myeloid leukemia patients.

Mariachiara Abbenante,1 Mariachiara Fontana,2 Giovanni Marconi,1 Giorgia Simonetti,1 Antonella Padella,1 Elena Tenti,1 Eugenia Franchini,2 Anna Ferrari,2 Sarah Parisi,2 Emanuela Ottaviani,2 Nicoletta Testoni,2 Viviana Guadagnuolo,3 Chiara Sartor,2 Silvia Lo Monaco,2 Cristina Papayannidis,1 Giovanni Martinelli2. 1 _Istituto L. e A. Seragnoli, bologna, Italy;_ 2 _Istituto L. e A. Seragnoli, Bologna, Italy;_ 3 _Cellply, Italy_.

Introduction PI3P is a molecule that regulate cell growth and mediates cell proliferation via PI3K/AKT/mTOR in response to various growth signals. Abnormal activation of genes in its pathway is associated to oncogenic activity and poor Overall Survival (OS). AMPK plays a role as a regulator of cellular energy homeostasis. Aims The aim of the this study is to define the role of PI3P pathways and AMPK pathway in AML.

Methods In this work we analyzed 208 consecutive newly diagnosed non M3 AML patients, screened for TP53, FLT3, NMP1, IDH1, IDH2, and DNMT3A mutations. Remission status was assessed with bone marrow biopsy. We performed Microarray-based Comparative Genomic Hybridization with Affymetrix SNP array 6.0 or Cytoscan HD in all the patients; we performed Whole Exome Sequencing (WES)in 80/208 patients. Survival data were collected prospectively, with a median follow-up of 18 months. Survival analysis was performed with Kaplan Meyer method using log rank test. Univariate and multivariable regression and Cox Hazard Ratio(HR) model was performed. Correlation between variables was assessed with Fisher's exact test.

Results We selected genes in pathways basing on literature and GO data. Alterations in these pathways involved 103/209 patients (48%). We analyzed the gene in two different pathways. PI3K/AKT/mTOR pathway includes the following genes: pik3ca, cdkn1a, akt1, akt3, mtor and pten, pdk1,pik3r1 and irs1. The second one is AMPK pathway and it include: sesn, prkaa1, prkab1, prkag1, prkag3. Alterations in PI3K/AKT/mTOR pathway confer worst OS (p = .035) when compared with unaltered patient, but events in these pathways did not affect therapy response. Alterations in AMPK pathway confer worst OS (p<.001); Alteration of regulators in cAMP were related to lower CR% after induction in univariate (p<.001) and multivariable analysis with age, karyotype, secondary AML, TP53 mutation (p=0.009). AMPc pathway alteration was significantly associated with complex karyotype and TP53 mutation (p<.001). WES in a sub-cohort of patients did not found any significant mutation in genes we analyzed, according to literature.

Conclusions Our work investigates the role of PI3P and cAMP pathways in AML. Surprisingly, it showed that alterations in these pathways are associated with poor prognosis. Significantly, alterations in cAMP pathways were associated with therapy resistance. Acknowledgement: ELN, AIL, AIRC, PRIN, Progetto Regione-Università 2010-12, FP7 NGS-PTL project, HARMONY

#516

**High-throughput functional evaluation of variants of unknown significance in** EGFR **.**

Shinji Kohsaka,1 Masaaki Nagano,1 Toshihide Ueno,1 Yoshiyuki Suehara,2 Takuo Hayashi,2 Naoko Shimada,2 Kazuhisa Takahashi,2 Kenji Suzuki,2 Kazuya Takamochi,2 Fumiyuki Takahashi,2 Hiroyuki Mano1. 1 _Graduate School of Medicine, The University of Tokyo, Tokyo, Japan;_ 2 _Juntendo University Graduate School of Medicine, Tokyo, Japan_.

Since transforming mutations in epidermal growth factor receptor gene (EGFR) were first identified in non-small-cell lung carcinoma (NSCLC), advancement of diagnostics for such mutations, and evolution of targeted therapeutics against EGFR has driven unprecedented improvements in the management as well as outcome of patients with this lethal disease. However, next-generation sequencer (NGS)-driven extensive analyses on NSCLC have revealed a large number of variants of uncertain significance (VUS) in EGFR and other regions in the cancer genome that await further investigation. Here we present a mixed all nominated mutants in one (MANO) method to evaluate the transforming potential and drug sensitivity of VUS of oncogenes, and applied this method to 101 non-synonymous EGFR mutants in a high-throughput manner. The sensitivity of individual mutants to tyrosine kinase inhibitors (TKIs) against EGFR was shown diverged, ranging from relatively insensitive mutations such as missense mutations within exon 19 to a highly resistant mutation within exon 21. Our data thus support the importance of examining uncommon mutations within EGFR, and also of functional evaluation of such mutations. Our MANO method may become a novel foundation for in vitro and in vivo assessments of variants of cancer-related genes to deliver precision medicine to individual cancer patients.

#518

Synergistic effects of dual inhibition of YAP1 and TAZ in non-small cell lung cancer cells.

Elaine Shum,1 Huijie Liu,2 Liang Zhu,2 Roman Perez-Soler,1 Balazs Halmos,1 Haiying Cheng1. 1 _Montefiore Medical Center/Albert Einstein College of Medicine, Bronx, NY;_ 2 _Albert Einstein College of Medicine, Bronx, NY_.

Background: The Hippo signaling pathway is associated with regulation of cell growth and apoptosis. Downstream effectors of this pathway, YAP and its paralog, TAZ, interact with TEA domain (TEAD) transcription factors leading to cell proliferation and anti-apoptosis. As a result, YAP and TAZ have emerged as attractive anti-cancer therapeutic targets. Several studies, including our previous report, have noted the individual effects of YAP or TAZ ablation; however, consequences of dual inhibition have not yet been investigated in lung cancer. We hypothesize that dual inhibition of YAP and TAZ will lead to more pronounced cytotoxicity than each knockdown alone as a result of diminished compensatory mechanism by the remaining family member.

Methods: NSCLC cell lines, such as PC9 and A549, were transfected with individual YAP1, TAZ siRNA or a combination of both siRNAs. Western blotting was performed to determine the levels of YAP1 and TAZ proteins following knockdown. Quantitative RT-PCR was also performed to confirm effective YAP1 and TAZ knockdown. In addition, gene and protein expression of downstream targets, such as CTGF and Cyr61, were assessed. Clonogenic assays were performed to determine cell survival.

Results: Successful siRNA-mediated YAP and TAZ ablation were confirmed with Western blot and qRT-PCR. The expression of the Hippo-YAP/TAZ downstream targets, CTGF, Cyr61 and AXL, were also reduced accordingly. Moreover, dual knockdown of YAP and TAZ led to a more pronounced inhibition of cell survival/proliferation than ablation of individual genes alone. In addition, dual inhibition of YAP/TAZ exhibited significantly diminished levels of the mesenchymal markers, vimentin and N-cadherin, and increased levels of the epithelial marker, E-cadherin. EMT has been recognized as an essential process during lung cancer tumor migration and metastasis.

Conclusion: Our findings show that dual inhibition of YAP1 and TAZ are synergistic in blocking NSCLC cell survival signaling. Thus, dual inhibition may serve as a better therapeutic strategy in targeting the Hippo pathway than sole YAP1 blockade in the management of NSCLC.

#519

Antitumor activity of MET antibody emibetuzumab (LY2875358) in combination with EGFR inhibitors in erlotinib resistant (ER) xenograft mouse models.

Suzane L. Um,1 Victoria L. Peek,1 Jennifer R. Stephens,1 Jessica A. Baker,1 Holly K. Cannon,1 Joel D. Cook,1 Isabella H. Wulur,1 Roger Agyei,2 Sudhakar Chintharlapalli,1 Robert J. Evans,1 William J. Feaver,2 Lysiane Huber,1 Linda N. Lee,1 Ling Liu,1 Liandong Ma,1 Ruslan Novosiadly,2 Volker Wacheck,3 Sau-Chi Betty Yan1. 1 _Eli Lilly and Company, Indianapolis, IN;_ 2 _Eli Lilly and Company, New York, NY;_ 3 _Eli Lilly and Company, Vienna, Austria_.

Background: MET amplification (amp) is a resistance mechanism to EGFR TKI treatment. Emibetuzumab, a bivalent MET antibody (Ab) blocks HGF binding to MET and internalizes the receptor. Combination of emibetuzumab with EGFR TKIs (erlotinib, AZD9291, CO1686) or EGFR Ab (necitumumab, cetuximab) was evaluated in 3 ER xenograft models.

Methods: Model 1: ER cell line HCC827ERL with high focal MET amp, high pMET, EGFR ex19 del (no T790M) was created from parental HCC827 NSCLC (EGFR ex19 del, EGFR amp, no MET amp) by increasing concentration of erlotinib in vitro over 7 months. Model 2: ER cell line HCC827-A8 was derived from HCC827 parental xenograft tumor serially passed in vivo with long term treatment of gefitinib and erlotinib. HCC827-A8 cells express high focal MET amp, high pMET/AXL (Western blot) while retaining EGFR ex19 del (no T790M). Model 3: LU0858 was an ER patient-derived NSCLC xenograft tumor, with focal MET amp, EGFR L858R (no T790M). MET amp and EGFRmt was determined by FISH and LNA-PCR sequencing respectively. Compound dosing: emibetuzumab 20 mg/kg qw; necitumumab 4 mg/kg or 20 mg/kg biw; cetuximab 4 mg/kg biw; erlotinib 25 mg/kg qd; 5 mg/kg AZD9291 qd; 30 mg/kg CO1686 bid.

Results: EGFR inhibitors, but not emibetuzumab showed significant single agent anti-tumor effect in xenograft tumors derived from non-MET amp HCC827 parental cells. In MET amp ER models, single agent emibetuzumab resulted in tumor growth inhibition in Model 1 (T/C= 51.7%-61.0%, p<0.05) and 3 (T/C=2.8%, p<0.05)] but no tumor regression, and no anti-tumor effect in Model 2. Where evaluated, EGFR inhibitors showed no anti-tumor effect in the 3 ER models as monotherapy, except necitumumab (20 mg/kg) in Model 1 (T/C = 36.2%, p<0.05). However, combination of emibetuzumab with AZD9291, CO1686, necitumumab (20 mg/kg), or erlotinib resulted in 80.4%, 58.2%, 44.4%, 69.1% tumor regression respectively (p<0.001) in Model 1, while emibetuzumab + cetuximab (4 mg/kg) resulted in tumor stasis (T/C=0.2%, p<0.05). In Model 2, emibetuzumab + AZD9291 resulted in tumor stasis (T/C = 12.9%, p<0.05). In Model 3, emibetuzumab + necitumumab (20 mg/kg) resulted in 80.1% tumor regression (p<0.001).

Conclusion: The three erlotinib resistant models with MET amp and retaining sensitizing EGFRmt (ex19 del or L858R), and no acquired T790M were found resistant to other EGFR inhibitors (Abs and TKIs). Emibetuzumab in combination with either EGFR TKI or Ab showed anti-tumor activity in MET amp ER xenograft models including tumor regression in 2 out of 3 models. The combination of emibetuzumab with erlotinib is being evaluated in NSCLC patients with EGFR activating mutation (NCT01897480).

#520

Trim44 is overexpressed in colorectal cancer with amplification, promoting degradation of SHP-1 and activating JAK/STAT pathway by ubiquitin modification.

Jie Hong, Qian Liang, Haoyan Chen, Jing-Yuan Fang, Hua Xiong. _Shanghai Jiaotong Univ School of Med Renji Hospital, Shanghai, China_.

Tripartite motif 44 (Trim44) was found to be expressed in several cancers, but the exact roles of Trim44 in colorectal cancer (CRC) remain unclear. The objective of this study was to determine the functional and prognostic implications of Trim44 in colorectal cancer. The Trim44 gene is recurrently amplified and upregulated in CRC. Trim44 overexpression and amplification are associated with poor survival in patients with CRC and the prognostic significance was confirmed by independent patient cohorts. High expression of Trim44 associates with activation of the JAK/STAT3 pathway in CRC tissues.

TRIM44 knockdown reduced proliferation, and invasion of colorectal cancer cells, as well as growth and metastasis of xenograft tumors in nude mice; overexpression of TRIM44 had the opposite effects. TRIM44 interacted physically with Shp-1, increasing its ubiquitination and degradation. Increased

levels of TRIM44 in human colorectal tumors correlated with reduced expression of SHP-1 and increased phosphorylation of STAT3. In a word, The putative ubiquitin ligase TRIM44 is up-regulated in human colorectal tumors compared with nontumor tissues. Levels of TRIM44 correlate with tumor progression and patient survival times. TRIM44 interacts with SHP-1, promoting its ubiquitination and degradation, and TRIM44 might promote colorectal malignant transformation via this mechanism.

#521

Dual-targeting of IGF-1R and ErbB3 pathways in Ewing's Sarcoma cellular models with istiratumab (MM-141), a bispecific, tetravalent monoclonal antibody.

Isabel Yannatos, Adam Camblin, Zhenhua Li, Michael Curley, Gege Tan, Chrystal U. Louis, Vasileios Askoxylakis, Greg Finn, Birgit Schoeberl, Rachel Nering. _Merrimack Pharmaceuticals, Cambridge, MA_.

Ewing's sarcoma family tumors (ES) are aggressive tumors that often present as metastatic in bone and soft tissue and predominantly affect adolescents and younger adults. Current treatment for ES includes surgical resection followed by loco-regional radiotherapy and chemotherapy. Survival rates for patients with metastatic disease continue to offer a particularly difficult clinical challenge, with a five-year survival rate of 20-30% for these patients. ES is primarily a genetic disease caused by fusion between the 5' segment of the Ewing sarcoma breakpoint region 1 gene (EWSR1) on chromosome 22 and the 3' portion of Friend leukemia virus integration site 1 (FLI1) on chromosome 11. Fusions of EWSR1 and other ETS transcription factors result in dis-regulated transcription factors which promote malignant progression of ES tumors. Recent studies have shown that most ES cell lines and clinical samples express IGF-1R. Importantly, an activated IGF-1R pathway appears to be a prerequisite for malignant transformation by the EWS-FLI1 translocation, presumably via activation of the PI3K-AKT and MAPK pathways. These findings have led to the preclinical and clinical evaluation of multiple IGF-1R-targeted therapeutics with varying results. Clinical experience with anti-IGF1R targeting therapies has demonstrated striking anticancer activity in minor subsets of patients with ES. Importantly, the paucity of a clinically useful biomarker to select patients continues to hinder IGF-1R drug development in ES. Istiratumab is an investigational, bi-specific, monoclonal antibody that acts as a tetravalent inhibitor of PI3K/AKT/mTOR, a major pro-survival pathway tumor cells use as a resistance mechanism to anticancer therapies. Istiratumab is designed to interfere with this pathway by blocking ligand-induced signaling through the IGF-1R and ErbB3 receptors, based on findings that ErbB3 activation mediates resistance to the IGF-1R blockade. We will present data in multiple ES models demonstrating the importance of both IGF-1R and ErbB3 in this disease as a mechanism of growth and resistance. Furthermore, preclinical xenograft studies demonstrate that the combination of istiratumab with an irinotecan-based chemotherapy regimen offers significant benefit over chemotherapy alone. These studies suggest that clinical evaluation of istiratumab in ES is warranted.

#522

Expression and functional analysis of signal peptidase complex 18 in bladder cancer.

Yoshinori Shigematsu, Yohei Sekino, Naoya Sakamoto, Kazuhiro Sentani, Naohide Oue, Tetsutaro Hayashi, Jun Teishima, Akio Matsubara, Wataru Yasui. _Hiroshima Univ., Hiroshima City Hiroshima, Japan_.

Urothelial carcinoma of the bladder is a common malignancy that causes approximately 150,000 deaths per year worldwide. So far, no molecularly targeted agents have been approved for treatment of the disease. In the present study, we built an in-house oligonucleotide array, on which 394 genes were selected based on our SAGE data and previously reported array data, in order to identify the genes of most relevance to gastric carcinogenesis. Among these genes, we focused on SEC11A, because it is frequently overexpressed in gastric cancer (GC). SEC11A encodes the SPC18 protein, which is one of the subunits of the signal peptidase complex (SPC). Most secretory proteins contain amino terminal- or internal signal peptides that direct their sorting to the endoplasmic reticulum (ER). From the ER, proteins are transported to either the extracellular space or the plasma membrane through the ER-Golgi secretory pathway. The ER signal peptides are then cleaved by the SPC. In GC, SPC18 contributes to progression via EGFR pathway associated with TGF-a secretion. However, the expression and function of SPC18 have not been investigated in bladder cancer (BC). In this study, we analyzed the expression and distribution of SPC18 in human BC. Expression of SPC18 was observed in 44 (54.3%) out of 81 BC cases that was associated with T category (p=0.0002), Grade (p=0.0425), vascular invasion (p=0.0112) and CK5/6 positive/ CK20 negative basal type BC (p=0.0004). The univariate analysis indicated that expression of SPC18 (HR, 3.14; 95% CI, 1.47-7.47; P=0.0027) was associated with overall survival. In the multivariate model, SPC18 expression was an independent prognostic indicator (HR, 3.46; 95% CI, 1.35-10.00; P=0.0087). BC cells KMBC2 transfected with SPC18 expression vector significantly induced the cell growth (p<0.01) and invasion (p<0.01) activity. Western blot showed the overexpression of SPC18 induced the phosphorylation of EGFR, Akt and Erk. Real-time reverse transcription PCR analysis revealed the SPC18 forced expression cells induced mesenchymal character. These results suggest that SPC18 might contribute to the progression of BC.

#523

HGF mediated upregulation of PHLDA2 is associated with apoptosis in gastric cancer.

Sung Ae Koh, Kyung Hee Lee, Jae Ryong Kim, Sang Woon Kim. _Yeungnam University Hospital, Daegu, Republic of Korea_.

Pleckstrin homology-like domain family A member 2 (PHLDA2), a homolog of the mouse TDAG51 gene, was the first apoptosis-related gene shown to be imprinted and expressed from the maternal allele in normal development. It is located within the tumor suppressor region of 11p15, and its expression was reported to be suppressed in several cases of malignant tumors. But, the apoptotic regulatory mechanism and its role is not fully known in gastric cancer. The purpose of the this study is to identify of function of PHLDA2 associated with apoptosis in gastric cancer.

We used cell culture, western blotting, RT-PCR, MTT assays, PHLDA2 knock-down with short hairpin RNA (shRNA). First, we confirmed that the expression level of PHLDA2 was up-regulated by HGF(hepatocyte growth factor). To identify associated pathway of HGF-induced LCN2, the cells were treated with PI3-kinase inhibitor (LY294002) or MEK inhibitor(PD098059) or p38 inhibitor(SB203580) and then analyzed by Western blotting. The HGF-mediated PHLDA2 protein level was only decreased with LY294002. The role for PHLDA2 with was determined by knock down cell of PHLDA2. PHLDA2-sh RNA cells showed a decreased level of P53 and a increased level of pAKT. We also examined to confirm the role of HGF-mediated PHLDA2. HGF-mediated cell proliferation and in vitro invasion was increased in PHLDA2 knock down cell. Also, HGF-mediated cell apoptosis decreased in PHLDA2 knock down cell.

Our study showed that PHLDA2 upregulated HGF is regulated through PI3K/AKT pathway in gastric cancer, and PHLDA2 have a role in cell apoptosis in gastric cancer, which may be a possible target for developing gastric cancer therapy.

#524

**Characterizing the** in vitro **and** in vivo **effects of the PIM kinase inhibitor HS140 in triple-negative human breast** **cancer.**

Michael Cobb,1 Lucas Hunter,2 David Carlson,3 David Darr,2 Timothy Haystead,3 Antonio T. Baines1. 1 _North Carolina Central Univ., Durham, NC;_ 2 _University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 3 _Duke University, Durham, NC_.

Triple-negative breast cancer (TNBC) lacks expression of the estrogen receptor (ER), the progesterone receptor (PR) and the ERBB2 (also known as HER2) receptor, making it the worse subtype of all breast cancers. Currently, there are no molecular targeted therapies for this cancer and chemotherapy is only successful in a limited number of patients. Recent studies suggest that members of the oncogenic PIM kinase family, especially PIM-1, play a significant role in the growth of TNBC. However, there is very limited information on the role other PIM family members have in the growth and development of TNBCs. As an attempt to address this concern, we treated a panel of TNBC cell lines with HS140, a PIM-2 kinase inhibitor developed in the laboratory. Using cytotoxicity assays, we were able to demonstrate a decrease in anchorage-dependent growth of cell lines at different concentrations of HS140. Also, a triple negative breast cancer GEMM (C3TAg) was used for an efficacy study. Mice (FVB/N background) have C(3)SV40 T-antigen resulting in inactivation of p53 and Rb. Nine mice received HS140 treatment (80mg/kg BIW IP) and 14 left untreated for control (non-treatment [NT]). All mice were monitored for weight loss and timespan to tumor development 5 times weekly. Tumor volumes at 21 days were significantly (Mann-Whitney, p=0.0002) reduced in the treated cohort with a mean of 109mm3 (range of 0-5000) compared to untreated 1393mm3 (0-239). We observed no toxicities with body mass stable at the treatment dose of 80mg/kg. Additionally, three mice from untreated and three mice from treated cohorts were chosen for blood sampling pre- and post-treatment via a submandibular bleed; no significant differences were noted in WBC, RBC, PLT, or HGB between untreated and treated cohorts. Overall, these results suggest that other members of the PIM kinase family, including PIM-2, have an important function in the growth and development of TNBC and may serve as a potential molecular target for future therapeutics.

#525

Role of CLCA2 in proliferation of certain growth-factor receptor dependent cancers.

Yufang Yin, Aarushi Sharma, Randolph C. Elble. _Southern Illinois Univ. School of Medicine, Springfield, IL_.

The CLCA2 gene is downregulated in most adenocarcinomas, suggesting it is a tumor suppressor gene. However, we have observed that CLCA2 is upregulated in squamous carcinomas and Her2+ breast cancers. Such cancers also upregulate the calcium-activated chloride channel Ano1 along with EGFR or Her2. Others have reported that Ano1 enhances EGFR signaling and that Ano1 knockdown confers sensitivity to EGFR inhibitors. Both EGFR and Her2 are known to transduce their mitogenic signal in part by causing release of intracellular calcium. We have found that CLCA2 activates Ano1 by upregulating intracellular calcium. Moreover, knockdown of CLCA2 reduced EGFR activation and cell proliferation while it enhanced sensitivity to the tyrosine kinase inhibitor lapatinib. These observations suggest that EGFR/Her2, Ano1, and CLCA2 comprise a feed-forward loop that amplifies growth factor signaling, so that certain cancer cell types maintain expression of all three.

#526

NFκB p65 overexpression promotes bladder cancer migration via FBW7-mediated degradation of RhoGDIα protein.

Yang Li, Junlan Zhu, Jingxia Li, Chuanshu Huang. _NYU Nelson Institute of Environmental medicine, Tuxedo Park, NY_.

Since bladder cancer (BC) is one of the most lethal urological malignant tumors worldwide, understanding the molecular mechanisms that trigger the migration, invasion and metastasis of BC has great significance in reducing the mortality of this disease. RelA/p65, a member of the NF-kappa B transcription factor family, is increasingly recognized as a crucial player in many steps of cancer initiation and progression. In this study, we unexpectedly found that p65 protein expression increased in the bladder carcinogen N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced high invasive bladder tumor tissues, and in human bladder cancer cell lines. We also observed that p65 overexpression promoted bladder cancer cell migration by inhibiting RhoGDIα expression. We further demonstrate that the regulatory effect of p65 on RhoGDIα expression is mediated by its upregulation of FBW7, which specifically interacted with RhoGDIα and promoted RhoGDIα ubiquitination and degradation. Mechanistic studies revealed that p65 stabilizing the E3 ligase FBW7 protein was mediated by its attenuating Pten transcription. Taken together, p65 overexpression results in Pten downregulation, and FBW7 protein degradation, and in turn leading to RhoGDIα protein degration and promotings human BC cell migration. The identification of p65/PTEN/FBW7/RhoGDIα axis provides a significant insight into understanding the nature of BC migration.

#527

Integrated genomic cfDNA/cfRNA profiling for AR-V7 liquid biopsy test in prostate cancer.

Shidong Jia. _Predicine Inc, Irvine, CA_.

The growth and survival of prostate cancer tumors relies primarily on the functioning of the androgen receptor (AR) signaling pathway. Recent studies suggested that the presence of AR-V7, a c-terminal truncated form of AR, in circulating tumor cells (CTCs) of patients with castration-resistant prostate cancer (CRPC) is associated with inherent and/or acquired resistance to enzalutamide and abiraterone, the stand of care androgen deprivation therapies in prostate cancer. A series of data also indicate that AR-Vs (eg, including AR-V7, ARv567es, AR-T878A, AR-F876L etc) may drive resistance in CRPC. Expression of AR-V7 has been shown to correlate with disease progression and shortened survival. Considering that truncated AR splicing variants with C-terminal loss lack a functional ligand-bingding domain (LBD) and are constitutively active, C-terminal AR-directed therapies may not be effective for patients with AR splicing variants. Circulating tumor cell (CTC)-based AR-V7 tests are currently being tested in the clinic. However, nearly half of the CRPC patients do not have enough CTCs for AR-V7 test, raising the request for a complementary, non-CTC platform to detect AR variants as well as other resistance markers in circulation.

Here we report the development, validation and clinical application of PrediSeq-Prostate, a non-invasive next generation sequencing-based diagnostics platform that offers integrated genomic cell-free DNA (cfDNA) and cell-free RNA (cfRNA) profiling of prostate cancer, identifying known (such as AR-V7, AR-V9, AR-V2, etc) and novel AR splicing variants, point mutation, copy number, and translocation (such as TMPRSS2-ERG) using a single tube of blood. Using plasma samples from mCRPC patients that developed resistance to enzalutamide and/or abiraterone, PrediSeq-Prostate NGS test identified AR-V7, AR-V2, AR-V3, AR-V4 and other splicing variants that impact diagnosis and therapeutic selection. We also developed a Bio-Rad digital PCR assay measuring AR-V7 and AR-FL (full length) simultaneously. Collectively, PrediSeq NGS and ddPCR assays offer comprehensive genomic profiling of both cfDNA and cfRNA in all patients with prostate cancer, regardless of their status of CTC enumeration. The successful development and clinical validation of these tests has potential to enable precision medicine in prostate cancer.

#528

Evaluation of single agent merestinib (LY2801653) or emibetuzumab (LY2875358) and the combination in a xenograft tumor model bearing MET exon 14 skipping.

Sau-Chi Betty Yan,1 Suzane L. Um,1 Victoria L. Peek,1 Jennifer R. Stephens,1 Wei Zeng,1 Bruce W. Konicek,1 Ling Liu,1 Volker Wacheck,2 Richard A. Walgren1. 1 _Eli Lilly and Company, Indianapolis, IN;_ 2 _Eli Lilly and Company, Vienna, Austria_.

Background: MET ex14 skipping, present in ~3% of lung cancer, is a strong oncogenic driver which is further evidenced by case reports of patients response to MET TKI treatment. ~15% of tumors in patients that harbor MET ex14 skipping also have MET amplification (amp). Merestinib is a type II MET kinase inhibitor1. Emibetuzumab, a bivalent MET Ab, internalizes MET receptor. Each single agent and the combination were evaluated in the Hs746t gastric cancer line bearing MET ex14 skipping and MET amp. Methods: Each agent was evaluated in vitro for inhibition of Hs746t cell proliferation and pMET levels. In vivo study in Hs746t-derived xenograft mouse model (n=7 mice/ arm, 28 day dosing) initiated when tumors were 150-350mm3: merestinib at 6 mg/kg (suboptimal dose - insufficient target coverage for 24 hrs) or 12 mg/kg (optimal dose) qd orally, emibetuzumab at 10 mg/kg qw by IP. Results: Merestinib inhibited Hs746t cell proliferation with IC50=34 nM and totally eliminated pMET at 65-100 nM. Emibetuzumab slightly inhibited Hs746t cell proliferation (IC50>100 nM), reduced 10-20% cell surface MET, and no effect on pMET expression (at 130-650 nM). In the Hs746t xenograft model, merestinib (12 mg/kg) treatment resulted in 91.8% tumor regression after 21 day dosing, while 6 mg/kg merestinib provided transient tumor regression followed by re-growth while on treatment with T/C=18.3% after 21 day dosing. No tumor re-growth was observed in 6/7 mice in the 12 mg/kg merestinib cohort during the 5 weeks post-treatment. Emibetuzumab treatment provided transient tumor regression (37.7%) after 3 doses, but tumors re-grew while on treatment. Combination of 6 mg/kg merestinib and 10 mg/kg emibetuzumab resulted in 85% tumor regression for the duration of the 28 day dosing period and the treatment was well tolerated. Tumors in animals re-grew upon termination of this combination treatment. Conclusion: Merestinib (12 mg/kg) treatment resulted in durable and complete response in 6/7 mice bearing Hs746t tumors with MET ex14 skipping and MET amp. When used singly, merestinib (6 mg/kg) or emibetuzumab (10 mg/kg) resulted in only transient tumor regression in this model, while the combination resulted in substantial tumor regression while on treatment. This combination treatment was however, not as durable as was observed with single agent 12 mg/kg merestinib. Single agents and the combinations were well tolerated. As a type II MET inhibitor, merestinib may provide a therapeutic option to treatment naïve patients or those who have progressed on type I MET inhibitor treatment, whose tumors have MET exon 14 skipping and/or MET amplification 2,3. Data in this study support a clinical evaluation of merestinib in patients with MET exon 14 skipping (NCT02920996). 1 \- Yan et al. Invest New Drugs 2013;31:833-844 2 \- Ou et al. J Thorac Oncol. 2016; PMID:27666659 3 \- Heist et al. J Thorac Oncol. 2016;11:1242-1245

#529

**The significance of activated PI3K/AKT pathway in** FGFR3-TACC3 **fusion positive cervical cancer.**

Ryo Tamura,1 Kosuke Yoshihara,1 Tetsuya Saito,2 Ryosuke Ishimura,2 Emmanuel Martínez-Ledesma,3 Yutaro Mori,1 Kaoru Yamawaki,1 Kazuaki Suda,1 Tatsuya Ishiguro,1 Yoichi Aoki,4 Seiya Sato,5 Hiroaki Itamochi,5 Masaaki Komatsu,2 Roeland Verhaak,3 Takayuki Enomoto1. 1 _Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan;_ 2 _Department of Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan;_ 3 _Department of Genome Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX;_ 4 _Department of Obstetrics and Gynecology, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan;_ 5 _Department of Obstetrics and Gynecology, Tottori University School of Medicine, Tottori, Japan_.

Uterine cervical cancer is one of the most common cancer in women worldwide, and the prognosis in advanced or recurrent cases remains poor because of no effective molecular target therapies for this disease. The aim of our study is to identify and validate therapeutically targetable gene fusions in uterine cervical cancer, leading to the development of new therapeutic strategies. We have analyzed RNA sequencing data of 253 TCGA cervical cancer samples to search for gene fusions by using PRADA algorithm, and validated recurrent fusions in our Japanese dataset (n = 100) by RT-PCR and sanger sequencing. In TCGA samples, we detected 358 fusion transcripts and extracted 3 kinds of recurrent in-frame fusion transcripts (FGFR3-TACC3, ARL8B-ITPR1 and NXN-ABR). We focused on FGFR3-TACC3 fusion as a candidate of therapeutic kinase fusion and identified FGFR3-TACC3 fusion in 2 of 253 (0.8%) TCGA samples and 2 of 100 (2%) Japanese samples. All of fusion positive samples, which were histologically diagnosed as squamous cell carcinoma, showed high expression of both FGFR3 and TACC3 genes. To evaluate the possibility as therapeutically targetable fusions in uterine cervical cancer, we transfected FGFR3-TACC3, wild type FGFR3 and TACC3 transcripts to normal immortalized cervical keratinocytes derived from ectocervix (Ect1/E6E7) and four uterine cervical cancer cell lines (SiHa, ME180, HeLa, CaSki). Continuous expression of FGFR3-TACC3 fusion transcript led to anchorage-independent growth in Ect1/E6E7 and the dramatically alteration of cell proliferation in all cancer cell lines. On the other hand, no obvious phenotype change was observed in FGFR3 or TACC3 transfected cells. Western blotting analysis demonstrated that MAPK pathway was activated in all the fusion transfected cell lines but PI3K/AKT pathway was activated only in ME180 and CaSki harboring PIK3CA mutation. Fusion transfected cell lines exhibited high sensitivity to fusion specific siRNA and FGFR inhibitor compared to control. Although PI3K/AKT activated cell lines (ME180 and CaSki) showed less effective against FGFR inhibitor compared to PI3K/AKT neutral cell lines (SiHa and HeLa), combined inhibition of FGFR and AKT had a synergic effect in PI3K/AKT activated cell lines. Our findings suggest FGFR3-TACC3 fusion gene is an oncogenic driver event and therapeutic target in a fraction of cervical cancer. When FGFR3-TACC3 fusion positive cervical cancer is treated by FGFR inhibitor, genomic background such as PI3K/AKT status should be considered.

#530

The role of EGFR in c-fos-dependent osteosarcoma formation.

Markus Linder,1 Elisabeth Glitzner,1 Sriram Srivatsa,1 Parastoo Shahrouzi,1 Latifa Bakiri,2 Monika Dumanic,1 Markus Mitterhauser,1 Erwin F. Wagner,2 Maria Sibilia1. 1 _Medical University of Vienna, Vienna, Austria;_ 2 _Spanish National Cancer Research Center (CNIO), Madrid, Spain_.

The Epidermal Growth Factor Receptor (EGFR) is overexpressed or mutated in human carcinomas and glioblastomas, which are tumors of epithelial and glial origin, respectively. Some publications indicated that EGFR overexpression could occur in human osteosarcomas (OS). Using EGFR knockout mice (Egfr-/-), we have recently shown that EGFR plays a role in bone development and osteoblast function, raising the possibility that EGFR is also involved in OS formation. Here we show that Egfrf/f Runx2-Cre mice (EgfrΔOb), lacking the EGFR in osteo-chondroprogenitor cells, develop an increased zone of hypertrophic chondrocytes in long bones and decreased bone formation. When bred to c-fos transgenic mice (H2-c-fosLTR) that develop osteosarcomas with 100% penetrance, EgfrΔOb mice show reduced tumor incidence and burden. At the molecular level, tumors from EgfrΔOb mice exhibit decreased expression levels of c-Fos and Cyclin D1. In vitro experiments in primary bone tumor cells further show that EGFR inhibition leads to reduced proliferation and increased apoptosis. Importantly, Egfr and c-Fos also play an important role in human OS, as co-expression of both proteins in tumor sections correlated with significantly reduced survival in patients suffering from primary OS. Taken together our data suggest an essential role of EGFR signaling during both development and progression of c-Fos-dependent OS and newly identified molecular targets currently under investigation will be presented.

#531

Activity of the selective FGFR 1, 2 and 3 inhibitor INCB054828 in genetically-defined models of triple-negative breast cancer.

Phillip C.C. Liu,1 Brian D. Lehmann,2 Bruce Ruggeri,1 Darlise DiMatteo,1 Johanna M. Schafer,3 Jin Lu,1 Sang Hyun Lee,1 Luping Lin,1 Timothy C. Burn,1 Melody Diamond,1 Alla Volgina,1 Liangxing Wu,1 Gregory Hollis,1 Reid Huber,1 Jennifer A. Pietenpol,2 Peggy Scherle1. 1 _Incyte, Wilmington, DE;_ 2 _Vanderbilt University Medical Center, Nashville, TN;_ 3 _Vanderbilt University, Nashville, TN_.

Activation of the Fibroblast Growth Factor (FGF)-FGF Receptor (FGFR) signaling axis occurs in many human cancers. In preclinical models, cell lines with genetic aberrations in FGF/FGFR genes are preferentially inhibited by compounds that selectively target the FGFR kinase. INCB54828 is a potent, selective, and reversible inhibitor of FGFR1, 2 and 3 that is currently in Phase 2 clinical trials for advanced malignancies characterized by FGF-FGFR alterations. In this study, we investigated the efficacy of INCB054828 in models of triple-negative breast cancer (TNBC).

FGFR1 and FGFR2 are amplified in approximately 4% and 5% of TNBC, respectively, and oncogenic fusion proteins including FGFR3-TACC3 have also been identified in some TNBC specimens. To profile the activity of INCB054828, we screened a panel of diverse TNBC cell lines that are representative of each of the four subtypes of TNBC. Three human TNBC lines MFM223, SUM185 and SUM52PE were highly sensitive to INCB054828 in viability assays. Each of these responsive cell lines has a known alteration in FGFR, whereas TNBC lines lacking any aberrations in FGF/FGFR genes were refractory to growth inhibition. Inhibition of cell viability was associated with suppression of growth promoting pathways including Ras-MAPK. To confirm this association in vivo, four PDX models of TNBC were tested: two chemo-refractory models with FGFR1 amplification (CNV = 4 and 6) and two without any known FGF/FGFR alterations. Both of the models with FGFR1 copy number gain showed a response to INCB054828 as monotherapy with 36 and 78% tumor growth inhibition that was statistically significant vs vehicle control (P<0.05 and p<0.001, respectively). At the maximally efficacious dose of 1 mg/kg daily, neither PDX model lacking FGF/FGFR alteration responded to the treatment. Finally to assess the effect of the microenvironment on drug sensitivity, mouse 4T1 breast cancer cells were orthotopically implanted into the mammary fat pad; under these conditions, 4T1 tumors retained sensitivity to a standard dose of INCB054828. In summary these results demonstrate that the FGFR1/2/3 inhibitor INCB054828 is highly active against models of TNBC with genetic alterations in FGFR genes, and confirms the importance of patient stratification strategies for clinical trials with FGFR targeted therapies.

#532

Identification of unpaired cysteine-mediated gain and loss of function CSF3R extracellular mutations.

Haijiao Zhang,1 Sophie Means,1 Anna R. Schultz,1 Kevin Watanabe-Smith,1 Bruno C. Medeiros,2 Tim Kükenshöner,3 Oliver Hantschel,3 Daniel Bottomly,1 Beth Wilmot,1 Shannon K. McWeeney,1 Jeffrey W. Tyner1. 1 _Oregon Health & Science University Knight Cancer Institute, Portland, OR; _2 _Stanford University School of Medicine, Stanford, CA;_ 3 _School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland_.

Mutations in the CSF3R extracellular domain have been reported so far only in neutropenia patients. These extracellular mutations are loss of function mutations, which interrupt ligand binding. Here we screened CSF3R extracellular domain variants in a variety of hematological malignances and described for the first time the identification of an activating CSF3R extracellular mutation W341C in a leukemia patient. Functional assays revealed that this mutation conferred Ba/F3 cell cytokine-independent growth and induced constitutive JAK-STAT activation.

We further characterized that cysteine substitutions at other amino acid positions (342, 356 and 477) and other amino acid substitutes of W341 (A/G/K/R/S) did not transform cells, indicating that both the amino acid position and cysteine substitution are essential for the transforming capacity. In agreement, increased dimer formation of W341C was observed in the co-immunoprecipitation assay and non-reducing condition immunoblot, which could be abrogated in an immunoblot run under reducing conditions, highly suggesting that dimerization is mediated by the formation of intermolecular disulfide bonds. Surprisingly, W356C demonstrated loss of function properties, however, showed increased dimer formation similar to W341C, indicating that only the increased dimerization is not sufficient for transforming capacity. Computational modeling based on IL6ST showed opposite directions of W341 and W356, which may orientate the cytoplasmic domain towards or away from one another.

Interestingly, a CSF3R cytoplasmic truncation mutation at amino acid W791 was found to be on the same allele as W341C in this patient. The W341C/W791X compound mutation transformed Ba/F3 with faster kinetics comparing to the W341C single mutation. Furthermore, the compound mutation, but not W341C alone demonstrated delayed receptor degradation, indicating enhanced oncogenic potential. Notably, the primary patient sample and the Ba/F3 cells transformed by W341C or the compound mutation were all sensitive to JAK inhibitors. The patient harboring these CSF3R mutations displayed myelodysplastic morphology with BCOR mutation at disease diagnosis, and showed good response to Azacytidine treatment. However her white blood count (mature neutrophils in particular) increased after 15-18 months' treatment, which was concomitant with the acquisition and expansion of CSF3R W341C and W791X and disease progression. We further investigated the oncogenic potential of disrupting original cysteine pairs in the CSF3R extracellular domain. Increased dimers are observed in these mutations, whereas two functional consequences were observed: loss of function and constitutive activity. This, therefore, represents the first characterization of unpaired cysteines that mediate both loss and gain of function phenotypes. This paradigm may apply to other cytokine receptors.

#533

XIAP BIR domain suppresses miR-200a expression and subsequently promotes EGFR protein translation and anchorage-independent growth of bladder cancer cell.

Xin Liao,1 Chao Huang,2 Jingxia Li,2 Chuanshu Huang2. 1 _New York University School of Medicine and Wuhan Tongji Hospital, Tuxedo Park, NY;_ 2 _New York University School of Medicine, Tuxedo Park, NY_.

The X-linked inhibitor of apoptosis protein (XIAP) is a well-known potent apoptosis suppressor, and also participates in cancer cell biological behaviors, therefore attracting great attentions as a potential antineoplastic therapeutic target for past years. We found here that lacking of XIAP expression resulted in a remarkable suppression of EGFR expression, consequently leading to the deficient of anchorage-independent cell growth. Further study demonstrated that BIR domain of XIAP was crucial for regulating the EGFR translation by suppressing the transcription and expression of miR-200a. Mechanistic studies indicated that BIR domain activated the PP2A activity by decreasing the phosphorylation of PP2A at Tyr307 in its catalytic subunit, PP2A-C. Such activated PP2A prevented the deviant phosphorylation and activation of MAPK kinases/MAPKs, their downstream effector c-Jun, and in turn inhibiting transcription of c-Jun-regulated the miR-200a. Collectively, our study uncovered a novel function of BIR domain of XIAP in regulating the EGFR translation, providing significant insight into the understanding of the XIAP overexpression in the cancer development and progression, further offering a new theoretical support for using XIAP BIR domain and EGFR as targets for cancer therapy.

#534

PDGFRB **gain-of-function mutations in multifocal infantile myofibromatosis: Implications for diagnosis & therapy.**

Florence A. Arts,1 Raf Sciot,2 Bénédicte Brichard,1 Marleen Renard,2 Laura A. Noël,1 Amélie I. Velghe,1 Christine Galant,1 Maria Debiec-Rychter,2 An Van Damme,1 Miikka Vikkula,1 Raphaël Helaers,1 Nisha Limaye,1 Hélène A. Poirel,1 Jean-Baptiste B. Demoulin1. 1 _University of Louvain, Brussels, Belgium;_ 2 _KU Leuven, Leuven, Belgium_.

Myofibromas and infantile myofibromatosis are among the most prevalent soft tissue tumors of infancy and childhood. They are characterized by the presence of solitary or multiple nodules in the skin, subcutaneous soft tissues, bones, muscles and viscera. Multifocal myofibromatosis with visceral lesions is associated with a poor prognosis. The pathogenesis of sporadic myofibromatosis is unknown. A few familial cases have been linked to mutations in various genes including PDGFRB, which encodes a receptor tyrosine kinase that is highly expressed in fibroblasts, pericytes and other cells of mesenchymal origin. In the present study, we investigated whether the sporadic form of the disease may also be associated with PDGFRB mutations. We sequenced the whole coding sequence of PDGFRB in 20 cases of myofibromatosis or solitary myofibroma using the Ion AmpliSeq technology at high coverage (Life Technologies). Six different mutations in the coding sequence of PDGFRB were identified in seven patients, six of whom had the sporadic multicentric form of the disease, with a median age at diagnosis of 13 months [range: 0 to 7 years]. Mutations were located in four different exons: the classical hotspot exons 12 and 14, encoding the juxtamembrane and the kinase domains, but also the exons 11 and 18, encoding the transmembrane domain and the activation loop respectively. The percentage of mutated reads varied between 4 and 33%, suggesting that the mutations were somatic, except for one patient who had a germline R561C substitution and a somatic second hit. Two patients had the same mutation in multiple separated lesions, suggesting an early post-zygotic mutation in a progenitor cell. By contrast, a third patient had three different PDGFRB mutations in the three nodules analyzed. We showed that these mutations constitutively activated receptor signaling in the absence of ligand and stimulated fibroblast proliferation in vitro. Furthermore, the mutant receptors were sensitive to the tyrosine kinase inhibitor imatinib, except D850V, which was inhibited by dasatinib and ponatinib, suggesting a treatment for severe myofibromatosis. In conclusion, we identified activating PDGFRB mutations in 66% of sporadic multifocal infantile myofibromatosis cases, shedding light on the mechanism of disease development. Our results provide a genetic test to facilitate diagnosis, and preclinical data for development of molecular therapies.

#535

Paving the path to metastasis/invasion through GIV: Identification of a polarized ex(o)citing machinery for cancer invasion.

Cristina C. Rohena, Nina N. Sun, Nicolas Aznar, Pradipta Ghosh. _University of California San Diego, San Diego, CA_.

Polarized exocytosis is a fundamental process involving the delivery of membrane and cargo proteins to target sites the plasma membrane (PM) and it is essential for a wide range of biological functions such as cell growth, morphogenesis, and polarized cell migration. Polarized exocytosis requires precise spatial control of vesicle tethering to target sites at the PM. This is mediated by the octameric exocyst complex that is conserved from yeast to man. How the exocyst complex imparts polarity during exocytosis remained a mystery until recent insights emerged from studies on budding yeast. A functional interaction between exocyst component Exo70p and the yeast polarity determinant scaffold protein Bem1p was reported, and it was shown that Bem1p regulates the targeting of Exo70p to polarized exocytic sites. Despite these insights, the counterpart of Bem1p in higher species remained elusive. Here we demonstrate that a novel polarity-determinant protein called GIV (Gα-interacting vesicle associated protein/Girdin) fulfills the key criteria and functions of its yeast counterpart Bem1p. GIV provides an evolutionary upgrade to the process of polarized exocytosis by making it responsive to growth factors and other external cues and by modulating it through G protein signaling that is unique to mammalian cells. GIV was originally identified in a yeast-2 hybrid screen as a binding partner of EXOC1, and subsequently validated by us as a PI4P-binding protein that is a direct binding partner of mammalian Exo70. Biochemical assays have confirmed that the mechanism of GIV:Exo70 interaction bears close semblance to the Bem1p:Exo70p interaction. Sequence alignment studies led to the identification of single point mutants in Bem1p and GIV that are selectively defective in binding to Exo70. Using genetic manipulation of cancer cells we have observed that GIV is essential for polarized tethering of Exo70-coated vesicles at the cell periphery. Consistent with the previously described prometastatic role of GIV, we found that GIV is required for EGF-stimulated exocytosis of matrix metalloproteases that aid in cancer invasion in MDA-MB-231 breast cancer cells and that specific point mutants of GIV that interrupt the interaction with Exo70 cause a reduction in the ability of these breast cancer cells to invade and degrade components of the extracellular matrix. Furthermore, GIV interacts specifically with TC-10●GTP[active], a monomeric GTPase that regulates fusion of exocytic vesicles at the plasma membrane. Insights gained continue to provide an in-depth understanding of how GIV affects a fundamental process of exocytosis, and what impact that may have on cancer invasion and metastasis.

#536

Novel identification of STAT1 as a crucial mediator of ETV6-NTRK3-induced tumorigenesis.

Jinah Park,1 Junil Kim,2 Poul H. Sorensen,3 Seong-Jin Kim1. 1 _Seoul National University, Gyeonggi-do, Republic of Korea;_ 2 _Perelman School of Medicine, Philadelphia, PA;_ 3 _British Columbia Cancer Research Centre, Vancouber, British Columbia, Canada_.

Chromosomal rearrangements that facilitate tumor formation and progression through activation of oncogenic tyrosine kinase are frequently observed in cancer. The ETV6-NTRK3 (EN) fusion has been implicated in various cancers, including infantile fibrosarcoma, secretory breast carcinoma and acute myeloblastic leukemia, and has exhibited in vivo and in vitro transforming ability. In the present study, we analyzed transcriptome alterations using RNA-Seq in EN-transduced NIH3T3 fibroblasts to identify the mechanisms that are involved in EN-mediated tumorigenesis. Through functional profile assessment of EN-regulated transcriptome alterations, we found that EN up-regulated genes mainly associated with cell motion, membrane invagination, and cell proliferation, while down-regulated genes are involved in cell adhesion, which described the transforming potential and increased proliferation in EN-transduced cells. Notably, KEGG pathway analysis identified the JAK-STAT signaling pathway with the highest statistical significance. Moreover, Ingenuity Pathway Analysis and gene regulatory network analysis identified the Stat1 transcription factor and its target genes as top EN-regulated molecules. We further demonstrated that EN enhanced STAT1 phosphorylation but attenuated STAT1 acetylation, thereby inhibiting the interaction between NF-κB p65 and acetylated STAT1. Consequently, nuclear translocation of NF-κB p65 and subsequent anti-apoptotic NF-κB activity were increased by EN. Taken together, here we report, for the first time, STAT1 as a significantly EN-regulated transcription factor and a crucial mediator of EN-induced tumorigenesis. [This work was supported by a National Research Foundation grant of Korea (NRF-2014M3A9B5073918) funded by the Korea government.]

#537

TRAF3 serves as a tumor suppressor in HPV-associated head and neck squamous cell carcinomas.

Tony W. Chen, Jialing Zhang, Xinping Yang, Zhong Chen, Carter Van Waes. _NIH/NIDCD, Bethesda, MD_.

The molecular mechanisms that control transformation and tumorigenicity of human papillomavirus-associated (HPV+) head and neck squamous cell carcinomas (HNSCCs) are currently being investigated. The number of HPV+ HNSCC cases has increased sharply in recent years, especially in tonsil and oropharyngeal cancers. These HPV+ HNSCCs often respond differently to treatments. Recently, The Cancer Genome Atlas (TCGA, 2015) identified novel loss-of-function genomic alterations of TNF receptor-associated factor 3 (TRAF3) in HPV+ HNSCCs. TRAF3 is a ring-finger E3 Ubiquitin Ligase which inhibits downstream alternative NF-κB signaling and promotes anti-viral immunity by promoting degradation of ubiquitinated proteins. To assess TRAF3's role in HPV+ HNSCCs, we identified cell lines with lower level of TRAF3 protein, consistent with deficient TRAF3 expression identified in TCGA data. Functional studies showed that TRAF3 expression led to decreased steady-state protein levels of the alternative NF-κB pathway components RELB and NF-κB2/p52, as assessed by Western blot, reporter assays and immunofluorescence. Additionally, TRAF3 expression led to decreased cell proliferation, tumorigenic activity and migration, and increased sensitivity to chemotherapy agent cisplatin. Interestingly, TRAF3 increases the steady state protein level of the classical tumor suppressors RB and p53 in HPV+ HNSCC cell lines as assessed by Western blot. Further in vitro characterization of TRAF3's function in HPV+ HNSCCs was assessed using clinically identified TRAF3 loss-of-function mutagenic isoforms, mimicking both TCGA data and HNSCC cell line data for TRAF3 defects. In contrast to wtTRAF3, these mutant forms of TRAF3 do not as strongly restore p53 or inhibit RELB and NF-κB2/p52 protein levels. One specific mutation which causes a frameshift at residue 210 greatly inhibits TRAF3 function as assessed by Western blot. To further assess effects of wt or TRAF3 loss-of-function mutants in vitro and in vivo, an inducible HPV+ HNSCC line expressing TRAF3 using the TET-ON system, and TRAF3 knockout in HPV+ HNSCC line and Human Oral Keratinocyte (HOK) line using CRISPR-Cas9 genomic editing are generated. In conclusion, the ring-finger E3 ubiquitin ligase TRAF3 inhibits the pro-survival alternative NF-κB signaling pathway and restores TP53 and RB, thereby serving as a tumor suppressor in HPV+ HNSCCs.

(Supported by NIDCD intramural project ZIA-DC-000016, 73 and 74)

#538

The cytosolic domain of a disintergrin and metalloprotease (ADAM) 15 promotes non-small cell lung cancer (NSCLC) anti-apoptosis ability.

Hsin-Han Hou, Chong-Jen Yu. _National Taiwan University Hospital, Taipei, Taiwan_.

Emerging evidence has indicated that proteins of a disintergrin and metalloprotease (ADAM) family contribute to cancer progression and metastasis. One member of this family, ADAM15, has been shown to be upregulated in multiple cancers, including gastric, lung, breast, and prostate cancers, and the enzymatic activities of its extracellular metalloprotrease domain promote breast cancer proliferation and migration through mediating ErbB signaling pathway. The patients with ADAM15 high-expressing lung tumors have shorter survival time and ADAM15 has been proved to enhance synovial fibroblasts anti-apoptosis ability via focal adhesion kinase signaling pathway. We firstly demonstrated other than extracellular enzymetic activity, the longest isoform of ADAM15 (ADAM15 i6), which contains the most cytoplasmic Src homology 3 (SH3) binding motifs, significantly upregulated in primary lung cancer tissues and promoted NSCLC proliferation via growth factor receptor-bound protein 2 (Grb2) and Src homolog 2 domain containing (Shc) association. In this study, we further explore the roles of ADAM15 cytosolic domain in NSCLC apoptosis resistance. Overexpression of ADAM15 i6 promoted CL1-0 cell anti-apoptosis ability according to the trypan blue inclusion assay. Ablation of nephrocystin (NPHP1) attenuated the ADAM15 i6-promoted anti-apoptosis ability. Thus, we identified a novel mechanism of the ADAM15 cytoplasmic domain in NSCLC tumor progression, which will shed light on the molecular mechanisms of ADAM proteins, and facilitate development of novel therapy in NSCLC.

#539

MAEL promotes colorectal cancer cell growth and migration by activating EGFR pathway.

Lulu Liu, Zhou Tong, Weiqin Jiang, Yi Zheng, Peng Zhao, Weijia Fang. _The First Affiliated Hosipital, Zhejiang University, Hangzhou, China_.

Colorectal cancer (CRC) is one of the most common malignant tumors of the digestive system. It is generally accepted that CRC pathogenesis is a long-term process involving accumulation of multiple genetic alterations. Herein, we aim to explore the molecular biological mechanisms of CRC initiation and progression. We have been committed to investigating MAEL function in digestive neoplasms and have found MAEL is a new candidate oncogene. Previously, overexpression of MAEL was frequently detected in CRC tissues and significantly associated with poor 5-year outcome. By MTT assay and transwell assay, functional study demonstrated that overexpression of MAEL promoted cell growth and cell migration in DLD1 and THC8307 cell lines. Furthermore overexpression of MAEL in DLD1 cell line could promote tumor formation in nude mice. Further study found that MAEL overexpression in DLD1 and THC8307 elevated the protein expression of p-EGFR, p-Raf-1, p-ERK 1/2, p-PI3K and p-AKT. These data indicated that MAEL enhanced the activity of EGFR/Ras/Raf/MEK/ERK1/2 and EGFR/PI3K/Akt pathways. However, more effort is needed to investigate MAEL function and how MAEL activate EGFR pathways.

## BIOINFORMATICS AND SYSTEMS BIOLOGY:

### Bioinformatics and Analysis for Therapy and Immune System

#540

Deep learning to determine breast cancer estrogen receptor status from nuclear morphometric features in H&E images.

Rishi R. Rawat, Daniel Ruderman, David B. Agus, Paul Macklin. _Ellison Institute for Transformative Medicine of USC, Los Angeles, CA_.

We hypothesized that the spatial arrangement of nuclei in a hematoxylin and eosin (H&E) stained image contains a vast quantity of latent information that is currently underutilized. We tested this hypothesis on the classification of breast cancer biopsies as estrogen receptor (ER) positive or negative. While determining ER status typically requires immunohistochemistry (IHC) staining, we aimed to recapitulate this step by predicting ER status directly from H&E stained slides. By training a deep neural network on nuclear-morphometric features, we demonstrate the power of machine learning to provide insights about cancer biology, and aim to scale precision oncology for the developing world.

We constructed a learning pipeline that segments nuclei from H&E stained slides scanned into digital images, extracts shape and orientation descriptors, and uses a deep neural network to learn spatial features to predict ER status. After training the pipeline on 57 tissue cores of invasive ductal carcinoma (IDC), we were able to predict ER status in patients with IDC in a statistically significant manner (AUC=0.72, 95%CI=0.55-0.88, n=56) and ductal carcinoma in situ (AUC=0.82, 95%CI=0.74-0.89, n=106).

To our knowledge, this is the first work to show that nuclear morphometric information can be used to predict the molecular status of a breast cancer tumor. An initial analysis of the features learned by the deep neural network provides a glimpse into the mechanistic differences between ER+ and ER- cancers: suggesting the importance of spatial heterogeneity in distinguishing between ER+ and ER-.

We anticipate that further analysis will provide deeper insights about the morphological differences between ER+ and ER- cancers, and that this type of analysis can be applied to other markers and cancers. Moreover, an optimized version of this pipeline may serve patients in the developing world by providing a means to predict marker status directly from H&E images.

#541

A comprehensive analysis delineating the immunotherapeutic terrain of cancer-related clinical trials.

Cara M. Statz, Sara E. Patterson, Taofei Yin, Susan M. Mockus. _The Jackson Laboratory for Genomic Medicine, Farmington, CT_.

Technological innovations have facilitated a greater understanding of how the tumor microenvironment contributes to cancer, leading to rapid FDA approval of four immunotherapies. To assess how these therapies are being further investigated in combination with other therapies and in tumor types outside of the current FDA approval, we performed a comprehensive analysis of the curated clinical trials in the JAX Clinical Knowledgebase (JAX-CKB). In brief, clinical trials investigating Atezolizumab, Nivolumab, Pembrolizumab, and Ipilimumab, curated from clinicaltrials.gov, were queried in the JAX-CKB and then analyzed for comparison. Further analyses were executed to illustrate possible unmet needs within the field of cancer therapeutics. Of the four immunotherapies, Pembrolizumab was identified with the greatest number of clinical trials overall, with 305 compared to Atezolizumab, 79, Nivolumab, 183, and Ipilimumab, 126. Of these trials the number of trials investigating Pembrolizumab, Atezolizumab, Nivolumab, or Ipilimumab in combination with another therapy was higher than those investigating one of the four immunotherapies as a monotherapy. Phase II trials for both single therapy and combinatorial therapies were greater than both Phase I and Phase III for the same groups, regardless of therapy. On average, 12% ± 3.8% of the combined trials for all four drugs included any advanced solid tumor. Nivolumab combined with Ipilimumab demonstrated the greatest number of trials (61) investigating an immunotherapy in combination with another immunotherapy. Among those, 30 were Phase II trials, 16 were Phase I, and 14 were Phase III. Across five cancer indications (lung, pancreatic, ovarian, prostate, and colon), lung cancer was most commonly indicated in the trials, among all four drugs. Prostate was indicated in the least number of trials, with Ipilimumab ranking the highest (10). The recent success with immunotherapies has garnered significant interest in understanding how these therapies will perform in different tumor types and whether specific combinations will have a greater impact. Interrogation of the clinical trial terrain in the JAX-CKB provides a basis for determining additional investigations that might be warranted.

#542

Genomic and clonal characterization of immunogenic epitope landscape across The Cancer Genome Atlas.

HoJoon Lee,1 Stephanie Greer,1 Susan M. Grimes,1 Jae-Ho Cheong,2 Hanlee P. Ji1. 1 _Stanford Univ., Stanford, CA;_ 2 _Yonsei University, Seoul, Republic of Korea_.

Recent advances in cancer immunotherapy and genomic sequencing technologies have created promising opportunities for precision cancer medicine. Somatic mutation in coding regions of cancer genomes may lead to amino acid alterations that generate immunogenic peptides, called neoantigens. These novel peptides are tumor-specific. An individual's own MHC genotypes restricts the presentation of these tumor-specific peptides and thus there are a limited number of candidate epitopes for immune cellular recognition. Current studies have not comprehensively studied how MHC-restricted neoantigens are present across tens of thousands of cancer. Nor have these studies determined how somatic mutations and their associated neoantigens are represented among different subclonal populations existing within individual tumors. To address these limitations, we have mapped the complex immunogenomic topology of neoantigen epitopes across 8,000 tumors representing 18 different cancer types. This neoantigen landscape accounts for how neoepitope candidates are distributed among the various clonal subpopulations existing within any given tumor.Our study utilized genomic data from the Cancer Genome Atlas (TCGA). Our automated pipeline provides results from 8,000 samples from 18 cancer types that have somatic variant calls, copy number variation, whole exome sequence, and RNA-Seq data. Identification of somatic mutations that lead to highly immunogenic antigens involved five different steps; i) in-silico translation of identified mutations, ii) expression as measured per RNA-Seq, iii) patient's own major histocompatibility complex genotype, iv) binding affinity specific to the MHC alleles for any given patient and v) occurrence of clonal subpopulations as demonstrated by intratumoral genetic heterogeneity. We mapped the neoantigen landscape with quantitative representation of the genetic clonal diversity existing within individual tumors. In average, 2% of missense mutations from a patient are detected in RNA-Seq, with binding affinity to own HLA genotypes, and from the dominant clonal subpopulation.In summary, our study demonstrates the various relationships among the number of optimal antigens, the number of clonal subpopulations, the number of mutations, and clinical phenotypes such as tumor stage. Our automated bioinformatics process facilitates the use of exome and RNA-Seq as potential diagnostic analytic process for precision immunotherapy.

#543

Harnessing synthetic lethality to predict clinical outcomes of cancer treatment.

Joo Sang Lee,1 Avinash Das,1 Livnat Jerby-Arnon,2 Seung Gu Park,1 Matthew Davidson,3 Dikla Atias,4 Arnaud Amzallag,5 Chani Stossel,4 Ella Buzhor,4 Welles Robinson,1 Kuoyuan Cheng,1 Joshua J. Waterfall,6 Paul S. Meltzer,6 Sridhar Hannenhalli,1 Cyril H. Benes,5 Talia Golan,4 Emma Shanks,3 Eytan Ruppin1. 1 _University of Maryland, College Park, MD;_ 2 _Tel Aviv University, Tel Aviv, Israel;_ 3 _Beatson Institute, Glasgow, United Kingdom;_ 4 _Sheba Medical Center, Ramat Gan, Israel;_ 5 _Harvard Medical School, Boston, MA;_ 6 _National Institute of Health, Bethesda, MD_.

Significance: The identification of Synthetic Lethal interactions (SLi) has long been considered a foundation for the advancement of cancer treatment. The rapidly accumulating large-scale patient data now provides a golden opportunity to infer SLi directly from patient samples. Here we present a new data-driven approach termed ISLE for identifying SLi, which is then shown to be predictive of clinical outcomes of cancer treatment in an unsupervised manner, for the first time.

Methods: ISLE consists of four inference steps, analyzing tumor, cell line and gene evolutionary data: It first identifies putative SL gene pairs whose co-inactivation is underrepresented in tumors, testifying that they are selected against. Second, it further prioritizes candidate SL pairs whose co-inactivation is associated with better prognosis in patients, testifying that they may hamper tumor progression. Finally, it eliminates false positive SLi using gene essentiality screens (testifying to causal SLi relations) and prioritizing SLi paired genes with similar evolutionary phylogenetic profiles.

Results: We applied ISLE to analyze the TCGA tumor collection and generated the first clinically-derived pan-cancer SL-network, composed of SLi common across many cancer types. We validated that these SLi match the known, experimentally identified SLi (AUC=0.87), and show that the SL-network is predictive of patient survival in an independent breast cancer dataset (METABRIC). Based on the predicted SLi, we predicted drug response of single agents and drug combinations in a wide variety of in vitro, mouse xenograft and patient data, altogether encompassing >700 single drugs and >5,000 drug combinations in >1,000 cell lines, 375 xenograft models and >5,000 patient samples. Of note, these predictions were performed in an unsupervised manner, reducing the known risk of over-fitting the data commonly associated with supervised prediction methods. Our prediction is based on the notion that a drug is likely to be more effective in tumors where many of its targets' SL-partners are inactive, and drug synergism may be mediated by underlying SLi between their targets. Most importantly, we demonstrate for the first time that an SL-network can successfully predict the treatment outcome in cancer patients in multiple large-scale patient datasets including the TCGA, where SLis successfully predict patients' response for 75% of cancer drugs.

Conclusions: ISLE is predictive of the patients' response for the majority of current cancer drugs. Of paramount importance, the predictions of ISLE are based on SLi between (potentially) all genes in the cancer genome, thus prioritizing treatments for patients whose tumors do not bear specific actionable mutations in cancer driver genes, offering a novel approach to precision-based cancer therapy. The predictive performance of ISLE is likely to further improve with the expected rapid accumulation of additional patient data.

#544

Hypoxia response signaling is linked to TACE resistance in hepatocellular carcinoma (HCC) patients.

Valerie Fako,1 Joyce Lee,2 Tan-To Cheung,2 Irene O. Ng,2 Xin W. Wang1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _University of Hong Kong, Hong Kong, Hong Kong_.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and outcome is dismal, due to tumor heterogeneity and lack of effective treatment options for patients with later stage disease. Transcatheter arterial chemoembolization (TACE) is the gold standard of therapy for patients with intermediate to locally advanced tumors. TACE delivers a high dose of chemotherapy directly to the tumor via the hepatic artery, followed by an embolizing agent to restrict tumor blood supply. However, tumor hypoxia is linked to alterations in metabolism, such as increased glycolysis (the Warburg effect), and can lead to enhanced cell survival. Several randomized control trials (RCTs) showed a survival benefit with TACE, but only with strict patient selection criteria. In Asia, TACE is also commonly used as adjuvant therapy after surgical resection, yet RCTs evaluating adjuvant TACE have shown conflicting results, likely due to patient selection and stratification. We hypothesize that tumor gene expression is predictive of response following TACE, and that differential cellular metabolism resembling a hypoxic phenotype prior to treatment is responsible for TACE resistance. We retrospectively analyzed gene expression data in treatment-naive tumor tissue from a cohort of Chinese patients who received TACE. Using hierarchical clustering, followed by class comparison and survival risk prediction, we identified a 14-gene signature that is predictive of response vs. non-response to TACE, as measured by overall survival, independent of other clinical variables. We found that hypoxia- and glycolysis-related genes are enriched among differentially expressed genes in TACE Responders vs. Non-Responders, and that hypoxia master regulator HIF-1α and hypoxia target gene VEGF are significantly up-regulated in Non-Responders. We determined that a key glycolysis gene is up-regulated in Non-Responders, and conversely, two rate-limiting genes involved in gluconeogenesis, the pathway opposing glycolysis, are up-regulated in Responders. We also examined metabolomic data from the TACE cohort, and found an enrichment of glycolysis-related metabolites in Non-Responders, and gluconeogenesis-related metabolites in Responders. Further investigation will be required to connect altered glucose metabolism to TACE resistance and to determine driver genes linking hypoxia and metabolism, which, together with our 14-gene signature, may serve as a stratification tool to guide personalized treatment modalities for HCC patients.

#545

Prognostic alternative mRNA splicing signature in non-small cell lung cancer.

Yuan Li,1 Nan Sun,2 Zhiliang Lu,1 Shouguo Sun,1 Jianbing Huang,1 Zhaoli Chen,1 Jie He1. 1 _National cancer center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China;_ 2 _Department of Clinical Cancer Prevention, the University of Texas MD Anderson Cancer Center, Houston, TX_.

Alternative splicing provides a major mechanism to generate protein diversity. Increasing evidence suggests a link of dysregulation of splicing associated with cancer. However, alternative splicing in non-small cell lung cancer (NSCLC) is largely unstudied. In this study, seven types of alternative splicing were profiled in 491 lung adenocarcinoma (LUAD) and 471 lung squamous cell carcinoma (LUSC) patients in TCGA using RNA sequencing data. Prognostic predictors for LUAD, LUSC and merged NSCLC patients were built by integrated survival analysis. Gene network and splicing correlation network analyses were also conducted. Overall, we detected 45,062 and 50,732 alternative splicing events in LUAD and LUSC, respectively, almost half of them were exon skipping events. A total of 3,691 and 2,403 alternative splicing events were significantly associated with patient survival in LUAD and LUSC, respectively. Gene network analysis uncovered important hub genes or nodes such as EGFR, CD44, PIK3C3, RRAS2, MAPKAP1 and FGFR2. The area under the curve (AUC) of the receiver-operator characteristic (ROC) curve for prognostic predictors in LUAD and LUSC were 0.960 and 0.940 at 2000 days of overall survival. Most relevant for clinical application, we also built the prognostic predictor for NSCLC patients with high performance. The AUC of ROC curve in NSCLC was 0.817 which were also over 0.8 in LUAD and LUSC, separately. Moreover, expression of 49 and 9 survival associated splicing factors were significantly correlated with 75 survival associated alternative splicing events in LUAD and LUSC, respectively. Interestingly, splicing correlation networks uncovered that "oncogenic" splicing factors were up-regulated in LUAD while "tumor suppressor" splicing factors were down-regulated in LUSC. Survival associated splice factors might have opposite roles in the regulation of RNA splicing in LUAD and LUSC. In conclusion, we created prognostic predictors based on alternative splicing events with high performances for risk stratification in NSCLC patients and uncovered interesting splicing networks in LUAD and LUSC which could be underlying mechanisms.

#546

Supporting neoantigen identification for personalized cancer vaccines trough analytical validation of an augmented content enhanced (ACE) transcriptome.

Jennifer L. Yen, Sean Boyle, Ravi Alla, Jason Harris, Martina Lefterova, Richard Chen. _Personalis, Menlo Park, CA_.

The identification of neoantigens has become a critical step in the development of neoantigen-based personalized cancer vaccines and other immunotherapy applications. Since neoantigens can be generated from tumor specific mutations in any expressed gene, the first step in neoantigen identification typically involves deep sequencing of the tumor exome and transcriptome combined with exome sequencing of the matched normal. As personalized vaccines enter clinical trials, there is a growing need for strong analytical validation of these platforms.

To address this, we have developed the ACE Exome (~200X) and Transcriptome platforms for neoantigen identification which harness an augmented exome approach specifically designed to increase sensitivity for neoantigens in low complexity, difficult to sequence regions. To enable this platform for neoantigen based personalized cancer vaccines, we have performed a validation of both our ACE Exome (tumor and normal) and ACE transcriptome platforms for detecting DNA-based SNVs and Indels, as well as for RNA based small variant and fusion calls, variant types that are especially important for neoantigen identification. In this abstract, we describe the ACE Transcriptome validation.

To validate the ACE Transcriptome, we assessed analytical sensitivity (AS) and positive predictive accuracy (PPA). AS was calculated from a reference set of 894 SNVs and 19 indels across 11 tumor cancer cell lines with matched normals. The reference set was constructed based on previously observed variants in CCLE, COSMIC or had been validated by Sanger sequencing. PPA and limit of detection (LOD) were calculated in a series of tumor-normal dilutions representing allele frequencies ranging from 10% to 100%. For fusions, we further selected an additional 10 cell lines to test the detection of 16 previously characterized fusion events including several clinically actionable fusions (ALK and BCR-ABL1).

We report an analytical sensitivity for SNVs of >99%, and a PPA of >96% for small variants with >=10% minor allele frequency (MAF). For fusion events, we report an analytical sensitivity of >99%, with the detection of all 16 fusion events supported by at least 5 reads.

We demonstrate that our ACE Transcriptome assay and RNA cancer pipeline is a highly sensitive and robust platform for detecting small variants and fusions in the RNA.

#547

Novel algorithms for spatial modeling of cellular interactions in the tumor microenvironment.

Yiyi Yan,1 Alberto Santamaria-Pang,2 Michael Gerdes,2 Fiona Ginty,2 Anup Sood,2 Svetomir N. Markovic,1 Alexey Leontovich1. 1 _Mayo Clinic, Rochester, MN;_ 2 _GE Global Research, Niskayuna, NY_.

Background: Understanding the spatial interactions between tumor and immune cells in the heterogeneous tumor microenvironment is pivotal to improve clinical outcomes of immunotherapy. Various multiplexed imaging platforms have been recently developed to visualize the different immune cell subtypes with various distribution patterns that can impact the antitumor immunity. However, the lack of tools that allow for the spatial pattern modeling remains a major barrier for systematic analysis of cellular interactions in the TME.

Methods: We used multiplexed immunohistochemistry (mIHC) to understand the cellular interplays in metastatic melanoma TME using FFPE section. This platform allows in situ quantitative single cell analysis with high-level of multiplexing while preserving the tumor heterogeneity. Single cell spatial data (position and location) in the context of TME can be obtained during mIHC, enabling further spatial modeling to characterize the cellular interactions between tumor immune cells. Novel spatial analysis algorithms are developed taking advantages of these spatial data.

Results: Using R programming language, we developed two novel analytical approaches to reveal the spatial distributions of various immune cell subtypes relative to tumor cells. The cell neighborhood analysis algorithm uses the "applynbd()" function to traverse through every cellular point on a two-dimensional plane and creates neighborhoods of 24um in diameter centered by a tumor cell. This algorithm captures the counts of a specific type of immune cells that are in direct contact with a tumor cell, allowing the visualization and statistical analysis of cellular attraction/repulsion patterns. The cell aggregate analysis algorithm uses a sliding-window based approach to capture cell aggregates with certain densities and the areas within the TME covered by specific cell aggregates. Combined with the Ripley K-function, the cell aggregate algorithm captures the aggregation vs. segregation pattern between different cell types, enabling the clustering and distance analysis of immune cells relative to tumor cells. Our algorithms demonstrate that tumor cells either attract CD3+ tumor-infiltrating T cells or favor the aggregation of CD20+ B cell in peri-tumor areas depends on the levels of tumor cell human leukocyte antigen-1 (HLA-1) expression.

Conclusion: Our novel spatial analysis algorithms enable the modeling of different interaction patterns between tumor and immune cells (direct contact vs. aggregation) at both single cell and TME level. It demonstrates that tumor cells with heterogeneous properties can impact the immune cells' distributions in the TME with various biological outcomes. It also establishes tools that are necessary for systematic analysis of the TME, allowing the elucidation of the "homogeneous patterns" within the heterogeneous TME.

#548

RNAseq analysis of infiltrating immune cells in liver cancer.

Krithika Bhuvaneshwar,1 Coleman I. Smith,2 Alexander H. Kroemer,2 Aiwu Ruth He,2 Yuriy Gusev1. 1 _Georgetown University, Washington, DC;_ 2 _Medstar Georgetown University Hospital, Washington, DC_.

Hepatocellular carcinoma (HCC) has emerged as second most common cause of cancer deaths worldwide. During the last 10 years, there has been a clear delineation of landscape of genetic alterations in HCC and deregulated pathways in HCC. However, the treatment for patients with advanced HCC is limited despite of great effort developing therapeutic targeting the deregulated pathways in HCC. Recent studies reveal a direct causal relationship between cancer & immune dysfunction, whereby tumor cells and their microenvironment are able to evade immune attack by exploiting various immunoregulatory mechanisms in a process termed cancer immune editing.

Methods In this poster, the objective is to perform exploratory analysis of TCGA liver cancer data to see if immune infiltrates matter, and if they offer anti-tumor immunity to a cell. For this purpose, we analyzed RNA-seq data for a cohort of 75 liver cancer samples from TCGA collection. We obtained the gene expression data from a pre-selected group of specific markers for infiltrating lymphocytes (several subtypes), and explored the association of expression of these markers with clinical outcome.We downloaded raw RNA-seq data from the TCGA Liver cancer collection from 75 patients. These included 25 patients who had Hepatitis B virus (HBV), 25 patients who had Hepatitis C virus (HCV) and 25 patients who had both viruses. After processing of raw data, we extracted isoform expression (TPM values) from specific markers for infiltrating lymphocytes. This data was stratified into 'high' and 'low' expression based on median cutoff.We compared the 'high' and 'low' expression groups of patients by performing differential expression & pathway analysis, to see if the differentially expressed results were linked to immune pathways. We then performed survival analysis tests (Log rank, Cox regression) and Kaplan Meier (KM) survival graphs to explore the association with overall survival outcome.

Results We found 14 of 75 HCC cases expressed CD8B isoform, while 61 of 75 HCC cases did not express CD8B isoform. The Log Rank survival test showed a significant association between the expression of CD8B isoform and overall survival (Chisq= 5.2 on 1 degrees of freedom, p= 0.0222). The survival test using a Cox model on the same CDBB isoform, showed that samples that expressed CD8B isoform were at higher risk of event compared to those that did not express the isoform. Other markers that showed good separation of survival curves included CD3 & CD8A.

Conclusion Additional immune cell subtype specific transcripts are being tested. Based on our preliminary analysis, we saw that most of the affected pathways were highly relevant to lymphocyte signaling & immune response and infiltration. Hence, exploring infiltrating lymphocytes can give evidence of immune surveillance against HCC. Testing immune cell specific transcript in tumor samples may service as predictor to treatment targeting immune evasion in cancer patients.

#549

Clustering analysis of next-generation sequencing T cell repertoire data in sipuleucel-T treated prostate cancer patients.

Li Zhang,1 Sounak Chakraborty,2 Jason Cham,1 David Oh,1 Nadeem Sheikh,3 Lawrence Fong1. 1 _University of California, San Francisco, San Francisco, CA;_ 2 _University of Missouri-Columbia, Columbia, MO;_ 3 _Dendreon Pharmaceuticals Inc, Seattle, WA_.

Immunotherapy has demonstrated significant clinical benefit in different cancers. T cells are a crucial component of the adaptive immune system and mediate anti-tumoral immunity. Antigen-specific recognition by T cells is via T cell receptor (TCR) which is the product of somatic V(D)J gene recombination with the addition/subtraction of nontemplated bases at recombination junctions. Next generation TCR sequencing effectively profiles the TCR repertoire. Currently TCR analyses quantify diversity across single clones, however, due to the low overlap of clones across samples, such analyses are limited to a single sample. Here we extend our previous analysis pipeline to track and examine TCR repertoire across time by focusing on V and J gene segments, overcoming the limitation of contemporary analytical approaches and thus obtain statistical inference across subjects directly. The published data of TCR profiling of serial peripheral blood mononuclear cells (PBMC) from three healthy subjects and five prostate cancer patients enrolled in an open-label, Phase II clinical trial of neoadjuvant sipuleucel-T (sip-T) were used as the basis of this analyses. After consolidating the clonal abundance for each combination of V and J gene segments, we calculated Pielou's evenness, which demonstrates that sip-T treated subjects displayed greater clonal expansion compared to healthy subjects (P<0.001). The Circos table viewer was used to circularly visualize the distribution of the combination of V and J gene segments. Sip-T treated subjects were successfully distinguished from healthy subjects with 12 V and J gene combinations explaining the majority of variance of the patients by applying principle components analysis (PCA). Furthermore, we developed a customized clustering workflow to cluster the combination of V and J gene segments based on the change in abundance over time, with gap statistics employed to estimate the optimal number of clusters and k-means algorithm used for partitioning. We found that for all the prostate cancer patients assessed, TCRBV06/TCRBJ01, TCRBV05/TCRBJ02 and TCRBV06/TCRBJ02, which were in the same cluster with the highest frequencies, expanded after first treatment and were maintained at high frequencies at later time points. The results demonstrate that in the setting of immune modulation after sip-T treatment, the V(D)J recombination is not entirely random, and that this non-randomness can be used to distinguish T cell repertoires from sip-T treated subjects versus healthy subjects. The use of additional information of V and J gene segments enables to investigate the profiling of TCR repertoire from a different angle and add another layer of understanding of TCR repertoire. The application of PCA and the customized clustering complete our initial workflow for TCR sequencing data.

#550

**Predictive value of co-expression network consisting of** FOXM1 **and its neighbor genes in cancers.**

Bic-Na Song, In-Sun Chu. _Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea_.

Purpose: Forkhead box protein M1 (FOXM1) is a key regulator of tumorigenesis. Previous studies demonstrated that FOXM1 expression was strongly correlated with poor prognosis of various cancers. The aim of this study is to define a signature consisting of FOXM1 and its associated genes and assess its prognostic potential in cancers including hepatocellular carcinoma (HCC) and others.

Experimental procedures: We analyzed a gene expression profile of 100 patients with HCC to identify FOXM1-correlated genes (the FOXM1 signature) associated with the prognosis of HCC patients. The validity of the FOXM1 signature was verified in an independent HCC cohort (n = 242). Using the signature, we also analyzed different cancer types including pancreatic adenocarcinoma, lung adenocarcinoma, breast carcinoma, and bladder urothelial carcinoma to verify the association between the FOXM1 signature and prognosis. Various statistical methods were applied to signature finding and validation. Upstream regulator and gene-to-gene network analyses were performed to identify potential key mediators of the FOXM1 signature.

Results: Through a gene expression profiling in 100 patients with HCC, we identified a gene set consisting of FOXM1 and its co-expressed genes for predicting poor prognosis. A predictive value of the signature was validated in an independent cohort containing 242 HCC patients. In multivariate analysis, the FOXM1 signature showed the most significant prognostic value (HR = 1.706, 95% CI = 1.176-2.475, P = 0.005). By directly applying the signature to four data sets in different cancer types, our signature showed a consistent prognostic significance, regardless of tumor types. Finally, upstream regulator and gene-to-gene network analyses found an interconnection of network hubs composed by FOXM1, MYC, and E2F1 that might be common key mediators of patient survival in cancer.

Conclusions: The FOXM1 signature represents a promising common diagnostic tool to identify high-risk cancer patients and may extend treatment options in various cancers.

#551

Assessment of computational approaches for quantification of immune cell infiltration from gene expression profiles of complex biological samples.

Brendan P. Hodkinson, Michael E. Schaffer, Michael Gormley. _Janssen Research & Development, Spring House, PA_.

Anticancer immunotherapies target immune cells to block immune suppression and/or promote immune activation in order to eliminate cancer cells. Ipilumumab and Nivolumab, targeting CTLA4 and PD1 respectively, have demonstrated dramatic responses in melanoma and lung cancer (Hodi et al. 2010; Risvi et al. 2015). However, poor response rates in other types of cancer underscore the need to better understand immunomodulatory mechanisms (Topalian et al. 2012). Examination of immune cell specific signals, in either the tumor microenvironment or the periphery, has proven to be a useful tool in developing prognostic biomarkers (Chi et al. 2014; Gentles et al. 2015), inferring mechanisms of action (Krejcik et al. 2016), and discerning immune-based predictors of drug response (Tumeh et al. 2014). In this study we evaluate various gene expression-based computational approaches for inferring immune cell identity in complex cellular mixtures in order to rank their relative utility for illuminating the details of the tumor microenvironment and potentially revealing new biomarkers for prognosis and response prediction.

Immune cell identity has been inferred by previous researchers using a variety of mathematical approaches, including least squares (Abbas et al. 2009), quadratic programming (Gong et al. 2011; Zhong et al. 2013), maximum likelihood (Qiao et al. 2012; Liebner et al. 2013), machine learning (Newman et al. 2015), and enrichment type approaches (Angelova et al. 2015). We used publically-available gene expression microarray data from blood, normal tissue, and tumor samples to assess the effectiveness of these methods in sample types relevant to oncology research. The results from comparisons of these different methods demonstrate that certain approaches are significantly more robust to noise, and are therefore more suitable for complex cellular mixtures such as tumor samples. The discerning use of methods to infer immune cell type proportions from gene expression profiles may lead to improved prognosis, predictive biomarkers for immunotherapy to assist in patient stratification, and new immunoncology targets by indicating immunosuppressive mechanisms.

#552

Multi-scale omics integration using parallel heatmap clustering for the systemic analysis and biomarker discovery of drug sensitivity in lymphoma cell lines.

Ivo Kwee,1 Andrea Rinaldi,2 Alberto J. Arribas,2 Eugenio Gaudio,2 Chiara Tarantelli,2 Filippo Spriano,2 Petra Hillmann,3 Francesco Bertoni2. 1 _Institute of Oncology Research & Dalle Molle Institute for Artificial Intelligence, Bellinzona, Switzerland; _2 _Institute of Oncology Research - IOR, Bellinzona, Switzerland;_ 3 _PIQUR Therapeutics AG, Basel, Switzerland_.

Background. Pharmacogenomics studies the role of genomics in drug response. By measuring the individual genome, it is hoped that pharmaceutical drug treatments can deviate from a "one-dose-fits-all" approach to a more "personalized" treatment. To achieve this goal, we need to explain which genomic differences cause the lack of response and to be able to predict the response from baseline omics data, that is from data available before starting treatment. Here, we present an integrated omics approach to analyze drug sensitivity based on in vitro experiments.

Methods. We measured the drug response in 61 lymphoma cell lines for a number of anti-cancer drugs using a standard MTT cell proliferation assay. The baseline genomics of these cell lines were fully profiled for gene expression, copy number and methylation. Pathway and gene set signatures were computed using GO, KEGG, Biocarta and lymphoma databases to provide gene set level features. Additionally, we computed high level "biological concept" features. These multi-scale features were directly correlated with drug sensitivity but also correlated between the multiple data types. Using a novel parallel clustering approach, we integrated our data for the different omics types and at different scales: gene, gene set and concept level. Finally, a shortest path algorithm extracted the most probable explanation between genotype and response phenotype.

Results. As a proof of concept, we first applied our method to the classification of activated B- cell like (ABC) and germinal center B (GCB) subtypes in diffuse large B-cell lymphomas (DLBCL). In accordance to previous knowledge, our method showed the MYD88/CD40/NFKB axis as strongly upregulated in the ABC, while, albeit less pronounced, the PI3K/MTOR and NOTCH signalling pathways were more enriched in the GCB subtype. We then applied our method to the drug sensitivity data of our lymphoma cell line panel. The analysis showed that drug response was largely driven by differential methylation rather than copy number aberrations. We found that B cell receptor signaling (BCR) and CD40 signalling were among the most correlated to drug sensitivity in a drug-specific manner. Genes in these pathways (such as SYK, SPIB and CD79A) appeared as good biomarkers for drug-specific response in lymphoma.

Conclusion. Using an integrated analysis of multiple omics data at multiple scales, we successfully identified functional modules related with drug sensitivity in lymphoma cell lines. The same method can be applied to study the sensitivity to other drugs in either hematological and/or solid cancer types.

#553

Microsatellite genotyping enables precision lung cancer risk classification.

Robin T. Varghese,1 Karthik Raja Velmurugan,1 Natalie C. Fonville,2 Harold R. Garner1. 1 _Edward Via College of Osteopathic Medicine (VCOM), Roanoke, VA;_ 2 _Riverside Law, LLP, VA_.

There remains a large discrepancy between the known genetic contributions to cancer and that which can be explained by genomic variants, both inherited and somatic. Recently, understudied repetitive DNA regions called microsatellites have been identified as genetic risk markers for a number of diseases including various cancers (breast, ovarian and brain). In this study we demonstrate an integrated process for identifying and validating microsatellite based risk markers for lung cancer using data from the cancer genome atlas (TCGA) and the 1000 genomes project. Comparing whole exome germline sequencing data from 488 TCGA lung cancer samples to germline exome data from 390 control samples from the 1000 genomes project, we identified 119 potentially informative microsatellite loci. These loci (risk markers) were found to be able to distinguish between cancer and control samples with sensitivity and specificity ratios over 0.8. Then these loci, supplemented with additional loci from other cancers and controls, were evaluated using a custom target enrichment kit and sample-multiplexed nextgen sequencing. Thirteen of the 119 risk markers were validated using high-depth (579x±315) nextgen sequencing of 30 lung cancer and 89 control samples, resulting in sensitivity and specificity ratios were 0.90 and 0.94, respectively. When 8 loci harvested from the bioinformatic analysis of other cancers are added to the classifier, then the sensitivity and specificity rise to 0.93 and 0.97, respectively. Analysis of the genes harboring these loci revealed two genes (ARID1B and REL) and two significantly enriched pathways (chromatin organization and cellular response to stress) which suggest that the process of lung carcinogenesis is linked to chromatin remodeling, inflammation, and tumor microenvironment restructuring. We illustrate that high-depth nextgen sequencing enables a high precision microsatellite-based risk classifier.

#554

Accurately identifying neoantigens utilizing both DNA and RNA somatic variants in an enhanced platform.

Sean M. Boyle, Jason Harris, Gabor Bartha, Ravi Alla, Mirian Karbelashvili, Steve Chervitz, Aldrin Montana, Craig Rowell, Patrick Jonganeel, Scott Kirk, Rena McClory, John West, Rich Chen. _Personalis, Menlo Park, CA_.

The identification of neoantigens is a crucial step in the development of neoantigen-based personalized cancer vaccines and other immunotherapies. Accurately predicting which neoantigens are likely to be immunogenic remains a key challenge owing to the complex processes involved in determining neoantigen immunogenicity including the antigen presenting machinery, likelihood of MHC class I and II binding, similarity to self, and ability to interact with the TCR.

We have developed a neoantigen detection pipeline built upon our analytically validated Accuracy and Content Enhanced (ACE) Exome and Transcriptome sequencing platform and somatic variants calling pipeline through combined DNA and RNA analysis. The analytical performance of these pipelines is greater than >97% sensitivity for small variants (RNA and DNA) with a specificity of >98% (DNA) and a fusion sensitivity of >99% (RNA).

Within our neoantigen pipeline, variants that are detected by our DNA and RNA cancer analysis pipelines are processed for antigen identification, including SNVs, indels, and fusion events. Importantly, both in-frame and out-of-frame events are accurately considered by transcript, allowing for detection of a wealth of candidate neoantigens. Our pipeline includes assessment of important immunologic components including HLA prediction, MHC binding (class I and II), immunogenicity, similarity to self, and similarity to known antigens. Additionally, peptides are evaluated for variant allele frequency in both the RNA and DNA of the tumor sample and gene expression level is considered. Collectively, our ImmunoID product provides a comprehensive assessment of features that may be used for identifying and ranking potentially immunogenic neoantigens.

To assess the effectiveness of this pipeline in predicting immunogenic neoantigens, we assembled a gold-set of 23 known, previously experimentally-validated immunogenic neoantigens from the literature. We spiked in these neoepitopes into exome data and assessed the ability of our neoantigen pipeline to find and rank these immunogenic known neoantigens. Preliminary results show our neoantigen pipeline is able to accurately identify 22 out of 23 (~96%) of the spiked in neoantigens as being potentially immunogenic.

#555

Examination and evaluation of MR radiomics features for characterization of dominant intraprostatic lesions.

Hassan Bagher-Ebadian, Janic Branislava, Chang Liu, Milan Pantelic, David Hearshen, Indrin Chetty, Mohamed Elshaikh, Benjamin Movsas, Ning Wen. _Henry Ford Hospital, Detroit, MI_.

Purpose: This pilot study investigates a set of radiomics features extracted from fast relaxation fast spin echo (FRFSE) T2 pulse sequences for normal tissue and Dominant Intraprostatic Lesions (DILs) in twenty prostate cancer patients.

Material and Methods: Twenty patients with prostate cancer were studied. All patients had axial FSRFSE T2 scans using a 3 Tesla scanner. A radiologist interpreted MR examinations, and contoured the suspicious DIL and the contralateral section of the prostate gland (normal) on the T2 weighted MR images. Patients underwent a 14-core transrectal Ultrasound Guided Biopsy and localization of positive cores, Gleason score and clinical tumor stage were recorded. 167 radiomics features were extracted from normal and DIL zones. These features were categorized into 8 different sets as following: Intensity Histogram Based (IHB), Gray Level Run Length (GLRL), Law's Textural Information (LAWS), Discrete Orthonormal Stockwell Transform (DOST), Local Binary Pattern (LBP), Two Dimensional Wavelet Transform (2DWT), Two Dimensional Gabor Filter (2DGF), and Gray Level Co-Occurrence Matrix (GLCM) with 8, 7, 18, 18, 6, 48, 40, and 22 features in each category respectively. A Welch's test and the Fisher method were used to test for significant differences among the 167 radiomics features and their subcategories. For all patients, correlation coefficients between the extracted features in the normal and DIL zones were also calculated.

Results: According to the Fisher combined p-values, among the eight categories of radiomics features, only 5 feature categories showed a significant difference (IHB, GLRL, DOST, LBPF and GLCM with pFisher= 2.0x10-6, 0.02, 12 x10-4, 3.7 x10-3, and 1.5 x10-6 respectively). Among all 167 features, only 7 showed a significant difference (D=100x[DIL/NP-1]) and small correlation between normal and DIL zones: IHB-Skewness

(r=0.19, p=0.03, and D=50.3%), GLCM-Contrast (r=0.12, p=0.03, and D=-67.5%), GLCM-Dissimilarity (r=0.12, p=0.01, and D=-67.5%), GLCM-Entropy (r=0.07, p=0.01, and D=-67.1%), GLCM-Difference-Variance (r=0.12, p=0.01, and D=-67.1%), GLCM-Difference-Entropy (r=0.10, p=0.01, and D=-60.4%), and GLCM-Information-Measure-of-Correlation (r=0.25, p=0.01, and D=-65.1%).

Conclusion and Discussion: This pilot study demonstrates the feasibility of using radiomics features from MR images to characterize DILs in prostate cancer patients. Among 167 radiomics features extracted from axial MR T2 FRFSE, 7 features were shown to be potentially significant for distinguishing normal tissue from DILs. This research supports an integrated decision making system, combining clinical factors and radiomics features extracted from MR images, for increasing the DIL detection performance in prostate cancer studies.

#556

CDD: functional insights into orphan GPCRs via subfamily domain architectures.

James S. Song, Noreen R. Gonzales, Roxanne A. Yamashita, Stephen H. Bryant, Aron Marchler-Bauer. _National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD_.

NCBI's Conserved Domain Database (CDD, https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) is a resource that aims at classifying ancient conserved domain families and capturing what is known about their functional diversity. It is a collection of multiple sequence alignments (MSAs) and derived position-specific score matrices (PSSMs). RPS-BLAST allows for the rapid identification of conserved protein domain footprints in protein sequences along with the location of annotated functional sites. CDD is a redundant collection, it imports external domain model collections (Pfam, COGs etc.) and supplement these with manually-curated domain models that are organized into hierarchical classifications, which facilitate annotation of functionally distinct protein families and subfamilies. More recently CDD has been curating information on conserved domain architectures to support functional labeling of proteins that fall into characterized families: SPARCLE (Subfamily Protein ArCHitecture Labeling Engine) is both a curation tool and a public service available at https://www.ncbi.nlm.nih.gov/sparcle. Architecture summaries provide interactive interfaces to lists of proteins that share the same characteristic domain architecture, and to their assigned names, functional labels, and supporting evidence. Here we present the functionally annotated, evolutionary hierarchical classification of the seven-transmembrane G-protein coupled receptors (7TM GPCRs), which includes the largest family of promising therapeutic targets, with more than 140 human orphan GPCRs of unknown function. Across the three kingdoms of life, GPCRs are involved in a wide array of critical physiological processes, and have been implicated in tumor initiation, tumor progression, angiogenesis and metastasis. Orphan subfamilies with unknown endogenous ligand/function are often found to have poor sequence similarity to canonical GPCRs. They have been assigned putative functions with predicted ligand-binding sites, downstream interacting partners, and/or the location of 7TM helices annotated by inference from the molecular and physiological functions of known related GPCR proteins, utilizing more than 25 available 3D-structures, phylogenetic relationships, and the published literature. We hope that this updated classification, together with NCBI's software tools (Cn3D/CDTree and SPARCLE), will aid researchers in the discovery of molecular therapeutic targets for cancer treatment by providing insights regarding as-yet-unidentified intra-and intermolecular interactions as well as functional mechanisms of GPCR signaling.

Acknowledgement: This research was supported by the Intramural Research Program of the National Library of Medicine, NIH.

#557

SPOP mutant subclass prediction and its impact on prostate cancer prognosis.

Deli Liu. _Weill Cornell Medicine, New York, NY_.

SPOP mutations define a distinct and key molecular class of prostate cancer, but there is no signature to identify SPOP mutant subclass based on transcriptional data, and its impact on prognosis and clinical outcomes of prostate cancer patients is unknown.

We developed the SPOP mutant transcriptional signature with high sensitivity and specificity based on transcriptional data. After we predicted SPOP mutant subclass from a large cohort project with transcriptional signature, we studied its association with clinicopathologic variables and prognosis in prostate cancer patients. On univariate analysis, SPOP mutant subclass was associated with lower preoperative serum prostate-specific antigen, extraprostatic extension and older patients (p<0.05). On multivariate analysis, SPOP mutant subclass was associated with metastasis (p<0.05). Combined SPOP mutant status and CAPRA-S models have slightly better prediction for metastasis. On Kaplan-Meier analysis, we found the significant association of SPOP mutant subclass with better patient outcomes of metastasis, and patients without radiation therapy with even better metastatic outcomes.

The SPOP mutant subclass could be predicted by using our SPOP mutant signature purely based on transcriptional data. Clinical and prognosis difference were found among SPOP mutant subclass.

#558

Computational analysis of clinically actionable genomic features: precision heuristics for interpreting the alteration landscape (PHIAL).

Brendan Reardon,1 Nathanael Moore,2 Eliezer VanAllen1. 1 _Dana-Farber Cancer Institute, Broad Institute, Harvard Medical School, Boston, MA;_ 2 _Dana-Farber Cancer Institute, Broad Institute, Indiana University School of Medicine, Harvard Medical School, MA_.

Background: PHIAL (Precision Heuristics for Interpreting the Alteration Landscape) was developed as a heuristic clinical interpretation algorithm for cancer genomic data to inform treatment decisions at the point of care and provide researchers with rapid assessment of tumor actionability. This approach used somatic whole exome sequencing data and a database of tumor alterations relevant for genomics driven therapy (TARGET). However, PHIAL was limited to first order genomic relationships, could not distinguish relative actionability given multiple actionable variants, did not maximize the richness of somatic-germline interactions, and could not leverage both exome and transcriptome data to move towards feature-based actionability. Towards that end, we developed a new interpretation methodology to address these areas and improve clinical actionability algorithms.

Methods: We revised PHIAL to predict actionable alterations based on the presence of SNVs (in the context of allele specific expression from RNA-seq), indels, SCNAs, fusions, and global features (e.g., context-specific mutational burden) that imply actionability. Additionally, we refined and expanded the TARGET database to enable PHIAL to produce scores on multiple dimensions and reflect newly discovered relationships between genomics and clinical actions. Predictive implication values were assigned to reflect the validities of TARGET's drug sensitivity, drug resistance, and prognostic claims.

Results: We applied both the original (PHIAL1) and an updated version of PHIAL (PHIAL2) to a 255 patient cohort with whole exome/transcriptome sequencing data (146 castration-resistant prostate cancer and 109 metastatic melanoma samples). PHIAL1 identified 1,342 clinically actionable/biologically relevant events across the cohort with a median of 3 events per patient and 95% of patients having at least one event. PHIAL2 identified 2,508 events, with a median of 6 events per patient and 98.5% of patients harboring at least one event. Of these events, 8.12% were associated with an FDA-approved therapy and 2.09% with a clinical trial. PHIAL2 identified events in 9 patient samples that PHIAL1 associated with no events.

Conclusion: PHIAL2 was able to identify and rank more putatively actionable alterations than PHIAL1, and effectively transitioned from a variant-based to a feature-based approach. This strategy may inform the utility of point-of-care whole-exome/transcriptome sequencing in larger contexts as these data emerge in clinical settings, and may bridge towards machine learning based approaches as patient outcomes are linked to genomic and transcriptomic features. Finally, PHIAL2 may ultimately provide a deeper understanding of, and suggest clinical actions for, cases in which there is no clear single genomic alteration associated with oncogenesis.

#559

Estimation of immune cell content in bulk tumour tissue using reference profiles from single-cell RNA-seq data.

Max Schelker,1 Jinyan Du,1 Sonia Feau,1 Edda Klipp,2 Birgit Schoeberl,1 Gavin MacBeath,1 Andreas Raue1. 1 _Merrimack Pharmaceuticals, Inc., Cambridge, MA;_ 2 _Humboldt-Universität zu Berlin, Germany_.

Although therapeutics that modulate the immune system provide remarkable benefit for many cancer patients, predicting who will respond remains an unsolved problem. As interactions between the immune system and cancer are governed by a complex network of cell-cell interactions, knowing the specific immune cell composition of a solid tumour may be essential in predicting response to immunotherapy. Here, we describe how to derive the cellular composition of a solid tumour from bulk gene expression data by mathematical deconvolution, using consensus cell type-specific gene expression profiles from recently published tumour-derived single-cell RNA sequencing data. Notably, successful deconvolution depends on these new data, as previously-available profiles from peripheral blood are insufficient. The presented method makes the problem of obtaining a patient's tumour immune cell composition from existing databases like The Cancer Genome Atlas as well as in the clinical setting computationally tractable.

#560

A pan-inflammatory and precancerous disease analysis reveals key biological characteristics in chronic inflammatory diseases with high oncogenic risk.

Chi Zhang,1 Fang Yao,2 Sha Cao,1 Tao Sheng,1 Sen Liang,3 Wei Du,3 Ying Xu1. 1 _Univ. of Georgia, Athens, GA;_ 2 _College of Life Science and Oceanography, Shenzhen, China;_ 3 _Jilin University, Changchun, China_.

It has long been observed that cancer development tends to be associated with chronic inflammation. Recent epidemiology studies have suggested chronic inflammation may be causally linked to a larger number of sporadic cancers. In this work, we have conducted a Pan-inflammatory and precancerous diseases analysis by comparing the 173 transcriptomics data sets consisting 4073 disease and 2669 normal human tissue samples of 10 cancer prone and 8 cancer independent inflammatory diseases, 11 precancerous diseases or benign tumors and 20 cancer types, aiming to identify fundamental differences between cancer-prone chronic inflammations and cancer-independent chronic inflammations and what biological characteristics cause cancer initiation or increased oncogenic risk. By applying differential gene expression, co-expression network, our in-house immune cell deconvolution and machine learning based hypoxia/oxidative stress prediction methods on the collected data, we have discovered the cancer prone chronic inflammatory diseases consistently have: (1) elevated relative proportion of CD4+ T, Macrophage, and Neutrophil cells, (2) decreased CD8 + T, adipocyte, monocyte and B cells, (3) highly disorder tissue repair phase I - IV related pathways, (4) increased oxidative stress level associated with Macrophage and Neutrophil proportions, (5) elevated iron ion metabolism, (6) over expressed extracellular glycosaminoglycan metabolism and (6) suppressed mitochondrial activity. These analyses results strongly suggest that there is a necessary pathway for the majority or all chronic

inflammatory diseases to become cancerous, namely consecutively dysregulated tissue repair process, increased reactive oxygen species producing immune cells, damage of extracellular matrix, Fenton reaction and production of hydroxyl radical and dysregulated mitochondrial activity. We believe these new insights could provide highly useful information in guiding future studies of inflammation associated cancers.

#561

Does molecular staging trump clinical staging: a pan-cancer, big-data analysis of TCGA data.

Nicholas Shannon, Melissa Ching Ching Teo, Narayanan Gopalakrishna Iyer. _National Cancer Centre Singapore, Singapore, Singapore_.

True integration of clinical staging and molecular prognostic biomarkers has been hampered by a lack of comprehensive data sets with adequate power. In the last two years, The Cancer Genome Atlas (TCGA) has generated and made available a repository of rich, high volume -omics datasets including molecular profiling together with matched clinical data.

Methods: Clinical and transcriptomics data were downloaded for TCGA datasets (38 projects) representing a total of 14,043 patients. Matched expression and clinical data was available for 34 cohorts, covering 10554 patients (median n=270, range 15-1091) and survival data was available for 24 cohorts (7854 patients, median 294 per cohort), with a median follow-up of 10.7 months (range 1.9 to 61 months). Prediction models were constructed based on the latest AJCC staging guide, a novel five gene molecular prognostic signature specifically derived for each cancer type, and a combined model utilising both. Accuracy of survival prediction was calculated as the area under the receiver operator characteristic curve (AUC), based on 2-year overall survival prediction.

Results: Pan-cancer analyses demonstrate accuracy of clinical staging from 0.55-0.91 (median 0.72), while molecular prognostication varies from 0.7-0.99 (median 0.84). The combined model is consistently more accurate, 0.71-0.99 (median 0.89, p<0.01), where the benefit of adding molecular data improved accuracy (median +0.13, +0.03-0.36). The absolute benefit of adding molecular data to clinical staging correlated more closely with strength of clinical prediction (R2=0.35) than strength of molecular prediction (R2=0.08). When clinical staging was more less accurate (clinical prediction < 0.7), there was a greater contribution of molecular data, compared to cancers with more accurate clinical staging (mean +0.15 vs +0.07, respectively; p=0.015). Interestingly, of the six tumours with poor clinical prediction (AUC < 0.7), three were squamous cell carcinomas (SCC) arising from the head and neck, cervix or lung respectively. The fact that there were no datasets representing SCC in the good clinical prediction group (AUC > 0.7) suggests an important need for more accurate prognostic models in this subtype.

Conclusion: The potential role for molecular staging as an adjunct to clinical staging remains undefined. In this study, we demonstrate for the first time the importance of molecular prognostication across a range of cancer sub-types, but especially where conventional staging performs poorly. The latter sub-group is further enriched for squamous cell cancers, where molecular information offers dramatic improvement in accuracy (+0.15 to AUC), and hence, should be prioritized for assessment of molecular signatures. The lack of integrating molecular data could also reflect limitations in our understanding of tumour biology, which in turn needs to be translated into potential actionable targets and therapeutic benefit.

#562

Oncovirus detection and integration analysis from human tumor samples using targeted massively parallel sequencing.

Robert T. Burns,1 Samuel S. Hunter,2 Matthew D. Ducar,1 Aaron R. Thorner,1 James A. Decaprio,1 Paul Van Hummelen,1 Alexander Frieden,1 Anwesha Nag,1 Haley A. Coleman,1 Michael K. Slevin,1 Andrea Clapp,1 Samantha D. Drinan,1 Suzanne R. McShane,1 Edwin Thai,1 Priyanka Shivdasani,1 Joshua Bohannon,1 Johann Hoeftberger,1 Reuben Jacobs,1 Bruce M. Wollison,1 Neil A. Patel,1 Monica D. Manam,1 Phani Davineni,1 Matthew Meyerson,1 Laura E. MacConaill1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _University of Idaho, ID_.

Viruses are a major contributor to oncogenesis, causing 10-15% of human cancers. Molecular pathways involved in malignant transformation are frequently activated by genetic alterations, including but not limited to, somatic mutations, copy number aberrations, structural variants, and oncoviruses. Precision cancer medicine aims to classify tumors by site, histology, and molecular tests to determine an "individualized" profile of cancer alterations. However, clinical tests for these various alterations are sequential, time consuming, and use a lot of material, which is often quite limited (e.g., biopsies). Moreover, tests for the presence of viral sequence are generally performed separately to tests (such as massively parallel sequencing) to detect human genomic alterations. Here we present a hybrid capture and massively parallel sequencing approach to detect viral infection concurrently with targeted genomic analysis, which may decrease assay costs, increase sensitivity and scalability, and detect many types of alterations, thereby providing a more complete tumor genetic profile all from a single sample.

We have created a custom hybrid capture probeset for targeted Illumina sequencing to determine whether oncoviruses are present in tissue samples and also determine if the virus has integrated into the host's genome. We have created both 'detection' and 'integration' baits for several oncoviruses, including polyomaviruses, human papilloma viruses, Epstein-Barr virus, human cytomegalovirus, Kaposi sarcoma herpesvirus, human T-lymphotropic virus, and hepatitis B virus. To distinguish between different strains of a single virus, strain-specific detection baits were created to bind to variable regions of viral genomes. The integration bait was designed to bind to regions of the viral genomes that are commonly integrated into the human genome. This baitset can also be combined with other capture panels targeting oncogenes to simultaneously determine infection and integration statuses, as well as somatic mutations, copy number and structural variants.

To detect virus presence, reads were aligned to a hybrid reference of both the human, and targeted virus genomes. Viral integration status and integration loci were determined by leveraging discordant read pairs that aligned to both the human genome and a viral genome. We have tested our techniques on tissue samples that were infected with either Merkel Cell Polyomavirus or Epstein-Barr virus, as determined using quantitative polymerase chain reaction (qPCR) or immunohistochemistry (IHC) techniques, and have successfully detected these viruses and identified viral integration loci. Overall, this viral hybrid capture probeset provides the ability to simultaneously determine a tissue sample's infection and viral integration status alongside other somatic genomic analyses, saving both time and sample material.

#563

Quantitative EMT expression score for predicting survival outcome.

Jason T. George, Mohit K. Jolly, Herbert Levine. _Rice University, Houston, TX_.

Tumor aggressiveness and subsequent metastasis still remain as major limitations to curative treatments for cancer patients. The epithelial to mesenchymal transition (EMT) is a process that occurs naturally during embryogenesis and has been linked to metastasis in many cancer types. EMT is characterized by phenotypic changes that allow for tissue extravasation and migration of cancer cells into the bloodstream. These changes may be linked to specific patterns in the gene expression signature of cells undergoing EMT. Recent theoretical efforts have predicted the existence of a stable, hybrid (E/M) phenotype which has also been observed experimentally in single cells. However, the effects of this hybrid phenotype on cancer patient survival have not been well characterized. The ability to quantify a patient's EMT status via a simple test involving a small collection of prognostic genes would provide an important tool for clinical risk stratification and treatment in the context of many cancer types. Here, we apply iterative statistical methods to generate an EMT score based on gene expression samples. We use this score to characterize the degree of the hybrid phenotype signature present in test samples. Predictions from our model are verified against cell lines with known EMT status. Lastly, we apply our model to clinical samples in order to assess survival differences in various EMT groups. We demonstrate that in many cases, EMT status successfully classifies patients into groups with statistically significant differences in survival, which is of immediate clinical relevance.

#564

Binary classification of superparamagnetic relaxometry data for cancer screening.

Javad Sovizi, Sara L. Thrower, David Fuentes, Wolfgang Stefan, John D. Hazle, Kelsey Mathieu. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Introduction: Superparamagnetic relaxometry (SPMR) is an emerging technology that holds potential for use as a second-line screening modality to improve early cancer detection. During SPMR scanning, targeted superparamagnetic iron oxide nanoparticles (SPIONs) specifically bind to cancer cells and their spatial distribution can be characterized by measurement of the magnetic field relaxation following a brief excitation pulse. Highly sensitive superconducting quantum interference devices (SQUIDs) detect relaxation of clusters of SPIONs bound to small tumors. Challenges inherent to the SPMR technology include measurement noise, as well as the competing influence of SPION uptake by healthy organs (namely the liver), which also contributes to the overall SPMR signal. Hence, manual and stand-alone classification of the SPMR data into positive (i.e., the subject has cancer) or negative (i.e., the subject does not have cancer) screen results can be erroneous.

Methods: We employed a data-driven approach based on Gaussian process (GP) formulation tailored to SPMR datasets to systematically quantify the probability of cancer. In silico, we simulated the SPION uptake process and generated SPMR signals that closely resembled experimental data collected in mouse models of cancer. We investigated the classification accuracy for different amounts of SPION accumulation within the tumor, as well as different levels of measurement noise (coefficient of variation (CV)). In a phantom study, a mouse liver was simulated by clustering together nine cotton swabs containing a total of 150 μg of immobilized SPIONs, while a mouse tumor was simulated by a single cotton swab containing either 9.4 μg or 14.4 μg of immobilized SPIONs. An additional nine cotton swabs containing 32.3 μg of immobilized SPIONs (<5 μg per phantom) were evenly distributed within the scan plane to represent background SPIONs not bound to the tumor or liver. For each of the tumor phantoms, 18 datasets were collected using a magnetic relaxometry device (Senior Scientific LLC) by moving the phantom to 18 different locations. Moreover, 10 datasets were collected without using the tumor phantom to represent the expected signal from healthy mice. In each iteration, the background SPION phantoms were randomly relocated within the scan plane.

Results: Our in silico analysis for tumor accumulations of 3% and 5% of the injected SPION dose achieved 87% and 97% classification accuracies, respectively, when CV=0 and 75% and 93% when CV=0.015. Similarly, in our phantom study, classification accuracies of 87.5% and 96.4%, respectively, were reported for the 9.4 μg and 14.4 μg tumor phantoms.

Conclusion: Using a data-driven GP model, tumor-status classification accuracies of up to 96.4% were achieved in SPMR phantom datasets. In the future, we plan to evaluate the accuracy of our classifier in preclinical settings using animal datasets.

#565

Analytical performance of TruSight® Tumor 170 in the detection of gene fusions and splice variants using RNA from formalin-fixed, paraffin-embedded (FFPE) solid tumor samples.

Tingting Du, June Snedecor, Jennifer S. LoCoco, Xiao Chen, Laurel Ball, Allan Castaneda, Danny Chou, Katie Clark, Brian Crain, Anthony Daulo, Manh Do, Sarah Dumm, Yonmee Han, Mike Havern, Chia-Ling Hsieh, Tingting Jiang, Suzanne Johansen, Scott Lang, Rachel Liang, Jaime McLean, Yousef Nassiri, Austin Purdy, Jason Rostron, Jennifer Silhavy, Natasha Talago, Li Teng, Kevin Wu, Clare Zlatkov, Chen Zhao, Ali Kuraishy, Karen Gutekunst, Sohela De Rozieres, Matthew Friedenberg, Anne C. Jager, Han-Yu Chuang. _Illumina, San Diego, CA_.

Recent studies have highlighted the importance of gene fusions and splice variants in solid tumor profiling1. Next-generation sequencing can be an effective means of detecting these alterations in FFPE samples using RNA rather than DNA, as a single chimeric RNA transcript could result from numerous alterations in DNA2. To that end, Illumina developed TruSight® Tumor 1703, a comprehensive, hybrid capture-based NGS assay targeting 170 key cancer genes. Along with a DNA workflow, the assay includes a RNA workflow for the identification of splice variants and gene fusions. Following sequencing on the NextSeq® or HiSeq® instruments, TruSight® Tumor 170 offers an analytical pipeline which initiates variant calling. These algorithms were first optimized against the simulated read data from >350 fusions and splice variants reported in the RNA content of the gene panel. A hybrid approach of read alignment and assembly was used to enhance the fusion calling sensitivity. Deliberate filters were designed to reduce false positive calling from sequence homologs, polymerase read-through, or FFPE artifacts. For splice variant calling, a panel of FFPE non-cancerous samples were used to capture false positive mutation calls. With endogenous RNA splicing in cellular physiology, exon-boundary probes were added in the hybrid capture to enhance enrichment efficiency. To the best of our knowledge, there is not yet a standard definition for the limit of detection (LoD) in detecting gene fusions and splice variants from NGS data. We propose to define the LoD of a fusion calling and splice variant NGS panel as the lowest molecule count of a chimeric transcript that could be reliably detected with a sufficient number of supporting sequencing reads. To determine the LoD of TruSight® Tumor 170 using this definition, we mixed cell lines expressing a panel of known fusions and splice variants to measure the copy number of each chimeric transcript. Using these samples we examined the ability of the assay to confidently detect the alterations using 40 ng of RNA input. To demonstrate the analytical sensitivity and specificity of this NGS based assay, we compiled a panel of 49 mixed samples and validated the molecule count to be near the LoD of 5 copies per ng RNA input by PCR. The sensitivity was >98% for fusions and 100% for splice variants. For understanding the limit of blank (LoB) of the assay, another panel of 40 samples not harboring fusions and splice variants was also assessed by TruSight® Tumor 170. These samples demonstrated a ~97% specificity for fusion calling and >95% specificity for splice variant calling. These results indicate that the TruSight® Tumor 170 panel analysis can identify lowly expressed fusions and splice variants from a small amount of compromised RNA from solid tumor samples at high analytical sensitivity and specificity.

1 Klijn et al. (2015) 2 Maher et al. (2009)

3 For Research Use Only.

#566

A pathway based drug selection for cancer precision medicine.

Varshini Vasudevaraja, Lijun Cheng, Sai Mounika Inavolu, Milan Radovich. _Indiana University, Indianapolis, IN_.

Background and Purpose:

Precision medicine tailors the right therapy to the right patient based on his/her tumor molecular profiles. In clinic and ongoing clinical trials, drug-targeted selection schemes depends more or less on drug targets' mutations or expression status. However, there is a lack of systematic pathway based method to connect individual genomics information with properly drug target and treatment in precision medicine. The paper aims to integrate various knowledge-bases, including cancer drugs, drug-targets and gene-gene regulatory pathway, to construct a drug target and properly drug selection algorithm for individual patient in precision medicine.

Materials and Methods:

In the drug selection algorithm, drug targets and properly drugs recommendation are based on the patient specific molecular profile data, including copy number variation, mutation, and gene expression. Our target and drug selections are based on whether these drug targets act as hub genes that either regulate or control the signaling pathways for many other genes in the biological pathways. Only those patients whose molecular profiles do not show targets, the usual chemotherapy treatment will be recommended. This novel algorithm is applied to individual Pancreatic Adenocarcinoma (PAAD) for drug target and drug selection. All of patients' genomic data obtained from the Cancer Genome Atlas (TCGA). The pathway information is from Pathway Commons and FDA approved cancer drugs and their targets is from DrugBank. The Cancer Cell Line Encyclopedia (CCLE) 46 pancreases cancer cell line are used to validate the algorithm result reliability.

Results:

Our algorithm identifies targets, such as ERBB2, CDK2, SRC, CDK9, SMAD2, CDK4, HDAC1, PPP1CA, AKR1B1, EGFR, IGF1R, AKT and MEK, for pancreatic adenocarcinoma cancer patients. In which, they cover the clinic first line effective drug targets, such as Gemcitabine for the Akt-mTOR signaling pathway (AKT) and CDK4/6 inhibitor and Erlotinib for HER2 Kinase Family (ERBB2, EGFR) activation inhibitor. In addition, new inhibitors include Src inhibitors (dasatinib, saracatinib and bosutinib), TGF beta inhibitor for target SMAD2 (galunisertib) and IGF-1R/insulin receptor inhibitors (ceritinib, brigatinib) are recommended accordingly and validated in pancreatic cancer cells.

Conclusion:

This novel algorithm might act as a better source for off-label drug selection and further cell line validations may help in providing a better treatment strategy in precision medicine.

## IMMUNOLOGY:

### Checkpoints 1

#567

CD38 blockade overcomes the immune resistance to anti-PD-L1 therapy by boosting CD8 T cell response.

Limo Chen, Lixia Diao, Yongbin Yang, Xiaohui Yi, Jaime Rodriguez, Youhong Fan, Leticia Rodriguez, Jared Fradette, Christin Ungewiss, Jonothan Roybal, Jingfen Zhu, Jing Wang, Lauren Byers, Stephen Ullrich, Ignacio Wistuba, John Heymach, Xiao-Feng Qin, Don Gibbons. _UT MD Anderson Cancer Ctr., Houston, TX_.

Although strategies incorporating immune checkpoint inhibition, e.g. PD-1/PD-L1 blockade, are achieving unprecedented successes and increasingly becoming incorporated into standard of care regimens for cancer patients, high rates of resistance still limit the potential efficacy. Therapeutic improvement requires a thorough understanding of the biological process of resistance. To date there have been few studies reporting mechanisms of resistance to PD-L1 blockade. We have explored the resistance mechanisms to functional PD-L1 loss in preclinical lung cancer models by using pharmacological and genetic approaches (PD-L1 blocking antibody treatment or CRISPR/Cas9-mediated deletion of PD-L1 on tumor cells). The molecular and immune profiles of the tumor microenvironment were evaluated in mutant K-ras/p53 (KP) GEM lung cancer models and multiple immunocompetent syngeneic models (both KP and Lewis lung cancer). Additionally, to determine the applicability of the results to patients with lung cancer, we analyzed 259 patient tumor specimens with IHC staining and evaluated the immune markers in TCGA datasets (adenocarcinoma and squamous) and the MD Anderson PROSPECT dataset. We observed that lung tumors gained resistance to anti-PD-L1 antibody treatment over time, and that the up-regulation of CD38 on tumor cells accounted for the treatment resistance. We also observed the same resistance mechanism caused by CD38 up-regulation in PD-L1 KO mice bearing PD-L1 KO Lewis lung tumors generated with the CRISPR/Cas9 system. Manipulation of CD38 on a panel of lung cancer cell lines, demonstrated that CD38 inhibits CD8+ T cell proliferation, antitumor cytokine secretion, and tumor cell killing capability in vitro and in vivo. Furthermore, to test whether CD38 blockade might be therapeutically efficacious to counter anti-PD-L1 resistance, we applied the combination therapy of anti-CD38 and anti-PD-L1 in lung cancer animal models and demonstrated dramatic therapeutic benefit on primary tumor growth and metastasis. Bioinformatic analyses of the patient tumor databases revealed a strong correlation between CD38 expression and an immune suppressive inflammatory signature. Finally, in 259 lung cancer specimens, 18.5% of cases exhibited positive staining for CD38 on tumor cells. Based upon our studies, we conclude that the up-regulation of CD38 on tumor cells is a major mechanism of resistance to anti-PD-L1 therapy, and that CD38 is a novel immune checkpoint that inhibits CD8+ T cell function. The blockade of CD38 and PD-L1 is a rational combination to prevent immune resistance and increase the response rate for lung cancer patients.

#568

Regulatory T cell recruitment limits the effectiveness of checkpoint inhibition for claudin-low breast cancer.

Sarah C. Vick, Nicholas A. Taylor, Michael D. Iglesia, W June Brickey, Lisa A. Carey, Bentley R. Midkiff, Karen P. McKinnon, Shannon Reisdorf, Joel S. Parker, Charles M. Perou, Benjamin G. Vincent, Jonathan S. Serody. _University of North Carolina, Chapel Hill, NC_.

Introduction: Breast cancer can be separated into five intrinsic subtypes based on differences in the transcriptome of the tumor. We propose that the intrinsic differences of specific tumor subytpes lead to extrinsic differences in the tumor microenvironment.

Methods: We utilized human clinical and genetically engineered mouse model (GEMM) samples of the intrinsic subtypes luminal A, basal-like, and claudin-low breast cancers to evaluate the immune landscape in the tumor microenvironment by histology and microarray analysis. Our claudin-low GEMM was derived from BRCA-/-/p53-/- mice. The HER-2 overexpressing, basal-like, and luminal A models have been previously descried. We utilized the FoxP3-DTR transgenic mouse model as a method of regulatory T cell (Treg) depletion to evaluate their function in these GEMMs.

Results: The claudin-low human tumors were heavily infiltrated with immune cells, with CD4+ T cells being the most prominent, when compared to the luminal A subtype (P = 0.01). There were also increased focal areas of Tregs in human claudin-low tumors. To evaluate the mechanism for these findings, we utilized a GEMM of claudin-low tumors in addition HER-2 overexpressing, basal-like, and luminal A models. Mice with claudin-low tumors recruited elevated numbers of immune cells to the tumor microenvironment when compared to other breast cancer subtypes (P=0.01). Additionally, there was increased expression of multiple chemokine ligands in the tumor microenvironment among claudin-low tumors, with CXCL12 being the most highly overexpressed. Because the claudin-low tumors were heavily immune infiltrated, we hypothesized that blockade of the inhibitory checkpoint receptors programmed death-1 (PD-1) and cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) could delay tumor growth and improve anti-tumor immune response. Surprisingly, we saw no delay in tumor growth in the claudin-low model using checkpoint inhibition. To investigate if presence of Tregs limited the function of checkpoint inhibitors, mice with claudin-low tumors were treated with AMD3100, a CXCR4 inhibitor. This decreased Treg infiltration into the tumor but did not alter tumor growth. We then utilized the FoxP3-DTR transgenic mouse model, where depletion of Tregs alone resulted in a very modest decrease in tumor growth, while depletion of Tregs plus checkpoint inhibition significantly improved survival (P = 0.03) and increased cytokine production by CD8+ T cells.

Conclusion: We found that an effective anti-tumor immune response in claudin-low tumors is inhibited by the recruitment of Tregs to the tumor microenvironment. These data highlight early Treg recruitment as a possible mechanism for the lack of response to immune checkpoint inhibition therapy for claudin-low breast cancer.

#569

Dexamethasone inhibits T-cell proliferation through a CTLA-4 mediated pathway.

Marsha-Kay N. Hutchinson, Amber J. Giles, Heather M. Sonnemann, Caitlin M. Reid, Deric M. Park, Mark Gilbert. _National Institute of Health, Bethesda, MD_.

BACKGROUND: The use of corticosteroids for therapeutic benefit has to be weighed against the risks of adverse consequences associated with these drugs. Brain tumor patients in particular, are routinely prescribed dexamethasone (a glucocorticoid) to reduce edema associated with their lesion. Checkpoint blockade, a type of immune therapy, is currently being investigated as a potential treatment for brain tumors. However, glucocorticoid signaling has been shown to attenuate the immune response through several mechanisms including the repression of transcription of genes controlling pro-inflammatory cytokines and chemokines.

HYPOTHESIS: Here, we propose that dexamethasone's ability to upregulate inhibitory T-cell molecules such as CTLA-4 and PD-1 might be an additional immunosuppressive mechanism.

METHODS: Healthy donor T cells were tested for response to dexamethasone. T cell proliferation, cell cycle analysis, apoptosis, and protein expression were assessed with flow cytometry. Protein expression was also measured with Western blots. Transcriptional changes were assessed with qPCR. A monoclonal antibody, ipilimumab, was used to block CTLA-4 binding.

RESULTS: Unexpectedly, dexamethasone did not elicit a direct lymphotoxic effect on T cells as measured by absolute cell number. However, we found that dexamethasone significantly reduced T cell entry into the cell cycle, but did not impact cells already undergoing mitosis. Checkpoint molecules CTLA-4 and PD-1 were increased with dexamethasone treatment when cells are stimulated. Blockade of CTLA-4 with Ipilimumab resulted in a substantial reversal of cell cycle entry inhibition that was induced by dexamethasone.

CONCLUSIONS: These results suggest that dexamethasone impairs T cell expansion by inhibiting cell cycle entry. Upregulated CTLA-4 expression contributes to cell cycle entry blockade which is reversed by inhibiting CTLA-4 with ipilimumab. These findings indicate that administration of ipilimumab before dexamethasone diminishes the negative proliferative effect on anti-tumor T cells suggesting that when needed, corticosteroids can be used after immune checkpoint blockade has been established.

#570

Transparent tumor tomography (T3): 3D spatial immunoanalysis for PD-L1 immune checkpoint blockade therapy.

Steve Seung-Young Lee, Vytautas P. Bindokas, Stephen J. Kron. _The University of Chicago, Chicago, IL_.

Untangling the complexity of programmed death-ligand 1 (PD-L1) expression within a heterogeneous tumor microenvironment is an urgent challenge in PD-1/PD-L1 immune checkpoint blockade therapy. Here, we address this challenge with a method, termed transparent tumor tomography (T3), facilitating three-dimensional (3D) visualization and spatial analysis of distributions of multiple biomarkers regarding to cancer cells, vasculature, and immune cells in context in the tumor microenvironment. With T3 analysis of transgenic mouse mammary tumors immunostained against Her2, CD45, Ki-67, CD31, and PD-L1, we reveal that PD-L1 expression within the tumor microenvironment is highly adaptable for efficiently preventing immune cell infiltration into the tumor. Stronger correlation of Her2 and PD-L1 expression in the tumor periphery where has a high CD45+ immune infiltrate density is determined by tumor-wide analysis. Also, tomographic analysis shows blood vessels expressing PD-L1 in the tumor core, where PD-L1 expression is lower. Furthermore, high-resolution T3 image localizes PD-L1 expression to a region between the endothelium and the surrounding smooth muscle cells in blood vessels. We investigate spatial pharmacokinetics of anti-PD-L1 antibody in the whole mouse mammary tumor in the context of hypoxia, CD31+ blood vessels, and target PD-L1+ cells. The 3D anti-PD-L1 antibody distribution is fit to a two-compartment pharmacokinetic model, yielding estimated distribution half-life of 4.7 min and terminal half-life of 2.5 days. We also evaluate anti-tumor immune responses after PD-L1 blockade therapy using T3. We observe broad distribution of tumor infiltrating CD3+CD8+ cytotoxic T cells in 3D tumor section following combination therapy of radiation and anti-PD-L1 antibody compared to PBS, anti-PD-L1 antibody alone, or radiation alone treatment group. Moreover, we apply T3 for immunoanalysis of whole core needle biopsy. We spatially map PD-L1 expression and granzymeB-producing CD3+CD8+ cytotoxic T cells in pre-treatment and in-treatment core needle biopsies at cellular resolution and in three dimensions. Meanwhile, T3 analysis is nondestructive, allowing secondary analysis by IHC and/or IF. We anticipate that T3 can be applied broadly to facilitate preclinical studies of immunotherapy and also find use in spatial, multiparameter analysis of patient biopsies, particularly to improve predictive testing and analysis of immune responses to tumor immunotherapy.

#571

Meta-analysis of genomic predictors of response to immune checkpoint therapy in metastatic melanoma.

Diana Miao,1 David Liu,1 Daniel Keliher,1 Sachet Shukla,1 Bastian Schilling,2 Claire Margolis,1 Alicia Smart,1 Levi Garraway,1 Stephen Hodi,1 Dirk Schadendorf,2 Eliezer M. Van Allen1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _University Hospital Essen, Essen, Germany_.

Introduction: Immune checkpoint therapies benefit a subset of patients with metastatic melanoma, but ability to predict clinical outcomes is limited. This meta-analysis of genomic predictors of outcomes to aPD1 and aCTLA4 in melanoma combines 220 sequenced tumors from 3 published cohorts, aiming to validate existing hypotheses regarding response to immune checkpoint therapies and discover new relationships with greater power.

Methods: Genomic data and clinical annotations from published cohorts were analyzed with standardized pipelines for somatic variant calling, mutational signature deconvolution, and neoantigen prediction. Patients were stratified into clinical benefit (CB) and no clinical benefit (NCB) as described in Van Allen et al. 2015. Analyses were repeated using two other published response metrics (CB=PFS>6 months; CB=CR or PR).

Results: Nonsynonymous mutational burden was significantly higher in CB vs. NCB using all 3 response metrics, though significance was less pronounced using PFS alone (p<0.01 vs. p<0.0001; Wilcoxon rank sum), partially due to 3 patients with high mutational burden who experienced PR for <6 months, potentially representing early acquired rather than intrinsic resistance. To assess the impact of mutational processes contributing to overall mutational burden, we used a non-negative matrix factorization framework to infer mutational activity in tumors from 6 signatures previously seen in melanoma: aging (S1), T>C substitutions (S5), mismatch repair (S6), alkylating agents (S11), UV (S7), and T>G substitutions (S17). The proportion of mutations in S7 or S11 was positively correlated with mutational burden (Spearman's rho=0.66), while S5 and S1 were anti-correlated (rho=-0.62). In a multivariate logistic model, S7 and S11 activity were independent predictors of CB adjusting for mutational load (p<0.05), with the sum of S7 and S11 activity being a strong predictor (p<0.001). Of the patients with low mutational burden (<median) with CB, a large majority (23/29) had >1/2 of mutations in S7 or S11, compared to only 36/71 of low-mutation NCB (p<0.01; Pearson's chi-squared). Neoantigen burden was strongly correlated with mutational burden, and did not improve ability to predict CB. In examining mutations in specific genes, >500 genes were mutated more frequently in either CB or NCB (p<0.05, Fisher's exact). Restricting analysis to genes recurrently mutated in cancer and correcting for patient mutational burden by permutation, nonsynonymous mutations in ACSL3 and MET and truncating alterations in ARID2 were significantly enriched in CB.

Conclusions: In this meta-analysis of 220 patients, harmonized clinical and whole exome analysis confirmed that mutational burden correlates with CB from aPD1 and aCTLA4 therapy, while mutational signatures and alterations in specific genes potentially provide additional predictive power.

#572

Inhibition of IDO1 with epacadostat enhances anti-tumor efficacy of PD-1 blockade in a syngeneic glioblastoma (GBM) model.

David A. Reardon,1 Prafulla C. Gokhale,1 Sarah R. Klein,1 Kristen L. Jones,1 Paul T. Kirschmeier,1 Maria Speranza,1 Holly Koblish,2 Peggy Scherle,2 Lance Leopold,2 Robert Newton,2 Gordon J. Freeman1. 1 _Dana-Farber Cancer Inst., Boston, MA;_ 2 _Incyte Corporation, Wilmington, DE_.

Purpose: To determine if epacadostat, an oral indoleamine 2,3-dioxygenase (IDO1) inhibitor has therapeutic benefit against GBM when administered as single agent and with PD-1 blocking antibody.

Methods: An initial survival experiment was performed to assess efficacy and was followed by an identical repeat experiment for validation. 1X105 luciferized GL261 cells, a murine GBM tumor line derived from intracerebral methylcholanthrene implantation, were stereotactically implanted intracranially in albino syngeneic C57BL/6 mice. Mice with increasing bioluminescence on days 3 and 6 were randomized (n=8/group) to receive treatment beginning on day 6: anti-PD-1 (332.8H3, mouse IgG1; 500 μg intraperitoneal (IP) on day 6, 250 μg q 3 days X 7); epacadostat (Incyte Corporation, orally dosed at 300 mg/kg/day for 5 days on/2days off for 3 weeks); anti-PD-1 + epacadostat; and control therapy (isotype IgG antibody IP and 0.5% methocel in water). Tumor response assessments were performed by quantifying bioluminescence and survival. A re-challenge experiment was performed in long-term survivors to assess for tumor immune responses capable of preventing relapse. All long-term surviving mice (defined as ≥ 100 days) from the efficacy experiment were injected with 1X105 GL261 cells in the contralateral hemisphere and followed for survival.

Results: In both preclinical efficacy experiments, median survival in the epacadostat monotherapy group did not differ from controls (approximately 30 days). Four of 8 mice (50%) treated with anti-PD-1 were long-term survivors in both efficacy experiments. In the epacadostat plus anti-PD-1 combination group, 81% of the mice were long-term survivors (7 of 8 in experiment 1 and 6 of 8 in experiment 2). Of note, none of the long-term surviving mice developed evidence of tumor; thus the median survival among the anti-PD-1 and epacadostat plus anti-PD-1 combination groups were both > 100 days. In the re-challenge study, all of the mice who underwent GL261 re-inoculation survived > 100 more days with no evidence of tumor recurrence.

Conclusions: IDO1 inhibition with epacadostat increased the eradication rate of anti-PD-1 therapy in an orthotopic syngeneic GBM model and long term survivors rejected tumor following orthotopic re-challenge. Further combinatorial studies incorporating IDO inhibitor therapy for GBM, including mechanistic studies, are warranted.

#573

Metabolism as checkpoint: Induction of anti-tumor immune response with the novel glutamine antagonist JHU-083.

Robert D. Leone, Judson M. Englert, Min-Hee Oh, Chih-Hsien Cheng, Rana Rais, Barbara Slusher, Jonathan D. Powell. _Johns Hopkins Hospital, Baltimore, MD_.

The metabolic characteristics of the tumor microenvironment (eg, hypoxia, acidity, nutrient depletion, elevated adenosine) present a significant hurdle for immunotherapy. Dysregulated glutamine metabolism plays a crucial role in establishing this environment. We previously demonstrated that the novel glutamine antagonist, JHU-083, profoundly alters tumor metabolism and inhibits growth with minimal toxicity. Further studies have demonstrated that JHU-083 can reprogram the immune contexture of the TME leading to decreased regulatory T cells (Treg), increased CD8:Treg ratio, decreased MDSCs, suppressed adenosine, and decreased acidification and hypoxia. We now report that JHU-083 not only markedly enhances antitumor response to anti-PD-1 and adoptive cellular therapy (ACT) but also generates potent immune-mediated responses as a single agent. In an MC38 tumor model C57BL/6 mice treated from day 10 with concurrent JHU-083 and low-dose anti-PD-1 therapy showed robust antitumor activity with complete rejection and normal lifespan in 9/10 mice (vs. 0/10 complete responses with anti-PD-1 monotherapy). The combination regimen was well tolerated, causing only minimal, transient weight loss. In other studies, a therapeutic ACT model with OVA-expressing B16 melanoma cells was employed and showed that low-dose JHU-083 and adoptive transfer of activated OVA-specific T cells led to markedly suppressed tumor growth and increased survival versus vehicle treated controls. In the course of these studies we found that incrementally increasing the dose of single agent JHU-083 generated 100% complete response rates (5/5) with 60% of mice rejecting tumor upon rechallenge, strongly suggesting an immune-mediated rejection had been established. Indeed, subsequent tumor studies in WT and RAG-/- mice confirmed that anti-tumor response to single agent JHU-083 was critically dependent on the adaptive immune system. This was unexpected as glutamine is known to be essential for T cell expansion. To this end, we used a vaccinia-OVA model and found that low-dose JHU-083 administered in the peri-vaccination period had minimal suppressive effect on OVA-specific CD8 expansion, but importantly, promoted generation of robust memory T cells with higher T-Bet expression and increased mTORC1 activity upon rechallenge. These studies demonstrate that, while the dosing and schedule of JHU-083 employed can disrupt tumor metabolism and markedly inhibit tumor growth, these same doses can promote long-lived changes in T cells that enhance memory response. Overall, our studies indicate that glutamine antagonism within tumors ameliorates critical checkpoints to immune function, dissipating metabolic suppression of immune-mediated anti-tumor responses. As such, targeting tumor metabolism can "unleash" endogenous anti-tumor responses as monotherapy and can markedly enhance anti-PD1 checkpoint blockade and adoptive cellular therapy.

#574

Phosphatidylserine targeting antibody in combination with tumor radiation and immune checkpoint blockade promotes anti-tumor activity in mouse B16 melanoma.

Sadna Budhu,1 Olivier De Henau,1 Roberta Zappasodi,1 Rachel Giese,1 Luis F. Campesato,1 Christopher Barker,1 Bruce Freimark,2 Jeff Hutchins,2 Jedd D. Wolchok,1 Taha Merghoub1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Peregrine Pharmaceuticals, Inc., Tustin, CA_.

Phosphatidylserine (PS) is a phospholipid that is exposed on surface of apoptotic cells, tumor cells and tumor endothelium. PS has been shown to promote immunosuppressive signals in the tumor microenvironment. Antibodies that target PS have been shown to reactivate anti-tumor immunity by polarizing tumor associated macrophages into a pro-inflammatory M1 phenotype, reducing the number of MDSCs in tumors and promoting the maturation of dendritic cells into functional APCs. In a mouse B16 melanoma model, targeting PS in combination with immune checkpoint blockade promoted greater anti-tumor activity than either agent alone. This combination was shown to enhance CD4+ and CD8+ T cell infiltration and activation in the tumors of treated animals. Radiation therapy (RT) is an effective focal treatment of primary solid tumors, but is less effective in treating metastatic solid tumors as a monotherapy. There is evidence that RT induces immunogenic tumor cell death and enhances tumor-specific T cell infiltration in treated tumors. The abscopal effect, a phenomenon in which tumor regression occurs outside the site of RT, has been observed in both preclinical and clinical trials when RT is combined with immunotherapy. In this study, we show that irradiation treatment of B16 melanoma causes an increase in PS expression on the surface of viable tumor and immune infiltrates. We subsequently examined the effects of combining RT with an antibody that targets PS (mch1N11) and immune checkpoint blockade (anti-PD-1) in B16 melanoma. We found that treatment with mch1N11 synergizes with RT to improve anti-tumor activity and overall survival in tumor bearing mice. In addition, the triple combination of mch1N11, RT and anti-PD-1 treatment displayed even greater anti-tumor and survival benefit. Analysis of local immune responses in the tumors of treated animals revealed an increase in tumor-associated macrophages with a shift towards a pro-inflammatory M1 phenotype after treatment with RT and mch1N11. In addition, analysis of the systemic immune responses in the spleen and tumor draining lymph nodes revealed an increase in CD8 T cell activation, effector cytokine production and differentiation into effector memory cells in the triple combination. This finding highlights the potential of combining these three agents to improve outcome in patients with advanced-stage melanoma and other cancers and may inform the design of clinical studies combining PS-targeting antibodies with RT and/or checkpoint blockade.

#575

PD-L1 positive tumor-infiltrating lymphocytes and mutational load in breast cancer.

Marcelo Sobral-Leite,1 Koen Van de Vijver,1 Magali Michaut,1 Hugo M. Horlings,1 Tesa M. Severson,1 Philip C. Schouten,1 Rianne van der Linden,1 Kelly Kersten,1 Anna Marie Mulligan,2 Nayana Weerasooriya,2 Joyce Sanders,1 Ashley Cimino-Mathews,3 Dennis Peters,1 Gerrit K. Hooijer,4 Erik Hooijberg,1 Annegien Broeks,1 Rene Bernards,1 Sabine Linn,1 Irene L. Andrulis,2 Marc J. van de Vijver,4 Lodewyk F. Wessels,1 Marleen Kok,1 Karin E. de Visser,1 Marjanka K. Schmidt1. 1 _The Netherlands Cancer Institute, Amsterdam, Netherlands;_ 2 _University of Toronto, Toronto, Ontario, Canada;_ 3 _The Johns Hopkins Hospital, Baltimore, MD;_ 4 _Academic Medical Center, Amsterdam, Netherlands_.

Background: PD-1 blockade has emerged as an effective treatment for a subset of cancer patients. Studies have shown that PD-L1 expression is associated with likelihood of response to PD-1 blockade. In order to select the right breast cancer patient for immunotherapy, characterization of the immune landscape of breast tumors is required. Therefore, we assessed PD-L1 expression and tumor-infiltrating lymphocytes (TILs) in different breast tumor subtypes and the link with prognosis. We also sequenced a panel of genes to assess the mutational load in triple negative tumors (TNBC) and investigate the association with PD-L1 positive TILs.

Material and methods: We analyzed 438 tumor samples from breast cancer patients of all ages treated between 1986 and 2007 with surgery, with or without adjuvant therapy. PD-L1 was stained using whole slide specimens (E1L3N® antibody) after methodological validation. Pathologists quantified TILs based on International TILs Working Group recommendations and scored PD-L1 based on the percentage of positive (tumor and/or immune) cells; as negative if 0%, positive if ≥1%, and high if >50%. Mutational load was assessed based on DNA kinome sequencing. Associations were measured by Cox/logistic regression model, including pathological variables. Multiplex imaging of 20 immune-infiltrated areas from four ER negative tumors were performed using the Vectra® system based on immunofluorescence staining panel of: CD4, CD68, CD8, FOXP3 and PD-L1.

Results: PD-L1 expression and TILs were higher in ductal (compared with lobular), high grade and estrogen receptor (ER)-negative tumors (p<0.001). TILs (density ≥5%) were significantly associated with worse distant metastasis-free survival (DMFS) only in ER-positive tumors (n=204): HR=2.72; 95%CI: 1.07-6.94. PD-L1 positivity (≥1%) followed the same trend: HR=1.66; 95%CI: 0.87-3.15. However, in ER-negative tumors (n=171), high PD-L1 expression (>50%) was significantly associated with better DMFS: HR=0.51; 95%CI: 0.27-0.98. TNBC with high PD-L1 expression of TILs (>50%) showed an association with increased mutation load (p=0.019) and a trend for better DMFS (HR=0.41; 95%CI: 0.16-1.04) compared with tumors lacking TILs. Further characterization of PD-L1 positivity in the immune-infiltrated cells was conducted by a multiplex imaging analysis. Preliminary results indicated that PD-L1 is expressed in CD68+, CD4+, FOXP3+ and CD8+ immune-cells.

Conclusion: Our findings suggest that PD-L1 positive TILs are associated with worse prognosis in ER-positive breast cancer and with better outcome in ER-negative group. In TNBC, high mutational load correlates with high PD-L1 positive TILs.

#576

Neoadjuvant immunotherapy improves efficacy of image-guided thermal ablation to generate curative responses in a murine breast cancer model.

Matthew T. Silvestrini, Elizabeth S. Ingham, Lisa M. Mahakian, Azadeh Kheirolomoom, Yu Liu, Brett Z. Fite, Sarah M. Tam, Samantha Tucci, Katherine D. Watson, Andrew W. Wong, Arta M. Monjazeb, Neil E. Hubbard, William J. Murphy, Alexander D. Borowsky, Katherine W. Ferrara. _UC Davis, Davis, CA_.

Magnetic resonance-guided focused ultrasound (MRgFUS) facilitates local tumor control via thermal ablation, however, the anti-tumor immune effects induced are weak and unable to consistently generate robust objective responses in distant lesions. Here, we set out to optimize a therapeutic approach for employing immunotherapy with thermal ablation for systemic cancer treatment. We assessed the efficacy of implementing MRgFUS ablation with blockade of the PD-1/PD-L1 axis (anti-PD-1) and activation of TLR9 (CpG oligonucleotide) under various protocols and in multiple models of murine cancer. Anti-PD-1 (200 µg, i.p., days 21 & 28) and CpG (100 µg, i.t., days 21, 24 and 28) were administered coincidentally with MRgFUS ablation (3 MHz central frequency, circular pattern with R=2 mm, 1 revolution per second, 65ºC for 1 min, days 21 and 28) over the course of a week in bilateral syngeneic neu deletion line (NDL), 4T-1 and B16 tumor bearing mice. Additionally, we evaluated the administration of immunotherapy prior to a course of thermal ablation (i.e., "primed ablation"), where anti-PD-1 (as above on days 21, 28 & 35), CpG (as above on days 21, 24, 28, 31, 38 and 45) and MRgFUS ablation (as above on days 31, 38 and 45) were administered in bilateral NDL tumor-bearing mice. Primed ablation generated a robust anti-tumor immune response in distant lesions two weeks after the start of treatment, where a threefold increase in tumor infiltrating leukocytes (reaching 40% CD8+ and 20% CD4+ T-cells) was observed. This led to a complete response in 80% of treated mice within 70 days after treatment commenced. This effect was also observed in animals with high tumor burden and when thermal ablation was performed sequentially at multiple independent sites; 80% of untreated lesions were eradicated at 50 days after the start of treatment. However, therapeutic efficacy was limited when thermal ablation was performed coincident with the first dose of immunotherapy; this protocol was not curative in any murine model. To elucidate the mechanism for this effect, we employed tumor histology and positron emission tomography immediately after MRgFUS ablation. We found that thermal ablation induced stromal inflammation, and the loss of cell-cell adhesion and local vascular integrity, which impacted the intratumoral transport of small molecules and proteins for 48 hours post treatment. These data suggest that tumor debulking using image-guided thermal therapy can be successfully incorporated within a curative protocol in which immunotherapy is initiated before ablation.

#577

Antagonism of the co-inhibitory receptor BTLA enhances efficacy of anti-PD-1 treatment in murine syngeneic tumor models.

Nathan R. Miselis,1 Douglas Linn,1 Clifford Restaino,1 Toya Baral,2 Jane Xia,2 Roanna Ueda,2 Anandi Sawant,2 Jeanne Baker,2 Gopalan Raghunathan,2 Xinzhong Wang1,1 Edward Bowman,2 Selvakumar Sukumar2. 1 _Merck & Co., Inc.,, Boston, MA; _2 _Merck & Co., Inc.,, Palo Alto, CA_.

Anti-PD1 therapies show a remarkable improvement in response over many standard of care regimens, but there is a significant need to further increase patient responses. Recent clinical studies show improved response rate when combining anti-PD1 and anti-CTLA4 therapies. This provides "proof of concept" that the combination of two immunotherapies can enhance efficacy. The goal of our work was to evaluate the anti-tumor efficacy of B and T lymphocyte attenuator (BTLA) antagonism with anti-PD1 therapy in preclinical mouse tumor models. BTLA is an inhibitory co-receptor that modulates T cell function and is a marker of "exhausted" T cells. The inhibitory signal mediated by BTLA is initiated following engagement with herpesvirus entry mediator (HVEM), a ubiquitous receptor that is highly expressed on malignant cells. One of the challenges to evaluating BTLA in mouse models is the presence of strain-specific allelic polymorphisms. These sequence differences occur adjacent to the HVEM binding sites and can interfere with the ability of certain antibodies to recognize BTLA in different strains of mice or block the HVEM-BTLA signaling. C57BL/6-specific and strain agnostic BTLA binding antibodies are commercially available, but none antagonize HVEM signaling through BTLA in BALB/c mice. To more comprehensively evaluate the anti-BTLA/anti-PD1 combination regimen we generated a first-in-class BALB/c strain polymorphism specific BTLA antagonist monoclonal antibody (40E4) and identified clone PJ196 as a C57BL/6 strain polymorphism specific antagonist. We show that 40E4 and PJ196 not only block BTLA:HVEM interaction as measured by cell ELISA, but also reverse HVEM-mediated suppression in primary T cells from the appropriate mouse strain. An effector function reduced mouse IgG1 (D265A) chimeric form of 40E4 was generated for in vivo use. In combination with anti-PD1 (mDX400), 40E4 mIgG1 (D265A) shows improved tumor growth inhibition (TGI) over mDX400 alone in preclinical subcutaneous mouse models of breast cancer (EMT6) and colon cancer (CT26). To show that anti-BTLA enhancement of the anti-tumor response to mDX400 is not unique to BALB/c mice, we tested the combination of a C57BL/6 BTLA strain-polymorphism specific monoclonal antibody (PJ196) with mDX400. Using the subcutaneous MB49 model of bladder cancer, we show that treatment with PJ196 and mDX400 significantly improved TGI over mDX400 alone. This is the first report evaluating a BTLA antagonist monoclonal antibody in preclinical mouse tumor models and the first report to show BTLA antagonism enhances the effect of anti-PD1 therapy.

#578

Discovery and characterization of novel antagonistic antibodies that bind with high affinity to human, cynomolgus, and murine TIGIT, an immune checkpoint receptor.

Julia C. Piasecki,1 Kenneth Brasel,2 Robert Rosler,1 Kevin M. Klucher,3 Scott R. Peterson1. 1 _Cascadian Therapeutics, Inc., Seattle, WA;_ 2 _Unaffiliated, Seattle, WA;_ 3 _Unaffiliated, CA_.

TIGIT is a recently identified coinhibitory immune checkpoint receptor expressed on NK, effector T, and regulatory T cells. In the oncology setting, TIGIT is upregulated on tumor infiltrating immune cells and is co-expressed with exhaustion markers including PD-1, TIM-3 and LAG-3 on infiltrates. TIGIT binds at least two ligands, CD155 and CD112, which are expressed on antigen presenting cells and other tissues, including tumor cells. These ligands also bind the activating receptor CD226, often co-expressed with TIGIT, creating a network that modulates adaptive and innate immune response in a manner analogous to the CD28-CTLA4-CD80-CD86 network. The absence of TIGIT signaling, resulting from genetic deficiency or blockade, enhances anti-tumor immunity in murine models, suggesting that disruption of TIGIT signaling may have clinical utility. To explore this concept, yeast antibody display was used to identify fully human, anti-TIGIT antibodies that block binding to ligands. Multiple rounds of selection with human and mouse TIGIT protein were performed to promote species cross-reactivity, diversity and affinity. A pool of 695 unique clones were screened for binding to TIGIT protein; 65 clones were then selected for further evaluation. Of the 65, 63 competed with CD155 for binding to TIGIT in a ForteBio screen. Fifty-three clones bound cyno TIGIT and 25 bound TIGIT from all three species (human, cyno, mouse). Antibodies bound endogenous TIGIT on primary T cells and blocked binding of ligands to cell surface expressed TIGIT in a dose dependent manner. Twelve clones showed functional activity in a TIGIT blockade bioassay and showed synergy with anti-PD-1 antibody in a PD-1/TIGIT combination bioassay. Activity in the bioassays correlated with affinity for recombinant and cell surface expressed TIGIT. Based on species cross-reactivity, binding affinity and activity in the bioassays, a lead candidate antibody was selected and produced as mouse IgG1 and IgG2a chimeras for testing in mouse tumor models. The chimeric antibodies behaved similarly to the parent clone in vitro exhibiting high affinity for TIGIT, competition with ligand for binding to TIGIT, and functional blockade of CD155-TIGIT interaction. Evaluation of the chimeric anti-TIGIT candidates alone and in combination with anti-PD-1 antibody in mouse syngeneic tumor models is ongoing, and results will be reported at the meeting. Antibody mediated blockade of coinhibitory immunoreceptors has proven clinically efficacious and supports the development of antibodies that target TIGIT. The unique human, non-human primate, and murine cross-reactive TIGIT-specific antibodies described here offer a simplified preclinical development path and the functional activity of these molecules supports their consideration as candidates for therapeutic development.

#4600

**Potent and selective C-C chemokine receptor (CCR4) antagonists potentiate anti-tumor immune responses by inhibiting regulatory T cells (T** reg **).**

Oezcan Talay, Lisa Marshall, Cesar Meleza, Maureen K. Reilly, Omar Robles, Mikhail Zibisky, Abood Okal, Lisa Seitz, Jenny McKinnell, Scott Jacobson, Erin Riegler, Emily Karbaz, David Chian, Angela Wadsworth, Paul Kassner, David Wustrow, Jordan S. Fridman. _FLX Bio, Inc., South San Francisco, CA_.

Naturally suppressive CD4+ Foxp3+ Treg are essential for immune tolerance. Although Treg-mediated suppression of effector cells is important to control inflammation and prevent autoimmune diseases, the presence of Treg in the tumor microenvironment (TME) has been shown to dampen anti-tumor immune responses. Human Treg express CCR4, the receptor for the chemokines CCL17 and CCL22. These chemokines are produced by tumor cells, tumor-associated macrophages and dendritic cells, as well as by effector T cells (Teff). Preclinical and clinical data supports a role for CCR4-mediated recruitment and accumulation of Treg in the TME which can be associated with poor prognosis. Further, recent longitudinal studies in patients receiving IO agents demonstrate an influx of Treg in responding patients which may dampen optimal anti-tumor responses. Therefore, CCR4 is an ideal target to selectively block Treg recruitment into the TME.

We have developed structurally unique series of small molecule antagonists of CCR4. These antagonists have cellular potencies in multiple assays (e.g. chemotaxis of primary human Treg in 100% serum) in the low double-digit nM range. Representative compounds are selective against other chemokine receptors, GPCRs and ion channels, including the hERG channel, and lack inhibition of common human CYP450 enzymes. Moreover, compounds have excellent in vitro and in vivo ADME properties, consistent with convenient oral dosing. In preclinical syngeneic tumor models, these CCR4 antagonists block Treg migration and support expansion of activated Teff. In contrast to the non-selective approach of depleting anti-CCR4 antibodies, our compounds reduce Treg in the tumor, but not in peripheral tissues such as blood, spleen or skin. In preclinical efficacy studies, CCR4 antagonists potentiate the anti-tumor effects of various checkpoint inhibitors and immune stimulators such as anti-PD-L1 and anti-CD137 antibodies. We observe enhanced tumor growth inhibition and increased tumor regressions when these agents are combined with CCR4 antagonists, without any gross toxicity. Further characterization of these CCR4 antagonists and their anti-tumor effects will be described.

#580

Nivolumab treatment of metastatic renal cancer patients impairs Tregs and potentiates NK function: The role of CXCR4 inhibition ("REVOLUTION Trial").

Sara Santagata, Anna Maria Trotta, Maria Napolitano, Luigi Portella, Sabrina Rossetti, Sisto Perdona, Sandro Pignata, Stefania Scala. _National Cancer Inst. Pascale, Naples, Italy_.

Despite encouraging results, Nivolumab response is not as wide as expected in renal cancer ( RCC). Among mechanisms of immunoresistance T-regulatory cells(Tregs) activity plays a central role. We previously showed that tumoral Tregs in RCC patients are more suppressive than healthy donors Tregs. Since tumoral Tregs express high level of CXCR4, the receptor antagonism was evaluated on patients Tregs revealing that CXCR4 antagonism reverted Treg suppressive activity( Santagata et al, submitted manuscript). To identify biomarkers informative and predictive of nivolumab efficacy Treg function, tumoral access and NK interactions was determined in nivolumab treated mRCC patients (" Revolution" trial). At today eleven patients were analyzed for Tregs and NKs function at time 0-2-4 weeks-3-6 months of treatment. 2 patients died for unrelated causes, 5 Patients showed PR, 4 SD and 1 PD at six months. At time 0 Tregs function, evaluated as inhibition of T-effector (Teff) proliferation, varied among patients; two patients showed anergy reversed by Nivolumab treatment. Overall, increase in Teff proliferation, compatible with a decreased Tregs activity, was revealed during nivolumab treatment and, while the total peripheral Tregs was unaffected, a decrease in CD4+CD25+127lowFOXp3 highCD45RA - (suppressive Tregs) was detected. NK activity, evaluated as CD107a exposure, increased with nivolumab treatment with a concomitant reduction in the inhibitory receptors CD158a, PD-1 and CXCR4. Moreover patients Tregs and NKs were evaluated in vitro for the efficacy of CXCR4 inhibition in affecting cell function. CXCR4 antagonists suppressed the Treg mediated inhibition of Teff proliferation. In conclusion the evaluation of 11 patients enrolled in the REVOLUTION study revealed detectable variations on Tregs and NKs function relatively to Nivolumab treatment to be correlated with clinical outcome. Tregs suppressive function was impaired by inhibition of CXCR4 receptor.

#581

Discovery and development of COM701, a therapeutic antibody targeting the novel immune checkpoint PVRIG.

Ofer Levy,1 Chris Chan,2 Gady Cojocaru,1 Spencer Liang,2 Eran Ophir,1 Sudipto Ganguly,3 Maya Kotturi,2 Tal Friedman,1 Benjamin Murter,3 Liat Dassa,1 Ling Leung,2 Shirley Greenwald,1 Meir Azulay,1 Sandeep Kumar,2 Zoya Alteber,1 Xiaoyu Pan,3 Andy Drake,2 Ran Salomon,1 Arthur Machlenkin,1 John Hunter,2 Zurit Levine,1 Drew Pardoll,3 Mark White2. 1 _Compugen Ltd., Holon, Israel;_ 2 _Compugen Inc, USA, SSF, CA;_ 3 _Bloomberg~Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University, Baltimore, MD_.

Background: While blockade of the CTLA4 and PD1 pathways has emerged as an effective treatment of cancer, the majority of patients do not derive long term benefit. This provides a rationale for identifying and targeting additional checkpoints. Employing our unique computational algorithms, we identified PVRIG, a new member of the B7/CD28 family. We report here the expression pattern, functional characterization, and anti-tumor activity of blocking antibodies targeting PVRIG as well as characterization of PVRIG KO mice.

Materials and Methods: PVRIG is expressed by T and NK cells within the tumor microenvironment. We identified PVRL2 as its counterpart and characterized the PVRIG-PVRL2 interaction. Antibody discovery was carried out with phage display and hybridoma platforms and antibodies against the human protein were screened for their ability to enhance T-cell activity in vitro, while surrogate antibodies targeting the mouse protein were assessed in syngeneic models for effects on tumor growth. PVRIG -/- KO mice were generated and characterized including phenotyping and anti-tumor immune response.

Results: PVRIG is expressed on different T cell subsets and on NK, NKT and γδ T-cells. Within T cells, memory subsets possess the highest level of PVRIG and its expression is induced upon long term activation with different stimuli. Within tumor microenvironment, PVRIG was found to be expressed on NK and CD8+ T cells in multiple cancers. A high affinity lead Ab was selected, COM701, for further clinical development and demonstrated blockade of the interaction of PVRIG with PVRL2 as well as enhancement of activation of both primary and tumor-derived effector immune cells through a PVRL2-dependent mechanism. Moreover, COM-701 showed notable enhancement of T cell function in-vitro when combined with PD1 or TIGIT Ab blockade. The lead antibody, COM-701, is currently in preclinical development. A surrogate antibody, that blocks PVRIG-PVRL2 interaction, was shown to inhibit growth of colon carcinoma and melanoma in syngeneic models upon combined treatment with anti-PDL1 antibody. Comparative analysis of PVRIG KO versus WT derived T cells revealed enhanced reactivity of PVRIG null T cells upon polyclonal activation in presence of PVRL2-Ig. Accordingly, MC38 tumors grew slower in PVRIG KO than in WT mice and ex vivo analysis pointed to the quantitative and functional differences in anticancer immunity developed in these mice.

Conclusion: We describe the identification of PVRIG as a novel T cell immune checkpoint. We further demonstrate that antibody blockade of the PVRIG-PVRL2 interaction has the potential to be efficiently combined with PD1 or TIGIT blockade for enhancing anti-tumor immunity. COM-701 is a high affinity antagonistic antibody that is currently in preclinical development. Taken together, these data demonstrate the utility of targeting PVRIG in addition to other B7 family checkpoints for the treatment of cancer.

#582

Circulating tumor DNA (ctDNA) variant allele frequencies are reduced in responders to durvalumab and low baseline variant allele frequencies are associated with improved overall survival in NSCLC patients.

Michael A. Kuziora, Brandon W. Higgs, Philip Brohawn, Rajiv Raja, Koustubh Ranade. _Medimmune, Gaithersburg, MD_.

Mutations can influence patient responses to treatment in non-small cell lung cancer (NSCLC). Monitoring such mutations can be critical to guiding treatment decisions, however, accessing sufficient biopsy material for mutation analysis can be challenging. Circulating tumor DNA (ctDNA) in the plasma offers a robust and highly sensitive approach to identifying mutations. Further, variant allele frequencies (VAFs) in individual mutations represent the number of cancer clones harboring a mutation, thus providing an indicator of tumor burden. Here we evaluated baseline mutation spectrum in genes implicated in cancer for association with clinical outcomes to durvalumab, an anti-PDL1 molecule, and examined changes in VAFs, as a surrogate for tumor burden, after treatment with durvalumab in NSCLC patients.

CP1108/NCT01693562 was a nonrandomized phase 1/2 trial evaluating durvalumab in patients with advanced NSCLC or other solid tumors. By 29APR2016, 304 NSCLC pts received 10 mg/kg Q2W of D ≤12 months with median 18.8 months follow up. A panel of 70 genes was assayed for nonsynonymous mutations and copy number (CN) variants using the Guardant360 cancer panel in plasma ctDNA from 115 NSCLC patients pre-treatment and 28 patients pre and 8 weeks post-treatment with durvalumab (Q2W). The mean VAF of all mutations harbored by a patient was correlated with clinical outcomes. Objective response rate (ORR) was calculated according to RECIST v1.1 and a Cox proportional hazards model was calculated adjusting for baseline ECOG, gender, age, smoking status, therapy lines, histology, and number of metastases.

Partial responders (PRs) showed a significant decrease (Δ= -2.7%, p=0.005) in ctDNA mean VAF post-treatment with durvalumab (i.e. reduction in tumor burden) compared to an increase in mean VAF (i.e. increase in tumor burden) in progressive disease (PD) patients (Δ=+1.7%, p=0.17). This correlation was also observed in total mutation count in PR (Δ=-5.3, p=0.037) compared to PD patients (Δ=+2.7, p=0.003). In a PD patient, the VAF of T790M EGFR mutation doubled after 6 weeks of durvalumab treatment. Patients with VAFs below the median VAF or no CN gains had longer median overall survival (15.7 months, 95% CI=[11.2,not reached] ) compared to those with VAFs above the median or CN gains (5.1 months 95% CI=[3.8,9.4]; HR=0.29; p=0.0005), even after adjustment for clinical covariates.

CtDNA VAFs were consistently reduced in responders but not non-responders after eight weeks of durvalumab. Below median pre-treatment VAFs or no CN gains in ctDNA correlated with longer overall survival in patients treated with durvalumab. Because we examined oncogenes and tumor suppressors, increased VAF in ctDNA of these genes may help to identify aggressive, and difficult to control tumors

#583

The CDK4/6 inhibitor abemaciclib induces synergistic immune activation and antitumor efficacy in combination with PD-L1 blockade.

Jack Dempsey,1 Lysiane Huber,1 Amelie Forest,2 Jennifer R. Stephens,1 Thompson N. Doman,1 Jason Manro,1 Andrew Capen,1 Robert S. Flack,1 Gregory P. Donoho,1 Sean Buchanan,1 Alfonso De Dios,1 Kyla Driscoll,2 Michael Kalos,2 Ruslan Novosiadly,2 Richard P. Beckmann,1 David A. Schaer2. 1 _Eli Lilly, Indianapolis, IN;_ 2 _Eli Lilly, New York, NY_.

Targeting cyclin dependent kinases 4 and 6 (CDK4/6) with inhibitors such as abemaciclib has shown promise in early and late phase clinical trials in both breast cancer and NSCLC. While there is evidence that patients benefit from single-agent abemaciclib, combination strategies leveraging this compound together with immunotherapy are of interest for the treatment of these and other cancers. Consequently, it is important to understand if and how a cell cycle inhibitor can be combined with immunotherapy. However, because most preclinical studies have been performed using xenograft tumors in immune-compromised mice, the potential immunomodulatory effects of abemaciclib have not been adequately ascertained. To investigate the immune combinatorial potential of abemaciclib, we studied the effects of treatment alone and in combination with checkpoint immunotherapy in a murine syngeneic tumor model sensitive to abemaciclib using immuno-competent mice. Abemaciclib monotherapy of established murine CT26 tumors, which harbor KRAS G12C mutation and CDKN2A deletion, caused a dose-dependent delay in tumor growth. Surprisingly, gene expression analysis showed that treatment was associated with an increase in intra-tumor immune inflammation without major alteration in immune subset frequencies. Testing of various dosing regimens in this preclinical model found that monotherapy abemaciclib pretreatment followed by combination with anti-PD-L1 antibody therapy, induced an enhanced anti-tumor response compared to abemaciclib and anti-PD-L1 monotherapies. Optimal combination therapy exhibited superior anti-tumor efficacy, resulting in complete tumor regression (CR) in 50-60% of mice in a setting where anti-PD-L1 monotherapy showed little or no efficacy (0% CRs). Mice which maintained CRs after cessation of combination therapy were able to resist later CT26 rechallenge, demonstrating that abemaciclib in combination with anti-PD-L1 enabled the generation of an immunologic memory. Examination of intra-tumor gene expression during treatment found that combination therapy further amplified the immune/T cell activation signature compared to both monotherapies. Intra-tumoral suppression of cell cycle genes, which are indicative of inhibition of CDK4/6, was also greater during the combination therapy, suggesting that the effects anti-PD-L1 therapy may augment the cell cycle arrest induced by abemaciclib. Although it was uncertain if agents that inhibit cell proliferation could be combined with immunotherapy, these preclinical results demonstrate that it is possible to combine CDK4/6 inhibition by abemaciclib with checkpoint immunotherapy to improve tumor efficacy. The synergistic responses observed in terms of tumor efficacy, immune activation, and cell cycle control provides support for the clinical investigation of this combination.

#584

From code to cure: Computational discovery of novel immune checkpoints.

Yair Benita, Amit Novik, Gady Cojocaru, Itamar Borukhov, Assaf Wool, Yossef Kliger, Tomer Zekharya, Zurit Levine, Sergey Nemzer, Ofer Levy, Amir Toporik. _COMPUGEN, Holon, Israel_.

Antibody blockade of CTLA4 and PD-1 immune checkpoints emerged as an effective treatment modality for cancer. However, the majority of patients do not achieve sustained long term benefit, suggesting a need for targeting of additional immune checkpoints. To identify additional B7/CD28 immune checkpoint targets, we developed a unique compendium of computational algorithms that identified multiple novel targets including TIGIT in 2008, which was an unknown protein at the time of discovery [Proc Natl Acad Sci U S A. 2009 Oct 20;106(42):17858-63], and PVRIG which we recently disclosed. Since their initial discovery, these targets have been functionally validated and anti-tumor activity was demonstrated with antibodies that target them.

In this presentation, we will describe the computational algorithms that led to the discovery of these novel immune checkpoints. These algorithms combine two complementary aspects: (i) endogenous immune checkpoint function prediction and (ii) prediction of immuno-modulatory activity in cancer. Immune checkpoint function was predicted based on gene structure similarity to B7/CD28 family members that is reminiscent of ancient common evolutionary origins. A gene structure alignment tool was developed to identify functional homologs of B7/CD28 genes even in the absence of sequence similarity. Next, the expression profile of these candidates was modeled and compared to profiles of known immune checkpoints in normal and cancer tissues. We will review the details of TIGIT and PVRIG discovery, which were among the immune checkpoints predicted in this process.

Our approach demonstrates the powerful ability of computational biology to translate genomic knowledge into rational and reliable drug target discovery.

#585

Chromosome 9p24.1 deletions as a determinant of tumor immune surveillance and immune checkpoint blockade therapy in non-small cell lung cancer.

Tao Shen, Jie Wu. _Stephenson Cancer Center, OUHSC, Oklahoma City, OK_.

Anti-PD-1/PD-L1 immune checkpoint blockade therapy requires the presence of PD-L1/PD-L2 and MHC class I antigens on the tumor cell surface. We previously found JAK1 truncating mutations in endometrial cancer cells that impairs JAK1/JAK2-STAT1-IRF1-mediated signaling pathway, which regulates MHC class I antigen presentation. To determine the potential role and genetic defects in the IFN-γ-IRF1 pathway in non-small cell lung cancer (NSCLC), we analyzed the Cancer Genome Atlas (TCGA) data. Expression of IRF1 correlated with cytolytic activity markers GZMA and PRF1. Loss-of-function (LOF) genetic alterations in the IFN-γ-IRF1 pathway genes were found in 64 cases (6.3%) among 1016 patients. These genetic defects occurred prevalently in the JAK2 gene (33 cases) and often through deletions (29 cases) of chromosome 9p24.1. JAK2 gene deletions were frequently associated with deletions of CD274 and PDCD1LG2 genes that encode PD-L1 and PD-L2, respectively, whereas CD274 and PDCD1LG2 deletions were always accompanied by JAK2 deletions. Chromosome 9p is frequently deleted in NSCLC. This has been attributed to CDKN2A/CDKN2B and PTPRD tumor suppressor genes located at the chromosome 9p21.3-9p24.1. Interestingly, JAK2, CD274, and PDCD1LG2 genes are co-localized at chromosome 9p24.1 near the CDKN2A/CDKN2B and PTPRD genes. JAK2, CD274, and PDCD1LG2 deletion often but not always coincided with CDKN2A/CDKN2B and PTPRD deletions. IFN-γ induced IRF1 expression and cell surface HLA-ABC levels in A549 cells that contain wildtype JAK2 but not in H1573 cells that had a truncating JAK2. Deletion of JAK2 or inhibition of JAK2 kinase activity in A549 cells resulted in loss of IFN-γ-regulated IRF1 and cell surface HLA-ABC, whereas expression of exogenous JAK2 in H1573 cells restored IFN-γ responses. These findings reveal a previously unrecognized significance of chromosome 9p deletion in NSCLC and point to chromosome 9p24.1 as a determinant of immune checkpoint therapy.

#586

Ionizing radiation-induced PD-L1 upregulation in glioma: a crucial role for the molecular chaperone FKBP5.

Paolo D'Arrigo, Michele Russo, Elia Guadagno, Roberto Pacelli, Maria Laura Del Basso De caro, Anna Rea, Martina Tufano, Stefania Staibano, Gennaro Ilardi, Maria Fiammetta Romano, Simona Romano. _University of Naples Federico II, Naples, Italy_.

Glioblastoma can avoid immune surveillance and induce tumor tolerance, through inhibitory molecules, e.g. PD-L1. Ionizing radiation (IR), used to treat this tumor, is known to increase tumor expression of PD-L1, thus inducing resistance mechanisms. Finding molecular determinants involved in IR-induced PD-L1 may provide a target for preventing such an effect and improve radiotherapy outcomes. We demonstrated that the short isoform of the cochaperone FKBP51 (FKBP51s) regulated PD-L1 expression in melanoma. In glioma, FKBP51s was expressed at high levels, together with PD-L1 and its silencing reduced PD-L1 levels. Conversely, overexpression of FKBP51s increased PD-L1. Different PD-L1 isoforms were observed by immunoblot. A lower band (~37 kDa) corresponding to the naïve protein and two upper bands (~50, ~68 kDa) ascribable to post-translationally modified isoforms. FKBP51s was found mainly bound to the heaviest bands of PD-L1, reasonably mature protein, while the canonical isoform FKBP51 appeared to bind only the naïve protein. Mature PD-L1 protein consists in carbohydrates addition, the principal chemical modification to most plasma membrane proteins, and, particularly, N-glycosylation. Treatment of immunoprecipitated PD-L1 protein with PNGaseF produced a decrease of the highest band and the appearance of a lower band, corresponding to the naïve PD-L1, in accordance with the concept that the heaviest band of PD-L1 is a glycosylated form. Moreover, following subcellular fractionation to obtain extracts from ER and Golgi compartments, we found that naïve 37 kDa PD-L1 was detectable in the ER, but not in the Golgi. The PD-L1 glycosylated band was expressed in ER in a small proportion and mostly in the Golgi. FKBP51s, but not the canonical FKBP51, was found in ER. Co-IP of FKBP51s and PD-L1 from ER extract confirmed the two proteins interacted each other in ER. Our results show that naïve PD-L1 colocalized in the ER of glioma cell complexed with FKBP51s, while the PD-L1 glycosylated form was measured in the Golgi apparatus. Treatment of glioma cell with increasing doses of IR upregulated PD-L1 expression, in a dose-response manner. Particularly, we found a significant increase in PD-L1 expression at 4 and 8 Gy, in comparison with unirradiated glioma cell. Moreover, IR induction of mature PD-L1 was efficiently counteracted by FKBP51s silencing. Subcellular fractionation of glioma cell subjected to IR in kinetics showed an early and transitory decrease in FKBP51s ER levels at 3hrs, in line with a reduction of the glycosylated band in the whole lysate. After 8 hrs from IR, FKBP51s rose up again in the ER inducing a full maturation of PD-L1. These findings suggested that FKBP51s has a role in catalyzing PD-L1 folding, an essential step to glycosylation, through which it controls the affinity for PD1. This study identifies FKBP51s as an essential element that regulates PD-L1 expression on glioma cell, which is exploited by the tumor to resist to IR.

#587

HMBD002, a novel neutralizing antibody targeting a specific epitope on the co-inhibitory immune checkpoint receptor VISTA, displays potent anti-tumor effects in pre-clinical models.

Piers J. Ingram, Dipti Thakkar, Jerome D. Boyd-Kirkup. _Hummingbird Bioscience, Singapore_.

V-domain immunoglobulin (Ig)-containing suppressor of T-cell activation (VISTA) also known as PD1 homolog (PD1H) is a co-inhibitory immune checkpoint receptor. VISTA is predominantly expressed on hematopoietic cells, particularly myeloid derived suppressor cells and antigen presenting cells, and at lower levels on CD4+ and CD8+ T cells and Foxp3 Tregs. Multiple studies have demonstrated VISTA can strongly suppress human T-cell activation, and the presence of high VISTA expressing cells in the tumor microenvironment has been postulated as playing a critical role in tumorigenesis and resistance to immunotherapy. VISTA neutralizing antibodies have been previously observed in pre-clinical models to increase the abundance of tumor infiltrating effector T cells as well as their effector functions, resulting in enhanced control of tumor growth, even in the absence of detectable expression of VISTA on the tumor cells, a potential advantage over PD1 or PDL1 blockade. There is an urgent need to develop the most clinically effective anti-VISTA antibodies however the limited characterization of the VISTA pathway and the uncertainty around the cognate VISTA ligand has limited the extent to which in vitro studies can support the selection of optimal antibodies for development. Hummingbird Bioscience's proprietary Rational Antibody Development platform to the design of neutralizing antibodies against specific epitopes on VISTA. Extensive in silico analyses of the VISTA structure and comparative structural modeling against other B7 protein family members has enabled the prediction of key binding sites where antibody binding will inhibit function, while preserving necessary safety and developability profiles. Subsequently, a directed evolution strategy was used to isolate monoclonal antibodies specifically targeting these key predicted functional domains on the native folded VISTA protein. Hummingbird's anti-VISTA antibodies bind human and murine VISTA protein with high affinity (kD <5nM) and with high specificity. Antibody HMBD-002, targeting a region on VISTA predicted to be critical to ligand binding, was given as single dose monotherapy to wildtype BALB/C mice, 3 days after inoculation with CT26 syngeneic colon cancer cells. Induction of cytokine release was observed in the mouse sera (up to 4 fold increase in IL-2, IL17 and IFNγ measured at 24 and 72 hrs after dosing), confirming robust activation of the immune response. This was associated with significant effects on tumor progression including delay in progression free survival and > 60% inhibition of tumor growth (after 15 days) with no observable toxicity. Further in vitro and in vivo validation experiments in other cancer models are ongoing, both as monotherapy and in combination with other immune therapies. The first in-human trial of HMBD-002 is planned for 2018.

#588

Advanced molecular characterization of severe autoimmune toxicities associated with checkpoint inhibitor therapies.

Justin M. Balko,1 Daniel Y. Wang,1 Yu Wang,1 Rami Al-Rohil,1 Margaret Compton,1 Jeffery A. Sosman,2 Igor Puzanov,3 Bret Mobley,1 Robert D. Hoffman,1 Yaomin Xu,1 Javid J. Moslehi,1 Chanjuan Shi,1 Douglas B. Johnson1. 1 _Vanderbilt University Medical Center, Nashville, TN;_ 2 _Northwestern University, Chicago, IL;_ 3 _Roswell Park Medical Institute, Buffalo, NY_.

Immune checkpoint inhibitors (ICIs) have made a profound impact on the treatment of a variety of cancers. However, as with any systemic treatment, toxicities are inevitable. With most classes of cancer therapies, toxicities are relatively predictable based on clinical trial safety data and therefore can be handled with prophylactic or supportive care measures. However, ICIs are unique in their ability to cause rare but severe auto-immune toxicities. The molecular underpinnings of these toxicities, as well as unique features of the patient, tumor, or affected tissue, have not been extensively explored. We recently reported a small case series of two patients with myocarditis resulting in death arising following combination ICI therapy (Johnson et al, N Engl J Med, 2016). High lymphocytic infiltration, coupled with PD-L1 expression was present in the affected myocardium and skeletal muscle. Common T cell clones were identified between the affected tissue and tumor, and abnormal expression of muscle-specific transcripts was identified in the associated tumor, suggesting release of peripheral tolerance to tumor-expressed self-antigens.To expand upon our reported study, we collected healthy and afflicted tissue from a series of cancer patients with immune-related colitis, myocarditis (MC), and encephalopathy following ICI treatment. We hypothesize that molecular analysis of these tissues will identify causal factors in the etiology of these toxicities, and how to better predict, prevent, and treat them. Thus, we performed molecular characterization of the immune infiltrate and diseased tissue microenvironment. A total of 20 affected (colon, cardiac, brain) and non-diseased control specimens were examined by spatial digital profiling (nanoString). This process generates a spatial heatmap of digital counts of 20 selected immunology and cellular markers and proteins across each specimen. Using this technology, the landscape of inflammation in ICI-affected organs can be resolved for insights into the mechanism whereby ICI-mediated auto-immunity occurs. Targeted RNAseq for selected immuno-oncology mRNA targets was also performed. In initial RNA sequencing analyses of MC cases, affected myocardium, skeletal muscle, and patient-matched tumors all demonstrated expression of immune activation markers (e.g. interferon-gamma and granzyme B), expression of PD-L1, and muscle-specific genes. In the expanded population, including colitis, digital spatial profiling analyses and targeted NGS (RNAseq) are underway. Although data analyses are incomplete at the time of this abstract, this work will be the largest and most comprehensive analysis of the molecular underpinnings of ICI-mediated auto-immune toxicity reported to date. These data should offer clarity in the mechanisms and features of these adverse events, how to prevent or predict them with precision medicine, and how to treat them when they do occur.

#589

Hedgehog signaling augments PDL-1 expression in cancer cells under hypoxic condition to inhibit antitumor effects by activated lymphocytes.

Hideya Onishi, Akiko Fujimura, Yasuhiro Oyama, Makoto Kawamoto, Akio Yamasaki, Takashi Morisaki. _Kyushu University, Fukuoka, Japan_.

[Background] Hedgehog (Hh) signaling is activated under hypoxic condition in cancerous tissue. This is thought to be one of the mechanisms of the induction of malignant phenotype of cancers. We have shown that Hh inhibitor decreases proliferation, invasiveness, and tumorigenesis. We have also revealed that Hh signaling plays pivotal roles for the maintenance of function in activated lymphocytes and dendritic cells under hypoxia. Recently, immune checkpoint inhibitor takes much attention, however, response rate is still limited. The mechanisms regulating PDL-1 expression also remains unclear. In this study, to increase reponse rate of immune checkpoint inhibitor, we investigated whether hypoxia-induced activation of Hh signaling contributes to PDL-1 expression in cancer and whether it affects the antitumor function of activated lymphocytes.

[Materials and Methods] Pancreatic cancer cell line, Panc-1, gallbladder cancer cell line, NOZ and GBd, and lung small cell cancer cell line, SBC-5 were used as target cancer cells. Human lymphocytes derived from PBMC were activated by using anti CD3 mAb and IL-2, and were used as effector cells. For normoxic conditions, cells were cultured in 5% CO2 and 95% air. For hypoxic conditions, cells were cultured in 1% O2, 5% CO2, and 94% N2 using a multigas incubator. For Hh signaling inhibition, cyclopamine, polysaccharide-K (PSK), and small interfering RNA targeting Gli1, Smo, MAML3, and RBPJ were used. Cell numbers were counted by light microscope. Expression of cell surface molecules was estimated by FACS.

[Results] 1) Hypoxia augmented PDL-1 expression in all 4 cancer cell lines. 2) Inhibition of Hh signaling using MAML3 siRNA, cyclopamine and PSK reduced PDL-1 expression under hypoxia in all 4 cancer cell lines. 3) When activated lymphocytes were cocultured with cancers treated with a Hh inhibitor, activated lymphocyte cell numbers significantly increased under hypoxia. 4) In contrast, this increase was abrogated when cancer cells were treated with a PDL-1 neutralizing antibody. 5) When activated lymphocytes were cocultured with cancers treated with a Hh inhibitor and/or anti-PDL-1 Ab, the percent of CD8+ lymphocytes decreased in both the cyclopamine- and anti-PDL-1-treated groups under hypoxic conditions, while there was no significant change in CD3 expression. 6) NKG2D expression increased on activated lymphocytes in anti PDL-1Ab-treated group.

[Conclusion] These results suggest that Hh signaling is one of regulatory pathways of PDL-1 expression under hypoxia and that inhibiting Hh signaling may induce lymphocyte antitumor activity. Therefore, Hh inhibition could be a promising drug target, not only as a direct tumor suppressor agent, but also as a PDL-1 inhibitor.

#590

A new standardized CD8 and PD-L1 dual assay.

Florence Monville,1 Emmanuel Prestat,1 Nadia Yessaad,2 Marine Villard,1 Luciana Batista,1 Jerome Galon,3 Julien Adam,4 Jacques Fieschi1. 1 _HalioDX, France;_ 2 _Mi-mAbs-CIML, France;_ 3 _INSERM, France;_ 4 _Institut Gustave Roussy, France_.

PD1/PD-L1 pathway blockade results in a durable clinical response in a fraction of the non-small cell lung cancer (NSCLC) patients. Today, the expression of PD-L1, detected by immunohistochemistry (IHC) at the surface of tumor or tumor-infiltrating immune cells, is used to select patients that may respond to immune checkpoint inhibitors (ICI). However the predictive value of that biomarker alone is questioned. In order to better stratify NSCLC patients, we have developed a new dual-staining IHC assay of PD-L1+ and CD8+ cells (TILs) present in the tumor microenvironment on a single slide prepared from FFPE tissue.

The assay was optimized and fully automated on the Benchmark XT platform (Roche Ventana) with anti-PD-L1 (HDX3) and anti-CD8 (HDX2) monoclonal primary antibodies, respectively revealed with DAB and fast red substrates. Stained slides are analyzed by a pathologist like any other existing IVD test together with the TILs information. In addition, after digitization, the samples are analyzed to quantify brown and red cells with a newly developed digital Pathology (DP) tool.

The following parameters are reported by the DP tool: (1) CD8+ cell density (cells/mm²) ; (2) PD-L1+ cell density (cells/mm²) ; proximity between CD8+ and PD-L1+ cells, either centered on CD8+ or on PD-L1+ cells and finally cluster indexes for CD8+ cells and PD-L1+ cells. HalioDx DP analysis tool can calculate these parameters on any Regions Of Interest (ROI) defined by the user.

Accuracy of the automated numeration of CD8+ and PD-L1+ cells was validated by an expert pathologist. Cell-to-cell distances were validated with an independent DP tool. Variability was assessed for all parameters using adjacent dual-stained slides on complete tissue sections. The concordance with main IVD approved PD-L1 methods was established on a representative set of 55 NSCLC tumours.

HalioDx has developed an innovative assay based on the dual staining of CD8+ and PD-L1+ cells associated to a DP tool to standardize the evaluation of the tumor microenvironment. This assay could have higher predictive performance than existing IVD tests used to identify ICI resistant NSCLC patients.

#591

Efficacy of ALKS 4230, a novel immunotherapeutic agent, in murine syngeneic tumor models alone and in combination with immune checkpoint inhibitors.

Heather C. Losey, Jared E. Lopes, Reginald L. Dean, Michael R. Huff, Rosemarie A. Moroso, Juan C. Alvarez. _Alkermes, Waltham, MA_.

ALKS 4230 is a novel immunotherapeutic agent being tested in an ongoing phase 1 study to evaluate safety and tolerability in the treatment of patients with refractory solid tumors. ALKS 4230 is a selective agonist of the intermediate-affinity interleukin 2 (IL-2) receptor that when administered to mice results in a significant increase in numbers of memory CD8+ T cells and NK cells with no expansion of CD4+ regulatory T cells (Tregs). The selectivity achieved by ALKS 4230 is due to the fact that the molecule is a fusion of circularly permuted IL-2 to the extracellular portion of the IL-2 receptor α, and the resulting fusion protein is sterically prevented from binding to the high-affinity IL-2 receptor expressed on Tregs. The efficacy of ALKS 4230 was compared to recombinant human IL-2 in a B16F10 lung tumor metastasis model. ALKS 4230 treatment resulted in dose-dependent reduction of lung tumor colonization, with 100% inhibition at the highest dose tested. In contrast, while IL-2 was able to reduce lung tumor colonization, the maximal level of inhibition achieved was 60-70% at multiple dose levels such that increasing doses did not result in greater inhibition. Thus, the activation and expansion of effector cells without expansion of Tregs in response to ALKS 4230 treatment correlates with its improved efficacy over IL-2, which non-selectively expands both effector cells and Tregs. The antitumor efficacy mediated by ALKS 4230 was further evaluated in several murine subcutaneous syngeneic tumor models, including B16F10, MC38 and EMT6. Treatment with ALKS 4230 or its murine ortholog resulted in inhibition of tumor growth and improved survival in multiple models. When dosed in combination with anti-CTLA-4 or anti-PD-1 antibodies, ALKS 4230 resulted in further improvement of tumor growth inhibition and survival. These results demonstrate the murine antitumor efficacy of ALKS 4230 alone and in combination with immune checkpoint inhibitors and support the ongoing clinical evaluation of ALKS 4230 as an immunotherapy for cancer.

#592

Distinct cellular mechanisms mediate anti-CTLA-4 and anti-PD-1 checkpoint blockade.

Spencer C. Wei,1 Jacob H. Levine,2 Dana Pe'er,2 James P. Allison1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Sloan Kettering Institute, New York, NY_.

Checkpoint blockade is able to achieve durable responses in a subset of patients, however the biological variables that distinguish responders from non-responders are not well understood. Furthermore, we lack a satisfying comprehension of the underlying mechanisms of anti-CTLA-4 and anti-PD-1 induced tumor rejection. Given that PD-1 and CTLA-4 attenuate T cell activity through distinct mechanisms that are separated spatially and temporally, we hypothesized that responses to anti-CTLA-4 and anti-PD-1 are driven by distinct mechanisms. To address this hypothesis we utilized mass cytometry to comprehensively profile the effect of checkpoint blockade on tumor immune infiltrates in murine tumor models. This approach allows for the interrogation of greater than 40 analytes at single cell resolution. We demonstrate that high dimensional mass cytometry analysis enables unsupervised identification of biologically relevant tumor infiltrating immune populations with high sensitivity and specificity.

Using this approach we analyzed immune infiltrates of MC38 and B16BL6 murine tumors in mice treated with anti-CTLA-4, anti-PD-1, or control antibodies. In both tumor models we identify 15 distinct T cell populations with 0.5% or greater frequency. The T cell populations identified in MC38 and B16BL6 tumors were highly congruent. Notably, some but not all of these T cell populations were responsive to checkpoint blockade. A subset of tumor infiltrating CD8 T cell populations expanded following both anti-CTLA-4 and anti-PD-1. Conversely, a subset of regulatory T cell populations contracted following both anti-CTLA-4 and anti-PD-1. Interestingly, we observed expansion of a Th1-like CD4 effector T cell population only in response to anti-CTLA-4 treatment. Thus, we find that anti-PD-1 predominantly engages subsets of tumor infiltrating CD8 T cells whereas anti-CTLA-4 engages both the CD4 and CD8 effector compartments.

Our findings indicate that anti-CTLA-4 and anti-PD-1 utilize distinct cellular mechanisms to induce tumor rejection. These findings highlight the importance of expanding our mechanistic understanding of immunotherapeutic approaches for the rational design of combinatorial therapeutic approaches. Furthermore, these results demonstrate that mass cytometry analysis can be utilized to identify biologically relevant tumor infiltrating T cell populations.

We acknowledge the MDACC core facility NCI Support Grant P30CA16672.

#593

**Cross-platform integrative analysis of NSCLC reveals association between PD-L1 expression patterns and tumor genetic profile** **.**

Robin Edwards, Patrik Vitazka, Peter Szabo, Han Chang, John Cogswell, Hao Tang, Kaushal Desai, Darren Locke, Qiuyan Wu, Joseph Szustakowski, Steven Bernstein, Michele Cleary. _Bristol-Myers Squibb, Princeton, NJ_.

Background: Immunotherapy with PD-1 inhibitors is associated with increased survival in NSCLC. While PD-L1 expression enriches for clinical benefit, additional predictive biomarkers are critically needed to further define patient subsets associated with specific response/resistance profiles and to develop optimal combination strategies. We hypothesized that integration of pathology features derived from H&E and PD-L1 immunohistochemistry with genetic/genomic data from NSCLC could identify phenotypic/genotypic associations that highlight specific immunosuppressive mechanisms. Towards that end, we present data from an initial pilot analysis.

Methods: Twenty-nine NSCLC (23 adenocarcinoma; 6 squamous cell carcinoma) were included. H&E-stained sections were scored for tumor grade (1-3), relative proportion of tumor stroma (stroma score 1-3), percent necrosis and intensity of chronic inflammatory infiltrate (1-3+). PD-L1 immunohistochemistry was performed using the DAKO 28-8 antibody. PD-L1 expression was scored in tumor cells by modified H-score and by the predominant pattern observed - diffuse, heterogeneous, tumor-stroma interface or negative, and in immune cells using a semi-quantitative intensity scale (1-3+). DNA and RNA extracts from FFPE tissue were subjected to whole exome sequencing and RNAseq from which mutation load, oncogene/tumor suppressor genotypes and inflammation signatures were derived.

Results: The highest PD-L1 H-scores were associated with diffuse expression patterns and were observed in high grade tumors (Grade 3). Deleterious mutations in TP53, STK11, KEAP1, KRAS, EGFR and MET occurred at expected frequencies. These mutations occurred across multiple PD-L1 expression patterns except for STK11 which was restricted to PD-L1 negative tumors (p=0.077, Fisher exact test). STK11-mutant tumors also displayed a lower PD-L1+ inflammation score (p=0.046, Fisher exact test). Trends toward an increase in mutation load with increasing levels of chronic inflammation on H&E stain as well as with PD-L1+ chronic immune infiltrates were noted. Inflammation signatures derived from RNAseq showed an association between diffuse PD-L1 expression by IHC and the highest levels of inflammation mRNA signatures comprising T-cells (CD8, Tregs), B-cells, macrophages and MDSCs.

Conclusions: The potential association between tumor grade, PD-L1 expression, intensity of the immune infiltrate and mutation load raise the possibility that tumor morphology predicts mutation load and associated immune response. The finding that STK11 mutation is restricted to PD-L1 negative tumors suggests an immunosuppressive mechanism is invoked in this setting. This dataset is currently being expanded to establish the significance of these findings.

#594

Dual targeting of TGFb and PD-L1 promotes potent anti-tumor efficacy in multiple murine models of solid carcinomas.

Karin M. Knudson,1 Sofia R. Gameiro,1 Kin-Ming Lo,2 Jeffrey Schlom1. 1 _NCI-CCR, Bethesda, MD;_ 2 _EMD Serono, Billerica, MA_.

Tumors evade host immune surveillance through multiple mechanisms, including promoting a tumor microenvironment able to effectively suppress immune effector functions. Secretion of cytokines such as TGFb and upregulation of immune checkpoint molecules are two main contributors to immune evasion and tumor progression. TGFb is an important immunosuppressive cytokine with pleiotropic actions in cancer, including promotion of epithelial to mesenchymal transition and metastasis. It promotes tumor immune evasion by impairing T and NK cell maturation, recruitment, and function. Tumors also inhibit antitumor immune responses through tumor cell-immune cell interaction, in which the negative regulatory checkpoint PD-L1/PD-1 interaction plays a major role. Therapies that independently target TGFb signaling or PD-L1/PD-1 interaction demonstrate limited clinical efficacy.

Anti-PD-L1/TGFbTRAP (M7824) is a novel immunotherapeutic agent designed to simultaneously block the PD-L1 and TGFb immunosuppressive pathways. It is comprised of the extracellular domain of human TGFbRII (TGFbTRAP) linked to the C-terminus of human anti-PD-L1 heavy chain (anti-PD-L1). M7824 has shown potent anti-tumor activities in preclinical models1 and some evidence of clinical efficacy in a phase 1 study2. Here, we demonstrate that anti-PD-L1/ TGFbTRAP neutralizes murine TGFb-induced signaling and binds to murine PD-L1 in multiple murine breast and colon carcinoma models.

In non-tumor bearing mice, anti-PD-L1/TGFbTRAP increases the number of CD8+ T cells in the lymph nodes, and elicits splenic CD8+ T and NK cells with a more active phenotype. Initial examination of peripheral immune subsets in tumor-bearing mice indicates that anti-PD-L1/TGFbTRAP induces CD8+ T cells and NK cells with a more active, less exhausted phenotype. Furthermore, dual targeting of PD-L1 and TGFb increases tumor expression of MHC-I, MHC-II, and PD-L1, suggesting additional immune effects in the tumor microenvironment.

Importantly, anti-PD-L1/TGFbTRAP displays potent anti-tumor efficacy against various murine models of breast and colorectal carcinomas. This response is durable, preventing relapses up to 7 months. Anti-PD-L1/TGFbTRAP also decreases spontaneous metastasis in a murine model of triple-negative breast cancer.

Taken together, these findings support the preclinical proof of concept for dual targeting of TGFb and PD-L1/PD-1 pathways with anti-PD-L1/ TGFbTRAP, its mode of action, and its potential clinical use as a monotherapy or in combination with other immunotherapies or standards of care.

1 Lan Y et al, Preclinical evaluation of M7824 (MSB0011359C), a novel bifunctional fusion protein targeting the PD-L1 and TGFb pathways. Keystone Symposium, Jan 2017.

2 Strauss J et al, Phase I trial of M7824 (MSB0011359C), a bifunctional fusion protein targeting PD-L1 and TGFb, in advanced solid tumors. Keystone Symposium, Jan 2017.

#595

Heterogeneity of immune checkpoint expression in lung cancer identified through rapid tissue donation.

Theresa Boyle, Andrea Shaffer, Janella N. Hudson, Luisa D. Arevalo, Matthew Schabath, Teresita Muñoz-Antonia, Christie Pratt, Gwendolyn P. Quinn, Eric B. Haura. _Moffitt Cancer Center, Tampa, FL_.

Background: Translational research in advanced lung cancer is hindered by the limited availability of specimens for advanced molecular techniques. Although helpful, the standard practice to biopsy a small amount of tissue from a single site of cancer provides limited information. We launched a thoracic rapid tissue donation (RTD) program to enable lung cancer research with collection of tissue at multiple tumor sites within hours after death. Many patients chose to enroll in the RTD program as an opportunity to contribute to cancer research. RTD tissue will support multiple research projects, such as studying differential expression of immune checkpoint proteins in immune oncology or understanding resistance to targeted agents.

Methods: The RTD program for patients with stage IV lung cancer was approved in June 2015 by the Moffitt Cancer Center institutional review board. Tissue specimens from multiple tumor sites from consented donors are collected rapidly (aim < 24 hours) after death and are frozen and/or preserved in formalin. Hematoxylin and eosin (H&E) staining is performed with evaluation of the histopathology and quality of the specimens. Immunohistochemistry is performed to evaluate expression of immune checkpoint biomarkers (PD-L1, CTLA4, LAG3, TIM3, BTLA, A2aR and iNOS).

Results: Between June 26, 2015 and November 16, 2016, 18 patients with stage IV lung cancer consented to the RTD program. Post-mortem tissue has been collected from multiple tumor sites for three cases. H&E staining of 25 tissue blocks from the first case showed minimal evidence of post-mortem tissue damage by autolysis, confirming high RTD tissue quality. Biomarker studies, such as immunohistochemistry for immune checkpoints, such as PD-L1, CTLA4, LAG3, TIM3, BTLA, A2aR and iNOS are ongoing. Preliminary analysis of PD-L1 expression in the first patient revealed heterogeneous PD-L1 expression within and between eight tumor sites (both lungs, liver, both kidneys, left adrenal gland, mediastinal and retroperitoneal lymph nodes).

Conclusions: Rapid tissue donation with collection of ample post-mortem tumor tissue is feasible and valuable for cancer research. A heterogeneous pattern of PD-L1 immune checkpoint expression was observed between multiple sites of tumor. Post-mortem tissue collection from multiple tumor sites facilitates understanding changes in tumor behavior and biomarker expression at metastatic sites, especially in the context of treatment failures.

Acknowledgements: NIH Sponsor #R21 CA194932-01 and Tissue Core Facility at the H. Lee Moffitt Cancer Center & Research Institute (P30-CA076292).

#596

eFT508, a potent and highly selective inhibitor of MNK1/2 regulates immune checkpoint and cytokine expression promoting anti-tumor immunity.

Kevin R. Webster,1 Vikas K. Goel,1 Jocelyn Staunton,1 Craig R. Stumpf,1 Rajesh Sharma,1 Ivy N. Hung,1 Gregory S. Parker,1 Jolene Molter,1 Gary G. Chiang,1 Christopher J. Wegerski,1 Samuel Sperry,1 Vera Huang,2 Joan Chen,1 Peggy A. Thompson,1 Chinh Tran,1 Justin T. Ernst,1 Paul A. Sprengeler,1 Siegfried H. Reich1. 1 _eFFECTOR Therapeutics, San Diego, CA;_ 2 _Molecular Stethoscope, San Diego, CA_.

Dysregulated translation of messenger RNA (mRNA) plays a role in the pathogenesis of multiple solid tumors and hematological malignancies. MNK1 and MNK2 integrate signals from several oncogenic and immune signaling pathways (including RAS, Toll-like receptors and T cell receptor) by phosphorylating eukaryotic initiation factor 4E (eIF4E) and other key effector proteins including hnRNPA1 and PSF. Phosphorylation of these RNA-binding proteins by MNK1 and MNK2 selectively regulates the stability and translation of a subset of cellular mRNA that control tumor/stromal cell signaling, the tumor microenvironment and immune cell function. eFT508 is a potent and highly selective inhibitor of both MNK1 and MNK2. Ribosome profiling has demonstrated that inhibition of MNK1 and MNK2 by eFT508 selectively regulates the translational efficiency and mRNA stability of a subset of genes that include inflammatory cytokines/chemokines, regulators of stress response, and effectors of anti-tumor immune response. Given the importance of MAPK signaling and translational control to immune cell activation and differentiation, the immunological effect of eFT508 was further evaluated in both normal human immune cells in vitro and immunocompetent syngeneic cancer models in vivo. eFT508 treatment of normal donor T cells has no deleterious effect on αCD3/αCD28 stimulated IL-2 production, T cell proliferation or T cell viability. However, eFT508 selectively down regulates the induction of IL-10 and specific immune checkpoint receptors, including PD-1 and LAG3. Further evaluation of the mechanism of translational regulation has shown LAG3 mRNA contains specific sequence elements in the 5'-untranslated region (UTR) that confer sensitivity to eFT508. In addition, IL-10 mRNA is destabilized upon treatment with eFT508 leading to significant inhibition of IL-10 production in activated T cells. Furthermore, eFT508 treatment results in upregulation of MHC class II molecules on tumor cells, macrophage and dendritic cells through an IL-10/MARCH1 dependent mechanism. The in vivo antitumor effect of eFT508 was assessed in the CT26 BALB/C syngeneic tumor model. CT26 mouse tumor cell proliferation and survival are insensitive to eFT508 in vitro. In vivo, daily oral treatment with 1 mg/kg eFT508 results in significant anti-tumor activity, modulation of tumor infiltrating lymphocytes and establishment of immune memory. In addition, combination of eFT508 with either anti-PD-1 or anti-PD-L1 monoclonal antibodies results in marked efficacy, significantly increasing the percentage of responder animals. eFT508 is currently under evaluation in two phase I/II clinical trials for patients with advanced solid tumors and patients with advanced lymphoma respectively. These findings support further clinical evaluation of eFT508 in combination with checkpoint blockade.

### T-cell Immunity to Cancer: New Progress

#597

Bispecific anti-CD123 x anti-CD3 ADAPTIR™ molecules for redirected T-cell cytotoxicity in hematological malignancies.

Michael R. Comeau, Danielle Mitchell, Rebecca Gottschalk, Lynda Misher, Mollie Daugherty, Lara Parr, Peter Pavlik, Brian Woodruff, Hang Fang, Megan Aguilar, Jeannette Bannink, Starrla Johnson, Gary Li, Robert E. Miller, Robert Bader, Nicole Zhang, Toddy Sewell, Maria Dasovich, Gabriela H. Hoyos, John W. Blankenship, Catherine McMahan, David Bienvenue, Jane A. Gross. _Aptevo Therapeutics, Seattle, WA_.

Introduction: CD123 is a component of the IL-3 receptor expressed in several hematological malignancies including AML, ALL, HCL, and MDS. CD123 is a compelling target in AML due to its overexpression on AML blasts as well as leukemic stem cells, which are thought to be resistant to chemotherapy and may be responsible for relapse of disease following treatment. While CD123 is expressed by some normal leukocyte populations in circulation and hematopoietic progenitor cells in the bone marrow, the low frequency of expression on normal cell types provides a therapeutic window for targeting CD123 in tumor settings with the potential for durable response and reversible side effects. We have developed bispecific anti-CD123 x anti-CD3 ADAPTIR molecules APVO436 and APVO437 for redirecting T-cell cytotoxicity to CD123-expressing tumor cells. Results are presented that examine the in vitro and in vivo activity of these molecules in preclinical models of AML.

Methods: APVO436 and APVO437 proteins were expressed in CHO cells. Affinity SPR studies were performed using recombinant CD123-ectodomain. In vitro functional studies were conducted with CD123+ AML tumor cell lines and primary human and cynomolgus macaque T-cell populations. Cytotoxic activity was determined using chromium release assays. On-cell binding, T-cell activation and proliferation were assessed using multi-color flow cytometry. Pharmacokinetic parameters were determined in BALB/c mice using a single IV dose of approximately 10 mg/kg. In vivo studies to examine tumor growth inhibition activity were performed with NOD/SCID mice co-implanted subcutaneously with AML tumor cells and human T-cells followed by treatment with APVO436 or APVO437. Tumor growth was assessed by measuring tumor volume and Bioluminescent Imaging.

Results: APVO436 and APVO437 bound human CD123 protein with high affinity and binding to CD123 and CD3 expressing cell lines was confirmed by flow cytometry. Both APVO436 and APVO437 induced concentration-dependent lysis of CD123+ AML cell lines with primary human effector T-cells, accompanied by T-cell activation and proliferation. Comparable redirected T-cell cytotoxicity function was observed using primary cynomolgus macaque T cells. These activities were dependent on the expression of CD123 by the tumor target cells. APVO436 and APVO437 demonstrated an extended elimination half-life in mouse serum, typical of molecules capable of binding the neo-natal Fc receptor. In vivo, growth of AML tumor cells was inhibited by treatment with low doses of APVO436 and APVO437, significantly improving host survival.

Conclusion: Taken together these data demonstrate potent in vitro and in vivo activity of APVO436 and APVO437 against CD123 expressing tumor cells and are supportive of further investigation of this approach as a potential treatment option for AML and other hematological malignancies.

#598

APOBEC mutagenesis: a link between innate immunity and cancer.

A Rouf Banday, Olusegun O. Onabajo, Krizia-Ivana Udquim, Adeola Obajemu, Ludmila Prokunina-Olsson. _NCI/NIH, Bethesda, MD_.

Introduction: Cytidine deaminase activity of APOBEC3 enzymes generates mutations that restrict viral infection and eliminate tumor cells but also contribute to viral and tumor evolution. For example, human immunodeficiency virus (HIV) is hypermutated by APOBEC3G (A3G), and human papilloma virus (HPV) can be hypermutated by APOBEC3s, including A3A and A3B. In many human tumor types explored by The Cancer Genome Atlas (TCGA), C to T or G substitutions in the TCA or TCT motifs are predominantly attributed to the activity of A3A and A3B enzymes. In bladder tumors more than 60% of all exonic mutations are of the APOBEC-signature type, with nearly 100% of all bladder tumors carrying these mutations. Previously, we reported that two germline genetic variants – a single nucleotide polymorphism (SNP) rs1014971 and a 30Kb germline deletion that eliminates A3B and creates an A3AB chimera, are the strongest factors associated with bladder and breast cancer risk, expression of A3A and A3B, and APOBEC mutagenesis in human tumors. Other factors affecting the source of APOBEC mutagenesis in human tissues are not well understood. Because some APOBEC3s can be induced in the course of antiviral response, we hypothesized that APOBEC mutagenesis might be a misfired innate immune response.

Methods: We used TaqMan-based expression analysis for selected APOBECs (A3A, A3B, and A3G) to explore expression in bladder (HT-1376, HTB-9, and RT-4) and breast cancer (MCF-7, MDA-MB-231, and T-47D) cell lines infected with Sendai virus (SeV). SeV is a murine RNA virus that results in self-limiting infection in a wide range of human cells, efficiently controlled by innate immune response of the host. The strong induction of APOBEC3s in RT-4 and MDA-MB-231 was followed-up by global transcriptome analysis with RNA-seq and pathway analysis. Cell lines were also treated with IFN-α, IFN-γ, IFN-λ3 and IFN-λ4 and tested for induction of A3A, A3B and A3G by TaqMan assays.

Results: A3A, A3B and A3G are interferon-stimulated genes (ISGs) because they were induced in the course of innate antiviral response to SeV infection and by IFNs in some conditions. Specifically, A3A was most strongly induced by SeV, with 32, 51 and 12,000-fold induction in 3 breast cancer cell lines, and 4, 5 and 167-fold induction in 3 bladder cancer cell lines. A3B was also induced by SeV but only in the range of 1-5 fold in all cell lines tested.

Conclusion: APOBEC3s can be strongly induced in the course of innate immune response even to transient and self-limiting viral infections. In the presence of single-stranded DNA (ssDNA), the APOBEC3 substrate generated by different cellular and environmental conditions, strong induction of some nuclear APOBEC3s, such as A3A and A3B, may result in mutation of human genomic DNA. In turn, accumulation of these mutations can lead to tumor initiation and evolution, particularly in individuals with germline APOBEC3 risk variants.

#599

Pharmacodynamic biomarkers for anti-TIGIT treatment and prevalence of TIGIT expression in multiple solid tumor types.

Fiore Cattaruzza, Pete Yeung, Min Wang, Alayne Brunner, Erwan Le Scolan, Jennifer Cain, Gretechen Argast, Gilbert O'Young, YuWang Liu, Belinda Cancilla, Austin Gurney, Tim Hoey, John Lewicki, Ann Kapoun. _OncoMed Pharmaceuticals, Redwood City, CA_.

TIGIT (T cell immunoreceptor with Ig ITIM domain) is a co-inhibitory receptor and its signaling axis inhibits T cell and Natural Killer (NK) cell activity in the healthy immune system. In tumors TIGIT is highly expressed on a subset of dysfunctional T and NK cells and of highly suppressive regulatory T cells (Treg). Loss of TIGIT signaling enhances NK cell activity, CD4+ T cell priming and CD8+ T cell effector functions, suggesting a role in anti-tumor immunity. We have developed an anti-TIGIT blocking antibody that shows potent antitumor efficacy in in multiple syngeneic mouse models, including CT26WT colon, B16F10 melanoma and 4T1 breast cancer. Dose-dependent (12.5-0.1 mg/Kg) single agent efficacy was demonstrated in established tumors, and pharmacodynamic (PD) biomarkers in blood and in tumors were identified.

Consistent with TIGIT's role as a co-inhibitory receptor, anti-TIGIT promoted a dose-dependent increase in activation of CD8+ and CD4+ tumor infiltrating leukocytes (TIL) and NK cells, as shown by increased staining of IFN-γ and CD69 by flow cytometry, when compared to controls. Anti-TIGIT also increased CD4 and CD8 T cell frequency in the tumor, measured by immunohistochemistry (IHC). Furthermore, anti-TIGIT caused an increase in splenic NK cell cytotoxicity, which correlated with dose and efficacy.

To identify gene expression biomarkers in tumor and in blood, we used microarray analysis, and found similar immune gene changes between the two tissues. As expected, anti-TIGIT increased the expression of genes associated with CD8+ T cells, CD4+ T cells, and NK cells. Markers indicative of cytotoxic activity and Th1 response were also induced by anti-TIGIT. The microarray data were validated by qRT-PCR and results were consistent with flow cytometry and cytotoxicity results, and underlie the mechanism of action of anti-TIGIT.

We have developed an IHC assay to evaluate TIGIT expression patterns in tumor and the associated stroma and TILs. Using this assay, we profiled 17 tumor types to evaluate patterns of TIGIT expression. Expression was high on immune cells in the stroma and on TILs in 8 tumor types, while it was generally low on tumor cells. TIGIT expression was also low on tumor cells in a panel of 27 PDX models. Consistent with the IHC results, analysis of 33 tumor types in the TCGA by RNA-Seq showed a good correlation of the expression levels of TIGIT and T cell markers, suggesting that TIGIT is mostly expressed on immune cells in tumors.

In conclusion, we have identified PD biomarkers for anti-TIGIT in tumors and in surrogate tissues in syngeneic mouse models. Analysis of the biomarkers demonstrates activation of T cells and NK cells upon inhibition of TIGIT signaling. These biomarkers can be used in the clinic to demonstrate target engagement. In addition, we have profiled the expression of TIGIT in multiple solid tumor types and characterized prevalence of TIGIT-positive cells.

#600

A cellular platform enables targeted brain delivery of T cells to glioblastoma.

Heba Samaha,1 Antonella Pignata,2 Tiara Byrd,2 Kristen Fousek,2 Fong Lam,2 Thomas Shum,2 Fabio Stossi,2 Sean Marrelli,1 Micheal Mancini,2 Meenakshi Hedge,2 Wayne smith,2 Nabil Ahmed1. 1 _Baylor College of Medicine, Texas Children's Hospital, Children's Cancer Hospital Egypt (CCHE 57357), Houston, TX;_ 2 _Baylor College of Medicine, Texas Children's Hospital, Houston, TX_.

BACKGROUND: Cancer renders the endothelium anergic, abrogating T cell homing and hindering cell therapy for central nervous system (CNS) malignancies. Activated Leukocyte Cell Adhesion Molecule (ALCAM) is a pathological adhesion molecule upregulated in the endothelium of Infiltrative CNS diseases. ALCAM in these conditions leads to overexpression of Intercellular Adhesion Molecule-1 (ICAM-1) and vascular CAM-1 (VCAM-1); mediating firm leukocyte anchorage . Antibodies blocking ALCAM decrease leukocyte BBB access and are currently in a clinical trial for Multiple Sclerosis (MS).

METHODS/RESULTS: We studied the difference in the dynamic signature of adhesion molecules GBM tumor endothelium. ALCAM is expressed on primary glioblastoma (GBM) endothelium, while ICAM-1 and VCAM-1 are paradoxically downregulated. We saw induction of vascular ALCAM alone under tumor condition. Thus, GBM endothelium fails to launch the second wave of adhesion molecules necessary for BBB transmigration. Then, we mapped the ALCAM minimal binding region and created an artificial molecule with the intent of creating a novel cellular platform to reverse endothelial anergy, through ALCAM specific anchorage. The exodomain is composed of domain 3 (D3) of CD6 , a stalk and a CD6 endodomain. D3 on T cells crosslinked to ALCAM in proximity ligation assay (PLA; <40nm) during TEM. Under shear stress, D3 T cells showed a global improved trafficking kinetics: higher capture, more rolling with slower velocity, arrest and eventually better TEM. This was not seen on ALCAM (-) normal endothelium or with non-transduced (NT) control T cells. In an ex vivo model of BBB, D3 T cells exhibited higher transmigratory ability over NT T cells. Signaling through the D3 endodomain phosphorylated ZAP70 recruiting Talin that enables LFA-1 open confirmation, mediating TEM. D3 T cells had higher F-actin/FAK colocalization using TIRF microscopy at its interface with ALCAM. In an orthotopic model of GBM, D3 T cells homed significantly more and accumulated at the tumor site. This was confirmed by the detection of D3 T cells in brain explants.

CONCLUSION: We created a cellular platform that enables targeted brain delivery of T cells. This platform serves as a gateway to the effective cellular therapeutics for brain malignancies but potentially as a delivery system for complex biologics.

#601

Oncolytic and immune effects of RAPTOR: a novel oncolytic Herpes Simplex Virus 1 expressing PTEN-L for brain tumor therapy.

Luke Russell,1 Alexander Sprague,1 Alena Cristina Jaime-Ramirez,1 Chelsea Bolyard,2 Yeshavanth Banasavadi-Siddegowda,1 JiYoung Yoo,1 Pin Yi Wang,3 Timothy Cripe,3 Balveen Kaur1. 1 _Ohio State University, Columbus, OH;_ 2 _OhioHealth Research Institute, Columbus, OH;_ 3 _Nationwide Childrens Hospital, Columbus, OH_.

Glioblastoma (GBM) is a World Health Organization grade IV astrocytoma with poor patient survival, characterized by extreme heterogeneity and invasiveness, and by resistance to radiation and chemotherapeutic drugs. Brain metastases are 2-3x more frequent than GBM, with breast cancer among the top 3 brain metastasizing tumor types. The tumor suppressor phosphatase and tensin homolog on chromosome ten (PTEN) is mutated or deleted in ~50% of all high grade gliomas and is lost in 33-49% of all breast cancers. We propose to target the dysregulated PTEN pathway in brain tumors to enhance patient survival.

Oncolytic viruses are engineered to destroy tumor cells while sparing non-neoplastic cells, and can be designed to incorporate payloads that enhance their oncolytic effects. PTEN-L is a recently discovered, longer form of PTEN which is secreted and membrane permeable, and retains its phosphatase activity after secretion and re-entry into cells. We have generated a novel oncolytic herpesvirus (RAPTOR) that secretes PTEN-L from infected cells for the treatment of GBM and breast cancer brain metastases (BCBM). In vitro assays to evaluate virally mediated tumor cell killing, virus replication, transgene expression, and flow cytometry to evaluate intracellular pAkt and cell surface programmed death ligand 1 (PD-L1) were performed. In vivo studies using mice bearing orthotopic human GBM (nude mice) or murine BCBM (nude and FVB/N) were used to evaluate brain immune infiltrates and survival.

Western blot analyses of infected cell lysates and conditioned media show that RAPTOR produces functional secreted PTEN-L, as confirmed by intracellular pAkt FACS. Tumor cell killing and replication were equivalent between RAPTOR and control HSVQ. In vivo, treatment of mice bearing established orthotopic BCBM revealed improved anti-tumor efficacy of RAPTOR. Significantly, 9/10 RAPTOR treated mice were long term survivors in immunocompetent FVB/N mice vs. 4/10 HSVQ treated mice (p=0.044). Treatment of nude mice bearing the same tumors did not produce long term survivors, implicating the development of antitumor immunity in RAPTOR treated animals. Flow cytometry revealed significant down regulation of the T-cell repressor PD-L1 on RAPTOR vs. HSVQ treated cells. Consistent with this, FACS of virus treated tumor bearing brain hemispheres revealed RAPTOR induced a significant influx in antigen presenting, NK, and CD8 T cells 7 days post treatment.

Our findings demonstrate that RAPTOR enhances survival of brain tumor bearing mice using a two-pronged approach: lytic tumor cell death and immune cell education and activation against the brain tumor. RAPTOR inhibits virally induced PD-L1 expression on tumor cells, and could pave the way for a paradigm shift in immune therapy where immune checkpoint inhibition occurs locally within the tumor rather than globally, overriding toxicity of neutralizing antibodies.

#602

A novel TGF-β/IL-12R signal conversion platform that protects CAR T cells from TGF-β-mediated immune suppression and concurrently amplifies effector function.

Benjamin Boyerinas, Sara Miller, Ryan Murray, Stacie Seidel, Geoffrey Parsons, Kathy Seidl, Kevin Friedman, Richard Morgan. _bluebird bio, Inc., Cambridge, MA_.

Numerous immune-suppressive mechanisms exist within the tumor microenvironment that may hinder chimeric antigen receptor (CAR) T cell efficacy. One such mechanism is mediated by TGF-β, a cytokine secreted by tumor cells and infiltrating suppressive immune cells that directly inhibits effector T cell activity. Effector T cells express the TGF-β receptors TGFBR1 and TGFBR2, and exposure of T cells to TGF-β induces phosphorylation of the major TGF-β signal mediators SMAD2 and SMAD3. Phosphorylated SMAD proteins (pSMADs) induce a suppressive transcriptional program that ultimately leads to reduced cytokine production, reduced cytotoxicity, and a failure to proliferate in response to antigen stimulation. A dominant negative receptor version (DNR) of TGFBR2 that does not contain signaling domains protects T cells from the impacts of TGF-β by blocking the ability of TGF-β to induce pSMADs. Here, we report the development of a novel TGF-β signal conversion platform that provides a T cell stimulatory signal upon exposure to TGF-β. This platform utilizes co-expression of chimeric variants of TGFBR2 and TGFBR1 where the TGF-β-binding domain of each receptor is fused to the transmembrane and intracellular signaling domains of the T cell simulating IL-12 receptors IL-12R-β2 and IL-12R-β1, respectively. Using a single lentiviral vector encoding both chimeric TGF-β receptors (CTBR) and a CAR, we demonstrated that CAR-CTBR T cells were completely protected from TGF-β-mediated SMAD phosphorylation. In addition, CAR-CTBR T cells generated significant amounts of pSTAT4 and pSTAT5 in response to TGF-β exposure, a response that mimics the T cell stimulation effects of IL-12. To further demonstrate successful signal conversion, we evaluated the impact of TGF-β exposure on the secretion of IFNγ, a major downstream target of IL-12 signaling. CAR-CTBR cells secreted significantly greater amounts of IFNγ than either control CAR T or CAR-DNR T cells following activation in the presence of TGF-β. Lastly, we utilized a serial restimulation assay to expand CAR, CAR-DNR, and CAR-CTBR T cells in the presence or absence of TGF-β. As expected, TGF-β exposure resulted in a significant inhibition of T cell proliferation in control CAR T cells. By contrast, both CAR-DNR and CAR-CTBR cells were protected from TGF-β-mediated inhibition of expansion. Gene expression analysis following 21 days of weekly antigen-driven expansion revealed specific TGF-β-mediated gene expression changes in CAR-CTBR cells consistent with increased T cell potency, including significant upregulation of IFNγ, IL10, IL18RAP, IL18R1, IL21R and CD62L transcripts. These data demonstrate the successful development of a TGF-β signal conversion platform that transforms the inhibitory effects of TGF-β exposure into an IL-12R-like T cell stimulatory signal that has the potential to produce superior CAR T cell responses in vivo.

#603

Evaluation of CD19 targeted T cells in relapsed or refractory ALL patients unable to afford allogenic bone marrow transplant in India.

Alka Dwivedi,1 Sushant Kumar,1 Terry Fry,2 Gaurav Narula,3 Rahul Purwar1. 1 _Indian Institute of Technology Bombay (IIT Bombay), Mumbai, India;_ 2 _National Cancer Institute (NCI), National Institute of Health (NIH), Bethesda, MD;_ 3 _Tata Mamorial Hospital, Mumbai, India_.

CAR-T cell therapy has demonstrated remarkable success in long-term remission of relapsed or refractory B-ALL. Unlike western countries, most of the relapsed/refractory B-ALL patients in India are unable to afford allogeneic stem cell transplant (allo-SCT), and simply choose palliation. To assess the potential use of CAR-T cells as a first line therapy, it is critical to evaluate CAR-T cell efficacy in patients unable to undergo allo-SCT. As a first step, a novel anti-CD19 CAR was designed and CD19-vector was produced using lentiviral mediated gene delivery system. To generate CD19+CAR T cells, T cells from healthy subjects were transduced and expanded either with rIL-2 or rIL-15. Almost 20-50% transduced T cells express CD19-CAR on their surface as analysed by flow cytometry. Next, efficacy of CD19-CAR-T cells was examined by cytotoxicity assay using CD19+ malignant B cell lines (Raji cells) and autologous B cells. CAR-T cells were able to kill majority of CD19+B cells effectively (100% killing). In addition, CAR-T cells secreted high levels of IFNΓ and IL-2. The CAR-T cells expanded in presence of rIL-15 showed fewer Treg compared to CAR-T cells expanded using rIL-2, however, there were no differences in numbers of T stem cells (TSC) and central memory T cells (TCM). In addition to data on healthy volunteers reflected above, results of above tests in relapsed/ refractory B-ALL patients is ongoing and being collated for inclusion in the final analysis. In conclusion, our data demonstrate the successful development of an indigenous CAR-T cell platform for subsequent use in clinical trials of CD19+B cell malignancies.

#604

Accurate identification and prioritization of candidate neoantigens from integrated cancer exome and transcriptome sequencing of FFPE samples.

Marián Novak, Sam Angiuoli, Luis A. Diaz, Andrew Georgiadis, Sian Jones, Peter R. Loverso, Sonya Parpart-Li, Maria Sevdali, Victor E. Velculescu, Ellen L. Verner, James White, Theresa Zhang, Mark Sausen. _Personal Genome Diagnostics, Inc., Baltimore, MD_.

Precise identification and characterization of candidate neoantigens is important for the development of effective cancer vaccines, adoptive T-cell transfer, and prediction of response to checkpoint inhibitors. The candidate tumor neoantigens are actionable only when expressed, however, current prediction methods lack the capacity to evaluate neoantigen expression. Sequencing both DNA and RNA from a patient's tumor tissue enables identification of mutations and evaluation of their expression leading to accurate identification of putative neoantigens. The purpose of this study was to develop and validate a methodology for co-extraction and sequencing of DNA and RNA from formalin-fixed paraffin-embedded (FFPE) samples to enable a robust neoantigen prediction protocol that integrates whole exome and transcriptome data to identify and prioritize tumor neoantigens for application in immuno-oncology research and clinical trials. In order to prepare high-quality sequencing libraries from FFPE specimens, the tissue was macrodissected to enrich for tumor-specific material, and improve the overall accuracy of next-generation sequencing for detection of somatic alterations. Total DNA and RNA was co-extracted and purified. The DNA was used to prepare whole exome sequencing (WES) libraries, while the co-extracted RNA was ribosome-depleted, and reverse-transcribed to prepare RNA sequencing (RNAseq) libraries. The WES and RNAseq data was then analyzed using a multi-algorithm HLA typing and neoantigen prediction protocol (ImmunoSelect-RTM). ImmunoSelect-R evaluates somatic genomic alterations identified from WES of tumor and matched normal tissue to ensure appropriate prediction of candidate neoantigens. The process of neoantigen prediction was then refined by integration of patient tumor-matched RNAseq data, which allowed for removal of non-expressed putative neoantigens. To further validate the approach, we applied the methodology to a set of experimentally validated neoantigens. In this setting, ImmunoSelect-R correctly classified 18 out of 19 as strong neoantigen candidates, suggesting a sensitivity of greater than 90%. Moreover, in a set of 10 patients, ImmunoSelect-R consistently ranked experimentally validated neoantigens within the top 20% of all neoantigen candidates derived from whole exome sequencing. In summary, our combined tissue processing, macrodissection, co-extraction, and neoantigen prediction methodology is able to identify and prioritize candidate neoantigens. Our approach is unique in combining high-fidelity sequencing (WES) and expression (RNAseq) data to accurately inform the selection of actionable tumor neoantigens for immuno-oncology applications.

#605

Potency of Gp96-Ig/Fc-OX40L cell-based combination vaccine in cancer immunotherapy.

Xin Xu, Louise E. Gonzalez, George J. Fromm, Suresh de Silva, Louise Giffin, Jason Rose, Taylor H. Schreiber. _Heat Biologics, Inc., Durham, NC_.

The breakthrough discoveries of checkpoint inhibitors in the field of tumor immunology have driven the clinical success of immunotherapies for cancer, despite their beneficial efficacy in only a small portion of patients. This is due in part to immuno-evasive mechanisms and the inability of the immune system to recognize tumor antigens as foreign.

As a therapeutic approach to effectively present these tumor antigens in order to elicit an anti-tumor immune response, we previously designed and characterized an allogenic, gp96-Ig secreting, cell-based vaccine (ImPACT); currently being assessed in a phase II study in non-muscle invasive bladder cancer and a phase Ib study in non-small cell lung cancer – the latter, in combination with the PD-1 antagonist Nivolumab.

We recently characterized a 'next-generation' vaccine (ComPACT) that combines the tumor antigen chaperone Gp96-Ig along with the T cell costimulator Fc-OX40L, which are both secreted from the same cell (Fromm et. al. Cancer Immunology Research. 2016). In preclinical assays, ComPACT is effective at stimulating CD4+ and CD8+ antigen-specific T cell expansion, the programing of a durable memory T cell phenotype, and the elimination of melanoma and colon tumors. This anti-tumor efficacy is enhanced when ComPACT is combined with checkpoint inhibition (anti-PD1 or anti-PDL1).

To support manufacturing and clinical efforts of both ImPACT and ComPACT, in anticipation of phase III expansion and/or new trial initiation, we have developed novel potency assays to quantify the biologically active form of Gp96-Ig and the in vitro activity of Fc-OX40L on T cell costimulation.

It has been shown that gp96 can interact with toll-like receptors (TLR) and that this interaction results in the activation of the NF-κB pathway. Since THP1 cells express abundant TLR2/4, we engineered a THP1 cell line to express luciferase that is regulated by NF-κB response elements. Furthermore, we utilized the human T cell line; Jurkat, as host cells in which we also express NF-κB-luciferase, to quantify Fc-OX40L costimulation. Jurkat/NF-κB-luciferase cells primed with either CD3/CD28 or TNFα, and subsequently cultured with ComPACT-secreted Fc-OX40L, results in a dose dependent increase in NF-κB (luciferase) expression.

Our current data in both assays shows a linear correlation with the input of Gp96-Ig and Fc-OX40L, and may serve as an effective potency assay to facilitate the manufacturing of our vaccine product as it transitions into more advanced cancer immunotherapy clinical studies.

#606

Targeting multiple immune checkpoints and their ligands using a humanized mouse model of ovarian cancer.

Ruea-Yea Huang, Kunle Odunsi. _Roswell Park Cancer Inst., Buffalo, NY_.

We have recently reported that multiple immune checkpoints are expressed in the TILs (tumor infiltrating T lymphocytes) and TALs (tumor associated T lymphocytes) from a murine ovarian cancer model and that blockade of one checkpoint pathway induces an upregulation of the other unblocked checkpoints. We hypothesized that resistance to single agent blockade (e.g. anti-PD1) is resulted from the compensatory upregulation of the other pathways (e.g. CTLA4 or LAG-3). To test this hypothesis in human ovarian cancer, we first examined the level of several immune checkpoints and their ligands in TILs or TALs from ovarian cancer patients. In addition to the highly expressed PD-1 and CTLA4, other checkpoint molecules such as TIM3, LAG3, VISTA, and TIGIT are also elevated in TALs as compared with control PBMC. The levels of multiple ligands for the immune receptors in the tumor samples were also analyzed. To translate the results from mice to humans, we developed several aggressive human ovarian cancer cell lines from patient derived xenografts to test the combinatorial checkpoint blockade strategies. Using a humanized mouse model of ovarian cancer we observed that adaptive transfer of autologous T cells in combination with checkpoint blockade of PD1 mildly delays ovarian tumor growth. The effect of blocking PD-1 and CTLA-4 pathways on tumor growth and the upregulation of other checkpoints are currently underway.

#607

PDL1 expression associated with triple negative breast ductal carcinomas in African American women.

Farhan Khan,1 Yasmine Kanaan,1 Luisel J. Ricks-Santi,2 Rabia Zafar,1 Hagos Aymut,1 Tammey Naab1. 1 _Howard University Hospital, Washington, DC, DC;_ 2 _Hampton University Hospital, Hampton, VA_.

Background: Tumor cells avoid host immune response through expression of inhibitory T cell regulator,

Programmed cell death ligand (PDL1).Our objective was to evaluate PDL1

expression by immunohistochemistry in the four major subtypes of breast

carcinoma (Luminal A, Luminal B, HER2, and Triple Negative) in a population of

202 African-American (AA) women with other clinicopathological factors.

Design: Tissue microarrays

(TMAs) were constructed from FFPE tumor blocks from primary ductal breast

carcinomas in 202 African-American females.

Two separate 1 mm cores represented each case. Five micrometer sections were stained with rabbit

monoclonal antibody against PDL1.

The sections were evaluated for the percentage and intensity of membrane

staining in both tumor and immune cells. Cut off was > 1%. Bivariate

analysis was done via χ2 analysis and survivability data was calculated via the

generation of Kaplan-Meier curves (SPSS v19). Statistical significance was

assumed if p < 0.05.

Results: PDL1 expression in

only tumor cells, only immune cells and in combination was associated with ER negative

(p<0.0001), PR negative (p<0.0001), Triple negative subtype (p<0.0001)

and high grade (p<0.0001) breast ductal cancers.

Conclusion: Our study finding of selective expression of PDL1 in triple negative breast ductal cancers (TNBC) in AA women suggests that inhibition of adaptive immune response is involved in the progression of these cancers. PDL1/PD1 is the inhibitory check point of immune response resulting in decreased T cell activation and cytokine production. Some studies have shown that PDL1 expression is associated with loss of PTEN and phosphatide inositol 3 kinase pathway (PI3K) activation. Our previous study showed loss of PTEN in TNBC. Combining PDL1 inhibitors with PI3K inhibitors may be useful therapy for aggressive TNBC.

#608

Augmenting and broadening T cell responses against mutated tumour neo-antigens.

Bruce W. Robinson,1 Shaokang Ma,1 Jonathan Chee,1 Craig Rive,1 Paula Van Miert,1 Rob A. Holt,2 Jenette Creaney1. 1 _University of Western Australia, Western Australia, Australia;_ 2 _University of British Columbia, Vancouver, British Columbia, Canada_.

Cytotoxic T lymphocytes (CTLs) recognize mutated tumor proteins (neo-antigens) and are important for anti-tumor immunity, especially in the context of immune checkpoint blockade immunotherapy (ICPB). Positive outcomes to ICPB are associated with high neo-antigen loads and with neo-antigen specific CTL responses. However only around 20% of patients respond to ICPB.

In order to examine ways in which the non-responders might become responders we examined several strategies to improve response rates, using anti-CTLA4 initially as the ICPB therapy in BALB/c mice in which Uqcrc2 has been defined as a DNA/RNAseq-identified neo-antigen in AB1 tumor lines induced by a relevant human carcinogen(1).

a) We first examined therapy-induced changes in T-cell responses against Uqcrc2. Anti-CTLA4 alone increased the magnitude of responses against Uqcrc2. Anti-CTLA4 combined with anti-GITR induced determinant spreading, unmasked responses against a new neo-antigen UNC45A that was undetectable during normal tumour growth. Immunogenic chemotherapy also unmasked responses against UNC45A, suggesting that subdominant neo-antigens can be unmasked by the appropriate immunotherapy.

b) We then evaluated neo-antigen vaccination strategies. Uqcrc2 vaccination only protected against tumor growth when administered in combination with partial Treg depletion (Foxp3.DTR mice), suggesting that neo-antigen vaccination will only be maximally effective when administered in combination with therapies that modulate existing immune restraints, including Tregs.

c) We determined optimal anatomical location for tracking neo-antigen CTL responses and identified the draining lymph node as an optimal, though not exclusive, location for response testing compared to blood or tumor.

We are currently examining these observations using sequencing-defined neo-antigens in our patients and will present data on changes in human neo-antigen responses to therapy. These observations have important translational implications for identification of key neo-antigens, choice of therapy and monitoring of anti-tumor responses.

1.

J. Creaney et al., Strong spontaneous tumor neoantigen responses induced by a natural human carcinogen. Oncoimmunology 4, e1011492 (2015).

#609

Overcoming host histocompatibility barrier to create a renewable source of off-the-shelf effector lymphocytes for adoptive immunotherapy.

Raedun L. Clarke, Matthieu Bauer, Ryan Bjordahl, Jeffrey Sasaki, Brian Groff, Svetlana Gaidarova, Tom Tong Lee, Weijie Lan, Michelle Burrascano, Ramzey Abujarour, Greg Bonello, Megan Robinson, Stewart Abbot, Scott Wolchko, Daniel Shoemaker, Bob Valamehr. _Fate Therapeutics, Inc., San Diego, CA_.

Encouraging clinical outcomes in autologous cellular immunotherapy have garnered hope and excitement. However, limitations of patient-derived cancer immunotherapies remain to be addressed to deliver reliable and efficacious therapies with broader applicability. Induced pluripotent stem cells (iPSCs) are a unique, renewable source for the continuous generation of cellular therapeutics and represent a highly promising approach for overcoming many of the limitations of autologous therapy. To advance the promise of iPSC technology as an "off-the-shelf" (OTS) source of cellular therapeutics, several considerations need to be addressed. Ensuring the persistence of allogeneic OTS therapies after adoptive cell transfer across histocompatibility barriers is a key requirement. Establishing a master cell line from genetically engineered clonal iPSC lines with the capacity to continuously generate homogenous populations of highly functional effector cells will also be necessary. Here we demonstrate a comprehensive approach for the generation of immune tolerant effector cells derived from a genetically engineered iPSC master cell line. We successfully combined deletion of classical human leukocyte antigen molecules with expression of immunosuppressive proteins to generate clonal iPSC lines with the ability to escape immune rejection. Utilizing in vitro quantitative live cell analysis we show that OTS-iPSCs elicit a significantly decreased cytotoxic response from both peripheral blood (PB)-NK cells (47.9 vs. 91.4% survival at 3:1 E:T ratio) and PB-T cells (>2.7-fold greater number of OTS-iPSC derived cells remaining at 88 hrs). Additionally, mixed lymphocyte reactions employing unfractionated PB mononuclear cells resulted in significantly decreased activation and proliferation of CD8+ T cells (63.4 vs. 29.6%), CD4+ T cells (70.9 vs. 17.3%) and NK cells (46.8 vs. 11.6%). In preclinical mouse models we demonstrate that OTS-iPSCs exhibit improved persistence in vivo. Bilateral engraftments were established in non-conditioned, fully immune-competent recipient mice using luciferized wildtype and OTS-iPSCs. Daily bioluminescence imaging revealed a significant increase in persistence of OTS-iPSCs during the 48-196 hour post injection window (>5.5 fold greater luminescence at 96 hrs). Using our potent chemically-defined stage-specific monolayer hematopoietic differentiation platform, we demonstrate that OTS-iPSC derived CD34 expressing hematopoietic cells are reproducibly scaled and readily give rise to functional lymphocytes carrying the engineered targeted modality in a homogenous manner (95 +/- 5%). The outlined preclinical data illustrate that iPSCs are an ideal renewable source for OTS hematopoietic cell-based immunotherapies and represent a potentially exponential advancement in adoptive immunotherapy.

#610

Blockade of tumor expression of PD-1 promotes lung cancer growth in a murine model.

Shisuo Du,1 Qing Guan,2 Adam P. Dicker,1 Bo LU1. 1 _Thomas Jefferson University, Philadelphia, PA;_ 2 _Fudan University, Shanghai, China_.

Lung cancer remains one of the most prevalent and fatal malignancies worldwide. There are substantial evidences that immunotherapies offer significant benefits among lung cancer patients. Pembrolizumab, a humanized programmed death receptor-1(anti-PD-1) antibody is now approved by FDA as a first-line treatment of patients with metastatic non-small cell lung cancer (NSCLC). Recent data demonstrate that PD-1 is not only expressed by immune cells, but also by human melanoma cells. However, little is known about the biological significance of PD-1 pathway in cancer including NSCLC. Thus, we examined PD-1 expression in a series of established murine lung cancer cell lines. RT-PCR amplification and sequencing of the full coding sequence of the murine PD-1 (PDCD1) gene revealed the presence of PDCD1 mRNA in the M109 lung cancer cells. Flow cytometry and immunofluorescence analysis demonstrated PD-1 protein expression in these murine lung cancer cells. To determine the molecular function of tumor PD-1 in lung cancer survival, we treated M109 cells in culture with either anti- mouse PD-1(100ug/ml) or isotype control antibody and found that PD-1 inhibition significantly enhanced cancer cell viability as measured by MTT and clonogenic assay. Alternative PD1 inhibition by either knockdown of PDCD1 via siRNA or knockout of PDCD1 by CRISPR-Cas9 all lead to increased survival of M109 cells. Furthermore, PDCD1-knockout M109 cells had no survival difference in the presence of anti-PD1 compared to its PD1-expressing parental cells. Conversely, treatment with recombinant PD-L1 fusion protein exhibited significantly increased apoptotic rates (7.5±3.2% vs 2.1±1.2%) suppressing the tumor cell growth in the wild-type M109 in culture. To validate these results in in vivo models, NSG mice were used to determine the direct impact of anti-PD1 on M109 xenografts without the interplay of host immune system. M109 xenografts treated with antiPD-1 (10mg/kg i.p.) antibodies showed accelerated growth compared to those treated by IgG control (2487±473 vs 1499±292mm3 at days 21 after tumor inoculation, p=0.013). M109 subcutaneous tumors in the PD-1 knockout mice also manifested more rapid growth than the same tumors implanted in wild-type mice. Taken together, our results uncover a novel function of PD-1/PD-L1 axis in lung cancer survival and implicate possible tumor-promoting effects from anti-PD-1 therapy upon PD1-expressing lung cancer in the absence of effective immune response. We plan to interrogate bio-specimens collected through NRG trials to establish the translational potential of these findings.

#611

Reversion of mesenchymal features in NSCLC cells using M7824, a first-in-class bi-functional immunotherapeutic agent targeting PD-L1 and TGF&#9461 (TGFβ&#946 TRAP).

Justin M. David,1 Jeffrey Schlom,1 Yan Lan,2 Claudia Palena1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _EMD Serono, Billerica, MA_.

Background: Evasion of antitumor immunity is widely regarded as a hallmark of cancer. Tumor cells can avoid immune destruction by upregulating the immune checkpoint molecule PD-L1, and antibodies targeting the PD-1/PD-L1 axis have demonstrated clinical efficacy in several tumor types. Antitumor immunity can also be suppressed by TGFβ, a pleiotropic cytokine that reduces innate and adaptive antitumor immune responses. Additionally, TGFβ impacts tumor cells by activating a differentiation program called epithelial-mesenchymal transition (EMT), in which tumor cells lose epithelial features and gain mesenchymal characteristics. The gain of mesenchymal traits is associated with metastasis, stemness, and resistance to multiple therapeutic modalities, including chemotherapy and radiation. The objective of this work is to investigate the therapeutic potential of M7824 in non-small cell lung cancer (NSCLC). M7824 is a first-in-class bi-functional immunotherapeutic agent designed to target two negative regulatory pathways in immunosuppression. It is a novel fusion protein comprised of the extracellular domain of human TGFβRII covalently linked to the C-terminus of the Fc domain of the fully human anti-PD-L1 IgG1 antibody avelumab.

Methodology: In vitro assays were conducted using NSCLC cell lines treated with TGFβ1 alone or in combination with avelumab or M7824 to assess the ability of M7824 to revert features of tumor cell EMT, including molecular marker expression, cell proliferation, and response to various chemotherapies. The ability of M7824 to mediate antibody-directed cellular cytotoxicity (ADCC) of TGFβ1-treated cells in vitro was also assessed.

Results: Treatment with TGFβ1 induced mesenchymal marker expression, reduced cell proliferation, and enhanced resistance to docetaxel, paclitaxel, and gemcitabine. Treatment with M7824, but not avelumab, was able to both prevent and revert these effects of TGFβ1. M7824 also exhibited the ability to mediate ADCC of TGFβ1-treated cells in vitro.

Conclusions: M7824 effectively antagonizes TGFβ1-mediated tumor cell mesenchymalization, including resistance to chemotherapy. These results suggest that M7824 has the potential to relieve immunosuppression and block treatment-resistance induced by TGFβ1 in patients with NSCLC.

#612

Methylation changes in DNA of CD8 T cells following CD137 costimulation.

M. Angela Aznar,1 Sara Labiano,1 Angel Diaz-Lagares,2 Manel Esteller,2 Juan Sandoval,3 Ignacio Melero1. 1 _Center for Appplied Medical Research University on Navarra (CIMA), Pamplona, Spain;_ 2 _Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Spain;_ 3 _Medical Research Institute La Fe, Valencia, Spain_.

Introduction: CD137 ligation imprints long term changes in the behavior of costimulated T-cells (1). There is not a satisfactory mechanistic explanation yet.

Experimental procedures: to determine the specific DNA methylation changes occurring upon CD137 costimulation, purified human CD8+ T cells from three healthy donors were activated in vitro for 5 days with anti-CD3 monoclonal antibody and either with an anti-CD137 monoclonal antibody or its corresponding isotype (huIgG4). Activated lymphocytes were left 5 days in culture with huIL-7 without further stimulation. Such back-to-resting CD8+ lymphocytes were reestimulated with anti-CD3 for 12, 24 and 36h to validate the expression of the genes differentially methylated upon primary stimulation at mRNA and protein levels.

DNA methylation profiles of both activated and resting cell subsets were characterized with Infinium 450K DNA methylation array (Illumina). To further confirm our observations, identical experimental procedure was performed with a different anti-CD137 agonist antibody (mIgG1 isotype) in a CD8+ T-cell donor.

Differentially methylated genes between anti-CD3+anti-CD137 versus their corresponding control counterparts were validated by pyrosequencing on activated and resting CD8+ from independent group of healthy donors (n=8 for hIgG anti-CD137 and n= 11 for mIgG anti-CD137). Expression changes were confirmed by qRT-PCR and flow cytometry in activated, rested and reestimulated CD8+ lymphocytes.

Results: 853 genes were differentially methylated in hIgG4 anti-CD137-treated CD8+ T cells as compared with their controls, 52 of which were shared with mIgG1 anti-CD137-costimulated CD8+ T lymphocytes. A number of differentially methylated genes are involved in i) T cell migration, ii) T cell activation, survival and homeostasis and iii) regulation of gene expression including key T-cell transcription factors.

Conclusions: CD137 costimulation induces CD8+ T lymphocytes that are poised to respond more effectively to a second antigen exposure. These acquired functions are imprinted in the genomic DNA of the CD8+ T cells by DNA methylation changes upon CD137 co-stimulation, and involve key genes for CD8+ T cells.

References: 1.

Hendriks, J., Y. Xiao, J.W. Rossen, K.F. van der Sluijs, K. Sugamura, N. Ishii, and J. Borst, During viral infection of the respiratory tract, CD27, 4-1BB, and OX40 collectively determine formation of CD8+ memory T cells and their capacity for secondary expansion. J Immunol 2005, 175: p. 1665-76.

#613

Development of FPA154, a novel tetravalent anti-GITR antibody, for the treatment of solid tumors.

Susannah D. Barbee,1 Amanda Chen,1 Susan Johnson,1 David I. Bellovin,1 John C. Timmer,2 Nebiyu Wondyfraw,1 Mikayel Mkrtichyan,1 Amir S. Razai,2 Kyle S. Jones,2 Chelsie Y. Hata,2 Denise Gonzalez,1 Quinn Deveraux,2 Brendan P. Eckelman,2 Luis Borges1. 1 _Five Prime Therapeutics, South San Francisco, CA;_ 2 _Inhibrx, San Diego, CA_.

Glucocorticoid-induced TNFR-related (GITR, TNFRSF18) is a member of the TNFR superfamily with pleiotropic T cell modulatory activity. We are developing a novel anti-GITR antibody with enhanced agonist activity for the treatment of solid tumors. Our candidate molecule, FPA154, is constructed with single-domain antibodies (sdAbs) in a tetravalent format, with an effector-competent IgG1 Fc domain. Both FPA154 and the mouse-reactive surrogate molecule (cmFPA154, mIgG2a isotype) bind to cell-surface GITR with high affinity. GITR is most highly expressed in vivo on activated and intratumoral Treg, and our data indicates that FPA154 and cmFPA154 potently mediate ADCC activity against Treg expressing high levels of GITR. In contrast, activated effector T cells express modest levels of cell-surface GITR, and FPA154 and cmFPA154 drive GITR-induced NF-κB activation. This activity is independent of Fc-mediated crosslinking, which is normally required for bivalent GITR antibodies to induce GITR signaling. cmFPA154 has potent antitumor activity in several syngeneic mouse tumors, both as a monotherapy treatment and in combination with anti-PD-1. In summary, FPA154 is a promising candidate with multiple mechanisms of action that contribute to generation of an antitumor immune response mediated by different T cell subsets.

#614

VB4-845 tumor cell killing in a combination study with the anti-PD-1, Nivolumab.

Rachelle Lee Dillon,1 Shilpa Chooniedass,1 Arjune Premsukh,1 Glen MacDonald,2 Jeannick Cizeau,1 Gregory A. Adams3. 1 _Viventia Bio Inc., Winnipeg, Manitoba, Canada;_ 2 _Eleven Biotherapeutics Inc., Winnipeg, Manitoba, Canada;_ 3 _Eleven Biotherapeutics Inc., Philadelphia, PA_.

VB4-845 is a Targeted Protein Therapeutic (TPT) comprised of a scFv fragment specific for the Epithelial Cell Adhesion Molecule (EpCAM) genetically fused to a truncated form of Pseudomonas exotoxin A (ETA), via a flexible linker. Due to the inherent immunogenicity of the ETA moiety, VB4-845 is only used for the treatment of loco-regionally accessible tumors and is currently in a Phase III clinical trial for the treatment of high grade, non-muscle invasive bladder cancer. In a Phase I study in late stage squamous cell carcinoma of the head and neck, direct injection of VB4-845 led to shrinkage of the principal injected tumor, as well as non-targeted tumors in some patients, suggesting the activation of a T cell-mediated anti-tumor response through cross-priming. Only cells undergoing immunogenic cell death, or ICD, are capable of stimulating cross-priming. ICD is characterized by distinct "eat-me" signals such as the release of ATP and HMGB1, and cell surface translocation of calreticulin, an endoplasmic reticulum chaperone protein. To evaluate whether VB4-845-mediated cell killing results in ICD events, treated tumor cell lines were assessed for these distinct signaling molecules. In vitro studies using flow cytometry and ELISA showed both translocation of calreticulin and release of ATP and HMGB1 by VB4-845-treated tumor cells. Immune T cell activation via an ICD pathway suggests a complimentary outcome when combined with checkpoint inhibitors. A study was performed in PDX tumor-bearing NOG mice reconstituted with a human immune system to assess the combination of intratumoral injection of VB4-845 with an anti-PD1 antibody, Nivolumab, given i.p. VB4-845 showed growth suppression of the injected tumor whereas the growth delay of the contralateral, non-injected tumor was more pronounced with the addition of Nivolumab. In summary, the data presented suggests that VB4-845 mediates tumor cell killing by an ICD pathway and that the resulting cross-priming effect can enhance the anti-tumoral activity of immune checkpoint inhibitors.

#615

Local irradiation with systemic anti-PD1 antibody may effectively suppress the micrometastasis in distant organ through the induction of abscopal effects.

Hidenori Tsukui, Rihito Kanamaru, Ai Sadatomo, Daishi Naoi, Tetsuichiro Shimizu, Makiko Tahara, Katsusuke Mori, Homare Ito, Mitsuaki Morimoto, Yoshihiko Kono, Yoshiyuki Inoue, Hiroyuki Maruyama, Koji Koinuma, Hisanaga Horie, Yasunaru Sakuma, Yoshinori Hosoya, Naohiro Sata, Joji Kitayama. _Jichi Medical University, Shimotsuke Tochigi, Japan_.

Background: Tumor shrinkage in response to radiotherapy (RT) is partially dependent on host immunity. Recent clinical results have suggested that PD-1/PD-L1 blockade markedly augments the antitumor immune response especially in case of numerous genetic mutations. Here, in this study, we evaluated if anti-PD-1 mAb can induce abscopal effects of local RT in murine model.

Method: Murine colon cancer cell, LuM-1, is a highly metastatic clone of colon 26. After subcutaneous (s.c) inoculation of LuM-1, micro- and macro- metastases were observed in bilateral lung at 1 and 2 weeks, respectively. Then, LuM-1 (2×105 per mouse) was s.c implanted subcutaneously in the left flanks of BALB/c mice. At day 8, mice were divided into 4 (control, RT, anti-PD-1 mAb, RT+anti-PD-1 mA) groups with each group containing 5 mice. In RT group, mice received local RT at day 9 and 17. In brief, anesthetized mice were held in the decubitus position, with the whole body except left flank tumor protected by a lead shield and the ionizing irradiation (X-ray, 8Gy) was selectively delivered to s.c. tumor using MBR-1505R2 (Hitachi Medical Co.) Anti-PD-1 mAb (J43) or control hamster mAb (0.5mg) was given at day 9,11,13 and 17,19,21 At day 28, mice were sacrificed and s.c tumor and lung metastases were quantified with tumor size and lung weight, respectively.

Results: S.c tumor was significantly reduced by RT and/or PD-1 mAb with additive effects. The whole lung weight in PD-1 mAb group was 120±64.0 gr, which was significantly less than control mAb group (306±38.4 gr, p<0.05) RT alone did not affect the lung weight (298±40.0 gr), however, RT if combined withPD-1 mAb, tended to further reduce the lung metastases (69.0±31.5gr,. p=0.12).

Conclusion: Local RT and PD-1 mAb is effective to prevent the growth of micrometastasis in non-irradiated sites through the augmentation of abscopal effects. Combination of RT and immune checkpoint inhibitors can be a new treatment strategy for locally advanced rectal cancer.

#616

The novel α-Gal-based immunotherapy AGI-134 invokes CD8+ T cell-mediated immunity by driving tumor cell destruction, phagocytosis and tumor-associated antigen cross-presentation via multiple antibody-mediated effector functions.

Jenny L. Middleton,1 Oliver Schulz,2 Amber Charlemagne,1 Sascha A. Kristian,1 Stephen Michael Shaw1. 1 _Agalimmune Limited, Sandwich, United Kingdom;_ 2 _The Francis Crick Institute, London, United Kingdom_.

Background: AGI-134 is a fully synthetic α-Gal (Galα1-3Galβ1-4GlcNAc-R) glycolipid that is being developed for the treatment of solid tumors. The α-Gal epitope is not expressed in humans, who, as a result of constant antigenic stimulation by α-Gal-bearing commensal bacteria, develop high titer natural antibodies to α-Gal. We have previously demonstrated that AGI-134 recruits anti-Gal antibodies to tumor cells in vitro, activating complement and driving phagocytosis by antigen presenting cells. AGI-134 also confers systemic protection from distal lesion development in a mouse model of melanoma and synergizes with anti-PD-11. Here we present further data characterizing the multiple pathways activated by the anti-Gal subclasses to drive AGI-134-mediated anti-tumor immunity.

Results: Using quantitative methods, we demonstrate that human anti-Gal is composed of a diverse repertoire of effector antibodies in a panel of serum samples, with IgM, IgG1 and IgG2 being the major subclasses. Polyclonal anti-Gal IgG purified from human IVIG was, like human serum, comprised mainly of IgG1 and IgG2. When AGI-134 treated cells were incubated with human serum, binding of all anti-Gal subclasses was detected by flow cytometry, demonstrating that all anti-Gal subclasses can interact with AGI-134 treated cells and are available to activate downstream effector functions. When human serum was depleted of IgG, AGI-134 stimulated complement dependent cytotoxicity (CDC) was still effective, indicating that anti-Gal IgM is primarily responsible for deposition of complement on AGI-134 treated cells. AGI-134 treated cells incubated with purified polyclonal anti-Gal IgG activated FcγRIIIA on a reporter cell line and promoted natural killer cell-mediated antibody-dependent cell-mediated cytotoxicity (ADCC). Opsonization of AGI-134 treated human cancer cells with human anti-Gal and complement stimulated their phagocytosis by human monocyte-derived macrophages. Ovalbumin-expressing cells treated with AGI-134 and then incubated with human serum to initiate CDC were specifically phagocytosed by murine CD8α+ dendritic cells and the immunodominant antigen of ovalbumin, SIINFEKL, was cross-presented to CD8+ T cells. In conclusion, AGI-134 stimulates adaptive anti-tumor immunity through immune activation and antigen cross-presentation, which is driven by the diverse repertoire of anti-Gal antibodies.

1. Shaw, S. et al. Abstract 4862: AGI-134: a fully synthetic alpha-Gal glycolipid that prevents the development of distal lesions and is synergistic with an anti-PD-1 antibody in a mouse melanoma model. [abstract]. AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4862.

#617

Evaluating the in-vitro effects of tumor treating fields on T cell responses.

Gil Diamant, Ilan Volovitz, Zvi Ram. _Tel Aviv Sourasky Medical Center, Tel Aviv, Israel_.

BACKGROUND: Tumor Treating Fields (TTFields) are low-intensity electrical fields that target proliferating cells by hindering formation of mitotic spindle and translocation of charged organelles. TTFields have been recently approved for the treatment of both recurrent and newly diagnosed Glioblastoma (GBM). Immunotherapeutic approaches for treatment of GBM are considered promising, and multiple strategies are currently being evaluated in basic research and clinical trials. Combining TTFields and immune-based therapies is a rational approach as they possess markedly different mechanisms of action (MOA). Conversely, TTFields may potentially abrogate various cellular functions required for effective T cell responses. We performed an in-vitro evaluation on the effect of TTFields on select human T cell functions that are pivotal for an effective anti-tumoral response. The study objective was to evaluate the potential compatibility between immune-based therapies and TTFields. METHODS: Peripheral blood mononuclear cells were isolated from healthy donors. Cells were cultured under normal versus TTFields conditions using the inovitro TTFields system, either with or without Phytohemaglutenin (PHA - a super antigen). Cellular responses were monitored using an 8-color flow cytometry panel that concurrently evaluated proliferation (CFSE dilution), cytokine secretion (IFNγ), cytotoxic degranulation (CD107a surface presentation), and T cell activation/ exhaustion (PD1 expression). The effect on T cell viability was assessed in a separate assay, by comparing the live-to-dead ratio of cells cultured in normal versus TTFields settings.

RESULTS: TTFields did not alter the functionality of non-activated T cells. Viable PHA-activated T cells cultured under TTFields exhibited no change in PD1 up-regulation, IFNγ secretion and CD107a surface-expression. The T cells exhibited reduced proliferation, which is in line with the known MOA of TTFields. As the presence of polyfunctional T cells is associated with effective anti-tumoral responses, a single-cell level polyfunctionality analysis of activated T cells was performed. The analysis demonstrated that under TTFields conditions non proliferating cells retained all other combinations of immune functions. TTFields were found to have a minor effect on the viability of un-activated T cells. In activated cells, there was a moderate effect on cells that did not attempt to proliferate, but TTFields substantially reduced the viability rate of cells that had proliferated. These findings were true for both helper and cytotoxic T cells.

CONCLUSIONS: Pivotal T-cell response parameters, but not proliferation, were found to be unhindered by TTFields. Our current data suggests that the integration of TTFields with various immunotherapeutic approaches may be a rational strategy to explore for the treatment of brain tumors.

#618

Toll-like receptor 2 costimulation potentiates the antitumor efficacy of CAR T cells.

Peng Li,1 Xin Du,2 Yun Xin,1 Jianyu Weng,2 Peilong Lai2. 1 _GIBH, CAS, Guangzhou, China;_ 2 _Guangdong General Hospital, Guangzhou, China_.

Chimeric antigen receptor (CAR) T cell immunotherapies have shown unprecedented success in treating leukemia but lack efficacy in solid tumors. Here, we generated 1928zT2 and m28zT2, targeting CD19 and mesothelin, respectively, by introducing the Toll/interleukin-1 receptor (TIR) domain of Toll-like receptor 2 (TLR2) to 1928z and m28z. T cells expressing 1928zT2 or m28zT2 showed enhanced effector function, expansion and persistency against CD19+ leukemic or mesothelin+ lung cancer cells in vitro and in vivo. In a patient with relapsed B cell acute lymphoblastic leukemia, a single dose of 5×104/kg 1928zT2 T cells resulted in robust expansion and leukemia eradication and led to complete remission. Hence, our results demonstrate that TLR2 signaling is a contributing component to CAR T cells for both leukemia and solid tumors and is capable of increasing the efficacy of CAR T cells and facilitating low dose clinical usage.

#619

Effect of anti-PD-1 therapy on immune cells in the peripheral blood of non-small cell lung cancer patients.

Eleni-Kyriaki Vetsika,1 Galatea Kallergi,2 Despoina Aggouraki,1 Zaharoula Lyristi,1 Aristeidis Koukos,1 Despoina Kourougkiaouri,1 Filippos Koinis,1 Vassilis Georgoulias,1 Athanasios Kotsakis1. 1 _Laboratory of Translational Oncology, University of Crete, Heraklion, Greece;_ 2 _Department of Biochemistry, University of Crete, School of Medicine, Heraklion, Greece_.

Background: Programmed cell death-1 (PD-1), an inhibitory immune check-point, plays a pivotal role in tumor immune escape. The interaction of PD-1 with its ligand (PD-L1) results in T cells exhaustion, and the blockade of this interaction can partially restore T cell function. Recently, antibodies targeting PD-1 and PD-L1 have been approved for treatment of advanced Non Small Cell Lung Cancer (NSCLC). In this pilot study, we aimed to investigate the effect of anti-PD1 treatment or chemotherapy on the frequencies of circulating PD-1+ T cells and PD-L1+ immunosuppressive cells in NSCLC patients. Patients & Methods: Peripheral blood samples were collected from 35 advanced NSCLC patients before initiation of treatment and after 3 cycles. Twelve treatment-naïve patients received front-line chemotherapy, whereas 23 patients received anti-PD1 treatment in the second-line setting. Flow cytometry was used to quantify PD-1- and PD-L1-expressing immune cells. Changes in the frequencies of these cells were compared between the two settings and correlated with the clinical outcome. Results: Chemotherapy had no effect on the percentages of PD-1+CD4+ and PD-1+CD8+ T cells after 3 cycles, whereas there was a significant decrease in PD-1+CD4+ and PD-1+CD8+ T cells in patients who received 3 administrations of anti-PD1 antibody (p=0.007 and p=0.05, respectively). Moreover, the levels of PD-1-CD4+ (p=0.009) and PD-1-CD8+ (p=0.009) were augmented in response to anti-PD-1 therapy. The frequencies of both peripheral CD4+ Tregs (CD3+CD4+CD25highCD127-/lowCD152+FoxP3+) and granulocytic MDSCs (G-MDSC; CD14-CD15+CD33+CD11b+HLA-DR-Lin-) expressing PD-L1 were decreased following anti-PD1 therapy (p=0.01 and p=0.02, respectively). In contrast, chemotherapy affected only the PD-L1+CD4+ Tregs, but not the PD-L1+G-MDSC, by increasing their levels after 3 cycles (p=0.04). Anti-PD-1 treatment induced a superior reduction of the PD-1+CD4+, PD-1+CD8+ T cells, PD-L1+CD4+ Tregs and PD-L1+G-MDSCs percentages compared to the effect of first line chemotherapy (p=0.04, p=0.05, p=0.002 and p=0.01, respectively). Furthermore, a significant decrease in PD-1+CD8+ T cells, PD-L1+CD4+ Tregs and PD-L1+G-MDSCs after 3 doses of anti-PD-1 was observed in patients who experienced stable disease compared to baseline (p=0.006, p=0.05 and p=0.03, respectively). At the time of response evaluation to chemotherapy, the percentage of the PD-L1+CD4+ Tregs after 3 cycles was significantly inferior compared to baseline, in disease progressors (p=0.04). Conclusion: These data indicate that although chemotherapy affected the levels of PD-L1+CD4+ Tregs, anti-PD1 therapy seems to exert an effect on both PD1+ T cells and PD-L1\+ immunosuppressive cells. Additional studies are needed in a larger cohort in order to document its impact on their clinical relevance in NSCLC patients. This study is ongoing and updated data will be presented at the meeting.

#620

Tumor microenvironment heterogeneity is not identified across multiple histologically similar tumors from the same patient.

Carl D. Morrison,1 Jeffrey Conroy,1 Sean Glenn,1 Blake Burgher,1 Sarabjot Pabla,1 Maochun Qin,1 Antonios Papanicolau-Sengos,1 Jon Andreas,1 Vincent Giamo,1 Mary Nesline,1 Shipra Gandhi,2 Manu Pandey,2 Nischala Ammannagari,2 Kunle Odunsi,2 Marc Ernstoff,2 Mark Gardner1. 1 _OmniSeq, LLC, Buffalo, NY;_ 2 _Roswell Park Cancer Institute, Buffalo, NY_.

Introduction: Tumor heterogeneity has been well documented for mutational analysis in virtually all types of tumors and is accepted as a true finding. Heterogeneity of the tumor microenvironment (TME) in the context of response to checkpoint inhibitors has not been well studied; the belief is that variation will be identified across multiple tumors from the same patient. The expectation is that multiple tumors from a single patient would demonstrate extensive TME heterogeneity driven by the neoplasm.

Methods: We validated and utilized a targeted RNA-seq immune panel of >350 genes to interrogate the TME of 49 different tumors from 17 unique patients. These samples for one patient represented primary and metastatic tumors that were separated by multiple years. Prior to this study we built a reference database of RNA-seq immune results for this panel of 167 samples. An in-depth analysis of genes associated with checkpoint inhibition (CPI) and tumor infiltrating lymphocytes (TILs) were the focus of the comparative analysis. Unsupervised analysis and gene rank by RNA-seq were the primary modes of comparison.

Results: For more than one-half of these patients the different tumors for a single patient separated by multiple years more closely resembled the other tumors from that patient than the reference population by unsupervised clustering. When ranked by LOW, MODERATE, or HIGH expression of genes associated with TILs or CPI the results for the majority of patients were highly concordant: LOW TILs / LOW CPI associated gene expression.

Conclusion: Our results support a paradigm shift in the influence of the host on TME heterogeneity with evidence that the host and not the neoplastic cells are the primary determining factor. TME heterogeneity is not identified across multiple tumors of the same histology collected from different sites across time points from the same patient. This study does not evaluate multiple primary tumors from the same patient, but is an additional study we have planned. | |  | |  | |  | |

---|---|---|---|---|---|---|---|---

Subject ID | M-Path | T-Path | PMR | Specimen Year | TILs Score | TILs Interpretation | CPI Score | CPI Interpretation

1 | Melanoma | Lymph node NOS | Metastatic | 2009 | 45 | LOW | 13 | LOW

1 | Melanoma | Subcutaneous tissue NOS | Metastatic | 2010 | 8 | LOW | 2 | LOW

2 | Bladder cancer | Urinary bladder NOS | Primary | 2012 | 37 | LOW | 16 | LOW

2 | Bladder cancer | Urinary bladder NOS | Primary | 2013 | 10 | LOW | 11 | LOW

2 | Bladder cancer | Soft Tissue | Metastatic | 2014 | 14 | LOW | 23 | LOW

3 | Melanoma | Brain NOS | Metastatic | 2010 | 58 | MODERATE | 37 | LOW

3 | Melanoma | Duodenum NOS | Metastatic | 2012 | 51 | MODERATE | 41 | LOW

4 | Melanoma | Soft Tissue NOS | Metastatic | 2005 | 98 | HIGH | 88 | HIGH

4 | Melanoma | Soft Tissue NOS | Metastatic | 2005 | 22 | LOW | 3 | LOW

5 | Melanoma | Mesentery NOS | Metastatic | 2012 | 45 | LOW | 35 | LOW

5 | Melanoma | Peritoneum NOS | Metastatic | 2014 | 87 | HIGH | 86 | HIGH

6 | Bladder cancer | Urinary bladder NOS | Primary | 2009 | 5 | LOW | 4 | LOW

6 | Bladder cancer | Lung NOS | Metastatic | 2012 | 43 | LOW | 41 | LOW

7 | Melanoma | Skin of Trunk NOS | Metastatic | 2011 | 4 | LOW | 4 | LOW

7 | Melanoma | Chest wall NOS | Metastatic | 2012 | 2 | LOW | 1 | LOW

7 | Melanoma | Soft Tissue | Metastatic | 2012 | 25 | LOW | 25 | LOW

8 | Lung adenocarcinoma | Lung right upper lobe of, NOS | Primary | 2009 | 99 | HIGH | 88 | HIGH

8 | Lung adenocarcinoma | Lung right upper lobe of, NOS | Primary | 2016 | 88 | HIGH | 80 | HIGH

9 | Melanoma | Lymph node NOS | Metastatic | 2009 | 2 | LOW | 5 | LOW

9 | Melanoma | Small intestine NOS | Metastatic | 2011 | 0 | LOW | 1 | LOW

10 | Melanoma | Skin of Lower Extremity NOS | Metastatic | 2012 | 16 | LOW | 8 | LOW

10 | Melanoma | Lymph node NOS | Metastatic | 2012 | 43 | LOW | 31 | LOW

10 | Melanoma | Skin of Lower Leg NOS | Metastatic | 2013 | 4 | LOW | 13 | LOW

11 | Melanoma | Lung left lower lobe of, NOS | Metastatic | 2012 | 14 | LOW | 5 | LOW

11 | Melanoma | Skin of Scalp NOS | Primary | 2010 | 22 | LOW | 20 | LOW

12 | Lung adenocarcinoma | Lung NOS | Primary | 2010 | 89 | HIGH | 77 | HIGH

12 | Lung adenocarcinoma | Brain NOS | Metastatic | 2014 | 41 | LOW | 13 | LOW

13 | Melanoma | Liver NOS | Metastatic | 2011 | 66 | MODERATE | 51 | MODERATE

13 | Melanoma | Lymph node NOS | Metastatic | 2011 | 47 | LOW | 13 | LOW

14 | Renal cell carcinoma | Lung right lower lobe of, NOS | Metastatic | 2013 | 95 | HIGH | 87 | HIGH

14 | Renal cell carcinoma | Adrenal gland NOS | Metastatic | 2014 | 65 | MODERATE | 71 | MODERATE

15 | Lung adenocarcinoma | Lung right middle lobe of, NOS | Primary | 2014 | 88 | HIGH | 75 | HIGH

15 | Lung adenocarcinoma | Brain NOS | Metastatic | 2015 | 26 | LOW | 1 | LOW

16 | Melanoma | Tonsil NOS | Metastatic | 2014 | 62 | MODERATE | 53 | MODERATE

16 | Melanoma | Soft Tissue | Metastatic | 2015 | 13 | LOW | 1 | LOW

17 | Melanoma | Dura Mater NOS | Metastatic | 2015 | 53 | MODERATE | 16 | LOW

17 | Melanoma | Lymph node NOS | Metastatic | 2015 | 99 | HIGH | 88 | HIGH

#621

Tumor suppressor TUSC2 immunogene therapy is synergistic with anti-PD1 in lung cancer syngeneic mouse models.

Ismail M. Meraz, Mourad Majidi, RuPing Shao, Meng Feng, Xiaobo Cao, David Rice, Boris Sepesi, Lin Ji, Jack Roth. _UT MD Anderson Cancer Ctr., Houston, TX_.

TUSC2, a pro-apoptotic tumor suppressor gene whose expression is lost or decreased in most lung cancers, activates the innate immune system through initiation of broad spectrum cytokine secretion and natural killer (NK) cell activation. TUSC2 delivered systemically by nanovesicles has mediated tumor regression in metastatic non-small cell lung cancer clinical trials. We studied the effect of TUSC2 on immune cell populations and the anti-tumor activity of TUSC2 in combination with anti-PD1 checkpoint blockade in two syngeneic mouse models: C57BL/6 mice subcutaneously injected with murine lung adenocarcinoma cell line CMT/167-luc cells (KrasG12V mutation) and 344SQ (KrasG12D allele and a knock-in Trp53R172HΔG allele) adenocarcinomas which metastasize to the lung in 129S2 mice. Tumor growth was monitored by scoring ex-vivo luminescence using the IVIS Imaging System 200. Multi-color flow cytometry was used for immune profiling of circulating immune cells after nanovesicle mediated TUSC2 intravenous injection. Cytokine gene expression in response to TUSC2 in sorted immune subpopulations was determined by real-time PCR. Tumor growth was significantly reduced with TUSC2 treatment compared with no treatment in both subcutaneous and metastatic mouse models. Synergistic anti-tumor activity was observed when TUSC2 was combined with anti-PD1 verified in five independent experiments. In the lung metastasis model, mice treated with TUSC2 + anti-PD1 lived significantly longer than with single agent treatment. Circulating NK cells increased three fold following TUSC2 nanovesicle intravenous injection both in tumor free and tumor bearing mice which correlated with tumor regression and survival. Cytotoxic T lymphocyte responses were increased whereas Tregs and MDSCs decreased with TUSC2 alone and TUSC2+anti-PD1 treatment. The levels of T cell checkpoint markers PD1, CTLA-4, LAG-3, and TIM-3 evaluated by flow cytometry were decreased after TUSC2 treatment. TUSC2 anti-tumor response was abolished when NK cells were depleted indicating NK cells are important mediators of the TUSC2 treatment effect. Single cell suspension analysis by flow cytometry showed high numbers of NK cells infiltrating lung tumor metastases after TUSC2 treatment. The number of tumor nodules in the lung was significantly less following treatment with TUSC2 nanovesicles compared with control. IL-15 gene expression which mediates NK cell proliferation, was increased by TUSC2. In conclusion, systemic TUSC2 nanovesicle immunogene therapy combined with checkpoint blockade showed synergistic anti-tumor efficacy and activated the immune system through upregulation of NK cells and CTL and downregulation of regulatory cells.

#623

**Identifying candidate antigens for a ductal carcinoma** in situ **vaccine that are essential to breast cancer survival across multiple subtypes.**

Sasha Elizabeth Stanton, Erik Ramos, James Annis, Andrew Timms, Tessa Rue, Mary L. Disis. _Univ. of Washington, Seattle, WA_.

Vaccine therapy may be ideal to destroy ductal carcinoma in situ (DCIS) and prevent recurrence. A vaccine can induce type 1 T-cells against DCIS antigens that could migrate from the circulation to invade and destroy the tumor as well as generate immunologic memory to provide long lasting protective immunity. One of the limitations to developing a DCIS vaccine is identifying antigens that target all subtypes of DCIS. DCIS has many of the same molecular abnormalities as invasive breast cancer (IBC), including overexpressed proteins that may be drivers of progression to invasive disease. Eliminating cells that are overexpressing driver proteins by vaccination may prevent progression to IBC if such proteins could be determined.

In this study, we identified proteins that were overexpressed from normal breast in both DCIS and IBC across fifteen Geo and Array Express data sets. From 68 candidate proteins, we identified twelve whose expression was necessary for cancer cell survival across breast cancer subtypes. We selected overexpressed proteins necessary for cancer survival using a high throughput siRNA screen and chose candidates that increased apoptosis and decreased cell survival in HER2 positive (HER2), triple negative (TN), and hormone receptor positive HER2 negative (HR) human breast cancer cell lines with decreased expression of the target protein. For example, decreased expression of NDC80 caused decreased cell survival to 56±3% in HR, 46±3% in TN, and 77±2% in HER2 breast cancer cell lines and increased apoptosis by 1.4±0.03 fold in HR and 1.2±0.03 fold in TN breast cancer cell lines. Decreased expression of RRM2 caused decreased cell survival to 66±3% in HR and 89±2% in HER2 breast cancer cell lines and increased apoptosis by 1.6±0.08 fold in HR, 1.4±0.07 fold in TN, and 2.4±0.3 fold in HER2 breast cancer cell lines. Twelve proteins (AURKA, KIF11, NDC80, RRM2, SDC1, UBE2C, HJURP, CENPA, CENPF, HIST2H2AA3, HIST1H2BD, and TOP2A) were essential for cancer cell survival in at least 2 breast cancer subtypes.

These protein targets are immunogenic in patients with DCIS. In the sera of women without breast atypia (n=36), autoantibodies to NDC80 were detected with a mean of 5.3±1.8 ng/mL while in women with fibroadenoma (n=36) autoantibodies were detected with a mean of 8.3±1.5 (p=0.05). In the sera of women without breast atypia (n=36), autoantibodies to RRM2 were detected with a mean of 0.16±0.7 ng/mL while in women with fibroadenoma (n=36) autoantibodies were detected with a mean of 7.2±2.1 (p=0.05) and in women with DCIS (n=59) autoantibodies were detected with a mean of 2.6±0.6 ng/mL (p=0.0003). These proteins represent DCIS antigens of biologic importance in tumor growth and, potentially, progression to IBC and are candidate immunogens for a vaccine to treat DCIS.

#624

Characterization of PD-L1 expression and the immune cell microenvironment in hepatocellular carcinoma (HCC) and non-cirrhotic liver tissue adjacent to HCC.

Christian Ihling,1 Sienna Yoast,2 Yue Zhang,3 Bartholomew Naughton,3 Miriam Urban,1 P. Alexander Rolfe,3 Eveline Frick-Krieger,1 Isabelle Dussault3. 1 _Merck KGaA, Darmstadt, Germany;_ 2 _Dako North American, Carpinteria, CA;_ 3 _EMD Serono Research and Development Institute Inc., Billerica, MA_.

Still-emerging evidence suggests that HCC is at least moderately responsive to therapeutic agents that target tumor immune suppression pathways, specifically the programmed death 1/programmed death ligand 1 (PD-L1) pathway. Prior studies raise the possibility that PD-L1 is a prognostic biomarker in HCC, where its expression levels have been reported to correlate with tumor aggressiveness and recurrence following surgical resection. The purpose of this study was to evaluate the expression of PD-L1 in non-cirrhotic liver tissue adjacent to HCC and HCC itself in 68 procured tissue samples, as well as elucidate the immune cell composition of the HCC tumor microenvironment. All cases showed the typical morphology of HCC and were classified as low- to high-grade trabecular, pseudoglandular, or solid with the common cytoplasmic features. Immunohistochemical (IHC) staining for PD-L1 in tumor-free liver (TFL) revealed PD-L1 staining in sinusoidal lining cells (macrophages [Mφ] and endothelial cells [EC], as confirmed by double labeling for CD68 and CD31), although there was considerable heterogeneity in the extent of PD-L1 staining. More specifically, in TFL, some but not all CD68+ Mφ were also PD-L1+, whereas most CD31+ EC were also PD-L1+. Furthermore, we evaluated PD-L1 staining qualitatively and semiquantitatively in HCC. Only few tumor cells displayed membranous PD-L1 staining, and 62/68 of the cases (91.2%) were categorized as PD-L1- vs 6/68 (8.8%) PD-L1+ (≥1% and <25% = PD-L1+); instead, PD-L1 expression within the tumor microenvironment predominantly emanated from immune cell infiltrates (as confirmed by PD-L1/pan-cytokeratin double labeling). Then we assessed the immune milieu in HCC tissue specimens using quantitative IHC. We found considerable interspecimen variation in the number of CD68+ Mφ, CD8+ cytotoxic T lymphocytes, and FoxP3+ regulatory T lymphocytes. Comparing the number of immune cells in different tumor compartments showed that the prevalence of CD68\+ cells (p=.0005), CD8\+ cells (p=.0004), and FoxP3\+ cells (p=.05) was significantly higher within the invasive margin vs the center of the tumor. Quantitative analysis of the immune cell content also showed that there was no correlation between T lymphocyte infiltration (CD8+/FoxP3+) and Mφ infiltration in any compartment. Taken together, our findings confirm the intertumoral heterogeneity of the immune cell microenvironment in HCC; however, future studies are needed to correlate these findings to the clinical setting with immunooncologic treatments.

#625

Eradication of cancer cells by T-cell receptor-engineered T cells targeting neoantigens/oncoantigens.

Tatsuo Matsuda,1 Taigo Kato,1 Yuji Ikeda,1 Matthias Leisegang,2 Sachiko Yoshimura,3 Tetsuro Hikichi,3 Makiko Harada,1 Makda Zewde,1 Jae-Hyun Park,1 Hans Schreiber,1 Kazuma Kiyotani,1 Yusuke Nakamura1. 1 _University of Chicago, Chicago, IL;_ 2 _Charité Campus Buch, Germany;_ 3 _OncoTherapy Science Inc, Japan_.

Cytotoxic T lymphocytes (CTLs) play critical roles in cancer-immune responses, and functional characterization of CTLs and their cancer-specific antigens will facilitate cancer immunotherapies. Immunogenic peptides, which can be derived from oncogenic proteins specifically expressed in cancer cells but not expressed in normal organs except testis (oncoantigens), or from peptides with somatic nonsynonymous mutations (neoantigens), are known as good targets for CTLs to eradicate cancer cells. In this study, we aimed to establish a method to efficiently identify oncoantigen/neoantigen-specific CTLs. Firstly, we screened candidate HLA-A2402-restricted oncoantigen/neoantigen peptides by in silico prediction of their binding affinity to MHC class I molecules. We conducted an in-vitro stimulation of CD8 lymphocytes carrying HLA-A24:02 allele by each peptide, and then confirmed clonal expansion of the peptide-specific CTLs by TCR repertoire sequencing analysis, interferon-γ enzyme-linked immunospot (ELISPOT) and/or peptide-HLA multimer assays. After identification of TCR alpha-beta pairs, we conducted retroviral transduction and prepared the TCR-engineered T cells to evaluate their cytotoxic activities against cancer cells. As oncoantigens, we isolated the CTLs for FOXM1 and UBE2T from healthy donors, and found these CTLs showed strong cytotoxicity against HLA-A2402-positive cancer cells expressing target proteins, but not against HLA-unmatched cancer cells. Similarly, the TCR-engineered T cells for FOXM1 and UBE2T showed killing effects for only HLA-A2402-positive cancer cells. Neoantigen-specific TCR-engineered CTLs also exhibited the mutated peptide-specific response, but did not cross-react to the nonmutated peptide. In addition, neoantigen-specific cytotoxicity was observed against HLA-A2402-positive cancer cells expressing the proteins with target somatic mutations. In conclusion, we developed the pipeline to screen possible onconatigens/neoantigens and establish antigen-specific TCR-engineered CTLs from peripheral blood lymphocytes. Our approach provides a promising strategy to develop personalized immunotherapies using onconatigen/neoantigen-reactive TCR-engineered T cells to treat cancer.

#626

BGB324, a selective small molecule inhibitor of receptor tyrosine kinase AXL, abrogates tumor intrinsic and microenvironmental immune suppression and enhances immune checkpoint inhibitor efficacy in lung and mammary adenocarcinoma models.

Katarzyna Wnuk-Lipinska,1 Kjersti Davidsen,2 Magnus Blø,1 Agnete Engelsen,2 Jing Kang,2 Linn Hodneland,1 Maria Lie,2 Sebastien Bougnaud,2 Kristina Aguilera,1 Lavina Ahmed,1 Agata Rybicka,1 Eline Milde Nævdal,1 Paulina Deyna,1 Anna Boniecka,1 Straume Oddbjørn,3 Salem Chouaib,4 Rolf Brekken,5 Gro Gausdal,1 James B. Lorens2. 1 _BerGenBio AS, Bergen, Norway;_ 2 _University of Bergen, Bergen, Norway;_ 3 _Haukeland University Hospital and University of Bergen, Bergen, Norway;_ 4 _Institut Gustave Roussy, Université Paris-Sud, Paris, France;_ 5 _UT Southwestern Medical Center, Dallas, TX_.

The AXL receptor tyrosine kinase is associated with poor overall survival in a wide spectrum of cancers including lung and breast adenocarcinomas. AXL signaling is an important regulator of tumor plasticity related to epithelial-to-mesenchymal transition (EMT) and stem cell traits that drive metastasis and drug resistance. Signaling via AXL is also a key suppressor of the anti-tumor innate immune response, and AXL is expressed on several cells associated with the tumor immune microenvironment including natural killer (NK) cells and tumor-associated macrophages. Hence AXL resides uniquely at the nexus between tumor and microenvironmental anti-tumor immune suppression mechanisms. We report that BGB324, a selective clinical-stage small molecule Axl kinase inhibitor, enhances the effect of immune checkpoint blockade in aggressive adenocarcinoma models with limited immunogenicity by targeting both tumor intrinsic and microenvironmental immune suppression. Immune therapy with anti-CTLA4/PD1 in the 4T1 model increased AXL and EMT-marker expression correlating with a lack of response. Combination with BGB324 resulted in durable primary tumor clearance versus anti-CTLA4/PD1 alone. In a separate study, BGB324 + anti-CTLA4 treatment resulted in significant long-term primary tumor clearance while no response was observed with anti-CTLA4 treatment alone. The extensive metastasis to the lung, liver and spleen characteristic of the 4T1 model was not detected in animals responding to the combination treatment. Importantly, responding animals rejected orthotopic 4T1 tumor cell re-challenge, demonstrating sustained tumor immunity. In the LL2 Lewis Lung model, BGB324 in combination with anti-PD1/PDL1 significantly prevented tumor growth compared to treatment with anti-PD1/PDL1. Tumors from mice treated with BGB324 in combination with immune checkpoint inhibitors displayed reduced EMT traits, altered cytokine expression, enhanced tumor infiltration of effector cells and decreased number of mMDSC. Also, BGB324 significantly reduced IL10 secretion by isolated human macrophages and enhanced human NK-cell mediated NSCLC tumor cell lysis. Collectively these results support a prominent role for AXL in resistance to immune therapy and support clinical translation of combining BGB324 with immune checkpoint inhibitors to improve cancer treatment. 

### Tumor Antigens and Adaptive Immunity

#627

NLRC5 **co-mutations are associated with impaired antigen presentation and immune exclusion in** KRAS **-mutant lung adenocarcinoma.**

Ferdinandos Skoulidis, Taghreed Hirz, Xiang Dong Lee, Jaime Rodriguez Canales, Edwin R. Parra, Pan Tong, Carmen Behrens, Vassiliki A. Papadimitrakopoulou, Jing Wang, Ignacio Wistuba, John V. Heymach. _UT MD Anderson Cancer Ctr., Houston, TX_.

Background: We recently reported that co-occurring genetic events constitute major determinants of the molecular diversity of KRAS-mutant lung adenocarcinoma (LUAC) (Skoulidis et al., Cancer Discovery, 2015). However, comprehensive evaluation of the functional impact of KRAS co-mutations on key cancer hallmarks is thus far lacking. Here, we find that inactivating mutations in NLRC5, a major transactivator of MHC class I molecules, are significantly enriched in KRAS-mutant LUAC and examine the impact of NLRC5 loss on the composition of the tumor immune microenvironment.

Methods: Our cohorts consist of 513 LUACs from the TCGA (145 KRAS-mutant), 152 chemotherapy-naïve surgically resected LUAC from the PROSPECT cohort, 20 platinum-refractory KRAS-mutant LUAC from the BATTLE-2 clinical trial, as well as a panel of 31 KRAS-mutant NSCLC cell lines. Analysis of immune cell sub-population was performed using automated IF-based enumeration. Antigen presentation score was defined as the geometric mean mRNA expression of HLA-A, HLA-B, HLA-C and β2M.

Results: In an unbiased analysis for genes significantly co-mutated with KRAS in LUAC (TCGA cohort) we identified NLRC5 (NLR family, CARD domain containing 5), encoding a recently discovered major transactivator of MHC class I genes (~11% of KRAS-mutant LUAC, odds ratio 2.99, P=0.0197).The spectrum of NLRC5 somatic mutations includes several nonsense and frameshift mutations, as well as missense mutations, many of which are predicted to abrogate normal NLRC5 function. In the TCGA cohort, KRAS/NLRC5 co-mutated tumors exhibited lower antigen presentation score compared to KRAS-mutant NLRC5 wild-type tumors (P=0.0369, t-test). Among KRAS-mutant LUAC from the TCGA, PROSPECT, BATTLE-2 cohorts expression of NLRC5 mRNA correlated tightly with the expression of core antigen presentation pathway components including HLA-A, HLA-B, HLA-C, β2M, TAP1, TAP2, PSMB8 and PSMB9 [in BATTLE-2: HLA-A r=0.7616, P=9.572e-05, HLA-B r=0.834, P=4.884e-06, HLA-C r=0.8029, P=2.036e-05, TAP1 r=0.8189, P=1.009e-05 ]. Similar results were obtained in a panel of 31 KRAS-mutant NSCLC cell line. Thus, both mutational and non-mutational mechanisms can account for NLRC5 inactivation. Finally, in a tumor microarray encompassing surgically resected, chemotherapy naïve LUAC (PROSPECT), NLRC5-low tumors (lower tertile for NLRC5 mRNA expression, N=34), exhibited reduced density of infiltrating CD3+ (P<0.0001, Mann-Whitney U test), CD8+(P<0.0001, Mann-Whitney U test) as well as PD-1+ cells (P<0.0001, Mann-Whitney U test) and PD-L1 Histo-score (P=0.0036, Mann-Whitney U test) compared to NLRC5-high tumors (N=41).

Conclusions: Co-mutations in NLRC5, are enriched in KRAS-mutant LUAC and are associated with immune exclusion. KRAS co-mutations can shape the tumor immune micro-environment and may therefore predict for response - or lack thereof- to immunotherapy.

#628

Neoantigens predicted by clonal mutation analysis in lung adenocarcinoma patients.

Dapeng Zhou,1 Weijing Cai,1 Deng Pan,1 Tan Wen Ling,1 Jiaqian Wang,1 Caichun Zhou,1 Yanyan Lou2. 1 _Shanghai Pulmonary Hospital affiliated with Tongji University School of Medicine, Shanghai, China;_ 2 _Mayo Clinic, Jacksonville, FL_.

Mutant peptides presented in cancer are superior vaccine candidates than self peptides. The efficacy of mutant K-Ras, P53 and EFGR peptides have been tested as cancer vaccines in pancreatic cancer, colorectal cancer, and lung adenocarcinoma. However, the occurrence of these mutations in cancer is limited. Further more, these mutant peptides can only be presented by certain MHC alleles. Neoantigens expressed by passenger mutations which are not involved in common proliferative molecular pathways are potential candidates for personalized vaccine design. Such passenger mutations have drawn attention by the recent clinical data that colorectal cancer with a large number of somatic mutations due to mismatch-repair defects are more susceptible to immune checkpoint blockade by PD1 antibody therapy. In this study, we analyzed the next generation sequencing data of 147 lung adenocarcinoma patients from The Cancer Genome Atlas and predicted neoantigens presented by MHC Class I and Class II molecules. We found 18175 expressed clonal somatic mutations, with an average of 124 per patient. Based on such somatic mutations, we predicted 8731 neo-peptides, among which 3888 strong-binders and 4843 weak-binders to MHC Class I molecules were found by NetMHCPan. For HLA-A*02:01, 778 strong binding and 822 weak binding neo-peptides were found. In average, 11 neo-antigen peptides of mutations were predicted per individual patient with HLA-A*02:01 allele. Smokers expressed 5.73 fold higher mutant neo-antigen peptides than non-smokers. The 20 most commonly mutated genes with predicted neo-antigens are TTN, RYR2, MUC16, KRAS, ZFHX4, TP53, USH2A, NAV3, KEAP1, ANK2, DNAH9, ADAMTS12, CSMD1, CSMD3, SPTA1, FAT3, ZNF804A, PAPPA2, PCLO, EGFR. As a positive control, the frequency of known proliferation-related mutations (KRAS, P53, and EGFR) were determined in 147 patients, which are consistent with previously next generation sequencing studies of lung adenocarcinoma. Mutation hotspots were found in proliferation-related mutations (KRAS), but not in passenger mutations. 6950 neo-peptides, including 259 strong binders and 6691 weak binders were found for HLA DRB1, an MHC Class II molecule. INDEL mutation type of neo-epitopes were found in 119 of 147 patients, with an average of 4.34 INDEL mutations presented by MHC Class I and 0.86 INDEL mutations presented by MHC Class II molecule HLA DRB1 in every patient. Our results support the feasibility of discovering individualized mutant peptides as candidates for immunodiagnosis and immunotherapy of lung adenocarcinoma. Neo-epitopes which can be presented by both MHC Class I and MHC Class II molecules are attractive targets for designing long-peptide vaccines.

#629

An integrated genomic and proteomic analysis of human tumors reveals key factors in neoantigen identification and enables epitope prediction for cancer immunotherapy.

Brendan Bulik-Sullivan, Jennifer Busby, Matthew Davis, Andrew Clark, Tyler Murphy, Michele Busby, Fujiko Duke, Jessica Smith, Jason Hudak, Adnan Derti, Josh Francis, Roman Yelensky. _Gritstone Oncology, Inc, Cambridge, MA_.

Background: Immunotherapy has become essential in the treatment of advanced cancer, with the advent of drug approvals in multiple indications and functional cures for a minority of patients. However, overall survival gains from immunotherapy have been limited and substantial progress remains. The discovery of tumor-specific neoantigens as principal targets of T cells unleashed by immune checkpoint blockade was a key breakthrough in immunotherapy research. Although example neoantigens have been reported, the impact of genomic features on HLA presentation and neoantigen identification has not been comprehensively assessed. Moreover, in silico methods for predicting which mutations lead to neoantigens are currently inadequate for most therapeutic applications, with a false positive rate of >95%.

Methods: We performed deep tumor/normal exome sequencing (Illumina-based, average unique depth 557x/149x), tumor transcriptome sequencing (avg. 111M unique reads), and mass spec HLA class I peptide sequencing (Thermo Lumos, avg. 3,482 peptides) on fresh-frozen primary tumor specimens from 23 patients (22 NSCLC, 1 OC). Somatic mutations were called with a custom GATK-based pipeline, gene expression assessed with RSEM, and peptides matched to mass spectra using COMET/Percolator. We examined the effect of NGS parameters on neoantigen identification, analyzed HLA presentation as a function of genomic features, and created a novel deep learning model for epitope prediction.

Results: We identified 6,252 nonsynonymous somatic mutations across the cohort (avg. 272 per patient) and found 3,874 (62%) transcribed in mRNA. Down-sampling tumor DNA NGS data to a more typical average unique coverage of ~200x and RNA to ~60M reads revealed 3,275 transcribed mutations, an overall 15% loss of candidate neoantigens and >25% loss in low tumor content samples, demonstrating the importance of sequencing depth. Gene expression measurement at the RNA level predicted HLA presentation more strongly than expected, with peptides from genes detected in RNA found presented >20X more often than peptides from non-detected genes. With this and public datasets we trained a model to predict HLA presentation and compared it to standard binding affinity prediction (e.g., NetMHC or MHCflurry) at the strong-binder 50nM threshold for well-characterized alleles. Whereas binding affinity-based prediction gave a positive predictive value (PPV) of only 3%, our integrated deep learning approach showed a PPV of >25% at equivalent recall, a near ten-fold gain.

Conclusions: A novel genomic and proteomic analysis of human tumors establishes NGS requirements for neoantigen identification, characterizes key correlates of HLA presentation, and enables prediction for personalized cancer immunotherapy research.

#630

Inactivation of DNA repair triggers dynamic neoantigen evolution and impairs cancer growth.

Giovanni Germano, Simona Lamba, Giuseppe Rospo, Alessandro Magrì, Federica Maione, Mariangela Russo, Nabil Amirouchene Angelozzi, Ludovic Barault, Monica Montone, Federica Di Nicolantonio, Enrico Giraudo, Alberto Bardelli. _Candiolo Cancer Institute, Candiolo (TO), Italy_.

Colorectal, ovarian, endometrial and other tumors carrying defects in DNA mismatch repair often show favorable prognosis and indolent progression. The genomes of these tumors -also known as microsatellite unstable (MSI) cancers- bear hundreds of thousands of somatic mutations, a feature which fosters cancer progression and rapid evolution of resistance to targeted therapies. The molecular bases for the favorable outcome of MMR deficient cancers have long remained a mystery. Recent evidences that a subset of MSI tumors respond prominently to anti-immune checkpoint blockade led to the seminal hypothesis that the presence of high number of somatic mutations may be responsible for effective immune-surveillance. However, several reports indicate that a relevant fraction of hyper-mutated tumors have unfavorable prognosis and do not respond to immune-modulators. Intrigued by these contradictory findings, we used the CRISPR system to genetically inactivated MutL homolog 1 (MLH1) in colorectal, breast and pancreatic mouse cancer cells. The growth of MMR deficient cells was comparable to their proficient counterparts in vitro and upon transplantation in immune-compromised mice. Strikingly however, isogenic MMR deficient colorectal, breast and pancreatic cancer cells were largely unable to form tumors when injected subcutaneously or orthotopically in syngeneic mouse models. MMR deficient tumors initially established in immunodeficient mice continued to grow exponentially when transplanted in syngeneic animals but regressed promptly when immune checkpoint inhibitors (anti PD-1 and anti CTLA-4) were administered. Exome sequencing of MMR proficient cells revealed mutational loads and neo-antigen profiles that were stable over time. MMR inactivation further increased the mutation burden and led to highly dynamic mutational profiles, resulting in persistent renewal of neoantigens. These results led us hypothesize that enforced increase of the number of mutations in cancer cells could be -paradoxically- beneficial for therapeutic purposes. We therefore performed a pharmacological screen to identify agents capable of permanent inactivation of MMR in colorectal, breast and PDAC cancer cells. We found that temozolomide triggers MLH1 inactivation and leads to rapid clonal evolution and dynamic neoantigen profiles. Temozolomide-treated cells were unable to form tumors in syngeneic animals, while cells treated with other alkylating agents did. Genomic analysis of these tumor models revealed that fluctuating levels of neoantigens, rather than the absolute number of mutations is critical to provoke immune surveillance. These results provide the rationale for developing innovative anticancer therapies that target DNA repair proteins.

#631

Dual-specific T cells are highly effective in eradicating solid tumors.

Clare Y. Slaney, Bianca von Scheidt, Phillip K. Darcy, Michael H. Kershaw. _Peter MacCallum Cancer Centre, Melbourne, Australia_.

Chimeric antigen receptor (CAR) T cell therapy is a novel form of adoptive cellular therapy and has recently generated remarkable effects in patients with hematological cancers. However, the success against solid cancers has been modest. The major challenges are the hostile tumor microenvironment and the low efficiency of CAR T cells infiltrating the tumor. Here, we present a major advancement in CAR T therapy that eradicated large established solid cancers, some in excess of 150 mm2, in immunocompetent mice.

We hypothesized that a vaccine composed of a recombinant poxvirus could be used as an antigen delivery vehicle to specifically activate CAR T cells through their T cell receptor (TCR) and simultaneously change the tumor microenvironment, allowing the recruitment and activation of CAR T cells. The approach involves adoptive cell transfer incorporating vaccination (ACTIV) therapy. We generated dual-specific T cells expressing a CAR specific for the tumor antigen Her2 and a TCR specific for the melanocyte protein (gp100). Injection of T cells, together with recombinant vaccinia virus expressing gp100, induced durable complete remission of a variety of Her2+ tumors and established metastases, some in excess of 150 mm2, in immunocompetent mice expressing Her2 in normal tissues, including the breast and brain. Tumor destruction mediated by dual-specific T cells occurred rapidly over a period of seven days and was associated with an extensive proliferation and infiltration of the dual-specific CAR T cells. Mice that had rejected tumors were resistant to rechallenge with the same Her2+ tumor cells and partially resistant to rechallenge with Her2- tumor cells, indicating the formation of immune memory and epitope spreading. This mouse model study supports the view that it is possible to design a highly effective CAR T cell therapy for solid cancers and metastases, even when the target antigen is also expressed in vital tissues.

To explore the translational potential for using the dual specific CAR T cell strategy, we established methods to transduce the T cells from human peripheral blood with both a TCR specific for gp100 and a CAR for Her2. From as little as 1 ml of human buffy coat, we could generate more than 109 dual-specific CAR T cells, which is sufficient for a course of treatment. The human dual-specific CAR T cells were functional in secreting IFN-γ and killing human cancer cells when co-cultured with the gp100 or Her2 expressing human cancer cells. The stimulation of gp100 through TCR enhanced the dual-specific CAR T cell proliferation, secretion of IFN-γ and killing of Her2+ human cancer cells in vitro. These characteristics were identified to be important for eradicating tumors in the mouse models. Taken together, our data provide valuable information for the development of CAR T cell therapies for patients with solid cancers.

#632

Genome-scale neoantigen screening using ATLAS™ prioritizes candidate antigens for immunotherapy in a non-small cell lung cancer patient.

Lila Ghamsari,1 Emilio Flano,1 Judy Jacques,1 Biao Liu,1 Zheng Yan,1 Aula Alami,1 Christine Kelley,1 Theresa Zhang,1 Jonathan Havel,2 Vladimir Makarov,2 Taha Merghoub,2 Jedd D. Wolchok,2 Matthew Hellman,2 Pamela Carroll,1 Timothy Chan,2 Jessica B. Flechtner1. 1 _Genocea Biosciences, Cambridge, MA;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Despite the unprecedented efficacy of checkpoint blockade (CPB) therapy in treating some cancers, the majority of patients fail to respond. Several lines of evidence support that the combination of CPB and neoantigen vaccine prolongs survival curves in cancer patients. Capitalizing on neoantigens derived from non-synonymous somatic mutations is a good strategy for therapeutic immunization. Current approaches to neoantigen prioritization involve mutanome sequencing, in silico epitope prediction algorithms, and experimental validation of cancer neoepitopes. Even the best in class in silico epitope prediction algorithms lack the accuracy necessary for efficacious personalized cancer vaccines.

We sought to circumvent some of the limitations of currently available prediction algorithms by prioritizing neoantigens empirically using ATLAS™, a technology developed to screen T cell responses from any subject against their entire complement of potential neoantigens. Exome sequences were obtained from peripheral blood mononuclear cells (PBMC) and tumor biopsies from a non-small cell lung cancer patient who had been successfully treated with pembrolizumab. The tumor exome was sequenced and somatic mutations were identified. Individual DNA sequences (399 nucleotides) spanning each mutation site were built, cloned and expressed in E. coli co-expressing listeriolysin O. Polypeptide expression was validated using a surrogate T cell assay or by Western Blotting. Frozen PBMCs, collected pre- and post-therapy, were used to derive dendritic cells (MDDC). Both CD4+ and CD8+ T cells were enriched and expanded using microbeads. The E. coli clones were pulsed onto MDDC in an ordered array, then co-cultured either with CD8+ or with CD4+ T cells overnight. T cell activation was detected by analyzing cytokines in supernatants. Antigens were identified as clones that induced a cytokine response that exceeded three standard deviations of the mean of all negative control wells, then their identities compared with T cell epitopes predicted using previously described algorithms.

We found biological evidence for neoantigens that were specifically responsive to peripheral CD8+ and CD4+ T cells, derived from the patient's tumor, pre- and post-CPB intervention. Some of these neoantigens were identified as a T cell target both pre- and post-CPB therapy. We identified neoantigens for which no epitopes were predicted by in silico methods. These data represent evidence that multiple patient-specific neoantigens can be identified through functional evidence of T cell response from peripheral blood without epitope prediction. By profiling natural and CPB-enhanced immunity to neoantigens, a broad catalog of T cell targets can be identified for development of immunotherapies that engage T cells against cancer to improve outcomes for patients for whom current therapies are ineffective.

#633

Induction of ADCC resistance profoundly alters tumor cell phenotype and stress response.

Dalal S. Aldeghaither,1 Joseph C. Murray,2 Sarah M. Roth,1 Elana J. Fertig,2 Shaojun Tang,1 Sandra A. Jablonski,1 Louis M. Weiner1. 1 _Georgetown University, Washington, DC;_ 2 _Johns Hopkins University, Baltimore, MD_.

Antibody dependent cellular cytotoxicity (ADCC) is one mechanism by which monoclonal antibodies (mAb) work. In addition to ADCC, mAbs act on their target by causing signal perturbation and complement activation. Cetuximab is an EGFR targeting mAb used to target EGFR overexpression and amplification seen in many types of cancer. While mechanisms of resistance to EGFR targeted therapy have been extensively studied, resistance to ADCC has not, mainly due to the lack of ADCC-resistance models for study. To address this limitation we established a model system for anti-EGFR ADCC using NK92-CD16V effector cells, cetuximab, and the high EGFR-expressing squamous cell carcinoma cell line A431. Continuous A431 exposure to ADCC yielded an ADCC resistant phenotype (ADCCr) that exhibits a stable phenotype in the absence of continued ADCC selection. We have explored: ADCCr cell gene expression profile, NK cell activation, metabolic signature and subsequent RPPA analysis to further understand the causes and properties associated with this resistance. This ADCCr cell line has a distinctive transcriptional profile highlighted by overexpression of histone- and interferon-related genes, reduced sensitivity to antimetabolites, DNA-intercalating and ABC transporter-regulated cytotoxic agents. Intense ADCC selection causes epigenetic modification and stress response characterized by the transcriptional overexpression of PCAF (KAT2B), which initiates histone hyper-acetylation and epigenetic changes, inducing DNA replication arrest, DNA damage and stress responses that activate checkpoint signaling in the cell cycle. The pharmacologic inhibition of KAT2B reverses the ADCCr phenotype. Immune checkpoints such as PD-L1 do not modulate ADCC in this model system. These results shed light on new mechanisms of ADCC resistance and inform future combinatorial treatments for mAb therapy. We are exploring the possibility that stress response mechanisms are responsible for resistance for diverse selection pressures imposed by immune synapse-mediated cytotoxic attack.

#634

RNAi-mediated β-catenin inhibition promotes T-cell infiltration and anti-tumor activity in combination with immune checkpoint blockade.

Shanthi Ganesh, Serena Shui, Weimin Wang, Bob D. Brown, Marc Abrams. _Dicerna Pharmaceuticals, Inc, Cambridge, MA_.

Recent research strongly suggests an important role for Wnt/β-catenin signaling in mediating cancer immune evasion and resistance to immune checkpoint therapy, including anti-PD-1, anti-PD-L1, and anti CTLA-4 antibodies. The mechanism by which Wnt/β-catenin signaling causes checkpoint resistance is believed to involve blocking of specific cytokines which trigger immune cell recruitment to the tumor, resulting in the phenomenon of T-cell exclusion and rendering the tumor to a non-inflamed state. Inhibition of β-catenin may be an effective strategy for increasing the low response rate to these effective medicines in numerous cancer populations. DCR-BCAT is a potent and specific chemically-optimized RNA interference (RNAi) trigger targeting CTNNB1, the gene which encodes β-catenin, formulated in a tumor-selective EnCore lipid nanoparticle. In syngeneic mouse tumor models, β-catenin inhibition promoted T-cell infiltration and significantly improved the sensitivity of the tumors to checkpoint inhibition. The combination of DCR-BCAT and IO therapy yielded significant tumor growth inhibition compared to monotherapy in B16F10 melanoma, 4T1 mammary carcinoma, Neuro2A neuroblastoma and Renca renal adenocarcinoma. Importantly, a significant increase in CD8+ T-cells after DCR-BCAT monotherapy was observed in all models evaluated, including genetically-driven spontaneous MMTV-Wnt1 tumors. These data support clinical development of these combination approaches for this first-in-class RNAi therapeutic.

#635

CD8+ tumor-infiltrating T cells before and after neoadjuvant therapy correlates with the pathological response in rectal cancer.

Shinji Matsutani, Masatsune Shibutani, Kiyoshi Maeda, Hisashi Nagahara, Tatsunari Fukuoka, Ryosuke Amano, Hiroaki Tanaka, Kazuya Muguruma, Kosei Hirakawa, Masaichi Ohira. _Osaka City University Graduate School of Medicine, osakasi, Japan_.

Background: Recently, neoadjuvant therapy (ie. neoadjuvant chemotherapy, neoadjuvant chemoradiationtherapy) for locally advanced rectal cancer has been generally performed. Although in unresponsive cases it may have disadvantages such as tumor progression or delaying surgery, factors predicting the clinical response to neoadjuvant therapy have not been adequately defined. Meanwhile CD8+ Tumor-infiltrating lymphocytes (TILs) have been reported to have a crucial effect in tumor progression and outcome as primary host immune response in various types of cancer, and antitumor immune effect has been reported to contribute to the response to radiotherapy and chemotherapy. The aim of this study was to elucidate the correlation between the local immune status and the effectiveness of the neoadjuvant therapy for locally advanced rectal cancer.

Patients and methods: A total of 51 patients who underwent curative operation for locally advanced rectal cancer after neoadjuvant therapy were enrolled. We retrospectively examined the number of CD8+ tumor-infiltrating lymphocytes (TILs) using immunohistochemical staining of pretreatment biopsy samples and resected specimens, and assessed the correlation with pathological response. The grade of tumor response was evaluated according to the definitions in the Japanese Classification of Colorectal Carcinoma. Grade0-1a were defined as "poor response" and Grade1b-3 were defined as "good response". We set each median value of the number of CD8+ TILs as the cut-off value.

Results: For the 26 patients with pretreatment biopsy samples, we classified the the patients into the poor response group (n=14) and the good response group (n=12). Then we set 6.0 as the cut-off value and classified the patients into the high pretreatment CD8+ TILs group (n=14) and the low pretreatment CD8+ TILs group(n=12). Low pretreatment CD8+ TILs were associated with poor response to neoadjuvant therapy (p=0.036). For resected samples (n=51), we classified the patients into the poor response group (n=25) and the good response group (n=26). Then we set 10.8 as the cut-off value and classified the patients into the high posttreatment CD8+ TILs group (n=28) and the low posttreatment CD8+ TILs group(n=23). Low posttreatment CD8+ TILs were also associated with poor response to neoadjuvant therapy (p<0.001). Additionally, the number of pretreatment CD8+ TILs tend to be related to the number of posttreatment CD8+ TILs.

Conclusion: In locally advanced rectal cancer patients, T lymphocyte-mediated immune reactions play an important role in tumor response to neoadjuvant therapy, and the quantitative measurement of CD8+ TILs in pretreatment biopsy samples may be a predictor of the clinical effectiveness of neoadjuvant therapy for locally advanced rectal cancer. Moreover, low posttreatment CD8+ TILs were associated with poor response to neoadjuvant therapy.

#636

Comprehensive analysis of T cells responsive to neoantigens derived from tumor-specific genetic mutations.

Tetsuro Sasada, Junya Ohtake, Satoshi Wada, Erika Yada, Shintaro Yoshida. _Kanagawa Cancer Center, Yokohama, Kanagawa, Japan_.

Neoantigens derived from tumor-specific genetic mutations can be recognized as foreign by the host immune system, and might be suitable as target for cancer immunotherapy possibly due to their higher immunogenicity. In this study, to really know the immunogenicity of tumor-specific neoantigens, we comprehensively investigated T cell responses against neoantigens derived from genetic mutations in gastric cancer. Using next-generation sequencing, 156 missense mutations were identified in tumor cells from two gastric cancer patients. From them, we selected 30 potentially immunogenic amino acid sequences, which were derived from the mutations and predicted to potentially bind to HLA-class I (A*0201, A*0206, or A*2402) by an epitope prediction server, IEBD. We synthesized 30 kinds of 27mer long peptides, in which the mutated sequences were located in the center, and then cultured peripheral blood mononuclear cells (PBMC) from healthy donors in the presence of the synthetic peptides to evaluate whether they could really induce antigen-specific T cell responses. In the analysis with PBMC from 18 healthy donors, 27/30 (90%) synthetic peptides showed an ability to induce antigen-specific T cell responses in at least one donor, assessed by cytokine production assay. Among them, 15 peptides were immunogenic in more than one donor. The antigen-specific T cell responses were detected more frequently in CD4+ T cells (70%) than in CD8+ T cells (43%). The specificity of T cell responses to mutated sequences, but not to the corresponding wild type sequences, were confirmed in 5 of 8 (63%) peptides examined. In addition, antigen-specific T cell responses induced by mutated peptides were shown to be much higher than those induced by the corresponding wild type peptides. These findings clearly demonstrated high immunogenicity and specificity of neoantigens derived from tumor-specific genetic mutations. Further studies would be recommended to develop a novel immunotherapeutic approach, "personalized cancer vaccination", targeting mutation-derived neoantigens.

#637

The class I HDAC inhibitor mocetinostat augments checkpoint inhibitor therapy via direct up regulation of antigen presentation transcriptional programs in tumor cells and increased T-cell clonality in tumors.

David M. Briere, Niranjan Sudhakar, Peter Olson, Jamie Christensen. _Mirati Therapeutics, San Diego, CA_.

Mocetinostat is a spectrum-selective class I/IV histone deacetylase (HDAC) inhibitor that augments checkpoint inhibitor therapy through enhanced antigen presentation capacity and a pro-immunogenic shift in the tumor microenvironment (TME). Mocetinostat up-regulated tumor antigen presentation machinery (i.e. major histocompatibility complex (MHC) genes and co-presentation molecules) and programmed cell death-ligand 1 (PD-L1) expression in a panel of non-small cell lung cancer (NSCLC) cell lines. Surprisingly, some of the most highly up regulated genes following mocetinostat treatment were MHC class II genes (~20 fold), which are normally expressed by antigen-presenting cells, but are silenced in most epithelial tissues and solid tumors. To elucidate the molecular mechanisms whereby mocetinostat regulates MHC class I and class II transcriptional programs in tumor cells, we performed chromatin immunoprecipitation DNA sequencing (ChIP-Seq). HDAC2 was detected at the promoters of many mocetinostat-regulated immune pathway genes. Further, mocetinostat treatment increased histone 3 lysine 27 acetylation and histone 3 lysine 4 trimethylation indicating an induction of active transcription at these loci. Class II transactivator (CIITA) is an interferon gamma (IFNγ)-sensitive regulator of MHC class II gene expression and was one of the top HDAC2/mocetinostat target genes based on ChIP-Seq data. In addition, CIITA and a representative MHC class II gene, HLA-DRA, were synergistically up regulated following mocetinostat and IFNγ treatment. To investigate the impact of Class I HDAC inhibition on the TME, syngeneic mouse tumor models were utilized. Mocetinostat treatment decreased intratumoral immune suppressive T regulatory cells (Tregs) and increased intratumoral CD8+ T cells. To gain additional insight into the functional effects of mocetinostat in the TME, we performed deep sequencing of the T-cell receptor in vehicle, mocetinostat, PD-L1 or combination-treated tumors. Mocetinostat-treated tumor cohorts exhibited increased T-cell clonality which likely reflects expansion of a subset of activated, antigen-specific T cells. Together, these data provide molecular insight into the mechanism whereby mocetinostat augments checkpoint inhibitor therapy by directly regulating tumor antigen presentation and through functional effects on the T cell repertoire.

#638

Epigenetic reprograming of immune cells through selective inhibition of HDAC6 reduces suppressive phenotypes and augments anti-tumor properties of T-cells.

Andressa L. Sodre,1 David M. Woods,1 Amod Sarnaik,2 Brian C. Betts,2 Steven Quayle,3 Simon Jones,3 Jeffrey Weber1. 1 _NYU Langone Medical Center, New York, NY;_ 2 _Moffitt Cancer Center, Tampa, FL;_ 3 _Acetylon Pharmaceuticals, Boston, MA_.

Alteration of the epigenetic landscape of immune cells can modify the pattern of gene expression, thus resulting in phenotypic and functional changes. Small molecule inhibitors targeting epigenetic modifiers, such as histone deacetylases (HDACs), have been shown to reduce tumor growth. Besides promoting direct anti-tumor effects, HDAC inhibitors also target immune cells and alter their gene regulation. Here, we demonstrate that the HDAC6 selective inhibitors ACY-241 and ACY-1215 (ricolinostat) decrease the function of myeloid derived suppressor cells (MDSC) and T regulatory (Treg) cells, maintain an effector phenotype by CD8+ T cells, and do not reduce viability of immune cells. First, peripheral blood mononuclear cells derived from melanoma patients were treated with ACY-241, and the phenotype of MDSCs was assessed. Expression of the suppressive molecules ARG1 (p<0.01) and NOS2 (p<0.05) was decreased in CD14+HLA DR lo CD11b+ and CD14+HLA DR lo CD33+ MDSC populations, suggesting less potent MDSCs. To gain insight into other suppressive populations, we evaluated the phenotype and function of Treg cells derived from melanoma patients. Cultures of CD3+ T-cells treated with ACY-1215 or ACY-241 resulted in decreased expression of FOXP3 and reduced frequency of Tregs (CD4+CD25+CD127-; p<0.001). ACY-1215 pre-treated Tregs exhibited less suppressive activity against responding conventional T-cells in standard assays, compared to Tregs pre-treated with DMSO control (p<0.01). CD3+ T-cells exposed to ACY-241 or ACY-1215 were activated via CD3/CD28 co-stimulation to assess effects on cytokine production. Selective HDAC6 inhibition shifted T-cell differentiation towards Th1-type (Tbet+) over Th2-type (GATA3+), compared to DMSO (p<0.05). Additionally, Th2 cytokines (e.g. IL-4, IL-5, IL-6, IL-10, IL-13) were significantly decreased (p<0.05). In accordance with decreased Th2 differentiation, mTORC signal transduction, including phosphor-SGK1, was similarly reduced (p<0.05). Targeted inhibition of mTORC signaling (i.e. SGK1 inhibitor) recapitulated decreased Th2 cytokine production (p<0.05). In order to address effector immune function, tumor infiltrating lymphocytes (TILs) were harvested from melanoma biopsies and expanded in vitro in the presence of ACY-1215 or ACY-241, and IL-2. Measuring CD45RA, CD45RO, CD62L and/or CCR7, an accumulation of central memory CD4+ and CD8+ T-cells was observed (p<0.05). Activated TILs treated with ACY-241 or ACY-1215 displayed higher expression of IFN-gamma and CD107a. Collectively, these data indicate that epigenetic reprograming of immune cells by HDAC6 selective inhibitors may decrease the function of suppressive subsets (e.g. Tregs, MDSCs), while enhancing the accumulation of anti-tumor memory and effector T-cell subsets.

#639

Morphological changes in mitochondria induced by CD137 (4-1BB) co-stimulation on CD8 T cells.

Sara Labiano, Álvaro Teijeira, Arantza Azpilikueta, Ángela Aznar, Elixabet Bolaños, Ignacio Melero. _Center for Applied Medical Research, Pamplona, Spain_.

Metabolism has emerged as a fundamental biological process in the control of immunological functions of T cells, playing essential roles in survival and differentiation of these immune cells. Mitochondria are key organelles in controlling these processes by performing fundamental steps in aerobic respiration, fatty acid metabolism and in the control of apoptosis. Recently, Carl June and colleagues showed that CARs engineered with the cytoplasmic tail of 4-1BB were able to promote oxidative phosphorylation in T cells as a result of enhanced mitochondrial biogenesis. CD137 (4-1BB) is an immunostimulatory receptor of the TNFR family expressed on the surface of antigen-activated T cells. Treatment with agonist monoclonal antibodies targeting 4-1BB is showing promising results for immunotherapy of cancer.

In this work, we observed that stimulation of 4-1BB with agonistic mAbs was able to raise the mitochondrial membrane potential on human CD8 T cells. 4-1BB co-stimulated CD8 T cells showed enlarged mitochondria (up to 3μm diameter) with a round shape when observed by confocal or transmission electron microscopy. We observed that CD8 T cells from DLNs (drainig lymph nodes) of tumors in mice treated with agonistic anti-4-1BB antibodies also contained enlarged mitochondria.

By double fluorescence labeling of mitochondria prior and after activation, we could ascertain that enlarged mitochondria were a product of preexisting mitochondria rather than de novo generated organelles. This observation prompted us to study the regulation of mitochondrial dynamics by 4-1BB receptor. 4-1BB stimulation induced an increase in expression of the mitochondrial fusion protein OPA-1. By Super-resolution microscopy with AiryScan detectors we observed more pronounced co-localization of OPA-1 with mitochondrial membrane in 4-1BB co-stimulated T cells than in T cells activated only by CD3 crosslinking.

Our results indicate an important role of 4-1BB in the regulation of mitochondrial morphology and performance in such a way that can be relevant for various T cell functions.

#640

Subsets of HLA alleles are capable of binding neoantigens derived from mutations within cancer driving genes such as KRAS and EGFR.

Andrew Nguyen, J Zachary Sanborn, Charles J. Vaske, Shahrooz Rabizadeh, Kayvan Niazi, Patrick Soon-Shiong, Steve Benz. _NantOmics, Santa Cruz, CA_.

Background: Immunoncology has shown great promise as a low toxicity tool to combat several cancers. Use of checkpoint inhibitors against PD1 or CTLA4 unlocks the immune system's ability to recognize tumor antigens and, more specifically, neoantigens caused by random mutations within cancers. The vast majority of neoantigens consist of private mutations unique to a patient's tumor genome, but several cancers harbor recurrent mutations. Mutations in the KRAS gene, such as p.G12V, occur in roughly 25% of colorectal cancers. Mutations in EGFR occur in 10% and 35% of patients with non-small cell lung cancer in the US and East Asia, respectively. Even more prevalent are mutations within the TP53 tumor suppressor gene, with roughly 23000 unique protein variants reported to date. If these mutations in cancer driving genes are so prevalent in cancers, why are neoantigens against these targets not more readily available?

Results: We collected recurrent mutations across a variety of cancer driving genes such as KRAS, EGFR, TP53 and MYC and performed binding analysis using netMHC 3.4 to see which HLA alleles are capable of binding specific cancer mutations such as KRAS p.G12V. Using this method, we report all possible HLA alleles capable of binding these recurrent mutations within cancer genes. We further performed 3-dimensional modeling to determine whether complexes created by the HLA alleles and cancer neoepitopes are stable.

Conclusions: Several HLA alleles are capable of binding recurrent cancer mutations. These include both MHC Class 1 and Class 2 alleles. The variation in alleles capable of binding commonly mutated genes such as EGFR may explain the difference in prevalence of these mutations between geographic populations. Determining whether a certain HLA allele confers resistance to common cancer gene mutations may lead to identification of immune cells within these populations that can recognize neoantigens from commonly mutated cancer genes.

#641

**PEGylated recombinant hyaluronidase PH20 (PEGPH20) enhances tumor infiltrating CD8** \+ **T cell accumulation and improves checkpoint inhibitor efficacy in murine syngeneic breast cancer models.**

Renee Clift, Jisook Lee, Curtis B. Thompson, Yujun Huang. _Halozyme Theraputics, Inc., San Diego, CA_.

Hyaluronan (HA) is an extracellular glycosaminoglycan that accumulates in the tumor microenvironment (TME) of many solid tumors and is associated with rapid tumor progression and poor prognosis. In preclinical studies, enzymatic degradation of TME HA by intravenous PEGylated recombinant human hyaluronidase PH20 (PEGPH20) remodels the TME, reduces tumor interstitial fluid pressure, decompresses tumor blood vessels, and facilitates delivery of chemotherapeutics. We have previously shown that PEGPH20-mediated HA degradation enhances anti-PD-L1 and anti-PD1 efficacy in an HA accumulating murine pancreatic tumor model (Rosengren, AACR 2016 poster #4886). Accordingly, we aimed to extend these checkpoint blockade studies into orthotopic breast cancer models; while further elucidating the effect of PEGPH20-mediated HA degradation on modulating tumor infiltrating lymphocytes (TILs). The mammary fat pads of BALB/c mice were inoculated with either EMT-6 cells or 4T1/HAS3, a 4T1 cell line engineered to over-express hyaluronan synthase-3. During tumor progression, both EMT-6 and 4T1/HAS3 mammary carcinomas accumulate high levels of HA (average 730 ng/mg and 1408 ng/mg, respectively). When treated with PEGPH20 alone (0.0375mg/kg), EMT-6 tumor growth was inhibited by ≥30% (p=0.01). Additional EMT-6 studies were conducted to evaluate PEGPH20 in combination with anti-PD-L1. PEGPH20 increased anti-PD-L1 efficacy by 38% relative to anti-PD-L1 alone (86% vs 62.4% tumor growth inhibition (TGI), p=0.0024). Comparable tumor growth experiments were conducted in the 4T1/HAS3 model. TILs were evaluated by flow cytometry. PEGPH20-mediated HA removal enhanced both checkpoint efficacy and CD8+ T cell recruitment. Specifically, PEGPH20 alone (1mg/kg) increased anti-PD-L1 efficacy by 411% relative to anti-PD-L1 alone (93% vs 18.2% TGI, p<0.0001) and increased the accumulation of CD8+ TIL by 176% (p=0.0025). Taken together, the data suggest that tumor HA accumulation may act as a barrier to immune cell access and negatively modulate CD8+ TILs, and that PEGPH20-mediated HA reduction facilitates increased access of CD8+ T cells. This increased recruitment may contribute to the enhanced anti-PD-L1 efficacy observed when combined with PEGPH20. A Phase 1 trial is ongoing to evaluate PEGPH20 plus checkpoint blockade in patients with advanced or metastatic non-small cell lung cancer and in patients with locally advanced or metastatic gastric adenocarcinoma (NCT02563548).

#642

Detection of circulating antibodies against KRAS in patients with advanced cancers.

Ed Kheder,1 Helen J. Huang,1 Alice Wu,2 David S. Hong,1 Sarina A. Piha-Paul,1 Daniel D. Karp,1 Siqing Fu,1 Vivek Subbiah,1 Apostolia M. Tsimberidou,1 Aung Naing,1 Adi Diab,1 Milind Javle,1 Scott Kopetz,1 Anil K. Sood,1 Jonathan M. Kurie,1 Funda Meric-Bernstam,1 Martin Gleeson,2 Filip Janku1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Genalyte INC, San Diego, CA_.

Introduction: KRAS gene plays a major role in tumorigenesis, cell proliferation and survival. Yet, there has been no effective targeted therapy against KRAS mutation-mediated tumors. We hypothesized that KRAS can induce humoral-mediated immune response.

Methods: Plasma or serum samples from patients with progressing advanced cancers with or without KRAS mutations were tested for the presence of circulating KRAS antibodies. We used the Maverick Detection System (Genalyte, San Diego, CA), which can perform real-time detection of macromolecules in crude samples using biologically functionalized silicon photonic biosensors lithographically printed on disposable silicon chips.

Results: We collected serum or plasma samples from 213 patients with advanced cancers (KRAS-mutant, N=100; KRAS wild-type, N=113) and 50 (23%) were found to have circulating KRAS antibodies. There was no association between KRAS antibodies and tumor KRAS mutation status (21/100, 21% for KRAS-mutant vs. 29/113, 26% for KRAS wild-type; P=0.52). In addition, there was no difference in detection of KRAS antibodies in colorectal cancer (21/89, 24%) compared to other cancers (29/124, 23%; P=1.00). There was no difference in the median survival in patients with KRAS antibodies compared to patients without KRAS antibodies (9.0 months vs. 10.1 months; P= 0.825). Similarly, there was no difference in the median survival according to the presence of circulating KRAS antibodies in subgroups of patients with tumor KRAS mutations (P= 0.96) and without tumor KRAS mutations (P=0.63). On the contrary, the median survival of patients with tumor KRAS mutation was shorter compared to patients with KRAS wild-type (7.2 months vs. 11.5 months; P<0.001).

Conclusion: Circulating KRAS antibodies can be detected in 23% of patients with advanced cancers. Biological implications of circulating KRAS antibodies remain to be understood.

#643

Induced MHCII expression on breast cancer cells broadens the responding T cell repertoire, delays tumor-specific T cell exhaustion, and impairs tumor growth.

Tyler R. McCaw,1 Mei Li,1 Selene Meza-Perez,1 Donald J. Buchsbaum,1 Dmytro Starenki,2 Sara Cooper,2 Andres Forero,1 Troy D. Randall1. 1 _University of Alabama at Birmingham, Birmingham, AL;_ 2 _HudsonAlpha Institute for Biotechnology, Huntsville, AL_.

We recently reported that the aberrant expression of Major Histocompatibility Class II (MHCII) molecules on human triple negative breast cancer (TNBC) cells correlates with prolonged progression-free survival and increased tumor infiltrating lymphocytes. We hypothesized that the expression of MHCII enhances the intratumoral CD4+ T cell response, thereby bolstering the tumor-specific CD8+ T cell response, resulting in more effective tumor control. To test our hypothesis, we created both MHCII-expressing and MHCII-negative tumor cells by transfecting murine breast cancer (TS/A) cells with the human class II transcriptional activator (hCIITA) or empty vector, respectively. Transfected cells were then injected into BALB/c mice and the resulting immune response analyzed by flow cytometry at four time points. We found that hCIITA-expressing tumors grew slower than control tumors in immunocompetent recipients, but that this difference was nullified in immunocompromised and markedly reduced in CD4+ T cell depleted mice. CD4+ T cells isolated from hCIITA-transfected tumors produced more IFNγ, IL-17A, and surprisingly granzyme B for longer times than their counterparts in control tumors. Similarly, CD8+ T cells isolated from hCIITA-transfected tumors displayed a more activated phenotype and produced more IFNγ and granzyme B for longer times. Nevertheless, both CD4+ and CD8+ T cells eventually became exhausted in both groups. In addition to enhanced effector functions, TCR repertoire analysis demonstrated that both the breadth and magnitude of expansion of responding T cell clones were increased in hCIITA-transfected tumors. Interestingly, TS/A-hCIITA tumors harbored more regulatory T cells (Tregs) with a more suppressive phenotype than Tregs from control tumors. Finally, we show that the histone deacetylase inhibitor (HDACi) Entinostat is capable of robust and dose-dependent induction of MHCII on tumor cells in vivo, an effect that correlates with dramatic reduction in tumor size. These results suggest that the clinical benefit associated with MHCII expression on TNBC cells is mediated by a delay in T cell exhaustion and increased intratumoral CD4+ T cell activation, which enhances the cytotoxic capacity of CD8+ T cells. Entinostat, and potentially other epigenetic modifying agents, may enable induction of MHCII expression on TNBC cells clinically and allow more patients to benefit from an augmented T cell response. These effects may be magnified by combinatorial therapy with checkpoint inhibitors to promote durable anti-tumor immune responses.

#644

CA125/MUC16 suppresses antibody-dependent cellular cytotoxicity of IgG1-based therapies via perturbation of antibody Fc-Receptor engagement on immune effector cells.

J. Bradford Kline, Rina Kennedy, Shawn Fernando, Jennifer McDonough, Earl Albone, Elizabeth B. Somers, Wenquan Wang, Charles Schweizer, Luigi Grasso, Nicholas C. Nicolaides. _Morphotek, Inc., Exton, PA_.

Background: Human cancers employ a number of mechanisms to evade host immune responses. Here we report the effects of the tumor-shed antigen CA125 (MUC16) on suppressing IgG1-mediated antibody-dependent cellular cytotoxicity (ADCC). This evidence stems from prespecified subgroup analysis of an 1100 patient Phase 3 clinical trial testing the investigational agent farletuzumab, a monoclonal antibody (mAb) to folate receptor alpha, plus standard-of-care in patients with recurrent ovarian cancer. In this study, patients with low levels of CA125 [no greater than 3X the upper limit of normal (< 3X ULN)] treated with farletuzumab compared to placebo demonstrated improvements in both progression free survival (HR 0.49, p = 0.0028) and overall survival (HR 0.44, p = 0.0108). Farletuzumab's pharmacologic activity is mediated in part through ADCC. Here we show that CA125 inhibits farletuzumab's ADCC activity by suppressing antibody interaction with the CD16a Fc-Receptor on effector cells.

Methods: Functional assays employing Jurkat-hCD16 reporter cells as well as primary NK cells were used to measure ADCC activity. CA125 isolates from various patients and cell lines were used in ADCC assays to monitor CA125 effects on immune-effector function. Multiple isogenic tumor cells expressing endogenous CA125 and their CA125-null counterparts were also used to monitor CD16a-mediated ADCC activity. CD16a+ cell binding assays were used to measure the effects of CA125 inhibition on CD16a Fc-Receptor interaction and activation. Recombinant CD16a and humanized mAbs were used in ELISA format to measure molecular binding.

Results: Functional assays showed that exogenously added tumor-shed CA125 was able to suppress ADCC mediated by IgG1-type mAbs against several target cell lines. This CA125-mediated ADCC suppression was also observed in cells naturally expressing membrane-bound CA125 but not in isogenic CA125-null cells. Molecular studies suggest that CA125-mediated immunosuppression is caused by the perturbation of antibody interaction with the CD16a Fc-Receptor and that this effect is specific to IgG-type mAbs.

Conclusions: Here we demonstrate that the tumor-shed antigen CA125 can elicit immunosuppression by blocking the interaction of tumor-targeting mAbs and CD16a Fc-Receptor on immune effector cells. This effect appears to be through a direct interaction of CA125 with antibody-CD16a proteins. These findings support a biological mechanism for the clinical observation that relapsed ovarian cancer patients with low CA125 blood levels have improved clinical responses to the experimental agent farletuzumab. These findings also provide new insights on the potential biological mechanisms for which tumors produce tumor-shed antigens such as CA125 to suppress host anti-tumor immune responses.

#645

Immunological landscape of non-small cell lung cancer (NSCLC): PD-1 expression in lung TILs correlate with co-expression of other checkpoint inhibitor molecules.

Se W. Jeong, Timothy Bullock. _University of Virginia, Charlottesville, VA_.

Lung cancer is the leading cause of annual cancer related mortality in the United States. More than 150,000 people in the U.S. will die from lung cancer this year which will lead to roughly as many deaths as breast, prostate, colon, and pancreatic cancers combined. Lung cancer is also of global concern as tobacco and cigarette use, a significant known risk factor, in developing countries have risen significantly. Stage II and III non-small cell lung cancer (NSCLC) patients often have a 5-year disease free survival of less than 50% even after surgical resection and post-operative chemotherapy and a dismal 5% overall survival for patients with stage IV metastatic disease. Even with the advent of PD-1/PDL-1 checkpoint blockade immunotherapies, only about 20% of patients are responsive to the treatment. This limited scope of success may be due to our lack of understanding the immune landscape of NSCLCs. Therefore, we set out to characterize the immune composition from surgical lung resections via flow cytometry. We found that NSCLCs have a diverse set of checkpoint inhibitor and co-stimulatory molecules expressed by T cells, including PD-1, TIM-3, and TIGIT. Interestingly, even though PD-1 was elevated compared to a normal donor, the expression level was lower than either breast or melanoma tumor samples. We also discovered that PD-1 heavily correlates with co-expression of other checkpoint inhibitor molecules. In our regulatory T cell analysis, FoxP3+ populations have a profound presence in these lung tumors suggesting there is a highly suppressive environment for tumor infiltrating lymphocytes (TILs). Our lung resection screenings furthermore suggests a dynamic range of both frequency and proportion of CD3-CD56+ natural killer (NK) cells. Finally, our data indicates there is a large population of CD11b+CD33+ myeloid-derived suppressor cells (MDSCs) in the tumors, with varying degrees of arginase-1 expression, a functional marker for MDSCs. Altogether, these findings in concert suggest there are potential goldmine of opportunities to target molecules on different immune cell populations. This multi-modal approach, directing combination of immunotherapies that influences multiple immune compartments yet complement each other, may have significant implications for future clinical studies.

#646

Targeting MHC class I molecules and immune checkpoints as key immune evasion strategies in EBV-associated nasopharyngeal carcinoma.

Sharie Pui-Kei Siu,1 Ka Fai To,1 Grace Tin-Yun Chung,1 Vivian W.Y. Lui,1 Yvonne Y. Li,1 Peter S. Hammerman,2 Kwok Wai Lo1. 1 _The Chinese University of Hong Kong, Hong Kong, Hong Kong;_ 2 _Dana-Farber Cancer Institute, Boston, MA_.

Nasopharyngeal carcinoma (NPC) is a distinct type of head and neck cancer that is consistently associated with intensive lymphocytes infiltration and Epstein-Barr virus infection. This study aims at elucidating the mechanism contributing to immune evasion in NPC. During NPC tumorigenesis, multiple intrinsic mechanisms are acquired to escape cellular immune response for the tumor and viral antigens. By genome sequencing, somatic alterations of multiple MHC class I genes including NLRC5, HLA-A, HLA-B, HLA-C and B2M were detected in 30% of primary NPC. Significant correlation of MHC class I gene alterations and poor survival rate in the patients was also shown. The finding suggested that the deficiency of antigen presentation mechanism represents a common strategy for immune evasion in NPC and favors rapid tumor progression. Losses of HLA-A, HLA-B and NLRC5 expression were validated in a panel of NPC tumor lines and selected primary tumors. The role of NLRC5 as key transcription regulator of HLA-A and HLA-B expression was demonstrated in the nasopharyngeal epithelial cells. To further evaluate the mechanisms contributed to immune evasion in NPC, we have elucidated immunosuppressive molecules involved in immune surveillance in Epstein Barr Virus (EBV)-associated NPC and its association with EBV-encoded LMP1 in an independent cohort of primary NPCs. By immunohistochemistry, the expression of EBV-encoded LMP1, MHC class I genes and various immunosuppressive molecules including PDL1, PDL2 and IDO1 was examined in 98 primary tumors. PDL1 and PDL2 were found to be over-expressed in 50% and 33.7% of NPC cases while IDO1 was found to be expressed in 28.8% of cases. Primary NPC cases expressing either PDL1 or PDL2 accounted for 64.3%. A significant association between LMP1 and PDL1 and PDL2 expression was also found. The correlation of PDL1, PDL2 and/or MHC class I molecule expression with the outcome of the patients was determined. The high incidence of MHC class I deficiencies and PDL1/PDL2 overexpression imply that EBV-associated NPC escape from the host immune response by targeting both antigen presentation mechanisms and immune checkpoints. Our findings also suggested that the expression of MHC class I molecules and PDL1/PDL2 is a potential biomarker for immunotherapy in NPC patients.

#647

**Bortezomib enhances CD8** + **T Lymphocyte antitumor effector function: Potential mechanism(s) via notch regulation.**

Ariana N. Renrick,1 Menaka C. Thounaojam,2 Portia Thomas,1 Samuel T. Pellom,1 Anil Shanker1. 1 _Meharry Medical College, Nashville, TN;_ 2 _Medical College of Georgia, Augusta, GA_.

The immunosuppressive tumor microenvironment disturbs host antitumor immunity by multiple mechanisms including interference with the Notch system, which is important for various metazoan cell fate decisions and hematopoietic cell differentiation and function. We observed that treatment with the proteasome inhibitor bortezomib in mice bearing various solid tumors resulted in an upregulated expression of various Notch signaling components in lymphoid tissues, thereby increasing CD8\+ T lymphocyte IFNγ secretion and expression of effector molecules, perforin and granzyme B, as well as the T-box transcription factor eomesodermin. Of note, bortezomib reversed tumor-induced downregulation of Notch receptors, Notch1 and Notch2, as well as increased the levels of cleaved Notch intracellular domain (NICD) and downstream targets Hes1 and Hey1 in tumor-draining CD8\+ T cells. Moreover, bortezomib promoted CD8\+ T cell nuclear factor-κB (NFκB) activity by increasing the total and phosphorylated levels of the IκB kinase and IκBα as well as the cytoplasmic and nuclear levels of phosphorylated p65. Even when we blocked NFκB activity by Bay-11-7082, or NICD cleavage by γ-secretase inhibitor, bortezomib significantly increased expression of Notch Hes1 and Hey1 genes as well as perforin, granzyme B and eomesodermin in activated CD8\+ T cells. Data suggest that bortezomib can rescue tumor-induced dysfunction of CD8\+ T cells by its intrinsic stimulatory effects promoting NICD-NFκB crosstalk. We are also elucidating components of microRNA regulation affecting NICD-NFκB crosstalk. Our preliminary data suggest that bortezomib is also able to positively regulate miR-155 expression in CD8+ T cells from mice bearing tumor. As well as, miR-155 suppression downregulates bortezomib-induced increase in Notch target genes in T cells. We are currently investigating alternative proteasome inhibitors in order to understand whether bortezomib's effect on miR-155 expression in CD8\+ T cells is specific to bortezomib or primarily conducive to a proteasome inhibition effect. These findings provide novel insights on using bortezomib not only as an agent to sensitize tumors to cell death, but also to provide lymphocyte-stimulatory effects, thereby overcoming immunosuppressive actions of tumor on antitumor T cell functions.

#648

Interferon-gamma induces PD-L1 expression via IFNGR-JAK-STAT pathway in ovarian cancer.

Tat-San Lau, Loucia Kit-Ying Chan, Tak-Hong Cheung, So-Fan Yim, Jacqueline Ho-Sze Lee, Joseph Kwong. _The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong_.

The purpose of this study is to investigate underlying mechanism of how cytokine interferon-gamma (IFN-γ) regulates PD-L1 expression in ovarian cancer cells. We treated a panel of human and mouse ovarian cancer cell lines with recombinant human/mouse IFN-γ. Our data showed that IFN-γ up-regulated mRNA and protein expression of PD-L1 significantly in a majority of ovarian cancer cells. The functional IFN-γ receptor is comprised of two ligand-binding IFNGR1 chains associated with two signal-transducing IFNGR2 chains and associated signaling machinery. Here we found that the mRNA expression levels of IFNGR1 and IFNGR2 were abundant in all human ovarian cancer cell lines being tested, while their expressions were not affected by IFN-γ treatment. After knocking down the expression levels of IFNGR1 and IFNGR2 in a ovarian cancer cell line by target gene-specific siRNA, our data showed that the IFN-γ-mediated induction of PD-L1 were diminished in the ovarian cancer cells when compared to those with non-targeting scrambled siRNA controls, indicating the induction of PD-L1 by IFN-γ is dependent on the presence of IFN-γ receptors in the ovarian cancer cells. Although abundant expression of IFNGR1 and IFNGR2 were found in all human ovarian cancer cell lines being tested, the IFN-γ-mediated induction of PD-L1 was not detected in a few of the human ovarian cancer cell lines (namely IGROV-1, TOV21G and SKOV3). We further investigated the integrity of IFN-γ signaling in the human ovarian cancer cell lines by examining activation of STAT1 protein and induction of IRF-1 gene in human ovarian cancer cell lines after IFN-γ treatment. Our data showed that phosphorylated-STAT1 protein and IRF-1 gene expression were up-regulated significantly in a majority of human ovarian cancer cells after IFN-γ treatment, except IGROV-1 and TOV21G cells. These results suggested that IGROV-1 and TOV21G cells might harbor defects in intracellular JAK-STAT1 signaling. We then examined the presence of JAK1 truncating mutations in human ovarian cancer cell lines by Sanger sequencing, and confirmed that IGROV-1 and TOV21G cells, but not the others, have JAK1 truncating mutations. Since our data showed that SKOV3 cells have wild type JAK1, we further investigated other possible defects in IFN-γ signaling in SKOV3 cells. We investigated the IFN-γ-induced STAT3 protein activation in human ovarian cancer cell lines, and defects were found in Y705 STAT3 phosphorylation in SKOV3 as well as in IGROV-1 and TOV21G cells. To summarize, our results showed that IFN-γ induces PD-L1 expression in ovarian cancer cells via IFNGR-JAK-STAT pathway. The failure of IFN-γ-mediated induction of PD-L1 in a minority group of human ovarian cancer cell lines is due to defective IFN-γ signaling, including JAK1 truncating mutations and impaired STAT3 activation. This work is supported by Hong Kong Research Grants Council General Research Fund (467713 and 14109515).

#649

Emigrant pre-REP tumor infiltrating lymphocytes profoundly differ from remnant T-cells.

Michelle R. Simpson-Abelson, Christopher Mosychuk, Maria Fardis, Michael T. Lotze. _Lion Biotechnologies, Tampa Bay, FL_.

Adoptive T cell therapy with autologous tumor infiltrating lymphocytes (TIL) provides up to 56% objective response rates and a complete response in 24% of patients with metastatic melanoma. The process of generating TIL from resected tumor involves morcellating the tumor into 1-3 mm3 fragments and expanding TIL in the presence of Interleukin 2 (IL-2) in a pre-Rapid Expansion Protocol (pre-REP). During the 'pre-REP', tumor-resident immune cells emigrate (eTIL) and proliferate. The length of the pre-REP varies between 11-21 days, depending on cell growth. Residual tumor fragments (remnants) are discarded and the expanded eTIL are subjected to a Rapid Expansion Protocol (REP) with irradiated PBMC feeders, anti-CD3 and IL-2. Viable cells remaining in the tumor remnants (rTIL) following the pre-REP were investigated to assess their function and phenotype. We evaluated and compared the rTIL and eTIL in melanoma, breast, renal, pancreatic, lung and colorectal tumors (n=9). Tumor rTIL are consistently phenotypically distinct from eTIL, as determined by differential expression of various markers (Table 1). The fundamental differences in rTIL were: Increased CD69+ (7 fold MFI in CD4+) (p<.001); diminished LAG3 (2 fold MFI in CD8) (p<.05); TIM3 (3 and 2 fold MFI in CD8 and CD4 respectively) (p<.05/.01); CD154 (3 fold MFI in CD4) (p<.01); and CD56 (5%) (p<.05). Surprisingly, a REP of rTIL and eTIL resulted in comparable expansion. The phenotypic signature of TIL was sustained post-REP with fidelity of the individual expression of LAG3, Tim3, and CD28. These studies have identified significant differences in the biology of cell populations in terms of tissue-resident T cells and the signals associated with emigration and retention. These data provide additional insights on the individual TIL populations that could be utilized for adoptive T-cell therapy in patients and raise important questions about the nature of tissue-resident T cells in sites of chronic inflammation such as tumor.

Table 1: Tumor resident remnant T cells are phenotypically distinct from emigrating T cells (N=9)

---

Marker Expression | LAG3 (CD8/ CD4) MFI | Tim3 (CD8/ CD4) MFI | PD-1 (CD8/ CD4) % | CD69 (CD8/ CD4) MFI | CD154 (CD8/ CD4) MFI | CD28 (CD8/ CD4) MFI | CD57 (CD8/ CD4) % | CD56

%

eTIL | 507/ 144 | 2832/ 1756 | 36.95/ 47 | 1320/ 1543 | 1498/ 3751 | 1163/ 5036 | 18.76/ 19.6 | 5.615

rTIL | 209/ 106 | 877/ 742 | 42.8/ 48 | 3437/ 223.4 | 1034/ 1167 | 458.3/ 2795 | 9.16/8.5 | 1.027

*P-values(CD8/ CD4)  | 0.05/ 0.21 | 0.05/ 0.01 | 0.38/ 0.89 | 0.11/ 0.001 | 0.55/ 0.01 | 0.05/ 0.11 | 0.05/ 0.06 | 0.05 _______

#650

B lymphocytes promote upregulation of an IL-1-NfkB dependent signaling and increase invasiveness of triple negative breast cancer.

Nicole Flynn,1 Rajasekharan Somasundaram,2 Jennifer Sims-Mourtada3. 1 _University of Delaware, Newark, DE;_ 2 _The Wistar Institute, Philadelphia, PA;_ 3 _Helen F. Graham Cancer Ctr., Newark, DE_.

Triple negative breast cancer (TNBC) is an aggressive form of breast cancer that progresses quickly from a non-invasive carcinoma in situ to an invasive state. Chronic inflammation associated to humoral immune responses has been found to promote aggressiveness in a number of solid tumor types. In breast cancer, B lymphocytes are associated with microinvasive disease and correlate with expression of inflammatory genes. The purpose of our work is to study the impact of B lymphocytes on the tumor microenvironment and increased invasiveness of TNBC cells. Through real-time PCR, we demonstrate that co-culture of B lymphocytes and TNBC cells leads to increased mRNA levels of IL1β and its downstream target interleukin 8 (IL8) in both B lymphocytes and in TNBC cells. Western Blot analysis shows that co-culture also leads to increased phosphorylation of p65, indicating IL-1 β activation of NFκB signaling. Additionally, co-culture of B lymphocytes with TNBC cells leads to increased expression of matrix metalloproteinases (MMPs) and cellular invasion through a matrigel invasion chamber. Gelatin zymography reveals increased functional MMP2 and MMP9 in tumor cell supernatant following co-culture with B lymphocytes. To complement our in vitro studies, we examined the presence of CD20+ B cells and expression of inflammatory cytokines IL-1β and IL-8 by immunohistochemistry in serial sections of tissue microarrays from patients with estrogen receptor (ER) positive and negative ductal carcinoma in situ (DCIS) and invasive carcinoma. Large areas of densely populated B cells were observed in ER-DCIS and in invasive TNBC compared to ER+ DCIS. Furthermore, in ER- DCIS and TNBC, both B lymphocytes and tumor cells are found to express IL1β whereas IL8 is found more specifically to be expressed by tumor cells. Our findings support the hypothesis that B lymphocytes promote a chronic inflammatory environment leading to increased invasion of TNBC cells.

---

#651

Evaluation of the expression and function of TIM-3 relative to PD-1 in human tumors.

Kristen McEachern,1 Srimoyee Ghosh,1 Yonghong Zhao,1 Qiyao Zhang,2 Norman Zhang,2 David W. Jenkins1. 1 _TESARO, Waltham, MA;_ 2 _Wuxi AppTec, China_.

While immunotherapies directed against PD-1 and PD-L1 have proven effective across multiple indications, there is still a large unmet medical need for therapies for patients who do not respond or who develop acquired resistance during the course of treatment. There are several emerging hypotheses to explain the lack of response, including the overall presence and localization of immune cells, as well as the up-regulation of additional T-cell checkpoints, including TIM-3 in tumor infiltrating lymphocytes. To investigate the potential role of TIM-3, we set out to evaluate the expression and function of TIM-3 and the relationship to PD-1 in several systems. Firstly, we developed a flow cytometry methodology to enumerate T-cell, including CD4 and CD8 positive cells, as well as other immune cell populations in a panel of human tumor samples, including non-small cell lung cancer (NSCLC). In these samples, we investigated the expression profiles of TIM-3 and PD-1 in both T-cell and non-T-cell populations. In addition, we performed complementary genomic studies to explore gene expression profiles of not only the bulk tumor samples but also of isolated PD1 positive/TIM-3 negative versus PD1 and TIM-3 double positive cell populations. The profiling identified tumors with a range of tumor infiltrating lymphocyte content and also differences in PD1 and TIM-3 expression. It was found that in addition to T-cells, TIM-3 was expressed on myeloid cell populations and furthermore, that there were distinct differences in the gene expression profiles of PD1 single positive versus PD1 and TIM-3 double positive cells. Building on the expression studies, the functional role of Tim-3 in T-cells and also on myeloid-derived cells was explored. Taken together, these studies provide further evidence for TIM-3 as an important immunological checkpoint and as a relevant therapeutic target for the treatment of cancer with the potential for biological effects in both the T-cell and myeloid compartments of the tumor microenvironment.

#652

PD-L1 and MHC class 1 expression levels are modulated by MEK inhibition on head and neck squamous cell carcinoma.

Seong-Ho Kang,1 Bhumsuk Keam,2 Soyeon Kim,1 Miso Kim,2 Yong-Oon Ahn,1 Tae Min Kim,2 Dong-Wan Kim,2 Dae Seog Heo2. 1 _Cancer Research Institute, Seoul National University, Seoul, Republic of Korea;_ 2 _Department of Internal Medicine, Seoul National University Hospital, Seoul, Republic of Korea_.

Background: To overcome immunosuppression, multiple pathways can be targeted by counter strategies, including elimination of suppressive signals (such as PD-L1 and CTLA-4) and promotion of antigen presentation from tumor cells, and of T cell infiltration into tumor sites. The purpose of this study is to investigate the change in expression of PD-L1 and MHC class I as well as of T cell chemoattractants, CXCL9 and CXCL10, in human head and neck squamous cell carcinoma (HNSCC) cell lines after treatment with MEK inhibitor. In addition, for evaluation of immunologic effects of MEK inhibitor in vivo, we employed SCC VII mouse squamous cell carcinoma (SCC) model.

Methods: Six human HNSCC cell lines (SNU-1041, SNU-1066, SNU-1076, Detroit 562, FaDu, and HN31) and a mouse squamous cell carcinoma cell line (SCC VII) were used. Trametinib was purchased from Selleckchem. We conducted cell viability assay using these cell lines after 72 h incubation with MEK inhibitor trametinib. PD-L1 and MHC class I expression levels were analyzed by flow cytometry after treatment with trametinib and/or interferon-gamma (IFN-γ). Expression of PD-L1, p-Erk1/2, and p-STAT1/3 were analyzed by western blot. STAT3 was knocked down by siRNA transfection. To determine the levels of CXCL9 and CXCL10 transcripts after trametinib treatment, reverse-transcription PCR was carried out.

Results: The growth inhibition by trametinib treatment was variable between cell lines with moderate sensitivity (10 nM < IC50 < 100 nM in five of seven cells). IC50 values were over 10 μM in Detroit 562 and FaDu. All the cell lines have no upstream RAS or RAF mutation. Although trametinib treatment was not shown to be enough for inhibiting growth of these cell lines, it up-regulated MHC class I expression in all cells either alone (> 2-fold) or when combined with IFN-γ (> 2-fold of IFN-γ alone). At the same time, we observed PD-L1 expression was elevated by trametinib treatment when combined with IFN-γ in all cells except SNU-1066, in which trametinib treatment down-regulate IFN-γ-induced increase of PD-L1 expression. Furthermore, we observed that trametinib alone and/or with IFN-γ enhanced mRNA expression of CXCL9 and CXCL10 in all human cells. Mechanistically, trametinib treatment enhanced STAT1 phosphorylation by IFN-γ in five of six human cells, and directly phosphorylated STAT3. When we silenced STAT3, enhanced expression of MHC class I by trametinib treatment was not observed in SNU-1041 cell.

Conclusion: Our results suggest that MEK pathway is closely related with modulating the expression of PD-L1 and MHC class I as well as of CXCL9 and CXCL10 in HNSCC cell lines. Up-regulated levels of MHC class I and T cell-recruiting chemokines imply that MEK inhibitor may mediate potentiating T cell responses. Moreover, simultaneous increase of PD-L1 expression suggests the possible synergistic role of MEK inhibitor when combined with anti-PD-1/PD-L1 immunotherapy in HNSCC.

#653

Inhibition of MMP9 improves anti-tumor immunity by changing the tumor microenvironment to promote T cell trafficking and activation.

Vladi Juric, Amanda Mikels-Vigdal, Chris O'Sullivan, Andrew Greenstein, Erin Stefanutti, Vivian Barry-Hamilton, Igor Mikaelian, Ted Sullivan, Erik Huntzicker, Jeremiah Degenhardt, Peng Yue, Victoria Smith. _Gilead Sciences, Foster City, CA_.

Background: Matrix metalloproteinase 9 (MMP9) acts via diverse mechanisms to promote tumor growth, invasion, and metastasis. It activates growth factors and signaling pathways, promotes angiogenesis, and impedes anti-tumor immune responses. We developed a monoclonal antibody that inhibits mouse MMP9 (AB0046) and assessed its mechanism of action in immunocompetent mouse tumor models and in vitro assays.

Methods: We examined MMP9 expression in a variety of human tumor tissues via immunohistochemistry. Human monocytes were differentiated in vitro and protein expression was assessed via enzyme-linked immunosorbent assay. Primary tumor growth in orthotopic, syngeneic tumor models was examined following anti-MMP9 antibody treatment. RNA sequencing, immunohistochemical and flow cytometry analyses were performed on tumor tissues to assess gene expression, stromal remodeling and macrophage polarization in response to AB0046 treatment.

Results: MMP9 levels are elevated in human tumors compared to healthy tissues. The protein is expressed predominantly in stromal cells, including macrophages and neutrophils, with more occasional heterogeneous expression in tumor epithelia. Differentiation of human monocyte-derived macrophages in vitro revealed that M2 polarization is associated with increased expression of MMP9 and Th2 markers CCL18 and TGFβ. Anti-MMP9 treatment in three independent mouse tumor models (HC11-NeuT, CT26, Lewis lung carcinoma (LLC)) resulted in decreased primary tumor growth (p=0.001 and p=0.018 for HC11-NeuT and CT26 respectively) and increased animal survival (p=0.024 for LLC). Gene expression profiling of tumors from the various models demonstrated that inhibition of MMP9 resulted in elevated expression of genes associated with immune cell activation pathways (Hallmark Interferon Gamma Response, p<0.05, FDR<0.001). Additional analysis in the HC11-NeuT model revealed a significant decrease in M2 macrophages in the tumor microenvironment (p < 0.05) with AB0046 treatment, as well as reductions in tumor-associated fibrillar collagen as assessed by Picrosirius red staining.

Conclusions: These analyses show that MMP9 is expressed in a variety of human tumors. Our data suggest that inhibition of MMP9 promotes anti-tumor immunity and enhances a Th1 immune response. GS-5745, a humanized anti-MMP9 inhibitory antibody, is being evaluated in gastric cancer in phase 3 and 2 studies with chemotherapy and nivolumab, respectively (NCT02545504, NCT02864381).

#654

The epigenetic landscape of T cell exhaustion.

Debattama Sen. _Dana Farber Cancer Institute/Harvard Medical School, Cambridge, MA_.

Exhausted T cells in cancer and chronic viral infection have distinctive patterns of gene expression, including sustained expression of the inhibitory receptor PD-1, but the regulation of gene expression in exhausted T cells is poorly understood. Here we define the accessible chromatin landscape in mouse and human exhausted CD8+ T cells and show that it is profoundly different from functional memory CD8+ T cells. Exhausted CD8+ T cells in a mouse model of chronic viral infection acquire an extensive, state-specific pattern of enhancers, which are organized into functional modules. One enhancer, -23.8kb from the Pdcd1 locus, is found only in exhausted T cells and other lymphocytes with sustained PD-1 expression. Genome editing shows it to be required for high PD-1 expression. Cas9-mediated in situ saturation mutagenesis of the enhancer pinpoints critical minimal sequences that correspond to bound transcription factor motifs for RAR, T-bet and Sox3 in exhausted CD8\+ T cells. State-specific enhancer profiles identified in mouse exhausted CD8+ T cells are conserved in human exhausted antigen-specific CD8+ T cells responding to HIV and HCV infection. Detailed functional enhancer maps of T cell exhaustion reveal state-specific regulatory sequences and offer targets for genome editing that could alter gene expression preferentially in exhausted CD8+ T cells.

## CLINICAL RESEARCH:

### Checkpoint Inhibitor and Prognostic Biomarkers

#655

Development of IHC staining protocols for assessment of PD-L1 expression in cytological samples.

Christine Boyiddle,1 Mark Ruboyianes,1 Ed Del Valle,1 Lukas Bubendorf,2 Kerstin Trunzer,3 Judith Pugh,1 Jennifer Boone1. 1 _Ventana Medical Systems, Oro Valley, AZ;_ 2 _University Hospital Basel, Basel, Switzerland;_ 3 _F. Hoffmann-La Roche, Basel, Switzerland_.

Immunohistochemistry (IHC) staining of non-small cell lung cancer (NSCLC) samples for programmed cell death ligand 1 (PD-L1) can help identify patients that may benefit from anti-PDL1 therapy. However, resection or biopsy samples cannot be obtained for some patients with NSCLC. Fine needle aspiration (FNA) is a less invasive method for obtaining samples in such patients, and it is yet to be determined if PD-L1 can be assessed using cytological samples. In this study we had three objectives 1. Develop staining protocols on the VENTANA BenchMark ULTRA automated staining platform for cytology samples fixed with the most common methods using cell lines as a model. 2, Determine the optimal cytology fixation method for VENTANA PD-L1 (SP142) IHC Assay (PD-L1 (SP142)) staining, and 3. Assess PD-L1 expression in tumor cells (TC) and tumor-infiltrating immune cells (IC) in cytological samples prepared from NSCLC patients. KARPAS 299 cell line was used as a model system for the fixation studies. Liquid-based preparations (LBPs) fixed in PreservCyt, conventional smears (unstained and pap-stained) fixed in 95% ethanol, and paraffin embedded cell blocks (CBs) fixed in 95% ethanol, 10% neutral buffered formalin (NBF), PreservCyt, and SurePath preservative were optimized on the BenchMark ULTRA using PD-L1 (SP142) antibody and OptiView DAB Detection and Amplification Kits. NSCLC FNA CBs fixed in 10% NBF (N=69) and a subset of FNAs with matched resections (N=20) were stained with the optimized protocol. FNAs and resections were assessed for percentage of TC and IC with PD-L1 staining. Staining parameters were optimized on the BenchMark ULTRA for all sample types tested, and they all produced a range of moderate to strong PD-L1 expression. CBs fixed in 95% ethanol and 10% NBF produced the highest percentage of staining, with expression in 90% of cells. CBs fixed in PreservCyt and SurePath preservative had a lower percentage of staining, 40% and 75% respectively. LBPs had 25% cells staining and smears ranged from 20%-60%. NSCLC FNA CBs fixed in 10% NBF produced interpretable results when stained with PD-L1 (SP142). IC staining was seen in 8.7% (6/69) of samples, TC staining was seen in 8.7% (6/69) of samples, both IC and TC staining was seen in 14.5% (10/69) of samples. The subset of FNAs with matched resections showed concordance with PD-L1 IC staining in 60% (12/20) of samples and TC staining in 75% (15/20) of samples. The discordant cases showed that FNAs were negative for IC when the resection was negative and positive for TC in 5% of cases when the resection was negative. The VENTANA PD-L1 (SP142) Assay staining parameters were found to be optimal for staining NSCLC FNA CB fixed in 10% NBF. PD-L1 staining was detected in both TC and IC in FNA samples and concordance of FNA and matched resections was high for both IC and TC. A larger study is necessary to validate the use of FNAs for assessment of PD-L1 expression in a clinical setting.

#656

PD-L1 expression in primary lesions vs metastatic sites and by demographics in advanced urothelial carcinoma samples.

M Zajac,1 A M. Boothman,1 Y Ben,2 A Gupta,3 X Jin,3 J Antal,3 A Sharpe,1 M Scott,1 M Rebelatto,3 J Walker1. 1 _AstraZeneca, Cambridge, United Kingdom;_ 2 _AstraZeneca, Gaithersburg, MD;_ 3 _MedImmune, Gaithersburg, MD_.

Background: Determination of programmed cell death ligand-1 (PD-L1) expression levels in tumors may help physicians understand which patients (pts) are most likely to respond to anti-PD-1/PD-L1 therapies in urothelial carcinoma (UC). Understanding the impact of different sample types and demographics on PD-L1 expression is important to determine suitability of tumor biopsies for testing.

Methods: As of July 24, 2016, 363 pts screened in the UC cohort of Study CD-ON-MEDI4736-1108 (NCT01693562) had tissue available for analysis and 47 pts had provided paired primary and metastatic samples. FFPE samples were tested in a central laboratory with the VENTANA PD-L1 (SP263) Assay using a BenchMark ULTRA instrument. Pts were classified as having either PD-L1 high (PD-L1 expression ≥25% either on tumour cells [TC] or immune cells [IC]) or PD-L1 low/negative (<25% on TC and IC) tumors. PD-L1 high prevalence was reported in primary vs metastatic sites, and by age, sex and race.

Results: PD-L1 status was evaluable for 332/363 (91.5%) pts (175/332 [52.7%] PD-L1 high and 157/332 [47.3%] PD-L1 low/negative) whose UC specimens were tested (intent to diagnose [ITD] population). Overall percentage agreement between paired primary and metastatic samples, based on combined TC/IC scoring ≥25%, was 74.5% (95% CI 59.7 - 86.1%). In the ITD population, using only the samples from which patient PD-L1 expression status was determined, PD-L1 high prevalence in primary and metastatic samples was 57.1% and 50.9% respectively (p=0.343, not significant). The proportion of pts with PD-L1 high status was not enriched in any demographic group (Table).

Conclusions: Initial data from UC pts in Study 1108 showed similar PD-L1 high prevalence in primary and metastatic lesions and good concordance between paired primary and metastatic samples. These results build optimism that samples obtained from either location could be used to determine PD-L1 status. Further data are needed to confirm these findings.

Patients screened for UC cohort with evaluable PD-L1 result - ITD population (n = 332)

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Parameter | PD-L1 high, n (%) | P value

Age, years | <65 (n=131) | 73 (55.7%) | 0.438

≥65 (n=201) | 102 (50.8%)

Sex | Male (n=236) | 127 (53.8%) | 0.610

Female (n=96) | 48 (50.0%)

Race | Asian (n=52) | 23 (44.2%) | Asian vs White: 0.406

Black or African American (n=10) | 6 (60.0%)

White (n=218) | 113 (51.8%)

Other (n=9) | 4 (44.4%)

#658

Association of tumor mutational burden (TMB) with DNA repair mutations and response to anti-PD-1/PD-L1 therapy in non-small cell lung cancer (NSCLC).

Andrew A. Davis, Young Kwang Chae, Sarita Agte, Alan Pan, Nisha Mohindra, Victoria Villaflor, Kirtee Raparia, Francis J. Giles. _Northwestern University, Chicago, IL_.

Background:

Identifying optimal biomarkers for response to anti-PD-1/PD-L1 therapies in non-small cell lung cancer (NSCLC) is critical. Tumor mutational burden (TMB) is a potential biomarker of genomic instability and neoantigen binding sites to activated effector T cells. The goal of this study was to examine the association of TMB with overall survival for patients treated with checkpoint blockade and to correlate TMB with mutational status, including DNA repair mutations.

Methods:

We retrospectively examined TMB using next-generation sequencing (FoundationOne) for 82 patients with NSCLC in our institution. TMB included coding base substitutions and indel alterations, but excluded potentially functional mutations. TMB high versus low was stratified based on 15 mutations per megabase pair. We correlated TMB with DNA repair mutations, mutational status, and smoking history. In addition, survival data were obtained for 35 patients who were treated with anti-PD-1/PD-L1 therapy.

Results:

TMB was associated with significantly longer overall survival for patients treated with checkpoint blockade (Table 1, p < 0.04). In addition, TMB was independently correlated with direct and indirect (Chae et al. 2016) DNA repair mutations (p < 0.03). TMB was also significantly associated with smoking, number of coding region mutations, and treatment with at least two prior lines of therapy (p < 0.02). A trend toward lower TMB was found for patients with mutations in EGFR, ALK, or KRAS (p = 0.06).

Conclusions:

Higher TMB was associated with improved survival for patients treated with anti-PD-1/PD-L1 therapies. In addition, TMB was correlated with DNA repair mutations, number of coding mutations, prior treatment, and smoking. Our results indicate that TMB can be used as a biomarker for response to checkpoint blockade in NSCLC. | |  | |

|

---|---|---|---|---|---

Table 1. Tumor mutational burden (TMB) by patient characteristic and mutation status

Patient Characteristic | TMB (mutations per megabase) | P

|  | TMB (<15) | TMB (≥15) | All

|

Smoking History | Minimal/Never (N) | 25 | 1 | 26 | P = 0.011+

|

Current/Former (N) | 38 | 18 | 56

|

|

All (N) | 63 | 19 | 82

|

Number of Treatment Lines Prior to Checkpoint Blockade

(Excluding Targeted Therapy) | 0-1 (N) | 20 | 4 | 24 | P = 0.017+

|

2+ (N) | 4 | 7 | 11

|

|

All (N) | 24 | 11 | 35

|

Presence of EGFR/ALK/KRAS Mutations | Yes | 27 | 3 | 30 | P = 0.061+

|

No | 36 | 16 | 52

|

|

All (N) | 63 | 19 | 82

|

Number of Potentially Functional Direct + Indirect DNA Repair Mutations | Mean (N) | 0.76 (N=63) | 1.26 (N=19) | 0.88 (N=82) | P = 0.027*

Number of VUS Direct +

Indirect DNA Repair Mutations | Mean (N) | 1.30 (N=63) | 2.26 (N=19) | 1.52 (N=82) | P = 0.011*

Number of Direct DNA Repair Mutations | Mean (N) | 1.08 (N=63) | 2.42 (N=19) | 1.39 (N=82) | P = 0.001*

Number of Total DNA Repair Mutations | Mean (N) | 2.06 (N=63) | 3.53 (N=19) | 2.40 (N=82) | P = 0.005*

Number of Potentially Functional Mutations | Mean (N) | 4.79 (N=63) | 8.00 (N=19) | 5.54 (N=82) | P = 0.002^

Number of VUS Mutations | Mean (N) | 9.13 (N=63) | 25.00 (N=19) | 12.80 (N=82) | P < 0.001^

Total Reported Mutations | Mean (N) | 13.92 (N=63) | 33.00 (N=19) | 18.34 (N=82) | P < 0.001^

Overall Survival | Kaplan-Meier Log-Rank Test | P = 0.020

|

Cox Proportional Hazards Regression Model | HR = 0.19

|  | 95% CI: [0.04 - 0.88]

|  | P = 0.034

VUS, Variant of Unknown Significance; HR, Hazard Ratio; + Pearson's Chi-Squared; * Logistic regression; ^ Welch's two-sample t-test

#659

Multiplex cocktails for immunotherapy targets: PD-L1 with tumor specific transcription factors.

David Altree-Tacha, Wei Yuan, George Yang. _Biocare Medical, Concord, CA_.

Introduction

Blocking the interaction between the programmed cell death (PD)-1 protein and one of its ligands, PD-L1, has been reported to generate impressive antitumor responses. Therapeutics targeting PD-L1 in several cancers are currently in clinical trials, and the U.S. FDA has granted accelerated approval for Keytruda (pembrolizumab) to treat patients with advanced (metastatic) non-small cell lung cancer (NSCLC). Various pathology scoring and interpretation methods have been put forth to assay PD-L1 expression, and variability of non-PD-L1 staining across tumors may have important prognostic aspects. PD-L1 expression on tumor cells can be upregulated via activation of CD8+ cytotoxic T lymphocytes. PD-L1 expression is also associated with certain subtypes of tumor-associated lymphocytes, macrophages and dendritic cells. In certain cancers, the cross reactivity of PD-L1 can make interpretation and scoring difficult. Thus, a strategy using IHC multiplex stains could help resolve challenging cases by cocktailing PD-L1 with target tissue transcription factor antibodies along with other potential markers.

Method and Materials

Formalin-fixed paraffin-embedded tissues and tissue microarrays for various tumor types including lung, bladder and melanoma were processed and cut at 4-5 microns. PD-L1 rabbit monoclonal antibody was cocktailed with the following antibodies: TTF-1 (lung cancer); CD163 (lung cancer); p40 + GATA3 (bladder cancer); and SOX10 (melanoma). Cocktails were detected with double stain detection systems using brown, red and blue chromogens for visualization. Sections were counterstained with a standard hematoxylin or Wiegert's iron hematoxylin.

Results

PD-L1 and TTF-1 with or without CD163 (macrophage) and PD-L1 + CD163 were used in lung cancers. The nuclear staining of TTF-1 in lung adenocarcinoma (blue chromogen) helped define tumor positive PD-L1 (DAB) positive cells and CD163 (Fast Red) marked macrophages. PD-L1 combined with p40 and GATA3 was deployed for bladder cancers. PD-L1 membranous expression in tumor cells and co-expression of p40 and GATA3 gave robust nuclear staining in bladder tumor cells. Finally, SOX10 nuclear staining was observed in most melanoma cells and could be easily separated from PD-L1 membrane staining.

Conclusion

PD-L1 cocktailed with strategic nuclear or cytoplasmic antibodies can help discriminate tumor cells from non-tumor cells, and may facilitate quantitation or scoring methods for more accurate assessment of this key immunotherapy marker.

#660

Pre-symptomatic detection and early treatment of mammary cancer with anti-PD-L1 in a mouse mode.

Hu Duan, Stephen Albert Johnston, Shen Luhui. _Biodesign Institution, ASU, Tempe, AZ_.

Cancer kills ~8M people/year and the WHO predicts an epidemic of cancer in the developing world over the next 20 years due to increased longevity. Development of a simple method to detect and treat cancer early could be a solution to this challenge. Here we test this approach in principle in a transgenic mouse mammary tumor model by immunosignature (IMS) diagnostics. IMSs are profiles of antibodies in the blood displayed on a high density peptide array. As previous published data showed, the IMS can distinguish different diseases based on the disease specific antibody profile in the patient, including different cancers and infectious diseases. According to the immune surveillance theory, the immune system, including the humoral immune response, starts to interact with cancer cells as soon as they are transformed. First, we tested if the IMS is sensitive enough to detect the antibody profile change caused by the early tumor development in the FVB-NeuN (MMTVneu/202Mul/J) mammary cancer model. In the FVB-NeuN model, the MMTV promoter activates the wild type rat ERBB2 gene in mammary cells, and every mammary cell potentially can convert into a cancer cell. The transition from normal mammary glands to hyperplasia of FVB-NeuN in mouse starts at 8.6 to 15 weeks after birth. The mice develop the first palpable tumor in one of the ten mammary glands by 19-45 weeks of age with an average age of 32±1.2 weeks in our hands. We collected serum every 4 weeks from the FVB-NeuN mice and the wild type FVB mice for the IMS analysis. The IMS can distinguish the FVB-NeuN mouse from the age matched wild type FVB mouse as early as 12 weeks (Stage-1) before the onset of the first palpable tumor. We also analyzed the IMSs from the Stage-2 (8 weeks before the palpable tumor and so on) to Stage 4. Different IMS of each stage has only partial overlap with the other stages. The cross-stage prediction analysis showed the accuracy of the cancer detection by using different IMS is from 75% to 93%. Second, we determined if early treatment at the time of diagnosis with the checkpoint blockade, anti-PD-L1, would inhibit the tumor growth. A course of 3 injections at the time of diagnosis significantly retarded tumor growth relative to untreated controls. The early treatment elicited long-term protection which could restrict the tumor growth even four months after the treatment. The treated mice presented a specific IMS at 28 days post treatment and 35 days post the first palpable tumor, compared to the untreated mice. This indicates that the early treatment elicited an anti-tumor immune response that could inhibit tumor growth. This preliminary experiment suggests that early diagnosis combined with systemic treatment might be a method to control or eliminate cancer.

#661

Evaluating "harmonization" of PD-L1 assays using image analysis.

Nathan T. Martin, Joshua C. Black, Zachary Pollack, Famke Aeffner, Joseph Krueger. _Flagship Biosciences, Westminster, CO_.

The current diagnostics landscape for therapeutics that target the PD1/PD-L1 pathway is highly complex. Four different companion or complementary diagnostics have been developed for pembrolizumab in NSCLC, nivolumab in NSCLC and melanoma, and atezolizumab in urothelial carcinoma. The need to reconcile diagnostics for this class of targeted therapies has been recognized by the creation of the FDA-AACR-ASCO "PD-L1 Blueprint" working group to explore means to "harmonize" PD-L1 testing in tissue based IHC assays. The results reported from this working group noted similarities, but also several important discrepancies, between the current assays. Since each test uses a specific interpretation for each assay and indication, this creates a highly complex diagnostic landscape, which is likely to continue to increase in complexity as more PD-1/PD-L1 therapeutics and potentially novel diagnostics continue to be approved in additional indications.

To address the need for adaptive, sustainable harmonization for PD-L1 diagnostics, Flagship Biosciences evaluated the utility of image analysis-based methods to harmonize multiple PD-L1 tests. We executed a proof-of-concept study utilizing a cohort of serial tissue sections from the same NSCLC patients, stained with the FDA approved Dako PD-L1 tests (28-8 and 22C3 clones), and our in-house PD-L1 assays (SP142 and E1L3N clones) for comparison. We digitized the tissue slides using a whole slide scanner, and evaluated the samples with our tissue Image Analysis (tIA™) technology. As expected, the patient samples stained with the separate PD-L1 assays yielded differences in staining and, thus, the reported scores for PD-L1 expression based on each test used, despite serial sections being derived from the same patient. To attempt to harmonize the scoring approaches for each test, we leveraged our computational Tissue Analysis (cTA™) platform to create a mathematically-derived "virtual slide score" for each sample, which enabled calibration of the various tests to deliver cross PD-L1 comparative scores. Based on the proof-of-concept demonstrated in this study, the cTA™ approaches could enable harmonization of the various PD-L1 tests through use of a digital pathology platform. The data presented provides a foundation for potential application of the cTA™ platform in the clinical laboratory setting to achieve harmonization of multiple PD-L1 tests.

#662

The differential association of PD-1, PD-L1, and CD8+ cells with response to pembrolizumab and presence of Merkel cell polyomavirus (MCPyV) in patients with Merkel cell carcinoma (MCC).

Nicolas A. Giraldo,1 Genevieve J. Kaunitz,1 Tricia R. Cottrell,1 Sneha Berry,1 Joel C. Sunshine,1 Peter Nguyen,1 Haiying Xu,1 Aleksandra Orgutsova,1 Candice D. Church,2 Natalie J. Miller,2 Jennifer H. Yearley,3 Evan J. Lipson,1 Ludmila Danilova,1 Paul T. Nghiem,2 Suzanne L. Topalian,1 Janis M. Taube1. 1 _Johns Hopkins Medical Institutions, Baltimore, MD;_ 2 _University of Washington, Seattle, WA;_ 3 _Merck Research Labs, Biologics Discovery, Palo Alto, CA_.

We recently reported a 56% objective response rate in patients with advanced MCC receiving pembrolizumab (anti-PD-1) as first-line therapy. However, a tumor biomarker predicting clinical response was not defined. The purpose of this study was to determine potential associations of anti-PD-1 response (RECIST 1.1, analysis 8/1/16) and the presence of MCPyV, with the density and distribution of CD8+, PD-1+ and PD-L1+ cell populations in the tumor microenvironment (TME). Pretreatment FFPE tumor specimens were stained for CD8 (n=23) and PD-L1 (n=16, 22C3 assay) with immunohistochemistry (IHC). Immunofluorescence (IF) was used to detect PD-1 (n=16). Intratumoral (IT), peritumoral (PT, 100 um zone) and total (PT+IT) densities of PD-1+ and CD8+ immune cells (IC) were determined with digital image analysis. PD-L1+ cases had >1% tumor cells (TC) expressing PD-L1. Tumors from patients who responded to anti-PD-1 showed higher densities of PD-1+ cells in the IT, PT, and IT+PT regions when compared to non-responders (Mann-Whitney test, p-value=0.03, 0.06, 0.03, respectively). There was no significant association of response with CD8+ IC densities (IT, PT or total) or TC PD-L1 expression. Similarly, when we quantified the number of PD-1+ IC located within 15 um from a PD-L1+ cell (TC or IC), an association was observed between PD-1 and PD-L1 proximity and clinical response (Mann-Whitney test, p-value =0.04), but not CD8 and PD-L1 proximity. Because viral neoantigens can elicit a strong immune response, we also studied these TME factors for their potential associations with MCPyV. We found the presence of virus was significantly associated with increased densities of PT CD8+ cells (Mann-Whitney test, p-value=0.008) and TC PD-L1 expression (Fisher's test, p-value=0.04), but not PD-1+ IC. A more extensive multiplex IF panel (CD8, PD-1, PD-L1, CD68, FoxP3, NSE) analysis was performed on select pre-treatment specimens using the Vectra multispectral imaging system (Perkin Elmer) to explore this divergence. We found that only ~60% of observed total PD-1 expression was displayed by CD8+ cells. Although preliminary, our results suggest a relationship between PD-1+ cells and response to anti-PD-1 therapy and highlight that lymphocyte subsets other than CD8+ T-cells may contribute to the observed response. Further studies in larger cohorts are needed to validate these results.

#663

Clinical performance and utilization of 3 PD-L1 immunohistochemical assays.

Steven M. Anderson,1 Bruce Horten,2 Bryan McCune,3 Ronald Paler4. 1 _Covance, Durham, NC;_ 2 _Integrated Oncology, New York, NY;_ 3 _LabCorp, Research Triangle Park, NC;_ 4 _Covance, Phoenix, AZ_.

Three different PD-L1 immunohistochemical (IHC) assays have recently been approved to help guide treatment decisions regarding anti-PD-1 (Keytruda®, Opdivo®) and anti-PD-L1 (Tecentriq®) immuno-oncology based therapies. The three assays differ in the primary monoclonal antibodies used, the assay platform (Dako, Ventana), scoring and interpretation criteria, and intended use. Our clinical laboratories currently offer all 3 assays (pharmDx™ 22C3, pharmDx™ 28-8, and SP142) because they are associated with different intended uses, companion and complementary diagnostics for non-small-cell lung cancer (NSCLC), and complementary diagnostic for urothelial carcinomas (UC). In the NSCLC samples evaluated in the first year since the approval of the two pharmDx™ assays, we have noted a higher percentage of positive samples with the pharmDx™ 28-8 assay (50%) compared to the pharmDx™ 22C3 assay (30%). This difference is mainly due to the use of different assay cutoffs associated with the two assays, as there is good correlation between the overall staining patterns, including the distribution of the percentage of tumor cells exhibiting positive staining. For the SP142 assay tested on UC samples, 22% of the cases were considered positive. All 3 assays are robust, with a limited number of samples considered inconclusive (<10%), mainly due to an inadequate number of tumor cells. The companion diagnostic PD-L1 assay (pharmDx™ 22C3) has had the greatest utilization of the 3 assays, as it is required for the use of the Keytruda® in NSCLC samples. The performance features of the 3 different PD-L1 assays in the clinical laboratory setting has shown that all assays are robust and can be readily scored and interpreted by the pathology team. The utilization of the 3 assays correlates with respective intended use, tumor type and companion versus complementary diagnostic application, for each assay.

#664

Analytical validation and clinical utility of an immunohistochemical PD-L1 diagnostic assay for treatment with durvalumab in urothelial carcinoma patients.

M Zajac,1 A M. Boothman,1 Y Ben,2 A Gupta,3 J Antal,3 X Jin,3 A Nielsen,4 G Manriquez,4 C Barker,1 P Wang,4 P Patil,4 N Schechter,4 M Rebelatto,3 J Walker1. 1 _AstraZeneca, Cambridge, United Kingdom;_ 2 _AstraZeneca, Gaithersburg, MD;_ 3 _MedImmune, Gaithersburg, MD;_ 4 _Ventana Medical Systems Inc., Tucson, AZ_.

Background: A high quality programmed cell death ligand-1 (PD-L1) diagnostic may help to identify patients (pts) most likely to respond to anti-PD-L1/programmed cell death-1 (PD-1) therapy. Here we describe a PD-L1 immunohistochemical (IHC) diagnostic test developed for urothelial carcinoma (UC) pts treated with durvalumab.

Methods: The IHC assay uses an anti-human PD-L1 rabbit mAb optimized for detection of both tumor cell (TC) and tumor-associated immune cell (IC) PD-L1 expression with the OptiView DAB IHC Detection Kit on the automated VENTANA BenchMark ULTRA platform. The assay was validated for intended use in UC formalin-fixed, paraffin-embedded samples in a series of studies that addressed sensitivity, specificity, robustness and precision and implemented in Study CD-ON-MEDI4736-1108 (NCT01693562). Pts were evaluated using the VENTANA PD-L1 (SP263) Assay at a prespecified PD-L1 expression cut-off. Efficacy was analyzed in pts with PD-L1 low/negative (defined as TC <25% and IC <25%) UC and in pts with PD-L1 high (defined as TC ≥25% or IC ≥25%) UC.

Results: The VENTANA PD-L1 (SP263) Assay met all the predefined acceptance criteria (average positive agreement and average negative agreement >85%), showing analytical specificity, sensitivity and precision. It demonstrated ≥97% and ≥85% inter-reader precision agreement for TC and IC respectively. For intra-reader precision, it demonstrated >96% and >87% agreement for TC and IC respectively. For intra-day performance, the assay demonstrated ≥96% agreement for TC and IC and for inter-day performance, it demonstrated ≥98% and 100% agreement for TC and IC respectively. Precision studies for inter-antibody lot, inter-detection kit lot and intra-platform demonstrated >97% agreement for both TC and IC. Inter-laboratory testing was performed at 3 external laboratories and demonstrated an overall agreement rate of 92.3%. The VENTANA PD-L1 (SP263) Assay was implemented in Study CD-ON-MEDI4736-1108 and durvalumab demonstrated clinical activity and durability of response in both PD-L1 high and PD-L1 low/negative subgroups, yet with different response rates. In addition, given the high negative predictive value of the assay, it is especially helpful in evaluating the likelihood of response to durvalumab; pts who were classified as PD-L1 high with the VENTANA PD-L1 (SP263) Assay tended to have a higher objective response rate per RECIST v1.1 than pts who were PD-L1 low/negative.

Conclusions: These data show that determination of PD-L1 expression in TC and IC in UC pts using the VENTANA PD-L1 (SP263) Assay is precise and highly reproducible and highlight the utility of the assay in a clinical setting. The VENTANA SP263 Assay is especially helpful in informing pts and physicians on the likelihood of response to durvalumab, but not for the purpose of restricting treatment to only PD-L1 high pts.

#665

Molecular and clinical analyses of patients with gynecologic malignancies treated with PD-1 directed immunotherapy.

Young Kwang Chae,1 Sabina Murshudova,1 William H. Bae,2 Jonathan F. Anker,1 Mario J. Pineda,1 Wilberto Nieves-Neira,1 Daniela E. Matei,1 John R. Lurain,1 Shohreh Shahabi,1 Francis J. Giles1. 1 _Northwestern University, Chicago, IL;_ 2 _Yonsei University College of Medicine, Seoul, Republic of Korea_.

Long term responses are seldom seen in treatments of advanced or recurrent gynecologic cancers. In contrast, immunotherapeutic strategies targeting immune checkpoints such as programmed cell death-1 (PD-1) are currently in active development in platinum resistant gynecologic malignancies with promising durable responses observed in subset of patients.

We analyzed all 14 patients with gynecology malignancies that were treated with PD-1 directed immunotherapy, nivolumab or pembrolizumab at the developmental therapeutics program (DTP) clinic in Northwestern University during 2015 till 2016. Seven patients had ovarian/fallopian/peritoneal cancer; six, endometrial cancer; 1, squamous cell carcinoma (SCC) of cervix that are refractory to standard-of-care chemotherapies. Comprehensive genomic profiling including microsatellite instability (MSI) status and tumor mutational burden (TMB) analyses was performed using next generation sequencing (NGS) (FoundationOne).

Among 7 patients with ovarian/fallopian/peritoneal cancer (median age, 62 years), objective response rate (ORR) was 20%. One patient had partial response (PR), another one had stable disease (SD), and 3, progressive disease (PD). Two patients were uable to be evaluated. In a patient with PR, CA 125 level normalized from 426.2 to 10.8 U/mL. Her tumor had very low TMB (1/Mbp). In all patients, TMB level varied from very low to low (1-5 mut/Mbp) with MSI status being stable. Progression free survival (PFS) ranged from 14 to 196 days; overall survival (OS) varied from 60 to 199 days.

Among 6 patients with endometrial cancer (median age, 68 years), ORR was 20%. One patient had PR while four experienced PD. One patient whose disease could not be assessed due to early death from an event unrelated to treatment had a remarkable chemical response with a decrease in CA125 level from 5,889 to 182.1 U/mL after one single dose. Her tumor had MSI-stable status and intermediate TMB (6 mut/Mbp). In a patient with PR, CA125 level decreased from 1,216 to 226.5 U/mL. Her tumor had MSI-high status with unknown TMB. Of note, one of four patients with PD had MSI-high status and intermediate level of TMB. Other patients had tumor with MSI-stable status and low level of TMB (3-4 mut/MB). PFS ranged from 23 to 258 days; OS varied from 73 to 265 days.

One patient with HPV positive SCC of cervix (34 year old) experienced PD. Her tumor had MSI-stable status with unknown TMB. PFS was 88 days; OS, 132 days.

In summary, patients with ovarian and endometrial cancer treated with PD-1 directed immunotherapy demonstrated ORR of 20%. One additional patient with endometrial cancer had an exceptional chemical response. None of the tumors had high TMB (>6/Mbp). No clear associations between genomic traits including TMB and MSI staus and responses were found. Further larger prospecitve studies are warranted to explore potential biomarkers for response with immunotherapy in gynecologic malignancies.

#666

CD37 tetraspanin as a novel biomarker for PD-1 blockade in diffuse large B-cell lymphoma.

Zijun Y. Xu-Monette, Ken H. Young. _UT MD Anderson Cancer Ctr., Houston, TX_.

PD-1 immune checkpoint blockade reconstituting antitumor immunity has changed the cancer treatment paradigm. PD-1 blockade has been successful in many types of solid tumors and Hodgkin lymphoma, but not yet for diffuse large B cell lymphoma (DLBCL), the most common aggressive B-cell lymphoma. We found several biomarkers including loss of CD37 tetraspanin expression in DLBCL correlated with increased PD-1 expression suggesting potential sensitivity of these DLBCL subsets to PD-1 blockade. CD37 (TSPAN26) is a member of the tetraspanin superfamily, widely expressed on normal and malignant mature B-cells and downregulated in plasma cells. It has been documented that CD37 plays important roles in T-cell-B-cell interactions, B-cell humoral response triggered by B-cell receptor cross-linking, and a balance between immune responses and tolerance. Interestingly, in a large cohort of DLBCL patients, we found that loss of CD37 expression in DLBCL predicts strikingly decreased overall and progression-free survival rates, and that PDCD1 gene expression was upregulated in CD37-negative activated B-cell-like (ABC) DLBCL by gene expression profiling, whereas the costimulatory molecule ICOSLG was upregulated in CD37+ germinal center B-cell-like (GCB) DLBCL. Using the new fluorescent multiplex technology, we further measured PD-1 and PD-L1 expression at the protein level on lymphoma cells and immune cells in the tumor microenvironment, and found that PD-1 protein levels were increased on both cytotoxic and helper T-cells infiltrating in CD37-negative DLBCL either of GCB or ABC subtype. These novel discoveries suggest that CD37 is important for sustained antitumor adaptive immunity, that immune dysregulation plays an important role for poor clinical outcomes in DLBCL, and that CD37-negative DLBCL may be sensitive to PD-1 blockade. In summary, loss of a CD37 tetraspanin was found to correlate with PD-1 overexpression in DLBCL clinical samples, and CD37 may serve as a novel biomarker for anti-PD-1 immunotherapy clinical trials in DLBCL.

#667

Immunoprofiling circulating blood as a means to early detection of solid tumors.

Amit Kumar,1 Dimitry Gabrilovich,2 Frank J. Rauscher,2 George Dominguez,2 Cyrus Sholevar,1 John Roop,1 Anthony Campisi,1 Alexander Polo2. 1 _Itus Corporation, Los Angeles, CA;_ 2 _Wistar Institute, Philadelphia, PA_.

Our goal was to evaluate whether profiling Myeloid Derived Suppressor Cells (MDSCs) in circulation correlated with the existence and stage of multiple, biopsy-verified solid tumor types and to evaluate whether such analyses could enable early detection. The tumor micro-environment (TME) is replete with numerous immune cells, and the type and concentration of such cells can provide prognostic information. The link between high concentrations of MDSCs and poor prognosis is most likely due to the immuno-suppressive effects of such cells. Some fraction of these cells spill into the blood stream. We utilized flow cytometry to phenotypically quantify subsets of MDSCs and other leukocytes in the circulation of biopsy-verified cancer patients, as well as in age and sex matched healthy donors. Our results indicate a marked increase in MDSC levels in the circulation of tumor patients relative to healthy donors. We have analyzed patients presenting over a dozen solid tumor types (lung, breast, ovarian, colon, melanoma, liver, thyroid, pancreatic, uterine, osteosarcoma, appendiceal, leiomyosarcoma, liposarcoma, and vulvar) and found notable differences in the immune-profiles of circulating blood in these patients. It appears that the immune response, as measured by our flow cytometry technique, is general for most tumor types. We will present correlations and inter-relations between different cell types and evidence of tumors. While our studies to date have been performed in an unblinded manner, we will present performance data (specificity and sensitivity) for our approach.

#668

Evaluation of α-1-acid glycoprotein as a prognostic marker and as a predictive biomarker for severe neutropenia induced by docetaxel in esophageal carcinoma patients.

Yusuke Sasaki,1 Ken Kato,2 Hidekazu Hirano,2 Hirokazu Shoji,2 Yoshitaka Honma,2 Satoru Iwasa,2 Atsuo Takashima,2 Tetsuya Hamaguchi,2 Kengo Nagashima,3 Narikazu Boku2. 1 _National Cancer Center Hospital, Hakodate Central General Hospital, Tokyo, Japan;_ 2 _National Cancer Center Hospital, Tokyo, Japan;_ 3 _Chiba University Graduate School of Medicine, Chiba, Japan_.

Background: Serum α-1-acid glycoprotein (AAG) level is an independent predictor of response and a prognostic factor of survival in patients with non-small cell lung cancer treated with docetaxel chemotherapy. However, whether AAG is associated with the outcomes of esophageal cancer patients treated with docetaxel remains unclear.

Methods: Between August 2009 and April 2014, pretreatment serum samples were obtained from patients with clinical stage II/III esophageal cancer. Individuals were subsequently treated with neoadjuvant DCF or CF followed by surgery. DCF consisted of docetaxel and cisplatin on day 1, and continuous infusion of 5-fluorouracil on days 1-5, with this regimen repeated every 3 weeks for up to three cycles. CF consisted of cisplatin and 5-fluorouracil. Patients were divided into groups based on the median value of baseline AAG levels. Response to chemotherapy, survival, and the most severe grade of neutropenia were compared between the high and low AAG groups at each regimen.

Results: A total of 129 patients were enrolled (44 received neo DCF and 85 neo CF). The median serum AAG level was 95 g/L (range 57-228 g/L) and 95 g/L (range 46-197 g/L) in the patients with neo DCF and CF, respectively. For all patients, overall survival (OS) was significantly shorter in the high AAG group (AAG ≧ 95 g/L) than the low AAG group (AAG < 95 g/L) (HR 2.18, p=0.03). Both in the neo DCF and neo CF patients, there was a similar trend in OS between individuals with high and low AAG levels. Multivariate analysis demonstrated that AAG level (HR 1.98) and performance status (HR 2.20) were independent prognostic factors. However, the relationship between response to chemotherapy and AAG level were similar both in DCF and CF patients. In addition, grade 4 neutropenia was observed in 50% of patients with low AAG level and 23% with high AAG (p = 0.06) in DCF patients. A correlation analysis revealed a statistically significant correlation between serum AAG level and nadir absolute neutrophil count caused by DCF chemotherapy. However, in CF patients, there was no difference in the frequency of severe neutropenia between individuals with high and low AAG level, and there was no correlation between AAG and nadir neutrophil count.

Conclusions: Serum AAG level may be a prognostic marker for survival in stage II/III esophageal cancer patients treated with neoadjuvant chemotherapy. In addition, low serum AAG level is a potential predictive biomarker of docetaxel-induced severe neutropenia.

#669

Quantification of estrogen (ER) and progestin receptor (PR) as well as HER2/neu proteins and gene expression improves discrimination of clinical behavior by triple positive breast carcinomas.

Zohair R. Hameed, Michael W. Daniels, D. Alan Kerr, James L. Wittliff. _University of Louisville, Louisville, KY_.

IHC analyses of ER, PR and HER2 proteins are used as clinical biomarkers for breast cancer management. However, IHC provides semi-quantitative results sometimes complicated by variation in methods and interpretation. Our goal was to ascertain clinical use of these three analytes to predict breast cancer recurrence when analyses for gene expression and protein products were quantified.

Procedures: We examined ER and PR protein in 1059 de-identified carcinoma biopsies previously quantified by radio-ligand binding and/or enzyme immunoassay (EIA) using FDA approved reagents and HER2 oncoprotein determined by EIA to assess relationships between biomarker profiles and disease-free (DFS) and overall survival (OS) for 123 patients. Our comprehensive, IRB-approved Database also contained de-identified Microarray results obtained for ~ 22,000 genes from LCM-procured breast carcinoma cells of 247 primary breast biopsies. ESR1, PGR and ERBB2 gene expression levels were validated previously in 278 cancers by qPCR.

Results: Generalized Pair Plots of the three protein biomarkers indicated a relationship only between ER and PR supporting evidence that ER complexed with estrogenic ligands promotes synthesis of PR. Of the 8 possible combinations of the 3 protein biomarkers, only breast cancers exhibiting ER+PR+HER2+ (triple positive breast cancer, TPBCa) exhibited increased OS, whereas those with triple negative breast cancers (TNBCa) had decreased OS. TPBCa was exhibited by 32.5 % of specimens compared to TNBCa, which represented 7.3% of biopsies when biomarker proteins were quantified. Number of TPBCa was less when expression of the 3 genes (ESR1+/PGR+/ERBB2+) was measured by microarray or by qPCR while those of TNBCa increased. Furthermore, when TPBCa were identified by either microarray or by qPCR measurements, collectively increased ESR1, PGR and ERBB2 expression was associated with increased PFS and OS of patients. In contrast, biopsies that were ESR1-/PGR-/ERBB+ (14.1%) correlated with poor prognosis and overall survival using qPCR data, while those exhibiting ESR1-PGR+ERBB2- (14.1%) profiles by microarray results exhibited decreased PFS and OS. Influence of patient menopausal status on biomarker profiles was examined.

Conclusions: Quantitative measurements of ER, PR and HER2 proteins as well as microarray and qPCR assessment of ESR1, PGR and ERBB2 gene expression were correlated with prediction of clinical outcomes of breast carcinomas exhibiting Triple Positive Breast Cancers (TPBCa). Results reinforce importance of assessing levels of the three biomarkers in a quantified fashion to enhance their use in breast cancer management and prediction of risk of recurrence.

#670

Integrative genomic analysis of alterations driving anti-androgen treatment resistance in vitro.

Dong Shen,1 Brendan Hodkinson,1 Deborah Ricci,1 Karin Verstraeten,2 Michael Schaffer,1 Michael Gormley,1 Shibu Thomas1. 1 _Janssen Research & Development, Spring house, PA; _2 _Janssen R &D BE, Beerse, Belgium_.

Prostate cancer is one of the most frequently diagnosed cancers in the world. It is the second most common type of cancer and the fifth leading cause of cancer death in American men. Enzalutamide (MDV) and apalutamide (ARN-509) are selective competitive antagonists of the androgen receptor (AR). Abiraterone acetate (ABI) is an androgen biosynthesis inhibitor which is used in combination with prednisone in metastatic castration-resistant prostate cancer. Although the anti-androgen and AR inhibitors provide breakthrough treatments of metastatic castration-resistant prostate cancer, approximately a third of patients fail to show PSA response (>50% decline from baseline after 12 weeks of treatment), while another third of patients progress after a few cycles of treatment. Recently, the acquired F876L mutation of the androgen receptor (AR) was identified in advanced prostate cancer cells with acquired resistant to enzalutamide and apalutamide. To systematically identify and understand the mechanisms of resistance, we evaluated 22RV1 and LNCaP prostate cancer cells which were resistant to the treatment of enzalutamide (MDV), apalutamide (ARN-509), and abiraterone acetate (ABI). We isolated the DNA and RNA from both resistant cells and their parent cells. We did RNAseq for gene expression analysis, whole exome sequencing to identify the DNA alterations, and array CGH for copy number change analysis. We confirmed the acquired F876L mutation of the AR identified in LNCaP cells resistant to apalutamide. We identified the novel G644R and R630Q mutations of AR, R213* mutation of TP53, N372H mutation of BRCA1, and E2621G mutation of ATM in LNCaP cells which were resistant to the treatment of enzalutamide and apalutamide. For copy number alterations, we identified acquired copy number gain on chromosome 3, 8, and 18 on 22RV1 resistant cells, and acquired copy number loss on LNCaP resistant cells. Besides acquired DNA alterations, we identified 16 up-regulated and 24 down-regulated genes which are common in both 22RV1 and LNCap cells resistant to enzalutamide and apalutamide. Through pathway enrichment analysis, we identified the Wnt/B-catenin, cAMP signaling, and TREM1 signaling pathways were up-regulated in 22RV1 and LNCap cells resistant to enzalutamide and apalutamide. Detection of acquired genetic alterations in cells resistant to anti-androgen treatment provides us with important insights into the mechanisms of their resistance. Further testing of clinical samples collected at disease progression after treatment with these agents is required to validate these findings.

#671

Prognostic importance of EGFR expression and Kras gene mutations in gallbladder carcinoma.

Anjali Singh,1 Abul Kalam Najmi,1 Pramod Mishra,2 Majid Talikoti1. 1 _Jamia Hamdard, New Delhi, India;_ 2 _MAMC, Delhi University, New Delhi, India_.

Gallbladder carcinomas involve multiple cascades of oncogenes like EGFR and Kras. Activation of Kras downstream pathways predict the sensitivity to anti EGFR treatment. In 2012, for the first time it was established that GBC and cholangiocarcinomas have their unique somatic genomic landscape and thus be studied independently. This study evaluates whether EGFR is a prognostic factor in GBC and identifies the frequency of codon 12 and codon 13 Kras mutations along with its association with subject survival. Seventy two GBC curative resections from North India were immunostained with anti EGFR monoclonal antibody and assessed for codon 12 and 13 KRAS mutation by real time PCR. Strong EGFR expression was observed in 25.4percent cases. About 28/72; G12C, 17/72; G12R, 10/72; G12D and 18/72; G13D cases harboured point mutations in Kras gene, which was significantly higher as compared to control tissues. Multivariate analysis revealed that EGFR expression (p=0.01; HR=3.14; 95percent CI=1.28-7.71) and codon-13 mutation (p=0.001; HR=38.34; 95percent CI=4.90-300.01) had a significant impact on survival and were independent prognostic factors in GBC. Our study suggests that EGFR expression and codon 13 mutations are independent prognostic factors in the selected GBC cohort and showed a high frequency of Kras codon 12 mutations as well.

#672

Baseline serum albumin as a prognostic factor in patients with stage I and II colon adenocarcinoma.

Luis F. Onate-Ocana. _Instituto Nacional de Cancerologia, Mexico City, Mexico_.

Background: Patients with stage I or II colon carcinoma (CC) have a significant risk of recurrence after adequately performed curative resection. Adjuvant chemotherapy has not improved recurrence or survival in such patients. In this study, a prognostic model in patients with stage I or II CC is presented.

Methods: Consecutive cases with CC treated at a cancer center in Mexico City from January 2008 to December 2014, with diagnosis of adenocarcinoma by colonoscopy and biopsy were included in this cohort. Patients were treated according to standard guidelines. Follow-up continued until June 2016; the Kaplan-Meier method and the Cox model were used to analyze the association of prognostic factors and overall survival (OS).

Results: 1,259 cases of CC were treated; only 496 patients have stage I and II CC and were included in the database: 234 women (47.2%) and 262 males (52.8%) and their mean age was 59.9 years (SD 14.9); 96, 339, 31 and 30 cases were pTNM stages I, IIa, IIb and IIc, respectively. Multivariate analysis of prognostic factors is depicted below. Predicted survival functions using this model, defined 4 risk groups that showed better prognostic value than simple TNM stages I-IIc.

Conlusion: BSA is an independent, significant, simple, cheap and widely available prognostic factor in patients with CC. BSA and other clinical factors add significant prognostic information to TNM classification. Feasibly, it can be used to develop multivariate prognostic models with clinical impact defining the use of adjuvant chemotherapy. The use of histopathological and immunohistochemical markers could improve the prognostic information of this model.

Multivariate análisis of prognostic factors

---

|

Beta | SE | p | HR | 95% CI

|  | |  | |

Male gender | 0.355 | 0.166 | 0.032 | 1.426 | 1.031-1.973

Hemoglobin | -0.088 | 0.038 | 0.02 | 0.916 | 0.85-0.986

Platelet count | -0.002 | 0.001 | 0.001 | 0.998 | 0.996-0.999

Neutrophil count | 0.082 | 0.028 | 0.004 | 1.085 | 1.027-1.147

Albumin | 0.782 | 0.228 | 0.001 | 2.185 | 1.397-3.417

Albumin/Globulin | -1.624 | 0.488 | 0.001 | 0.197 | 0.076-0.513

BMI | -0.042 | 0.022 | 0.051 | 0.958 | 0.918-1.0

TNM 0/I | |  | 0.054 | 1

|

TNM IIa | 0.554 | 0.255 | 0.03 | 1.74 | 1.056-2.869

TNM Iib | 0.734 | 0.404 | 0.069 | 2.083 | 0.944-4.597

TNM IIc | 0.95 | 0.367 | 0.01 | 2.586 | 1.26-5.305

Chemotherapy | -0.765 | 0.2 | <0.0001 | 0.465 | 0.315-0.688

R0 | |  | <0.0001 | 1

|

R1 | 0.267 | 0.475 | 0.574 | 1.306 | 0.515-3.311

R2 | 0.936 | 0.348 | 0.007 | 2.551 | 1.29-5.044

No Resection | 1.936 | 0.288 | <0.0001 | 6.932 | 3.944-12.18

Platelet/Lymphocute ratio | 0.001 | 0.001 | 0.08 | 1.001 | 1.0-1.002

#673

Exploration of tissue morphologies in breast cancer samples using unsupervised machine learning.

Riku Turkki,1 Dmitrii Bychkov,1 Nina Linder,1 Jorma Isola,2 Heikki Joensuu,3 Johan Lundin1. 1 _Institute for Molecular Medicine Finland, Helsinki, Finland;_ 2 _BioMediTech, University of Tampere, Tampere, Finland;_ 3 _Translational Cancer Biology Research Program, Helsinki, Finland_.

We applied a machine learning approach for exploration of tissue morphology in hematoxylin and eosin (H&E) stained breast cancer tissue microarray (TMA) samples. We then investigated whether the morphological categories produced were associated with clinically relevant molecular biomarkers and 10-year overall survival.

The data set comprises digitized (0.22 µm/pixel) and H&E stained TMA spots from tumor samples of 490 women who were diagnosed with primary breast cancer within a Finnish breast cancer database (FinProg) collected in 1991 and 1992. In order to quantitatively describe the tissue morphologies of the TMA spots, we divided the tissue images into rectangular sub-images (224x224 pixels), and extracted features with a pre-trained convolutional neural network. We then clustered the sub-images (n=147,266) with a non-linear data embedding algorithm that creates a two-dimensional mapping of the tissue morphologies. Lastly, we defined a quantitative profile for each tumor, describing the morphologies within the tissue spot image by dividing the two-dimensional map of morphologies into 128 separate clusters with k-nearest neighbor clustering.

Visual inspection of the two-dimensional embedding of tissue spot images verified that the morphologies clustered coherently, i.e. similar looking sub-images formed distinct clusters in the map. Interestingly, some morphological patterns were strongly associated with tumor estrogen receptor content, progesterone receptor content, human epidermal growth factor receptor 2 status, and the proliferation marker Ki-67 status (p<0.0001 for each comparison). In exploratory analyses we identified one morphological category that was associated with a favorable 10-year overall survival with a risk ratio of 0.68 (CI95% 0.53-0.89, p=0.002, power = 0.87).

Our work demonstrates that unsupervised machine learning can be applied to explore and better understand the role of morphological patterns in breast cancer. Methods that quantitatively assess the morphology of cancer tissue may complement molecular biomarkers and potentially reveal novel prognostic and predictive factors.

#674

HPV16 was the preponderant type of HPV infection in cutaneous squamous cell carcinoma.

Gang Shi,1 Anne C. Fischer,2 Jonathan M. Zenilman,1 Patti E. Gravitt,3 Max Fischer,4 Inbal B. Sander,1 Jonathan D. Cuda,1 Janis M. Taube,1 Mohammed Lilo,1 W P Andrew Lee,1 Anthony P. Tufaro1. 1 _Johns Hopkins University School of Medicine, Baltimore, MD;_ 2 _Florida Atlantic University, Boca Raton, FL;_ 3 _Johns Hopkins Bloomberg School of Public Health, Baltimore, MD;_ 4 _University of Maryland School of Medicine, Baltimore, MD_.

Purpose: Human papillomavirus (HPV) has been well established as a causative factor in mucosal oropharyngeal cancer and anogenital surface malignancies. However, its etiological role in cutaneous squamous cell carcinoma (cSCC) is still debated. The aim of this study is to identify the presence of HPV types in cSCC and identify their associations with cSCC.

Methods: 77 cSCC blocks from 54 patients underwent DNA isolation and a broad spectrum of HPV PCR tests to detect the presence of HPV genotypes by the use of a universal primer pair GP5+/GP6+. The resultant PCR products were cloned to T Vectors and subsequently underwent DNA sequencing and Nucleotide BLAST search to identify the HPV types in cSCC. To ensure the accuracy of the HPV PCR test, 16 blocks from 10 head and neck SCC (oropharyngeal) patients were also included in the HPV presence and type analyses. Cutaneous SCC patients' demographics, pathology and clinical parameters were compared and analyzed statistically in conjunction with the HPV test results.

Results: HPV DNA was found in 37 of 77 (48.1%) blocks representing 32 of 54 (59.3%) cSCC patients. HPV16 was the preponderant type of HPV Infection, accounting for 28 of 32 (87.5%) and 30 of 37 (81.08%) HPV positive patients and blocks. Other HPV genotypes found included HPV18, 57, 10, 2, accounting for 4 of 32 (12.5%), 2 of 32 (6.25%), 2 of 32 (6.25%), 2 of 32 (6.25%) of the HPV positive patients and 4 of 37 (10.81%), 2 of 37 (5.41%), 2 of 37 (5.41%), 2 of 37 (5.41%) of the HPV positive blocks, respectively. HPV infection was significantly associated with the cSCC tumor size in this cohort of patients. Tumors with a size of ≤20mm were more frequently associated with HPV. Moreover, the average tumor size of the HPV positive group was also significantly smaller than that of the HPV negative group.

Conclusions: HPV infection is very commonly found in cSCC patients and HPV16 is the major type of HPV involved in cSCC oncogenesis, accounting for >80% of HPV infections. HPV is a contributing factor, which appears to be contributing to a smaller size of cSCC, which may improve the prognostic classification of patients to provide a new insight into tumorigenesis.

#675

The interaction between hedgehog and EMT pathway in non-small cell lung cancer and its prognostic implication.

Ho Jung An,1 Tae-Jung Kim,2 Young Jo Sa,2 Chang Suk Kang2. 1 _St. Vincent's Hospital, The Catholic University of Korea, Suwon, Republic of Korea;_ 2 _Yeouido St. Mary's Hospital, The Catholic University of Korea, Seoul, Republic of Korea_.

Introduction: The Hedgehog (Hh) signaling plays essential role in transcription regulation and embryonic development. In many cancers including lung cancer, it is related to carcinogenesis and poor prognosis. This signaling also mediates epithelial to mesenchymal transition (EMT) by paracrine mechanism in various diseases. We aim to study the interaction between Hedgehog and EMT pathway and its clinical implication in non-small cell lung cancer (NSCLC).

Method: Total 271 cases of NSCLC who received surgical resection in Yeouido St. Mary's Hospital between 1997 and 2011 were included. For immunohistochemical analysis of Hh and EMT related proteins, 4um sections were sliced from tissue microarray. Hh signaling proteins including Sonig hedgehog (Shh), Patched (Ptch), Smoothened (Smo), Glioma-associated oncogene (Gli-1) and Suppressor of fused (Sufu), and EMT associated molecules including E-cadherin and vimentin were analyzed. Staining intensity (IS) was estimated as follows 0 (no staining), +1 (weak), +2 (distinct), +3 (strong). Intensity score (IS) ≥1 was defined as positive. Medical record was retrospectively reviewed. The study was approved by the institutional review board the hospital.

Result: The median age was 63 (18-84) years, and 65.3% were male. Squamous cell carcinoma was diagnosed in 113 (41.7%), adenocarcinoma in 153 (56.5%), and mixed type in 153 (56.5%). Stage 1 and 2 were 46.9% and 30.6%, respectively, and 8 cases had distant metastasis. The Hh proteins showed positive correlation each other. For EMT, the expressions of Hh protein was significantly correlated with tumoral and stromal positivity of vimentin, and inversely with positivity of E-cadherin (P = 0.002 and P = 0.028, respectively). The expression of Hh proteins were significantly associated with lymph node metastasis along with large tumor size (P = 0.021 and P = 0.035, respectively). In survival analysis, expressions of Shh and Gli1 were independent predictor of disease free survival and cancer-specific survival (P = 0.007 and P = 0.025, respectively).

Conclusion: Expression of Hh protein is associated with epithelial-mesenchymal transition and associated with poor prognosis in NSCLC.

#676

Nuclear expression of insulin receptor (InsR) adds prognostic information in primary breast cancer patients with high BMI.

Sofie Björner, Ann H. Rosendahl, Maria Simonsson, Andrea Markkula, Karin Jirström, Signe Borgquist, Carsten Rose, Christian Ingvar, Helena Jernström. _Lund University, Lund, Sweden_.

Aim and introduction The aim of this study was to elucidate the prognostic importance of nuclear expression of the insulin receptor (nucInsR) in primary breast cancer. The InsR is important for the metabolism, for the insulin-like growth factor (IGF) signaling network and is implicated in breast cancer. Insulin and InsR are interlinked with obesity and body mass index (BMI), which are also associated with breast cancer. The InsR is present both in the membrane and cytoplasm and can additionally translocate to the nucleus. The role of nucInsR is not well understood and the prognostic importance is unclear.

Methods The tumor-specific expression of nucInsR was evaluated by immunohistochemistry on invasive breast cancer tissue microarrays from 984 primary breast cancer patients included in a population-based cohort in Sweden. Patients were followed for up to 11 years, the median follow-up for patients still at risk was five years. nucInsR in relation to prognosis was analyzed using Cox regression, adjusted for age, invasive tumor size, axillary lymph node involvement, histological grade, estrogen receptor (ER) status, BMI, tumor storage time, and treatment.

Results Status for nucInsR was available for 900 patients (91.5%). nucInsRpos staining was present in 214 patients (23.8%) and was associated with lower age (P=0.004), and lower waist-to-hip ratio (P=0.006), but not with BMI, ER or PR status. During follow-up, 107 patients had a first breast cancer event. nucInsR had no prognostic significant impact among all patients. However, there was a significant effect modification of BMI on nucInsR regarding event-free survival (Pinteraction=0.006). Patients with nucInsRpos-expressing tumors and higher BMI (≥25 kg/m2) had lower risk for any breast cancer event, adjusted HR (0.49; 95% CI 0.26-0.93), while patients with nucInsRpos-expressing tumors and normal BMI (<25 kg/m2) were associated with a non-significant higher risk of events, adjusted HR (1.72; 95% CI 0.93-3.17).

Conclusions These results indicate that nucInsR status may add prognostic information in primary breast cancer depending on BMI.

#677

Progranulin/GP88 tumor tissue expression further stratifies survival outcomes of patients classified by their Nottingham Prognostic Index.

Ginette Serrero,1 Douglas Hawkins,2 Olga Ioffe,3 Pablo Bejarano,4 Binbin Yue1. 1 _A &G Pharmaceutical, Inc., Columbia, MD; _2 _University of Minnesota, Minneapolis, MN;_ 3 _University of Maryland Greenebaum Comprehensive Cancer Center, Baltimore, MD;_ 4 _Cleveland Clinic Florida, Weston, FL_.

Background: The Nottingham Prognostic Index (NPI) combines nodal status, tumor size and histological grade and is used to provide predictive value information on survival for patients with primary breast cancer. Attempts to improve the performance of NPI have been carried out by inclusion of other factors, such as biomarker expression as well as addition of other structural features such as vascular invasion. In the present study, we investigated whether expression of the survival factor GP88 (progranulin), known to be overexpressed in breast cancer, would improve NPI's predictive value. GP88/Progranulin is an 88 kDa autocrine growth and survival factor that is overexpressed in estrogen receptor positive (ER+) invasive ductal carcinoma (IDC) in association with resistance to anti-estrogens and aromatase inhibitor while the cells remain ER+. Previous training and validation pathological studies totaling 600 cases of ER+ IDC had demonstrated that GP88/Progranulin tissue expression was an independent prognostic marker od recurrence and mortality for early stage ER+ IDC.

Methods: We examined by immunohistochemistry the GP88 tumor tissue expression of 508 formalin fixed paraffin embedded cases of ER+ IDC with known clinical outcomes and for which NPI had been determined. GP88 IHC expression was scored by two board certified pathologists and classified into two score groups of GP88 < 3+ (0, 1+, 2+) and GP88 = 3+. The correlation between GP88 scoring, NPI and disease-free (DFS) and overall survival outcomes (OS) was then examined by Kaplan Meier analysis, Cox proportional Hazard ratio and Pearson's Χ2 test.

Results: Patients were categorized into three NPI groups (< 3.4, 3.4-5.4, >5.4). Kaplan-Meier survival graphs of cases categorized by their NPI scores and GP88 expression showed that for each NPI subgroup, patients having tumors with a high GP88 expression (GP88 IHC score of 3+) had a worse DFS than patients within the same NPI subgroup with low GP88 expression (GP88 IHC score < 3+). When adjusted for NPI, high GP88 score was highly significantly associated with recurrence. Its hazard ratio is 3.30 (95% CI 2.12 to 5.14).

Conclusions: These data suggest that the determination GP88 tumor expression at time of diagnosis for early stage breast cancer patients could provide additional survival information than the ones provided by NPI alone that maybe useful for improving the risk management of patients.

#678

A strategy to overcome the interfering problem of tissue embedding OCT compound in activity based multiplex profiling of tyrosine kinase substrates.

Stephan Arni, Claudio Caviezel, Walter Weder, Sven Hillinger. _University Hospital Zürich, Zürich, Switzerland_.

Introduction: The analysis of clinically relevant human tissue preserved in optimal cutting temperature (OCT) medium with activity based proteomic approaches are promising for the discovery of novel druggable disease biomarkers for the diagnosis, prognosis and prediction of response to therapeutic interventions. Nonetheless, and for many different proteomic approaches, there are important signal interferences observed in the presence of the OCT compound.

Methods: We tested activity based multiplex profiling tyrosine kinase substrates in a large batch of neoplastic and non-neoplastic lung resection specimen embedded with or without OCT. We observed significant differences in overall phosphorylation levels and searched for reasons explaining such a large effect.

Results: We ruled out the implication of either short versus long storage time after sample extraction or of nonhomogeneous batch processing of samples. We documented that the clear downward shift in overall phosphorylation levels coincided with the introduction of OCT as an improved embedding medium for resection specimen. For all the kinomes extracted, we developed a corrective procedure where a median centering was performed on the values of each peptide, separately for the with or without OCT samples.

Conclusions: We applied corrective filtering of data to the multiplex profiling approach of well characterised tyrosine kinase substrates obtained in lung resection specimen embedded with or without OCT. With the OCT correction parameters applied, the quantitation of molecular prognosis signature based on tyrosine kinase activity differences found in lung adenocarcinoma resection specimens may result in the identification of novel targets for future anti-lung cancer therapies.

#679

Serine/threonine kinase inhibitors on improving symptoms of neuroendocrine tumor.

I-Hua Liu, Yusian Ding, Chiung-Wen Liou, Jia-Ming Chang. _Development Center for Biotechnology, New Taipei City, Taiwan_.

Recently, neuroendocrine tumors (NETs) are increased and mostly occurred in gastrointestinal tract and lung in the worldwide. It secretes functional hormones and non-functional neural transmitters to affect patients unwell so called carcinoid syndrome. In clinically, surgical removal and somatostatin treatment are used to ease the syndrome by NETs. A new strategy for advanced NETs is used by targeting mTOR signaling and neovascularization. In 2011, Everolimus, an mTOR inhibitor, was approved for the treatment of progressive NETs of pancreatic origin in patients with unresectable, locally advanced or metastatic disease, but safety and effectiveness of everolimus have not been established. In this study, low dose of mTOR inhibitor was investigated for capability in modifying the carcinoid syndrome. In an NCI-H727 NET xenograft model, the level of chromogranin A (CgA) was determined in accordance with tumor volume. With multiplexing the serum hormones, alpha-melanocyte-stimulating hormone (alpha-MSH) and beta-endorphin were lowered in tumor-bearing mice, but they were rebound when animals treated with Everolimus. Another mTOR inhibitor INK 128 had no apparent tumor inhibition in the same model with doses ranged from 0.25-1 mg/kg. There was no change in the level of CgA in INK128-treated tumor-bearing mice but alpha-MSH level was restored compared with normal mice at a dose-dependent manner. In conclusion, we discovered that low dose of mTOR inhibitor could modify the carcinoid syndrome, where alpha-MSH and beta-endorphin may serve as biomarkers for prognoses of mTOR treatment.

#680

Prognostic impact of OPN and DKK1 in patient of hepatocellular carcinoma after hepatectomy.

YunSung Seo, Hye Rim Byeon, In Hae Park, Seung Duk Lee. _National Cancer Center, Goyang-si, Gyeonggi-do, Republic of Korea_.

The new biomarkers are essential for improving the survival and prognosis of hepatocellular carcinoma(HCC) patients. Alpha-fetoprotein(AFP) is the most widely used biomarker. But the low sensitivity and specificity limits its clinical application. Recent study validated the diagnostic capability of osteopontin(OPN) and dickkopf-1(DKK1) and assessed the combination of AFP, DKK1, and OPN as a panel for the diagnosis of HCC. Based on these previous studies, we hypothesized that combination of OPN and DKK1 can be used to as a marker for prognosis to patient of HCC after hepatectomy. From January 2006 to December 2008, patients undergoing hepatectomy for hepatocellular carcinoma were screened serum that has been stored in the Bank of tumor. To target the selected patient, it was confirmed that the remaining tissue specimen is stored after diagnosis. Serum of the patients was used to investigate the OPN and DKK1 by ELISA. In the paraffin block were prepared unstained slide and OPN and DKK1 level checked by IHC. It examined the correlation between prognosis and biomarkers through statistical analysis. AFP, OPN(serum level) and DKK1(serum level) are an independent prognostic factor for overall survival(OS) in HCC after hepatectomy(n=60, P=0.0204, 0.0167 and 0.0455 respectively). New biomarkers combinations based on the AFP existing biomarker are showed a falling curve of the overall survival(OS) and disease-free survival(DFS) in Kaplan Meier curve. In conclusion, combination of OPN, DKK1 and AFP as a biomarker could support the correct diagnosis for HCC after hepatectomy.

#681

Methylomics profiling reveals epigenetically silenced HS3ST2 sensitize IL6 signaling and confers poor prognosis of ovarian cancer.

Rui-Lan Huang,1 Hsiang-Ju Chan,2 Lin-Yu Chen,3 Yaw-Wen Hsu,3 Tai-Kuang Chao,4 Po-Hsuan Su,1 Yu-Chun Weng,1 Chiou-Chung Yuan,1 Hung-Cheng Lai1. 1 _Taipei Medical University-Shuang Ho Hospital, New Taipei, Taiwan;_ 2 _Taiwan International Graduate Program Molecular and Cell Biology, Ph.D. Program, Academia Sinica, New Taipei, Taiwan;_ 3 _Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan;_ 4 _Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan_.

The attempts of target therapy in ovarian cancer therapy mostly fail. There is a need of biomarkers for better patient stratification in clinical trials. Epigenetics is a driving force for cancer development and may serve as a molecular marker of disease. The present study was to analyze the clinical relevance of methylation phenotypes and figure out the mechanism in ovarian cancer. We integrated the analysis using public dataset of epigenomics and transcriptomics, and our hospital-based methylomic database and tissue arrays of patients diagnosed with epithelial ovarian cancers (EOC). Bioinformatics results were verified and validated in cell lines and clinical samples. From 25 genes, we identified even genes, ADRA1A, CD248, HS3ST2, NEFH, IGSF21, POU4F2, and TWIST1, of which hypermethylation was significantly related to the poor overall survival (OS). The association of NEFH and HS3ST2 hypermethylation and poor 5-year OS was validated in our independent dataset and TCGA database. The NEFH/HS3ST2 hypermethylated signature was an independent risk factor for 5-year OS (adjusted Hazard ratio = 2.93, 95% confidence interval = 1.25 to 6.85, P = 0.013). In vitro experiment re-expressing HS3ST2 repressed migration and invasion, and suppressed IL-6 signaling pathways. The combination of hypermethylation of HS3ST2 and high expression of IL-6 confers the worse outcome. Our data suggested that methylation of NEFH/HS3ST2 constituted a poor prognostic signature of ovarian cancer. The sulfation status of proteoglycan modified by HS3ST2 implicates an intrinsic sensitivity of ovarian cancer cells to IL-6, which shed a new light on the application of HS3ST2 as a biomarker for personalized medicine of targeting IL-6.

#682

Novel Mesenchyme Homeobox2-target transcription axes are involved in cancer-drug resistance, overall survival and therapy prognosis in lung cancer patients: a functional epigenome wide study.

Federico Avila Moreno,1 Leonel Armas lopez,1 Patricia Piña-Sánchez,2 Oscar Arrieta,3 Enrique Gúzman de Alba,4 Abril Marcela Herrera-Solorio,1 Blanca Ortiz-Quintero,4 Patricio Santillán-Doherty,4 David C Christiani,5 Joaquín Zúñiga4. 1 _Universidad Nacional Autónoma de México (UNAM), Mexico City, Mexico;_ 2 _Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico;_ 3 _Instituto Nacional de Cancerología (INCAN), Mexico City, Mexico;_ 4 _Instituto Nacional de Enfermedades Respiratorias (INER) Ismael Cosío Villegas., Mexico City, Mexico;_ 5 _Harvard Medical School, Boston, MA_.

Lung cancer remains the most progressive malignant disease strongly resistant to oncological therapies including platinum-derived cancer drugs and Epidermal Growth Factor Receptor (EGFR)-Tyrosine Kinases Inhibitors (TKIs). Homeobox-related gene (HOX) transcription factors as Mesenchyme HOX-2 (MEOX2) have previously been associated with cancer-drug resistance, progression and/or clinical prognosis in lung cancer patients. However, transcriptional mechanisms epigenetically modulated have not totally been elucidated in lung cancer therapy resistance.

Here an epigenomic strategy was conducted to identify a novel MEOX2 gene sequence-promoter targets profile, associated or involved in therapy resistance mechanisms in human lung cancer. For that chromatin from human non-small cell lung carcinomas (NSCLC), MEOX2 versus RNA Pol II immunoprecipitation and hybridization assays using gene promoter tiling-arrays and bioinformatics analyses were performed, while a set of quantitative and functional assays with clinical-outcome prognosis validation predictions analyses, were assessed.

Stringent bioinformatics results identified a common profile of 13 gene promoter sequences, which included in others Sonic Hedgehog-GLI-1, ALDH1A2, MMP24, RUFY3 and ZEB1 (FDR≤0.1) in NSCLC patients with different clinical outcome data. GLI-1 gene promoter-sequences upstream -2,192 to -109 quantitatively validated, were occupied by MEOX2 and RNA Pol II in both NSCLC cell lines and NSCLC patients, as well as consistently enriched with the histone activation marks H3K27Ac and H3K4me3, in addition, confirmed by the ENCODE database bioinformatics analyses. Furthermore, a set of genetic silencing functional assays validated a novel transcriptional MEOX2-GLI1 axis in a cisplatinum dose-dependent manner, involved in cellular migration, invasion, and proliferation capacity. Finally, MEOX2-GLI1 axis expression was clinically validated and analyzed using Kaplan-Maier survival analyses on an independent cohort of 90 NSCLC patients, identifying a significant MEOX2-dependent GLI-1 overexpression statistically associated with clinical poorer overall survival prognosis and treatment response into cisplatinum-based first-line therapy and/or second-line EGFR-TKIs target therapy protocols.

In conclusion, a chromatin-immunoprecipitation and epigenome-wide analysis based on the MEOX2-transcriptional occupation study, identified novel MEOX2-transcriptional gene promoter axes involved in embryonic development, oxidoreductase activity, matrix metalloprotease, cellular polarity, epithelial-mesenchyme phenotype, cancer cell migration and invasion pathways. Promoting cisplatinum-based resistance mechanisms and EGFR-TKIs based therapy response prognosis in human lung cancer.

#683

Quantification of rare PD-L1 expressing leukocytes and CTCs in peripheral blood of cancer patients.

Adam Jendrisak, Jiyun Byun, Mahipal Suraneni, David Lu, Rachel Krupa, Sarah Orr, Ryon Graf, Yipeng Wang, Mark Landers, Ryan Dittamore. _Epic Sciences, San Diego, CA_.

Background: Expression of PD-L1 on tumor tissue is associated with improved response to PD-1 and PD-L1 checkpoint inhibitors. Additionally, expression of PD-L1 on infiltrating T cells is associated with immune exhaustion. Currently, PD-L1 analysis as well as measures of infiltrating T cells are examined in surgically removed or biopsied samples, usually taken long before clinical decision points. Additionally, given tumor heterogeneity, metastatic lesions are likely to be under-sampled. We sought to examine expression of PD-L1 on circulating tumor cells (CTCs) and leukocytes cell populations with a non-invasive liquid biopsy. Examining dynamic biomarker changes in longitudinal samples could enable the development of novel diagnostic tools for response prediction and pharmacodynamics studies related to immunotherapy.

Methods: Blood samples from cancer patients were received and shipped to Epic Sciences. Contrived blood samples were also developed utilizing spike-in of cancer cell line controls into healthy blood samples. RBCs were lysed, and nucleated cells plated onto glass slides utilizing the Epic Sciences process. Slides are stained with an IF cocktail (CK, DAPI, CD45, PD-L1) and scanned. Approximately 3 million nucleated cells are examined through advanced Digital Pathology pipelines to detect rare CTCs (CK+/-, cancer morphology, CD45-) and leukocytes (CK-, CD45+, leukocyte morphology) and analyze for PD-L1 expression.

Results: A limit of detection of 1 cell/mL of blood was analytically validated. Using Epic's rare cell detection platform, we could detect a wide range of PD-L1+ leukocyte counts in a subset of peripheral blood of cancer patients, observed as low as 0.003% (110 of 3,192,263) of total leukocytes. PD-L1+ CTCs were observed in the presence of both high and low counts of PD-L1+ leukocyte populations.

Conclusions: Epic Sciences CTC platform's low limit of detection coupled with ability to archive patient blood samples allowed precise quantification of PD-L1 expression on CTCs and leucocytes retrospectively. In addition, we have previously demonstrated the platform's ability to detect leucocyte subtypes such as CD3, CD4 and CD8 cells, which will allow us to further characterize PD-L1 expression in T-lymphocytes and other immune cell types. Development of a liquid biopsy based platform that is capable of simultaneously measuring immune biomarkers in CTCs as well as leucocytes will allow real time assessment of response to immune checkpoints inhibitors.

#683A

Mechanism of liver metastasis induced systemic suppression of checkpoint inhibitor response.

James C. Lee, Adil Daud, Jeff Bluestone. _UCSF, San Francisco, CA_.

Introduction: In stage IV melanoma and NSCLC, PD-1 checkpoint inhibitor therapy is effective and now FDA approved as first-line therapy. However, despite these rationally designed strategies to manipulate immune cells, curative responses remain rare, and the majority of metastatic cancer patients ultimately succumb to their disease. Emerging potential biomarkers of response have required specialized technologies such as next-generation sequencing or flow cytometry, which are difficult to access, costly, and not immediately clinically applicable. To this end, we have previously reported that presence of liver metastasis was significantly associated with the lack of antitumor immune infiltrates at distant biopsy sites as well as poor clinical response to PD-1 blockade. Here, we sought to study the mechanism of liver metastasis related non-response to PD-1 blockade using a syngeneic immunocompetent murine tumor model.

Methods: C57BL/6 mice were implanted with a "primary" subcutaneous tumor as well as a secondary "metastatic" tumor in the liver or the lungs (control), and given systemic PD-1 blockade therapy to mimic the clinical observation and study the effects of liver tumor involvement on systemic antitumor immunity. Animals are monitored for survival and tumor growth. The immune infiltrates of both the primary and metastatic sites are phenotypically analyzed.

Results: Animals with liver tumor involvement appear to have significantly shortened survival as well as difficulty rejecting tumors, both at the primary and secondary site of implantation, when compared to non-liver involved controls. The lack of response to PD-1 blockade in the liver cohort is correlated with a significant decrease in CTLA4+/PD-1+/CD8+ "exhausted" antigen experienced T cells (TEx) within the tumor microenvironment of the subcutaneous and liver-embedded sites. The deleterious effect on TEx appears to be liver-specific and not tumor cell-line dependent, as delivery of different tumor cells to the liver reproduces the same effect while delivery of tumor cells to other organs does not change the quantity of TEx at the subcutaneous site. Lastly, liver associated deletion of TEx appears to be a an antigen dependent process, as delivery of mismatched tumor cell line to the liver abrogates this effect and restores TEx within the tumor microenvironment at the distant subcutaneous site.

Conclusions: Using an immunocompetent checkpoint blockade animal tumor model, we recapitulated the clinically observed difference in response to PD-1 blockade treatment that is associated with liver metastasis. Our studies suggest that the liver is capable of suppressing effective T cell mediated antitumor response systemically, rendering checkpoint blockade less effective. The findings here propose a novel mechanism for which non-responsiveness to PD-1 blockade therapy could occur and may impact the treatment decision for cancer patients with liver metastasis.

### Childhood Cancer Clinical Translational Research

#684

Integrative proteomics and transcriptomics to define the cell surface landscape of neuroblastoma.

Amber K. Weiner,1 Simone Sidoli,1 Tina Glisovic-Aplenc,2 John M. Maris,2 Benjamin A. Garcia,1 Sharon J. Diskin2. 1 _Univ. of Pennsylvania School of Medicine, Philadelphia, PA;_ 2 _Children's Hospital of Philadelphia, Philadelphia, PA_.

BACKGROUND. Neuroblastoma (NB) is an embryonal tumor of the sympathetic nervous system that accounts for 12% of childhood cancer deaths. Despite intense multimodal therapy, survival probability for high-risk NB patients remains below 50% and relapsed NB is essentially incurable. To date, the cell surface landscape of NB at diagnosis and relapse is currently unknown. An unbiased survey of these proteins and their isoforms will greatly facilitate the identification of candidate immunotherapeutic targets for preclinical validation.

METHODS. To identify proteins on the cell surface of NB, we performed plasma membrane protein extraction utilizing an optimized sucrose density gradient methodology followed by nano-liquid chromatography coupled to mass spectrometry (nLC-MS/MS) in NB cell lines. Proteomic data is integrated with RNA-sequencing data to assess the correlation between these data types, determine differential expression between NB (n=2242) and normal tissues (n=7859), and investigate NB-specific isoforms. In parallel, we have developed a software tool to aid in proteomic data analysis and interpretation.

RESULTS. To date, we have applied our proteomic approach to nine NB cell lines stratified by MYCN amplification. This methodology has yielded on average 66% (range: 60-68%) membrane protein enrichment with high reproducibility between biological replicates (80%; range: 78-84% ). This has allowed us to confirm known cell surface proteins that are currently being developed as immunotherapeutic targets (ALK, GPC2 and NCAM1). We are currently integrating the mass spectrometry based proteomics data, where we identified 4826 membrane proteins, with RNA-sequencing data to evaluate proteins expressed uniquely in neuroblastoma and those that correlate with MYCN amplification. We are further determining how RNA and protein levels correlate on the cell surface and will utilize these data to detect NB-specific isoforms. In parallel, we have also created GiaPronto, an efficient tool for data analysis and visualization. After normalization, our program outputs several plots and figure legends that assess data quality, regulated or abundant proteins, and Gene Ontology.

CONCLUSION. We have developed robust methodology for cell surface protein isolation and quantification along with a software package to facilitate downstream analyses. Parallel proteomic and transcriptomic studies in patient derived xenografts (PDX) and patient tumors are ongoing to define the cell surface landscape in both diagnostic and relapsed NB.

#685

GPC2 is an oncogene and immunotherapeutic target in high-risk neuroblastoma.

Kristopher R. Bosse,1 Pichai Raman,1 Maria Lane,1 Robyn T. Sussman,1 Jo Lynne Harenza,1 Daniel Martinez,1 Sabine Heitzeneder,2 Zhongyu Zhu,3 Komal Rathi,1 Michael Randall,1 Laura Donovan,4 Sorana Morrissy,4 Doncho V. Zhelev,3 Yang Feng,3 Jennifer Hwang,3 Yanping Wang,3 Bruce Pawel,1 Tricia Bhatti,1 Mariarita Santi,1 Javed Khan,3 Michael Taylor,4 Dimiter S. Dimitrov,3 Crystal Mackall,2 John M. Maris1. 1 _Children's Hospital of Philadelphia, Philadelphia, PA;_ 2 _Stanford University, Stanford, CA;_ 3 _National Cancer Institute, MD;_ 4 _Hospital for Sick Children, Toronto, Ontario, Canada_.

Background: GD2-directed immunotherapeutic strategies have improved outcomes in neuroblastoma; however, the majority of patients treated suffer relapse and GD2 expression on pain fibers causes dose-limiting toxicities.

Methods: To identify alternative cell surface immunotherapeutic targets, we compared high-risk neuroblastoma (n=126 tumors) and normal tissue RNA sequencing data (GTEx; n=7859 samples from 31 normal tissues) and prioritized genes by differential and absolute expression and cell surface prediction. Genes were further surveyed for somatic copy number gain and correlative expression with MYCN amplification. Differential protein expression and localization were confirmed in neuroblastoma primary tumors (n=98), patient-derived xenografts (n=32; PDXs), cell lines (n=23), and normal pediatric tissues (n=36). Cell lines were subjected to candidate gene gain and loss of function studies (n=11). Additional pediatric tumor RNA sequencing data was surveyed followed by confirmatory immunohistochemistry (IHC). Finally, candidate specific antibodies were isolated from a human Fab phage library and utilized for antibody-drug conjugate (ADC) engineering followed by cytotoxicity studies.

Results: We identified 33 differentially expressed cell surface molecules from which we prioritized glypican-2 (GPC2) for validation given GPC2's robust differential expression (log-fold change tumor vs. normal tissue = 1.71-9.22; p=1.99 x 10-9-1.88 x10-300), high-level absolute RNA expression (median FPKM=60), and frequent DNA copy number gain associated with higher GPC2 expression (35%, n=182 tumors; p<0.005). GPC2 expression was also higher in MYCN amplified neuroblastomas (p<0.05), MYCN binds the GPC2 promoter shown by chromatin immunoprecipitation (ChIP) sequencing and reporter assays, and MYCN depletion resulted in decreased GPC2 expression. Immunoblot, flow cytometry, immunofluorescence, and IHC analysis of primary tumors, PDXs, and cell lines confirmed dense cell surface GPC2 expression. Medulloblastomas (n=62) were also found to have high GPC2 expression that positively correlated with MYC, MYCN, and GPC2 loci gain (p<0.0001). Pediatric normal tissues had very restricted cell surface GPC2 expression, with only low levels found in the esophagus and skin. GPC2 depletion in neuroblastoma cell lines resulted in apoptosis and growth inhibition and GPC2 forced over-expression increased neuroblastoma cell proliferation (p<0.001 for all assays). Finally, a human GPC2 antibody, D3-GPC2-Fab, was developed and shown to bind GPC2 with high affinity and specificity. D3-GPC2-IgG1 induced internalization of GPC2 and was conjugated to pyrrolobenzodiazepine (PBD) dimers to form an ADC which induced potent and specific cytotoxicity to GPC2 expressing neuroblastoma cells (IC50 = 1.7-11 pM).

Conclusions: GPC2 is an oncogene and immunotherapeutic target in neuroblastoma and potentially other cancers.

#686

**onTARGET: a new computational strategy to predict** in vivo **relevant targets against neuroblastoma.**

Elin Almstedt, Ingrid Lönnstedt, Cecilia Krona, Sven Nelander. _Uppsala University, Uppsala, Sweden_.

One of the main bottlenecks of anticancer drug development is the assessment of the in vivo relevance of emerging therapies. Previously, drugs that suppress the in vivo progression of neuroblastoma have been hard to identify. To address this problem, we propose a new computational technique, onTARGET, that enables researchers to select, with good accuracy, compounds that are likely to induce changes in cellular pathways that are consistent with specific clinical outcomes and subgroups.

onTARGET prediction is based on an integration of massive expression and other data resources, including the NIH LINCS dataset (with 1,300,000 expression profiles), the NIH TARGET and R2-AMC neuroblastoma cohorts, and our own in-house databases. In a first step, the datasets are preprocessed by factor analysis to remove systematic bias in these large data sets. In a second step, Bayesian statistics are applied to prioritize compounds whose cellular responses match clinically relevant differences between patient subsets, such as MYCN status, survival hazard ratio, or histopathological assessment.

To evaluate the method, we used onTARGET prediction to identify compounds and gene targets relevant for clinical outcomes in the NIH-TARGET (n=249) and R2-AMC (n=98) neuroblastoma cohorts. The algorithm confirmed existing targets and made interesting predictions. Of 50 the top-ranking compounds associated with a survival outcome, agents targeting the mTOR/PI3K axis are highly prevalent. The algorithm correctly predicts targeting of the MYC pathway, and predicts possible modulators of MYC signaling, such as TRRAP, AURKA and BMP2. In addition to nominating agents with a favorable outcome, the algorithm can also flag compounds that might worsen prognosis, further facilitating the prioritization of targets for evaluation.

In summary, onTARGET may guide target selection for neuroblastoma studies and can be adapted for other cancers as well. The many targets that were identified immediately suggest an opportunity for continued evaluation in cells and in vivo models. This work has been initiated in collaboration with researchers at Lund University.

#687

Targeting tumor-promoting neuroblastoma microenvironment: inhibiton of tumor development and progression by targeting mPGES-1 expressed by cancer associated fibroblasts.

Anna Kock,1 Karin Larsson,2 Nina Eissler,1 Filip Bergqvist,2 Joan Rauf,2 Marina Korotkova,2 John-Inge Johnsen,1 Per-Johan Jakobsson,2 Per Kogner1. 1 _Women's and Children's Health, Stockholm, Sweden;_ 2 _Medicin, Stockholm, Sweden_.

Background: High-risk neuroblastomas present a tumor promoting microenvironment with infiltrating cancer associated fibroblasts (CAFs) expressing the mPGES-1 enzyme, essential for prostaglandin E2 (PGE2) synthesis regulating tumor inflammation and immune suppression, angiogenesis, genetic instability, tumor progression and therapy resistance. We investigated the impact of novel therapy targeting the COX/mPGES-1/PGE2 pathway.

Methods: Human neuroblastomas were investigated for immunosuppressive microenvironment and expression of the COX/mPGES-1/PGE2/EP-receptor pathway. High-risk in vivo models, human 11q-deleted xenografts and transgenic MYCN-driven tumors, were treated with a novel specific mPGES-1 inhibitor. Tumor-fibroblast co-cultures examined cell migration. Inflammatory lipid mediators were analyzed by LC-MS/MS. Tumor tissues were analyzed by immunohistochemistry, immunofluorescence and FACS.

Results: Tumor microenvironment in human high-risk neuroblastomas and both 11q-deleted xenografts and MYCN-driven transgenic mice displayed mPGES-1 expression in PDGFRb+ cancer associated fibroblasts. MPGES-1 expression correlated with high-risk neuroblastoma prognosis and infiltration of tumor-promoting macrophages with M2-polarization markers CD163 and CD206. The inflammatory regulator STAT3 was active in mPGES-1 expressing CAFs. Expression of the inflammatory COX/mPGES-1/PGE2/EP-receptor pathway in experimental tumors resembled high-risk primary human neuroblastomas. Targeting mPGES-1 with a novel compound decreased PGE2, induced M1 polarization of macrophages, decreased cancer associated fibroblasts and reduced angiogenesis significantly in treated tumors. Tumor development in the xenograft model was delayed and growth of established xenografts and transgenic tumors was significantly decreased by non-toxic treatment in vivo when compared to neuroblastoma tumors in untreated animals. Tumor cell stimulated CAF migration and infiltration was inhibited by targeting mPGES-1.

Conclusions: Tumor-promoting inflammation and suppression of anti-tumor immunity in neuroblastoma is mediated through prostaglandin E2 and STAT3 expression in cancer associated fibroblasts in the tumor microenvironment. Early targeting of mPGES-1 may inhibit CAF infiltration and tumor development. This novel tumor treatment targeting mPGES-1 decreases inflammatory mediators, modulates tumor-promoting microenvironment and inhibits significantly aggressive tumor growth and progression. We conclude that treatment targeting non-malignant cells in the neuroblastoma microenvironment may constitute a novel clinical therapeutic approach.

#688

**Meta-[** 211 **At]astatobenzylguanidine ([** 211 **At]MABG) is a potent alpha particle emitting systemic targeted radiotherapeutic in preclinical models of neuroblastoma.**

Vandana Batra,1 Jimmy Elias,1 Mehran Makvandi,2 Matthew Tsang,1 Pietro Ranieri,1 Catherine Hou,2 Yimei Li,1 Daniel A. Pryma,2 John M. Maris1. 1 _Children's Hospital of Philadelphia, Philadelphia, PA;_ 2 _University Of Pennsylvania, Philadelphia, PA_.

BACKGROUND The alpha particle emitting radiotherapeutic [211At]MABG theoretically has superior radiobiological properties for anti-tumor efficacy compared to the currently used agent ([131I]MIBG). Specifically, [131I]MIBG does not target microscopic deposits due to the long path length of the beta particles, while alpha particles have both a short path length and higher linear energy transfer to induce clustered double strand breaks. Here we sought to define the anti-tumor activity of [211At]MABG in preclinical models of human neuroblastoma (NB).

METHODS 211At was synthesized using a bismuth target via the 209Bi(α,2n)211At reaction and used for solid-phase radiosynthesis of [211At]MABG (Ultratrace, Progenics, NY). We determined NET (SLC6A2) mRNA and protein expression in 35 NB cell lines and created isogenic pairs by overexpression of NET in 5 NB cell models. We performed uptake, cytotoxicity and biodistribution studies using these models with [131I]MIBG and [211At]MABG and extrapolated human dosimetry. Additionally, dose escalation studies with [211At]MABG (n=10 at each dose, range 10-100 uCi) were performed to determine toxicity in SCID mice. Therapeutic in vivo trials were conducted with NET transfected xenografts and patient derived xenograft (PDX) models injected intravenously with escalating doses of [211At]MABG, [131I]MIBG or vehicle (n=10 respectively).

RESULTS We now routinely synthesize [211At]MABG with a radiochemical yield of 50-70% and radiochemical purity >99%. NET-overexpressing cell lines showed 4-10 fold higher uptake of NET ligands than parental isogenic lines, and tumor-specific [211At]MABG uptake (tumor-muscle ratios of 7.37). Estimated dosimetry confirmed the potential to deliver therapeutic doses to tumors. Intravenous [211At]MABG was well tolerated in murine models at doses of 10 and 25 µCi except for transient thrombocytopenia (nadir at 6 weeks; p=0.001 and p=0.0005 respectively) while doses higher than 50 uCi caused significant weight loss. [211At]MABG was a potent cytotoxic agent in vitro (EC50's ranged from 0.0006-0.1 uCi/ml compared to 0.25-46 uCi/ml with [131I]MIBG). Single dose therapeutic trials showed significant regression of established NET overexpressing SKNSH xenograft and COG-N-453x PDX models (p<0.0001) sustained for a median of 14.5 days, comparable to single dose [131I]MIBG therapy and prolonged survival was noted in mice receiving [211At]MABG.

CONCLUSIONS The biodistribution and uptake of [211At]MABG is similar to [131I]MIBG and there was no unanticipated toxicity. [211At]MABG is more potent than [131I]MIBG in vitro and the two agents showed similar activity in bulky xenograft models, remarkable due to the several log difference in half-life (7.2 hours for [211At]MABG, 8.04 days for [131I]MIBG). Additional studies exploring fractionated dosing of [211At]MABG are ongoing and will be reported.

#689

Immune checkpoint targeting to improve immunotherapy in neuroblastoma.

Myrna L. Ortiz-Ruiz. _H. Lee Moffitt Cancer Ctr. & Res. Inst., Tampa, FL_.

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Current treatments accounts only for at the most 40% of survival rate in high-risk NB patients. Hence, there is a need to develop new strategies that disrupt immunosuppressive signals in the tumor microenvironment in order to achieve robust anti-tumor immune responses. Co-inhibitory signals through receptors such as PD-1 can lead to the inactivation of T cells. On the other hand, co-stimulatory signals through receptors such as 41BB can enhance T cell activity against tumors. These receptors can serve as tools to develop strategies to optimize and enhance T cell anti-tumor functions. In this study, we used 9464D NB tumor mouse model to evaluate antibody treatment as a possible immunotherapy strategy. We further examine the infiltration and function of T cells after targeting checkpoints in 9464D NB tumor bearing mice. Blockade of various receptors in mice bearing 9464D tumor showed that anti-PD1 and anti-41BB delayed tumor growth. Combination therapy with anti-PD1 and anti-41BB antibodies significantly delayed tumor growth compared to single antibody treatment. This combination therapy led to a higher infiltration of CD8+ cells within the tumor. Splenocytes from 9464D tumor bearing mice treated with anti-PD1 and anti-41BB are tumor specific. In conclusion, our findings intend to design the best complement therapy to improve survival of patients with advanced NB. Our data demonstrates that tumor reactive T cells in NB tumors may express different checkpoint receptors including PD1 and 41BB. 9464D tumor bearing mice that received a combination of anti-PD1 and anti-41BB antibodies have smaller tumor size compared to single antibody therapy. Future direction includes examine T cells activation after combination therapy of BMT and immunecheckpoint targeting. Also we want to test adoptive transfer of T cells in tumor bearing mice after BMT. Further we want to measure the infiltration of other immune cells, such as myeloid cells and Tregs, in the tumor environment and possible target immune suppressive populations to see the effects in tumor growth.

#690

Identifying the genetic basis of stage 4S neuroblastoma.

Zalman Vaksman, Lee D. McDaniel, Maura Diamond, John M. Maris, Sharon J. Diskin. _Children's Hospital of Philadelphia, Philadelphia, PA_.

Introduction: The 5 year mortality rate for stage 4 metastatic neuroblastoma (NBL) is ~50%; however, the stage 4S subset of NBL is typically classified as low-risk with tumors exhibiting spontaneous regression. To date, the underlying genetic mechanisms influencing this distinct phenotype remain unknown.

Method: To identify genetic determinants specific to the 4S phenotype, we performed genome-wide association study (GWAS) analyses. Blood DNA samples from 6,195 NBL patients and 11,384 non-NBL controls were genotyped using Illumina SNP arrays. European ancestry subjects were selected for inclusion in a discovery [stage 4S (n=148), stage 4 (n=996), non-NBL controls (n=9,923)] or replication cohort [stage 4S (n=76), stage 4 (n=608), non-NBL controls (n=1,461)]. Following imputation using 1KG Phase 3, we conducted case:case comparisons including stage 4S vs. 4 and stage 4S vs. 1, followed by comparisons of NBL cases in each group to matched non-NBL controls. Variants uniquely associated with 4S NBL were defined as those meeting the following criteria: (1) stage 4S vs. 4 meta P < 5.0 x 10-5 and P < 0.005 in the discovery cohort (2) 4S vs. non-NBL control P < 0.05 and (3) either not significant (P > 0.05) or odds ratio in the opposite direction in 4 vs. non-NBL controls.

Results: 4S-specific variants that met our criteria spanned multiple regions with several harboring genes important to neuronal function including 9q34 (SARDH/DBH; rs2502746), 1p32.2 (KCNH1; rs12122529), 1p34 (KCNQ4; chr 1:41317634), 7q34 (CNTNAP2; rs79403134) and 2q24.1 (KCNJ3; rs78829625). At the 9q34 locus, rs2502746 showed further evidence as an expression quantitative trait locus (eQTL) for Dopamine Beta-Hydroxylase Antisense RNA1( DBH-AS1 P = 1.9 x 10-5 sun-exposed skin) and Sarcosine Dehydrogenase SARDH expression (P = 2.5 x 10-6 whole blood). Other 4S-specific loci observed include 3q27.3 (rs113207181), 8p21.3 (rs12548388), and 15q12 (rs62189512). Notably, none of the previously identified NBL susceptibility loci were found to be associated with 4S NBL in the 4S vs. non-NBL control comparison, suggesting a unique genetic basis for this enigmatic subset.

Discussion: Although a unifying mechanism underlying 4S NBL has yet to be identified, our results suggest a potential role for ion-channel and neuronal function genes. Ongoing efforts include investigating the role these genes and associated pathways play in NBL, including an integrative genomic, epigenomic and transcriptome analysis of stage 4 and 4S NBL.

#691

DFMO synergizes with BET inhibitors targeting ODC and MYCN to impede neuroblastoma cell proliferation and tumor initiation.

Sarah DeCou, Ping Zhao, Tracey Avequin, Abhinav Nagulapally, Jeffrey Bond, Giselle Saulnier Sholler. _Helen DeVos Children's Hospital, Spectrum Health, Grand Rapids, MI_.

Background: Neuroblastoma (NB) is the most common extracranial solid pediatric tumor and is associated with MYCN amplification. MYCN is a regulator of ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine biosynthesis. Inhibition of this pathway in MYCN-amplified NB tumors has been shown to be a target for treatment. Alpha-difluoromethylornithine (DFMO) inhibits ODC and is currently being used in a Phase II clinical trial for NB. BET inhibitors JQ1 and OTX-015 have been shown to be effective against MYCN-amplified cancers; it is hypothesized that they downregulate MYCN as well as cancer stem cell (CSC) signaling. We propose that inhibiting MYCN with a BET inhibitor, coupled with inhibition of ODC with DFMO, will result in enhanced inhibition of NB growth and tumor-initiating properties.

Methods: Single and combination drug treatments were conducted on BE(2)-C, SMS-KCNR, CHLA90, and one patient-derived cell line. CellTiter-Glo Luminescent Cell Viability Assay was used to determine cell viability in 96-well plates. IC50 values were calculated with a four-parameter variable-slope dose response curve using GraphPad Prism v.5 software. Drug combination studies were conducted in MYCN amplified tumors BE(2)-C and SMS-KCNR using ray designs to evaluate for synergism. IncuCyte ZOOM Live-Cell Imaging system was used for kinetic monitoring of cytotoxicity and apoptosis in NB cells. Western blots measured protein levels of apoptosis markers (cleaved caspase 3 and cleaved PARP) and CSC markers (Nanog, Sox2, NF-kB, CXCR4, Lin28B, and MYCN). Neurosphere assays were used to evaluate tumor initiation via sphere formation.

Results: Cell viability of MYCN amplified NB showed an IC50 range of 1.48-1.69 μM for JQ1 and 839.3 nM-2.36 μM for OTX-015; MYCN non-amplified NB showed an IC50 range of 4.22-11.58 μM for JQ1 and 2.99-11.03 uM for OTX-015. Combination treatment in MYCN NB had a synergistic effect, based on Loewe-additivity as the null hypothesis, for cell viability suppression by ray design experiments for BE(2)-C and SMS-KCNR. Western blots showed greater expression of apoptosis markers and decreased expression of CSC markers in combination relative to single drug treatments. A greater than 50% decrease in neurosphere formation with combination treatment in BE(2)-C, SMS-KCNR, and the patient cell line provides evidence for reduced CSC activity. IncuCyte imaging showed an increase in cell death with time post-treatment.

Conclusion: The combination of DFMO with BET inhibitors has a synergistic effect in treating MYCN amplified NBs as shown by a decrease in cell viability. This combination targets CSC pathways and decreases tumor-initiating ability. Given the lack of effective treatment options for children with relapsed or refractory high-risk NB, this combination may be a promising novel therapy.

#692

**Pappalysin-1 is a suitable target for T cell receptor transgenic T cells to kill Ewing sarcoma** in vivo **and** in vitro **.**

Uwe Thiel,1 Andreas Kirschner,1 Melanie Thiede,1 Thomas GP Grünewald,2 Rebeca Alba Rubio,2 Günther Richter,1 Thomas Kirchner,2 Dirk Busch,3 Poul Sorensen,4 Stefan Burdach1. 1 _Laboratory for Functional Genomics and Transplantation Biology, Munich, Germany;_ 2 _Institute of Pathology of the LMU, Munich, Germany;_ 3 _Technische Universität München, München, Munich, Germany;_ 4 _BC Cancer Research Centre, Vancouver, British Columbia, Canada_.

Pregnancy-associated plasma protein-A (PAPPA), also known as pappalysin, is a member of the insulin like growth factor (IGF) family. PAPPA acts as a protease, cleaving IGF inhibitors, i.e. IGF binding proteins (IGFBPs), thereby releasing IGFs from IGFBPs. The insulin/IGF-axis is involved in cancer in general and in Ewing sarcoma (ES) in particular. ES is a highly malignant bone tumor characterized by early metastatic spread. PAPPA stimulates normal bone growth, and is also associated with various cancers. In particular, PAPPA is overexpressed and required for proliferation in ES. We isolated HLA-A*02:01+/peptide restricted T cells from A*02:01\- healthy donors directed against PAPPA, generated by priming with A*02:01\+ PAPPA peptide-loaded dendritic cells. After T cell receptor (TCR) identification, retrovirally TCR transduced CD8+ T cells were assessed for their in vitro specificity and in vivo efficacy in human ES bearing Rag2-/-γc-/- mice. Engraftment in mice and tumor infiltration of TCR transgenic T cells in the mice was evaluated. The TCR transgenic T cell clone PAPPA-2G6 demonstrated specific reactivity towards HLA-A*02:01+/PAPPA+ ES cell lines. We furthermore detected circulating TCR transgenic T cells in the blood in Rag2-/-γc-/- mice and in vivo engraftment of the in bone marrow. Tumor growth in mice with xenografted ES was significantly reduced after treatment with PAPPA-2G6 TCR transgenic T cells compared to controls, and tumors from treated mice revealed tumor infiltrating PAPPA-2G6 TCR transgenic T cells. In summary, we demonstrate that PAPPA is a promising target for TCR based immunotherapy of ES. We demonstrate that TCR transgenic T cells recognize this target, home to the tumor, and causes tumor regression in a preclinical mouse model.

#693

Antibody-based targeting of the cell surface receptor tyrosine kinase FGFR4 in rhabdomyosarcoma and other cancers.

Martin Skarzynski,1 Nitya Shivaprasad,1 Baskar Subramanian,1 David Azorsa,2 Zhongyu Zhu,3 Dimiter Dimitrov,3 Javed Khan1. 1 _Oncogenomics Section, Genetics Branch, National Cancer Institute, Bethesda, MD;_ 2 _The Translational Genomics Research Institute (TGen), Scottsdale, AZ;_ 3 _Protein Interactions Section, Cancer and Inflammation Program, National Cancer Institute, Frederick, MD_.

Fibroblast Growth Factor Receptor 4 (FGFR4) is a receptor tyrosine kinase that is highly expressed in rhabdomyosarcoma (RMS), an aggressive soft tissue sarcoma originating from skeletal muscle in children and adolescent young adults. FGFR4 is expressed during development in myoblasts, but not in terminally differentiated skeletal muscle. Of note, FGFR4 is directly induced by the chimeric fusion oncogene PAX3-FOXO1 found in the most aggressive fusion-positive RMS and is mutationally activated in approximately 10-13% of the remaining fusion-negative RMS. High FGFR4 expression is associated with adverse outcome and is a driver oncogene in RMS. Metastatic RMS has a dismal outcome in the clinic with <30% survival. Novel treatments are needed for this devastating disease of children and young adults. Given that FGFR4 is functionally important and differentially overexpressed in RMS, we therefore hypothesized that FGFR4 is an ideal target for antibody-based therapeutics. To test our hypothesis, we first generated 15 binders against FGFR4: 3 murine monoclonal antibodies (mAbs), 2 rabbit mAbs and 10 fully human mAbs. We then assessed the specificity and affinity of these binders for FGFR4 by ELISA and surface plasmon resonance. Using flow cytometry and confocal microscopy, we next evaluated whether the mAbs internalized upon binding FGFR4. We then selected the internalizing binders from our panel for development of antibody-drug conjugates (ADCs). To investigate the ability of internalizing anti-FGFR4 mAbs to deliver cytotoxic payloads to RMS cells, we used a rapid screening method which employs drug-conjugated secondary antibodies (2°ADC), and thus circumvents the need for direct conjugation of cytotoxic small molecules to mAbs. When the anti-FGFR4 primary antibody and 2°ADC are co-administered, target cells internalize both antibodies in complex with the antigen. In addition to rapid screening of binders, this method facilitates comparison of various cytotoxic payloads, such as DNA alkylating and tubulin polymerization inhibiting agents. From the internalization and cytotoxicity screening data, we identified two murine mAbs that bound FGFR4 with low nanomolar affinity, readily internalized and killed RMS cells when paired with an anti-murine 2°ADC carrying MMAF or DMDM. In summary, we have determined that our anti-FGFR4 ADCs can deliver cytotoxic payloads to target cells through receptor-mediated endocytosis. We will next convert the two internalizing murine mAbs into mouse/human chimeric mAbs, directly conjugate them with a cytotoxic payload and test them in vitro and in vivo against RMS cell lines and RMS patient derived cells. The ultimate goal of these studies is to demonstrate the potential for clinical utility of FGFR4 targeting and develop novel therapeutics that can make a clinical impact on RMS and other FGFR4 overexpressing cancers, such as hepatocellular carcinoma, breast and lung cancers.

#694

TK-216: a novel, first-in-class, small molecule inhibitor of EWS-FLI1 in early clinical development, for the treatment of Ewing Sarcoma.

Saravana P. Selvanathan,1 Eric Moseley,1 Garrett T. Graham,1 Katti Jessen,2 Brian Lannutti,2 Aykut Üren,1 Jeffrey A. Toretsky1. 1 _Georgetown University, Lombardi Comp. Cancer Center, Washington, DC;_ 2 _Oncternal Therapeutics, Inc., San Diego, CA_.

One of the most significant challenges in creating more potent, less toxic treatments for patients is to identify new cancer therapeutic targets that distinguish the malignant from normal cells. EWS-FLI1 is a well-established Ewing sarcoma (ES) oncogene that has the potential to be an ideal therapeutic target by directly impacting malignant cells. We have previously reported the discovery and characterization of YK-4-279, an enantiomer-specific inhibitor of EWS-FLI1, which has been demonstrated to induce apoptosis, inhibit EWS-FLI1 transcription, block RNA helicase A co-immunoprecipitation with EWS-FLI1, and result in alternative splicing to mimic EWS-FLI1 knockdown. Continuous efforts in structure-guided medicinal chemistry has yielded TK-216, an analog of YK-4-279 inhibitor of EWS-FLI1, which is 3-4 fold more potent with excellent drug-like properties. TK-216 potently inhibits the proliferation of ES cells. Induces apoptosis in a dose -dependent manner as measured by caspase-3 activity in multiple ES cell lines with distinct translocation variants. The effects of TK-216 on alternative splicing (AS) were further validated using genes including ARID1A, CLK1, CASP3, PPFIBP1 and RUNX2. The splicing pattern was similar between TK-216 and YK-4-279. In addition to the in vitro activity of TK-216 , we show that TK-216 displays anti-tumor activity in a number of ES xenograft models. In summary, TK-216, a novel, first-in-class therapeutic which directly inhibits EWS-FLI1, offers a promising approach for the treatment of Ewing Sarcoma and is currently in Phase 1 clinical trials in patients with relapsed or refractory Ewing Sarcoma (clinicaltrials.gov - NCT02657005).

#695

Targeted nanoparticle therapy for poor prognosis Ewing's sarcoma.

HyungGyoo Kang,1 Jon Nagy,2 Julia Samulak,1 Timothy Triche1. 1 _USC/Children's Hospital Los Angeles, Los Angeles, CA;_ 2 _Nano Valent Phamaceuticals, Bozeman, MT_.

Targeted nanoparticles have shown the potential to deliver anticancer drugs to cancer cells selectively and thus to overcome unexpected cytotoxicity and limited efficacy of chemotherapy caused by non-selective delivery to normal cells. However, the optimal formulation of targeted nanoparticles for actual translational applications has not been established. By refining early formulations and after extensive testing, we have created an optimized targeted delivery system, HPLNs (Hybrid Polymerized Liposomal Nanoparticles) and demonstrated that this new, targeted nanoparticle technology overcomes many of these deficiencies. This technology has tremendous potential for treating virtually any type of cancer. The goal of our present study was to determine the efficacy and toxicity of this novel targeted nanoparticle in the treatment of Ewing's Sarcoma, a rare and highly lethal cancer of children and young adults. The efficacy of this targeted delivery system was compared to 1) administration of the free drug and 2) the drug in untargeted nanoparticle form. Our studies have shown that HPLN-Dox targeted with human antiCD99 antibodies can successfully inhibit the growth of Ewing's Sarcoma in murine models without any detectable off-target toxicity. The deliverable for our study was to demonstrate that the effectiveness of doxorubicin in the treatment of recurrent Ewing's sarcoma could be dramatically improved when encapsulated in human antiCD99 antibody targeted HPLNs. At the same time, toxic side effects normally seen with free drug was not detectable. Success with human anti-CD99 antibodies targeted HPLNs containing Doxorubicin was then easily adapted to other tumor types (Leukemia, Prostate or Pancreatic cancers) and other payloads such as irinotecan, EWS-FLI1 specific ASOs, siRNA and CRISPR/Cas9 targeting the ETS domain of the chimeric EWS-ets gene found in all Ewing sarcoma. In conclusion, our study showed a potential of targeted HPLN as a selective delivery vehicle of cytotoxic payloads to cancers and this technology is then easily adapted to other tumor types and virtually any small molecule therapeutic.

#696

Inhibition of Ewing sarcoma cell growth by targeting Sp1 and survivin with the small molecule clotam.

Sagar Shelake,1 Umesh T. Sankpal,1 W. Paul Bowman,1 Matthew Wise,2 Anish Ray,3 Riyaz Basha1. 1 _University of North Texas Health Science Center, Fort Worth, TX;_ 2 _Texas College of Osteopathic Medicine, Fort Worth, TX;_ 3 _Cook Children's Medical Center, Fort Worth, TX_.

Ewing sarcoma (ES) is the most common soft tissue and bone tumor in adolescents and young adults. The survival rate of patients with metastatic disease is poor suggesting an urgent need for the development of more effective therapeutic options. Our aim was to investigate novel targets and identify less toxic agents to improve therapeutic efficacy in ES patients. Specificity protein 1 (Sp1) transcription factor regulates critical genes involved in cell proliferation. Sp1 also mediates the expression of survivin, an inhibitor of apoptosis protein. Overexpression of Sp1 and survivin is linked to aggressiveness and poor prognosis in several cancers; however, their precise association is not adequately evaluated in ES. Previously, we showed that tolfenamic acid (TA) inhibits leukemia, medulloblastoma and neuroblastoma cell proliferation by targeting Sp1. In this study, we investigated the anti-cancer activity of this small molecule using human ES cell lines. CHLA-9 and TC-32 cells were treated with vehicle (DMSO) or TA (2.5-20 µg/ml) and cell viability was measured at 24, 48 and 72 h post-treatment using CellTiter Glo kit. ES cells were treated with vehicle or TA (15 µg/ml) for 48 h. The mRNA and protein expression of Sp1 and survivin was determined by quantitative polymerase chain reaction (qPCR) and Western blot analysis respectively. Flow cytometry was used to measure apoptotic cell population using Annexin-V staining and cell cycle phase distribution with propidium iodide. Markers of apoptosis (caspase 3/7 activity using caspase Glo kit and cleaved PARP by Western blot) and cell cycle arrest (Cyclin D1 and p21 expression by Western blot) were also determined. Sp1 DNA-binding was evaluated by gel shift assay. TA treatment inhibited ES cell viability in a dose and time-dependent manner which is accompanied by the inhibition of Sp1 and survivin protein expression and a 2-3 fold increase in apoptotic markers, caspase 3/7 activity, Annexin-V staining, and c-PARP expression. Interestingly, TA inhibited mRNA expression of survivin but not Sp1, suggesting it has post-translational effects, perhaps proteasome-dependent degradation of Sp1. Gel shift assay revealed a decrease in Sp1 DNA-binding, indicating that TA could directly disrupt the binding of Sp1 with its consensus binding site in the promoter of target genes. Cyclin D1 is crucial for moving the cells from G0/G1 to next phases and induction of p21 is known to inhibit Cyclin D1. In this study, TA up-regulated p21, reduced Cyclin D1 expression and accumulated cells in G0/G1 phase. These results demonstrate that TA induces apoptosis and causes cell cycle arrest in ES cells by targeting Sp1 and survivin.

#697

**Stage 1** in vivo **evaluation of multi-receptor tyrosine-kinase inhibitor lenvatinib in osteosarcoma patient derived mouse xenograft models.**

Wendong Zhang,1 Michael E. Roth,2 Jonathan Gill,2 Sajida Piperdi,1 E. Anders Kolb,3 David S. Geller,4 Bang Hoang,4 Rui Yang,4 Richard Gorlick2. 1 _Albert Einstein College of Medicine, Bronx, NY;_ 2 _Albert Einstein College of Medicine/Children's Hospital at Montefiore, Bronx, NY;_ 3 _Nemours/A.I. duPont Hospital for Children, Wilmington, DE;_ 4 _Albert Einstein College of Medicine/Montefiore Medical Center, Bronx, NY_.

Background: Osteosarcoma (OS) is the most common primary bone tumor in children and young adults. Over the past three decades improvement in outcomes for children with OS have remained stagnant. Novel therapies are needed to improve outcomes for these patients. Lenvatinib is an oral small-molecule tyrosine-kinase inhibitor (TKI), targeting multiple receptors including VEGFR1-3, FGFR 1-4, PDGFRα, RET, and c-kit. Prior studies have shown that PDGFR-VEGF/VEGFR and FGFR3 are overexpressed in human OS cell lines. Lenvatinib is FDA-approved for the treatment of differentiated thyroid cancer, and, in combination with mTOR inhibitor everolimus, for the treatment of renal cell carcinoma. The current study assessed the in vivo efficacy of lenvatinib in osteosarcoma xenograft models.

Materials and Methods: Single agent lenvatinib was tested against 5 well-established osteosarcoma patient derived mouse xenograft models. Lenvatinib was administered using a dose of 7.5mg/kg given by oral route daily (5/7 days) for 6 weeks or until study endpoints were met. Tumor volume and mouse body weight were measured bi-weekly. Tumor volume was compared to baseline tumor volume to calculate the relative tumor volume (RTV). The RTV was compared between the control and experimental groups utilizing the student t-test. P-values <0.05 were considered statistically significant. Additional statistical analyses were conducted as per the Pediatric Preclinical Testing Program analysis plan.

Results: Lenvatinib was generally well tolerated in vivo, without noted toxicity in any of the treated animals. Lenvatinib treatment led to delayed tumor progression in 4/5 models tested. Relative tumor volumes in the treatment group compared to the control group at week 3 in xenografts OS1, OS2, OS17, OS31, and OS33 were 0.77 (p=0.013), 0.88 (p=0.147), 0.49 (p<0.001), 0.66 (p<0.001), and 0.40 (p<0.001), respectively. Despite delayed progression observed in the OS xenografts treated with lenvatinib, all groups experienced progressive disease by the end of the study period.

Conclusions: Single agent lenvatinib demonstrates consistent anti-tumor activity in osteosarcoma patient derived mouse xenograft models. Preclinical studies, as well as clinical trials, have demonstrated the efficacy of tyrosine kinase inhibition in combination with inhibition of mTOR in a variety of malignancies. The current study, along with prior data, suggests that targeting receptor tyrosine kinases, in addition to mTOR, may lead to more effective inhibition of osteosarcoma cell growth. Ongoing studies are assessing the efficacy of lenvatinib, administered in combination with mTOR inhibitor, everolimus, in osteosarcoma patient derived mouse xenografts.

#698

Functional outcome and socio-psychological problems for bone & soft tissue sarcoma patients in childhood & AYA generation.

Kanya Honoki,1 Toshiaki Shinomiya,1 Hiromasa Fujii,1 Takashi Ishihara,1 Shinji Tsukamoto,1 Shingo Kishi,1 Yumiko Kondo,1 Akira Kido,1 Toshifumi Tsujiuchi,2 Midori Shima,1 Yasuhito Tanaka1. 1 _Nara Medical Univ., Nara, Japan;_ 2 _Kindai Univ., Higashiosaka, Japan_.

[Objective] To elucidate the problems between functional and sociological outcomes in patients of bone and soft tissue sarcomas in childhood and adolescent/young adult (AYA) generation.

[Method] Clinical (prognostic and functional) and socio-psychological outcomes were retrospectively analyzed in 50 patients with bone and soft tissue sarcomas under 29 years old treated in our institute from 1998 to 2014.

[Results] Twenty-two patients under 15 and 28 of AYA generation (15 to 29 years old) (28 male and 22 female) were subjected, including 35 cases of bone and 15 cases of soft tissue sarcomas, and 39 of extremity cases. Most common diagnosis was osteosarcoma of 27 cases. Limb-sparing surgery was performed in 30 cases, including 14 of prosthetic and 8 of biological reconstruction for bone tumors. Overall Survival rates were 79% for 2 year and 61% for 5 year follow-up periods. 70% of the patients experienced limited activities; 50% in high school and college students experienced drop-out or delay, suspension and repeating the same class; 56% faced to their job lost or limitation of job search; 20% experienced changes of relationship with families and partners (Table). Most taken palliative care intervention was the counseling for family members and caregivers, besides pain management and psychological care. Correlation coefficient failed to show a significant correlation between functional score and sociological disability score both in all limb sparing cases (R2 = 0.2664) and in osteosarcoma cases ((R2 = 0.1757) .

[Conclusion] The sociological problems for patients in this particular group could be focused on school life, job search and relationship with families and partners. QOL is a multidimensional measure with various domains, such as physical, psychological, social well-being, spiritual health as well as functional ability, therefore, multi-disciplinary care for socio-physiological and psychological disadvantage must be taken for this group of patients at earliest convenience.

Reletionship between physiological and socio-psychological problems

---

Subject | All patients | Limb-spared patinets

Limitation of activities | 70% (30/43) | 75% (21/28)

School dropout/delay in high-schoolers & college students | 50% (14/28) | 32% (8/25)

Lost jobs/job search difficulties | 56% (9/16) | 33% (3/9)

Changing social reletions with families & partners | 20% (8/40) | 21% (3/28)

#699

Clinical feasibility of chemotherapy monitoring for bone sarcoma patients with diffuse optical spectroscopic imaging.

Hannah M. Peterson,1 Bang H. Hoang,2 David Geller,2 Richard Gorlick,2 Rui Yang,2 Jeremy Berger,2 Janet Tingling,2 Michael Roth,2 Jonathan Gill,2 Darren Roblyer1. 1 _Boston University, Boston, MA;_ 2 _Montefiore Medical Center, Bronx, NY_.

Background: Sarcomas account for almost 15% of all childhood cancers and 40-60% of these patients develop locally recurrent or metastatic disease. Currently in many sarcomas, the only clinically accepted prognostic marker in patients with localized disease is the percent of tumor necrosis at the time of surgery. Diffuse Optical Spectroscopic Imaging (DOSI) is an emerging near-infrared (NIR) imaging technique that non-invasively measures quantitative functional information and may provide a new means to monitor and predict chemotherapy response in sarcoma which could impact treatment decisions. Here we evaluate the feasibility of measuring pediatric sarcomas with DOSI.

Methods: Patients diagnosed with either Ewing's Sarcoma or osteosarcoma underwent DOSI measurement at multiple time points during their course of neoadjuvant chemotherapy. A handheld probe was used to take point measurements in a 1 cm spaced grid at tumor and contralateral normal locations. The measurement area was approximately 6 x 7 cm and a single point measurement typically took 15-30 s, which includes the time it takes to position the probe, ensure good contact between the probe and the skin, and acquire data. The optical properties, and absolute concentrations of oxyhemoglobin (ctHbO2), deoxyhemoglobin (ctHb), water (ctH2O), and lipids were compared between tumor and normal measurements.

Results: To date, one osteosarcoma and two Ewing's sarcoma patients have been measured. At baseline and early in treatment, all subjects had a statistical difference in total hemoglobin concentration between the affected and contralateral normal tissues (p < 0.05 for Student's t-test, Kolmogorov-Smirnov test, and Mann-Whitney rank test). Statistical differences also appeared in oxyhemoglobin, deoxyhemoglobin, oxygen saturation, water, lipids, scattering amplitude, and scattering power although longitudinal trends varied across patients.

Conclusions: DOSI can be utilized to measure optical properties and to obtain functional information in bone sarcoma patients. It can detect differences between affected and normal tissue. A better understanding of the optical properties in this patient population is needed. Larger data sets are needed to characterize and quantify sarcoma optical properties and treatment response dynamics, ideally finding a functional biomarker associated with pathologic response. Additional accrual of primary bone sarcoma patients and normal volunteers is ongoing.

#700

Characterization of the mode of action of Fenretinide treatment in alveolar rhabdomyosarcoma cells.

Eva Brack, Marco Wachtel, Beat W. Schaefer. _University Children's Hospital Zurich, Zurich, Switzerland_.

Alveolar rhabdomyosarcoma (aRMS) is a highly malicious childhood malignancy characterized by a specific chromosomal translocation encoding the oncogenic transcription factor PAX3-FOXO1. As aRMS cells are addicted to the tumor-specific fusion protein, it may serve as an ideal therapeutic target. Previously, we have identified from a large drug library screen the compound Fenretinide (retinoic acid p-hydroxyanilide), which is already in clinical use, to affect both PAX3-FOXO1 expression as well as aRMS cell viability. The aim of this study was therefore to characterize the mode of action of Fenretinide in more detail. First, we were able to show that Fenretinide induced the generation of reactive oxygen species (ROS) in mitochondria. A more detailed characterization revealed that the Fenretinide-induced ROS derived from an interaction of Fenretinide around complex II of the mitochondrial respiratory chain, leading to the production of superoxides. ROS scavenging as well as complexing of iron ions completely abolished cell death.

To identify the mode of cell death involved, we then used a range of pharmacological inhibitors of specific cell death pathways including Z-vad (pan -caspase inhibitor), Necrostatin-1 (necroptosis pathway inhibitor (RIP-1 kinase inhibitor)), 3-Methyadenine (3-MA) (autophagy pathway inhibitor (phosphatidylinositol 3-kinase inhibitor)) and Ferrostatin (ferroptosis pathway inhibitor) during Fenretinide treatment. Surprisingly, none of these inhibitors alone was able to prevent cell death and even different combinations were not sufficient to completely inhibit cell death. CRISPR/Cas9 mediated depletion of key players in the apoptotic and necroptotic pathway (Bak, Bax and RIPK1) confirmed the pharmacological data. We therefore conclude that other, less characterized cell death pathways or a combination of several pathways including apoptosis and necroptosis might be crucial. Interestingly, electron microscopic examination of cells pointed towards an excessive accumulation of vacuoles to be characteristic.

Taken together, our data show that Fenretinide shows high potential for the treatment of aRMS, inducing several forms of cell death mediated through the production of ROS. These properties open the search for additional compounds acting in a combinatorial manner.

#701

Pazopanib synergizes with clofilium tosylate in the treatment of malignant rhabdoid tumors.

Celine Chauvin,1 Aurianne Lescure,1 Wilfrid Richer,1 Arnault Tauziède-Espariat,2 Amaury Leruste,1 Zhi-Yan Han,1 Didier Surdez,1 Stefano Cairo,3 Elaine Del Nery,1 Olivier Delattre,1 Franck Bourdeaut1. 1 _Curie Institute, Paris, France;_ 2 _Sainte-Anne Hospital, Paris, France;_ 3 _XenTech, Evry, France_.

Background : Rhabdoid tumors (RTs) are rare, highly aggressive pediatric malignancies which occur in kidney, soft-parts and brain of young children. They are characterized by a complete inactivation of the SMARCB1 tumor suppressor gene encoding a core subunit of the chromatin remodeling SWI/SNF complex. Prognosis for children with RTs is poor as, in many instances, these tumors are resistant to conventional type chemotherapy. Hence, new therapies should be designed and biologically-driven strategies targeting the SMARCB1-deficiency or its biological consequences have to emerge.

Methods : For this purpose, we realized a high-throughput screening by using the Prestwick Chemical Library of 1200 FDA-approved drugs on two rhabdoid tumor cell lines and one SMARCB1-reexpressing cell line. Cell count was assessed by automatic cell counting using DAPI.

Results : Among 10 drugs that showed cytotoxic activity specifically in SMARCB1-deficient cell lines, we identified vatalanib, a broad receptor tyrosine kinase (RTK) inhibitor. This prompted us to analyse the response to a wider number of 9 RTK inhibitors on 7 SMARCB1-deficient cell lines compared to 6 SMARCB1-expressing cell lines. This "supervised" screening confirmed the activity of vatalanib but showed that pazopanib had an even greater cytotoxic ability. Interestingly, the screening also identified a potassium channel inhibitor (clofilium tosylate), that showed among the strongest cytotoxic activity as a single agent, and also synergistically reduced viability and induced apoptosis, when combined with vatalanib and pazopanib. With in vivo experiments on two patient-derived xenograft (PDX) models of rhabdoid tumor, we notably showed that pazopanib is effective in reducing tumor growth and that combination with clofilium tosylate enhanced the anti-tumor activity of pazopanib in the two PDX models. Interestingly, we also highlighted that pazopanib combined to conventional chemotherapies is even more efficient than chemotherapy alone. Finally, to identify candidate kinases that confer pazopanib sensitivity to rhabdoid tumor cell lines, we performed a phosphoarray experiment and found PDGFRa, PDGFRb and FGFR2 as pazopanib targets, RTK that were among the most highly expressed in our expression profiling dataset.

Conclusion : To conclude, our findings demonstrate that targeting PDGF and FGF axis is an effective means of therapy against rhabdoid tumors. We therefore propose that pazopanib should be tested in children with rhabdoid tumors in which there is a dire need for an effective therapy.

#702

**A novel cell-penetrating ATF5 antagonist peptide CP-d/n-ATF5 exerts** in vitro **and** in vivo **anti-tumor effects in a broad spectrum of pediatric cancers.**

Debarshi Banerjee,1 Shuobo Boboila,1 Cherease Street,1 Shunpei Okochi,1 Filemon S. Dela Cruz,2 Eileen Connolly,1 Angela Kadenhe-Chiweshe,1 Darrell Yamashiro3. 1 _Columbia Univ. Medical Ctr., New york, NY;_ 2 _Columbia Univ. Medical Ctr, New york, NY;_ 3 _Columbia Univ. Medical Ctr., New York, NY_.

Purpose: Activating transcription factor 5 (ATF5), a member of the ATF/CREB family transcription factor, has been implicated in the pathogenesis of glioblastoma and other adult tumors. Recently, a novel cell penetrating (CP-d/n-ATF5) peptide has been developed to antagonize ATF5 function. The goal of the current study is to test the efficacy of CP-d/n-ATF5 in several children tumors including neuroblastoma, hepatoblastoma, Ewing sarcoma, and rhabdoid tumor, in vitro and in vivo.

Methods: A panel of neuroblastoma cell lines: SK-N-Be(2)C, SK-N-DZ, NGP, IMR-32, NGP, SHEP-21N, KELLY, CHP-212, CHLA-20, CHLA-15 and SK-N-SH; hepatoblastoma cell lines HUH 6 and Hep-G2; Ewing sarcoma cell lines A673, SKNMC, SKNEP1, and TC32; and the rhabdoid cell G401, were treated with vehicle or 50, 100 and 200 μM of CP-d/n-ATF5. Cell viability and apoptosis were assessed after 72 hr by WST-8 and TUNEL assays, respectively. To test in vivo efficacy, SK-N-Be(2)C kidney xenograft tumors were treated with the peptide at dose 50mg/kg, IP injection once per day for first three days and then twice per week. Tumor growth was monitored by bioluminescence imaging and mice were sacked when flux reached a threshold value. Organ metastases were determined by ex vivo imaging. A patient-derived xenograft (PDX) model of rhabdoid tumors was employed where PDX tumors, at 150-200 mm3 size, were enrolled in the penetratin (control) and CP-d/n-ATF5 treatment (same dose as above). Tumors were measured biweekly with a calipers and mice were sacrificed when tumor volume reached a threshold (1500 mm3).

Results: CP-d/n-ATF5 exerted cytotoxicity or apoptosis, in a dose dependent manner, across a wide panel of pediatric tumor cell lines. In vitro, tumor cell viabilities were reduced 40-70% (P<0.05) and apoptosis was increased 50-80% at 200 μM of CP-d/n-ATF5. In vivo, CP-d/n-ATF5 significantly inhibited SK-N-Be(2)C xenograft growth in nude mice, with a median survival of 35 days for control against 21 days for CP-d/n-ATF5, P=0.0013. CP-d/n-ATF5 also reduced SK-N-Be(2)C metastatic burden in the liver (P<0.05) and bone marrow (P<0.01). In the rhabdoid PDX model, there was a significant inhibition of tumors treated with CP-d/n-ATF5 as compared to Penetratin treatment, with a mean tumor volume of control 1283 ± 266.6 mm3 (n=5) vs CP-d/n-ATF5 234.2 ± 50.66 mm3 (n=6), at day 10 post treatment. Penetratin treated tumors showed a median post-treatment time of 13 days to reach threshold volume. None of the CP-d/n-ATF5 treated tumors reached the threshold after 28 days of treatment with some tumors demonstrating regression, indicating a profound anti-tumor effect of the peptide.

Conclusion: Our study shows that a novel ATF5-targeting peptide CP-d/n-ATF5 has broad and profound cytotoxic and apoptotic effects in several pediatric tumors in vitro and in vivo. Our study also indicates that CP-d/n-ATF5 has the potential to act as an anti-metastatic agent.

#703

Combinatory treatment for pediatric low grade glioma with the dual mTORC1/2 inhibitor TAK228 and MEK inhibitor Trametinib.

Antje Arnold,1 Fausto Rodriguez,1 Charles George Eberhart,1 Eric Hutton Raabe2. 1 _Johns Hopkins University School of Medicine; Division of Neuropathology and Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD;_ 2 _Johns Hopkins University School of Medicine; Division of Neuropathology and Sidney Kimmel Comprehensive Cancer Center; Division of Pediatric Oncology, Baltimore, MD_.

Pediatric low grade glioma (PLGG) is one of the most common childhood tumors. If the tumor is located in a region of the brain that is not accessible for surgical resection or if the tumor recurs after surgery, additional therapies are needed. Recent studies highlighted the important role of mTORC and MEK-activation in PLGG. The dual mTORC1/2-inhibitor, TAK-228, and the FDA approved MEK-inhibitor, Trametinib, have good brain penetration and are promising candidates for targeted therapy PLGG. We hypothesized that TAK-228 and Trametinib would show synergistic effects both in vitro and in vivo in PLGG models. We treated the PLGG derived cell lines Res186 and Res259 with TAK-228, Trametinib, and vehicle. Cell growth was investigated using MTT-assay over different days and compared to the treatment with the vehicle. DNA replication was measured through bromodeoxyuridine (BrdU) incorporation assay and apoptosis was evaluated through cleaved caspase 3 (CC-3) staining. Cells were analyzed and counted with ImageJ. Activation of MAPK pathway was detected via Western Blot by phosphorylated pERK compared to total pERK, and β-actin. Treatment of Res186 and Res259 with TAK-288 or Trametinib reduces cell growth and proliferation in a dose and time depended manner. Res186 have a significant reduction in cell growth after 4 days treatment (TAK-288: 20nM, **p<0.01; Trametinib: 20nM, **p<0.01). Res259 showed a significant cell reduction at the same time point, but with a higher drug dose (TAK-288: 50nM, *p<0.05; Trametinib: 50nM, **p<0.01). IC50 values for TAK-288 was 15nM for Res186 and 20nM for Res259 cells on day 4. IC50 values for Trametinib was 50nM for Res186 and 100nM for Res259 cells. Staining for CC-3 showed a significant increase for apoptosis in Res259 cells after treatment with TAK-288 or Trametinib (**p < 0.01). No positive CC-3 staining was detected in Res186 cells after drug treatment. MAPK pathway was activated in a dose-dependent manner as determined by phosphorylated pERK Western Blot after TAK-288 and inactivated after Trametinib treatment. No change in total ERK concentration was detected, suggesting that cells attempt to compensate for loss of mTOR signaling by upregulating MAP kinase signaling. Our preliminary results show that the PLGG-derived cell lines are sensitive to TAK-228 and Trametinib treatment. All cell lines showed decreased proliferation at various doses of either inhibitor. The increased MAP kinase activity we identified after TAK-228 treatment suggests a compensatory mechanism that may render these cells especially sensitive to treatment with both TORC1/2 and MEK inhibitors. We will now investigate both drugs in vivo. Evidence of activity in murine models will be necessary to provide a pre-clinical rationale for combination therapy of these agents in aggressive PLGG.

#704

Therapeutic potential of Delta24-ACT, a novel immunostimulatory oncolytic adenovirus, for the treatment of pediatric solid tumors: Initial study in pHGG, DIPG and osteosarcoma.

Marc Garcia Moure,1 Naiara Martínez-Vélez,1 Enric Xipell,1 Marisol González-Huarriz,1 Ana Patiño,1 Oren J. Becher,2 Cande Gómez-Manzano,3 Juan Fueyo,3 Marta M. Alonso1. 1 _University Hospital of Navarra, Pamplona, Spain;_ 2 _Duke University School of Medicine, Durham, NC;_ 3 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Pediatric High Grade Glioma (pHGG), including Diffuse Intrinsic Pontine Glioma (DIPG), and osteosarcoma are amongst the most common and lethal pediatric solid tumors. Despite improvements in surgery, radiotherapy and chemotherapy, the outcome for children affected with these malignancies remains dismal since the current treatments are ineffective and inflict severe side effects. Thus, it is critical to implement novel and radically different therapeutic approaches to improve the survival and quality of life of these children. Delta24 is a modified adenovirus with a tumor dependent replication, and its antitumor effect has been tested in preclinical and clinical models of both high grade glioma and osteosarcoma. The adenovirus destroys the tumor mass as it replicates within the tumor. Additionally, the adenovirus infection triggers the recruitment of lymphocytes to the tumor. However, tumors are often characterized by an immunosuppressive environment that hampers the synergistic effect mediated by the virus and the immune system. Here we describe the generation of a new oncolytic adenovirus Delta24-ACT, based on the Delta24 platform, encoding an immunostimulatory protein. For this project we used relevant immunocompetent pediatric solid models. The toxicity of the virus was assessed by dose-escalation experiments. Delta24-ACT was well tolerated and did not lead to important adverse effects. The antitumor effect of Delta24-ACT was evaluated in immunocompetent mice bearing orthotopic supratentorial glioma, DIPG or local osteosarcoma that spontaneously metastasize to lungs, obtaining significant tumor shrinkage or complete elimination in some of them. Delta24-ACT also promoted an increased T-cell infiltration within the tumor and an enhanced antitumor immune response. Of importance, treatment with Delta24-ACT led to a significant increase in median survival in all the tumor models tested and resulted in long-term survivors free of disease. Furthermore, in our osteosarcoma model local treatment of primary tumors led to the regression of spontaneous lung metastases, which are usually the leading cause of death in most of the patients affected by osteosarcoma. Currently, we are performing re-challenge experiments to evaluate a possible memory effect mediated by the virus and mechanistic studies to elucidate the mechanism of action. Altogether, these results demonstrate the potential therapeutic benefit of Delta24-ACT adenovirus in the treatment of pediatric solid tumors such as pHGG, DIPG and osteosarcoma, representing an important milestone in the fight against cancer.

#705

Elucidating pediatric brain tumor pathophysiology by assessing signal transduction pathway activation.

Laurent Holtzer, Wim Verhaegh, Anja Van de Stolpe. _Philips Research, Eindhoven, Netherlands_.

A major problem in pediatric neuro-oncology is lack of insight into underlying tumor biology, making treatment with targeted therapy challenging for this group of patients. To enable targeted therapy choice, understanding of underlying signal transduction pathway activity is required, as well as availability of appropriate diagnostic tests to predict therapy response. We have developed a novel method to identify activity of the ER, AR, HH, Wnt, TGFbeta, NFκB, and PI3K signal transduction pathways in an individual tissue sample. Our method is based on measuring mRNA levels of the pathway transcription factor target genes and interpreting these expression levels using a knowledge-based Bayesian network computational model which infers the probability of activity of the respective transcription factor (Verhaegh et al, Cancer Res. 2014). Public patient sample datasets from the GEO database were used to investigate pathway activation in pediatric brain tumors. In normal brain tissue from medulla, cerebellum, cerebral cortex and thalamus (GSE50161), pathway analysis showed no significant oncogenic pathway activation.

Observed pathway activity in medulloblastoma was strongly correlated with known pathway-activating mutations, which was used as initial validation of these models for use in pediatric brain tumor diagnostics. In ependymoma (GSE66354), three distinct subtypes were analyzed: infratentorial posterior fossa A (PFA), PFB and supratentorial. These tumors are known to have a subtype-dependent prognosis; mainstay of therapy is surgery and radiation. Tumor driving pathway identification in pediatric ependymoma using mutation analysis is usually not helpful because epigenetic mechanisms prevail (Mack et al, Brain Pathology 2013).

Using our pathway activity analysis, we find remarkable differences between different types of ependymoma with respect to signal transduction pathway activity. For PFA we find high NFκB and Wnt pathway activity. The dominant NFκB activity is in agreement with the described inflammatory phenotype. On the other hand, for PFB we find low NFκB and Wnt pathway activity. Finally, for supratentorial ependymoma we find almost all samples to have an active Hedgehog and Wnt pathway. We also analyzed multiple other pediatric tumors for pathway activity, for which data will be presented.

The above pathway activity results provide highly interesting leads for targeted therapy selection, and we expect that such pathway analysis will have clinical utility in a neoadjuvant setting to reduce tumor size prior to surgery, and possibly also in an adjuvant setting to prevent recurrences.

#706

Combination ionizing radiation and oncolytic Seneca Valley virus injection enhances tumor cell killing of pediatric gliomas.

Yuchen Du. _Baylor College of Medicine, Houston, TX_.

Background and Purpose: Radiation therapy is the traditional and only therapy that offers benefits to patients with diffuse intrinsic pontine gliomas (DIPG) which are highly aggressive pediatric central nervous system tumors without known curative therapies. However, unfortunately, efficacy of radiation therapy is transient. Seneca Valley virus-001(SVV-001) is a novel non-pathogenic oncolytic piconavirus that can be systemically administered and pass through brain blood barrier (BBB). Phase I clinical trial of SVV has proven its efficacy in adults patients with cancers with neuroendocrine features and in pediatric patients with non-central nerve system (CNS) tumors. We sought to examine if combining ionizing radiation with an oncolytic virus SVV-001 would lead to synergistically enhanced tumor cell killing and significantly improve therapeutic efficacy in vivo in DIPG patient tumor-derived intra-brain stem orthotopic xenograft mouse models (PDOX).

Methods: By infecting PDOX derived tumor cells with green fluorescent protein (GFP)-coupled SVV (SVV-GFP) or SVV-001, in vitro virus infection and anti-tumor activity of SVV were examined by cell viability assay, Western Blotting for apoptosis and autophagy-related protein and immunohistochemistry (IHC) staining or Flow cytometry for cell mitochondria content. In vivo therapeutic efficacy was evaluated by systemic administration of SVV into PDOX derived DIPG xenografts alone or in combination with irradiation.

Results: We first had established a novel panel of nine PDOX derived orthotopic xenograft mouse models of DIPG. Our data confirmed that SVV can infect and efficiently kill DIPG tumor cells in vitro by inducing apoptosis and autophagy, leading to improved animal survival in a subset of DIPG xenograft models. We found that the lack of mitochondria in the xenograft tumor cells in vivo impaired the intracellular replication of SVV-001 and subsequently compromised its oncolytic cell killing of tumor cells in vivo. Radiation induced elevated mitochondria biogenesis in tumor cells therefore boosted SVV oncolysis of tumor cells. Moreover, combining fractionated radiation with single i.v.injection of SVV led to significant improvement of animal survival in a subset of DIPG models.

Conclusion: SVV-001 killed DIPG xenograft cells in vitro. Radiation activated mitochondria content of tumor cells in vivo, which restored impaired SVV oncolytic function.Combining radiation with single systemic administration of SVV synergistically enhanced animal survival in a subset of DIPG xenograft models.

#707

Personalization of dexamethasone in acute lymphoblastic leukemia.

Rosanna K. Jackson,1 Ali Alhammer,1 Zach Dixon,2 Gareth J. Veal,1 Julie AE Irving1. 1 _Newcastle University, Newcastle, United Kingdom;_ 2 _Bristol University, Bristol, United Kingdom_.

Synthetic glucocorticoids, such as dexamethasone (Dex), are pivotal in the treatment of childhood acute lymphoblastic leukemia (ALL) but are associated with significant variability, both in terms of toxicity and efficacy. We aimed to investigate three key variables to better understand how Dex personalization may be achieved: pharmacokinetics (PK), intracellular Dex accumulation, and cellular response, following Dex binding to the glucocorticoid receptor (GR) in ALL cells.

For Dex PK studies, blood samples were collected post oral administration on one of the first three days of induction chemotherapy in 99 patients on the UKALL 2011 trial receiving either 6mg/m2 for28 days (standard arm) or 10mg/m2 for 14 days (short arm). Plasma Dex levels were analysed using a validated LC/MS method, and a non-compartmental pharmacokinetic analysis. To assess intracellular Dex levels, cell lines, primagraft (n=9) and primary patient samples (n=6) were studied. The plasma Dex LC/MS method was optimized to quantify Dex in ALL cell lysates. Dex accumulation was also assessed using flow cytometric analysis of Dex conjugated to FITC. Cellular Dex sensitivity was assessed using Alamar Blue assays.

There was a wide Dex PK variability, with AUC0-12h, and Cmax significantly higher on the short compared to the standard arm; 564 (202-1606) versus 408 (142-1009), median (range), p=0.0003 and 0.0006, respectively. However there was substantial overlap between the two arms, with a number of patients on the standard arm exhibiting higher exposures than those on short therapy.

Dex GI50 values ranged from 37 - > 1000 nM and 2 - > 1000 nM in cell lines and patient samples respectively. Western blotting indicated wildtype GR in all samples, with R3D11 and REH cell lines serving as hemizygous deleted and GR negative controls. Dex accumulation in cell lines was 2.1 and 1.8 (range 1.2 - 2.1) pmol/106 cells in PreB697 and Dex resistant sub-lines, respectively. While patient samples exhibited greater variability, Dex accumulation was not significantly different between sensitive and resistant cells; mean of 1.0 versus 1.4 (range 0.1-2.3, 0.4-4.4 pmol/ 106 cells, p=0.17). Flow cytometry Dex FITC accumulation confirmed these data, with a mean fluorescence intensity of 4.2 versus 4.1 (range 1.5-5.9, 2.0 - 9.1, respectively; p=0.97).

These data suggest that while PK and cellular response are hugely variable, variations in drug accumulation do not appear to play a key role in Dex response in ALL cells. Importantly, 62% of patient cell samples had Dex GI50 values greater than plasma concentrations observed in any patient, on both arms on the UKALL 2011 trial. A combined approach incorporating PK assessments and cellular response in ALL cells should be further investigated, to allow a comprehensive understanding of Dex pharmacology with a view to optimizing its clinical utility.

#708

Prohibitin is a prognostic marker of treatment failure and therapeutic target to block chemotherapy resistance in Wilms tumor.

Michael Vincent Ortiz,1 Melissa Burns,2 Amy Eisenberg,3 Saima Ahmed,2 Lyvia Gaewsky,2 Gary Bradwin,2 Paolo Cifani,1 Anton Henssen,1 Ian Macarthur,1 Michael LaQuaglia,1 Anthony Letai,4 Arlene Naranjo,5 Samantha Gadd,6 Yueh-Yun Chi,5 Jeffrey Dome,7 Elizabeth Perlman,6 Elizabeth Mullen,2 Hanno Steen,2 Alex Kentsis1. 1 _Memorial Sloan Kettering Cancer Center, NY;_ 2 _Boston Children's Hospital, MA;_ 3 _University of California, Berkeley, CA;_ 4 _Dana Farber Cancer Institute, MA;_ 5 _University of Florida, FL;_ 6 _Lurie Children's Hospital of Chicago, IL;_ 7 _Children's National Medical Center, DC_.

Wilms tumor (WT) is the most common kidney tumor of children. Over the last three decades, clinical trials employing multi-modality therapies have resulted in overall survival of greater than 90% for patients with low-risk disease. In spite of these advances, treatment of patients with advanced, anaplastic, and relapsed Wilms tumors remains challenging, with substantial rates of treatment failure and death. To improve risk stratification and identify novel therapeutic targets, we used high-accuracy mass spectrometry urine proteomics to identify urine tumors markers associated with relapsed WT and non-WT renal tumors. We measured urine proteomes at diagnosis of 54 patients with renal WT, clear cell sarcoma, rhabdoid tumor, and age-matched controls, leading to the quantitation of 6,519 urine proteins. In particular, we identified specific urine WT markers, including those that were enriched in patients with relapsed WT, such as mitochondrial regulators prohibitin and DAD1, β-catenin antagonist DACT2, and DNA repair factor SUN1. Using a specific enzyme-linked immunosorbent assay (ELISA) developed to measure urine prohibitin in an independent cohort of 139 WT and control samples, we found that urine prohibitin concentrations over 1000 ng/mL were significantly associated with the risk of disease relapse, with an odds ratio of relapse of 153 and receiver operating characteristic area under the curve of 0.77 (95% confidence interval of 0.64-0.99). Immunohistochemical tumor analysis revealed that prohibitin was highly expressed in primary Wilms tumor specimens. Importantly, using loss- and gain-of-function genetic experiments, we found that prohibitin was required for the growth and survival of Wilms tumor cells, and its overexpression conferred concomitant resistance to vincristine, doxorubicin and actinomycin D. Consistent with prohibitin's functions in mitochondria, we are using BH3 profiling to elucidate specific intrinsic apoptotic dependencies in distinct subsets of refractory Wilms tumors, as a prelude to rational combination blockade of chemotherapy resistance, such as blockade of BCL2 dependence using venetoclax. In all, the use of urine prohibitin measurements may improve initial therapy stratification, and enable monitoring of response to therapy and early detection of relapse. In addition, therapeutic targeting of chemotherapy resistance induced by prohibitin overexpression may offer improved therapies for patients with relapsed or refractory Wilms tumors.

#709

Incidence and mortality of diethylstilbestrol-related clear-cell adenocarcinoma of the vagina and cervix: 40 years of long-term follow-up.

Dezheng Huo, Diane Anderson, Arthur L. Herbst. _Univ. of Chicago, Chicago, IL_.

Objective: Women exposed to diethylstilbestrol (DES) in utero are at increased risk for the development of vaginal and cervical clear cell adenocarcinoma (CCA), and possibly of breast cancer, diabetes, and cardiovascular diseases as well. A peak in the age-incidence curve in DES- related CCA has been documented at ages 19 to 24. It is unknown if a second peak will occur in later life, the ages when CCA developed spontaneously in the pre-DES era. In addition, the long-term prognosis and health impact for adolescents and young adults who have DES-related CCA are unknown.

Methods: 720 cases of CCA were reviewed from the CCA Registry at the University of Chicago through 2014. Rates and cumulative risks for CCA were calculated based on white women born in the U.S. from 1948 through 1971. Kaplan-Meier method was used to estimate survival probability among women with CCA. Cox models were used to identify independent prognostic factors. To gauge the relative impact of young-onset clear-cell adenocarcinoma on mortality, we compared their mortality hazards with mortality hazards of the U.S. female population, and calculated standardized mortality ratio (SMR).

Results: In 420 CCA cases there was documented evidence of prenatal DES exposure. 80% patients had a CCA between ages 15 and 31 but some occurred as late as age 55. A small second peak occurred around age 40. The risk of U.S. born, white DES-related CCA was highest in the 1951-1956 birth cohort, and this birth cohort effect correlated with DES use in the U.S. By age 50, the cumulative risk of CCA was 1 per 750 exposed women. A total of 695 CCA patients were included in the survival analysis. After a median follow-up of 22.7 years (interquartile range: 10.8-35.1 years), 219 patients died. The 5-year, 10-year, and 20-year survival probabilities were 83%, 75%, and 69%, respectively. As expected, tumor stage was a strong prognostic factor for survival among DES-related CCA cases. Tubular-cystic histology was associated with better survival compared with papillary, solid, or a combination of these histological patterns. Adolescent patients (<18 years old) had worse survival than young adult patients. Compared with general U.S. female population, women with DES-related CCA had 27-fold increased risk of death between 10 and 35 year of age (SMR 27.06, 95% CI: 23.04-31.58), 5-fold increased risk between 35 and 50 year of age (SMR 5.07, 95% CI: 3.59-6.95), 2-fold increased risk after age 50 (SMR 2.03, 95% CI: 1.22-3.18).

Conclusion: Our study drew a relatively complete epidemic curve of DES-related CCA. There existed late-onset CCA among women with prenatal exposure of DES. DES-related CCA increased mortality not only during early life among exposed women, but also impacted life after age 50. These results suggest continued close health follow-up of this population. 

### Early Detection

#710

Identification of autoantibody to ECH1 & HNRNPA2B1 as potential biomarkers in the early detection of lung cancer.

Liping Dai,1 Jitian Li,2 Jun-Chieh J. Tsay,3 Xiao Wang,1 John S. Munger,3 Harvey Pass,3 William N. Rom,3 Eng M. Tan,4 Jian-Ying Zhang1. 1 _Zhengzhou University, Zhengzhou, China;_ 2 _the University of Texas at El Paso, El Paso, TX;_ 3 _New York University, New York, NY;_ 4 _The Scripps Research Institute, San Diego, CA_.

Background: Identification of biomarkers for early detection of lung cancer (LC) may lead to more effective treatment and reduction of mortality.

Methods: Serological proteome analysis (SERPA) was used to identify proteins around 34 kDa, which had been previously recognized by autoantibody in sera from LC patients. We have validated autoantibody response in sera from 90 LC patients, 89 normal controls by using immunoassay. Another independent cohort of 25 LC patients with 219 serial serum samples and 56 matched normal controls were examined to evaluate whether the autoantibody can be detected in the preclinical stage.

Results: The proteins with molecular weight of 34 kDa were identified as ECH1, GAPDH and HNRNPA2B1. In the validation study, autoantibody to ECH1 achieved an area under the curve (AUC) of 0.799 with sensitivity of 62.2% and specificity of 95.5% in discriminating LC from normal individuals, and showed negative correlation with tumor size (rs=0.-256, p=0.023). Autoantibody to HNRNPA2B1 performed an AUC of 0.874 with sensitivity of 72.2% and specificity of 95.5%, and showed negative correlation with lymph node metastasis (rs=0.-279, P=0.012). By using longitudinal preclinical samples, autoantibody to ECH1 showed an AUC of 0.763 with sensitivity of 60.0% and specificity of 89.3% in distinguishing LC with matched normal controls, and elevated autoantibody levels could be detected greater than two years prior to LC diagnosis.

Conclusions: ECH1 and HNRNPA2B1 are autoantigens that elicit autoimmune responses in LC and can be used as potential biomarkers for the early detection of LC.

Funding support: This work was supported by the National Natural Science Foundation of China (81672917, 81372371) and the National Institutes of Health (SC1CA166016 and U01CA086137).

#711

Mechanistic implications of COL1A1 as a prostate cancer biomarker.

Andrej Jedinak,1 Camille Vuichoud,2 Andrew El-Hayek,2 Katherine Kaplan,2 Jason Savage,3 Sarah Prophet,4 Adam S. Feldman,4 Kevin A. Camphausen,3 Kevin R. Loughlin,5 Marsha A. Moses1. 1 _Boston Children's Hospital, Harvard Medical School, Boston, MA;_ 2 _Boston Children's Hospital, Boston, MA;_ 3 _National Cancer Institute, Bethesda, MD;_ 4 _Massachusetts General Hospital, Boston, MA;_ 5 _Brigham and Women's Hospital, Boston, MA_.

Prostate cancer (PCa) is the second most frequently diagnosed form of male cancer and shares similar symptoms with BPH (Benign Prostate Hyperplasia), a disease characterized by prostate enlargement. Elevated levels of prostate-specific antigen (PSA) can be observed with either benign or malignant growth of the prostate and therefore cannot effectively discriminate between these two prostate diseases. Currently, a test that sensitively and accurately distinguishes between BPH and localized prostate cancer does not exist creating an urgent need for novel biomarkers that can successfully distinguish between these two prostate diseases. The goal of this study was to identify and validate non-invasive urinary biomarkers that distinguish between BPH and PCa. Our previous proteomic study identified elevated levels of several proteins, including EGF (epidermal growth factor), HE-4 (human epididymis protein 4), COL1A1 (collagen, type I, alpha 1) and other proteins in the urine of PCa patients compared to urine samples from patients with BPH. In this current study we have analyzed and validated the presence of EGF, HE-4 and COL1A1 by enzyme-linked immunosorbent assay (ELISA). Our ELISA experiments revealed that COL1A1 was significantly (P < 0.002) elevated in the urine of patients diagnosed with early or localized PCa vs. BPH. In vitro experiments performed on seven different prostate cell lines identified cells from the tumor microenvironment that secrete the highest levels of COL1A1. Expression of COL1A1 by these cells was confirmed by immunohistochemistry (IHC) using prostate tissue microarrays (TMA). In addition, protein array experiments identified elevated levels of several proteases in the urine of PCa patients, including MMP-9, uPA, ADAM-TS1 and several cathepsins. Substrate gel electrophoresis (zymography) revealed elevated activity of both MMP-9 and MMP-2 in urine from PCa patients. These data suggest that MMP-9 and MMP-2 may participate in the cleavage of collagen type 1, resulting in elevated levels of COL1A1 in urine of PCa patients. We are currently testing this hypothesis. In summary, COL1A1 may represent a novel non-invasive urinary biomarker that can effectively discriminate between BPH vs. localized PCa. (Supported by The Ellison Foundation)

#712

Detection of lung cancer by assay of novel methylated DNA markers in plasma.

Hatim T. Allawi,1 Maria Giakoumopoulos,1 Evan Flietner,1 Austin Oliphant,1 Carla Volkmann,1 Brian Aizenstein,1 Tamara Sander,1 Drew Eckmayer,1 Ashley Poenitzsch Strong,1 Melissa Gray,1 Barry Berger,1 Tracy Yab,2 William Taylor,2 Douglas Mahoney,2 John B. Kisiel,2 David E. Midthun,2 David A. Ahlquist,2 Graham P. Lidgard1. 1 _Exact Sciences Corporation, Madison, WI;_ 2 _Mayo Clinic, Rochester, MN_.

Purpose: Lung cancer is the leading cause of cancer deaths worldwide. Most present symptomatically at late stage with high lethality. Early detection reduces mortality but accurate and readily accessible tools for population screening are limited. By whole methylome sequencing, we have identified novel methylated DNA markers (MDMs) for lung cancer in tissue (Giakoumopoulos et al. ASCO 2016). Using top candidate MDMs in the present study, we now explore their clinical accuracy for lung cancer detection when assayed from plasma.

Experimental Procedures: Archival plasmas from two independent study groups were tested in blinded fashion. Lung cancer cases and controls (apparently healthy smokers) for each group were balanced on age and sex (Group 1: 64 cases, 231 controls; Group 2: 23 cases, 80 controls). Using multiplex PCR followed by QuARTS (Quantitative Allele-Specific Real-time Target and Signal amplification) assay, a post-bisulfite quantification of MDMs on DNA extracted from plasma was performed. We selected 31 MDM candidates for initial evaluation in Group 1 (1 ml plasma/patient); top individual MDMs were subsequently tested in Group 2 to identify optimal MDM panels for lung cancer detection (2 ml/patient).

Results: From Group 1 analyses, 13 high performance MDMs were selected for further testing (CYP26C1, SOBP, SUCLG2, SHOX2, ZDHHC1, NFIX, FLJ45983, HOXA9, B3GALT6, ZNF781, SP9, BARX1, EMX1) with individual areas under the receiver operator curve (AUCs) ranging from 0.593 to 0.939. Discrimination by individual MDMs was corroborated in Group 2 in which data was analyzed using two methods: a logistic regression fit and a regression partition tree approach. The logistic fit model identified a 4-marker panel (ZNF781, BARX1, EMX1, and SOBP) with an AUC of 0.96 and an overall sensitivity of 91% and 90% specificity. Analysis of the data using a regression partition tree approach identified 4 markers (ZNF781, BARX1, EMX1, and HOXA9) with AUC of 0.93 and an overall sensitivity of 96% and specificity of 94%. For both approaches, B3GALT6 was used as a standardizing marker of total DNA input.

Conclusion: A panel of MDMs assayed in plasma achieved high sensitivity and specificity for all types of lung cancer. Further clinical evaluation and validation of this promising panel in larger patient groups are clearly indicated.

#713

Proteomic early detection biomarkers for ER+ breast cancer to improve mammography screening.

Carly B. Garrison,1 Tracey Marsh,1 Matthew Buas,2 Yuzheng Zhang,1 Margaret Pepe,1 Paul D. Lampe,1 Christopher I. Li1. 1 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 2 _Roswell Park Cancer Institute, Buffalo, NY_.

There are clear clinical and public health needs to improve the early detection of breast cancer in order to save lives. Breast cancer is the leading cause of cancer death in women worldwide and is the second leading cause of cancer mortality in women in the United States. Although mammography is widely used to screen for breast cancer, it suffers both from false negative and false positive readings resulting in later diagnoses or unnecessary follow-up (e.g., biopsies), respectively. Improvement of breast cancer early detection may be possible through the measurement of minimally invasive, easy to perform blood based biomarkers.

Our studies were performed on pre-clinical plasma samples collected up to 12.5 months before breast cancer diagnosis from cases and matched controls obtained from the Women's Health Initiative (WHI) Observational Study (a prospective cohort of 93,676 post-menopausal women). We utilized high-density antibody microarray methods for both discovery and preliminarily validation on distinct sample sets for early detection biomarkers of estrogen receptor positive (ER+) breast cancer (around 70% of all breast cancers are classified as ER+). Antibody microarray features (i.e., spots) identified as having ≥30% sensitivity at 80% specificity were considered to have met the cutoff criteria and identified as potential markers. A total of 129 features, corresponding to 101 proteins, were identified as potential markers in one WHI dataset (90 cases vs. 90 controls). Of the 129 features (101 proteins), 47 (34 proteins) validated by our cutoffs and demonstrated over or under-expression in cases compared to controls consistent with the first dataset in the second set of WHI samples (121 cases vs. 121 controls).

Given this level of reproducibility, we believe these biomarkers could be used to improve early detection of ER+ breast cancer for these clinical scenarios: 1. Informing timing of a subsequent mammogram in women with a negative screening mammogram; 2. Informing continuation of mammographic screening among women 75-79 years; 3. Prioritizing women who should be screened with mammography in areas with limited resources.

We are currently performing further validation of the protein markers in Early Detection Research Network (EDRN) specimen reference set from patients at the time of diagnosis. We plan to test our markers on a pre-diagnostic cohort from the Cardiovascular Health Study using a different, more clinically applicable platform. We used the same high-density antibody microarray platform to measure autoantibody-autoantigen complexes and sialyl Lewis-A and –X modifications in one of the WHI sample sets and are now testing these markers in the EDRN set for validation. Given the strength of our biomarker candidates, the high quality sets of biospecimens we used, the study designs employed, and the clearly delineated clinical applications proposed, we believe these markers could have near-term clinical impact.

#714

Human transcriptome alterations in pre-cancer and cancer epithelium identify candidate biomarkers of progression to pancreatic cancer.

Elena V. Komissarova, Jorge Sepulveda, Sarawut Kongkarnka, Maryam Shirazi, Brynn Levy, Claudia Cujar, Antonia R. Sepulveda. _Columbia University, New York, NY_.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most common cancers in the United States. The five-year survival rate for patients with PDAC remains dismal. Identification of biomarkers for early diagnosis of PDAC and pre-cancer pancreatic intraepithelial neoplasia (PanIN) lesions with risk of progression to PDAC is critically needed. We hypothesized that differentially expressed genes and regulatory pathways in PanIN and PDAC compared to normal duct epithelium (ND) may represent biomarkers of development of malignancy. We used Affymetrix Human Transcriptome Arrays 2.0 to establish gene expressionprofiles in ND, low-grade PanIN, and PDAC epithelium. Total RNA was isolated after laser capture microdissection (LCM) of frozen tissue sections and then used for producing hybridization-ready DNA. Hybridization quality control was performed with Expression Console 1.4 software and background corrected/normalized data were analyzed with Transcriptome Analysis Console (TAC) 3.1 and the ASSIGN algorithm. We tested RNA from 22 LCM samples (9 PDACs, 5 PanINs, and 8 ND), including 4 matched trios of ND, PanIN and PDAC from the same patients. Differential expression analysis with one-way between subject ANOVA revealed over 2000 genes differentially expressed in PDAC and PanIN vs. ND group (filter criteria up/down >2; ANOVA p<0.05). The most frequent alteration in PanIN compared to ND samples was upregulation of 433 genes and in PDAC compared to ND downregulation of 566. We found 60 (40 coding) upregulated genes and 750 downregulated genes (filter criteria up/down >1.5; ANOVA p<0.05) in both PanIN and PDAC vs. ND epithelium. Signaling pathway analysis of WikiPathways showed a number of significantly altered pathways in PDAC and PanIN compared to ND including the Gastric Cancer Network 1 with upregulated S100P in both PanIN and PDAC whereas other genes including CENPF, KIF20B,TPX2, and UBE2C were upregulated in PDAC only. Using the ASSIGN algorithm and the Kruskal-Willis test for analysis of difference in pathway activity, we found additional regulatory pathways with altered activity including Nuclear Receptor meta-pathway with reduced overall score in PDAC compared to PanIN and ND samples. In summary, over 400 genes were significantly up-regulated in pre-cancer PanIN lesions compared to normal duct epithelium, whereas gene down-regulation was the most frequent alteration in PDAC. Sixty genes, including 40 coding genes were up-regulated in both PDAC and PanIN. The altered pathways associated with the differentially expressed genes may represent an approach for integrated biomarker testing of neoplastic progression.

#715

The AroCell TK 210 ELISA may complement Pro PSA and the Prostate Health Index in differentiating pre cancerous and cancerous conditions in prostate cancer.

Jagarlamudi Kiran Kumar,1 Staffan Eriksson,1 Zupan Moca,2 Kumer Kristina,3 Josko Osredkar,3 Fabjan Teja,3 Hlebic Gregor,4 T Smrkolj4. 1 _Swedish Agricultural University, Uppsala, Sweden;_ 2 _Blood transfusion unit, Lubjliana, Slovenia;_ 3 _Institute of Clinical Biochemistry, Lubjliana, Slovenia;_ 4 _University Medical Centre, Lubjliana, Slovenia_.

Background: Thymidine Kinase 1 (TK1) is a cytosolic enzyme that plays an important role in DNA precursor synthesis and TK1 enzyme activity in serum is an established biomarker for prognosis and treatment monitoring, particularly for haematological malignancies. The AroCell TK 210 ELISA determines TK1 protein levels, in contrast to TK1 activity, and it has demonstrated superior sensitivity in distinguishing between subjects with solid tumours and controls.

Pro PSA, free PSA and the prostate health index (PHI) have been proposed as more discriminating tests than PSA in distinguishing between benign and cancerous prostate changes. The purpose of this study was to compare the AroCell TK 210 ELISA with PSA, free PSA, pro PSA and PHI in men with pre-cancerous conditions, and patients with confirmed prostate cancer.

Experimental Procedures: Serum samples from 94 patients with known PSA values (in the range 2 to 10 µg/L) that were under investigation for prostate cancer were collected at the University Medical Centre, Ljubljana. TK1 protein levels were determined using the AroCell TK 210 ELISA. PSA, fPSA and pro PSA levels were analysed with commercial assays (Hybritech PSA, Hybritech Free PSA and Access 2pPSA - Beckman Coulter USA) analysed on the Access 2 Beckman Coulter analyzer.

Results: Overall, 16/94 patients had serum TK1 protein levels above the estimated cut-off value for age-matched healthy men (0.45µg/L). 65/94 men had pre-cancerous prostate conditions, including benign prostate hyperplasia, prostatitis and high grade prostatic intraepithelial neoplasia, while 29 patients had confirmed prostate cancer (PCa). Only 7 /65 in the pre-cancerous group (11%) had TK1 protein levels above the cut-off, whereas in the prostate cancer group 9 out of 29 (31%) were positive. Moreover, the TK1 protein values in the PCa group (mean=0.41µg/L) differed significantly from those with pre-cancerous conditions (mean=0.31µg/L, P=0.01). Further analysis showed that pro PSA and PHI also differentiated the prostate cancer group from those with pre-cancerous conditions (P= 0.01 and P<0.001, respectively). This was not observed for fPSA and PSA. Overall, the AroCell TK 210 ELISA showed significant correlation with PHI (r=0.32, P=0.0017) and pro PSA (r=0.21 P=0.044) but not with fPSA or PSA.

Conclusions: This preliminary study has demonstrated that serum TK1 assayed with the AroCell TK 210 ELISA can differentiate between pre-cancerous and prostate cancer patients with a similar accuracy to that of pro PSA. Further clinical studies will establish the capacity of the AroCell TK 210 ELISA to complement pro PSA and PHI in distinguishing between pre-cancerous and prostate cancer, potentially providing another tool in prostate cancer management.

#716

Genomic classification of longitudinally observed small colorectal polyps.

Meta C. van Lanschot,1 Beatriz Carvalho,1 Charlotte J. Tutein Nolthenius,2 Christian R. Rausch,1 Ernst J. Kuipers,3 Jaap Stoker,2 Evelien Dekker,2 Gerrit A. Meijer1. 1 _Netherlands Cancer Institute, Amsterdam, Netherlands;_ 2 _Academic Medical Center, Amsterdam, Netherlands;_ 3 _Erasmus Medical Center, Amsterdam, Netherlands_.

Introduction:

Colorectal polyps are very common in elderly people with a prevalence of 15-30% reported in screening series. However, only a minority of 5% of these lesions is estimated to develop into invasive cancers, whereas others will remain stable or regress over time. In standard practice all polyps observed during colonoscopy are removed by polypectomy and therefore, knowledge about their natural history is limited. In this study, small (6-9mm) colorectal polyps were followed over time and subsequently resected during colonoscopy.

Aim:

The prevalence of DNA copy number alterations (CNAs), a feature associated with adenoma to carcinoma progression, was compared in polyps that morphologically progressed versus those that did not.

Methods:

In the CT-colonography (CTC) arm of the COCOS-trial1 small 6-9mm polyps were left in situ. After a three-year surveillance interval, growth according to CTC of 95 small polyps was measured. Based on overall volumetric change on CTC, polyps were classified as either progressed (>30% growth), stable (30% to -30% growth) or regressed (<-30% growth). From 65 resected polyps, formalin-fixed paraffin-embedded material was retrieved and reviewed by an expert pathologist (GAM). Of these polyps, 48% (31/65) progressed, 41% (27/65) remained stable and 11% (7/65) regressed. Seventy-two percent (47/65) were tubular adenomas, 14% (9/65) tubulovillous adenomas, 3% (2/65) sessile serrated lesions and 11% (7/65) hyperplastic polyps. Subsequently, the samples that yielded sufficient quality DNA were analyzed with low-coverage whole genome sequencing (HiSeq, Illumina, San Diego, CA, USA). DNA copy number status was called with the R-package QDNAseq.

Results:

From 50/68 resected polyps, originating from 38 individuals (mean age of 66.8 years (s.d. 7.0); 63% male), sufficient DNA was available. The mean surveillance interval of these polyps was 3.27 years (s.d. 0.29).

The mean number of CNAs per sample was 0.8 (range 0-8), the most frequently observed was 13q gain (20%). No significant differences in number, or regions of CNAs were observed between polyps that progressed and the ones that did not change or diminished in size (stable or regressed). Larger polyps showed more CNAs, of which specifically 13q gain was more often present (43% in ≥10mm and 8% in <10mm polyps). Of note, no serrated lesions were ≥10mm. No differences in CNAs were found between the different histological subtypes.

Conclusions:

In a series of colorectal polyps left in situ for an average 3.27 years, no difference in prevalence of DNA copy number alterations was observed between polyps that volumetrically progressed and those that did not. Of the CNAs observed, 13q gain was most prevalent and found in 43% of the ≥10mm polyps.

1. Stoop, E. M. et al. Participation and yield of colonoscopy versus non-cathartic CT colonography in population-based screening for colorectal cancer: A randomised controlled trial. Lancet Oncol. 13, 55-64 (2012).

#717

Diagnosis of basal like breast cancer using DNA methylation markers at the promoters of long noncoding RNAs.

Yoo J. Han, Sonja M. Boatman, Jing Zhang, Albert C. Yeh, Yonglan Zheng, Olufunmilayo I. Olopade. _University of Chicago, Chicago, IL_.

Background: Patients with basal-like breast cancer (BLBC) have poor overall survival and suffer a high rate of metastasis to the brain or lung within three to five years of initial presentation. Absence of a cure for advanced BLBC warrants early detection of BLBC, which might save more lives, especially those of women of African ancestry who are disproportionately affected by young onset BLBC. Aberrant DNA methylation is frequently observed in BLBC. DNA methylation is the most robust epigenetic marks and can be analyzed using clinical specimens including FFPE, tumor biopsies and liquid biopsies. Because expression of long non-coding RNAs (lncRNAs) is controlled temporally in response to neoplastic stimuli, we investigated the potential for lncRNA promoter methylation marks to be used for detection and prediction of BLBC development and progression in African Americans.

Methods: To identify lncRNAs dysregulated in BLBC, we performed a Ribo-Zero RNA-seq or microarray analysis on breast tissues isolated from African American (n=63) and European American patients (n=14). Differentially methylated and hydroxymethylated regions of lncRNA genes were identified in African American samples (n=30) using our chemical methods to assay 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). The Cancer Genome Atlas (TCGA) datasets were utilized to validate our results. To functionally annotate lncRNAs, we knocked down lncRNAs using LNA-antisense oligonucleotides (ASO) and performed Caspase 3/7 assays, flow cytometry, and qRT-PCR. We also utilized the CRISPR-Cas9 editing tools to knock out lncRNAs.

Results: LncRNAs displayed distinct expression patterns between tumors and normal breast tissues. Out of hundreds of lncRNAs specifically expressed in BLBC tumors, we selected two lncRNAs, lnc19 and lnc98, which represent, respectively, a significantly increased and decreased lncRNA in BLBC tumors, compared to normal breast tissues and other subtype tumors. Lnc19 is highly up-regulated (a 39-fold increase) whereas lnc98 is dramatically down-regulated (a 59-fold decrease) in BLBC tumors. Methylation analysis showed significantly lower levels of promoter methylation for lnc19 and higher levels for lnc98 in BLBC tumors. A significant inverse correlation between methylation and expression of lnc19 was observed. Depletion of lnc19 resulted in rapid cell death of BLBC cells, with increased sensitivity to chemotherapy drugs. The data suggests that chemo-resistance of BLBC might be partly due to lnc19 overexpression, which is mediated through epigenetic control.

Conclusion: We identified two epigenetically dysregulated lncRNAs in BLBC tumors, which contribute to chemo-resistance of these tumors. Our findings on the regulation of lncRNAs by cytosine methylation raise the possibility of new epigenetic biomarkers for diagnosis and prognosis of aggressive BLBC tumors.

#718

Biomarker monitoring by quantitative MALDI imaging; application to the tryptophan-kynurenine pathway in immuno-oncology.

Rima Ait Belkacem,1 Vanessa Bol,2 Gregory Hamm,1 Stefan Linehan,1 Bruno Gomes,2 Jonathan Stauber1. 1 _Imabiotech, Loos, France;_ 2 _iTeos Therapeutics SA, Gosselies, Belgium_.

Tryptophan is an essential amino acid for cell proliferation and survival that can be metabolized through different pathways, a major route being the kynurenine pathway. The first and rate-limiting enzyme of this pathway is the indoleamine-2,3-dioxygenase 1 (IDO1), that is a natural endogenous molecular mechanism of immune suppression acting through modulation of the Trp degradation pathway. Inhibition of T-cell functions, activation of the regulatory T-cells, and inhibition of Natural Killer cells are among the important immunosuppressive effects of IDO1. IDO1 enzyme is proposed to have a therapeutic potential in immunodeficiency-associated abnormalities, including cancer. IDO1 over expression has been described in multiple cancer indications and IDO1 inhibitors are being developed to stimulate the anti-tumor immune response. The existing and standard quantitation methods are based on the total level of Trp and its metabolites determined by LC-MS/MS analysis in human plasma, cerebrospinal fluid and brain. We describe the setup, development and application of a new method based on MSI to detect, localize, and quantify Trp and Kyn in biological tissues. This method allows both the study of the sub-tissular localization and the detection/quantitation of metabolites of interest in tumor tissues. In the present study, an experimental tumor model overexpressing IDO1 and its wild-type counterpart were implanted in mice. Tissue sections of different tumors were realized and used for MSI analysis. MALDI FTICR high resolution imaging followed by data analysis enabled an absolute on tissue quantitation. Internal standards of tryptophan (Trp-d5) and kynurenine (Kyn-d4) metabolites were used for normalization. As expected, our results showed an increase of Kyn in parallel to a decrease of Trp amount in IDO1-positive tumors. Following, immunostainings of IDO1 and Trp-depletion sensor pathways were carried. Overlaying images between the immunostainings and the molecular MS images allowed co-localization studies and underlined both the biology and the tumor heterogeneity. This study allowed us to highlight key metabolites of the Trp pathway that are responsible for the immunosuppressive tumor microenvironment. It illustrated the heterogeneity of tumor immune areas.The development of immunotherapies such as IDO1 inhibitors requires a deep understanding of the interplay between the immune system and cancer cells, these immune endogenous metabolites can now be followed by QMSI and as biomarkers towards enhancing immunotherapies efficiency.

#719

Pathobiological implications of Trefoil Factors in the progression and metastasis of pancreatic cancer.

Rahat Jahan, Sukhwinder Kaur, Muzafar A. Macha, Yuri Sheinin, Lynette Smith, Jane Meza, Surinder K. Batra. _University of Nebraska Medical Center, Omaha, NE_.

Background: Pancreatic cancer (PC) is a highly lethal malignancy and the disastrous statistics warrants an urgent need to develop new diagnostic and prognostic marker(s) and therapeutic target(s) for its better management. Trefoil factors (TFF 1/2/3) are a family of small, stable molecules observed to co-express with mucin and secreted by mucus-producing cells that facilitate mucosal repair after injury. Recent studies have shown that TFF1 and TFF3 can differentiate between molecular subtypes of PC. Based on this, we hypothesized that differential expression of TFF1 and TFF3 could predict pancreatic intraepithelial neoplasia (PanIN) as well as the early stage of PC. The present study explores the diagnostic and functional significance of TFF in PC.

Material and Methods: Immunohistochemistry was used to determine the expression status of TFF1 and TFF3 in PC tissue microarray containing normal pancreatic (n=40), pancreatic intraepithelial lesion 1, 2, 3 (PanIN 1- 3, n=13) and well, moderately and poorly differentiated PC tissues (n=149). Sandwich ELISA was used to detect TFF1 levels in serum. The diagnostic significance of TFF1 was evaluated in the sample set (n=331) comprising of benign control (BC, n=104), chronic pancreatitis (CP, n=47), early stage PC (EPC, Stage 1 and 2, n=78) and late PC (LPC, Stage 2-3, n=69). RT-PCR and ELISA were carried out to profile levels of TFF1 and TFF3 in PC cell lines and cell supernatants respectively. Recombinant treatment with TFF3 was performed to observe the impact on of transmembrane (MUC4) and secreted mucins (MUC5AC) stability and expression.

Results: Differential expression of TFF3 was observed in PanIN precursor lesion, pancreatic cancer tissue in comparison to normal pancreatic tissue (p < 0.0003). Furthermore, the TFF3 expression was significantly higher for well-differentiated tumor in comparison to poorly differentiated cases (p = 0.006). For TFF1, expression was significantly higher in PanIN lesions compared to normal pancreas (p < 0.0001), and PC versus normal pancreatic tissue (p < 0.0001). In serum, significantly increased expression of TFF1 was observed in EPC serum samples compared to BC (p-value = 0.035) and CP (p-value=0.0058) cases. RT-PCR analysis revealed positive expression of TFF1 and TFF3 in the majority of the PC cell lines. Treatment of PC cells with recombinant TFF3 lead to differential expression of MUC4 and MUC5AC.

Conclusion: Overall, both tissue and serum based studies show that TFF1 and TFF3 are differentially expressed in early PanIN lesions as well early stage of PC tissues compared to normal pancreas. Overall, these results suggest a potential implication of TFF1 and TFF3 in PC pathogenesis and provide a basis for functional impact of TFF 1 and 3 on mucins stability and downstream signaling in PC pathogenesis.

#720

Systematic discovery and evaluation of tumor-associated antigens (TAAs) as biomarkers in cancer immunodiagnosis.

Jianxiang Shi,1 Lu Zhang,1 Peng Wang,1 Hua Ye,1 Liping Dai,1 Pei Li,2 Chenglin Luo,2 Chuhua Song,1 Kaijuan Wang,1 Xiao Wang,1 Jun Ouyang,1 Zhenyu Ji,1 Jianying Zhang1. 1 _Zhengzhou University, Zhengzhou, China;_ 2 _The University of Texas at El Paso, El Paso, TX_.

Proteins aberrantly over-expressed in tissues from nineteen types of cancers have been identified from the Human Protein Atlas (HPA) database. Among these proteins, some were overexpressed in several types of cancers while certain proteins were found only overexpressed in certain type of cancers. It is demonstrated that aberrantly overexpressed proteins might shed into the blood and trigger the immune system to produce autoantibodies during the process of oncogenesis. Autoantibodies are more stable and often have higher titer compared to autoantigens. Therefore, we hypothesize that autoantibodies against these aberrantly overexpressed proteins may have the potential to serve as diagnostic biomarkers in certain type of cancers. In current study, we have selected 11 proteins, including EDNRB, KRT4, PLAT, MSLN, WFDC2, F5, FOXA1, AURKC, STC1, CAB39L, and SFTPA1, to further evaluate the diagnostic values of their corresponding serum autoantibodies in five types of cancers, including hepatocellular carcinoma, lung cancer, gastric cancer, breast cancer and ovarian cancer. Sera from 470 patients (94 per cancer type), 94 hepatitis patients and 192 age- and gender-matched normal controls were used in current study. Serum autoantibodies were detected by ELISA. Receiver operating curve (ROC) analysis was used to evaluate the diagnostic value of serum autoantibodies against these potential TAAs. The data indicates that several anti-TAA autoantibodies can be found to have diagnostic value for certain types of cancers. Autoantibodies against PLAT can distinguish breast and ovarian cancer while autoantibodies against SFTPA1 and CAB39L can distinguish cancers both in male and female. However, none of these serum anti-TAAs was found to be specific to a single type of cancer. In summary, HPA database has made systematic discovery of cancer biomarkers possible. Whether these in silicon biomarkers can be used to distinguish normal and cancer still needs to be evaluated in more serum samples from normal controls and cancer patients.

#721

Minimally invasive test and composite biomarker for early detection of serous ovarian carcinoma.

Keren Bahar-Shany,1 Georgina D. Barnabas,2 Limor Helpman,3 Ariella Yakobson-Siton,4 Tamar Perri,1 Ram Eitan,4 Jacob Korach,1 Tamar Geiger,2 Keren Levanon1. 1 _Sheba Medical Center, Ramat Gan, Israel;_ 2 _Tel Aviv University, Tel Aviv, Israel;_ 3 _Meir Medical Center, Kfar Saba, Israel;_ 4 _Rabin Medical Center, Petach Tikva, Israel_.

Background: Current screening programs for early detection of high grade epithelial ovarian cancer (HGOvC) among high-risk populations have failed to show improvement in HGOvC mortality, therefore these women are offered risk-reducing bilateral salpingo-oophorectomy (RRBSO) at 35- 40 years. Stratification of high-risk population, especially BRCA mutation carriers, may enable personalized risk counseling and individualization of timing of RRBSO. In most cases, the precursor lesions of HGOvC arise in the epithelium of the fallopian tube (FT) fimbriae rather than intra-peritoneally. It is therefore plausible that proteins, RNA or DNA from early-stage tumor cells may be identifiable in fluid samples obtained from the lumen of the gynecological tract, thus making it possible to identify curable, early stage lesions.

Aims: (1) Test the feasibility of uterine lavage as a minimally invasive test for early detection of ovarian cancer, and (2) Identify novel early-detection biomarkers in the uterine lavage fluid (UtLF).

Methods: We developed a method for sampling of gynecologic tract fluid termed uterine lavage fluid (UtLF), which is a simple, reproducible, low-cost office procedure that can be performed routinely during gynecologic follow-up visits. We have already collected UtLF from 140 HGOvC patients and control women undergoing gynecologic surgical procedures for non-malignant indications. Deep proteomic profiling of UtLF is performed by isolation of microparticles from body fluids, followed by solubilization, trypsin digestion and high resolution mass spectrometric (MS) analysis (on the Q-Exactive MS). Machine learning algorithms have been used to extract a classifier that can predict the diagnosis of ovarian cancer.

Results: Uterine lavage appears to be a feasible, low burden procedure. The MS approach has identified thousands of proteins in each UtLF specimen, in a high throughput manner. The label-free quantification algorithm (MaxQuant) enables a quantitative comparison between samples from cases and controls. We have derived a 20-protein classifier with an area under the curve (AUC) of Receiver Operating Characteristics (ROC) curve of 0.91 at 20% error. The composite biomarker has been applied to an independent validation set with a negative predictive value (NPV) of 92% and positive predictive value (PPV) of 45%.

Conclusions: A minimally invasive technique of uterine lavage to collect unique diagnostic samples, coupled with state-of-the-art proteomics methods, results in a highly sensitive and specific composite biomarker which may be developed in to a screening tool for early detection of serous ovarian cancer in high-risk populations.

#722

Circulating EBV microRNA BART2-5p helps to diagnose nasopharyngeal carcinoma &lt screen for high risk individuals precisely.

Chen Jiang,1 Lei Li,2 Li Jiangchao,3 Shang-hang Xie,2 Su-mei Cao,2 Xin-yuan Guan1. 1 _The University of Hong Kong, Hong Kong, China;_ 2 _Sun Yat-Sen University Cancer Center, Guangzhou, China;_ 3 _Guangdong Pharmaceutical University, Guangzhou, China_.

Nasopharyngeal carcinoma is an EB-virus associated malignancy which is highly prevalent in Southeast Asia. If diagnosed early, the 5 year disease-free survival rate is about 90%. However, most patients have already developed local or regional invasion when diagnosed, which results in poor prognosis. So it is essential to develop biomarkers to screen high risk individuals or diagnose the disease at early stages. As nasopharyngeal carcinoma is closely related to EBV infection and microRNAs encoded by BART region (BamHI A Rightward Transcripts) is abundant in NPC, we propose to identify biomarkers from those BART microRNAs. To quantify the copy number of microRNAs in serum samples, we used Taqman Probe-based qPCR. After adding Spiked-in Control (Mixture of Cel-39,Cel-54, Cel-238) to each 100ul serum sample, we extracted total RNA and reverse transcribed microRNA into cDNA with corresponding Taqman primers. Meanwhile, microRNA mimics with known copy number were serial diluted and reverse transcribed along with the samples. qPCR was performed to quantify the level of microRNAs in these samples with diluted cDNA and Taqman Probe. All data was normalized by Spiked-in controls and exact copy number of each microRNA was calculated through the standard curve generated by the serial diluted microRNA mimics. Here, we screened 17 BART microRNAs in discovery stage and found BART 2-5p as a candidate biomarker. In training cohort consisting of 266 patients with nasopharyngeal carcinoma and controls from Hong Kong, the sensitivity, specificity and AUC is 93.2%, 89.8%,0.976 respectively. The results are similar in validation cohort 1 constituted by 376 patients and controls from Guang Zhou. To evaluate the ability of BART 2-5p to distinguish preclinical NPC patients from healthy high risk individuals, we established a nested case-control study with serum samples prospectively collected from 10 NPC patients at least one year prior to their clinical diagnosis and 92 matched healthy high risk controls from a screening scheme conducted in South China. The sensitivity and specificity is 90.0% and 31.5%. Taken together, EBV microRNA BART2-5p is a promising biomarker to improve NPC diagnosis and target preclinical patients more precisely than serologic test.

#723

Improved detection of cancer specific serum exosomal aspartyl (asparaginyl) beta hydroxylase (HAAH).

Mark A. Semenuk, Anokhi S. Cifuentes, Eleanor R. Ghanbari, Michael S. Lebowitz, Hossein A. Ghanbari. _Panacea Pharmaceuticals, Inc., Gaithersburg, MD_.

By means of an ELISA, we have found the pan-cancer biomarker HAAH to be closely associated with serum exosomes primarily in cancers of the breast, lung, colon, and prostate. This new understanding of the HAAH target has led to a dramatically improved serological detection assay, and in turn diagnostic reagent kit. In keeping with the multivalent particulate nature of exosomes, appropriately reformatting the ELISA assay has yielded a simplified so-called simultaneous-homologous format. Currently in this format, a single monoclonal anti-HAAH antibody (FB50) serves both as microplate capture and as a biotinylated detector. All reagents, including peroxidase-streptavidin, are co-incubated in the FB50- coated microplate simultaneously with serum samples, in the absence of intervening sequential steps.

This considerably shorter exosome-enabled ELISA format requires half the time and yields generally on average a two-fold or higher increase in detection of HAAH. This format was evaluated with over 100 banked serum samples from several early stage cancers. While increasing the sensitivity for HAAH in cancer samples, the improved detection and performance has not caused any undesirable increases in false positives among more than 30 normal serum samples from healthy subjects. The reagent kit components include recombinant HAAH calibrators, positive /negative controls, vialed detection reagents and a Mylar packaged FB50 pre-coated microplate. The simplified reagent kit format has yielded close inter-operator and day to day trending consistency with a limit of detection (LOD) of 3 ng/mL.

Recognizing the exosomal nature of the HAAH target has resulted in some changes in blood sample procurement, sample processing, and sample shipping. Such field testing of the HAAH reagent kit was recently done with serum samples from 48 high risk or mildly symptomatic volunteers with general concerns about cancer. Upon testing these samples 9 had an HAAH level greater than 3.0 ng/ mL (range 4.2 to 116.7) and 39 had less than 3.0 ng/ mL. Based upon the post-analysis diagnostic determination, there were two false negatives and one false positive, hence an overall accuracy of 93.8%.

#724

Clinical impact of modified telomerase-specific adenovirus-based identification of viable-peritoneal tumor cells in peritoneal lavage fluid in patients with potentially resectable pancreatic cancer.

Masahiro Tanemura,1 Kenta Furukawa,1 Soichiro Mori,1 Masahisa Otsuka,1 Youzo Suzuki,1 Mitsuyoshi Tei,1 Toru Masuzawa,1 Kentaro Kishi,1 Yasuo Urata,2 Hiroki Akamatsu1. 1 _Osaka Police Hospital, Osaka, Japan;_ 2 _Oncolys BioPharma Inc., Tokyo, Japan_.

Pancreatic cancer (PC) is a highly aggressive disease with dismal prognosis. Although only a surgical resection can offer the chance of a cure, the 5-year survival rate after a potentially curative resection have been reported to be a low as 10-30 %. In PC, the presence of peritoneal carcinomatosis preclude the possibility of surgical cure, irrespective of the resectability of the primary tumor. Peritoneal lavage cytology (CY) is used widely in the diagnosis and staging of gastric and pancreatic cancer. Positive CY findings (CY+) in PC is defined as stage IV disease, however, the true value of CY+ for the patient's prognosis remains controversial. We rise the question of whether CY+ status has predictive value for survival and early intraperitoneal recurrence. The aim of this study was to evaluate use of a new genetically modified telomerase-specific replication-selective adenovirus, expressing GFP (TelomeScan F35) in rapid detection of viable peritoneal tumor cell (v-PTC) dissemination of PC. This human clinical trial sought to determine if the presence of virally-detected, rare v-PTC predict peritoneal recurrence and patient outcome. This study was approved by the Osaka Police Hospital IRB. Patients with resectable cytologically or histologically proven ductal adenocarcinoma of the pancreas were enrolled. Peritoneal lavage fluid was harvested just after a laparotomy in 27 patients with PC. Half of the fluid was examined by cytology with papanicolau staining and MOC-31 immunostaining and the remaining half was used to detect v-PTC with TelomeScan F35. To distinguish between leucocyte and cells with epithelial origin, cells were stained with anti-CD45 Ab. To further distinguish cells with primary tumor origin, cells were labeled with anti-CEA and anti-CA19-9 Abs. GFP-positive and CD45-negative, and either CEA- or CA19-9-positive cells were counted as v-PTC. Patients were followed after surgery to evaluate its clinical significance. Among 27 patients aged 57-91 years (16 males and 11 females), 3 were cytologically positive (CY+), other 3 were virally positive by TelomeScan F35 (v-PTC+). All 27 patients underwent a surgical resection (PD/DP/TP/H-PD=13/7/6/1). One patient was double positive (CY+/v-PTC+), and postoperative peritoneal recurrence early occurred at 5 month after resection despite adjvant chemotherapy. 2 were CY+, but v-PTC-, and no recurrence in the abdominal cavity were observed (0%). On the other hand, other 2 were CY-, but v-PTC+, and one of these 2 patients occurred local recurrence in the abdominal cavity (50%). Remaining 22 patients (CY-/v-PTC-) were observed with neither local recurrence nor distant metastasis. In conclusion, the TelomeScan F35-based v-PTC detection may be an independent prognostic factor in patients with resectable PC and had close association with local or peritoneal recurrence.

#725

Feasibility and evaluation of exosomal RNAs as novel diagnostic biomarkers for high grade serous epithelial ovarian cancer.

Emily N. Prendergast, Xianzhi Lin, Rosario I. Corona, Dennis J. Hazelett, Beth Y. Karlan, Kate Lawrenson. _Cedars-Sinai Medical Center, Los Angeles, CA_.

Introduction: High-grade serous ovarian cancer (HGSOC) survival has been essentially unchanged over the last ten years despite new therapeutic strategies. In order to make a large-scale impact on overall survival, early detection strategies are imperative. Exosomes are cell-derived vesicles derived from multivesicular bodies or the plasma membrane that carry a variety of biomolecules, including protein, DNA, and RNA. Exosomal contents represent a novel and underexplored source of tumor biomarkers, in particular for HGSOC. This study aimed to first evaluate approaches to exosomal RNA purification and to then compare the exosomal RNA profiles from serum of patients with benign ovarian disease and HGSOC to determine if unique profiles exist, and to identify biomarkers useful for early detection.

Methods: Cell free serum was obtained from patients in each of the following categories: HGSOC with early (n=24) or late stage (n=24) disease, benign ovarian masses (n=24) and healthy age-matched controls (n=24). We compared ultracentrifugation and a commercial precipitation solution to enrich and isolate exosomes. Exosomal enrichment was confirmed by both transmission electron microscopy (TEM) and western blot using anti-CD63 antibodies. RNA was extracted for each and deep sequencing analysis was performed for 12 candidates within each group. A second cohort of 48 samples will be used for validation of the best performing candidate biomarkers from the discovery cohort via RT-qPCR.

Results: Enrichment of exosomes was confirmed via qualitative assessment using TEM and Western blot analysis. RNA quality was superior when RNA isolations were performed using the QIAgen RNeasy kit compared to the AllPrep kit. Mean RNA yield for each group ranged from 10.3-28.6ng/µl and RNA yield and quality was independent of sample age. RNAseq analyses are ongoing to identify biomarkers specific to HGSOC that can be detected at the earliest stages. The top differentially RNAs for the ovarian cancer groups will be validated in an independent cohort of specimens. A panel will then be generated in combination with CA125 to determine the clinical utility of an integrated -omics approach in early stage detection of HGSOC.

Conclusions: Efficient isolation of exosomes and RNA can be achieved from archived specimens >10 years old. Evaluation of exosomal RNA in HGSOC provides a novel method for biomarker discovery.

#726

Ultrasensitive quantification of promoter methylation in cell-free circulating DNA for early detection of lung cancer.

Delphine Lissa. _NIH/NCI, Bethesda, MD_.

Lung cancer is the leading cause of cancer-related deaths worldwide. Early detection of lung cancer using Low Dose Computed Tomography (LDCT) screening has been shown to decrease the mortality rate. However, most nodules found are deemed to be benign upon further invasive testing. Thus, complementary minimally-invasive tests are being sought that will help discriminate malignant from benign nodules. Molecular biomarkers are increasingly becoming part of routine clinical practice for the diagnosis, prognosis or prediction of treatment response, with improved disease management and survival outcome. Cell-free circulating DNA (cfDNA) in body fluids, including serum, plasma and urine, has recently emerged as a surrogate for tumor DNA. In addition to providing a minimally-invasive source of tumor DNA, cfDNA reflects molecular alterations and tumor heterogeneity. Epigenetic changes, including DNA methylation, occur early in carcinogenesis. Cancer cells are characterized both by global hypomethylation and hypermethylation of CpG islands in gene promoter regions. Analysis of tumor-specific DNA methylation in cfDNA is a promising strategy for applying epigenetic biomarkers to the detection of cancers at an early-stage. In a prior genome-wide analysis of DNA methylation, we identified a locus methylated de novo in fresh frozen tumor tissues resected from stage I lung cancer patients, that had high discriminatory power to distinguish tumor from non-tumor tissues in multiple patient cohorts. High promoter methylation was also associated with shorter cancer-specific survival. The present study aims at evaluating the diagnostic significance of promoter methylation in cfDNA, and the prognostic value in formalin-fixed paraffin-embedded (FFPE) tissues. We developed a methylation-specific droplet digital PCR (ddPCR) assay to quantify rare methylation events. DNA was subjected to bisulfite treatment to convert unmethylated cytosine residues to uracil. We designed specific primers and probe containing 7 CpGs to only amplify the methylated promoter. Experimental conditions were first optimized using fully methylated and unmethylated control DNAs, DNA extracted from lung cancer cell lines, germline cells and lung tissues (paired tumor and non-tumor). The ddPCR assay has a limit of detection of 30 haploid genomes equivalent of methylated promoter DNA, and a limit of quantification of a single methylated allele present at 0.2% (i.e., 1 methylated copy among 500 unmethylated copies). We demonstrated the assay linearity, reproducibility and specificity for the methylated locus. Differences in methylation levels between tumors and adjacent tissues were also observed. We have thus established a robust and ultrasensitive method for standardized determination of promoter methylation status in cfDNA. We are currently evaluating its potential value for noninvasive diagnosis and prognosis of lung cancer patients.

#727

Prospective lung cancer diagnostic screening using whole, unstimulated saliva versus stool.

Yosef Y. Tobi,1 Fadi Antaki,2 MaryAnn Rambus,2 Martin Tobi3. 1 _R &D Detroit VA Medical Ctr, Detroit, MI; _2 _Detroit VA Medical Ctr., Detroit, MI;_ 3 _Saginaw VA Medical Ctr., Saginaw, MI_.

Current screening for lung cancer (LC) reduces mortality but tests are not ideal. Low-dose CT scans are expensive, incur radiation exposure, and high false positive rates. Although sputum cytology is unhelpful, saliva biomarker testing is promising but no prospective data are available.

Methods: In conducting a study using stool markers in a high risk colorectal neoplasia population we prospectively collected saliva and stool samples. Subsequently, 11 with available saliva; 8 with stool specimens but without significant personal or family history of GI neoplasia or symptoms, had contracted LC. We used 3 monoclonal antibodies to test reactivity in saliva (Adnab-9, BAC 18.1, COX-2). These are biomarkers of innate immune system, cell-mediated immunity, and inflammation, respectively. We used ELISA with either alkaline phosphatase (ALP) or immunoperoxidase (IMP) substrate and contrasted reactivity in LC patients and asymptomatic controls with no advanced polyps, and chemiluminescent dot blots with manual or Bel-blotter 96-well replicating tool.

Results: Saliva IMP testing was positive in 73% of 11 LC patients and 50% of 8 controls contrasted with ALP ELISA for stool Adnab-9 in 75% of 8 LC patients and 32% in 34 controls (OR 6.27:CI1.09-36.25;p<0.05).Specificity was 68%. Sensitivity for Adnab-9 for IMP manual and bel-blotting was 73 and 9% respectively; specificities were 37 and 64% respectively. BAC 18.1 sensitivities were 73 and 55% respectively; specificities 13 and 29%. COX-2 sensitivity for bel-blotting only was 27% and specificity was 71%. Inherent salivary peroxidase activity (OD<0.1/1μg protein) was negative in all 7 LC versus 9 of 23 (39%) of non-LC patients (p=0.07). The peroxidase absorbance means[SD] were significantly different (0.077[0.014] versus (0.116[0.056];p<0.007). Equivalent inherent alkaline phosphatase of saliva samples was negative in both groups and means were not significantly different. The approximate time from saliva collection to diagnosis of LC was 3.76 years and 3.89 for stool.

Conclusions: Adnab-9 sensitivity was moderate but promising due to the ability to make an early preclinical diagnosis. While this was only significantly different from controls in stool ALP ELISA, inherent IMP activity could be blocked to improve specificity. Significantly suppressed inherent peroxidase activity in LC saliva may explain the insensitivity of the Bac18.1 and Cox2 inflammatory biomarkers. The Bel-blotter volume capacity is 4-10μl/blot and may explain the lower sensitivity using this tool. A battery of tests, including Adnab-9 in an ALP ELISA format may allow for early disease intervention.

#728

Development and validation of ColoScape™ - a new colorectal cancer mutation detection assay.

Michael J. Powell, Elena Peletskaya, Anne Vallerga, Qing Sun, Larry Pastor, Aiguo Zhang. _DiaCarta, Richmond, CA_.

Introduction: Colorectal cancer is a highly preventable disease as early detection increases rates of patient survival to near 100%. Herein we report the development and validation of a novel multigene mutation biomarker real-time PCR based assay for qualitative detection of colorectal cancer associated biomarkers that comprise tumor specific mutations in the following genes: APC (Exon 15), KRAS (Exon 2), BRAF (Exon 15) and CTNNB1 (Exon 3) called ColoScapeTM . The assay allows the sensitive detection of the presence or absence of mutations in the targeted regions of the genes interrogated.

Methodology: The high sensitivity of this multigene biomarker assay is achieved due to xenonucleic acid (XNA) probe technology. XNA probes are novel backbone modified oligomers having natural nucleoside bases (A,T,C and G) that hybridize by Watson-Crick base pairing to natural DNA and RNA with much higher binding affinity than natural deoxyribonucleic acid oligomers of the same sequence. XNA probes are designed that bind to the selected wild-type sequences at the respective genetic loci in the target genes. These XNA probes have a much higher Tm than the primer annealing temperature and suppress amplification of WT DNA templates and only allow amplification of the target mutant DNA templates in the sample. Both single nucleotide polymorphisms (SNP's) and insertion/deletions (indels) mutations can be detected. For each of the selected mutation sites primers and FAM-labeled TaqMan hydrolysis probes were designed and tested together with the selected XNA oligomers and analytical assay performance confirmed. An internal PCR control amplicon selected in the Human β-Actin (ACTB) gene was employed utilizing a HEX-labeled TaqMan probe. Performance parameters of the assay were established on DNA of colorectal cancer patients extracted from FFPE as well as reference DNA materials (synthetic and cell line derived DNA). The assay demonstrates high sensitivity and specificity in detection of colon cancer and adenoma samples based on the set of biomarkers involved in colorectal cancer neogenesis and disease progression.

Conclusion: The ColoScapeTM Colorectal Cancer Mutation Detection assay is shown to be a sensitive tool intended to facilitate research in colon cancer development, early detection, disease monitoring and therapeutic interventions.

#729

A novel cancer-stem-cell biomarker, DCLK1-S, traffics to nuclei of colon cancer cells: potential use as a biomarker for assessing colon cancer risk after screening colonoscopy.

Shubhashish Sarkar, Vsevolod L. L. Popov, Malany O'Connell, Heather L. Stevenson, Brian S. Lee, Robert A. Obeid, Pomila Singh. _UT Medical Branch, Galveston, TX_.

DCLK1 expression is critically required for maintaining growth of human colon cancer cells (hCCCs). We and others recently reported that the 5'promoter of hDCLK1-gene gets hypermethylated and silenced in human colorectal tumors (hCRCs) during adenoma-carcinoma sequence of colon carcinogenesis (Reviewed by Singh et al. SCI, 2016). We also made the discovery that hCRCs and hCCCs express a novel short isoform of DCLK1 (DCLK1-S) (isoform2) from β-promoter in intron V of hDCLK1-gene, while normal-colons express the canonical long-isoform (DCLK1-L) (isoform1) from 5'(α)-promoter, suggesting that DCLK1-S, and not DCLK1-L, marks cancer-stem-cells (CSCs) (O'Connell et al, Sci Rep, 2015). Even though DCLK1-S differs from DCLK1-L by only six amino-acids, we succeeded in generating a mono-specific DCLK1-S-Antibody (PS41014), which does not cross-react with DCLK1-L, and specifically detects DCLK1-S in several assay platforms, including westerns and IHC. Sub-cellular localization of S/L isoforms was examined by immune-electron-microscopy (IEM). Surprisingly, besides plasma membrane and cytosolic fractions, S/L also localized to nuclear/mitochondrial fractions, with pronounced localization of S-isoform in the nuclei and mitochondria of hCCCs. Sporadic CRCs develop from adenomas (Ads). Screening colonoscopy is used for detection/resection of growths, and morphological/pathological criteria are used for risk assessment and recommendations for follow-up colonoscopy. But, these features are not precise and majority of the patients with adenomas will never develop CRC. We hypothesized that antibody-based assay(s), which identify CSCs, will significantly improve prognostic value of morphological/pathological criteria. We conducted a pilot retrospective study with PS41014-Ab, by staining archived adenoma specimens from patients who developed (High-risk) or did not develop (Low-risk) adenocarcinomas within 10-15 years. PS41014-Ab stained adenomas from initial and follow-up colonoscopies of high-risk patients, at significantly higher levels (3-5X) than adenomas from low-risk patients, suggesting that PS41014-Ab could be used as an additional tool for assessing CRC-risk. We previously reported that CRC-patients, whose colonic tumors were positive for relatively high levels of DCLK1-S expression (by qRT-PCR), had a worse overall survival and disease free interval than low-expressers (O'Connell et al, Sci Rep 2015). We now report that DCLK1-S-Antibody may help to identify patients at high-risk for developing CRCs within 10-15 years, at the time of index/screening colonoscopy, and thus serve as a useful biomarker at early time points of colon carcinogenesis, unlike the currently available fecal/blood tests.

#730

High sensitive detecting procedure of circulating repetitive RNA as novel early marker of pancreatic cancer.

Takahiro Kishikawa, Motoyuki Otsuka, Kazuhiko Koike. _University of Tokyo, Tokyo, Japan_.

Pancreatic ductal adenocarcinoma (Pdac) is one of the most intractable malignancies due to difficulties in early detection. Although promising biomarkers are increasingly reported, such methods are not yet easy to apply clinically, mainly due to their low reproducibility or technical difficulties. In this study, we developed a convenient and sensitive method for quantifying aberrantly expressed satellite repeat RNAs in sera, which can be used to efficiently detect patients with Pdac. It is difficult to correctly quantitate repeat arrays because its repetitive nature makes it difficult to establish appropriate primers that amplify a single product using simple PCR procedures. Here, we introduce a Tandem Repeat Amplification by nuclease Protection (TRAP) method combined with droplet digital PCR (ddPCR) to detect human satellite II (HSATII) RNAs, which are specifically expressed in human Pdacs at greater levels than normal tissues. HSATII RNA core sequence levels in sera were significantly higher in Pdac patients compared with non-cancer patients (median copy number: 14.75 and 3.17 per µl in the training set and 17.35 and 2.9 in the validation set, respectively). In addition, patients with intraductal papillary mucinous neoplasm (IPMN), a precancerous lesion of Pdac, could also be efficiently detected. This method can be routinely applied to screen patients with Pdac and high-risk patients, facilitating the development of preventive medicine for this disease.

#731

Stool DNA testing of Fusobacterium nucleatum for detection of colorectal tumors.

Yutaka Suehiro, Kouhei Sakai, Toshihiko Matsumoto, Takahiro Yamasaki. _Yamaguchi Univ. Graduate School of Medicine, Ube, Japan_.

Background: Accumulating evidence shows an overabundance of Fusobacterium nucleatum (Fn) in colorectal tumor tissues. Although stool DNA testing of Fn might be a potential marker for the detection of colorectal tumors, the difficulty in detecting Fn in stool by conventional methods prevented further explorations. Therefore, we developed a droplet digital PCR assay for detecting Fn in stool and investigated its clinical utility in the management of colorectal tumors in a Japanese population.

Methods: Feces were collected from 60 healthy subjects (control group) and from 11 patients with colorectal non-advanced adenomas (non-advanced adenoma group), 19 patients with colorectal advanced adenoma/carcinoma in situ (advanced adenoma/CIS group), and 158 patients with colorectal cancer (CRC) of stages I to IV (CRC group). Absolute copy numbers of Fn were measured by droplet digital PCR.

Results: The median copy number of Fn was 17.5 in the control group, 311 in the non-advanced adenoma group, 122 in the advanced adenoma/CIS group, and 317 in the CRC group. In comparison with that in the control group, the Fn level was significantly higher in the non-advanced adenoma group, the advanced adenoma/CIS group, and the CRC group.

Conclusions: This study illustrates the potential of stool DNA testing of Fn by droplet digital PCR to detect individuals with colorectal tumors in a Japanese population.

#732

Potentials of some serum proteins and urinary molecular biomarkers for early diagnosis of prostate cancer in Nigeria patients.

Oluyemi Akinloye,1 Aniebietabasi S. Obot,2 Taiwo A. Adewole2. 1 _University of Lagos, Lagos, Nigeria;_ 2 _Ladoke Akintola University of Technology, Osogbo, Nigeria_.

Background: Prostate cancer (PCa) an adenocarcinoma is the most common cancer diagnosed in African men today. At present, the only widely accepted screening tools for prostate cancer are prostate specific antigen (PSA) and digital rectal examination. There is controversy regarding the appropriate level of serum PSA that should trigger a biopsy. This study is aimed at finding a better marker/panel of markers for prostate cancer.

Methods: 150 consented patients requiring a prostate biopsy and 100 age matched controls were recruited for this study. Genetic materials were found in 132 (88%) of the samples. Serum Total PSA (TPSA) , and free PSA were assayed using ELISA method, % free/total PSA(%f/tpsa) was obtained statistically, prostatic volume was determined using TRUS. In addition, selected urinary RNA's were assayed; transmembrane serine protease (TMPRSS2:ERG and TMPRSS2:ETS) fusion genes, PSA gene and PCA3 (prostate cancer antigen 3) , using standard polymerase chain reaction (PCR) protocols.

Results: TMPRSS2:ERG was detected in 9 (7 %) of the samples and limited to biopsy positive for PCa. Similarly, TMPRSS2:ETS was found in only 4 (3%) of the samples and also restricted to biopsy positive for PCa. PCA3 score had the best discriminatory accuracy in diagnosing PCa amongst patients with serum Total PSA in the range of 4 - 10 ng/ml with AUC of 0.705 compared to Total PSA, f/t PSA ratio, and PSA Density which were 0.365, 0.695, and 0.541 respectively. At the cut off value of 24.6, PCA3 score yielded its best sensitivity of 0.615 and specificity of 0.630. At the cutoff of 18, free/total PSA ratio (0.615, 0.77), at the cutoff of 0.14 PSAD yielded its best (0.538, 0.704.) respectively. Direct logistic regression was performed to access the predictability of PCa using different models comprising of three (3) covariates, the model comprising PCA3 Score, f/t PSA ratio and PSAD had the best discriminating accuracy in the subgroup with TPSA range of 4 - 10ng/ml, with the sensitivity, specificity, positive predictive value, and negative predictive values of 0.59, 0.93, 71.4% and 75.8% respectively, over models comprising PCA3 Score ,TPSA and %f/t PSA, and PCA3 Score, TPSA and PSAD with these values (0.23, 0.85, 42.9 % and 70.1%), (0.39, 0.89, 62.5%, and 75 %) and ( 0.15, 0.85,66.7% and 67.6%) respectively.

Conclusions: In predicting PCa amongst patients with serum total PSA in the grey area of 4 - 10 ng/ml, the model comprising PCA3 Score, f/t PSA ratio and PSAD had the best discriminating accuracy.

#733

Detection of HCC-derived major HBV integration junctions in urine and their implications for driver identification.

Selena Y. Lin,1 Jamin D. Steffen,2 Yih-Ping Su,3 Surbhi Jain,2 Ting-Tsung Chang,4 Wei Song,1 Ying-Hsiu Su3. 1 _U-Screen Dx, Inc., Doylestown, PA;_ 2 _JBS Science, Inc, Doylestown, PA;_ 3 _The Baruch S Blumberg Institute, Doylestown, PA;_ 4 _National Cheng Kung University Medical College, Tainan, Taiwan_.

Chronic Hepatitis B Virus (HBV) infection is a major etiology of hepatocellular carcinoma (HCC), a leading cause of cancer mortality worldwide. Integration of HBV DNA into the host genome occurs during the course of chronic infection. Integrated HBV DNA is observed in most of HBV-associated HCC. The integration junctions derived from the original tumor cell become abundant (referred to as major integration junctions) in the infected liver because of clonal expansion during tumor development. Detection of circulating DNA containing these tumor-derived integration junctions may therefore be useful for cancer detection. We have adapted a method known as Primer Extension Capture (PEC) to enrich integrated HBV DNA for next-generation sequencing (NGS). Our initial studies using this approach to enrich the HBV DR1-2 region (a common site for HBV integrated breakpoints) identified major integration junctions from HBV-HCC tissue samples and matched urine. Further analysis revealed that most recurrently targeted integrations from these HCC tumors have previously reported involvement in cancer. This suggested that identification of recurrently targeted genes is applicable for driver identification. Interestingly, we show how HBV targets the TERT promoter in a localized region even though no two TERT junctions examined are identical. We have further developed this PEC to enrich for the entire HBV genome and applied it to (i) liver tissue DNA from 20 matched HCC and adjacent non-HCC samples, and (ii) DNA from urine of 20 hepatitis, 20 cirrhosis and 20 HCC patients. The HBV enriched libraries were sequenced by NGS and the integration events were analyzed using the in-house developed software. The complexity of HBV junction sites in HCC and non-HCC tissue and urine derived circulating DNA is reported. Our approach has potential to be used for liquid biopsies to study the complexities of HBV integrations in chronic HBV infection and carcinogenesis, and to identify HCC-related DNA modifications for early detection and disease management.

#734

Improved detection of salivary glands' RNA markers in saliva samples.

Adrian D. Schubert, Evgeny Izumchenko, Piotr T. Wysocki, David Sidransky, Mariana Brait. _Johns Hopkins University, School of Medicine, Baltimore, MD_.

RNA based liquid biopsy in saliva could be part of the diagnostic process and surveillance in patients with Salivary Gland Tumors. There is no established approach for saliva markers in salivary gland tumors. These tumors occur approximately in one out of 100,000 adults per year in the USA. We aim to develop a method to identify RNA shed specifically from the salivary glands.The two most common salivary gland malignancies harbor frequent gene fusions, which may be candidate RNA based markers. In mucoepidermoid carcinoma, the MECT1-MAML2 fusion. In adenoid cystic carcinoma, either a MYB-NFIB or MYBL1-NFIB fusion. We identified collection and processing methods to be tested on healthy individuals' saliva samples. We compared three saliva stimulation methods: Chewing Gum, Tabasco and Vitamin C powder with unstimulated collected saliva; and also utilized different processing and RNA extraction procedures: Trizol based: QIAzol method (Qiagen), RNEasy saliva protecting reagent (Qiagen), OrageneRNA (DNA Genotek), and mirVana Kit (ThermoFisher). We evaluated the RNA´s quantity and quality with Spectrophotometer and Agilent's 2100 Bio analyzer. We identified two salivary gland specifically highly expressed genes (HTN3 and CA6) to be analyzed by quantitative RT-PCR. We have collected and processed 221 samples and analysis is ongoing. Bio analyzer showed that RNA from the unstimulated collected saliva has a higher concentration than stimulated saliva. Unstimulated saliva is expected to contain mucosa cells, bacteria and rests of food, therefore those could also be RNA sources. When saliva's RNA is protected by either of the buffers tested, the RIN scores (RNA Integrity Number) obtained by Bio analyzer are higher. Based on our preliminary data, stimulation with either Tabasco, Chewing Gum or Vitamin C followed by RNA stabilization with the OrageneRNA kit and extraction with Trizol resulted in the highest quality and specificity of the RNA for its salivary gland origin. Unstimulated collection followed by the same processing generates a greater RNA yield. This saliva RNA is likely contaminated with RNA form squamous cells, bacteria and food residues, which may decrease the sensitivity of the specific gene expression of HTN3 and CA6. Once established how to best collect and protect saliva for the extraction of nucleic acids originated from the salivary glands, we anticipate accelerating the path to clinically highly demanded kits for the surveillance of salivary gland cancer patients. We plan to prospectively collect samples from Salivary gland tumor patients with the chosen method and evaluate tumor related markers. The detection of tumor markers in bodily fluids will open a new avenue for the diagnosis and the clinical management of patients with this type of tumor. Moreover, our work creates the possibility of a liquid biopsy-based detection of several disease-specific alterations identified in different types of salivary gland tumors. 

### Molecular Diagnostics

#735

HER2 protein quantification in multiple cancer indications identifies candidates for HER2 targeted therapies.

Shankar Sellappan,1 Sarit Schwartz,1 Ellen Wertheimer,1 Fabiola Cecchi,1 Steven W. Mamus,2 Daniel VT Catenacci,3 Todd Hembrough1. 1 _NantOmics, Rockville, MD;_ 2 _Cancer Center of Sarasota-Manatee, Sarasota, FL;_ 3 _University of Chicago, Chicago, IL_.

Background: Anti-HER2 therapy in cancer indications other than breast and gastric cancers is the subject of ongoing clinical trials. We previously used mass spectrometry to identify HER2 protein expression level cutoffs that correlate with standard measures of HER2 positivity (740 and 750 amol/µg in breast and gastric cancers, respectively). These studies also demonstrated that trastuzumab-treated patients whose tumors express high levels of HER2 (≥ 2200 and ≥ 1825 amol/µg in breast and gastric cancers, respectively) survived much longer than patients with lower HER2 levels. We hypothesized that targeted proteomics would reveal high HER2 levels in indications other than breast and gastric cancers, thus identifying patients likely to benefit from anti-HER2 therapy.

Methods: We summarized results from samples processed in our CAP/CLIA-certified laboratory. Tumor areas from FFPE tissue blocks (N=3828) representing multiple cancer indications were marked by a pathologist, microdissected, and solubilized to tryptic peptides. In each liquefied tumor sample, HER2 and other protein targets were quantified with mass spectrometry-based proteomic analysis.

Results: HER2 superexpression (> 2200 amol/µg) was found in 0.64% (12/1891) of patients with non-breast, non-gastroesophageal cancers. Among indications with > 50 patients tested, the highest rates of HER2 superexpression were in gynecological cancers (3/124; 2.4%) and bladder cancer (1/51; 2.0%). Treatment and outcome data are largely unavailable, but we are aware of 3 anecdotes: In a 74-year-old male with invasive adenocarcinoma of the gallbladder who had disease progression on gemcitabine + cisplatin, proteomic testing found high HER2 protein expression (3105 amol/µg). The patient responded to trastuzumab + FOLFIRI for 5 months. In a uterine cancer patient whose HER2 status was equivocal by genomic analysis, proteomics found high HER2 expression (4995 amol/µg). Proteomics also revealed the absence of a resistance marker for taxane (TUBB3) and high levels of the response marker for antifolate agents (FRalpha = 10500 amol/µg). The patient responded to trastuzumab + taxol for 9 months before developing resistance and responding to trastuzumab + antifolate. Lastly, a cervical cancer patient whose disease had progressed on chemotherapy showed HER2 superexpression (11322 amol/µg). She was treated with anti-HER2 combinations for > 12 months.

Conclusions: Patients with high HER2 protein expression as measured by targeted mass spectrometry in multiple cancer types have benefitted from anti-HER2 therapy. Only small numbers of patients with non-breast, non-gastric tumors have HER2 protein levels indicative of survival benefit from anti-HER2 therapy. However, multiplexed targeted proteomics offers simultaneous, precise quantification of other biomarkers (eg, ERCC1, TUBB3, FRalpha) to guide therapy selection for multiple cancer types.

#736

Cell lineage-oriented clinical sequencing unveils distinct clonal ontogeny of acute myeloid leukemia with myelodysplasia-related changes.

Kazuaki Yokoyama,1 Nozomi Yusa,2 Sousuke Nakamura,3 Mika Ito,3 Asako Kobayashi,3 Masayuki Kobayashi,3 Rika Kasajima,4 Hiroaki Yui,4 Eigo Shimizu,4 Atushi Niida,4 Rui Yamaguti,4 Tsuneo Ikenoue,5 Seiya Imoto,6 Yoichi Frukawa,2 Satoru Miyano,7 Arinobu Tojo8. 1 _Department of Hematology/Oncology, Research Hospital, The Institute of Medical Science, The University of Tokyo (IMSUT), Tokyo, Japan;_ 2 _Department of Applied Genomics, Research Hospital, IMSUT, Japan;_ 3 _Division of Molecular Therapy, Advanced Research Center, IMSUT, Japan;_ 4 _Human Genome Center, ISMUT, Japan;_ 5 _Division of Clinical Genome Research, ISMUT, Japan;_ 6 _Health Intelligence Center, IMSUT, Japan;_ 7 _Human Genome Center, ISMUT, Tokyo, Japan;_ 8 _Department of Hematology/Oncology, Research Hospital, IMSUT, Japan_.

Acute myeloid leukemia (AML) is characterized by unregulated clonal expansion and maturation arrest of myeloid committed progenitors (MP). AML generally represents de novo onset or evolves from preceding myelodysplastic syndrome (MDS), which is defined by refractory cytopenias, clonal hematopoiesis, and/or multi-lineage dysplasia. The WHO classification 2008 includes this entity as "AML with myelodysplasia-related changes (AML-MRC)", and currently, diagnosis of AML-MRC is based on either previous history of MDS, multi-lineage dysplasia, or MDS-related cytogenetic abnormality. However, AML-MRC often represents de novo onset without these MDS-compatible clinical features. Considering that AML-MRC exhibits rather poor prognosis with refractoriness to conventional chemotherapy against AML, more accurate and objective diagnostic approach is requisite to unveil hidden "MDS signatures" in patients with apparently de novo AML. A certain set of gene mutations is specific and recurrent in MDS. Given the pre-existing "MDS signatures", the founder gene mutations might be detected in not only blast cells but also neutrophils and/or T cells in AML-MRC. To test this hypothesis, we performed FACS sorting of neutrophils, T cells, and blasts fractions, respectively, followed by mutation screening using targeted deep sequencing, namely, cell lineage-oriented sequencing (CLS). Genomic DNA both from each cell fraction and buccal swab was subjected to screening mutations in 54 genes which are tightly involved in MDS and AML. Pair-end deep sequencing was performed on an Illumina MiSeq, using library prepared by TruSight Myeloid Panel (Illumina, San Diego, CA). Bioinformatic analysis was performed by in-house pipeline. We performed CLS of clinically diagnosed AML-MRC (n=7), suspected AML-MRC (n=2), de novo AML (AML with t(15;17) or AML with inv16, n=4), MDS (RAEB-1 and RAEB-II, n=3), and familial MDS (n=1). As expected, in a familial MDS case, overlapping germline RUNX1 driver mutation was demonstrated in granulocytes, blast cells and T cells, supporting that it would be originated from a hematopoietic stem cell. Notably, in MDS, AML-MRC, and suspected AML-MRC cases with no germ-line mutations, the founder mutations present in neutrophils were also retained in the AML blast cells, irrespective of a history of MDS, suggesting that these are derived from a myeloid progenitor cell. In marked contrast, there were no overlapping driver mutations between blast cell and neutrophil fractions in de novo AML characterized by recurrent chromosomal abnormalities. In summary, CLS revealed that founder mutations are shared by neutrophils and AML blast cells in AML-MRC, but not in de novo AML. Although our data should be validated in a larger cohort of AML cases, CLS is a promising approach to molecular diagnosis of latent AML-MRC which require distinct therapeutic options from de novo AML.

#737

Signficant DLX4 expression in inflammatory breast cancer tumors from African American patients.

Jaehong Jeong,1 Tammey Naab,2 Martin Ongkeko,1 Kepher Makambi,1 Farhan A. Khan,2 Jan Blancato1. 1 _Georgetown Univ., Washington, DC;_ 2 _Howard Univ., Washington, DC_.

Purpose: To examine protein expression characteristics of Distal-less homeobox4 (DLX4) in inflammatory breast cancer (IBC) cases from an African-American population to determine if a) DLX4 over-expression occurs in IBC vs. normal mammary tissue and b) over-expression is associated with clinico-pathologic features such as ER, PR, HER2.

Experimental Design: Twenty nine blocks of formalin-fixed paraffin-embedded (FFPE) tissues from well-characterized human IBC cases were used for immuno-histochemical staining (IHC). Tumor tissues were from African American subjects seen at Howard University Hospital. Normal breast tissues from 30 mammoplasties were controls. IHC results were assigned intensity and percentage scores based upon intensity of positive staining and % of stained cells. Percentage scores were assigned as 0, 1 (0%-25%), 2 (26%-50%), 3 (51%-75%) or 4 (76%-100%) and intensity scores were assigned 0, 1+, 2+ or 3+. For the analysis of the IHC, a percentage score of 3 or 4 was considered high and an intensity score of 2+ or 3+ was categorized as high. Chi-square and Fisher's exact tests were used for analysis.

Results: The staining pattern for the categorized cohort in which 82.8% (24 out of 29) of IBC cases showed high percentages of positive cells staining for DLX4 protein, while 40.0% (12 out of 30) normal breast tissues demonstrated DLX4 expression (P=0.001). In terms of staining intensity, 75.9% (22 out of 29) of IBC cases showed a high level of intensity, compared to 20.0% (6 out of 30) of normal breast tissues (P<0.001). In terms of the association between breast cancer characteristics and IHC staining, intensity of DLX4 was higher in HER2- (87.0%, 20 out of 23) than HER2+ (33.3%, 2 out of 6; P=0.018). Other associations were not statistically significant in this pilot study.

Conclusions: DLX4 expression is significantly higher in this pilot study of IBC cases from AA patients than in normal breast tissue cases. In addition, HER2- is associated with high intensity of DLX4 expression in IBC.

Associations DLX4 protein expression (IHC) In IBC and normal breast tissues

---

|

Low | High | P-value

Percent positivity | n (%) | n (%)

|

Controls (n=30) | 18 (60.0) | 12 (40.0) | 0.001

IBCs (n=29) | 5 (17.2) | 24 (82.8)

Staining intensity | n (%) | n (%)

|

Controls (n=30) | 24 (80.0) | 6 (20.0) | <0.001

IBCs (n=29) | 7 (24.1) | 22 (75.9)

#738

Highly multiplexed and precisely calibrated reference materials for copy number variation detection.

Catherine Huang, Yves Konigshofer, Jessica Dickens, Bharathi Anekella. _Seracare Life Sciences, Gaithersburg, MD_.

Introduction: Genomic instability is a hallmark of cancer, and copy number variation (CNV), including gene duplication and deletion, can be a key driver of oncogenesis. Detection of CNV is needed for personalized treatment, and may involve microarray analysis or Next Generation Sequencing. Often, DNA from patient-derived cell lines is used as positive control material in these assays. However, these materials are often poorly characterized, may change over time, and have additional confounding genetic alterations. Here we describe the development of new reference materials that are highly characterized by digital PCR and can aid in the validation and calibration of new diagnostic assays for CNV.

Methods: The GM24385 reference cell line has been extensively characterized by the Genome in a Bottle project1 and originates from a participant in the Personal Genome Project. DNA from this cell line was used to make a library of full-length gene inserts. The library had >12X coverage of the GM24385 genome and an average insert size of ~135 Kb. PCR based screening was used to identify and isolate full length MYC, FGFR3 and ERBB2 genes. Next Generation Sequencing, using Illumina Nextera library preparation kits and a MiSeq instrument, was used to verify the gene of interest. The genomic DNA and gene inserts were then formulated into reference materials, in purified DNA format to amplify the copy numbers of these key genes. Digital PCR was used to control the mixing and verify the number of copies of genes and/or mutations within the reference material.

Results: The sequence confirmed genes were formulated into GM24385 genomic DNA at levels consistent with 5X and 10X amplification. Testing on the ArcherDx VariantPlex™ Solid Tumor Kit indicated that each amplification could be detected at the expected level.

Conclusions: Highly characterized and consistently manufactured reference materials for copy number detection are currently lacking. The described Seraseq CNV Reference Materials provide proof of concept for a biosynthetic, engineered material that uses digital PCR as an orthologous reference method and provides a "ground truth" copy number level for key oncogenes.

#739

Orthogonal validation of oncomine cfDNA panel data with digital PCR using TaqMan Rare Mutation Assays.

Vidya Venkatesh, Marion Laig, Kamini Varma, Yanchun Li, Kelli Bramlett, Dalia Dhingra, Richard Chien. _Thermo Fisher Scientific, South San Francisco, CA_.

The discovery of circulating tumor DNA (ctDNA) in the blood, urine and other bodily fluids of cancer patients has led to a new type of non-invasive method of characterizing cancer-causing mutations, the liquid biopsy. With NGS technologies becoming increasingly sensitive, down to a 0.1% Limit of Detection (LOD), they are rapidly gaining traction as a valid assay for cancer genotyping and have potential to direct cancer treatment plans. The wide-angle view provided by NGS panels, combined with dPCR's zoomed-in precision detection of DNA provide a comprehensive picture of a cancer's genetic makeup. By applying these complementary techniques at the appropriate time based on the disease type and stage, cancer treatment will become quicker, more precise and more cost-effective in the future. NGS and digital PCR together provide a complete picture of the cancer genome. As part of our research, we wet-lab tested a subset of Rare Mutation Assays corresponding to the Oncomine cfDNA panel for next generation sequencing. Synthetic plasmid (GeneArt) carrying the mutation was spiked into wild-type genomic DNA to reflect a mutation rate of 0.1%. Wild-type genomic DNA was used as negative control. Thermal cycling was performed according to protocol for digital PCR using the QuantStudio 3D. In this study, we tested a pilot set of samples using AcroMetrix Oncology Hotspot Control and Horizon cfDNA Reference Standard with both NGS using Oncomine cfDNA panel and digital PCR with Rare Mutation Assays. Comparison of NGS and digital PCR results for the same sample showed excellent correlation in the low mutation range around 0.1%. This study confirms that digital PCR using QuantStudio 3D and Rare Mutation Assays is effective as a method for orthogonal validation of NGS data using the Oncomine cfDNA panel. Additionally, TaqMan Rare Mutation Assays offer a sensitive and precise solution for downstream mutation tracking over a time course. For Research Use Only. Not for use in diagnostic procedures.

#740

Rapid detection of IDH1/2 mutations using RNaseH2 dependent, multiplex quantitative PCR Assays.

Yun Bao,1 Aymen Baig,2 Yu Wang,1 A. John Iafrate,2 Caifu Chen,1 Darrell R. Borger2. 1 _Integrated DNA Technologies, Redwood City, CA;_ 2 _Massachusetts General Hospital Cancer Center, Boston, MA_.

Isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) are enzymes that normally catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate during glucose metabolism. Heterozygous mutations in the IDH1 and IDH2 genes have been identified in a specific subset of cancers. Mutations typically affect the enzyme active sites at codon R132 in the IDH1 gene, and R140 or R172 in the IDH2 gene. This confers neomorphic enzyme activity that produces the oncometabolite 2-hydroxyglutarate that is involved in cancer initiation and progression. The detection of IDH1/IDH2 mutations has important diagnostic, prognostic, and therapeutic implications and testing for these variants is being incorporated in clinical settings. In fact, the identification of IDH1 or IDH2 gene mutations is now required for the classification of tumors of the central nervous system under the 2016 WHO guidelines. Therefore, there is a significant need for an IDH1 + IDH2 test that can be performed in a rapid manner to support clinical diagnosis and treatment decisions. A strategy utilizing allele-specific quantitative PCR (qPCR) is well suited for evaluating a small number of hotspot variants in IDH1 and IDH2 from clinical tissue samples in a matter of few hours. Using RNase H2-dependent PCR (rhPCR) and a universal reporter system, we have developed new multiplex qPCR assays that effectively discriminate between closely related sequences that differ by only a single nucleotide base at the same position or adjacent position in a single tube reaction. In addition, the rhPCR mechanism of the assays also greatly reduces the impact of primer-primer interactions, further improving the multiplex capability of the assays. During initial testing, specificity, accuracy, and sensitivity were evaluated. Mutations present in patient samples at frequencies as low as 2% were identified with high reproducibility. The assays have been tested across 49 patient samples that included FFPE tissue, blood, and bone marrow aspirate with results that are concordant to those found using an orthogonal method of next generation sequencing. Our study showed that this assay provides a rapid method for detection of multiple IDH1 or IDH2 mutations in single rhPCR reactions with high accuracy, specificity, and sensitivity.

#741

**Rapid detection of necrosis in breast cancer with** ex vivo **and** in situ **mass spectrometry analysis methods.**

Arash Zarrine-Afsar,1 Bindesh Shrestha,2 Alessandra Tata,1 Michael Woolman,1 Manuela Ventura,3 Nicholas Bernards,1 Milan Ganguly,3 Howard Ginsberg,4 Jinzi Zheng,3 Emma Bluemke1. 1 _Techna Institute for the Advancement of Technology for Health, University Health Network, Toronto, Ontario, Canada;_ 2 _Waters Corporation, Milford, MA;_ 3 _University Health Network, Toronto, Ontario, Canada;_ 4 _St. Michael's Hospital, Toronto, Ontario, Canada_.

Necrosis is a form of cell death that is often associated with highly aggressive forms of cancer, is of prognostic value in treatment planning. Mass Spectrometry (MS) is a highly sensitive analytic platform capable of providing a molecular profile of cancer on the basis of mass to charge (m/z) ratio of tissue constituent molecules. MS analysis of ex vivo tissue slices from metastatic murine xenograft tumors from LM2-4 cell line with Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) allowed direct comparisons with histology images to determine the molecular profile of necrotic tissues. The necrotic tissue is characterized by the presence of a ceramide absent from the viable cancer regions. The spatial distribution of this ion fully correlated to necrotic areas from pathology in additional independent tumor samples examined. The same ion was detected from in situ necrotic tissue using tissue aerosols generated by hand-held ablation probes coupled to evaporative ionization interface in only a few seconds of sampling. These developments further establish MS as a novel tool for rapid pathology that is highly complementary to current histology based methods widely used in characterization of cancer in both imaging mode (to provide spatial information on cancer border) and profiling mode (to provide information on cancer type and subtype); all based on unique molecular profile associated with each cancer type and subtype. Current efforts in creating cancer molecular profile libraries will facilitate translation.

#742

Comprehensive detection of all major classes of MET deregulation by Anchored Multiplex PCR and next-generation sequencing.

Brian A. Kudlow,1 Josh Haimes,1 Marc Bessette,1 Namitha Manoj,1 Laura M. Griffin,1 Danielle Murphy,2 Robert Shoemaker,2 Jason Amsbaugh,1 Joshua Stahl1. 1 _ArcherDX, Boulder, CO;_ 2 _Ignyta, San Diego, CA_.

Introduction: Deregulation of the proto-oncogene, MET, confers an aggressive phenotype in a variety of human cancers, promoting proliferation, invasive growth and angiogenesis. MET deregulation can be driven by gene amplification, overexpression, exon 14 skipping, gene fusions and single nucleotide variants (SNVs), such as kinase-activating point mutations. MET is a target of intensive drug development efforts, although the various mutated forms of MET exhibit unique drug sensitivities. Therefore, detection of these mutations has an important role in the development of drugs targeting MET, and has the potential to guide treatments for cancers driven by MET deregulation.

Next-generation sequencing (NGS) enables comprehensive detection of all mutation types from whole genomes and transcriptomes. However, low detection sensitivity, high input requirement and high costs render these approaches impractical for routine detection of mutations from low-input clinical sample types. We developed a targeted NGS assay based on Anchored Multiplex PCR (AMP™) to detect all types of mutations driving MET deregulation from a single sample.

Methods: AMP only requires a single gene-specific primer for amplification, enabling open-ended capture of DNA and cDNA fragments for NGS-based detection of known and unknown mutations. We developed AMP-based Archer® VariantPlex™ and FusionPlex® library preparation assays to detect mutations from DNA and RNA, respectively. AMP probes were designed to cover the MET gene for detection of copy numbers variants (CNVs) and SNVs from DNA, and known and novel fusions, exon skipping and expression levels from RNA.

Results: We show that the VariantPlex assay enables NGS-based detection of MET amplifications from DNA in concordance with FISH results. Further NGS analysis of RNA from the same sample using the FusionPlex assay revealed the resulting overexpression of MET. We also demonstrate that AMP-enabled open-ended capture of cDNA fragments allows for reliable detection of exon 14 skipping in FFPE samples and in cells, consistent with RT-PCR results. Parallel analysis of DNA from the cell samples revealed splice site mutations that have been previously reported to drive exon 14 skipping. Furthermore, this open-ended capture also permitted identification of a novel GTF2I:MET gene fusion in a patient-derived xenograft model. Finally, we detected an kinase-activating point mutation in MET, p.Y1253D, by analysis of genomic DNA with the VariantPlex NGS assay.

Conclusions: These results show that AMP-based VariantPlex and FusionPlex Assays enable comprehensive detection of multiple mutation types from low-input clinical sample types, such as FFPE specimens. As MET deregulation can be driven by many different genetic aberrations, this allows for NGS-based characterization of MET deregulation from a single sample.

#743

Detection of TERT C228T and C250T promoter mutations in melanoma tumor and plasma samples using novel mutation-specific droplet digital PCR assays.

Broderick Corless,1 Gregory Chang,1 Samantha Cooper,2 Mahrukh Syeda,1 Iman Osman,1 George Karlin-Neumann,2 David Polsky1. 1 _New York Univ. School of Medicine, New York, NY;_ 2 _Bio-Rad Laboratories, Pleasanton, CA_.

Purpose: Detecting mutations in the plasma of patients with solid tumors is becoming a valuable method of diagnosing and monitoring cancer. Mutations in 1 of 2 hot spots in the TERT promoter sequence are found in several cancers, including up to 85% of melanomas and the majority of cases that lack BRAF or NRAS mutations (about one-third of melanomas). Due to the high G-C content of the TERT promoter sequence these mutations can be difficult to detect using NGS approaches. We developed novel droplet digital PCR (ddPCR) assays to detect these 2 mutations with high sensitivity and specificity, and demonstrate the application of these assays in melanoma clinical samples.

Methods: Assays were optimized using cell lines with Sanger sequencing-confirmed mutations: glioblastoma A172 (C228T), and melanoma NYU12-126 (C250T). We varied assay designs and amplification conditions to optimize probe-based detection using the Bio-Rad QX-200 ddPCR system. Assay sensitivities and specificities at various DNA input levels were determined using serial dilutions with 3 replicate wells for each condition. Sensitivity is defined as the lowest mutant allele dilution for which the confidence interval did not overlap with that of the 0% mutant wells. We used normal and cancer-derived DNA sources of different quality (e.g. normal human DNA (Promega), cancer cell lines, plasma and FFPE-derived DNAs) with and without the mutations, and compared the efficiency of detection of amplicons of 88, 113 and 163 base-pairs. We compared efficiencies to assays of similar size for RPP30, a housekeeping gene. Patient-matched metastatic melanoma tumors and plasma samples were analyzed to explore the clinical utility of these assays.

Results: The assays showed greater sensitivity when higher amounts of DNA were analyzed. For C228T the limit of detection (LOD) of the mutant allele was 1%, 0.25% and 0.1% for 6.6ng/well, 33ng/well and 66ng/well respectively; for C250T the LODs were 0.25%, 0.05% and 0.05% respectively. Using normal human DNA, the efficiency of the TERT assays averaged approximately 90% of that for RPP30 across assays of similar size, and no decrease in assay efficiency was observed as amplicon length increased. In contrast, whereas amplicon size had only a modest effect on assay efficiency in plasma cfDNA, it gave a more pronounced effect on FFPE DNA's, decreasing to 38% for the 163bp amplicon. We observed 100% concordance between TERT mutation detection by SNaPshot and ddPCR in 10 FFPE tumor samples, and in plasma samples from 4 metastatic melanoma patients with matching tumor samples.

Conclusion: We developed robust ddPCR assays to detect TERT promoter mutations with high sensitivity and specificity. Mutated TERT DNA can be detected and quantitated in the plasma of patients with metastatic melanoma, and is likely to be present in the plasma of other cancer patients in whom TERT mutations occur.

#744

ctDNA use in molecular diagnostics.

Gilda Magliacane, Greta Grassini, Ilaria Francaviglia, Elena DalCin, Chiara Lazzari, Alessandra Bulotta, Monika Ducceschi, Vanesa Gregorc, Lorenza Pecciarini, Claudio Doglioni, Maria Giulia Cangi. _San Raffaele Scientific Inst., Milano, Italy_.

The analysis of circulating cell-free tumor DNA (ctDNA), which can be obtained from plasma by non-invasive procedures, is expected to provide useful biomarkers in the management of non-small-cell lung cancer (NSCLC) patients. Indeed, several studies have assessed ctDNA prognostic and predictive value as source of key data for therapeutic targets and drug resistance in carcinoma patients. The expanding number of targeted therapeutics for NSCLC always expects a real-time wider tumor genotyping, and the use of ctDNA as either a complement or an alternative to tumor tissue DNA (ttDNA) could be a valid option for Next-Generation Sequencing (NGS) of key cancer genes.

However, isolation and enrichment of ctDNA is a big challenge because of its high degree of fragmentation and its low concentration against the normally occurring background of cell-free DNA derived from healthy cells. Therefore standardized methods for ctDNA extraction and analysis are crucial aspects in the setting-up of a molecular diagnostic approach.

In this study we aimed to evaluate the ctDNA use for molecular profiling, also analyzing the impact of both pre-analytical and analytical variables on DNA yield and mutation detection.

Matched ttDNA and ctDNA from 30 NSCLC patients were extracted, quantified and quality-controlled, and then investigated by different standard methods (real-time PCR, digital PCR, Mass Spectrometry genotyping) for EGFR, KRAS, BRAF, PIK3CA status. Mutational screening of ctDNA samples by IonTorrent NGS (Oncomine™ Lung cfDNA Assay, ThermoFisher) was also performed. Further, we extended ctDNA evaluation to additional 30 lung cancer patients with no available tumor sample.

We found that cell free DNA concentration in plasma correlated with both stage and number of metastatic sites. Analyzing matched ttDNA and ctDNA by standard methods, we identified mutations in EGFR, KRAS, PIK3CA genes, with an overall concordance of 77%. Interestingly, Oncomine Lung cfDNA assay detected these same mutations with the same allelic frequency of standard methods; mutations in TP53 and ALK genes were also found.

In the 30 patients with no available tumor sample, we found EGFR (10%) and KRAS (7%) mutations on ctDNA by standard methods; NGS analysis is under evaluation.

This study evaluated the use of multiple different methods to detect mutations in NSCLC and showed that ctDNA can be a feasible option for clinical monitoring of lung cancers, including for those patients who cannot undergo invasive diagnostic procedures, due to either comorbidities or absence of biopsable tumor lesions.

#745

Interleukin-13 conjugated quantum dots to identify glioma initiating cells and exosomes.

Achuthamangalam B. Madhankumar,1 Oliver Mrowczynski,1 Brad Zacharia,1 Michael Glantz,1 Lichong Xu,1 Becky Webb,1 Christopher Siedlecki,1 Akiva Mintz,2 James R. Connor1. 1 _Penn State Univ. College of Medicine, Hershey, PA;_ 2 _Wake Forest University Baptist Medical Center, Winston Salem, NC_.

High grade brain tumors like glioblastoma multiforme (GBM) and certain other brain metastatic cancers possess high tumor invasive and infiltrating properties making them harder to detect. Identification of cancer stem cells and exosomes secreted by them in the biofluids like cerebrospinal fluid (CSF) will be an ideal way of detecting the presence of residual cancer after therapy. In the current approach, a ligand conjugated quantum dot (QD) was utilized to specifically label the cancer stem cells and exosomes to identify the presence of IL13Rα2 a tumor invasive marker. Human glioma initiating cells were cultured in adherent form and were exposed to interleukin-13 conjugated quantum dots (IL13QD), which binds the glioma initiating cells expressing IL13Rα2 receptor. Similarly the exosomes isolated from cancer stem cell condition media and CSF was briefly incubated with IL13QD, before analyzing their complexing pattern. Exosomes that were isolated from glioma stem cells and patients CSF were demonstrated to express IL13Rα2 receptor. Fluorescent microscopy, transmission electron microscopy and flow cytometry were performed on the exosomes after complexing with IL13QD. IL13QD was demonstrated to specifically bind glioma stem cells both in the monolayer and spheroid culture. The binding affinity of the exosomes to the quantum dots were quantitatively and qualitatively confirmed by atomic force microscopy (AFM). The morphology and size of exosomes before and after complexing with exosomes were confirmed by electron microscopy and AFM. The density plot from the flow cytometry experiments with IL13QD-exosomes complex indicates a possibility to identify the tumor associated exosomes. Our experiment infers that tumor targeted quantum dots can selectively bind with glioma stem cells and extracellular vesicles (exosomes) that are secreted by cancer cells. This binding profile could be utilized to identify the expression of certain tumor promoting receptors and antigens on the surface of exosomes present in the CSF and cancer stem cells from the glioma patients. Experiments are in progress to identify the tumor initiating cells in an orthotopic mouse glioma tumor model after intravenous administration of wild type IL13 and certain high affinity mutated IL13 conjugated quantum dots, by non-invasive way.

#746

TaqMan Rare Mutation Assays targeting the TERT promoter region.

Marion Laig, Kamini Varma, Vidya Venkatesh. _Thermo Fisher Scientific Inc., South San Francisco, CA_.

The enzyme Telomerase maintains telomeres at the ends of chromosomes. The Telomerase Reverse Transcriptase (TERT) gene codes for the enzyme's catalytic domain and is not expressed in normal somatic cells. As a consequence, normal cells acquire senescence by shortening of their telomeres during cell division and eventually undergo apoptosis. In contrast to normal somatic cells, expression of TERT is reinstated in cancer cells causing escape from senescence and apoptosis by maintaining the telomeres. It has recently been shown that mutations in the TERT promoter region play a key role in regulating and reinstating TERT expression. 90% of cancers carry a mutation in the TERT promoter region. Mutations like C228T and C250T create new binding site for the E26 transformation-specific (ETS) transcription factor that regulates TERT expression. Experimental evidence showed that the ETS factor GA-binding protein, alpha subunit (GABPA) binds to the de novo ETS motif and activates TERT transcription in cancer cells.

We undertook a project designing TaqMan Rare Mutation assays addressing mutations in the TERT promoter. These assays are TaqMan SNP Genotyping assays that are optimized for use in digital PCR with the Applied Biosystems QuantStudio 3D. In digital PCR, partitioning the sample into many individual reaction wells facilitates detection and quantification of rare mutant alleles. TaqMan SNP Genotyping Assays ensure reliable discrimination of mutant and wild-type allele. This will enable easy and sensitive detection of TERT promoter mutations in cancer research samples. These assays are suitable for detection in liquid biopsy applications with cell free DNA (cfDNA).

Assay design proved to be challenging due to high GC content and repetitive elements in this region of the TERT gene and required varying both the assay design and experimental cycling conditions. Template for wet-lab testing was synthetic plasmid carrying the mutation spiked into wild-type genomic DNA and wild-type genomic DNA control.

The result was a TaqMan Rare Mutation assay detecting the C228T mutation in the TERT promoter region. We showed that designing TaqMan Rare Mutation Assays for digital PCR is feasible for the challenging TERT promoter region. Work addressing additional mutations in this region is ongoing.

For Research Use Only. Not for use in diagnostic procedures.

#747

An improved fixative for histomorphology, recovery of nuclic acids and proteins.

Stephen M. Hewitt,1 Joon-Yong Chung,1 Candice Perry,1 Kris Ylaya,1 William M-A Smith,1 Robert A. Star2. 1 _National Cancer Inst., Bethesda, MD;_ 2 _National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD_.

Neutral Buffered Formalin (NBF) has been the primary diagnostic fixative for histopathology for nearly a century. The limitations of NBF are well known, especially with reference to the analysis of RNA and proteins. Alternative fixatives have been proposed, but not been widely adopted. Recently, the US Environmental Protection Agency (EPA) classified formaldehyde as a carcinogen, further tilting the balance away from the continued use of NBF. We recently described a fixative based on 70% ethanol and phosphate buffered saline, called BE70. Using BE70 as the base fixative, we have developed a modular fixative in which different chemicals can be supplemented for "fit for purpose" applications. The addition of guanidinium salts to BE70 , termed BE70G results in a fixative in which both proteins and nucleic acids are improved in quantity and quality after extraction from the paraffin block. The guanidinium replaces non-freezable water, and prevents hydrolysis and oxidation of the biomolecules. Histomorphology is not disrupted at lower concentrations, with only alterations of hematoxylin intensity at higher concentrations. Studies are underway to evaluate the stability of RNA and protein in the tissue block as well as on cut sections. This fixative, BE70G has the potential to address a number of challenges facing the development of precision biomarkers, while preserving histomorphology, and improving the safety of pathology staff. Other formulations are currently being evaluated that may support applications for cytology and electron microscopy. The goal is a well characterized fixative, in which modifications to the chemistry are defined and can be applied to obtain the optimal pathology specimen for both histomorphologic and molecular analysis.

#748

**A rapid and accurate nucleic acid amplification and detection method for** KRAS **mutation testing in colorectal cancer specimens.**

Choong Eun Jin, Seung-Seop Yeom, Yong Shin, Seok-Byung Lim. _University of Ulsan College of Medicine, Seoul, Republic of Korea_.

Colorectal cancer (CRC) is one of the most common type of cancers both men and women in worldwide. Fortunately, overall death rates of CRC have been decreasing for the last two decades due to the improvement of screening test assays that detect early-stage cancer and pre-cancerous polyps. Nevertheless, the most common treatment for CRC is surgery, because it may completely eliminate the cancer region. In case of the cancer with systemic metastasis, chemotherapy is required before or after surgery for primary or metastatic lesions. Among the regimens for the chemotherapy, both monoclonal antibodies (Cetuximab and Pantitumumab) against the epidermal growth factor receptor have been shown to improve survival for only patients with lack of RAS mutations. Thus, the KRAS gene mutations (codons 12 and 13) in CRC patients have been extensively studied as a strong negative predictive biomarker to indicate whether a CRC patient responds to the treatment. Therefore, testing the KRAS mutational status of tumor samples is becoming an essential tool for managing patients with CRCs. Although a myriad of nucleic acid testing methods have been developed to analyze the mutation status in the key regions of the KRAS gene of CRC, several obstacles still remain related to low sensitivity, time consuming, and required large instruments including thermal cyclers. Here, we present a novel nucleic acid amplification and detection method for KRAS mutations (G12D and G13D) testing that enable rapid and accurate detection. This method is based on combination of isothermal DNA amplification method and bio-photonic silicon sensor that can be detected the mutations in a label-free and real-time manner. The proposed method can detect the mutant cell present at 1% in a mixture of wild type cells, while both PCR and sequencing can detect the mutations in a sample containing approximately 30% of mutant cells. We used 60 tissue samples from CRC patients (22 samples with G12D mutations, 23 samples with G13D mutation, and 15 samples with no mutation) to compare the clinical utility of three methods including PCR, Sanger sequencing and the proposed method. The proposed method with both G12D and G13D showed a value of 100% and 100% for sensitivity and specificity, respectively. One the other hand, the sensitivity and specificity of PCR (90.5% and 100%) and sequencing (95% and 100%) were lower than that of the proposed method. Therefore, the proposed method was found to be a rapid (< 30 min), highly sensitive and specific method for KRAS mutation testing. We believe that this rapid and accurate method will enable proper treatment for CRC patients.

#749

**Novel** ALK **specific mRNA** in situ **hybridization assay for non-small cell lung carcinoma.**

Noriko Hirai, Takaaki Sasaki, Yoshinobu Ohsaki. _Asahikawa Medical Univ., Asahikawa, Japan_.

A recent technical advance in the mRNA in situ hybridization (mRNA-ISH) assay provides simultaneous signal amplification and background suppression with a unique probe design to achieve single molecule visualization. We assessed the utility of the mRNA-ISH assay as a diagnostic tool to detect anaplastic lymphoma receptor tyrosine kinase (ALK) mRNA in non-small cell lung carcinoma. We compared the mRNA-ISH assay with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). The study included 279 surgically resected lung adenocarcinomas and 44 transbronchial biopsied (TBB) adenocarcinomas. mRNA-ISH was conducted using the RNAscope 2.0 system which includes pre-designed detection probes for the tyrosine kinase domain of ALK mRNA. IHC was conducted on all of the 323 samples using ALK-specific antibodies. mRNA-ISH was performed on 279 surgical samples and 6 TBB samples. Break-apart FISH was used to assess cases that were mRNA-ISH or IHC positive. ALK protein expression was detected in 11/279 (3.9%) specimens. ALK mRNA was also detected by mRNA-ISH in these cases, and 9 of the 11 specimens (81%), were also positive by ALK FISH. Using the IHC results as a reference, the sensitivity and specificity of mRNA-ISH was 100%. In the TBB cohort, ALK protein expression was observed in 3/44 (6.8%) of specimens, in which ALK mRNA expression was also detected. ALK mRNA-ISH data were highly correlated with the IHC data, and ALK mRNA-ISH was able to identify every FISH-positive sample. We conclude that mRNA-ISH could play an alternative or complementary role in ALK genetic diagnosis in NSCLC.

#750

Detection of mutations in single circulating tumor cells using MALDI-TOF mass spectrometry.

Jackie L. Stilwell,1 Ryan T. Birse,2 Arturo Ramirez,1 Melinda Duplessis,1 Brennan Enright,1 Darryl Irwin,2 Eric Kaldjian1. 1 _RareCyte, Inc., Seattle, WA;_ 2 _Agena Bioscience, Inc., San Diego, CA_.

Background: As cancer therapeutics are increasingly selected based upon molecular genetic information, having reliable, accurate, rapid and inexpensive methods for mutational analysis are extremely desirable. Circulating tumor cells (CTCs) allow non-invasive "liquid biopsy" access to intact cells for molecular analysis. Here we demonstrate the successful detection of mutations in model CTCs individually isolated from blood (AccuCyte® - CyteFinder® system, RareCyte) using MALDI-TOF Mass Spectrometry (MassARRAY, Agena Bioscience).

Methods: Breast (MDA-MD-231) and lung (NCI-H1975) cancer cells with a set of known mutations were spiked into blood and processed by AccuCyte onto microscope slides and stained on an automated immunostainer. Slides were imaged using the CyteFinder digital fluorescence scanning microscope and mCTCs were identified by positive nuclear, EpCAM, and cytokeratin staining, and negative CD45 staining. mCTCs and white blood cell (WBC) negative controls were picked from the slides and put into PCR tubes using the CytePicker® module. DNA from individual or small pools of cells (3-5) was amplified using the PicoPLEX® (Rubicon) whole genome amplification (WGA) kit; alternatively cells were lysed and directly entered into the ensuing iPLEX® Pro workflow. Specific regions surrounding 5 different mutations in each of the mCTC lines were amplified from the WGA product or the lysed cells and the products were detected and scored for the mutations using a single PCR reaction iPLEX® Pro panel that includes a combination of 10 common lung and breast cancer mutations using the MassARRAY® platform.

Results: Five point mutations in four different genes (CDKN2A, EGFR, PIK3CA, and TP3) were measured in the NCI-H1975 lines and four point mutations in four genes (BRAF, KRAS, NF2, and TP53) and a deletion in one gene (CDKN2A) were measured in the MDA-MB-231 cells by iPLEX® Pro chemistry on the MassARRAY® system. All mutations were accurately detected in the WGA single and pooled cell samples and most were also detected in cells that did not undergo WGA before PCR with with iPLEX Pro panel. Allelic frequency observed was consistent with known zygosity of the mutation.

Conclusions: MassARRAY successfully detected mutations in single model CTCs that were individually picked from a blood sample processed by the AccuCyte - CyteFinder system. Integrating CTC isolation with MassARRAY may be a practical way to identify and monitor known cancer mutations non-invasively.

#751

Characterization of STAT3 activation in human prostate cancer.

Marco A. De Velasco,1 Yuji Hatanaka,1 Yurie Kura,1 Naomi Ando,1 Kazuko Sakai,1 Koichi Sugimoto,1 Masahiro Nozawa,1 Kazuhiro Yoshimura,1 Kazuhiro Yoshikawa,2 Kazuto Nishio,1 Hirotsugu Uemura1. 1 _Kindai University Faculty of Medicine, Osaka-Sayama, Japan;_ 2 _Aichi Medical University, Nagakute, Japan_.

Signal transducers and activators of transcription (STATs) were initially associated with cytokine signal transduction pathways but are now also recognized as key modulators of key survival processes in in various cancers. Activation of STAT3 occurs by the binding of various cytokines to its receptors leading to the activation of the JAK/ STAT3 signaling pathway. Interleukin-6 (IL-6) has been implicated in regulating growth of various malignant tumors. Activated IL-6 has also been shown to be elevated in the sera from patients with metastatic prostate cancer, and persistent activation of STAT3 is a common feature. To better characterize the potential role of JAK/STAT3 as a therapeutic target for advanced prostate cancer, we examined expression patterns of activated STAT3 in 111 cases of localized prostate cancer from patients who underwent radical prostatectomy. Association studies were conducted with clinicopathological characteristics and biochemical recurrence. We also analyzed the expression of IL-6/KAK/STAT3 genes from RNA-sequencing data form the Cancer Genome Atlas Prostate Adenocarcinoma (TCGA-PRAD) data set and its association to long-term outcomes. Immunohistochemical analysis revealed that 11.7%, 42.3% and 45.9% of patients had high, moderate or low/negative expression of phosphorylated-STAT3 (Tyr705). Cancerous glands tended to have higher STAT3 expression compared to paired normal adjacent tissue, P=0.088. No correlations were observed between STAT3 expression and clinicopathological parameters. Patients with moderate-high p-STAT3 expression tended to experience shorter times to biochemical recurrence (median time, 15 months) compared to patients with low/negative STAT3 activity, (median time to recurrence not reached, P=0.064). Cluster analysis suggested a trend for decreased disease-free survival for patients with high IL-6/KAK/STAT3 gene expression signature. Our data suggests an association between STAT3 signaling and prostate cancer recurrence and provides clinical evidence to support JAK/STAT3 as a potential therapeutic target for patients with advanced prostate cancer.

#752

**Evaluation of VENTANA BenchMark ULTRA and Roche cobas** ® **z 480 analyzer for detection of EGFR mutations.**

Lorraine Stewart, Chunyan Liu, Judith Pugh, Heather Gustafson. _Ventana Medical Systems, Tucson, AZ_.

Research has shown non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for 85% cases. Exon 19 deletion and L858R mutation in exon 21 account for approximately 90% of EGFR mutations in adenocarcinoma NSCLC1. Accuracy and sensitivity in detection of these markers aids the selection of patients with NSCLC for therapy with an EGFR tyrosine kinase inhibitor (TKI). This study demonstrates how validated Immunohistochemistry (IHC) and real-time PCR testing can be used to aid one another in detection of epidermal growth factor receptor (EGFR) mutations in formalin-fixed, paraffin embedded biopsies in our CAP/CLIA laboratory.

Evaluation of IHC detection was completed on the VENTANA BenchMark ULTRA Automated IHC/ISH slide stainer and PCR testing was performed using the Roche cobas® z 480 analyzer. The IHC diagnostic markers selected were the two most common EGFR mutations, E746-A750del (SP111) and L858R (SP125). Sample preparation and real-time PCR testing was done using the cobas® DNA Sample Preparation Kit and cobas® EGFR Mutation Test v2 utilizing a custom, automated plate preparation program on the Perkin Elmer NGS Express Platform.

Analytical data from the BenchMark ULTRA platform staining and the cobas® z 480 analyzer correlated in the majority cases. Out of 32 specimens tested, we observed a 100% concordance between IHC and PCR for L858R exon 21 mutation, whereas EGFR exon 19 deletion concordance between IHC and PCR was 90%. This decrease in concordance was due to 3 specimens with indeterminate IHC staining results with weak staining in a small tumor area. Use of a custom automated workflow for PCR plate preparation demonstrated successful facilitation of sample preparation and tracking.

Evaluation of results indicate that not only is >10% tumor content important for detection of mutations via PCR (as directed by the IVD package insert), but heterogeneity within the tumor can further decrease sensitivity of PCR. However weak or low IHC staining in only small areas of the tumor may prove to have less clinical relevance. For the most comprehensive results, IHC staining in addition to molecular testing provides better characterization of tumor profiles. Preliminary results suggest that automated dissection of tumor with the Roche Automated Dissection instrument prior to PCR increases sensitivity. In conclusion, IHC and real-time PCR should be used together to detect not only the array of mutation available in the PCR test, but also to confirm the two most common mutations by IHC and increase sensitivity of testing which may be strongly impacted by heterogeneity.

1. - Marc Ladanyi and William Pao: Lung adenocarcinoma: guiding EGFR-targeted therapy and beyond. Modern Pathology (2008)21, S16-s22; doi:10.1038/modpathol.3801018

#753

Human epidermal growth receptor-2 immunohistochemistry in gastric cancer.

Babatunde M. Duduyemi,1 Mary Y. Afihene,1 Emmanuel A. Asiamah2. 1 _Kwame Nkrumah University of Science and Technology, Kumasi, Ghana;_ 2 _Komfo Anokye Teaching Hospital, Kumasi, Ghana_.

Introduction: Gastric cancer is the second most common GI cancers and third leading cause of cancer-related deaths worldwide. In Ghana, gastric cancer contributes significantly to cancer morbidity and mortality. The recent development on the benefit of targeted therapy in the management of gastric cancer using HER-2 monoclonal antibody in combination with chemotherapy has greatly improved the overall survival of patients. This study therefore aims at determining the frequency of Her-2 over-expression in gastric carcinomas in Kumasi, Ghana and comparing with other prognostic factors.

Methodology: Demographic data and histological diagnosis were retrieved from the surgical day book of the Department of Pathology, Komfo Anokye Teaching Hospital, Kumasi. The slides were retrieved and reviewed to confirm diagnosis, grading and classification of gastric cancer. Immunohistochemistry was done with anti-HER-2 antibody to confirm HER-2 over-expression. The data was analysed with SPSS version 17. Results: Of the 99 cases of gastric cancer seen over the 8 year period, there were 57 males and 42 females, adenocarcinoma (91), GIST (5) and Non Hodgskin Lymphoma (3). The age range of the study population was 8-90 years with modal age group in the 6th decade. There were more biopsies (63) than resections (36). Of the adenocarcinomas, 45 were poorly differentiated, 38 moderately differentiated and 8 well differentiated. Diffuse type is most common with 47 cases followed by intestinal 41cases and mixed type 3 cases. Three of the 4 patients under the age of 30 years had lymphoma. HER-2 over-expression was seen in 14 out of the 47 tested and all were intestinal type.

Conclusion: HER -2 over-expression was seen a small cohort of our patients with gastric cancer especially those with intestinal type.

#754

**Treatment decision-making of rare** ERBB2 **(HER2) mutations in lung cancer; a role for multidisciplinary molecular tumor boards.**

Arja ter Elst,1 Nils A. 't Hart,1 Anthonie J. van der Wekken,1 Wim Timens,1 Lucie B. Hijmering-Kappelle,1 Geke A. Hospers,1 Hilde Jalving,1 Elise M. van der Logt,1 Leon C. van Kempen,1 Sjoukje F. Oosting,1 Matthew R. Groves,2 T Jeroen Hiltermann,1 Anke van den Berg,1 Harry J. Groen,1 Ed Schuuring1. 1 _University Medical Center Groningen, Groningen, Netherlands;_ 2 _University Medical Center Groningen, University of Groningen, Groningen, Netherlands_.

Introduction:

Breakthroughs in cancer research have resulted in mutation-specific targeted therapies (precision medicine). Most of these new drugs are only effective in patients with an actionable molecular profile. Thus, predictive molecular testing enables oncologists to select individual patients for the most appropriate (targeted) therapy and to reduce the burden of overtreatment. The number of clinically relevant predictive markers that are routinely analyzed is growing rapidly, resulting in the identification of rare mutations, mutations with unknown relevance and coexistence of two or more mutations in the same sample. Incorporating these into the optimal treatment for the individual patient can be complex.

Methods: A total of 2461 sequential tumor biopsies were analyzed at our institute using targeted next generation sequencing (Ion Torrent platform). 230 of these patients were discussed at a weekly Molecular Tumor Board (MTB) meeting. Cases involved 170 lung and 21 colorectal carcinomas, 24 melanomas, 1 GIST and a range of other malignancies with uncommon and rare mutations. The board is composed of pulmonologists, medical oncologists, pathologists and clinical scientists in molecular pathology. The goal of the MTB is to discuss the biological and clinical relevance of rare mutations or uncommon profiles and to suggest treatment options based on registered, off-label or trial-based drugs presently available in the Netherlands.

Results: In this abstract we report on four patients with an ERBB2 exon 20 mutation and 1 patient with ERBB2 amplification received anti-HER2 treatment after an MTB consensus decision. Two patients with an insertion in exon 20 of ERBB2: (c.2313_2324dup; p.(Y772_A775dup)) received first line therapy with afatinib and showed a partial response and stable disease respectively. One patient with a c.2524G>A; p.(V842I) mutation received afatinib and showed stable disease for 3 months. A patient with another ERBB2 exon 20 insertion (c.2326_2327insTAT:p.(G776delinsVC)) received afatinib but had progressive disease within two months. One patient with an ERBB2 amplification by FISH and high (3+) HER2(ERBB2) expression, showed a partial response to trastuzumab. All patients had stage IV and would without genomic knowledge been treated with chemotherapy.

Conclusion: Lung cancer patients with sporadic ERBB2 mutations might benefit from targeted ERBB2 therapy. For an optimal treatment decision, patients with rare mutations in general, may benefit from discussion in a multidisciplinary molecular tumor board. In the future, both the considerations for targeted therapy as well as treatment response and toxicity should be registered in an open-access database and shared with other national and international Molecular Tumor Board initiatives to allow comparison with traditional treatments.

#755

Supporting precision cancer treatment decision with functional evaluation of cancer gene mutations and variants.

Jingjing Jiang,1 Zhongguang Luo,2 Jia Wei,3 Guanglei Zhuang,4 Song Yi,5 Ying Yan,6 Tengfei Yu,6 Wei Du,6 Tingting Tan,6 Ling Qiu,6 Jiali Gu,6 Xin K. Ye,6 Jie Liu,2 Zhenyu Gu6. 1 _GenenDesign USA Co, Ltd., Menlo Park, CA;_ 2 _Huashan Hospital, Shanghai, China;_ 3 _Nanjing University, Nanjing, China;_ 4 _Shanghai Jiao Tong University, Shanghai, China;_ 5 _MD Anderson Cancer Center‎, Houston, TX;_ 6 _GenenDesign Co, Ltd., Shanghai, China_.

Precision oncology requires identifying and understanding of cancer genome changes in a patient tumor tissue and finding the best cancer therapy targeting the changes. Although many cancer gene targets have been validated so far, next-generation genomic profile analyses have uncovered much more cancer gene variants with unconfirmed functions. Developing methods to functionally evaluate mutations/variants and understand their roles in cancer development and drug responses, such as drug resistance or synthetic lethality, will be critical in cancer treatment decision support. In addition, in some clinical cases, multiple treatment choices such as multiple drug combinations exist. Developing cancer models which can test multiple treatments will provide direct comparison of those therapies and select the best options. At GenenDesign, we have performed drug tests on mouse "avatars", which are also known as Patient-Derived Xenograft (PDX) models. They are personalized cancer models derived from patient tumor samples with cancer mutation profiles and drug responses very similar to the corresponding cancer patients. Drug screenings were carried out in avatars by testing chemotherapies or targeted drugs against specific cancer gene mutations and variants. Selected drugs or drug combinations from avatar studies have been applied to corresponding patients with highly consistent treatment outcome. From genomic profile analysis of our near 1500 PDX tumor models in cancer types such as lung, colorectal, gastric, liver, and esophageal, we are able to identify a large number of cancer gene mutations/variants, gene fusions, as well as gene copy number and RNA expression changes in major cancer signal pathways such as EGFR, Her2, c-Met/ALK, Ras/Raf, FGFRs, PI3K/Akt, Wnt, Notch, DNA repair, cell cycle regulation, angiogenesis. Many of these gene aberrations are potential drug targets and have been functionally tested in PDX models with approved drugs or clinical drug candidates. The mutation/variant information and drug response information generated from PDX models have been organized into our Precision Cancer Information Lab database. Patient tumor DNA test results have been used for searching genetically matched PDX models in our database. Once matched PDX models are identified, the available drug response information can be used as evidence for clinical treatment decision. In addition, the matched PDX models can also been used to test more treatment options such as different combinations and new clinical drug candidates.

#756

Molecular characterization of triploid and tetraploid urine FISH samples.

Lorenza Pecciarini, Valeria De Pascali, Ilaria Francaviglia, Anna Talarico, Chiara Iacona, Massimo Freschi, Maria Giulia Cangi, Claudio Doglioni. _San Raffaele Hospital, Milano, Italy_.

Urothelial carcinomas (UC) are characterized, among other aberrations, by chromosome 3, 7, 17 gains and 9p21 (p16) loss. Since UC cells readily exfoliate into the urine, molecular cytogenetics techniques have been used to detect cells consistent with UC diagnosis and follow-up: a multitarget Fluorescence In Situ Hybridization (FISH) test, composed of 3, 7 and 17 centromeres and 9p21 locus multicolor probes, has been proved useful for atypical cells identification in both void urine and lower and upper urinary tract samples before morphological changes are apparent through cytological and histological examination. To date, there are no uniform criteria for urine FISH scoring though the test is commonly considered positive following the UroVysion Bladder Cancer Kit criteria (Abbott Molecular): 4 or more cells with a gain of 2 or more chromosomes (polysomy), and/or 12 or more cells with 9p21 homozygous deletion. This definition of polysomy includes trisomy and tetrasomy classes, but, as tetraploidy may represent cells at S/G2 phase, this criterion may lead to false-positives; moreover it has been described that also inflammation induces tetraploidy, and that small percentages of normal urine cells show trisomy/tetrasomy. On the other hand, triploidy and tetraploidy may have a direct role in tumorigenesis, as an intermediate state in the development of cancer aneuploidy. At the moment little is reported about the clinical correlations of urine FISH cases showing only trisomy/tetrasomy and more data are needed to clarify the diagnostic value of triploid and tetraploid urothelial cells. Aiming to better classify patients with a trisomy/tetrasomy urine FISH result, we extracted genomic DNA from 50 fixed urine cell suspensions, previously tested by urine FISH, and investigated them for the presence of fibroblast growth factor receptor 3 (FGFR3) and telomerase reverse transcriptase (TERT) promoter mutations, which constitute the most recurrent somatic alterations in BC. In particular frequent mutations in exons 7, 10 and 15 of FGFR3 characterize non-muscle invasive UC while mutations in the core promoter region of TERT gene, mainly at -124 and -146 positions from the ATG start site, have emerged as the most frequent somatic genetic alteration in both non-muscle invasive and muscle invasive UC. All specimens were subjected to PCR amplification and Sanger sequencing for FGFR3 exon 7 and 10, and for the C228T and C250T TERT promoter mutations. A preliminary analysis of 20 urine FISH cases showed 4 samples with trisomy/tetrasomy, and 1 of these 4 had a mutation of the TERT promotor. Complete results of the FGFR3 and TERT promoter molecular analysis will be presented and correlated to both FISH results and patients clinical data, in order to evaluate if the presence of FGFR3 and/or TERT promoter mutations in urine samples with FISH detected trisomy/tetrasomy may help to identify patients with a higher risk of recurrence and progression.

#757

**Development and validation of the ActionSeq** TM **test system.**

Samantha Helm, Vanessa Spotlow, Aleksandra Ras, Kevin Kelly, Guruprasad Ananda, Sara Patterson, Honey V. Reddi. _Jackson Laboratory for Genomic Medicine, Farmington, CT_.

Introduction In the constantly changing field of oncology precision medicine, it is exceedingly important to keep diagnostic and therapeutic assays clinically relevant. Next generation sequencing (NGS) panels in oncology are greatly impacted by new findings in clinical actionability. In order to ensure that cancer panels continue to provide the most beneficial results to patients, they must be regularly updated. In keeping with this idea, JAX has launched a new 212 oncology gene panel which focuses on genes and variants with documented actionability, referred to as ActionSeq.

Methods Development of ActionSeq included the optimization of a new targeted capture assay. This process included running multiple batches of samples through the assay to determine appropriate DNA input, ligation times, PCR cycles, and pooling conditions. The fully optimized assay was then validated using 24 uncharacterized FFPE samples. The validation was executed in 5 phases: (1) confirm that assay optimizations yielded sufficient wet lab results; (2) LOD & sensitivity (3) inter-assay concordance; (4) intra-assay concordance; (5) specificity and accuracy.

Results During development, the standard protocol was optimized using a 200ng input, 30 minute ligation period, 5 cycles of pre-PCR, and the pooling of 4 samples per hybridization reaction. Wet lab processing results of the first validation batch can be seen in Table 1. The inter- and intra-assay concordances were found to be ≥ 96% for variants and 100% for CNVs. The sensitivity was calculated to be 98.92% at a LOD of 3% for SNVs, 100% at a LOD of 8% for INDELs, 100% at a LOD of 6 copies for CNV amplifications, and 100% at a LOD of 0 copies for CNV deletions. The specificity and accuracy were found to be 100% for all mutation types.

Conclusion Based on the success of this validation ActionSeq has been incorporated into the JAX clinical test menu. This addition accomplished the goal of providing a more clinically relevant (actionable) somatic tumor profiling assay to patients and clinicians. | |  | |  | |

---|---|---|---|---|---|---

Sample | Post Prep ng/ul | Capture Pool ng/ul | Average Base Pair Size | Mean Target Coverage | Percent Duplication

1 | 27 | 1.41 | 309 | 511 | 23%

2 | 24 | 1.41 | 309 | 594 | 23%

3 | 24 | 1.52 | 302 | 659 | 22%

4 | 29 | 1.52 | 302 | 652 | 21%

5 | 21 | 2.22 | 290 | 535 | 16%

6 | 35 | 2.22 | 290 | 560 | 13%

7 | 29 | 2.23 | 299 | 530 | 17%

8 | 72 | 2.58 | 304 | 1196 | 14%

9 | 65 | 2.58 | 304 | 732 | 14%

10 | 43 | 2.58 | 304 | 571 | 13%

11 | 42 | 2 | 303 | 722 | 18%

12 | 37 | 2.0 | 303 | 611 | 16%

13 | 27 | 2.0 | 303 | 735 | 18%

14 | 42 | 1.9 | 309 | 731 | 20%

15 | 40 | 1.9 | 309 | 683 | 19%

16 | 67 | 1.9 | 309 | 675 | 16%

17 | 48 | 1.9 | 309 | 573 | 20%

18 | 53 | 1.89 | 301 | 655 | 19%

19 | 31 | 1.89 | 301 | 763 | 22%

20 | 82 | 1.89 | 301 | 761 | 17%

21 | 56 | 1.89 | 301 | 877 | 19%

22 | 34 | 1.99 | 329 | 771 | 18%

23 | 48 | 1.99 | 329 | 886 | 16%

24 | 78 | 1.99 | 329 | 841 | 15%

Established Quality Control Cut Offs:

Post Library Prep ng/ul: | ≥ 20

Post Capture Pool ng/ul: | ≥ 0.5

PC Average Base Pair Size: | 250-350

Mean Target Coverage: | ≥ 500

Percent Duplication: | ≤ 25%

Table 1: This table displays the wet lab processing results for the 24 samples used to confirm the efficiency of the developmental optimizations made to the assay prior to beginning the clinical validation.

#758

Capture and characterization of circulating tumor cell clusters in patients with metastatic castrate-resistant prostate cancer.

Samantha L. Savitch,1 Stephanie S. Yee,1 Devon Soucier,1 Denis Smirnov,2 Chandra Rao,2 Steve Gross,2 Ravi K. Amaravadi,1 David J. Vaughn,1 Naomi B. Haas,1 Erica L. Carpenter1. 1 _Division of Hematology and Oncology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA;_ 2 _Janssen Pharmaceutical, Johnson & Johnson, Huntington Valley, PA_.

Introductory Sentence indicating purpose of study: Circulating tumor cell (CTC) clusters have been shown to have higher metastatic potential than single CTCs in breast, pancreatic, and other cancers, yet these clusters have not been extensively described in metastatic castrate-resistant prostate cancer (MCRPC). Here we sought to determine the feasibility of capturing and characterizing CTC clusters in prostate cancer patients.

Description of Experimental Procedures: Fifty-five blood samples from 29 MCRPC patients, ages 50 to 81 (median age 68), were obtained prior to the patient starting or switching to androgen receptor inhibitor or 17 alpha lyase inhibitor therapies. Eighteen patients received enzalutamide and 11 received abiraterone. The majority of patients had a Gleason score > 7 (22; 75.9%), bone metastases (19; 65.5%), and an ECOG status of 0 (21; 72.4%). All patients had previously undergone, or were receiving at time of enrollment, androgen deprivation therapy. Eleven patients (37.9%) had been on a prior 2nd generation anti-androgen therapy. CTC single cells and clusters (2 or more cells together in one image) were enumerated using the CellSearch system and stained to detect expression of androgen receptor (AR), glucocorticoid receptor (GR), and neuroendocrine (NE) markers.

Summary of Data: Five or more single CTCs, a measure which has previously been associated with an unfavorable prognosis, were detected in 13 of 29 patients (44.8%), and in 19 of 55 blood samples (34.5%). Altogether, a total of 282 CTC clusters was detected, with 1 or more clusters found in 10 patients (34.5%) and 13 samples (23.6%). The number of clusters per 7.5ml of blood ranged from 0-150, and clusters contained anywhere from 2 to 16 cells. Most CTC clusters (268; 95.0%) contained only CTCs and no leukocytes. Just over half the detected clusters (162; 57.4%) contained only 2 CTCs. Cluster staining patterns were fairly homogenous with 29.4% of clusters having uniform expression of either AR, GR, or NE markers, i.e., all CTCs in the cluster expressed the marker of interest. Most clusters (67.7%) were uniformly marker negative and the remaining 2.8% demonstrated a mix of marker positive and marker negative CTCs. Serum Chromogranin A levels, as determined by standard of care clinical blood testing, were found to be positively associated with the number of CTC clusters per 7.5ml of blood (p<0.0001).

Statement of Conclusions: The capture and characterization of CTC clusters in the blood of MCRPC patients can be successfully performed using the CellSearch system. Further investigation into the clinical implications of these clusters is warranted, including whether cluster characteristics are associated with more aggressive disease.

#759

Highly-sensitive detection of EGFR mutation in non-small cell lung cancer using mutant-enrichment method.

Hung-Chi Chien. _Industrial Technology Research Institute, Hsinchu, Taiwan_.

Detection of mutations in EGFR in non-small cell lung cancer is important for the clinical decision-making process of molecularly targeted therapies, including Iressa®, Tarceva®, and Tagrisso®. In this study we developed an assay allowing the detection of EGFR gene mutations being conducted in the routine diagnostic procedure and its analytic and clinical performance were evaluated using different types of specimens. The EGFR mutation assay in this study allows for detecting EGFR exon 19 deletion (Del), exon 20 insertion (Ins), and mutation in T790M, L858R, S768I and L861Q of rare amounts in a normal genetic background. Enhancement of detection sensitivity was achieved with the developed mutant enrichment method which combines mutation-specific primers and 3'-phosphorylation oligonucleotides as blockers for inhibiting wild-type gene from DNA amplification. Analytic performance for each mutant was assessed using the control plasmids containing the mutant genes of known amounts as well as the genomic DNA extracted from mutant cell lines and Horizon FFPE samples. The assay performance were also evaluated using clinical samples in parallel with the FDA-approval companion diagnostic product therascreen® EGFR RGQ PCR Kit . The sensitivity of the assay evaluated using control plasmids for Del, T790M , L858R, S768I, L861Q mutation and Ins was 5%, 0.5%, 1%, 0.5%, 0.1% and 0.5% respectively, and was 1%, 0.5%, 0.5%, 0.5%, 0.1% and 0.5% respectively when applying on HDx reference standards. Diagnostic results for the 116 clinical samples for identifying mutations of del, T790M, L858R, S768I, L861Q and Ins was 92.2%, 100%, 99.1%, 100%, 97.4% and 100% respectively concordance with the therascreen® EGFR RGQ PCR Kit, including 17 cases of multiple mutations. The overall diagnostic results for all mutations evaluated with PPA (positive percent agreement), NPA (negative percentage agreement) and OPA (overall percentage agreement) were 83.3%, 98.2% and 90.5%, respectively. The three samples firstly diagnostic as false positive were retested and confirmed to be Exon 19 del by DNA sequencing. The assay demonstrated good sensitivity when compared with commercial product. More clinical studies are being performed to establish relationship between drug efficacy and different mutation combinations.

#760

Detection of EGFR T790M mutation by ddPCR in untreated NSCLC patients: Correlation with clinical outcome.

Monica Ganzinelli,1 Eliana Rulli,2 Elena Tamborini,1 Adele Busico,1 Giuseppe Lo Russo,1 Giulia Corrao,1 Milena Vitali,1 Marina Chiara Garassino,1 Massimo Broggini,2 Mirko Marabese2. 1 _Fondazione IRCCS Istituto Tumori Milano, Milan, Italy;_ 2 _IRCCS - Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy_.

Background: Activating EGFR mutations are associated with response to first generation tyrosine kinase inhibitors (TKI) that significantly increased PFS and OS in NSCLC patients. However, almost invariably, patients relapse while in treatment with TKI, mostly for the presence of EGFR T790M mutation. New second and third generation EGFR inhibitors active against T790M are now under development with very promising results. To date it is not known if the presence of T790M (and its amount) at diagnosis influences clinical outcome of patients with activating EGFR mutation.

Methods: We analysed 50 cases with NSCLC. 48 patients harbour TKI sensitizing mutation in either exon 19 (N=24) or 21 (N=19). 2 EGFR wt patients (blinded to the investigator) were analysed in parallel. These patients were screened by ddPCR for the presence of T790M mutation using Biorad QX200 Droplet Digital PCR System. We used rough DNA extract from FFPE sections after deparaffinization and proteinase K digestion, as available in pathology unit for daily diagnosis.

Results: 12 samples failed to amplify, maybe due to the poor quality of DNA. 13 samples produced results, but were excluded for the analysis because of too low number of calls retrieved. T790M mutation was detected in a significant proportion of remaining cases. Among the latter, 4 patients were sampled after TKI treatment and here the percentage of T790M were higher compared to those patients not treated [0.65% - 28% vs 0.019% - 1.65%, respectively]. One out four of these patients was not detected by routinely method of sequencing (Sanger or RT-PCR). T790M positive samples harboured mutations affecting exon 19 or 21 in equal manner.

Conclusions: ddPCR has proven to be a sensitive method to detect T790M mutation, although the quality of DNA could affect the results. The system has been challenged for specificity an sensitivity and the results proved to be clinically relevant. The correlation between the T790M positivity and clinical outcome will be available and we plan to define a ddPCR threshold value which can help in selecting those patients who are likely to have early recurrence under first generation TKI and that could therefore be directly shifted to second or third generation inhibitors.

#762

**A concordance study of the ArcherDX Reveal** TM **ctDNA 28 NGS panel and Biocept's Target Selector** TM **mutation assay using ctDNA collected in Biocept CEE-sure** TM **blood collection tubes.**

Jason C. Poole,1 Brian Kudlow,2 Jill Stefanelli,2 Skyler Mishkin,2 Anh Pham,1 Jeff Chen,1 Veena M. Singh,1 Josh Stahl,2 Lyle J. Arnold1. 1 _Biocept, Inc., San Diego, CA;_ 2 _ArcherDX, Inc., Boulder, CO_.

Biocept's Target Selector technology is a targeted hotspot mutation panel designed to enrich for mutant targets in a large excess of WT DNA. The test is specific to small regions of interest, is highly sensitive, validated down to 7 mutant copies in a background of 14,000 WT at >98% sensitivity. The ArcherDX Reveal ctDNA test is a 28 gene NGS panel that targets key oncogene activating mutations, drug resistance mutations, in addition to full coverage of TP53. The Reveal ctDNA assay utilizes Anchored Multiplex PCR to enrich, tag, and efficiently capture short ctDNA fragments. Both technologies are designed specifically for use with plasma associated ctDNA. We undertook to evaluate the feasibility of using the ArcherDX Reveal ctDNA 28 NGS panel with the DNA extracted from plasma collected with Biocept's patented blood collection tube which has been validated at room temperature for 4 days for circulating tumor cells and 8 days for ctDNA. The ability to build complex, targeted libraries, free of chemically induced mutations is of major importance for the detection of the vanishingly rare mutations that can be present in the plasma of cancer patients. We found that high quality NGS libraries were produced from plasma collected and stored in the Biocept blood tube, indicating that little damage occurred to the DNA during preservation. In addition, the two methods were found to be highly concordant and complementary when using both mutation positive and negative patient samples. The Target-Selector assay was the more sensitive, however, the ArcherDX panel revealed several additional mutations not targeted by the Biocept Target Selector assay.

#763

**Analytical validation of Ki67/CD8 duplex IHC assay using computational tissue analysis (cTA** TM **).**

Staci J. Kearney, Joshua C. Black, Benjamin J. Landis, Sally Koegler, Brooke Hirsch, Roberto Gianani. _Flagship Biosciences, Inc., Westminster, CO_.

Chromogenic multiplex immunohistochemistry (IHC) assays enable investigation of the spatial relationships between tumor and immune cells, which is thought to be important for understanding and predicting therapeutic response. Development and analytical validation of multiplex IHC assays enables the use of such assays to simultaneously investigate multiple biomarkers as predictors of clinical response. In this study, we analytically validated a chromogenic duplex IHC assay that quantifies Ki67 and CD8 in formalin-fixed, paraffin-embedded non-small cell lung cancer tissues. Five performance criteria were selected and evaluated based on Clinical Laboratory Standards Institute guidelines: reportable range, analytical sensitivity, analytical specificity, accuracy, and precision. Similar to analytical validation studies for monoplex IHC assays, this study utilized a reference method and multiple days of staining. The percentage of cells positive for Ki67 nuclear staining and/or CD8 membrane staining were quantified using our computational Tissue Analysis (cTATM) platform. Performance of the Ki67/CD8 chromogenic duplex IHC assay was considered acceptable for the five criteria evaluated. Once the performance of the assay was established, additional exploratory cTA-based endpoints were examined, including the quantification of each biomarker in the tumor compartment and the tumor microenvironment, and analysis of the spatial arrangement of immune cells relative to tumor cells. In conclusion, Flagship's cTA platform allows for more consistent quantification of individual analytes on dual-stained tissue sections, enabling investigation of complex biological questions that cannot be achieved with traditional tissue-based manual endpoints.

### Special Populations / Biostatistics in Clinical Trials

#764

Survival analysis for the elderly esophageal cancer patients (≥80 years) with surgery and non-surgery treatments.

Dan Feng Du,1 Dan Feng Du,2 Fu You Zhou,3 Lian Qun Zhang,3 Xue Na Han,1 Yuan Yuan,3 Zong Min Fan,1 Jian Po Wang,3 Yao Wen Zhang,3 Tai Jiang Liu,4 Guo Hua Liu,4 Yi Jun Qi,5 Yan Rui Zhang,6 Li Dong Wang1. 1 _The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China;_ 2 _Basic Medical College of Zhengzhou University, Zhengzhou, China;_ 3 _Anyang Tumor Hospital, China;_ 4 _Huojia Red Cross Hospital, China;_ 5 _The First Affiliated Hospital of Henan University of Science and Technology, China;_ 6 _Henan Provincial People's Hospital, Zhengzhou, China_.

With the aging of population, the elderly esophageal cancer patients are increasing. However, the optimal treatment methods have not been well characterized for the elderly esophageal cancer patients because of that most randomized studies have excluded the elderly. The aim of this study was thus to analyze the outcomes of surgery and non-surgery therapy on the elderly patients aged 80 years or older with esophageal squamous cell carcinoma (ESCC) to provide more evidences for optimal treatment design on the elderly patient. The enrolled 1,143 ESCC cases in this study were from the ESCC database in Henan Key Laboratory for Esophageal Cancer Research. Of the 1,143 patients, there were 614 males with a mean age of 82.45±2.64 and 529 females with a mean age of 82.44±2.61. The age ranged from 80 to 96 years old at diagnosed. All the patients were confirmed as ESCC by histopathology. The patients were classified into four groups based on treatment methods, i.e., radical esophagectomy (group I), esophagectomy plus radiochemotherapy (group II), radiochemotherapy only (group III), and symptomatic treatment (group IV). Radical esophagectomy was performed on 304 patients (26.6%), including 134 males and 97 females. In addition, of the 304 patients with radical esophagectomy, there were 73 patients who were received radichemotherapy after surgery. 518 patients received only radiochemotherapy (45.3%). Radiochemotherapy included 50-60 Gy of radiation concurrent with or without 5-fluorouracil alone or combined with cisplatin. Moreover, there were 321 patients (28.1%) who did not receive either surgery or radiochemotherapy, but only with symptomatic treatment. The Kaplan-Meier analysis indicated that there was no significant difference in gender, age or histology between patients in the different groups. The overall 1-, 3- and 5-year survival rates were 56%, 31% and 19%, respectively, with a median survival time of 2.5 years. In group I, the overall 1-, 3- and 5-year survival rates were 68%, 48% and 30%, respectively, with a median survival time of 3.8 years. In group II, the overall 1-, 3- and 5-year survival rates were 48%, 32% and 14%, respectively, with a median survival time of 1.9 years. In group III, the overall 1-, 3- and 5-year survival rates were 58%, 28% and 18%, respectively, with a median survival time of 2.5 years. In group IV, the overall 1-, 3- and 5-year survival rates were 39%, 18% and 0.08%, respectively, with a median survival time of 1.6 years. The log rank survival analysis showed that the surgery group had better prognosis than surgery plus radiochemotherapy or radiochemotherapy alone group (P<0.002) and the symptomatic treatment group (P<0.000). The present results demonstrate the benefit of esophagectomy on the elderly ESCC patients in terms of survival. [Supported by the Joint Funds of NSFC (U1301227), Natural Science Foundation of Henan Province (20161110) and Correspondence to: Li Dong Wang, Email: ldwang2007@126.com]

#765

Prognostic impact of Philadelphia chromosome in elderly acute lymphoblastic leukemia patients ineligible for hematopoietic stem cell transplantation.

Ja Min Byun. _Seoul National University Hospital, Seoul, Republic of Korea_.

Elderly acute lymphoblastic leukemia (ALL) patients are often unfit for neither dose-intensive chemotherapy nor stem cell transplantation, thus their clinical outcomes have been dismal. The increased incidence of BCR-ABL translocation with increasing age also contributed to poor prognosis but, it was our conjecture that the introduction of potent tyrosine kinase inhibitors (TKI) targeting ABL, BCR-ABL translocation might not deliver negative prognostic impact in elderly ALL. Considering that many elderly Ph- ALL patients do not tolerate cytotoxic chemotherapy required for effective tumor control, we supposed that efficacy of ABL TKIs and their combination strategy with low dose cytotoxic chemotherapy would result in better survival for elderly Ph+ ALL compared to their Ph- counterparts.

This study was a multicenter longitudinal cohort study of elderly de novo ALL (≥ 60 years) recruited from 11 hospitals in Korea from January 2005 to December 2015. We considered Ph+ if it was positive in at least one of three techniques: conventional cytogenetics, fluorescence in situ hybridization, or quantitative real-time polymerase chain reaction (qRT-PCR).

Among the 96 patients enrolled, there were 50 Ph- ALL patients (52.1%) and 46 Ph+ ALL patients (47.9%). There were no differences between the 2 groups regarding age, sex distribution, performance status, and induction regimens. For treatment outcomes evaluation, the 5 Ph+ ALL patients not receiving TKI were eliminated and 41 Ph+ ALL patients treated with TKI were compared with 50 Ph- ALL patients (Table). Complete remission (CR) was attained in 27 (54.0%) of patients in the Ph- group versus 35 (85.4%) in the Ph+ group (P=0.001). The median RFS was significantly longer in the Ph+ group (3.1 months for Ph- vs. 8.9 months for Ph+, P=0.017). Induction death and induction failure occurred significantly more often in the Ph- group. The median OS was 6.3 months for Ph- group versus 10.3 months for Ph+ group (P=0.033).

In conclusion, using real world data, this study reports the possible favorable impact of Ph+ in TKI-era for elderly patients.

#766

Comorbidities limiting recruitment of non-small cell lung cancer (NSCLC) patients in early phase trials.

Narjust Duma, Yucai Wang, Aaron Mansfield. _Mayo Clinic, Rochester, MN_.

Background: Early phase trials (EPCT) provide significant value by evaluating the possible benefits of new agents. The eligibility criteria in EPCT are usually rigorous and may exclude many patients (pts) commonly seen in clinical practice. Our objective was to identify the most common comorbidities excluded in EPCT for NSCLC.

Methods: ClinicalTrials.gov was queried on August 1st of 2016. 299 interventional drug trials were extracted from 2010 to 2016. We studied the trial characteristics including: experimental therapy, location, funding and eligibility criteria. Exclusion of CNS metastasis was treated as a binary variable (strict exclusion vs. allowed). Logistic regressions were used for statistical analysis.

Results: Of the 299 trials, 164 (55%) were phase II, 81 (27%) phase I and 54 (18%) phase Ib/II. 164 (55%) were conducted in the United States (US), 45 (15%) in Asia, 39 (13%) in Europe and 51 (17%) were international. 171 (57%) trials were funded by the industry, 54 (18%) by government agencies and 74 (25%) were investigator initiated. Survival was an end-point in 97 (32%) trials. 70 (23%) trials required a repeat lung biopsy. 54 (18%) trials excluded pts >75 years of age. 99 (33%) trials required creatinine levels <1.5 mg/dl, 132 (44%) liver enzymes <2.5 and total bilirubin <1.5 of the upper limit of normal. 88 (29%) trials required Hgb>9 g/dl and 141 (47%) excluded pts with platelets <100,000/dl. In terms of comorbidities, 84 (28%) trials excluded pts with atrial fibrillation, 94 (31%) with any anticoagulation therapy and 120 (40%) HIV positive pts. CNS involvement was a significant exclusion factor, 123 (41%) trials excluded pts with CNS metastasis. On univariate analysis, the odds of CNS metastasis exclusion were significantly higher in immunotherapy trials (OR 1.72, 95%CI 1.2-2.4, p<0.01) and lower in European trials (OR 0.76, 95%CI 0.6-0.9, p<0.005). In multivariate analysis, US based trials had higher odds of exclusion of CNS metastasis (OR: 3.2, 95%CI 1.4-7.5, p<0.05).

Conclusions: Many of the EPCTs excluded pts with advanced age, cytopenias and other common comorbidities, eliminating the possibility of many of our aging pts from participation. Regarding CNS disease, our analysis suggests the type of agent and location may influence the trial design about CNS metastasis inclusion. Investigators should review whether sufficient justification exists for every exclusion criterion before their incorporation in future trial protocols.

#767

Breast cancer in sub-Saharan Africa: a histopathologic review from a tertiary hospital in Northern Nigeria.

Saad A. Ahmed, Balarabe Kabir, Nasiru Abubakar. _Ahmadu Bello University, Zaria, Nigeria_.

Introduction: Breast cancer is the commonest cancer in women world-wide, however, there are discrepancies in the incidence among different geographical regions. The highest incidence rate is in North American, Northern and Western European, while the lowest rate is found among Asian and sub-Sahara African women. Breast cancer is undoubtedly heterogeneous in terms of its presentation, morphology and molecular profiles. Studies from the developing countries suggested that there are differences between the biology of BC in the developing and developed countries.

Methods: This is a retrospective histopathologic review of breast cancer cases between 1st January 2003 and 31st December 2015 from the Ahmadu Bello University Teaching Hospital Zaria - Nigeria.

Results: A total of 784 breast cancer cases were seen during the study period and majority of the cases (99.4%) were females. The prevalence of breast cancer was found to be 12.6%. Triple-negative breast cancers (TNBC) constitute 67.7% of cases. The mean age was 45.8 ±12.0 years and the youngest patient was 18 years while the oldest was 90 years. Out of the 784 cases, 208 were mastectomies while the remaining were incisional, trucut needle and excisional biopsies. Infiltrating ductal carcinoma NST was the most common histologic variant seen (94.7%) and most of these cases (59.1%) were Grade II tumors. The Nottingham Prognostic Index (NPI) shows that 91.4% of the cases have NPI Score of ≥3.5 with attendant poor 5-year survival rate.

Conclusion: This study found that breast cancer is common and affects mostly women in fifth decade of life. There is a high TNBC among the population studied and therefore androgen receptor profile and genomic studies may be useful to determine the course of therapy.

#768

The utility of a breast cancer registry for improved cancer management in developing countries.

DeAnna McGarity,1 Brandon Epps,1 Alexis Maynor,2 Tania Anderson,2 Ernest Alema-Mensah,1 Delroy Fray,3 Maung Aung,3 Kisha Holden,1 James Lillard,1 Brian Rivers,1 Derrick Beech1. 1 _Morehouse School of Medicine, Atlanta, GA;_ 2 _Tuskegee University, Tuskegee, AL;_ 3 _Western Regional Health Authority and Cornwall Regional Hospital, Montego Bay, Jamaica_.

Background

Breast cancer is one of the leading causes of cancer related deaths in the island nation of Jamaica, West Indies. Cancer and other non-communicable diseases have increased in many developing nations in the Caribbean as well as Central and South America. The health systems of these developing countries continue to provide care to patients with limited resources. The use of a breast cancer registry can assist in care coordination of these patients and allow targeted screening and treatment programs in under-resourced environments.

Methods

In collaboration with the Western Regional Health Authority, a breast cancer registry database was developed in RedCap to provide a comprehensive analysis of breast cancer in under resourced environment. 53 patients from Atlanta were compared to 78 patients from Jamaica These patients were entered into the breast cancer registry from June 1, 2014 to March 31, 2015 by the attending physician and de-identified. Demographics, tumor related factors, treatment, survival and health behavior information was collected by the attending physician. The findings were then analyzed using Statistical Analysis System (SAS) and Data Analysis and Statistical Analysis (STATA): Fisher Exact test, chi-square and multiple regressions.

Results

The median of patient ages were 61 years old (range 38 - 81 years) for the U.S. patients and 53 years old (range 26 - 84 years old) for Jamaican women. For the U.S. patients, 84% were Black, 4% White, and 12.24% were Latin or Asian. The majority of the patients were diagnosed with stage 1 invasive ductal breast cancer. In contrast, the majority of the Jamaican patients were diagnosed with late-stage breast cancer. Utilizing the data from the breast cancer registry, we were able to identify parishes (counties) with disproportionately high rates of late-staged cancer at the time of diagnosis.

Conclusion

The use of a disease specific registry can improve care coordination and identify actionable target areas for enhanced screening and treatment. It is our hope to frequent review the dashboards associated with the registry data by providers, medical centers and health systems. This will optimize care coordination and allow timely improvements in cancer diagnosis and management in developing countries.

#769

Matched-pair comparisons of chemotherapy plus bevacizumab versus chemotherapy alone as third-line treatment or beyond for advanced non-small cell lung cancer.

Binbin Hu,1 You Lu,1 Xiaojuan Zhou,1 Qian Li,2 Mengmeng Xiang2. 1 _West China Hospital, Sichuan University, Chengdu, China;_ 2 _West China School of Medicine, Sichuan University, Chengdu, China_.

Background: The addition of bevacizumab to chemotherapy has proven efficacy as first-line treatment for advanced NSCLC. Whether this combination still be efficient as salvage treatment for NSCLC patients who have not received bevacizumab previously remains unclear. This retrospective matched-pair study was designed to compare the efficacy and safety of chemotherapy plus bevacizumab with chemotherapy alone as third-line treatment or beyond for NSCLC patients.

Methods: Patients with stage IV NSCLC who had received single-agent chemotherapy plus bevacizumab after failure of at least two prior regimens were enrolled in this study. Each of them was matched with two patients who had received chemotherapy alone (1:2) by age (within ±5 years), sex, smoking history, histology, EGFR mutation status and number of prior regimens. Tumor response rate, survival and toxicity analyses were performed.

Results: Between January 2011 and June 2016, a total of 40 patients who had received chemotherapy plus bevacizumab (chemotherapy-bevacizumab group) were matched with 80 patients who had received chemotherapy alone (chemotherapy-alone group) as third-line treatment or beyond. Objective response rate (ORR) and Disease control rate (DCR) were improved for chemotherapy-bevacizumab compared with chemotherapy alone (ORR, 22.5% v 6.2%, respectively, p= .014; DCR, 62.5% v 40.0%, respectively, p= .032). Progression-free survival was prolonged for chemotherapy-bevacizumab compared with chemotherapy alone (median, 3.9 v 2.5 months, respectively; HR, 0.64; 95%CI, 0.43 to 0.97; p = .032). Incidence of severe (≥ grade 3) adverse events was low and similar across groups.

Conclusion: The combination of chemotherapy and bevacizumab can be an efficient alternative salvage treatment for NSCLC patients who have not received bevacizumab previously. But taking this combination as first-line treatment for advanced NSCLC still should be a priority.<!--EndFragment-->

#770

Colorectal cancer screening in rural community health clinics using the fecal immunochemical test (FIT).

Connie L. Arnold,1 Terry C. Davis,1 Alfred W. Rademaker,2 James Morris,1 Glenn Mills1. 1 _LSU Health Sciences Center - Shreveport, Shreveport, LA;_ 2 _Northwestern University, Chicago, IL_.

Introduction: Colorectal cancer (CRC), the second leading cause of cancer death in the United States, can be significantly reduced if it is detected early. Although overall CRC screening rates have increased significantly, disparities persist among low income individuals, adults with low literacy and those living in rural areas. These groups all have screening completion rates below 50%.

Objectives: To assess patient knowledge, beliefs, and self-efficacy about CRC screening and compare the effectiveness of two health literacy informed telephone follow-up strategies to improve initial and annual repeat screening with Fecal Immunochemical Test (FIT) in rural community clinics.

Methods: A two-arm, randomized controlled trial is being implemented in four community clinics. Clinics reported CRC baseline screening rates of 1% to 3%. Eligible patients, age 50-75, are recruited at the clinic prior to a scheduled appointment. A research assistant (RA) conducts a baseline structured interview measuring CRC screening knowledge, beliefs, and self-efficacy. The RA then recommends screening and gives brief literacy and culturally appropriate education using a pamphlet (4th grade level), the FIT kit with pre-addressed envelope, simplified instructions (3rd grade level) and a demonstration of how to use it, At four weeks patients who have not returned their kit receive either 1) a personal follow-up call (PC) from a central prevention coordinator using motivational interviewing skills and reminding them to complete and mail FIT kits; or 2) an automated follow-up call (AC) using plain language and motivational messages encourages patients to complete and mail the FIT. Outcomes include FIT completion after intervention, and again at 12 and 24 months. 650 patients will be enrolled.

Results: To date 599 patients not up-to-date with screening have been enrolled; 64% African American, 56% women; 40% inadequate literacy. Although 90% reported having heard of CRC, only 64% knew a test to check for CRC. 70% reported a provider had recommended CRC screening in the past and 91% reported they would want to know if they have CRC. Self-efficacy was high with over 90% indicating they would return the kit to the lab. Screening results: 599 patients enrolled to date (AC: n=300 / PC: n=299); 412 (69%) completed tests (AC: n=210 (70%) / PC: n=202 (68%)). Follow-up calls for Unreturned Kits - AC: 113 people called - 26 returned FIT (23% of people called completed FIT). PC: 115 people called - 22 returned FIT - (19% of people called completed FIT). 2nd Year Screening: 129 Second kits mailed out to-date. AC (n=61): n=29 (48%) completed kits. PC (n=68): 28 (41%) completed kits.

Discussion: Implementing literacy and culturally appropriate CRC education and screening strategies using the FIT and phone reminders has the potential to increase CRC screening rural community clinics and address public health disparities.

#771

Cervical cancer: Our experience in Sudan.

Kamal Eldein H. Mohamed, Dr Ammar Ashmeig Ahmed Ashmeig. _Faculty of Medicine, Univ of Khartoum, Khartoum, Sudan_.

Introduction: Cervical cancer is the second most prevalent cancer in Sudanese Women; breast cancer forms between 29 - 35% of female cancers while Ca Cervix forms 12 - 16%. There are limited screening activities in the country using VIA, and Pap smears in Obs. and Gyn. clinics. With the main emphasis on health education programs, teaching women the early symptoms of ca cervix and early detection. Due to lack of awareness the majority of women don't know anything about screening, so there is limited demand.

Method: This is a retrospective study of 405 Cases of Ca Cervix treated during the period between 2000—2013 at the Radiation and Isotopes Center of Khartoum, RICK. Data was collected from patients records.

Results: Age distribution showed,<40 years =8 = 2 %,41 -50 = 94 =23.2%,51—60 = 187 = 46.2%,61—70 = 102 = 25.2%,>70 14= =3.5%. Age range =28—79,mean age =47 years.

Residence distribution was as follows, western Sudan = 48 %,central Sudan = 41.%,North = 7%,East = 4%.

Length of duration of symptoms, range between 1—10 months, mean of 5 months.

FIGO Stage distribution: Stage 1= 6%, Stage 2 A =13%.Stage 2 B =23%,Stage 3 A=18%,Stage 3B =34%,Stage 4 A,B =7%,.

Pathology: poorly dif. Squamous ca 38%, moderately dif. Squamous 26%,well dif 25%,Adenocarcinoma 9%,others 2%.

Treatment: external Radiotherapy,45-50 Gy in 20—25 fractions, by 2 or 4 fields, using Co 60 or lineacs 6,9 Mv, concurrent chemo radiotherapy was given to patients with stage 2B, 3A, 3B, and 4A. Using weekly Cisplatinum, this was given to 32% of

patients. 60 %% of the patients had Brach therapy, 34% with Low Dose Rate, LDR, Manual Brachy therapy, 35Gy to point A,and 66% had High Dose Rate Brachy, HDR, 9 Gy x 2 sessions, using a cobalt source.

Results: 175 patients (43.3%) survived for 5 years and more, mostly patients with stage one and two 158 = 39% survived for 3-4 years after treatment,39 patents = 9.6 %, mostly patients with stage four, died during follow up due to the disease and 33 patients = 8.2% disappeared after treatment. The poor outcome is attributed to the advanced stages at presentation, lack of screening and awareness about cervical cancer, poverty, illiteracy, the large size of the country and the poor distribution of the limited medical resources. The patients treated with LDR or HDR have equal outcome and morbidity.

Conclusion: Most of Ca Cervix Patients present with advanced disease, hence the poor prognosis, due to lack of awareness and screening, illiteracy and poverty, the majority are poorly differentiated squamous cancers. There is a need for increasing public awareness about ca cervix and its early symptoms, and provision of a pap smear and VIA services in the primary health care facilities.

#773

Optimizing drug regimens in oncology by clinical trial simulations: Why and how.

Jocelyn Gal,1 Gerard Milano,1 Julien Viotti,1 Renaud Schiappa,1 Audrey Dugue,2 Agnes Paquet,3 Sylvie Chabaud,4 Jean-Marc Ferrero,1 Emmanuel Chamorey1. 1 _Ctr. Antoine Lacassagne, Nice, France;_ 2 _Ctr. Francois Baclesse, Caen, France;_ 3 _Molecular and Cellular Pharmacology Institute, Sophia Antipolis-Valbonne, France;_ 4 _Léon Bérard Cancer Center, Nice, France_.

Introduction: In therapeutic research, the safety and efficacy of pharmaceutical products are necessarily tested on humans via clinical trials after a very long and expensive development period. Alternative methodologies such as computer modeling and Clinical Trial Simulation (CTS) are a valuable option to reduce animal and human assays. The relevance of these methods is well recognized in pharmacokinetics and pharmacodynamics from the pre-clinical phase to post-marketing. However, they are seldom used and are poorly regarded for drug approval, despite FDA and EMA recommendations. We propose to discuss the principle behind, and the interest of, CTS approaches in drug development in oncology. Our work is based on a systematic review using two electronic literature databases (Medline and Web of Science).

Key components of CTS: We will explain why and how to successfully develop CTS.

Why developing CTS?

To obtain a better knowledge of pharmacology and/or better prediction of safety and efficacy

To evaluate disease progression dynamics and to test the potential impact of biomarkers

To analyze subpopulations and extend the post-marketing authorization to help in the choice of the best outcome

To optimize experimental designs in order to better anticipate the progress of the study

How to develop CTS?

CTS project must follow three steps:

1/The constitution of pharmacometricians team who elaborates clinical model and simulation plan that must contain three components:

Input/output model: How do covariates impact outcomes?

Covariate distribution Model: How can we generate virtual patients?

Execution model: What can happen during the clinical trial?

2/Obtaining data for simulations. We will present the contribution of machine learning for CTS

3/The use of specialized software to realize simulations

Limitations of CTS: We will present the features that can slow down or accelerate the development of CTS. The generalization of CTS could be greatly facilitated by the availability of software for modeling biological systems, by the clinical trials studies and hospital databases. Data-sharing and data-merging raise legal, policy and technical issues that will need to be addressed.

CTS challenges and prospects: Development of future molecules will have to use CTS for faster development and thus enable better patient management. Drug activity modeling coupled with disease modeling, optimal use of medical data and increase of the computing speed should allow this leap forward. The realization of CTS requires not only the bioinformatics tools to allow interconnection and global integration of all clinical data, but also a universal legal framework to protect the privacy of every patient. CTS will have to be used to provide quantitative support for drug development decisions but will never replace real clinical trials. This in silico medicine opens the way to the 4P medicine: predictive, preventive, personalized and participatory. 

## TUMOR BIOLOGY:

### Angiogenesis and Vascular Biology 1

#774

Notch pathway activation predicts resistance to bevacizumab therapy in glioblastoma.

Norihiko Saito, Kazuya Aoki, Nozomi Hirai, Satoshi Fujita, Junya Iwama, Masashi Ikota, Haruo Nakayama, Morito Hayashi, Keisuke Ito, Takatoshi Sakurai, Satoshi Iwabuchi. _Toho University Ohashi Medical Center, Tokyo, Japan_.

Glioblastoma, the most common adult glioma, is associated with a dismal prognosis. Treatment with bevacizumab has not significantly prolonged overall patient survival times. Glioblastoma resistance to angiogenesis inhibitors is attributed to multiple interacting mechanisms. We have thus embarked on a comprehensive effort to detecting expression signatures that are associated with response to the therapy and these signatures may allow prospective selection of patients with high likelihood of responding to therapy. Notch signaling pathway is an evolutionarily conserved pathway that plays an important role in multiple cellular and developmental processes including cell fate decision, differentiation, proliferation, survival, angiogenesis and migration. Analysis of The Cancer Genome Atlas expression dataset identified a group (43.9%) of tumors with proneural signature showing high Notch pathway activation. In this study, we compared CD133, Notch, and VEGF expressions in histological sections of primary and recurrent glioblastomas after radiotherapy and chemotherapy. Tumor samples were collected from 27 patients at the time of tumor recurrence. We used immunohistochemical techniques to compare expression of CD133, Notch-1 and VEGF. Expressions of CD133-, Notch-1-, and VEGF-positive glioma cells were higher in recurrent glioblastoma after radiotherapy and chemotherapy. To determine the clinical importance of Notch-1 expression in glioblastoma, we analyzed 15 patients who had received bevacizumab therapy followed by a second surgery at recurrence. OS was significantly longer in cases with Notch-1 negativity (8.8 months) than in those with Notch-1 positivity (6.8 months). Electron microscopic observation of two autopsy cases revealed the effects of blood vessel normalization in Notch-1 positive glioblastoma. Electron microscopic images confirmed the presence of pericytes surrounding the vascular endothelium. Autopsied tumors exhibited marked proliferation of Notch-1 and VEGF positive cells around vessels. In tumor angiogenesis, vascular endothelial growth factor and Notch signaling induce sprouting angiogenesis and recruitment of vascular endothelial cells such as tip cells, stalk cells, and phalanx cells. Fully mature phalanx cells are in close contact with pericytes. These findings indicate that bevacizumab treatment promotes vascular normalization by recruiting mature pericytes and associated with resistance to bevacizumab therapy in glioblastoma with high Notch pathway activation.

#775

Identification of activated signal transduction pathways and molecules in the HMGB-1 induced angiogenesis.

Yujin Kwon, Sujin Shin, Won Kyu Kim, Hoguen Kim. _Yonsei Univ. College of Medicine, Seoul, Republic of Korea_.

High-mobility group box-1 (HMGB-1) is a highly conserved protein playing various roles both in the nucleus and cytosol and known to be expressed in almost all types of mammalian cells. Secreted HMGB-1 has been most intensively studied and reported as an immune cell derived cytokine regulating systemic inflammation, signal transduction, and activation of endothelial cells (EC). Especially, HMGB-1 over-expression is now considered as a hallmark in cancers based on its involvement in cell proliferation, inflammatory microenvironment, invasion, metastasis, and sustained angiogenesis. Numerous studies have shown that secreted HMGB-1 directly or indirectly promotes angiogenesis by activation of ECs. However, mechanisms of HMGB-1 mediated angiogenesis have not yet been fully understood, especially, in terms of genomic changes caused by HMGB-1 treatment. Moreover, differences between vascular endothelial growth factor (VEGF) mediated and HMGB-1 mediated angiogenesis have not been studied.

To explore the roles of HMGB-1 in angiogenesis, we first confirmed whether HMGB-1 treatment renders ECs angiogenic by evaluating proliferation, migration, wound-healing, and tubule formation of ECs after HMGB-1 treatment. In addition, we performed gene expression microarray to clarify how HMGB-1 treatment induced EC activation at various time points and subsequent angiogenic features according to time-dependent gene expression changes.

We show that HMGB-1 treatment induces proangiogenic features to ECs such as stimulation of tubule formation, promotion of proliferation, and accelerated wound healing. By analysis of microarray data, we found that VEGF and HMGB-1 commonly promoted the activation of MAPK, cell proliferation, and wound healing pathways which directly regulate angiogenesis. On the other hand, axon guidance, gap junction, P53 signaling, cell cycle, and cell differentiation pathways were specifically up-regulated by HMGB-1. Taken together, HMGB-1 leads to angiogenesis via upregulation of both angiogenic and nonangiogenic genes.

#776

Acquired platinum resistance enhances tumour angiogenesis through activation of vascular mimicry.

Aya El Helali,1 Nuala McCabe,1 Naomi Dickson,1 Lara Dura Perez,1 Denis P Harkin,2 Richard Wilson,1 Richard Kennedy1. 1 _Centre for Cancer Research and Cell Biology, Belfast, United Kingdom;_ 2 _Almac Diagnostics, Craigavon, United Kingdom_.

Introduction:

Platinum resistant High Grade Serous Ovarian Cancer (HGSOC) has a poor outcome with limited treatment options. Angiogenesis is a key pathological feature of ovarian cancer and anti-angiogenics have dominated the field of drug development in EOC, particularly in the second-line setting (Marachini et al 2013). In this study we asked if platinum resistance could be associated with an improved response to anti-angiogenic agents.

Method:

A review of phase III anti-angiogenic clinical trials was used to investigate the association between platinum resistance and response to anti-angiogenic agents. To investigate the effect of chemotherapy on predefined ovarian cancer molecular subgroups (Gourley, et al. J Clin Oncol 32:5s, 2014), we analysed 35 matched pre- and post-chemotherapy samples by gene expression. Novel isogenic cisplatin-resistant HGSOC cell lines were established to study the mechanisms of cisplatin section pressure and shift to an angiogenic phenotype. This was further validated in novel ascites-derived primary cell lines from HGSOC patients with known outcomes following platinum-based chemotherapy.

Results:

Critical review of 22 phase III anti-angiogenic trials suggested that there was a better response to anti-angiogenics following previous platinum-based chemotherapy. Our analysis demonstrated that 67% of treatment naïve tumours that were initially classified as non-angiogenic shifted to an angiogenic biology, which was associated with platinum resistance. Additionally we found that cisplatin resistant cancer cell lines demonstrated hallmarks of vascular mimicry and an associated increase in vessel density using an angiogenesis Matrigel plug assay in Athymic nude mice (p-value=<0.0001). In addition, cell lines established from platinum-resistant patients as well as cell lines made platinum resistant in-vitro, demonstrated overexpression of VEGFa which would be expected to stimulate angiogenesis.

Conclusion:

We have demonstrated that platinum-resistance in HGSOC is associated with angiogenic biology supporting the use of anti-angiogenic agents in this setting.

#777

ELTD1/ADGRL4, a novel adhesion GPCR regulator of tumour angiogenesis, suppresses lipid metabolism in endothelial cells, and is upregulated in breast cancer endothelium and epithelium.

David M. Favara,1 Madhulika Nambiar,2 Helen Sheldon,1 Massimo Masiero,1 Demin Li,1 Ali Jazayeri,2 Alison H. Banham,1 Adrian L. Harris1. 1 _University of Oxford, Oxford, United Kingdom;_ 2 _Heptares Therapeutics, Welwyn Garden City, United Kingdom_.

Background: We identified ELTD1/ADGRL4, an orphan GPCR belonging to the adhesion GPCR family (aGPCR), as a novel regulator of angiogenesis and a potential anti-cancer therapeutic target. ELTD1 is normally expressed in both endothelial cells & vascular smooth muscle cells. Expression in the tumour vasculature is significantly increased. Our aims were to analyse ELTD1's function in endothelial cells & its role in breast cancer.

Method: After ELTD1 silencing, mRNA array profiling was performed on primary human umbilical vein endothelial cells (HUVECs) & validated with qPCR & confocal microscopy. We investigated ELTD1 signalling by applying the aGPCR 'Stinger/tethered-agonist Hypothesis'. For this, truncated forms of ELTD1 & peptides analogous to the proposed tethered agonist region were designed. FRET-based 2nd messenger (Cisbio IP-1; cAMP) & luciferase-reporter assays (NFAT; NFKB; SRE; SRF-RE; CREB) were performed to establish canonical GPCR activation. To investigate ELTD1 in breast cancer, a panel of cell lines representative of all molecular subtypes were qPCR screened. Furthermore, primary human breast cancers (n=245) & matched primary & nodal secondary breast cancers (n=79) were stained for ELTD1 expression. Staining intensity was then scored & compared with survival.

Results: HUVEC mRNA expression profiling after ELTD1 silencing showed upregulation of SLC24A1, which transports citrate from the mitochondria to the cytoplasm & ACLY, which converts cytoplasmic citrate to Acetyl CoA, feeding fatty acid and cholesterol synthesis, and acetylation. We validated this at RNA & protein expression level & showed that ELTD1 inhibited lipid droplet formation. Signalling experiments revealed that unlike other aGPCRs, ELTD1 does not couple to any canonical GPCR pathways (Gαi; Gαs; Gαq; Gα12/13). In breast cancer, we found that no representative cell line screened expressed ELTD1. Breast cancer immunohistochemistry revealed higher intensity vascular ELTD1 staining within the tumour stroma contrasted to normal stroma & expression within tumour epithelial cells (15%). Higher ELTD1 expression in both the tumour stroma vasculature (n=241; HR=0.68; p=0.04) & within the subset of tumour positive cases (n=24; HR=0.3; p=0.02) correlated with improved relapse free survival (RFS).

Conclusion: ELTD1's regulation of lipid synthesis through suppression of ACLY & SLC25A1 highlights ELTD1's role as a novel regulator of endothelial metabolism. Unlike other aGPCRs, ELTD1 does not signal through canonical G protein pathways. The good prognosis of ELTD1 expression may be related to inducing a quiescent endothelial population. It will be of interest to relate this to activity of anti-angiogenic therapy. Nevertheless, a relevant fraction of patients expressing ELTD1 still relapsed & this may be a suitable target for this population.

#778

Identifying, subtyping and classifying tumor associated circulating endothelial cells in patients with solid tumors.

Daniel L. Adams,1 R. Katherine Alpaugh,2 Steven H. Lin,3 Jeffrey R. Marks,4 Raymond Bergan,5 Stuart S. Martin,6 Sarany Chumsri,7 Massimo Cristofanilli,8 Cha-Mei Tang,9 Steingrimur Stefansson10. 1 _Creatv MicroTech, Inc., Monmouth Junction, NJ;_ 2 _Fox Chase Cancer Center, Philadelphia, PA;_ 3 _MD Anderson Cancer Center, Houston, TX;_ 4 _Duke University, Durham, NC;_ 5 _Oregon Health and Science University, Portland, OR;_ 6 _University of Maryland, Baltimore, MD;_ 7 _Mayo Clinic Cancer Center, Jacksonville, FL;_ 8 _Northwestern University, Chicago, IL;_ 9 _Creatv MicroTech, Inc., Potomac, MD;_ 10 _HeMemics Biotechnologies, Inc., Rockville, MD_.

Background: Tumor endothelial cells (ECs) are a population of stromal cells required for tumor growth that cooperate with tumors to form angiogenic structures. In blood, circulating ECs (CECs) are normal constituents of healthy individuals, although a Cancer Associated Vascular Endothelial cell (CAVE) subtype has been observed in cancer patients. The CAVE population has been isolated and identified using their large size or multicellular clustering and a pooled mixture of classical EC markers (i.e. CD31 and CD146). However, there has been no attempt to differentiate CAVEs from the many EC subtypes. This is not surprising as in-depth phenotyping of ECs requires an array of biomarkers that until recently has not been feasible. A multi-phenotypic screening of EC markers was tested on CAVEs from 116 blood samples in 3 types of solid tumors. This data suggests that CAVEs exist as a common and diverse subtype of tumor derived CECs that may express cytokeratin (CK) and various EC biomarkers, correlating to disease stage.

Methods: Peripheral blood samples from 116 cancer patients (stage I-IV) were drawn from 2012-2014 including breast (n=42), lung (n=39) and prostate (n=35), as well as blood from 34 healthy controls. Blood was processed by an established filtration approach, i.e. the CellSieveTM microfiltration technique (Creatv MicroTech), filtering blood by size exclusion and staining cells for CK 8, 18 & 19, EpCAM and CD45. After identification and imaging, the QUAS-R (Quench, Underivatize, Amine-Strip and Restain) technique was used to remove fluorescence signal and restain all cells with CD31, CD146, CD144, & DAPI. After reimaging, QUAS-R was again used to remove fluorescence and restain the cells for CD14, CD105, CD34, & DAPI.

Results: Out of 116 patient samples, we identified CAVEs in 63 patients (54%) based on positivity of CD31, CD144 or CD146, but none were found in healthy controls. CAVEs per 7.5mL sample in patients averaged 5.1 (breast), 5.6 (prostate) and 7.9 (lung). Presence of CAVEs appeared related to stage with 26% in stage 1, 61% in stage 2, 68% in stage 3, and 74% in stage 4 patients. No CAVEs were positive for CD14 or CD45. CD31 was the most present marker, found on 93% of CAVEs, followed by CD144 (85%), CD34 (64%), CD146 (45%), & CD105 (4%). In contrast with the previous study on this topic, CK was found in 67% of CAVEs, but was not a universal marker.

Conclusions: It has been reported that CK+ and CD45- CECs are isolated from the blood of cancer patients in colon and lung cancers, prompting some to classify them as circulating tumor cells. However, subtyping these CECs is incomplete when characterized with only 3-4 biomarkers. A multi-phenotypic subtyping technique was used to properly identify and subtype these CECs in cancer patients. This data suggest that a subset of CECs, e.g. CAVEs, are found in circulation as CK+/CD45-, but exist as a heterogeneous population of cancer specific circulating cells that require further study.

#780

In vitro **screening of individual human neuroendocrine tumors for their angiogenic response to tyrosine kinase inhibitors.**

Tanja Milosavljevic,1 Elise J. Chouest,1 Catherine E. Anthony,2 Ariana Dirige,1 Yi-Zarn Wang,1 Philip J. Boudreaux,1 Thiagarajan Ramcharan,1 Eugene A. Woltering1. 1 _LSU Health New Orleans, New Orleans, LA;_ 2 _Merkey Cancer Center, University of Kentucky, Lexington, KY_.

Background: Human neuroendocrine tumors (NETs) are highly vascular in nature and reliant on multiple receptor tyrosine kinases (TKs) for their neovascularization, growth, and, metastasis. These tumors most commonly originate in the small bowel (SB) and frequently metastasize to the lymph nodes and other organs. Current treatment of metastatic NETs involves a variety of approaches including antiangiogenesis therapies. In this study we tested effectiveness of six TK inhibitors (TKIs) [Dovitinib lactate, Regorafenib, Erlotinib, Imatinib, Vatalanib, and Sunitinib] on individual NETs angiogenic response in vitro.

Methods: Specimens were obtained from NET patients who underwent removal of their primary tumor (small bowel/pancreas/stomach), nodal, and organ (i.e. liver/ovary/omentum/mesentery) metastasis. Fresh tumors were minced, embedded in a fibrin-thrombin clot and supplemented with nutrient culture media per the in vitro human tumor angiogenesis model protocol. Neovessels were visually scored and evaluated for angiogenic parameters: percent initiation (%I), angiogenic growth (AI), and overall angiogenic response (OAR). All TKIs were prepared consistent with manufacturer's instructions and their effective concentration was determined by dose response experiments. The selected TKI dose reflected the clinically achievable plasma level. A large group of NETs was tested for their antiangiogenic response to six TKIs [Dovitinib lactate (D): n=164, Regorafenib (R): n=163, Erlotinib (E): n=35, Imatinib (I): n=51, Vatalanib (V): n=163, Sutent (S): n=164)]. Paired samples t-test was used to compare TKI to control results for each angiogenic parameter, and independent samples t-test to compare TKI-response of primary and metastatic sites (MedCalc).

Results: Each selected dose [D: 82nM, R: 1100nM, E: 100µM, I: 2.5µM, V: 20µM, S: 188nM] achieved statistically significant inhibition of OAR (D:94.23%, R:35.58%, E:52.07%, I:59.77%, V:76.65%, S:64.79%) in all NETs (p<0.0001). This is accomplished by the simultaneous statistically significant decrease of %I by at least 3.47% (p=0.0164) and AI by at least 34.03% (p<0.0001). Comparison of OAR between the primary and metastatic tumor sites revealed no differences in their response to each tested TKI (p>0.2119). In all NETs, TKIs inhibited both mechanisms of angiogenesis, but preferentially targeted growth (all TKIs: p<0.0001) over %I (D, I, V, S: p<0.0001; E: p=0.0072; R: p=0.0164).

Conclusions: In vitro screening of individual tumors revealed that TKIs effectively inhibited all parameters of angiogenesis in all NETs, primary and metastatic tumors. Selected TKIs preferentially inhibited angiogenic growth rather than initiation in all NETs. Our preclinical results show that Dovitinib inhibits angiogenesis most effectively in human NETs compared to other TKIs in vitro.

#781

Functional consequence of the p53 codon 72 polymorphism in colorectal cancer.

Venkat R. Katkoori,1 Upender Manne,2 Harvey Bumpers1. 1 _Michigan State Univ., East Lansing, MI;_ 2 _University of Alabama at Birmingham, Birmingham, AL_.

Background: The codon 72 polymorphism in p53 has been implicated in colorectal cancer (CRC) and CRC health disparities. In this study, we determine the functional consequence of this polymorphism in CRC following both in-vitro cell based and in-vivo mouse model studies.

Experimental Design: Plasmids (pCMV6) that express different phenotypes of p53 [p53 wild type (wt) at codon 72 (R72wt), R72wt with mutation at codon 273 cysteine (R72273Cys), p53 mutation at codon 72 (P72wt) and P72wt with mutation at codon 273 (P72273Cys)] were constructed. We selected a CRC cell line Caco2, which does not express p53 for in vitro studies. Severe combined immunodeficient mice were inoculated with CRC cells (HT29, SW480, and LS174) to establish tumor xenografts (tumors). Tumor angiogenesis was assessed in tumors by immunostaing for CD31. Sequencing analysis for codon 72 polymorphism of p53 was performed using genomic DNA purified from tumors. Western blot (WB) or immunostaining analyses for proteins of signaling mechanisms were performed to assess the functional consequence of P72 phenotype of p53.

Results: Our study demonstrated that P72 tumors had well established vascularity, while R72 tumors had very poor vascularization. Indeed, the mean micro vessel density was higher in P72 tumors than in R72 tumors. WB analyses revealed that P72wt or mutant phenotypes effectively induced the activation of p38 and RAF/MEK/ extracellular signal-regulated kinase (ERK) MAP kinases. Up-regulation of phosphorylated SEK1/MKK4, an upstream kinase of p38 MAPK was associated with P72wt or mutant phenotypes. This activation was accompanied by up-regulation of phosphorylated-MAPKAPK-2, -Hsp27, and -CREB, downstream targets of p38 MAPK. Increased activation of CREB was found to be higher in tumors that exhibit P72 phenotype. Metastatic lesions of CRC expressed more phospho-CREB than non-metastatic lesions. Furthermore, suppression of RAF/MEK/ERK activation was significantly higher in cells that express R72wt phenotype compared to cells that express P72wt or mutant phenotypes. The expression of P72wt or mutant phenotypes displayed decreased expression of E cadherin and/or an increased expression of vimentin, fibrinectin, CD44, thereby promoting CRC metastasis.

Conclusions: These findings offer significant novel insights into the mechanism by which P72 contributes to the aggressiveness of CRC. Because P72 is over-expressed in CRC, specifically in African-American patients, these studies suggest a role for P72 in cancer health disparities. This work was supported by NIH/NCI Workforce Diversity Grant R21-CA171251.

#782

Revisiting the angiogenic switch: Host genetic modifiers induce non-productive angiogenesis and inhibit breast cancer.

Michael Flister, Cody Plasterer, Shirng-Wern Tsaih, Angela Lemke, Dana Murphy, Amit Joshi, Peter LaViolette, Carmen Bergom. _Medical College of Wisconsin, Milwaukee, WI_.

Purpose: Multiple aspects of the tumor microenvironment (TME) impact breast cancer risk, yet the genetic modifiers of the TME are largely unknown, including those that modify tumor vascular formation and function.

Methods: To discover host TME modifiers, we developed a system called the Consomic/Congenic Xenograft Model (CXM). In CXM, human breast cancer cells are orthotopically implanted into genetically-engineered consomic/congenic xenograft host strains that are derived from two parental strains with different susceptibilities to breast cancer. Because the host strain backgrounds are different, whereas the inoculated tumor cells are the same, any phenotypic variation is due to TME modifier(s) on the substituted chromosome (i.e., consomic) or subchromosomal region (i.e., congenic) of the host's germline DNA. Here, we assessed TME modifiers on rat chromosome 3 (RNO3) that impact growth, angiogenesis, vascular function, and hematogenous metastasis of tumors implanted in the SSIL2Rγ and SS.BN3IL2Rγ CXM strains.

Results: Breast cancer xenografts implanted in SS.BN3IL2Rγ (consomic) had significant inhibition of tumor growth and hematogenous metastasis compared with SSIL2Rγ (parental control), despite a paradoxical increase in the density of blood vessels in the SS.BN3IL2Rγ tumors. We hypothesized that decreased growth of SS.BN3IL2Rγ tumors might be due to nonproductive angiogenesis. To test this possibility, SSIL2Rγ and SS.BN3IL2Rγ tumor vascular function was examined by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), micro-computed tomography (micro-CT), and ex vivo analysis of primary blood endothelial cells; all of which revealed altered vascular function in SS.BN3IL2Rγ tumors compared with SSIL2Rγ. Gene expression analysis also revealed a dysregulated vascular signaling network in SS.BN3IL2Rγ tumors, among which DLL4 was localized to RNO3 and downregulated on the vasculature of SS.BN3IL2Rγ tumors compared with SSIL2Rγ. CXM congenic mapping confirmed that the DLL4 modifier allele is physically linked with breast cancer inhibition by inducing non-productive angiogenesis. Finally, using whole genome sequencing of the germline DNA, we have identified multiple polymorphisms in the DLL4 promoter and a proximal regulatory lncRNA that likely alter DLL4 expression and function.

Conclusions: Collectively, these data suggest that DLL4 can function as a heritable modifier of non-productive angiogenesis that inhibits breast cancer growth and metastasis.

#783

Cancer cell-derived extracellular vesicles stimulate tumor angiogenesis by delivering VEGF to endothelial cells.

Song Yi Ko, Honami Naora. _UT MD Anderson Cancer Ctr., Houston, TX_.

Angiogenesis is essential for tumor growth and metastasis, and is orchestrated by a repertoire of growth factor signaling pathways that stimulate endothelial cell growth, migration and vessel formation. Recent studies have shown that angiogenesis can also be regulated by cell-derived extracellular vesicles. Exosomes are small endosomal-derived membrane vesicles that contain various biomolecules such as RNA and proteins, and are increasingly thought to play important roles in transferring informational cargo between cancer cells and stromal cells. In the majority of studies to date, the effects of cancer cell-derived exosomes have been attributed to their RNA cargo. In this study, we identified that exosomes derived from ovarian, colon and renal cancer cells contain abundant vascular endothelial growth factor (VEGF). Cancer cell-derived exosomes were found to activate the VEGF signaling pathway in endothelial cells and to stimulate endothelial cell migration and tube formation. Furthermore, our studies using inhibitors of VEGF signaling demonstrated that the stimulatory effects of cancer cell-derived exosomes on endothelial cells depend on the presence of VEGF in exosomes and are mediated via the VEGF signaling pathway. These findings indicate that cancer cell-derived exosomes promote tumor angiogenesis by delivering VEGF to endothelial cells.

#784

Pharmacodynamic biomarkers in metronomic chemotherapy: Multiplex cytokine measurements in gastrointestinal cancer patients.

Paloma Valenzuela,1 Karla Parra,1 Derrick Oaxaca,1 Luis Reza,1 Jose Lopez,1 Montserrat Garcia Arreguin,1 Diana Garcia,1 Georgialina Rodriguez,1 Alfredo Falcone,2 Giacomo Allegrini,2 Teresa Di Desidero,2 Guido Bocci,2 Robert Kirken,1 Giulio Francia1. 1 _UT El Paso, El Paso, TX;_ 2 _University of Pisa, Pisa, Italy_.

Metronomic chemotherapy has shown promising antitumor activity in a number of malignancies. For example, we previously reported (Allegrini et al., Angiogenesis (2012) 15(2):275-86) a phase II clinical trial of metronomic UFT (a 5-fluorouracil prodrug; 100 mg/twice per day p.o.) and cyclophosphamide (CTX; 500 mg/mq2 i.v. bolus on day 1 and then 50 mg/day p.o.) plus celecoxib (200 mg/twice a day p.o.) in 38 patients with advanced refractory gastrointestinal tumors. The mechanisms of action of metronomic chemotherapy include upregulation of the angiogenesis inhibitor Thrombospondin-1, the suppression of bone marrow derived endothelial progenitor cells and, at least for drugs such as CTX, activation of the immune system. To further evaluate the latter, we carried out an immune system multiplex 14-cytokine profiling of plasma samples that were available (for day 0, day 28, and day 56) from 31 of the 38 patients in the above noted (Allegrini et al) clinical trial. Our results show that pre-treatment plasma level cut-offs of interferon-gamma (>12.84pg/ml), sCD40L (<2168pg/ml), interferon-alpha2 (>55.11pg/ml), and IL-17a (<15.1pg/ml) were predictive markers for those patients with better progression-free survival (p<.05 for each cytokine). After 28 days of metronomic therapy, the plasma levels of sCD40L, IL-17a, and of IL-6 (<130pg/ml) could serve as predictors of improved progression-free survival, as could levels interferon-gamma and sCD40L after 56 days of therapy. We observed minimal changes in cytokine profiles, from baseline, as a consequence of the metronomic therapy, with the exception of an elevation of IL-6 and IL-8 levels 28 days (and 56 days) after treatment started (p<0.05). Our results indicate that a selective cytokine elevation, involving IL-6 and IL-8, following metronomic chemotherapy administration. In addition, interferon-gamma and sCD40L may be potential biomarkers for gastrointestinal cancer patients that are likely to benefit from metronomic chemotherapy. Our study contributes to our understanding of the mechanisms of action of metronomic chemotherapy, and may guide future patient selection criteria for metronomic chemotherapy for gastrointestinal cancers.

#785

Molecular profile of sunitinib resistance in clear-cell renal cell carcinoma.

Oscar Reig Torras,1 Mercedes Marin-Aguilera,2 Natalia Jiménez,2 Paré Laia,2 Patricia Galvan,2 Carme Mallofre,1 Aleix Prat,1 Begoña Mellado1. 1 _Hospital Clínic de Barcelona, Barcelona, Spain;_ 2 _Fundació Clínic per a la Recerca Biomèdica, Barcelona, Spain_.

Background: Sunitinib (SU) is a tyrosine kinase inhibitor used in the first line setting in metastatic clear-cell renal cell carcinoma (ccRCC). Recently, nivolumab, a checkpoint inhibitor, has been approved as a second line therapy. The identification of biomarkers of resistance to these drugs may be useful to select patients' treatment. In this study we investigated the molecular and immune profile associated to SU resistance.

Methods: Forty-four patients (pts) with metastatic ccRCC treated with SU were included. A global transcriptome analysis (Affymetrix Human Gene 2.0 ST array) on 6 extremely sensitive (ES; progression-free survival (PFS) > 24 months) and 8 refractory (R; ; progression disease as best response) ccRCC pts treated with SU was performed. Differentially expressed genes were tested in the whole cohort by qPCR. In silico validation using the microarray data from 53 SU-treated pts (Beuselinck et al, Clin Cancer Res 2015) was performed. In addition 730 immune-related genes were tested in the same cohort and in 10 additional pts treated with anti-PD1/PD-L1 therapies as an exploratory analysis.

Results: Differential expression analysis between R and ES pts showed 330 differentially expressed genes: 148 were overexpressed and 182 underexpressed in R pts. Top two deregulated networks were centered in LYN and NR3C1 (Network 1), and in chemokine ligands and receptors (e.g. CCL5, CCRL1 and CXCL10) (Network 2). In Beuselinck dataset, 9 genes were differentially expressed between R and ES pts. The expression of 7 genes (BST2, CD44, ELK1, FADS2, LYN, NKAP and VEGFC) was correlated with a lower PFS in both cohorts (Table 1). A different immune-related molecular profile was shown between resistance to SU and to anti-PD1/PD-L1 therapies. TIM-3 was overexpressed in SU-sensitive pts and in anti-PD1/PD-L1 refractory pts.

Conclusions: We described a gene expression profile of SU resistance in ccRCC pts. The value of TIM-3 as a potential biomarker in ccRCC merits further exploration.

Survival analysis of selected genes in the training and validation set. *High vs low expression.

---

|

HR (Training set) | P | Median PFS, months* | HR (Validation set) | P | Median PFS, months*

ELK1 | 2.66 (1.1 - 6.2) | 0.022 | 5.2 vs 17.2 | 2.47 (1.2 - 5.1) | 0.014 | 12 vs 24

BST2 | 4.87 (1.5 - 15.7) | 0.008 | 4.6 vs 10.6 | 2.22 (1.1 - 4.6) | 0.031 | 11 vs 15

CD44 | 2.55 (1.2 - 5.6) | 0.023 | 5 vs 18.9 | 2.47 (1 - 6) | 0.046 | 9 vs 14

FADS2 | 2.84 (1.1 - 7.7) | 0.039 | 4.2 vs 6.9 | 2.1 (1.1 - 4) | 0.025 | 11 vs 19

LYN | 5.38 (1.6 - 18.1) | 0.006 | 4.5 vs 17.2 | 2.35 (1.1 - 4.9) | 0.024 | 8.5 vs 16

NKAP | 3 (1.3 - 7.1) | 0.012 | 5.1 vs 17.2 | 2.27 (1.1 - 4.8) | 0.031 | 11 vs 16

VEGFC | 3.02 (1.2 - 7.5) | 0.016 | 4.5 vs 17.2 | 1.97 (1 - 3.8) | 0.045 | 12 vs 25

#786

Vascularity and vascular maturity in breast cancer: a comparative analysis of the different molecular subtypes.

Nehad M. Ayoub,1 Ahmed Y. Al Husban,1 Hussein M. Al Husban,2 Abdul Rahman N. Al Khatib,1 Alaa O. Al-Mesleh,1 Ekhlas A. Al-Majali2. 1 _Jordan University of Science and Technology (JUST), Irbid, Jordan;_ 2 _Royal Medical Services, Amman, Jordan_.

Currently, angiogenesis inhibitors lack considerable activity and did not result in survival

advantage in breast cancer. The goal of this study is to evaluate vasculature in terms of

vascularity and vascular maturity among breast cancer subtypes. Archived tumor

samples for breast cancer patients were retrieved from Pathology Department at Royal

Medical Services in Jordan. Tissue sections were stained for the expression of

basement membrane laminin using immunohistochemical analysis. Endothelial cell and

pericyte were identified and counted based on the shape of the nuclei as visualized with

DAPI stain. Demographic, clinical, and pathological data were retrieved from patient

medical records at time of diagnosis. Vascularity was obtained by counting the number

of blood vessels and maturity of blood vessels is represented by the ratio of the average

number of endothelial cell to average number of pericytes. A total of 60 patients were

enrolled in this study in which 34 patients (56.7%) have hormone-dependent disease, 14

patients (23.3%) determined to have HER2-positive disease, and 12 cases (20.0%) were

classified as triple-negative. Results showed a significant difference for the mean

number of blood vessels (p=0.008) and endothelial cell to pericyte ratio (p=0.000)

among the different molecular subtypes of breast cancer. Post hoc analysis indicated

statistically significant difference in mean number of blood vessels between hormone-

dependent and HER2-positive tumors (p=0.015). In addition, ratio of endothelial cells to

pericytes was significantly different between hormone-dependent cases and each of

HER2-positive and triple-negative cases (p=0.000 and 0.014, respectively). Size of

tumor was significantly correlated to endothelial cell to pericyte ratio for the population

examined (r=0.345, p=0.007). A significant correlation was found between the number of

blood vessels and number of involved lymph nodes among all patients (r=0.271,

p=0.038). Independent samples t–test analysis comparing mean number of blood

vessels and endothelial to pericyte ratio according to hormone receptor status showed

significant difference between hormone receptor negative and positive cases for both

estrogen and progesterone receptors (p<0.05). No significant difference was found for

the vascular parameters tested according to tumor stage, grade, and lymphovascular

invasion (p>0.05). Collectively, these findings showed that the degree of tumor

vascularity and vascular maturity are not uniform among breast cancer subtypes.

Vascular maturity was remarkably higher for hormone-dependent breast tumors

compared to both HER2-positive and triple-negative cases. It is critical that a careful

assessment of tumor vasculature is being considered in breast cancer patients in order

to determine responsiveness to chemotherapy and anti-angiogenic treatment among the

different molecular subtypes.

#787

Vascular mimicry mediated mechanisms drive therapy resistance in glioblastoma.

Kartik Angara, Mohammad Rashid, Thaiz Borin, Bhagelu Achyut, Meenu Jain, ASM Iskander, Roxan Ara, Ali Arbab. _Augusta University, Augusta, GA_.

Therapy resistance in solid tumors is of growing concern due to the failure of multiple therapeutic approaches. Targeting tumor cells alone with chemotherapy, tumor vasculature with antiangiogenic therapies (AAT) and tumor-infiltrating myeloid cells with CSF1R inhibitor have all lead to the development of refractory tumors with greater relapse rates. There is an urgent need to understand molecular mechanisms of therapy resistance in cancer. There could be tumor cell extrinsic and intrinsic mechanisms in the tumor microenvironment. We focused our study to investigate tumor cell intrinsic pathways using glioblastoma (GBM) as a model tumor and AAT (antiVEGF-VEGFR) as a model therapy. The benefits of AAT are transient with increased relapse owing to adaptive responses by the GBM. Our preclinical study and in vitro data, for the very first time, identified that AAT induces transdifferentiation of tumor cells into endothelial-like cells, capable of forming functional blood vessels in the growing tumors, termed as vasculogenic mimicry (VM). We observed that anti-VEGFR2 (Vatalanib) induced VM vessels are positive for periodic acid-Schiff (PAS) matrix but devoid of any endothelium on the inner side and lined by tumor cells on the outer-side. The PAS+ matrix is positive for basal laminae (laminin) indicating vascular structures. Vatalanib treated GBM displayed various stages of VM such as initiation (mosaic), sustenance, and full-blown VM. In addition to this, vatalanib treated tumors show significantly increased Laminin positive loops characteristic of VM in tumor center as well as at the periphery. A positive correlation was observed between the VM-like structures and the tumor size. We also performed in vitro tube formation assays with AAT treated GBM cells alone and HUVEC cells (co-culture) to confirm the role of GBM cells in the formation of mosaic vessels in normoxic conditions. Interestingly, tumor cells are incorporated into the tubes formed by HUVEC cells. We found a higher number of complete tube like structures with AAT treated tumor cells as compared to control. Cytokine array with the condition media from tumor cells treated with AAT showed a significant upregulation in the levels of IL8. We observed a significant increase in the CXCR1+ and CXCR2+ endothelial-like GBM cells following treatment with AAT. Ongoing investigations are focused on study of IL8-CXCR1/2 pathway in VM regulation using loss or gain of function approaches. The study will identify critical mediators of VM in GBM. In clinics, discovering novel targets causing VM associated therapy resistance is essential for identifying subset of patients that could be treated with alternate regimens.

#788

Less is more: macrophage depletion via CSF1/CSF1R pathway improves anti-VEGF therapy after adaptive resistance.

Yasmin A. Lyons, Sunila Pradeep, Jean M. Hansen, Michael J. Wagner, Robert L. Dood, Sherry Y. Wu, Rebecca A. Previs, Wei Hu, Robert L. Coleman, Anil K. Sood. _University of Texas MD Anderson Cancer Center, Houston, TX_.

Objectives: Anti-angiogenesis therapy shows clinical benefit in patients with high-grade serous ovarian cancer (HGSC), but adaptive resistance typically occurs. Thus, approaches to overcome such resistance are needed. Here, we systematically assessed immune cell populations enriched during adaptive resistance and identify novel therapeutic avenues.

Methods: A series of in vitro and in vivo (immune competent and nude mice) experiments were carried out. Animals were treated with anti-VEGF antibody continuously until resistance emerged, at which point full immune profiling was performed. Based on these results, efficacy of AC708 (CSF1R inhibitor to target tumor-associated macrophages) was tested in the adaptive-resistance models. F4/80 antibody was used as a macrophage marker.

Results: On the basis of full immune profiling, we detected significantly increased macrophage infiltration in tumors with anti-VEGF antibody resistance compared to tumors from sensitive mice (p<0.0001). Given the dominant role of CSF1R in macrophage function and overexpression of CSF1R in HGSC, we added AC708 following emergence of adaptive resistance to anti-VEGF antibody. Mice treated with AC708 after anti-VEGF antibody resistance demonstrated complete response upon completion of the experiment, while those that did not receive AC708 still had abundant tumor. To mimic treatment with the AURELIA regimen, we next treated mice with anti-VEGF antibody and paclitaxel until resistance emerged, and then AC708 was added. The addition of AC708 restored response to anti-angiogenesis therapy, resulting in 82% lower tumor burden compared to treatment with anti-VEGF antibody and paclitaxel alone (p < 0.0001), and a substantial decrease in macrophages (p<0.0004).

Conclusions: The addition of CSf1R inhibitor to anti-VEGF therapy and taxane chemotherapy results in robust anti-tumor effects. To confirm these findings, a clinical trial at our institution is nearing activation. The REDIRECT (RandomizEd Induction DIscontinuation TRial of EmaCTuzumab) trial will randomize patients to continue weekly paclitaxel and biweekly bevacizumab with or without a CSF1R inhibitor (emactuzumab) following an induction phase.

#789

**Establishment of an** in vitro **model for the study of vasculogenic mimicry in ovarian and gastrointestinal cancer cells.**

Andres Valdivia,1 Dusan Racordon,2 Raul Aravena,3 Gabriel Mingo,2 Alejandra Sandoval,1 Maria Loreto Bravo,4 Mauricio A. Cuello,2 Sumie Kato,2 Rafaela Erices,2 Carolina Ramirez,2 Pamela Gonzalez,2 Beatriz Sanchez,2 Alejandro H. Corvalan,5 Gareth I. Owen6. 1 _Pontificia Universidad Catolica de Chile, Advanced Center of Chronic Diseases (ACCDis), Santiago, Chile;_ 2 _Pontificia Universidad Catolica de Chile, Santiago, Chile;_ 3 _Pontificia Universidad Catolica de Chile, Universidad Santo Tomas, Santiago, Chile;_ 4 _Pontificia Universidad Catolica de Chile, Center UC Investigation in Oncology, Millennium Institute on Immunology and Immunotherapy, Santiago, Chile;_ 5 _Pontificia Universidad Catolica de Chile, Center UC Investigation in Oncology, Advanced Center of Chronic Diseases (ACCDis), Santiago, Chile;_ 6 _Pontificia Universidad Catolica de Chile, Center UC Investigation in Oncology, Millennium Institute on Immunology and Immunotherapy, Advanced Center of Chronic Diseases (ACCDis), Santiago, Chile_.

Introduction: A key step in cancer progression is tumor irrigation. The process of angiogenesis can be complemented in sub-sets of highly aggressive cancers by the process of vasculogenic mimicry, which is the formation of tubular structures by tumor cells. Herein, we sought to establish an in vitro assay of vasculogenic mimicry and determine the percentage of ovarian and gastrointestinal primary cultures capable of undergoing this process.

Materials & Methods: Gastric cancer cell lines, AGS and Hs746T, and the ovarian cancer cell lines SKOV-3 and HEY were used in conjunction with gastrointestinal and ovarian primary cultured cancer cells extracted from peritoneal fluid. Cells were seeded on matrigel and monitored for a week. Hollow channels formation was evaluated by fluorescent dye microinyection, periodic acid Schiff staining, confocal microscopy and X-Ray microtomography (Micro-CT).

Results: SKOV-3, HEY and AGS cell lines underwent the process of vasculogenic mimicry. Both confocal microscopy and Micro-CT reconstruction were able to show the presence of a lumen of the structures in 3D. In 22 primary cancer cultures (Ovarian, Colon and Gastric) only 38% could underwent vasculogenic mimicry.

Discussion: We have standardized an in vitro assay to assay and quantify vasculogenic mimicry. Regardless of origin, only a low percentage of cultures have the ability to undergo vasculogenic mimicry. An understanding of this process could shed light on new therapies for this sub-set of highly aggressive cancers.

Funding: FONDECYT 1120292, 3150028 & 1140970. CORFO L2 13IDL2-18608, BMRC 13CTI 21526-P6, IMII P09/016-F, CONICYT-FONDAP #1513001

#790

Regulation of tumor angiogenesis by low dose Aspirin.

Jinia Chakraborty,1 Gargi Maity,2 Snigdha Banerjee,2 Sushanta K. Banerjee2. 1 _VA Medical Center/Blue Valley West High School, Kansas City, MO;_ 2 _VA Medical Center, Kansas City, MO_.

Background and Objective: Tumor angiogenesis is a pathophysiological process in which new blood vessels are formed in the primary tumor site or distant organs for the nourishment of cancer cells and metastatic growth. Thereby, targeting tumor angiogenesis is an important area of research for cancer therapy. Abnormal structure of blood vessels (i.e. leakiness due to abnormal lining of pericytes on the microvessels) is one of the critical features of tumor angiogenesis that sensitizes vascular cells to cytokines and helps circulating tumor cells to metastasize to distant organs. Our long term goal is to repurpose the drugs that may prevent tumor angiogenesis or normalize the vessels by repairing leakiness via recruiting pericytes or both. Our recent studies found that Aspirin (ASA) has the potency to inhibit breast cancer growth and metastasis, as well as reprogram the mesenchymal to epithelial transition (MET). Given the importance of ASA, we tested whether ASA may be able to regulate tumor angiogenesis.

Methods: To do so, we determined the effect of low dose ASA (1mM, which is equivalent to 80mg human dose), ASA-treated (2.5mM) conditioned media (231-CMASA) or vehicle-treated conditioned media (231-CMVT) of MDA-MB-231 cells on different endothelial cell physiology. These include endothelial cells' migration towards serum using 3D modified Boyden chamber assay, in vitro capillary-like structure formation on Matrigel, cell permeability using in vitro endothelial permeability assay and interactions of pericytes-endothelial cells. We also determined the effect of ASA on various angiogenic factors associated with tumor angiogenesis. Finally, we determined the effect of ASA on in vivo tumor angiogenesis using in vivo Angiogenesis Assay (Trevigen)

Results and Conclusions: We found that 231-CMASA significantly blocks in vitro migration, the formation of in vitro capillary-like structures parallel with leakiness via incomplete interaction of pericytes and endothelial cells as compared to 231-CMASA. The antiangiogenic effect of ASA was also documented in in vivo assays. Mechanistically, ASA treatment blocks several angiogenic factors including VEGF-A that are associated with these three events, implicating a low dose of Aspirin is potentially therapeutic for breast cancer via blocking and normalizing tumor angiogenesis.

#791

Inhibitory effects of theaflavin-3, 3′-digallate (TF3) on ovarian cancer cells.

Yi C. Chen,1 Gary O. Rankin2. 1 _Alderson Broaddus Univ., Philippi, WV;_ 2 _Marshall University, Huntington, WV_.

Theaflavin-3, 3'-digallate (TF3) is a black tea polyphenol produced from polymerization and oxidization of green tea ployphenols epicatechin gallate and (−)-epigallocatechin-3-gallate (EGCG) during fermentation of fresh tea leaves. TF3 has been reported to have anti-cancer properties. However, the effect of TF3 on tumor angiogenesis and the underlying mechanisms are not clear. In the current research, TF3 was verified to inhibit tumor angiogenesis. Compared with EGCG, TF3 was more potent. TF3 inhibited human ovarian carcinoma OVCAR-3 cells induced angiogenesis in human umbilical vein endothelial cell model and in chick chorioallantoic membrane model. TF3 reduced tumor angiogenesis by down-regulating HIF-1α and VEGF. One of the mechanisms was TF3 inactivated Akt/ mTOR/ p70S6K/ 4E-BP1 pathway and Akt/ c-Myc pathway. Besides, TF3 suppressed the cleavage of Notch-1, subsequently decreased the expression of c-Myc, HIF-1α and VEGF, and finally impaired cancer cells induced angiogenesis. Nevertheless, TF3 didn't have any influence on MAPK pathways. Taken together, these findings suggest that TF3 might serve as a potential anti-angiogenic agent for cancer treatment.

#792

Development of high-throughput cell-based co-culture angiogenesis assay system using hTERT immortalized cells.

CHAOZHONG ZOU,1 Chia-Wen Hsu,2 Menghang Xia,2 Metewo S Enuameh1. 1 _ATCC, Gaithersburg, MD;_ 2 _National Institutes of Health, Bethesda, MD_.

Angiogenesis is a multi-step physiological process which is involved in a large number of normal and disease state processes; In vitro angiogenesis models provide very useful tools to study these processes, one of which is the analysis of tubule formation. Tubules formed in co-culture assays were significantly more heterogeneous and more closely resembled capillaries than Matrigel® tubules. Current co-culture models using primary cells have donor variability, and inconsistent results due to lot to lot variation. In this study, we established an in vitro co-culture model system consisting of an assay ready mixture of an aortic endothelial cell line TeloHAEC-GFP (hTERT immortalized human aortic endothelial cell line) and a hTERT immortalized adipose-derived mesenchymal stem cell line (hTERT-MSCs) in a specially formulated medium containing VEGF supplement (Angio-Ready™ Angiogenesis Assay System). Both cell lines were immortalized by hTERT (human telomerase reverse transcriptase) alone and have been well-characterized showing that the cells retain the most important characteristic of their parental counterparts. The new co-culture system forms functional tubular structures in less than 7 days, and in addition, the hTERT-MSC cells which surround the tubular structures have undergone transformation indicated by elevated positive αSMA staining (a marker of smooth muscle cells), indicating that the system has physiological relevance. Notably, our results showed the co-culture system has minimal lot-to-lot variation indicated by the treatment of three lots with the anti-cancer drug, Ramucirumab (Cyramza®), which also targets the VEGF pathway. Next, we tested the new system with compounds that impact angiogenesis, results demonstrated that the angiogenesis system responds positively to elevated doses of VEGF and negatively to increasing concentrations of suramin; more importantly, the tubular formation efficiency is reduced or blocked by well-known anti-cancer drugs such as Sunitinib (SUTENT®) and Bevacizumab (Avastin®), both of which target the VEGF pathway. Finally, we used the Angio-Ready™ system validated 4 HIF-1(hypoxia inducible factors-1) inhibitors which have anti-angiogenic properties identified by high-throughput screening methods; data showed the results of the new system match with other screening methods including a system screening time as short as 3 days. Therefore, the co-culture model developed by using hTERT-immortalized cell lines described in this report provide a consistent and robust in vitro system for studying cardiovascular biology, drug screening and tissue engineering.

#793

Human endothelial progenitor cells: A new target for anti-vascular therapy.

Ghada Ben Rahoma, Neha Tuli, Rachana Maniyar, Sanjukta Chakraborty, Sarnath Singh, Abraham Mittelman, Raj K. Tiwari. _New York Medical College, Valhalla, NY_.

Breast cancer affects one in eight women in the USA. Early diagnosis and newer treatment modalities have rendered breast cancer manageable. However, triple negative breast cancer is still difficult to treat and warrantes a search for newer targets. One strategy that has emerged in cancer research involves targeting of tumor associated blood vessels which provide growing tumors with oxygenated blood and growth factors necessary for maintenance and metastasis. Antiangiogenic drug therapy is transient and has not been able to gain mainstream therapeutic modality. We discovered that endothelial progenitor cells (EPCs) are mobilized from the bone marrow to the tumor site and contribute to the development of breast tumor vessel formation in an estrogen dependent manner. Therefore, characterization of tumor associated endothelial progenitor cells in breast cancer may provide a more specific antivascular therapy. Using the highly proliferative human umbilical cord blood derived EPCs, having the phenotype (CD133+, CD34+, VEGFR-2+), the effect of growth factor and chemokine rich EPCs conditioned medium (CM) was assessed in luminal (MCF-7), and post-EMT (MDA-MB-231) breast carcinoma cell lines. We observed an initial halt in cellular proliferation in MCF-7 followed by a significant increase in proliferation after forty eight hours of treatment. On the other hand, MDA-MB-231 showed decreased proliferation even after forty eight hours of treatment. Treating the EPCs with breast cancer conditioned medium resulted in morphological and cellular growth changes in the EPCs. MDA-MB-231 CM resulted in an increase of the EPCs proliferation and differentiation by increasing the number of spindle shaped attaching cells, and MCF-7 CM resulted only in an increase in the differentiation rate by increasing the number of cell clusters. This increase in EPCs proliferation and differentiation associated with MDA-MB-231 CM treatment might explain the invasiveness of this breast cancer cells through the increase in the tumor associated neovascularization. The analysis of the paracrine interaction between breast cancer cells and EPCs along with the associated cellular changes will facilitate identification of the interactive mediators and subsequent development of effective antivascular therapy.

#794

Potential role of DLL4 in uveal melanoma vascular mimicry.

Julia Escandon, Matthew G. Field, Stefan Kurtenbach, Jeffim Kuznetzov, Christina L. Decatur, J William Harbour. _Univ. of Miami, Miami, FL_.

Uveal melanoma is the most common malignancy of the eye. Thanks to gene array analysis it is possible to classify uveal melanoma in Class 1 (low metastasis risk) and Class 2 (high metastasis risk) tumor. This classification will ultimately determine the tumor treatment, risk of metastasis and patient surveillance. Progression to metastasis remains by far the greatest problem in uveal melanoma and is associated with loss of BAP1 tumor suppressor. Bioinformatic analyses of RNA-Seq indicated that pro-angiogenic genes such as DLL4, VEGFA, VEGFC and HIF1a are overexpressed in Class 2 compared to Class 1 uveal melanoma while angiogenic inhibitors such as ZFP36L1, HIF1AN, VEGFB, VHL and HIF3A are downregulated. Further, we found that DLL4 is among the 5 most highly overexpressed genes associated with BAP1 loss in clinical specimens and in uveal melanoma cell lines induced to deplete BAP1. DLL4 is a Notch ligand known to regulate endothelial cells, bone marrow endothelial cell progenitors and angiogenesis. We hypothesize that DLL4 contributes to vascular mimicry in uveal melanoma. To test this hypothesis, we will test uveal melanoma cell lines induced to deplete BAP1 using shRNA in cell culture-based and in vivo models. The results of this research have the potential to elucidate the mechanism by which vascular mimicry occurs in uveal melanoma.

### Cell Culture and Animal Models of Cancer 1

#795

**The capacity of high-grade serous ovarian cancer cells to form spontaneous multicellular structures (SMCS)** in vitro **predicts their** in vivo **tumorigenicity.**

Alicia A. Goyeneche, Zu-hua Gao, Carlos M. Telleria. _McGill University, Montreal, Quebec, Canada_.

High grade serous ovarian cancer (HGSOC) is the most frequent histopathological subtype among epithelial ovarian cancers (OC). While an increase in concentration of CA125 in the blood anticipates clinical remission following standard of care, we lack biomarker/s to diagnose early disease stage or predict progression speed. In this work we question whether the capacity of HGSOC cells to form Spontaneous Multi-Cellular Structures (SMCS) when incubated under culture conditions that promote, not prevent, adherence to a plastic surface, correlates with their degree of tumorigenicity. We studied 3 HGSOC cell lines developed from the same patient's ascites: longitudinally along disease progression—established at platinum-sensitive relapse (PEO1); from further progressive disease 10 months (mo.) later (PEO4); and after failure to respond to high-dose cisplatin 3 mo. later (PEO6). We noticed all cell types developed an adherent phenotype and a differential capacity to form SMCS. This capacity was more evident in PEO6 cells having high SMCS forming ability, followed by PEO4 cells denoting some SMCS forming capacity, while PEO1 cells depicted no apparent capability to form SMCS. Next, 2x106 PEO1, PEO4 or PEO6 cells were implanted into the abdominal cavity of nude mice; the animals were sacrificed either after having met an end-of-wellness endpoint criterion, or after a maximum of 14 mo. if no such criteria were met. PEO6-injected mice reached the humane endpoint due to accumulation of ascites 6-7 mo. following injection, and presented discrete yet visible solid tumors in the omentum, pancreatic-spleen region, liver base and diaphragm. Animals injected with PEO4 or PEO1 cells, however, did not develop any apparent disease 14 mo. following injection. Yet, when a 10-fold higher load of PEO4 cells but not PEO1 cells (i.e., 20x106) were injected, animals met euthanasia criteria due to ascites accumulation 6 mo. later and displayed macroscopic disease similar to that caused by 1/10 of the load of their successors (PEO6 cells) within the same timeframe. Furthermore, the anatomical localization and histopathological aspect of the lesions generated with PEO6 cells were similar from that generated with PEO4 cells, suggesting that the more aggressive PEO6 cells were likely present, although in fewer quantities, within the less aggressive PEO4 cell population. Our data demonstrate, then, that the in vitro SMCS forming capacity of HGSOC cells has a positive correlation to their capacity to sicken the animals. Moreover, this study suggests that the in vitro SMCS forming capacity of epithelial OC cells obtained from ascites of patients diagnosed with HGSOC may be used to predict their aggressiveness and, consequently, guide prognosis.

#796

Reproductive hormones modulate cardioprotection from doxorubicin-induced cardiomyopathy in female spontaneously hypertensive rats.

Kaytee Pokrzywinski,1 Elliot T. Rosen,1 Julia L. Bonanno,1 Baikuntha Aryal,1 Thomas Biel,1 Delaram Moshkelani,1 Steven Mog,2 Ashutosh Rao1. 1 _FDA, Silver Spring, MD;_ 2 _FDA, College Park, MD_.

Doxorubicin (Dox) causes cardiomyopathy, partially driven by the excess generation of mitochondrial reactive oxygen species (ROS). The incidence of Dox-induced cardiac stress is greater in prepubescent and postmenopausal females compared to reproductively mature women, suggesting that reproductive hormones may mediate cardioprotection. We investigated the role of exogenous hormone replacement therapy (HRT) and naturally cycling reproductive hormones in providing cardioprotection from Dox chemotherapy. Mammary adenocarcinoma SST-2 cells were implanted in spontaneously hypertensive rats (SHRs) that were either ovariectomized and received HRT, or left reproductively normal and maintained natural hormone cycling. HRT animals received time-release pellets containing 17-β-estradiol (E2), progesterone (P4), Tamoxifen (Tam) or combinations thereof, and were subsequently treated with Dox. Naturally cycling animals were treated with Dox, the iron chelator Dexrazoxane (Drz), or Dox+Drz. Tumor size was used to evaluate anticancer activity. Vaginal cytologies and circulating E2 and P4 levels were obtained. Serum cardiac troponin I (cTnI), histopathology and echocardiograms were used to assess cardiac health. Dox treated animals supplemented with E2 and P4 (alone) showed cardioprotection relative to the Dox treated animals. Dox+/-Drz exhibited sustained anti-tumor activity independent of endocrine status. After 13 days with Dox, all naturally cycling animals arrested in diestrus (lowest hormone levels). These results demonstrate that E2 and P4 have a role in providing cardioprotection from Dox therapy in SHRs, and that estrous cycle stage may provide a safety treatment window, potentially reducing the risk of cardiomyopathy in reproductively normal females. We are also currently analyzing RNA sequencing data obtained from heart tissues to achieve a better understanding of the molecular mechanisms of cardioprotection.

#797

Glioblastoma animal model using CRISPR-Cas9 technology.

Da Eun Jeong,1 Kee Hang Lee,1 Sung Soo Kim,1 Yoon Kyung Bae,1 Hyun Nam,2 Ji Yoon Hwang,3 Hee Jang Pyeon,2 Hye Jin Song,2 Kyeung Min Joo1. 1 _SAIHST, Sungkyunkwan University, seoul, Republic of Korea;_ 2 _Sungkyunkwan University, seoul, Republic of Korea;_ 3 _Samsung Medical Center, seoul, Republic of Korea_.

Current in vivo model system poses limitation on fully recapitulating genomic characteristics of a tumor due to high complexity and poor understanding of the heterogeneous microenvironment conditions in cancer pathogenesis. In an effort to address such issues, strategic models are required. In present study, we propose that the most representative cancer models have consistent tumor microenvironments and genomic mutations. The Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is a powerful genome editing tool for efficient and precise genome engineering. Here, we employed CRISPR-Cas9 system in vivo to generate Cre-dependent Cas9 knock-in mouse (B6;129-Gt(ROSA)26Sortm1(CAG-cas9*,-EGFP)Fezh/J, Jackson lab.). The Cre-dependent Cas9 mouse models harbor combinations of genomic alterations including well-established oncogenes such as EGFRviii, c-MET, PDGFRa, IDH1 R132H and KRAS, EGFR, ALK, BRAF in Brain and Lung cancer models, respectively. While, they also consist of tumor suppressor genes including PTEN, NF1, Ink4a/ARF, Rb, TP53 and TP53, PTEN, NKx-1, APC in both Brain and Lung models, respectively. Cre-dependent model allows us to study in-depth into the tumor initiation and progression, while able to follow up in the role of tumor microenvironment in cancer maintenance. A better understanding of cancer models for preclinical research including their uses, as well as their limitations, may aid future potential studies regarding the development and implementation of new immune targeted therapies and potential validation of novel therapeutic biomarkers.

#798

Label-free enrichment and detection of circulating tumor cells in metastatic breast cancer patients show 82% of cohorts have detectable targets.

Yu-Jen Chang,1 Chen-Lin Chen,1 Wei-Fan Hsu,1 Meng-Ze Li,1 Wei-Yuan Ma,1 Ken-Chao Chen,1 Guan-Syuan Huang,1 Wai-Sang Wong,1 Jhan-Yu Syu,1 Thomas, Yo-Yan Huang,2 Ching-Hung Lin,3 Andrew M. Wo,1 Chiun-Sheng Huang3. 1 _National Taiwan University, Taipei, Taiwan;_ 2 _University of California, San Diego, CA;_ 3 _National Taiwan University Hospital, Taipei, Taiwan_.

Circulating tumor cells (CTC) are believed to be the culprit of metastasis and studies have shown that their enumeration has prognostic value in wide range of solid tumors. Although much progress has been made in CTC technology, their rarity in blood and their inherent heterogeneity still provide much challenge towards maturity of the technology. This paper presents semi-automated enrichment of CTC via density-based approach in a novel microfluidic disk, followed by multi-step on-disk immunofluorescence staining (pan-CK, EpCAM, Hoechst and CD45), fluorescence microscopy for image capture, and software image analysis. To characterize the performance of the system, we spiked 1 to 300 cells (mean 105, median 125) from six cell lines (DLD-1, Huh-7, MCF7, PC3, MDA-MB-231 and PC-9) from five cancers into whole blood from healthy donors. The recovery rate of the system is 87.4%±3.7% (R2=0.958) regardless of EpCAM expression levels of the cell lines. To interrogate the limitation of the technology, this data set included ultra-low cell counts spiked of 1 to 13 cells (mean 5.7, median 5) which might be indicative of CTC from metastatic breast cancer (MBC) patients. The recovery rate for this low cell count is 90%±10%. Notably, one single cell was spiked into healthy whole blood, processed via the technology, and one target cell was detected. This test was repeated in triplicate with all three tests recovered one cell each. Furthermore, the microfluidic disk technology enables operation over a range of blood volume (from 2 to 7.5ml) with no statistically significant difference in recovery rate. Recovered MCF-7 cells spiked in blood were subsequently cultured for 6 days and showed good viability with cell proliferation.

We tested the technology in MBC patients along with CA15-3 and CT imaging. A total 34 of blood samples were collected from 21 patients over the course of their systemic treatment. Results showed CTC were detected in 28 samples (82%). The target CTC detected ranges from 0 to 138 (mean 16, median 4 CTCs per 7.5mL). 15 out of 34 samples (44%) had CTC number ≥ 5/7.5mL). In one patient with triple negative diagnosis, CTC count, CA15-3 and CT imaging were monitored during the course of chemotherapy. The CTC count remained zero at the end of the first and second treatment course, elevated to 24 CTCs at the end of the third course, and continued its elevation to 31 by the end of the fourth treatment course. During this entire treatment course, CA15-3 did not vary significantly. However, CT image confirmed the metastasized liver tumor grew from 6.15cm at the beginning of the second chemo course to 8.09cm at the beginning of the fourth course.

Taken together, our label-free CTC enrichment technology has high analytical sensitivity (87%) over a wide range of cancers, able to handle a flexible blood volume (2mL to 7.5mL), and amenable to detect a high percentage (82%) of CTC in MBC patients.

#799

**Tumour micro-environmental factors and the development of mammary calcifications: Recent findings from an** in-vitro **model.**

Shane O'Grady, Maria P. Morgan. _RCSI, Dublin 2, Ireland_.

The presence of microcalcifications on mammographic images represents a highly valuable tool in the early detection of breast cancer, often allowing radiographers to diagnose mammary tumours at an early, in-situ stage. The presence of microcalcifications has also been linked to a number of unfavourable prognostic factors including decreased survival and an increased probability of relapse. Despite their long history of use in the clinic, the precise mechanisms by which microcalcifications are formed remains poorly understood. Pathological soft-tissue calcification is often considered the result of an imbalance between pro- and anti-calcifying factors. However, the possible impact of such an imbalance has not been studied in the context of breast calcification. We have established an in-vitro model of microcalcification formation using the triple-negative adenocarcinoma cell line MDA-MB-231. When cultured in the presence of the osteogenesis-promoting reagents β-glycerophosphate, ascorbic acid and dexamethasone, calcified deposits begin to form by Day 14, as verified by Alizarin Red staining and the quantitative o-cresolphthalein assay. Following the establishment of our model, we set out to identify the underlying molecular triggers initiating the calcification process, in particular the role of micro-environmental factors. Altered magnesium homeostasis has been suggested as an important mediator of tissue calcification. We found that a slight increase in magnesium concentration almost completely blocked calcium deposition in our model. Previous studies in other tissues have shown this protective effect to be dependent on the cation channel TRPM7 which is increased in breast cancer patients with calcifications compared to those without. Unexpectedly, inhibition of the TRPM7 channel by two separate compounds (2-APB and NS-8593) not only failed to reverse the inhibition of microcalcification formation by exogenous magnesium, but actually further decreased calcium deposition by Day 28, suggesting that calcium influx via the TRPM7 channel may be promoting development of mammary calcifications. Finally, we investigated the effect of several tumour associated cytokines on the rate of calcium deposition and found that IL-1β and TNF-α blocked mineralisation whilst IL-6 and BMP2 lead to an increase. Interestingly, co-administration of IL-6 alongside a soluble form of its receptor (sIL-6R) was observed to promote mineralisation even in the absence of dexamethasone, which had previously been essential to the formation of calcifications in our model. To date, our model has yielded a number of important insights into the formation of calcifications in breast cell lines, many of which recapitulate observations from patient studies. It is hoped that this work will contribute to our understanding of the origin of these pre-invasive diagnostic clues.

#800

Enhancing tumor killing abilities of NK cells by targeting CD16 shedding, and ADAM17.

Hemant K. Mishra,1 Jianming Wu,1 Dan S. Kaufman,2 Bruce Walcheck1. 1 _University of Minnesota, St. Paul, MN;_ 2 _University of California, San Diego, CA_.

CD16 consists of two isoforms (CD16a and CD16b) encoded by two highly homologous genes that differ by only 6 amino acids in their extracellular regions. CD16b is expressed by human neutrophils and CD16a by human natural killer (NK) cells. The ectodomain regions of both CD16 isoforms are cleaved proximal to the cell membrane by a proteolytic process referred to as ectodomain shedding. We demonstrate that the membrane metalloprotease ADAM17 cleaves CD16 in isolated leukocytes and in human patients. By mass spectrometry analysis, we determined 3 adjacent cleavage sites in neutrophil CD16b and one cleavage site in NK cell CD16a that occurred at the same location as the predominant cleavage site in CD16b, which is interesting considering CD16b is linked to the plasma membrane via a GPI anchor and CD16a is a transmembrane protein. Antibody-dependent cell cytotoxicity (ADCC) by NK cells is a key mechanism in the anti-cancer effects of therapeutic antibodies, and CD16a exclusively recognizes tumor-bound antibodies. Surface levels of CD16a are rapidly down-regulated upon NK cell activation by cytokines, target cell interaction, and tumor infiltration, which is associated with impaired ADCC. Thus blocking this process has important clinical significance. We contend that maintaining high surface levels of CD16a during NK cell-based immunotherapy will enhance their killing of antibody-bound tumor cells. In ongoing studies, we are examining pharmaceutical and gene-targeting means of preventing CD16a cleavage as a novel therapeutic strategy to enhance the anti-cancer effects of NK cells.

#801

**A zebrafish model of** NF1 **-mutant melanomas that lack activating mutations of** BRAF **or** NRAS **.**

Shuning He,1 Marc R. Mansour,2 Hillary M. Layden,1 Scott J. Rodig,3 E. Elizabeth Patton,4 A. Thomas Look1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _University College London, London, United Kingdom;_ 3 _Brigham and Women's Hospital, Boston, MA;_ 4 _University of Edinburgh, Edinburgh, United Kingdom_.

Cutaneous melanoma is the most lethal type of skin cancer, with ~76,380 newly diagnosed melanoma and ~10,130 melanoma-associated deaths per year in the US. Thus, there is a need for improved understanding of the molecular pathogenesis and more effective targeted therapies for this devastating disease. The recent work of The Cancer Genome Atlas Network has defined melanoma as an RTK/RAS-driven solid tumor that can be classified into four genomic subtypes: BRAF-mutant, RAS-mutant, NF1-mutant, and triple-wild-type. This landmark study highlighted the important role of the previously understudied NF1 tumor suppressor in melanoma pathogenesis, especially for the 9% of melanoma patients who have acquired inactivating NF1-mutations, but lack hotspot mutations that activate BRAF or RAS. To date, animal models have not been developed for the NF1-mutant subtype of melanoma, which has significantly impaired the development of novel therapeutic strategies for this subtype. Here we report the first zebrafish model for NF1-mutant melanoma, which we generated by combining the loss of nf1 with loss of both pten and p53. The resultant compound mutant zebrafish develop aggressive melanomas from the age of 7 weeks and the tumor penetrance is 80% by the age of 18 weeks. We demonstrate further that these high-risk zebrafish melanomas were exclusive of hotspot mutations of braf and nras. Sustained inhibition of both MEK and PI3K suppressed tumor progression in vivo, whereas inhibition of MEK or PI3K alone was insufficient to suppress the growth of these tumors. Surprisingly, single agent therapy with rapamycin, an MTOR inhibitor, proved even better for short- and long-term suppression of tumor cell growth in nf1/pten-mutant melanomas. Thus our model appears ideal for the testing of drugs that will prove uniquely active for the significant subset of NF1-mutant, BRAF/NRAS-wildtype human melanomas.

#802

Loss of function of Arid1a synergizes with MYCN in neuroblastoma pathogenesis.

Hui Shi, Ting Tao, Cigall Kadoch, Thomas Look. _Dana-Farber Cancer Inst., Boston, MA_.

ARID1A is a key component of the SWI/SNF-A (BAF) chromatin-remodeling complex that is known to modulate chromatin structure and gene transcription. Recent exome and whole-genome resequencing has identified SWI/SNF as the most frequently mutated chromatin remodeler in human cancers, and ARID1A as the most highly mutated component among the SWI/SNF subunits, primarily in solid tumors. In high-risk neuroblastoma, ARID1A is mutationally inactivated in one allele in ~1% of patient tumors. In addition, ARID1A is deleted in one allele in at least 85% of cases with chromosome 1p deletions, which is the most common deletion found in neuroblastoma. Thus, ARID1A appears to function as a haploinsufficient tumor suppressor in a substantial fraction of high-risk neuroblastomas. The arid1a gene is duplicated in zebrafish, and the two genes are hence named arid1aa and arid1ab. We targeted each allele of the two genes using CRISPR/Cas genome editing system and recovered stable lines with loss-of-function mutations in each arid1a gene. arid1ab but not arid1aa homozygous mutation showed embryonic lethality. We bred these lines with a transgenic zebrafish line expressing high levels of MYCN in the peripheral sympathetic nervous system driven by the dopamine beta-hydroxylase promoter. Loss of one copy of either arid1aa or arid1ab dramatically accelerated the onset and increased the penetrance of MYCN-induced neuroblastoma. Loss of additional copies of arid1aa or arid1ab accelerated the tumor onset even futher, with the most rapid onset in compound heterosygotes of both genes. The compound heterosygotes showed increased fraction of sympathoadrenal cells in active cell cycle compared to the MYCN transgenic fish with wild type arid1a allales. Our long-term goal is to use the zebrafish model to elucidate the mechanisms underlying the highly dose-dependent tumor suppressor role of this component of the BAF chromatin remodeling complex in neuroblastoma, as a first step toward designing targeted therapies synthetic lethal with reduced expression levels of ARID1A.

#803

Targeting β-catenin/CBP signaling in OSCC.

Khalid Alamoud,1 Khikmet Sadykov,1 Vinay Kartha,2 Stefano Monti,2 Anna Belkina,2 Jennifer Snyder-Cappione,2 Sara Pai,3 Maria Kukuruzinska1. 1 _Boston University Henry M. Goldman School of Dental Medicine, Boston, MA;_ 2 _Boston University School of Medicine, Boston, MA;_ 3 _Massachusetts General Hospital, Boston, MA_.

Objectives: Oral squamous cell carcinoma (OSCC) is an aggressive malignancy characterized by molecular heterogeneity and locoregional spread associated with high morbidity. Aggressive cancers are thought to arise from populations of cancer initiating cells (CICs) that exhibit the properties of stem cells and drive tumor development, recurrence and resistance to therapy. The transcriptional regulator, β-catenin, has been implicated in OSCC CICs. Nuclear β-catenin has been shown to recruit the chromatin remodeling CREB binding protein (CBP) to drive expression of proliferation and survival genes, as well as genes that maintain stem-like phenotypes. We hypothesized that targeting β-catenin-CBP interaction will inhibit CICs in oral tumors and restore an epithelial phenotype.

Methods: To test tumor aggressive potential of OSCC CICs, we used zebrafish as a model system. We isolated CD44+CD24hiCD29hi cells fom aggressive HSC-3 OSCC cells by FACS and assayed their ability to drive tumor growth and metastases in zebrafish compared to unsorted and CD44+CD24lowCD29low cells. In addition, we examined the role of the β-catenin/CBP axis in the aggressive phenotype of these cells. We also assessed whether the β-catenin/CBP axis affected CICs in tumors from immune competent HPV+ mice.

Results: Zebrafish injected with subpopulation of cells co-expressing CD44+CD24hiCD2hi primitive cell surface markers drove rapid tumor growth and metastases, followed by unsorted and sorted CD44+CD24lowCD29low. Treatment of CD44+CD24hiCD29hi cells with a small molecule inhibitor of the β-catenin-CBP interaction, ICG-001, interfered with tumor growth and metastases in zebrafish. Further, ICG-001 inhibited tumor growth in immunocompetent HPV+ murine model. On a cellular level, ICG-001 promoted membrane localization of β-catenin, enhanced E-cadherin adhesion and restored epithelial phenotype. Significantly, ICG-001 gene signatures tracked with reduced overall patient survival in the cancer genome atlas, TCGA. Conclusion: Our studies indicate that the β-catenin/CBP axis promotes OSCC CICs and that ICG-001 may be an effective therapeutic agent for this malignancy.

Support: Evans Center for Interdisciplinary Biomedical Research ARC funding AU 5303015 8000000.

#804

Zebrafish as a model organism to study human pancreatic CSC's.

Laura E. Sanchez,1 Pablo Cabezas Sainz,1 Bruno Sainz,2 Laura Muinelo,3 Rafael Lopez4. 1 _University of Santiago de Compostela, Lugo, Spain;_ 2 _Autonoma de Madrid University, Madrid, Spain;_ 3 _IDIS, Santiago de Compostela, Spain;_ 4 _University of Santiago de Compostela, Santiago de Compostela, Spain_.

Zebrafish is establishing itself as a powerful animal model for understanding human disease, including cancer. Xenotransplantation of human cancer cells into zebrafish is becoming an attractive research tools for studying cancer behaviour, both for established lines and primary tumours, however, its utility as a model for cancer stem cells (CSCs) is still in its infancy. In addition, only when the differences between primary tumours and established cancer cell lines are minimized will we be truly one step closer to developing biologically relevant personalized medicine platforms for cancer. To address these two limitations, we set out to achieve a deeper behavioral understanding of pancreatic CSCs inside the zebrafish embryo. The main methodology of this study was focused on the xenotransplantation of human primary pancreatic ductal adenocarcinoma (PDAC) cells (185 scd line) into zebrafish embryos using two different conditions: adherent cells (non-CSCs) vs sphere-derived cells (CSCs). Marked with a lipophilic dye, cells were injected into zebrafish embryos and incubated at 36ºC during 48h/72h to assess the proliferation of the injected cells. Self-developed Matlab software was used to analyze the intensity and area of the injected cells in order to give a 'proliferation ratio'. Results obtained indicated that the sphere-derived cells proliferated and migrated at a higher rate than adherent cells. This is likely due to the different CSC content present between the two conditions tested: adherent pancreatic 185 cells contain 1% CSCs versus the 15% enrichment of CSCs in sphere-derived 185 cells. Apart from the differential CSC content, we also hypothesize that the 3D environment afforded by the yolk sac of the zebrafish embryo additionally favors the expansion of CSCs. Importantly, similar results were obtained with CSCs isolated via FACsorting, using autofluorescence as a marker for CSCs. This work not only contributes to the behavioral understanding of pancreatic CSCs using the zebrafish model organism but more importantly concludes that 1) PDAC CSC proliferation and migration can be studied in zebrafish, 2) the zebrafish model represents a potentially powerful surrogate model for the study of PDAC CSC biology in vivo, and 3) this model can potentially be adapted for to test chemotherapeutics that specifically target PADC CSCs, the cell type considered to be the most chemoresistant and tumorigenic in pancreatic cancer.

#805

**KRAS** G12D **and TP53** R167H **cooperate to induce pancreatic carcinoma in conditional transgenic** Sus scrofta **.**

Daniel Principe,1 Nana Overgaard,2 Andrew Diaz,3 Carolina Torres,3 Ronald McKinney,3 David Dawson,4 Laurie Rund,5 Regina Schwind,3 Paul J. Grippo,3 Lawrence Schook5. 1 _University of Illinois-Rockford, Rockford, IL;_ 2 _Technical University of Denmark, Denmark;_ 3 _University of Illinois-Chicago, Chicago, IL;_ 4 _University of California-Los Angeles, Los Angeles, CA;_ 5 _University of Illinois-Urbana Champaign, Urbana, IL_.

Although survival has improved in recent years, pancreatic ductal adenocarcinoma (PDAC) remains remarkably virulent with a median survival time of only four months. As there is no effective strategy for the management of PDAC, there is a need for the development of new preclinical strategies. The majority of this work is being done in genetically modified mice, which faithfully recapitulate a variety of pancreatic cancer histotypes. While mice have allowed for unprecedented insight into pancreatic cancer etiology, due to fundamental differences in anatomy and physiology, mouse models of PDAC often fail to accurately predict responsiveness to therapy. As the pig pancreas has more similar anatomical orientation and localization to humans than that of rodents, pigs may serve as a more relevant model for PDAC and other malignancies of the pancreas. As KRAS and TP53 mutations are observed in approximately 95% and 70% of PDAC patients, respectively, we generated Cre/lox transgenic Sus scrofta expressing a LSL-KRASG12D-TP53R167H cassette and administered an Adeno-Cre particle to the pancreas gland parenchyma. This resulted in a mixed histoype of metastatic leiomysarcoma and neoplastic disease of the pancreas. To produce a similar pancreas histoype while avoiding the leiomyosarcoma, we next restricted the Adeno-Cre administration to the main pancreatic duct, which resulted in both PDAC and neuroendocrine carcinoma with no abnormality of the smooth muscle. This serves as the first large animal model of pancreatic carcinogenesis, and given the anatomical/physiological similarity of pigs and humans, may allow for insight into new avenues of research not before possible in rodents.

#806

MGA is a potential tumor suppressor in acute myeloid leukemia.

Qiaoyang Sun,1 Lingwen Ding,1 Kar-Tong Tan,1 Wenwen Chien,1 Xinyi Loh,1 Jinfen Xiao,1 Anand Mayakonda,1 Dechen Lin,1 Yanyi Jiang,1 Henry Yang,1 Sigal Gery,2 H. Phillip Koeffler1. 1 _Cancer Science Institute of Singaore, Singapore, Singapore;_ 2 _Cedars-Sinai Medical Center, Los Angeles, CA_.

MGA is an incompletely studied gene with a high mutation frequency in MLL-PTD AML (9%) and in core bind factor AML (8%). This gene encodes a MAX-interacting protein and is believed to act as a transcription factor that suppresses MYC binding to its target. By in silico analysis, we found that MGA is expressed in normal myeloid hematopoietic cells and AML, and the expression level is comparable with TET2 or DNMT3A. Further data mining of TCGA revealed a high frequency of inactivating mutations of the MGA gene in a variety of cancers such as various adenocarcinomas. To interrogate functionally its role in leukemogenesis, lentiviral constructs containing either shRNA or CRISPR-sgRNA targeted to different regions of the MGA gene were generated. MGA expressing AML cell line EOL-1 was silenced by shRNA or CRISPER system. Silencing was confirmed by western blot (shRNA) and Sanger Sequencing (sgRNA). An increase of methylcellulose colony number (~30%) was observed in MGA silenced cell lines. Control EOL-1 cells or EOL-1 cells silenced with MGA CRISPR sgRNAs were injected into both flanks of NSG mice, and tumor masses were harvested 21 days after injection. Silencing of MGA by CRISPR-sgRNA consistently enhanced in vivo xenograft cell growth. In addition, western blot analyses revealed silencing of MGA in EOL-1 cells increased protein levels of Cyclin E1 and phos-RB (S807 phosphorylation inhibits the ability of RB to target protein allowing cell cycle progression), indicative of a proliferative advantage conferred by the silencing of MGA.

MGA may be a potential regulator of the MYC pathway. We, therefore, examined whether silencing of MGA alters MYC transcriptional activity. Luciferase reporter assay was carried out in 293FT cells stabilized with either scramble or shRNA- targeting MGA. Luciferase activities were measured 48 h after transfection of cells with MYC activity reporter pMyc4ElbLuc and normalized to the corresponding co-transfected Renilla luciferase activity. A fourfold increase in luciferase activity was observed in MGA silenced cells when compared with non- targeting shRNA controls. Furthermore, Kaplan–Meier survival analysis was performed in the TCGA-AML patients by comparison of cases with highest versus lowest expression of MGA. P-values were calculated by log-rank test. MGA expression data and patient survival data were retrieved from TCGA-AML patients RNA seq, or microarray (70 AML patients). The MGA expression 'high' and 'low' groups were defined by 15% higher than the median or 15% lower than the median, respectively. AML patients with lower levels of MGA in their leukemic samples had a worse outcome compared with those whose leukemic cells expressed higher levels of MGA. Collectively, our results suggest that MGA may function as a potential tumor-suppressor in AML.

#807

A novel immunodeficient rat for modeling human cancer.

Fallon K. Noto, Angela Arey, Christopher McClain, Wei Zhang, Tseten Yeshi. _Hera BioLabs, Lexington, KY_.

Animal models of human cancer offer the potential to study human tumor growth kinetics, genetic variance among human cancers, and provide in vivo platforms for drug efficacy testing. In particular, immunodeficient mouse models have been invaluable in modeling a wide range of human cancers. However, some cancer lines don't grow well in the available mouse models or show variability in growth kinetics from mouse to mouse, making drug efficacy studies difficult due to differences in tumor size at the onset of treatment. These challenges are also seen in patient derived xenograft (PDX) models, in addition to long timeframes to obtain sufficient mice with PDX tissue growth for drug efficacy studies. Mice are also limited in tumor growth potential with regard to humane endpoints and small size also limits the volume of blood that can be collected for analysis. An immunodeficient rat model could provide a solution to some of these issues. A rat model would allow for larger tumor size, easier surgical manipulation, and greater volume of tissue and blood sampling for downstream analysis. In addition, large tumors from rats could be serially transplanted into mice for drug efficacy testing and could provide a large number of transplanted mice in a shorter period of time compared with serially transplanting from mouse to mouse.

We have created an immunodeficient rat model with a functional deletion of the Rag2 gene. This knockout, created using spermatogonial stem cells, lacks mature B and T cells. To assess the capability of the Rag2 knockout rat to accept human xenografts, we transplanted 2 commercially available human cancer cell lines into our animals. The human REH acute lymphocytic leukemia cell line was transplanted via intravenous injection and the human glioblastoma cell line U87MG was transplanted subcutaneously. Both cell lines survived in the Rag2 knockout rat and resulted in the growth of tumors comprised of human cells. Studies are underway to characterize the Rag2 knockout rat's ability to grow other human cell lines, including those that do not grow well in mice, and PDX tissues.

#808

PDGF-A overexpression and p53 depletion in rat neural precursor cells induces large brain tumors that resemble human glioblastoma.

Nina P. Connolly,1 Craig S. Schneider,1 Amol Shetty,1 Su Xu,1 Tatsuya Ozawa,2 Anthony J. Kim,1 Jeffrey A. Winkles,1 Eric Holland,2 Graeme F. Woodworth1. 1 _University of Maryland, Baltimore, Baltimore, MD;_ 2 _Fred Hutchinson Cancer Research Center, Seattle, WA_.

One of the major obstacles in developing new therapeutics for glioblastoma is testing these agents in reliable models that recapitulate the tumor biology of human disease. The RCAS/tv-a system enables spatial, temporal, and cell-type specific control of oncogenic transformations in the brain. In this study, we sought to develop a rat RCAS-TVA model of glioblastoma to eventually facilitate translational studies using magnetic resonance imaging (MRI), targeted radiation, focused ultrasound, and local drug delivery strategies. We developed a high copy number nestin-promoter driven tv-a (Ntv-a) transgenic Sprague-Dawley rat line. To initiate tumors, RCAS PDGF-A and p53 shRNA constructs were injected intracranially. The tumors were followed over time using MRI and MR proton spectroscopy. Animal survival was monitored and histopathology and gene expression analyses were performed. All animals (n=8) developed tumors that could be visualized with MRI throughout the tumor formation process. Early stage tumors showed relatively homogenous characteristics with minimal mass effect. Later stage tumors demonstrated large heterogeneous lesions with evidence of necrosis, increased vascularity, and significant mass effect. MR proton spectroscopy revealed increases in choline to creatinine ratio (Cho/Cr) and decreases in NAA, consistent with aggressive tumor progression. Immunohistochemistry revealed pseudopallisading necrosis, brain invasion, and vascular proliferation, all key features of human GBM. Immunohistochemistry confirmed a high proliferative index within the tumor core as well as neovascularization as evidenced by positive Ki67 and Smooth Muscle Actin (SMA) respectively. Gene expression analysis revealed approximately 1000 differentially expressed transcripts between the normal and tumor tissue. In addition to typical markers such as Ki67 and PDGFR-A overexpression, SPP1 and POSTN both of which are linked to glioma and tumor-associated macrophages, were found to be differentially expressed in the tumor. Comparisons between the rat gene expression profile and published human TCGA data indicated that the RCAS/tv-a tumors appear to align with the proneural GBM subtype. In summary, transgenic Ntv-a rats generate reproducible brain tumors following combined PDGF-A and p53 genetic alterations. The tumor progression process from low-grade tumor to high grade malignancy can be visualized with MRI. Histopathological features strongly resemble human GBM, and gene expression analysis suggests these tumors correspond to the proneural subtype.

#809

A novel woodchuck model of hepatic artery infusion (HAI) with FACT complex targeted drug CBL0137.

Minhyung Kim, Leslie I. Curtin, Colin A. Powers, Sandra Sexton, Katerina V. Gurova, Andrei V. Gudkov, Renuka V. Iyer. _Roswell Park Cancer Institute, Buffalo, NY_.

Background: The liver has unique anatomy in that most blood flow to normal hepatocytes is derived from the portal venous system, while liver tumors obtain their nutrient blood supply exclusively from the hepatic artery. Hepatic artery infusion (HAI) concentrates chemotherapy agents delivering them directly to the tumor bed with the first-pass effect. Although, HAI chemotherapy as a regional therapy has been used clinically for decades in the treatment of both primary and metastatic cancers of the liver, significant toxicity remains a concern, and clinical results have been mixed. The purpose of this study was to establish preclinical HAI model in a woodchuck and administer the FACT (facilitates chromatin transcription) complex targeted drug CBL0137.

Material and methods: Woodchuck hepatitis virus infected Eastern Woodchucks (Marmota monax) were used for the study. Hepatocellular carcinoma (HCC) developed spontaneously about 18 months after birth. The size of HCC was monitored with ultrasound every 2 weeks, and 4 woodchucks bearing over 2 cm tumors were used for the experimental group. Prophylactic cholecystectomy was performed first, and then a 2Fr. (ID=0.012", OD=0.025") vascular access port (VAP) was implanted in the gastroduodenal artery (GDA) after collateral vessel ligations. HAI was performed at room temperature via a VAP using 15mg/kg of CBL0137 at a volume of 3 mL/kg for 30 minutes, and tumor responses were investigated with MRI on day 0 (the surgery day of HAI port) and day 7 after HAI.

Results: Anatomy variations existed in the size and location of the supraduodenal artery or pancreatico-duodenal artery. 4/4 success rate of a port implantation was achieved without mortality, but 1/4 animals suffered post-operative bleeding at the port head site, attributed to coagulopathy secondary to hepatic dysfunction. And, 4/4 animals were partially anorexic for up to 7 days post-surgery. Decreased tumor volume and Ktrans (a measure of capillary permeability) were detected 7 days after single HAI with CBL0137.

Conclusion: Although the anticancer effect of CBL0137 on HCC was not clear because of a lack of the control group, we successfully implanted a VAP into the GDA of 4 woodchucks bearing unresectable HCC for HAI. The described woodchuck model may serve as a platform for identifying novel drugs and optimal conditions for the treatment of primary and metastatic liver tumors.

#810

Establishing patient derived preclinical in vitro and in vivo models of pediatric brain cancers.

Sridevi Yadavilli, Madhuri Kambhampati, Jamila Gittens, Eshini Panditharatna, Mojca Stampar, Lindsay B. Kilburn, Suresh Magge, Roger J. Packer, Javad Nazarian. _children's National Health System, Washington, DC_.

The recent surge in understanding genomic aberrations of some of the deadliest childhood brain cancers has highlighted the need for robust preclinical models. Such models will allow for robust drug screening and preclinical evaluation of efficacy, toxicity, and tumor penetrance in vivo. Given the rarity and importance of patient derived specimens, handling and processing methods are perhaps the most critical steps for successful establishment of viable and reproducible in vitro and in vivo models. Since specimen source varies (biopsy, autopsy, or cryo-preserved), processing methods should be refined to allow for optimal extraction of maximum numbers of viable cells from each specimen type. We have developed standardized procedures for handling and processing of tissue samples obtained from biopsy, autopsy, or cryo-preserved specimens as well as necropsy tissue obtained from existing xenograft models. Two processing methods for generating viable cell suspensions are described. The first method, which uses collagenase-DNAse mediated digestion of the tissue is efficient with bulky samples and can be used with tissues obtained at autopsy. The second method uses a commercially available enzymatic dissociation kit optimal for small volume samples such as biopsy, cryo-preserved and mouse necropsy specimens. We show that obtaining viable cell suspension from precious tumor tissue by these methods results in successful generation of pre-clinical in vitro and in vivo models of DIPG, pilocytic astrocytoma and medulloblastoma that represent the exact genetic makeup of the original patient tumor. We further demonstrate intracranial injections of these cells into P2 mice for generating orthotopic xenograft models of brainstem or cortical tumors. Our methods and results allow for rapid establishment of preclinical models using rare and valuable childhood brain tumor specimens. These pre-clinical models serve as valuable tools for understanding the molecular mechanisms of the disease, identifying targetable molecules, and screening of novel therapeutics.

#811

Histological heterogeneity contributes to sunitinib resistance in clear cell renal cell carcinoma.

Zsuzsanna Lichner, Rola Saleeb, Henriett Butz, Roy Nofech-Mozes, Sara Riad, Mina Farag, Andras Kapus, George Yousef. _Keenan Biomedical Research Centre, St Michael's Hospital, Toronto, Ontario, Canada_.

Introduction: The receptor tyrosine kinase (RTK) inhibitor sunitinib is the first line treatment for advanced clear cell renal cell carcinoma (ccRCC). Sunitinib inhibits angiogenesis via blocking signaling through VEGFR. About 80% of patients develop resistance after a drug-sensitive period. Molecular changes early in treatment may impact drug resistance, but are poorly understood.

Experimental Procedures: ACHN, 786-O and Renca cell lines were treated with 1 µM sunitinib. NSG mice were s.c. xenografted with the model cell lines and were treated with sunitinib at 40 mg/kg/day dose. mRNA expression was screened using Illumina HT-12 bead chip array and miRNA expression was assessed by Nanostring nCounter assay. R statistical packages were used for data processing. Reactome and miRPath softwares were used for downstream analysis.

Results: Sunitinib treatment of ccRCC xenografts led to several early changes in tumor histology, such as the emergence of live tumor areas within the necrotic spaces. These areas showed membranous staining for E-cadherin, and β-catenin, while the rest of the tumor and vehicle-treated tumors were negative. In vitro model cell lines developed cancer spheroids when treated with sunitinib. Cancer spheroids were highly tumorigenic and metastatic, and expressed several established cancer stem cell markers. ccRCC cancer spheres, but not the 2D adherent cells, showed membranous staining for E-cadherin and β-catenin; similarly to the live tumor areas observed in

in vivo sunitinib treatment. In vitro inhibition of E-cadherin by EGTA or by siRNA, interfered with viability of sunitinib treated ccRCC cell lines.

Conclusions: Sunitinib treatment causes early phenotypic changes of the tumor in vivo and in vitro. The formation of highly metastatic and tumorigenic cancer spheres in model cell lines is the most prominent effect in vitro. We provide preliminary evidence that sunitinib induced in vitro cancer spheres and the live tumor areas that survive within necrotic patches of the sunitinib-treated xenografts, are related. Finally, membranous expression of E-cadherin enhances the survival of ccRCC cell lines under sunitinib treatment.

#812

Multiplatform modeling of pancreatic cancer using patient-derived cells: A new approach for defining drug resistance mechanisms.

Erika Maria Parasido,1 Praathibha Sripadhan,1 George Avetian,1 Richard Schlegel,1 Jonathan Brody,2 Jordan Winter,2 Charles J. Yeo,2 Michael J. Pishvaian,1 Erik Glasgow,1 Stephen Byers,1 Christopher Albanese1. 1 _Georgetown University, Washington, DC;_ 2 _Thomas Jefferson University, Philadelphia, PA_.

Background: Pancreatic adenocarcinoma (PA) is the fourth leading cause of cancer-related death in the USA. Current treatments utilize Gemcitabine alone or in combination with other drugs, such as nab-paclitaxel, however, a growing number of patients have shown resistance to these regimes. In order to enhance clinical response, it is important to better understand the mechanisms of drug resistance. To date, one of the main limitations in PA research has been the reliance on a few commercially available PA cell lines or a limited number of genetically engineered or PDX mouse models for in vitro and in vivo studies. It was therefore imperative to develop more accurate cellular models that better represent a given patient's tumor. In our current study, we present the use of patient-derived primary PA cells as a model system for basic and translational research, and for personalized medicine.

Methods: Patients' biopsies were collected after surgery, and long-term cultures of PA cells were established using the conditional reprogramming of cells (CRC) approach we developed. KRAS and p53 sequencing verified the PA origin of both the patient samples and the matched CRC lines. The IC50s for Gemcitabine and Abraxane were determined and used to establish drug resistant clones. Our two dimensional (2D) CRC cultures have shown to be adaptable to different platforms, such as 3D spheroid cultures as well as zebrafish- and mouse- PDX models.

Results: We established KRAS-mutant primary cell lines derived from patients' PA samples. From two different parent cell lines we created five nab-paclitaxel and two Gemcitabine resistant clones. The clones were 3-1000 times less sensitive to the drugs as compared with the parent lines. Drug resistance ratios between parent and resistant clones were confirmed both in 3D cultures and our zebrafish PDX platform. The injection of both parent and resistant clones into NCI/nude mice induced tumor masses in 100% of the cases. Unlike the parent cell-derived tumors, the nab-paclitaxel clone-derived tumors exhibited a metastatic phenotype. Histological analyses of these PDX models recognized the presence of morphological features of human PA, including ductal acinar structures comprised of pleomorphic epithelial cells with extensive stromal desmoplasia surrounding the tumors, and thereby represent a fundamentally important advancement in the PDX modeling field.

Conclusion: Overall, the ease of culture, the genetic stability and the high-throughput ability to identify differences between patients sensitivity to FDA approved drugs confirm the power of this technology for on-demand in vitro use in PA research. The in-vivo approach demonstrated the accuracy of our model system in recapitulating the patient tumor. Our method now enables the high-resolution experiments necessary to better understand the resistance mechanisms that directly affect clinical outcomes

#813

Development of a porcine model for pancreatic cancer.

Neeley A. Remmers,1 Jesse L. Cox,1 Paul M. Grandgenett,1 James A. Grunkemeyer,1 Laurie A. Rund,2 Lawrence B. Schook,2 Michael A. Hollingsworth,1 Mark A. Carlson3. 1 _University of Nebraska Medical Center, Omaha, NE;_ 2 _University of Illinois, Urbana, IL;_ 3 _University of Nebraska Medical Center; NE-Western IA VA, Omaha, NE_.

Pancreatic ductal adenocarcinoma (PDAC) remains a lethal disease with a 5-year survival rate of 4%. Without significant advances in early detection and treatment, PDAC is estimated to become the second leading cause of cancer deaths in the United States by 2020. Many advancements in PDAC have come from murine models and retrograde studies of patient samples. A supplemental pig model would allow such studies as noninvasive image-guided technologies, radiation oncology, drug metabolism, surgical experimentation/training, and early detection screening to be more readily performed. Here, we sought to develop an orthotopic model of porcine pancreatic cancer using transformed pancreatic tumor cells to help address some of these issues.

We isolated primary porcine epithelial cells from pancreatic ducts harvested from a normal pig. We isolated epithelial cells from fibroblasts by serial dilutions and stained our final population with cytokeratin 19 (specific for pancreatic ductal epithelium), E cadherin, alpha-SMA, and collagen I to show that our population of primary cells were indeed of epithelial origin and not mesenchymal. We used these cells to generate 3 different tumorigenic cell lines: PGKP (contains mutated Kras and p53), PGKPS (PGKP + SMAD4 shRNA), and PGKPSC (PGKPS + p16 shRNA). We tested these cell lines using in vitro assays to test their tumorigenic properties. The PGKP cells outperformed the primary cells in proliferation, population doubling time, and soft agar growth with p-values <0.03. The additional hits in the PGKPS and PGKPSC cells generated more pronounced invasion through matrigel, migration rate, anchorage-independent growth, proliferation capacity, and population doubling times with p<0.01 when compared both to primary cells and the PGKP cells.

We used all three cell lines in a subcutaneous injection model in nude mice to determine if they could produce tumors in vivo. We injected 5x106 cells into the hind flanks of mice with groups of 10 mice/cell line. Of the 30 mice injected, 28 grew tumors that were ≥1.8 cm2 in size within 5 weeks. All of the tumors had large necrotic centers, were hypervascular, and mucous producing. Upon H&E analysis, all tumors displayed an undifferentiated morphology with mild desmoplasia. Representative tumor sections were stained for the same markers as used above in the primary cell isolates with the addition of vimentin. These stains indicate that our tumorigenic cells formed undifferentiated carcinomas with mild desmoplasia.

Here, we demonstrate for the first time development of porcine pancreatic tumor cells that can induce tumors in vivo. The expression of vimentin in our tumor slides indicates that our cells may have the potential to recapitulate metastatic disease when orthotopically injected into the pancreas of syngeneic pigs. We feel that this model could greatly supplement the existing murine models of pancreatic cancer to help improve imaging, surgical, and future diagnostic techniques.

#814

NOTCH1 **regulates head and neck cancer cell growth and invasion.**

Andrew C. Birkeland, Megan Ludwig, Jacqueline Mann, Samantha Devenport, Rebecca C. Hoesli, Chloe Matovina, Matthew E. Spector, Carol R. Bradford, Thomas E. Carey, Mark E. Prince, J. Chad Brenner. _University of Michigan, Ann Arbor, MI_.

Introduction: NOTCH1 has recently been identified as a frequently mutated gene in head and neck squamous cell carcinoma (HNSCC). However, its role in HNSCC is poorly understood. Creation of genetic models investigating the role of NOTCH1 in HNSCC behavior will be crucial to further characterize its function, and to potentially modify treatment paradigms in NOTCH1 mutant HNSCCs. To date, no syngeneic HNSCC cell line knockout models of NOTCH1 have been created to specifically study the effects of NOTCH1 in HNSCC phenotypes.

Methods: We transfected a NOTCH1 gRNA-CRISPR-Cas9 lentiviral construct into a HNSCC cell line with wildtype NOTCH1 (UMSCC-49) and selected knockout clones. Whole exome sequencing, qRT-PCR, and Western blotting were performed to validate the cell line knockout. Invasion chamber and growth assays were performed to assay for cell line phenotypes. Immunofluorescence staining and proteomic analysis were performed to study expression changes in various invasive markers.

Results: We successfully created a NOTCH1 knockout cell line (UMSCC-49N1KO) with an otherwise identical genetic architecture as its parent line (UMSCC-49). Knockout status was verified via exome sequencing, qRT-PCR and Western blotting. Analysis of UMSCC-49N1KO suggested an increased aggressive and invasive phenotype. Cell proliferation assays demonstrated an increased cell proliferation in UMSCC-49N1KO. Invasion chamber assays demonstrated increased invasion in the NOTCH1 deficient cell line. Proteomic analysis demonstrated increased expression of proteins associated with invasion and cell proliferation and decreased expression of cell differentiation markers in UMSCC-49N1KO, consistent with an aggressive and invasive phenotype in the NOTCH1 deficient line. These phenotypes were validated with subsequently generated UMSCC-49 NOTCH1 knockout lines.

Conclusions: NOTCH1 acts as a key tumor suppressor gene in HNSCC and regulates cell proliferation and invasion. We have created a useful genetic model to study effects NOTCH1 in tumorigenesis, proliferation and metastasis with a wildtype NOTCH1 parent (UM-SCC49) and mutant NOTCH1, otherwise syngeneic cell line (UM-SCC49N1KO). NOTCH1 mutant status correlates with worse outcome in HNSCC patients. Further characterization of the role of NOTCH1 in cell proliferation, invasion and metastasis may provide insight into treatment stratification paradigms, targetable options and predictive biomarkers for patients with NOTCH1 mutant HNSCCs.

#815

Using CRISPR/Cas9 to generate isogenic cell lines and reference standards for applications in cancer diagnostics.

Xin Cheng, Nga Nguyen, Qi Zheng, Mi Shi, Andrew Hilmer, Huanyu Jin, Ruby Yanru Chen-Tsai, Ling-Jie Kong. _Applied StemCell, Inc., Milpitas, CA_.

We are entering an era of precision medicine, in which there are significant initiatives to employ companion diagnostics and molecular profiling to inform clinical decisions in therapeutic treatment. In order for these initiatives to work as effectively as possible, there is a need for molecular reference materials that can help facilitate precision assay development. Cell-line based reference standards are ideal for such applications because they represent a reproducible and renewable source of control materials that can be employed for assay development, quality control, or lead compound discovery. With this in mind, we have used the CRISPR/Cas9 platform to engineer series of isogenic cell lines that feature 51 recurrent mutations in the MAPK signaling pathway. These cell lines contain footprint-free, homozygous mutations in the kinase domains of the EGFR, KRAS, and BRAF genes in HCT116 and RKO cancer cell lines, with 32 mutations in EGFR, 12 mutations in KRAS, and 7 mutations in BRAF. The mutations include precise deletions, point mutations and small fragment insertions, and these engineered cell lines have been further employed for the development of derivative reference materials, including genomic DNA (gDNA) and FFPE cell line blocks. Such materials can be employed as positive and negative controls in diverse assay platforms, including next generation sequencing (NGS), quantitative PCR (qPCR), in situ hybridization (ISH), and others. We present quality control data for these reference materials, which demonstrate excellent consistency and uniformity, as well as examples of the application of these materials in molecular assay validation. We conclude that using CRISPR/Cas9 technology to generate molecular reference standards is a very efficient and precise method in generating footprint-free, biorelevant mutations for companion diagnostic assays.

#816

Molecular and genomic characterization of a newly established male breast cancer cell line.

Tereza Vaclova,1 Sarah Maguire,1 Matthew Pugh,1 Peter Barry,2 Nick Orr1. 1 _ICR, London, United Kingdom;_ 2 _The Royal Marsden NHS Foundation Trust, London, United Kingdom_.

Although the majority of breast cancers affect women, approximately 1% of cases occur in men. At present comparatively little is known about the molecular mechanisms that influence male breast cancer predisposition and tumorigenicity. This is largely due to the current unavailability of an established cell line model in which to study male breast cancer. Development of in vitro models of the disease is therefore warranted, not least because growing evidence indicates that it is sufficiently different from female breast cancer such that extrapolating knowledge from one to the other may be misleading.

Here we report the establishment and characterisation of a male breast cancer cell line derived from a primary tumor arising in a 61-year old male patient. We used 3T3-J2 mouse fibroblast co-culture in the presence of a ROCK inhibitor to conditionally reprogram epithelial cells from freshly resected tumor tissue. Once established, the cell line showed a rapid proliferation rate and growth in full conditioned media without the support of mouse fibroblasts. Immunohistochemical profiling demonstrated expression of epithelium-specific antigens AE1/AE3, breast epithelial marker CK7 and confirmed estrogen receptor positivity in concordance with the primary tumor. Germline sequencing detected no pathogenic germline predisposition mutations in either BRCA1 or BRCA2. There was no evidence of somatic mutation in any of the established female breast cancer driver genes. Somatic whole genome sequencing of early and late passages indicated a paucity of structural aberration and little evidence for obvious accumulation of gross genetic alterations.

In conclusion, we have established a novel in vitro model for studying male breast cancer. That the cell line genome displays minimal evidence of significant acquired genetic changes arising due to reprogramming suggests that it will be of value in future studies of the biology of male breast cancer.

#817

Unbiased discovery of exosome-associated biomarkers using xenograft models.

Milcah C. Scott,1 John R. Garbe,1 Hirotaka Tomiyasu,1 Alicia Donnelly,1 Brad A. Bryan,2 Subbaya Subramanian,1 Jaime F. Modiano1. 1 _Univ. of Minnesota, Minneapolis, MN;_ 2 _Texas Tech University Health Sciences Center, El Paso, TX_.

We developed a novel, genome-wide method for biomarker discovery that is able to separate intrinsic markers of disease from markers of host response. Specifically, we implanted cell lines derived from two primary canine osteosarcoma tumors with different biological behavior into the tibiae of athymic nude mice. Both cell lines formed orthotopic tumors, and each recapitulated the biological behavior of the original primary tumor in terms of growth rate and metastatic potential (1,2). We also developed a cross-species hybrid genome that allowed us to identify separate canine and mouse transcripts in tumor xenografts (2). In other words, we were able to determine the contribution of canine mRNA sequences (derived from the implanted tumor cells) and mouse mRNA sequences (derived from infiltrating stroma) to define tumor-intrinsic features and host-specific features that contribute to osteosarcoma progression. When we applied this analysis method to exosome-derived mRNAs, we found canine-derived transcripts that were only present in exosomes of tumor-bearing mice that were associated with functions such as protein kinase A (PKA) and actin cytoskeleton signaling, and with upstream transcriptional regulators such as FOXF1, ESR1, and TP53. We also found tumor-specific, canine-derived transcripts that were associated with functions such as caveolae-derived endocytosis, energy utilization and metabolism, immune interactions, and G-coupled receptor signaling, and with upstream transcriptional regulators such as HNF1A, ESR1, NIPR1, and HNF4A. This method has the potential to reduce the statistical uncertainty that arises from the use of pre-selected molecules among the background of thousands of genomic features in patient samples. Using genome-wide analyses, we are able to combine multiple, highly correlated genes to manage this problem, increase markers' robustness, and avoid random correlations. In conclusion, this technology is useful to identify nucleic acids that serve as cancer biomarkers with high precision, and inter-species sequence conservation can be used to further improve its predictions.

References

1. Scott MC, Sarver AL, Tomiyasu H, Cornax I, Van Etten J, Varshney J, et al. Aberrant Retinoblastoma (RB)-E2F Transcriptional Regulation Defines Molecular Phenotypes of Osteosarcoma. J Biol Chem 2015;290(47):28070-83.

2. Scott MC, Tomiyasu H, Garbe JR, Cornax I, Amaya C, O'Sullivan MG, et al. Heterotypic models of osteosarcoma recapitulate tumor heterogeneity and biological behavior. Disease Models & Mechanisms 2016, doi: 10.1242/dmm.026849.

#818

Unravelling the role of androgen targeted therapies in epithelial plasticity during prostate cancer progression.

Ellca Ratther, Katrina G. Sweeney, Nataly Stylianou, Qiuhua Hu, Gregor Tevz, Colleen C. Nelson, Elizabeth D. Williams, Brett G. Hollier. _Queensland University of Technology, Institute of Health & Biomedical Innovation, Australian Prostate Cancer Research Centre – Queensland, Translational Research Institute, Brisbane, Australia_.

Recent evidence suggests that the use of potent androgen receptor (AR) antagonists have contributed to the increased prevalence of prostate cancer (PCa) tumors exhibiting therapy resistance. Androgen targeted therapies (ATT) inhibit the androgen/AR axis which PCa cells rely on for growth. ATT are initially effective however most men progress to metastatic castration resistant prostate cancer (mCRPC). Mechanisms of resistance include programs of epithelial plasticity (EP) such as epithelial-to-mesenchymal transition (EMT) and neuroendocrine transdifferentiation (NEtD). EMT enables PCa cells to become more motile and invasive. PCa cells that undergo NEtD gain neuroendocrine (NE)-like traits. Patients with NE prostate cancer (NEPC) usually present with visceral metastasis, are refractory to ATT and have short response to chemotherapies. We aim to characterize models of therapy-induced NEtD (tNEPC) and to investigate the role of tumor plasticity in the progression to mCRPC.

To recapitulate ATT in vitro, androgen-dependent LNCaP cells were treated with either enzalutamide (Enz) (10μM) or shRNA targeting AR in culture medium containing fetal bovine serum (FBS) or charcoal stripped serum (CSS) with or without 10nM dihydrotestosterone (DHT). To model EMT, LNCaP cells were engineered to express doxycycline (Dox) inducible overexpression of EMT transcription factor Snail. Androgen deprivation in vitro induced expression of NE markers in LNCaP cells compared with FBS treated cells (mean fold increase ± SEM): neuron specific enolase (ENO2) (6.0 ± 2.5), neural cell adhesion molecule (NCAM) (10.4 ± 6.8) and midkine (MDK) (13.2 ± 2.4). Cells co-treated with Enz and DHT had increased expression of NE markers compared to DHT alone: ENO2 (2.7 ± 0.6), NCAM (2.9 ± 0.3) and MDK (2.3 ± 1.8). AR knockdown resulted in a potent induction of NE markers compared with control cells: ENO2 (8.3 ± 1.4), NCAM (8.3 ± 2.4) and MDK (9.0 ± 2.2). Dox-induced Snail enhanced androgen deprivation-mediated upregulation of NE markers compared with no-Dox cells: ENO2 (1.3 ± 0.1), NCAM (8.3 ± 2.4) and MDK (9.0 ± 2.2). To more accurately model a preclinical setting, LNCaP tumors were grown subcutaneously in severe combined immune deficiency mice. Once tumors grew to 200mm3, mice were castrated and upon tumor recurrence mice were treated with Enz (10mg/kg). At the ethical endpoint, expression of EMT and NE markers will be assessed.

Patients treated with ATTs show an increase in NE-like traits. Thus, there is a need to identify molecular mechanisms involved in this adaptive response. In vitro, we have modelled the acquisition of NE-like features in PCa cells using ATTs, EMT drivers and AR knockdown. Further investigation using these models may lead to the development of biomarkers and therapeutic targets for the clinical management of advanced PCa.

#819

Transcriptomic profiling of two canine mammary cancer cell lines for translational oncology studies.

Heidge Fukumasu,1 Yonara G. Cordeiro,1 Pedro L. Xavier,1 Arina L. Rochetti,1 Pamela A. Alexandre,1 Claudia C. Mori,2 Silvio H. Freitas,1 Ricardo F. Strefezzi1. 1 _Univ. of São Paulo, Pirassununga - São Paulo, Brazil;_ 2 _Univ. of São Paulo, São Paulo, Brazil_.

Dogs develop spontaneous cancers as humans and shares a variety of features including histology, genomic alterations, molecular targets, biological behavior and response to conventional therapies. Although dogs can be considered good models for translation of new cancer treatments, there is a lack of established preclinical in vitro models of canine cancers. Therefore, our aim here was to establish two canine mammary cancer cell lines and characterize their transcriptomic profile to speed the translation of new cancer treatments from dogs to humans. Mammary cancer samples were obtained from female dogs that underwent surgery. Pieces of the tumors were FFPE for histopathology and the other fragments were washed with PBS, minced and dissociated with hyaluronidase and collagenase. Cells were filtered and put in culture with DMEM-F12, 5%FBS and 1% pen-strep. After 5 passages, FBS was increased to 10%. After passage 15, cells were characterized for doubling time, karyotype, immunocytochemistry for cytokeratin, vimentin and α-smooth muscle actin, matrigel-invasion assay and tumor growth in nude mice. At last, RNA-seq was performed on total RNA extracted with TRIZOL for functional enrichment analysis of differentially expressed (DE) genes from cancer cell lines. From 49 tumor samples, we characterized 2 cell lines named M5 and M25, originally from a comedocarcinoma and a mixed carcinoma, respectively. Both cell cultures showed spindle-shape cells and were positive for cytokeratin, vimentin and α-smooth actin, suggesting myoepithelial origin. The doubling time was 26.0h for M5 and 42.8h for M25 cells. Karyotyping analysis showed moderate aneuploidy for both cell cultures. M25 cell line presented significant higher invasion potential than M5 cells and formed tumors in mice but the latter didn't. Histopathology of these tumors resembled the original tumor in the dog. An average of 16.2 million paired-end 100 bp reads were sequenced per replicate (three for each cell culture) with an average of 81.1% alignment to the reference genome. A total of 11,260 and 11,476 genes with more than 1 count per million were detected in M5 and M25 cell lines. M5 cell line have 404 and 394 up- and downregulated genes in comparison to M25 cell line, respectively. Gene ontology of DE genes showed the more malignant cell (M25) significantly enriched for pathways related to drug metabolism, ECM-receptor interaction, cell cycle, DNA replication, focal adhesion and cell differentiation. In conclusion, M25 cell line is more malignant than the M5 which is supported by their transcriptome. Thus, it is possible to use these cells as a comparative translational model for drug development especially if one wishes to work with molecular targets as described above.

#820

Potential pro-proliferative role of SIRT6 in melanoma.

Liz Garcia-Peterson, Mary A. Ndiaye, Chandra K. Singh, Wei Huang, Nihal Ahmad. _University of Madison Wisconsin, Madison, WI_.

Melanoma is one of the deadliest form of skin cancer that can rapidly metastasize to become lethal, if not diagnosed early or left untreated. In 2016, approximately 76,380 new melanoma cases and 10,130 melanoma-related deaths are predicted in the United States. Current preventive and therapeutic strategies have not been sufficiently effective in the management of melanoma. Therefore, novel molecular targets and treatments are required for an effective management of this neoplasm. In our laboratory, we are assessing the role and functional and therapeutic significance of sirtuin family of proteins in melanoma. Sirtuins (SIRTs) have been conserved through evolution from prokaryotic to eukaryotic cells. SIRTs are nicotinamide adenine dinucleotide (NAD+) dependent protein deacetylases and belong to class III of the histone deacetylase (HDAC) family. Seven members of the mammalian SIRT family are known to date, and despite structural similarities, each SIRT has their own biological niche, performing unique functions via regulating critical mechanisms in the cell. The role of SIRTs in cancer is somewhat controversial, as they have exhibited conflicting functions (tumor promoter vs. tumor suppressor) depending on cell and tissue contexts. The sirtuin SIRT6, a predominantly nuclear protein, has been shown to conduct ADP-ribosyl transferase and histone deacetylase activities. SIRT6 plays key roles in DNA repair, inflammation and metabolic diseases such as cancer. Currently, the role of SIRT6 in melanoma is not known. The objective of this study was to determine the role of SIRT6 in melanoma. Using a panel of human melanoma cell lines (A375, Hs 294T, G361, SK-MEL-2, SK-MEL-28, SK-MEL-31, WM115 and WM35) differing in genetic complexity and disease progression stage, and normal human epidermal melanocytes (NHEMs), we determined the endogenous expression levels of SIRT6. We found that compared to NHEMs, SIRT6 is significantly upregulated in melanoma cell lines, at mRNA as well as protein levels, as shown by quantitative Real-Time PCR and western blot analyses. Further, employing a human tissue microarray (TMA) coupled with quantitative Vectra™ analysis, we determined the expression profile of SIRT6 protein in human melanoma and melanocytic nevus tissues. Our data demonstrated that SIRT6 is significantly overexpressed in human melanoma tissues when compared to nevi. Furthermore, lentiviral short hairpin RNA-mediated knockdown of SIRT6 in human melanoma cells was found to result in a marked anti-proliferative response in melanoma cells. Taken together, our data suggest that SIRT6 overexpression could potentially be a contributing factor in melanoma progression. Further detailed studies are underway to understand the functional significance of SIRT6 in melanoma development and progression.

#821

Loss and retention of mutations in cell culture model systems.

Pamela S. Shaw,1 Rick Nicoletti,1 Naghmeh Salimi,1 Helen L. Yang,1 Abigail E. Witt,1 Agoston T. Agoston,2 Elin S. Agoston1. 1 _Cellaria Biosciences, Cambridge, MA;_ 2 _Brigham and Women's Hospital, Boston, MA_.

Although relevant, the genomic and proteomic alterations between original tumor tissue and newly established cell lines are frequently uncharacterized. In developing a novel cell model (Powder) of high grade serous carcinoma (HGSC), we followed the status of clinically relevant biomarkers to identify changes taking place between the tissue and cultured cell population. Understanding these changes enables proper utilization of new models and continued model optimization. Using a validated panel, we analyzed 592 genes in the Powder cell model and tumor from which it was derived, detecting several classes of genomic alterations. This included point mutations, indels, copy number variations, fusions, and variant transcripts. p53 mutation status of Powder was matched to a panel of previously reported HGSC cell lines. We further subdivided the panel by p53 protein expression level by reanalyzing previously published reverse phase protein array (RPPA) data. Significance of differential expression between groups was determined with a Welch's t-test. A majority (14/21 or 67%) of the mutations detected in the HGSC tumor were conserved in the Powder cell model. Two additional mutations, undetected in the tumor, were found in the cell model. The tumor exhibited moderate to strong diffuse positive p53 immunostaining, and p53 mutations were measured in 79% of alleles. While the p53 mutation did not carry to the cell model, the cells were positive for CK7 and PAX8, consistent with ovarian cancer cells. In the panel of HGSC cell lines, we examined p53 mutation status and protein level, identifying 3 groups: (1) p53-mut/high protein, (2) p53-mut/low protein and (3) p53-WT/low protein. Powder identified with the p53-WT/low protein group. Previous work identifies the p53-WT/low protein group of cell models as part of a larger set of novel ovarian cell models with a stem-like molecular profile and higher drug resistance. Analysis of differential protein expression revealed upregulation in the p53 low protein group of both normal cell cycle genes and of notable cancer-related proteins. This included p21 and BAD, which are upregulated in cells with functional p53, eIF4E, a candidate cancer therapeutic target, and BOP1, which is disregulated in multiple solid cancers, including ovarian. These results underscore the need for further characterization of the Powder cell model to clarify its applications to specific research questions. Continued examination of Powder and other p53-WT cell models derived from HGSC tumors may uncover independent pathways driving disease. Additionally, understanding which mutations are underrepresented by cell models is necessary for ongoing methods development.

#822

**Simple and easy monitoring of tube formation and migration assays with the CytoSMART** TM **Live Cell Imaging System.**

Stefanie Buesch,1 Sabine Schaepermeier,1 Theresa D'Souza,2 Bodo Ortmann,1 Claudia Schwartz,1 Jenny Schroeder1. 1 _Lonza Cologne GmbH, Cologne, Germany;_ 2 _Lonza Walkersville Inc., Walkersville, MD_.

Movement of cells plays a critical role in the development of cancer. Analyzing the motility of cells in appropriate cell culture models is therefore an important tool for cancer researchers. Live cell imaging is particularly well suited to capture dynamic processes in cell culture.

The CytoSMART™ Device is an easy-to-use, small and affordable live cell monitoring system suitable for the label-free analysis of cell motility within a standard cell culture incubator. Label-free approaches offer the benefit of cell analysis without potential cytotoxic or other side-effects of the used markers or dyes on the cells. This presentation shows the suitability of the CytoSMART™ Device for the analysis of different cancer-relevant assays.

The formation of new blood vessels is required to ensure sufficient nutrient and oxygen supply and to allow solid tumors to grow beyond a certain size. This process can be mimicked in cell culture models in so-called tube formation assays. In this study, Human Umbilical Vein Endothelial Cells (HUVEC) were seeded on Engelbreth-Holm Swarm Sarcoma derived Basement Membrane Extract (BME). The resulting formation of endothelial tubes was monitored with the CytoSMART™ Device. Subsequently, the average length of the formed tubes as well as the number of closed tube circles was quantitatively determined. In addition, the impact of Suramin on tube formation was evaluated as an example for a tube formation inhibiting compound.

The migration of cancer cells is also required for the growth and in particular the metastasis of tumors. In a first example, the closure of a so-called wound or scratch in a confluent monolayer of cancer cells was monitored with the CytoSMART™ Device. Determining the migration speed of the cells by measuring the speed of wound closure is a simple assay to determine the migration potential of cancer cells. Modifying the cancer cells, e.g. by knocking down specific genes with siRNA, can help to identify genes that play a role in cell migration. Compounds that are expected to reduce cell motility and therefore reduce the metastasis potential of cancer cells can be easily tested.

In a second example, the invasion of cancer cells into a three-dimensional (3D) matrix was analyzed. Tumor cells were embedded in a cancer-relevant matrix and their invasion into the 3D matrix was documented with the CytoSMART™ Device. While this type of model is slightly more difficult in set-up and analysis compared to simple scratch assays, it may reflect better the in vivo situation where solid tumors develop within three-dimensional tissues. The images captured with the CytoSMART™ System were quantitatively analyzed using appropriate software.

Overall, the CytoSMART™ System is an easy-to-use, small and affordable live cell imaging system suitable for the label-free analysis of different cancer-relevant assays.

#2799

Development of a mouse model of KRAS mutated colorectal cancer tumorigenesis.

Radhashree Maitra,1 Madhu Kumar Venkatesh,2 Titto Alby Augustine,1 Carol Chandy,1 Qiang Liu,1 Sanjay Goel1. 1 _Montefiore Medical Center, Bronx, NY;_ 2 _Albert Einstein College of Medicine, Bronx, NY_.

Introduction Development of a preclinical animal model that mimics KRAS mutated colorectal cancer disease etiology is essential for validation of new therapeutic interventions. Colorectal cancer patients with tumors harboring KRAS mutation are excluded from receiving anti EGFR monoclonal antibodies, and currently have no alternate FDA approved treatment options available. Ongoing research requires validations of different modalities on a robust preclinical study platform that will resemble the disease phenotype and genetic profile. In this context we have developed viable animal mouse model of colorectal cancer bearing KRAS mutation that authentically serves as a model for the disease.

Methodology: The C57 BL/6 mice with truncated APC floxed allele was crossed with heterozygous KRAS floxed outbred mice to generate APC f/f KRAS +/f mouse colony. In another set of breeding APC floxed mice were crossed with CDX2-Cre-ERT2 mice and selected for APCf/f CDX2-Cre-ERT2 after second round of inbreeding. The final model of the disease was generated by the cross of the two aforementioned colonies when viable APC f/f KRAS +/f CDX2-Cre-ERT2 were genotyped and characterized. The model animals were tamoxifen induced to generate tumors. Micro-PET scan was used to detect and measure tumor volume. H & E staining was done to establish neoplasm and immunohistochemistry was performed to determine histological similarities with human FFPE biopsies. The MSI/MSS status was also determined.

Results Ø The newly developed animal model develops colonic tumors upon induction with Tamoxifen. Ø The tumors were malignant as confirmed by H and E staining. Ø The tumors showed a higher radioactive FDG uptake (SUV) in micro-PET scan Ø The tumors resembled human colorectal cancer tissue Ø Once tumor is generated the animals died of cachexia and rectal bleeding

Conclusion: Heterozygous mutation of KRAS oncogene along with deletion mutation of Adenomatous polyposis coli (APC) gene is embryonically lethal in animal models. We have successfully developed a viable animal model of KRAS mutated colorectal cancer with tamoxifen inducible tumors harboring KRAS and APC mutations. The tumors mimic the human disease type both genetically and immunohistochemically. This model can serve as a robust preclinical platform for understanding the disease mechanism at the molecular level as well for evaluation of various therapeutic interventions.

### Cellular Responses to Ionizing Radiation

#823

Inhibition of focal adhesion kinase (Fak) leads to sensitization of p53 mutant head and neck cancer to genotoxic stress.

Manish Kumar, Liang Yang, David Molkentine, Jeffrey Myers, Raymond Meyn, Curtis Pickering, John Heymach, Heath Skinner. _UT MD Anderson Cancer Center, Houston, TX_.

Head and neck squamous cell carcinoma (HNSCC) is most commonly treated with DNA-damaging therapies such as cisplatin and/or radiation. However, a significant proportion of HNSCCs are resistant to this therapy. Recently, we have identified focal adhesion kinase (Fak) as a targetable biomarker of therapeutic resistance in this disease. Additionally, utilizing in vivo shRNA screening of druggable targets in a panel of HNSCC cell lines, Fak was one of the most significant genes related to radiosensitization. Additional investigations utilizing a combination of in vivo and in vitro assays have shown that inhibition of Fak either chemically or via shRNA leads to dramatically increased sensitivity to DNA damage. This effect is primarily found in p53-mutant tumors and cell lines, which are known to be resistant to DNA-damaging therapies. DNA damage due to radiation appears to be potentiated in p53 mutant HNSCC cell lines following Fak inhibition, but less so in matched p53 wild type HNSCC. Moreover, basal Fak activity is higher in HNSCC cell lines engineered to express missense p53 mutations compared to either wild type or p53 null cells. We also found that several forms of mutant p53 bind to Fak and may alter its activity directly. Taken together, our data suggest a targetable nexus of mutant p53 and Fak that can be directly modulated to improve the efficacy of DNA-damaging therapy.

#824

Reduction in prostate tumor growth and induction of anti-tumor immunity after combined treatment with oncolytic VSV and radiation.

Thirupandiyur S. Udayakumar, Dillon Betancourt, Glen Barber, Anis Ahmad, Brian Marples, Alan Pollack. _University of Miami, Miami, FL_.

Androgen deprivation therapy (ADT) is a mainstay of treatment for high-risk non metastatic and metastatic prostate cancer. Patients initially respond to ADT, but eventually develop resistance to treatment. To overcome these limitations, we have used oncolytic virotherapy which has the potential to influence both local and systemic disease through direct cytotoxic and immune mechanisms. Here, we investigated the effectiveness of radiation therapy (RT) combined with Vesicular Stomatitis Virus (VSV) vector expressing interferon β (IFNβ) in VSV resistant prostate xenografts. In initial cell culture experiments, microarray analysis indicated that PC3 cells treated with VSV-hIFNβ and RT displayed several genes known to directly inhibit VSV replication (e.g., MX2, OAS2, TRIM25, IFITM1) were downregulated compared with VSV-hIFNβ therapy alone. Additionally, many apoptotic pathway-associated genes (e.g. IL24, GADD45B, KLF4&10, TNF, EGFR) were upregulated several fold during combination therapy. Gene expression changes were confirmed by RT-PCR. Further, immunoblot analysis demonstrated expression levels of IFNAR, BCL-XL, phosphorylated AKT, JAK2 and STAT3 were also markedly decreased when VSV-hIFNβ was combined with RT. However, RIG-I was not significantly altered after VSV-hIFNβ+RT treatment. A marked decrease in MAVS, NFKB (p50), IRF7 and antiviral protein PKR was seen after combined treatment. The role of IFN pathway in viral response was investigated using an interferon-inducible promoter ISRE-luciferase (ISRE-Luc) reporter construct. A significant difference (>600 fold) in the ISRE-Luc activation was seen for PC3 compared to LNCaP cells, indicating that the IFN pathway is highly active in PC3 cells after VSV infection alone. The activation of ISRE-mediated transcription of luciferase was significantly attenuated (p<0.05) in both the cell lines after combined treatment with VSV-hIFNβ and RT. In subsequent in vivo studies, we determined that radiation treatment combined with intratumorally-delivered VSV-mIFNβ function synergistically to induce oncolysis in both PC3 and LNCaP xenografts and a syngeneic RM9 murine model. In the syngeneic model, combination therapy resulted in a robust anti-tumor immune response with CD8 and CD4 T-cell expansion and resistance to tumor rechallenge compared to either therapy alone. These studies demonstrate that two mechanisms are involved in combined RT and VSV-mediated oncolysis. These are, (1) the attenuation of the antiviral response which increases the direct oncolytic effect of VSV on the tumor cells and (2) improved generation of an adaptive immune response earmarked by CD8+ lymphocyte expansion and anti-tumor activity. Our study demonstrates that the combined strategy of RT plus VSV-IFNβ, affects tumor cell death that are resistant to VSV through direct and systemic activity that includes a pronounced enhancement of anti-tumor immunity.

#825

Cetixumab enhanced apoptosis effects of radiation on human prostate cancer cell line PC-3.

Chia-lun Chang, Szu-Yuan Wu. _Wan Fang Hospital, Taipei, Taiwan_.

Prostate cancer is a leading cause of cancer death in men in developed countries. Radiotherapy is the main treatment to prostate cancer. Dose escalation of irradiation with image guide rasdiotherapy and intensity modulated radiotherapy were used with more and more evidences in prostate cancer patients and bring in longer PSA-free survival time. Although the evolution of radiation techniques bring better outcomes, but modern modalities were not avaliable in non-western world. Radiation enhancement is still an improtant issus in prostate cancer in non-western world, especially in androgen independnt prostate cancer. The epidermal growth factor receptor (EGFR) network has rich targets for prostate cancer killing. The chimeric monoclonal antibody Cetuximab (IMC-A12, Erbitux®) binds to EGFR and prevents its intracellular signaling. Currently, it is approved for treatment of wild-type KRAS colon and head and neck cancer. The effect and mechanisms of Erbitux and radiotherapy are still not clear. In this study, we evaluated the effects of combining the EGFR inhibition and radiation on PC-3 prostate cancer cells. PC-3 cells were treated with various doses(1,2.5,5 and 10 ug/ml) of anti-EGFR antibody (CETUXIMAB) and irradiation(5, 7.5 Gy). The results revealed that 5 and 10 ug/ml of CETUXIMAB significnatly reduced the colony formation. The cell cycle analysis revealed that CETUXIMAB significnatly enhanced the radiation induced G2/M phase arrest and subG1 peak increasing. The Hoechst staining and DNA laddering analysis revealed radiation and CETUXIMAB combined use will induced more apoptotic bodies or DNA fragmentation than radiation alone use. By using a apaptosis protein array, we found that several apaptosis related protein (Catalase, cIAP-1, Clusterin, Fas, HIF-1α, HSP27, HSP60, HSP70, SMAC and Survivin) were significantly downregulated in radiation and CETUXIMAB combined group. Taken together, our results demonstrated that Erbitux enhanced apoptosis effects of radiation on PC-3.

#826

Nrf2 confers radioresistance by regulating cancer stem trait in lung cancer cells .

Xiaoqing He,1 Alex Chi,1 Liying Wang,2 Yon Rojanasakul1. 1 _West Virginia Univ., Morgantown, WV;_ 2 _National Institute for Occupational Safety and Health, Morgantown, WV_.

Lung cancer is the leading cause of cancer-related death accounting for more than 1.3 million deaths worldwide annually. Despite advances in treatment, the five-year survival rate is 2% if the cancer has metastasized to other organs. The major cause of poor prognosis of advanced lung cancer is multi-resistance to chemo/radio therapy. Therefore, it is crucial to understand the underlying mechanisms of chemo/radio resistance and develop therapeutic strategies to overcome the resistance. Transcription factor Nrf2 is a key regulator of cytoprotective genes which facilitate cellular defense against harmful insults. We found that Nrf2 and its downstream target NQO1 are highly upregulated in lung cancer patient tumor tissues as well as in lung cancer cell lines compared to adjacent normal tissues or normal cell lines. We hypothesized that the upregulated Nrf2 may be responsible for radio/chemo resistance and malignancy in lung cancer. RNA interference studies showed that knockdown of Nrf2 substantially increased the sensitivity of human lung cancer H460 and A549 cells to radiation-induced cell death. Knockdown of Nrf2 also decreased tumor sphere formation, suggesting its role in cancer stem cell (CSC) regulation which may be responsible for the radioresistance. Analyses of CSC markers including ALDH and Oct4 in control and Nrf2-knockdown H460 and A549 cells support this finding. Together, our results indicate an essential role of Nrf2 in the control and regulation of CSCs and radioresistance. Since CSCs are widely believed to be responsible for cancer chemoresistance and relapse, our novel finding on the role of Nrf2 in radiation resistance may aid in the design of novel therapeutic strategies for resistant cancers.

#827

Statistical analyses of stable housekeeping gene expression in cancer post-irradiation.

Albert R. Wang, Gopal Iyer, Sean Brennan, Shay Bourgeois, Eric Armstrong, Pari Shah, Paul M. Harari. _University of Wisconsin-Madison, Madison, WI_.

Ionizing radiation (IR) can induce DNA damage in human cells and result in changes in gene expression. The changes could also include some of the commonly used housekeeping genes (HKG), and thus making them unstable over the time period after being exposed to radiation. Normalization of housekeeping genes is critical for understanding gene expression post-irradiation. Toward the validation of stable HKGs, cancer cell lines from head and neck, non-small cell lung and pancreas were irradiated to 2, 4 and 6 Gy IR doses. Statistical analysis of RNA expression of fourteen HKGs measured at 5 min to 48hrs post-irradiation reveal tradition HKGs such as beta-actin (ACTB) and glucose-6-phosphate-dehydrogenase (G6PD) to be unstable, while TATA-Box Binding Protein (TBP) and Importin 8 (IPO8) was identified as stable HKGs in non-small lung and pancreas cell across all IR doses and time. Interestingly in head and neck cancer, TBP is also found stable across all IR doses and time. The statistical framework used for identification and validation of HKGs can serve as a reliable metric for quantifying gene expression post-irradiation.

#828

Radiosensitization of glioblastoma stem-like cells by Selinexor, a selective inhibitor of nuclear export (SINE) compound.

Amy Wahba, John W. O'Neill, Kevin Camphausen, Philip J. Tofilon. _NIH/NCI, Bethesda, MD_.

Radiation therapy remains a primary treatment modality for glioblastoma (GBM). Recently, radiation-induced translational control has been implicated in regulating processes involved in the cellular radioresponse of GSCs. Reactome analysis disclosed that one major pathway under translational control after irradiation in GSCs is the nuclear export pathway, specifically by the nuclear export receptor Exportin 1 (XPO1, also referred to as CRM1). XPO1 transports over 200 cargo proteins from the nucleus to the cytoplasm via the nuclear pore complex, including several tumor suppressor proteins and it is dysregulated in multiple types of cancers. Selinexor (KPT-330), a SINE compound, is an XPO1 inhibitor that has shown antineoplastic activity in preclinical and clinical models, both alone and in combination with other treatments. In this study, we investigated the effect of XPO1 inhibition by Selinexor in combination with radiation on GSCs. To determine whether XPO1 could serve as a target for GSC radiosensitization we focused on the GSC cell line NSC11. Based on clonogenic survival assay, addition of Selinexor 1h before irradiation significantly enhanced the radiosensitivity of NSC11 cell as compared to controls. To begin to investigate the mechanism behind Selinexor-mediated radiosensitivity, γH2AX foci dispersal, a surrogate marker for DNA double strand breaks (DSB) was evaluated. NSC11 cells treated with Selinexor 1h before irradiation (2Gy) and collected 1-24h later for analysis of γH2AX demonstrated a significant delay in DNA DSB repair 24h after irradiation. Because XPO1 is responsible for export of various cargo proteins out of the nucleus, including several ribosomal subunits, it is predicted to play a role in global translation. To evaluate the effects of XPO1 inhibition on GSCs, the polysome profiles of NSC11 treated with Selinexor were evaluated. Polysome profiles of NSC11 cells demonstrated significant attenuation of the polysome portion of the profile as early as 1h after treatment with Selinexor. After 24h of Selinexor treatment, polysomes were almost undetectable with a decrease in translational efficiency (TE) by more than half. This attenuation is not accompanied by a change in the monosomal peaks, suggesting that poly-ribosome binding to mRNA is specifically affected by radiation. Taken together, these data suggest that XPO1 plays a role in translation as well as in cellular radioresponse, and inhibition of XPO1 by Selinexor enhances GSC radiosensitivity, possibly by inhibiting global translation.

#829

MicroRNA regulation of radiation sensitivity in colorectal cancer.

Shushan Rana, Katherine Kelley, Rebecca Ruhl, Charles Thomas, Liana Tsikitis, Sudarshan Anand. _Oregon Health & Science University, Portland, OR_.

Background Patients with locally advanced rectal cancer receive neoadjuvant chemoradiation therapy (CRT) and subsequent surgery. While 10-25% of patients have complete response to CRT, the remaining patients undergo extensive tumor excision resulting in significant quality of life issues. Response to CRT is an independent predictor of overall survival highlighting the importance of improving CRT response rates. Several tumor intrinsic factors govern responses to CRT including specific gene expression programs. Emerging evidence suggests that microRNAs (miRs) modulate gene expression programs in response to radiation and have been implicated in several pathological processes associated with colorectal cancer progression. In this context, we hypothesized that differential expression of miRs regulates colorectal cancer radiation sensitivity and can be used as a biomarker to predict radiation efficacy.

Methods To investigate the differences in miR profiles between rectal cancer patients that had either a pathological partial response (PR) or no response (NR), we isolated RNA from FFPE biopsies using the miRvana microRNA isolation kit (Life Technologies). We used the Nanostring miR profiling platform and obtained absolute counts for >700 human miRs. We performed in vitro gain and loss of studies with candidate miR transfections in human CRC cell lines and used a luminescence-based assay for proliferation (Cell titer glo, Promega). Surviving fraction assays were performed by seeding cells and counting colonies stained with Crystal Violet. Eight distinct miR bioinformatic target prediction algorithms initiated miR target identification, and a RISC-Trap assay was implemented to confirm miR-target interactions. The Cancer Genome Atlas (TCGA) datasets were accessed to acquire provisional survival data on 639 colorectal adenocarcinoma patients.

Results We identified 17 miRs that were differentially expressed, and miR-451a, among the most upregulated miRs, inhibited proliferation and colony formation in 2D and 3D assays in the presence of radiation. Target prediction algorithms highlighted CAB39, EMSY, EREG, and MEX3C as prominent miR-451a targets in colorectal cancer and/or radiation. TCGA subset anaylsis found CAB39 and EMSY protein levels were found to be upregulated in 14% and 6% of cases, respectively, and upregulated co-expression of these genes significantly reduced 3 year overall survival (69% vs 78%, p < 0.05). miR-target interaction was confirmed via the RISC-Trap assay with miR-451a mimic transfection resulting in robust 2.4-, 1.2-, and 2.8-fold enrichment of EREG, CAB39, and EMSY, respectively. In our partial responders patient cohort possessing upregulated miR-451a, there was significant downregulation of CAB39 and EMSY mRNA and protein compared to non-responders.

Conclusions miRs alter cell survival networks affecting radiation sensitivity and serve to identify pathways amenable to alternative therapeutic modulation

#830

MiRNA-106a and LITAF are novel modulators of prostate cancer radioresistance.

Christianne Hoey,1 Jessica Ray,1 Paul Boutros,2 Stanley K. Liu3. 1 _University of Toronto, Toronto, Ontario, Canada;_ 2 _Ontario Institute for Cancer Research, University Health Network, Toronto, Ontario, Canada;_ 3 _Sunnybrook Research Institute, Toronto, Ontario, Canada_.

Prostate cancer (PCa) is the second most prevalent cancer affecting men worldwide, with radiotherapy being a primary treatment modality. PCa recurrence is a major clinical problem with up to a 40% biochemical recurrence rate at five years after external beam radiotherapy (ionizing radiation, IR). Therefore, there is an unmet need to characterize radioresistance in order to improve therapy and patient outcomes. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression. MiRNAs are aberrantly expressed in cancer, specifically, miR-106a has been found to be overexpressed in various cancer types. The purpose of this study is to determine whether miR-106a confers IR resistance and tumor aggression in PCa.

Utilizing The Cancer Genome Atlas Data Portal, bioinformatics analysis discovered that miR-106a was significantly overexpressed in PCa samples relative to normal prostate. This suggests that miR-106a is involved in prostate carcinogenesis. Clonogenic survival assays revealed that cells overexpressing miR-106a had increased survival after IR treatment compared to control cells. Proliferation assays showed that miR-106a cells had a higher proliferation rate than control cells in both unirradiated and IR-treated cells. Affymetrix gene array analysis was used to identify possible targets of miR-106a. Combining results from the gene array, in silico prediction algorithms, and in vitro analyses, we identified lipopolysaccharide-induced TNFα factor (LITAF) as a target of miR-106a. In addition, LITAF knockdown lead to increased proliferation and clonogenic survival following IR, recapitulating the phenotype of miR-106a overexpression. Cells were stained for β-galactosidase expression following IR to assess senescence, as a mode of cell death. We found that both miR-106a overexpression and LITAF knockdown resulted in significantly fewer senescent cells post-IR. Upon examination of critical DNA damage response genes, we found that miR-106a overexpression and LITAF knockdown increased expression of ATM mRNA and protein. Upregulation of ATM is associated with IR resistance, elucidating the mechanism by which miR-106a overexpression and LITAF knockdown are involved in radioresistance. Tumor xenograft experiments, using a PCa cell line stably overexpressing miR-106a, confirmed that miR-106a increases proliferation compared to control tumors before and after IR.

Thus, our results strongly suggest that miR-106a is involved in PCa aggression and confers a radiation-resistant phenotype, by targeting the novel radiation response gene LITAF.

#831

TGFβ controls the DNA damage response via miR-182 regulation of BRCA1 and ATM.

Qi Liu,1 Haydeliz Martinez-Ruiz,2 John Murnane,1 Simon N. Powell,3 Mary Helen Barcellos-Hoff1. 1 _University of California, San Francisco, San Francisco, CA;_ 2 _Eli Lilly, Indianapolis, IN;_ 3 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Transforming growth factor β (TGFβ) is a well-documented tumor suppressor; a poorly studied aspect of TGFβ biology is its control of genomic stability. Our prior work showed that TGFβ compromises ATM (ataxia telangiectasia mutated) kinase activity, which mediates recognition and repair of DNA damage. Consequently, inhibiting TGFβ following ionizing radiation increases clonogenic death in vitro and tumor control in vivo in breast, brain, and lung cancer preclinical models. As yet unknown is how TGFβ controls ATM kinase. We recently determined that TGFβ regulates mammary lineage commitment by post-transcriptional control of BRCA1 mRNA and protein by its suppression of miR-182 (Martinez-Ruiz et al., Science Signaling, in press). miR-182-mediated downregulation of BRCA1 impacts DNA repair and sensitivity to PARP inhibitors in cancer cell lines (10.1016/j.molcel.2010.12.005) Here we used two cell culture models to investigate the effect of TGFβ regulation of BRCA1 and miR-182 on DNA damage response. Spontaneously immortalized Tgfb1 heterozygote and wild type fibroblasts from primary mammary epithelial preparations were synchronized in S-phase and exposed to UV radiation before fixation to detect unrepaired DNA damage by quantitative image analysis of 53BP1 foci. Consistent with compromised BRCA1 function, more than twice as many cells with 53BP1 foci were evident in Tgfb1 heterozygote fibroblasts compared to wild type cells (p<0.01). The second model consisted of non-malignant human breast epithelial cell line, MCF10A, stably transduced with a scrambled or miR-182 antagomir, grown under serum free conditions and treated with a small molecule inhibitor of TGFβ type I receptor kinase, LY364937. FOXO3 is a target of miR-182 and thus also regulated by TGFβ. and FOXO3 association with ATM is required for its kinase activity (10.1038/ncb1709). As reported, FOXO3 and ATM co-immunoprecipitated, but was eliminated by TGFβ inhibition and this effect was lost in MCF10A cells expressing a miR-182 antagomir. The impaired DNA repair phenotype induced by TGFβ inhibition can be fully rescued by antagonizing miR-182. These studies support TGFβ as stringent regulator of the DNA damage response via suppression miR-182, which directly targets BRCA1 and indirectly affects ATM activity via FOXO3. We predict that cancers that have lost TGFβ signaling capacity will be genomically unstable due to defective DNA damage control, which is consistent with our studies showing that TGFβ inhibition sensitize cancers to radiation therapy.

#832

In vitro **characterization and clinical correlation of BRCAness as a personalized biomarker for radiosensitization with homologous recombination-directed therapies.**

Jennifer Ma,1 Andrew C. Bell,1 Jeremy Setton,1 Justin Haseltine,1 Marcher Thompson,1 Rachna Shah,1 Benjamin H. Lok,1 Robert Delsite,1 Rebecca Aft,2 Nadeem Riaz,1 Simon Powell1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Washington University, School of Medicine, St. Louis, MO_.

Introduction: BRCA1 and BRCA2 are involved in double-strand break (DSBs) repair via homologous recombination (HR). Clinically, BRCA1/2 mutant tumors show sensitivity to cisplatin, a cross-linking agent, but only mild sensitivity to ionizing radiation (IR). The addition of cisplatin to IR has been one of the largest advances in improving patient outcomes with radiotherapy in the past 50 years. Recently, BRCAness has been described wherein sporadic deficits in HR are observed in the absence of germline BRCA1/2 mutations. We hypothesize that BRCAness tumors also exhibit sensitivity to the combination of cisplatin + IR, and are a significant driver of the observed clinical benefit of combination therapy.

Methods: Sensitivity to cisplatin + IR was examined in vitro and correlated with clinical data. Functional HR deficiency (HRD) was assessed in isogenic BRCA1 (H1299; lung) and BRCA2 (DLD1; colon) knockout pairs using 3 assays: the Rad51 foci assay, a flow-based DRGFP assay (DSB reporter assay) and clonogenic survival assays (CSA) with cisplatin +/- IR. Cancer Cell Line Encyclopedia (CCLE) data was used to identify breast cancer cell lines with sporadic, non-BRCA1/2 mutations. Functional HRD in 4 breast cancer cell lines was assessed with the same 3 assays used for the BRCA1/2 isogenic pairs. We also examined 14 triple-negative breast cancer (TNBC) patients treated with neoadjuvant cisplatin + IR. Nine of the 14 had available tissue, which was stained for Rad51 foci.

Results: Functional HRD is observed in the BRCA1/2 isogenic pairs, as demonstrated by the Rad51 foci and DRGFP assay results. Increased sensitivity to cisplatin + IR is observed in the BRCA1/2 knockout lines in CSA results compared to isogenic controls. A genomic scar score, LST (large scale transition), was generated for each line based on CCLE data, with higher LST scores suggesting increased genomic instability and therefore HRD. Four cell lines were selected for further study. Similar functional HRD and cisplatin + IR sensitivity is observed in 2 breast cancer cell lines with high LST scores, as compared to 2 with low LST scores. Epistasis was observed upon siBRCA1 knockdown in the 2 cell lines with high LST scores. Half of the 14 TNBC patients demonstrated pathological complete response to neoadjuvant cisplatin + IR. Nine of the 14 patients had available tissue, of which 4 demonstrated functional HRD by the Rad51 foci assay.

Conclusions: Multiple in vitro assays suggest that HRD tumors are much more sensitive to the widely used combination of cisplatin + IR as compared with HR proficient tumors. Data from our small cohort of triple-negative breast cancer patients appear concordant with these findings. Reliable determination of "BRCAness" tumors should identify a subpopulation of non-germline BRCA1/2-mutated breast cancer patients who may benefit from targeted therapy with cisplatin + IR.

#833

Alginate hydrogels for three-dimensional culture and real-time monitoring of cancer cell metabolism and radiation response.

Aparna H. Kesarwala, Jenna L. Carter, Graham H. Read, Natsuko Miura, Kazutoshi Yamamoto, James B. Mitchell, Murali C. Krishna. _National Cancer Institute, Bethesda, MD_.

The purpose of this study was to characterize a three-dimensional calcium alginate culture system for real-time monitoring of cancer cell metabolism and radiation response. HCT116 colorectal cancer cells were encapsulated in 2.5% calcium alginate using a custom electrostatic rig adapted from a previously published system. Bead diameter was a function of voltage and alginate concentration: 4 x 107 cells/mL encapsulated at 4 and 8 kV resulted in mean diameters of 880 and 584 µm, respectively. The distribution of bead radii for all voltages showed positive excess kurtosis (4 kV = 1.6, 8 kV = 3.0) and 72.9% of 4 kV and 77.2% of 8 kV bead diameters fell within one standard deviation of the mean. Encapsulated cell viability was assessed by staining with Trypan Blue and clonogenic survival assays (CSAs). The baseline viability of cells immediately after encapsulation was 93.2%, which decreased 2 days after a single 10 Gy dose (90.7%, p<0.05). After two weeks of continuous culturing, 84.1% of non-irradiated and 73.8% of irradiated encapsulated cells remained viable (p<0.003). Pimonidazole staining demonstrated the presence of hypoxic cores proportional to bead size. CSAs of 8 kV beads showed a dose modifying factor (DMF) of 1.06 relative to cells grown in a monolayer, while larger 4 kV beads showed a DMF of 1.58. Mean oxygen consumption rate rose with increasing number of 8 kV beads/well: 1 bead, 35.5; 2 beads, 97.4; 3 beads, 175.5; 4 beads, 209.9; 2.0 x 104 monolayer cells, 92.6 (all pmol/min, ~8.2 x103 cells/bead, p<0.005). Hyperpolarized 13C-NMR spectroscopy of 400 uL of 8 kV beads containing 1.2 x 108 cells/mL showed a detectable conversion of [1-13C]-pyruvate to [1-13C]-lactate; 1 hour after a single 10 Gy dose, the lactate:pyruvate ratio decreased by 25%. Unpaired 2-tailed Student's t-tests were used to determine significance between and one-way ANOVA was used to determine significance among groups with α set at p≤0.05. These data demonstrate the versatility of alginate hydrogels for real-time metabolic and radiation response studies which are non-invasive, higher throughput, and lower cost compared to in vivo systems. Future directions include additional metabolic flux analysis and hyperpolarized 13C-NMR spectroscopy to further investigate the cancer cell metabolic response to drug and/or radiation therapy.

#834

**Formation of DNA double-strand breaks in colon tumors after targeted alpha therapy with** 211 **At-mAb.**

Sophie E. Eriksson,1 Erika Elgström,1 Sture Lindegren,2 Tom Bäck2. 1 _Lund University, Lund, Sweden;_ 2 _Univeristy of Gothenburg, Gothenburg, Sweden_.

Introduction: Targeted alpha therapy has shown promising results in preclinical and clinical studies. Alpha particle irradiation gives a high fraction of DNA double-strand breaks (DSB), as shown in vitro, resulting in a high probability of cell death. We have previously examined the therapeutic effects of 211At on solid colon carcinoma tumors (diameter approximately 1 cm), with tolerable activities (5 MBq/animals) resulting in non-palpable tumors within one week p.i. The aim of the present study was to investigate the formation of DNA DSB during tumor regression after radioimmunotherapy with 211At-mAb in a syngeneic rat colon carcinoma model. Methods: 18 rats bearing solid colon tumors (1 cm in diameter) between peritoneum and the abdominal muscle were injected intravenously with 5 MBq/animal 211At-BR96. Tumors were excised and paraffin-embedded after 10 min, 2 h, 8 h, 18 h, 24 h, and 48 h p.i. (3 tumors per time point). 53BP1 was stained by immunohistochemistry and used as a marker for DNA DSB. Untreated tumors were used as controls (n=9). DNA DSB were counted in central and peripheral tumor areas selected at random. Results: A few DNA DSB were detected in untreated tumors. Already 10 min p.i., the number of DNA DSB had increased slightly in peripheral tumor tissue. The number peaked 8 h p.i., when the number of DNA DSB had increased 50 times in the tumor periphery and 24 times in the tumor center. The number of DNA DSB then declined, but the difference between center and periphery remained, as expected considering the intratumoral distribution of radioimmunoconjugate. This correlates with the 211At half-life of 7.2 h. Conclusion: DNA DSB are formed early after injection of 211At-mAb and follows the intratumoral distribution of mAbs.

#835

Sensitivity of PTEN deficient non-small cell lung cancer to ionising radiation through inhibition of ataxia terangiectasia related 3 kinase (ATR).

Victoria Louise Dunne, Kelly Redmond, Caroline Coffey, Karl Butterworth, Kevin Prise, Gerard Hanna. _Centre for Cancer Research and Cell Biology, Queens University, Belfast, United Kingdom_.

This study sought to determine the therapeutic efficacy of AZD6738, a small molecule inhibitor of ataxia telangiectasia related 3 kinase (ATR), in combination with ionising radiation (IR) for the treatment of PTEN deficient non-small cell lung cancer (NSCLC).

An isogenic PTEN deficient cell model were generated in H460 and A549 cell lines by HuSH PTEN shRNA constructs in pGFP-V-RS vectors from Origene. Radiosensitivity was determined at various concentrations of AZD6738 by clonogenic assay following IR with 225 kV X-rays. Target validation and mechanistic evaluation of the ATR radiation treatments was performed by western blotting. Cell cycle distribution was obtained using propidium iodine flow cytometry analysis which was performed 48 hours following treatment with AZD6738 alone or in combination with IR. In vivo efficacy was determined using cell line derived xenografts in female SCID mice treated with AZD6738 at a concentration of 25 mg/kg per day by oral gavage for 28 days. Animals were exposed to size fraction and hypofractionated radiation dose under CBCT image guidance using the small animal radiotherapy research platform (SARRP). Radiation induced toxicity in normal lung tissue effects was investigated in a model of radiation induced fibrosis in C57BL6/J mice.

In-vitro, combined treatment with AZD6738 and IR selectively targets PTEN-deficient tumour cells causing a significant reduction in clonogenic survival for both H460- and A549-PTEN KO cell models (p= <0.05 at 8Gy). Following 2 Gy IR exposure there were significantly higher mean foci per cell in both H460-KO and A549-KO cells in comparison to WT cells (p= <0.05 at 2, 4 and 8 hours following treatment). Both PTEN-KO cell lines exhibited increased sub G0/G1 and G2/M populations in comparison to PTEN-WT cell lines when treated with AZD6738 and IR. Minimal toxicity was observed in both normal HBE and BEAS-2B cell lines.

Currently in vivo tumour response experiments are underway to investigate IR and ATR kinase inhibitor AZD6738 using H460-WT and H460-PTEN KO cell models as well as determining the effects of AZD6738 in a radiation induced model of lung fibrosis.

Approximately 10% of NSCLC patients have mutations in PTEN. This research therefore elucidates PTEN loss as a therapeutic target for combined IR with pharmacological inhibition of ATR in NSCLC. Targeting ATR inhibition in PTEN-deficient NSCLC may serve to increase radiation sensitivity in an in vivo model thus highlighting its clinical relevance as a potential personalised medicine approach for patients with PTEN deficient NSCLC.

#836

ATR inhibitor BAY 1895344 shows potent anti-tumor efficacy in monotherapy and strong combination potential with the targeted alpha therapy Radium-223 dichloride in preclinical tumor models.

Antje Margret Wengner, Gerhard Siemeister, Ulrich Luecking, Julien Lefranc, Philip Lienau, Gesa Deeg, Eleni Lagkadinou, Li Liu, Sven Golfier, Christoph Schatz, Arne Scholz, Franz von Nussbaum, Michael Brands, Dominik Mumberg, Karl Ziegelbauer. _Bayer Pharma AG, Berlin, Germany_.

The integrity of the genome of eukaryotic cells is secured by complex signaling pathways, known as DNA damage response (DDR). Recognition of DNA damage activates DDR pathways resulting in cell cycle arrest, induction of DNA repair, or cell death. Proteins that directly recognize aberrant DNA structures recruit and activate kinases of the DDR pathway, such as ATR (ataxia telangiectasia and Rad3-related). ATR responds to a broad spectrum of DNA damage, including double-strand breaks (DSB) and lesions derived from interference with DNA replication as well as increased replication stress. Therefore, inhibition of ATR kinase activity could be the basis for a novel anti-cancer therapy in tumors with increased DNA damage, deficiency in DNA damage repair or replication stress. Radium-223 dichloride (Xofigo®) is the first and only approved targeted alpha therapy so far. It is indicated for the treatment of patients with castration-resistant prostate cancer (CRPC), symptomatic bone metastases and no known visceral metastatic disease, based on improvement of overall survival. It exhibits strong cytotoxic effects on adjacent cells via the induction of DNA DSB.

Here, we disclose for the first time the structure and functional characterization of the novel ATR kinase inhibitor BAY 1895344. In vitro, BAY 1895344 is a selective low-nanomolar inhibitor of ATR kinase activity, potently inhibiting proliferation of a broad spectrum of human tumor cell lines (median IC50 of 78 nM). A clear separation between highly sensitive (IC50 <10 nM) and less sensitive cell lines was observed. The majority of the sensitive cell lines are characterized by mutations affecting the ATM (ataxia telangiectasia mutated) pathway. In cellular mechanistic assays BAY 1895344 inhibited hydroxyurea-induced H2AX phosphorylation demonstrating the anticipated mode of action. BAY 1895344 is an ATR inhibitor that exhibits strong in vivo anti-tumor efficacy in monotherapy in a variety of xenograft models of different indications that are characterized by DDR deficiencies, inducing stable disease in ovarian and colorectal cancer or even complete tumor remission in mantle cell lymphoma models. In addition, we could demonstrate that combination treatment with BAY 1895344 and Radium-223 exhibits clear synergistic anti-tumor activity in a bone metastases xenograft model of CRPC.

Our findings validate the concept of synthetic lethality of genetically determined DNA repair deficiency and ATR blockade by demonstrating strong monotherapy efficacy of the highly potent ATR inhibitor BAY 1895344 in a variety of tumor indications. Furthermore, the mechanism-based combination potential of DNA damage induction by Radium-223 with BAY 1895344 creates a powerful new treatment option for CRPC patients with bone metastases.

The start of clinical investigation of BAY 1895344 is planned early 2017.

#837

PSMB8 as a candidate marker of responsiveness to preoperative radiotherapy in rectal cancer patients.

Yejin Ha,1 Ka hee Tak,1 Chan wook Kim,1 Seon Ae Roh,1 Eun Kyung Choi,1 Dong Hyung Cho,2 Jeong-Hwan Kim,3 Seon-Kyu Kim,3 Seon-Young Kim,3 Yong Sung Kim,3 Jin-Cheon Kim1. 1 _Asan Medical Ctr., Seoul, Republic of Korea;_ 2 _Kyung Hee University, Yongin, Republic of Korea;_ 3 _Korea Research Institute of Bioscience & Biotechnology, Daejeon, Republic of Korea_.

Prediction of individual responsiveness is urgently needed, specifically in locally advanced rectal cancer (LARC) patients who underwent preoperative chemoradiotherapy (CRT). The present study, RNA-Seq was used to compare the basal expression profile between responders and non-responders to preoperative CRT, in correlation with the tumor regression grade (TRG) among 22 LARC patients. Eight differentially expressed genes (B3GALT4, HSPA1B, KRBOX1, PPBP, PPP1R18, PSMB8, SLC39A7, and TAP2) associated with preoperative CRT responses were primarily identified among the 22 LARC patients by RNA-Seq (p<0.0005 and >16-fold difference). Among these genes, PSMB8 and SLC39A7 were upregulated in the responsive group in the other 40 LARC patients (p<0.05). PSMB8 overexpression significantly reduced colony formation and increased apoptosis-inducing molecules like cleaved caspase-3 and cleaved PARP in irradiated CRC cells after 6 Gy irradiation. PSMB8 knockdown increased colony formation and decreased caspase-3 activation and cleaved PARP levels after irradiation. In mice treated with ionizing radiation, tumor growth suppression was significantly greater in HCT116/PSMB8-xenografts (81%) than in HCT116/vector-xenografts (53%) (p=0.001). However, SLC39A7 overexpression had no significant effect on irradiated CRC cells. These results suggest that PSMB8 appears to predict radiosensitivity in LARC patients with preoperative CRT, although further clinical validation is needed in a larger cohort.

#838

Effect of tumor treating fields on cell proliferation and synergistic antitumor efficacy in combination with ionizing radiation.

Eun Ho Kim,1 Yunhui Jo,2 Jiwon Sung,2 Hyo Sook Song,2 Myonggeun Yoon2. 1 _Korea Institute of Radiological & Medical Sciences, Seoul, Republic of Korea; _2 _Korea University, Seoul, Republic of Korea_.

Alternating electric fields at an intermediate frequency (100~300 kHz), called tumor treating fields (TTFs), are reported to interrupt the process of mitosis via apoptosis and to act as an inhibitor of cell proliferation. Although the presence of an antimitotic effect of TTFs has been widely reported, the efficacy of TTFs is still controversial issue among medical experts. To resolve this controversial issue, the comparison study between the effects of TTFs and the effect of other conventional cancer treatment techniques need to be carried out. In this study, experimental evidence of the synergistic antimitotic effect of TTFs in combination with ionizing radiation (IR) is reported. For these experiments, aggressive brain cancer cells, glioblastoma multiforme (GBM) cells (U373 and U87), were treated either with TTFs alone, with IR alone or with TTFs followed by ionizing radiation (IR). The percentages of cell apoptosis, DNA damage, and mitotic abnormalities were increased by the application of TTFs alone and its quantity was markedly increased when TTFs were combined with IR. Our results also suggested that radiotherapy together with TTFs may be clinically beneficial since the combination treatment seemed to synergistically suppress both cell migration and invasion, based on the inhibition of MMP-9 and vimentin.

#839

Network-driven analytics of published tissue-based biomarkers to predict response to neoadjuvant therapy in rectal cancer.

Liam R. Poynter,1 Kirill Veselkov,1 Dieter Galea,1 James Kinross,1 Alexander Mirnezami,2 Jeremy Nicholson,1 Zoltan Takats,1 Reza Mirnezami,1 Ara Darzi1. 1 _Imperial College London, London, United Kingdom;_ 2 _University of Southampton, United Kingdom_.

Introduction Neoadjuvant chemoradiotherapy (nCRT) plays a central role in the management of locally advanced rectal cancer. For many, nCRT leads to clinically meaningful tumour regression. However, up to 20% exhibit little to no response and, in this group, nCRT results in unnecessary delays to definitive treatment. There is a critical need for development of robust molecular methods to predict response to nCRT, to allow for more precise treatment stratification. Although numerous molecular pathways and biomarkers have been implicated in radiosensitivity, the lack of a unifying interpretation of these findings has restricted translational deployment. The aim of the current study was to develop a 'knowledge network' with which to visualise and interpret published, quantitative, biomarker data relating to radiosensitivity in rectal cancer, beyond the conventional format of a systematic review.

Methods Existing data on predictive biomarkers were retrieved by way of a systematic review of electronic bibliographic databases. Biomarkers were classified according to biological function and built into a hierarchical Gene Ontology tree. Significance was binarized based on p-values or multivariate statistics. An interactive, direct acyclic graph was developed using the Dagre-D3 JavaScript library. Nodes were sized by number of studied biomarkers and color-coded according to their significance scores. The scores reflect the ratio of significant versus non-significant evidence across studied biomarkers. A negative score range indicates more non-significant biomarker findings for that ontological term (node). Weightings were applied to reflect those biomarkers confirmed as significant across two or more studies. p-values of 0.05 or less (adjusted for multiple comparative analysis where appropriate) were considered to be statistically significant.

Results 72 individual biomarkers were identified through review. On highest order classification, the domains of response to stress and factors inhibiting apoptosis were found to be most significant (aggregate significance scores across identified biomarkers, 0.75 and 0.714 respectively). A predictive power was not reached for the majority of prognostic biomarkers; rather, the levels of their statistical significance were assessed.

Conclusions Building a knowledge-based network analysis of published data identifies promising areas for further research into cellular mechanisms, which may aid in biomarker discovery. Regarding significant node clusters within a network of published data on predictive biomarkers, modifications in cellular metabolic responses to the insult posed by nCRT appear to hold promise in developing a panel of biomarkers with a predictive capacity for response. Network-based analytics takes into account the complex nature of response to therapy, and is a novel way of presenting results obtained from a systematic review.

#840

Polymorphism in DNA repair gene XRCC1 leading to nonconservative amino acid substitution and its role in susceptibility to lung cancer risk.

Gh Rasool Bhat, Rakesh Kumar. _Shri Mata Vaishno Devi University, Katra, India_.

Lung cancer is a heterogeneous disease and major global health concern accounting for more than a million annual deaths worldwide. Histologically, 85% of lung cancers are non-small cell lung cancer (NSCLC) and the other 15% are small cell lung cancer (SCLC). In India, it is the most common cause of cancer-related mortality in both men and women. According to the Indian Council of Medical Research cancer registry, 57,795 new cases were reported in 2010, which is projected to increase up to 67,000 new cases annually by the year 2020. Studies revealed that in Srinagar (Jammu & Kashmir) the incidence of lung cancer among males is highest and recent investigation by Global Adult Tobacco Survey (GATS) declared Jammu & Kashmir as the lung cancer capital of India with 26.6% smokers. Lung cancer remains the biggest health concern and challenge for society· Various factors being responsible for lung cancer, but among them smoking is the main cause of lung cancer. However, not all of cigarette smokers develop this disease. This depicts the significance of genetic makeup in predicting individual's susceptibility towards lung carcinoma. This genetic susceptibility may result from the inherited polymorphisms in genes responsible for DNA damage repair mechanism and carcinogen metabolism. DNA repair mechanism plays a very critical role in protecting the genome from insults of cancer causing agents and maintaining genomic integrity. XRCC1 (X-Ray Repair Cross Complementing group 1) is a major DNA repair gene, acts as a scaffold of different activities involved in repair by interacting with components of base excision repair (BER) at the site of damage. Functional polymorphisms in XRCC1 gene, codon 399 (Arg/Gln), 194 (Arg/Trp) have been known to effect the repair efficiency which might predispose an individual to lung cancer risk. However, such studies are completely lacking in the region of Jammu & Kashmir. With this background, we elected to screen these functional variations in Patients and Healthy Individuals in a case-control based study design in population group from Jammu and Kashmir. In the pilot study, we have observed that both the variations are present in the population group of Jammu & Kashmir and hence can be screened for the case-control association. Further, preliminary analysis showed a difference in the frequency distribution of the variations in Cases and controls. However, a larger sample size is required to reach a conclusion regarding the association. We are working on increasing the sample size of the patient and double the number of controls, from where a real picture of the association can emerge.

#841

UTR analysis identifies Musashi1 as a translational regulator of radiation-induced Golgi-related gene expression.

Stacey L. Lehman, John W. O'Neill, Kevin Camphausen, Philip J. Tofilon. _National Cancer Institute, Bethesda, MD_.

The cellular radioresponse is regulated by post-translational modification of constitutively expressed proteins and by changes in gene expression. Although the role of constitutively expressed proteins, such as those involved in the DNA damage response and cell cycle checkpoints, is well-understood in the context of the radioresponse, radiation-induced changes in gene expression are not as clearly defined. We have previously demonstrated that glioblastoma stem-like cells (GSCs) alter their gene expression at the translational level in response to ionizing radiation (IR). In particular, the NSC11 GSC line demonstrated thousands of genes differentially translated at one hour after a dose of 2 Gy, while considerably fewer were modified at the transcriptome level. Furthermore, there was little overlap between the transcriptome and the translatome. Genes undergoing translational regulation in response to IR fell into distinct functional categories, including mitochondrial structure and function, Golgi function, and translational regulation. Here, we aimed to elucidate the mechanism by which IR induces translational changes in the NSC11 GSC line. NSC11 cells were either exposed to 2 Gy or mock irradiated, and polysomes were isolated by sucrose gradient fractionation one hour later. Polysome-bound mRNA was purified and analyzed by microarray. The 5' and 3' untranslated regions (UTRs) of the top 75 up- and down-regulated genes were analyzed for the presence of regulatory elements. The most frequently occurring element in the UTRs of both the top up- and down-regulated genes was the Musashi1 binding site. Musashi1 is an RNA binding protein that typically binds to 3' UTRs to regulate mRNA translation. It is a neural stem cell marker, and its expression is correlated with the grade of malignancy in glioma. GO term analysis of known Musashi1 target genes that were translationally regulated in response to IR revealed an enrichment of genes involved in Golgi function and vesicular trafficking. Knockdown of Musashi1 prevented IR-induced Golgi dispersal in NSC11 cells. Musashi1 knockdown also radiosensitized NSC11 cells, indicating a role for the protein in the cellular radioresponse. Together, these data demonstrate that in the NSC11 GSC line, Musashi1 contributes to radiation-induced translational regulation, specifically in the context of Golgi-related genes, and suggests that Musashi1 is a novel target for radiosensitization. 

### Epithelial to Mesenchymal Transitions in Metastasis

#842

Breast cancer: Relationship between GPC3 and EMT process.

Lilian F. Castillo, Gisela V. Novack, Elisa Bal de Kier Joffé, María G. Peters. _Instituto de Oncologia Angel H. Roffo, CABA, Argentina_.

Epithelial-mesenchymal transition (EMT) is a process whereby epithelial cells acquire mesenchymal fibroblast-like properties and display reduced intracellular adhesion and increased motility.

Glypican-3 (GPC3) is a heparan sulfate proteoglycan associated with cancer. We have established that GPC3 regulates the EMT process underwent by breast cancer cells. However, cellular and molecular events underlying this modulation are not completely described.

At the present work we analyzed the effect of GPC3 on cell adhesion and on the expression of molecules associated with this property. In addition, molecular regulators of EMT were also studied. We employed a murine breast cancer cell line (LM3: GPC3-) as well as human lines (MDA-MB231: GPC3- and MCF-7: GPC3+). We have blocked GPC3 expression in MCF-7 cells, while it was overexpressed in MDA-MB231 and LM3 ones.

It is know that EMT depends on a reduction in cell adhesion. We determined that GPC3 overexpression induced an increase in the homotypic adhesion of MDA-MB231 and LM3 cells, although GPC3 silencing did not lead significant changes in the organization of MCF-7 spheroids. GPC3 was also able to modulate the adhesion to extracellular matrix components. GPC3 overexpressing cells were more adherent to fibronectin than controls, but no changes were observed in their adhesion to laminin.

E-Cadherin, a molecule involved in homotypic adhesion, is considered a suppressor of invasion and growth of many epithelial cancers. Here, we have corroborated by WB that GPC3 overexpression induces the E-Cadherin upregulation in LM3 and MDA-MB231 cells, whereas GPC3 silencing inhibits the expression of this epithelial marker in MCF-7 cells. We also confirmed the called "cadherin switch", since N-Cadherin was downregulated in GPC3 overexpressing cells and upregulated in GPC3 silenced ones.

β-Catenin is a key downstream effector in the Wnt pathway. We confirmed that GPC3 expressing breast cancer cells exhibit a canonical Wnt signaling inhibition. β-Catenin also activates Slug. Similar to Snail, Slug binds to the E-Cadherin

promoter to repress transcription. By WB and qPCR we demonstrated that GPC3 induces a decrease in Slug and Snail levels. Surprisingly, Twist expression was higher in GPC3 expressing cells.

It was reported that Slug is able to represses integrins expression. However, β1-Integrin levels were decreased in GPC3 overexpressing MDA-MB231 cells, while they were increased in GPC3 blocked MCF-7 sublines.

Our results suggest the key role of GPC3 in the EMT regulation. GPC3 would modulate cell adhesion and expression of E-Cadherin, N-Cadherin and β1-Integrin. In addition, GPC3 would regulate the expression of different EMT molecules (as Slug, Snail and Twist) as well as signaling pathways involved in that process (like canonical Wnt). The set and balance of these regulations would result in the reversal of EMT, reinforcing the function of GPC3 as an inhibitor of mammary tumor progression.

#843

CLDN1 mediates tumor invasion in HNSCC by regulation of EMT through AMPK-TGF-β signalling.

Jae Won Chang,1 Seung-Nam Jung,1 Lihua Liua,1 Jin Man Kim,1 Myung Jin Ban,2 Yeon Soo Kim,3 Bok-Soon Lee,4 Yang Eun Kim,4 Ho Ryun Won,4 Chul-Ho Kim,4 Bon Seok Koo1. 1 _Chungnam National Univ. College of Medicine, Daejeon, Republic of Korea;_ 2 _Soonchunhyang University College of Medicine, Cheonan, Republic of Korea;_ 3 _Konyang University College of Medicine, Daejeon, Republic of Korea;_ 4 _Ajou University School of Medicine, Suwon, Republic of Korea_.

Background: Claudin-1 (CLDN1), a major component of tight junction complexes in epithelium, maintains cellular polarity and plays critical roles in cell-to-cell communication and epithelial cell homeostasis. Although, the role of CLDN1 in cancer has been generally studied in cancers, the exact regulatory mechanisms and downstream pathways in cancer have been controversial and are not completely understood.

Materials and Methods: After, analysed mutational profiling of tumor/non-tumor paired tissue in the patients with head and neck squamous cell carcinoma (HNSCC) by next-generation sequencing (NGS), CLDN1 expression levels were measured in human HNSCC cell lines and tissues from HNSCC patients using next-generation sequencing. For functional in vitro analysis, cell proliferation testing using a WST-1 assay, an invasion assay, and Western blotting was performed after CLDN1 expression regulation. In vivo validations were performed using nude mouse and patient-derived tumor xenograft (PDTX) model.

Results: Mutational profiling of tumor/non-tumor paired HNSCC tissue by NGS revealed that HNSCC tumor tissues and cell lines had relatively high levels of CLDN1 expression. Although, CLDN1 regulation did not affect proliferation of HNSCC cell lines, knock down of CLDN1 inhibited invasive characteristics (migration/invasion) with down-regulation of EMT associated proteins (vimentin and slug). We also found that CLDN1 negatively correlated with AMPK phosphorylation and AMPK activator (AICAR) showed the same effect with CLDN1 suppression on HNSCC cells without change in CLDN1 expression which lead to inhibition of TGF-β-induced Smad phosphorylation and their transcriptional activity, suggesting that CLDN1/AMPK/TGF-β cascade. Then, we confirmed antitumorigenic effects of suppression of CLDN1 in HNSCC in a nude mouse and PDTX model.

Conclusions: Our findings suggest that CLDN1 acts as oncogenic driver in HNSCC via regulating EMT through AMPK/TGF-β signalling and if validated in further studies, a CLDN1-targeting agent partnered with an existing anti-TGF-β modality could be a novel tactic to overcome HNSCC.

#844

PPARD downregulation in cancer cells suppresses metastases by inhibiting tumor angiogenesis and EMT.

Xiangsheng Zuo,1 Weiguo Xu,1 Min Xu,1 Rui Tian,1 Micheline J. Moussalli J. Moussalli,1 Fei Mao,1 Mihai Gagea,1 Daoyan Wei,2 Anil K. Sood,1 Imad Shureiqi2. 1 _UT MD Anderson Cancer Ctr., houston, TX;_ 2 _UT MD Anderson Cancer Ctr., Houston, TX_.

PPARD is upregulated in many major human cancers, but the role that its expression in cancer cells has in metastasis remains poorly understood. Here, we show that specific PPARD downregulation or genetic deletion of PPARD in cancer cells significantly repressed metastasis in various cancer models in vivo. Mechanistically, PPARD promoted angiogenesis via interleukin 8 in vivo and in vitro. Analysis of transcriptome profiling of HCT116 colon cancer cells with or without genetic deletion of PPARD and gene expression patterns in The Cancer Genome Atlas colorectal adenocarcinoma database identified novel pro-metastatic genes (GJA1, VIM, SPARC, STC1, SNCG) as PPARD targets. PPARD expression in cancer cells drastically affected epithelial-mesenchymal transition, migration, and invasion, further underscoring its necessity to metastasis. Clinically, high PPARD expression in various major human cancers (e.g., colorectal, lung, breast) was associated with significantly reduced metastasis-free survival. Our results demonstrate that PPARD, a druggable protein, is an important molecular target in metastatic cancer.

#845

A clinically validated multiplex immunofluorescence assay for the quantitative assessment of changes in EMT phenotypes in FFPE tumor tissues in response to cancer therapeutics.

Tony Navas,1 Melinda G. Hollingshead,2 Suzanne Borgel,3 John P. Carter,3 Angelena Millione,3 Brad A. Gouker,4 Donna Butcher,4 Susan Holbeck,5 Apurva K. Srivastava,1 Robert J. Kinders,1 Donald P. Bottaro,6 Shivaani Kumar,5 Alice Chen,5 James H. Doroshow,5 Ralph E. Parchment1. 1 _Clinical Pharmacodynamics Program, Applied/Developmental Directorate, Leidos Biomedical Research, Frederick National Laboratories, Frederick, MD;_ 2 _Biological Testing Branch, Developmental Therapeutics Program Directorate, NCI-Frederick, Frederick, MD;_ 3 _Biological Testing Branch, Developmental Therapeutics Program Directorate, Leidos Biomedical Research, Frederick National Laboratories, Frederick, MD;_ 4 _Pathology/Histotechnology Laboratory, Animal Sciences Program, Leidos Biomedical Research, Frederick National Laboratories, Frederick, MD;_ 5 _Division of Cancer Treatment and Diagnosis and Center for Cancer Research, NCI, Bethesda, MD;_ 6 _Urologic Oncology Branch, Center for Cancer Research, NCI, Bethesda, MD_.

Epithelial-mesenchymal transition (EMT) is a critical process implicated in metastasis. Despite extensive research, the clinical significance of EMT remains unclear, and a quantitative evaluation of this process in human tumors has yet to be demonstrated. We previously reported a validated EMT immunofluorescence assay (EMT-IFA), that utilizes β-catenin as a tumor segmentation marker to delineate tumor tissue from surrounding stroma in FFPE tumor biopsies. The assay accurately quantifies individual expression and co-localization of E-cadherin (E) and vimentin (V) in tumor cells (Navas et al. NCI-EORTC 2015). We used this assay to measure changes in both the total number and the corresponding EMT phenotypes of tumor cells in xenograft tissues following treatment with various anticancer agents currently in clinical trials at the NCI. Daily treatment for 14 days with the multikinase inhibitor pazopanib caused significant tumor regression and delayed regrowth in the epithelial (E+) gastric cancer xenograft model MKN45, and the tumor cells remaining after treatment were significantly shifted toward a mesenchymal (V+) phenotype. In contrast, pazopanib (QDx15) had less anti-tumor efficacy in the mesenchymal gastric cancer xenograft model SNU5. In another instance, daily treatment of the MDA-MB-468 breast cancer xenograft model with the BCR-Abl kinase inhibitor nilotinib for 19 days did not demonstrate any significant anti-tumor efficacy or change in the predominance of the E+ phenotype, whereas a cycle of treatment with the anti-tubulin agent paclitaxel (Q3Dx4) led to MDA-MB-468 tumor regression and delayed tumor regrowth after completion of the treatment cycle. Furthermore, the combination of nilotinib with paclitaxel not only significantly diminished the total MDA-MB-468 tumor cell number compared to single-agent arms, but also effectively transformed the EMT phenotype of the tumor, with only a subset of cancer cells surviving by the last day of treatment, which were mostly CD44+CD133+ mesenchymal cells and potential cancer stem cells. The changes in EMT phenotype brought on by effective drug treatments occurred via preferential killing of cells with E+ phenotype, suggesting that V+ and V+E+ tumor cells may be more resistant to therapy. The EMT-IFA provides a much-needed analysis tool suitable for clinical investigation of the proposed role of EMT in tumor progression, metastasis, and acquired drug resistance, and is able to detect changes in EMT signature which may serve as early indicators of treatment efficacy and tumor resistance. Funded by NCI Contract No. HHSN261200800001E.

#846

A novel high-throughput 3D screening system for EMT inhibitors.

Kazuya Arai,1 M Mamunur Rahman,2 Manabu Itoh,1 Norio Masuda,1 Takanori Eguchi,3 Tetsuya Nakatsura,4 Stuart K. Calderwood5. 1 _ORGANOGENIX, Inc., Kanagawa, Japan;_ 2 _MBL international, Woburn, MA;_ 3 _Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan;_ 4 _National Cancer Center, Chiba, Japan;_ 5 _Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA_.

EMT (epithelial-mesenchymal transition) is a significant event in tumor metastasis and malignancy. Therefore, inhibition of EMT is considered to enable controlling of malignant transformation. In this study, we introduce an innovative three-dimensional (3D) high-throughput (HTS) system that leads to an identification of EMT inhibitors. At first, we established 3D-HTS EMT model with NanoCulture Plate (NCP) that provided a gel-free micro-patterned scaffold for cells forming spheroids naturally. In the NCP-based 3D cell culture system, A549 lung cancer cells migrated, gathered, and then formed multiple spheroids within 7 days. Live cell imaging experiments showed that an established EMT-inducer TGF-β promoted peripheral cells around the core of spheroids to acquire mesenchymal spindle shapes, loss of intercellular adhesion, and migration from the spheroids. Along with such morphological change, EMT-related gene expression signatures were altered, particularly alteration of mRNA levels of ECAD/CDH1, NCAD/CDH2, VIM and ZEB1/TCF8. These EMT-related phenotypic changes were blocked by SB431542, a TGF-βreceptor I (TGFβR1) inhibitor. Inside of the spheroids were highly hypoxic; in contrast, spheroid derived peripheral migrating cells were normoxic, revealed by visualization and quantification using Hypoxia Probe. Thus, TGF-β-triggered EMT caused spheroid hypoplasia and loss of hypoxia. Spheroid EMT inhibitory (SEMTIN) activity of SB431542 was calculated from fluorescence intensities of the Hypoxia Probe, and then was utilized in a drug screening of EMT-inhibitory small molecule compounds. In a pilot screening, 9 of 1,330 compounds were above the thresholds of the SEMTIN activity and cell viability. Finally, two compounds SB-525334 and SU9516 showed SEMTIN activities in a dose dependent manner. SB-525334 was a known TGFβR1 inhibitor. SU9516 was a cyclin-dependent kinase 2 (CDK2) inhibitor, which we showed also had an EMT-inhibitory activity. Taken together, it was shown that this 3D NCP-based HTS system was useful for screening of EMT-regulatory drugs.

#847

Anti-cancer effects of selective Cox-2 and EP2 inhibition through suppression of EMT and the clinical implications of overexpression of Cox-2 and downregulation of E-cadherin in pharyngeal squamous cell carcinoma.

Yoshihiro Watanabe,1 Yorihisa Imanishi,2 Hiroyuki Ozawa,3 Kaori Kameyama,3 Koji Sakamoto,3 Ryoichi Fujii,3 Seiji Shigetomi,3 Noboru Habu,3 Kuninori Otsuka,3 Yoichiro Sato,3 Mariko Sekimizu,3 Fumihiro Ito,3 Yuichi Ikari,3 Shin Saito,3 Kaoru Ogawa3. 1 _Keio University, School of Medicine/Tokyo Saiseikai Central Hospital, Tokyo, Japan;_ 2 _Keio University, School of Medicine/Kawasaki Municipal Kawasaki Hospital, Tokyo/Kawasaki, Japan;_ 3 _Keio University, School of Medicine, Tokyo, Japan_.

Background: Overexpression of cyclooxygenase 2 (Cox-2), an inducible prostaglandin (PG) synthetase, is assumed to promote cancer progression through its multifaceted function, including induction of angiogenesis, stimulation of cell proliferation, restraint on apoptosis, and immunosuppression. Furthermore, recently its inverse relationship with E-cadherin expression was reported. The epithelial-to-mesenchymal transition (EMT) accompanied by the downregulation of E-cadherin is supposed to promote metastasis. However, neither the anti-cancer effect of selective Cox-2 inhibitors on the regulation of E-cadherin nor its specific mechanism has been examined in pharyngeal squamous cell carcinoma (PhSCC). In addition, the anti-tumor effect of EP2 antagonist, a possibly more selective inhibitor of PGE2, remains to be elucidated.

Methods: We used quantitative real-time PCR to examine the effects of a selective Cox-2 inhibitors, Celecoxib, and a selective EP2 antagonist, PF-04418948, on the gene expressions of CDH-1, its transcriptional repressors (snail, ZEB1, twist), and vimentin in the human PhSCC cell lines BICR6 and FaDu. Alteration in E-cadherin expression on the cell surface was evaluated by immunofluorescent staining. In vitro proliferation and migration assays were performed to assess cellular behaviors. Clinicopathological factors and immunohistochemical expressions of Cox-2 and E-cadherin were evaluated using surgical specimens from 54 patients with PhSCC who underwent transoral resection.

Results: Both Celecoxib and PF-04418948 upregulated the CDH-1 expression and the membranous E-cadherin expression and downregulate the vimentin expression in the PhSCC cells through the suppression of snail, ZEB1, and twist expression. The extent of this effect depended on the baseline expression levels of both E-cadherin and Cox-2 in each cell line. Additionally, the cell migration was attenuated by both Celecoxib and PF-04418948, whereas the cell proliferation was not affected. Univariate analysis showed that differentiation, overexpression of Cox-2, and decreased expression of E-cadherin were significantly correlated with lymph node metastasis. Multivariate logistic regression revealed that overexpression of Cox-2 (odds ratio [OR]=53.49, P<0.001) and decreased expression of E-cadherin (OR=0.06, P=0.016) were the independent risk factors affecting lymph node metastasis.

Conclusions: These findings suggest that the appropriately selective administration of the Cox-2 inhibitor and EP2 antagonist may have an anti-metastatic effect through suppression of EMT by restoring E-cadherin expression. In addition, overexpression of Cox-2 and downregulation of E-cadherin may be closely implicated in lymph node metastasis in PhSCC.

#848

Netropsin blocks EMT of murine BRAF mutated melanoma cells.

Juliano Freitas, Fenfei Leng, Lidia Kos. _Florida International University, Miami, FL_.

Melanoma is the most aggressive and deadliest type of skin cancer due to its high propensity to metastasize. Despite current progress in the identification of promising drugs for treating melanoma, drug resistance has emerged as a serious issue indicating that novel therapeutic strategies may be required. It is well documented that the process of converting the epithelial tumorigenic cell to a mesenchymal phenotype, the epithelial-mesenchymal transition (EMT), is responsible for conferring an invasive phenotype to cancer cells. An EMT-like process has been shown to occur during melanoma progression. High-mobility-protein AT-hook 2 (HMGA2) is a multi-function transcription factor linked to oncogenesis and EMT in a variety of cancers. HMGA2 is a small DNA-binding protein with three "AT-hook" DNA-binding motifs that specifically recognize the minor groove of AT-rich DNA sequences. HMGA2 is significantly upregulated in human primary melanoma and metastases, including those with BRAF mutations. We accessed the potential application of netropsin, a potent inhibitor of HMGA2-DNA interactions, as an EMT blocking agent of BRAF mutated melanoma cells. Three murine melanoma cells lines that carry mutations in the BRAF gene and show resistance to vemurafenib (D4M, YUMM1.1, and YUMM1.7) were used. Cells were treated with EMT Inducing Media Supplement along with Netropsin and the expression of E-cadherin as well as their migratory behavior were evaluated. Netropsin treatment of EMT induced cells was not cytotoxic, prevented changes in cell morphology, maintained high levels of E-cadherin expression and decreased migratory behavior. All three cell lines express HMGA2 which continues to be expressed in the tumors resulting from subcutaneous injections in mice. YUMM1.1 cells showed the highest levels of expression of HMGA2 amongst the three cell lines. Interestingly, in our preliminary study only YUMM1.1-derived tumors generated overt metastasis in the lung 30 days after the injections. Altogether our data showed that Netropsin is capable of blocking EMT of murine BRAF mutated melanoma cells. The results of this study will contribute to the development of strategies to prevent EMT that may result in the implementation of more effective therapies to treat melanoma.

#849

Molecular characterization of primary tumor & the paired liver metastatic biopsies of colorectal cancer reveals a critical role of immunosuppression, EMT & angiogenesis in cancer metastasis.

Jiangang Liu,1 Yong Beom Cho,2 Hye kyung Hong,3 Song Wu,1 Philip J. Ebert,1 Steven M. Bray,1 Swee Seong Wong,1 Jason C. Ting,1 John N. Calley,1 Catherine F. Whittington,1 Shripad Bhagwat,1 Emma Bowden,1 Amit Aggarwal,1 Christoph Reinhard,1 Robert Wild,1 Do-Hyun Nam,2 Woo Yong Lee,2 Sheng-Bin Peng1. 1 _Eli Lilly and Company, Indianapolis, IN;_ 2 _Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea;_ 3 _Samsung Biomedical Research Institute, Seoul, Republic of Korea_.

Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality worldwide. In United States alone, it was estimated that nearly 137,000 people were diagnosed, and more than 50,000 were dead from the disease each year. CRC primary tumors often metastasize to liver which accounts for most of CRC death. The molecular mechanism of tumor metastasis is complicate and remains poorly understood. In this study, we have collected 79 human CRC samples, and 60 of them are paired primary tumor and liver metastatic surgically resected samples. Molecular characterization of these samples was conducted by exome and RNA sequencing, as well as SNP6.0 analysis. Genetic analysis revealed no significant difference in genetic alterations including common oncogenic mutations, whole genome mutations and copy number variations between primary and their matched liver metastatic tumors. To further characterize the molecular mechanism of metastatic progression, we have assembled gene correlation networks by utilizing a genome-wide interrogation of co-regulated networks based on RNA sequencing data of these CRC samples. Computational analysis of these correlation networks has identified gene signatures of immune-suppression, epithelial-mesenchymal transition (EMT) and angiogenesis as the key events and potentially synergistic drivers associated with CRC metastasis. Further independent cohort validation using published datasets verified that these specific gene networks of tumor microenvironment were progressively up-regulated throughout the carcinogenesis, and represented distinct biological processes. These gene networks were capable of discriminating the previously categorized CRC molecular subclasses. In addition, we also showed an association of type I interferon network with good clinical outcome of CRC, and gene networks of EMT, angiogenesis, immune-suppression and T cell exhaustion are closely associated with the poor patient outcome. We further demonstrated that the networks of EMT and angiogenesis were related to innate anti-PD-1 resistance, and the networks of immunosuppression and T cell exhaustion were associated with resistance to radiation and checkpoint blockade. Overall, we conclude that a genome-wide interrogation of co-regulated networks utilized in this study represents a valuable strategy to identify molecular mechanisms of cancer metastasis, and gene networks of immune-suppression, EMT and angiogenesis are among the key events associated with CRC metastasis.

#850

The Pi3K/Akt pathway mediates epithelial-mesenchymal transition (EMT) and malignant progression in BRCA-defective epithelial ovarian cancer.

Mehida Alexandre, Z Ping Lin, Elena S. Ratner. _Yale University School of Medicine, New Haven, CT_.

Objectives: Despite improvement in therapy, the majority of patients with ovarian cancer relapses and eventually succumbs to the disease. Therefore, there is an urgent need for further understanding the development and progression of ovarian cancer. It is well established that the phosphatidylinositol 3-kinase (Pi3k/Akt) pathway is activated in 70% of ovarian cancers. We have previously shown that in AKT activity is up regulated in BRCA1 and BRCA-2 deficient cancer. Epithelial-mesenchymal transition (EMT), a process by which cells acquire motility and lose their intercellular adhesions, plays an important role in normal development. Cancer cells have been shown to exhibit similar EMT-like process during invasion and metastasis. Recently, the activation of the Pi3k/Akt pathway is emerging as a central feature of EMT. This study aims to investigate whether activation of the Pi3k/Akt pathway is necessary for promoting EMT and malignant progression in BRCA-defective ovarian cancer.

Methods: BRCA2-mutated PEO1 and BRCA2-wild type PEO4 epithelial ovarian cancer cell lines were derived from the same patient at first and second relapse, respectively, following platinum-based chemotherapy. Non-targeted siRNA control (NTC) and BRCA1-knockdown (BRCA1-kd) SKOV3 cell lines were previously established in our laboratory. Western blot analysis and luciferase reporter assay were performed to determine EMT markers (snail, slug, fibronectin) in BRCA defective ovarian cancer cells. Scratch wound assays were conducted to determine the ability of cells to migrate. Clonogenic and apoptosis assays were carried out to determine cell survival in response to various concentrations of carboplatin and olaparib with or without MK-2206, a small molecule inhibitor of AKT.

Results: PEO1 cells exhibited an increase in mesenchymal markers Snail and fibronectin, whereas PEO4 cells exhibited an increase in the epithelial marker E-cadherin. PEO1 cells also exhibited an increase in cell migration compared with PEO4 cells. In the luciferase reporter assay, BRCA2- mutated PEO1 and BRCA-1 Kd SKOV3 cells exhibited lower levels of the epithelial maker, E-cadherin compared to their BRCA-wild type counterparts, PEO4 and SKOV3 NTC, respectively. Treatment with MK-2206 caused increased susceptibility of BRCA1-kd SKOV3 cells to olaparib compared with BRCA1-wild type SKOV3-NTC cells.

Conclusions: BRCA2-mutated PEO1cells manifested an increase in mesenchymal markers and invasive phenotypes. BRCA1-Kd SKOV3 cells manifested a decrease in the epithelial trait, E-cadherin activity. In addition, AKT inhibition heightened susceptibility of BRCA-1Kd SKOV3 cells to DNA damaging agents such as olaparib. These findings suggest that BRCA-defective cells rely on the induction of EMT via activation of AKT to survive DNA damage and develop therapeutic resistance during malignant progression.

#851

Reversion of epithelial to mesenchymal transition under androgen deprived conditions promotes chemoresistance in prostate cancer.

Akanksha Upadhyaya,1 Nataly Stylianou,1 Anthony M. Davies,2 Sarah-Louise Ryan,2 Brett G. Hollier,1 Elizabeth D. Williams1. 1 _Australian Prostate Cancer Research Centre – Queensland, Institute of Health and Biomedical Innovation, School of Biomedical Sciences, Queensland University of Technology and Translational Research Institute, Australia, Brisbane, Australia;_ 2 _Australian Prostate Cancer Research Centre – Queensland, IHBI, School of Biomedical Sciences, Queensland University of Technology and Translational Research Institute, Australia, Translational Cell Imaging – Queensland, Brisbane, Australia_.

Introduction:

Metastatic prostate cancer (PC) is one of the leading causes of cancer related male deaths in Australia. Radical prostatectomy followed by androgen deprivation therapy (ADT) upon recurrence of disease is the conventional form of treatment. Upon relapse, administration of androgen targeted therapy (ATT) and subsequent chemotherapy improves quality of life and overall survival of patients. However, it is not curative and the disease ultimately progresses. Epithelial to mesenchymal transition (EMT) is a process that can facilitate tumour growth and progression to metastasis and has also been associated with chemoresistance. We therefore aim to determine how EMT and resultant reversal of this state (MET) influences the chemoresistance profile of PC cells and further determine if and how specific genetic regulators are involved in this process.

Methods:

Doxycycline hyclate (Dox) inducible Snai1 and Zeb1, LNCaP EMT models were used to determine the effect of the EMT status in response to a clinically relevant panel of drugs using cellular and molecular assays. All assays were performed in both fetal bovine serum (FBS) and charcoal stripped serum (CSS) supplemented culture media. CSS media was used to mimic an ADT-like environment. Using 2D monolayer and 3D spheroid [Happy Cell©] formats. Cells were induced for 5 days of EMT in the presence of Dox and were allowed to revert for 14 days of MET (absence of Dox) before the drug assays were carried out over a period of 72 hours. Live/dead cell staining was used to determine viability percentage in both models. Flow cytometry was used for 2D assays and 3D assays were analysed using either a GE IN Cell Analyzer live cell imaging system or confocal microscopy.

Results:

Induction of Snai1 and Zeb1 induced an elongated cell shape and increased mRNA and protein expression of mesenchymal markers such as vimentin during EMT. This was associated with a decrease in expression of epithelial markers E-cadherin and EpCam,. An eventual drop in the expression levels of vimentin at around 14 days of dox removal along with an increase in epithelial markers was observed. These changes were also associated with a gradual reversal in the phenotype of these cells. However, this reversal in phenotype and expression of EMT markers was not as striking in cells grown in CSS compared to cells in FBS media (both 2D and 3D). Mitoxantrone concentration-response assays in a 2D format showed that cells that undergo a subsequent MET are more chemoresistant than uninduced or EMT cells when grown in CSS media. This was however, not observed in cells undergoing similar transitions in the FBS media.

Conclusions:

The chemoresistance profile of prostate cancer cells is altered by their EMT status. This effect is enhanced in the context of ADT. Cells that revert back to a more epithelial phenotype after undergoing an EMT exhibit higher resistance towards chemotherapy drugs.

#852

Bromodomain and extraterminal proteins regulate the epithelial-mesenchymal transition in breast cancer.

Guillaume Andrieu, Gerald V. Denis. _Boston Univ. School of Medicine, Boston, MA_.

The epithelial-mesenchymal transition (EMT) is a developmental program that cancer cells often activate to acquire a highly plastic phenotype that promotes invasion, metastasis, but also chemoresistance and cancer stem cell generation. As readers of epigenetic marks, bromodomain and extra-terminal (BET) proteins BRD2, BRD3 and BRD4 participate in the regulation of multiple transcriptional programs implicated in cancer progression. We sought to unravel the roles of BET proteins in EMT in breast cancer.

Despite their homology, we report that BET proteins differentially regulate EMT. Based on an EMT PCR array, we identify a BRD2-specific transcriptional profile that promotes EMT, whereas BRD3 and BRD4 signatures repress this program. These individual signatures are unidentifiable upon pan-BET inhibition using JQ1, reinforcing the necessity to target each BET member separately to better understand their functions. Upon BRD2 depletion, basal-like breast cancer cells, which present a mesenchymal phenotype, exhibit a reduced expression of mesenchymal markers (N-cadherin, vimentin) and re-express epithelial markers (E-cadherin, cytokeratins). Moreover, a large panel of EMT master transcription factors is downregulated in BRD2-depleted cells, including the Snail and ZEB families or Twist. Interestingly, we found that BRD3 or BRD4 depletion leads to the opposite phenotype: an increase of mesenchymal marker expression and repression of the epithelial markers. In luminal A breast cancer cells which present an epithelial phenotype, BRD2 overexpression leads to the expression of mesenchymal markers. Similar results were obtained by depleting BRD3 or BRD4 in these cells, confirming the differential roles of BET proteins in EMT regulation.

Taken together, our results establish that BRD2 positively regulates EMT, whereas BRD3 and BRD4 repress this program. BET proteins possess separate and opposite biological functions, reinforcing the relevance of an individual targeting instead of a pan-BET inhibition using JQ1. We hypothesize that BET proteins modulate EMT through the regulation of its master transcription factors. We propose that the balance of BET proteins presence at the promoters of the EMT genes is a novel mechanism of regulation of this program in breast cancer cells.

#853

C1orf116, a gene with unknown function, is a novel driver of epithelial phenotype in epithelial-to-mesenchymal transition in human cancer.

Sarah R. Amend, James Hernandez, Princy Parsana, Kenneth J. Pienta. _Johns Hopkins Univ., Baltimore, MD_.

Metastatic prostate cancer is an incurable lethal disease and is the cause of death for 28,000 men in the US annually. In order for a cancer cell to metastasize from the primary tumor, it must move. In a critical early event of metastasis, a rare population of cells in the primary tumor undergoes a phenotype switch from a proliferating epithelial cell to gaining the migratory capacity of a mesenchymal cell, a process, termed epithelial-to-mesenchymal transition (EMT). To identify previously overlooked regulators of EMT, we undertook a multi-study gene expression array analysis (15 published gene expression studies; 95 total samples; 6 cancer types) and identified C1orf116 as a candidate driver of the epithelial phenotype in EMT across multiple cancer types.

Of note, C1orf116 is an unnamed gene of unknown function that is largely uncharacterized in any setting, including in cancer biology or in EMT.

To substantiate its potential role in EMT, we queried the NCI-60 cell line collection gene expression dataset and found that C1orf116 shared a similar expression pattern with epithelial marker genes (ESRP1, ESRP2, OVOL1, OVOL2) and a converse expression pattern to mesenchymal marker genes (CDH2, SNAI2, VIM). Moreover, we found that C1orf116 expression is decreased in metastatic prostate cancer tumors compared to localized disease (Oncomine), suggesting the potential for clinical translation.

To specifically interrogate the role of C1orf116 in EMT, we utilized the PC3 cell line, an in vitro model of prostate cancer EMT. We found that C1orf 116 expression was higher in an epithelial PC3 cell line (PC3-epi) compared to PC3 cells that had undergone a stable EMT (PC3-emt). Moreover, shRNA-mediated knockdown of C1orf116 expression in PC3-epi cells resulted in decreased gene expression of canonical epithelial markers (OVOL1, ESRP1, CDH1) and elevated expression of standard mesenchymal markers (CDH2, ZEB1, VIM), suggesting a role as a driver of the epithelial phenotype.

Interestingly, the sole publication specifically studying C1orf116 describes it as Specifically Androgen Regulated Gene (SARG). The authors demonstrate the presence of an androgen response element upstream of the C1orf116 start site the in androgen-responsive prostate cancer cell line LNCaP. Our data indicate a role for C1orf116 in the androgen receptor-negative prostate cancer cell line PC3, suggesting a non-androgen-mediated role for C1orf116 in prostate cancer.

In addition to elucidating its role in EMT, work is underway to characterize the RNA and protein products of C1orf116. Alternative splicing results in two transcripts of varying length that likely give rise to at least two protein product sizes. RNA sequencing and mass spectrometry will reveal transcript and protein identity and provide insight into potential isoform switching regulating EMT and other processes.

#854

Human tissue Kallikrein 6 enzyme activity regulates epithelial-mesenchymal transition in colon cancer.

Hwudaurw Chen,1 Earlphia Sells,2 Haiyan Cui,1 Ritu Pandey,2 George Pampalakis,3 Georgia Sotiropoulou,3 Thomas Doetschman,2 Natalia A. Ignatenko2. 1 _Univ. of Arizona Cancer Ctr., Tucson, AZ;_ 2 _Univ. of Arizona, Tucson, AZ;_ 3 _Univ. of Patras, Patras, Greece_.

Background: Kallikrein-related peptidase 6 (KLK6) belongs to the family of human tissue kallikrein genes, majority of which are shown to be differentially expressed in cancers. Clinical studies have demonstrated that upregulation of KLK6 in primary colorectal tumors and lymph nodes correlates with serosal invasion, liver metastasis and indicative of poor prognosis in patients. It has been reported that KLK6 protein is involved in regulation of the epithelial-mesenchymal transition (EMT) program in an organ-specific context. The aim in this study was to investigate contribution of KLK6 enzyme in the EMT during neoplastic transformation in the colon.

Results: We expressed enzymatically active or inactive KLK6, using pcDNA3.1(+)preproKLK6 and pcDNA3.1(+)preproKLK6 Ser197Ala mutant plasmids, in Caco-2 colon cancer cell line, which has been characterized before as a very low KLK6 expresser with an undetectable secreted KLK6. Stable isogenic clones were selected and further evaluated for their ability to migrate and invade using in vitro assays and to metastasize in vivo using SCID orthotopic mouse model. We found no effect of KLK6 enzyme activity on migration of Caco-2 cells, expressing the empty vector (Caco-2 mock), and Caco-2 cells, expressing an enzymatically active KLK6 (Caco-2 KLK6 wt) or inactive KLK6 (Caco-2 KLK6 mut). But Caco-2 KLK6 wt cells demonstrated the invasive phenotype in Matrigel invasion assays (p<0.001, compared to Caco-2 mock and Caco2 KLK6 mut cells). The Caco-2 mock and Caco-2 KLK6 mut cells, injected in SCID mice orthotopically, developed primary colon tumors but no metastatic lesions were identified. In contrast, Caco-2 KLK6 wt cells formed primary colon tumors and metastasized locally, although they failed to form the distant metastasis (lung and mesentery). Animals, growing the Caco-2 KLK6 wt tumors, displayed a significant decrease in their survival rates, compared to other groups (p=0.02). In Caco-2 KLK6 wt cells TGF-β protein expression and secretion was induced, which resulted in activation of TGF-β-SMAD2/3 signaling pathway. This phenotype was associated with the elevated expression of known regulator of the EMT, zinc-finger protein Snail. In addition, the expression of a high-mobility group AT-hook 2 (HMGA2) protein was induced in Caco-2 KLK6 wt cells. The HMGA2 expression is implicated in the EMT program, acting through the TGF-β signaling pathway and is associated with a poor survival in colorectal cancer.

Conclusion. These findings demonstrate that KLK6 enzyme activity is required for colon cancer progression via induction of the EMT program. We identified the TGF-β- signaling pathway as a mechanism driving the EMT in colon cancer cells expressing KLK6 enzyme.

#855

Ube2v1 promotes epithelial mesenchymal transition and metastasis in colorectal cancer by epigenetically transcriptional suppression of autophagy.

Tong Shen,1 Dong Ling Cai,1 Hong Yu Liu,2 Juan Wen Gan,1 Ming Li,1 Ru Jing Wang,1 Da Peng Guo,1 Qun Zhou,1 Xing Xing Lu,1 Shi Li,1 Na Li Sun,1 Ming Jian Li1. 1 _Soochow University Medical School, Suzhou, China;_ 2 _Baoan Hospital, Shenzhen, China_.

Ubiquitination is one of the basic post-translational modifications for cellular homeostasis. The conjugating enzyme (E2) family plays as a bridge linking the first step mediated by E1 with the final step mediated by E3 in the ubiquitin-proteasomesystem. However, the role of Ube2v1, one of Ubiquitin-conjugating E2 enzyme variant proteins (Ube2v), in colorectal cancer (CRC) and autophagy is unclear. Here, we found that Ube2v1 is elevated in tumor samples of CRC patients and correlated with poorer survival of CRC patients. Furthermore, Ube2v1 promotes migration and invasion of CRC cells in vitro and boosts tumor growth and metastasis of CRC cells in vivo. Interestingly, Ube2v1 suppresses autophagy program and promotes EMT and metastasis of CRC cells in an autophagy dependent pattern in vitro and in vivo. Moreover, Rapamycin attenuates the enhanced in vitro migration and invasion and in vivo lung metastasis of Ube2v1 overexpression by inducing the autophagy pathway. Mechanistically, Ube2v1 increases histone H4 lysine 16 acetylation by down-regulating expression of Sirt1 and epigenetically suppresses gene expression of autophagy genes in colorectal cancer. In conclusion, Ube2v1 functions as a globalregulator for autophagy by epigenetically transcriptional suppression of autophagy, and consequently promotes EMT and metastasis of CRC. Our study functionally links Ube2v1, one member of E2 family in the ubiquitin-proteasome system toautophagy program in CRC progression and metastasis.

#856

Notch-1 promotes a mesenchymal phenotype in colon cancer cells via Notch-3 and Smad-3 activation.

George Sigounas,1 Fred E. Bertrand,2 Douglas A. Weidner,1 Kaitlyn E. Vinson,1 Alexander G. Clark,1 Azeem Khan1. 1 _East Carolina Univ. School of Medicine, Greenville, NC;_ 2 _University of Alabama at Birmingham, Birmingham, AL_.

A role for cancer initiating cells (CIC) in tumor growth, drug resistance, relapse, and metastasis via epithelial to mesenchymal transition (EMT) has been supported by experimental evidence. It is well-documented that Notch-1 receptor signaling participates in stem cell maintenance and lineage commitment in various organs, including the colon, and can regulate EMT. The function of Notch signaling in colorectal cancer (CRC) is not clear. We have reported that Notch-1 activation in the human colon tumor cell line HCT-116 resulted in upregulation of Jagged-1, Smad-3, CD44, and Slug proteins. These changes were accompanied by acquisition of a mesenchymal cell phenotype that included increased anchorage independent growth and migratory activity. Experiments with a γ-secretase inhibitor (DAPT) and soluble Jagged1-Fc protein demonstrated that Notch-1 signaling activates CD44 and Slug via other Notch receptors. In subsequent experiments, we observed an increase in Notch-3 expression in the presence of activated Notch-1. Herein, we tested the hypothesis that the stem cell-like mesenchymal phenotype induced by Notch-1 signaling in colon cancer cells is mediated by the Notch-3 receptor and Smad-3. The human colon tumor cell line HCT-116 was transduced with constitutively active Notch-1, and a Notch-3 shRNA to produce a Notch-3 null cell line with activated Notch-1 (ICN1-shN3 cells). ICN1-shN3 cells exhibited a 2.5-fold lower plating efficiency than the HCT-116/ICN1 cell line (p<0.01) that expresses constitutively active Notch-1 in the presence of Notch-3. In addition, Notch-3 null cells with constitutively active Notch-1 (ICN1-shN3 cells) were slower by 29% in completing wound healing as compared with the HCT-116/ICN1 cell line (p<0.05). Consistent with the lower plating efficiency, a 37% decrease in colosphere formation was observed with ICN1-shN3 cells as compared with the HCT-116/ICN1 cell line (p<0.001). These data indicated that loss of Notch-3 abrogated the Notch-1 induced mesenchymal cell phenotype. Western blot analysis demonstrated that the HCT-116/ICN1 cells with intact Notch-3 exhibited increased levels of the EMT associated proteins Smad-3, CD44 and Slug as compared to the parental HCT-116 cell line. Expression of CD44 and Slug in Notch-3 receptor null ICN1-shN3 cells was highly reduced compared with HCT-116/ICN1 cells and was similar to that detected in the parental HCT-116 cells. Treatment of HCT-116/ICN1 cells with a Smad-3 inhibitor resulted in significant reduction of the CD44 and Slug proteins. Meanwhile, Notch-3 receptor null ICN1-shN3 cells were also more sensitive to chemotherapy treatment than the HCT-116/ICN1 cell line (p<0.05). Collectively, our data provide evidence for a novel pathway in which Notch-1 signaling in colon tumor cells promotes a mesenchymal phenotype via activation of Jagged-1, Notch-3 and Smad-3, followed by expression of CD44 and Slug.

#857

ELF3 modulates mesenchymal to epithelial transition through regulation of CD44 variant isoform expression in pancreatic cancer cells.

Ayano Kabashima-Niibe,1 Hiromasa Takaishi,2 Takanori Kanai,2 Hideyuki Saya,2 Gregory J. Gores,1 Hajime Higuchi3. 1 _Mayo Clinic, Rochester, MN;_ 2 _Keio University School of Medicine, Tokyo, Japan;_ 3 _International University of Health and Welfare, Tokyo, Japan_.

BACKGROUND: Epithelial to Mesenchymal Transition (EMT) and Mesenchymal to Epithelial Transition (MET) may participate in pancreatic ductal adenocarcinoma (PDAC) metastasis. CD44 proteins are cell membrane proteins which function as co-receptors regulating many cellular functions. Some reports have shown that isoform switching from CD44 variant form (CD44v) to CD44 standard form (CD44s) takes place during the EMT. (Brown et al., J. Clin. Invest, 2011, Yae et al., Nat Commun., 2012). The AIM of this study was to explore the regulation of CD44 isoform switching in PDAC cells.

Methods: The expression levels of CD44v and CD44s in PDAC cell lines were assessed by immunohistochemistry and immunoblot analysis. PDAC cell lines were implanted into immunodeficiency mice and treated with sulfasarazine for 14 days. Sulfasarazine, which inhibits glutamate-cysteine transport, reduces CD44v-dependent cell growth. CD44v or CD44s positive cells were isolated using flow cytometry and induced to undergo the EMT by TGF-β treatment (7.5ng/mL) for 72 hours. A doxycycline inducible system was employed to overexpress Slug, which promotes the EMT phenotype. To induce MET, the cells were released from TGF-β treatment and cultured for an additional 72 hours. E74-like ETS Transcription Factor 3 (ELF3) expression was knocked-down by siRNA transfection.

RESULTS: Immunohistochemistry analysis revealed that CD44v protein is expressed specifically by PDAC with ductal structures within the complex tumor environment of these cancers. However, in contrast, CD44v expression was not observed in solitary infiltrating cancer cells. PANC-1, AsPC-1 and SUIT-2 PDAC cell lines expressed both CD44v and CD44s isoforms. Tumor mass in implanted PANC-1, AsPC-1 and SUIT-2 cells were reduced 69%, 50% and 36% respectively by sulfasarazine treatment following 2 weeks of therapy. However, CD44v null HPAC cells were resistant to sulfasarazine therapy following implantation. E-cadherin was expressed in CD44v positive cells but not in CD44s positive cells, and both CD44v and E-cadherin protein were reduced by TGF-β treatment. Slug over expressed cells were resistant to sulfasarazine treatment compared to non-Slug-induced cells. Removal of TGF-β restored both CD44v and E-cadherin expression levels which consistently occurs with MET. However, restoration of CD44v expression was diminished when ELF3 expression was reduced by siRNA transfection.

CONCLUSION: The isoform switching between CD44v and CD44s was reversible. The expression of CD44v isoform appears to be controlled by ELF3 during MET.

#858

The circadian clock of glioma cells undergoing epithelial-mesenchymal transition.

Arpan De, Dilshan Harshajith Beligala, Vishal Premdev Sharma, Benjamin Ryan Fry, Michael Eric Geusz. _Bowling Green State University, Bowling Green, OH_.

Highly invasive gliomas contain many cells that have changed to an enhanced migratory state through an epithelial-mesenchymal transition (EMT). These cells have the phenotype of glioma stem cells (GSCs). Similar to GSCs, glioma cells undergoing EMT show phenotypic heterogeneity, altered gene expression, and resistance to anticancer drugs along with increased invasiveness. Circadian rhythms in tumor cells influence the progression and severity of cancer and appear to regulate cell division cycles. Increased cancer incidence and progression have often been linked to disruption or deregulation of the molecular mechanism of the circadian clock. One of the major clock proteins, PER2, has been shown to play a regulatory role as a tumor and EMT suppressor in metastatic breast cancer cell lines. GSCs in tumorsphere cultures contain circadian clocks that may regulate their cancer properties. The possibility of a regulatory role for circadian rhythms in EMT was examined here. We used a standard method to induce EMT in the C6 rat glioma cell line that has known circadian rhythms in gene expression generated by the circadian clocks within its cells. EMT was induced by exchanging the cell culture medium with a serum-free stem cell medium (SCM) containing growth factors (EGF, FGF2, PDGF-alpha-beta). Cell cultures were imaged continuously with a microscope and digital camera in a cell incubator to monitor cell shape, cell death, migration, and apoptosis. During EMT, cells changed from an extended flat state to rounded and spindle shapes, ceased proliferating, and expressed EMT markers ZEB1 and vimentin. At the end of two days in SCM, 33.4% of the cells were ZEB1-positive and only 1.26% were GFAP-positive (n=3 cultures). Cell diameters after EMT were within the size range of C6 GSCs described as Hoechst-negative cells positive for stem cell markers nestin and CD133. Following EMT, small tumorspheres began to form. After initiating EMT, the rounded cells were counted at hourly intervals for up to four days after the medium exchange. As the number of post-EMT cells increased, the population size oscillated, and when examined by Lomb-Scargle periodogram analysis, four cultures had a significant period within the circadian range, 19-29 hours, (average 22.20 ±2.45 SD, p<0.05). One had a 16-hour period and one lacked a significant rhythm. Rhythms were present even when circadian clocks in the cultures were not synchronized with an initial forskolin treatment. We conclude that EMT may be timed by endogenous circadian oscillators in gliomas that favor larger numbers of post-EMT cells at a particular time of day. These results suggest that pharmacological treatments that suppress EMT or target GSCs would be more effective when delivered at particular times during the circadian cycle of the tumor.

#859

Epigenetic regulation of epithelial-mesenchymal transition in esophageal squamous cell carcinoma.

Chen Chen,1 Ming Zhao,1 Xiaojie Huang,1 Xiang Wang,1 Wenliang Liu,1 Mingjiu Chen,1 Bangliang Yin,1 Zhi Li,2 Yunchang Yuan,1 Qianjin Lu,1 Fenglei Yu1. 1 _The Second Xiangya Hospital of Central South University, Changsha, China;_ 2 _Beijing Genomics Institute at Shenzhen, Shenzhen, China_.

Epithelial-mesenchymal transition (EMT) is a developmental program, which is associated with tumor progression and metastasis. Here we investigated the DNA methylation and histone modifications in the regulation of EMT in esophageal squamous cell carcinoma (ESCC). The RNA-Seq analysis identified a total of 6150 differentially expressed genes (3423 up-regulated and 2727 down-regulated). The GO terms showed that these genes belonged to several molecular functions and biological pathways. The abnormal expression of key EMT genes, some of Sox family genes and EZH2 significantly related to patient survival. The MeDIP-Seq analysis identified differentially methylated regions (DMRs) on the whole genome level with focal hypermethylation and widespread global DNA hypomethylation. The gene ontology analysis showed that the DMRs related genes belonged to several different ontological domains, such as EMT progression, cell cycle, adhesion, proliferation, and apoptosis. The results of the bisulfite-sequencing confirmed the EMT gene DMRs identified by MeDIP-Seq. However, further analyses showed that the EMT gene DMRs only related to gene expression, but not the patient survival. ChIP-Seq was then performed to observe the relationship between histone methylation and gene expression. A total of 295 genes were found to correlate with H3K27 tri-methylation (H3K27me3) , and a certain number of key EMT genes, such as ZEB1/2,AREG, GATA4,and CDH8 were identified. Further study showed that Ezh2-mediated H3K27me3 regulate the expression of key EMT genes. Ablation of Ezh2 expression prevents EMT, whereas forced expression of Ezh2 restores EMT. Sox4 gene could directly regulate Ezh2 expression, tri-methylated H3K27me3 for EMT gene repression. Taken together, our results suggest that both of the DNA methylation and H3K27me3 modification involved in the regulation of EMT in ESCC. Sox4-Ezh2-mediated H3K27me3 marks associate with key EMT gene regulation, representing an important epigenetic EMT signature.

#861

Olfactomedin 4 downregulation is associated with enhancing epithelial-mesenchymal transition in human benign prostate cells and prostate cancer cells.

Hongzhen Li,1 Wenli Liu,1 Jianqiong Zhu,1 Kay Chin,1 Jaime Rodriguez-Canales,2 Griffin P. Rodgers1. 1 _NIH-NHLBI, Bethesda, MD;_ 2 _UT - MD Anderson Cancer Center, Houston, TX_.

We have previously reported that lost or reduced olfactomedin 4 (OLFM4) expression is caused by deletions within the OLFM4 gene in 10-25% of all human prostate-cancer samples, and that its expression is lost in more than 58% of advanced prostate-cancer samples. Therefore, we sought to study the role of epigenetic factors in the regulation of OLFM4 gene expression in normal prostate, as well as prostate-cancer, cell lines. Pyrosequencing of genomic DNA extracted from normal prostate or prostate-cancer tissue cells demonstrated that three of the eight CpG sites in the OLFM4 gene promoter were significantly hypermethylated in prostate cancer compared with normal tissue. Exposure to the methylation inhibitor 5-azadeoxycytidine (5-Aza 5 μM) significantly increased OLFM4 expression in RWPE1 benign prostate cells. Knockdown of OLFM4 expression in RWPE benign prostate cells reduced autophagy and enhanced the epithelial-mesenchymal transition (EMT). Restoration of OLFM4 expression in metastatic prostate-cancer cells lacking OLFM4 inhibited cell growth in both in vitro culture models and an in vivo xenograft tumor model, and also inhibited EMT marker gene expression in these cells. 5-Aza induced OLFM4 expression in PC-3 prostate cancer cells and OLFM4 shRNA transfected PC-3 cells affects 5-Aza mediated cell proliferation compared to control-shRNA transfected PC-3 cells. Taken together, our results suggest that OLFM4 inhibits the EMT program in both normal prostate and prostate-cancer cells, and may play a critical role in regulating the EMT program during the progression of cancer. They also suggest that modulation of OLFM4 expression might be a novel molecular target for advancing prostate-cancer therapies.

#862

Atypical protein kinase c inhibitors can repress epithelial to mesenchymal transition (type III) in malignant melanoma.

Wishrawana Sarathi Ratnayake, Mildred Acevedo-Duncan. _Univ. of South Florida, Tampa, FL_.

Melanoma is a type of cancer occurs in melanocytes. Approximately 90% of melanoma occurs in skin (cutaneous melanoma) but can rarely arise from the mucosal surfaces or areas which neural cells migrate. Examples are eye, intestine and mouth [Eur. J. Cancer, 69: 39-42 (2016)]. 76,380 of new cases and 10,130 number of deaths are expected in 2016 in the USA due to melanoma [http://seer.cancer.gov/statfacts/html/melan.html (11/05/2016)]. Atypical PKCs contains two structurally and functionally distinct isozymes in human which are PKC-ι (iota) and PKC-ζ (zeta). They are believed to be involved in cell cycle progression, tumorigenesis, cell survival and cell migration. We believe that atypical PKCs play an important role in cell motility of melanoma by involving the signaling pathways which induces EMT-type III (Epithelial to Mesenchymal Transition). In normal melanocytes, PKC-ζ was found in low levels and PKC-ι was not detected. But both proteins are detected in very high levels in malignant melanoma [Melenoma Res. 12:201-209 (2002)]. In the current study, we have investigated the effects of novel atypical PKC inhibitors [4-(5-amino-4-carbamoylimidazol-1-yl)-2, 3-dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1) which is specific to PKC-ι and 8-hydroxy-1, 3, 6-naphthalenetrisulfonic acid (Compound-50) which is specific to PKC-ζ on the cell proliferation, apoptosis and cell migration of two malignant melanoma cell lines (SK-MEL-2 and MeWo) compared to a normal melanocyte cell line (PCS-200-013). We showed that both inhibitors can decrease the levels of total and phosphorylated levels of PKC-ζ and PKC-ι. Furthermore, both inhibitors increased the levels of E-cadherin and decreased the levels of Vimentin which is a mesenchymal marker associated with EMT. Treatments with inhibitors altered the levels of CD44, a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion, migration and tumor cell homing during metastasis. These results suggest that both PKC-ι and PKC-ζ are involved in signaling pathways which upregulate EMT and which can be effectively suppress using ICA-1 and Compound-50. Furthermore we established that treatment with ICA-1/Compound-50 induced apoptosis as shown by increasing Caspase-3 levels and decreasing Bcl-2 levels.

#863

Neuronal splicing regulatory factors are upregulated in models of epithelial-mesenchymal transition.

Yevgenia L. Khodor,1 Julia Froese,2 Frank Gertler,1 Robert A. Weinberg,2 Christopher B. Burge1. 1 _MIT, Cambridge, MA;_ 2 _Whitehead Institute, Cambridge, MA_.

Epithelial-mesenchymal transition (EMT) is an essential developmental program through which epithelial cells lose their polarity and junctions with neighboring cells and acquire migratory and invasive properties. Cancer cells hijack at least a portion of the EMT program to increase their invasive properties, acquire resistance to senescence, and metastasize. Major changes in cell signaling, transcriptional and post-transcriptional regulation occur during EMT. To study post-transcriptional changes we characterized gene expression and splicing across EMT in 3 cell culture models of EMT: the luminal epithelial MCF7ras breast cancer cell line with EMT induced by SLUG+SOX9 and in basal human mammary epithelial (HMLEras) cells, with EMT induced by either SNAIL or ZEB1. RNA-seq was performed at 2-day intervals across these transitions. We observed broad overlaps between these systems, with 821 genes that changed significantly in all three. In all three time courses, genes associated with cell cycle and growth decreased in expression, while genes associated with signaling, cell motion, adhesion, extracellular matrix organization, and differentiation increased. Intriguingly, genes associated with neurogenesis and angiogenesis were also induced in the MCF7 and HMLER-Snail time courses. Those include known axon guidance and angiogenic factors in the Robo and Slit families. We also found many alternative splicing events shared in all 3 systems, including 272 skipped exons. Sequence analysis of regions around these exons revealed binding motifs for canonical splicing regulators, including ESRP1 and ESRP2 and the RBFOX protein family, which are known to play a role in EMT. While most genes encoding RNA binding proteins decreased in expression in EMT, a small subset consistently increased in expression, including some that matched our motif analysis. Specifically, several splicing factors associated with nervous system development were upregulated, including factors of the QKI and NOVA families. We have validated the increased expression of QKI and NOVA1 proteins during EMT in vitro, and of QKI and NOVA2 in tumor sections. Further, we found that perturbation of QKI expression affects the motility of mesenchymal cancer cells in culture. Together, our results point to the existence of a program of alternative splicing shared between neuronal and mesenchymal cells.

#864

Alternated activation of TGFβ pathway and differential expression of epithelial to mesenchymal transition transcription factors in relation to lymph node metastasis in two subtypes of breast cancer tumors.

Victoria Ortega-Hernandez,1 Patricia Gajardo-Meneses,1 Wanda Fernandez,2 Pilar Carvallo1. 1 _Pontificia Universidad Catolica de Chile, Santiago, Chile;_ 2 _Hospital San Borja Arriaran, Santiago, Chile_.

Breast cancer is the leading cause of cancer death among women worldwide, being distant metastases the main cause of death. Epithelial to mesenchymal transition (EMT) has been implicated in promoting cancer invasion and metastasis. This process can be induced by TGFβ through the expression of transcription factors: TWIST, SNAIL, SLUG and ZEB1, which expression may be promoted by different signaling pathways such as SMAD2/SMAD3, PI3K/AKT and ERK/MAPK. The expression of EMT transcription factors has been previously studied in several cancer cell lines, although no studies have been performed in breast cancer tumors. In this study, we analyzed by immunohistochemistry the expression of the four transcription factors, and the signal transduction key proteins p-SMAD2 (ser465/467), p-SMAD2L (ser245/250/,255) and p-AKT (ser473), in 107 breast cancer tumors. We found expression of at least one transcription factor in all triple negative tumors (n=24), while only 70% luminal tumors showed expression of at least one of these proteins. Expression of ZEB1, SNAIL and SLUG was observed in tumors with metastases to lymph node, whereas the majority of tumors expressing TWIST were negative for lymph node status. All triple negative tumors with an active state of the signal transduction pathway TGFβ/SMAD showed expression of at least one transcription factor, whereas 65% of luminal tumors with an active state of the same pathway showed no expression of these transcription factors. Active state of PI3K/AKT pathway was observed only for luminal tumors as it has been described. Sixty-three percent of tumors having this pathway active showed expression of at least one transcription factor. In conclusion we found differential expression of EMT transcription factors in relation to lymph node status and a differential active state of signaling pathways in relation to tumoral subtype of breast cancer tumors. Our results suggest a different mechanism of EMT induction by TGFβ in relation to two tumor's subtypes and lymph node metastasis.

#865

Cytoplasmic polyadenylation element binding protein 2 splice variants have opposing functions in the epithelial to mesenchymal transition pathway.

James T. DeLigio, Charles E. Chalfant, Margaret A. Park. _Virginia Commonwealth University School of Medicine, Richmond, VA_.

Alternative splicing of cytoplasmic polyadenylation element binding protein 2 (CPEB2) generates two isoforms, CPEB2B and CPEB2A, through inclusion/exclusion of exon 4, respectively, in mature mRNA. We previously reported that CPEB2B is essential to establish anoikis resistance (AnR), and promotes metastasis using in vivo orthotopic models. In contrast, CPEB2A decreases AnR and metastatic transformation. In the current studies, we examined the function of CPEB2 isoforms using next generation sequencing analysis of downstream signaling pathways. We found that CPEB2B expression upregulated AnR in breast cancer cells and induced key genes/proteins in the epithelial to mesenchymal transition (EMT) and hypoxic response (hypoxia inducible factor 1α (HIF1α)-dependent) pathways. Inhibition of either TWIST1 or HIF1α reduced the CPEB2B-mediated AnR in cells ectopically expressing CPEB2B, confirming a link between CPEB2 alternative splicing and the induction of the EMT/hypoxia pathways. We then extended our findings, and demonstrated that CPEB2 alternative splicing induced de novo translation of both HIF1α and TWIST1, whereas CPEB2A decreased translation of these factors. Furthermore, CPEB2 isoforms were found to modulate differential polyadenylation site selection of the TWIST1 3'UTR and competed for binding to HIF1α and TWIST1 3'UTR cytoplasmic polyadenylation element (CPE) site sequences. These results demonstrate that CPEB2A and CPEB2B modulate the translation of factors in the EMT/hypoxia pathways in an opposing fashion via differential polyadenylation site selection and subsequent translation.

#866

The epithelial-to-mesenchymal transition regulates E-selectin ligand activities of breast cancer cells.

Grady E. Carlson, Alexander O. Ostermann, Venktesh S. Shirure, Fabian Benencia, Monica M. Burdick. _Ohio University, Athens, OH_.

The epithelial-to-mesenchymal transition (EMT) leads to specific stem-like and invasive properties in breast cancer cells. However, it remains unclear whether the EMT modifies sialofucosylated ligands that circulating tumor cells use to attach and adhere to vascular endothelial E-selectin during metastasis. To address this issue, immunohistochemistry and E-selectin microsphere dynamic adhesion assays were initially used to assay 110 cases of breast cancer for EMT biomarkers and E-selectin ligand activity. The expression of E-cadherin, but not N-cadherin or vimentin, directly correlated with E-selectin ligand activity. Thus, it was hypothesized that the EMT, and in turn the mesenchymal-to-epithelial transition (MET), regulate expression of functional E-selectin ligands in breast cancer cells. The ectopic expression or shRNA knockdown of transcription factors (i.e., Snail and Twist) in target cell lines were used to induce EMT or MET, respectively. Shear flow adhesion assays and flow cytometry demonstrated that cells with epithelial phenotypes had greater E-selectin ligand activities and greater expression of sialofucosylated carbohydrates compared to cells with mesenchymal phenotypes. Moreover, glycoprotein E-selectin ligand activities of breast cancer cells were more affected by the EMT compared to glycolipid E-selectin ligand activities. Assessing expression of α1,3- and α1,4- fucosyltransferases (FUT) at the RNA level using qRT-PCR revealed that FUT3 and FUT6 gene expression were decreased via the EMT, corresponding to the lowered E-selectin ligand activities observed in mesenchymal phenotype cells. Altogether, the results of this investigation demonstrate that the EMT, as well as the MET, mechanistically regulate the expression of functional E-selectin ligands expressed by breast cancer cells.

#867

Circulating lung tumor cells capture extracellular vesicles conferring resistance phenotype by the occurrence of epithelial-mesenchymal transition reprogramming.

Josephine Zangari,1 Marius Ilie,2 Simon Heeke,1 Véronique Hofman,2 Baharia Mograbi,1 Sophie Raynaud,3 Salome Lalvee,2 Nathalie Yazbeck,1 Charles-Hugo Marquette,2 Sylvie Leroy,2 Patrick Brest,1 Paul Hofman2. 1 _Université Côte d'Azur, Nice, France;_ 2 _Université Côte d'Azur/FHU Oncoage, Nice, France;_ 3 _CHU Nice, Nice, France_.

Background: The overall prognosis of lung cancer patients remains dismal, in particular at advanced stages. Only complete surgical resection of early-stage tumors improves the prognosis of certain non-small cell lung cancer (NSCLC) patients. However, migration of circulating tumor cells (CTCs) into the blood stream is probably an early event of carcinogenesis inducing metastases. The fact that some of these CTCs could survive within this "liquid environment" suggest that they acquire phenotypic changes that would confer them improved resistance against anoikis. In this context, we looked if immune blood cells could transfer to CTCs some factors associated with this increased resistance.

Materials and Methods: Lung cancer cells (A549 cell line) were incubated with different sub-population of blood cells (PBMCs, granulocytes, platelets) and serum to look for potential transfer of extracellular vesicles (EVs) into the cancer cells. To assess the inter-cell transfer we looked for the presence of a validated blood cell specific marker the miR-223 that should not be expressed in lung cancer cells. By using a direct method for CTCs detection and characterization, we looked for these biomarkers within CTCs of lung cancer patients with early and late stages. Moreover the quantification of these biomarkers was correlated with the expression of vimentin, cytokeratin and E-cadherin

Results: First, A549 cells were able to quickly capture microRNA (in particular miR-223) within the control blood samples. We then identified neutrophils as a major source of these miRNA. The inter-cell transfer was EVs dependent and promoted improved resistance and survival of A549 cells cancer cells. In NSCLC patients with different stages, the presence of some specific miRNAs for blood cells was detected in a subpopulation of CTCs showing epithelial to mesenchymal phenotype, in particular an increase of vimentin expression and loss of E-cadherin expression.

Conclusion: These results tend to prove that CTCs may capture extracellular vesicles from immune blood cells within the bloodstream and this phenomenon may be associated with a more aggressive phenotype. Inhibition of specific EVs transfer could be on interest for development of new targeted immunotherapies.

#868

GRK5 promotes a mesenchymal transition in prostate cancer.

Joseph B. Black, Iqbal Mahmud, Hamsa Thayele Purayil, Yehia Daaka. _University of Florida, Gainesville, FL_.

Locally advanced and metastatic prostate cancer is incurable, despite decades of research targeting the activity of the androgen receptor (AR). Our lab previously identified G protein-coupled receptor 5 (GRK5) for its ability to regulate PCa progression, independent of AR activity. GRK5 partially partitions to the nucleus, wherein it has been shown to regulate the transcriptome in non-PCa models. To globally elucidate the mechanistic impact of GRK5 on PCa progression, we are investigating the impact of GRK5 on the PCa transcriptome. We hypothesize that GRK5's regulation of the PCa transcriptome promotes tumor progression. To assay the effect of GRK5 on the PCa transcriptome, RNA sequencing was performed in two cell lines: PC3 Control (PC3 shGFP) and PC3 GRK5 Knockdown (PC3 shGRK5). Ontologic analysis identified the epithelial–mesenchymal transition (EMT) as being affected when GRK5 is depleted. The epithelial–mesenchymal transition (EMT) is highly associated with promoting PCa progression and chemoresistance, thus we selected this pathway for further investigation. Confirmatory western blot analysis and quantitative PCR (qPCR) validated that depleting GRK5 suppresses the expression of the mesenchymal markers Vimentin and N-Cadherin. Similarly, overexpression of GRK5 increases the expression of these mesenchymal markers. Stable overexpression of GRK5 promotes cells to develop a spindle-like morphology, indicative of a mesenchymal state. GRK5 mediates this mesenchymal transition through increasing the expression of the EMT transcription factor, Twist1. Further analysis identifies that GRK5 activity promotes in vitro invasion. Cell lines overexpressing GRK5 demonstrate an increase in resistance to docetaxel, the mainstay chemotherapy for advanced PCa. Overexpression of mutated forms of GRK5 that are relegated to the nucleus are able recapitulated all aforesaid changes, arguing that the nuclear activity of GRK5 mediates this effect. Collectively, this data presents a novel mechanism promoting PCa progression, independent of AR activity. Future work seeks to assay the ability of GRK5 to promote metastasis in vivo in mouse models.

#869

Mesenchymal identity of breast cancer promotes brain metastases and therapeutic resistance through MLK4/NFkB signaling Pathway.

Heba Allah M. Alsheikh, Mutsuko Minata, Shinobu Yamaguchi, Ichiro Nakano. _University of Alabama at Birmingham, BIRMINGHAM, AL_.

Patients with breast cancer brain metastases (BCBM) suffer from a devastating outcome despite recent progress in a variety of therapeutic modality. Among the four major molecular subtypes, breast cancers that are either human epidermal growth factor receptor (HER) 2- positive or triple-negative breast cancer (TNBC) patients have a higher risk of BCBM compared with the luminal cancer patients. The mechanisms by which the disseminated cancer cells overcome the multistep defense mechanism in the brain, adapt the microenvironment, and subsequently initiate metastasis remain elusive. Recently, we identified a set of genes that contribute to initiate Glioblastoma (GBM) and at least some of them are functionally essential for GBM establishment and the therapy resistance. Mixed lineage kinase 4 (MLK4) is a relatively poorly characterized serine/threonine kinase, which is largely expressed in various cancers including GBM. We recently found a critical role of MLK4 in controlling the mesenchymal identity in GBM stem cells and plays a role in GBM initiation through direct activation of NF-kB signaling. Our preliminary data indicates MLK4 is also highly expressed in patient-derived hormone receptor (HR)-negative breast cancer cells in comparison with the other HR-positive counterparts. For therapeutic development purpose, we performed the structure-based drug design, which has led to identify two small molecule MLK4 inhibitor candidates to test efficacy on a set of patient-derived breast cancer cells in vitro and their xenografted tumors in vivo. Results will be presented in the conference.

### Imaging Cancer Immunotherapy, Tumor Microenvironment, and Other Aspects of Tumor Biology

#870

Radiation-induced PD-L1 upregulation can be detected by Zr-89-PD-L1 PET/CT in the tumor micro-environment of murine HPV positive HNSCC model and melanoma model.

Masahiro Kikuchi, Raghvendra M Srivastava, David A Clump, Julio A Diaz-Perez, Lingyi Sun, Dexing Zeng, W. Barry Edwards, Carolyn J Anderson, Robert L Ferris. _University of Pittsburgh, Pittsburgh, PA_.

Background: Radiation therapy (RT) can induce upregulation of programmed death ligand 1 (PD-L1) on tumor cells or myeloid cells, which might affect the response to RT with or without anti-PD-1/PD-L1 blockade. RT-induced PD-L1 expression during therapy could be a predictive marker for the therapy response, however, serial biopsies to monitor its distribution may be unreliable because its expression is heterogeneous. Therefore, non-invasive imaging of tumor PD-L1 expression could be more helpful.

Materials and Methods: Two different murine tumor models (MEER and B16F10) were established in two locations, at the back of the neck and at the right flank of C57BL/6 mouse. Then fractionated RT (2Gyx4 or 2GyX10) with or without anti PD-1 therapy was delivered only to the neck tumor. PD-L1 expression was measured by PET/CT and biodistrubtion with Zr-89-DFO-anti-mouse PD-L1 monoclonal Ab (clone 10F.9G2) and the results were corroborated by flow cytometric analysis and immunohistochemistry. PET/CT imaging and biodistributions were performed 48 or 96 hours after tracer-injection. The change of PD-L1 expression on irradiated neck tumor was evaluated using non-irradiated flank tumor as a control. Endothelial cell morphology in tumor vessels was also analyzed by CD31 staining to determine whether RT-induced increased permeability of tumor vessels might result in increased non-specific tracer accumulations in the irradiated tumors.

Results: PET/CT imaging and biodistribution study of ex-vivo tracer uptake values demonstrated significant increased tracer uptake in irradiated neck tumor compared to non-irradiated flank tumor in either case of MEER and B16F10. Tracer uptakes in the spleens were significantly decreased if high dose of non-labeled anti-mouse PD-L1 monoclonal Ab was given in advance before tracer-injection, which indicates the tracer is fully functional. Flow cytometry and immunohistochemistry showed PD-L1 upregulation in both irradiated tumors corroborating PET/CT imaging of RT-induced PD-L1 upregulation. Flow cytometry also revealed RT-induced PD-L1 upregulation on myeloid cells is more prominent than that of tumor cells in both MEER and B16F10. CD31-positive staining in the irradiated tumors was not different from that of non-irradiated tumors, which shows RT did not induce alterations in tumor blood vessels. PD-L1 upregulation of MEER was not seen unless it was performed 2GyX10 RT, however, that of B16F10 could be seen at an earlier time-point of 2GyX4. Combination therapy of anti PD-1 with RT did not show further PD-L1 upregulation compared to RT without anti PD-1 therapy.

Conclusion: RT-induced PD-L1 upregulation in different tumor models of MEER and B16F10 was identified by PET/CT using Zr-89 labeled PD-L1 monoclonal Ab and its validity was corroborated by flow cytometric analysis and immunohistochemistry.

#871

**[18F]BMS-986192 as a novel PET imaging agent for assessment of PD-L1 expression** in vivo **.**

Ralph A. Smith,1 David Donnelly,1 Paul E. Morin,1 Dasa Lipovsek,2 Jochem Gokemeijer,2 Daniel Cohen,1 Joonyoung Kim,1 Adrienne Pena,1 Olufemi Adelakun,1 Xi-Tao Wang,1 Patrick Chow,1 Samuel J. Bonacorsi,1 Wendy Hayes1. 1 _Bristol-Myers Squibb, Princeton, NJ;_ 2 _Bristol-Myers Squibb, Waltham, MA_.

Objectives Inhibition of the Programmed Death Ligand-1 (PD-L1)/PD-1 interaction allows for potent anti-tumor activity and antibodies that disrupt this interaction have been approved for the treatment of multiple cancer types. PD-L1 expression has been investigated clinically as a potential biomarker to predict response to anti-PD-1/PD-L1 therapy. BMS-986192, an Adnectin with high affinity and specificity for human PD-L1, was selected in vitro from a complex library. Here we report the discovery and first preclinical evaluation of [18F]BMS-986192 as a PET imaging agent to detect PD-L1 expression in vivo.

Methods [18F]BMS-986192 was radiolabeled via copper-free click chemistry and assessed for its ability to detect PD-L1 expression. Tracer binding to human L2987 (PD-L1+) and HT-29 (PD-L1-) xenografts as well as human non-small cell lung cancer (NSCLC) tissue samples was assessed by autoradiography (ARG). Tracer binding was compared to PD-L1 expression assessed independently with anti-PD-L1 immunohistochemistry (IHC). In vivo performance of the tracer was also assessed by PET imaging in mice bearing bilateral L2987 and HT-29 xenografts, and tracer biodistribution was further assayed in these animals ex vivo by gamma counter. Finally, initial in vivo biodistribution and radiation dosimetry was measured by PET in cynomolgus monkey.

Results ARG studies showed increased [18F]BMS-986192 total binding to PD-L1(+) L2987 xenograft compared to PD-L1(-) HT-29 xenograft tissue. Radiotracer binding was higher in all tested human NSCLC tissue samples compared to xenografts. Dose-dependent blockade was seen in all PD-L1(+) tissues co-incubated with cold BMS-986192, and binding was unaffected by co-incubation with cold non-PD-L1 binding control. Visual comparison of tracer binding aligns closely with PD-L1 IHC both spatially as well as in intensity. Preferential accumulation of [18F]BMS-986192 was noted in PD-L1(+) L2987 compared to PD-L1(-) HT-29 xenografts in tumor-bearing mice. PET studies in cynomolgus monkeys confirmed binding to PD-L1(+) tissue (e.g. spleen) with minimal nonspecific background signal exclusive of primary clearance organs. Radiation dosimetry of [18F]BMS-986192 indicates an estimated single administration dose limit of 6.2 mCi for an average human subject.

Conclusions ARG, PET studies, and ex vivo measurements in rodent and cynomolgus monkey demonstrated sensitive and specific [18F]BMS-986192 binding to PD-L1. Low background signal in cynomolgus monkey in the context of endogenous PD-L1 expression further supports the potential of this tracer for sensitive detection of PD-L1(+) lesions in vivo. Radiation dosimetry suggests that [18F]BMS-986192 can be safely administered in human trials, with estimated absorbed radiation doses well within safe parameters for human administration. [18F]BMS-986192 has potential as a sensitive PD-L1 imaging agent for same-day imaging in patients.

#872

Noninvasive monitoring of endogenous Pdl1 expression by using a dual reporter system.

Min Zhou, Xinjian Liu, Fang Li, Chuan-Yuan Li. _Duke University Medical Center, DURHAM, NC_.

Programmed death-ligand 1 (Pdl1) is a key immune checkpoint protein facilitating tumor escape from host immune system. Clinical approval of anti-PD1 and anti-PDL1 antibodies for the treatment of a variety of malignancies underscores the promise of immune checkpoint therapy. Further progress in this promising area of research is likely to come from better understanding of the mechanisms for regulation of immune checkpoint proteins such as PDL1. In the present study, we designed a dual reporter imaging system to monitor Pdl1 expression by use of the CRSPR-Cas9 technology. A fusion reporter that consists of eGFP and Luc2 was introduced into downstream of the endogenous Pdl1 gene, right before the stop codon, through CRISPR-Cas9 mediated homologous recombination. A P2A self-cleaving motif is inserted between the fusion reporter gene (eGFP-Luc2) and Pdl1. The reporter system was introduced into two murine tumor cell lines, 4T1 and B16F10. In vitro, an increase in eGFP intensity and bioluminescence of the reporter cells was observed after exposure to ionizing radiation or IFN-gamma, which has been reported to upregulate the expression of Pdl1. In vivo, we were able to observe radiation induced increase in bioluminescence signals from tumor established by use of the reporter cells. Our study therefore provided proof of principle for the use of our endogenous Pdl1 reporter imaging system to monitor the expression levels of Pdl1 in vitro and in vivo noninvasively and serially.

#873

Evaluating immunotherapy effects using preclinical molecular imaging tools for quantitative immune cell tracking.

Marie-Laurence Tremblay,1 Zoe O'Brien-Moran,2 Christa Davis,1 Kimberly Brewer1. 1 _IWK Health Center, Halifax, Nova Scotia, Canada;_ 2 _Dalhousie University, Halifax, Nova Scotia, Canada_.

Immunotherapies are a promising class of cancer therapeutics, but clinical translation is often hampered by a lack of understanding regarding optimal therapy administration, combination, and reliable biomarkers of success. Traditional metrics such as RECIST, and modern metrics like irRC and PERCIST used for monitoring cancer therapy outcome, have limitations for immunotherapy evaluation and are not always reflective of the underlying immune mechanisms. We aim to better characterize and monitor these immunotherapies, with focus on combination therapy optimization, by tracking immune cell migration in response to these therapies using preclinical magnetic resonance imaging (MRI). MRI is used to obtain anatomical tumor changes, detect and quantify superparamagnetic iron oxide (SPIO)-labeled cells in vivo.

Goal: To link immune cell migration to early prognostic biomarkers for immunotherapy success.

Methods: C57/BL6 mice (n=40) received an implant of 5x105 C3 cancer cells in the left flank. Mice (n=10/group) were i) untreated or treated with ii) 200µg of anti-PD1/day on days 7, 9, 11, 21 and 25, iii) the peptide-based vaccine DepoVaxTM (DPX) on day 15, or iv) with anti-PD1 and DPX. CD8+ cytotoxic T cells (CD8) and suppressive regulatory T cells (Tregs) were isolated from diseased-matched & treated donor mice for expansion in culture before labeling with SPIO for adoptive cell transfer into mice receiving scans. PET/MRI Data: Anatomical and qualitative SPIO data is collected using a balanced steady-state free precession (bSSFP) sequence. Iron quantification is done using R2* maps from a multi-echo single point imaging sequence (TurboSPI). Tumor metabolism was assessed by 18F-fluorodeoxyglucose uptake during simultaneous acquisition of positron emission tomography (PET) with MRI. Imaging was done 21 and 28 days post-implant.

Results: CD8 and Treg cells are consistently recruited to both the tumor and vaccine draining inguinal lymph nodes. CD8 T cells are primarily recruited to the tumor periphery and do not always penetrate the tumor core. Positive therapy outcomes are correlated with an increasing CD8/Treg ratio in the tumor, particularly at earlier time points (21 vs 28 days). In certain cases, CD8 T cells were found within the fat pad between the tumor and lymph node. As expected, DPX & anti-PD1 combination therapy resulted in the best prognosis. Simultaneous acquisition of PET/MRI demonstrated large areas of necrosis in tumor cores. Using TurboSPI, we have begun quantifying CD8 and Tregs cells, evaluating if volumetric tumor changes due to pseudoprogression correlate with Treg or CD8 T cell migration and comparing results to pre-existing biomarkers.

Conclusions: Using MRI/PET with quantitative immune cell tracking results in more in-depth, longitudinal, characterization of immunotherapies at the preclinical level, which can be used to optimize therapy combinations.

#874

Lifting the iron curtain: Imaging cellular barriers to combination chelation-immune checkpoint therapy.

Avigdor Leftin, Suresh Veeraperumal, Huiyong Zhao, Sadna Budhu, Elisa de Stanchina, Jedd Wolchok, Taha Merghoub, Jason Koutcher. _Memorial Sloan Kettering Cancer Center, New York, NY_.

Iron accumulation in cancer cells and macrophages can result in a tumor microenvironment that is unfavorable for checkpoint immune therapy. Chelation is a safe approach of reducing cellular iron accumulation that while affecting all cells biochemically, has been shown to also regulate tumor growth via specific "off-label" effects on macrophages that can hypothetically support an immune therapy responsive microenvironment. To further investigate the connection between infiltration of macrophages, iron chelation therapy, and synergy with immune therapy, we used established orthotopic Myc-CaP and TRAMP-C2 prostate cancer models and quantitative iron and cellular MRI, histology, and FACS to evaluate pre-treatment and post-chelation macrophage/immunological status, and MRI to monitor tumor growth. Quantitative assessment of cellular iron from MRI and histology of tumors, livers, and spleens revealed differences in tissue iron levels between host mouse strains, and enabled us to correlate these differences with extent of iron-macrophage infiltration in the tissues. To see if we could reduce these cells metabolically we administered the oral chelator deferiprone at tumor onset which resulted in reductions in endpoint tumor volume in both prostate tumor models with the largest response to chelation being observed in low-iron C57BL6 model backgrounds; a result subsequently confirmed in MMTV-PyMT orthotopic breast cancer models. Even in cases of complete tumor growth inhibition, systemic iron-load was not significantly affected, indicating that chelation achieved these therapeutic gains without harmful side-effects. Reduction of iron-macrophages and tumor volume accompanied shifts in macrophage surface receptor presentation and T-cell repertoire indicating that pro-tumor (M2) macrophage de-polarization to anti-tumor (M1) phenotypes occurred with chelation therapy. As these changes can potentially support improved action of checkpoint PDL-1 inhibitors, we then tested the combination chelation-checkpoint therapy approach in the prostate tumor models. These trials showed a modest reduction in tumor growth with either chelation or immune checkpoint therapy alone. A notable synergistic effect was observed using combination chelation-checkpoint therapy that accompanied a shift in macrophage polarization suggesting that chelation was sufficient to enhance checkpoint response. These therapeutic imaging studies demonstrate how tumor infiltration of iron-macrophages can be used as a biomarker of the immune-microenvironment, and how by using iron-chelation immunotherapeutic response can be enhanced.

#875

Improved resection and prolonged overall survival with PD-1-IRDye800CW fluorescent probe-guided surgery and PD-1 adjuvant immunotherapy.

Yang Du, Jie Tian. _Institute of Automation, Chinese Academy of Sciences, Beijing, China_.

Purpose: An intraoperative technique to accurately identify microscopic tumor residuals could be applied to decrease the risk of positive surgical margins. Several lines of evidence support the expression and immunotherapeutic effect of PD-1 in breast cancer. Here, we sought to develop a fluorescence labeled PD-1 probe for in vivo breast tumor imaging and image-guided surgery. The efficacy of PD-1 monoclonal antibody (mAb) as adjuvant immunotherapy after surgery was also assessed.

Experimental Design: A near-infrared dye labeled PD-1-IRDye800CW probe was developed and examined for its application in tumor imaging and image-guided tumor resection in an immunocompetent 4T1 mammary mouse tumor model that recapitulates clinical metastatic cancer. Fluorescence molecular imaging (FMI) was performed to monitor probe biodistribution and intraoperative imaging. Bioluminescence imaging (BLI) was carried out concurrently to monitor tumor growth and evaluate post-surgical tumor residuals, recurrences, and metastases.

Results: The PD-1-IRDye800CW probe exhibited a specific and stable signal at the tumor region compared to the IgG control, and exhibited the highest tumor-to-background ratio (TBR) 8 h post-injection (11.62 ± 0.79), approximately 5 fold higher than the IgG control (2.13 ± 0.30). Furthermore, PD-1-IRDye800CW guided surgery combined with PD-1 adjuvant immunotherapy inhibited tumor regrowth and microtumor metastases, as determined by BLI imaging, and thus improved survival rate with no obvious side effects.

Conclusions: Our study demonstrated the feasibility of using PD-1-IRDye800CW for breast tumor imaging and image-guided tumor resection. Moreover, we found that PD-1 mAb adjuvant immunotherapy reduces cancer recurrences and metastases emanating from minimal tumor residuals.

#876

Imaging the interaction of leukemia and bone marrow microenvironment in murine model of ALL.

Karine G. Harutyunyan,1 Saradhi Mallampati,1 Anna Zal,1 Mateusz Rytelewski,1 Michael C. Gutkin,2 Jason M. Butler,2 Tomasz Zal,1 Marina Konopleva1. 1 _MD Anderson Cancer Ceter, Houston, TX;_ 2 _Weill Cornell Medical College, New York, NY_.

Interactions of leukemia and the bone marrow (BM) microenvironment are known to play a key role in the survival and growth of leukemic cells, and we have shown that HIF-1α stabilization in BM stromal cells facilitates leukemia homing and progression (Chen et al. Blood 2012, 119:4971). Leukemic cells have been shown to hijack the homeostatic mechanisms of normal hematopoietic stem cells (HSCs) and take refuge within the BM niche. This mechanism is pivotal during chemotherapy and contributes to disease relapse. In this study, we aimed to characterize the time-dependent progression of BM hypoxia involving both acute lymphocytic leukemia (ALL) cells and components of the BM niche, using multiphoton intravital microscopy (MP-IVM) . We generated a transplantable, fluorescent leukemia model by retrovirally transducing C57Bl6-Ai14 murine BM cells that express red fluorescing tdTomato with the p190-Bcr/Abl oncogene (KG Harutyunyan et al, Blood 2014 124:2396). The resulting p190-Bcr/Abl tdTomato cells caused rapid development of ALL in non-irradiated C57Bl6 immunocompetent mice, manifested by infiltration of multiple organ and BM sites, followed by death within 14-18 days. We utilized Col2.3-GFPemd transgenic mice as recipients of leukemia to highlight the osteoblastic niche, and visualized vasculature by injection of TRITC-dextran. We showed the dynamic of homing and engraftment of ALL leukemic B cells (LBC) in OB-GFP recipient mice, with homing in the vicinity of blood vessels visualized by MP-IVM, followed by proliferation and leukemia expansion. This was accompanied by invasion of both vascular and osteoblastic components of BM microenvironment. Longitudinal assessment of hypoxia utilizing pimonidazole staining showed progressive development of BM hypoxia starting from Day 10 p.i., paralleling leukemia progression, despite the abundant vascularization of the BM. To assess the integrity of the BM vascular niche during leukemia progression the experimental mice were intravitally injected with low doses of an Alexa-Fluor-647-conjugated VE-cadherin antibody (MG. Poulos et al, Epub 2013 Sep 5) to visualize the number and morphology of the perfused vessels and to analyze the architecture of the hematopoietic compartment. We observed that at late stage of leukemia progression (day 12-14 p.i) BM vessels are disorganized as a result of expansion of leukemic B cells. Ongoing longitudinal imaging experiments will characterize the cellular origin of hypoxic niche cells and the dynamic of vasculature alteration in ALL. In summary, these findings demonstrate rapid development of intra-BM hypoxia that parallels leukemia progression and involves interactions between leukemia cells and BM niche cells, as well as disordered vasculature.

#877

**Real-time imaging of adherent and non-adherent cell interactions: utility of an automated microfluidic trap platform to recapitulate** in vivo **cell culture microenvironment.**

Rekha Kannan, Victor Yeh, James Helton, Amedeo Cappione. _Millipore Sigma, Hayward, CA_.

The study of dynamic cell processes and their interactions is of crucial importance to understand the complexities of tissue microenvironments. Simulation of in vivo cell culture microenvironments can greatly improve biologic relevance of cell-cell interaction studies, and so there is an ongoing demand for heuristic optimization of cell co-culture devices and protocols. One particularly challenging task is establishing real-time image analysis while co-culturing non-adherent cells in contact with adherent cell layers under precisely controlled environmental parameters. To address this challenge, we present a novel microfluidic approach to trap two different cell types in precise locations in order to image and study their interactions over time. The platform utilizes a well plate format that contains multiple microfluidic units. Each unit consists of a 3 mm x 3 mm x 40 µm (L x W x H) chamber for culture, and an array of microscopic trap areas tailored to the dimensions of the cells of interest, typically ~15 µm. The micro-scale geometries of the traps physically confine non-adherent cells, and co-cultures can be achieved by trapping the non-adherent cells over a monolayer of cells. The microfluidic plate is integrated with a system which enables perfusion-based nutrient supply along with gas and temperature control for long-term cell culture. Each chamber within the plate can be addressed by programmable and on-demand perturbation of up to 6 reagents, enabling uninterrupted real-time live cell imaging assays. We present here the use of this microfluidic platform to replicate tumor microenvironment by maintaining adjacent co-cultures of cancer and immune cells. We have successfully imaged and cultured monolayers of tumor cell lines for 7 days followed by subsequent loading and trapping of immune (non-adherent) cells. In conclusion, the microfluidic platform enables unique co-cultures with environmental control and real-time imaging to facilitate the investigation of cell-cell interactions in a wide range of applications such as drug response and screening in tumor microenvironments, invasion, evasion pathways and other mechanisms governing cell-cell interactions.

#878

A new SOX2/OCT4 stem cell biosensor reveals the mechanism of cancer stem cell dissemination in human breast cancer.

Ved P. Sharma,1 Sonia Voiculescu,1 Yarong Wang,1 George S. Karagiannis,1 Binwu Tang,2 Lalage Wakefield,2 David Entenberg,1 Sumanta Goswami,3 Maja Oktay,1 John Condeelis1. 1 _Albert Einstein College of Medicine, Bronx, NY;_ 2 _National Cancer Institute, Bethesda, MD;_ 3 _Yeshiva University, New York, NY_.

There is increasing consensus that cancer stem cells (CSC) play an important role during metastatic progression of breast cancer. Previous studies have shown that stem cells display a pro-invasive phenotype in breast cancer. However, the phenotypes specific to breast cancer stem cells have not been evaluated at single cell resolution in vivo. Here, we employ high-resolution intravital two-photon microscopy in both orthotopic xenograft tumors, and their metastases, formed from human breast cancer cell lines expressing a previously characterized SOX2/OCT4 transcription-based fluorescent stem cell biosensor (SORE6). Using this high resolution imaging technology we found that SORE6+ stem cells: (a) constitute a minority population of the primary mammary tumors, (b) move approximately ten times slower than non-stem breast cancer cells (0.1 vs 1.1 µm/min, respectively), (c) are migratory toward blood vessels and, (d) compared to non-stem cells, they are enriched for invasive cellular protrusions called invadopodia. This is important because we have shown that these phenotypes are specifically associated with the disseminating population of tumor cells in the primary tumor site and, in addition, invadopodia are required for transendothelial migration during intravasation. Stem cells also have a three-fold higher incidence of direct contact with macrophages compared to non-stem cells. In fact, stem cells were frequently seen to be part of the tripartite macrophage-tumor cell- endothelial cell complex called TMEM, which was previously shown to be the doorway for intravasation of tumor cells in primary mammary tumors and is validated as a prognostic of metastasis in human breast cancer patients. Furthermore, we followed breast cancer stem cell dissemination to the lung by using a novel lung window for high resolution imaging of the lung (WHRIL), which allows visualization of the same lung tissue in a single mouse, serially, over days to weeks. Using WHRIL, in combination with intravital multiphoton microscopy and the above biosensor, we visualized the arrival, extravasation and outgrowth of spontaneously arriving breast tumor cells in the lung. We report here for the first time, the kinetics and stemness state of the spontaneously metastasizing tumor cells and, the record at single cell resolution, of their fate and metastatic outgrowth in the lung over time.

#879

Multi-scale time-lapse intravital imaging of soft tissues to map single cell behavior.

Jessica M. Pastoriza,1 Maria Soledad Sosa,2 Kathryn Harper,2 Julio Aguirre-Ghiso,2 Maja H. Oktay,1 David Entenberg,1 Yarong Wang,1 John S. Condeelis,1 Aviv Bergman,1 Mihaela Skobe,2 Benedicte Lenoir2. 1 _Albert Einstein College of Medicine, Bronx, NY;_ 2 _Icahn School of Medicine at Mount Sinai, New York, NY_.

After more than 10 years of research into the tumor micro-environment and the sources of tumor micro-heterogeneity, it is becoming increasingly clear that in addition to driver mutations, the tumor microenvironment determines tumor metastatic phenotype. This research has led to a new understanding of the impact of tumor microenvironments and their heterogeneity upon tumor cell proliferation, dissemination, dormancy, and survival. A full understanding of this heterogeneity, both temporally and spatially, how it supports tumor cell dissemination, dormancy and eventual further metastatic growth, and how it responds to therapeutic interventions, is crucial. Traditional single parameter studies have not been as productive as hoped in revealing the above relationships and have highlighted the need to understand tumors as integrated systems of genes, gene networks, and intracellular interactions, particularly with regard to the interplay between cells and their immediate microenvironment. To accomplish this, we have developed a new technology in the form of large volume intravital imaging using multiphoton intravital microscopy (MIVM) where images of large tumor volumes are stitched together to form a comprehensive record of the genes, gene networks, and intracellular interactions that occur throughout many tumor microenvironments at single cell resolution. Here, we report a protocol for obtaining large area high-resolution mosaic imaging in living animals. The protocol is composed of surgical techniques for the stabilization of soft tissues including mammary gland, lymph node, liver, and lung and the acquisition of multiple high-magnification tiles that are stitched together to form a large-area low-magnification image. We have developed specific protocols for the surgery as well as tools for tissue stabilization and the acquisition and stitching of the images to generate very large MVIM data sets. We have interfaced these very large MVIM image data sets with support vector machine (SVM) classification, a nonlinear, multiparametric classification algorithm suitable for analysis of systems with arbitrary distributions and/or non-linear parameter's correlations. To define the combinations of microenvironment parameters where tumor cell phenotypes of interest are likely to occur, we used these microenvironment parameters as an "input" for the SVM classifier to identify associated tumor cell phenotypes as the classifier's "output". Using this approach at widely varying temporal and spatial scales (from minutes to weeks and from sub-cellular to tissue wide) and at different stages (early carcinoma on to metastasis), has resulted in a catalog of associations between microenvironment conditions and tumor cell phenotypes associated with progression. These associations resulted in new insights into the mechanisms of metastasis.

#880

A computational and statistical approach for interpreting real-time in-vitro gene reporter data.

Alexander T. Pearson,1 Dan Sun,2 Zhengda Li,2 Jacques Nor,3 Qiong Yang,2 Laura Buttitta2. 1 _Univ. of Michigan Health System, Ann Arbor, MI;_ 2 _Univ. of Michigan, Ann Arbor, MI;_ 3 _Univ. of Michigan Dental School, Ann Arbor, MI_.

We have developed a new computational approach to track and analyze data from cells engineered with fluorescent cell cycle gene reporters. Gene reporters are a rapidly growing tool to gather information captured with time-lapse imaging, and can give insights to real-time metabolic conditions, cell cycle state, cancer stem cell state, and many other applications. Gene reporters may reflect cellular conditions that are cyclical rather than monotonic. Currently, the primary mechanism for interpretation of time lapse gene reporter data is reporting the mean fluorescence of images over time. This simplistic analysis discards information such as the cycle length and variation between of gene reporters between cells, and can be biased due to the overall fluorescence shift from population growth. More nuanced data acquisition and analysis approaches have been limited due to requirement of laborious hand-on data analysis. We have implemented a high-throughput computational pipeline for interpretation of cell cycle information using a combination of commercial and newly developed software to partition the activities of individual cells. The fluorescence information for each cell is smoothed and the cycle states for each cell are assigned adaptively. Our approach captures and quantifies the total time each individual cell spends in each presumptive state, and then analyses state information from the cohort of observed cells using censored survival time data methods such as the Cox Proportional Hazards Model to account for cell death or movement out of the observed area. As a proof of concept for our pipeline, we engineered 3T3 fibroblast cells with a cell cycle reporter construct including mCherry-CDT1 and mVenous-p27K- reporters which can discriminate cells in G0 phase from G1 phase. We calculated the sample size required for statistical power of 80% to detect a hazard ratio (HR) of 0.8 between groups at α = 0.05 at 386 tracked cells per group. As our treatment model, we used the Protein Phosphatase 2A inhibitor Okadaic Acid (OA) an agent known to promote departure from G0. We captured a distinct total of 601 vehicle cells and 479 treatment cells in a 24 hour monitoring period with 20 minute capture intervals. We were able to discern subtle differences between control and treatment G0 time, including a subtle, statistically significant decrease in G0 time after treatment with OA (p=0.0166, HR = 0.84). Here we have developed a computational pipeline with the capability for high-throughput image-stack data processing and nuanced quantification. Our approach assigns cycling cell states adaptively and the time until each cell departs its current state is tallied and interpreted statistically with survival time statistical analysis. The cell-level analysis affords a high degree of statistical power to discriminate group differences. Our pipeline is highly adaptable and has a multitude of applications in cancer biology research.

#881

CD44-specific supramolecular hydrogels for fluorescence molecular imaging of EMT induced BRAF <V600E> mutant thyroid cancer cells.

Hyung Kwon Byeon,1 Minhee Ku,1 Yeon Ju Yang,1 Min Hee Cho,1 Yoojung Oh,1 Jae Wook Kim,2 Myung Jin Ban,2 Ji-Hoon Kim,3 Da Hee Kim,1 Joo Hyun Kim,1 Jaemoon Yang,1 Yoon Woo Koh1. 1 _Yonsei University College of Medicine, Seoul, Republic of Korea;_ 2 _Soonchunhyang University College of Medicine, Seoul, Republic of Korea;_ 3 _Yonsei University Wonju College of Medicine, Wonju, Republic of Korea_.

Previously, the authors have identified that the acquired drug resistance to BRAF inhibitor, PLX4032 in BRAF (V600E) mutant anaplastic thyroid cancer promotes not only tumor progression and proliferation, but also migration and invasion of cancer through upregulated epithelial-to-mesenchymal transition (EMT). The underlying mechanism to the acquired resistance to BRAF inhibition involves c-Met-mediated reactivation of PI3K/AKT pathway. Therefore combinatorial dual targeted therapy of BRAF and c-Met inhibition has shown to reverse EMT and show maximal antitumor effect. Previously, the authors have developed a novel in vivo imaging strategy using CD44-targetable near-infrared (NIR)-sensitive supramolecular hydrogels (NIRSHs) for the recognition of CD44-expressing cancer cells. In the present study, we applied this NIR-sensitive molecular imaging probe in detecting the upregulated EMT changes in PLX4032-treated 8505C cells. The CD44-targetable NIRSHs were fabricated by polyplexing Cy5.5-conjugated polyethyleimine and hyaluronic acid in an aqueous medium. Ectopic xenograft mouse models were prepared by injecting 8505C cells at the flank of male athymic nude BALB/c mice, aged 6 weeks. After confirming tumor formation at 3 weeks post-injection, the mice were randomly divided into four groups and were each treated under different conditions; DMSO, PLX4032, PHA665752, PLX4032 and PHA665752. After 3 weeks, the pre-established NIRSH probes were injected and confirmed by IVIS imaging. The injected NIRSH probes showed highest uptake in the PLX4032 single treatment group and lowest uptake in the PLX4032 and PHA665752 combination group. Sizes of tumor were verified by MRI which showed correlations with the NIRSH fluorescence imagings. The results suggest that CD44-targetable NIRSHs imaging shows potential as a non-invasive in vivo imaging tool in detecting the increased invasion potential of cancer cells and monitoring appropriate therapeutic effects.

#882

Interpreting glioma MR imaging and somatic mutations in a cancer hallmark context.

John Graf,1 Mirabela Rusu,1 Yunxia Sui,1 Dattesh Shanbhag,2 Uday Patil,2 Jeffrey Kiefer,3 Jill Barnholtz-Sloan,4 Michael Berens,3 Fiona Ginty,1 Sandeep Gupta,1 Chinnappa Kodira,1 Lee Newberg,1 Anup Sood1. 1 _GE Global Research, Niskayuna, NY;_ 2 _GE Global Research, Bangalore, India;_ 3 _Translational Genomics Research Institute, Pheonix, AZ;_ 4 _Case Comprehensive Cancer Center, Cleveland, OH_.

Extracting biologically relevant data from radiology images can enable better monitoring of disease progression and therapy response. The field of radiogenomics is providing new approaches for such genomic/radiology correlations. However, there are several challenges in validation and clinical translation in that few DNA mutations are shared between tumors from different individuals and the differences in scale between imaging and genomic features can limit interpretation of underlying mechanisms. The goals of this work were to i) analyze correlations between low grade glioma (LGG) DNA somatic mutations, using a novel DNA impact scoring approach, and MRI derived imaging features; and ii) to interpret results in context of cancer hallmarks1. Multi-parametric MRI and corresponding DNA data from 32 LGG patients were extracted from The Cancer Genome Atlas (TCGA) and The Cancer Imaging Archive (TCIA). The cohort included 18 males (56%), with mean age of 44 years (range: 21-74 years). An expert radiologist outlined the normal and tumor regions of interest using ITK-Snap tool. The normal region was used as a reference to normalize image intensities in the tumor region. Tumor mean intensity and mean variance were computed from Apparent Diffusion Coefficient (ADC), T1 enhancement ratio (derived from T1 pre- and post- contract MRI), and Fluid-Attenuated Inversion Recovery (FLAIR) images. A novel algorithm was used to compute DNA impact scores for each somatic mutation. The score represents the probability of a DNA variant being pathogenic vs. nonpathogenic. First, the scoring algorithm computes a score for nucleotide base insertions, deletions, or single base changes and then computes the consequence of such changes on amino acid coding, binding sites, splice sites and protein phosphorylation sites. An impact score was then computed based on the individual DNA impact scores of mutations within the gene. Finally, an average DNA impact score was computed at the Cancer Hallmark level using a gene-cancer hallmark map. At gene level, significant positive correlations were found between the ATRX (p=0.0002), TP53 (p=0.02) and ADC mean intensity. At pathway level, regulation of TP53 expression and degradation, and DNA damage response, signal transduction by p53 class mediator, and DNA translocase activity were found to be enriched with genes that correlated with ADC and FLAIR. These pathways also contained genes that were enriched in the following cancer hallmarks: replicative immortality, evading growth suppression and genome instability. The ATRX gene is a member of all three hallmarks and TP53 a member of two. Since ADC is a measure of water diffusion and hence an indirect measure of cellularity, these findings demonstrate that mutations in replication and repair pathways are contributing to imaging features at the tumor level.1 Hanahan, D. and Weinberg, R.A. (2011). Hallmarks of cancer: the next generation. Cell 144(5):646-74.

#883

**Elucidating cancer hallmark context from glioma MR imaging and RNA expression data** **.**

Yunxia Sui,1 Mirabela Rusu,1 Dattesh Shanbhag,2 Uday Patil,2 Jeffrey Kiefer,3 Jill Barnholtz-Sloan,4 Michael Berens,3 Fiona Ginty,1 Graf John,1 Sandeep Gupta,1 Chinnappa Kodira,1 Lee Newberg,1 Anup Sood1. 1 _GE Global Research, Niskayuna, NY;_ 2 _GE Global Research, Bangalore, India;_ 3 _Translational Genomics Research Institute, Pheonix, AZ;_ 4 _Case Comprehensive Cancer Center, Cleveland, OH_.

Radiogenomics or radiomics is an emerging field where tumor genomic data is correlated with radiology image features, thereby potentially providing more biological information about the tumor phenotype. A central challenge is the potential for model over-fitting due to analysis of many thousands of genomic data-points with hundreds of corresponding patient image features. Biological interpretation of the imaging feature correlations is also challenged by overlapping pathways and common gene effects. Our goals were: i) to explore correlations between gene expression and corresponding Magnetic Resonance (MR) Apparent Diffusion Coefficient (ADC) derived imaging features in low grade glioma (LGG); ii) to classify significant gene and imaging correlates by cancer hallmark1. RNA expression data from 32 LGG patients were extracted from The Cancer Genome Atlas (TCGA) and matched with corresponding MR image data from The Cancer Imaging Archive (TCIA). Among 32 patients, 18 were males (56%), and ages ranged from 21 to 74 years (mean age 44). Tumor and normal regions in the MR images were annotated by an expert radiologist using ITK-Snap. The normal reference region was used normalize image intensities in corresponding tumor regions. Tumor texture features were computed on ADC Maps at each voxel location within the disease region (including first and second order statistics, Run Length and co-occurrence matrix derived measures features. The voxel features were finally aggregated within the tumor region using statistical measures of mean, variance, median, kurtosis, and skewness. ADC imaging features (n=310) were correlated with each single gene expression value (11614 genes after MAD>0.4 filtering). Only image features and genes with pairwise correlations higher than 0.68 (0.68 is the 3-standard deviation above average correlation) and FDR (False Discovery Rate) <0.1 were used for follow-up analyses. Significant genes and MR image features were aggregated into 3 groups based on gene expression and correlated with cancer hallmarks. Seven Haralick image features (reflecting the average level of image intensity heterogeneity) were independently, significantly correlated with the Angiogenesis Hallmark (FDR all < 0.001). Three Haralick image features (reflecting asymmetric distribution of intensity) were significantly correlated with the Activating Invasion and Metastasis Hallmark (FDR all < 0.001). Validation of these findings in additional LGG cases with additional imaging protocols and features is ongoing. Radiogenomics informed by genomic profiling may usher in processes to infer cancer hallmarks to aid treatment planning and prognosis of glioma patients.1 Hanahan D and Weinberg RA (2011). Hallmarks of cancer: the next generation. Cell 144(5):646-74.

#884

Detection of intratumoral heterogeneity using MR-defined tumor habitats in breast cancer model under melatonin treatment.

Bruna V. Jardim-Perassi,1 William Dominguez-Viqueira,2 Asmaa El-Kenawi,2 Dominique F. Abrahams,2 Mikalai Budzevich,2 Epifanio Ruiz,2 Suning Huang,2 Pedro M. Enriquez-Navas,2 Gary Martinez,2 Debora Aparecida Pires de Campos Zuccari,1 Robert J. Gillies2. 1 _Faculdade de Medicina de São José do Rio Preto, São José do Rio Preto, Brazil;_ 2 _Moffitt Cancer Center, Tampa, FL_.

Breast cancer shows high intratumoral heterogeneity, which can be visualized as localized sub-populations of tumor cells with different malignancy potential and treatment responsiveness. These regions are like "habitats" that contain different combinations of environmental selection forces, such as hypoxia, acidosis, or nutrient availability. Thus, agents that can perturb these forces may be useful in limiting cancer progression. Studies have shown that melatonin can affect tumor metabolism and we hypothesize that this may be a mechanism by which the disruption of circadian melatonin production by light exposure at night (LAN) promotes breast cancer initiation and progression. The aim of this study was to evaluate the effect of melatonin on intratumoral heterogeneity measured by non-invasive MR imaging in a syngeneic mouse model of breast cancer (4T1). To visualize habitats, clusters were generated from combined MR images generated by different pulse sequences. These were co-registered with histology using tumor specific 3-D printed cradles to classify these tumor habitats. Methods: Mice were exposed to LAN (Control) or LAN plus melatonin. The melatonin metabolite 6-sulfatoxymelatonin (6-SMT) was assessed in urine as a measure of melatonin production. Mice were imaged in a 7T MRI system with: 1) anatomical T2 images, 2) T2*, 3) diffusion-weighted MRI (DWI); and 4) T1 weighted dynamic contrast enhanced (DCE). DCE maps were obtained through semi-quantitative analysis of the pre- and post-contrast agent bolus time-series on a pixel-by-pixel basis. These were combined to the corresponding T2, T2* and Apparent Diffusion Coefficient (ADC) values to generate parameter maps. Parameter maps were used to classify multiple clusters, based on a Gaussian mixture model. Following euthanasia, a 3D printed mold based on the T2 tumor isosurface was created in order to co-register the MRI and histology. Tumors were stained with H&E and for pimonidazole (hypoxia) by immunohistochemistry. Results: Melatonin treatment increased the 6-SMT in urine of mice exposed to LAN and statistically reduced tumor growth by day 15 (p<0.05). Distinct hypoxic habitats have been observed by MRI. Habitat clusters with low values in ADC (high cellularity) and low contrast enhancement were associated with positive staining of pimonidazole. Initial results suggest that the melatonin-treated mice had more homogeneous tumors, although additional numbers will be needed to reach statistical significance. Conclusion: These data indicate that the spatial heterogeneity of breast tumors, corroborated by histological features, may be used as a non-invasive imaging method for monitoring the changes in intratumoral heterogeneity following cancer therapy.

#885

Live-imaging reveals the correction of skin deformities within two tumor-associated mutational models.

Cristiana Pineda, Samara Brown, Valentina Greco. _Yale University, New Haven, CT_.

Healthy tissues are frequently subject to oncogenic mutations that fail to produce malignancy. Such mutations can lead to aberrant cellular behaviors such as abnormal differentiation or hyperproliferation and the subsequent formation of aberrant tissue growths and cancer. However, it remains unclear how phenotypically normal tissue is capable of harboring mutations and raises the question as to how the switch from normal to malignant is initiated. One challenge to understanding the initiating events towards malignancy is the inability to follow the same cells over time in an intact mammal. Specifically, this roadblock hinders the ability to understand both the role of specific cells and how their location contributes to their growth, whether that growth be normal or cancerous. To overcome this, we have established a novel live imaging approach to perform in vivo lineage tracing of the same mutant cells over time within two distinct mutational systems in the skin epithelium.

To begin, we turned to the pro-differentiation pathway Wnt/β-catenin, which when mutated is known to drive benign growths in the skin, and activated this mutant form in skin stem cells. We show that the resulting growths that deform the skin tissue architecture spontaneously regress, irrespective of their size. Endogenous behaviors such as differentiation and apoptosis as well as ectopic cell extrusions are employed to eliminate mutant cells from the tissue and dismantle the aberrant structures. Following regression, the remaining structures are either completely eliminated or converted into functional skin appendages in a niche-dependent manner. While these results demonstrate the remarkable capacity to reverse aberrant phenotypes, the question remained whether this phenomenon was Wnt/β-catenin specific. To address this, we next turned to mutant Hras, which is well established to cause a hyperproliferative, pro-growth advantage and frequently found mutated in malignant skin tumors such as cutaneous Squamous Cell Carcinoma. Activation of mutant Hras in the skin epithelium produced a range of phenotypes from hyperthickening to follicular structural deformities to macroscopic skin growths, all of which demonstrated the capacity to correct. Currently, we are utilizing a combination of extensive biochemical characterization together with targeted cellular ablations to investigate the mechanisms at play both within the mutant pool and the surrounding normal tissue that leads to the reversibility and integration observed within the Hras model. Altogether, this study reveals an unanticipated plasticity of adult skin epithelium when faced with mutational insult and elucidates the dynamic cellular behaviors employed for its return to a homeostatic state.

#886

An integrative platform for three-dimensional quantitative analysis of spatially heterogeneous metastasis landscapes.

Ian H. Guldner, Lin Yang, Fang Liu, Danny Z. Chen, Siyuan Zhang. _University of Notre Dame, Notre Dame, IN_.

Metastatic microenvironments are spatially and compositionally heterogeneous. This seemingly stochastic heterogeneity provides researchers great challenges in elucidating factors that determine metastatic outgrowth. Herein, we develop and implement an integrative platform that will enable researchers to obtain novel insights from intricate metastatic landscapes. Our two-segment platform begins with whole tissue clearing, staining, and imaging to globally delineate metastatic landscape heterogeneity with spatial and molecular resolution. The second segment of our platform applies our custom-developed SMART 3D (Spatial filtering-based background removal and Multi-chAnnel forest classifiers-based 3D ReconsTruction), a multi-faceted image analysis pipeline, permitting quantitative interrogation of functional implications of heterogeneous metastatic landscape constituents, from subcellular features to multicellular structures, within our large three-dimensional (3D) image datasets. Coupling whole tissue imaging of brain metastasis animal models with SMART 3D, we demonstrate the capability of our integrative pipeline to reveal and quantify volumetric and spatial aspects of brain metastasis landscapes, including diverse tumor morphology, heterogeneous proliferative indices, metastasis-associated astrogliosis, and vasculature spatial distribution. Collectively, our study demonstrates the utility of our novel integrative platform to reveal and quantify the global spatial and volumetric characteristics of the 3D metastatic landscape with unparalleled accuracy, opening new opportunities for unbiased investigation of novel biological phenomena in situ.

#887

Magnetic relaxometry detection of stealth, antibody-targeted micellar iron oxide nanoparticles in-vivo.

Rebeca Romero Aburto,1 Konstantin Sokolov,1 Adam M. Kulp,1 Erika C. Vreeland,2 Zhen Lu,1 Robert C. Bast,1 John D. Hazle,1 Kelsey B. Mathieu1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Senior Scientific LLC, Albuquerque, NM_.

Magnetic relaxometry (MRX) has the potential to provide unprecedented sensitivity in early detection of cancer by sensing changes in magnetic relaxation of iron oxide (Fe3O4) nanoparticles targeted to cancer biomarkers and is expected to exceed the detection limits of established clinical modalities. MRX uses superconducting quantum interference device (SQUID) sensors to measure Neél relaxation of bound particles. Our strategy was to develop molecular specific Fe3O4 nanoparticles using amphiphilic functionalized phospholipids that allow for clinical translation of the MRX technology. To accomplish this, we used automated, controlled rate, direct infusion of an organic phase mixture of phospholipids and nanoparticles into water to produce monodisperse micellar nanoparticles with a mean diameter of 75±12 nm and surface charge of -10mV. The particles were determined to be stable in various biological media, including human plasma, for more than 24 hours with no detectable formation of a protein corona. Furthermore, in-vivo studies in healthy mice showed blood circulation times of more than 2 hours, as well as minimal MRX signals during this time. Additionally, we developed maleimide conjugation chemistry for epidermal growth factor receptor (EGFR) antibody attachment to micellar nanoparticles. We have achieved molecular specific labeling of cancer cells over-expressing EGFR. In the future, we will evaluate the MRX signal impact from injecting EGFR-conjugated nanoparticles into tumor-bearing mice.

#888

Volumetric reconstruction of targeted nanoparticles for superparamagnetic relaxometry.

Sara L. Thrower,1 Kelsey Mathieu,2 Wolfgang Stefan,2 Zhen Lu,2 Robert C. Bast,2 Javad Sovizi,2 David Fuentes,2 John D. Hazle2. 1 _The University of Texas Graduate School of Biomedical Sciences at Houston; The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Superparamagnetic relaxometry (SPMR) is an emerging technology that uses the unique magnetic properties of superparamagnetic iron oxide nanoparticles (SPIONs) to detect cancer cells. In order to estimate tumor locations from raw MRX data, we developed an L1 reconstruction algorithm under the assumption that early stage disease is sparsely distributed throughout the anatomy. The approach was previously validated in phantom datasets of known signal locations. Advantages of our method are that the solver does not require the user to input prior information regarding the expected number of tumors or their approximate locations. Additionally, the solver reconstructs a volumetric distribution of detected sources within the field of view. To validate the algorithm for use in preclinical settings, SPMR was performed on SKOV3 ovarian tumor bearing mice (n = 3) with the MRX device over time following an intratumoral injection of anti-Her2 antibody-conjugated 25nm SPIONs (Senior Scientific LLC). The SPMR data was reconstructed with our sparse solver and was found to be highly correlated (r = 0.9978) with the results generated by the commercial software that accompanies the MRX instrument (MSA). Additionally, segmentation of the reconstruction revealed a strong signal (2.0·106 pJ/T) in the area of the tumor and almost no signal in areas outside of the tumor (0.077 pJ/T) at four hours after injection. This result was consistent with our prior observations which have revealed that a large fraction of intratumorally-injected nanoparticles remain localized within the tumor for several hours after injection. Furthermore, these results were consistent with SPMR data collected by measuring tissue samples excised 24 hours after injection, of which the tumor had the highest signal. Thus, our sparse reconstruction algorithm was able to return the expected results without prior information regarding the location of nanoparticles. Future work will focus on quantifying the uncertainty in our reconstruction method, as well as characterizing its stability with increasingly complex nanoparticle distributions and detectability limits. In conclusion, this work represents an important advancement of the SPMR technology by allowing for volumetric reconstructions of bound nanoparticles from in vivo data.

#889

A comparison between quantitative ultrasound and histology in heterogeneous tumor structure.

Jerome Griffon,1 Delphine Le Guillou-Buffello,1 Oumeima Laifa,1 Alexandre Dizeux,1 Micheal Oelze,2 Lori Bridal,1 Michele Lamuraglia1. 1 _Laboratoire d'Imagerie Biomedicale (UPMC, CNRS, INSERM), Paris, France;_ 2 _Bioacoustics Research Laboratory, University of Illinois at Urbana-Champaign, Urbana-Champaign, IL_.

Background: To investigate how QUS (Quantitative Ultrasound) maps reflect regional variations in heterogeneous tumor microstructure, we compared like-regions of QUS effective scatterer diameter (ESD, μm) and effective acoustic concentration (EAC, dB/cm) maps to whole-slice, virtual histology sections stained for vascular, cellular and other microstructural features.

Methods: An ectopic model of murine colorectal carcinoma (CT26) was implanted in 7 mice (BALB/c). US radiofrequency data were acquired for the longitudinal plane of tumors in vivo (SSI, Aixplorer, SL15-4 probe, research mode) on days 6 and 14 after start of therapy with an anti-angiogenic (AA: n = 3) or placebo (P: n = 4). The AA used is Pazopanib, an oral tyrosine kinase inhibitor of VEGF receptors 1/-2/-3, platelet-derived growth factor receptors (PDGFR) -alfa/-beta, and c-KIT. A daily dose of 2 mg of AA, diluted in 100µl of solution, was administered per os. Average backscattered power spectra were calculated (FFT, 512 pts, Rectangular gated RF), normalized with respect to a reference phantom and corrected for attenuation (0.4 dB/cm/MHz). ESD and EAC were mapped (2 x 2 mm blocks, with 75% axial and lateral overlap). Tumors were excised and marked to conserve orientation and approximate position relative to US imaging plane. Whole-slice, histological sections were prepared with fluorescent immunohistochemical markers at D6 and D14 for invasive assessment of cell nuclei, apoptosis and vascular endothelium. The Number of Nuclei (NU), the % area of Apoptosis (AP) and % area of Vascular (VA) marker were calculated with image analysis processing for each 333µm by 333µm region of histological sections. QUS and histological maps of each tumor were manually segmented and coregistered using an affine registration method so that matched regions could be compared.

Results: The absolute value of the non-parametric Spearman correlation coefficient (RS) was computed separately for each mouse to study the correlation between histological parameters and QUS parameters. Two mice of the AA group showed a moderate correlation (0.4<=RS<0.6) between NU and EAC. Only one mouse in the P group showed strong correlation (0.6<=RS) for [AP vs. ESD], [AP vs. EAC], [VA vs. ESD] and [VA vs. EAC]. The other tumors (1 from AA group and 3 from P group) showed poor correlation (RS<0,4). Additional data is available from 14 other mice (7 from AA group and 7 from P group) and will be analyzed to more fully investigate correlation between the QUS parameters and the histological parameters.

Conclusions: This original technique to compare QUS parameter maps and whole slice histology analysis shows a promising evolution for this non-invasive imaging technique. Complete results on 21 mice will be presented in April. 

### Migration and Invasion 1

#890

The role of calpastatin isoforms in breast cancer progression.

Bhudsaban Sukkarn, Sarah Storr, Ian O. Ellis, Kirsty Jewell, Tim Parr, Stewart G. Martin. _The University of Nottingham, Nottingham, United Kingdom_.

Calpastatin is the endogenous inhibitor of the ubiquitously expressed m-calpain and μ-calpain. Both of them are important regulators of various cellular pathways including proliferation, apoptosis, adhesion, and migration. Dysregulation of the calpain system is associated with a wide range of pathologies including tumour invasion and metastasis. In breast cancer, down-regulation of calpastatin mRNA, in particular an exon 3 containing variant, is associated with lymphatic vessel invasion which is the predominant form of lymphovascular invasion (LVI) (96%), a critical initial step of metastases. The current project seeks to determine the role that individual calpastatin isoforms play in regulating breast cancer cell migration and endothelial interactions to understand processes involved in regulating LVI.

Seven breast cancer cell lines were assessed for calpain system protein expression (calpain-1, -2, -4 and calpastatin) and calpastatin (CAST) mRNA expression (total CAST, CASTexon3, CAST I, II and III) by Western blotting and qRT-PCR, respectively. T47D and MDA-MB-231 cell lines showed low calpastatin but high calpain expression. From haptotaxis cell migration data, T47D migrated more slowly than MDA-MB-231 (35.49±6.14 and 80.34±5.52 percent wound closure at 24 hours post-scratch respectively) (P value=0.0233). These cell lines, that represent different subtypes of breast cancer (luminal and basal respectively), were therefore selected to study the role of differential calpastatin type/isoform expression.

GFP-tagged XL and Leader domains of calpastatin type I, II and III, with or without exon 3 were overexpressed in each cell line. The different calpastatin types show differential subcellular localization. In MDA-MB-231, type I, III, IΔ3, IIIΔ3 were expressed generally in cytoplasm whereas type II and IIΔ3 were located as punctate nuclear invaginations. To assess if overexpression of the different calpastatin types cause phenotypic changes, full length HA-tagged calpastatin type I, II and III were transfected into MDA-MB-231 and T47D and stable clones produced. Thus far stable type II overexpression in MDA-MB-231 and type III in T47D have been obtained with others under single cell selection. The localization of full length type II in MDA-MB-231 was similar to the localization of the truncated form that showing a strong single perinuclear signal. In T47D, the full length and truncated type III also show similarly localization that expressed throughout the cytoplasm. For the effect of calpastatin types on cell proliferation, calpastatin type II in MDA-MB-231, and type III in T47D, had no significant effect on cell doubling time when compared to respective controls (P value=0.6400 and 0.8874, respectively). According to preliminary studies, calpastatin type II seems to have no effect on clonogenicity of MDA-MB-231. Such effect on T47D and other phenotypic changes involved in regulating LVI of transfected cell are being examined.

#891

Annexin A2 (ANXA2) promotes migration and invasion of esophageal cancer cells via stabilizing c-Myc and promoting HIF-1α transcription.

Sai Ma, Yan-Yi Jiang, Li-Fei Wu, Jia-Jie Hao, Yu Zhang, Xin Xu, Yan Cai, Ming-Rong Wang. _State Key Laboratory of Molecular Oncology Cancer Institute, National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China_.

Purpose:

We previously revealed that the expression of Annexin A2 (ANXA2) was increased in ESCC by two-dimensional electrophoresis and MALDI-TOF and LC-ESI-IT MS. This study aims to investigated the implication of Annexin A2 in ESCC.

Annexin A2 (ANXA2) promotes migration and invasion of esophageal cancer cells via stabilizing c-Myc and promoting HIF-1α transcription.

Experimental Design:

Immunohistochemistry was performed to analyze the expression of ANXA2 in esophageal squamous cell carcinomas (ESCC). siRNA knockdown, transwell assay and wound-healing assays were used to investigate the function of ANXA2 in cells. Quantitative reverse-transcription polymerase chain reaction, Western blot analysis were used to seek the potential effector molecule of ANXA2. Chromatin immunoprecipitation, and reporter gene assays were used to confirm the relationship between c-Myc and HIF-1 alpha. Co-immunoprecipitation assay was performed to demonstrate the relationship between ANXA2 and c-Myc. Immunofluorescence was used to reveal the location of ANXA2.

Results:

Knockdown of Annexin A2 significantly inhibited the migration and invasion of ESCC cells. At the molecular level, VEGF, MMP-2, MMP-9, p-MEK and p-ERK were down-regulated in ANXA2-siRNA cells. The HIF-1 alpha mRNA and protein levels were significantly lowered after knocked down the Annexin A2. HIF-1 alpha is the effector molecule of ANXA2 in ESCC cells, and that ANXA2 affects the expression of HIF-1 alpha at the transcription level because when knocking down HIF-1 alpha, the level of VEGF, MMP-2, MMP-9, p-MEK and p-ERK reduced as well. c-Myc is a transcription factor for HIF-1 alpha. ANXA2 has a direct protein interaction with c-Myc and knockdown of ANXA2 down-regulated the expression of c-Myc. The Tyr 24 phosphorylation form of Annexin A2 (Y24D-ANXA2) was located in the nucleus, and the non-phosphorylation form (Y24A-ANXA2), in the cytoplasm and cell membrane. Transfection of pcDNA3.1-ANXA2-Y24D enhanced the migration and invasion as compared with that of pcDNA3.1-ANXA2-Y24A in esophageal cancer cells. A treatment with the Annexin A2 phosphorylation inhibitor, Dasatinib, changed the location of Annexin A2 and lowered the expression of c-myc and HIF-1 alpha.

Conclusions:

These results suggest that in esophageal cancer cells, phosphorylated Annexin A2 could enter into nucleus, which stabilizes c-myc and promotes the transcription of HIF-1 alpha, and the activates MAPK pathway, increases matrix metalloproteinase (MMP) activity and vascular endothelial growth factor (VEGF) expression, which results in the enhancement of cell invasion and migration.

#892

ROCK dependent signalling pathways contribution to collective invasion of colorectal carcinoma.

Fotine Libanje, Joel Raingeaud, Zoé ap Thomas, Fatiha Sangar-Mavouna, Anne Chauchereau Chauchereau, Fanny Jaulin. _Gustave Roussy, Villejuif, France_.

Metastatic progression of cancer, which is responsible for 90% of patients death, results from tumor cells dissemination out of the primary tumor throughout the body. During the first step of this process -that consists in invasion of the peritumoral stroma- cancer cells can adopt 1) a single cell mode of invasion, in which cell have lost cell-cell junctions to move individually, 2) or a collective mode of invasion where cells maintain their cell-cell junctions to move as a cohort in which Leader cells at the front drag the follower cells at the rear. Although tumour histology data from cancer patients show that invasion occurs predominantly in a collective manner, this mode of cell invasion remains underinvestigated. My work aims at identifying the molecular and cellular mechanisms underlying colorectal carcinoma (CRC) collective invasion. I use 3D organotypic models of CRC: Caco-2 cell lines or organoids generated from CRC patients derived xenografts (PDX) and assess invasion in collagen-I based gels using microscopy approaches on fixed or live samples. Knowing its central role in the cytoskeleton dynamics which is the motor of cell motility, we hypothesize that RhoGTPases signaling pathways could control the collective mode of invasion. We therefore performed a siRNA based screen targeting the 98 effectors of the pathway and found ROCK to be an anti-invasive protein. Although it had been described as a proinvasive protein in the single cell mode of invasion, we confirmed using pharmacological inhibitors (Y27632 and H1152), that ROCK activity inhibition triggered collective invasion. Using a ROCK2 dominant negative mutant specifically targeting ROCK2 isoform but not ROCK1, I demonstrated that ROCK2 inhibition was sufficient to induce leader cell formation leading to collective invasion. In contrast, depletion of MyosinII -ROCK's most common effector- was not sufficient to induce efficient protusive leader cells. However I found RAC1 to be necessary and in a 2nd siRNA based screen targeting GEFs, we identified FARP2, a GEF for Rac1, as the mediator of ROCK-RAC1 crosstalk in collective invasion. Even though the activation of FARP2 alone or RAC1 alone were not sufficient to induce leader cell formation, the concomitant inhibition of MyosinII recapitulated the collective invasion induced by ROCK inhibition. Altogether these results show a new anti-invasive role of ROCK2 kinase in collective invasion as it controls the formation of leader cells, through 1) the negative regulation of RAC1 and its GEF FARP2, and 2) the positive regulation of MyosinII.

#893

Systematic assessment of spliceosome components as drivers of breast cancer progression.

Esmee Koedoot, Sylvia Le Dévédec, Bob van de Water. _Leiden University, Leiden, Netherlands_.

Accumulating evidence indicates that RNA splicing is involved in different steps of carcinogenesis. Although the critical role of a limited number of splicing factors has been demonstrated in several cancer studies, the function of many spliceosome components is still unknown. In breast cancer samples higher expression levels of a subset of spliceosome components consistently correlated with more aggressive tumor types in patient microarray as well as RNA sequencing data from The Cancer Genome Atlas (TCGA). We further systematically evaluated the effect of siRNA-mediated knockdown of 244 individual splicing factors on both cell migration and proliferation in two aggressive breast cancer cell lines MDA-MB-231 and Hs578T using high throughput microscopy. Primary candidates were validated with four single siRNAs and classified based on their effects on migration and proliferation. Knockdown of validated spliceosome factors such as SNRPG and SNRPA1 affected the levels of small nuclear RNAs, the main catalyzers of the spliceosome. Moreover, high expression of these key candidate hits is associated with poor breast cancer metastasis free survival. To investigate the downstream mechanisms and splicing patterns controlled by our candidate genes we performed RNA sequencing on both control and siRNA mediated knockdown cells. Interestingly, knockdown of our hits affect extracellular matrix pathways that are essential in tumor cell migration and invasion. Altogether our data indicate a key role for spliceosome factors in the control of breast cancer progression which provide new leads for future therapeutic intervention.

#894

Heparanase-induced shedding of syndecan-1 promotes cancer cell invasion : prevention by inhibitory synstatin peptide.

Oisun Jung, Alan Rapraeger. _University of Wisconsin-Madison, Madison, WI_.

Syndecan-1 (Sdc1, CD138) is highly expressed in multiple myeloma (MM). Heparanase (HPSE), an enzyme that cleaves heparan sulfate (HS) chains on Sdc1, induces shedding of the Sdc1 ectodomain; this is associated with poor prognosis in this disease. However, the link between Sdc1 shedding and poor prognosis remains unclear. We now show that HPSE-induced shed Sdc1 (sSdc1) engages α4β1 integrin (VLA-4) and vascular endothelial cell growth factor receptor-2 (VEGFR2) to form a ternary receptor complex on the cell surface. This coupling of VEGFR2 to clusters of VLA-4 via sSdc1 leads to autoactivation of VEGFR2 and the subsequent activation of protein kinase A (PKA) bound to the VEGFR2 cytoplasmic domain. PKA activation proceeds via VEGFR2-mediated stimulation of the G-protein coupled cytokine receptor CXCR4 and the subsequent generation of cAMP by its activation of adenylate cyclase 7. This mechanism is prevented by SSTNVEGFR2, a peptide that competes for the VEGFR2 docking site in the Sdc1 ectodomain, thereby disrupting VEGFR2 binding to sSdc1. VLA-4 has been shown to mediate the polarized invasion of a variety of cells due to α4 integrin phosphorylation on Ser988 by PKA, followed by Rac GTPase activation, a process that is restricted to the leading edge of the cell. But how this is confined at the leading edge is wholly unknown. We find that HPSE-mediated shedding of Sdc1 promotes an invasive phenotype by re-localizing VLA-4 and VEGFR2 to the leading edge of migrating myeloma cells as well as human T-acute lymphoblastic leukemia, melanoma, and endothelial cells where VEGFR2-dependent PKA activation causes α4 integrin phosphorylation. These results reveal a novel mechanism in which the expression of HPSE, acting by causing Sdc1 shedding, potentially regulates angiogenesis and the extravasation and invasion of cancer cells and identifies SSTNVEGFR2 as a promising cancer therapeutic.

#895

Fucosylation inhibits invadopodia formation and melanoma invasion.

Tyler S. Keeley,1 Eric Lau,2 Shengyu Yang1. 1 _Penn State University, Hershey, PA;_ 2 _Moffitt Cancer Center, Tampa, FL_.

Background: Melanoma is the most deadly skin cancer and metastasis is essentially responsible for all melanoma related death. Recently, fucose treatment has been shown to inhibit melanoma cell migration and invasion in vitro, and melanoma metastasis in a mouse model, suggesting that protein fucosylation could be exploited to prevent or suppress melanoma metastasis. Invadopodia are proteolytic actin-rich protrusions used by metastatic melanoma cells to degrade extracellular matrix and to promote invasion and metastasis. In this study, we investigated the mechanisms by which fucosylation regulate melanoma invasion and invadopodia formation

Method: The melanoma cells were treated with L-fucose-supplemented medium or infected with fucokinase-expressing lentivirus to increase protein fucosylation. shRNAs were also employed to knockdown fucokinase and to inhibit fucosylation in melanoma cells. The effects of fucosylation on invadopodia formation and extracellular matrix degradation were determined by phalloidin staining and degradation of fluorescently labeled gelatin thin film.

Results: Here, we show that the addition of L-fucose to complete media inhibits the ability of melanoma cells to form invadopodia in a dose-dependent manner. The overexpression of hFUK in WM793 cells showed a similar result as L-fucose treatment, where invadopodia formation was significantly reduced. In addition, we observed a significant decrease in the percentage of cells that have invadopodia during L-fucose supplementation or hFUK overexpression. In an invadopodia precursor assay, the hFUK-overexpressing cell line exhibited delayed FBS-stimulated invadopodia formation.

Conclusions: Our data suggest that fucose or the overexpression of hFUK reduces melanoma invasion by inhibiting invadopodia formation. Given that invadopodia formation was hindered within an hour after stimulation, we believe that invadopodia initiation is affected by increased fucosylation, resulting in fewer invadopodia and slower extracellular matrix degradation.

#896

Long term exposure to Oroxylin A inhibits cell migration via suppressing CCL2 in oral squamous cell carcinoma cells.

Jiun-Jia Tung,1 Tony Jer-Fu Lee,2 Wei-Ting Ku,1 Kuo-Chu Lai1. 1 _Tzu Chi University, Hualien, Taiwan;_ 2 _Buddhist Tzu Chi General Hospital, Hualien, Taiwan_.

Oral squamous cell carcinoma (OSCC) is the sixth most common cancer in the world. Like other cancers, the major causes of OSCC-related death are cervical node and distant metastasis. Therefore, it is a critical need to identify new potential therapeutic agents against OSCC migration, invasion, and metastasis. Oroxylin A, a main bioactive flavonoids extracted from Scutellaria radix, has been reported to inhibit migration in breast and non-small-cell lung cancer. In this study, we explored the anti-migration effects of oroxylin A on OSCC cells and investigated its underlying mechanisms. A 24-hour exposure (short-term) of OSCC cells to oroxylin A at noncytotoxic concentrations (5-20 μM) significantly suppressed cell migration using the wound healing assay. Furthermore, a 30-days exposure (long-term) to oroxylin A (20 μM), which still had no cytotoxic effect on OSCC cells, but significantly suppressed more cell migration than that of short-term oroxylin A exposed OSCC cells. To uncover the molecular mechanisms involved in the inhibitory effect of oroxylin A on OSCC migration, cDNA microarray technique and Ingenuity software were used. There were 468 differentially expressed genes, including 112 upregulated and 356 downregulated genes identified in long-term oroxylin A exposed cells compared to untreated OSCC cells. Among them, 77 genes have been reported to be associated with cancer cell migration. Consistent with results from cDNA microarray, we revealed that the expressions of several cell migration-related genes, such as CCL2, S100A9, LCN2, and THBS1 were significantly decreased in long-term oroxylin A exposed OSCC cells using QPCR assay. Western blotting and ELISA results also demonstrated that CCL2 expressions at mRNA and protein levels were significantly decreased in long-term oroxylin A exposed OSCC cells compared to untreated OSCC cells. In the meantime, the expressions of downstream targets of CCL2, including p-ERK, NFκB, MMP2, and MMP9 were also decreased in long-term oroxylin A exposed OSCC cells. These results suggest that long-term oroxylin A treatment inhibits migration may via suppressing CCL2 in OSCC cells.

#897

PEA-15 regulates epithelial-mesenchymal transition and invasive behavior through its phosphorylation in triple-negative breast cancer.

Jihyun Park,1 Evan N. Cohen,2 Gaurav Chauhan,1 Jangsoon Lee,1 Naoto T. Ueno,1 Debu Tripathy,1 James M. Reuben,2 Chandra Bartholomeusz1. 1 _Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX_.

Triple-negative breast cancer (TNBC) is an aggressive subtype with no proven active targeted therapies available. Patients with TNBC have a very poor prognosis because the disease often metastasizes. Newer approaches to preventing metastasis and inhibiting tumor growth, are crucial to improving prognosis for these patients. Previously it has been shown that PEA-15 binds to ERK, preventing ERK from being translocated to the nucleus and hence blocking its activity. Our previous studies showed that overexpression of wild type PEA-15 inhibited ERK activity and reduced tumor volume in a TNBC xenograft model. We also showed that PEA-15 being unphosphorylated at both Ser104 and Ser116 inhibited ovarian cancer cell tumorigenicity. However, the function and impact of PEA-15 phosphorylation on TNBC is not well understood. From these observations, we hypothesized that unphosphorylated PEA-15 will prevent metastasis in TNBC through the inhibition of EMT. To study the effect of the phosphorylation status of PEA-15 on metastasis in TNBC, we established stable cell lines overexpressing nonphosphorylatable (PEA-15-AA) and phosphomimetic (PEA-15-DD) mutants in MDA-MB-468 cells. The clonogenic growth of PEA-15-AA was significantly reduced by 80% compared with vector control, PEA-15-V. Anchorage-independent growth of PEA-15-AA was inhibited by 60% compared with PEA-15-V (P<0.05). PEA15-AA upregulated the expression of E-cadherin, a key epithelial molecular marker and decreased expression of vimentin suggesting that PEA-15-AA reverses EMT. Moreover, compared with PEA-15-V, migration and invasion of PEA-15-AA were significantly reduced. To determine the mechanism of how PEA-15 regulates EMT and metastasis, we performed RT-PCR immune and metastasis arrays. We found that expression levels of IL-6, IL-8 and PDGF-BB were greatly decreased in PEA-15-AA. Upon stimulation of the PEA-15-AA with these cytokines, mesenchymal characteristics were partially rescued indicating that PEA-15-AA may inhibit the expression of these cytokines, thereby reversing EMT. Further, to determine the in vivo effect of PEA-15-AA, we injected stable transfectants of MDA-MB-468 cells into the mammary fat pad of NOD/SCID mice. The PEA-15-DD-injected group showed an increase (P<0.05) in tumor volume compared with PEA-15-V and PEA-15-AA groups. Additionally, immunohistochemical data revealed that vimentin and p-β-catenin (Ser675) expression was greatly decreased in PEA-15-AA tissues, suggesting that PEA-15-AA has antitumor effects. Further studies are warranted to evaluate the impact of PEA-15 phosphorylation on metastasis in vivo using mice bearing highly metastatic human breast tumor and determine the mechanism underlying decreased cytokine expression in PEA-15-AA. Together, this study highlights the potential for overexpression of unphosphorylated PEA-15 as an approach for TNBC-targeted therapy.

#898

**Elucidation of the molecular mechanism of Mena** INV **expression, invadopodium maturation and tumor cell intravasation during breast cancer dissemination by TMEM.**

Maxwell D. Weidmann,1 Chinmay R. Surve,1 Jeanine Pignatelli,1 Javier J. Bravo-Cordero,2 George S. Karagiannis,1 Maja H. Oktay,1 John S. Condeelis1. 1 _Albert Einstein College of Medicine, Bronx, NY;_ 2 _Icahn School of Medicine at Mount Sinai, Manhattan, NY_.

Numerous clinical studies have identified Tumor MicroEnvironment of Metastasis (TMEM) and MenaCalc as distinct but functionally interrelated prognostic indicators of distant metastasis in breast cancer patients. TMEM sites are the only sites where tumor cells intravasate in mammary tumors. TMEM score is calculated histologically as the density of tripartite microanatomical structures involving a perivascular macrophage, a Mena-expressing tumor cell and an endothelial cell, all three in direct physical contact. On the other hand, MenaCalc represents the pattern of Mena splice-isoforms present in a tumor sample. MenaINV is the key metastasis-promoting Mena splice-isoform driving tumor cell migration toward blood vessels, intravasation and dissemination. However, the precise molecular mechanisms relating TMEM formation and function and MenaINV expression in these critical steps of the metastatic cascade have not been elucidated.

Here we show that MenaINV promotes invadopodium-based proteolysis, which is required for tumor cell invasion and transendothelial migration, by preventing the localization of the phosphatase PTP1B to invadopodia. Interestingly, PTP1B regulates invadopodium maturation by limiting cortactin phosphorylation at a key residue (Y421) that is necessary for actin polymerization during invadopodium maturation. Additionally, we demonstrate that MenaINV expression, invadopodium activity, and subsequent transendothelial migration are induced in tumor cells via Notch1-mediated signaling induced by contact of tumor cells with macrophages. Knock-down of MenaINV expression in tumor cells leads to a proportional decrease in mature invadopodium formation. Complete knock-out of Mena in mouse mammary tumors (PyMT-MMTV) abolishes TMEM assembly and TMEM functions, including TMEM-dependent vascular permeability, circulating tumor cells and lung metastases.

In summary, our work shows that macrophage contact of TMEM-associated tumor cells during TMEM assembly stimulates Notch1 to drive expression of MenaINV in tumor cells. MenaINV expression then inhibits PTP1B at invadopodia to promote cortactin phosphorylation at invadopodium precursors, driving invadopodium maturation and promoting transendothelial migration. These findings provide, for the first time, an integrative molecular mechanism for two clinically validated prognostic indicators of metastatic risk, TMEM and MenaCalc, and identify new drug targets for limiting the metastatic spread of breast cancer.

#899

CD99 plays an important role in glioblastoma cell migration.

Lais C. Cardoso,1 Antonio M. Lerario,2 Suely K. Marie,1 Roseli S. Soares,1 Sueli M. Oba-Shinjo1. 1 _School of Medicine of University of São Paulo, São Paulo, Brazil;_ 2 _University of Michigan, Ann Arbor, MI_.

Glioblastoma (GBM) is the most common and aggressive malignant brain tumor in adults and the standard treatment consists of surgical resection of the tumor followed by radiation and chemotherapy with temozolomide. A combination of standard therapy with other biologically-based therapies is necessary to improve the survival of patients with GBM, which currently stands only to 15-17 months. In this sense many studies have been developed in pursuit of expressed membrane proteins in GBM, which are potential targets for immunotherapy. Our laboratory demonstrated increased CD99 mRNA and protein expression in GBM samples. CD99 has a key role in several biological processes, including cell adhesion, migration, apoptosis, differentiation of T cells and thymocytes, diapedesis of lymphocytes to inflamed vascular endothelium, maintenance of cellular morphology and regulation of intracellular membrane protein trafficking.

In this study, the transcriptome of two GBM cell lines (U87MG and A172) transfected with siRNA for CD99 were analyzed in relation to the non-target control (NTC). A total of 728 genes (hypoexpressed with fold change ≤-1.5 relative to NTC or hyperexpressed with fold change ≥1.5 relative to NTC) were observed in both cell lines. An enrichment analysis by MetaCoreTM revealed the following processes as the most significant: (1) Cytoskeleton_Regulation of cytoskeleton rearrangement, (2) Cell adhesion_Cell-matrix interactions (3) Development_Blood vessel morphogenesis. Further, both cell lines were silenced for CD99 expression by shRNA (two clones) to perform functional assays, as wound healing (migration assay), adhesion, invasion and apoptosis. The CD99 knockdown reduced migration in both cell lines, with the highest decrease of the migration observed in the highest CD99 knockdown. Adhesion assay was performed using fibronectin as an extracellular matrix substrate. U87MG cells showed greater adhesion to fibronectin than the control (scrambled shRNA). On the other hand, A172 cells knocked down for CD99 presented lower adherence to fibronectin than the control. Apoptosis analysis with shCD99 U87MG cells showed a tendency of cells entering into late apoptosis when treated with temozolamide for 48 hours, but the same was not observed in shCD99 A172 cells. Additionally, CD99 (RPE) and phaloidin (Alexa Fluor 488) colocalized at lamellipodia, suggesting that CD99 is related to cytoskeleton rearrangement in both GBM cell lines. The different phenotypic behavior of migration and apoptosis observed in these two GBM cell lines with different somatic mutation background may be due to distinct signaling activation. Migration/invasion is the major characteristics of GBM which limits the surgical tumor resection, and consequently leads to tumor recurrence. Therefore, further analysis of CD99 activating pathways in the context of cell migration is worthwhile to unveil new therapeutic strategies to halt GBM progression.

#900

Tumor Treating Fields (TTFields) affect human glioma cell migration, invasion and adherence properties in vitro.

Dario Garcia-Carracedo,1 Rosa S. Schneiderman,2 Einav Zeevi,2 Anna Shteingauz2,2 Moshe Giladi,2 Tali Voloshin,2 Yaara Porat,2 Mijal Munster,2 Roni Blat,2 Eilon D. Kirson,2 Uri Weinberg,3 Yoram Palti2. 1 _Novocure, New York, NY;_ 2 _Novocure, Haifa, Israel;_ 3 _Novocure, Luzern, Swaziland_.

TTFields, an approved treatment modality for glioblastoma, are delivered via continuous, noninvasive application of low intensity, intermediate frequency alternating electric fields. The antimitotic effects of TTFields have been extensively investigated. We investigated other processes that may be affected by TTFields such as human glioma cell migration and cellular invasion properties. Four human glioma cell lines were treated with TTFields using the inovitro system. Cell migration rates were measured using wound healing assays. Invasion assays were performed using a modified Matrigel coated Boyden chamber. Cell adhesion assays were performed during TTFields treatment and compared to untreated controls. At the end of TTFields' treatment, adhered cells were trypsinized and counted. A cells de-adhesion assay was performed following 24 and 72 hours of TTFields application with the outcome being the number of cells removed after varying times of trypsinization. Application of TTFields in-vitro led to a significant reduction in cell migration velocity compared with untreated control cells. Furthermore, comparison of the anti-migratory efficacy of TTFields applied from 2 directions vs. a single direction revealed that the single direction approach applied perpendicularly to the course of migration was more effective than TTFields applied in 2 directions or in a single parallel direction. Glioma cells invasion was significantly reduced compared to untreated cells in all tested cell lines. Cell adhesion to the substrate (fibronectin) was significantly reduced when exposed to TTFields. On the other hand, cell de-adherence following TTFields treatment took significantly longer time of trypsinization. Our results suggest that human glioma cell motility is impaired by exposure to TTFields. Both alterations in cells adherence and de-adherence during exposure to TTFields may contribute to reduction in cell motility. Further studies are needed to elucidate the mechanism by which TTFields disrupts cellular motility in glioma cancer cells.

#901

The mechanobiome of pancreatic cancer: a viable, targetable drug space.

Alexandra Surcel, Eric S. Schiffhauer, Dustin Thomas, Qingfeng Zhu, Robert Anders, Douglas Robinson. _Johns Hopkins Univ. School of Medicine, Baltimore, MD_.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with an annual mortality rate of 37,000 in the US alone. Its 5-year survival rate of ~6% remains relatively unchanged over the past 4 decades. Because PDAC has elevated rates of de novo and acquired resistance to traditional chemotherapies, we are exploring new drug spaces that target metastatic hepatic disease, the primary mortality factor for patients. Specifically, we have identified the cluster of proteins that sense and respond to mechanical stimuli - collectively known as the mechanobiome. This cytoskeletal machinery is responsible in large part for endowing metastatic cells with the ability to navigate through different tissue types. Based on their mechanoresponsiveness profile, we have predicted and identified via western blot and immunohistochemistry proteins in the mechanobiome that are upregulated in patient-derived pancreatic tissues. These proteins include non-muscle myosin IIA and IIC, alpha-actin-4, and filamin B. We are performing knockdown and overexpression studies of these isoforms on 2D and 3D behaviors - cell mechanics, migration, invasion, and tissue sphere formation and are poised to begin mouse studies on the metastatic sufficiency in altering the expression of these proteins. In addition, we are testing 4-HAP, a small molecule mechanics modulator that we previously identified that targets myosin IIC, thus affecting PDAC mechanics in PDAC murine models. Limited studies reveal that 4-HAP reduces metastasis. These data suggest that direct targeting of the mechanobiome may eventually expand treatment strategies for pancreatic cancer.

#902

Cancer-stem-cell (CSC) marker, DCLK1-S, enhances invasive potential of cancer cells by phosphorylating/activating NFATc2: role of COL3A1 and SPARC in mediating metastatic effects of DCLK1-S/NFATc2.

Malaney O. Connell, Shubhashish Sarkar, Heidi Spratt, Steven Widen, Thomas G. Wood, Pomila Singh. _UTMB, Galveston, TX_.

DCLK1 expression is critically required for colon carcinogenesis in mice, and for maintaining tumorogenic potential of human colon cancer cells (hCCCs) (Singh et al, 2016). Down-regulation of DCLK1, combined with chemoprevention, eliminates CSCs, and avoids colon cancer relapse (Kantara et al, 2014). We recently discovered that long (L) and Short (S) isoforms of DCLK1 (DCLK1-L/DCLK1-S) are transcribed by two separate promoters (5'(α)/IntronV(β)) in the hDCLK1-gene (O'Connell et al, 2015). During adenoma-carcinoma sequence of colon-tumorigeneses, L-isoform becomes silenced by DNA-methylation while S-isoform gets upregulated by many fold (O'Connell et al, 2015). S-isoform specifically imparts invasive potential to cancer cells, unlike L-isoform (Singh et al, 2016); others have similarly reported metastatic potential of DCLK1 expressing cancer cells (Ito et al, 2016). Thus the goal of our studies was to evaluate molecular/genetic pathways mediating invasive effects of DCLK1-S in cancer cells. Isogenic clones of HCT116-cells, wild-type or down-regulated for DCLK1-S (HCT-C/HCT-D), were subjected to next generation sequencing and pathways analysis. SPARC and COL3A1 emerged as two candidate genes/proteins, which were decreased/increased by several fold in response to loss/overexpression of DCLK1-S, respectively. We present data confirming a critical role of COL3A1 and SPARC in mediating metastatic effects of DCLK1-S expression in hCCCs. We additionally discovered that DCLK1-S functions as a specific kinase for the transcriptional factor, NFATc2, and phosphorylates 53SPPS56 motif of NFATc2, resulting in activation of NFATc2 and increased expression of COL3A1. Conclusions. Our novel findings, suggest for the first time, that DCLK1-S expression by colonic tumors in humans, mediates invasive potential of colon cancer cells by phosphorylation/activation of NFATc2, resulting in up-regulation of COL3A1/SPARC; the latter proteins re-model extracellular matrix, assisting unhindered invasion of colon cancer cells.

#903

CXCR4 and CXCR7 play distinct and overlapping roles in prostate cancer dissemination to bone.

Sounak Roy,1 Kenneth C. Valkenburg,2 Kenneth J. Pienta2. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _Johns Hopkins School of Medicine, Baltimore, MD_.

Approximately 28,000 men die of prostate cancer (PCa) each year in the US, and 90-100% of them will be due to bone metastasis. It has been demonstrated that PCa cells detach from the primary tumor and "home" to bone. Osteoblasts (bone-forming cells) secrete the chemoattractant stromal-derived factor 1 (SDF-1, also known as CXCL12), and PCa cells express the receptors for SDF-1, C-X-C chemokine receptors 4 and 7 (CXCR4 and CXCR7). PCa cells use these receptors to enter the chemoprotective bone microenvironment, specifically, the hematopoietic stem cell (HSC) niche in the endosteum. While small molecule inhibition of CXCR4 can mobilize PCa cells from the bone marrow microenvironment, it is unknown whether CXCR7 plays a compensatory role when CXCR4 is antagonized. SDF-1 is known to bind with greater affinity to CXCR7 compared to CXCR4 indicating a possible role of CXCR7 in tumor migration to the endosteal niche. Therefore, it is crucial to determine the individual and combined roles of CXCR4 and CXCR7 in the formation and treatment of PCa bone metastases. To answer these questions, first we assessed CXCR4 and CXCR7 expression across a panel of prostate cancer cell lines. We then transiently overexpressed and knocked down CXCR4 and CXCR7 in PCa cells and found CXCR4 and CXCR7 expression in vitro to directly correlate to cell viability and migration in PC3 PCa cells. To further test dissemination and metastasis in bone, we are making stable overexpression and knockout cells and will perform in vivo metastasis experiments. We have developed a novel immunofluorescence protocol for detecting and quantifying tumor cells in murine blood and bone marrow and will use this technique to determine the ability of PCa cells to disseminate to bone in the presence or absence of CXCR4 and/or CXCR7. This will be quickly translatable because we are currently running a first-in-prostate cancer clinical trial to determine whether small molecule inhibition of CXCR4 to mobilize PCa tumor cells from the bone marrow, combined with docetaxel, will benefit metastatic PCa patients. If we determine that CXCR7 plays a compensatory role in dissemination and/or metastasis, we will need to add CXCR7 inhibition to our treatment strategy to obtain the most efficient benefit for patients.

#904

Podocyte-specific SMPDl3b modulates radiation-induced renal dysfunction.

Anis Ahmad,1 Alla Mitrofanova,1 Saba Ansari,1 Thirupandiyur Udayakumar,1 Jacek Bielawski,2 Alan Pollack,1 Alesia Fornoni,1 Brian Marples,1 Youssef Zeidan1. 1 _UM, Miami, FL;_ 2 _MUSC, Charleston, SC_.

Background: The underlying mechanisms responsible for the renal failure and proteinuria in radiation nephropathy remain largely unknown. Radiotherapy with or without chemotherapy may result in radiation-induced kidney injury in pelvic malignancies such as gynecologic cancers, lymphomas, gastrointestinal cancers, sarcomas of the upper abdomen and during total body irradiation. The current study investigates the role of sphingolipids in radiation-induced podocytopathy using a murine model. The molecular and functional effects of kidney irradiation were evaluated after single-dose exposures.

Material/Methods: In cell culture, SMPDL3b expression post radiation (8Gy) was determined by real-time PCR (RT-PCR) and Western blotting. Morphological changes and DNA damage were detected post radiation using immunofluorescence microscopy. Wild-type C57BL/6 male and female mice (age 10-14 weeks) were irradiated with a single dose of X-ray (14 Gy) using an image guided small animal arc radiation treatment system (iSMAART). Rituximab/IgG was administered (50 mg/kg, intraperitoneal injection) 30 min before the single dose of irradiation. Functional kidney parameters, kidney histology, and gene expression were analyzed at 20, 30 and 40 weeks after irradiation.

Results: Following irradiation, SMPDL3b expression at protein level was significantly reduced in vitro and in vivo. However, no significant changes were observed at the transcriptional level. Podocyte number also decreased significantly post radiation in vivo. iSMAART dynamic contrast enhanced (DCE) imaging data analysis showed reduced glomerular filtration rate post radiation. In agreement with the functional data, hematoxylin and eosin staining of kidney sections showed a multifocal increase in the number of pericytes, tubular atrophy, and glomerular damage. Periodic Acid-Schiff (PAS) staining showed an increase in glomerular mesangial matrix accumulation post radiation. Sirius red staining showed diffuse intertubular fibrosis, especially in the renal cortex post radiation. These histological changes were paralleled by the change in serum Creatinine, urine albumin. Rituximab pretreatment to mice, improved kidney functional parameters, vascular structure, normalization of pericyte coverage, suppress the development of fibrosis and tubular damage post irradiation.

Conclusion: This study shows that rituximab pretreatment protects mice against radiation-induced nephrotoxicity, which may have therapeutic implications for radiation-induced injuries in cancer patients.

#905

Lysyl oxidase is required for chemotaxis.

Haoran Tang, Nathalie Dhomen, Richard Marais. _CRUK Manchester Institute, Manchester, United Kingdom_.

Purpose: Lysyl oxidase (LOX) increases extracellular matrix stiffness by cross-linking collagen. A stiffer matrix drives integrin activation, and therefore LOX is believed to promote cancer cell invasion through enhanced integrin signaling. However increased matrix stiffness does not necessarily lead to a more permissive environment for invasion. Fibrillar collagen matrix orientation, density and porosity also determine how well cells can invade. Assays based on collagen gel-covered transwell systems have previously been used to show that LOX is required for invasion. These systems are driven by a chemoattractant gradient, and thus a role for LOX in chemotaxis could not be excluded. In this study we aimed to clarify whether LOX was required for matrix invasion or chemotaxis.

Experimental procedures: To set up a 3D invasion assay that was not driven by a chemoattractant gradient, spheroids from multiple LOX expressing cancer cell lines were embedded into a thick 3D collagen gel. Cells were then allowed to randomly invade into the surrounding collagen. Alternatively, cancer cells were allowed to invade a fibroblast-modified 3D collagen gel in an organotypic assay. In this assay the invasion was driven by a strong chemoattractant gradient through a dense collagen gel, with cancer cells cultured atop the collagen gel at the air-medium interface. A Dunn chemotaxis chamber or a transwell tissue culture insert was used to investigate chemotaxis towards serum or EGF. Small interfering RNA (siRNA) or short hairpin RNA (shRNA) specific to human LOX was used to deplete LOX expression in the cancer cells, to test potential LOX functions in the above assays.

Results: LOX depletion in parental MDA-MB-231 cells, MDA-MB-231 brain or bone metastatic sub-population cells, U87 and U118 cells did not impact 3D collagen gel invasion. LOX inhibition by BAPN also did not impact collagen gel invasion in MDA-MB-231, LN229, U87, U118 and U138 cells. The data indicated that LOX was not required for invasion through a thick 3D collagen gel in vitro. However, in the chemotactic-driven organotypic assay, loss of LOX in MDA-MB-231 cells completely abolished invasion. When the chemotactic potential towards serum and EGF of LOX depleted MDA-MB-231 and U87 cells was tested using Dunn chemotaxis chambers, we observed strong chemotactic defects in both cell lines. The chemotactic defects of these cells were also observed when a transwell based chemotaxis assay was used. Random cell migration of MDA-MB-231 and U87 cells were otherwise not affected by LOX depletion.

Conclusions: LOX is not required for collagen matrix invasion per se. Instead LOX is important for cells to sense chemoattractants and to maintain directional cell migration. The molecular mechanisms underlying these observations are now the subject of further investigation.

#906

PATZ1 promotes migration and invasion of thyroid cancer cells through upregulation of the activity of plasminogen activator and matrix metalloproteinases.

Asumi Iesato,1 Teruo Nakamura,1 Tsuyoshi Uehara,1 Hiroto Izumi,2 Ken-ichi Ito1. 1 _Shinshu University School of Medicine, Matsumoto-city, Nagano-Prefecture, Japan;_ 2 _University of Occupational and Environmental Health, Kitakyusyu-city, Japan_.

Background: PATZ1, a transcription regulator, has been reported to function as oncogene or tumor suppressor in several human malignant neoplasms. The aim of the study was to examine involvement of PATZ1 in carcinogenesis and progression of thyroid cancer.

Materials and methods: PATZ1 expression in 165 clinical thyroid specimens obtained from 87 patients were evaluated by IHC. We examined the function of PATZ1 using an immortalized normal thyroid follicular epithelial cell line (Nthy-ori 3-1), and four thyroid cancer cell lines; TPC-1 (papillary cancer), FTC-133 (follicular cancer), FRO and ACT-1 (anaplastic cancer). Knockdown of PATZ1 in Nthy-ori 3-1 and thyroid cancer cell lines was performed with siRNA-PATZ1, and overexpression of PATZ1 in thyroid cancer cell lines was performed with pcDNA3-FLAG-PATZ1.

Results: In the IHC analysis of clinical specimens, nuclear PATZ1 expression in anaplastic carcinoma was significantly less frequently than that in normal thyroid tissue or well-differentiated carcinoma (Table 1). Knockdown of PATZ1 in Nthy-ori 3-1 and differentiated thyroid cancer cell lines (TPC-1, FTC-133) induced morphological change of the cells and significant increase of cell proliferation and migration as well as moderate increase of invasion. Furthermore, the expression of u-PA, MMP2 and MMP11 was increased by inhibition of PATZ1 in TPC-1 and FTC-133. On the other hand, overexpression of PATZ1 in anaplastic cancer cell lines (FRO and ACT-1) decreased proliferation and migration of the cells along with the decreased expression of u-PA, MMP2, MMP9 and MMP11.

Conclusion: Our study suggests that PATZ1 might play an important role as a tumor suppressor in thyroid follicular epithelial cells and might be involved in progression of thyroid cancer.

Expression of nuclear PATZ1 in thyroid tumor

---

type | Normal thyroid/ goiter (N=68) | Follicular adenoma (N=5) | Papillary cancer (N=39) | Follicular cancer (N=8 ) | Poorly differentiated cancer (N=12) | Anaplastic cancer (N=28) | Medullary cancer (N=5)

positive (%) | 68(100%) | 4(80.0%) | 35(89.7%) | 5(62.5%) | 7(58.3%) | 3(10.7%) | 4(80.0%)

negative (%) | 0(0%) | 1(20.0%) | 4(10.3%) | 3(37.5%) | 5(41.7%) | 25(89.3%) | 1(20.0%)

#907

Uptake of lung cancer exosomes induces migratory and invasive phenotypic changes in lung epithelial cells in an oncogene context dependent manner.

Yoshihisa Shimada, Paul Yenerall, Kimberly Avila, Hyunsil Park, Brenda Timmons, Kenneth Huffman, Boning Gao, Dhruba Deb, Maithili Dalvi, John D. Minna. _University of Texas Southwestern Medical Center, Dallas, TX_.

Cancer-derived exosomes are intracellular signaling organelles that can act via inter-cellular communication to help cancer cells invade neighboring tissues and prime metastatic sites for cancer cell spread. Lung cancer is a highly metastatic disease, with metastases often occurring when tumors are in a clinically early stage. Little is known of how lung cancer exosomes influence the migratory/ invasive phenotype of lung cancer cells. We studied migration and invasion mechanisms by transferring cancer-derived exosomes to recipient cells including cancer cells, human bronchial epithelial cells (HBECs) cells, and HBECs that we had modified with common lung cancer oncogenic changes. Exosomes isolated from lung cancer line H1299 cells (p53null, NRAS mutant) and non-malignant, immortalized HBEC3-KT cells were characterized for exosome content and type by nanoparticle tracking analysis, electron microscopy, western blotting with antibodies directed at HSP70 and CD63, and generation of H1299 and HBEC3-KT derivatives stably transfected with CD63-GFP, releasing green fluorescence exosomes for tracking purposes. Exosomes were isolated using a variety of published methods and the amount quantified: 106 H1299 and HBEC-3KT cells produced 9 x 109 and 7 x 109 exosomes of similar size (125.4nm and 129.8nm). Effect of exosomes from cancer and HBECs were tested on: 1. cell motility and invasiveness of HBECs and their oncogenic derivatives determined by transwell chamber migration assay and scratch assay; 2. vascular leakiness properties in the lung by evaluating mouse lung endothelial permeability after exosomes were injected (an early event in metastatic spread for the migrated cells); and 3. xenograft tumor formation. We found CD63-GFP exosomes from H1299 and HBEC3-KT are actively incorporated by all of the cell types. However, only H1299 exosomes (but not HBEC3-KT exosomes) induced migratory/invasive phenotypic and morphologic changes and they did this in a concentration-dependent manner in lung cancer cells and HBECs with oncogenic changes (HBEC sh-p53+KRASv12 and sh-p53+KRASv12+c-MYC manipulated HBECs) but not in the parental HBEC3-KT cells. Also, H1299 exosomes but not HBEC3-KT exosomes enhanced the lung endothelial permeability as evaluated by the extravasated dextran leaking. NSCLC H1299 cells when injected with its own exosomes produced better xenograft takes compared to H1299 cells alone. Lung cancer-derived exosomes compared to exosomes derived from immortalized but non-malignant human bronchial epithelial cells induced an increased migratory/invasive phenotype with lung vascular leakiness and higher xenograft tumor take rates. However, they induced these changes only in the context of key oncogenic changes. These results suggest understanding and targeting the mechanism of such lung cancer derived exosome behavior could lead to novel therapeutic strategies.

#908

A role for MST4 in organelle organization and breast cancer cell vascular invasion.

Eric B. Berens,1 Mara Gilardi,2 Matteo Moretti,3 Anna T. Riegel,1 Anton Wellstein1. 1 _Georgetown University, Washington, DC;_ 2 _University of Milano-Bicocca, Milan, Italy;_ 3 _IRCCS Istituto Ortopedico Galeazzi, Italy_.

There exist 5 Mammalian Sterile20-like (MST) kinases that are broadly distributed into two subgroups. While MST1 and MST2 have established roles in the canonical Hippo signaling pathway, MST3, MST4, and YSK1 are known to regulate the actin cytoskeleton and cell polarity. Here we clarify distinct roles for MST3 vs. MST4 in breast cancer cell lines. Stable knockdown of MST4, but not MST3, enhances the ability of breast cancer cells to disrupt an endothelial monolayer as well as invade from an endothelial network formed within a microfluidic device. In addition, depletion of MST4 in MDA-MB-231 cells alters their morphology, induces abnormalities in nuclear structure, and affects the organization of cellular organelles like the Golgi apparatus. We conclude that, despite their relative homology, MST4 versus MST3 uniquely impacts the intracellular organelle status and vascular invasive behavior of breast cancer cells.

#909

Tracking of migration using digital holography to monitor drug-induced phenotypic shifts in breast cancer cells.

Sofia Kamlund, Stina Oredsson. _Lund University, Lund, Sweden_.

This study aims to investigate the migration behaviour of individual JIMT-1 breast cancer cells during the cell cycle. Mechanisms behind cell migration are important to unravel in the context of cancer relapses caused by distant metastases. JIMT-1 cells were cultivated in normoxia or hypoxia and treated with 0.5 µM of the cancer stem cell inhibiting compound salinomycin. The cells were imaged in a digital holography microscope placed in a regular cell culturing CO2 incubator (normoxia) or in a hypoxia chamber (1% O2). In digital holography, a laser beam is split into two beams - the reference beam and the object beam. The object beam is directed through the sample, where the difference in refractive index between air, the cell medium, and the cell body induces a phase shift in the laser beam. After having passed the object, the beam is merged with the reference beam giving an interference pattern, which is used to construct a three-dimensional image of the cells in a software program. The laser is non-phototoxic and therefor it can be used for long-term studies. In our study, images were acquired every 5th minute during a 72-hour incubation period. After acquisition, the individual cells in the images were tracked throughout the time lapse. Cell movement is described by three parameters: motility, which is the length of the total cell movement path, migration, which is the shortest distance from the start to the end position, and migration directness, which is migration divided by motility. When cells were exposed to hypoxia and/or treated with salinomycin, the cell cycle time increased compared to non-treated cells in normoxia. When the cell cycle time increased, motility increased as well. However, migration directness decreased despite increase in motility implying increased local movement of cells treated with salinomycin compared to control cells. We have previously shown that salinomycin-treated JIMT-1 cells obtain an epithelia phenotype (Huang et al., 2016). Thus, we decided to investigate a breast cancer cell line with epithelial traits i.e, the MCF-7 cells. The MCF-7 cells behaved like the JIMT-1 cells treated with salinomycin i.e. the motility was high and the migration directness was low. Thus, we suggest that cells with mesenchymal and epithelial traits can be distinguished using digital holographic imaging based on motility and migration directness and that drug-induced phenotypic shifts can be studied using this technique. References: Xiaoli Huang, Björn Borgström, Sebastian Kempengren, Lo Persson, Cecilia Hegardt, Daniel Strand and Stina Oredsson. Breast cancer stem cell selectivity of synthetic nanomolar-active salinomycin analogs. BMC Cancer (2016) 16:145. DOI 10.1186/s12885-016-2142-3

#910

Phloridzin docosahexaenoate (PZ-DHA) inhibits breast cancer cell invasion and angiogenesis.

Wasundara Fernando,1 Emma MacLean,1 Krysta Coyle,1 Paola Marcato,1 David W. Hoskin,1 H. P. Vasantha Rupasinghe2. 1 _Dalhousie University, Halifax, Nova Scotia, Canada;_ 2 _Dalhousie University, Truro, Nova Scotia, Canada_.

In our previous studies, we showed the selective cytotoxicity, in vitro anti-proliferative and anti-metastatic activities of phloridzin docosahexaenoate (PZ-DHA). PZ-DHA combines phloridzin (PZ), a dihydrochalcone found in apple peels, and docosahexaenoic acid (DHA), an ω-3 fatty acid found in fish oil, through an acylation reaction. Significant suppression of tumor growth resulted from the intratumoral administration of PZ-DHA to non-obese diabetic severe combined immunodeficient (NOD-SCID) female mice xenografted with MDA-MB-231 breast cancer cells. Examination of haematoxylin and eosin-stained tumor sections confirmed that PZ-DHA caused tumor cell death. PZ-DHA also suppressed the invasion of MDA-MB-231 cells through membranes coated with extra cellular matrix proteins, fibronectin and gelatin, in a Boyden chamber assay. The effect of PZ-DHA on the mRNA transcription of the gelatinase, matrix metalloproteinase-2 (MMP-2,) was measured in RT-qPCR analysis, which showed that MMP-2 mRNA expression was inhibited at 12 h and 24 h following PZ-DHA treatments. Furthermore, suppression of MMP-2 protein expression was shown by western blot analysis of cell lysates of PZ-DHA-treated MDA-MB-231 cells. Intraperitoneal administration of PZ-DHA suppressed the growth of primary tumors and metastasis of 4T1 and green fluorescence protein (GFP)-tagged-MDA-MB-231 cells to lungs of BALB/c and NOD-SCID female mice, respectively. Anti-angiogenic activity of PZ-DHA was investigated using human umbilical vein endothelial cells (HUVECs). PZ-DHA suppressed the metabolic activity of HUVECs in MTS assays and the cell death was confirmed using flow cytometric analysis of 7-AAD and Annexin-V-FLUOS/propidium iodide-stained HUVECs. Anti-proliferative activity of PZ-DHA was shown using Oregon Green 488 stained-HUVECs by flow cytometry. PZ-DHA treatment arrested the HUVEC cell cycle at S-phase. PZ-DHA also suppressed the migration of HUVECs in a gap closure assay and inhibited HUVEC tubule formation on a matrigel matrix. For comparison purposes, PZ and DHA were included in all experiments, but in all cases were less effective than PZ-DHA. Taken together, these findings reveal the potential of PZ-DHA to inhibit triple-negative breast cancer progression. We are currently working on identifying potential phase I and II metabolites of PZ-DHA and understanding its pharmacokinetic parameters.

#911

Destabilizing β-catenin and Ras could be an ideal therapy for treating metastatic colorectal cancer.

Yong-Hee Cho, Jeong-Soo Yoon, Kang-Yell Choi. _Yonsei University, seoul, Republic of Korea_.

INTRODUCTION Aberrant activations of Wnt/β-catenin and Ras signaling pathways by APC (90%) and K-Ras (40-50%) mutations are closely interacted and accelerate CRC tumorigenesis. Protein level of β-catenin as well as Ras is increased by APC loss via inhibiting GSK3B-mediated poly-ubiquitination dependent Ras degradation through recruitment of β-TrCP E3 linker. Moreover, additional K-Ras mutation results in the liver metastasis by secondary activation of the Wnt/β-catenin signaling pathways via Ras-ERK and -AKT pathways in addition to the initial activation by APC loss. Consequently, both β-catenin and Ras were highly increased in human CRC patient tissues, especially in metastatic tumor.

EXPERIMENT PROCEDURES To investigate the synergistic effects of aberrant Wnt/β-catenin and Ras signaling pathways on tumor development to metastasis, we established the APCMin/+/K-RasG12DLA2 compound mouse that harbor APC and K-Ras mutations in animal model. We also analyzed Ras protein level using human CRC patient tissues of normal mucosa and adenocarcinoma (N=24) and metastatic adenocarcinoma (N=26) and tumor budding (N=10). To recapitulate the metastatic tumor environment which have enriched receptor tyrosine kinases (RTKs), we maintained and experimented with EGF (20ng/ml) in every in vitro studies.

RESULTS We observed that both β-catenin and Ras were increased in adenocarcinoma and metastatic adenocarcinoma of human CRC compared with normal mucosa. Interestingly, both β-catenin and Ras were most significantly increased in tumor budding regions which histopathologically represent epithelial mesenchymal transition (EMT). In murine model, the EMT phenomenon was effectively induced and invasion of small intestinal tumors were occurred in APCMin/+/K-RasG12DLA2 with increased level of β-catenin and Ras compared with APCMin/+. However, EMT phenomenon and its following events such as migration and invasion of CRC cells induced by APC and K-Ras mutations were abolished by destabilization of β-catenin and Ras.

CONCLUSION Our results show that destabilizing β-catenin and Ras could be effective therapeutic targets for inhibiting metastasis in the early stage of CRC development

#912

ADAM9-plectin interaction involved in prostate cancer malignancy progression.

Che-Ming Liu,1 Chia-Ling Hsieh,2 Shian-Ying Sung2. 1 _China Medical University, Taiwan;_ 2 _Taipei Medical Univ., Taipei, Taiwan_.

Introduction: To identify a specific biomarker for malignant prostate cancer progression and therapeutic target, we had demonstrated the correlation of ADAM9 expression and cancer progression. The aim is to clarify the association of ADAM9 and interactive proteins in malignant phenotypic progression.

Methods: Knockdown of ADAM9 expression was conducted to explore its role in cell motility and metastasis activities. Pull-down assays were performed to identify the proteins that interact with ADAM9 during cancer cell migration. Proximity ligation assay (PLA) was carried out to validate the protein-protein interaction. Transmission electron microscope (TEM) analysis to confirm ADAM9 and plectin interaction. Protein degradation test was conducted to determine the regulation pathway.

Results: Our study demonstrated that ADAM9 play an important role in the regulation of prostate cancer migration, invasion and metastasis in the in vitro assay and xenograft animal models. The combination of ADAM9 pull-down assays and mass spectrum analysis demonstrated hemidesmosome components, plectin, ladinin, and cytoskeleton were pull-down with ADAM9. However, the key element of hemidesmosome, integrin α6β4, did not pull-down together. Both TEM and PLA confirmed the interaction of ADAM9 and plectin in the protrusion end of migrating cells. This interaction can be strengthened after knockdown integrin β4; and vice verse, knockdown of ADAM9 increased interaction of integrin β4 and plecin. These results indicated ADAM9 and integrin β4 compete for plectin binding. Furthermore, shADAM9 attenuated the degradation of integrin β4. Clinical evaluation of 96 patient samples in TMU demonstrated increased of ADAM9 and plectin interaction in tumor region when compared to the benign part of the same patient.

Conclusions: Our studies demonstrated that ADAM9 promotes cancer metastasis through alteration of hemidesmosome dynamic by enhancing of integrin β4 degradation. Therefore, these results indicated possible to use ADAM9-plectin interaction to predict prostate cancer malignancy progression.

#912A

Cadherins control Src kinase induced fibroblast cell motility.

Stephanie A. Sheehan, Edward P. Retzbach, Yongquan Shen, Gary S. Goldberg. _Rowan University, Stratford, NJ_.

Cancer associated fibroblasts (CAFs) are a part of the tumor microenvironment, and help remodel the extracellular matrix to pave the way for tumor cells to forge invasive fronts. Src tyrosine kinase activity is often increased in the tumor microenvironment, and has been shown to induce cell motility and disrupt adherens junctions. Adherens junctions are formed by cadherin proteins that mediate intercellular adhesion and control cell migration. Previous studies have found correlations between cadherin expression and cancer cell motility. However, effects of cadherin expression on CAF motility have not been thoroughly elucidated. We are investigating how junctions formed by E-cadherin (ECAD) and N-cadherin (NCAD) affect individual and collective migration of fibroblasts expressing different levels of Src kinase activity. We are utilizing fluorescently tagged ECAD or NCAD to study the localization and effects of cadherin proteins in real time by live cell imaging along with wound healing and Transwell migration assays. Results from these studies indicate that ECAD and NCAD have different effects on migration of cells depending on Src kinase activity. Src kinase activity did not affect collective cell migration, but increased individual cell migration by over 4 fold (p<0.0001 by t-test) in the absence of forced cadherin expression. In addition, our data indicate that ECAD increases collective cell motility, while NCAD decreases collective cell migration in the absence of strong Src kinase activity. Collective migration by ECAD expressing cells was over 4 fold higher than cells expressing NCAD or control transfectants (p<0.0001 by t-test) in the absence of strong Src activity. Conversely, collective migration of cells expressing NCAD was over 5 fold lower than control transfectants, and over 30 fold lower than cells expressing ECAD (p<0.005 by t-test) without strong Src kinase activity. In contrast, both cadherins decrease individual cell motility promoted by Src kinase activity. ECAD and NCAD transfected cells migrated over 4 fold less than control transfectants with high levels of Src kinase expression (p<0.0001 by t-test). Interestingly, human oral squamous cell carcinoma (OSCC) cells that express high levels of both ECAD and NCAD exhibit collective migration equal to cells with low Src kinase activity, but individual migration over 5 fold higher than nontransformed cells (p<0.0001 by t-test). This system should prove useful to elucidate how cadherins and Wnt signaling pathways affect motility of CAFs and tumor cells. For example, western blotting analysis suggest that both ECAD and NCAD may protect β-catenin from destruction initiated by Src kinase activity. Taken together this work should elucidate how cadherins affect the ability of the Src kinase to increase CAF motility, and identify mechanisms that can be exploited to identify potential chemotherapeutic targets.

### Stem Cell Heterogeneity

#913

Wnt signaling promotes mouse and human gastric stem cell proliferation.

Elise Susan Demitrack, Linda C. Samuelson. _University of Michigan, Ann Arbor, MI_.

Background: The Wnt signaling pathway is required for proliferation of intestinal stem cells and activating mutations in the Wnt pathway can promote colorectal cancer. The requirement for Wnt to support gastric stem cell function is much less clear, despite recent reports showing that activating Wnt pathway mutations occur in human gastric cancer. Here we tested the role of Wnt to support stem cell proliferation in the mouse stomach and in human stem cells cultured from control or gastric cancer tissues. Methods: Wnt signaling in the mouse stomach was detected using TCF/Lef:H2B-GFP reporter mice, and Wnt+ gastric cells were identified via GFP immunostaining. Wnt function in SOX2+ gastric stem cells was tested with Sox2-CreER; Apcfl/+ mice, 1-month post-Apc deletion. Wnt regulation of human gastric stem cells was tested in organoid cultures derived from paired control (non-cancer) and gastric cancer tissues. Established organoids were treated with the Wnt inhibitor IWR-1-endo and growth was assessed by quantification of organoid size. Results: GFP+ cells were observed in the gastric epithelium of TCF/Lef:H2B-GFP mice, located throughout corpus glands and at the gland base in antral glands. Interestingly, GFP+ epithelial cells were largely post-mitotic although in the antrum, a subset of GFP+ cells at the gland base co-localized with the antral stem cell marker LGR5. In both corpus and antrum, a subset of GFP+ epithelial cells co-stained with the pan-endocrine marker Chromogranin A and in the antrum, a subset also co-stained with the deep mucous cell marker GSII, suggesting Wnt may regulate gastric cell fate. Analysis of Sox2-CreER; Apcfl/+ mice revealed that Wnt pathway activation in SOX2+ gastric stem cells led to increased epithelial cell proliferation in both corpus and antrum. In the antrum, this incease in proliferation was also accompanied by increased expression of the cancer stem cell marker CD44 at the gland base. Wnt activation also perturbed epithelial cell differentiation in the gastric antrum, with expansion of GSII+ deep mucous cells. Wnt inhibition reduced growth of human gastric organoids derived from non-cancer and gastric cancer tissues. Interestingly, organoids derived from gastric cancer tissue were significantly larger than control organoids and required a 5-fold increase in IWR-1-endo to achieve significant growth inhibition, implying that gastric cancer organoids are somewhat resistant to Wnt inhibition to slow growth. Conclusion: Wnt signaling is a critical pathway for regulation of mouse and human gastric stem cell homeostasis, promoting cellular proliferation. Thus, Wnt may represent an effective therapeutic target for gastric cancers with Wnt-activating mutations.

#914

Age-associated neoplastic transformation of mammary stem cells.

Xiang Gu, Hakim Bouamar, Kyle Pressley, LuZhe Sun. _University of Texas Health Science Center San Antonio, San Antonio, TX_.

Incidence rate of breast cancer increases significantly with age. However, the mechanism of age-associated mammary tumorigenesis is unknown. Although mammary stem/progenitor cells have been proposed as the cells of origin for breast cancer, the mechanism remains largely elusive. Previously we discovered that in the old (28 to 30 month-old) mouse mammary gland, the luminal cells in the hyperplastic lesions elevated expression of CD49f, which is a basal cell marker. This led to an increased percentage of mammary stem cells (MaSCs) marked by CD49fhi in the old in comparison to the young (3 to 6 month-old). Similarly, old (>60 yr) human mammary ducts in the adjacent normal tissues from patients with breast cancer showed more hyperplastic lesions and CD49fhi luminal cells than young (<40 yr) tissues. Regenerated mammary glands by the old CD49fhi MaSCs through in vivo transplantation in cleared mammary fat pads showed more hyperplastic and dysplastic lesions than those regenerated by the young CD49fhi MaSCs, suggesting the old CD49fhi MaSCs contain transformed MaSCs. Moreover, gene set enrichment analysis of whole genome transcriptome data showed that they also have elevated expression of luminal cell specific genes and reduced expression of basal cell specific genes. Through large scale screening with flow cytometry guided by bioinformatics analysis, we found that Lin- CD24med CD49fhi CD13\+ CD73- mammary epithelial cells had increased number of MaSCs. By comparing young (3 to 6 months old) and old Balb/C mice, we found that the frequency of this specific MaSCs was significantly increased by four folds in old mammary epithelial cells (young: 4%, old: 16%). In contrast, relative percentage of Lin- CD24med CD49fhi CD13- CD73+ cells significantly decreased (young: 46%, old: 16%) during aging while the CD13 and CD73 double positive or double negative subpopulations remained unchanged. CD13\+ CD73\- cells generated significantly many more mammospheres (around 40 spheres per 1,000 cells) in suspension culture than other three types of cells (around 2 spheres per 1,000 cells). However, there was no significant difference between the young and old in terms of sphere formation efficiency for any of the four subpopulations marked by CD13 and CD73. We are studying whether anyone of these four subpopulation from CD49fhi old cells will generate more hyperplastic and dysplastic lesions in regenerated glands than the other subpopulations as well as than the corresponding young subpopulation. Our study show that CD13 and CD73 may be the markers for the identification of tumor-initiating MaSCs.

#915

Thrombomodulin expression regulates the tumor stroma differentiation of mesenchymal stem cells for controlling tumor growth.

Tsung-Hsien Shih,1 Jhy-Ming Li,1 Guey-Yueh Shi,2 Hua-Lin Wu2. 1 _Chang Gung University, Taoyuan City, Taiwan;_ 2 _Cheng Kung University, Tainan City, Taiwan_.

Introduction: Thrombomodulin (TM), a thrombin receptor on endothelial cell surface, acts as an anti-coagulant factor in thrombin-catalyzed protein C activation and inhibits the pro-coagulant functions of thrombin. Owing to TM's widely expression patterns on various cell types, our studies have demonstrated that beside anti-coagulation, TM also participates in controlling cell-cell adhesion, tumor growth, inflammation, angiogenesis, and atherosclerosis. However, the significance of TM expression in mesenchymal stem cells (MSCs) has never been investigated. The non-hematopoietic MSCs could specifically home into the developing tumors and becomes the active components of tumor stroma, which affects tumor's growth, immunity, progression, and therapeutic resistance. Recently, we investigated the role of TM on the tumor microenvironment differentiation of MSCs for establishing the active stroma that assists tumor growth.

Materials & methods: Normal and TM gene-targeting transgenic mice-isolated bone marrow-derived MSCs were used as the research models to investigate the expressing significance of TM on MSC's biological activities and differentiations during mouse melanoma B16F10 cell's conditioning in vitro and in vivo.

Results: Our data showed that MSC's TM expression was low in the quiescent state but was up-regulated while treating with B16F10 cell's conditioned medium in the mechanism of plate-derived growth factor signaling dependence. Accordingly, using conditional gene knockout strategy, under the condition of tumor cell other than normal culture resulted in that TM gene knockout significantly affected MSC's proliferation, migration, interleukin-6 secretion, angiogenic activity, and tumor-associated fibroblast differentiation in vitro. Furthermore, TM knockout notably reduced the capacity of MSCs on regulating the growth, vasculogenesis, and active stroma establishment of B16F10 melanoma xenograft in vivo.

Conclusion: These results indicate that the secretion of tumor-associated plate-derived growth factor is a critical event for MSC's TM expression that controls MSC's stroma differentiation for developing active tumor microenvironment, which promotes tumor progression. This study further suggests that the modulation of TM expression on MSCs has the therapeutic potential via diminishing the establishment of functional tumor stroma that sensitizes tumor for the therapies.

#916

Asymmetric cell division regulator prevents hyperproliferation in glioma cell-of-origin.

Mathieu Daynac, Claudia K. Petritsch. _UCSF, San Francisco, CA_.

In the past, we demonstrated that adult oligodendrocyte progenitor cells (OPC) undergo asymmetric cell division (ACD) to self-renew and generate functional cells by unequally distributing active EGFR and the OPC-specific marker NG2. Moreover, premalignant OPC switch from an asymmetric to symmetric, self-renewing division mode at premalignant stages, and exhibit aberrant proliferation and impaired differentiation (Sugiarto S et al Cancer Cell 2011). OPC are the cell-of-origin of glioma in genetically engineered mouse models (Persson A et al Cancer Cell 2010) and OPC-like glioma cells have high tumor-initiating potential in some glioma types. These data contribute to the emerging evidence that asymmetric cell division (ACD) has tumor suppressive activity. Whether the loss of ACD directly promotes tumor initiation is unclear, because the detailed mechanisms of mammalian ACD have not been elucidated (Gomez-Lopez S et al. Cell Mol Life Sci 2014). Lethal giant larvae 1 (Lgl1) was initially identified as a tumor suppressor in Drosophila and has been implicated in the asymmetric localization of cell fate determinants in mammalian neural progenitor cells (Klezovitch et al., Genes Dev, 2004). Others have shown also that human Lgl1 is inhibited by PTEN loss, which is common in glioblastoma, and Lgl1 depletion in human glioblastoma cells increases their invasive properties (Gont et al, Oncotarget, 2013 and 2014). Whether Lgl1 restricts proliferation, promotes differentiation and prevents neoplastic transformation of OPC, by regulating their asymmetric division, is unknown. We developed a flow cytometry method to isolate proliferative and differentiation-committed OPC (COP) and showed that Lgl1 expression is elevated in differentiation. Using a Cre-conditional knock-out allele (Klezovitch et al., Genes Dev, 2004), we show that Lgl1 knockout OPC proliferate more and differentiate less when compared with Lgl1 wildtype OPC. Lgl1 depletion increases symmetric, self-renewing divisions and reduces the frequency of ACD. Lgl1 appears to control the expression of several conserved ACD regulators, as revealed by comparative Taqman expression analyses of Lgl1 knockout versus control OPC. Ongoing studies are aimed to investigate the link of Lgl1 depletion, altered ACD regulator expression and observed OPC phenotypes. Data derived from this work are expected to provide further insights into the extent to which defective ACD contributes to neoplastic transformation in all cancers.

#917

CD82/KAI1 maintains the dormancy of long-term hematopoietic stem cells through interaction with DARC-expressing macrophages.

Jae-Il Choi,1 Jin Hur,1 Hwan Lee,1 Pniel Nham,1 Cheong-Whan Chae,1 Young-Eun Choi,1 Taewan Kim,1 Ga-Young Lee,1 Sung Hee Baek,2 Hyo-Soo Kim1. 1 _Seoul National University Hospital, Seoul, Republic of Korea;_ 2 _Creative Research Initiative Center for Chromatin Dynamics, Seoul, Republic of Korea_.

Hematopoiesis is regulated by crosstalk between long-term repopulating hematopoietic stem cells (LT-HSCs) and supporting niche cells in the bone marrow (BM). Here, we examine the role of CD82/KAI1 (CD82 hereafter) in niche-mediated LT-HSC maintenance. We found that CD82 is expressed predominantly on LT-HSCs and rarely on other hematopoietic stem-progenitor cells (HSPCs). In Cd82-/- mice, LT-HSCs were selectively lost as they exited from quiescence and differentiated. Mechanistically, CD82-based TGF-b1/Smad3 signaling leads to induction of CDK inhibitors and cell-cycle inhibition. The CD82 binding partner DARC/CD234 is expressed on macrophages and stabilizes CD82 on LT-HSCs, promoting their quiescence. When DARC+ BM macrophages were ablated, the level of surface CD82 on LT-HSCs decreased, leading to cell-cycle entry, proliferation, and differentiation. A similar interaction appears to be relevant for human HSPCs. Thus, CD82 is a functional surface marker of LT-HSCs that maintains quiescence through interaction with DARC-expressing macrophages in the BM stem cell niche. Finally, we hope that further development of our study will allow us to improve leukemia treatment by awakening not only "good" stem cells to keep beneficial stem cell functions, but also "bad" stem cells (e.g. cancer stem cells) in order to increase the sensitivity to treatment and avoid relapse.

#918

Cdc42 is crucial for intestinal stem cells survival by regulating Wnt and YAP signaling.

Xiao Zhang,1 Nan Gao2. 1 _Rutgers Newark, Harrison, NJ;_ 2 _Nan Gao, Harrison, NJ_.

Strictly defined architecture of intestinal epithelial cells and the gradient of signaling cues along the villus-crypt axis ensure the maintenance of adult intestinal stem cells, as well as proper regeneration and differentiation of all intestinal epithelial cell types. The Rho family small GTPase Cdc42 controls the polarized division of intestinal stem cells, and affects subsequent fate-determination of the progenitor cells. As we previously described in Cdc42 Villin-Cre knockout mice, loss of Paneth cells from crypt bottom and emergence of Goblet cell-like intermediate cells at the villus indicate possible compromise of the canonical Wnt signaling during Paneth cell differentiation. Here we further characterized the underlying molecular mechanism of Cdc42 regulating intestinal stem cell survival, proliferation, and differentiation. IHC of the intestinal tissue in Cdc42 Villin-Cre knockout mice suggests greatly reduced Olfm4 level in the stem cells, which are abnormally positioned in the crypts, along with a reduction of both transcription co-factors beta-Catenin and YAP. However, the Notch signaling, which is required for stem cells and the differentiation of absorptive linage, seems intact. The autonomous regulation of Cdc42 on Yap signaling is probably through its specific role in stabilizing cell-cell junction and actin filaments in the epithelium, both of which are known regulatory inputs of the Hippo-YAP pathway. Both Wnt/beta-Catenin and Yap signaling are required for the initiation and progression of colorectal cancer, and the regeneration of stem cells post radiation. Given that Cdc42 is overexpressed in multiple types of cancers, especially colorectal cancer, our finding that Cdc42 regulates both Wnt and YAP pathways in the homeostasis of adult intestinal tissue sheds new light on the molecular mechanism of its pathological involvement, and potential therapeutic target.

#919

Identification of stem cells in stomach corpus and antrum.

Junichi Matsuo, Akihiro Yamamura, Khay Guan Yeoh, Motomi Osato, Yoshiaki Ito. _National University of Singapore, Singapore, Singapore_.

Gastric cancer is still relatively poorly understood, because of the lack of knowledge on stem cells in stomach. By radio-labeling experiments and electron microscopy, rapidly proliferating stem cells have been suggested in isthmus of both corpus (which is just below pit cell zone) and antrum (which is a quarter above the base) (1). In addition, there have been reports indicating the proliferation capacity under certain conditions in a subset of chief cells present at the base in corpus (2). We reported earlier that 270 bp Runx1 enhancer element (termed aR1) that drives the expression of Runx1 in hematopoietic stem cells (3). We found that eR1 also marks the cells in these three locations of stomach (4). In antrum, the stem cell marker, Lgr5, has been reported to mark the cells at the base of antrum but not in corpus (5). eR1+ and Lgr5+ stem cell in antrum appears to be different. It will be interesting to find out the difference between these two types of stem cells. Transgenic mouse was generated that harbors eR1 linked to heterologous promoter and Cre recombinase (eR1-CreERT2). By treating the mouse with tamoxifen, we showed lineage tracing from eR1+ cells in the isthmus of both corpus and antrum that replenish entire glands. We expressed K-rasG12D in eR1+ cells in isthmus and observed antralization of corpus epithelium that is similar to precancerous lesion in human stomach after H pylori infection. On the other hand, eR1+ chief cells rarely multiply. However, they robustly proliferate after tissue damage or oncogene expression. When K-rasG12D is activated in eR1+ chief cells, they show retrodifferentiation to show SPEM (spasmolytic polypeptide expressing metaplasia) phenotype. eR1+ chief cells appear to be reserve stem cells that can replace cells after tissue damage. We now have capability to activate or inactivate genes of interest in the stomach stem cells. We are examining stepwise carcinogenesis in stomach. From eR1+ cells in isthmus of both corpus and antrum, we are generating organoids. We will describe interesting difference in the properties of organoids generated from eR1+ cells from corpus and those from antrum. <Reference> (1) Karam and Leblond., The Anatomical Record 1993; 236: 259-273. (2) Goldenring et al., Gastroenterology 2011; 138: 2207-2210. (3) Ng et al., Stem Cells 2010; 28: 1869-81. (4) Matsuo et al., Gastroenterology 2016; PMID: 27670082. (5) Barker et al., Cell Stem Cell 2010; 6: 25-36.

#920

ICAM-1(CD54) is involved in the osteogenic differentiation of mesenchymal stem cells and affects the progression of osteosarcoma cells.

Yidan Zhang,1 Sajida Piperdi,1 Nikolas Zaphiros,1 Pratistha Koirala,1 Tingting Ren,2 Michael Roth,1 Jonathan Gill,1 Bang Hoang,1 David Geller,1 Rui Yang,1 Wendong Zhang,1 Xiuquan Du,1 Jinghang Zhang,1 Richard Gorlick1. 1 _Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, NY;_ 2 _Peking University People's Hospital, Beijing, China_.

Purpose: The aim of this study is to validate the relationship between the expression of ICAM-1(CD54), a cell surface glycoprotein, and osteogenic differentiation of mesenchymal stem cells (MSCs) and the role of ICAM-1 in the progression of osteosarcoma (OS) cells.

Experimental Design: To compare the gene expression profiles between MSCs and MSCs after osteogenic induction, a microarray analysis was first performed. The differences in expression of specific genes including ICAM-1 were confirmed using western blot and flow cytometry. MSCs were sorted into two groups of cells by CD105+CD44+CD54+ and CD105+C44+CD54- and properties of these cells were compared. Expression of ICAM-1 among tumor cell lines and xenograft OS cells were tested at the protein level. ICAM-1 was stably, constitutively overexpressed in multiple cell types. Differences in osteogenic differentiation potential between normal cell lines and transformed cell lines were studied using alizarin red S staining. Motility, proliferation and invasion as well as the metastatic potential of the transformed and normal cells were evaluated.

Results: The initial microarray analysis revealed that ICAM-1 expression was significantly increased in MSCs after osteogenic induction for 15 days (p=0.042). Consistent results were achieved in western blot and flow cytometry analysis. Further microarray analysis between CD105+CD44+CD54+ and CD105+C44+CD54- groups determined that NF-kB signaling pathway was differentially activated. As for OS cells, ICAM-1 is not expressed at high level among OS tumor cell lines and is inconsistently expressed in xenograft OS cells. Enhanced osteogenic differentiation potential was observed in ICAM-1 overexpressing cells. Although there were no major difference in in vitro motility, proliferation or invasion tests, substantially less lung nodules occurred with transformation as compared to the normal control.

Conclusions:

ICAM-1 is involved in the osteogenic differentiation of mesenchymal stem cells and its down regulation may be involved in the progression of osteosarcoma cells.

#921

Cdyl2 is a chromodomain protein involved in the maintenance of pluripotency of stem cells.

Naoko Hattori,1 Kana Kimura,1 Jumpei Taguchi,2 Toshio Imai,1 Yasuhiro Yamada,2 Toshikazu Ushijima1. 1 _National Cancer Center Research Institute, Tokyo, Japan;_ 2 _Kyoto University, Kyoto, Japan_.

Reader proteins of histone modifications are required to translate the information of histone marks into the cellular phenotypes, including pluripotency and malignancy. For example, a reader protein BRD4 has its target specificity and becomes a target of cancer therapy. Here, we aimed to identify reader proteins involved in the pluripotency of stem cells. Using the UCSC database, 11 genes coding chromo- or PHD-domain proteins were isolated as actively transcribed in mouse ES cells (ESCs). Among the 11 genes, Cdyl2, chromodomain protein Y-like2, was found to be down-regulated upon ESC differentiation using RT-qPCR. Immunofluorescence and ChIP assay revealed that Cdyl2 recognized H3K27me3 of the specific regions, including differentiation-associated genes. Cdyl2-knock-out ESCs could not survive after differentiation induced by LIF removal or retinoic acid because of the induction of apoptosis, indicating that Cdyl2 is important for normal differentiation of ESCs. ESC expressing exogenous Cdyl2 showed incomplete down-regulation of Oct-4 and Nanog by retinoic acid, and generated teratomas with an abnormal composition of three germ layers, showing perturbation of differentiation ability. Pathologically, aberrant expression of human CDYL2 was observed in breast cancer cell lines and primary breast cancers. Two breast cancer cell lines (MDA-MB-231 and MDA-MB-468) expressing exogenous CDYL2 showed enhanced attachment ability along with the up-regulation of integrin alpha-6 and integrin beta-1. The population of cancer stem cells, defined as ALDH positive cells, were increased in CDYL2-expressing MDA-MB-468 cells, showing that human CDYL2 is involved in proliferation of cancer stem cells. From these data, we conclude that, between self-renewal and differentiation of stem cells, mouse and human Cdyl2/CDYL2 is necessary for the implementation of the initial step of differentiation.

#922

Delta133p53 represses p53-inducible senescence genes and enhances the generation of human induced pluripotent stem cells.

Izumi Horikawa,1 Kye-yoon Park,2 Han Li,3 Kazunobu Isogaya,1 Yukiharu Hiyoshi,1 Katsuhiro Anami,1 Ana I. Robles,1 Abdul M. Mondal,1 Kaori Fujita,1 Manuel Serrano,3 Curtis C. Harris1. 1 _National Cancer Inst., Bethesda, MD;_ 2 _NIH, Bethesda, MD;_ 3 _Spanish National Cancer Research Center, Madrid, Spain_.

p53 functions to induce cellular senescence and apoptosis, which can be incompatible with self-renewal of pluripotent stem cells such as induced pluripotent stem cells (iPSC) and embryonic stem cells (ESC). On the other hand, p53 regulates DNA damage response and repair and thus plays an essential role in maintaining genomic integrity and suppressing malignant transformation in iPSC and ESC. It remains to be elucidated whether and how p53 and its regulators contribute to balanced regulation between the self-renewing capacity and the genomic and functional integrity in these pluripotent stem cells. We hypothesized the involvement of Δ133p53, a physiological p53 protein isoform that inhibits the activity of full-length p53 (FL-p53), and here examined 12 lines of human iPSC and their original fibroblasts, as well as 3 human ESC lines, for endogenous protein levels of Δ133p53 and FL-p53, and mRNA levels of p53 target genes of different functions. While FL-p53 levels in iPSC and ESC widely ranged from below to above those in the fibroblasts, all iPSC and ESC lines expressed elevated levels of Δ133p53. The p53-inducible genes that mediate cellular senescence (e.g., p21WAF1 and miR-34a), but not those for apoptosis and DNA repair, were downregulated in iPSC and ESC. Consistent with these endogenous expression profiles, overexpression of Δ133p53 in human fibroblasts preferentially repressed the p53-inducible senescence mediators and significantly enhanced their reprogramming to iPSC. The iPSC clones derived from Δ133p53-overexpressing fibroblasts, when injected into immunodeficient mice, formed well-differentiated, benign teratomas, suggesting that Δ133p53 overexpression is non- or less oncogenic than total inhibition of p53 activities. Overexpressed Δ133p53 prevented FL-p53 from binding to the regulatory regions of p21WAF1 and miR-34a, providing a mechanistic basis for its dominant-negative inhibition. This study supports the hypothesis that upregulation of ∆133p53 is an endogenous mechanism that facilitates human somatic cells to become pluripotent without malignant transformation.

#923

Human embryonic stem cells exhibit altruistic cell death that release death signals having potent anti-cancer activity.

Bidisha Pal,1 Seema Bhuyan,2 Jaishree Garhyan,1 Herman Yeger,3 Bikul Das1. 1 _The Forsyth Institute, Cambridge, MA;_ 2 _KaviKrishna Laboratory, Indian Institute of Technology (IIT), Guwahati, India;_ 3 _Hospital for Sick Childern, Toronto, Ontario, Canada_.

Background: We recently described the altruistic stem cell (ASC) phenotype in human embryonic stem cells (hESCs) as well as in mesenchymal stem cells (1, 2). The ASC phenotype was characterized by altered state of p53/MDM2 feedback system that permitted the cells to transiently acquire an unstable state of low p53 allowing the cells to survive in extreme microenvironment (1). Interestingly, we noted that ASCs spontaneously returned to its basal state of high p53 after a period of two weeks (1, 2). In this study, we speculate that this return of p53 levels to basal state could be a safety mechanism to prevent malignant transformation of ASCs. We also speculate that ASCs may release soluble factors like high mobility group protein B1 (HMGB-1) that could selectively target the cells exhibiting abnormal p53 e.g. cancer cells. Here, we further characterized ASC phenotype and its fate.

Methods: hESC, BG01 cells were exposed to extreme hypoxia followed by reoxygenation and then ABCG2+/SSEA3+ cells were flow cytometry sorted. This specific subpopulation is enriched in ASCs and could be cultured in vitro for two weeks (1). The post- hypoxia treated ABCG2+/SSEA3+ cells were maintained in serum free media, and subjected to apoptosis, and senescence assays. The conditioned media (CM) of these cells and subjected to HMGB-1 measurement by ELISA. To measure bystander apoptosis, teratocarcinoma cells (Tera-2) were treated with the CM followed by measurement of apoptosis.

Results: Here we confirmed that p53 and MDM2 in ABCG2+/SSEA3+ cells exhibited return of oscillation between days 19-24. This was associated increase in apoptosis, and senescence of these cells. Apoptosis/senescence was decreased when treated with either Pifithrin α, an inhibitor of p53, or by siRNA silencing of p53, suggesting p53-dependent apoptosis. Additionally, treatment with Nutlin-3, an inhibitor of MDM2 (1) led to a 10-fold increase in apoptosis indicating that high MDM2 was required to prevent the apoptosis of ASCs. Thus, ASCs underwent spontaneous apoptosis (altruistic cell death) due to the return of p53/MDMD2 oscillation. We suggest that this return of p53 and associated apoptosis could be a safety mechanism to prevent malignant transformation of ASCs. Next, we found that the CM of ABCG2+/SSEA3+ cells contained high levels of HMGB-1 compared to the parental BG01 cells. Importantly, CM treatment led to p53 dependent apoptosis of Tera-2 cells. Furthermore, CM treatment also inhibited the growth of Tera-2 xenografts in NOD/SCID mice.

Conclusion: Our results indicate that ASCs undergoes altruistic cell death, and releases death signal mediated by HMGB1 that can target neighboring cells exhibiting abnormal p53 including cancer cells. We suggest that this unique property of ASCs could be exploited as a novel cancer therapeutic.

1. Das B et al, Stem Cells, 2012; 30(8):1685-95.

2. Pal B et al, Cancer Research, 2016; Volume 76, Issue 14 Supplement, pp. 251

#924

Dedifferentiation into polyploid blastomere-like cancer stem cells via formation of polyploid giant cancer cells.

Na Niu, Jinsong Liu. _UT MD Anderson Cancer Ctr., Houston, TX_.

We have recently shown that polyploid giant cancer cells (PGCCs) are capable of tumor initiation and acquisition of embryonic-like stemness and thus represent a novel type of cancer stem cells. However, two important questions remain to be answered from this surprising finding: (1) how PGCCs acquire such stemness; (2) on which stage of normal development PGCCs correspond to. Here, we tracked the fate of single PGCCs induced via mitotic failure by paclitaxel. Morphologically, early spheroids derived from PGCCs were indistinguishable from human embryos at the polyploid blastomere, compaction, morula, and blastocyst-like stages by scanning electron microscopy. PGCCs showed time- and space-dependent activation of expression of the embryonic stem cell markers OCT4, NANOG, SOX2, and SSEA-1 and lacked expression of Xist. PGCCs also showed time-dependent activation of expression of the germ layer-specific markers alpha-fetoprotein, smooth muscle actin, and β3-tubulin and were capable of redifferentiation into three germ layers in vitro. PGCCs-derived daughter cells showed attenuated invasive ability and increased resistance to paclitaxel. PGCCs-derived spheroids grew into a wide spectrum of human neoplasms, including malignant dysgerminoma and embryonic carcinoma, poorly differentiated or well-differentiated carcinomas, and benign squamous tissue. We also observed PGCCs in ovarian cancer from patients treated with chemotherapy. Thus, our data demonstrated that PGCCs acquired novel cancer stem cell properties and capability to redifferentiate into different tumors including the germ cell tumors, which are at the topmost developmental hierarchy. Our studies, for the first time, link PGCCs to the polyploid blastomere-like cancer stem cells and thus offer a new paradigm for the origin of cancer.

#925

Tumorigenesis of murine iPS cell is prevented by iron depletion with downregulation of stemness markers.

Yuki Katsura,1 Toshiaki Ohara,1 Hajime Kashima,1 Hiroaki Sato,1 Takuya Kato,1 Takayuki Ninomiya,1 Kazuhiro Noma,1 Yasuko Tomono,2 Hiroshi Tazawa,1 Shunsuke Kagawa,1 Yasuhiro Shirakawa,1 Toshiyoshi Fujiwara1. 1 _Okayama Univ., Okayama, Japan;_ 2 _Shigei Medical Research Institute, Okayama, Japan_.

Background: Iron plays a crucial role in the various metabolic pathways and it is essential for life. However excess of iron is known to cause cancer. There are many reports that iron depletion treatment indicates antitumor effect. Previously, we have confirmed that iron depletion treatment indicates antitumor effect against Cancer stem cell (CSC) model, miPS-LLCcm, which was converted from murine induced pluripotent stem cell (miPS cell) in Okayama university (Chen at el. PLOS ONE 2012), and also suppresses CSC markers. Many tumors contain phenotypically and functionally heterogenous cancer cells due to pluripotency of CSC. Although there are various theories about the origin of CSC, miPS has a pluripotency and regarded as the top of the hierarchy, it can be the candidate of the origin of CSC. miPS cell is known to form teratoma when inoculated into nude mouse because of its undifferentiated status. We hypothesized if we could suppress the stemness and tumorigenicity of miPS cell by iron depletion treatment, it can be applied to the therapy for CSC and undifferentiated status cancer cell. In this study, we investigated the stemness and tumorigenecity of miPS cell with iron depletion treatment.

Methods: We used miPS cell(cell name : iPS-MEF-Ng-20D-17) purchased from Riken Cell Bank(Japan). Deferasirox (DFX), an popular commercial available oral iron chelator was used for iron depletion treatment. Western blot analysis was done to examine the expression of the stemness markers including Nanog, Oct3/4, Sox2, Klf4, c-Myc. Subcutaneous model of miPS cell and miPS cell treated with DFX (50μM) were used in vivo study. Tumors were harvested for immunohistochemistry.

Results: Stemness markers of miPS cell were suppressed strongly by DFX in western blot analysis. DFX also suppressed the subcutaneous tumor growth. The average tumor volume of the control group was 665.3±430.8mm3 while that of the DFX treated group was 204.3±76.2 mm3(p<0.05) on day 20. Immunohistochemistry of the tumor revealed the suppression of the stemness markers in the DFX treated group compared with the control group.

#926

Generation of a potential breast cancer stem cell model from induced pluripotent stem cells.

Neha Nair,1 Anna Sanchez Calle,1 Maram Hussein Zahra,2 Aung Ko Ko Oo,1 Arun Vaidyanath,1 Shinobu Masuda,3 Tomonari Kasai,1 Masaharu Seno1. 1 _Okayama University, Okayama, Japan;_ 2 _Menoufia University, Shebin El-Koam, Egypt;_ 3 _Nihon University School of Medicine, Tokyo, Japan_.

The role of cancer stem cells (CSC) in the present scenario of breast cancer research is perceived as indispensable in the development and progression of the disease. The CSC niche maintains a heirarchy of heterogeneous cells, which facilitates the inception of the tumor until its invasion. Therefore, our study focuses on the development of an effective breast cancer stem cell model from mouse induced pluripotent stem cells (miPSCs).

Taking into consideration the intertumor heterogeneity of breast cancer, three human breast cancer cell lines, namely BT549, SKBR3 and T47D representing three major hormone subtypes were used to derive respective conditioned medium. The miPSCs were treated with these conditioned media separately for a period of one month following the protocol previously established by our laboratory. The resulting survived cells were subcutaneously and orthotopically transplanted into Balb/c nude mice. Within a short span of 15-20 days, both subcutaneous and orthotopic tumors were developed. Serial transplantations in to nude mice also generated malignant tumors with same vigor. Histopathological studies of these tumors confirmed a malignant infiltrating ductal carcinoma with apparent desmoplasia in orthotopic tumor and Her2 expression in comparison with subcutaneous ones, all of which confirmed us the conversion of miPSCs to CSCs. Presence of lung metastatic nodes further ascertained the invasiveness of this in vitro generated CSCs. Marked expression of prominent CSC markers namely CD44, Nanog, Sox2 and CD49f were observed in primary cells generated from these tumors. Tumor sphere formation assay further confirmed the presence of a good population of self renewing CSC population in the tumor tissues.

This study attempts to recapitulate human breast cancer disease in mice without any genetic manipulation, but by exploiting the tumor microenvironment in the form of conditioned medium. This should pave the way for the establishment of personalized therapy and a model to assess effective therapeutic interventions.

#927

Cancer stem cell proliferation in human prostate cancer cells utilizing a new defined 3D spheroid culture system.

Nick Asbrock, Vi Chu, Kan Saito. _MilliporeSigma, Temecula, CA_.

Solid tumors grow in a three-dimensional (3D) spatial conformation, resulting in a heterogeneous exposure to oxygen and nutrients as well as to other physical and chemical stresses. To mimic the 3D spatial conformation, 3D in vitro culture models have been used in cancer research since the diffusion-limited distribution of oxygen (hypoxia), nutrients, metabolites, and signaling molecules is not mimicked in conventional two-dimensional (2D) monolayer cultures. One of the 3D in vitro culture models, the tumorsphere culture is an emerging model for studying and expanding the cancer stem cell (CSC) population. E006AA cell line is a spontaneously immortalized cell line derived from a Gleason 6 localized prostate cancer in a hormone-naive prostate cancer patient of African American descent. This cell line expresses androgen receptor and was repeatedly reported to be non-tumorigenic in nude mice. However this cell line forms continuously growing tumor in NOG-SCID triple-deficient mice (i.e., NOG-SCID mice not having NK, B, and T-cells) and an establishment of highly tumorgenic subline E006AA-hT was reported suggesting this cell line has a potential for CSC proliferation by 3D sphere cultures. Here we report tumorsphere cultures of E006AA cells by a new 3D spheroid culture media system. The cells showed continuous proliferation supported during serial passage of 3D tumorsphere cultures. In addition to the stable proliferation, increases of Aldehyde Dehydrogenase (ALDH) expressing CSC population were observed with the increase of passages of 3D tumorsphere cultures.

#928

**Combined treatment by manuka honey and metformin inhibited growth and inducted apoptosis in CD133** + **and CD90** + **subpopulation hepatocellular carcinoma cancer stem cells.**

Amira S. Fyala, Ahmed S. Sultan. _Faculty of Science, Alexandria University, Alexandria, Egypt_.

Manuka honey is well known for its anti-bacterial and wound healing properties but its anticancer role against cancer stem cells (CSC) in hepatocellular carcinoma (HCC) cell lines and the underlying molecular mechanisms remain uninvestigated. Recently, we identified a CSC subpopulation in HCC cell lines characterized by their CD133+ and CD90+ phenotype. The CD133+/ CD90+cells demonstrated a more aggressive phenotype than the CD133-/ CD90- counterpart and formed metastatic lesions in the lung of immunodeficient mice. In order to evaluate the effect of Manuka honey alone or in combined treatment with Metformin, a biguanide that has been widely used to treat type 2 diabetes, on cell viability and apoptosis induction in human HCC cell lines, isolated and identified CD133+/ CD90+subpopulation CSC were used. Cell viability was monitored using an MTT assay. In addition, apoptosis induction was labeled by Annexin V-fluorescein isothiocyanate/propidium iodide and measured using flow cytometry. Western blotting was used to examine the protein expression of p53, Bcl-2, Bax, Bcl-2-associated death promoter (Bad), Bcl-2 homologous antagonist/killer (Bak) and cleaved-caspases-3 and -9. The results of the present study revealed that Manuka honey alone or in combined treatment with Metformin suppressed the cell viability of CD133+/CD90+cells in a concentration-dependent manner and the maximum effect was detected in combined treatment after 24 h. In addition, Manuka honey alone or in combined treatment with Metformin induced a significant apoptosis in the tested cells. Furthermore, it was demonstrated that combined treatment increased the expression of proapoptotic proteins, including p53, Bax, Bad and Bak, while it decreased the protein level of antiapoptotic protein Bcl-2 compared to control, Manuka honey or Metformin alone. Furthermore, the combined treatment strongly inhibited stemness characteristics of CSC subpopulation, colony formation and dramatically inhibited CD133+/CD90+tumor growth in vivo. Our data demonstrated that combined treatment induced apoptosis in CD133+/CD90+ subpopulation through G1 arrest in the cell-cycle and regulating p53/Bcl-2/caspase-3 signaling pathway. In conclusion, our results suggested that Monuka honey alone or combined treatment with Metformin may be a potential treatment for apoptosis induction in CSC and may provide a novel therapeutic model for HCC.

#929

Dissecting heterogeneity and population dynamics in breast cancer stem cells.

Michael D. Brooks, Max S. Wicha. _University of Michigan, Ann Arbor, MI_.

The CSC hypothesis posits that there is a subset of tumors cells driving tumor growth and metastasis and that by targeting this population, tumors can be more efficiently eradicated. Recently it was shown that there are two types of CSCs in breast cancer, mesenchymal CSC's characterized as CD44+/CD24- and an epithelial type marked by ALDH expression. The cellular plasticity of these CSC states may contribute to their metastatic capacity. However, little is known about the heterogeneity of these CSC populations or how they are regulated by the tumor microenvironment. To address these issues we have utilized Drop-Seq to generate single cell RNA-Seq data from thousands of cells across patient derived xenografts from various molecular subtypes of breast cancer. In order to monitor real-time state transitions between the determined cellular populations, we have also designed and constructed fluorescent reporter constructs for genes specific to critical cell populations and cancer stem cells. We used CRISPR/Cas9 to introduce these reporters into the native loci of the genes in order to recapitulate native gene expression patterns. Single cell transcriptomics will help to characterize the molecular heterogeneity of CSC populations. The addition of fluorescent reporters will help to elucidate the mechanisms that regulate breast CSC's as well as their interaction with the tumor microenvironment and to identify potential therapeutic targets.

#930

Role of DLK1 in sarcoma stem cells.

Ilkyu Han, Sun Hee Ahn, Han-Soo Kim. _Seoul National Univ. Hospital, Seoul, Republic of Korea_.

Sarcoma is a rare form of cancer that comprises about 1% of all malignancies. A large proportion of deaths by sarcomas are caused by tumor metastasis, of which the biological basis remains obscure. A significant body of evidence suggests that only a small population of sarcoma cells possess the ability to initiate and maintain tumors. These tumor-initiating cells have the ability to self-renew, recapitulating the cellular heterogeneity of the original cancer, and thus are often referred as cancer stem like cells (CSLCs).Delta-like factor 1 (DLK1) was cloned in the preadipocyte cells 3T3-L1 cDNA library. Previous studies reported on the role of DLK1 in mesenchymal stem cell differentiation, adipogenesis, myogenesis, and osteoblastogenesis. It was reported that DLK1+ liver cancer cells have characteristics similar to those of CSLCs and that DLK1 could be a potential therapeutic target against the liver CSLCs. However, little is known about role of DLK1 in CSLCs of sarcoma. The mRNA (real time PCR) and protein (Western blot) expression of DLK1 from 13 sarcoma cell lines was examined. CSLCs from sarcoma cell lines were isolated using FACS with CD133 and sphere forming assay. The effect of DLK1 on sarcoma tumorigenesis was examined using cell proliferation and cell invasion assays by DLK1 siRNA treatment.Real-time PCR showed the increased expression of DLK1 mRNA in sarcoma cell lines. In particular, DLK1 mRNA was highly expressed in U2OS (osteosarcoma), SW872 (liposarcoma), A204 (rhabdomyosarcoma), and HS-SYII (synovial sarcoma) cells. We observed a reduction in cell proliferation and invasion in U2OS, A204, and sw872 cells treated with DLK1 siRNA. CSLCs were isolated from U2OS, A204, and sw872 by the FACS sorting method using the CD133, a CSLC marker in sarcoma. The number of active sarcoshperes in CD 133+ cells were significantly higher than that in CD133- cells by sphere forming assay. Soft agar assays revealed the presence of many more colonies in CD133+ cells than in CD133- or wild type cells. To investigate the effect of DLK1 on sarcoma CSLCs, sw872 cells were categorized into four groups: wild type, CD133-, CD133+ and spheres. Real-time PCR and western blot were used to detect the DLK1 gene in all four groups. We observed that DLK1 expression in sw872 CSLCs (CD133+ and spheres) were relatively low. We found that suppression of DLK1 by siRNA inhibited the invasiveness of CSLCs although it inhibited more effectively to activation of invasive cells as well as cell proliferation in sw872 non-CSLCs.These results suggest that DLK1 may play a role in tumorigenesis of CSLCs and cancer cells in various sarcomas.

#931

Extracellular matrix stimuli regulate cancer stem cell population and migratory potential in glioblastoma.

Jee-wei E. Chen,1 Jan C. Lumibao,1 H Rex Gaskins,1 Jann N. Sarkaria,2 Brendan A. Harley1. 1 _University of Illinois at Urbana-Champaign, Urbana, IL;_ 2 _Mayo Clinic, Rochester, MN_.

Introduction Glioblastoma (GBM) is the most common and lethal form of brain cancer. Poor survival is linked to diffuse infiltration throughout the brain, suggesting the need to examine the effect of the tissue environment at the margins of the GBM tumor on processes of GBM invasion. Further, the presence of GBM cancer stem cells (GSCs) in primary tumor specimens is believe to play a significant role in GBM spreading, recurrence, and therapeutic resistance. While biophysical properties of the tumor microenvironment, such as spatial gradients in matrix stiffness and hyaluronic acid (HA) content as well as regional hypoxia, may affect GBM invasive phenotypes, limited tools are available to rigorously examine the relationship between tissue microenvironment and tumor growth, invasion, and therapeutic outcomes. This work focuses on the integration of HA-functionalized gelatin hydrogels with 3D cell invasion assays to investigate the impact of tissue microenvironment on GBM invasion and GSC population.

Methods Hydrogels are generated from methacrylamide functionalized gelatin (GelMA) and methacrylated HA. Prepolymer mixture was crosslinked under UV light in the presence of a photoinitiator. GBM cells, either U87 cell lines or patient-derived xenografts (PDX), were mixed with prepolymer solution prior to polymerization and collected for up to 7 days in culture. To assess invasion, cells were seeded onto dextran beads overnight, with populational invasion metrics calculated after 3 and 7 days, while live cell tracing via confocal microscopy was employed for single cell invasion metrics. Samples were cultured with 5% CO2 and ~20% O2 (normoxia) or 1% O2(hypoxia).

Results The invasion of U87 and U87viii (constitutively active EGFR) cells increased significantly in GelMA hydrogels lacking HA, consistent with our previous findings that compensatory secretion of soluble HA in the absence of matrix-bound HA was associated with upregulated invasion. Heightened U87 invasion was observed in the presence of hypoxia for all hydrogel compositions, and was associated with upregualted markers of invasion (gene expression: MMP-2, VEGF; protein expression: ERK1/2) while PI3K was downregulated, suggesting cells respond to hypoxia by alternating invasion and proliferation behaviors. We subsequently examined the effect of matrix biophysical properties and hypoxia on the expansion of GSC populations within PDX cells (a gift of Jann Sarkaria, Mayo Clinic). Here, the expansion of the GSC population in EGFRviii mutant GBM6 was a function of hypoxia as well as matrix-bound HA.

Conclusions This work demonstrates that GBM cells respond dynamically to their matrix biophysical (Stiffness, HA content, hypoxia) properties. Using PDX cells, we provide evidence that the expansion of GSC is related with both metabolic environment (O2%) and matrix composition (+/- HA). Ongoing work seeks to understand how GSCs contribute to the invasive phenotype of GBM.

#932

Wnt antagonist, secreted frizzled-related protein 4 (SFRP4) and peptides from its associated domains, increases chemotherapeutic response of cancer stem cells of malignant mesothelioma cell lines.

Vanathi Perumal,1 Senthil Kumar,2 Frank Arfuso,3 Simon Fox,1 Arunasalam Dharmarajan2. 1 _School of Pharmacy, Curtin University, Perth, Australia;_ 2 _School of Biomedical Sciences, Curtin University, Perth, Australia;_ 3 _Curtin University, Perth, Australia_.

Malignant mesothelioma (MM) is a highly aggressive cancer associated with past asbestos exposure that is characterized by rapid progression, late metastases and poor prognosis. There is a need for much improved and novel therapies for this cancer through improved biological understanding of the disease.Previous studies have shown that SFRPs in general and SFRP4 in particular are down regulated in mesothelioma tissue and cell lines 1-4. Based on our own study and previously demonstrated a role for sFRP4 in making chemoresistant tumours amenable to chemotherapeutics 5-16. We have now progressed our work into the effects of peptides derived from the cysteine-rich domain and the netrin-like domain of sFRP4 on tumour initiating cells derived from MM cell lines.We examined the effect of secreted frizzled-related protein 4 (sFRP4), a Wnt signaling antagonist and its associated peptides, in chemosensitizing the MM cell line ONE 58 and JU77 and MM cancer stem cells (CSCs) enriched from these two cell lines to chemotherapeutics.We found tumorspheres exposed to sFRP4 and peptides alone and in combination with cisplatin induced cell death and decreased CD133 and ALDH1 expression.We thus identified for the first time that sFRP4 and associated peptides could help to sensitise to chemotherapeutics and destroy cancer stem cells of MM cell line, which would lead to effective treatment regimen to combat malignant mesothelioma. Our initial in vitro data exemplify the ability of these peptides in diminishing the numbers of therapy-resistant Tumor initiating cells (TICs).

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#933

Bcl-xL overexpression promotes tumor aggressiveness.

Maria Grazia Tupone, Daniela Trisciuoglio, Marianna Desideri, Marta Di Martile, Chiara Gabellini, Simonetta Buglioni, Laura De Salvo, Gabriele Alessandrini, Simona D'Aguanno, Donatella Del Bufalo. _Regina Elena National Cancer Institute, Rome, Italy_.

Bcl-xL is an antiapoptotic protein highly expressed in cancer and contributes to tumor initiation and progression by promoting cell survival. It also promotes tumor angiogenesis and metastasis. By using M14 melanoma and ADF glioblastoma cell lines and their derivative bcl-xL overexpressing clones, in the current study, we investigated the role of bcl-xL in aggressive features of melanoma and glioblastoma models. We found that in both models, bcl-xL overexpression increased in vitro cell migration and invasion and facilitated tumor cells to form de novo vasculogenic structures. Furthermore, bcl-xL overexpressing cells exhibited significantly higher tumorsphere formation capacity, reflecting the self-renewal activity, and expressed high levels of some stem cell markers, supporting the concept that bcl-xL plays essential roles in the maintenance of cancer stem cells phenotype. Forced expression of bcl-xL in melanoma cells also increased vascular endothelial growth factor (VEGF) and matrix metalloprotease-2 (MMP-2) expression, and the activation of hypoxia-inducible factor 1 (HIF-1), a key pathway involved in melanoma vascularization and aggressiveness. On the contrary, no differences were observed after bcl-xL overexpression in glioblastoma model, in terms of HIF-1 expression and activity, and VEGF protein secretion. Immunohistochemical analysis revealed that the expression of the vascular markers, and the vasculogenic mimicry were upregulated in the bcl-xL overexpressing xenografts derived from both tumor histotypes. The work presented here brings further support to the understanding of the malignant actions of bcl-xL both in melanoma and glioblastoma, and in particular to the concept that bcl-xL contributes to the aggressiveness of these two tumor histotypes by promoting invasion, migration, vasculogenic mimicry and cancer stem cells phenotype. It also indicates a cell type-specific effect of bcl-xL on HIF-1/VEGF axis.

#934

EGFR induces ILF3 and G9a expressions to maintain Oct4-mediated stemness property in lung cancer.

Chun-Chia Cheng,1 Jungshan Chang,2 Huan-Chau Lin,1 Ai-Sheng Ho,3 Ken-Hong Lim,1 Chun-Chao Chang,4 Yi-Fang Chang,1 Cheng-Wen Wu5. 1 _Mackay Memorial Hospital, New Taipei City, Taiwan;_ 2 _Taipei Medical University, Taipei, Taiwan;_ 3 _Cheng Hsin General Hospital, Taipei, Taiwan;_ 4 _Taipei Medical University Hospital, Taipei, Taiwan;_ 5 _National Yang-Ming University, Taipei, Taiwan_.

Cancer stem cells survive and lead to tumor recurrence in tumor therapeutic treatments. Lung cancer cells highly express epidermal growth factor receptor (EGFR), which is critical for tumor progression. We hypothesized that EGFR may play a pivotal role in the induction of cancer stemness cells (CSCs). The aim of this study attempted to investigate the molecular mechanism of EGFR-mediated cancer stemness, and to discover the potent therapeutic agents. A high expression level of cancer stemness markers, ALDH1, CD133, Oct4, and Nanog was observed in tumorspheres derived from HCC827. External EGF further elevated the expression of Oct4 in HCC827 cells. Moreover, chemical inhibition and genetic knockdown of EGFR reduced the Oct4 expression and also tumorsphere formation. We found that inhibitors targeting to EGFR (afatinib), ILF3 (YM155), and G9a (UNC0642) are potential to reduce the formations of tumorspheres. The results revealed that EGFR triggered the formation of tumorspheres through elevating the activations of G9a-mediated stemness genes. In addition, YM155 effectively inhibited tumor growth and formation of tumorspheres as an anti-lung cancer agent blocking the EGFR-G9a cell initiating program pathway. This study demonstrated that EGFR participated in maintain of cancer stemness property through expressing ILF3 and G9a expressions. YM155 inhibited the EGFR-G9a stemness pathway, and reduced the formation of tumorspheres as a potent anti-lung cancer agent.

#935

**CD133** + **cancer stem cells promoted by VEGF accelerate the recurrence of hepatocellular carcinoma.**

Dexi Chen,1 Kai Liu,1 Meijun Hao,1 Yabo Ouyang,1 Jiasheng Zheng,1 Kishore Babu. Challagundla2. 1 _Beijing You An Hospital, Capital Medical University, Beijing Institute of Hepatology, Beijing, China;_ 2 _Children's Center for Cancer and Blood Diseases and The Saban Research Institute, CA_.

The function of cancer stem cells (CSCs) on inducing HCC recurrence after radiofrequency ablation (RFA) is still unclear. Here, 19 RFA-treated primary HCC patients were enrolled. We identified a dramatic increase of plasma VEGF in some patients (termed type II, n=9) after RFA, who suffered early HCC recurrence; the other patients (termed type I, n=10) had no increased plasma VEGF but had longer interval for HCC recurrence. Moreover, the expression of CSCs marker, CD133, was dramatically increased in recurrent HCC tissue of Type II in comparison with type I, suggesting induction of CD133+ CSCs may contribute to early HCC recurrence. In vitro studies demonstrated that VEGF stimulus enhanced the level of CD133+ CSCs and their self-renewal ability by inducing Nanog dependently on activation of VEGFR2. In vivo studies further demonstrated that CD133+ CSCs with VEGF stimulus formed larger tumor size in comparison with non-stimulus; and VEGF stimulus increased the tumorigenic cell frequency of primary HCC cells dependently on the presence of Nanog and VEGFR2. In recurrent HCC tissue of type II, but not type I, almost all CD133+ cells were Nanog and p-VEGFR2 positive cells, suggesting that activation VEGFR2 is critical for RFA-induced tumor stemness in HCC. Taken together, RFA-induced VEGF promotes tumor stemness and accelerates tumorigenesis in HCC dependently on Nanog and VEGFR2, which is valuable for the prediction of HCC recurrence after RFA and development of novel therapeutics.

#936

Single cell analysis reveals cancer stem cell heterogeneities in hepatocellular carcinoma.

Hongping Zheng, Yotsawat Pomyen, Maria Hernandez, Caiyi Li, Ferenc Livak, Tim Greten, Anuradha Budhu, Xin Wang. _NIH, Bethesda, MD_.

Tumor heterogeneity represents a major obstacle to effective cancer treatment and personalized medicine. Hepatocellular carcinoma (HCC) is clinically and biologically heterogeneous, which is partly attributed to the presence of hepatic cancer stem cells (CSCs). CSC surface makers can have overlapping expression or exclusive expression of different subpopulations of hepatic CSCs, and these cells may contain different oncogenic changes. The existence of various CSC surface-marked subpopulations might pose a problem for CSC targeted therapeutics. Little is known about whether there are shared or distinct pathways in different subpopulations. Moreover, it remains unknown whether heterogeneity exists within certain subpopulations. Thus, we propose to profile the global transcriptome of surface-marked CSCs (EpCAM+, CD133+ and CD24+) at the single-cell level using index flow cytometric sorting and single-cell RNA sequencing technology.

We statistically compared mRNA transcriptomes of individual single cells of triple positive (EpCAM+/ CD133+ /CD24+) CSCs and triple negative (EpCAM-/ CD133- /CD24-) cells. Further, we compared the triple positive and the triple negative cells at the single cell level in terms of their self-renew capability in both normoxic and hypoxic conditions. Our results show that at the single cell level, the transcriptomic profiles show a high-degree of heterogeneity in HCC cell lines, meanwhile, there is a dramatic difference between triple positive and triple negative cells in their transcriptomic profiles. There is a high-degree of intra-tumor and inter-tumor heterogeneity observed in single cell transcriptomes of HCC patients' samples. Triple positive CSC single cells show continuous rather than discrete stemness-related gene expression patterns. In parallel, the self-renewal capability of triple positive cells also exhibits heterogeneity, while triple negative cells show little self-renewal capability. Thus, our single cell analysis study reveals molecular and biological heterogeneity of cancer stem cells in HCC, which may provide insight into the underlying mechanisms of how CSCs contribute to tumor heterogeneity and underscore key pathways and novel targets for hepatic CSC therapy.

#937

Plasticity in heterogenous cancer stem cells promotes glioblastoma radioresistance.

Mutsuko Minata,1 Alessandra Audia,2 Junfeng Shi,3 Songjian Lu,4 Marat Pavlyukov,1 Yancey Gillespie Gillespie,1 Krishna Bhat,2 Ichiro Nakano1. 1 _University of Alabama at Birmingham, Birmingham, AL;_ 2 _The University of Texas, M.D. Anderson Cancer Center, Houston, TX;_ 3 _The Ohio State University, Columbus, OH;_ 4 _University of Pittsburgh School of Medicine, Pittsburgh, PA_.

Some cancers including Glioblastoma (GBM) have hierarchical cellular architecture with cancer stem cells (CSCs) as a minor tumorigenic subpopulation. While CSCs have been touted to contribute to initiate recurrence, post-surgical therapy-induced phenotypic changes in CSCs remain elusive. Here we report that treatment of GBM cells with ionizing radiation (IR) promotes a wound-healing mesenchymal signature, accompanied by conversion of naïve CSCs expressing CD133 to post-treatment more radioresistant CSCs expressing the GPI-anchored, TGFb-inhibitory protein CD109 in an ATM, NF-kB, CEBP-b dependent manner. In post-IR GBM, these therapy-escaped CD133-/CD109+ GBM cells, but not the rest of the tumor, uniquely retain clonogenic, tumorigenic, and multi-potent capacities both in vitro and in vivo. CD109 silencing unexpectedly eliminates cancer stemness via controlling YAP/TAZ as the downstream target and this CD109-YAP/TAZ signaling axis is evolutionally conserved between drosophila and human brain tumors. Clinically, both CD133 and CD109 are independently associated with poorer prognosis of GBM patients, while the poorest prognosis group of GBM expressed both CD133 and CD109 at higher rates. These data indicate a possibility of the post-IR CSC plasticity and subsequent heterogeneity, and the activation of the CD109-YAP/TAZ signaling axis as previously unidentified therapeutic targets for post-IR recurrent GBM.

#938

Estrogen induced NRF1 signaling is a molecular mechanism underlying the generation of different breast cancer stem cell subpopulations leading to intratumoral heterogeneity.

Jayanta Kumar Das, Deodutta Roy. _Florida International University, Environmental and Occupational Health, Bruce W Carter VA Medical Center, Research Service Laboratory, Miami, FL_.

Life time exposure to elevated levels of estrogen (E2) is a major risk factor for breast cancer. Among women with ER/PR positive tumors, only 50 to 60% of women respond to endocrine therapy. Intra-tumoral heterogeneity of breast cancer cells may be one of the reason for unresponsiveness to endocrine therapy in more than 40% of ER-positive breast cancer cases. In this study we examined that heterogeneous breast cancer stem cells (BCSCs) are produced by exposure to E2 and/or ectopic expression of molecular risk factor - nuclear respiratory factor 1 (NRF1). To elucidate this, we used single cell confocal imaging, flow sorting, ChIP and qRT-PCR approaches. We also measured the key regulators of pluripotency, epithelial-mesenchymal transition (EMT), stemness, cell apoptosis and cell cycle regulation. The functional assay for cancer stem cells enrichment employed were selection of cells in B27 medium as spheroids, anchorage-independent growth and mammosphere formation assays. Carcinogenic treatments of E2 to MCF10 A CD24- cells induced formation of ~1.46% CD44+/CD24+ and 26.02% of CD44+/CD24- subpopulations, which are a typical phenotypes associated with human breast tumor-initiating cells (BTICs) or BCSCs. These subpopulations are capable of forming self-renewing mammospheres. In contrast, CD24+ or CD24-/CD44- cells did not form mammospheres. Stable NRF1 overexpression induced formation of 2.36% CD44+/CD24+ and of 64% CD44+/CD24- subpopulations. Carcinogenic treatments of E2 to stable NRF1 overexpressing MCF10 A cells, induced to acquire 21.54% CD44+/CD24+ and 44.54% CD44+/CD24- subpopulations. E2 treatment to NRF1 overexpressing BSCSs markers significantly increased mammosphere forming capability, compared to NRF1 alone. The dominant negative form of NRF1 diminished the effects of E2 and/or NRF1 induced acquiring of BCSCs antigen markers and their capability of forming mammospheres. E2 induced BTICs/BCSCs were heterogeneous. Each subpopulation was characterized by a different transcriptional and biomarker profiles of CD24, CD44, CD49f, CD133, ALDH1A1, CXCR4, and NRF1. The expression of different markers for pluripotency (OCT4, SOX2 and Nanog), EMT (E-cadherin, N-cadherin and Vimentin), stemness (CD24, CD44, CD39f, CD133, ALDH1A1), cell cycle (p16,CDC2,CDC25C, cyclinB1) and metastasis (CXCR4), were associated with BTICs produced by E2 and/or NRF1. In summary, we observed new roles of NRF1 in contributing to acquire breast tumor initiating stem-like cells and in regulating EMT and invasiveness of BCSCs, thus opens a new direction of NRF1's role in breast cancer research. A better understanding of how E2 dependent breast neoplasm heterogeneity depends on NRF1 network may open new avenues for therapeutic strategy against breast cancer. This work was supported partly by VA MERIT Review (VA BX001463) grant to DR.

#939

Single cell mRNA expression profiling reveals heterogeneity of normal and malignant breast stem cell populations.

Shamileh Fouladdel,1 Justin Colacino,2 Ebrahim Azizi,1 Max S. Wicha1. 1 _University of Michigan, Department of Internal Medicine, Division of Hem/Onc, and Comprehensive Cancer Center, School of Medicine, Ann Arbor, MI;_ 2 _University of Michigan, Department of Environmental Health Sciences, School of Public Health, Ann Arbor, MI_.

The normal breast contains epithelial populations which are hierarchically organized, a profile recapitulated in breast cancer. At the apex of these hierarchies are "stem like" cells defined by their capacity to self-renew as well as to generate more differentiated progeny. In breast cancer these "cancer stem cells" (CSCs) have been shown to be important mediators of tumor metastasis and treatment resistance. Although the phenotypes of normal rodent and human mammary stem cells have been characterized, CSC populations are operationally defined by their capacity for tumor initiation. In addition, putative breast CSC markers such as CD44+/CD24low/- and or ALDH+ have proven useful in enriching for cells capable of tumor initiation and previous studies have suggested that these markers identify alternative mesenchymal and epithelial stem cell states, respectively. However, it remains unclear whether CSCs exist in discrete states or whether these cells actually represent a continuum reflected in heterogeneity of cell populations defined by expression of these markers. To address this question we utilized single cell mRNA expression profiling of normal and malignant breast stem cells. We first generated a panel of 96 genes representing stem cell and developmental pathways and analyzed expression of these 96 genes utilizing Fluidigm's C1 and BioMark HD technologies and TaqMan gene expression assays. We also utilized Fluidigm's Polaris to selectively isolate single cells that were then analyzed by illumina RNA-Seq method. Utilizing these advanced technologies, we demonstrated that both normal and malignant breast stem cells characterized by CD44+/CD24low/- and or ALDH+ expression are highly heterogeneous. For example, the ALDH isoforms ALDH1A1 and ALDH1A3 were each expressed in some single cells and co-expressed in others. ALDH1A1 expressing single cells appeared to express a unique gene pattern characterized by SLIT/ROBO, PLXNA2, RHOU and HNF1a. These results suggest that at the level of mRNA expression, normal and malignant breast stem cells display a greater heterogeneity than has previously been reported. These results have important implications for breast carcinogenesis as well as for the development of therapeutic strategies designed to target CSCs in breast cancer.

#940

Altering intratumoral heterogeneity in HER2+ breast cancers with PTEN inactivation and trastuzumab therapy.

Joseph P. Burnett, Shamileh Fouladdel, Nathan Truchan, Ebrahim Azizi, Max Wicha, Duxin Sun. _University of Michigan, Ann Arbor, MI_.

Within the context of HER2+ breast cancer, PTEN mutation has been associated with poor patient survival, trastuzumab resistance, and induction of the epithelial to mesenchymal transition. Accumulating evidence supports the theory that breast cancers are organized in a hierarchical manner with the presence of cancerous stem, lineage restricted progenitor, and differentiated-like cells. Recent literature has also indicated that CSCs may exist in distinct epithelial and mesenchymal states which retain unique properties. In order to understand the influence of PTEN inactivation within the context of a hierarchical model of cancer, and examine which populations themselves are targeted by trastuzumab, we have performed analysis of traditional CSC markers and single cell lineage tracing experiments. Here, a lentiviral vector containing shPTEN and dsRed reporter are introduced into HER2+ cell line BT474. Following bulk sorting of dsRed+ cells, long term culture of parent and shPTEN cells with and without trastuzumab for 4 weeks reveal no obvious changes in traditional EMT-CSC markers CD44+/CD24- and only modest changes in Aldefluor + cells. In order to take a CSC-marker agnostic approach we performed a single cell colony formation assay and identify that trastuzumab enriches for rare cells with colony forming potential. The majority of single cell derived colonies exhibited distinct morphologies stable with long term culture which are similar regardless of PTEN knockdown or trastuzumab therapy. However, quantification reveals PTEN knockdown results in a higher frequency of colonies which exhibit distinctly mesenchymal morphology and less of those with epithelial morphology. Interestingly rare colony forming cells could recapitulate all morphologies present in the parental cell line, suggesting they were derived from a true CSC. Single cell multiplex RT-qPCR of 96 target genes was performed on mixed and distinct morphology colonies using Fluidigm's C1 and BioMark HD instruments and TaqMan gene expression assays. Principal component and t-SNE analysis was capable of separating morphologically distinct colonies and revealed multiple cell clusters within each colony. Using hierarchical clustering it is evident that a minority of cells which appear as unique clusters in PCA analysis are those which are proliferative and express distinct lineage markers reflective of the colony itself. Taken together these results suggest that PTEN knockdown shifts the equilibrium from primarily epithelial lineage differentiation to a more mesenchymal differentiation while trastuzumab enriches for stem and progenitor cells. 

### Tumor Microenvironment 1

#941

CXCL1-CXCR2 signaling might recruit the bone marrow-derived mesenchymal cells to the carcinoma-associated fibroblasts of gastric cancer.

Masakazu Yashiro, Tomohisa Okuno, Hiroaki Kasashima, Yuichiro Miki, Hirohisa Nakamae, Kisyu Kitayama, Toshiyuki Kawashima, Takaharu Hatano, Heishiro Fujikawa, Tsuyoshi Hasegawa, Takahiko Nakane, Masayuki Hino, Kosei Hirakawa, Masaichi Ohira. _Osaka City Univ. Grad. School of Medicine, Osaka, Japan_.

Background & Aims: Activated fibroblasts in the tumor stroma, also termed carcinoma-associated fibroblasts (CAFs), play a critical role in the progression of cancer. In our previous studies, we identified CAFs as playing an important role in cancer progression in the development of gastric cancer. However, it remains unclear which factor recruit and promote CAFs. The aim of this study is to clarify from where CAFs originate and which factor recruits them into the gastric cancer microenvironment.

Methods: In vitro study analyzed the chemotaxis-stimulating factor from gastric cancer cells using bone marrow-derived mesenchymal cells (BM-MCs). The nude mice with bone marrow transplantation from CAG-EGFP mice were used for the in vivo gastric cancer experiments. The influences of chemokine (C-X-C motif) receptor 2 (CXCR2) inhibitor on the migration of BM-MCs were examined in vitro and in vivo.

Results: BM-MCs migrated into tumor stroma of in vivo gastric cancer. The number of BM-MCs was much greater in the gastric tumor than in normal tissue from three days after inoculation of cancer cells. C-X-C motif ligand 1 (CXCL1) from gastric cancer cells significantly (p<0.001) stimulated the chemoattractant ability of BM-MCs in vitro. Anti-CXCL1 antibody and CXCR2 inhibitor significantly (p<0.05) decreased the migration-stimulating activity of gastric cancer cells. A CXCR2 inhibitor, SB225002, decreased tumor size and lymph node metastasis of gastric tumor, and significantly (p=0.039) prolonged survival of gastric-tumor bearing mice. The histologic findings indicated that SB225002 decreased the recruitment of BM-MCs into the tumor stroma, resulting in the reduction of CAFs.

Conclusions: BM-MSCs recruit into gastric tumor and differentiate into CAFs. CXCL1 from gastric cancer cells stimulates the recruitment of BM-MCs into gastric cancer locus via CXCR2 signalling. Inhibition of BM-MCs' recruitment appears a promising therapy for gastric cancer.

#942

CXCR2 acts as a checkpoint regulator of the pancreatic stellate cells activity within pancreatic cancer tumor microenvironment.

Mohammad Awaji, Michelle Varney, Abhilasha Purohit, Surinder K. Batra, Rakesh K. Singh. _University of Nebraska Medical Center, Omaha, NE_.

Pancreatic cancer (PC) is a grotesque disease featured by an inflamed complex tumor microenvironment (TME) that contribute to advancing tumor progressing. Oncogenic K-ras, the most common mutation in PC, amplifies inflammation within TME through overexpression of multiple immune factors such as CXCR2 and its ligands. K-ras induced overexpression of CXCR2 axis, made CXCR2 seem intuitively a good target for PC treatment. In KC, a spontaneous PC mouse model with oncogenic K-ras, genetic ablation of CXCR2 caused anti-tumor events such as increased apoptosis and decreased angiogenesis; and also presented pro-tumorigenic events; most notably was the increased fibrosis and metastasis to the liver. These observations have shed light on the possible role the CXCR2 on the other TME component especially pancreatic stellate cells (PSCs). The aim of this study is to determine the differential effect of K-ras status and CXCR2 chemokines expression in the PC cells on their interaction with PSCs. Unidirectional segregated co-culture studies were conducted using conditioned media (CM). CM collected from PSCs were used to grow multiple PC cell lines, and CM obtained from different PC cell lines were used to culture PSCs. Cellular proliferation and gene expression were analyzed. Furthermore, PSCs proliferation potentials were assessed as those cells treated with exogenous CXCL1 and CXCL8, or PC cell lines derived-CM in the presence or absence of CXCR2 antagonists. Co-culture studies revealed that PSC-derived CM had increased proliferation of PC cell lines positive to oncogenic K-ras as well as increasing their expression of multiple chemokines such as CXCL1, CXCL5, and CCL2. On the other hand, PC cell lines with a wildtype K-ras were inhibited by PSC-derived CM treatment. PSCs proliferation potentials were decreased with CM derived from oncogenic K-ras positive PC cell lines and increased with CM derived from PC cell lines with wildtype K-ras. Furthermore, CM from oncogenic K-ras PC cell lines has increased the expression of multiple pro-tumorigenic genes in the PSCs including cytokines such as IL-10, IL-4 and IL-13, and chemokines such as CXCL2 and CXCL7. Also, treating PSCs with exogenous CXCL1 and CXCL8 exhibited a similar proliferation inhibition to that observed with oncogenic K-ras PC derived CM treatment. CXCR2 antagonists have shown rescued inhibition or enhanced proliferation of PSCs when incorporated with CXCR2 chemokine or PC cell line derived CM treatment. These observations suggest that K-ras status of PC dictates their interaction with PSCs within the TME. We have demonstrated that oncogenic K-ras through increased production of CXCR2 chemokine would dampen PSCs proliferation and further orient them to support tumor progression by increased expression of pro-tumorigenic genes. Besides, we observed that CXCR2 inhibition in PSCs would increase their proliferation which may further their fibrogenic activity.

#943

RelA regulates CXCR5/CXCL13 transcription and associated immune response in breast cancer.

Sougata Roy Chowdhury, Subir Biswas, Gunjan Mandal, Arindam Bhattacharyya. _University of Calcutta, Kolkata, India_.

It is evident from research outcomes that metastases-associated deaths are predominant in breast cancer. Recent developments on early diagnosis using mammographic screening and the implementation of adjuvant therapies may have reduced breast cancer associated deaths in decent numbers, although new markers for prognosis are of utmost importance for patients with higher risk of developing metastases or recurrence.

Chemokines are the key messenger of cellular immune response and can be targeted to reduce the intratumoral regulatory T cells (Treg) for antitumor immunity. Targeting either specific immunomodulators and/or intervening molecular mechanisms is thought to be a potential therapeutic option. In our previous study, we have found that co-expression of CXCR5 and CXCL13 is significantly associated with epithelial to mesenchymal transition (EMT) of cells and lymph node metastasis (LNM) during breast cancer progression. In this study, we are aiming to investigate how the transcriptional regulation of this receptor-ligand pair directs the process of disease development. Interestingly, it was found that RelA (p65), a subunit of NFkB protein, promoted the transcription of both CXCR5 and CXCL13. The putative RelA binding sites were validated using sequential deletion of respective promoter regions of CXCR5 and CXCL13. We observed that expression of CXCR5 significantly (p<0.05) increased with RelA and Nrf2 overexpression and CXCL13 co-stimulation in MDA-MB-231 cell line. RelA was also found to induce the expression of CXCL13 in T-47D and MDA-MB-231 cell line. Significantly, SNAIL, a key EMT regulatory element, was found to be highly expressed in RelA-transfected MDA-MB-231 cell line with or without CXCR5 and CXCL13 co-stimulation. We also observed CXCR5+ T follicular helper (Tfh) and Treg sub-population within the tumor microenvironment, although numbers varied with stages and molecular subtypes. In chemotaxis assay, CXCR5\+ Treg cells were found to be predominant among migratory T-cell subsets against RelA overexpression and/or CXCL13 stimulation. Simultaneously, CCL2, a pro-inflammatory chemokine, induced macrophages to secrete CCL22, which in turn, might attract CXCR5\+ Treg and Th2 cells into the tumor microenvironment. Study in 4T1-BALB/c breast cancer mouse model demonstrated significant (p<0.05) increase in CXCR5 and CXCL13 expression levels upon immunostimulation.

The study may help to understand the prognosis value of CXCR5 and CXCL13 considering the impact of this receptor-ligand pair on the regulation of Tfh/Treg ratio, EMT and LNM. We believe that this investigation may lead to a comprehensive prediction of the tumor fate as well as to explore possible markers for breast cancer prognosis and future chemotherapy with more precision.

#944

Chemokine receptor signaling as a new tool to improve lung cancer diagnostics and therapy.

Katalin Nagy-Major,1 Jörg Sänger,2 Harshad R. Kulkarni,2 Peter Fix,2 Almut Kunze,2 Amelie Lupp,3 Reiner Bonnet,2 Werner Seeger,4 Richard P. Baum,2 Patricia Grabowski,1 Rajkumar Savai4. 1 _Charité Campus Benjamin Franklin, Berlin, Germany;_ 2 _Zentralklinik Bad Berka, Bad Berka, Germany;_ 3 _Jena University Hospital, Jena, Germany;_ 4 _Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany_.

Background: The tumor microenvironment plays a critical role in cancer development, progression, and control. We demonstrated in vitro and in vivo that a bidirectional crosstalk between tumor-associated macrophages and cancer cells via CCR2-CCL2 and CX3CR1-CXCL1 signaling is fundamental for lung cancer growth and metastasis offers new treatment concepts.

Aim: Our working hypothesis is to develop a new platform for specific chemokine receptor (CCR2) PET imaging and a range of theranostic applications in lung cancer. For this purpose, we planned to define the CCR2 expression in NSCLC as well as in SCLC, tumor cells as well as immune/inflammatory cells.

Material and Methods: This is a multicenter trial with patients from Zentralklinik Bad Berka GmbH (n =100 NSCLC, n=100 SCLC). All patients gave informed consent according to the declaration of Helsinki. FFPE material was available in all cases. CCR2 CXCR4 and CX3CR1 expression was determined by immunohistochemistry with the avidin-biotin-peroxidase-complex.

Results: Analyzing 96 NSCLC (47 adenocarcinoma, 49 squamous cell carcinoma) and 45 SCLC tumor samples, we found CXCR4 and CCR2 chemokine receptor expression in tumor, immune and inflammatory cells. In NSCLC, co-expression between CCR2 and CXCR4 staining was very high concerning their expression on tumor cells rising with higher tumor stages, proliferation rate and grading. CCR2 expression in immune cells was restricted to lower stages; the same pattern could be observed in 45 SCLC tumor samples where CCR2 expression on immune cells was found especially in limited disease rather than in metastatic disease; CXCR4 expression in tumor cells was found to be high in all stages of SCLC. To evaluate the therapeutic efficacy of CCR2 antagonism in vivo, we injected LLC1 s.c. into WT mice treated with CCR2 antagonist (RS504393) or vehicle. Notably, tumor growth was significantly inhibited in CCR2 antagonist-treated mice as compared with vehicle-treated mice.

Discussion: Our preliminary results show a significant portion of CCR2 positive tumors - NSCLC as well as SCLC which would qualify for a CCR2-based imaging as well as - demonstrated in our mice models - CCR2 inhibitory strategies. This should be evaluated in further preclinical trials.

#945

Oligodendrocytes might up-regulate the Invasiveness of glioblastoma cells via angiopoietin-2 signaling.

Toshiyuki Kawashima. _Osaka city university school of medicine, osaka, Japan_.

Background & Aims: Glioblastoma (GBM, WHO Grade IV) is considered as the most lethal neoplasm of all solid cancers due to its inherent intensive invasiveness. GBM may arise de novo, or following progression from lower grade gliomas. Recently, solid tumor progression has been recognized as the product of an evolving crosstalk between the cancer cells and its surrounding glial cells. The intensive invasion activity of GBM cells might be regulated by surrounding normal cells such as oligodendrocytes or fibroblasts. In this study, we evaluated the interactive factors between GBM cells and normal glial cells to determine the role of oligodendrocytes in regulating the invasiveness of glioblastoma cells.

Methods: Two GBM cell lines, T98G and U251, were used. Two oligodendrocyte cell lines, ODC1 and ODC2, were derived one each from surgical tissues of patients with low-grade glioma (WHO Grade II). Two fibroblasts cell lines, GF1 and GF2, were derived one each from surgical tissues of patients with GBM. Oligodendrocytes and fibroblasts were confirmed by immuno-histochemical staining. The invasive property of GBM cells was analyzed in the presence or absence of conditioned medium from oligodendrocytes or fibroblasts by wound-healing assay and Boyden chamber invasion assay. The proliferation ability of GBM cells was examined by MTT assay. Subsequently Cytokine array was used to examine the cytokines and growth factors in the conditioned medium from oligodendrocytes or fibroblasts.

Results: Oligodendrocyte cells, ODC1 and ODC2 cells, significantly (p<0.01) increased the migration and invasion ability of GBM cells, T98G and U251. In contrast, fibroblasts, GF1 and GF2 cells, did not affect the migration and invasion ability of GBM cells. Cytokine array indicated that Angiopoietin-2 was found in the conditioned medium from oligodendrocytes, but not that from fibroblasts. Angiopoietin-2 significantly (p<0.01) increased the invasiveness of GBM, but not their proliferation ability.

Conclusion: Oligodendrocytes might up-regulation of the invasiveness of GBM cells via angiopoietin-2 signaling pathway. Hence, angiopoietin-2 can be considered as a promising target for the treatment of malignant gliomas.

#946

Characterize and identify secreted high molecular weight heparanase from PC3M conditioned medium.

Donghong Ju, Mary A. Kosir. _Barbara Ann Karmanos Cancer Inst., Detroit, MI_.

Introduction: Neutrophils, platelets, lymphocytes are typical sources of NAP-2 which is associated with heparanase activity. Heparanase degrades polymeric heparan sulfate molecules into shorter oligosaccharides, participating in extracellular matrix degradation. It is active under acidic conditions during tumor invasion and inflammatory processes. Our lab previously found that some cell lines like androgen independent prostate cancer cells (PC3M) can secrete a protein that reacts with anti-NAP-2 antibody, but has a much higher molecular weight(>50KD) than NAP-2 (MW~6-14 KD), and has heparanase activity. The purpose of this study is to characterize and identify this protein.

Procedures: Immunoprecipitation was performed using Pierce DirectIP kit according the manufacturer's instructions. Heparin binding affinity column used HiTrap™ Heparin HP column from GE Healthcare Bio-Sciences. The binding buffer was 10mM NaAcetate pH 5.0, the eluting buffers were 0.5M-2M NaCl in 10mM NaAcetate pH 5.0. The mass spectrometry was done by KCI Proteomics Core facility.

Data: The immunoprecipitation method confirmed that this higher molecular weight protein indeed can be pulled down by the NAP-2 antibody, and after using heparin binding affinity column to purify, we can still get the > 50KD band on the western blot for NAP-2, meaning it is a heparin binding protein.

Conclusions: A protein identified with NAP-2 antibody, but with higher molecular weight than native NAP-2 is found in the conditioned medium of PC3M cells that binds heparin, further confirming a potential role in tumor progression for prostate cancer.

#947

CXCR4 in combination with immune check point inhibition in ovarian cancer mouse model demonstrates potential for novel therapeutic strategies.

Kristen Starbuck, Robert McGray, Samar Masoumi-Moghaddam, Ariel Francois, Kunle Odunsi, Emese Zsiros. _Roswell Park Cancer Institute, Buffalo, NY_.

Chemokine CXCL12 and its receptor CXCR4 are the most highly expressed chemokine axis in serous ovarian cancer. CXCR4 promotes tumor growth, angiogenesis, and metastasis. High CXCR4 expression is associated with poor survival, making it an attractive therapeutic target. The objective of this study was to establish an immune competent murine ovarian cancer model with high CXCR4 expression and to test single agent CXCR4 blockade and combination therapy to optimize emerging treatment strategies for future clinical trials. CXCR4 expression was evaluated by flow cytometry in ID8, ID8-VEGF, IE9-MP1, BR5, BR5-Akt, BR5-C2k, and BR5-Kras mouse ovarian cancer cell lines as well as IE9-MP1 tumor samples. Subsequently an exploratory study was undertaken utilizing the ID8 immune competent mouse model to assess the efficacy of AMD3100, the only commercially available CXCR4 inhibitor, alone and in combination with bevacizumab and anti-PD-1. Immune analysis of circulating lymphocytes by flow cytometry was performed during treatment to monitor changes of immune cell subsets. Surface expression of CXCR4 receptor was minimal in ID8 (1.6%), ID8-VEGF (0.9%), IE9-MP1 (1.8%), BR5 (0.9%), BR5-Akt (1.2%), BR5-C2k (2.3%), and BR5-Kras (1.1%), however intracellular staining revealed widespread CXCR4 expression in ID8 (99.6%), ID8-VEGF (99.7%) BR5 (96.7%), BR5-Akt (94.3%), BR5-C2k (98.2%), and BR5-Kras (98.7%) cell lines. Using the ID8 intraperitoneal ovarian cancer mouse model in C57BL/6J mice, treatment with immunotherapy significantly increased cell surface expression of CXCR4 from 2% in the control group to 79.6-91.8% in the treatment group. Treatment with AMD3100 as a single agent showed no improvement in survival over control. However, AMD3100 in combination with anti-PD-1 therapy significantly improved survival. The addition of bevacizumab demonstrated no further survival benefit and bevacizumab as a single agent showed no improvement in survival over control, either alone or in combination with anti-PD-1. Analysis of circulating lymphocytes revealed a trend toward higher CD4 to CD8 ratio in mice who were treated with anti-PD1 therapy. Targeting the CXCL12-CXCR4 axis in combination with other immunotherapies such as immune checkpoint inhibitors is a novel therapeutic strategy which may provide benefit over single-agent immunotherapy and warrant investigation in a clinical trial.

#948

Characterization of the microenvironment of murine primary triple negative breast cancer.

Kassondra Balestrieri, Keith Pittman, Mohamed Ramez, Nasreen Vohra, Kathryn Verbanac. _East Carolina University/Brody School of Medicine, Greenville, NC_.

The purpose of this study was to investigate the role of inflammatory cells in the metastatic process using reliably metastatic murine models of Triple Negative Breast Cancer (TNBC). We have previously selected, developed and characterized two reliably metastatic TNBC mouse models for our investigations. The T11 (claudin-low) and 2225LM (basal-like) Balb/c-derived transplantable tumors display molecular profiles that mirror human TNBC. Here we report characterization of the inflammatory cells and microenvironment in these primary TNBC tumors and at metastatic sites in both heterotopic and orthotopic models. Tumors were implanted in the s.c. flank or mammary fat pad and resected 1-2 weeks later to promote the growth of seeded metastases, when volumes reached ~700 mm3 or 300 mm3, respectively. Both flow cytometric analysis and immunohistochemistry of primary tumors demonstrated high infiltration by myeloid-derived cells (CD11b+) and low levels of lymphoid cells (CD3+). Myeloid cells were also the major infiltrating cells in lung metastases and were significantly elevated compared to normal lung. The granulocytic subset (CD11b+Ly6G+) predominated, consistent with an immunosuppressive role for cells with this phenotype (N2, G-MDSC, etc.). Primary 2225LM and T11 tumors and metastatic lung tumor homogenates were assayed for immunomodulators and compared to normal breast and lung tissue from age-matched contemporary controls. G-CSF was significantly increased in tumors and levels (pg/mg protein) were correlated with tumor burden. VEGF was significantly elevated in all tumors, as were chemoattractants for granulocytic cells (KC/CXCL1, MIP-2/CXCL2), monocytic cells (MCP-1/CCL2), eosinophils (Eotaxin/CCL11) and T cells (MIG/CXCL-9). In the heterotopic model, lung levels of KC/CXCL1 and MCP-1/CCL2 were significantly increased prior to frank lung metastasis (5-30 days post-implant). These data suggest that TNBC-derived G-CSF may play a role in the mobilization of CD11b+Ly6G+ granulocytes and progenitors from the bone marrow and key chemokines may mediate migration of these cells to metastatic sites. Future studies will define phenotypic and functional characteristics of the G-MDSC that are promoting tumor progression in these murine TNBC models.

#949

Resistin potentiates stemness and chemoresistance of breast cancer cells through STAT3 activation: implications in breast cancer health disparity.

Sachin Kumar Deshmukh,1 Arun Bhardwaj,1 Sanjeev K. Srivastava,1 Nikhil Tyagi,1 Ahmed Al-Ghadhban,1 Ajay P. Singh,1 Donna L. Dyess,1 James E. Carter,2 Seema Singh1. 1 _Mitchell Cancer Institute, Mobile, AL;_ 2 _College of Medicine, Mobile, AL_.

Breast cancer (BC) is most frequently diagnosed cancer and remains the second leading cause of cancer-related deaths in women in the United States. African American (AA) women bear an additional burden of BC with early onset of disease, poorer prognosis, higher risk of recurrence, and worst clinical outcome as compared to Caucasian American (CA) women. Emerging evidence strongly argue the role of tumor-microenvironment (TME) in BC of AA and CA racial backgrounds. On the similar line, our earlier findings suggested that serum levels of resistin; an inflammatory cytokine are significantly elevated in AA BC patients compared to their CA counterparts. Furthermore, we demonstrated that resistin promoted growth and aggressiveness of BC cells. In the present study, we investigated the role of resistin in stemness and chemoresistance of BC cells. For this, two BC cells of CA (MDA-MB-231) and AA (MDA-MB-468) origin were treated with resistin and sphere-forming ability, a key characteristic of cancer stem cells, was determined. Data demonstrate that number of spheres is significantly enhanced in both the BC cells upon resistin stimulation. Immunoblot analyses reveal that resistin-induced sphere-forming ability is associated with enhanced levels of stemness-associated transcription factors (Nanog and KLF4) in BC cells. We next examined if resistin conferred chemoresistance to BC cells. Our data suggest that resistin-treated MDA-MB-231 and MDA-MB-468 BC cells develop greater resistance against doxorubicin-induced cytotoxicity. Notably, the effects conferred by resistin were more prominent in AA BC cells comparing to CA BC cells. Our mechanistic studies unveiled an important role of STAT3 activation in the resistin-induced stemness and chemoresistance of BC cells. Taken together, our findings provide novel insight into the role of resistin in BC biology and further strengthen its role in racially disparate clinical outcomes.

#950

Involvement of proinflammatory chemokine network between adipocytes and triple negative breast cancer cells.

Rosa Mistica Coles Ignacio,1 Hyeongjwa Choi,1 Carla Gibbs,1 Eunsook Lee,2 Samuel Adunyah,1 Deok-Soo Son1. 1 _Meharry Medical College, Nashville, TN;_ 2 _Florida A &M University, Tallahassee, FL_.

Obesity has become a global epidemic and causes a chronic low-grade inflammation. This poses a large health burden because excess adiposity can lead to chronic diseases such as type 2 diabetes, cardiovascular disease and some cancers. Obese individuals get higher incidence, progression and mortality of cancers compared to lean ones. Particularly, breast cancer (BC) in women is closely associated with obesity. Despite an epidemiological link between obesity and low cancer survival rates, little is known about the molecular mechanisms by which obesity promotes BC progression. Obesity-derived chronic low-grade inflammation involves alteration of a chemokine network that may contribute to the cancer progression and metastasis. Herein, we proposed the novel concept that the chemokine network between obesity and BC builds an inflammatory burden via proinflammatory chemokines to promote cancer progression. We employed mesenchymal BC cell PY8119 and epithelial-like BC PY230 for triple negative BC (TNBC) cell model, and mouse 3T3-L1 preadipocyte and adipocyte conditioned media (CM) to mimic lean and obese conditions in vitro. We examined the profiles of chemokine signature of PY8119 and PY230 treated with preadipocytes and adipocytes CM using PCR array. Both preadipocytes and adipocytes CM-treated PY8119 and PY230 cells absent or low levels in chemokine receptors. On the other hand, the chemokines CCL2, 5, 7 and CXCL10 showed higher expression in both preadipocyte and adipocyte CM-treated PY8119. Interestingly, adipocyte CM-treated PY8119 cells dominantly expressed CXCL1-3 compared to preadipocyte CM-treated cells. In addition, CCL2, 5, 7, 20, and CXCL1, 5, 10 were highly expressed in both preadipocyte and adipocyte CM-treated PY230 cells. Moreover, CXCL2 and 3 were significantly induced in PY230 cells after treatment with adipocyte CM compared to preadipocyte CM. Taken together, the results indicate that dominant chemokines CXCL1-3 expressed in adipocyte CM-treated TNBC cells promote a pro-tumor inflammatory network. Hence, these proinflammatory microenvironment augmented by adipocytes might contribute to TNBC progression.

#951

Induction of COX-2 and NFκB activation represent TLR3-independent undesirable aspects of poly-I:C activity.

Marie-Nicole Theodoraki,1 Ravi Muthuswamy,2 Jamie Voyten,2 Francesmary Modugno,2 Natasa Obermajer,2 Robert P. Edwards,2 Pawel Kalinski2. 1 _University of Pittsburgh, Pittsburgh, PA;_ 2 _University fo Pittsburgh, Pittsburgh, PA_.

Background: Infiltration of cytotoxic T-cells (CTL) in tumors is known to be associated with improved patient's clinical outcomes. In contrast, local infiltration with regulatory T-cells (Treg) predicts accelerated progression and shorter overall survival. The above observations highlight the need for new measures to promote selective accumulation of CTLs but not Tregs in tumor microenvironments (TME). Here we compare the TME-modulating impact of poly-I:C with a selective TLR3 ligand, rintatolimod showing an induction of CCL22, CXCL12 and expression of COX-2 and downstream suppressive factors, selectively by poly-I:C. Finally, we report on the optimized combinatorial adjuvant to reprogram tumor microenvironment.

Materials and Methods: Twelve ovarian cancer specimens were cultured in the presence of IFNα, indometacin (COX-1/2 inhibitor) or/and one of two synthetic TLR3 ligands, poly-I:C (non-selective activator of TLR3 and helicases) or rintatolimod (selective TLR3 ligand). Biopsies were harvested for mRNA measurements and culture supernatants were analyzed for CCL5, CXCL10 and CCL22 concentrations. Alternatively, established ovarian cancer cell lines, monocyte-derived macrophages, THP1 cells or adult fibroblasts were used in analogous experiments. Chemotaxis assays were performed using pre-activated CD8+ T-cells (top chamber) and supernatants from the differentially treated ovarian cancer specimens. Western blot experiments were used to evaluate the impact of NFκB. Blocking experiments using soluble TNFα receptor inhibitor were performed.

Results: Both, Poly-I:C and rintatolimod, induced CTL attractant CXCL10. Unexpectedly, poly-I:C but not rintatolimod promoted the expression of the MDSC/Treg attractant CXCL12, which was reversed by addition of indometacin. Furthermore, poly-I:C induced expression of COX-2 and COX-2-dependent suppressive factors (IDO, IL-10). Evaluating the COX-2 cascade, TNFα was found to be increased in the presence of poly-I:C but not rintatolimod. Additionally, Western Blot analysis showed I-κB degradation (as a marker of NF-κB activation), selectively in response to poly-I:C. We observed that the combination of both TLR-3 ligands with IFNα and indomethacin selectively induced the desirable chemokines CCL5 and CXCL10 and suppressed CCL22 in tumor samples with similar results in the macrophages and fibroblasts. The ovarian cancer cell lines showed only minimal expression of chemokines.

Conclusion: We demonstrate the feasibility of selective modulation of TME, using different clinically applicable factors and their combinations. We show for the first time, that poly-I:C, but not a selective TLR3 ligand, induces NFκB and undesirable COX2-dependent suppressive factors, which though can be eliminated through addition of a COX-1/2 inhibitor. These effects may be avoided using a selective TLR3 ligand.

#952

Modulation of immune cell trafficking into human colorectal cancer by gut microbiota.

Eleonora Cremonesi,1 Jesus G. Garzón,2 Valeria Governa,1 Valentina Mele,1 Francesca Amicarella,1 Elisabetta Padovan,1 Manuele Muraro,1 Paul Zajac,1 Daniel Oertli,3 Lubor Borsig,2 Giandomenica Iezzi1. 1 _Universitat Spital, Basel, Switzerland;_ 2 _Istitute of Phisiology, University of Zürich, Zürich, Switzerland;_ 3 _Department of Surgery, Universitat Spital, Basel, Switzerland_.

Introduction: Colorectal cancer (CRC) is a leading cause of cancer-related death. CRC infiltration by immune cells, including cytotoxic CD8+ T cells (CTLs), IFN-gamma-producing T-helper 1 cells (Th1), Foxp3+ regulatory T cells (Tregs) and CD16+ MPO+ neutrophils, is associated with favorable prognosis. However, chemokines driving these cell populations into the tumor site, their cellular sources and their microenvironmental triggers remain to be elucidated.

Aim: We investigated the chemokine/chemokine receptor network promoting CRC infiltration by immune cells associated to favorable prognosis.

Results: CRC infiltration by immune cells was associated with defined chemokine gene signatures, including CCL5, CXCL9 and CXCL10 for cytotoxic T lymphocytes and T-helper 1 cells, and CCL17, CCL22 and CXCL12 for T-helper 1 and regulatory T cells. Most of these chemokine genes were expressed by tumor cells upon exposure to gut bacteria in vitro and in vivo. Indeed, chemokine expression levels were significantly higher in orthotopic xenografts than in intraperitoneal tumors, and were drastically reduced by antibiotic treatment of tumor-bearing mice. Importantly, in human CRC samples, extents of chemokine production and immune cell infiltration was significantly associated with bacterial loads.

Conclusion: Gut microbiota stimulates chemokine production by CRC cells, thus favoring T cell recruitment into tumor tissues.

#953

Atypical chemokine receptor 1 (ACKR1/DARC) expressing tumors are associated with distinct recruitment of immune cells and increased pro-inflammatory chemokines.

Brittany D. Jenkins,1 Rupali Hire,1 Elizabeth Howerth,1 Michele Monteil,2 Rachel Martini,1 Melissa B. Davis1. 1 _Univ. of Georgia, Athens, GA;_ 2 _AU/UGA Medical Partnership, Athens, GA_.

Interactions between chemokines and their receptors can improve a host's anti-tumor response by influencing the targeted migration of immune cells via a chemokine gradient. Atypical Chemokine Receptor 1 (ACKR1/DARC), a genetically diverse transmembrane GPCR, acts as a decoy receptor for a variety of CXC and CC chemokines, including those with pro-malignant and pro-inflammatory effects, such as CCL2 (MCP-1, MCAF, JE) and CXCL8 (IL-8). The purpose of this study is to determine if the migration of tumor-associated immune cells is unique based on epithelial ACKR1 expression on breast cancer cells, and if this association is correlated to an increase in pro-malignant chemokines, better survival odds, and differences in race. Immunohistochemistry techniques were used to determine expression levels of ACKR1 on primary breast tumors, along with relative expression of T-cells, B-cells, dendritic cells, and macrophages. Concentrations of pro-inflammatory chemokines in circulation were determined using a Luminex-based immunoassay and matched patient peripheral blood samples. In silco analyses were performed to determine associations between ACKR1 tumor expression status, race, and survival. Finally, using human breast cancer cell lines and immunofluorescence techniques, co-localization between ACKR1 and selected pro-inflammatory chemokines was investigated. Results from these tests indicate that there is differential expression of immune cell types in tumors expressing ACKR1, and this difference was associated with the migration of B-cells and dendritic cells, which were not detected in ACKR1 negative tumors. Significantly increased circulating CCL2 and CXCL8 chemokine levels we also determined to be positively correlated with ACKR1 expression in primary breast tumors. Survival analyses showed a significantly increased relapse free survival in patients having tumors with high ACKR1 expression, while investigations into racial differences revealed a significant race effect, with Caucasians having higher ACKR1 levels on their tumors than African-Americans. Finally, co-localization between ACKR1 with CCL2 and CXCL8 is observed in cultured human breast cancer cells. Given that the data collected shows a tendency for those tumors positively expressing ACKR1 to have a more favorable prognosis, we suggest that a partial role of ACKR1 on breast tumor cells is to sequester pro-inflammatory chemokines in the tumor microenvironment, indirectly recruiting a distinct subset of tumor-associated immune cells.

#954

TGF-ß signaling inhibition counteracts myelofibrosis in MPN.

Matthias Bartenstein,1 Lanzhu Yue,2 Wanke Zhao,3 Wanting Tina Ho,3 Cem Murdun,2 Adam W. Mailloux,2 Ling Zhang,2 Anjali Budhakoti,1 Kith Pradhan,1 Franck Rapaport,4 Huaquan Wang,5 Zonghong Shao,5 Ulrich Steidl,1 Ross L. Levine,4 Zhizhuang Joe Zhao,3 Amit K. Verma,1 Pearlie K. Epling-Burnette2. 1 _Albert Einstein College of Medicine, Bronx, NY;_ 2 _Moffitt Cancer Center and Research Institute, Tampa, FL;_ 3 _Oklahoma University Health Sciences Center, Oklahoma City, OK;_ 4 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 5 _Tianjin Medical University General Hospital, Tianjin, China_.

Myelofibrosis (MF) is a common feature of the Philadelphia-Chromosome negative (Ph-) myeloproliferative neoplasms (MPN), a group that includes Primary Myelofibrosis (PMF), Essential Thrombocytosis (ET) and Polycythemia Vera (PV). Current treatment with Jak2 inhibitors reduces splenomegaly, but does not eliminate malignant clones nor improve bone marrow (BM) or spleen fibrosis, underlining the need for alternative therapies. One promising target is TGF-β1, a cytokine crucial for the development of myelofibrosis that is elevated in PMF patient plasma, progenitors and megakaryocytes (MK), and is known to promote fibrosis through stimulation of fibroblasts and their progenitors, mesenchymal stroma cells (MSC). Dysregulation of TGF-β1 signaling has been described in a number of mouse models of myelofibrosis exhibiting aberrant megakaryopoiesis, one of the hallmarks of MPNs. Preliminary data from 93 MPN patients showed overexpression of TGF-ß in PMF samples (p=0.001), with increased activation of the canonical target SMAD2 (p=0.005). We tested the efficacy of the orally available TGF-β1 signaling inhibitor Galunisertib (LY2157299) in counteracting fibrosis in two MF mouse models of common MPN oncogenes Jak2 and Mpn. Mice in the first model express the constitutively active human Jak2V617F mutant under the control of the ubiquitous hematopoietic promoter vav1 and show a PV-like phenotype at 6 weeks old, progressing to fibrosis of bone marrow (BM) and spleen around 25-30 weeks. Thirty week old mice were treated with Galunisertib for 4 weeks and showed a significant decrease in fibrosis compared to control mice. Similar results were observed in 50 week old mice treated with a murine antibody against TGFβRII (IMC-TR1,LY3022859) for 4 weeks.Our second model relies on transplantation of mice with BM cells transduced with MPLW515L, a mutant mpl variant that is active in the absence of TPO. These mice rapidly develop MF, myeloproliferation and splenomegaly. Flow cytometry showed an expansion of immature and mature megakaryocytes with overexpression of TGF-β1 in MPLW515L cells. Treatment from day 12 to day 26 after transplantation reduced both white blood cell counts (a measure of myeloproliferation) and fibrosis as measured by reticulin staining. In vitro, Galunisertib counteracted the pro-fibrotic effects of TGF-ß1 on both murine and human MSC by antagonizing its stimulatory effects on collagen I and III production. Analysis of MSCs from Galunisertib-treated mice also showed reduced collagen I and III production compared to controls.

In conclusion, we offer further evidence that aberrant megakaryopoiesis drives TGF-β1 overproduction in MPN by increasing megakaryocyte numbers and overexpressing TGF-β1 in mutant MKs. Galunisertib counteracts TGF-ß1 induced fibrosis in two MPN mouse-models and is a promising candidate for symptomatic treatment of myelofibrosis.

#955

LY3200882, a novel, highly selective TGFβRI small molecule inhibitor.

Huaxing Pei,1 Saravanan Parthasarathy,1 Sajan Joseph,1 William McMillen,1 Xiaohong Xu,2 Stephen Castaneda,2 Ivan Inigo,2 Karen Britt,1 Bryan Anderson,1 Gaiying Zhao,1 Scott Sawyer,1 Douglas Beight,1 Talbi Kaoudi,2 Chandrasekar Iyer,1 Huimin Bian,1 Amy Pappas,1 David Surguladze,2 David Schaer,2 Karim Benhadji,2 Michael Kalos,2 Kyla Driscoll2. 1 _Eli Lilly, Indianapolis, IN;_ 2 _Eli Lilly, New York, NY_.

The transforming growth factor β (TGFβ) signaling pathway is a pleiotropic cellular pathway that plays a critical role in cancer. In fact, aggressive tumors are typically associated with high ligand levels and thus associated with poor prognosis in various tumor types. Cancer cells use autocrine and paracrine TGFβ signaling to modulate tumor cells and the tumor microenvironment leading to a highly invasive and metastatic phenotype, inducing and increasing tumor vascularization, modulating the extracellular matrix in the stroma, and inhibiting immune surveillance and antitumor immunity. Clinical studies with galunisertib (aka LY2157299 monohydrate), a small molecule inhibitor targeting the TGFβ pathway, have provided proof of concept data supporting the role of TGFβ in cancer and the utility of targeting the TGFβ pathway.

Here we describe the identification of LY3200882, a next generation small molecule inhibitor of TGF-β receptor type 1 (TGFβRI). The molecule is a potent, highly selective inhibitor of TGFβRI embodied in a structural platform with a synthetically scalable route. It is an ATP competitive inhibitor of the serine-threonine kinase domain of TGFβRI.

Mechanism of action studies reveal revealed that LY3200882 inhibits various pro-tumorigenic activities. LY3200882 potently inhibits TGFβ mediated SMAD phosphorylation in vitro in tumor and immune cells and in vivo in subcutaneous tumors in a dose dependent fashion. In preclinical tumor models, LY3200882 showed potent anti-tumor activity in the orthotopic 4T1-LP model of triple negative breast cancer and this activity correlated with enhanced tumor infiltrating lymphocytes in the tumor microenvironment. Durable tumor regressions in the orthotopic 4T1-LP model were observed and rechallenge of congenic tumors resulted in complete rejection in all mice. In in vitro immune suppression assays, LY3200882 has shown the ability to rescue TGFβ1 suppressed or T regulatory cell suppressed naïve T cell activity and restore proliferation. Therefore, LY3200882 shows promising activity as an immune modulatory agent. In addition, LY3200882 has shown anti-metastatic activity in vitro in migration assays as well as in vivo in an experimental metastasis tumor model (intravenous EMT6-LM2 model of triple negative breast cancer). Finally, LY3200882 shows combinatorial anti-tumor benefits with checkpoint inhibition (anti-PD-L1) in the syngeneic CT26 model.

In conclusion, we have developed a novel potent and highly selective small molecule inhibitor of TGFβRI for the treatment of cancer.

#956

Novel oral small molecule CXCR4 inhibitor improves activity of immune checkpoint blockade in ovarian cancer mouse model.

Kristen Starbuck, Robert McGray, Samar Masoumi-Moghaddam, Ariel Francois, Kunle Odunsi, Emese Zsiros. _Roswell Park Cancer Institute, Buffalo, NY_.

Ovarian cancer continues to be the most lethal gynecologic malignancy with no real cure for patients presenting with advanced stage disease. Immune check point blockade showed modest clinical response in patient with recurrent ovarian cancer, thus additional therapeutic strategies for combination therapy are needed. As chemokines and their receptors drive both immune cell migration and tumor growth, angiogenesis and metastasis formation, they are an attractive target for combinatorial cancer therapy.

CXCR4 is the most highly expressed chemokine receptor in advanced stage high grade serous ovarian cancer, thus the objective of this study was to evaluate the efficacy of a novel oral small molecule CXCR4 inhibitor (X4-136) alone and in combination with immune checkpoint inhibition and the anti-angiogenic agent bevacizumab, and characterize the changes in circulating immune cells during treatment in murine ovarian cancer model. The ID8 cell line was used in C57BL/6J mice to establish an immune competent murine model and to compare single agent and combination therapy with oral X4-136 CXCR4 inhibitor, bevacizumab, and anti-PD1. During treatment blood sampling was performed and immune cells were analyzed by flow cytometry.

Our results demonstrated that single agent therapies alone with either drug had no significant effect on tumor progression or survival. Combination therapy with the CXCR4 inhibitor and anti-PD1 improved survival compared to control animals and the other combination therapy groups. The addition of bevacizumab to the dual combination did not further prolong survival. Analysis of circulating immune cells revealed elevations in CD11b+Ly6C+ and the ratio of CD11b+Ly6C+ to CD11b+Ly6G+ in groups treated with the CXCR4 inhibitor, indicating an increase in circulating myeloid cells. Bevacizumab had no activity in this mouse model as a single agent, and did not have synergistic effect in combination therapy.

For the first time we demonstrated that novel CXCR4 inhibitor X4-136 in combination therapy with anti-PD1 showed improved survival in murine ovarian cancer model. CXCR4 blockade increased the proportion of circulating myeloid cells during active treatment, thus further investigation into this novel therapeutic approach is warranted.

#957

Use of angiotensin system inhibitors is associated with longer overall survival in pancreatic ductal adenocarcinoma patients who underwent pancreatectomy.

Hao Liu,1 Matthias Pinter,1 Joao Incio,1 Hang Lee,1 William Ho,1 Jonathan Crain,1 Kamila Naxerova,1 Mengyang Di,2 Alex Jacobson,1 Daniella Dias Santos,1 Andrea Zanconato,1 Vikram Deshpande,1 Keith Lillemoe,1 Carlos Fernandez del Castillos,1 Michael Downes,3 Ronald Evans,3 James Michaelson,1 Cristina Ferrone,1 Yves Boucher,1 Jain K. Rakesh1. 1 _Massachusetts General Hospital, Boston, MA;_ 2 _Brown University, Providence, RI;_ 3 _Salk Institute, La Jolla, CA_.

Introduction: Angiotensin system inhibitors (ASI) are widely used to manage hypertension. Laboratory and retrospective clinical data suggests that ASIs can improve cancer prognosis. The aim of this study is to investigate the effect of ASIs on overall survival in pancreatic ductal adenocarcinoma (PDAC) patients.

Methods: We performed a retrospective review of the clinicopathologic records of patients with PDAC seen at the Massachusetts General Hospital (MGH) between 1/2006 - 12/2010. Patients on angiotensin converting enzyme inhibitors (ACEi) or angiotensin receptor blockers (ARB) were included as ASI users. We performed RNAseq and Gene Set Enrichment Analysis (GSEA) of primary tumor samples from patients with or without chronic ASI use. We also identified a surrogate signature of differentially expressed genes and we measured the extent of angiotensin inhibition in GEO and TCGA datasets. The extent of inhibition was used to correlate with survival. Statistical analysis was performed using Kaplan-Meier estimator and Cox proportional hazards ratio model.

Results: A total of 794 consecutive PDAC patients were included, of whom 297 (37.4%) were on ASIs and 183 (23.0%) were on non-ASI antihypertensive drug therapy. In resected patients, ASI users had a significantly longer overall survival on univariate (median OS: 36.3 vs. 19.3 months, p=0.011) and multivariate analysis (HR, 0.49; 95%CI, 0.333-0.734; p<0.001). In our sub-group analysis of resected hypertensive patients treated with chemotherapy, chronic ASI users had a significantly longer overall survival than ASI-naïve patients (p=0.048) (28.7 vs 12.3 months, p< 0.05). Gene Set Enrichment Analysis revealed that the ASI lisinopril down-regulated genes which stimulate the mitotic cell cycle, WNT and Notch signaling and the interaction between integrins and the extracellular matrix. Lisinopril also enhanced gene sets linked with oxidative phosphorylation, antigen processing and presentation and the cytotoxic activity of T cells. In unresected patients, the effect of ASI was only significant in patients with locally advanced disease in multivariate analysis (HR, 0.572; 95%CI, 0.386-0.847; p=0.005), but not in metastatic patients. The low expression of genes down-regulated by ACEi was also significantly associated with longer survival in the TCGA and GSE71729 datasets.

Conclusion: In patients with PDAC, ASI use is associated with longer overall survival in resected patients and may benefit patients with locally advanced disease. These findings suggest the need for a prospective study to determine the efficacy of ASI in PDAC patients.

#958

Blocking CD70+ cancer associated fibroblasts: Are we paving the way towards immunotherapy in colorectal cancer.

Julie Jacobs,1 Vanessa Deschoolmeester,1 Karen Zwaenepoel,2 Christophe Hermans,1 Christian Rolfo,2 Marc Peeters,2 Filip Lardon,1 Vasiliki Siozopoulou,2 Evelien Smits,3 Patrick Pauwels2. 1 _University of Antwerp, Wilrijk, Belgium;_ 2 _Antwerp University Hospital, Edegem, Belgium;_ 3 _Vaccine and Infectious Disease Institute, Edegem, Belgium_.

Introduction: Numerous studies have reported that tumor progression and invasiveness are determined not only by the malignant cancer cells themselves but also by the surrounding tumor microenvironment, including cancer-associated fibroblasts (CAFs). Although CAFs are implicated in tumor progression, their total depletion of CAFs has been demonstrated to induce more aggressive tumors, indicating that different CAF subpopulations have opposing tumor-promoting or tumor-inhibitory roles. Unfortunately, specific markers to target these subsets of CAFs are lacking. Expression of the immune checkpoint CD70 is normally tightly regulated and limited to cells of the lymphoid lineage only. Instead, tumors hijack CD70 to facilitate immune evasion by increasing the amount of suppressive regulatory T cells (Tregs), inducing T cell apoptosis and skewing T cells towards T cell exhaustion. Recently, a lot of clinical successes have been generated by the blockade of immune checkpoints. However, in colorectal cancer (CRC) the efficacy remains limited to a small subset of patients with mismatch repair-deficient (MSI) tumors which might be caused by the intense dialogue between stroma and malignant cells. Therefore, we have explored the expression patterns of the immune checkpoint molecule CD70 in CRC, with a particular focus on CAFs.

Methods: The prognostic value of CD70 was analyzed by immunohistochemistry on 51 CRC specimens. In addition, the relationship with Tregs and microsatellite instability was explored. Furthermore, primary CAF cell lines were successfully cultured from 20 different primary resection specimens. These cell lines were used to study the effect of CD70 on the tumor microenvironment in vitro.

Results: We revealed expression of CD70, not just on the malignant cells but on the majority of CAFs in invasive CRC specimens. Thereby, CD70-expression was significantly correlated with negative clinicopathological parameters such as metastasis (P=0.007), differentiation (P=0.053) and advanced stage (P=0.001). Moreover, CD70-positive CAFs proved to be a poor prognostic marker by univariate as well as multivariate analysis. We have also detected a significant association between elevated Treg amounts and CD70-expressing CAFs (P=0.012). In vitro data on the effects of CD70 on CRC behavior and immune escape are currently being analyzed.

Conclusion: We have identified a new targetable CAF subpopulation, marked by the expression of CD70 and equipped with strong tumor-promoting properties. Thereby, we have found evidence of a potential cross talk between CD70+ CAFs and Treg, paving the way towards immune escape. The lack of association of CD70 expression and MSI-status, which highlights the potential of this target in CRC subsets that do not benefit from immune checkpoint blockade. We believe that targeting CD70 holds great potential in CRC, especially in light of the limited immunotherapeutic options available.

#959

Targeting neuronal activity regulated neuroligin-3 dependency for high-grade glioma therapy.

Humsa S. Venkatesh, Lydia T. Tam, Pamelyn J. Woo, Michelle Monje. _Stanford University, Stanford, CA_.

Neuronal activity promotes high-grade glioma (HGG) growth. An important mechanism mediating this neural regulation of brain cancer is activity-dependent cleavage and secretion of the synaptic molecule and glioma mitogen neuroligin-3 (Nlgn3), but the therapeutic potential of targeting Nlgn3 in glioma remains to be defined. Here, we demonstrate a striking dependence of HGG growth on microenvironmental Nlgn3 and determine a targetable mechanism of secretion. Patient-derived orthotopic xenografts of pediatric glioblastoma (pGBM) and diffuse intrinsic pontine glioma (DIPG) fail to grow in Nlgn3 knock out mice. Using genetic mouse models, we illustrate that Nlgn3 is cleaved from both neurons and oligodendrocyte precursor cells via the ADAM10 sheddase. Administration of an ADAM10 inhibitor robustly blocks pGBM and DIPG xenograft growth via modulation of the tumor microenvironment. This work defines the therapeutic potential of and an effective strategy for targeting Nlgn3 secretion in the glioma microenvironment, which could prove transformative for treatment of HGG.

#960

Mechanisms underlying primary ibrutinib sensitivity in CLL.

Shih-Shih Chen,1 Priyadarshini Ravichandran1,1 Yasmine Kieso,1 Jacqueline C. Barrientos,1 Jan Burger,2 Kanti R. Rai,3 Nicholas Chiorazzi1. 1 _The Feinstein Institute for Medical Research, Manhasset, NY;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _Karches Center for Chronic Lymphocytic Leukemia Research, The Northwell Health System, Manhasset, NY_.

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib is efficacious in B cell malignancies including chronic lymphocytic leukemia (CLL). Although the majority of CLL patients respond to ibrutinib, CLL clones with unmutated IGHV gene (U-CLL) are significantly more sensitive to ibrutinib than clones with mutated IGHV (M-CLL). About 77% of U-CLL but only 33% M-CLL patients experience a partial or complete response. Since ibrutinib resistance occurs, understanding the molecular mechanisms underlying this difference will improve clinical practice. Here we hypothesized that the difference in ibrutinib sensitivity between U-CLL and M-CLL was due to a greater vulnerability of U-CLL B cells to the loss of environmental prosurvival signals.

We first evaluated 28 treatment naive CLL cases receiving ibrutinib. We defined not only the characteristic CLL B cell but also T cell lymphocytosis and the latter resolved faster in patients with good prognosis. After 1 treatment cycle, CLL T cells failed to proliferate upon mitogenic stimulation and to home to solid tissues after transfer into NSG mice. Without BCR signaling and T cell support, the CLL B cell proliferative fraction was diminished and the remaining B cells had elevated but dysfunctional surface membrane (sm) CXCR4 based on impaired recycling, internalization and signaling. Ibrutinib also inhibited CXCR4 phosphorylation at ser324/325/339 at a greater level in U-CLL.

We then investigated ibrutinib mediated changes in microenvironment in a xenograft mouse model using primary cells from 3 U-CLL and 3 M-CLL cases. Ibrutinib significantly inhibited CLL cell growth in spleen; the treated CLL cells developed elevated but dysfunctional smCXCR4. The inhibition on tumor growth and impaired CXCR4 occurred to much greater extent in U-CLL. Notably, ibrutinib also significantly inhibited T cell growth only in U-CLL cases. Thus, ibrutinib inhibited CXCR4 signaling and CLL B-T cell crosstalk essential for tumor growth in tissue niches, especially in U-CLL cases that has limited B-lymphocytosis.

Finally, molecules controlling the vulnerability of U-CLL B cells to death in the absence of environmental prosurvival signals were examined in 15 patients. Ibrutinib reduced BCL2 protein levels in U-CLL but not M-CLL; levels of BCL-XL and MCL-1 were unchanged. Consistent with these results, U-CLL but not M-CLL cell survival in vitro was promoted by T cell stimulation that led to upregulation of prosurvival BCL2. These findings suggest enhanced CLL B cell death by inhibiting BCL2 in U-CLL after the loss of environmental prosurvival signals.

Altogether, ibrutinib inhibits CLL-microenvironment crosstalk by blocking CXCR4 signaling and T cell support. U-CLL cells had a greater vulnerability to the loss of environmental prosurvival signals after ibrutinib inhibition. Our data demonstrate mechanisms underlying the differences in primary ibrutinib sensitivity between U- and M- CLL patients, supporting the use of inhibitors of BTK and BCL2 in CLL.

#961

Inhibitors of fibrosis and TGF-beta delay tumor xenograft growth and reduce ascites in ovarian cancer models.

Qing Zhang, Xiaonan Hou, Bradley J. Evans, John J. Weroha, William A. Cliby. _Mayo Clinic, Rochester, MN_.

Background: Most ovarian cancer (OC) patients present with advanced disease and ultimately develop chemo-resistant relapses. Stroma plays a significant role in OC behavior including invasiveness, fibrosis and chemoresistance. TGF-β is important for crosstalk between stroma and cancer cells, and stromal activation in cancer has similarities to matrix remodeling in fibrosis. We have shown that TGF-β and fibrosis-inhibitors can suppress OC cell proliferation, migration and invasion. Such inhibitors are available for clinical use, and we have begun to test their efficacy in OC in vivo.

Methods: Animal experiments were approved by Mayo Clinic IACUC. Intraperitoneal xenograft models were derived from OVCAR8 (5×106) cells (OV8-CDX) or 0.1 gm OC patient tumor tissue (PDX) in NSG mice. OV8-CDX and PDX PH003 (an aggressive, chemoresistant and ascites-prone model) (n=5-10 mice/arm) were treated with 2 agents and compared to untreated controls (C): 1) C vs. the fibrosis inhibitor Pirfenidone (P, 200 mg/kg, BID, provided by Genentech); 2) C vs. TGF-β receptor I inhibitor Galunisertib (G, 75 mg/kg, BID, provided by Eli Lily). Mice were pretreated for 2 days before xenografting and treated for 4 weeks with ultrasound (US) to measure tumor growth. At day (d) 28, we i) collected tumor (OV8-CDX); or ii) observed until progression (PH003, PH053). OV8-CDX tumors were used for target gene analysis.

Results: P treatment delayed tumor growth in OV8-CDX relative to control (C): tumors established in 0/5 (P) vs. 1/5 (C), and 1/5 (P) vs. 4/5 (C) of mice at d14 and d28, respectively. The reduced tumor weight (g) at d28 was also confirmed (0.20±0.10 vs. 0.54±0.23, p=0.02). Similarly, P treatment in PH003 inhibited the tumor growth (tumor area (cm2) at d28: 0.74±0.43 vs. 1.04±0.51, p=0.28), ascites development (volume (ml): 1.14±0.54 vs. 3.37±0.62, p=0.05) and prolonged survival time (p=0.04).

G treatment was also associated with delayed tumor establishment in OV8-CDX relative to C: tumors presented in 0/5 (G) vs. 1/5 (C), and 1/5 (G) vs. 4/5 (C) of mice at d14 and d28, respectively. Associated d28 tumor weight was significantly reduced (0.20±0.10 vs. 0.54±0.23, p=0.02) as well. For PH003, G treatment was associated with delayed tumor growth (tumor area at d28: 0.57±0.27 vs. 1.04±0.51, p=0.05), a marked reduction in ascites (volume: 1.25±0.56 vs. 3.37±0.62, p=0.05) and a prolonged survival time (p=0.04)

We identified down-regulation of TGF-β signaling-related genes (e.g. TGFB1, TGFBR2) and fibrosis-related biomarkers (e.g. FAP, COL11A1) following drug treatment confirming inhibition of relevant pathways.

Conclusion: We report that 2 currently available inhibitors targeting either fibrosis or TGF-β appear to have efficacy in vivo and delay tumor growth in OC PDX models. These results suggest promise for a strategy of targeting the stromal component of OC.

#962

Novel targeting of tumor-infiltrating TNFR2 Tregs: removing suppression with microenvironment-specific antibodies.

Heather Torrey, John Butterworth, Toshiyuki Mera, Yoshiaki Okubo, Limei Wang, Danielle Baum, Audrey Defusco, Sara Plager, Sarah Warden, Daniel Huang, Eva Vanamee, Rosemary Foster, Denise L. Faustman. _Massachusetts General Hospital & Harvard Medical, Charlestown, MA_.

Background: A recognized and potent subtype of regulatory T cells (Tregs) expresses tumor necrosis factor receptor 2 (TNFR2), a member of the tumor necrosis factor (TNF) superfamily. TNFR2 expressing Tregs are overabundant in human and murine tumors and are the most potent suppressors of host immune responses. In addition, recent data in diverse human tumors show that TNFR2-expressing host T cells are the dominant Tregs in the tumor microenvironment, expressed far in excess of OX40 and GITR. Unlike many tumor markers, TNFR2 has limited expression in the immune system and almost no expression on parenchymal cells. This makes TNFR2 Tregs an attractive target for anticancer therapy.

Methods: We produced monoclonal antibodies to human TNFR2 with the goal of inactivating the potent host Tregs that express TNFR2, competing with the agonism of TNF and inactivating infiltating Tregs of human tumors. Tregs from fresh ovarian ascites fluid were compared to Tregs from normal human blood donors for antibody potency.

Results: Using a 48-hr cell-based Treg assay, we showed that TNFR2 antagonistic antibodies can inhibit Treg proliferation, suppress soluble TNFR2 secretion on normal cells and allow T effector expansion. The antagonism is dominant, succeeding even in the presence of TNF, which is a TNFR2 agonist. Furthermore, the structural biology of dominant TNFR2 receptor antagonism uncovers a unique conformation that equally inhibits downstream NFkB signaling. We also showed that dominant TNFR2 antibodies in a dose dependent fashion kill Tregs in ovarian cancer infiltrates more strongly than Tregs from healthy donors. Treg killing in the cancer microenvironment was dependent on cell proliferation and turnover the Tregs.

Conclusion: In sum, blocking TNFR2 signaling on the most potent Tregs of the tumor microenvironment with a dominant antagonist antibody selectively heightens death of the immunosuppressive Tregs in the tumor microenvironment, at least in ovarian cancer. With the expanding knowledge that TNFR2 Tregs are the most potent host cells of the immune response, their dominance in diverse human tumor infiltrates and the natural low expression of this marker on normal lymphoid cells makes targeting of TNFR2 in the tumor microenvironment attractive.

#963

**Ovarian-dependent SSM2** ucd **mouse mammary carcinoma cells depend on** Cyp3a11 **epoxygenase activity for proliferation.**

Zhijun Guo,1 Patrick McGrarrah,1 Ted Bebi,2 Ashley Mooneyham,1 Alejandra Osorio,3 Cesar Herrera,4 Victor Arrieta,4 Sebastian Mohar,4 Irwin Hernandez,4 Robert Klink,1 Robert Cardiff,5 David A. Potter1. 1 _Univ. of Minnesota, Minneapolis, MN;_ 2 _Macalester College, Saint Paul, MN;_ 3 _Univ. of Central Florida, Olando, FL;_ 4 _Instituto Nacional de Cancerologia, Mexico City, Mexico;_ 5 _Univ. of California, Davis, CA_.

Introduction: Silencing of the human cytochrome P450 (CYP) monooxygenase enzyme CYP3A4 in the ER+ MCF-7 tumor cell line inhibits tumor growth in the mammary fat pad, but the relative roles of tumor cell intrinsic CYPs vs. vascular/microenvironment CYPs remain to be determined and a syngeneic animal model is needed to answer this question. The SSM2ucd ovarian dependent mouse mammary carcinoma cell line forms a transplantable estrogen-receptor positive (ER+) mouse mammary tumor in which growth dependence on cell intrinsic CYPs may be tested in vitro and in vivo. The SSM2ucd cell line is derived from 129SvJ: homozygous Stat1-null female mice, which develop ER+ mammary tumors (1,2). Expression profiling of spontaneously arising mouse mammary carcinomas in a TP53 KO model revealed that the CYP3A4 ortholog Cyp3a11 is up-regulated in these tumors.

Methods: Cyp3a11 bactosomes were used to assay for NADPH dependent biosynthesis of EETs from arachidonic acid using an LC-MS/MS method. siRNA silencing and CRISPR/Cas9 knock out of the Cyp3a11 exons 2 and 3 was performed and the cell lines were characterized for EET dependence using an MTT assay. The highly potent inhibitor and chemical probe of CYP epoxygenase activity, hexyl-benzyl-biguanide (HBB), was used to test dependence of SSM2ucd cells on CYP epoxygenase activity. Total cellular EETs in SSM2ucd cells exposed to HBB at the IC50were measured by the LC-MS/MS method.

Results: Cyp3a11 was shown to have robust NADPH dependent epoxygenase activity, while SSM2ucd cells were EET dependent for growth. Silencing of Cyp3a11 was demonstrated to inhibit proliferation of the SSM2ucd cell line, which was partly abrogated by EETs. CRISPR/Cas9 knock out of the Cyp3a11 in the SSM2ucd cell line was performed and exon 2 and exon 3 deleted cell lines were derived. SSM2ucd Cyp3a11 knock out cell lines were more sensitive to EET-induced proliferation under serum free condition than control lines. HBB potently inhibits the SSM2ucd cell line in vitro (IC50=25 uM) and reduces total cellular EET levels in this cell line at IC50. These results suggest that we may see inhibitory activity of HBB in a syngeneic 129/SvJ tumor transplantation model.

Conclusions: The Cyp3a11 gene can be silenced in the SSM2ucd cell line, without disrupting cell viability, allowing further study of the roles of EETs in mouse mammary carcinoma proliferation, survival and clonogenicity. The Cyp3a11 gene has been knocked out in the SSM2 ucd cell line, allowing in vitro and in vivo studies of the roles of Cyp3a11 in the growth of ovarian dependent mouse mammary carcinoma.

1. Breast Cancer Res. 2012 Jan 20;14(1):R16

2. PLoS One. 2015 Jun 15;10(6):e0129895]

#964

A quantitative flow cytometric assay for the simultaneous detection of PD-L1 in tumor and immune infiltrate present in NSCLC.

Ryan T. Rodriguez,1 Amanda Chargin,1 Rian Morgan,1 Randall K. Wetzel,2 Reginaldo Prioli,2 Bruce Patterson,1 Keith Shults1. 1 _IncellDx, Menlo Park, CA;_ 2 _Cell Signaling Technology, Danvers, MA_.

As our understanding of the inherent complexity of the immune checkpoints becomes clearer, we must understand the expression of immune modulators such as PD-L1 within the context of the tumor microenvironment. This includes measures of immune infiltrate, levels of expression of immune modulators, such as PD-L1, on lymphocytes and tumor cells, as well as DNA heterogeneity within tumor cells. We show here characterization of the rabbit anti-PD-L1 clone E1L3N compared to other commercial sources of antibody for use in a flow cytometric assay. First we demonstrate specific fluorescence and sensitivity in tumor derived cell lines and then combine cell lines and PBMCs to form a tumor microenvironment mimic. When E1L3N was combined with antibodies directed toward CD45, CD3, and CD8, the cellular subsets known to be present in the tumor micro environment, including cytotoxic lymphocytes, T- helper cells and CD45 negative tumor derived cells are readily detected. The use of sequential gating strategies allows the assay to detect PD-L1 on each of these subsets which, when combined with quantitative MESF beads (Bangs Laboratories) along with the known fluorescence-to-protein ratio of each antibody, allows the calculation of the average relative PD-L1 receptors bound for each cell type. This assay further segregates cells using the DNA-specific dye (DAPI) allowing the measurement of proliferation and delineation of the mutated aneuploid fraction. We demonstrate the assay's ability to measure quantitative PD-L1 expression, immune infiltrate, and tumor ploidy in NSCLC tumor samples. We believe this will be a valuable assay in the clinician's toolbox to make informed decisions for patients who may respond best to PD-1 pathway inhibitors.

#965

Two in one: nanotechnology based strategies for the treatment of ER+ breast cancer.

Maria Ines Diaz Bessone,1 Lorena Simón-Gracia,2 Pablo Scodeller,2 Tambet Teesalu,2 Marina Simian1. 1 _Universidad Nacional de San Martín, Buenos Aires, Argentina;_ 2 _University of Tartu, Tartu, Estonia_.

Understanding endocrine resistance mechanisms is key to develop new therapeutic strategies. We focus on the role the tumor microenvironment plays as a modulator of endocrine therapy resistance in breast cancer, in particular associated to Tamoxifen. Studies show that the use of nanoparticles (NPs) as antitumor drug delivery systems is a good strategy to improve the efficacy and decrease secondary effects of conventional chemotherapies. In this context we hypothesize that a therapeutic strategy based on the use of Tamoxifen carried in NPs coated with the tumor penetrating peptide iRGD, would be more effective than conventional Tamoxifen. Neuropilin-1, that mediates iRGD induced endocytosis, has been shown to be associated to breast cancer stem cells. Thus, we postulate that our multifunctional NPs would be effective in reducing this cell population, contrary to what is observed with free Tamoxifen. Moreover, iRGD blocks the interaction between integrin β1 and fibronectin, a mechanism we have previously shown induces Tamoxifen resistance (Pontiggia et al. 2012). NPs were synthesized with polyethyleneglycol and polycaprolactone and coated with the iRGD peptide. This peptide was constructed with a FAM fluorophore in order to track the NPs. To evaluate the cell uptake, MCF-7 cells were incubated for 5 h with iRGD-NP or FAM-NP as a control. NP entry was higher when the NPs were coated with iRGD, both in 2D and in 3D cultures. Cell viability experiments revealed that Tamoxifen encapsulated in NPs was more effective than the free drug (p<0,05), and NPs coated with iRGD had an even stronger effect (p<0,01). Moreover, resistance to Tamoxifen induced by fibronectin was reversed with the iRGD-coated NPs, contrary to what is observed with the free drug. Previously, we demonstrated that treatment with Tamoxifen enhances the stem cell population in estrogen receptor-positive breast cancer cell lines (Raffo et al. 2013). In this context iRGD coated NPs lead to a decrease in breast cancer stem cells as evaluated by mammosphere assays (p<0,01). Finally, in vivo tumor homing was evaluated to test whether the iRGD peptide affected the biodistribution of the NPs. Nude mice carrying MCF-7 breast tumors were treated with iRGD-NP and control NPs. Tumors were collected at 4 and 24 hrs post inoculation. Frozen sections were analyzed by confocal microscopy revealing that only the mice treated with the iRGD-NPs had high levels of NPs in the tumor, compared to the control group. Moreover, frozen sections of kidneys, livers, lungs and spleen showed very low levels of iRGD-NPs, confirming that the targeting was effective. In vivo experiments of the impact of iRGD-NPs on tumor growth, metastasis and the stem cell population are ongoing. These results suggest that iRGD-NPs carrying Tamoxifen could be an effective strategy to avoid the development of endocrine resistance in breast cancer.

#966

Phase II clinical trial patient responses to the macrophage activating agent RRx-001 correlate to TGF- β pathway activation and markers for fibrosis.

Saheli Jha,1 Thomas A. Summers,2 Karen Zeman,3 Christine Brzezniak,3 Corey Carter,3 Lindsey Ferry,3 Jan Scicinski,1 Bryan Oronsky,1 Scot Caroen,1 Jane B. Trepel,4 Pedro Cabrales,5 Regina Day2. 1 _Epicentrx, Mountain View, CA;_ 2 _USUHS, Bethesda, MD;_ 3 _Walter Reed National Military Medical Center , Bethesda, MD;_ 4 _NCI NIH, Bethesda, MD;_ 5 _UCSD, La Jolla, CA_.

Introduction: RRx-001 is a novel systemically non-toxic small molecule macrophage stimulating agent with promising activity in an ongoing clinical trial in small cell and non-small cell lung cancer and in neuroendocrine tumors. In preclinical studies, RRx-001 activated normally immune-suppressive M2-like Tumor Associated Macrophages (TAMs) to express a series of pro-inflammatory cytokines and chemokines such as transforming growth factor-β1 (TGF-β1), tumor necrosis factor-α TNF-α), inducible nitric oxide synthase (iNOS), and interleukins-10 and -12, a profile that resembles the M1 activated macrophage state. Due to its chemotactic activity for macrophages, TGF-β1 was hypothesized to be involved in the antitumor mechanism of RRx-001. We therefore compared expression patterns of TGF-β1 and TGF-βRI in patient-derived biopsy samples obtained at screening and post RRx-001. As TGF-β1 is closely associated with the induction of fibrosis, we also examined key fibrosis markers.

Methods: Tumor biopsies before and after treatment with RRx-001 were obtained from consented patients with NSCLC, SCLC, and neuroendocrine tumors in the QUADRUPLE THREAT Phase II clinical trial (NCT02489903). Post treatment biopsies were obtained following 6 weeks of once-weekly RRx-001 treatment co-incidental with the first on-study CT scan. Tumor samples were evaluated immune-histochemically with putative markers for TGFβ pathway activation (TGF-β1, TGF-βR1), fibrosis (alpha-smooth muscle actin [α-SMA], Matrix metallopeptidase-9 [MMP-9], and Collagen III deposition), and macrophage activation. Patients were subsequently followed for tumor progression.

Results: Positive immune-histochemical staining for TGF-β1 and TGF-βR1 was seen in all responding patients, and in none of the non-responding patients to date. In responders we also found that MMP-9 and α-SMA activity was down regulated post RRx-001. The down-regulation of these fibrosis markers is suggestive of less invasive malignancy. The number of tumor associated macrophages and their activation was related to the activity of RRx-001.

Conclusions: Our pre and post dose patient derived data to date indicate a correlation between TGF-β signaling activation and a response to RRx-001 that may also correlate with increased numbers of macrophages, and their activation status, in the vicinity of the tumor. These data suggest that activation of the TGF-β1 pathway, as evident by expression of TGF-β1 and TGF-βR1 by tumor cells, could be a predictive biomarker for RRx-001 treatment.

#967

Development of fluorescent-cell based high throughput assay to identify novel therapeutics of bladder cancer cells through upregulation of SPARC expression.

Chirayu M. Patel. _Wake Forest Health Sciences, Winston-Salem, NC_.

Bladder cancer is the most common malignancy affecting the urinary system with an estimated 76,960 new cases and projected 16,390 deaths in 2016 in the United States. The role of SPARC in various cancers is contextual and differs based on the tumor classification, malignancy and progression. We have recently reported the tumor suppressor effect of SPARC in urinary bladder cancer. We have shown that the loss of SPARC in bladder cancer resulted accelerated bladder carcinogenesis and metastasis multiple pathways involving cell cycle deregulation, inflammation and angiogenesis. We have also reported that SPARC protein expression significantly decreased in the cancerous compartment in advanced human bladder cancer as well as in carcinogen-induced murine urothelial cancer. However, the mechanism of the downregulation of SPARC expression in cancer in general and in bladder cancer in particular is still unraveled. The purpose of our study is to develop a robust, high throughput assay to identify therapeutic candidates as regulators of SPARC expression in bladder cancer. We cloned SPARC promoter in a third generation lentiviral vector coupled with fluorescent GFP or mCherry fluorescent protein to quantitatively measure SPARC expression. We generated stable bladder cancer cell lines expressing SPARC expression in bladder cancer cell lines. UMUC3 bladder cancer cells were transduced with lentiviral vector to generate SPARC-promoter reporter system to screen regulators of SPARC expression. Reporter cells were plated in 96 well plates and treated with 10uM of FDA-approved and natural product drug libraries. Real time monitoring of changes in SPARC expression was done by measuring GFP fluorescence intensity using IncuCyte® ZOOM Live Imaging Analysis System. We identified drugs that consistently exhibited dosage-dependent augmentation or down-regulation of SPARC expression in UMUC3 cells. Our screens identified novel up-regulators of SPARC expression that are both standard of care and novel bladder cancer therapeutics. Our efforts produced an efficient robust assay to identify novel therapeutics and regulators of the tumor suppressor SPARC in the bladder cancer ecosystem that can be used in the adjuvant and neoadjuvant settings.

#968

Podoplanin's diverse potential as a chemotherapeutic target for oral squamous cell carcinom.

Edward P. Retzbach,1 Harini Krishnan,1 Jhon A. Ochoa-Alvarez,1 Yongquan Shen,1 Evan Nevel,1 David J. Kephart,1 Evelyne Kalyoussef,2 Soly Baredes,2 Mahnaz Fatahzadeh,3 Kingsley Yin,1 Alan J. Shienbaum,1 Yukinari Kato,4 Lasse Jensen,5 Gary S. Goldberg1. 1 _Rowan University, Stratford, NJ;_ 2 _Rutgers New Jersey Medical School, Newark, NJ;_ 3 _Rutgers School of Dental Medicine, Newark, NJ;_ 4 _Tohoku University Graduate School of Medicine, Sendai, Japan;_ 5 _Linkoping University, Linkoping, Sweden_.

Oral cancer is diagnosed in over 300 thousand people, and kills over 100 thousand people, around the world each year. Current treatments rely on radiation and surgery procedures that often decrease the quality of life for oral cancer survivors. There is a clear need to improve treatments for these patients. Over 90% of oral cancers are oral squamous cell carcinoma (OSCC). Most OSCC cells express the transmembrane receptor podoplanin (PDPN), which has emerged as a promising target for OSCC treatment. The PDPN receptor promotes tumor cell invasion and metastasis which leads to the vast majority of cancer deaths. Here, we describe efforts to target PDPN in order to prevent and treat oral cancer. PDPN can be targeted with Maackia amurensis seed lectin (MASL) to inhibit tumor cell migration and viability. Recent evidence also suggests that PDPN may undergo cancer specific changes, which indicates another potential route for targeting PDPN in oral cancer. Taken together, these data indicate that PDPN can serve as a functionally relevant target to prevent and combat oral cancer.

#969

Expression of membrane-type matrix metalloproteinases (MT-MMPS) in head and neck squamous cell carcinomas, a target for prodrug development.

Rene Ankrah,1 Steve D. Shnyder,1 James A. McCaul,2 Phil A. Batman,1 Robert Falconer,1 Paul M. Loadman1. 1 _Univ. of Bradford, Bradford, United Kingdom;_ 2 _Royal Marsden Hospital and Northwick Park Hospitals, London, United Kingdom_.

Introduction: Head and Neck Squamous Cell Carcinomas (HNSCC), a group of highly aggressive heterogeneous epithelial malignancies, are considered the fifth most common cancer with the seventh highest cancer mortality. Despite advances in treatment, low five-year survival rates of around 30% still remain. Several studies have implicated the actions of matrix metalloproteinases (MMPs) in HNSCC progression. Their role in mediating extracellular matrix remodeling is paramount to tumour progression, as they facilitate invasion, angiogenesis and metastasis. In contrast, expression of active MMPs in normal tissues is largely absent. MT1-MMP (MMP14) expression is known to correlate with poor clinical response in several tumour types, but little is known about the expression of other MT-MMPs in HNSCC. We have assessed the protein expression of the entire MT-MMP complement in matched tumour and normal tissues of clinical HNSCC cases by immunohistochemistry, supported by Western Blot and gene expression data and will ultimately demonstrate translation of protein expression into MT-MMP-specific proteolytic activity. Differential expression would support the development of prodrugs specifically activated in the tumour by MMPs.

Methods: 32 clinical cases (matched normal & tumour tissues) were assayed for gene expression of the 6 members of the MT-MMP family (MMP14,15,16,17,24,25). Expression profiling was carried out using quantitative real-time reverse transcription-PCR analysis. Analysis of protein expression used IHC on fixed sections used with monoclonal antibodies specific for MT1 to MT6 MMP and Western blot analysis with monoclonal antibodies optimized for western blotting.

Results: The protein expression of all six MT-MMPs has been characterised in clinical HNSCC samples and related to matched normal tissue controls. A differential in gene and protein expression levels exists between the MT-MMP profiles across the normal and cancer tissues examined. MT1-, MT3- and MT6-MMP in particular were shown to be highly overexpressed at both gene and protein level in the majority of tumours. MT2, MT4 and MT5-MMP expression was low or undetectable in all normal tissue and low in tumour tissue

Conclusion: The existence and demonstration of differential MT-MMP protein expression in HNSCC relative to normal tissues is a valuable tool for prodrug development. MT1-, MT3- and MT6-MMP are highly expressed in the majority of tumour samples compared to matched normal control tissue. However, further work to elucidate the proteolytic activity of the expressed proteins and therefore the proteolytic capacity of tumours is needed to determine the suitability of HNSCC for prodrug therapy.

#970

Probing the expression and function of aldehyde dehydrogenases in prostate cancer using ALDH-affinic compounds and siRNA.

Maria Sadiq,1 Ali I. Ibrahim,1 Fiona Frame,2 Simon J. Allison,3 Mark Sutherland,1 Roger M. Phillips,3 Norman J. Maitland,2 Klaus Pors1. 1 _University of Bradford, Bradford, United Kingdom;_ 2 _University of York, York, United Kingdom;_ 3 _University of Huddersfied, Huddersfield, United Kingdom_.

The human aldehyde dehydrogenases (ALDHs) play a major role in detoxifying highly reactive aldehydes into carboxylic acids. Deregulation of ALDHs have implications in a number of cancers including prostate cancer. They play an important role as a cancer stem cell (CSC) marker due to high activity found in CSCs while high expression is also known to lead to resistance to drugs including docetaxel. Although the exact role of ALDHs is not fully understood, emerging information indicates several isoforms including ALDH1A3 and ALDH7A1 play a key role in cancer. To further elucidate the role of ALDHs in prostate cancer, we here report on the perturbation of ALDH expression and function using chemical probes and siRNA.

Primary prostate epithelial cells cultured from patient tissue were used for this study. Cancer samples were obtained from radical prostatectomies and benign samples from transurethral resection of the prostate.

qPCR analysis showed ALDH1A3 to be more highly expressed than ALDH7A1 in the primary prostate epithelial cultures in 18 patient samples. Expression of ALDH1A3 was 3-fold higher in the cancer samples compared to the benign samples. The RNA data correlates with protein expression in 6 patient samples by immunofluorescence. qPCR analysis also showed that knockdown of ALDH7A1 resulted in an increase in the expression of ALDH1A3 suggesting a compensatory mechanism. Trypan blue exclusion assay showed that knockdown of ALDH1A3, ALDH7A1 or a combination of both resulted in a reduction in cell numbers. Flow cytometry was used to study cell differentiation upon knockdown of ALDH1A3, ALDH7A1 or both. In all 7 samples studied there was a reduction in CD49b expression indicating cell differentiation. ALDH7A1 knockdown showed a higher level of cell differentiation in all cases. The colony forming ability of primary cells was also investigated post-transfection of siRNAs against ALDH1A3, ALDH7A1 or both using the colony formation assay which resulted in a lower number of colonies in all 7 samples tested. The effect was more pronounced in benign prostatic hyperplasia (BPH) than in malignant cancer samples and patient variability was observed. ALDH-affinic probe compounds (DEAB and three derivatives, Ali-9, Ali-14 & Ali-18) were tested against 5 patient samples to investigate if they have an effect on cell viability. All four compounds showed reduction in cell viability at the highest concentration while Ali-14 and Ali-18 showed a synergistic effect in combination treatment with docetaxel.

In conclusion, knockdown of ALDH1A3 and ALDH7A1 reduce cell number, induce cells to differentiate and reduce their colony forming ability. Novel ALDH-affinic probe compounds reduced cell viability alone and in combination with docetaxel, therefore these compounds may be of value both as single treatments and to provide a strategy to enhance taxane-based therapy such as docetaxel.

## BIOINFORMATICS AND SYSTEMS BIOLOGY:

### Computational Cancer Biology

#971

Genome directed diagnosis informs clinical cancer care.

Alexander Penson, Niedzica Camacho, Anna M. Varghese, Adam Abeshouse, Pedram Razavi, Aijazuddin Syed, Ahmet Zehir, Nikolaus Schultz, David B. Solit, David Hyman, Barry S. Taylor, Michael F. Berger. _MSKCC, New York City, NY_.

Cancer treatment is primarily guided by the organ of origin, which despite extensive histopathological and clinical evaluation, remains ambiguous in many cases and may be inaccurate in cases of occult primary. We determine the extent to which routine prospective tumour sequencing, combined with conventional histopathology, can be used to infer the tumor type and thereby direct clinical decisions.

Characteristic patterns of somatic mutations, broad and focal copy number alterations, structural rearrangements, mutational signatures, and other facets acquired from prospective tumor sequencing can inform the tissue of origin classification of patient disease. Such prospective sequencing of active cancer patients presents an opportunity to guide diagnosis and therapy beyond the identification of individual biomarkers of treatment response. Probabilistic classification allows for systematic combination of genome-directed diagnosis with conventional histopathology and clinical history in the course of disease management.

Using more than 10,000 tumors collected from advanced cancer patients at our institution and sequenced using a comprehensive cancer panel (MSK-IMPACT) encompassing 341 genes, we have developed a probabilistic classifier that infers the tumor type from nine broad categories of genomic aberrations. Scores from a RandomForest classifier are calibrated to correspond to the probability for each of the 22 interrogated tumor types. In 18% of all cases, a prediction is made with very high confidence (greater than 99%), of which just over 99% agree with the existing diagnosis. The classifier has been used to guide clinical decision making, including re-diagnoses that distinguish between a recurrence and a new primary, spare or encourage surgery, facilitate access to FDA-approved molecularly targeted drugs and prompt germline genetic testing.

We demonstrate that genome-directed diagnosis, based on alterations routinely identified from prospective sequencing, can inform clinical cancer care with the potential to improve patient outcomes.

#972

Improving pre-clinical cancer pharmacogenomics with novel drug sensitivity metrics based on growth rate inhibition.

Marc Hafner, Mario Niepel, Peter K. Sorger. _Harvard Medical School, Boston, MA_.

Drug sensitivity in growing cells is conventionally quantified by IC50, AUC, or Emax values, but these metrics suffer from a fundamental flaw: they highly depend on the division rate of cell lines. This dependency creates artefactual correlations between genetic alterations and drug sensitivity which impede biomarker discovery. To address this issue, we recently developed novel drug response metrics insensitive to the number of divisions occurring during the assay. These are based on estimating growth rate inhibition (GR) under treatment using fixed assays.

Here, we illustrate the flaws of using IC50 values for pharmacogenomic studies by reanalyzing a recently published large dataset of drug sensitivity and showing cases in which differences in division rates drive associations between IC50 values and tissue type or genetic alterations. Using GR50 values prevents these artificial correlations and restores known associations between drug resistance and genomic markers such as PTEN loss driving lapatinib resistance in breast cancer cell lines. We also show how use of GRmax values, a measure of drug efficacy, allows quantifying differences in the phenotypes and distinguishing cytostatic from cytotoxic response. For drugs that have a narrow range of GR50 values like taxanes, efficacy is the most relevant metric: in many ovarian BCL2-deleted cell lines, docetaxel induces a cytotoxic response (negative GRmax values), whereas wild-type lines elicit a cytostatic response (positive GRmax values). Because efficacy (GRmax) varies independently of potency (GR50), we conclude that both metrics are complementary for pharmacogenomics and should be studied jointly.

Adopting GR metrics requires only modest changes in experimental protocols and analysis is facilitated by our interactive website: GRcalculator.org. We expect GR metrics to improve the identification of reliable drug response biomarkers and enhance the reproducibility of large-scale sensitivity studies.

#973

Methylation accurately predicts age of cancer onset in patients with Li Fraumeni Syndrome.

Benjamin M. Brew,1 David Malkin,1 Lauren Erdman,1 Andrea Doria,1 Jason Berman,2 Adam Shlien,1 Tanya Guha,1 Ana Novokmet,1 Anna Goldenberg1. 1 _The Hospital for Sick Children, Toronto, Ontario, Canada;_ 2 _IWK Health Centre, Toronto, Ontario, Canada_.

Introduction: Li Fraumeni Syndrome (LFS) is a rare hereditary genetic cancer predisposition syndrome. Germline mutations of the TP53 tumor suppressor gene are the underlying cause in >80% of patients with LFS, and are associated with an increased risk of second tumors and a spectrum of early onset cancers, even in the absence of a family history of cancer. We have previously developed and implemented a comprehensive life-long clinical surveillance protocol for individuals with a germline TP53 mutation. We set out to make this screening process more targeted by building a predictive model of age of onset. We accomplished this goal by implementing machine learning methods on germline methylation data.

Methods: We made use of the Toronto Hospital for Sick Children (SickKids) LFS family cohort in our predictive model of age of onset. In all, we have 74 patients with germline methylation data, consisting of ~450,000 probe sites. We subset this data by identifying probes that fall into differentially methylated regions between LFS and cancer patients with wild-type TP53. The probes identified in these regions were used in our predictive model of age of onset. Because age of sample collection was highly correlated with age of onset (r2 ~ .90), we corrected for confounding using a strategy that is two-fold: (1) we extracted the variation of each probe that is independent of the age of sample collection (the residual after regressing on the age of sample collection) and use these as predictors in our model, and (2) we test our models on the task of predicting the age of sample collection for LFS patients that do not have cancer. The former provided us with more robust predictions while the latter verified that we are in fact predicting age of onset, rather than simply predicting age at which the sample was collected.

Results: Our machine learning model was able to achieve 86% correlation between true and predicted values of the age of onset. Additionally, we have tested the ability of our models to predict whether an individual will be diagnosed before or after the age of 4. Our classification machine learning model achieved 91% accuracy on average. We verified that our model does not simply predict age of sample collection by using our cohort of LFS patients that do not have cancer (n = 37). The distribution of the age of sample collection matched those of the patients used in our model. The model has no predictive power on the age of sample collection, thus confirming that our model is highly predictive of the age of cancer onset in LFS TP53 Mutation patients.

Conclusions: We identified two predictive models for age of cancer onset in LFS patients that achieve high accuracy, both when predicting the age of onset as a continuous variable (86% correlation) and whether cancer onset will occur before or after the age of 4 (91% accuracy). Our model will assist clinicians in targeting high risk patients for screening, lower the cost of treatment, and raise the likelihood of survival among LFS patients.

#974

Automating deconvolution of heterogeneous bulk tumor genomic data.

Theodore Roman, Brenda Xiao, Russell Schwartz. _Carnegie Mellon Univ., Pittsburgh, PA_.

Tumor heterogeneity has been linked to many measures of poor patient outcome. Despite numerous recent methodological advances, though, reliable estimates of clonal and subclonal architecture remain elusive. We present advances in computational deconvolution of tumor heterogeneity, aimed at better handling complex substructure, more automated inference, and increased flexibility to heterogeneous genomic data sources. Our methods make use of geometric models of mixture substructure, a specialized form of a strategy called manifold learning, to better resolve clonal substructure shared across genomic samples. We validate the methods on breast tumor dataset from the Cancer Genome Atlas (TCGA). We find statistically significant Spearman correlation between inferred mixture models and independently derived clinical subtype across DNA, RNA, and combined datasets. DAVID tissue term enrichment, applied to inferred amplified gene sets, further yields significant correlation (p< 0.01) with various ontological terms expected for breast tumors, for example, "Breast Carcinoma". We further verified the ability of the mixture modeling to classify tumors by progression state, showing cross-validated k-fold loss of 0.2538 in separating tumors by early (Stage I - II) vs. late (Stages III-IV) stage, corresponding to significant (p<0.001) separation by chi-squared test. The results demonstrate the effectiveness of our improved geometric deconvolution approach in resolving clonal structure to separate tumors by biologically relevant pathways and degrees of progression.

#975

A multiscale computational model for spatio-temporal tumor immune response.

Chang Gong,1 Oleg Milberg,1 Bing Wang,2 Paolo Vicini,3 Rajesh Narwal,4 Lorin Roskos,4 Aleksander S. Popel1. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _MedImmune, Mountain View, CA;_ 3 _MedImmune, Cambridge, United Kingdom;_ 4 _Medimmune, Gaithersburg, MD_.

When the immune system responds to tumor development, patterns of immune infiltrates emerge, highlighted by expression of immune checkpoint-related molecules such as PD-L1 on cancer cells and its receptor PD-1 on cytotoxic T cells. Pre-treatment tumor spatial heterogeneity could bear information on intrinsic characteristics of the tumor lesion for individual patient, and thus has the potential to comprise biomarkers for anti-tumor therapeutics. We developed a systems biology computational multiscale agent-based model to capture the interactions between immune cells and cancer cells during tumor progression. Cytotoxic T cells and cancer cells are modeled as free-moving agents in a 3-dimensional grid, where each cell acts in response to its local microenvironment and carries out functions such as division, apoptosis, cytotoxic killing and switching between states with different PD-1 or PD-L1 expression levels. Subsequently, we analyzed the emergent behavior of tumor progression by looking at all these local interactions as a whole. Using this model, we are able to reproduce temporal dynamics of cytotoxic T cells and cancer cells during general tumor progression, as well as 3-dimensional spatial distributions of these cells over the time course of the simulation. By varying the characteristics of the neoantigen profile of individual patients, such as mutational burden and immunogenicity, a spectrum of pre-treatment spatial patterns of PD-1/PD-L1 expression is generated in our simulations, resembling immune-architectures obtained via immunohistochemistry from patient biopsies. We evaluate potential prognostic biomarkers by correlating these spatial characteristics with in silico treatment results with immune checkpoint inhibitors. Simulation results demonstrate that the percentage of PD-L1 positive cancer cells which are not in close proximity of the tumor boundary or vasculature is more indicative of successful anti-PD1/anti-PD-L1 treatment. Our findings suggest that tumor spatial heterogeneity, especially its immune-architecture, reflects the course of tumor progression as well as patient-specific properties, and is thus likely to carry important information about tumor susceptibility to treatment such as with immune checkpoint inhibitors. We demonstrated how prognostic biomarkers could be realistically simulated in a general cancer scenario. The model is further refined for use to predict treatment/biomarker combinations in specific cancer types.

#976

ImmunoMap: a novel bioinformatics tool for immune cell repertoire analysis.

John-William Sidhom,1 Catherine A. Bessell,1 Jonathan J. Havel,2 Timothy A. Chan,2 Jonathan P. Schneck1. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _Memorial Sloan Kettering Cancer Center, New York City, NY_.

Background: There has been a dramatic increase in T-cell Receptor (TCR) sequencing spurred, in part, by the clinical demand in Immuno-oncology and technological advances in TCR sequencing. However, there has been little in the way of approaches to parse the data in a biologically meaningful fashion. The ability to parse this data to understand the T-cell repertoire in a structurally relevant manner has the potential to open new discoveries about how the immune system responds to insults such as cancer and infectious diseases.

Methods: Here we describe a novel method to visualize and quantify TCR repertoire sequence diversity. This method includes metrics such as visualization of repertoire with: 1) weighted phylogenetic trees that display relatedness and frequency of the sequences; 2) dominant motif analyses identifying clusters of highly homologous sequences that contribute significantly to response and; 3) TCR diversity score measuring the average relatedness (by sequence homology) of all TCR's in a sample. To demonstrate the power of the approach, we have applied it to understanding the CD8 T Cell response to model self (TRP2) and foreign (SIY) antigens in naïve and tumor-bearing (B16 melanoma) B6 mice. Additionally, this method was applied to tumor infiltrating lymphocytes, TIL, taken pre- and on-therapy, from patients undergoing Nivolumab (α-PD1) therapy for metastatic melanoma.

Results: Analysis of the naïve CD8 response demonstrated a highly conserved (measured by the TCR diversity score) and less clonal response to SIY whereas the response to TRP2 was less conserved and highly clonal. Dominant motif analysis demonstrated highly rich motifs consisting of many homologous sequences in the SIY response but few sequences per motif in the TRP2 response. This may reflect the outcome of tolerance mechanisms to self-antigens. Presence of tumor demonstrated differential immune pressure on the TRP2 vs SIY response. Tumor primed novel SIY motifs but constricted the number of dominant motifs in the TRP2 response while additionally altering the sequence of the motifs. In patients undergoing α-PD1 therapy, we identified signatures in pre- and post-therapy TCR repertoires that correlated with clinical outcome response. Prior to therapy, patients whose dominant motifs were rich with many sequences responded favorably to checkpoint inhibition over those with less rich motifs. After four week on therapy, patients whose TCR repertoires became more conserved responded more favorably to PD1 treatment while those who did not respond had no change in their TCR diversity score.

Conclusions: In summary, we have developed and demonstrated a novel method to meaningfully parse and interpret TCR repertoire data and have applied it to yield a novel insight of CD8 T Cell responses to different types of antigens in model systems as well as key characteristics of TIL repertoires from patients who respond clinically to α-PD1 therapy.

#977

Phenotypic analysis of single-cell breast cancer inhibition data reveals insights into EMT.

William S. Chen,1 Nevena Zivanovic,2 Dana Pe'er,3 Bernd Bodenmiller,2 Smita Krishnaswamy1. 1 _Yale School of Medicine, New Haven, CT;_ 2 _University of Zurich, Zurich, Switzerland;_ 3 _Columbia University, New York, NY_.

Background: A leading model of cancer metastasis is epithelial-to-mesenchymal transition (EMT). We sought to determine whether single-cell inhibition data targeting potential mediators of EMT could uncover mechanistic insights into the EMT process.

Methods: EMT was artificially induced on Py2T murine breast cancer cells by TGFb treatment. Additionally, a unique drug inhibitor was added to each well of a multiplexed CyTOF experiment. 37 transcription factors and cell surface markers were measured in each cell to assess epithelial and mesenchymal states, SMAD, AKT, and MAPK signaling activity, cell cycle regulation, and apoptosis pathway activation. The final single-cell dataset consisted of 300 inhibition and control conditions (cell populations), which we aimed to characterize in relation to one another with respect to effect on EMT.

Analyzing the similarity between drug inhibitions amounts to a novel type of clustering problem that involves computing the similarity between diverse cell populations generated by each inhibitor. Traditional methods for comparing cell populations are not robust to the intra-population heterogeneity we observed amongst cells undergoing EMT. Thus, we developed Phenotypic Earth Mover's Distance (PhEMD). This method for comparing cell populations leverages the insight that only a limited number of "cell subtypes" (e.g. mesenchymal, epithelial, transitional) are observed in unperturbed and perturbed EMT. By classifying each cell as one of these distinct subtypes using community-detection based clustering, PhEMD represents an inhibition or control condition as its relative abundance of each cell subtype. It then uses Earth Mover's Distance (EMD) to compare two relative abundance distributions (i.e. heterogeneous cell populations). PhEMD thus derives a single value representing the dissimilarity between two inhibition conditions. Using PhEMD as measure of dissimilarity between each pair of inhibition and control conditions, we constructed an inhibitor-inhibitor graph and used graph clustering to identify groups of inhibitors that had similar effects to one another.

Results: PhEMD analysis revealed that MEK, EGFR and Src inhibitors significantly halted EMT by generating far fewer mesenchymal cells and maintaining a large epithelial cell subpopulation. PhEMD also revealed that different mTOR, PI3K, and Akt inhibitors tended to have similar effects to one another and collectively generated a distinct, transitional cell subpopulation with altered pS6 expression. The pS6 dysregulation may be explained by the fact that ribosomal protein S6 is downstream of PI3K/AKT/mTOR, and the intermediate levels of both E-cadherin and vimentin suggest that this subset of cells may have been halted mid-transition. Finally, several Aurora Kinase and CDK inhibitors resulted in a relatively high percentage of apoptotic cells, suggesting these kinases may be important cell cycle regulators in the context of EMT.

## CANCER CHEMISTRY:

### Novel Therapeutic Targets, Molecules, and Approaches for the Treatment of Cancer

#978

Spatial-temporal delivery of OX40 agonist and PD-1 inhibitor using nanoparticles improves therapeutic efficacy of cancer immunotherapy.

Yu Mi, Christof C. Smith, Feifei Yang, Jonathan Serody, Benjamin Vincent, Andrew Z. Wang. _UNC Chapel Hill, Chapel Hill, NC_.

Background: Cancer immunotherapy is an exciting new approach to cancer treatment and there is strong interest in strategies to improve the long-term durable response rates of cancer immunotherapy. One approach is to combine checkpoint inhibitors such as aPD-1 with T cell activator such as OX40 agonist to further increase immune activation. We hypothesized that we can improve the therapeutic efficacy of this approach by temporally control the activation of OX40 and inhibition of PD-1 pathways. To accomplish this, we utilized nanoparticles that can deliver anti-OX40 and anti-PD1 antibodies simultaneously to T cells.

Methods: Agonist antibody (anti-OX40) and antagonist antibody (anti-PD1) were conjugated to PLGA-PEG-Maleimide nanoparticles (AANPs) with precise ratio control and quantified by ELISA. Their specific binding to the target proteins was shown in vitro by flow cytometry. The tumor inhibition efficiency was assessed on mice bearing different tumor models. Two tumors were inoculated subcutaneously (105 B16F10 cells) or on fourth mammary fat pad (105 4T1 cells) on both flanks of mice. One side of tumor was irradiated once and AANPs were injected twice every 3 days. In vivo depletion experiments were tested on C56Bl6 mice and CD11b/c mice. Different populations of T cells in tumor and spleen were analyzed by flow cytometry and by fluorescent IHC staining. T cell killing assay and IFN-γ ELISpot were studied. Co-localization was demonstrated with fluorescent labeled antibodies and the corresponding AANPs.

Results: AANPs showed a 30% cure rate, compared to 10% of free antibodies, 0% of anti-PD1 conjugated NPs, and 0% of anti-OX40 conjugated NPs in B16F10 melanoma model. We then re-challenged the cured mice with 2×105 B16F10 cells and none of the mice developed another tumor. In 4T1 breast cancer model, the survival rate on day 39 was 50% with AANPs treatment, compared to 22% in the mixture of anti-PD1 conjugated nanoparticles and anti-OX40 conjugated nanoparticles, and 0% of free antibodies. We demonstrated that AANPs led to a higher medium TCD8+/Treg ratio in tumors. The therapeutic effect was mediated by CD8+ T cells as elimination of these cells abrogated the therapeutic effects. In vitro study confirmed that AANPs were able to improve T cell stimulation compared to free antibodies by increasing IFN-γ excretion (2x). We further confirmed co-localization of antibodies with AANPs on tumor infiltration T cells in vivo. Our data demonstrated that spatial-temporal delivery of agonist and antagonist could improve T cell activation and cancer immunotherapy.

Conclusions: Our data demonstrates that spatial-temporal delivery of agonist and antagonist can improve T cell activation and cancer immunotherapy.

#979

Discovery of the JAK1 selective kinase inhibitor AZD4205.

Jason G. Kettle,1 Qibin Su,2 Neil Grimster,2 Sameer Kawatkar,2 Scott Throner,2 Richard Woessner,2 Huawei Chen,2 Geraldine Bebernitz,2 Kristen Bell,2 Erica Anderson,2 Linette Ruston,1 Jon Winter-Holt,3 Paul Lyne,2 Melissa Vasbinder,2 Claudio Chuaqui2. 1 _AstraZeneca, Stockport, United Kingdom;_ 2 _AstraZeneca, Waltham, MA;_ 3 _AstraZeneca, Cambridge, United Kingdom_.

Janus kinases are a family of four enzymes; JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2) that are critical in cytokine signalling, with constitutive activation of JAK/STAT pathways associated with a wide variety of malignancies. Elevated JAK/STAT signalling leading to increased activation of STAT3 is reported in a wide variety of cancers, including breast, liver, prostate, colorectal, head and neck, oesophageal, pancreatic, bladder, and non-small cell lung, and is implicated in the pathogenesis of diffuse large B-cell lymphoma and nasopharyngeal carcinomas. Overall, up to 70% of human tumours are linked to persistent elevated STAT3 activity which can be associated with poorer prognosis in many of these settings. In addition, elevated pSTAT3 is observed in response to chemotherapy treatment, and also in response to treatment with inhibitors of oncogenic signalling pathways such as EGFR, MAPK and AKT, and is associated with resistance or poorer response to agents targeting these pathways. In many of these cases, JAK1 is believed to be a primary driver of STAT3 phosphorylation and signalling, suggesting inhibition of JAKs as a therapeutic approach to treat these potential resistance mechanisms. The mixed JAK1/2 kinase inhibitor ruxolitinib is approved for the treatment of myeloproliferative neoplasms including intermediate or high risk myelofibrosis and polycythemia vera and has been tested in a variety of tumor settings. Since JAK2 is essential for the signal transduction downstream of erythropoietin, thrombopoietin and related receptors that control erythrocyte and megakaryocyte expansion, dosing of inhibitors that target JAK2 can be limited by toxicities such as thrombocytopenia and anaemia.

Starting from a non-kinome selective screening hit, structure-based design was used to optimise a series of aminopyrimidines that led to JAK1-selective candidate drug AZD4205. This compound demonstrates ATP competitive binding with IC50's in a high ATP concentration enzyme assay against JAK1 of 73 nM (Ki = 2.8 nM), with high selectivity against JAK2 and JAK3 with IC50's of 13,233 nM and >30,000 nM respectively. In addition it showed potent inhibition of p-STAT3 in a cell based assay of JAK1 activity with an IC50 of 128 nM and excellent selectivity across the kinome. In summary, AZD4205 is a highly potent JAK1-selective kinase inhibitor with excellent preclinical pharmacokinetics with potential for further clinical development. The optimization from screening hit to first disclosure of this candidate drug will be presented.

#980

BAY-299, a novel chemical probe for in-depth analysis of the function of the bromodomain proteins BRPF2 and TAF1.

Lea Bouche,1 Clara D. Christ,1 Stephan Siegel,1 Cynthia Tallant,2 Amaury E. Fernández-Montalván,1 Kilian V. Huber,2 Verra Pütter,1 Susanne Müller,2 Oleg Fedorov,2 Antonius ter Laak,1 Tatsuo Sugawara,1 Detlef Stöckigt,1 Julia Meier,3 Simon J. Holton,1 Ingo V. Hartung,1 Bernard Haendler1. 1 _Bayer Pharma AG, Berlin, Germany;_ 2 _Structural Genomics Consortium, Oxford, United Kingdom;_ 3 _Merck, Germany_.

With the exception of the bromodomain and extra-terminal domain BET subgroup, little is known about the role of bromodomain (BD) containing proteins in cancer so that there is a dire need for chemical probes addressing other family members. The bromodomain and PHD-finger (BRPF) family encompasses three paralogs, BRPF1, BRPF2 and BRPF3, which are all found in histone acetyltransferase (HAT) complexes. BRPF2 is a scaffold protein and its knock-out leads to embryonic lethality at E15.5, potentially due to its role in embryonic stem cell differentiation. Here we present the structure-activity relationship (SAR) and characterization of the first selective BRPF2 chemical probe BAY-299, with additionnal strong activity at TAF1, a major component of the basal transcription initiation complex TFIID. BAY-299 shows in vitro activity for BRPF2 (IC50 = 67 nM) and TAF1 second bromodomain (BD2; IC50 = 8 nM) in the TR-FRET assay, as well as in the cellular NanoBRET assay [IC50 (BRPF2) = 575 nM; IC50 (TAF1 BD2) = 825 nM]. To the best of our knowledge BAY-299 is the only disclosed inhibitor showing BRPF2 selectivity over its two paralogues BRPF1 and BRPF3. It belongs to the 1,3-benzimidazolone scaffold and bears a novel substitution which is responsible for its high BRPF2 selectivity and also for its inactivity on BET BDs. The dual inhibitory properties of BAY-299 against BRPF2 and TAF1 make it an ideal research tool for further investigation of these two proteins in physiological and pathological processes.

#981

The design and characterization of a selective TRIM24 degrader.

Lara N. Gechijian,1 Dennis Buckley,1 Matthew Lawlor,2 Thomas Scott,2 Joshiawa Paulk,1 Jaime Reyes,1 Georg Winter,1 Michael Erb,1 Chris Ott,1 Sirano Dhe-Paganon,2 James Bradner1. 1 _Harvard Medical School, Boston, MA;_ 2 _Dana Farber Cancer Institute, Boston, MA_.

A fundamental biological understanding of the individual contributions of functional domains within multidomain proteins is critical to inform therapeutic approaches to targeting mechanisms driving human disease. TRIM24 is a multi-domain protein that has been broadly characterized as a co-regulator of transcription. It is therapeutically relevant as it is implicated as a dependency in many human cancers, however, the potent and selective inhibitors of the TRIM24 bromodomain do not have well-characterized phenotypic consequences. Where inhibition of one activity of a protein does not appear to be efficacious, chemical knockdown tools allow for the acute depletion of the entire protein, therefore eliminating all protein activities. In this study, we have used chemical degradation as one strategy to target the entire TRIM24 protein, where we have shown that a bifunctional degrader molecule hijacks the ubiquitin ligase machinery for targeted TRIM24 degradation. We have shown that TRIM24 degradation is required to perturb the oncogenic state in leukemia. In this context, TRIM24 degradation rather than bromodomain inhibition alone is required to displace TRIM24 from chromatin. Using this probe, a further understanding of the contribution of TRIM24 domains to its transcriptional activation function will provide mechanistic insight as to how TRIM24 promotes a gene expression program permissive of the oncogenic state, as well as inform a therapeutic approach to target multidomain proteins, such as TRIM24, that are tightly linked to disease.

#982

Development of homogeneous dual-drug ADCs: Application to the co-delivery of auristatin payloads with complementary antitumor activities.

Matthew R. Levengood, Xinqun Zhang, Kim K. Emmerton, Joshua H. Hunter, Peter D. Senter. _Seattle Genetics, Inc., Bothell, WA_.

A common theme in treating cancer is the use of combination chemotherapy, where multiple drugs with different mechanisms of action are combined to elicit synergistic activity or overcome differential drug sensitivities. Antibody-drug conjugates (ADCs) have emerged as a powerful approach for treating cancer, combining the tumor targeting specificity of monoclonal antibodies with the potent cell-killing activity of cytotoxic drugs. Like other therapies, these agents are increasingly being tested in combination with unconjugated, clinically approved anticancer agents. In addition, emerging data demonstrates that insensitivity to a particular ADC can be overcome through delivery of a different payload using the same antibody. For these reasons, the development of ADCs that can deliver two complementary payloads to a tumor would likely be a significant advancement in ADC technology. To enable dual-drug conjugation, we utilized a multiplexing drug carrier that contains cysteine residues with orthogonal protecting groups and identified novel conditions for utilization of these protecting groups on a folded protein. Sequential cysteine unmasking enables discrimination between conjugation sites to allow for site-specific drug conjugation. This strategy provides homogeneous ADCs bearing 16 total drugs per antibody, split evenly between the two drug linkers. Importantly, this strategy is flexible, as it does not require engineered antibodies or custom enzymes for drug-linker conjugation. To demonstrate the potential benefits of ADC dual drug delivery, this strategy was applied to the construction of ADCs bearing two classes of auristatin drug linkers that have different physiochemical properties and complementary anti-cancer activities. Dual-auristatin ADCs were tested in cell line and xenograft models that have differential sensitivities to the individual auristatin components, including those with heterogeneous antigen expression or high levels of drug efflux transporters. The data from these studies demonstrate that the dual-auristatin ADCs were active on cells and tumors that are refractory to treatment with either of the individual component drugs. This work highlights the potential for delivering two synergistic or complementary payloads on a single ADC and presents a flexible method for constructing dual-drug ADCs with site-specific and homogeneous drug loading.

#983

Identification of potent, highly selective and orally available ATR inhibitor BAY 1895344 with favorable PK properties and promising efficacy in monotherapy and combination in preclinical tumor models.

Ulrich T. Luecking,1 Julien Lefranc,1 Antje Wengner,1 Lars Wortmann,1 Hans Schick,2 Hans Briem,1 Gerhard Siemeister,1 Philip Lienau,1 Christoph Schatz,1 Benjamin Bader,1 Gesa Deeg,1 Franz von Nussbaum,1 Michael Brands,1 Dominik Mumberg,1 Karl Ziegelbauer1. 1 _Bayer Pharma AG, Berlin, Germany;_ 2 _ASCA GmbH, Berlin, Germany_.

The integrity of the genome of eukaryotic cells is secured by complex signaling pathways, known as DNA damage response (DDR). Recognition of DNA damage activates DDR pathways resulting in cell cycle arrest, suppression of general translation, induction of DNA repair, cell survival or even cell death. Proteins that directly recognize aberrant DNA structures recruit and activate kinases of the DDR pathway, such as ATR (ataxia telangiectasia and Rad3-related). ATR responds to a broad spectrum of DNA damage, including double-strand breaks (DSB) and lesions derived from interference with DNA replication as well as increased replication stress (e.g. in oncogene-driven tumor cells). Therefore, inhibition of ATR kinase activity could be the basis for a novel anti-cancer therapy in tumors with increased DNA damage, deficiency in DNA damage repair or replication stress. Herein we report the identification of the potent, highly selective and orally available ATR inhibitor BAY 1895344 by a collaborative effort involving medicinal chemistry, pharmacology, DMPK and computational chemistry. The chemical structures of lead compound BAY-937 and clinical candidate BAY 1895344 as well as the main SAR trends within this novel class of naphthyridine derivatives will be disclosed for the first time. The novel lead compound BAY-937 revealed promising inhibition of ATR (IC50 = 78 nM) and high kinase selectivity in vitro. In cellular mechanistic assays BAY-937 inhibited hydroxyurea-induced H2AX phosphorylation (IC50 = 380 nM) demonstrating the anticipated mode of action. Moreover, BAY-937 was shown to inhibit proliferation of a variety of tumor cell lines with low- to sub-micromolar IC50 values. In initial xenograft studies, BAY-937 revealed moderate activity in monotherapy and in combination with cis-platin. However, BAY-937 also revealed low aqueous solubility, low bioavailability (rat) and activity in the hERG patch clamp assay. Extensive lead optimization efforts led to the identification of the novel, orally available ATR inhibitor BAY 1895344. In vitro, BAY 1895344 was shown to be a very potent and highly selective ATR inhibitor (IC50 = 7 nM), which potently inhibits proliferation of a broad spectrum of human tumor cell lines (median IC50 = 78 nM). In cellular mechanistic assays BAY 1895344 potently inhibited hydroxyurea-induced H2AX phosphorylation (IC50 = 36 nM). Moreover, BAY 1895344 revealed significantly improved aqueous solubility, bioavailability across species and no activity in the hERG patch-clamp assay. BAY 1895344 also demonstrated very promising efficacy in monotherapy in DNA damage deficient tumor models as well as combination treatment with DNA damage inducing therapies. The start of clinical investigation of BAY 1895344 is planned for early 2017.

#984

Identification of potent and highly selective PTEFb inhibitor BAY 1251152 for the treatment of cancer: from p.o. to i.v. application via scaffold hops.

Ulrich T. Luecking,1 Arne Scholz,1 Dirk Kosemund,1 Rolf Bohlmann,1 Hans Briem,1 Philip Lienau,1 Gerhard Siemeister,1 Ildiko Terebesi,1 Kirstin Meyer,1 Katja Prelle,1 Ray Valencia,2 Stuart Ince,2 Franz von Nussbaum,1 Dominik Mumberg,1 Karl Ziegelbauer,1 Michael Brands1. 1 _Bayer Pharma AG, Berlin, Germany;_ 2 _Bayer HealthCare Pharmaceuticals Inc., Whippany, NJ_.

PTEFb/CDK9 mediated transcription of short-lived anti-apoptotic survival proteins like Mcl-1 and Myc plays a critical role in cancer cell growth and survival in various tumor entities including AML. In addition, these survival proteins play important roles in the development of resistance to chemotherapy. We previously disclosed the preclinical profile of BAY 1143572, the first selective, orally available PTEFb/CDK9 inhibitor that entered clinical development [1-3]. BAY 1143572 had low nanomolar activity against PTEFb/CDK9, an at least 50-fold selectivity against other CDKs in enzymatic assays and broad anti-proliferative activity against a panel of tumour cell lines with sub-micromolar IC50 values. BAY 1143572 also showed single agent in vivo efficacy at tolerated doses in various xenograft tumour models in mice and rats upon once daily oral administration. To fully explore future treatment options using selective PTEFb/CDK9 inhibitors we initiated a follow-up program to identify novel PTEFb/CDK9 inhibitors for treatment of cancer with increased potency enabling i.v. treatment of patients. Extensive lead optimisation efforts, including various scaffold hops, led to the identification of BAY 1251152. In comparison to oral BAY 1143572, BAY 1251152 shows significantly increased biochemical (IC50 CDK9 = 3 nM) and cellular potency (IC50 MOLM13 = 29 nM), increased selectivity against CDK2 as well as high permeability and no efflux. The significantly reduced therapeutic dose and high solubility of BAY 1251152 enable the desired i.v. application. BAY 1251152 demonstrated excellent efficacy upon i.v. treatment in xenograft models (e.g. MOLM13) in mice and rats. BAY 1251152 is currently being evaluated in Phase I studies (NCT02635672; NCT02745743) to determine the safety, tolerability, pharmacokinetics and initial pharmacodynamic biomarker response in patients with advanced cancer. This presentation will highlight the key learnings from our PTEFb/CDK9 i.v. lead optimization program and disclose the structure of BAY 1251152 for the first time. [1]: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3022. [2]: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr DDT02-02. doi:10.1158/1538-7445.AM2015-DDT02-02. [3]: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2828. doi:10.1158/1538-7445.AM2015-2828

## CLINICAL RESEARCH:

### Cancer Survivorship and Quality of Life

#985

Adipose tissue inflammation in breast cancer survivors: Effects of a 16-week aerobic and resistance exercise intervention.

Christina M. Dieli-Conwright,1 Jean Hughes-Parmentier,2 Kyuwan Lee,1 Darcy Spicer,1 Wendy Mack,1 Fred Sattler,1 Steven D. Mittelman2. 1 _University of Southern California, Los Angeles, CA;_ 2 _Children's Hospital Los Angeles, Los Angeles, CA_.

Background and Purpose: Obesity is a leading modifiable contributor to breast cancer mortality due to its association with increased recurrence and decreased overall survival rate. There are over 2.5 million breast cancer survivors, 64% of whom are overweight/obese (BMI >25 kg/m2). A central mechanism by which obesity stimulates cancer progression is through chronic, low-grade inflammation in white adipose tissue, leading to accumulation of adipose tissue macrophages (ATMs), in particular the pro-inflammatory M1 phenotype macrophage. Exercise has been shown to reduce M1 ATMs, and increase the more anti-inflammatory M2 ATMs in obese adults. The purpose of this study was to determine whether a 16-week exercise intervention would positively alter adipose tissue inflammation by changing ATM phenotype and cytokine secretion in obese postmenopausal breast cancer survivors.

Experimental Design: Twenty obese postmenopausal breast cancer survivors were recruited from USC and randomized to either the exercise (EX) or control (CON) group. The EX group participated in 16 weeks of supervised exercise sessions 3 times/week. Sessions included total-body resistance training consisting of 8 exercises with a rest period of 45 seconds between each set of resistance exercise followed by 30 minutes of moderate-vigorous intensity (65-80% HRmax) aerobic exercise. The CON group was asked to maintain their current activity levels. Superficial subcutaneous abdominal adipose tissue biopsies were performed at baseline and following the 16-week study period. Adipose tissue samples were analyzed using fluorescence-activated cell sorting (FACS) to characterize ATM characterization (M1 vs M2). Portions (~100 mg) of each biopsy were incubated in media overnight to measure cytokine secretion. A 2x2 (group x time) repeated measures ANOVA was used to evaluate changes in adipose tissue and systemic inflammation.

Summary of Results: At baseline, there were no group differences (p>0.05) in age (55.1±5.2 yrs), BMI (34.4±7.5 kg/m2), percent body fat (36.2±4.9%), or ATM M1 (25.4±6.7%) and M2 (4.2±0.9%) levels. EX was associated with a significant decrease in ATM M1 (-18.8±7.3%) and increase in ATM M2 (9.6±1.6%; p<0.01). EX increased adipose tissue secretion of the anti-inflammatory cytokines, adiponectin (14.6±5.2%), IL-12 p40 (2.4±0.8), IL-12 p70 (2.8±1.8%) and decreased secretion of IL-6 (-4.8±2.0%), and TNF-a (-2.0±0.7; p<0.01). The CON group did not experience any changes in ATM characterization and cytokine secretion during the study period (p>0.01).

Conclusions: A 16-week aerobic and resistance exercise intervention attenuates adipose tissue inflammation in obese postmenopausal breast cancer survivors. Future large randomized controlled trials are warranted to investigate the impact of exercise-induced reductions in adipose tissue inflammation and breast cancer recurrence.

#986

Targeting nicotinic acetylcholine receptors for the prevention and reversal of chemotherapy-induced peripheral neuropathy.

S Lauren Kyte,1 Wisam Toma,1 Ganeshsingh Thakur,2 M Imad Damaj,1 David A. Gewirtz1. 1 _Virginia Commonwealth Univ., Richmond, VA;_ 2 _Northeastern University, Boston, MA_.

While chemotherapy has played a significant role in the survival of cancer patients, exposure to drugs such as the taxanes, platinum-based compounds, and bortezomib often results in chemotherapy-induced peripheral neuropathy (CIPN), a consequence of peripheral nerve fiber dysfunction or degeneration. CIPN is characterized by sensory symptoms such as numbness, burning pain, and allodynia, resulting in an overall decrease in quality of life. Paclitaxel (Taxol), a taxane that is commonly used to treat breast, lung, and ovarian cancers, has been found to cause CIPN in 59-78% of cancer patients. There is currently no effective preventative or therapeutic treatment for this side effect, which can be a dose-limiting factor or delay treatment. Our studies have established that the prototypical nicotinic acetylcholine receptor (nAChR) agonist, nicotine, is capable of preventing and reversing the development of paclitaxel-induced CIPN, and does not stimulate tumor growth or interfere with the cytotoxic properties of paclitaxel. Male C57BL/6J mice were treated with paclitaxel (8 mg/kg, i.p.) every other day for a total of four injections. Nicotine was either infused via a subcutaneous 7-day osmotic minipump (24 mg/kg/day) starting 48 hours prior to paclitaxel treatment, or acutely injected (0.9 mg/kg, i.p.) following paclitaxel treatment. Von Frey filament testing revealed that nicotine can prevent and reverse paclitaxel-induced mechanical allodynia; the latter effect of nicotine can be inhibited by the α7 nAChR antagonist methyllycaconitine (10 mg/kg, s.c.). Also, α7 nAChR knockout mice exhibit a greater sustained decrease in mechanical threshold after paclitaxel administration than wild-type mice, suggesting the involvement of this receptor in CIPN development and maintenance. This hypothesis was supported by the reversal of paclitaxel-induced mechanical allodynia by the α7 nAChR silent agonist R-47. While nicotine activates its receptors in the tumors cells, based on stimulation of the PI3K/Akt pathway, MTT/MTS colorimetric assays showed that concentrations of nicotine ranging from 0.1 to 10 µM fail to significantly increase the viability of A549 or H460 non-small cell lung cancer cells, murine Lewis lung carcinoma cells, or primary human lung cancer cells. Most importantly, the paclitaxel-induced decreases in H460 proliferation and LLC viability are not significantly attenuated by 1 µM nicotine. Moreover, 1 µM nicotine does not interfere with paclitaxel-induced apoptosis of A549 cells. Finally, R-47 also suppresses CIPN without increasing lung tumor cell viability and colony formation, or interfering with the cytotoxicity of paclitaxel. Our in vitro findings are supported by studies demonstrating that nicotine does not enhance tumor volume or cause premature death in tumor-bearing mice. These data suggest that nAChRs may be promising drug targets for the prevention and treatment of CIPN.

#987

Unanticipated cardiotoxicity due to targeted anti-cancer therapy in hematologic malignancies patients: Natural history and risk factors.

Chintan Shah, Yan Gong, Anita Szady, Qian Sun, carl J. Pepine, Taimour Langaee, Alexandra R. Lucas, Jan S. Moreb. _Univ. of Florida, Gainesville, FL_.

INTRODUCTION: Unanticipated cardiotoxicity is now identified as a significant clinical problem associated with new anti-cancer targeted agents. Risk factors and natural history are still poorly understood. We aim to determine potential clinical risk factors for cardiotoxicity among patients with hematologic malignancies (HM) who were treated with targeted therapies over a 10-year period.

METHODS: We used 114 diagnosis codes for HM and 17 codes for cardiac diseases in order to identify patients in our electronic medical records (EPIC) and identify patients with HM who met above criteria. Cardiotoxicity was defined mainly by left ventricular ejection fraction (LVEF) of < 50%, arrhythmias, or ischemic cardiovascular event that occurred after initiation of the drug of interest. The targeted agents of interest include tyrosine kinase inhibitors (TKIs), proteasome inhibitors, monoclonal antibodies, hypomethylating agents, and immunomodulatory agents. Multivariable logistic regression was performed to estimate the odds ratios (ORs) and 95% confidence intervals (95% CI) of the potential risk factors. Kaplan-Meier analysis and log-rank test were used to evaluate the effect of cardiotoxicity on the overall survival of the patients.

RESULTS: Of 820 patients with both HM and cardiac diagnosis, 29 patients developed cardiotoxicity after initiation of targeted therapies. We selected 70 matched controls based on type of targeted therapy. In the study group, the median time from exposure to cardiac event was 120 days (range, 1-1176). Significantly higher number of patients had prior exposure to anthracyclines in study versus control group (65.5% vs 42.8%, P=0.04), however, this was not significant in multivariable analysis. Multiple other variables, including traditional risk factors for heart disease, were analyzed and did not differ significantly between the two groups. Only two variables remained significant in the multivariable analysis, including prior history of DVT/PE (OR 4.88, 95% CI: 1.44-16.54, P=0.011), and Karnofsky score of ≥80% (OR 3.99, 95% CI: 1.51-10.6, P= 0.005). With median follow-up of 27 months (range, 1-120), 17 patients in the study group died, but only 2 of cardiac causes. Repeat echocardiograms showed worsening of LVEF in 4 patients while stable/improved in 23 patients, and 21 patients were able to receive further chemotherapy. There was a trend towards worse overall survival in the study group (P= 0.071).

CONCLUSIONS: About 3.5% of patients with HM experience unanticipated cardiotoxicity due to targeted anti-cancer agents with related mortality of 6.8 %. Most patients do well with stable compensated cardiac function and 35% have an objective improvement in LVEF. Risk of cardiotoxicity was significantly higher in patients with known history of DVT/PE. Future studies of possible underlying genetic predisposition will be of great importance.

#988

Effect of a supervised exercise intervention on sarcopenic obesity and metabolic syndrome in prostate cancer patients: A randomized pilot study.

Jacqueline L. Kiwata, Tanya B. Dorff, E. T. Schroeder, Christina M. Dieli-Conwright. _University of Southern California, Los Angeles, CA_.

Purpose: Prostate cancer survivors (PCS) on androgen deprivation therapy (ADT) experience adverse side effects such as skeletal muscle loss and adiposity gain, together called sarcopenic obesity, and changes in cardiometabolic factors that increase risk of metabolic syndrome (MetS). Resistance exercise can increase skeletal muscle mass, but no exercise interventions to date in PCS on ADT have concomitantly improved sarcopenic obesity and cardiometabolic risk factors. Utilizing a 12-week intervention of progressive resistance exercise designed to target skeletal muscle mass, this ongoing pilot trial investigates sarcopenic obesity and as a secondary analyses, MetS components, in PCS on ADT.

Methods: Eighteen PCS (65.6±8.8 yr) on current or previous ADT were recruited from the USC Norris Comprehensive Cancer Center and randomized to resistance training (RT; n=9) or a control stretching program (CS; n=9). Body composition, measured through dual-x-ray absorptiometry, and MetS outcomes, including waist circumference, blood pressure, fasting blood glucose, triglycerides and HDL, were assessed at baseline and after the 12-week intervention. Appendicular skeletal muscle index (ASMI), a common index of sarcopenia, was calculated from body composition. RT performed a supervised total-body resistance exercise and stretching program 3 times/week. CS performed home-based stretching 3 times/week. Baseline differences were tested with univariate ANOVA. Differences in all outcomes were tested with 2(group) x 2(time) ANOVA.

Results: No significant differences in ADT duration, Gleason score, body fat, skeletal muscle mass, or MetS components were found between groups at baseline (P>0.05). RT program compliance was 98.3%, while CS program compliance was 75.5%. Post-intervention, significant increases were observed in RT compared to CS for appendicular skeletal mass (mean±SE; 0.8±.4 kg; P=0.04) and ASMI (0.3±.1 kg/m2; P=0.041). A nonsignificant decrease in body fat (%) was observed in RT compared to CS (1.3±.7 %; P=.067; d=0.89). No differences were found in MetS components.

Conclusions: While 12 weeks of resistance exercise in PCS on ADT improved skeletal muscle mass, no changes in adiposity and MetS variables were observed. Future interventions are needed for PCS to determine the optimal exercise prescription to target both sarcopenic obesity and cardiometabolic risk factors.

#989

Do physician-reported toxicities accurately reflect patient-reported symptom burden? An analysis of ESAS and CTCAE for patients with lung cancer.

Bansi Savla,1 Thomas Dilling,2 Syeda Mahrukh Naqvi,2 Jae K. Lee,2 Hsiang-Hsuan M. Yu2. 1 _University of South Florida, Tampa, FL;_ 2 _Moffitt Cancer Center, Tampa, FL_.

Purpose/Objectives: Symptom adverse events can be quantitatively monitored by oncologists with the U.S. National Cancer Institute's Common Terminology Criteria for Adverse Events (CTCAE). Symptoms can also be reported by patients through the Edmonton Symptom Assessment Scale (ESAS). There is increasing recognition of the importance of assessing patient-reported symptoms as part of clinical care. The aim of this study is to examine correlation between patient-reported and physician-graded symptoms in patients with lung cancer who underwent external beam thoracic radiotherapy, in an attempt to identify gaps between these parameters.

Materials/Methods: Between August 2015 and July 2016, 265 patients with diagnosis of lung cancer had completed ESAS and CTCAE data obtained during weekly clinic visit while undergoing thoracic radiotherapy.The following stratification for symptom severity was used: ESAS (none, 0; mild 1-3, moderate 4-6, severe 7-10) and CTCAE scores (none, 0; mild, 1; moderate, 2; and severe, 3-4). Five associated symptoms were compared: tiredness, nausea, shortness of breath, and other (cough and dysphagia) from ESAS and fatigue, nausea, dyspnea, cough and esophagitis from CTCAE. Frequency tables and boxplots combined with the scatter plots were used to assess the distribution, correlation and to identify possible outliers. Spearman correlation coefficients were analyzed to evaluate rank-associated correlations between associated ESAS domains and CTCAE toxicities.

Results: Statistical analysis showed that the associated ESAS symptoms and CTCAE toxicity pairs (tiredness/fatigue, nausea, shortness of breath/dyspnea, dysphagia/esophagitis, cough) were highly correlated (p<0.05), However, analysis showed that ESAS reported by patients screens for more severe symptoms than the toxicities graded by physicians using the CTCAE; this includes fatigue (16.1% as opposed to 0.5%), nausea (5.0% as opposed to 0.0%), dyspnea (11.8% as opposed to 2.4%), cough (2.1% as opposed to 0.2%), and esophagitis (1.5% as opposed to 0.5%). ESAS collected additional symptom domains including overall wellbeing symptoms, depression, anxiety, and spiritual wellbeing, which are not included on CTCAE. ESAS detected 6.2% of patients reporting severe depression, 8.3% with severe anxiety, 9.7% marking severe for poor overall wellbeing, and 8.3% marking severe for poor spiritual wellbeing.

Conclusion: This studied demonstrated that while ESAS and CTCAE reports are correlated, patients reported more severe symptoms through ESAS compared to physician-graded toxicities from CTCAE in this group of lung cancer patients who underwent thoracic radiotherapy. Systematic acquisition of patient-reported symptoms is important to optimize clinical care and symptom management.

#990

Patterns of racial disparities in health-related quality of life among colorectal cancer patients and relationship with survival.

Michelle A. T. Hildebrandt, Alem A. Belachew, Monica E. Reyes, Yuanqing Ye, Xifeng Wu. _UT MD Anderson Cancer Center, Houston, TX_.

Distinct racial disparities are evident in CRC prognosis with Black patients experiencing worse outcomes than Hispanics and Whites. In a prior study of Health Related Quality of Life (HR-QoL) in a cohort of CRC patients, we observed that racial minority patients experienced lower HR-QoL scores compared to White CRC patients. Therefore, in this study, we focused on the identification of patterns of racial disparities in HR-QoL scores and relationship to differences in prognosis. White (N=450), Hispanic (N=366), and Black (N=316) CRC patients within 1 year of diagnosis at MD Anderson Cancer Center completed the SF-12 quality of life questionnaire to determine Mental Composite Summary (MCS) and Physical Composite Summary (PCS) scores. Participants also completed a questionnaire to collect epidemiology and sociodemographic variables. Vital status and histology information was obtained from the institutional tumor registry. Racial disparities were reported in HR-QoL with both Black and Hispanic patients reporting lower mean PCS and MCS scores compared to White patients, suggesting poorer HR-QoL in these populations. We observed differences in patterns of association between epidemiology and sociodemographic variables and poor HR-QoL by race. Hispanics who never married were at higher risk of poor physical HR-QoL (OR: 2.55(1.15-5.67), P=0.021) compared to married patients, which was not observed for White or Black CRC patients. Similarly, CRC patients with some college education was associated with a decreased risk of poor PCS, but only in Hispanics (OR: 0.26(0.13-0.52), P<0.0001). White females have about two-fold risk of poor PCS (P=2.00 x 10-4) and MCS (P=2.21 x 10-4) scores compared to White males. This relationship was also observed for Black females OR: 2.28(1.35-3.84), but not Hispanic females. Among CRC patients reporting poor PCS (<50), significant differences in median survival times (MSTs) were observed by race. Hispanic patients had the highest MST at 85.4 months followed by Blacks (47.8 months) and Whites (43.2 months). A similar relationship was observed for poor MCS (<50) stratified by race with MST times of 81.9 months for Hispanics, 40.8 months for Blacks, and 54.1 months for Whites. In conclusion, we identified patterns of racial disparities in epidemiology and sociodemographic factors that correspond to poor baseline HR-QoL in CRC patients. We also demonstrated that a prognostic correlation exists between baseline HR-QoL and patient overall survival, and that this relationship is influenced by race. The patterns of racial disparity identified in this study can be an important tool for assessing the underlying mediators of HR-QoL in CRC patients and to further identify those who are particularly at risk for poor prognosis.

#991

Association of high-sensitivity C-reactive protein with health-related quality of life among breast cancer survivors.

Hyun Jeong Cho,1 Zisun Kim,2 Hyun Jo Youn,3 Jihyoung Cho,4 Jung Eun Lee5. 1 _Sookmyung Women's University, Seoul, Republic of Korea;_ 2 _Soonchunhyang University Bucheon Hospital, Bucheon, Republic of Korea;_ 3 _Chonbuk National University Medical School, Jeonju, Republic of Korea;_ 4 _Keimyung University School of Medicine, Daegu, Republic of Korea;_ 5 _Seoul National University, Seoul, Republic of Korea_.

Backgrounds/Aims Decline in health-related quality of life (HRQoL) is associated with worse prognosis among patients with breast cancer. Increased risk of inflammation with cancer-related symptoms such as low physical function, depression and pain could be a potential mechanism. We aimed to examine whether high-sensitivity C-reactive protein (hs-CRP) were related to HRQoL among breast cancer survivors.

Methods This cross-sectional study of Korean breast cancer survivors included a total of 165 women aged 29 to 63 years who had been diagnosed with stage I to III breast cancer and survived six months or more after surgery. Post-diagnostic HRQoL levels were measured using the 36-Item Short Form Health Survey (SF-36) containing eight components: physical functioning, role-physical, body pain, general health, vitality, social functioning, role-emotional, and mental health. The higher scores indicated better HRQoL. Circulating levels of hs-CRP were measured using particle-enhanced immunoturbidimetric assay. We categorized hs-CRP levels to four groups; <0.4, 0.4-<0.6, 0.6-<1.0 mg/L (low risk), and ≥1.0 mg/L (average or high risk) on the basis of the Centers for Disease Control/American Heart Association criteria (low risk; <1.0 mg/L, average risk; 1.0-3.0 mg/L, and high risk; >3.0 mg/L). We calculated the least square means (LS-means) and 95% confidence intervals (CIs) using generalized linear models (GLM) to examine the associations of each component with categories of hs-CRP according to HRQoL levels. We adjusted for age at diagnosis, body mass index at diagnosis, weight change, income level, stage at diagnosis, time since surgery, tumor size and plasma vitamin D levels.

Results We found that increasing levels of physical functioning and role-physical components were associated with decreasing levels of hs-CRP; according to <0.4, 0.4-<0.6, 0.6-<1.0, and ≥1.0 mg/L of hs-CRP, LS-means (95% CIs) were 78.1 (73.2-83.0), 75.2 (69.6-80.8), 76.1 (69.5-82.7), 70.1 (64.5-75.7), respectively (p for trend = 0.05) for physical functioning and 77.5 (70.6-84.4), 81.0(73.2-88.8), 79.4(70.2-88.7), 66.6(58.7-74.4), respectively (p for trend = 0.05) for role-physical. Higher scores of mental components were also associated with lower levels of hs-CRP; LS-means (95% CIs) of <0.4 and ≥1.0 mg/L of hs-CRP levels were 84.6 (77.8-91.4) and 67.1 (59.4-74.9; p for trend = 0.002) for role-emotional, 87.4 (82.3-92.6) and 77.4 (71.5-83.4; p for trend = 0.03) for social functioning, 62.4 (56.4-68.4) and 53.3 (46.5-60.2; p for trend = 0.05) for vitality.

Conclusions High circulating levels of hs-CRP, an inflammation biomarker, were associated with lower levels of HRQoL among Korean breast cancer survivors. Funding information This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2014R1A2A2A01007794)

### Laboratory and Computational Tools to Enhance Clinical Decision Making

#992

Patient-derived tumor organoids of neuroendocrine prostate cancer.

Loredana Puca,1 Rohan Bareja,1 Reid Shaw,2 Wouter Karthaus,3 Dong Gao,3 Chantal Pauli,1 Juan Miguel Mosquera,1 Joanna Cyrta,1 Rachele Rosati,4 Rema Rao,1 Andrea Sboner,1 Carla Grandori,4 Giorgio Inghirami,1 Yu Chen,3 Mark A. Rubin,1 Himisha Beltran1. 1 _Weill Cornell Medicine, New York, NY;_ 2 _Cure First, seattle, WA;_ 3 _Memorial Sloan Kettering, New York, NY;_ 4 _Cure First, Seattle, WA_.

Background: The development of neuroendocrine prostate cancer (NEPC) is one mechanism of treatment resistance to androgen receptor (AR)-targeted therapies for a subset of patients with advanced prostate cancer. This is associated with transition from a prostate adenocarcinoma to small cell/NEPC histology, low AR signaling signaling, and expression of neuroendocrine markers as Chromogranin A (CGHA), Synaphophysin (SYP) and CD56). Patient derived preclinical models recapitulating the NEPC phenotype may be used to address NEPC pathogenesis and test emerging therapeutic targets.

Methods: Tumor organoids were developed according to protocols previously described (Gao et al, Cell 2015). Briefly the tissue biopsies (liver and bone biopsy) were washed, enzymatically digested and then seeded in Matrigel (BD) droplets. Organoids were characterized at genomic (WES), RNA and protein level (IHC) to confirm the expression of specific markers. Lentiviral infections were performed using shRNAs against EZH2 to knock down EZH2 in organoids. Organoids were also subcutaneously injected in NSG mice to generate patient derived xenografts (PDXs) for drug treatment in vivo.

Results: We developed and characterized two NEPC tumor organoids from tumor biopsies (liver and bone) of two patients both in vitro and in vivo (as PDXs). NEPC tumor organoid models retained the molecular and histological characteristic of their matched patient samples. We successfully manipulated the activity of the histone methyltransferase EZH2 by using a catalytic inhibitor and its expression by infecting organoids with shEZH2. We showed that the absence of EZH2 affects the expression of neuroendocrine-associated programs as stem cell and neuronal pathway. Moreover treatment with EZH2 inhibitor decreased tumor organoids viability and PDXs tumor volume. Drug screening approaches on NEPC organoids were used to discovery novel drug targets and combinations that could potentially benefit NEPC patients. Top single agent hits included previously identified targets such as EZH2, AURKA, as well as novel synergies.

Conclusions NEPC patient tumor organoids are clinically relevant tumor models to study the NEPC phenotype in advanced prostate cancer and may be used to elucidate novel drug targets.

#993

Diagnostic leukapheresis (DLA): Molecular characterisation and organoid culture of circulating tumor cells (CTC) from metastatic castration resistant prostate cancer (mCRPC).

Maryou B. Lambros,1 Veronica S. Gil,1 Mateus Crespo,1 Mariane S. Fontes,2 Rui N. Neves,3 Niven Mahra,1 Gemma Fowler,1 Berni Ebbs,1 Penny Flohr,1 George Seed,1 Wei Yuan,1 Joanne Hunt,2 Deirdre Moloney,2 Dionne Ayanda,2 Joost F. Swennenhuis,4 Kiki C. Andree,4 Semini Sumanasuriya,1 Matthew Clarke,1 Pasquale Rescigno,2 Zafeiris Zafeiriou,2 Joaquin Mateo,2 Diletta Bianchini,2 Nikolas H. Stoecklein,3 Leon W. Terstappen,4 Gunther Boysen,1 Johann S. De Bono1. 1 _The Institute of Cancer Research, London, United Kingdom;_ 2 _The Royal Marsden NHS Foundation Trust, London, United Kingdom;_ 3 _University Hospital of the Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany;_ 4 _University of Twente, Enschede, Twente, Netherlands_.

Introduction: CTC count is an independent predictor of overall survival in mCRPC. Isolation of CTC from peripheral blood (PB) for genomic and functional analysis is challenging, especially in patients (pts) with low CTC count. It has been shown that DLA increases CTC yield. However, it has yet to be proven whether CTC isolation from DLA can be used in complementary studies such as molecular characterization and growth of organoid culture for drug sensitivity studies. Here we present preliminary data of an on-going study, which evaluates DLA in mCRPC pts, focusing on safety, CTC enrichment, molecular characterization and feasibility for organoid culture. Methods: mCRPC pts considered for clinical trials were selected according to performance status (ECOG 0-1) and number of CTC found in 7.5ml PB (>20 cells/7.5mL). DLA products (200x106 cells) were processed using the CellSearch CTC kit (Janssen Diagnostics, LLC) according to manufacturer procedures. The contents of CellSearch cartridges were sorted into single cell by fluorescence activated cell sorting (FACS) and subsequently assessed by array comparative genomic hybridization (aCGH) for copy number aberrations (CNA). Enrichment of CTC for organoid culture was performed by density gradient of mononuclear cells followed by positive selection using magnetic beads. Results: Overall 12 mCRPC patients underwent DLA without any complication or toxicity. The mean CTC count was 90 CTC/7.5 ml peripheral blood (median = 31) and ranged from 20 to 324. CellSearch CTC count in the DLA yielded a mean of 466 (median=203) and ranged from 60 to 2496 with an up to 40-fold increase (mean = 13, median = 6) in CTC count separation when comparing 1mL of PB to 1mL of DLA. Molecular analyses of FACS single CTC from the DLA by aCGH showed that these CTC genomic profiles had the typical hallmarks of mCRPC with CNAs including AR and MYC locus (8q) amplification, and PTEN, RB1, TP53, CHD1 loss. Additionally, ex vivo culture of CTC-derived organoids was successfully achieved. aCGH of these organoids matched the genomic profile that of the CTC from the same patient. Conclusion: DLA from mCRPC pts was well tolerated and yields higher CTC capture than PB and may provide an alternative to tissue biopsy and routine blood volumes. Our strategy allowed us to isolate genomic DNA with good quality for molecular characterization and viable CTC for organoid culture and functional studies.

#994

Diagnosing leukemia structural variations via a novel genome mapping approach.

Christopher Pool, Jie Xu, Darrin Bann, David Goldenberg, Arati Sharma, David Claxton, Feng Yue, James Broach. _Penn State Hershey Medical Center, Hershey, PA_.

Identification of structural variants (SVs) is critical to the prognosis of AML. SVs are not readily identified by next generation sequencing methods and are currently diagnosed by cytogenetics, an intrinsically low resolution tool. We have adapted a new method for determining large SVs as a clinical tool for evaluating AML and other cancer genomes. The Bionano Irys images in microfluidic nanochannels large (>150 Kb) segments of genomic DNA that have been bar coded by site specific nicking and local incorporation of fluorescent label. The complement of individual molecules can be assembled through matching bar codes to create a de novo map of the genome from which, by matching to a reference genome, identifies and catalogs structural variants at resolution of ca. 1 Kb.

With this technology, we characterized structural variants signatures in breast cancer, renal clear cell carcinoma, and CML cell lines. Structural variants were then validated via PCR/Sanger sequencing, fluorescent in situ hybridization and whole genome sequencing. We identified a thorough compendium of consistent false positive rearrangements identified by this technology, most of which reflect errors in the reference human genome assembly. We then applied this methodology to ten AML patient samples, seven from archived blood samples and three directly obtained from newly diagnosed patients. Karyotyping on one archived sample identified a t(6:9) translocation in the majority of cells. We identified that rearrangement at a much higher resolution than cytogenetics, which allowed us to map the breakpoint via PCR/Sanger sequencing to nucleotide resolution and confirm a fusion of NUP214 to DEK, a known oncogene. We also discovered the reciprocal t(9:6) translocation, which had not been identified by cytogenetics. In the six other cases from archived samples, we have confirmed the translocation identified by cytogenetic karyotyping and in several cases found additional rearrangements. Moreover, in the first pediatric case, which we completed within five days of obtaining the sample, we have identified four distinct translocations, all of which have been associated previously with AML.

Overall, this is technology has the ability to quickly and accurately identify SVs in cancer cell lines and AML patient samples with higher resolution than cytogenetics, demonstrating its clinical usefulness in the diagnosis and subsequent treatment of leukemia.

#995

Impact of molecular subtypes on predicting chemotherapy response and survival in muscle invasive bladder cancer.

Woonyoung Choi,1 Debasish Sundi,1 Michael Metcalfe,1 i-ling Lee,1 Shanna Pretzsch,1 Jolanta Bondaruk,1 Elizabeth Plimack,2 Arlene Odelia Siefker-Radtke,1 Bogdan CzerniaK,1 Colin Dinney,1 Daivd McConkey3. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Fox Chase Cancer Center, Philadelphia, PA;_ 3 _Johns Hopkins, Baltimore, MD_.

Using whole genome mRNA expression profiling we reported the existence of molecular subtypes (basal, p53-like and luminal) characterized by distinct gene expression patterns and clinical outcomes in muscle invasive bladder cancer (MIBC). To further explore the potential clinical significance of the molecular subtypes, we measured the relative clinical impact of neoadjuvant chemotherapy (NAC) in each of the subtypes. We created a meta-dataset using 148 transurethral resection (TUR) specimens from patients who received NAC followed by radical cystectomy (RC) within the context of 5 separate Phase II clinical trials and another consisting of 127 TURs from patients who were treated with RC without NAC. We used TCGA's complete MIBC dataset as a validation cohort. We assigned the tumors to subtypes using a published one nearest neighbor (oneNN) classifier, and the relationship between subtype assignment and overall survival (OS) was assessed by Kaplan-Meier analysis with log-rank test. In the NAC cohort, chemotherapy was active as measured by an overall pathological downstaging rate (≤pT1 at cystectomy) of 47 % (70/148). Based on pathological downstaging, p53-like tumors were resistant to chemotherapy (response rate; 28%, p=0.009). Patients with basal tumors had the best survival (median OS: 211 months, p= 0.033) compared to p53-like (45 months) and luminal tumors (85 months). In contrast, in the RC without NAC cohort, patients with basal tumors had the shortest survival (17 months, p<0.001) compared to p53-like (110 months) and luminal tumors (108 months). When we compared the impact of NAC on OS within each subtype, patients with basal tumors obtained substantial clinical benefit (p<0.0001), whereas NAC produced no significant differences in OS in patients with p53-like or luminal tumors. These results were confirmed in independent analyses of TCGA's dataset, where patients with basal tumors were the only ones who received significant survival benefit from chemotherapy (median OS with chemotherapy-undefined; without chemotherapy -17 months, p=0.027). Our results demonstrate that the molecular subtypes of MIBC are associated with differential benefit from chemotherapy. If validated prospectively, the data suggest that basal tumors should be identified at diagnosis so that patients with these tumors can be aggressively managed with neoadjuvant chemotherapy. The molecular mechanisms that probably underlie the preferential impact of chemotherapy in basal tumors will be discussed.

#996

**A translational phosphoproteomic approach to study differences in** KRAS **signaling in pancreatic, colorectal and lung cancers.**

Adam Stewart,1 Elizabeth A. Coker,1 Anna Minchom,2 Sebastian Pölsterl,1 Alexandros Georgiou,2 Paul Huang,1 Bissan Al-Lazikani,1 Udai Banerji2. 1 _The Institute of Cancer Research, London, United Kingdom;_ 2 _The Institute of Cancer Research and The Royal Marsden, London, United Kingdom_.

Aims To understand any context-dependent differences in signaling pathways between pancreatic (PAN), colorectal (CR) and lung (LU) cancers with KRAS mutations using a targeted phosphoproteomic approach in cell lines and patient-derived cancer cells exposed to targeted anticancer drugs ex-vivo.

Materials and Methods We studied a panel of 30 KRAS mutant cell lines: 10 PAN, 10 CR and 10 LU cell lines. Cancer cells were also immuno-magnetically isolated from pleural effusions and ascites of patients with KRAS mutant CR and LU cancer and exposed to a DMSO control and clinically relevant concentrations of PI3K (pictilisib), AKT (AZD5363), mTOR (everolimus), EGFR (gefitinib), BRAF (vemurafenib), MEK (trametinib) and HSP90 (luminespib) inhibitors for 1 hr. Dynamic changes in a panel of 52 relevant phosphoproteins were studied using the Luminex 200 platform. Hierarchical clustering and logistic regression were used to find differences in dynamic changes in phosphoproteins between KRAS mutant, PAN, CR and LU cancer cells.

Results Supervised clustering studying exposure to different drugs revealed that when exposed to the PI3K inhibitor, pictilisib, KRAS mutant LU cancers did not significantly cluster together; p=0.008, p=0.104 following Benjamini-Hochberg correction. Independently, logistic regression showed significant differences in signaling of KRAS mutant cells when exposed to the PI3K inhibitor, pictilisib. PAN and CR cancers showed an increase in p-MEK while LU cancer cells did not; p=0.0195. LU cancer cell lines showed significantly more reduction of p-AKT compared to PAN and CR cell lines when exposed to the PI3K inhibitor; p=0.0423. As expected, exposure to vemurafenib increased p-MEK levels across the majority of the KRAS mutant cell lines, however compensatory reductions in p-mTOR levels were seen significantly more in PAN and CR cell lines and not in LU cell lines; p=0.0084. The dynamic phosphoprotein changes caused by pictilisib were validated in cancer cells isolated from serous effusions of 3 KRAS mutant LU and 4 KRAS mutant CR cancer patients. Validation of these findings using multiple other inhibitors and time-points is ongoing.

Interpretation/conclusions We hypothesise that the significantly greater reduction in p-AKT and less increase of compensatory p-MEK caused by PI3K inhibition in KRAS mutant LU cells compared to KRAS mutant PAN and CR cell lines represents preferential signaling of these cells through the PI3K pathway. Increase in p-MEK driven by BRAF inhibitors caused a reduction in p-mTOR in PAN and CR but not in LU cell lines also indicating preferential dependence of signaling in KRAS mutant lung cancer cells through the PI3K pathway. These findings are important while designing clinical trials of KRAS mutant cancers and more broadly to precision medicine where mutation status independent of tissue context is often used.

#997

Impact of a personalized medicine research program (PMRP), using targeted tumor profiling and a cloud based clinical trials matching platform, on clinical decision-making.

Thomas D. Brown,1 Paul D. Tittel,1 Philip J. Gold,1 Charles W. Drescher,1 John M. Pagel,1 J D. Beatty,1 Patra Grevstad,1 Desiree Iriarte,1 Shlece Alexander,1 Madeleine Brindle,1 Xiaoyu Liu,1 Donielle O'connor,1 Mariko Tameishi,1 Danbin Xu,2 Anna B. Berry3. 1 _Swedish Cancer Inst., Seattle, WA;_ 2 _CellNetix Pathology and Laboratories, Seattle, WA;_ 3 _Swedish Cancer Inst., CellNetix Pathology and Laboratories, Seattle, WA_.

Background: Cancer care is evolving to a model of precision medicine where genomic changes in a patient's tumor are used to inform individualized management (mgmt). The optimal approach and impact of tumor profiling on cancer care remain important research questions. We report the impact on clinical decision-making by results from a PMRP in a research practice.

Methods: A custom designed next generation sequencing (NGS) 68 gene alteration (GA) panel, covering clinically relevant genes and regions was developed in 2014. The NGS results were used to: 1) prioritize standard therapies; 2) match patients (pts) with clinical trials (CT); and 3) serve as a data mining resource. NGS testing was offered early in the course of mgmt. An Institutional Review Board approved prospective registration protocol (PMRP) was activated in 2014, with the objective of establishing a centralized longitudinal, molecular phenotypic, and research data repository. Primary endpoints include proportion of pts where NGS impacted mgmt, to include enrollment onto CT. A cloud-based informatics platform was developed to: manage PMRP; facilitate CT matching; perform quality assurance/quality improvement; pursue research initiatives.

Results: As of 11/15/2016, 869 pts gave informed consent, with 844 pts enrolled. The top primary sites included: breast (115); colorectal (111); central nervous system (103); lung (91); ovary (49); hematologic malignancies (46); pancreas (37); uterus (28); esophagus (25); skin (21). Of solid tumor pts with documented clinical stage, 130 (40%) pts had early stage cancer (I, II and III), and 193 (60%) pts had advanced stage (IV) cancer. NGS results: 739 (88%) pts with GA found; 27 (3%) pts without GA. Of pts with GA, 178 (24%) pts had actionable (on-label drugs) GA and 476 (64%) pts had applicable (off-label or CT) GA, for a total of 546 (74%) pts with actionable and/or applicable GA. The top actionable GA were: KRAS (125); PIK3CA (17); BRAF (13); EGFR (12); NRAS (11); AKT1 (3); TET2 (2); ERBB2 (2); HRAS (2). The top applicable GA, included: TP53 (225); TPMT (78); TYMS (78); PIK3CA (77); APC (56); PTEN (52); IDH1 (34); CDNK2A (22); CTNNB1 (18); TET2 (16). Care mgmt impact was reported by physicians for 508 pts with actionable/applicable GA. Physicians reported mgmt impact, at time of reporting, for 105 (21%) pts, to include: new treatment (Tx) in 30 (6%) pts; no Tx given in 18 (4%) pts; Tx changed in 12 (2%) pts; Tx stopped in 1 (<1%) pt; 6 (1%) pts enrolled onto CT. In 403 (79%) pts, physicians reported no mgmt impact, to include: insufficient evidence in 294 (58%) pts; drugs/CT access in 91 (18%) pts; refused Tx in 17 (3%) pts.

Conclusions: NGS profiling of tumors with this 68 GA panel has an impact on clinical decision- making in a minority, though substantial number, of pts. Impact on CT participation remains modest. Access to drugs and CT remains an important barrier.

#998

OPeN: the oncology precision network data sharing consortium.

Lincoln Nadauld,1 Derrick Haslem,1 Paul D. Tittel,2 Mariko Tameishi,2 Thomas Brown,2 James Ford3. 1 _Intermountain Healthcare, St. George, UT;_ 2 _Providence Health and Services, Seattle, WA;_ 3 _Stanford, Palo Alto, CA_.

Background: The advent of Next-Generation Sequencing (NGS), and other molecular diagnostic technologies, has enabled the use of genomic information to guide targeted treatment in cancer patients. While this precision oncology approach can yield exciting clinical outcomes, the innumerable genomic variants identified in individual tumors effectively establishes each case as a unique N=1 clinical presentation. This scenario is contrary to a basic dogma of medical practice where historical cases and treatment outcomes guide future management and therapeutic decisions. Aggregation of large data sets, on a multi-institutional basis, has the potential to overcome the N=1 paradox and yield management insights in the implementation of precision oncology.

Methods: We have formed the Oncology Precision Network (OPeN), an oncology data sharing consortium, to aggregate big data sets consisting of clinical, genomic, pharmacological, and treatment response data from diverse patient cases. Data from Intermountain Healthcare, Stanford University, and Swedish Cancer Institute-Providence St. Joseph Health, as well as other institutions, comprises the database and is derived from 79 hospitals, over 800 physician clinics and more than 50,000 annual cases.

Results: The OPeN database can be interrogated by variant type, specific therapeutics, clinical outcomes, and by grouped variables, in a structured data format. The overarching IT platform is a cloud based, open source, triple store precision oncology solution, Syapse. These data are yielding valuable insights, including tumor mutational burden (TMB) scores and their correlation to immunotherapy response, clinical response in various drug-gene combinations, and therapy-specific adverse events.

Conclusions: We anticipate this resource will be used by the Molecular Tumor Boards of contributing institutions for clinical interpretation, and by treating providers to overcome the N=1 challenge associated with precision oncology.

## ENDOCRINOLOGY:

### Estrogen Receptor in Cancer Progression and Therapies

#999

ESR1 mutations activate and confer hormone resistance via distinct mechanisms.

Weiyi Toy,1 Kathryn E. Carlson,2 Teresa A. Martin,2 Christopher G. Mayne,2 Sean W. Fanning,3 Pedram Razavi,1 José Baselga,1 Yang Shen,4 Geoffrey Greene,3 Benita Katzenellenbogen,2 John Katzenellenbogen,2 Sarat Chandarlapaty1. 1 _Mem. Sloan Kettering Cancer Ctr., New York, NY;_ 2 _University of Illinois at Urbana-Champaign, Urbana, IL;_ 3 _University of Chicago, Chicago, IL;_ 4 _Texas A & M University, College Station, TX_.

Mutations in the ligand-binding domain (LBD) of ESR1 have been identified in the tumors and plasma of hormone-resistant, metastatic ER+ breast cancer patients. The most prevalent alterations are Y537S and D538G, and both mechanistic and clinical investigations have focused on the implications of these 2 mutations. Through large-scale clinical sequencing efforts, we have examined the coding region of ESR1 from over 1000 cases of metastatic breast cancer and found a diversity of ESR1 mutations. These have included a number of never before reported alterations including mutations in the DNA binding domain and dimerization interface. The biochemical and biologic impact of many of these mutations is unknown. In this study, we have examined the impact of these alterations on ER conformation, transcriptional activity, breast cancer growth, and drug sensitivity. Using transcriptional assays, we found several classes of mutation including: (1) mutations that weakly promoted ligand-independent activity, (2) mutations that led to ligand-independent activity comparable to estradiol stimulation, and (3) mutations that impaired transcriptional activity. Among mutations that promoted some level of ligand-independent activation, we examined whether they had similar effects on receptor conformation in vitro. First, we characterized a subset using a FRET-based co-activator recruitment assay and found that, unlike Y537S and D538G, several mutants (e.g. E380Q and S463P) were unable to recruit SRC in the absence of estradiol. In keeping with this observation, trypsin digestion assays also revealed differences in the local structure within helix 11 between these mutants. To further characterize the molecular basis for these differences, we conducted molecular dynamic (MD) modeling of mutants and compared these with the MD and crystal structure models of apo Y537S and D538G mutants. These studies further revealed differences in overall receptor conformation including localization of H12. From a therapeutic point of view, we examined the effect of different mutants on sensitivity to various ER antagonists. Once again, we observed important differences between mutants, with several mutants of class 2 exhibiting reduced sensitivity to SERDs compared to mutants of class 1. Nevertheless, it appeared that all mutants could be effectively antagonized by more potent SERDs, implying a continued ability of the mutant ERs to become distorted into the antagonist conformation. Taken together, the data reveal distinct conformational restrictions on ER activity that can be relieved by different ESR1 mutations. These data also imply the value of more broad coverage of ER in clinical sequencing efforts in order to effectively capture all potential resistance alleles.

#1000

Estrogen receptor coactivator MED1 in breast tumorigenesis and therapeutic resistance.

Xiaoting Zhang. _University of Cincinnati, Cincinnati, OH_.

Recent studies have established Mediator Subunit 1 (MED1) as a key ER transcriptional coactivator for in both normal mammary gland development and breast cancer. Significantly, the MED1 gene is located at the chromosome 17q12 region, also known as the HER2 amplicon, and co-amplifies with HER2 in almost all instances. Importantly, we found that MED1 serves as a key crosstalk point for the HER2 and ER pathways in anti-estrogen resistance of breast cancer. Significantly, MED1 expression highly correlates with poor disease-free survival of breast cancer patients; and most recent studies have discovered increased frequency of MED1 mutations in circulating tumor cells of human patients following anti-estrogen and anti-HER2 treatments. To determine the role of MED1 in HER2-driven tumorigenesis, we have crossed the MMTV-HER2 mammary tumor model with our established MED1 mutant knockin and newly generated MED1 mammary specific overexpression mouse models. Collectively, our studies revealed critical roles for MED1 in tumor progression, metastasis, cancer stem cell formation and therapeutic resistance in HER2-mediated mammary tumorigenesis. These studies not only for the first time reported key roles for a HER2 amplicon co-amplified gene in HER2-driven tumorigenesis but also support MED1 as a potential therapeutic target. To test that, we have assembled highly innovative RNA nanotechnology-based pRNA-HER2apt-siMED1 nanoparticles. We found these RNA nanoparticles have a very high Tm value, and is ultra-stable under RNase A, 8 M urea, serum and PBS conditions. Importantly, pRNA-HER2apt-siMED1 nanoparticles could specifically target HER2+ human breast cancer, efficiently deplete the expression of MED1 and decrease ER-mediated gene transcription both in vitro and in vivo. Most significantly, pRNA-HER2apt-siMED1 nanoparticles not only greatly reduce the growth, metastasis and cancer stem cell formation of HER2+ breast cancer, but are also able to overcome their therapeutic resistance. (This work is supported by Susan G. Komen for the Cure Foundation Career Catalyst Grant, American Cancer Society Research Scholar Grant and NIH/NCI Grant Number R01CA197865).

#1001

ESR1 mutations confer novel metastatic functions in genome-edited breast cancer models.

Zheqi Li,1 Amir Bahreini,1 Peilu Wang,2 Kevin Levine,1 Nilgun Tasdemir,1 David Chu,3 Ben Ho Park,3 Adrian Lee,1 Steffi Oesterreich1. 1 _University of Pittsburgh, Pittsburgh, PA;_ 2 _Tsinghua University, Beijing, China;_ 3 _Johns Hopkins University, Baltimore, MD_.

Background: Estrogen receptor alpha (ERα), encoded by the ESR1 gene, is expressed in approximately 70% of breast cancers. Recent studies conducted by us and others have shown that somatic mutations in ESR1 gene play a key role in conferring endocrine resistance in ER+ breast cancer. These hotspot mutations mainly occur on the ligand binding domain of ERα, leading to poor outcomes in 25-30% of patients with ER+ metastatic breast cancer in clinic. The mechanisms behind the potential enhanced metastasis of these mutations have become an urgent issue to be addressed, but they are not well understood due to a lack of ESR1 mutant models.

Methods: We generated and characterized genome-edited T47D and MCF7 breast cancer cell lines with the two most common ESR1 mutations (Y537S and D538G), using CRIPSR/Cas9 and rAAV systems respectively. Multiple clones for each mutant were sorted and the mutation frequencies were detected using digital droplet PCR (ddPCR). We subsequently performed an RNA-sequencing to deeply differentiate the gene expression patterns in these mutants. The growth of these pooled mutant-cells was determined in both 2D and 3D plates. The cell-matrix adhesions were measured based on ECM array, and 84-ECM adhesion related genes were further tested by qPCR array. IncuCyte real-time image system was used to monitor the cell migration based on the wound-scratching assay.

Results: We first identified the robust mutation frequencies at both RNA and DNA levels in our cell models. The RNA-seq exhibits multiple ligand-independent genes overlapping between either cell lines or mutants, which were further conformed by qPCR. We also found that both Y537S and D538G mutants present ligand-independent growth in 2D and 3D ultra-low attachment plates. Using wound-scratching assay, we observed significant higher migration rate in D538G mutant of T47D cell lines on both matrigel and type I collagen, indicating a cell-line and mutant-specific phenotype. We also detected lower attachment of both mutants on type I collagen in both cell lines, and our qPCR array revealed that alterations in the MMP pathways could be one of the major mechanism causing this phenotype.

Conclusion: In sum, our study presents the first in-depth metastatic functional analysis of the biology of ESR1 mutations in genomic knock-in cell models, pointing out the enhanced migration and decreased cell-matrix adhesion as a potential novel gain-of-function of the Y537S and D538G mutant-cells. These findings suggest the potential role of enhanced metastasis of these ESR1 mutations through remodeling of transcriptional profiles, shedding lights towards the development of efficient therapies of ESR1 mutant breast cancer.

#1002

An epigenetic strategy to degrade the estrogen receptor in breast cancer.

Huili Li,1 Foteinos-Ioannis Dimitrakopoulos,2 Meredith Stone,1 Lauren Murphy,1 Stephen Baylin,1 Cynthia A. Zahnow1. 1 _Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD;_ 2 _University of Patras, Medical School, Rion, Greece_.

Epigenetic changes in DNA methylation can lead to altered gene expression and the development and progression of breast cancer. Luminal breast tumors are associated with increased DNA hypermethylation of CpG islands while basal, triple negative tumors more commonly have lower levels of DNA methylation. Increasing evidence suggests that aberrant epigenetic alterations in DNA methylation and chromatin structure may also contribute to endocrine resistance in luminal breast cancers. We thus asked whether epigenetic therapy using the demethylating agent, 5-Azacytidine (AZA) is an effective anti-tumorigenic agent in luminal breast cancers, what epigenetic mechanisms might be involved in the response and whether AZA could play a role in the sensitization of tamoxifen resistant cells.

We have demonstrated using 26 breast cancer cell lines that luminal B, ER+ breast cancer is more responsive to treatment with AZA than are Her2+ or triple negative (TN) breast cancers. Cells were treated in culture with low nanomolar doses of AZA for 72 hrs, followed by transplantation into mice and weekly palpation of tumors. Tumorgraft measurements show that 67% of luminal B, ER+ cells are sensitive to AZA, whereas only 22% of TN and 25% of Her2+ cells exhibit an anti-tumorigenic response to AZA in mice. Expression data showed that AZA leads to decreases in ER mRNA and protein expression in 50% and 67% of luminal B lines respectively. Further investigation showed that a longer AZA treatment led to lower ERα protein levels and stronger tumor growth inhibition. Protease inhibitor (MG132) treatment rescued ERα protein reduction in 50% of the luminal B cell lines, suggesting that an AZA-mediated, ubiquitin-26S proteasomal pathway may be responsible for the degradation of ER. Gene expression analysis demonstrated that the E1 ubiquitin-activating enzyme, (UBA7-UBE1L) was up-regulated at the mRNA and protein levels by AZA. UBA7 has been suggested to function as a tumor suppressor in lung cancer and elevations in UBA7 have been correlated with longer overall survival in breast cancer. The interaction of UBA7 with ERα protein was validated by immunoprecipitation and over-expression of UBA7 in ZR-75-1 cells led to a reduction in ERα protein. We further analyzed the UBA7 promoter region by Illumina 450K array and bisulfite sequencing in our cell lines and identified two CpG rich sequences in the promoter that may be important for AZA induced UBA7 promoter demethylation and gene re-expression. We also demonstrated in both tamoxifen sensitive and resistant cells that combination therapy with both AZA and tamoxifen was more effective at inhibiting tumor growth than the single agents. Studies are ongoing in our lab to better understand this response. Our data suggest that AZA may be clinically useful to inhibit the growth of some luminal breast tumors via its actions to increase expression of UBA7 and to degrade ER and may have efficacy in combination with anti-estrogen treatments.

#1003

Extranuclear ERα-mTOR signaling rewires cancer cell metabolism during obesity-associated breast cancer.

Zeynep Madak Erdogan, Yiru C. Zhao, Gianluigi Rossi, Kinga Wrobel, Eylem Kulkoyluoglu, Sung Hoon Kim, John A. Katzenellenbogen, Jodi Flaws, Rebecca Smith. _Univ. of Illinois at Urbana-Champaign, Urbana, IL_.

Obesity is a preventable risk factor for post-menopausal ERα-(+) breast cancer. We hypothesized that serum from obese post-menopausal women contain factors that would increase tumorigenicity of breast cancer cells and increase risk of ERα-(+) breast cancer. Using whole metabolite profiling and OLINK biomarker panel of about 400 proteins associated with cancer, inflammation and cardiovascular disease, we identified biomarkers that were differentially present in serum from 50 obese v.s. 50 non-obese postmenopausal women. Next, using in vitro cell based assays as proxy we identified certain free fatty acids (FFAs) as factors from serum that correlate with increased cell proliferation, motility and mTOR activation in ERα(+) breast cancer cells. We performed RNA-Seq, ERα ChIP-Seq and metabolomics analysis in breast cancer cells that are exposed to conditions that mimic serum from obese postmenopausal women. This integrative -omics approach enabled us to uncover ERα and mTOR pathway-dependent metabolic rewiring in breast cancer cells under these conditions. Pathway preferential estrogens (PaPEs), which target ERα and mTOR signaling, were able to block free fatty acid-dependent proliferation of breast cancer cells. In fact, efficient cancer cell killing by PaPEs was achieved only in the presence of FFAs, suggesting a role for obesity-associated metabolic rewiring in providing new vulnerabilities for the breast cancer cells. In summary, we uncovered a novel role for extranuclear-initiated ERα signaling in rewiring breast cancer cell metabolism in response to obesity-associated factors in the serum. Our findings provide a basis for preventing or inhibiting obesity-associated breast cancer by using PaPEs that would exploit new metabolic vulnerabilities of breast tumors in obese postmenopausal women.

#1004

LDLR knockdown reduces the growth of Her2 overexpressing breast cancer in mouse models of hyperlipidemia.

Emily J. Gallagher, Brian A. Neel, Zara Zelenko, Irini Markella Antoniou, Nathan Kase, Derek LeRoith. _Icahn School of Medicine at Mount Sinai, New York, NY_.

Women with higher circulating low density lipoprotein (LDL) cholesterol levels are more likely to have advanced HER2 positive breast cancers. The LDL receptor (LDLR) is the main receptor for cholesterol uptake into cells from circulating LDL and its expression is higher in HER2 positive breast cancers than other subtypes. The aim of our study was to understand the importance of the LDLR in the progression of HER2 positive breast cancer growth in the setting of hyperlipidemia. To study the role of hyperlipidemia in HER2 cancer progression, we used two mouse models with elevated LDL cholesterol: Apolipoprotein E knockout (ApoE-/-), and LDLR knockout (LDLR-/-) mice on an FVB/n background. ApoE-/-, LDLR-/- and control (WT) mice were placed on a western diet at 8-10 weeks of age. After 2 weeks on the diet, serum cholesterol concentrations were measured and demonstrated significantly higher cholesterol levels in the ApoE-/- and LDLR-/- mice compared with WT mice (WT: 208±19mg/dL; LDLR-/-: 488±37mg/dL; ApoE-/- 660±39mg/dL). We used the MCNeuA (MMTV-Neu derived) breast cancer cells to study the effect of hyperlipidemia on Her2/Neu positive breast cancer in this model, as the MCNeuA cells were found to have high levels of LDLR protein expression compared with other murine breast cancer cell lines. To study the importance of the LDLR in mediating the effects of hyperlipidemia, we knocked down the LDLR in the MCNeuA cells using shRNA, and selected two cell clones with successful gene knockdown confirmed by RNA and protein analysis. ApoE-/-, LDLR-/- and WT mice on the western diet were injected into the 4th mammary fat pad with 2 x 106 MCNeuA (MMTV-Neu derived) control shRNA or MCNeuA LDLR knockdown clone 1 or clone 2 cells (n=6-10 mice per group). Tumor growth was measured for 30 days. Control shRNA tumors in both hyperlipidemic mouse genotypes grew more rapidly than in the WT mice. 30 days after injection the control tumor volumes were: WT 514±66.5mm3; LDLR-/- 767±78.2mm3; ApoE-/- 1189±94mm3. The LDLR knockdown clones 1 and 2 had a reduction of tumor volume of 11% and 30% in the WT mice, 28% and 62% in LDLR-/- mice, and 26% and 53% in the ApoE-/- mice, respectively compared with the control shRNA cells. Western blot analysis of tumor protein lysates showed an increase in p19 cleaved caspase 3 in the tumors with LDLR knockdown, compared with the control cells. In vitro studies demonstrated a decrease in survival of MCNeuA cells with LDLR knockdown in serum starved conditions, and an increase in caspase 3 cleavage. These data demonstrate that hyperlipidemia promotes the growth of Her2/Neu overexpressing breast cancer cells and the LDLR plays an important role in protecting Her2 / Neu tumors from apoptosis. Therefore, targeting cholesterol uptake and metabolism may be an important strategy for women with HER2 positive breast cancers.

#1005

Interaction between the estrogen receptor and fibroblast growth factor receptor pathways in non-small cell lung cancer.

Laura P. Stabile,1 Natalie J. Rothenberger,1 Mariya Farooqui,2 Sanja Dacic,1 Jill M. Siegfried2. 1 _Univ. of Pittsburgh School of Medicine, Pittsburgh, PA;_ 2 _University of Minnesota, Minneapolis, MN_.

The estrogen receptor (ER) promotes cell proliferation in non-small cell lung cancer (NSCLC). Since fibroblast growth factors (FGFs) are known regulators of stem cell markers in ER positive breast cancer, we investigated whether a link between the estrogen pathway, FGFs, and stem cell markers could be demonstrated in NSCLC animal models, human cells lines and tumor tissue. In the lungs of female FVB/N mice exposed to the tobacco carcinogen 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone, NNK, treatment with the anti-estrogen fulvestrant and/or the aromatase inhibitor anastrozole blocked secretion of both FGF2 and FGF9, and reduced expression of the stem cell markers SOX2 and nanog. These effects were observed in the normal airway epilthelium as well as in both lung preneoplasias and lung adenomas. To investigate the effects of β-estradiol (E2), male mice were administered E2 in the drinking water during exposure to NNK for 4 weeks. In this mouse model, the incidence of carcinogen-induced lung preneoplasias increased 1.8-fold (p<0.05) with E2 exposure accompanied by increased expression of FGF2, FGF9, SOX2, and nanog in airway preneoplasias demonstrating a relationship between activation of the FGF pathway in the lungs, expansion of cells with a stem cell phenotype, and promotion of pre-cancerous changes. In FGFR1 normal copy number NSCLC cell lines that express multiple FGFR family members and secrete several FGFs, E2 treatment caused a significant increase (up to 4-fold; p<0.05) in release of FGF2, an effect that was completely blocked by fulvestrant. Treatment with fulvestrant also resulted in a 30% reduction in phosphorylation of fibroblast growth factor receptor substrate 2, the FGFR docking protein. Upon co-inhibition of ER and FGFRs using fulvestrant and the pan-FGFR inhibitor AZD4547, significantly enhanced (p<0.01) anti-proliferative effects were observed in FGFR1 normal copy number NSCLC cells that show low sensitivity to FGFR inhibitors as single agents. In NSCLC xenografts, the combination of AZD4547 (daily 12.5 mg/kg) and fulvestrant (30 mg/kg twice weekly) resulted in a significantly greater inhibition (67-85% decrease; p<0.05) of tumor growth and decreased expression of Ki67 and stem cell markers compared to each single agent treatment. Furthermore, tumor histology appeared more differentiated and up to a 25% decrease in malignant cellularity was observed with combination treatment compared to each single agent treatment. In NSCLC patient tumors, high ERΒ expression correlated with high FGFR1 expression (p<0.001). Taken together, these results suggest that interaction between the ER and FGFR pathways in NSCLC promotes a stem-like state. Combining an FGFR inhibitor with an ER pathway inhibitor could be exploited to increase the efficacy of FGFR inhibitors for NSCLC patients who lack FGFR genetic alterations. Supported by P50 CA090440 and the V-Foundation.

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

### Novel Mechanisms of Drug Resistance

#1006

Drug-tolerant persister cancer cells are vulnerable to GPX4 inhibition.

Matthew J. Hangauer,1 Vasanthi S. Viswanathan,2 Matthew J. Ryan,2 Dhruv Bole,1 Jake Eaton,2 Stuart L. Schreiber,2 Frank McCormick,1 McManus T. Michael1. 1 _University of California, San Francisco, San Francisco, CA;_ 2 _Broad Institute/Harvard University, Cambridge, MA_.

Acquired drug resistance prevents chemotherapy and targeted therapy from achieving stable and complete responses. Emerging evidence implicates a key role for nonmutational mechanisms during early stages of acquired drug resistance. Drug-tolerant "persister" cells are a model of nonmutational cancer drug resistance in which small fractions (<5%) of cells within cancer cell lines survive cytotoxic drug exposure despite lacking resistance-conferring mutations. These residual surviving cells are quiescent, occupy a unique chromatin state, and exhibit reversible drug resistance: removal of drug allows regrowth of cells which become resensitized to drug treatment. Importantly, this phenomenon has been observed across a wide range of cancer lineages with both chemotherapy and targeted therapy. Persister cells remain quiescent for weeks to months during constant drug exposure, however some persister cells eventually regrow and acquire irreversible drug resistance-conferring mutations that did not preexist. This model mirrors the common clinical scenario in which tumor response is followed by relapse with acquired resistance-conferring mutations. Targeting nonmutational resistance may therefore present a therapeutic opportunity to eliminate residual surviving tumor cells to prevent relapse. We have performed an unbiased functional genomics analysis of HER2+ breast cancer persister cells which survive HER2 targeted therapy. Utilizing RNAseq, pathway analysis, RNAi screens and chemical inhibitor screens, we discovered that persister cells undergo a switch to mesenchymal gene expression and are uniquely susceptible to inhibition of the phospholipid hydroperoxidase GPX4. This finding is not restricted to breast cancer, rather we have observed that all persister cells tested including those derived from melanoma, lung, ovarian and pancreatic cancer, from either targeted therapies or chemotherapies, are each specifically sensitive to loss of GPX4 function. Parental, drug naive cancer cells and normal human cells are insensitive to GPX4 inhibition. Mechanistically, we found that GPX4 dependency in persister cells is due to a disabled antioxidant program characterized by global downregulation of antioxidant genes, decreased levels of reducing cofactors glutathione and NADPH, and immediate ROS increase upon GPX4 inhibition. These findings point toward a GPX4 inhibition strategy to prevent tumor relapse by inducing ferroptosis in the residual pool of surviving persister cells.

#1007

SHP2 inhibition restores sensitivity to ALK inhibition in resistant ALK-rearranged non-small cell lung cancer (NSCLC).

Leila Dardaei,1 Hui Qin Wang,2 Paul Fordjour,2 Manrose Singh,1 Grainne Kerr,2 Satoshi Yoda,1 Jinsheng Liang,2 Yichen Cao,2 Yan Chen,2 Justin F. Gainor,1 Luc Friboulet,3 Ibiayi Dagogo-Jack,1 David T. Myers,1 Emma Labrot,2 David Ruddy,2 Melissa Parks,1 Dana Lee,1 Richard H. DiCecca,1 Susan Moody,2 Huaixiang Hao,2 Morvarid Mohseni,2 Matthew LaMarche,2 Juliet Williams,2 Keith Hoffmaster,2 Giordano Caponigro,2 Cyril H. Benes,1 Alice T. Shaw,1 Aaron N. Hata,1 Fang Li,2 Jeffrey A. Engelman2. 1 _Massachusetts General Hospital Cancer Center, Charlestown, MA;_ 2 _Novartis Institutes for BioMedical Research, Cambridge, MA;_ 3 _Gustave Roussy Cancer Campus, Paris, France_.

Despite development of highly potent and selective inhibitors (e.g., ceritinib, alectinib, lorlatinib) targeting anaplastic lymphoma kinase (ALK), resistance invariably develops and limits the efficacy of these inhibitors in the clinic. The major classes of resistance are on-target genetic alterations (e.g., secondary ALK kinase domain mutations) and activation of alternative or bypass signaling pathways. While most patients are responsive to sequential treatment with two or more ALK inhibitors, ALK-independent resistance eventually emerges and leads to failure of further ALK-directed monotherapy. We used a synthetic lethal pooled shRNA screen to discover loss-of-function events that could sensitize resistant patient-derived cell lines to ALK inhibition. In addition to identifying known bypass targets such as FGFR, EGFR and SRC, we also identified PTPN11 (which encodes SHP2, a non-receptor protein tyrosine phosphatase that modulates signaling downstream of growth factor receptors) as a common hit shared by cell lines exhibiting different mechanisms of bypass activation. In parallel with the shRNA screen, we also performed a high throughput combination compound screen in the same patient-derived models, and identified activation of the same bypass signaling pathways. We showed that the highly potent and selective small-molecule SHP2 inhibitor SHP099 could sensitize resistant cell lines to ALK inhibition. In biochemical studies, co-targeting of ALK and SHP2 overcame resistance mediated by ALK-independent bypass mechanisms by decreasing RAS-GTP loading potential of cells and inhibiting phospho-ERK rebound. These results suggest that dual ALK and SHP2 inhibition may represent a new therapeutic strategy for ALK-positive patients, whose lung cancers have evolved ALK-independent mechanisms of resistance, including activation of bypass signaling pathways.

#1008

Gain-of-function kinase library screen identifies FGFR1 amplification as a mechanism of resistance to antiestrogens and CDK4/6 inhibitors in ER+ breast cancer.

Luigi Formisano,1 Yao Lu,1 Valerie M. Jansen,1 Joshua A. Bauer,1 Ariella B. Hanker,1 Melinda E. Sanders,1 Paula González-Ericsson,1 Sunkyu Kim,2 Monica Arnedos,3 Fabrice André,3 Carlos L. Arteaga1. 1 _Vanderbilt University, Nashville, TN;_ 2 _Novartis Pharmaceuticals Corporation, NJ;_ 3 _Institute Gustave Roussy, France_.

The CDK4/6 inhibitor palbociclib was recently approved in combination with endocrine therapy for treatment of ER+ metastatic breast cancer. The goal of this study was to discover mechanisms of resistance to ER antagonists alone and in combination with CDK4/6 inhibitors. To achieve this goal, we used lentiviral vectors to individually express 559 human kinase open reading frames (ORFs) in ER+ MCF7 human breast cancer cells treated with fulvestrant ± the CDK4/6 inhibitor ribociclib (Novartis). In MCF7 cells treated with fulvestrant alone or with ribociclib, we identified 21 and 17 kinases, respectively, which induced a >30% increase in cell viability compared to control cells; 11 of these kinases overlapped in both treatment groups. In a secondary screen, MCF7 cells were stably transduced with V5-tagged lentiviruses expressing the positive 'hits' for treatment with fulvestrant/ribociclib. Five of 11 kinases (FGFR1, FRK, HCK, FGR, CRKL) were confirmed to induce resistance to fulvestrant/palbociclib and fulvestrant/ribociblib. Survey of TCGA for copy number alterations and/or expression of these five genes showed only FGFR1 to be amplified/overexpressed in 17% of ER+ breast cancers. Experiments in vitro showed that ER+/FGFR1-amplified (amp) MDA-134, CAMA-1 and HCC1500 human breast cancer cells and MCF7 cells stably transduced with FGFR1 were relatively resistant to estrogen deprivation, fulvestrant and fulvestrant/palbociclib compared to non-FGFR1 amp MCF7 cells. This resistance was abrogated by treatment with the FGFR tyrosine kinase inhibitor (TKI) lucitanib. Treatment with fulvestrant or palbociclib, each alone, modestly delayed growth of ER+/FGFR1-amp breast cancer patient-derived xenografts (PDX) established in nude mice. However, addition of the FGFR TKI erdafitinib to fulvestrant/palbociclib resulted in marked PDX regressions in all mice without associated toxicity. Treatment of FGFR-amp cells with FGF-2 strongly induced CCND1 (cyclin D1) expression. Downregulation of CCND1 with CCND1 RNAi oligonucleotides or kinase inhibition with erdafitinib restored sensitivity of FGFR1-amp cells to fulvestrant/palbociclib. Conversely, overexpression of CCND1 in MCF7 cells induced resistance to estrogen deprivation and fulvestrant ± palbociclib. At this time, we are examining whether FGFR1 amplification measured by FISH correlates with maintenance of proliferation (Ki67) in 110 patients with ER+/HER2- breast cancer treated with palbociclib for 14 days immediately before surgery (Arnedos et al. ASCO 2016). In summary, using a gain-of-function ORF kinome screen, we identified FGFR1 amplification as a mechanism of resistance to anti-ER therapies ± CDK4/6 inhibitors. Based on these data we propose FGFR inhibitors should be tested in combination with ER antagonists and CDK4/6 inhibitors in patients with ER+/FGFR amplified breast cancer.

#1009

**Comprehensive ctDNA sequencing reveals mechanisms of resistance to rociletinib in EGFR** **T790M-mutated NSCLC.**

Elena Helman,1 Andrew D. Simmons,2 Chris A. Karlovich,2 Thomas C. Harding,2 Mitch Raponi,2 Darya I. Chudova,1 Daniel A. Simon,1 Richard B. Lanman,1 AmirAli Talasaz1. 1 _Guardant Health, Redwood City, CA;_ 2 _Clovis Oncology, San Francisco, CA_.

Background: First and second-generation EGFR tyrosine kinase inhibitors (TKIs) have benefited patients with EGFR-mutated non-small cell lung cancer (NSCLC), but resistance invariably develops after a median of 9-16 months. In ~60% of patients, resistance is mediated by a second mutation in EGFR, namely T790M. Hence, third-generation EGFR TKIs such as osimertinib and rociletinib were developed to target both activating EGFR mutations as well as T790M. Unfortunately, patients also develop resistance to these therapies through mechanisms that have not yet been thoroughly explored. Since repeat tissue biopsies pose potential complications from invasive procedures, circulating tumor DNA (ctDNA) testing is increasingly used in the clinical setting to identify potentially targetable mechanisms of resistance.

Methods: Matched pre-treatment and progression plasma from 57 patients with EGFR-mutated NSCLC treated with rociletinib were profiled using a 70-gene ctDNA targeted next-generation sequencing panel (Guardant360) that detects somatic single nucleotide variants, short insertions and deletions, fusions, and copy number variants. Pre-treatment EGFR ctDNA allele fractions were also determined by BEAMing, a technique based on droplet digital PCR followed by flow cytometry. Pre-treatment tumor EGFR status was assessed by the therascreen EGFR test.

Results: In all 57 pre-treatment samples profiled, plasma-based ctDNA analysis detected the initial EGFR driver and T790M resistance mutations that were identified in the matched tumor. Interestingly, we found that 12% (7/57) of patients had evidence of compound EGFR driver mutations at baseline, including E709A-L858R, K860I-L858R, and L718V-L858R. EGFR T790M mutations in plasma were observed subclonally (present on average at 40% of the allele fraction of the driver mutation), suggesting tumor heterogeneity at baseline. The correlation coefficients (r) between Guardant360 and BEAMing for EGFR L858R, Exon19Del, and T790M were 0.90, 0.92, 0.95, respectively. Upon progression on rociletinib, 5% of patients (3/57) developed the EGFR C797S resistance mutation, 5% (3/57) developed focal MET amplification, and 2% (1/57) developed a NTRK1 fusion that were not present in the matched baseline plasma. Additionally, 4 deleterious BRCA1/2 alterations (2 germline and 2 somatic) were identified, with the somatic alterations emerging at progression. In 14% (8/57) of the patients, mutations in genes involved in the RAS/RAF signaling pathway, including KRAS Q61H, KRAS K117N and NF1 Q1822*, emerged or increased at progression.

Conclusions: Plasma ctDNA revealed heterogeneity and multiple mechanisms of resistance in rociletinib treated patients. Thus comprehensive ctDNA sequencing allows for the identification of potentially actionable alterations and may help inform the choice of next therapy for patients progressing on a third-generation EGFR TKI.

#1010

Genomic analysis of recurrent ovarian cancer in patient-derived xenografts treated with platinum and taxane chemotherapy.

Elizabeth Stover,1 Ali Amin-Mansour,2 Sangeetha Palakurthi,1 Sanam Dharma,1 Qing Zeng,1 Shan Zhou,1 Palak Desai,1 Levi Garraway,1 Ursula Matulonis,1 Joyce Liu1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Broad Institute, Cambridge, MA_.

Introduction: Many women with advanced ovarian cancer (OC) are initially sensitive to platinum and taxane chemotherapy, but subsequently develop recurrent disease which is often incurable. OC chemotherapy resistance mechanisms are incompletely understood. We used patient-derived xenografts (PDX) to model OC recurrence following chemotherapy and to analyze genomic evolution of recurrent tumors.

Approach: We leveraged PDX derived from human OC ascites that accurately represent high-grade serous OC. We used two PDX models: PDX-A derived from a chemotherapy-naive patient with WT BRCA1/2; PDX-B derived from a chemotherapy-resistant patient with a BRCA1 mutation. Using luciferase-labeled tumor cells, we generated intraperitoneal disease in multiple recipients. We treated cohorts of 5-10 animals with vehicle, carboplatin, paclitaxel, or carboplatin + paclitaxel (C/T) for 3 cycles followed by observation with bioluminescence imaging of tumor burden.

Results: While vehicle- and paclitaxel-treated animals exhibited rapid disease outgrowth, carboplatin- and C/T-treated animals showed disease regression, followed by recurrence after 90-300 days. Median survival was significantly increased in carboplatin- and C/T-treated animals. Recurrent tumors were histologically similar to vehicle-treated tumors. We performed whole-exome sequencing and whole-transcriptome sequencing on ascites cells from 4 animals in each treatment group. We used a decontamination algorithm to remove stromal mouse reads from our tumor samples, resulting in a highly pure (over 99%) human tumor sequence data. Mutations, insertions/deletions, and copy number alterations were identified and compared among treatment groups. Known TP53 mutations were present at 100% frequency across samples. Average mutation frequencies were similar across all groups. We identified several mutations and copy number variants present in the carboplatin or C/T recurrent tumors that were distinct from the vehicle groups, including putative alterations in chemoresistance pathways such as DNA repair (BRCA1/2), apoptosis (BCL2L1), drug transport (ABC transporters), and PI3K signaling (EEF2K). In model PDX-A, all four carboplatin and one C/T treated recurrent tumors exhibited a similar copy-number profile, suggestive of subclonal expansion, which included an increase in WT copies of PTEN relative to the vehicle tumors. Phylogenetic analysis and whole-transcriptome analysis are ongoing.

Conclusions: Our study models recurrent ovarian cancer in PDX and identifies potential features of in vivo chemotherapy resistance.

#1011

RNAseq analysis obtained from on-purpose tumor biopsies of patients in the MATCH-R trial allows the identification of potential mechanisms of acquired resistance to PD(L)1 therapies.

Loic Verlingue, Linda Mahjoubi, Sandrine Aspeslagh, Marion Pedrero, Giulia Buzzatti, David Brandao, Zsofia Balogh, Etienne Rouleau, Ludovic Lacroix, Rastislav Bahleda, Christophe Massard, Antoine Hollebecque, Anas Gazzah, Céline Lefebvre, Serge Koscielny, Jean Yves Scoazec, Eric Angevin, Fabrice André, Aurélien Marabelle, Jean Charles Soria. _Gustave Roussy, Villejuif, France_.

Background: MATCH-R is a prospective molecular characterization trial (NCT02517892) aiming at defining the molecular basis of acquired resistance to targeted agents and immune checkpoint blockers. RNA sequencing (RNAseq) has been used to identify mechanisms of secondary resistance to immunotherapy.

Patients and methods: Patients' metastatic tumors were multi-site biopsied at relapse under immunotherapies after a period of clinical benefit, defined by a partial response or a stable disease of more than 6 months. Genome-wide RNAseq counts were intra-patient normalized and a score of each gene's expression was computed in comparison to a cohort of 450 metastatic cancer patients with RNAseq available at the time of analysis.

Results: To date, 10 patients treated by immunotherapies have had a successful RNAseq in the MATCH-R trial. Five patients were treated with PD-1 inhibitors and 5 with PD-L1 inhibitors. Three patients had NSCLC, 2 MSI high endometrial carcinoma, 2 anal carcinoma, 2 urothelial carcinoma and 1 TNBC. Eight out off ten patients had an expression of IDO1 higher than the median expression of IDO1 in our 450 controls (p value = 0.005). A patient with endometrial carcinoma had one of the highest expressions of IDO1 in the cohort. Consistently, IDO1 activation has previously been reported as a mechanism of secondary resistance to immunotherapies. A 40 year old smoker NSCLC patient with a TP53 mutation has been treated during 11 months with anti-PD1. RNAseq analysis on the biopsy of a progressive lesion showed decreased expression of different actors of the JAK-STAT pathway (biopsy composed of 40% tumor cells and 60% microenvironment). Of the 78 genes signatures used (including 52 immunogenes signatures), the interferon gamma signature had the lowest expression (p value = 0.004), consistent with a previous report of JAK-STAT-induced resistance to immunotherapy. Two more patients had an altered immune profile that could be involved in resistance to immunotherapies, but was not yet reported in the litterature. Confirmation of the RNAseq analysis with immunohistochemistry is currently ongoing. The gene signatures of the 10 patients, composed of immunogenes, DNA repair genes and epigenes, were compared to the whole cohort in order to deduce corresponding false discovery rates. As such we could identify 2 gene clusters, one enriched in T cells, dendritic cells and macrophages, and the other enriched in epigenes and DNA repair genes. Analysis of more patients is currently ongoing in order to cluster the results with clinical characteristics.

Conclusion: Gene expression in the biopsy of patients that relapsed after initial benefit to immunotherapy is informative and helps to identify the mechanism of acquired resistance.

#1012

Patient-derived tumor microenvironment models uncover nonautonomous TKI resistance mechanisms in NSCLC.

Haichuan Hu, Hillary Mulvey, Sundus Noeen, Kodack David, Aaron Hata, Matthew Niederst, Cyril Benes, Jeffrey Engelman. _MGH Cancer Center, Charlestown, MA_.

Background: Tyrosine kinase inhibitors (TKI) have yielded great responses in non-small-cell lung cancer (NSCLC) with EGFR mutations and ALK translocations, however these and other targeted therapies are limited by intrinsic and acquired drug resistance. Previous study from our group was looking into tumor autonomous resistance mechanisms by developing patient-derived cancer models (PDCs). In this study, we aimed to decipher the non-autonomous resistance mechanisms via tumor microenvironment by developing patient-derived fibroblast (PDF) cell lines.

Method: Cancer-associated fibroblast cells are isolated directly from EGFR mutant and ALK translocated NSCLC biopsies. Over 30 PDFs models have been established, which represent different clinical features and response profiles.

Result: By co-culturing the PDCs with PDFs, we found that there is considerable variability in both models for their magnitude and mechanism by which the TKI treatment is desensitized. Both HGF dependent and HGF independent resistance mechanisms can be overcome by specific therapeutic combinations.

Conclusion: Together, our results indicate that PDFs are clinically relevant models for deciphering non-autonomous resistance mechanisms, that they are heterogeneous in protecting cancer cells from TKI treatment, and that the resistance mediated by PDFs can be overcome by specific therapeutic combinations.

## IMMUNOLOGY:

### Novel Insights into Mechanisms of Response to Immunotherapy

#1013

Location matters: unique functional and transcriptional, but not flow cytometric, characteristics of intratumoral FOXP3+ Tregs vs. Tregs from other sites of patients with lung tumors.

Wayne W. Hancock, Tatiana Akimova, Tianyi Zhang, Evgeniy Eruslanov, Sunil Singhal, Steven M. Albelda. _CHOP/U Penn, Philadelphia, PA_.

FOXP3+ Tregs are considered important to limiting antitumor immunity but are rarely characterized clinically. We studied 227 samples from 66 lung cancer patients using blood, lymph node (LN), tumor and distant lung samples, with a mean age of 67.8±1.1 years, 67% males, 62% adenocarcinoma, 31% squamous cell carcinoma, 7% miscellaneous tumors, mean tumor size of 3.4±0.3 cm, and 21% rate of metastases. In addition to quantitating FOXP3+CD4+ Tregs, we evaluated their expression of 35 markers by flow cytometry: CD15s, CD25, CD26, CD27, CD39, CD40L, CD45RA/RO, CD62L, CD69, CD101, CD120b, CD161, CCR4, CTLA4, GARP, GITR, Helios, HLA-DR, ICOS, LAP, neuropilin, PD-1, TIGIT, Tim3, CCR4, CCR5, CCR7, CXCR3, CXCR4 and CCR8, and tested Treg suppressive function. We used PrimeFlow to evaluate mRNA expression of target genes in 100% pure human Treg cells, gated on CD4+FOXP3+ cells. FOXP3+ Tregs (%) in the CD4 cels of tumors and LN were significantly increased (p<0.0001 Kruskal-Wallis) compared to other sites; tumors 19.2±8.5%, LN 14.7±8.5%, healthy PBMC donors 7.2±2.5%, PBMC lung cancer 7.2±2.7%, and lungs 6.6±3.2%. Tregs of lung tumors were remarkably suppressive vs. all other sites (p<0.0001 vs. PBMC & LN, p=0.0106 vs. lung Tregs). None of the 35 markers evaluated by flow cytometry were statistically significantly different for tumor Tregs vs. other sites. However, PrimeFlow showed tumor Tregs, but not FOXP3- T cells or Tregs from other sites, had upregulated mRNA expression of 4 transcription factors (TF): Eos, Irf4, Satb1 and Gata1. These 4 TFs plus LEF1 were recently described as a quintet of Treg self-locking signature TF; expression of any 2 TF plus FOXP3 promoted full Treg gene expression and function. Tumor Tregs also showed significant upregulation of FOXP3 mRNA and protein. Tumor and lung Tregs expressed more Eos, Irf4, Satb1 and Gata1 mRNA, and much higher levels of FOXP3 protein per cell, while in PBMC, LNs and healthy donor Tregs upregulation of TFs did not correspond with increase of FOXP3 protein, indicating the "Treg self-locking signature" is subject to regulation by local factors. Indeed, cultured tumor Tregs downregulated FOXP3 protein, mRNA and TF expression, while PBMC and LN Treg incubated in tumor-conditioned media upregulated FOXP3 mRNA and protein, and Treg TF expression, moving toward the tumor Treg-like phenotype. We found significantly increased numbers and suppressive function of FOXP3+ Tregs within lung tumors vs. other sites in the same patients. In addition, while large-scale flow cytometric studies were not useful in identifying key features of tumor Tregs vs. Tregs at other sites, PrimeFlow showed that tumor Treg have a unique phenotype with upregulated expression of FOXP3 mRNA and protein, as well as Eos, Irf4, Satb1 and Gata1 mRNAs. These features appear to be malleable and associated with features of the local tumor microenvironment.

#1014

Selective impairment of intratumoral regulatory T cells by targeting Ezh2 enhances cancer immunity.

David Q. Wang, Jason R. Quiros, Chien-Chun S. Pai, Lawrence H. Fong, Jeffrey A. Bluestone, Michel J. DuPage. _University of California, San Francisco, San Francisco, CA_.

High numbers of tumor infiltrating regulatory T (Treg) cells are indicative of poor outcome in several malignancies, including ovarian cancer, colorectal cancer, and melanoma. Thus, selectively abrogating intratumoral Treg cell function while maintaining systemic immune tolerance remains an attractive, albeit elusive, strategy for cancer immunotherapy. We have identified the epigenetic enzyme Ezh2, an H3K27 methyltransferase, to be a critical mediator of lineage stability and function in activated Treg cells. Consequently, deficiency of Ezh2 in Treg cells strongly impairs their function in non-lymphoid tissues. We hypothesized that this phenomenon might translate to a similar defect that is restricted to the tumor microenvironment. Here we tested this hypothesis in three transplantable syngeneic tumor models in mice (MC38 colon carcinoma, TRAMP-C2 prostate cancer, and B16F10 melanoma). We observed that constitutive deletion of Ezh2 in Treg cells resulted in potent anti-tumor activity, significantly impaired tumor outgrowth, and in many instances, complete tumor rejection. These results were also recapitulated when Ezh2 was temporally deleted in the vast majority of Treg cells at the time of tumor inoculation. Functional analyses at early time points after Ezh2 deletion revealed enhanced functionality of effector CD4 and CD8 populations, as evidenced by increased IFN-γ production. These effects were evident without reduction of intratumoral Treg cell frequencies and despite presence of residual wild type Treg cells. Mechanistic studies showed that Ezh2-deficient Treg cells were destabilized and produced pro-inflammatory cytokines IL-2 and IFN-γ selectively within the tumor microenvironment, suggesting a direct contribution of Ezh2-deficient Treg cells to tumor clearance. To more directly address this hypothesis, we generated mice that harbored both Ezh2-deficient and wild type Treg cells and observed anti-tumor activity similar to mice that exclusively harbored Ezh2-deficient Treg cells. In stark contrast to Ezh2 deficiency, mice whose Treg cells were completely depleted, using a Foxp3-driven diphtheria toxin receptor allele, were unable to reject tumors, further supporting a dominant role of Ezh2-deficient Treg cells in driving anti-tumor immunity. Finally, tumor protection in the presence of Ezh2-deficient Treg cells occurred without significant morbidity due to autoimmune pathologies, which were prevalent in mice with systemic Treg cell depletion. These results suggest that targeted deletion of Ezh2 in Treg cells drives a unique phenotype among tumor infiltrating Treg cells, reprogramming the tumor microenvironment and selectively augmenting the anti-tumor immune response. Cancer therapies that pharmacologically inhibit Ezh2 to target tumor cells are under investigation, going forward it will also be important to assess the potential for beneficial effects via modulating the immune response.

#1015

Directionality of T-cell-mediated cross-protection against the same tumor in lungs versus skin.

Jessica J. O'Konek, Elena Ambrosino, Anja Bloom, Liat Izhak, Jay A. Berzofsky, Masaki Terabe. _National Cancer Inst., Bethesda, MD_.

Immunotherapy is a new pillar of cancer therapy. One theoretical advantage of immunotherapy of cancer is that effector cells induced at one site should be able to kill metastatic cancer cells in other sites or tissues. On the other hand, it has been recognized that each tissue has unique immune components that play critical roles in protection against pathogens. However, very little is known whether effector T cells induced against tumors in one tissue can work against the same tumors in other tissues. To address this question, we compared the effect of effector cells induced against the same tumor cells growing in either the skin or the lung by using CT26 murine tumor models. Rejection of s.c. CT26 tumors was achieved by pretreatment with anti-CD25, which blocks the function of Treg cells. Both CD4 and CD8 T cells were necessary for the protection. When anti-CD25-pretreated mice challenged with s.c. CT26 were simultaneously inoculated i.v. with CT26, they also rejected tumors in the lung, while anti-CD25-pretreated mice without s.c. CT26 did not. This observation suggested that T cell mediated anti-tumor protective immunity induced against s.c. tumors can also protect against lung metastases of the same tumors. In contrast to Treg depletion which allowed for the induction of protective immunity in the s.c. tumor model, NKT cell-deficiency in CD1d KO mice induced significant CD8 T cell-mediated protection against lung metastasis of CT26 but had no effect on the growth of s.c. CT26 tumors. When CD1d KO mice rejecting i.v. CT26 were simultaneously challenged with s.c. CT26, the development of s.c. tumors was not affected, indicating that tumor rejection induced against the CT26 in the lung did not confer protection for the same tumor cells in the skin. Since the protection against CT26 in the lung in CD1d KO mice is mediated by CD8 T cells, we transferred T cells from CD1d KO mice inoculated i.v. with CT26 into RAG1 KO recipients, and challenged the recipient mice with the CT26 tumor cells. The recipient RAG1 KO mice were highly resistant to CT26 inoculated i.v. but not s.c. confirming that the CD8 T cells protective in the lung are not protective in the skin. These data

indicate the effector cells against the same tumor do not work in all tissues, and the induction site of the effector T cells is critical to control metastasis.

#1016

Evaluation of progression associated neoepitopes and immune contexture in pulmonary premalignancy.

Kostyantyn Krysan, Linh M. Tran, Brandon S. Grimes, Tonya C. Walser, William D. Wallace, Steven M. Dubinett. _UCLA David Geffen School of Medicine, Los Angeles, CA_.

Lung cancer is the leading cause of cancer death in the US and in the world. Over the past 30 years, the five-year survival rate for lung cancer has increased by only 5%. With the widespread implementation of screening programs, detection of premalignant and early stage disease is increasing. A better understanding of genomic alterations and the microenvironment along the spectrum of early disease could lead to identification of progression-associated mutations (PAMs), defined as those shared between premalignant lesions and invasive cancer, and their neoepitopes. Unleashing the immune response against pulmonary premalignancy could transform therapy and outcomes. FFPE tissue blocks from patients with resected lung adenocarcinoma (ADC) were obtained from the UCLA Lung Cancer Tissue Repository. For each patient, the following regions were dissected from distal airways utilizing Laser Capture Microdissection: a) normal airway epithelial cells (1-3 regions), b) premalignant atypical adenomatous hyperplasia (AAH, 2-4 regions), c) adenocarcinoma in situ (AIS, when present) and d) ADC, 1-3 regions followed by whole exome sequencing. Forty-one complete cases have been sequenced to date. Our data suggest that premalignant lesions from the same patient may a) have different mutational profiles and b) bear progression-associated mutations, common with the primary lung tumor. This inter-lesion heterogeneity suggests that a progression-associated mutational landscape could be defined in longitudinal studies of pulmonary premalignancy which will be the focus of future investigations. Next, utilizing the mutational data, we performed in silico neoantigen analysis to identify potential neoepitopes among the genes mutated in premalignant lesions. The neoantigen analysis demonstrated that among the top 11 peptides with high binding avidity for autologous MHC, 9 were derived from PAMs. This suggests that neoepitopes exist in premalignancy that could serve as targets for development of future vaccines. Finally, we performed the quantitative immunohistochemical (IHC) and immunofluorescence (IF) staining for Granzyme B, PD-1, PDL-1, CD4+, CD8+ and FOXP3 to evaluate cell-mediated immunity on the same samples that were utilized for WES. We found both infiltration of T effector cells as well as upregulation of checkpoints in premalignancy and detected a significant inter-lesional heterogeneity. Our studies lay the ground work for identification of neoepitopes that can be targeted before the development of invasive lung cancer, thus shifting the approach to disease interception through immunoprevention and treatment of the very earliest phase of the disease.

#1017

Lipid metabolic reprogramming drives resistance to PD1 blockage.

Maria A. Cortez, Sharareh Niknam, Efrosini Cuko, Jonathan E. Schoenhals, Hampartsoum Barsoumian, Ahmed I. Younes, Ailin Li, Jody V. Vykoukal, Cristina Ivan, George A. Calin, Patrick Hwu, James W. Welsh. _UT MD Anderson Cancer Ctr., Houston, TX_.

The mechanisms underlying immunosuppression and resistance to PD1 inhibitors in cancer are not well understood. We attempted to fill this gap with an integrated analysis of mRNA, microRNA, and protein expression in an anti-PD1-resistant lung adenocarcinoma mouse model. The model was created by in vivo passage of 344SQ murine lung cancer cells (p53R172HΔg/+K-rasLA1/+) in a syngeneic host repeatedly dosed with anti-mouse PD1 antibodies. Anti-PD1-resistant 344SQ (344SQ_R) and 344SQ parental (344SQ_P) cells were then inoculated into syngeneic 129Sv/ev mice, which were then dosed twice with anti-PD1 or control IgG antibodies. Tumor tissues were collected and analyzed as follows: transcriptome with Affymetrix; protein levels by reverse phase protein array analysis; signature enrichment by gene set enrichment analysis; metabolome by mass spectrometry; and lipid content with fluorescent probes Oil O rad and BODIPY. We also isolated tumor-infiltrating immune cells for flow cytometry and gene expression analyses. We identified lipid-related metabolic pathways as being the most highly enriched in anti-PD1-resistant tumors (344SQ_R) vs. their 344SQ_P counterparts; the resistant cells also had more lipid droplets than the 344SQ_P cells. The anti-PD1-resistant tumors overexpressed several genes involved in lipogenesis and fatty acid pathways (e.g., fatty acid binding proteins [FABPs], fatty acid synthase, acetyl-coA-acyltransferase 2, fatty acid elongases). Specifically, FABP overexpression promoted fatty acid uptake and lipid-droplet accumulation in resistant tumors. Lipid-sensitive targets linked to inflammation and insulin signaling (e.g,. stress-activated kinases such as JNK and NFκB) were altered in 344SQ_R vs. 344SQ_P tumors. Mechanistically, JNK downregulation by NFκB-regulated microRNAs protected PD1-resistant tumors from lipotoxicity caused by FABPs upregulation and fatty acid uptake. FABP levels were higher in plasma from 344SQ_R than from 344SQ_P tumors. Tumor-infiltrating macrophages from 344SQ_R tumors had 4 times the amount of FABP mRNA than parental tumors and a correspondingly higher percentage of M2-like macrophages. 344SQ_R tumors promoted immune suppressive cells by upregulating FABPs expression in M2-like macrophages, marked by increased fatty acid intake and fatty acid oxidation. Conversely, percentages of CD4+ and CD8+ tumor-infiltrating lymphocytes were reduced in the resistant tumors. These results suggest that lipid metabolic rewiring drives resistance PD1 inhibitors supporting the accumulation of immunosuppressive cells, including M2-like macrophages, preventing type I immune responses elicited by T cells. Collectively, these findings reveal new potential lipid-related targets for drug development or new treatments combining inhibitors of these targets with anti-PD1 therapy.

#1018

High-resolution microbiome profiling and genome wide arrays uncover bacteria driven alterations of oncogenic and immune pathways in head and neck cancer patients treated with surgery, chemo-radiation and PD-1 checkpoint blockade therapy.

Rafael E. Guerrero-Preston,1 James Robert White,2 Filipa Godoy-Vitorino,3 Herminio Gonzalez,3 Arnold Rodríguez-Hilario,3 Kelvin Navarro,3 Gustavo A. Miranda-Carboni,4 Christina Michailidi,1 Anne Jedlicka,5 Stephanie Hao,6 Sierra Canapp,1 Jessica Bondy,1 Amanda Dziedzic,5 Barbara Mora Lagos,7 Gustavo Rivera-Alvarez,8 Winston Timp,6 William Westra,1 Wayne Koch,1 Hyunseok Kang,1 Luigi Marchionni,1 Young Kim,9 David Sidransky1. 1 _Johns Hopkins Univ. School of Medicine, Baltimore, MD;_ 2 _Resphera Biosciences, Baltimore, MD;_ 3 _Inter American University of Puerto Rico, San Juan, PR;_ 4 _University of Tennessee Health Science Center, Memphis, MD;_ 5 _Johns Hopkins Univ. School of Public Health, Baltimore, MD;_ 6 _Johns Hopkins Univ. Whiting School of Engineering, Baltimore, MD;_ 7 _Universidad de La Frontera School of Medicine, Temuco, Chile;_ 8 _University of Puerto Rico School of Medicine, San Juan, PR;_ 9 _Vanderbilt University School of Medicine, Nashville, TN_.

Previous microbiome studies at the genus level have described altered microbiota in head and neck squamous cell carcinoma (HNSCC), both in terms of taxonomic composition and metabolic capacity. We applied high-resolution microbiome profiling (Resphera Insight) to analyze 16S rRNA sequencing data in saliva and tissue samples from HNSCC patients and healthy controls. DNA extraction and amplicon library preparation was performed for saliva samples from HNSCC (n=38) and controls (n=25), as well as tissue samples from HNSCC (n=25) and controls (n=8). Raw sequences were processed for quality and length, screened for chimeras and filtered for contaminant human and chloroplast DNA. High-quality passing sequences were submitted to Resphera Insight for species-level taxonomic assignments, followed-by differential abundance analysis with the DESeq package. Samples from a subset of HNSCC patients were significantly enriched with commensal species from the vaginal flora, including Lactobacillus gasseri/johnsonii (710x higher in saliva and 1990x higher in tissue) and Lactobacillus vaginalis (52x higher in saliva). These species were not observed in normal saliva or tissue samples from Hopkins patients (n=33) nor in normal saliva samples (n=292) from the Human Microbiome Project (HMP). Interestingly, both species were only observed in saliva from Human Papilloma Virus positive (HPV+) and HPV negative (HPV-) oropharyngeal cancer patients, and we confirmed their representation in vaginal samples from the HMP (n=249). We also found that Fusobacterium nucleatum (F.nucleatum), an oral cavity flora commensal bacterium linked to colon cancer, is enriched (600x higher in saliva and 51x higher in tissue) in a subset of HNSCC patients with advanced tumors (T3 or above). F. nucleatum was detected in samples obtained before and after treatment with chemo-radiation, but not with surgery alone. Interestingly, we identified upregulation of the oncogenic Wnt/Beta catenin pathway (Wnt7B, FZD6, SFRP4) and down regulation of immune system pathways (TLR10, IRF8) with genome-wide mRNA arrays (Affymetrix) in HNSCC samples enriched for F.nucleatum. Using fluorogenic quantitative PCR we confirmed that F.nucleatum and Fusobacterium spp. are enriched in saliva samples collected prior to treatment in another cohort of HNSCC patients, and in post-treatment saliva samples obtained from HNSCC patients treated with PD-1 checkpoint blockade. We also found enrichment of HPV+ oropharyngeal tumors with F.nucleatum and Fusobacterium spp., prior to therapy, while some recurrent HPV- oropharyngeal tumors are enriched only with Fusobacterium spp. Together, these results suggest that bacteria may impact therapy in HPV+ and HPV- oropharyngeal and oral cavity cancer by altering oncogenic and immune system pathways.

#1019

In vivo **CRISPR screening identifies Ptpn2 as a target for cancer immunotherapy.**

Robert T. Manguso,1 Hans W. Pope,1 Margaret D. Zimmer,1 Flavian D. Brown,1 Kathleen B. Yates,1 Brian C. Miller,1 Natalie B. Collins,1 Kevin Bi,1 Martin W. Lafleur,1 Vikram R. Juneja,2 Sarah A. Weiss,1 David E. Fisher,3 David E. Root,4 Arlene H. Sharpe,2 John G. Doench,4 W Nicholas Haining1. 1 _Dana-Farber Cancer Institute/Harvard Medical School, Boston, MA;_ 2 _Harvard Medical School, Boston, MA;_ 3 _Massachusetts General Hospital, Boston, MA;_ 4 _Broad Institute of Harvard and MIT, Cambridge, MA_.

Despite the dramatic clinical success of cancer immunotherapy with PD-1 checkpoint blockade, most patients don't experience sustained clinical benefit, suggesting that additional therapeutic strategies are needed. Functional genomic screens in cancer cells to discover new therapeutic targets are usually carried out in vitro where interaction with the immune system is absent. Here we report a pooled, loss-of-function genetic screening approach using CRISPR/Cas9 genome editing that is conducted in vivo in mouse transplantable tumors treated with vaccination and PD-1 checkpoint blockade. We tested 2,400 genes expressed by melanoma cells for those that synergize with or cause resistance to checkpoint blockade, and recovered the known immune evasion molecules, PD-L1 and CD47. Loss of function of multiple genes required to sense interferon-y caused resistance to immunotherapy. Deletion of Ptpn2, a pleotropic protein tyrosine phosphatase improved response to immunotherapy. In vivo, Ptpn2 deficient tumors showed increased infiltration of activated CD8+T cells. In vitro, Ptpn2 loss by tumor cells increased antigen presentation to T cells. Biochemical, transcriptional and genetic epistasis experiments demonstrated that loss of function of Ptpn2 sensitizes tumors to immunotherapy by enhancing interferon-y-mediated effects on the tumor cell. Thus, augmenting interferon-y signaling in tumor cells could increase the efficacy of immunotherapy. More generally, in vivo genetic screens in tumor models can identify new immunotherapy targets and rationally prioritize combination therapies.

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

### Chromatin Structure and Function

#1020

BRD9 defines a novel mammalian SWI/SNF (BAF) complex configuration which supports proliferation in AML.

Brittany C. Michel,1 Joshua Pan,1 Robin M. Meyers,2 Paola Grandi,3 Phillip G. Humphreys,4 Neil G. Garton,4 Rab K. Prinja,4 Cigall Kadoch1. 1 _Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA;_ 2 _Broad Institute of MIT and Harvard, Boston, MA;_ 3 _Cellzome, Heidlberg, Germany;_ 4 _GlaxoSmithKline, Hertfordshire, United Kingdom_.

Genes encoding subunits of the mammalian SWI/SNF (BAF) ATP-dependent chromatin remodeling complexes are mutated in over 20% of human cancer. Specific subunits are mutated in specific malignancies, highlighting their tissue-specific protective roles; moreover, synthetic lethal screens have uncovered genetic- and lineage-based features which confer dependence on specific mSWI/SNF subunits. As combinatorial complexity represents a major challenge, identification of specialized mSWI/SNF configurations, subunit-specific functions, binding restrictions, and exclusivity relationships is critical for understanding oncogenic mechanisms and for the selection of appropriate therapeutic agents targeting mSWI/SNF complex subunits.

Here, we discover that BRD9, a recently identified mSWI/SNF subunit, defines a novel complex configuration distinct from BAF and PBAF, which we term non-canonical BAF, or ncBAF. We used biochemical methods to isolate BRD9-containing complexes and find that BRD9 selectively marks a sub-stoichiometric group of mSWI/SNF complexes of smaller molecular weight that lack several members of canonical BAF complexes such as BAF47 and ARID1A. Moreover, chemoproteomics using a BRD9 inhibitor (GSK) isolates only ncBAF and does not resolve BAF-specific or PBAF-specific components, including the highly related bromodomain-containing subunit BRD7. We further identified regions of BRD9 and BRD7 that confer specificity of these subunits to ncBAF and PBAF complexes, respectively, resolving their mSWI/SNF binding domains.

Using genome-wide ChIP-seq and RNA-seq experiments, we determined that ncBAF complexes target a distinct subset of all mSWI/SNF complex target genes and, consistent with previous studies, maintain proliferation of AML cells. Finally, we applied a newly- generated approach to deriving functional relationships within and between protein complex families from shRNA and CRISPR-based genetic screening datasets across hundreds of cancer cell lines to explore ncBAF-specific subunits. We find that ncBAF-specific complex subunits form a distinct functional module, supporting biochemical studies and pointing to the specific and divergent functions of the ncBAF configuration. Cancers of hematologic origin collectively exhibit the most significant responses to perturbation of three ncBAF subunits including BRD9, substantiating previous small molecule screening efforts using BRD9 bromodomain inhibitors. These data demonstrate that ncBAF complexes represent a novel BAF complex composition with distinct function in cancer.

#1021

Mutations in SWI/SNF chromatin remodeling complex sensitize tumors to OXPHOS inhibition.

Yonathan Lissanu Deribe. _UT MD Anderson Cancer Ctr., Houston, TX_.

Recent large-scale cancer genomic studies have established a framework in which biological functions and genetic interactions of established and novel cancer genes can be explored. One of the major findings of these studies has been the discovery of frequent genetic alterations in chromatin and epigenetic regulators including inactivating mutations in components of the SWI/SNF chromatin remodeling complex. In lung adenocarcinoma in particular, a quarter of tumors have inactivating mutations in the SWI/SNF components Smarca4, ARID1A or the histone modifier SETD2. With the aim of understanding the mechanism of tumor development and identifying potential vulnerabilities of SWI/SNF mutant tumor, we developed a genetically engineered mouse model (GEMM) of lung adenocarcinoma by selectively ablating Smarca4 in the respiratory epithelium. We demonstrate that Smarca4 acts as a bona fide tumor suppressor and cooperates with p53 loss. Cross species integrative gene expression analyses revealed signatures of enhanced oxidative phosphorylation and reactive oxygen species (ROS) response in Smarca4 mutant murine and human lung tumors. We further show that Smarca4 mutant cells have increased oxygen consumption, enhanced basal and maximal respiratory capacity and production of ROS. This is primarily driven by activation of the mitochondrial master regulator, PGC1alpha. Finally, we show that Smarca4 and other SWI/SNF mutant lung cancer cell lines have exquisite sensitivity to inhibition of oxidative phosphorylation using a novel small molecule that is under development. These findings provide a mechanistic basis for further development of OXPHOS inhibitors as therapies in SWI/SNF mutant tumors.

#1022

Chromatin remodeler HELLS is an epigenetic driver for hepatocellular carcinoma progression.

Cheuk-Ting Law, Larry L. Wei, Felice HC Tsang, Iris MJ Xu, Robin KH Lai, Daniel WH Ho, Joyce MF Lee, Carmen CL Wong, Irene OL NG, Jack CM Wong, The State Key Laboratory for Liver Research. _The University of Hong Kong, Hong Kong Island, Hong Kong_.

Hepatocellular carcinoma (HCC) is the major type of liver cancer and the second leading cause of cancer death worldwide. Every year, more than six hundred thousand people are newly diagnosed. For the advanced HCC patients, only one FDA-approved drug is currently available. Therefore, there is an urge to delineate the molecular mechanism of HCC progression for developing new therapeutics. Increasing evidence showed that epigenetic alterations play an important role in the carcinogenesis process. The epigenetic regulatory mechanism is accomplished by DNA methylation, histone modification, and chromatin remodeling. Deregulation of DNA methylation and histone modifications have recently been characterized in HCC, but the significance of chromatin remodeling in liver carcinogenesis remains to be explored. In this study, we employed RNA-seq to investigate the expression of chromatin remodelers in human HCCs. We found that HELicase, Lymphoid-Specific (HELLS), a SWI2/SNF2 chromatin remodeling enzyme, was remarkably overexpressed in HCC. Overexpression of HELLS was correlated with more aggressive clinicopathological features and poorer patient prognosis. We further showed that up-regulation of HELLS in HCC was conferred by hyper-activation of transcription factor SP1. To investigate the functions of HELLS in HCC, we generated both gain- and loss-of-function models by CRISPR activation system, lentiviral shRNA, and CRISPR/Cas9 genome editing system. We demonstrated that overexpression of HELLS augmented HCC cell proliferation and migration. In contrast, depletion of HELLS reduced HCC cell growth and motility. Moreover, inactivation of HELLS induced apoptosis in HCC cells. Coherently, ablation of HELLS also mitigated tumorigenicity and lung metastasis in vivo as demonstrated with both subcutaneous and orthotopic tumor implantation models. Mechanistically, by using RNA-seq and MNase-seq, we revealed that HELLS controls the nucleosome occupancy at gene enhancer and transcription start site (TSS). Overexpression of HELLS increased nucleosome occupancy that obstructed the accessibility of enhancers and hindered the formation of nucleosome-free region (NFR) at TSS of its target genes, thereby blocks the binding of transcription factors for activating gene expression. Consequently, though this mechanism, up-regulation of HELLS mediated epigenetic silencing of multiple tumor suppressor genes including E-Cadherin, FBP1, IGFBP3, XAF1 and CREB-H in HCC. In conclusion, our data unravel that HELLS is a key epigenetic driver of HCC. By altering the nucleosome occupancy at NFR and enhancer, HELLS epigenetically suppresses numerous tumor suppressor genes to promote HCC progression.

#1023

Activation of proto-oncogenes by enhancer-hijacking in high‐risk neuroblastoma.

Daniel Dreidax,1 Moritz Gartlgruber,1 Sebastian Steinhauser,1 Larisa Savelyeva,1 Ron Schwessinger,1 Umut Toprak,1 Nati Ha,1 Dilafruz Juraeva,1 Martin Peifer,2 Matthias Fischer,2 Stefan Gröschel,1 Kai-Oliver Henrich,1 Young-Gyu Park,1 Benedikt Brors,1 Matthias Schlesner,1 Carl Herrmann,1 Frank Westermann1. 1 _German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 2 _University of Cologne, Cologne, Germany_.

Background: Neuroblastoma (NB), a neural crest-derived tumor of the sympathetic nervous system, is the most common extracranial solid tumor in children. We have previously shown that genomic rearrangements activate proto-oncogenic telomerase by juxtaposing active enhancer elements to the TERT gene in a large fraction of high-risk NBs. In the present study, we applied a global approach integrating whole genome sequencing (WGS), Chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) data of NB cells and tumors to identify further key oncogenes activated by enhancer-hijacking in NB.

Methods: WGS was applied to search for structural rearrangements in 120 NB tumors and five NB cell lines. Corresponding RNA-seq data were used to discover mono-allelic and/or outlier expression of candidate genes potentially involved in enhancer hijacking events. ChIP-seq of 34 NB tumors and 17 NB cell lines was applied to identify active enhancer elements in NB. Circular chromatin conformation capture sequencing (4C-seq) was used to confirm physical promoter-enhancer interactions in NB cell lines.

Results: WGS analyses revealed that chromosomal rearrangements are common events in NB tumors and cell lines and frequently affect regions harboring proto-oncogenes and lineage specific enhancers. ChIP-seq analyses of the chromatin mark histone 3 lysine 27 acetylation (H3K27ac), surrogate for enhancer activity, confirmed that these rearrangements recurrently juxtapose active enhancer elements to oncogenes including MYCN and MYC in NB. Intriguingly, quantification of H3K27ac ChIP-seq profiles uncovered that the enhancer elements translocated to MYC were among the most active ones within the respective epigenomes. 4C-seq analyses proofed physical interactions between translocated enhancer elements and promoters of the respective oncogenes, which is in line with their elevated expression in rearranged cases.

Conclusions: Our study reveals that structural rearrangements in high-risk neuroblastoma frequently juxtapose strong enhancers to key oncogenes, including MYCN and MYC, leading to physical promoter-enhancer interactions which likely drive overexpression of the oncogenes observed in rearranged cases. The common mechanism of oncogene activation by enhancer-hijacking may open a therapeutic window for epigenetic drugs including BET or CDK7 inhibitors in high-risk NBs.

#1024

Genome-wide examination of topological variability reveals cell type-specific mechanisms of oncogene activation.

Caleb Lareau, Martin Aryee. _Harvard University, MA_.

INTRODUCTION:

The organization of chromatin within the nucleus into hierarchical three-dimensional (3D) structures plays a key role in the regulation of gene expression. Differences in 3D architecture, such as the presence or absence of "loops" between specific enhancers and their target genes, are a major driver of transcriptional and phenotypic variation. By determining the accessibility of genes to transcriptional machinery and distal regulatory regions, mechanisms mediating oncogene activation can be discerned. Recent studies have implicated pathogenic alterations in genome topology with the activation of proto-oncogenes in a multitude of cancers.

METHODS:

To characterize the topological changes associated with different cancer states, we developed a computational suite of tools that facilitates the systematic discovery, annotation, and visualization of features in chromatin topology and assigns statistical confidence estimates to regions with variability between cellular phenotypes. Our framework facilitates the integration of heterogeneous genomic data, including gene expression, histone modification, DNA methylation, open chromatin, and genetic variation, to further characterize changes in 3D genome. Ultimately, we propose a joint model that functionalizes the effect of epigenetic modifications on transcription as mediated through hierarchical chromatin structure.

RESULTS:

We identify regions of topological variation unique to breast cancer, cervical cancer, glioblastomas with and without an IDH1 mutation, melanoma, and leukemia. Patterns of disrupted insulation and subsequent localization of enhancers to proto-oncogenes can be attributed to variation in the epigenome, including variation in DNA methylation and open chromatin, at distal regulatory regions. These 3D alterations provide a mechanism for activation of specific oncogenic pathways, including signaling mediated by p38, PLK3, and BRCA.

CONCLUSIONS:

Variable patterns of chromatin topology provide a unique signature and mechanism of oncogenes for the cancer phenotypes considered. Additionally, our framework provides a novel means for interpreting epigenetic variability, particularly at distal regulatory regions, and explaining transcriptional variation associated with cancer phenotypes as mediated through structural changes in the DNA.

#1025

Controlled and endogenous depletion of a synthetic lethal target in human cancer cell lines via SMASh degron engineering.

Florencia Rago, Matthew T. Dimare, Geoffrey Bushold, Avnish Kapoor, Hyo-eun C. Bhang, Zainab Jagani. _Novartis Institutes for Biomedical Research, Cambridge, MA_.

The SWI/SNF multi-subunit complexes play an important role in regulating processes such as gene transcription through ATP-dependent remodeling of chromatin. The importance of this role is highlighted by the prevalence of mutations in various SWI/SNF subunits across approximately 20% of cancers encompassing various lineages and subtypes. Functional genomics screening via pooled shRNAs has led to the discovery of distinct vulnerabilities of SWI/SNF mutant cancers to loss of mutually exclusive subunits. In particular, cancer cells with BRG1 loss become exquisitely dependent on the remaining BRG1 counterpart, BRM. To further understand the synthetic lethal relationship between BRM and BRG1, we have adapted a recently described degron, the small molecule assisted shutoff (SMASh) tag, showing for the first time the ability to apply this system to control endogenous levels of target protein, in this case BRM. We used the SMASh tag to target the endogenous BRM locus in both a BRG1 wild-type and mutant cancer cell line. Using these engineered cell lines, we were able to show that we can achieve tunable protein levels by varying the concentration of the effector molecule. The ability to tune BRM depletion allowed us to investigate the nature of the BRG1/BRM synthetic lethal relationship. Our data interestingly suggests that while modest depletion of BRM is sufficient to observe changes in the expression of a target gene regulated by SWI/SNF in a dose-dependent manner, a higher level of BRM depletion is required to elicit a growth arrest phenotype in the BRG1-mutant non-small cell lung cancer line. Together these results uncover a novel relationship between the catalytic SWI/SNF subunit and gene expression, where we find that the latter responds in a dose dependent manner to BRM levels. Additionally, we elucidate the level of BRM depletion required to observe the synthetic lethal phenotype, providing insight and informing on strategies for therapeutic targeting whether by protein degradation approaches, such as PROTAC, or other small molecule approaches. Finally, having provided a proof-of-concept, such tools can be readily adapted for application to endogenous degradation of other target proteins in different systems.

#1026

Identify BRD4 as a facilitator of replication stress response signaling.

Jingwen Zhang,1 Brandon Willis,1 Maureen Hattersley,1 Alan Lau,2 Corinne Reimer,1 Michael Zinda,1 Stephen Fawell,1 Gordon Mills,3 Austin Dulak,1 Huawei Chen1. 1 _AstraZeneca Pharmaceuticals, Waltham, MA;_ 2 _AstraZeneca Pharmaceuticals, Macclesfield, United Kingdom;_ 3 _The University of Texas MD Anderson Cancer Center, Houston, MA_.

BRD4 is a member of the BET (bromodomain and extraterminal domain) family of chromatin readers that recognize acetylated-lysines on histones and nuclear proteins. Previous reports have demonstrated that select cancers depend on BRD4 to regulate oncogenic gene transcriptional programs. However, whether BRD4 contributes to cancer malignancy through other mechanisms has not been extensively evaluated. Here, we show that BRD4 is important for maintaining an intact DNA replication checkpoint in cancers. Displacement of BRD4 by BET bromodomain inhibitor AZD5153 in cell lines under intrinsic or exogenous replication stress led to a time-dependent reduction in phospho-Chk1 first detected within 30 minutes, and reaching maximum phospho-Chk1 inhibition (~85%) at an hour after AZD5153 treatment in U2OS cells. The decrease in Chk1 phosphorylation was observed without a concomitant decrease in total Chk1, and this was not replicated by treatment with pan-transcriptional inhibitors, suggesting a non-transcriptional mechanism linked to BRD4. Furthermore, BRD4 interacts with the DNA pre-replication complex and inhibition of BRD4 leads to hyperactivation of the pre-replication complex and aberrant DNA replication re-initiation under replication stress conditions. Consistent with a role in S-phase signaling, BETi treatment sensitizes replicating cells to replication stress-inducing agents. Finally, we observed that ovarian cancer cell lines are highly-sensitive to the combined inhibition of BRD4 and ATR. Coadministration of AZD5153 and AZD6738 (ATR inhibitor) significantly inhibited tumor growth in OVCAR3 ovarian xenograft model (TGI after 21 day dosing: AZD5153, 52%; AZD6738, 46%; Combo, 84%). Sustained tumor growth delay was observed after combination treatment cessation. Together, our study uncovered a novel function for BRD4 in regulating DNA replication stress response, and provide mechanistic rational for combining BETi with DNA damage-targeted agents for cancer therapies.

### Oncogenes and Tumor Suppressors 1: Function and Therapeutic Vulnerabilities

#1027

Development of orthotopically grafted organoid models to study pancreatic cancer progression.

Chang-Il Hwang, Eunjung Lee, Brandon Da Silva, Kevin Wright, Youngkyu Park, David A. Tuveson. _Cold Spring Harbor Laboratory, Cold Spring Harbor, NY_.

Pancreatic ductal adenocarcinoma (PDA) is one of the most difficult human malignancies to treat. The 5-year survival rate of PDA patients is 7% and PDA is predicted to become the second leading cancer-related cause of death in the USA. A subset of potential tumor suppressor genes was identified by genome-wide analysis of human PDA and insertional mutagenesis in genetically engineered mouse models (GEMMs). Since the functional validation of these genes in GEMMs is time-consuming and labor-intensive, we have developed a rapid and efficient 'orthotopically grafted organoid' (OGO) models in conjunction with RNAi and CRISPR/Cas9 technology to study PDA progression. Previously, we showed that OGO model represents the full spectrum of PDA progression in vivo upon orthotopic engraftment. Here, as a proof-of-concept experiment, we demonstrate that ablation of Trp53 by viral introduction of shRNA or gRNA in PanIN-derived organoids accelerates PDA progression upon transplantation. In addition, we engineered tetracyclin-inducible shRNA against Trp53 in ColA1 locus by Flpe recombinase-mediated cassette exchange in organoids derived from Kras +/G12D ; Rosa26-rtTA ; ColA1-homing cassette mouse. Orthotopic transplantation followed by doxycycline administration resulted in rapid PDA progression with metastases. Isolated tumor organoids were re-transplanted to evaluate the effect of p53 restoration in PDA progression. Restoration of p53 by doxycycline withdrawal led to reduced liver metastases, although there was no difference in survival and primary tumor growth. This will provide a new insight how p53 regulates PDA metastasis. Therefore, OGO models should provide an excellent platform to study the functional role of genes in PDA progression in vivo.

#1028

Genetic disruption of Kras sensitizes lung cancer cells to Fas-mediated apoptosis.

Haiwei Mou,1 Jill Moore,1 Sunil Malonia,1 Yingxiang Li,1 Deniz Ozata,1 Soren Hough,1 Chunqing Song,1 Jordan Smith,1 Has Yin,2 Andrew Fisher,1 Daniel Anderson,2 Shipping Weng,1 Michael Green,1 Wen Xue1. 1 _Univ. of Massachusetts Medical School, Worcester, MA;_ 2 _David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Worcester, MA_.

Genetic lesions that activate KRAS account for ~30% of the 1.6 million annual cases of lung cancer. Despite clinical need, KRAS is still undruggable using traditional small molecule drugs/inhibitors. When oncogenic Kras is suppressed by RNA interference, tumors initially regress but eventually recur and proliferate despite suppression of Kras. Here we show that tumor cells can survive CRISPR-mediated knockout of oncogenic Kras, indicating the existence of Kras-independent survival pathways. Thus even if clinical KRAS inhibitors were available, resistance would remain an obstacle to treatment. Kras-independent cancer cells exhibit decreased colony formation in vitro but retain the ability to form tumors in mice. Comparing the transcriptomes of oncogenic Kras cells and Kras knockout cells, we identified 603 genes that were specifically upregulated in Kras knockout cells, including the Fas gene, which encodes a cell surface death receptor involved in physiological regulation of apoptosis. Antibodies recognizing Fas-receptor efficiently induced apoptosis of Kras knockout cells but not oncogenic Kras expressing cells. Increased Fas expression in Kras knockout cells was attributed to decreased association of repressive epigenetic marks at the Fas promoter. Concordant with this observation, treating oncogenic Kras cells with histone-deacetylase inhibitor and Fas-activating antibody efficiently induced apoptosis, thus bypassing the need to inhibit Kras. Our results suggest that activation of Fas could be exploited as an Achilles' heel in tumors initiated by oncogenic Kras.

#1029

MAPK-mediated immunomodulation in disseminated murine Emu-TCL1 chronic lymphocytic leukemia.

Aparna Lakshmanan, Yo-Ting Tsai, Amy Lehman, Ellen J. Sass, Minh Tran, Fabienne Lucas, Meixiao Long, Bonnie K. Harrington, Krista La Perle, Vincenzo Coppola, Gerard Lozanski, Natarajan Muthusamy, John C. Byrd, Michael R. Grever, David M. Lucas. _The Ohio State University, Columbus, OH_.

Work in melanoma and other solid tumors shows mutations activating MAPK pathway not only promote malignancy by promoting survival and proliferation but also are immunosuppressive than wild-type (WT) counterparts. However, relevance of MAPK-activating mutations to immune modulation in disseminated cancers such as leukemia is uncertain, and the mechanisms by which this occurs are likely to differ. BRAF activating mutation is found in nearly all hairy cell leukemia cases, 4% of chronic lymphocytic leukemia (CLL) and 9% CLL cases when including other MAPK-activating mutations such as KRAS and NRAS. To assess immunomodulatory effects of BRAF-mutant CLL cells, we co-cultured dox-inducible OSU-CLL cells transfected with WT or BRAFV600E constructs with healthy donor T cells incubated with anti-CD3/anti-CD28 antibodies, and T cell proliferation, cytokine production and expression of surface proteins were assessed by flow cytometry. We also employed a transgenic mouse model of BRAF B cell leukemia. CD19-Cre x BRAFV600E or CD19-Cre only mice were crossed with the well-characterized Emu-TCL1 model of CLL to obtain mice with spontaneous B cell leukemia expressing either WT BRAF (CD19-Cre xTCL1) or mutant BRAF (BRAFV600E xCD19-Cre xTCL1) under the native BRAF promoter. For adoptive transfer (AT) experiments, leukemia cells (2e7) from transgenic mice were engrafted intravenously into syngeneic healthy adult animals. Blood and spleen cells were examined by flow cytometry. OSU-CLL cells expressing BRAFV600E more strongly inhibited anti-CD3/CD28-induced proliferation of normal donor T cells. Transwell assays showed this effect was due both to soluble and contact-dependent factors. TNF levels were higher in BRAFV600E-expressing cells and reduced by vemurafenib, but a TNF neutralizing antibody did not alter the inhibitory effect of BRAFV600E-expressing cells on T cell proliferation. Proliferation was rescued by the BRAFV600E inhibitor dabrafenib, further supporting the contribution of mutant BRAF to this effect. Impact of BRAFV600E mutation on myeloid compartment was also evaluated using AT model. Mice were engrafted with BRAFWT or BRAFV600E leukemia cells, and upon achieving similar disease loads, BRAFV600E leukemic B cells caused 2.2-fold increase in PD-L1 expressing-peripheral myeloid cells (p<0.001; n=25 WT; 11 mutant). Additionally, there was an increase in F4/80\+ macrophages (11%; p=0.002), CD11b+Ly6CintLy6Ghi MDSCs (8.5%, p=0.014) and decrease in CD11b+Ly6CloLy6Glo patrolling monocytes (11%; p=0.031; n=33 WT; 40 mutant) in spleens of mice with BRAFV600E leukemia, indicating that this mutation influences the myeloid cell compartment as well. Ongoing studies are investigating the mechanism of these effects and the potential for pharmacologic reversal. Together, these results demonstrate immunosuppressive impact of BRAF in B-cell leukemia.

#1030

Metabolic reprogramming caused by mutations of the Fbw7 ubiquitin ligase in colorectal cancer.

Ryan J. Davis,1 Mehmet Gonen,2 David Hockenbery,1 Adam Margolin,2 Bruce E. Clurman1. 1 _Fred Hutchinson Cancer Research Ctr., Seattle, WA;_ 2 _Oregon Health Sciences University, Portland, OR_.

Fbw7 is an evolutionary conserved F box protein that functions as a substrate adapter for the Skp1-Cullin 1-F box (SCF) E3 ubiquitin ligase complex. Fbw7 is also a tumor suppressor gene that is mutated in diverse human cancers. Many Fbw7 substrates are oncogenic transcription factors, including c-Myc, Notch, and Jun. Cancer-associated Fbw7 mutations diminish its ability to interact with substrates, which results in inappropriate oncoprotein accumulation. One of the significant challenges for understanding the consequences of Fbw7 mutations in tumors is that Fbw7 coordinately regulates approximately two dozen broadly-acting transcription factors. To address this problem, we used novel computational methods to analyze TCGA gene expression datasets to derive transcriptional signatures that could predict a tumor's Fbw7 mutational status; these signatures were then analyzed for enrichment of genes involved in specific biological processes. We found that genes associated with mitochondrial function were highly predictive of Fbw7 mutations across several tumor types, suggesting that metabolic reprogramming is a crucial oncogenic consequence of Fbw7 mutations. We validated this prediction by creating isogenic colorectal cancer cells with normal or mutant Fbw7 alleles. We found that mitochondrial signature genes are elevated in Fbw7 mutant cells, which also exhibit increased oxidative metabolism. Moreover, metabolomic studies revealed deregulation of other metabolic pathways in Fbw7-mutant cells, including amino acid and nucleotide biosynthesis. These data suggest that metabolic vulnerabilities may represent novel therapeutic targets in Fbw7-associated cancers.

#1031

Nuclear relocalization of NPM1c induces terminal differentiation and cell growth arrest.

Lorenzo Brunetti,1 Michael C. Gundry,1 Anna G. Guzman,1 Ilaria Gionfriddo,2 Federica Mezzasoma,2 Francesca Milano,2 Maria Paola Martelli,2 Brunangelo Falini,2 Margaret A. Goodell1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _University of Perugia, Perugia, Italy_.

NPM1 mutated acute myeloid leukemia (AML) is a distinct entity in the WHO classification of hematopoietic cancers. It displays a specific phenotype characterized by favorable prognosis and upregulation of HOX cluster genes. NPM1 is a multifunctional nucleolar chaperone. Mutations in NPM1 result in cytoplasmic protein localization (NPM1c+) through the acquisition of a nuclear export signal (NES) at the C-terminus. The most frequent NPM1 mutation is a heterozygous 4bp insertion in exon 12 (mutA).

The role of NPM1 in leukemogenesis is still a matter of debate and there is no direct proof that cytoplasmic localization of mutant NPM1 is necessary for maintenance of leukemia. We recently developed a CRISPR strategy for highly efficient gene editing in hematopoietic cells. We hypothesized that NPM1 mutA could be specifically targeted due to its 4bp insertion, avoiding the WT allele. Using an sgRNA spanning the insertion site (NPM1c sgRNA), we aimed to introduce indels adjacent to the mutation and disrupt the C-terminal NES. To test our hypothesis, we used the NPM1 mutated OCI-AML3 cell line.

After transfection of OCI-AML3 with NPM1c sgRNA, while NPM1mutA allele showed 70-90% indel frequencies, the NPM1wt allele was intact. To determine whether the novel edited alleles generated NPM1 that was re-localized to the nucleus, we cloned the alleles into a GFP-NPM1 fusion construct and observed nuclear localization after transfection. Consistent with this finding, immunofluorescence revealed that edited OCI-AML3 cells had lost nearly all cytoplasmic NPM1. Return of NPM1 protein to the nucleus was followed by terminal differentiation and cell cycle arrest in G1 phase (controls 45±3%, NPM1c sgRNA 68±1.5%). Cell growth (3.7-4 fold decrease in cell counts in NPM1c sgRNA samples), colony forming ability (16-20 fold reduction in colonies in NPM1c sgRNA samples) and engraftment in xenograft models (significant loss of indels at NPM1mutA allele in engrafted cells) were also significantly impaired after NPM1mutA targeting. Furthermore, RNA sequencing on NPM1mutA-targeted and control OCI-AML3 cells revealed almost complete loss of expression of the HOXA and HOXB cluster genes as well as MEIS1 in treated cells (4.5 average fold reduction of HOXA9-A13; 5.6 average fold reduction of HOXB7-B13; 5 fold reduction of MEIS1).

Allele-specific editing is a powerful tool to probe mechanistic aspects of oncogene dependencies. By achieving nuclear re-localization of mutant NPM1, we demonstrated that cytoplasmic localization of NPM1c+ is necessary for OCI-AML3 cells to maintain their leukemic phenotype. Drugs promoting mutant NPM1 nuclear localization such as CRM1 inhibitors are attractive candidates for clinical success in NPM1-mutated AML.

#1032

Identification of Ran binding protein 6 as a novel negative regulator of EGFR and candidate tumor suppressor in glioblastoma.

Wan-Ying Hsieh,1 Barbara Oldrini,2 Hediye Erdjument-Bromage,3 Paolo Codega,1 Maria S. Carro,4 Igor Vivanco,5 Dan Rohle,1 Carl Campos,1 Craig Bielski,1 Barry Taylor,1 Paul Tempst,1 Massimo Squatrito,2 Ingo K. Mellinghoff1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Spanish National Cancer Research Centre, Madrid, Spain;_ 3 _New York University School of Medicine, New York, NY;_ 4 _Medical Center University of Freiburg, Freiburg, Germany;_ 5 _The Institute of Cancer Research, London, United Kingdom_.

Amplification and overexpression of the epidermal growth factor receptor (EGFR) are common in glioblastoma (GBM) and frequently associated with silencing of the phosphatase and tensin homologue (PTEN) tumor suppressor. PTEN silencing has been associated with clinical resistance to EGFR tyrosine kinase inhibitors, in part by raising EGFR levels. Here, we investigated the effect of PTEN on the EGFR signaling complex by EGFR affinity immunopurification and mass spectrometry with and without PTEN knockdown. We identified Ran binding protein 6 (RanBP6), a 125-kDa protein of previously unknown functions, as EGFR interacting protein in PTEN expressing, but not PTEN knockdown cells. Further studies of the effect of RanBP6 on EGFR revealed that RanBP6 depletion by shRNA or CRISPR/Cas9-mediated gene silencing resulted in increased EGFR mRNA levels and upregulation of EGFR promoter activity. Consistent with a model of a negative EGFR regulation by RanBP6, we observed an inverse correlation between RanBP6 and EGFR mRNA levels in PTEN wildtype but not PTEN altered cancer cells in a large panel of human cancer cell lines (Cancer Cell Line Encyclopedia). To further understand the mechanism of how RanBP6 negatively regulates EGFR mRNA level, we found that RanBP6 interacted with nuclear Ran-GTPase and repressed EGFR transcription by promoting nuclear import of Signal transducer and activator of transcription 3 (STAT3). Lastly, RanBP6 appeared to be frequently deleted on chromosome 9p in GBM. We showed that RanBP6 silencing raised EGFR levels and signal output and accelerated in-vivo glioma growth. Our results establish a novel function of RanBP6 as a link between EGFR signaling and the Ran-mediated nuclear import pathway, and identify RanBP6 as candidate tumor suppressor on chromosome 9p.

#1033

Estrogen receptor gene fusions drive endocrine therapy resistance in estrogen receptor positive breast cancer.

Jonathan T. Lei,1 Jieya Shao,2 Jin Zhang,3 Michael Iglesia,4 Doug W. Chan,1 Ryoichi Matsunuma,1 Xiaping He,4 Purba Singh,1 Yoshimasa Kosaka,5 Robert Crowder,2 Svasti Haricharan,1 Shyam Kavuri,1 Jeremy Hoog,2 Chanpheng Phommaly,2 Rodrigo Goncalves,6 Susana Romalho,7 Wei-Chu Lai,2 Oliver Hampton,1 Anna Rogers,2 Ethan Tobias,2 Poojan Parikh,2 Sherri Davies,2 Cynthia Ma,2 Vera Suman,8 Kelly Hunt,9 Mark Watson,2 Katherine A. Hoadley,4 Aubrey Thompson,10 Charles Perou,4 Chad J. Creighton,1 Chris Maher,3 Matthew J. Ellis1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Washington University School of Medicine in St. Louis, MO;_ 3 _Washington University in St. Louis, MO;_ 4 _University of North Carolina Chapel Hill, NC;_ 5 _Kitasato University School of Medicine, Japan;_ 6 _University of Sao Paulo School of Medicine (FMUSP), Brazil;_ 7 _State University of Campinas, Brazil;_ 8 _Mayo Clinic, Rochester, MN;_ 9 _MD Anderson Cancer Center, Houston, TX;_ 10 _Mayo Clinic, Jacksonville, FL_.

Dysregulation of estrogen receptor gene (ESR1) is an established mechanism of inducing endocrine therapy resistance. We previously discovered a chromosomal translocation event generating an estrogen receptor gene fused in-frame to C-terminal sequences of YAP1 (ESR1-YAP1) that contributed to endocrine therapy resistance in estrogen receptor positive (ER+) breast cancer models. This current study compares functional and pharmacological properties of additional ESR1 gene fusion events of both early stage (ESR1-NOP2) and advanced endocrine therapy resistant (ESR1-YAP1 and ESR1-PCDH11x) breast cancers. The YAP1 and PCDH11x fusions conferred estrogen-independent and fulvestrant-resistant growth in T47D, an ER+ breast cancer cell line in vitro and in vivo, in contrast to the NOP2 fusion which was sensitive to hormone deprivation. Immunohistochemical (IHC) staining of mouse lungs revealed significantly higher numbers of micrometastatic ER+ cells from the T47D tumors expressing the YAP1 and PCDH11x fusions than YFP control and NOP2 fusion. Estrogen response element (ERE) reporter and pull down assays revealed that although all ESR1 fusions studied bound EREs, only the YAP1 and PCDH11x caused ERE activation. Cell lines containing these "canonical" ESR1 fusions upregulated expression of ER responsive genes such as TFF1 and GREB1 in hormone deprived conditions. In contrast, the NOP2 fusion neither induced ERE activity nor upregulated TFF1 and GREB1 gene expression. The proliferative ability of canonical fusion-containing T47D cells was inhibited by palbociclib, a CDK4/6 inhibitor, in a dose-dependent manner. In vivo growth of patient-derived xenograft tumors naturally harboring the ESR1-YAP1 fusion (WHIM18) was significantly reduced in mice fed palbociclib-containing chow. Mice transplanted with WHIM18 also formed lung micrometastases, with an ER IHC staining pattern similar to lungs from YAP1 and PCDH11x fusion expressing T47D xenografts. In conclusion, in-frame ERE activating canonical fusions occur in end-stage, drug resistant, advanced breast cancer and can be added to ESR1 point mutations as a class of somatic mutation that may cause acquired resistance. Endocrine therapy resistant growth induced by these fusions can be treated with CDK4/6 inhibition, using an FDA approved drug, palbociclib, which could potentially improve outcomes in patients with ESR1 translocated tumors.

## TUMOR BIOLOGY:

### Animal Models of Human Oncogenesis

#1034

Role of KEAP1/NRF2 and TP53 mutations in lung squamous cell carcinoma development and radiation resistance.

Youngtae Jeong,1 Ngoc Hoang,2 Henning Stehr,1 Alexander Lovejoy,1 Andrew Gentles,1 Aadel Chaudhuri,1 Billy Loo,1 Ash Alizadeh,1 Maximilian Diehn1. 1 _Stanford University School of Medicine, Stanford, CA;_ 2 _San Francisco State University, CA_.

Although lung squamous cell carcinomas (LSCC) comprise a large fraction of non-small cell lung cancers (NSCLCs), their pathogenesis and cell of origin remain incompletely understood and biomarkers that predict therapeutic responses are lacking. Here we describe novel, clinically relevant murine LSCC models driven by inactivation of Trp53 with or without Keap1, both of which are frequently mutated in human LSCCs. Homozygous inactivation of Keap1 or Trp53 promoted airway basal stem cell (ABSC) self-renewal both in vitro and in in vivo, suggesting that Trp53 or Keap1 mutations lead to expansion of mutant stem cell clones. Deletion of Trp53 with or without Keap1 in ABSCs, but not more differentiated tracheal cells, produced tumors recapitulating histologic and molecular features of human LSCCs. However, deletion of Trp53 with or without Keap1 in type II pneumocytes (ATIIs) or bronchioalveolar stem cells (BASCs) produced tumors with the features of adenocarcinoma, indicating that ABSCs represent the likely cell of origin for LSCC in this model. Deletion of Keap1 promoted tumor growth, metastasis and resistance to oxidative stress. N-acetylcysteine (NAC) treatment enhanced tumorsphere formation and metastasis in Keap1WT LSCCs, but not in Keap1-/- LSCCs, suggesting that NRF2-ROS pathway activation is the main mediator of Keap1 loss. Finally, Keap1 deletion induced radioresistance in vitro and in vivo in both LSCCs and lung adenocarcinomas (LUADs). Congruous with these findings, KEAP1/NRF2 mutation status strongly predicted risk of local recurrence in NSCLC patients treated with RT and these mutations could be non-invasively identified in circulating tumor DNA. These data suggest that Trp53 and Keap1 mutations in ABSCs play important roles in LSCC initiation and progression and identify KEAP1/NRF2 mutations as predictive biomarkers that could be used for personalization of therapeutic strategies for NSCLCs, and likely other cancers in which they are recurrently mutated.

#1035

Direct evidence for a pro-tumor role of APOBEC3A in cancer initiation and progression in vivo: enhanced mutagenesis and immune suppression in a novel humanized autochthonous model of pancreatic cancer.

Sonja Woermann,1 Robert Cowan,1 Susan M. Ross,2 Andrew D. Rhim1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _University of Illinois, Chicago, IL_.

Introduction: The apolipoprotein B editing complex 3 (APOBEC3) family of enzymes are possible candidates for inducing mutations across a number of tumors, including pancreatic ductal adenocarcinoma (PDA; Alexandrov et al, 2013; Roberts et al, 2013). APOBEC3A (hA3A) is one of eight identified isoforms in humans and known to deaminate cytidines in genomic ssDNA at a specific sequence motif. Large scale sequencing studies demonstrate significant enrichment of this signature in a number of human cancers. Furthermore, hA3A is overexpressed in a variety of solid tumors, including PDA. Taken together, these data suggest that hA3A may catalyze point mutations in cancer and drive cancer initiation and progression. However, direct evidence to support this hypothesis in vivo are lacking. Here, we utilize a novel genetically engineered mouse model to determine the precise effects of hA3A on PDA initiation and progression.

Methods: As opposed to humans, mice only contain one APOBEC3 isoform which has limited to no deaminating activity on genomic DNA. Thus, to delineate the function of hA3A on PDA initiation and progression, we exchanged the murine APOBEC3 protein coding sequence for hA3A, leaving the murine 5' and 3' endogenous regulatory sequences intact to ensure physiologic expression (murine APOBEC3 and hA3A reflect similar expression kinetics during PDA development). We then bred these mice to a well-established GEMM of PC (Rhim et al., Cell 2012) to yield LSL-KrasG12D; p53fl/+; Pdx1-Cre; RosaLSL-YFP; hA3A+/- (KPCY;hA3A) mice. We compared these mice to KPCY mice. We genotyped tumors from both cohorts with Ilumina exome capture sequencing with variant calling by a custom caller based on VarScan.

Results: All KPCY; hA3A (n=11) mice developed tumors and expired significantly faster compared to KPCY (n=18) controls (3.9 v. 7.0 mo; p<0.01). One third of the KPCY; hA3A mice developed macrometastatic disease compared to 3/12 in the KPCY control. Interestingly, KPCY; hA3A tumors contained dramatic desmoplasia, far surpassing the KPCY controls. Moreover, comparative RNAseq and histologic analyses revealed dramatic differences in the number and distribution of various T-cell and B-cell subsets. Finally, we found that KPCY; hA3A mice contained significantly more single nucleotide variants compared to KPCY controls (51.3 v. 8.4; p<0.05).

Conclusions: These data show for the first time that physiologic expression of hA3A in vivo leads to increased mutations, altered immune response and more aggressive cancer. Future studies will address the precise mechanisms by which hA3A catalyzes tumorigenesis using our novel gain of function model. Furthermore, our data suggest that incorporating our humanized A3A germline allele may provide a more genomically recapitulative model of cancer.

#1036

Non-myogenic origin of embryonal rhabdomyosarcoma.

Catherine J. Drummond, Matthew R. Garcia, Daniel J. Devine, Jennifer Peters, Victoria Frohlich, David Finkelstein, Mark E. Hatley. _St Jude Childrens Research Hospital, Memphis, TN_.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Despite aggressive chemotherapy, radiotherapy and surgery, clinical outcomes for RMS have not improved for three decades, emphasizing the need to uncover the molecular underpinnings of the disease. RMS includes two histopathologic subtypes: alveolar RMS, driven by the fusion protein PAX3/7-FOXO1, and embryonal RMS (ERMS), which is genetically heterogeneous. RMS has been presumed to originate from derailed muscle progenitors based on the histologic appearance and gene expression pattern of the tumors. However, an origin restricted to skeletal muscle does not explain RMS occurring in tissues devoid of skeletal muscle such as the prostate, bladder, biliary tree and the omentum. Previously, we showed that activation of Sonic Hedgehog signaling through expression of a conditional, constitutively active Smoothened allele, SmoM2, under control of an adipocyte-restricted adipose protein 2 (aP2)-Cre recombinase transgene in mice gives rise to aggressive skeletal muscle tumors that display the histologic and molecular characteristics of human ERMS. In this model, tumorigenesis occurs with high penetrance (~80%), is early onset (by 2 months of age), and is restricted to the head and neck. Also, unlike previous RMS models, this model requires no additional background mutations, such as inactivation of p53, and results in only ERMS neoplasia. We illustrated that the gene expression signature of the aP2-Cre;SmoM2 tumors recapitulates both other mouse ERMS models as well as human ERMS. With the short latency and anatomic restricted tumor location, we sought to leverage this model to explore the cell of origin. Here, we use genetic fate mapping of aP2-cre with reporter mice to determine the effect of constitutive hedgehog activation on the identity and localization of aP2-Cre expressing cells. aP2-cre expressing cells are found in both brown and white adipose and to be localized within the muscle intersitium but not beneath the laminin sheath of the muscle fibers. These aP2-cre expressing cells are distinct from Pax7-positive skeletal muscle stem cells or satellite cells and do not contribute to myofiber formation. Instead these cells were confirmed to be endothelial by gene expression analysis. Colocalization between aP2-cre expressing cells and CD31 was also observed. When compared to aP2-Cre;R26-Tom mice, the addition of oncogenic SmoM2 (aP2-Cre;R26-Tom;SmoM2) resulted in embryonic expansion of these aP2-lineage interstitial muscle cells and formation of ERMS. Our findings suggest that non-skeletal muscle progenitors are a cell of origin for Sonic Hedgehog-driven ERMS.

#1037

Progression from melanocytic nevi to melanoma is associated with increased genomic mutations in a UV-induced mouse model of human melanoma.

Helen Michael, Chi-Ping Day, Howard Yang, Aleksandra Michalowski, Maxwell Lee, Glenn Merlino. _National Institutes of Health, Bethesda, MD_.

Melanoma is the deadliest form of skin cancer with approximately 132,000 cases worldwide each year. Benign melanocytic nevi are nearly universal, and although progression of nevi to melanoma is very rare, 20-50% of melanoma appear to arise from a pre-existing nevus. UV exposure, particularly childhood sunburn, is believed to play an important role in the development of melanocytic nevi and melanoma, but the exact mechanism is unknown. Alterations in MAPK pathway genes, especially NRAS and BRAF, are common in both benign nevi and melanoma, but approximately 1/3 of melanomas do not have an identified driver mutation. Studying nevus initiation and progression prospectively in the human population is impractical due to the long latency to progression and repeated UV exposures Our laboratory has developed a hepatocyte growth factor (HGF) genetically engineered mouse model with "humanized" junctional distribution of melanocytes on an iDCT-GFP background with melanocyte-specific GFP expression, allowing melanocytic lesions to be tracked through percutaneous GFP imaging. Following a single relevant dose of UV modeling childhood sunburn, HGF iDCT-GFP develop discrete, small melanocytic lesions consistent with nevi. Most nevi remain stable over the lifetime of the mouse, but around 1 in 30 progress to melanoma usually starting at 6-12 months. The melanocytic lesions are histologically similar to human nevi and melanoma, label with melanocyte markers and tumors are transplantable into syngeneic mice. Melanomas that arise in the model are heterogeneous and include radial growth phase and vertical growth phase tumors and sometimes metastasize to liver and lung. Exome sequencing of 28 nevi and melanomas show that vertical growth phase melanomas have approximately 3x more mutations than radial growth phase melanomas or nevi. The increased number of mutations in vertical growth phase tumors is due to an increase in C>T transitions despite the lack of additional UV exposure. Interestingly, melanocytic nevi and melanomas with DNA repair pathway mutations average 3x more mutations than lesions without mutations in these pathways. Melanomas sometime contain mutations in hotspot locations from human melanomas, including GNAQ, but most do not have a previously identified dominant driver. Genes potentially involved in the initiation of melanocytic lesions or progression to aggressive melanomas and relevant to human melanoma have been identified and are being functionally tested using CRISPR to introduce point mutations or knock out genes and in vitro skin reconstitution assays. Identification of novel drivers and pathways involved in non-BRAF, non-NRAS melanoma has the potential to uncover biomarkers and new therapeutic targets to improve clinical outcomes for melanoma patients.

#1038

The KIT-V654A second site mutation confers perinatal lethality and increased oncogenesis in a mouse model of GIST.

Jennifer Q. Zhang, Benedikt Bosbach, Cristina Antonescu, Peter Besmer, Ronald DeMatteo. _MSKCC, New York, NY_.

Tyrosine kinase inhibitors have revolutionized the treatment of gastrointestinal stromal tumor (GIST), which are mostly driven by mutations in the receptor tyrosine kinase KIT. However, resistance commonly develops, which is associated with second site mutations in KIT. Here, we created the first genetically engineered mouse model of the most common second site mutation, KIT-V654A (mouse KIT-V653A), to study in vivo its oncogenic properties and mechanisms of resistance in order to develop next-generation GIST therapies.

The knock-in strategy consisted of inserting into the endogenous murine Kit locus a targeting vector containing both the KitV558Δ exon 11 mutation and the KitV653A exon 13 mutation under the control of a floxed neomycin-resistance gene-expression cassette which acts similar to a loxP-STOP-loxP cassette. Treatments included imatinib 45 mg/kg i.p., sunitinib 40 mg/kg p.o., and cabozantinib 60 mg/kg p.o. Tumors were assessed by histology, immunohistochemistry, and western blot.

In contrast to our previously published GIST model with a single mutant KitV558Δ allele, the double knock-in KitV558Δ; V653A-neo allele caused perinatal lethality when activated in pre-implantation embryos with a germline EIIa-Cre approach. We therefore sought to restrict the induction of the KitV558Δ;V653A double mutation to the interstitial cells of Cajal (ICC), the cell of origin of GIST, via the essential ICC lineage survival factor ETV1. The KitV558Δ; V653A-neo/+ mouse was crossed with the Etv1Cre-ERT2/+ mouse expressing Cre recombinase under the Etv1-specific promoter. Systemic administration of tamoxifen at birth in KitV558Δ; V653A-neo/+; Etv1Cre-ERT2/+ mice resulted in cecal GIST development with full penetrance. The tumors were histologically similar to human GIST, stained positive for KIT, and displayed active p-KIT signaling. As expected, high-dose imatinib treatment did not inhibit p-KIT Y719 in these GISTs and did not induce a histologic response. Sunitinib and cabozantinib each had significant anti-tumor effects, as assessed by decreased Ki67 index and increased histologic response. Compared to single mutant KitV558Δ-neo/+; Etv1Cre-ERT2/+ mice, double mutant KitV558Δ; V653A-neo/+; Etv1Cre-ERT2/+ mice developed larger cecal GISTs and had decreased survival.

The KitV558Δ; V653A-neo/+; Etv1Cre-ERT2/+ mouse is the first in vivo model of the most common secondary mutation in GIST and the first in vivo demonstration that cell-autonomous expression of mutant KIT in the ICC lineage gives rise to GIST. We have found cabozantinib, an FDA-approved drug, to be an effective therapy in GISTs harboring the V653A mutation. Furthermore, our results suggest the addition of the KIT-V653A second site mutation leads to increased oncogenesis.

#1039

**Disruption of circadian clockwork in** in vivo **reprogramming induced mouse kidney cancer and human Wilms tumor.**

Munehiro Ohashi,1 Yasuhiro Umemura,1 Yoichi Minami,1 Hitomi Watanabe,2 Tomoko Tanaka,3 Tsuneharu Miki,4 Osamu Ukimura,4 Tatsuro Tajiri,3 Gen Kondoh,2 Yasuhiro Yamada,5 Kazuhiro Yagita1. 1 _Department of Physiology and Systems Bioscience, Kyoto Prefectural University of Medicine, Kyoto, Japan;_ 2 _Laboratory of Animal Experiments for Regeneration, Institute for Frontier Medical Science, Kyoto University, Kyoto, Japan;_ 3 _Department of Pediatric Surgery, Kyoto Prefectural University of Medicine, Kyoto, Japan;_ 4 _Department of Urology, Kyoto Prefectural University of Medicine, Kyoto, Japan;_ 5 _Center for iPS Research and Application, Kyoto University, Kyoto, Japan_.

Most organisms have evolved intrinsic circadian clock to optimize their behavior and physiology for diurnal environmental changes. The circadian clock exists not only in the organismal level but also in the peripheral organs and cellular levels, and regulates the various physiological aspects. Among these regulation systems, the relationship between circadian clock and cancer has been enthusiastically investigated in this decade. However, the mechanistic link connecting circadian clock and cancer has not been fully understood. Recently, we have clarified that circadian clock is tightly connected with cellular differentiation using the in vitro mouse embryonic stem cells (ESCs) differentiation model, and in addition, misregulation of differentiation leads to the circadian clock disruption via highly expression of KPNA2 and suppression of CLOCK protein. On the other hand, it was revealed that altered dedifferentiation leads to cancer development using in vivo reprogramming mouse model. So we considered that the viewpoint of dysdifferentiation can help to provide significant pathophysiological implications for the relationship of circadian clock and cancer. Firstly, we established Rosa26:M2-rtTA TetO:OSKM ES cells carrying circadian rhythm reporter mPer2:luc, and generated chimeric mice. The chimeric mice were treated with doxycycline (Dox) for transient in vivo reprogramming. The Dox treatment induced Wilms tumor-like kidney tumors and we sectioned the kidney tumor for monitoring real time bioluminescence signals, and examined the RNA and protein expression profiles. Here, we showed that the circadian clock was disrupted in the Wilms tumor-like mouse kidney tumor tissues, while the control mouse kidney exhibited the obvious circadian bioluminescence oscillation. And the gene expression signature of the circadian clock development correlated gene set in the tumor was similar to the dysdifferentiation-mediated circadian clock disrupted cells. Moreover, the highly expressed KPNA2 and suppression of CLOCK protein were also observed in the mouse tumor cells. Next, we examined human Wilms tumor gene and protein expression profiles. Astonishingly, the transcriptional signature of the circadian clock development correlated genes was similar to the mouse kidney tumor induced by dedifferentiation. Furthermore, the protein expression patterns of KPNA2 and CLOCK coincided with them. These findings suggest that the severely dedifferentiated cancers may lose their circadian clocks and the mechanism is common with the suppression mechanism during differentiation coupled circadian clock development. Moreover, circadian clock functionality may reflect the etiology of cancer development including dedifferentiation. In future prospects, a new view point of the circadian clock may help to evaluate the type of cancer cells, and it leads to more improved therapeutic strategy.

#1040

**LIN28B-mediated** let-7 **independent activation of AKT promotes neuroblastoma pathogenesis.**

Ting Tao,1 John T. Powers,2 Hui Shi,1 Pavlos Missios,2 Antonio R. Perez-Atayde,2 Shizhen Zhu,3 George Q. Daley,2 Thomas A. Look1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Boston Children's Hospital, Boston, MA;_ 3 _Mayo Clinic, Rochester, MN_.

LIN28 is well known as a RNA-binding protein and a suppressor of microRNA biogenesis, by selectively blocking the processing of let-7 precursors. It plays diverse functions in cellular reprogramming, development, metabolism and tumorigenesis. Many of these functions are executed through its ability to inhibit let-7 maturation. However, little is known about its function independent of let-7. Here we made zebrafish transgenic lines expressing high levels of either wild-type or mutant LIN28B (does not block the maturation of let-7) in the peripheral sympathetic nervous system driven by the dopamine beta-hydroxylase promoter. We bred these lines with a transgenic zebrafish line overexpressing the MYCN oncogene in these cells. Either wild-type or mutant LIN28B overexpression accelerated the onset and increased the penetrance of MYCN-induced neuroblastoma, despite the fact that only wild-type LIN28B blocked let-7 maturation compared to MYCN-only tumors. Mechanistically, both wild-type and mutant LIN28B overexpression enhanced MYCN-induced hyperplasia by increasing cell proliferation in the sympathoadrenal lineage. Further studies revealed that overexpression of either wild-type or mutant LIN28B resulted in hyperphosphorylation of AKT on both Ser473 and Thr308 in zebrafish tumors and also serum-deprived human neuroblastoma cell lines. Both wild-type and mutant LIN28B interacted with IGF2BP1, a known interactor that increases stability and translation of a subset of RNAs. We found IGF2 RNA levels to be increased in response to either form of LIN28B. Coincident with IGF2 overexpression, IGF1R was phosphorylated, providing an explanation for PI3K-AKT activation in LIN28B overexpressing cells. Finally, overexpression of a constitutively active, myristoylated murine Akt2 (myr-mAkt2) alone in zebrafish induced ganglioneuroma in the interregnal gland (the zebrafish equivalent of the human adrenal medulla), without a requirement for MCYN overexpression. Thus, our studies indicate that overexpression of LIN28B leads to AKT activation mediated through its interaction with IGF2BP1 and subsequent upregulation of IGF2. This pathway provides a mechanism underlying enhanced transformation in LIN28B overexpressing neuroblastomas, which is independent of the inhibitory activity of LIN28B on let-7 maturation.

### Niches and Routes of Metastasis

#1041

Binding of soluble DC-HIL to endothelial cell creates immunosuppression on premetastatic niches.

Vijay Ramani,1 Takahiro Teshima,2 Kyoichi Tamura,2 Jin-Sung Chung,1 Ponciano Cruz,1 Kiyoshi Ariizumi1. 1 _UT Southwestern Medical Center, Dallas, TX;_ 2 _Nippon Veterinary and Life Science University, Tokyo, Japan_.

Highly metastatic attribute of melanoma can be recapitulated in a mouse model by high potential of B16 melanoma-derived soluble factors that form more premetastatic niches (PMNs) in distal organs than those of LL2 lung carcinoma. Having shown that the T cell-inhibitory receptor DC-HIL is a potent promoter of B16 melanoma growth and that B16 cells secrete it into blood circulation as a soluble receptor (sDC-HIL), we hypothesized that sDC-HIL accounts for the metastatic nature. We tracked sDC-HIL in various organs of melanoma-bearing mice by immunoblotting and showed that sDC-HIL was expressed in particular organs; at highest levels in bone marrow (BM) and lung; and no-to-little in lymphoid organs and liver. We next examined effects of sDC-HIL on LL2 cells (no DC-HIL expression). In a model of spontaneous metastasis quantified by colonogenic assay, syngeneic mice implanted subcutaneously with LL2- transfected-sDC-HIL cells produced 6-fold more lung mets than mice with LL2-transfected-GFP. We obtained similar results in a model of experimental metastasis (EM), in which LL2 cells injected i.v. in tumor-bearing mice. To establish specificity, similar mice were treated i.v. with anti-DC-HIL mAb 2 h before and 24 h after infusion of LL2 cells: The mAb led to 80% reduction in lung mets in mice with sDC-HIL-LL2 tumor. Next we infected tumor-free mice with lentiviruses for sDC-HIL/GFP or GFP gene and then performed the EM assay: lung mets in mice expressing sDC-HIL were 5-fold greater than control. To determine whether sDC-HIL is responsible for the ability of B16-conditioned media (CM) to induce metastasis, tumor-free mice were injected i.p. daily with CM from LL2, B16, or DC-HIL-knocked down (KD) B16 cells and then assayed for EM. B16-CM induced significantly more lung mets than LL2-CM, with mets almost corresponding to that produced by KD-B16-CM. We tracked the fate of sDC-HIL in lung by immunofluorescence and showed sDC-HIL accumulated on particular (not all) endothelial cells (EC), at sites where LL2 and BM-cells migrated. Kinetic studies in BM-chimera mice showed that sDC-HIL arrives at lung sites before VEGFR1+ BM-progenitors, which initiate formation of PMNs. Since the mets-enhancing effect of sDC-HIL was not seen in immunodeficient mice, we asked whether lungs expressing sDC-HIL were immunosuppressive. CFSE-labeled CD8+ T cells were infused into mice with sDC-HIL- or GFP-LL2 tumors, and their proliferation assayed by FACS: T-cell proliferation in lungs expressing sDC-HIL was reduced significantly (28%) vs. control (81%). Because sDC-HIL by itself is not suppressive, we asked whether binding of sDC-HIL by EC makes immunosuppressive. CD8+ T cells were co-cultured with sDC-HIL-bound EC and then co-stimulated: sDC-HIL-bound EC completely suppressed the IFN-γ response. Thus, sDC-HIL critically contributes to the highly metastatic potential by making PMNs immunosuppressive even before the arrival of tumor cells.

#1042

CLIC4 is incorporated into extracellular vesicles of murine breast cancer cells and may influence metastatic burden.

Alayna B. Craig-Lucas, Vanesa C. Sanchez, Abigail Read, Ji Lou, Anjali Shukla, Stuart H. Yuspa. _NCI, Bethesda, MD_.

Chloride intracellular channel 4 (CLIC4) is an evolutionarily conserved, 29kD, dimorphic protein that contributes to TGF-β signaling by preventing the de-phosphorylation of phospho-SMAD2/3 upon nuclear translocation. In several cancer types, CLIC4 is excluded from the nucleus and downregulated in the cytoplasm of the tumor cells as the tumor progresses, suggesting that CLIC4 acts as tumor suppressor. In a parallel sequence, CLIC4 becomes upregulated in the stromal compartment, where it enhances tumor growth and invasion. Recent reports have suggested that CLIC4 is detectable in the serum of cancer patients and incorporated into extracellular vesicles, and has potential as a biomarker. We hope to gain a better understanding of the role that CLIC4 plays in the tumor stromal and epithelial compartments as well as their respective release of extracellular vesicles. Using in-vitro and in-vivo assays, we have conducted experiments using the FVB mouse MMTV-c-MYC 6DT1 breast cancer model. By CRISPR/ Cas9 system, CLIC4 was deleted from wild type 6DT1 cells. Following clonal selection, the loss of the CLIC4 protein at both the cellular and released vesicle level was validated. Both functional assays on CLIC4 deleted clones and evaluation of their extra-cellular vesicles were undertaken in order to further understand their tumorigenic and metastatic capabilities. In-vitro, CLIC4 was not necessary for vesicle biogenesis and its deletion did not have a significant effect on cellular proliferation. In vivo, selected clones were orthotopically injected into the 4th mammary fat pad of wild type FVB mice. Compared to wild type 6DT1 clones, CLIC4 deleted clones formed primary tumors that had greater mass but a fewer number of lung metastasis. Future studies are designed to isolate vesicles circulating in tumor bearing hosts to determine their stromal or epithelial origin and to provide a better understanding of the role that CLIC4 may play in tumor growth, creating a metastatic niche and as a potential serological biomarker.

#1043

CSF-1R inhibitor prevented pre-metastatic lung niches in metastatic mammary tumor.

Thaiz F. Borin, Kartik Angara, Mohammad Rashid, Adarsh Shankar, Asm Iskander, Roxan Ara, Meenu Jain, Bhagelu R. Achyut, Ali S. Arbab. _Augusta University, Augusta, GA_.

Exosomes are small vesicle cellular products originating from the endocytic pathway abundantly secreted by tumor cells. These exosomes have the ability to alter their immediate microenvironment (ME) through cell-cell interaction by fusion with plasma membrane and subsequent endocytosis or release of their cargo. Exosomes are critical modulators of pre-metastatic niche creation by increasing the recruitment of inflammatory cells. In breast cancer, tumor-derived exosomes recruit myeloid derived suppressor cells in the creation of an immunosuppressive pro-tumorigenic lung niches. Colony stimulating factor 1-receptor (CSF1R), which is a key regulator of myeloid cell proliferation, survival, and differentiation can be blocked by a selective inhibitor GW2580. Overexpression of CSF-1 has been implicated in the creation of increased number of metastatic niches in numerous cancers. In our study, we have demonstrated that the exosomes secreted under hypoxic conditions can initiate early pre-metastatic niche creation in lungs in a metastatic breast cancer model compared to normoxia-secreted exosomes. Exosomes were injected intravenously into Balb/c female mice three days after the implantation of 4T1 breast tumor and continued for a week with injections on alternated days. The animals were pre-treated with GW2580 the day before tumor implantation and continued for a week concomitantly with exosomes on alternate days. Lungs, bone marrow, spleen and brain tissues were collected and analyzed by flow cytometer to detect myeloid and angiogenic cells populations. GW2580 was able to prevent myeloid cell infiltration in lungs and bone marrow. Further, we observed significant increase in anti-tumorigenic M1-macrophage population in the lungs of exosome treated animals pre-treated with GW2580. However, these findings were not observed in the bone marrows of the same group. Vasculogenic leukocyte and angiogenic myeloid cell populations were significantly decreased in the lung of exosome treated animals pre-treated with GW2580. A similar decrease in these populations of cells was not seen in the lungs of animals pre-treated with GW2580 only. These surprising results have led us to hypothesize that GW2580 treatment can prevent the effects of exosomes, which causes infiltration of myeloid cells in the lung to create metastatic niche. These observations indicate a role of CSF1R inhibitor in preventing the distant metastatic niche formation.

#1044

Development of a miRNA-based signature to predict human cancer metastasis.

Lian Willetts, Konstantin Stoletov, Juan Jovel, Emma Woolner, John D. Lewis. _University of Alberta, Edmonton, Alberta, Canada_.

Metastasis is the primary cause of death for cancer patients, and invasive cancer cell migration is required at multiple steps during metastasis (e.g. intravasation and extravasation). Since microRNAs (miRNAs) have been implicated as key regulators of metastatic spread of cancer cells, we sought to develop a miRNA signature that can be used to predict cancer metastasis. We hypothesized that the miRNAs that are functionally required for invasive cell migration could serve as biomarkers to predict human cancer metastasis.

We developed an intravital imaging-based approach combined with NGS to screen for miRNAs that are required for invasive cell migration of human HT1080 fibrosarcoma cells. The screen-identified miRNAs were validated in a panel of in vitro and in vivo assays for invasive migration. Microarray analysis identified genes downregulated in transfected HT1080 cells. Publically available databases were used to correlate the expression of screen-identified miRNAs and the progression of multiple human cancers. We used a qRT-PCR approach combined with machine learning to develop a miRNA signature to predict metastasis in a 66-patient cohort of prostate cancer.

We identified over twenty novel miRNAs that regulate directional cancer invasion. Microarray analysis in HT1080 cells revealed that the altered expression of metastasis-regulating miRNAs is associated with 50% reduction in gene expression of migration and adhesion gene network components such as integrin a4, CDC42, and transgelin).

We evaluated the potential of screen-identified miRNAs to serve as biomarkers to predict cancer metastasis. Using publically available databases, we found that majority of screen-identified miRNAs are dysregulated in multiple human cancer types (e.g. prostate, breast, ovarian and lung) and the expression of these miRNAs correlate directly with patient disease progression. We analyzed the expression of the top two screen-identified miRNAs in plasma samples from the PCa patient cohort. A signature was generated using a weighted K-nearest neighbor algorithm that provided a ROC area under the curve of 0.79 for predicting metastatic disease.

We identified a panel of novel metastasis-regulating miRNAs that is functionally involved in human cancer metastasis. These miRNAs have the potential to serve as both biomarkers to predict metastasis and potentially as therapeutic targets to block metastasis.

#1045

Understanding breast cancer metastasis through circulating tumor cells.

Remi Klotz, Thomas Amal, Alan Wang, Matthew Mackay, Kathleen Heller, Lin Li, Maxwell Serowoky, Grace Lee, Jane Han, Andrew Smith, Min Yu. _USC, LOS ANGELES, CA_.

Due to the advance in technologies for rare cell isolation, circulating tumor cells (CTC) have recently received vast interest. During tumor progression tumor cells invade the primary tumor microenvironment and intravasate into blood vessels, where they are referred as CTCs. These CTCs disseminate to other organs and a subset of these cells will be able to form metastasis. The growing interest for CTC is confronted with the difficulty associated with their isolation and characterization. To address this challenge, our lab has recently optimized the ex vivo culture condition and was able to establish CTC lines from breast cancer patients. These CTC lines constitute a unique cell population in the metastatic process and gave us a rare opportunity to investigate the signal pathways involved in each step of the metastatic cascade. To assess the metastatic potential of breast cancer patient-derived CTC lines we utilized an experimental mouse model for metastasis by injecting CTCs directly into the left ventricle of the heart in female immuodeficient NSG mice. The ability of 4 CTC lines to arrest and colonize in organ was monitored by bioluminescence imaging once every 2 weeks for 5 months. CTC lines were capable of generating brain, lung, bone and ovary metastases. Most of those organs are common sites of metastases in breast cancer patients. Two patient-derived CTC lines have a high metastatic potential (over 80% of mice had metastases after 3 months) with generation of simultaneous metastases in multiple organs such as bone, lung and Ovary. These mice remained brain-metastases free for up to 8 months. However, two other patient-derived CTC lines demonstrated the brain as preferential site of metastasis despite their overall low metastatic potential. Interestingly, of 4 breast cancer patients where CTC lines were generated, one developed a metastatic brain tumor and her CTC line has the highest risk of brain metastases in our mouse model. We further investigated genetic and epigenetic determinants that regulate the organotropism of CTCs. We isolated metastatic variants corresponding to a subpopulation of CTCs with a preferential tropism for the brain, lung and bone. Gene expression analysis (RNA-seq) of these variants identified potential gene signatures of breast cancer metastasis. The pathways with the highest enrichment scores were glioblastoma multiform, ceramide biosynthesis and PCP pathway for the brain metastatic variants and were interferon and mTOR signaling for the lung metastatic variants. Next we used a method for assaying chromatin accessibility (ATAC-seq) and identified potential regulatory regions mediating the organ tropism in breast cancer. Together our data provide the evidence of a promising role of CTC as an early prognostic factor in metastasis. Additionally, we expect to develop novel organ tropism associated markers, which can be considered for potential therapeutic targets in breast cancers.

#1046

Conditional knockout of N-Myc and STAT Interactor disrupts normal mammary development and enhances metastatic ability of mammary tumors.

Hawley Christine Pruitt, Brandon J. Metge, Shannon E. Weeks, Dongquan Chen, Shi Wei, Lalita A. Shevde, Rajeev S. Samant. _Univ. of Alabama at Birmingham, Birmingham, AL_.

The process of development requires a delicate balance between plasticity and differentiation. This balance is disrupted in cancer initiation and progression, ultimately ending in metastasis and plummeting rates of patient survival. Recent findings from our lab have revealed that protein expression of evolutionarily conserved, N-Myc and STAT Interactor (NMI) is decreased in 70% of primary patient specimens with metastatic breast cancer. To study how Nmi loss facilitates metastatic behavior and gain insight into its role in normal mammary biology, we created a novel mammary specific Nmi knock out mouse model, which we then challenged with carcinogen and oncogene induced tumorigenesis. Our studies show that Nmi is induced at the onset of pregnancy and its expression remains throughout lactation. Furthermore, prolactin stimulation and differentiation of HC11 murine mammary epithelial cells is accompanied by up-regulation of Nmi. STAT5 is the downstream effector of prolactin and is essential for differentiation of secretory alveolar epithelium. NMI interacts with STAT5 and this interaction appears to be critical to normal differentiation of mammary epithelium. Indeed, loss of Nmi in vivo caused a decrease in STAT5 activity and a subsequent transcriptomic shift in mammary epithelial and breast cancer cells. Moreover, knockdown of Nmi in HC11 cells impedes the expression of beta-casein upon differentiation with prolactin. Concurrently, Nmi knock out alveoli exhibit an extensive presence of nuclear beta-catenin, a mediator of stem cell maintenance in the mammary gland. As a result, mice exhibit an increased number of alveoli and more proliferating mammary epithelial cells (MECs). The Nmi knock out pubertal ductal tree extends into the mammary fat pad at an accelerated rate and exhibits enhanced terminal end bud numbers, a phenotype that mirrors mice altered for Wnt signaling. Tumors derived from Nmi null mammary epithelium contain significantly more cells with stem and progenitor markers, indicating that these tumors are less differentiated than their littermate counterparts. In addition, Nmi null mammary tumors exhibit invasive morphology as well as enhanced distant metastasis. We observe that conditional Nmi loss disrupts differentiation in the mammary gland and promotes the progression of tumors with aggressive metastatic characteristics.

#1047

Role of endocytosis in NM23 mediated motility suppression.

Imran Khan, Patricia S. Steeg. _National Institute of Health (NIH), Bethesda, MD_.

NM23 has been identified as a metastasis suppressor gene and is known to inhibit motility of cancer cells and suppress metastasis in multiple in vivo model system. Biochemically, NM23 is a nucleoside diphosphate kinase (NDPK). As an NDPK, NM23 is first autophosphorylated on its histidine 118 before transferring its phosphate to an NDP. NM23 is also known to trans-phosphorylate other proteins to act as a protein histidine kinase (PHK) using same histidine H118 phosphorylation intermediate. In spite of many studies on NM23 in different cancers, the molecular mechanism of NM23 action has not been well worked out. Studies on Drosophila homologue of the NM23, awd, highlights its role in regulating cell differentiation and motility by influencing endocytosis. We now hypothesize that NM23 mediated motility/metastasis suppression could be due to increased endocytosis of cell surface receptors, needed for cellular motility. We over expressed NM23 (human H1 and H2 and murine h1) in MDA-MB-231T breast cancer cells and observed that the motility suppression phenotype (4 fold, p<0.0001) of NM23 correlates with increase in NM23 histidine auto phosphorylation, as observed by using a new anti-histidine 1-phosphate (1-pHis) antibody. NM23 over expressing cells were studied for the level of endocytosis using Transferrin-Alexa-594 and pHrodo-Red-EGF labelled dyes. It was observed that NM23 over expression causes increased endocytosis of both the above labelled ligands. Based on the literature, Dynamin is known to be involved in endocytosis and is also an interacting partner of NM23. We validated NM23 and Dynamin interactions by pulldown studies (using both anti-NM23 and anti-Dynamin antibodies) in NM23 over expressing cells. We also observed reduced endocytosis with Dynamin inhibitors. Further, when a series of Dynamin inhibitors (MiTMAB, OcTMAB, Dynole-34-2 and Iminodyn-22) were used in motility assays, they were found to be inhibitory to motility. However, no further decrease in motility was observed upon NM23 overexpression, highlighting the reversal of NM23 mediated motility suppression in absence of Dynamin. To further address the role of endocytosis in NM23 mediated motility suppression, two crucial mutants of NM23 namely; P96S (has NDPK activity with very low HPK activity) and H118F (has no NDPK and HPK activity) were made and stable cells were generated. We observed that the motility suppression phenotype of NM23 was reverted to normal in both the mutants, highlighting a possible role of HPK activity in motility suppression. Similarly, endocytosis studies on these cells shows loss of increased endocytosis in both the mutants. To date, the data indicate that NM23 phosphohistidine levels, motility suppression and increased endocytosis are well correlated and is being influenced by Dynamin signaling. Different approaches to further understand the physiological meaning of NM23-dynamin interactions are underway. 

# Monday, April 3, 2017

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

### Combination Strategies: Novel Agents and Standard Therapies

#1048

Reactive oxygen species-mediated synergistic and preferential induction of cell death and reduction of drug resistance in oncogenic H-Ras-expressing bladder cancer cells by combined romidepsin and cisplatin with gemcitabine.

Lenora A. Pluchino, Hwa-Chain R. Wang. _Univ. of Tennessee, College of Veterinary Medicine, Knoxville, TN_.

Human urinary bladder cancer is the fifth most commonly diagnosed cancer in the United States and is often associated with induction of oncogenic H-Ras. Long-term survival of patients is suboptimal with the current chemotherapeutic regimen of combined cisplatin and gemcitabine and others due to acquired drug resistance and recurrence. Thus, it is urgent to develop new regimens effective in control of tumor growth, drug resistance, and recurrence to reduce the morbidity and mortality of this disease. We used our cellular system, consisting of human urinary bladder cancer J82 cells paired with its derived oncogenic H-Ras-expressing J82-Ras cells, to investigate the efficacy of a novel combination regimen in treating bladder cancer cells. Our studies revealed, for the first time, the ability of a combination of cisplatin and romidepsin with gemcitabine to synergistically and preferentially induced cell death and reduced drug resistance in J82-Ras versus J82 cells. The Ras-ERK-Nox pathway played an essential role in mediating signals to elevate reactive oxygen species (ROS), leading to enhanced caspase activation, DNA damage, and DNA oxidation, as well as reduced glutathione, to synergistically increased cell death and reduced drug resistance in cells treated with combined romidepsin, cisplatin, and gemcitabine. Preferentially induced Ras-ERK-Nox-ROS pathway, caspase

activation, and DNA damage/oxidation, as well as reduced glutathione, contributed to the preferentially induced cell death and reduced drug resistance in J82-Ras versus J82 cells. Synergistically induced death and reduced drug resistance were also detected in human bladder cancer SW780 cells treated with combined romidepsin, cisplatin, and gemcitabine. Hence, our results lead us to suggest that a combination of romidepsin, cisplatin, and gemcitabine should be seriously considered as a new therapeutic regimen for controlling the development and recurrence of human urinary bladder cancer, especially Ras-ERK-activated cancers.

#1049

Combinatorial effects of thymoquinone on the anticancer activity of doxorubicin in adult T-cell leukemia.

Hala Gali-Muhtasib,1 Isabelle Fakhoury,1 Maamoun Fatfat,1 Rasha Mismar,1 Regine Schneider-Stock2. 1 _American University of Beirut, Beirut, Lebanon;_ 2 _University Erlangen, Nuremberg, Germany_.

Doxorubicin (Dox) is a clinically approved drug which suffers from drug resistance and cardiotoxicity. Recent studies have shown that thymoquinone (TQ) in combination with Dox can reduce Dox toxic side effects in vitro as well as in vivo. Both TQ and Dox have shown promising anticancer effects against aggressive adult T-cell leukemia (ATL), however, the anticancer potential of TQ and Dox combination treatment has never been tested against ATL. We hypothesized that co-treatment with TQ could enable the use of lower doses of Dox to achieve similar or enhanced anticancer activity. The effects of TQ and Dox combination on cell death and cell cycle were evaluated by trypan blue and propidium iodide. TUNEL assay was used to investigate the mode of cell death. The levels of reactive species (ROS) were determined using DCFH assay, and mitochondrial membrane potential was measured by rhodamine assay. The regulation of key proteins involved in cell cycle regulation and cell death was determined by Western blot. The results reveal that the human T-lymphotropic virus (HTLV-1) positive HuT-102 cells are more resistant to treatment with Dox alone, than the HTLV-1 negative Jurkat cells. However, treatment with high doses of TQ and low doses of Dox simultaneously, enhances cell death in both cancer cell lines as compared to treatment with Dox alone. TUNEL assay on Jurkat and HuT-102 cells further indicated that the combination of TQ and Dox caused cell death by apoptosis. An increase in ROS production was noted in response to treatment with TQ alone and

with TQ and Dox combination in both Jurkat and HuT-102 cells. The oxidative stress; however, was only shown to play a role in the disruption of the mitochondria of Jurkat cells. Similarly, caspase activation was involved in the disruption of the mitochondria of Jurkat cells. The expression levels of key regulatory proteins were modulated in response to treatment with the combination. In conclusion, the combination of TQ and Dox effectively inhibits ATL leukemic cancer cell growth at lower doses of Dox which can potentially lower the side effects of the drug. In vivo studies are still warranted to assess the adjuvant chemotherapeutic potential of TQ in combination with Dox.

#1050

Mefloquine enhances the cytotoxic effects of tyrosine kinase inhibitors in blast phase chronic myeloid leukaemia by lysosome membrane disruption.

Wei Xiang,1 Yi Hui Lam,1 Colin Sng,2 May Anne Cheong,1 Hein Than,1 William YK Hwang,1 Charles Chuah1. 1 _Singapore General Hospital, singapore, Singapore;_ 2 _Duke-NUS medical school, singapore, Singapore_.

Despite the remarkable clinical responses achieved with BCR-ABL tyrosine kinase inhibitors (TKIs) in the treatment of chronic phase-chronic myeloid leukaemia (CML), these TKIs have been less effective as single agents in blast phase (BP) CML. Identification of new therapeutic strategies is needed for the better clinical management of BP-CML. Given the known pharmacokinetics and toxicity profile, drug repurposing represents an alternative therapeutic strategy for CML. Mefloquine is a FDA-approved antimalarial drug that has been recently reported to have anti-cancer activities. In this work, we investigated the effect of mefloquine and its underlying mechanisms in CML.

We show that mefloquine inhibits proliferation and induces apoptosis of CML cells in a dose-dependent manner. In addition, mefloquine is also effective in targeting BP-CML CD34+ progenitor cells. It induces apoptosis, inhibits colony formation and self-renewal capacity of CD34+ cells derived from a TKI-resistant BP-CML patient. Mefloquine significantly enhanced anti-proliferative and pro-apoptotic effects of imatinib and dasatinib in CML cell lines (eg, K562, LAMA84 and KU812) as well as BP-CML CD34+ cells, suggesting that mefloquine augments the effects of BCR-ABL TKIs.

Mechanistically, mefloquine increases lysosome biogenesis in various CML cell lines. The action of mefloquine on lysosome is further confirmed by our observation that the lysosomal vacuolar ATPase bafilomycin A1 rescues mefloquine-induced CML cell death. Mefloquine also induces oxidative stress in CML cells as shown by the increased mitochondrial superoxide levels. Importantly, α-tocopherol (known to inhibit lysosomal lipid-mediated ROS production) but not N-acetyl-L-cysteine (NAC, known to inhibit thiol redox-mediated ROS production) protects against mefloquine-mediated cell death, suggesting that oxidative stress induced by mefloquine in CML cells is lysosome-mediated. It is known that the length of isoprenoid side chain of α-tocopherol is important for the stabilization of biological membranes. Interestingly, α-tocopherol analog Trolox, which differs from α-tocopherol by decreasing isoprenoid side chain fails to rescue mefloquine-induced cell death, suggesting that mefloquine may destabilize lysosome membrane in CML cells. In addition, we show that mefloquine decreases phosphorylation of S6 and increases LC3B II levels, suggesting that mTOR inhibition and autophagy induction might be involved in the action of mefloquine in CML cells.

Taken together, our work suggests that mefloquine has a potential to be repurposed for CML treatment, given its efficacy as a single agent and synergism as combination with BCR-ABL TKIs in CML primary progenitors and cell lines. Our work also highlights the therapeutic value of targeting the lysosome in the treatment of CML.

#1051

Preclinical analyses of synergistic effect of eribulin and paclitaxel for triple-negative breast cancer.

Takaaki Oba, Ken-ichi Ito. _Shinshu University, Matsumoto, Japan_.

Purpose:

There is an urgent need to develop novel therapeutic strategies for triple negative breast cancer (TNBC). To explore the antitumor effect of combination of eribulin (ERI) and paclitaxel (PTX) for TNBC, we conducted preclinical experiments.

Materials & methods:

Combinational effects of ERI and PTX were analyzed both in vitro and in vivo using two TNBC cell lines (MDA-MB-231, Hs578T).

Results:

When ERI was added simultaneously with PTX to TNBC cells, the sensitivity to PTX was increased synergistically. On the other hand, synergistic increase of sensitivity to ERI was observed in the presence of PTX. When the TNBC cells were treated with each drug subsequently, pretreatment with ERI for 96 hours enhanced the sensitivity to PTX administrated subsequently. In contrast, pretreatment of TNBC cells with PTX enhanced the sensitivity to the subsequently administrated ERI. When the TNBC cells were treated with ERI, the expression of epithelial marker, E-cadherin was increased, while the expression of mesenchymal markers, pSmad2, ZEB1, Slug, and vimentin were decreased. In contrast, when the cells were treated with PTX, the expressions of mesenchymal markers were increased. When epithelial-mesenchymal transition (EMT) was induced by TGF-β in the TNBC cells, the sensitivity to ERI was enhanced, while TGF-β receptor inhibitor (LY2157299) reduced the sensitivity to ERI. Furthermore, knockdown of ZEB1 by small interfering RNA conferred resistance to ERI in both TNBC cell lines. In the mouse xenograft model bearing MDA-MB-231, ERI plus PTX combination therapy significantly inhibited tumor growth compared with either PTX or ERI monotherapy. Furthermore, prior treatment with ERI significantly enhanced the antitumor activity of PTX in the mouse xenograft model.

Conclusions:

Our findings demonstrate an EMT-mediated synergistic antitumor effect by combination of two tubulin inhibitors. This combination therapy may serve as a novel therapeutic strategy for TNBC.

#1052

Aldose reductase inhibitor increases doxorubicin-sensitivity of colon cancer cells and decreases cardiomyopathy.

Himangshu Sonowal, Pabitra B. Pal, Satish K. Srivastava, Kota V. Ramana. _UT Medical Branch, Galveston, TX_.

Chemotherapy is one of the most common option for colorectal cancer (CRC), specifically at the advanced stages and after surgical resection of the tumor. Anthracycline drugs such as doxorubicin, daunorubicin are the most commonly used for the therapy of leukemia, lymphoma and breast cancer. However, doxorubicin is not a very effective drug to kill CRC cells since a high amount of doxorubicin is required for colon cancer therapy, which leads to unwanted side effects like cardiac toxicity and cardiomyopathy. Therefore, better adjuvant therapies are required to increase sensitivity of CRC to doxorubicin as well as to decrease the associated cardiomyopathy. Our recent studies indicate that aldose reductase (AR; AKR1B1) inhibitors such as fidarestat prevent CRC growth as well as metastasis in nude mice xenograft models. We now examined our hypothesis that AR inhibition increases the sensitivity of CRC to doxorubicin and decreases its cardiotoxicity by modulating oxidative stress-mediated signaling pathways. We have investigated the effect of co-administration of doxorubicin along with AR inhibitor fidarestat in in vitro and in vivo CRC cells growth and toxicity. Our results suggest that treatment of CRC cells with fidarestat increases the efficacy of doxorubicin-induced death of HT-29 and SW-480 colon cancer cells. Flow cytometric analysis indicates that in fidarestat + doxorubicin -treated CRC cells, the intracellular accumulation of doxorubicin is increased as compared to doxorubicin alone-treated cells. Further, AR inhibition prevents doxorubicin-induced increase in the expression of drug transporter proteins such as MRP-1 and ABCG-2 in CRC cells. In addition, combination of fidarestat with doxorubicin significantly prevented the growth of CRC cells in nude mice xenografts as compared to doxorubicin alone. Fidarestat also prevented the doxorubicin-induced cardiac toxicity as measured by serum troponin-I levels and inflammatory cytokines and chemokines in the serum and heart of doxorubicin-treated mice in combination with fidarestat. Most importantly, fidarestat protects the cardiac damage and dysfunction in doxorubicin-treated mice. Thus, our results suggest that fidarestat could be used as a novel adjuvant therapy in enhancing doxorubicin sensitivity of CRC cells and to reduce the doxorubicin-associated cardiotoxicity.

#1053

Synergistic anticancer effects of combined γ-tocotrienol and pterostilbene is associated with a suppression in Rac1/WAVE 2 signaling in highly malignant breast cancer cells.

Ibrahim G. Algayadh, Paul William Sylvester. _Univ. of Louisiana at Monroe, Monroe, LA_.

Breast cancer is the most common cancer and the second leading cause of cancer death in American women. Rho GTPases play a crucial role in regulating different cellular functions, including cell proliferation, gene expression, cell morphology, actin polymerization, cell motility, metastasis and apoptosis. Wiskott-Aldrich syndrome protein family verprolin-homologous protein2 (WAVE2) is a member within the Wiskott-Aldrich syndrome protein (WASP) family displays a significant role in actin polymerization and cytoskeletal organization which, are essential for cellular migration and invasion. γ-Tocotrienol is a natural isoform within the vitamin E family that displays anticancer activity against a variety of cancer cell types. Pterostilbene is a natural dimethyl ether analog of resveratrol that has several beneficial effects such as anticancer activity. Studies were conducted to examine the effects of combined γ-tocotrienol and pterostilbene on Rac1/WAVE 2 signaling in mouse +SA and human MDA-MB231 breast cancer cells and cellular migration. Results show that combined treatment of γ-tocotrienol and pterostilbene caused a synergistic inhibition of both mouse +SA and human MDA-MB231 breast cancer cells growth, and corresponding reduction in Rac1/WAVE2 signaling as characterized by a significant inhibition in the levels of phospho-Rac1/cdc42, WAVE2, Arp2, and Arp3 expression. Additional studies indicated that this combination resulted in a significant inhibition in mammary cancer cell migration. In summary, these findings strongly suggest that combined treatment of γ-tocotrienol with pterostilbene may provide great benefit as a therapeutic approach in the treatment of metastatic breast cancer. Supported by a grant from the Louisiana Cancer Foundation.

#1054

Nobiletin synergizes cytotoxicity of antimicrotubule agents by inhibiting Pin1 pathway.

Kazunori Kato, Takumi Yamazaki, Masaaki Honma. _Toyo Univ., Saitama, Japan_.

Nobiletin is a citrus polymethoxy flavonoid that suppresses cell proliferation, angiogenesis and metastatic properties in various cancer cells. In this study, we investigated the combined effects of nobiletin and various chemotherapeutic agents on the cytotoxicity of human colon and esophageal cancer cells. We cultured cancer cells at serial dilution of chemotherapeutic agents with or without nobiletin and assessed cell cytotoxicity at 5 days after drug treatment. The addition of a suboptimal dose of nobiletin did not alter the growth of cancer cells, however cytotoxic effect of antimetabolites (gemcitabine and 5-FU) and DNA-platinating agent (cisplatin) were markedly attenuated by nobiletin. By contrast, nobiletin markedly enhanced antitumor effect of antimicrotubule agents such as docetaxel, paclitaxel, vincristine and MMAE. Moreover, enhanced cytotoxicity by nobiletin was also verified in the combination with MMAE- or DM1-conjugated antibodies to EpCAM and EphA2. We found the inhibition of phosphorylation and nuclear translocation of Pin1 (peptidyl-prolyl isomerase) by nobiletin, suggesting a rational molecular target of nobiletin is Pin1. Overall, these data suggest that nobiletin might useful for the chemotherapeutic treatment of microtubule inhibitors.

#1055

Enhanced efficacy of trifluridine for ionizing radiation in human colorectal cancer cell lines.

Kazuaki Matsuoka,1 Mamoru Nukatsuka,1 Takashi Kobunai,2 Teiji Takechi2. 1 _Taiho Pharmaceutical Co., Ltd., Tokushima, Japan;_ 2 _Taiho Pharmaceutical Co., Ltd., Tokyo, Japan_.

Background: Trifluridine/tipiracil is a combination of trifluridine (FTD), a nucleoside metabolic inhibitor, and tipiracil, a thymidine phosphorylase inhibitor, in a molecular ratio of 1:0.5. Trifluridine/tipiracil is indicated for the treatment of patients with metastatic colorectal cancers refractory to standard chemotherapies. Chemotherapy can make radiation therapy more effective against some colorectal cancers. In this study, we investigated whether cytotoxicity was enhanced when FTD was sequentially used with ionizing radiation (IR).

Method: The colorectal cancer cell lines HT-29, HCT-15, and HCT 116, showing low, middle and high sensitivity for IR, respectively, were treated with the combinations of FTD and IR as follows, 1) sequence 1 (pre-radiation): exposure to 2-8 Gy radiation alone and no treatment for 24 h followed by exposure to 0, 2.0, or 4.0 µM FTD for 24 h, 2) sequence 2 (post-radiation): exposure to 0, 2.0, or 4.0 µM FTD for 24 h followed by 2-8 Gy radiation alone and no treatment for 24 h. Cells were irradiated using a cabinet X-ray irradiation system and all treatments were evaluated by the clonogenic survival assay. The dose modification factors (DMFs) were calculated from clonogenic survival curves as the ratio of radiation doses (control radiation dose divided by the FTD-treated radiation dose). To evaluate DNA strand-breaks, alkaline comet assays were performed and the comet tail moment was determined.

Results: DMFs of sequence 1 at 8 Gy for 2.0 µM FTD were 1.55, 1.24, and 1.03 and for 4.0 µM FTD were 1.75, 1.26, and 1.02 for HT-29, HCT-15, and HCT 116, respectively, and of sequence 2 at 8 Gy for 2.0 µM FTD were 1.82, 1.38, and 1.03 and for 4.0 µM FTD were 2.73, 1.41, and 1.09 for these cell lines, respectively. The DMFs increased in an FTD dose-dependent manner compared with IR-only treatment. In particular, FTD enhanced the efficacy of radiation in HT-29 and HCT-15 indicating low and middle sensitivity to IR. Compared with IR-only treatment, the median comet tail moments of sequence 1 at 8 Gy for 2.0 µM FTD, respectively, were 13.8-, 14.4-, and 2.0-fold and for 4.0 µM FTD were 16.6-, 12.6-, and 3.6-fold in HT-29, HCT-15, and HCT 116, respectively, and of sequence 2 at 8 Gy for 2.0 µM FTD were 4.8-, 4.3-, and 1.8-fold and for 4.0 µM were 7.5-, 14.3-, and 2.6-fold in these cell lines, respectively. Taken together, FTD promoted DNA strand-breaks and enhanced the efficacy of radiation in these three cell lines, regardless of both sequential treatments.

Conclusion: Sequential combination of FTD and radiation was more effective than radiation alone against colorectal cancer cell lines with different radiosensitivities. These results suggest that combination therapy of radiation with trifluridine/tipiracil might be useful for colorectal cancer even with low sensitivity to radiation alone.

#1056

Volasertib in combination with radiotherapy: The perfect match in non-small cell lung cancer.

Jolien Van den Bossche,1 Ines De Pauw,1 Hilde Lambrechts,1 Céline Merlin,1 Christophe Deben,1 Vanessa Deschoolmeester,1 Pol Specenier,2 Patrick Pauwels,2 Marc Peeters,2 Filip Lardon,1 An Wouters1. 1 _Antwerp University, Wilrijk, Belgium;_ 2 _Antwerp University Hospital, Edegem, Belgium_.

Background: Polo-like kinase 1 (Plk1), a master regulator of mitosis and the DNA damage response, is overexpressed in several human malignancies, making it a promising therapeutic target. Monotherapy of Plk1 inhibition has shown only a moderate effect in clinical trials, indicating the need to combine with other therapies. Remarkably, Plk1 inhibition arrests cancer cells in mitosis, which is the most radiosensitive cell cycle phase. Hence, we are the first to investigate the effect of volasertib, the lead agent in category of Plk1 inhibitors at the moment, on the radiosensitivity of a panel of non-small cell lung cancer (NSCLC) cell lines with a different p53 background under both normal and reduced oxygen conditions.

Material and methods:

Three isogenic NSCLC cell lines (i.e. A549 (p53 wt); A549-NTC (non template control, p53 wt); and A549-920 (p53 deficient)) and one TP53 mutant cell line (NCI-H1975, R273H mutation) were included. Cell survival after volasertib monotherapy (0-85 nM, 24h) was assessed using the sulforhodamine B assay. The effect of volasertib (0-20 nM, 24h) on cell cycle distribution was determined flow cytometrically using the Vindelov method. The clonogenic assay (24h 0-10 nM volasertib followed by 0-8 Gy irradiation) was performed to evaluate the radiosensitizing effect of volasertib. Cells were incubated under normoxia or hypoxia (1% O2) during the treatment period. Data analysis was done using WinNonlin and FlowJo software.

Results: Plk1 inhibition by volasertib established a dose-dependent growth inhibition in all cell lines under both normoxia and hypoxia. Cell survival was significantly influenced by the p53 status, with a reduced sensitivity to volasertib in p53 deficient/mutant cells compared to p53 wild type cells (p<0.001). Except for the TP53 mutant NCI-1975 cell line, IC50-values were significantly higher in hypoxic cells compared to their normoxic counterparts (p<0.001). Treatment with increasing concentrations of volasertib induced a strong G2/M phase block (p<0.001), which was most pronounced in p53 deficient A549-920 cells, accompanied by a significant decrease in the number of G1 and S phase cells (p<0.001). Under hypoxia, a mitotic arrest was only detected when high volasertib concentrations were used. Intriguingly, the radiosensitizing effect of the Plk1 inhibitor was more pronounced in p53 wild type cells than in p53 deficient cells. For example, the dose enhance factor (DEF) for volasertib treatment ranged from 1.60 to 2.13 in A549-NTC cells and from 1.18 and 1.22 in A549-920 cells, respectively. Importantly, radiosensitivity was retained when cells were treated and irradiated under hypoxia.

Conclusion: Our in vitro data confirm the therapeutic potential of volasertib in NSCLC cells. Pretreatment with the Plk1 inhibitor enhanced radiosensitivity, especially in cells with functional p53, suggesting a potential role for p53 in radiosensitization by volasertib.

#1057

Co-treatment with fenretinide and safingol induced p38 MAPK and/or FOXO3a activated pro-survival autophagy in glioblastoma multiforme cells.

Nikhil Vad, Dong Wang, Charlie Linch, Barry J. Maurer. _Texas Tech Univ. Health Sciences Ctr., Lubbock, TX_.

Cytotoxicity of the synthetic retinoid, fenretinide (4-HPR), is associated with increased reactive oxygen species and/or de novo synthesized D-erythro-dihydroceramides. Co-treatment with L-threo-sphinganine (safingol, S), synergistically increased fenretinide cytotoxicity in cell lines of many cancer types coincident with increase of safingol-derived, L-threo-dihydroceramides. Previously, we showed that 4-HPR+S induced ER stress, unfolded protein response (UPR), pro-survival autophagy, and mixed non-apoptotic and apoptotic cell death in glioblastoma multiforme (GBM) cells in vitro. However, the mechanisms regulating such activity remained unclear, including the role of ceramide-activated signal transducers, such as ASK1. We now report that 4-HPR+S activated stress kinase ASK1 via phosphorylation at Thr845 (+1-6 hrs), an event known to induce p38 MAPK phosphorylation. Furthermore, 4-HPR+S induced an increase in the nuclear localization of transcription factor FOXO3a coincident with decreased Akt signaling and increased phosphorylated levels of JNK1/2, p38 MAPK, ERK1/2, and autophagy regulator, AMPK (+1-6 hrs). 4-HPR+S caused a time-dependent (+6-24 hrs) increase of GRP78 protein levels, a key regulator of ER stress response and UPR, and increased levels of pro-apoptotic transcription factor, CHOP. GRP78 increase was p38 MAPK-dependent, as p38 MAPK inhibitor, SB203580, greatly reduced GRP78 increase. Interestingly, 4-HPR+S induced cytotoxicity was unchanged in the presence of an inhibitor of ERK 1/2 (PD98059) but significantly decreased when cells were concurrently treated with p38 MAPK inhibitor, SB203580, or JNK1/2 inhibitor, SP600125 (+24-72 hrs, p ≤ 0.05). Significantly, co-treatment with dorsomorphin, an AMPK inhibitor, increased and accelerated 4-HPR+S cytotoxicity (+24-72 hrs, p ≤ 0.05). Moreover, 4-HPR+S treatment induced FOXO3a Ser7 phosphorylation (+1-6 hrs) and a corresponding decrease in levels of nuclear E3-Ub protein ligase, Skp2. The decrease of SKp2 protein was coincident with increased levels of CARM1 protein, a known transcriptional activator of autophagy-related genes. Activation of ASK1, JNK1/2, p38 MAPK and ERK1/2 by 4-HPR+S occurred earlier, and to a greater extent, than single agent 4-HPR and S exposures at all time points (+1-24 hrs). Together, these results support that 4-HPR+S induced both pro-survival autophagy and cell death at least partly through activation of FOXO3a and ASK1-dependent activation of p38 MAPK, possibly in response to the known progressive increase of de novo synthesized D-erythro and L-threo dihydroceramides. A Phase I trial of intravenous 4-HPR+S in adult solid tumors (ClinicalTrials.gov Identifier:NCT0155307) is currently open in the Texas State-supported, South Plains Oncology Consortium (SPONC.org).

#1058

Synergistic activity of p97 inhibitors with histone deacetylase 6 inhibitors in mantle cell lymphoma.

Rekha M. Rao,1 Pratikkumar H. Vekaria,1 Trisha Home,1 Anusha Vallurupalli,1 Abdulraheem Yacoub,1 Frank Schoenen,2 Joseph McGuirk1. 1 _Univ. of Kansas Medical Ctr., Kansas City, KS;_ 2 _Univ. of Kansas, Lawrence, KS_.

p97, or valosin-containing protein (VCP), is an ATPase whose function is essential to restore protein homeostasis in cells. Working in concert with the ubiquitin proteasome system, p97 promotes the retrotranslocation of misfolded proteins from the endoplasmic reticulum and/or their degradation. Consequently, p97 inhibition has emerged as a novel therapeutic target in cancer cells, especially those that depend on high protein turnover and/or ER function. p97 is also present in a complex with many protein complexes including the HSP90, histone deacetylase (HDAC) 6 and heat shock factor 1 (HSF1)-also called the repressive complex. p97 participates in the disaggregation of the repressive complex to regulate HSP90 and HSF1 function under stressed conditions. Accumulation of misfolded polyubiquitylated proteins in the cytosol or on damaged organelles promotes the binding of p97 to polyubiquitylated proteins in an HDAC6-dependent manner and promotes their turnover by autophagic degradation. Given that perturbation of protein homeostasis and ER function induces apoptosis as well as autophagy in B cell malignancies such as multiple myeloma and mantle cell lymphoma (MCL), we hypothesized that inhibition of p97 function in combination with HDAC6 inhibitors would induce proteotoxic stress and/or apoptotic cell death in MCL cells. In this study, we report that the p97 inhibitors DBeQ, ML240 and NMS-873 induce a dose-dependent loss of cell viability in cultured and primary MCL cells. Treatment of MCL cells with ML240 induces the accumulation of polyubiquitylated proteins and induces markers of ER stress such as glucose regulated protein 78 (GRP78) and phosphorylated eukaryotic initiation factor 2 (eIF2) α as well as the autophagic markers LC3-B and p62 (protein whose accumulation is suggestive of reduced autophagic clearance of misfolded proteins). Co-treatment with ML240 and the HDAC6 inhibitor ACY-1215 induces more accumulation of polyubiquitylated proteins and markers of enhanced ER stress as well as autophagy, than either agent alone. Mechanistically we determined that co-treatment with ML240 and ACY-1215 inhibits the binding of p97 to polyubiquitylated proteins and HDAC6, resulting in the reduced clearance of cytotoxic protein aggregates in the cells. Co-treatment with ML240 and ACY-1215 also induces the accumulation of cytosolic polyubiquitylated proteins and their co-localization with LC-3B puncta as demonstrated by immunofluorescent microscopy. These observations are suggestive of enhanced initiation of autophagy but not its completion. Consequently, treatment of MCL cells with ML240 and ACY-1215 resulted in enhanced proteotoxic stress and synergistic apoptotic cell death in MCL cells. Collectively our studies create a strong rationale to test efficacy of the combination of p97 inhibitors in combination with HDAC6 inhibitors in MCL.

#1059

Enhanced anti-tumor efficacy of a checkpoint inhibitor in combination with the HDAC inhibitor belinostat in a murine hepato-cellular carcinoma preclinical model.

Diana Llopiz,1 Marta Ruiz,1 Perrine Pivette,2 Véronique Trochon-Joseph,2 Bérangère Vasseur,2 Caroline Lemarchand,2 Graham Dixon,2 Pablo Sarobe,1 Bruno Sangro3. 1 _CIMA (Centro de Investigación Médica Aplicada), Pamplona, Spain;_ 2 _Onxeo, Paris, France;_ 3 _Clínica Universidad de Navarra, Pamplona, Spain_.

Introduction Belinostat is an HDAC inhibitor currently marketed in the US for the treatment of PTCL. A new oral formulation is under development (positive PoC in preclinical PK study) and provides an increase potential to develop new indications of belinostat in combination with other drugs. It has been extensively demonstrated that some anti-tumor agents, besides their direct anti-tumor effect, may induce additional mechanisms involving activation of immune responses. Thus, combination of these drugs with other immunotherapeutic protocols may yield improved therapeutic benefits. The objective of the present study is the characterization of the therapeutic efficacy of a combination of checkpoint inhibitors (anti-CTLA-4 antibodies) with belinostat in a murine HCC model. In addition, immunoprofiling was performed in order to assess the associated immune response. Methods In vivo efficacy was performed in a Hepa 129 murine hepatocellular carcinoma model implanted subcutaneously in immune-competent C3H mice using anti-CTLA4 alone, belinostat alone or in combination. Treatments were optimized to be able to demonstrate positive effect of the combination. Tumor volume was the primary endpoint. Samples from the spleen were taken to analyze immune mechanism mediating the antitumor activity. Percent of CD4, CD8 T cells and regulatory T cells were determined by flow cytometry. Anti-tumor T cell response was measured by IFN-gamma ELISPOT assay. Results Belinostat improved anti-tumor therapeutic response induced by the anti-CTLA4 checkpoint inhibitor with a significant superior tumor growth inhibition compared to control groups. Treatment with the combination resulted in a complete cessation in tumor growth in all mice during the belinostat treatment period which continued for 1 week after the final dose. Mechanistic studies showed that the underlying immune response correlated with the observed therapeutic effect of the combination with enhancement of IFN-gamma production as antitumor T cell response and decrease in regulatory T cells in the spleens of treated animals. Conclusion These results provide a rational for using belinostat in combination with checkpoint inhibitors to reinforce therapeutic response. Currently only approx. 20% of patients respond to check point inhibitors alone. The oral formulation of belinostat will allow greater flexibility in dosing schedule and use in multiple clinical situations. Further studies are ongoing in order to fully characterize this finding and to facilitate its translation into patients.

#1060

Identification and validation of repurposed drugs for treating head and neck cancer.

Jennifer L. Bryant, Nikolaos Batis, Baksho Kaul, Margaret Hartley, Gordon B. Ryan, Rachel J. Spruce, Hisham Mehanna. _University of Birmingham, Birmingham, United Kingdom_.

Background: Head and neck squamous cell carcinoma (HNSCC) prevalence is rising and new treatments are urgently required. Repurposing existing, clinically approved drugs to be used for cancer therapy is appealing due to the reduced time and cost required to achieve patient benefit.

Aim: The aim of this study was to identify repurposed drugs for use in HNSCC and to validate these drugs and their targets in vitro and in vivo.

Methods: A library of 100 BNF-approved drugs were screened against HNSCC cell lines using high-throughput AlamarBlue proliferation assays and the drug combination IHN56 and IHN29 was identified. To validate this finding, AlamarBlue assays were repeated using a range of concentrations either alone or in combination with cisplatin. In addition, the ability of IHN56 + IHN29 to reduce colony formation and promote apoptosis of HNSCC cell lines was quantified using clonogenic survival assays and Annexin V/PI FACS analysis. To determine the mechanisms of action of IHN56 + IHN29, CAL27 cells were exposed to drugs for 24 hours and the expression of STAT3, pSTAT3-Y705, pSTAT3-S727, EGFR, pEGFR, p21, p53, LC-3 and Ki67 were visualized by Western blot. Finally, in vivo mouse models were employed to confirm our in vitro findings. 5 x 105 CAL27 cells were injected into male NSG mice and allowed to reach 150 mm3. Mice then received IP injections of 310 mg/kg INH29 on days 3-14 after escalating doses on days 1-2, 40 mg/kg INH56 days 3-14, 2 mg/kg cisplatin days 3, 5 and 7, or vehicle control days 1-14. Weights and tumors were measured at least 3 times a week.

Results: The initial screen identified two promising drugs in combination; INH56 and INH29, originally used to treat helminthiasis and epilepsy. The IC50 of INH56 in CAL27 cells utilising the AlamarBlue assay was 1.47 μM and reduced to 0.66 µM in the presence of 0.9 mM INH29. Clonogenic assays demonstrated the ability of INH56 to reduce colony formation by 55% at 2 µM and by 85% when combined with 0.9 mM INH29. Furthermore, a dose-dependent increase in apoptosis was observed with INH56 treatment, which was markedly increased following the addition of INH29. Western blot analysis supports these observations through decreased Ki67 (proliferation), reduced STAT3 activation (oncogenic signaling), increased p21 (apoptosis) independent of p53, and increased LC3 (autophagy) protein levels. In vivo, CAL27 xenograft tumor growth was significantly impaired by IP administration of INH29 alone (P=0.002) and in combination with INH56 (P=0.002) after 11 days, without any obvious toxicity.

Conclusion: The combination of INH56 + INH29 demonstrates strong promise as a novel treatment for HNSCC. This study reveals that INH56 + INH29 alone and in combination with standard treatment (cisplatin and irradiation) reduces proliferation, impedes colony formation and increases apoptosis in vitro, and reduces tumor growth in vivo. We are progressing to a phase I/II trial in HNSCC patients starting in the new year.

#1061

Modulation of death pathways by TRAILR2 agonist antibody and NF-κB pathway by NIK inhibitor in HPV-positive head and neck squamous cell carcinomas.

Yi An, Lillian Sun, Adeeb Derakhshan, Sophie Carlson, Rita Das, Zhong Chen, Carter Van Waes. _NIDCD/NIH, Bethesda, MD_.

Head and neck squamous cell carcinomas (HNSCC) induced by human papilloma virus (HPV) have increased recently in the US, and exhibit a different prognosis and response to therapies from HPV(-) cancers. Analysis of HNSCC TCGA datasets provide evidence for distinct alterations in expression of components of the NF-κB and death pathways in HNSCC with different HPV status. Previously, we have found that birinipant, a novel SMAC mimetic that inhibits inhibitor of apoptosis proteins (IAPs), sensitizes a subset of HPV(-) HNSCC cell lines to death agonists like TNF-α and TRAIL. In this study, we have observed that birinipant also sensitizes most HPV(+) cell lines to TRAIL and TNF-α in vitro. The IC50 of birinipant was under 50nM for HPV(+) UPCI-SCC-90 and UM-SCC-47 cell lines, when combined with TNF-α or TRAIL. To explore the therapeutic potential of enhancing TRAILR mediated death signaling in HPV(+) HNSCC cells, we investigated the effects of an agonistic polyclonal TRAILR2 antibody. Treatment of cells with TRAILR2 antibody alone shows little or no inhibitory effect on UPCI-SCC-90 and UM-SCC-47 cells in vitro. However, a combination of birinipant and TRAILR2 antibody, or triple combination of birinipant, TRAIL, and TRAILR2 antibody shows additive or synergistic effects to inhibit cell proliferation and induce cell death in a dose dependent manner. In addition, our preliminary data suggested that the non-canonical NF-κB pathway is predominately activated in HPV(+) HNSCC cells, and that NF-κB inducing kinase (NIK) is a key component of this pathway. When we tested the NIK inhibitor 4H-isoquinoline-1,3-dione in UPCI-SCC-90 cells, we found it reduced cell proliferation in a dose-dependent manner (IC50 =1.5 μM). Taken together, these results indicate that TNF-α, TRAIL, and TRAILR2 agonist antibody sensitized birinipant anti-tumor activity, and triple combination exhibited synergistic effects in HPV(+) HNSCC cell lines. NIK inhibitor alone inhibits cell proliferation of HPV(+) cells, supporting the hypothesis of aberrant activation of alternative pathway in HPV(+) HNSCC cells. (Supported by NIDCD intramural projects ZIA-DC-000016, 73, 74).

#1062

The combination of metformin and valproic acid induces synergistic apoptosis of prostate cancer cells via p53 activation and the intrinsic pathway.

Linh N.K. Tran, Ganessan Kichenadasse, Katherine L. Morel, Pamela J. Sykes. _Flinders University and Medical Centre, Adelaide, Australia_.

The anti-diabetic drug metformin (MET) and the anti-epileptic drug valproic acid (VPA), when used alone, have demonstrated anti-cancer effects in prostate cancer (PCa). We have demonstrated that the combination of MET and VPA (MET+VPA) can induce synergistic apoptosis in the human prostate cancer cell line LNCaP (p53+) but not in PC3 (p53-), and that the response is dependent on functional p53. Here, we aimed to (1) investigate the mechanism of synergistic apoptosis induced by MET+VPA in LNCaP and (2) propose patient subsets for clinical applications of MET+VPA based on the mechanism involved.

LNCaP and PC3 cells were treated with vehicle, 2.5 mM MET, 2.5 mM VAL, or 2.5 mM MET+VAL and proteins extracted from cell fractions. Western blots were used to confirm cytoplasmic and mitochondrial fractions by measuring the presence of β-actin, pyruvate dehydrogenase subunit E1-α, and ATP synthase subunit-α protein, as well as the release of cytochrome c from mitochondria to the cytoplasm. The mechanism of apoptosis via p53 activation is proposed based on our findings and current understanding from literature.

The release of cytochrome c from the mitochondria to the cytoplasm was significantly increased up to 12-fold (p<0.05) in LNCaP, but not in PC3, in response to MET+VPA compared to vehicle, MET alone, and VPA alone. This finding suggests that both MET and VPA are required to induce an intrinsic apoptosis response in LNCaP via p53. The mechanism proposed is that MET can inhibit the activity of mitochondrial complex 1 which alters the AMP/ATP ratio. This change can activate adenosine monophosphate protein kinase which phosphorylates p53 at Ser15 and leads to p53-dependent cell death. As a non-specific histone deacetylation inhibitor, VPA causes protein acetylation including p53 at lysine residues 373 and 382. By combining metformin and VPA, increased p53 protein activation can occur through phosphorylation of p53 at different sites driving p53-dependent cell apoptosis via the intrinsic pathway, as evidenced by release of cytochrome c into the cytoplasm.

Our results suggest that MET+VPA provides a useful treatment for PCa at a clinically localized stage where p53 is present in most patients or advanced stage where p53 has not been mutated. Additionally, MET+VPA is potentially an alternative to androgen deprivation therapy, particularly in tumors with functional p53. A phase I neo-adjuvant clinical trial will start in early 2017 to evaluate the response of high risk localized PCa to MET+VPA (ANZCTR trial ID: ACTRN12616001021460).

#1063

Benefit from combination cancer therapy arising from patient-to-patient variability rather than additive or synergistic drug action.

Adam C. Palmer, Peter K. Sorger. _Harvard Medical School, Boston, MA_.

Combination cancer therapy is an important means to increase rates and durability of drug response. Superior responses to combination therapies are commonly interpreted as evidence that individual tumors respond more strongly to a combination than to a monotherapy, and combinations are often developed based on pre-clinical observations of drug additivity or synergy. Here we explore an alternative hypothesis, namely that combination therapy can confer benefit simply by providing multiple chances of an above-average response to monotherapy.

The benefit conferred by two-drug combination therapy in human clinical trials and PDX animals was compared with a model of independent drug action postulating that net response in each patient equals the better of two potential responses to drugs given individually with no benefit from the second agent. Data were derived from clinical trials of drug combinations in melanoma, ovarian, colorectal, pancreatic, and breast cancers treated with immunotherapies, chemotherapies and inhibitors of kinases, growth factor receptors, angiogenesis, and DNA repair enzymes; as well as analysis of 4,500 drug response measurements in patient-derived tumor xenografts, and a first-principles model of tumor kinetics and variability in drug response.

A majority of approved combination cancer therapies analyzed confer benefits consistent with independent drug action. This benefit arises from high inter-subject variability, which can be assessed from clinical trials, and low correlation in responsiveness to different drugs, assessed from PDX studies. In a minority of cases responses exceed this expectation, sometimes dramatically, and are likely to represent true drug interaction. Therefore, independent drug action, without any assumption about additive or synergistic effects, provides a sufficient and more parsimonious explanation for the superiority of many clinically approved combination cancer therapies and changes how such data are interpreted mechanistically. Optimization of independent action also provides an alternative means for designing new drug combinations.

#1064

STAT3 NH2-terminal domain inhibition sensitizes medulloblastoma cells to chemotherapy.

Sutapa Ray,1 Don W. Coulter,1 Shawn D. Gray,2 Jason A. Sughroue,2 Nagendra K. Chaturvedi,3 Shantaram S. Joshi,3 Kishor K. Bhakat,3 Timothy R. McGuire,2 John G. Sharp3. 1 _Pediatrics, University of Nebraska Medical Center, Omaha, NE;_ 2 _Pharmacy Practice, University of Nebraska Medical Center, Omaha, NE;_ 3 _Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE_.

Medulloblastoma (MB) is the most common malignant brain tumor in children that arises from cerebellar neuronal progenitor cells. Despite aggressive treatment involving radiation and chemotherapy, the prognosis for high-risk MB remains poor and long-term complications from current therapies are common. Therefore, new effective therapies based on the molecular features of MB are needed to improve therapeutic outcomes. The STAT3 transcription factor is known to be constitutively activated in a variety of human cancers, including MB and functions as an oncoprotein, mediating cancer cell survival, proliferation, migration and drug-resistance. We have delineated the functional role of STAT3 NH2-Terminal Domain (NTD) in MB by using a cell permeable peptide derivative of the STAT3 second helix that specifically binds and perturbs the structure/function of STAT3 and interferes with tetramerization of STAT3 dimers, protein-protein interactions and target genes transcription. Herein, we report that treatment of MB cells with STAT3-NTD inhibitor (S3-NTDi) leads to growth inhibition, cell cycle arrest and apoptosis. The inhibition of STAT3 signaling was also confirmed by downregulation of its downstream targets, including MYC, CCND1, BCl2L1, BCL2, PIM1 and APEX1. Moreover, we observed that S3-NTDi exposure attenuated the migration of MB cells in a wound-healing assay, a prerequisite for tumor invasion and metastasis. We also found that S3-NTDi abrogated IL-6 induced epithelial-mesenchymal transition (EMT) marker protein expression and inhibition of EMT-related transcription factors SNAIL and TWIST. Most importantly, we observed that combination therapy with S3-NTDi and cisplatin significantly decreased the highly aggressive MYC-driven MB cell growth in a dose dependent manner and induced apoptosis by downregulating STAT3 regulated anti-proliferative and anti-apoptotic gene expression. To elucidate the mechanisms of S3-NTDi mediated inhibition, we showed that S3-NTDi upregulated expression of pro-apoptotic gene C/EBP-homologous protein (CHOP) and concomitantly decreased association of the STAT3 transcription factor to endogenous proximal promoter of CCND1 and BCL2 in chromatin immunoprecipitation assay. Furthermore, we determined that S3-NTDi mediated downregulation of miRNA-21 in MB cells, de-repressed Protein Inhibitor of Activated STAT3 (PIAS3), a negative regulator of STAT3 which, in turn, attenuated STAT3 signaling pathway. Overall, our results revealed an important role of STAT3 NTD and its downstream effector molecules in regulating MB pathogenesis and disruption of this pathway with S3-NTDi may serves as a promising new candidate for therapeutic interventions in MB therapy, thereby improving the outcomesin high-risk pediatric MB patients.

#1065

MMP sensitive liposomes followed by radiotherapy improves tumor control in head and neck cancer mice models.

Rikke Y. Brogaard,1 Allison Khoo,2 Rasmus Eliasen,1 Fredrik Melander,1 Awalpreet Chadha,2 Kathryn Aziz,2 Sunil Krishnan,2 Thomas L. Andresen1. 1 _Technical University of Denmark, Kongens Lyngby, Denmark;_ 2 _MD Anderson Cancer Center, Houston, TX_.

Many cancers at advanced stages require a combination of chemotherapy and radiotherapy. A big challenge with this approach is accumulated toxicity because both of these treatment modalities are non-selective and inevitably damage non-target tissues. Nanoparticles such as liposomes provide the ability to differentiate between healthy and cancerous tissues and thus offer efficient delivery of chemotherapy to cancerous tissues. Recently, we developed a matrix metalloproteinase (MMP)-sensitive liposomal platform to carry anticancer agents to the tumor. High local expression of MMPs within the tumor microenvironment ensures bioavailability of the carried agent by converting a polyethylene glycol (PEG)ylated anionic liposome into a dePEGylated cationic liposome that is readily internalized by cells. Using oxaliplatin-loaded MMP-sensitive liposomes, we previously demonstrated great in-vivo efficacy in mice bearing CT26 tumors.

In the present study, we assessed the combinatorial effect of MMP-sensitive liposomes and radiotherapy in head and neck squamous cell carcinoma models. Using a clonogenic cell survival assay, we evaluated MMP-sensitive liposomes combined with radiotherapy. A slightly sensitizing effect was produced by oxaliplatin-loaded MMP-sensitive liposomes while free oxaliplatin produced an additive effect. Similarly, levels of platinum intercalated into DNA were three times higher in cells treated with oxaliplatin-loaded MMP-sensitive liposomes than in those treated with free oxaliplatin. The in-vivo efficacy of oxaliplatin-loaded MMP-sensitive liposomes combined with radiotherapy was studied in the same tumor models, FaDu and HN5, where zymogram gels confirmed an MMP-positive tumor microenvironment. MMP-sensitive liposomes and radiation had a much greater anti-tumor effect than any of the monotherapies or free oxaliplatin combined with radiation. In FaDu, the mean tumor size between liposomes + radiation and liposomes only were significantly different (p<0.05). Thus, our preliminary results suggest that MMP-sensitive liposomes in combination with radiation hold a great potential for clinical translation. The MMP-sensitive liposome offers a valuable tool to deliver the payload in a controlled fashion into the tumor microenvironment, where radiation can potentially amplify the therapeutic index.

#1066

Imipridone ONC201 efficacy in refractory multiple myeloma via Erk1/2 inhibition and pro-apoptotic Bim upregulation: single agent and combinatorial approaches.

Yongsheng Tu,1 Jin He,1 Huan Liu,1 Richard Eric Davis,1 Robert Z. Orlowski,1 Varun Vijay Prabhu,2 Joshua Allen,2 Jing Yang1. 1 _The University of Texas M.D. Anderson Cancer Center, Houston, TX;_ 2 _Oncoceutics, Inc, Philadelphia, PA_.

Disease relapse and drug resistance occurs in the majority of multiple myeloma (MM) patients despite improvements offered by new treatments. There is an urgent unmet need for new therapies that can overcome drug resistance and prolong patient survival after failure of standard-of-care. ONC201, founding member of the impridone class of compounds, has robust preclinical efficacy in a variety of tumor types. A first-in-human ONC201 clinical study has demonstrated exceptional safety, therapeutic pharmacokinetics and evidence of tumor engagement that has led to several Phase I/II advanced cancer studies. Given the pronounced sensitivity of B-cell lymphomas to ONC201, we assessed the efficacy of ONC201 in MM preclinical models. We treated human MM cell lines and patient-derived cells isolated from bone marrow aspirates with ONC201 for 72 hours. ONC201 treatment decreased MM cell viability in CellTiter-Glo assays, with IC50 values that were 1 μM to 1.5 μM, even in high risk MM cell line RPMI8226. Consistent with prior reports, the efficacy of ONC201 was independent of TP53 status, as MM.1S or NCI-H929 cells with wild-type TP53 and OPM-2 or RPMI8226 with mutated TP53 had a similar sensitivity towards ONC201. Additionally, ONC201 was equally effective in paired MM cell lines with TP53 wild type or knockout. Western blot analysis showed increased apoptosis, cleavage of caspase-9, caspase-3, and PARP. We also found that ONC201 induced expression of the pro-apoptotic protein Bim in MM cells, which can occur downstream of ERK inactivation. shRNA knockdown of Bim expression in MM cells abrogated ONC201-induced apoptosis in annexin-V binding assays. Phosphorylation of Bim at Ser69 by Erk1/2 has been shown to promote proteasomal degradation of Bim. In accordance with this mechanism, we observed that ONC201 treatment reduced levels of phosphorylated Erk1/2, an indicator of Erk1/2 kinase activity, and downregulated Bim pSer69. In addition, ONC201 induced apoptosis in dexamethasome-, bortezomib-, and carfilzomib-resistant MM cell lines with the same efficacy as in wild-type cells. As a rational strategy to increase the efficacy of ONC201 by enhancing its inhibition of proteasome-mediated Bim degradation, we tested combinations of ONC201 with proteasome inhibitors. Synergistic reduction in cell viability and enhanced Bim expression and PARP cleavage was observed with ONC201 in combination with bortezomib or carfilzomib in MM cells. The combination of ONC201 and bortezomib enhanced the levels of Bim and cleaved PARP in MM cells. Overall, these findings demonstrate that ONC201 inhibits the Erk1/2 signaling pathway and induces Bim expression to induce apoptosis in MM regardless of p53 status and resistance to standard-of-care therapies. Our studies provide a strong rationale for clinical trials of ONC201 as a single agent or in combination with approved drugs in relapsed/refractory MM.

#1067

Anti-cancer efficacy of imipridones in pancreatic cancer: single agent ONC212 or combination of ONC201 with IGF1-R inhibition.

Avital Lev, Amriti R. Lulla, Jessica Wagner, David T. Dicker, Wafik S. El-Deiry. _Fox Chase Cancer Ctr., Philadelphia, PA_.

Pancreatic cancer is a highly chemo-resistant tumor type known to aggressively metastasize at an early stage with an overall five-year survival rate of ~6%. In this study we tested the efficacy of ONC201 and ONC212 in a panel of pancreatic cancer cell lines and patient-derived models in vitro and in vivo. ONC201 is the founding member of the imipridone class of small molecules with anti-proliferative and pro-apoptotic effects in various tumor types. Kline et al. and Allen et al. previously showed that ONC201 stimulates the integrated stress response by up-regulating ATF4, CHOP and DR5 followed by induction of apoptosis by up-regulating TRAIL. While the spectrum of ONC201 efficacy is broad among tumor types, pancreatic cancer cells are relatively insensitive. ONC201 exhibits low micromolar GI50 values in 3 out of 7 pancreatic cancer cell lines and 2 out of 9 patient-derived cells that were most sensitive among the pancreatic cell lines and samples tested. ONC201 induces the integrated stress response in both ONC201-sensitive and –insensitive pancreatic cancer cell lines. Interestingly, ONC201-treated resistant cells arrest in G1 and do not go through apoptosis. In order to address insensitivity to ONC201 in pancreatic cancer we explored two approaches. The first approach included treating with the ONC201 analogue ONC212, an imipridone that is currently under pre-clinical development. In vitro and in vivo studies consistently show a higher potency of ONC212 as compared to ONC201 in pancreatic cancer, especially in ONC201-insensitive models. We are currently investigating the mechanism of action of ONC212 in comparison to ONC201. The second approach to address ONC201-insensitive pancreatic cancer was combination treatment with the IGF1-R inhibitor AG1024. Western blot analysis of receptor tyrosine kinase expression levels in the panel of pancreatic cancer cell lines revealed a strong correlation between resistance to ONC201 and high expression of IGF1-R. Therefore we hypothesized that IGF1/IGF1-R might play a role in the resistance of pancreatic cancer cells to ONC201. Indeed, treatment of resistant pancreatic cancer cells with the IGF1-R inhibitor AG1024 sensitized the cells to ONC201. We are currently further exploring the involvement of IGF1-R in the ONC201 resistance mechanism. Furthermore, in-vivo studies are ongoing to validate the efficacy of the combination treatment. In summary, although pancreatic cancer is known to be refractory to many drugs, this study introduces two different approaches with imipridone small molecules ONC212 and ONC201 that show promising therapeutic potential for pancreatic cancer.

#1068

Driver pathway blockage synergizes with PLK1 inhibition in anaplastic thyroid cancer.

Daniela De Martino, Emrullah Yilmaz, Arturo Orlacchio, Antonio Di Cristofano. _Albert Einstein College of Medicine, Bronx, New York, NY_.

Anaplastic thyroid cancer (ATC) is one of the most lethal malignancies, with a median survival of less than 6 months from the time of diagnosis. Molecular changes that characterize ATC have been recently well defined and involve most often p53 mutations, and activation of PI3K, RAS and BRAF. PLK1, an essential mitotic regulator, has been found overexpressed in ATC.

Previous data from our lab have shown that PLK1 inhibitors are effective in ATC cell lines. However PLK1 inhibition often results in escape from growth arrest through mitotic slippage. This leads to the generation of polyploid, genetically unstable, cell populations.

We have tested the effect of combining PLK1 and PI3K inhibitors in ATC cell lines. Combined treatment with the PLK1 inhibitor BI6727 and the PI3K inhibitor BKM120 resulted in a significant synergistic effect in ATC cell lines with high levels of AKT activity, but not in those with undetectable pAKT. Combination of the two drugs enhanced the G2/M arrest at doses in which the single drugs showed no effect. In addition, it led to a massive reduction of the tetraploid cells population. Furthermore, combined treatment with BI6727 and BKM120 in PI3Khigh cell lines showed a significant induction of apoptosis in a time- and dose-dependent manner.

Combined inhibition of PI3K and PLK1 was extremely effective in inhibiting tumor growth in vivo, in an immunocompetent allograft model of ATC.

Our results show that combination of PLK1 and PI3K inhibitors is an effective treatment for ATC cells with high levels of PI3K signaling. This combination results in cell cycle arrest, inhibition of of tetraploid cell generation, and induction of apoptosis, suggesting a clear therapeutic potential.

#1069

Inhibition of the nuclear import receptor, KpnB1 synergizes with cisplatin toxicity in cervical cancer cells.

Ru-pin Chi,1 Wei Wei,2 Michael Birrer,2 Virna D. Leaner1. 1 _Univ. of Cape Town, Cape Town, South Africa;_ 2 _Harvard University, Cambridge, MA_.

Elevated expression of members of the nuclear transport protein family has been reported in multiple cancers and presents as novel anticancer therapeutic targets. Using a cervical cancer model system, we have previously shown that inhibition of the nuclear import receptor, KpnB1 by siRNA and a novel small molecule, Inhibitor of Nuclear Import (INI-43) resulted in cancer cell death via apoptosis. In this study, we investigated the cancer cell killing effects of KpnB1 inhibition in combination with Cisplatin (CDDP), a first-line chemotherapeutic agent used in the treatment of many cancers. KpnB1 siRNA and INI-43 treatment at sub-lethal concentrations enhanced cancer cells' sensitivity to CDDP. Our data shows that the combination treatment of INI-43 and CDDP significantly decreases CDDP IC50 compared to CDDP treatment alone. Increased PARP-cleavage was observed in combination treated cells and this correlated with increased γH2A.X, indicating increased apoptosis and DNA damage. Furthermore, INI-43 treatment reduced the nuclear import of NFκB, a stress-regulated response transcription factor known to be a KpnB1 cargo protein that promotes cancer cell survival. Decreased levels of downstream pro-survival and DNA-repair targets of NFκB were observed, including cyclinD1, c-Myc and XIAP, which correlated with increased DNA damage and apoptosis. Taken together, results suggest increased Cisplatin sensitivity of cervical cancer cells when nuclear import via KpnB1 is inhibited.

#1070

Role of FOXO1 in response of ovarian carcinoma cells to the XPO1/CRM1 inhibitor KPT-330/selinexor in combination with cisplatin.

Simone Stucchi,1 Michelandrea De Cesare,1 Cristina Corno,1 Nives Carenini,1 Emilio Ciusani,2 Nadia Zaffaroni,1 Laura Gatti,1 Paola Perego1. 1 _Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy;_ 2 _Fondazione IRCCS Istituto Neurologico C. Besta, Milan, Italy_.

Ovarian carcinoma is a major cause of cancer-related death in women. Besides late diagnosis, treatment often fails to produce a persistent disease control. The efficacy of the platinum drug-based therapy is limited by drug resistance. Thus, an ideal therapy for women with ovarian carcinoma is still missing. Because the karyopherin XPO1/CRM1 contributes to the regulation of the cellular localization of the transcription factor FOXO1 which participates in apoptosis regulation, the aim of this study was to examine if interference with XPO1 to improve FOXO1 nuclear localization may be exploited to kill efficiently ovarian carcinoma cells and to improve cisplatin efficacy. Here, we employed preclinical pharmacology approaches including growth inhibition assays, western blot analyses, gene knockdown by siRNA, quantitative Real-time PCR, immunofluorescence analyses and tests in in vivo models. The drug interaction was analyzed using the Chou and Talalay method. When cell sensitivity to KPT-330 of a panel of ovarian carcinoma cell lines was examined a marked sensitivity to the XPO1 inhibitor was found. The effect of the combination of cisplatin and KPT-330 was investigated in the IGROV-1 cells, using simultaneous or sequential schedules. According to the combination index values, when KPT-330 exposure followed cisplatin exposure the most favourable drug interaction was observed. In IGROV-1 cells, a modulation of proteins involved in apoptosis (p53, Bax) and in cell cycle progression (p21) was found, besides G1 and G2/M accumulation after exposure to KPT-330 and to the cisplatin/KPT-330 combination, respectively. KPT-330-treated cells exhibited FOXO1 nuclear staining, in keeping with the capability of the compound to inhibit FOXO1 nuclear export. Knock-down experiments by RNA interference indicated that FOXO1-silenced cells displayed a reduced sensitivity to KPT-330, but no significant changes in cisplatin sensitivity. FOXO1 silencing tended to reduce the efficacy of the drug combination at selected cisplatin concentrations. An analysis of the antitumor efficacy of KPT-330, indicated the capability of KPT-330 to significantly inhibit tumor growth when IGROV-1 cells were subcutaneously injected in immunodeficient mice or growth intraperitoneally. Our findings support that the XPO1 inhibitor KPT-330 is a promising agent for the treatment of ovarian carcinoma. The effect of the KPT-330-cisplatin combination appears to be dependent on the treatment schedule, as a synergistic interaction occurs when cells are treated with cisplatin followed by KPT-330. Such an interaction can be modulated by silencing of FOXO1, which results in reduced sensitivity to KPT-330. Our results suggest that therapeutic regimens selected on the basis of the molecular tumor features should be used to achieve a personalized therapy tailored to the specific characteristics of each patient.

#1071

Efficacy of trifluridine/tipiracil + anti-mouse PD-1 antibody combination on mouse colorectal cancer model and related tumor immunomodulatory effects.

Norihiko Suzuki, Hiroshi Tsukihara, Fumio Nakagawa, Takashi Kobunai, Teiji Takechi. _Taiho pharmaceutical CO., LTD., Tokushima, Japan_.

Background: Trifluridine/tipiracil (FTD/TPI) is an oral nucleoside antitumor agent that is composed of trifluridine and tipiracil hydrochloride at a molecular ratio of 1:0.5. Checkpoint-blockade immunotherapies are particularly effective in patients with tumor T cell infiltrations. In this study, the antitumor effects of FTD/TPI + anti-mouse PD-1 antibody combination were studied in a syngeneic mouse model and the tumor-infiltrating lymphocyte (TIL) subsets were evaluated. Method: The mouse colorectal cancer cell line CMT-93 was subcutaneously implanted into C57BL/6 mice. Vehicle (0.5% Hydroxypropyl methylcellulose, 10 mL/kg, p.o.), FTD/TPI (75, 100, and 150 mg/kg/day, twice daily, days 1-14, p.o.), anti-mouse PD-1 antibody (clone RMP1-14; 0.1 mg/body; once daily, days 1, 5, and 9; i.p.), and FTD/TPI + anti-mouse PD-1 antibody combination were administered, and inhibitory activity was evaluated according to tumor-volume changes. Single-cell suspensions were prepared from collected tumors. Based on cell marker expression, CD4+ T cells, CD8+T cells, and regulatory T cells (Tregs) were identified using antibodies against CD4, CD8, CD25, and Foxp3, and subsets of TILs were evaluated by flow cytometry. Results: Both anti-mouse PD-1 antibody and FTD/TPI monotherapies were effective in vivo. Tumor-growth inhibition by anti-mouse PD-1 antibody was 81.7% at 0.1 mg/kg/day and that by FTD/TPI was 33.4%, 46.1%, and 59.5% at 75, 100, and 150 mg/kg/day, respectively. FTD/TPI + anti-mouse PD-1 antibody combination using FTD/TPI (75, 100, and 150 mg/kg/day) with anti-mouse PD-1 antibody (0.1 mg/kg/day) inhibited tumor growth at significantly higher rates (P < 0.05; 91.8%, 95.7%, 98.4%, respectively) than monotherapy. Remarkably, the 150-mg/kg/day FTD/TPI + 0.1-mg/kg/day anti-mouse PD-1 antibody combination caused complete tumor regression in four/five mice without body-weight reduction or drug-related deaths; however, none of the monotherapies caused complete tumor regression. Flow cytometric analysis revealed a higher CD8+ T cell percentage among total lymphocytes and a lower Treg percentage in CD4+ T cells after combination therapy compared with the controls. Conclusion: FTD/TPI + anti-mouse PD-1 antibody combination was synergistically effective on CMT-93 mouse colon tumor. This combination increased CD8+ T cell percentages in whole lymphocyte and decreased Treg percentages in CD4+ T cell. This suggests that FTD/TPI + anti-mouse PD-1 antibody combination modulates tumor T cell populations and improves their antitumor activity.

#1072

Synergistic effect of gemcitabine and a Dclk1 inhibitor on pancreatic cancer cell survival.

Daichi Kawamura, Yoshihiro Takemoto, Arata Nishimoto, Toshiki Tanaka, Yukari Hironaka, Kumiko Yoshida, Junichi Murakami, Naruji Kugimiya, Eijiro Harada, Koji Ueno, Tohru Hosoyama, Kimikazu Hamano. _Yamaguchi University, Ube, Japan_.

Pancreatic cancer has the highest mortality rate of all major cancers and is one of the most lethal malignancies. There is a constant upward trend in the number of patients diagnosed with pancreatic cancer and the number of deaths due to pancreatic cancer. Gemcitabine (GEM) is often used in the treatment of pancreatic cancer (PDAC), but has limited effects. Doublecortin-like kinase 1 (Dclk1) is important in the progression of early pancreatic neoplastic lesions and PDAC. However, the functional role of Dclk1 in PDAC is unknown. To identify the substrate protein phosphorylated by Dclk1, we performed a protein microarray analysis on Dclk1 knockdown cells. The results of this analysis directed our studies toward Chk1, which is known to be a potential regulator of the cell cycle and experiences upregulation of phosphorylation after GEM treatment. In general, GEM treatment results in DNA damage to pancreatic cancer cells, an increase in phosphorylated Chk1 (p-Chk1), and arrests the cell cycle progression to repair the damaged DNA. On the basis of the preliminary data, we hypothesized that the decrease in Chk1 phosphorylation by Dclk1 inhibition circumvents cell cycle arrest and impairs the subsequent DNA repair. The aim of this study was to evaluate the synergistic effect of Dclk1 inhibition and GEM treatment on pancreatic cancer cell survival. We used the human pancreatic cancer cell line MIA PaCa-2 and LRRK2-IN-1 (LRRK) as the Dclk1 inhibitor for this study. First, we examined the effects of GEM or the Dclk1 inhibitor or both on cancer cell proliferation and the expression of p-Chk1. Significantly decreased cell proliferation was observed on co-treatment of GEM and LRRK compared to GEM treatment alone. In addition, the expression of p-Chk1 significantly decreased on co-treatment compared to GEM treatment alone. Second, we used flow cytometry to analyze the cell cycle after treatment with GEM and/or LRRK. Almost all cancer cells treated with GEM alone were arrested in the S phase of the cell cycle. The addition of LRRK allows the cell cycle to proceed in the same manner as untreated control cancer cells do. We also evaluated DNA damage by measuring the intensity of gamma-H2AX. Cancer cells that were co-treated experienced more DNA damage than with GEM treatment alone. The co-treatment induced apoptosis without the repair of DNA damage in the cancer cells. In conclusion, the combined treatment with GEM and a Dclk1 inhibitor decreased the cell survival rate compared to treatment with GEM alone through the suppression of p-Chk1. Targeting Dclk1 in combination with GEM might offer an excellent opportunity for future pancreatic cancer treatments.

#1073

Increased internalization and processing of the CD37-targeting antibody-drug conjugate, naratuximab emtansine (IMGN529), in the presence of rituximab leads to enhanced potency in diffuse large B-cell lymphoma models.

Stuart W. Hicks, Katharine C. Lai, Yong Yi, Prerak Shah, Cristina L. Gavrilescu, Joe Ponte, Callum M. Sloss, Angela Romanelli. _Immunogen, Inc., Waltham, MA_.

Naratuximab emtansine (IMGN529) is an investigational CD37-targeting antibody-drug conjugate (ADC) that has shown both preclinical and clinical activity in DLBCL . We have shown that rituximab, an anti-CD20 monoclonal antibody, enhances the preclinical activity of IMGN529. The combination of IMGN529 and rituximab is more active than either agent alone, and this benefit is associated with an increase in apoptosis and cell death, resulting in enhanced potency in DLBCL models. Efficacy of ADCs, such as IMGN529, relies on the internalization, intracellular trafficking and degradation of the ADC leading to the release of the cytotoxic catabolite. To explore the mechanism underlying the enhanced activity of the combination, we investigated the effect of rituximab treatment on the binding, internalization and processing of IMGN529 in vitro. To evaluate the effect on the internalization of IMGN529, DLBCL cell lines were incubated with fluorescently-labeled K7153A antibody (the CD37 targeting moiety of IMGN529) alone or in combination with rituximab or a non-targeting control antibody. K7153A internalization was similar whether incubated alone or with the non-targeting control. However, when cells were co-incubated with K7153A and rituximab, the internalization of K7153A significantly increased. This increase was also observed with other anti-CD20 antibodies suggesting an anti-CD20 class effect.

Radiolabeled processing studies were performed to determine if the observed increase in internalization led to a corresponding increase in antibody degradation, which is required for the release of the cytotoxic agent in IMGN529. By trace-labeling the K7153A antibody with tritiated propionate (3H-K7153A), we were able to follow binding, uptake, and degradation of the antibody component of IMGN529 in DLBCL cell lines. Following pulse exposure, the amount of 3H-K7153A degraded after 24 hours remained the same whether treated alone or in combination with other B-cell targeting antibodies. However, when combined with rituximab the percentage of degraded 3H-K7153A increased as much as 3-fold and a similar increase of 3H catabolite was generated. When compared, the combination of K7153A and rituximab produced considerably more catabolite (~6-fold, p-value <0.0001) than the combination of rituximab and a CD19-targeting antibody.

These findings suggest a novel mechanism where the increased potency of IMGN529 and rituximab can be explained by CD20 binding resulting in an increase in internalization and degradation of IMGN529, leading to generation of greater amounts of cytotoxic agent. Overall, these data provide a biological rationale for the enhanced activity of the rituximab plus IMGN529 combination, further supporting the clinical development strategy of this combination in DLBCL.

#1074

Combining forces: Synergy of erlotinib and crizotinib in a wild-type squamous non-small cell lung cancer cell line.

Nele Van Der Steen,1 Christian Rolfo,2 Patrick Pauwels,2 Godefridus J. Peters,1 Elisa Giovannetti1. 1 _VU University Medical Center, Amsterdam, Netherlands;_ 2 _Antwerp University Hospital, Antwerp, Belgium_.

Oncogenic drivers are often overexpressed in adenocarcinoma non-small cell lung cancer (NSCLC) patients, so this tumor type is often sensitive to targeted therapies. Sensitizing mutations in the epidermal growth factor receptor (EGFR) are good predictive biomarkers for response to EGFR-small molecule inhibitors, eg erlotinib. Amplification or exon 14 skipping of cMET are good biomarkers for cMET-small molecule inhibitors, eg crizotinib. In other subtypes of NSCLC, eg squamous, targeted therapy is not considered to be active.

We tested the combination of erlotinib and crizotinib on a panel of squamous NSCLC cell lines that were wild-type for both EGFR and cMET. The effect of this drug combination was tested with different assays. The sulforhodamine B-assay was used to determine growth inhibition and combination indexes (CI) were calculated with the method of Chou&Talalay. A CI< 0.8 was synergistic, 0.8<CI<1.2 was additive and CI>1.2 was antagonistic. With flow cytometry the cell cycle was studied. With the wound-healing assay we determined the effect on cell migration. A 3D spheroid assay was performed to determine the effect of limited nutrient availability and hypoxia. Protein phosphorylation was studied with a pathscan array and phospho-specific western blotting.

The LUDLU cell line gave a synergistic CI of 0.39±0.07, whereas the Calu1 (CI = 0.81±0.04), H520 (CI = 0.87±0.06) and SKMES1 (CI = 0.81±0.02) were additive and the H1703 cell line (CI = 1.20±0.15) was antagonistic. Cell cycle analysis revealed that erlotinib monotherapy caused a G1 phase arrest (38 to 76%) and crizotinib caused a G2/M (13 to 64%) phase arrest. In cells treated with the combination, the effect of crizotinib was dominant (13 to 52%). In the wound-healing assay the LUDLU cells were not migratory. The pathscan revealed a decrease in the phosphorylation of pERK1/2 (Thr202/204), pPRAS40 (Thr246) and pGSK3β (Ser9). Western blotting showed that the levels of pPRAS40 were decreased in the LUDLU cells after treatment with erlotinib or crizotinib, and almost gone when treated with the combination. In the SKMES1 cells only the combination decreased pPRAS40. H1703 cells showed no change in pPRAS40.

These changes in phosphorylation make us hypothesize that Her3 and PI3K/Akt signaling may be important in the interaction of both drugs. Her3 is a promiscuous receptor that is able to heterodimerize with EGFR and cMET. When blocking one of these receptors, Her3 is still able to continue downstream PI3K/Akt signaling. When both EGFR and cMET are blocked, Her3 and PI3K/Akt signaling is inhibited, leading to synergy. The expression of total-Her3 was lowest in the antagonistic cell line. The combination of both inhibitors lead to a complete inhibition of pHer3 (Tyr1289).

In conclusion: Blocking both EGFR and cMET signaling causes inhibition of Her3 downstream signaling through PI3K/Akt, leading to synergy in wild-type squamous NSCLC cells.

#1075

JQ1 induces DNA damage, inhibits expression of DNA repair proteins, and synergizes with PARP inhibitors in pancreatic cancer cells.

Aubrey Lynn Miller,1 Tracy Gamblin,1 Leona Council,1 Robert van Waardenburg,1 Eddy Yang,1 James Bradner,2 Karina Yoon1. 1 _Univ. of Alabama at Birmingham, Birmingham, AL;_ 2 _Novartis Institutes for Biomedical Research, Cambridge, MA_.

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer. PDAC is a highly aggressive tumor with a 5-year survival of <6%. Surgery remains the only curative treatment for PDAC patients, but only 20% of newly diagnosed patients have resectable disease. The remaining 80% present with locally advanced or metastatic disease, and receive systemic chemotherapy with gemcitabine. The median survival of this patient population is ~6 months. Development of more effective therapy for this chemorefractory disease is imperative.

The goal of the current study was to develop effective combination therapy for PDAC by identifying agents that might be combined with the BET bromodomain inhibitor JQ1, which we have shown to inhibit the growth in vivo of PDAC patient derived xenografts (PDX). Expression profile analysis of tumors from vehicle control and JQ1 treated mice revealed that JQ1 inhibited the expression of multiple gene products involved in DNA repair. Notably, JQ1 inhibited expression of DNA double-strand break (DSB) repair proteins BRCA2 and Ku80. Immunohistochemical staining confirmed down-regulation of expression of both proteins in tumors of mice treated with JQ1.

Further, immunoblot and immunofluorescence analyses demonstrated that decreased expression of BRCA2 and Ku80 was coincident with increased levels of DNA damage, as reflected by expression of the DNA DSB marker gamma-H2AX. Data generated in vivo in three independent PDX models corroborated in vitro data generated using pancreatic cancer cell lines BxPC3 and Panc1. The data suggest that JQ1 induces DNA damage by inhibiting DNA repair.

Because DNA repair deficiency sensitizes cells to PARP inhibitors, we hypothesized that JQ1-induced DNA repair deficiency would sensitize PDAC cells to PARP inhibitors. To address this hypothesis, we exposed Panc1 and BxPC3 to JQ1 or to a PARP 1/2 inhibitor (veliparib or olaparib) or to the combinations, and assessed the efficacy of each. Growth inhibition data, analyzed using Compusyn software and reported as combination indices, demonstrated that the combinations of JQ1 + veliparib or olaparib exert synergistic cytotoxicity. Further, the combination of JQ1 + a PARP inhibitor increased the accumulation of DNA damage in vitro, compared to either agent alone. We conclude that JQ1 induces DNA damage due at least in part to DNA repair deficiency, and propose that this mechanism sensitizes PDAC cells to PARP inhibitors.

#1076

Potent antitumor activity of artemisinin.

Mounir Tilaoui. _Faculty of Science and Technology Beni-Mellal, Béni Mellal, Morocco_.

Cancer is a leading cause of death worldwide and remains a therapeutic enigma. Phytochemical compounds are emerging as a new generation of anticancer agents with limited toxicity in cancer patients. The purpose of this study was to investigate the potential impact of artemisinin, the main active compound of the Chinese herb Artemisia annua L, on survival cancer cells and tumor growth in vivo. Exposure of tumor cells P815 (murine mastocytoma) and BSR (kidney adenocarcinoma of hamster) to increasing artemisinin concentrations resulted in a significant inhibition of viability, as revealed by the respective IC50 values (12 µM for P815 and 52 µM for BSR cells). Interestingly, no cytotoxic effect was observed of artemisinin on peripheral blood mononuclear cells. The in vitro cytotoxicity studies were complemented by the determination of apoptotic DNA fragmentation and Annexin V- Streptavidin-FITC assay. Our results provide evidence that artemisinin leading to apoptotic pathway on P815 but not on BSR cancer cell lines. Moreover, we demonstrate that artemisinin synergizes with microtubule - damaging agent vincristine to inhibit cellular viability. The in vivo anticancer activity of artemisinin was also evaluated using the DBA2/P815 (H2d) mouse model inoculated with the P815 mastocytoma cells. Our in vivo data clearly showed that the oral administration of artemisinin inhibited solid tumor development. In view of the available experimental findings, we contend that artemisinin could have clinical potential as an anticancer drug alone or in combination with chemotherapeutic agents such as vincristine.

#1077

Extra-virgin olive oil Met inhibitor oleocanthal-lapatinib: a novel synergistic combination for HER2-dependent breast malignancies.

Abu Bakar Siddique, Hassan Y. Ebrahim, Mohamed R. Akl, Mohamed M. Mohyeldin, Khalid A. El Sayed. _Univ. of Louisiana College of Pharmacy, Monroe, LA_.

Breast cancer (BC) is the most commonly diagnosed cancer in women, claiming the lives of hundreds of thousands of women each year. The Mediterranean diet is known to reduce BC and colon cancer incidence. Extra‐virgin olive oil (EVOO) represents a main ingredient in this diet. The EVOO‐derived (‐)‐oleocanthal was shown to target the Receptor tyrosine kinases (RTK) c‐Met. Oleocanthal exerted potent in‐vivo efficacy in an orthotopic athymic nude mouse breast cancer xenograft model much greater than its modest in‐vitro potency. Dysregulation of RTKs, specifically EGFR/HER‐2 pathways, correlates with poor prognosis and more aggressive breast cancer phenotypes. Dysregulation of hepatocyte growth factor (HGF) and its receptor c Met correlates with aggressive proliferation, invasive character, and pathological motility. c‐Met amplification correlates with escape from the anticancer effects of EGFR/HER2 inhibitors and cetuximab. (‐)‐Oleocanthal (OC) is a naturally occurring phenolic secoiridoid from EVOO showed significant in vivo activity against invasive breast cancers through targeting HGF/Met axis. The Dual EGFR/HER‐2 inhibitor lapatinib (LP) has already been in clinical practice for HER2‐amplified breast cancer, which occasionally develops resistance through c‐Met upregulation. Lapatinib's therapeutic dose induced significant hepatotoxicity. The combined use of OC and LP was hypothesized not only for therapeutic synergy but also to notably reduce LP's effective doses and therefore minimize its hepatotoxicity. Combined treatment of subeffective doses of lapatinib and OC caused significant in vitro and in vivo synergistic antiproliferative effects against the HER‐2‐dependent BT‐474 and SKBR‐3 BC cells. Interestingly, OC induced 4‐fold lapatinib dose reduction index with improved potency. Protein microarray and Western blot analyses of OC‐LP combination treatments synergistically reduced EGFR, HER‐2, FAK, JAK1, MEK, and c‐Met activation. These results propose OC use as dietary supplement to synergize the chemotherapeutic effects of LP, reduce its therapeutic dose to ¼ and therefore can minimize its morbidity. Combinatorial inhibition of c‐Met‐HER‐2‐EGFR is an effective strategy for the control of HER‐2 positive breast cancer. This study was supported by NIH/NCI project 1R15CA167475‐01. 

### Combination Therapy 1

#1078

Dinaciclib overcomes resistance to BKM120 in triple negative breast cancer patient-derived xenograft models.

Sandeep Rajput, Fang Guo, Li Shun, Cynthia Ma. _Washington University St. Louis, St. Louis, MO_.

Purpose: Triple negative breast cancer (TNBC) is one of the most lethal subtypes of breast cancer with limited therapeutic options. Development of molecularly targeted agents for TNBC is an unmet clinical need. The phosphoinositide 3-kinase (PI3K) pathway, which is a major cell growth and survival pathway, is frequently activated in TNBC as a result of genetic aberrations such as loss of the negative regulator PTEN or gain of function mutations in PIK3CA, therefore an attractive therapeutic target. However, single agent PI3K inhibitors have shown limited anti-tumor activity in both preclinical models and in clinical trials. One of the most important mechanisms of resistance to PI3K inhibitor is amplification of Myc. Dinaciclib, a potent inhibitor of cyclin-dependent kinases (CDKs)1, 2, 5 and 9, has shown to be particularly effective in Myc dependent tumors in preclinical studies and in TNBC. We therefore hypothesized that dinaciclib could overcome tumor cell resistance to PI3K inhibitors and improve the therapeutic efficacy in TNBC. In this study, we evaluated the anti-tumor and molecular effect of dinaciclib and BKM120 (Pan-PI3K inhibitor), either alone or in combination, in a panel of patient derived xenograft (PDX) models of TNBC.

Methods: Four TNBC PDXs models were selected for the study for in-vivo and ex-vivo response to vehicle, BKM120, dinaciclib, or the combination of BKM120 and dinaciclib. Tumor volume changes over time in each group were documented to calculate the percentage of tumor growth inhibition by either agent alone or in combination. Tumor tissues harvested post treatment were examined by immunohistochemistry for cleaved PARP to determine the extent of apoptosis and phospho-Histone H3 for G2 to M phase cell cycle progression. Western blot analysis and reverse protein phase array (RPPA) were also performed to determine treatment effect on PI3K downstream targets and cell cycle molecules.

Results: The combination of BKM120 and dinaciclib induced significantly greater growth inhibitory effect on tumor growth than either single agent alone in TNBC PDX models. This is accompanied by an enhanced apoptotic induction and reduced cell cycle progression. In addition, the combination of dinaciclib and BKM120 significantly reduced the level of cyclin B and the key anti-apoptotic protein survivin as well as significant downregulation of pAKT, and pS6.

Conclusions: These data suggest that dinaciclib and BKM120 combination is an effective approach in treating TNBC. Additional mechanistic investigation for the efficacy of this combination is underway.

#1079

Preclinical activity of the FGFRinhibitor BAY 1163877 alone or in combination with antihormonal therapy in breast cancer.

Oliver Politz,1 Peter Ellinghaus,2 Sebastian Bender,2 Sylvia Gruenewald,1 Franziska Siegel,1 Marie-Pierre Collin,2 Sabine Zitzmann-Kolbe,1 Dominik Mumberg,1 Karl Ziegelbauer1. 1 _Bayer AG, Berlin, Germany;_ 2 _Bayer AG, Wuppertal, Germany_.

BAY 1163877 is an orally available, highly potent and selective pan fibroblast growth factor receptor (FGFR) inhibitor. In an ongoing Phase 1 clinical trial (NCT01976741) BAY 1163877 showed clinical responses at exceptional tolerability in patients suffering from different tumor types including urothelial bladder carcinoma or lung tumors, which were selected based on elevated FGFR1-3 mRNA expression. In the preclinical phase, the compound demonstrated significant single agent anti-tumor activity in various tumor models with different FGFR alterations leading to FGFR overexpression (e.g. FGFR gene amplifications or mutations). Genetic alterations of FGFRs can also be found in breast cancer with 7.5 - 17% of all tumors harboring a FGFR1 gene amplification. Elevated FGFR1 mRNA levels can be found in up to 22% of breast cancer cell lines as well as clinical samples. Other FGFR alterations include FGFR2 or FGFR4 gene amplifications as well as elevated FGFR mRNA levels, which were reported in all breast cancer subtypes. We therefore investigated BAY1163877 monotherapy in various breast cancer models. Due to the favorable clinical safety profile of BAY1163877, we also examined a combination treatment with early line antihormonal therapies in hormone receptor positive breast cancer.

In vitro profiling of BAY 1163877 in a number of breast cancer cell lines showed a clear association of efficacy with expression levels of different FGFR isoforms. The efficacy was further investigated in several patient- or cell line-derived breast cancer in vivo models. For instance, BAY 1163877 alone dosed 38mg/kg twice daily induced tumor growth inhibition of greater than 90% in a subcutaneous mouse syngeneic 4T1 breast cancer model expressing elevated levels of FGFR2.

Resistance to endocrine therapy appears associated with FGFR1 gene amplification and may explain the poor prognosis of FGFR1 overexpressing tumors treated with adjuvant tamoxifen. We therefore investigated the combination of the panFGFR-inhibitor BAY 1163877 with the clinically used antihormonal compound fulvestrant in selected luminal breast cancer PDX models. Some of these models showed resistance to antihormonal treatment in monotherapy but improved in vivo efficacy in combined treatment using BAY 1163877 and fulvestrant.

These data may warrant further clinical investigation of BAY1163877alone or in combination with antihormonal therapy in patients with FGFR overexpressing breast cancer.

#1080

Targeting the PI3K-Akt and NF-κB pathways as a combination therapy in blocking prostate cancer progression.

Eswar Shankar,1 Rajnee Kanwal,1 Aditi Goel,1 Xiaoping Yang,2 Sanjeev Shukla,1 Gregory T. MacLennan,1 Pingfu Fu,1 Anant Madabhushi,1 Parameswaran Ramakrishnan,1 Sanjay Gupta1. 1 _Case Western Reserve Univ., Cleveland, OH;_ 2 _VA Medical Center, Cleveland, OH_.

Akt/protein kinase B and transcription factor NF-ĸB are important signaling molecules and key survival factors involved in the control of cell proliferation, apoptosis and oncogenesis. Although Akt and NF-ĸB are components of separate signaling pathways, Akt can induce cell survival signal through NF-κB activation. Previous studies from our laboratory have shown constitutive activation of NF-ĸB/p65 and PI3K-Akt in clinical prostate cancer specimens and in autochthonous mouse model of prostate cancer. In this study we investigated if these two signaling pathways converge and whether dual targeting is effective in blocking disease progression. Using tissue samples obtained during transurethral prostatic resection and paraffin-embedded sections of benign and malignant prostate tissue, we first analyzed nuclear levels of NF-ĸB/p65, native/activated Akt levels, and evaluated co-localization of these two proteins. Compared to benign tissue, cancer specimens exhibited constitutive Akt and NF-ĸB/p65 activation which was more pronounced in high-grade cancer (Gleason grade 7-10). Immunohistochemical analyses further confirmed a progressive increase in the activated form of Akt and NF-ĸB/p65 in the nucleus of cancer tissues compared to benign specimens, and exhibited co-localization of these proteins in a subset of aggressive cancer. Utilizing androgen-responsive prostate cancer LNCaP cells, with increased Akt activity, and androgen-refractory PC-3 cells harboring high levels of Akt and NF-ĸB. Individual treatment of cells with Akt Inhibitor VIII (0.075-1.8µM) and NF-ĸB inhibitor Parthenolide (0.32-60µM) for 24-72 h demonstrated partial suppressive effect in cell growth. Strikingly, concurrent blocking of Akt and NF-κB/p65 at 1:10 molar ratio resulted in potentiated toxicity with marked inhibition in proliferation and induction of cell cycle arrest. Interruption of Akt activation using dominant-negative approach resulted in significant inhibition in Akt-phosphorylation (Ser473) and induction of apoptosis in LNCaP cells, whereas ectopic expression of NF-κB/p65 increased doubling time and invasiveness in these cells, which was abrogated by combinational treatment. Furthermore, combination treatment decreased the expression of p-IKK (Ser176/180), p-NF-κB/p65 (Ser536), and p-Akt (Ser473) and downstream targets viz. cyclin D1, Bcl-2, VEGF and MMP9 in both cell lines. In vivo administration of Inhibitor VIII and Parthenolide at 1:10 ratio to PC-3 tumor xenograft for 8 weeks resulted in marked decrease in tumor volume and reduced expression of target genes, compared to individual treatments. These results suggest that nuclear co-localization of Akt and NF-ĸB/p65 may be developed as prognostic biomarker and concurrent targeting of these molecules by combination of pharmacological inhibitors is an effective approach and is more efficacious than use of single agent blocking prostate cancer progression.

#1081

APC regulation of breast cancer therapeutic resistance.

Anne Arnason,1 Monica VanKlompenberg,2 Jenifer R. Prosperi2. 1 _University of Notre Dame, Notre Dame, IN;_ 2 _Indiana Univ. School of Medicine-South Bend, South Bend, IN_.

Resistance to chemotherapy is one of the leading causes of death from breast cancer. Our lab discovered that Adenomatous Polyposis Coli (APC) loss in breast cancer cells results in an elevation of tumor-initiating cells (TICs) and resistance to chemotherapy-induced apoptosis. Given that TICs are often most resistant to standard chemotherapeutic compounds, we sought to understand the mechanism responsible for APC-mediated TIC enhancement. Our hypothesis is that the molecular mechanism involved in chemotherapeutic resistance parallels that promoting the TICs. APC-mutant cells have amplified activation of signal transducer and activator of transcription 3 (STAT3). Interestingly, inhibition of STAT3 with a small molecule inhibitor A69 decreases the TIC population and restores drug sensitivity. Studies are ongoing in the laboratory to investigate other molecular signaling pathways involved in APC-mediated enhanced TIC population and therapy resistance. These data have begun to reveal the molecular mechanisms of APC loss in breast cancer that can guide future treatment plans to counteract chemotherapeutic resistance.

#1082

MerTK promotes resistance to irreversible EGFR TKIs by activation of the PI3K-AKT pathway in NSCLCs expressing wild-type EGFR.

Dan Yan,1 Xiaodong Wang,2 Stephen V. Frye,2 Shelton H. Earp,3 Deborah DeRyckere,1 Douglas K. Graham1. 1 _Aflac Cancer and Blood Disorders Center, Atlanta, GA;_ 2 _Center for Integrative Chemical Biology and Drug Discovery, Chapel Hill, NC;_ 3 _UNC Lingerger Comprehensive Cancer Center, Chapel Hill, NC_.

Lung cancer is the leading cause of cancer-related death with poor survival rates worldwide. Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancers and 60% of these have overexpression of wild-type EGFR (wtEGFR), which portends a poor prognosis. In addition, in a recent clinical trial 6 of 12 lung cancer patients whose tumors acquired a resistance-conferring T790M EGFR mutation during treatment with an EGFR TKI reverted to wtEGFR after treatment with Rociletinib/CO-1686, an irreversible EGFR TKI that is selective for the T790M mutant. These data suggest that targeting wtEGFR may improve treatment outcomes; however, to date single agents that target wtEGFR have not been effective in large scale clinical trials and new ways to target wtEGFR in this context are needed. Our laboratory previously identified the MerTK receptor tyrosine kinase as a potential therapeutic target in NSCLC and developed MRX-2843 as a novel MerTK-selective small molecule tyrosine kinase inhibitor with favorable properties for clinical translation. Irrespective of driver oncogene status, treatment with a MerTK inhibitor yields potent anti-tumor effects in NSCLC cell culture models and blocks tumor growth in xenografts of the MerTK-dependent wtEGFR-expressing A549 cell line. In an attempt to more potently block A549 lung cancer cell proliferation, we screened a library of 378 kinase inhibitors that are in various stages of development and identified synergy between MRX-2843 and multiple irreversible EGFR TKIs, including CO-1686 and Osimertinib/AZD-9291. Further, we found that wtEGFR and MerTK were frequently co-expressed and co-immunoprecipitated with each other in NSCLC cancer cell lysates. Synergistic inhibition of cell expansion was observed in a spectrum of NSCLC cell lines with wtEGFR expression treated with the combination therapy, including H1299 (NRAS mutation), H157 (KRAS mutation), H3122 (ALK fusion), and Colo699 (FGFR1 overexpression). On a mechanistic level, combined treatment with 1μM CO-1686 and 100 nM MRX-2843 dramatically inhibited phosphorylation of both MerTK and EGFR and downstream signaling through the PI3K-AKT and MAPK-ERK pathways, while treatment with equivalent doses of either single agent did not efficiently inhibit MerTK, EGFR or downstream signaling. Additionally, CO-1686 mediated synergistic inhibition of A549 expansion in combination with a PI3K or AKT inhibitor, suggesting a role for PI3K-AKT activation downstream of MerTK contributing to the resistance of wtEGFR NSCLCs to irreversible EGFR TKIs. Taken together, our data provide rationale for a novel strategy for treatment of NSCLC with wtEGFR overexpression by combining MRX-2843 and an irreversible EGFR inhibitor.

#1083

Synergistic drug combination prediction through drug differential dependency network analysis.

Seungchan Kim, Gil Speyer, Harshil Dhruv, Jeff Kiefer, Michael Berens. _The Translational Genomics Research Institute, Phoenix, AZ_.

In an effort to discover strategies which identify effective drug combinations, we analyzed 39 of the 480 compounds screened in the Cancer Therapeutics Response Portal (CTRP) where combinations of two compounds were tested against 860 cancer cell lines; this enabled a comparison of the drug sensitivity of the combinations versus that of the individual compounds. More than half of the drug combinations (n=21) did not significantly improve the drug sensitivity, compared to individual compounds alone. In fact, some of the combinations showed reduced drug sensitivity. In EDDY-CTRP* analysis, the Cancer Cell Line Encyclopedia (CCLE) RNAseq data and CTRP compound response measurements were analyzed to discover both 1) pathways enriched with differential dependencies between sensitive and non-sensitive cell lines for each compound and 2) the mediators of cell line response to a drug. A mediator is a gene in a pathway that plays a significantly different role between sensitive and non-sensitive conditions. The significance is assessed for either essentiality, measured as a node's centrality change, or specificity, measured as the difference in condition specific edges. These drug-pathway-mediator connections are predicted to reveal crucial molecular determinants of drug sensitivity that otherwise are hidden in the complexities of the molecular networks of the cell (Speyer et al., PSB 22:497-508, 2017). We further investigated whether mediators identified for single compounds could predict sensitivity to drug combinations. This analysis revealed that if two single compounds share the same specificity mediators, i.e. the genes with the most significant re-wiring of gene dependencies between sensitive and non-sensitive cell lines, combination of these two compounds correlate with improved sensitivity. The converse was also found: compounds that do not share mediators rarely show synergy. Further analysis and empirical testing of predicted combinations promises to prioritize synergistic drug combinations. We believe that this methodology may predict synergistic drug combinations from cancer cell line drug screening data. Supported by NIH U01CA168397.

*available at http://biocomputing.tgen.org/software/EDDY/CTRP

#1084

Combination of eribulin and AURKA inhibitor prevents metastatic colonization and eradicates established metastases in breast cancer.

Elena N. Pugacheva, Varvara Kozyreva, Anna Kiseleva, Ryan Ice, Brandon Jones, Yuriy Loskutov. _WVU Cancer Institute, Morgantown, WV_.

The majority of cancer-related deaths (90%) are happened due to metastasis from the primary tumor site to distant organs. MLN8237 is a small, highly selective molecule inhibitor of Aurora A kinase (AURKA), which results in disruption of the mitotic spindle, chromosome segregation collapse, and inhibition of cell proliferation. Numerous studies have showed that levels of AURKA are elevated in many types of cancer, including breast cancer. Our previous studies indicated that MLN8237 is extremely potent against pulmonary metastasis, but not the primary tumor in orthotropic xenograft model. To further enhance MLN8237 based regiment, its combination with other therapeutic compounds can be used. As a potential partner we chose eribulin - fully synthetic macrocyclic analogue of the marine natural product halichondrin B. Eribulin belong to the class of non-taxane microtubule destabilizing molecules, currently used in clinic to treat taxol resistant metastatic breast cancer. In present study we have investigated the effect of MLN8237 and eribulin against breast cancer in vivo and in vitro. The results showed that combination of drugs possess a synergistic effect on both primary tumor and metastases, through inducing cytotoxic autophagy and apoptosis. This data clearly indicate great potential behind the MLN8237 based therapies and introduce a new hope for the eradication of metastatic breast cancer.

#1085

**Time dependent analysis for the combination of standardized** Annona cherimola **and** Solanum nigrum **extracts with 5-FU against head and neck cancer cells.**

Serag Eldin I. Elbehairi,1 Aly F. Mohamed,1 Nahla G. Mohamed,1 Mohammad I. Basuony,2 Mahmoud M. Salem,1 Metwally M. Montaser,2 Mohammad Y. Alfaifi,3 Ayman M. Noreddin,4 Ahmed M. Al-Abd5. 1 _Egyptian Organization for Biological Products and Vaccines (VACSERA), Giza, Egypt;_ 2 _Azhar University, Cairo, Egypt;_ 3 _King Khalid University, Abha, Saudi Arabia;_ 4 _Chapman University School of Pharmacy, Irvine, CA;_ 5 _National Research Centre of Egypt, Giza, Egypt_.

Phytochemicals of natural origin constitutes very initiative field of anticancer drug discovery. Annona sp. and Solanum nigrum are folk herbs with reported use for the treatment of several neoplasias. Natural herb products and folk medicine might not be potent anticancer remedies and are recommended for combination therapy. In the current work, we mathematically evaluated the influence of combining standardized extracts of A. cherimola and S. nigrum to the cytotoxic profile of 5-FU against head and neck cancer cells (HEP-2). Cytotoxicity assessment and combination analysis were calculated after MTT assays followed by fitting to Emax model and calculating the combination index (CI-value). After 24 h exposure, both A. cherimola and S. nigrum extracts were more potent than 5-FU with IC50 of 29.4±4.4, 7.3±1.3 and 3.4±4.4 µg/ml, respectively. Both A. cherimola and S. nigrum extracts significantly decreased the IC50 of 5-FU to 5.2±0.55 and 7.6±0.85 µg/ml, respectively; however the CI-values were indicative of additive and antagonistic interactions, respectively (0.91 and 2.49, respectively). After 48 h of exposure, the IC50 of 5-FU, A. cherimola and S. nigrum extracts were 3.1±0.45, 0.87±0.22 and 7.2±0.65 µg/ml, respectively. Similarly, combination of A. cherimola and S. nigrum extracts with 5-FU showed additive and antagonistic interaction with CI-values of 1.05 and 3.37, respectively. After 72 h of exposure, the IC50 of 5-FU, A. cherimola and S. nigrum extracts were 1.9±0.1, 2.1±0.24 and 2.1±0.1 µg/ml, respectively. Similarly, combination of A. cherimola and S. nigrum extracts with 5-FU showed additive and antagonistic interaction with CI-values of 1.2 and 2.21, respectively. Then, CnT model was used to assess the relative influence of drug concentration and exposure time on the cytotoxic profile of treatments under investigation. For S. nigrum and its combination with 5-FU, n-values were 0.9 and 0.63 which is indicative of relatively high influence of drug concentration. On the other hand, n-value for 5-FU treatment was 0.37; this indicates higher influence of drug exposure time compared to other treatment. The n-values of A. cherimola and its combination with 5-FU were 0.51 and 0.54, respectively; this indicate comparable influence of both drug concentration and exposure time. As a confirmatory measure, expression of the apoptotic gene, BAX, was increased after 5-FU combination with A. cherimola and decreased after combination with S. nigrum. In conclusion, some natural herb remedies such as A. cherimola might influence the cytotoxic profile of 5-FU and particularly after short exposure time; while on the other hand, some herb remedies such as S. nigrum significantly antagonizes its cytotoxic activity against head and neck cancer cells.

#1086

Vitamin D3 increases the response to cisplatin in bladder cancer through VDR and TAp73 pathway crosstalk.

Brittany L. Bunch,1 Donald Trump,2 Candace S. Johnson1. 1 _Roswell Park Cancer Institute, Buffalo, NY;_ 2 _Inova Dwight and Martha Cancer Institute, Falls Church, VA_.

Neoadjuvant cisplatin-based chemotherapy is used to treat muscle invasive bladder cancer (MIBC); however the relative 5-year survival of advanced disease is only 15%. The two major pitfalls of neoadjuvant chemotherapy are delaying cystectomy in patients with poor response to cisplatin, and the lack of available biomarkers to identify these patients. Previous studies in our lab showed that pretreatment of preclinical bladder cancer models with 1,25 dihydroxyvitamin D3 (1,25D3), the active metabolite of vitamin D3, enhances the apoptotic response to cisplatin. Mechanistic understanding can provide insight into potential markers for response to 1,25D3 and cisplatin combination therapy in patients. Greater than 50% of MIBCs harbor p53 mutations; however p53 status has not been linked to clinical response to cisplatin. We previously determined that TAp73, a pro-apoptotic p53 family member protein induced after DNA damage, is required for the response to 1,25D3 and cisplatin. In two bladder cancer cell lines with mutant p53, T24 and RT-112, we identified signaling crosstalk between the vitamin D receptor (VDR) and TAp73. Treatment with 1,25D3 and cisplatin, compared to either alone, results in greatest increase of TAp73 mRNA and protein, 2-fold (T24) and 15-fold (RT-112), as well as TAp73 transcriptional target BAX, 3-fold (T24), and 4-fold (RT-112). TAp73 and BAX induction is lost in cells transfected with VDR siRNA, indicating a requirement for VDR. Cells treated with 1,25D3 and cisplatin, compared to either alone, also show the greatest increase of VDR mRNA and protein, 3-fold (T24) and 4-fold (RT-112). CYP24A1, a VDR target gene, has the greatest induction after the combination treatment as well, with an increase of approx. 60-fold (T24), and 100-fold (RT-112). CYP24A1 induction requires TAp73, determined using TAp73 shRNA. These data demonstrate that VDR signaling is enhanced by cisplatin treatment, and TAp73 signaling is enhanced by 1,25D3. We expanded these findings in vivo by treating mice on a vitamin D3 sufficient diet (1,000 IU) or a vitamin D3 deficient diet (25 IU) with 5 mg/kg of cisplatin (IP, q.wk for 3 weeks). Initially, mice on both diets respond to cisplatin treatment, as evidence by a 30% reduction in tumor volume. After the second cycle of treatment, tumors in the deficient mice stopped responding and returned to their initial tumor volume within 24 days. In the sufficient mice, tumors continued to respond to cisplatin with a maximum reduction of 50%. This was maintained for approximately 40 days after treatment. Further study will determine the molecular response to confirm our in vitro mechanism. Our work suggests that vitamin D3 is important in and can increase the response to cisplatin, it also provides rationale for investigating TAp73 protein levels and serum vitamin D3 levels as potential markers to determine patients' likelihood of responding to cisplatin.

#1087

CPX-351 works synergistically in combination with FLT3 inhibitors against AML with FLT3-ITD.

David K. Edwards,1 Nathalie Javidi-Sharifi,1 Angela Rofelty,1 Max Gordon,1 Riley Roth-Carter,1 Paul Tardi,2 Lawrence Mayer,2 Jeffrey W. Tyner1. 1 _Oregon Health & Science University, Portland, OR; _2 _Jazz Pharmaceuticals, Vancouver, British Columbia, Canada_.

Introduction: CPX-351 (Vyxeos) is a liposomal combination of cytarabine and daunorubicin at a synergistic 5:1 molar ratio. Recently, CPX-351 has been shown to be significantly more effective than the 7+3 standard of care chemotherapy in treating high-risk AML patients, including patients with the FLT3-ITD mutation. We previously treated primary patient samples with CPX-351 ex vivo and found that FLT3-ITD+ samples were significantly more sensitive to CPX-351 and showed enhanced drug uptake. We hypothesized that dysregulated FLT3 signaling results in an activation of liposome uptake pathways, leading to increased sensitivity to CPX-351 and ultimately cell death. Furthermore, we examined the effect of combining CPX-351 with existing FLT3 inhibitors (e.g. quizartinib and midostaurin).

Methods: To examine drug uptake dynamics, we exposed AML cell lines (including MOLM-13 and MOLM14 that contain FLT3-ITD, and ME1 that contains an activating FLT3 mutation) to varying concentrations of CPX-351, with or without pre-treatment of quizartinib and midostaurin. We evaluated cell viability using a colorimetric assay and measured intracellular daunorubicin fluorescence, an indicator of drug uptake, by flow cytometry. Additionally, we analyzed the synergy of exposing these cell lines to CPX-351 and FLT3 inhibitors in combination and at different dose schedules, followed by measuring cell viability and daunorubicin fluorescence.

Results: We observed that cell lines containing FLT3-ITD or FLT3-activating mutation were more sensitive to CPX-351 and exhibited increased drug uptake compared to cell lines with other genetic abnormalities. Interestingly, we observed that pre-treatment with quizartinib for 16 hrs produced a population of cells (approximately 50% of the total population) that exhibited decreased daunorubicin fluorescence, suggesting that prolonged FLT3 inhibition may decrease CPX-351 uptake. Consistent with this, we observed robust synergy when combining CPX-351 with FLT3 inhibitors simultaneously or with CPX-351 exposure scheduled 24 hours prior to FLT3 inhibitor exposure. However, exposure to FLT3 inhibitors 24 hours prior to CPX-351 administration was less synergistic and even antagonistic at certain doses.

Conclusions: These data provide additional supportive evidence that FLT3 activation results in increased uptake of CPX-351. This is consistent with results from the CPX-351 Phase III trial in which FLT3-ITD+ patients survived significantly longer when treated with CPX-351 compared to 7+3 chemotherapy. We also show that combining CPX-351 with existing FLT3 inhibitors can elicit a synergistic response when administered in dosing regimens where FLT3 inhibition does not precede CPX-351 treatment. Cumulatively, our data support further testing of CPX-351 in combination with FLT3 inhibitors for treating AML patients with genetic dysregulation of FLT3 signaling.

#1088

Metronomic therapy of doxorrubicin and molecular iodine in canine mammary cancer.

Xochitl Zambrano Estrada,1 Brianda Landaverde,2 Andrés A. Dueñas,2 Marco A. De Paz,2 Gerardo A. Hernández,2 Benjamín Solorio,3 Manuel Trejo,3 Laura Pérez,3 Carmen Aceves1. 1 _Instituto de Neurobiología, Juriquilla, México, Mexico;_ 2 _Facultad de Estudios Superiores Cuautitlán, Estado de México, Mexico;_ 3 _Facultad de Ciencias Naturales, Juriquilla, México, Mexico_.

Introduction: Conventional chemotherapy is administered at the maximal tolerated dose (MTD), usually every 21 days, and the drug-free period allows the patient to recover from the adverse effects secondary to the treatment. However, this period also enables tumor cells to reinitiate growth through induced tumoral revascularization. In recent years, a new strategy known as metronomic chemotherapy explored the scheme of long-lasting low-dose administration of antineoplastic drugs, which results in the attenuation or interruption of tumor growth by inhibition of angiogenesis and/or reactivation of anti-tumoral immune system. Moreover, the use of combination therapies including cell re-differentiation messengers are currently being tested to provide a better quality of life. In this sense, it has been demonstrated that the supplement of molecular iodine (I2) induces apoptotic and cellular re-differentiation effects, and its combination with doxorubicin (DOX) promotes a cytotoxic effect and generates cardioprotection. In this study, canine mammary cancer is used as a model of spontaneous carcinogenesis.

Objective: Determine the efficacy of metronomic therapy of DOX in combination with I2 analyzing the clinical, pathological, and molecular effects involved in proliferation, invasion, and chemoresistance.

Methods: 27 female dogs with mammary cancer were given daily I2 (10 mg) supplements or placebo (colored water) in conjunction with four cycles of DOX (30mg/m2, intravenous). Two protocols were carried out: the standard protocol with a 21-day interval and 20-minute infusions, and the metronomic protocol with a 15-day interval and 60-minute infusions (DOX-M).

Results: The metronomic scheme (DOX-M) attenuated the severity of side effects, but it showed no difference in tumor response (size, gene expression). The I2 supplement significantly improved the quality of life in both schemes, and it exerted synergistic effects on the tumor response in DOX-M, decreasing the epithelial component as well as markers of chemoresistance (MDR1 and Survivin) and invasion (uPA). The antitumor immune response (lymphocytic infiltration) in this scheme was significantly activated, and all these responses were accompanied by a major increase in peroxisome proliferator-activated receptor type gamma (PPARy) expression. Endocrine evaluation showed that I2 supplements do not alter thyroid function or estrogen response.

Conclusions: Combination of DOX + I2 in a metronomic scheme exerts synergistic effects that prevent drug resistance and invasion and greatly improve the patient's quality of life. Studies are underway to decrease the dose of DOX. Key words: Molecular Iodine, Doxorubicin, Canine Mammary Cancer, Metronomic Therapy. The authors appreciate the technical support of Alexander Bontempo, Evangelina Delgado and María Juana Cardenas. Investigation supported by PAPIIT-UNAM IN200813 and 201516; CONACYT 245255.

#1089

Anti-tumor activity of selinexor is enhanced by palbociclib in preclinical models of HER2+ breast cancer.

Hua Chang, Sharon Friedlander, Trinayan Kashyap, Boris Klebanov, Christian Argueta, Oscar A. Gonzalez, Erkan Baloglu, Yosef Landesman, Sharon Shacham, Margaret Lee, William Senapedis. _Karyopharm Therapeutics, Inc., Newton, MA_.

Introduction: XPO1 (exportin-1/ CRM1) inhibitor selinexor (KPT-330) is the first-in-class, orally bioavailable, clinical stage SINE (Selective Inhibitor of Nuclear Export) compound with marked anti-tumor activity towards solid and hematological malignancies. This activity can be further enhanced by other therapeutic agents. We have previously shown strong synergistic preclinical activity of selinexor plus trastuzumab against HER2+ breast cancer. In cancer models of acquired resistance to HER2-targeted therapies, G1/S phase cell cycle regulators Cyclin D1 and CDK4/6 are inappropriately activated. We therefore investigated the combinatorial effect of selinexor plus palbociclib, a CDK4/6 inhibitor, in HER2+ breast cancer models as a treatment option for recurrent and relapsed HER2+ breast cancers.

Methods: The effects of selinexor or palbociclib single agents or in combination were tested in vitro with BT474 HER2+ breast cancer cell line. Total RNA and protein was extracted from cell lysates and analyzed by qPCR and immunoblots. In vivo, a subcutaneous BT474 xenograft mouse model was treated with selinexor (5 mg/kg or 15 mg/kg; qodx3) or palbociclib (50 mg/kg or 150 mg/kg; qd) single agents or in combination. Tumor growth and body weights were monitored for 60 days. Tumors were harvested and analyzed by immunohistochemistry (IHC).

Results: Selinexor plus palbociclib was highly effective in vitro and in vivo in BT474 breast cancer cells. In in vitro assays, selinexor or palbociclib single agents demonstrated inhibitory effects on cell proliferation and showed strong synergy when combined. In vivo, the combination resulted in significant survival benefit and enhanced tumor growth inhibition compared to vehicle or either single agent. IHC analysis of xenograft tumors showed synergistic inhibition of cell proliferation by selinexor plus palbociclib. The Ki67 proliferation index determined by IHC was 25% for vehicle control, 20% for selinexor, 7% for palbociclib and 2% for the combination. Based on IHC analysis, the synergistic anti-tumor activity of selinexor plus palbociclib was achieved at multiple levels of the CDK4/6 pathway. Selinexor treatment increased p21, p27 and Rb nuclear staining. Both p21 and p27 are inhibitors of CDK4/6 while Rb is a negative regulator of cell cycle progression. CDK4/6 phosphorylates and inactivates Rb, which allows cell cycle progression. In selinexor as well as palbociclib treated samples, phosphorylated Rb in the nucleus decreased, indicating a down-regulation of the CDK4/6 pathway.

Conclusion: Selinexor plus palbociclib shows synergistic inhibition of cell proliferation in vivo and in vitro in HER2+ breast cancer cell line BT474 by down-regulation of CDK4/6 pathway. This combination therapy warrants further investigation as an effective treatment option for recurrent and relapsed HER2+ breast cancer.

#1090

Preclinical evaluation of combination therapy with dasatinib and sorafenib for the treatment of brain metastases.

Qingyu Stephanie Zhou, Xiaofang Guo. _Univ. of South Florida College of Pharmacy, Tampa, FL_.

Brain metastases, the most common type of intracranial tumors in adults, carry a dismal prognosis with a median survival of less than 18 months regardless of primary status or treatment given. As part of an ongoing project, this study is concerned with the preclinical evaluation of targeted combination therapy for the treatment of brain metastases. The cell line used in this study was the H1915 (ATCC® CRL-5904TM) human lung cancer cell line, which was originally derived from a brain metastasis from primary lung adenocarcinoma. The cell line was stably transfected with a plasmid that allows expression of firefly luciferase (i.e., H1915-luc) so that the tumor growth in a mouse orthotopic model of brain metastases can be assessed in real time using the Xenogen in vivo bioluminescence imaging (BLI). Our previous study has demonstrated that the synergistic effect of combined dasatinib and sorafenib treatment was cell-line dependent. In this study, the cytotoxicity of dasatinib and sorafenib, when used alone or in combination at the fixed molar ratios of 3:1, 1:1 and 1:3 (dasatinib:sorafenib), was further examined in H1915-luc cells using the MTT assay. The results demonstrated that simultaneous and continuous exposure of H1915-luc cells to dasatinib and sorafenib at the fixed concentration ratio of 1:1 and 1:3 for 72 h showed synergism (the combination index value between 0.7 and 0.8) at effect levels of 40 and 80% inhibition of cell viability. For the in vivo study, an orthotopic mouse model of brain metastases was established by injecting H1915-luc cells into the right general carotid artery of individual athymic nude mice followed by permanent ligation. The preliminary study showed that following the intracarotid injection of H1915-luc cells, brain metastases were formed and readily to be detected by BLI. The obtained whole brain samples were fixed in OCT and stored at -80oC. The brain sections are to be subjected to the immunofluorescence double staining of CD31 and firefly luciferase to assess the potential effect of the dasatinib-sorafenib combination on microscopic brain metastasis formation and progression and vessel co-option. Given the fact that brain metastases usually have a poor response to chemotherapeutic agents in part due to the presence of blood-brain barrier, a pharmacokinetic study is underway to characterize the distribution of sorafenib and dasatinib in normal brain tissues and brain metastases using the serial blood sampling and brain microdialysis technique. Overall, results from the in vitro cytotoxicity study demonstrated the synergistic effect of dasatinib and sorafenib on H1915-luc human lung cancer cells, providing a rationale for the in vivo evaluation the potential of this combination for the treatment of brain metastases from lung carcinomas.

#1091

Paclitaxel-loaded microparticles in combination with gemcitabine-loaded microparticles decreases gemcitabine resistance and promotes cell death in pancreatic cancer cell lines.

Maria Munoz-Sagastibelza, Ariella Moshchinsky, Oluwafeyikemi Okome, Jenny E. Paredes Sanchez, Raavi Gupta, Laura Martello-Rooney. _SUNY Downstate Medical Ctr., Brooklyn, NY_.

Pancreatic cancer is the fourth leading cause of cancer death in the United States with only 7% of diagnosed patients surviving 5 years. Current systemic chemotherapies have not been very effective at decreasing tumor burden, but nonetheless expose patients to the adverse side effects of treatment. Poly(lactic-co-glycolic acid)-based microparticles (MPs) are a promising tool for localized drug delivery within the tumor due to their high biocompatibility, flexibility in drug encapsulation and extended drug release. A previous in vivo experiment from our laboratory demonstrated minimal penetration of systemic gemcitabine into the tumor resulting in low levels of apoptosis compared to locally injected gemcitabine-loaded microparticles (GMPs), which demonstrated much higher levels of intratumoral apoptosis. The present study investigated whether paclitaxel-loaded microparticles (PMPs), alone or in combination with GMPs, decreased gemcitabine resistance, colony formation and promoted cell death in two human pancreatic cancer cell lines, PANC-1 and MIAPaCa-2. In vitro studies with PANC-1 cells treated with PMPs or GMPs showed enhanced cell killing and a significant decrease in colony formation in the higher MPs volume groups. Moreover, the combination treatment of PMPs followed by GMPs, showed a greater decrease in colony formation in comparison with the single treatments. Subsequently, we tested the effect of single and combination treatments on two well-known resistance markers for gemcitabine, ribonucleotide reductase catalytic subunit M1 (RRM1) and cytidine deaminase (CDA), as well as on the promotion of cell death measuring cleaved caspase-3 (CC3). Interestingly, PANC-1 cells treated with PMPs alone showed an increase in CC3 expression and a significant decrease in the expression of both markers when treated with different volumes of PMPs. MIAPaCa-2 cells exhibited similar results. The combination treatment also showed a modulation in RRM1 and CDA proteins and an increase in CC3 expression. However, when PANC-1 cells were treated with GMPs alone, both markers went up suggesting an increase in resistance against gemcitabine. These experiments are in progress for the MIAPaCa-2 cells. In addition, preliminary drug release studies with GMPs demonstrated detection of gemcitabine up to 14 days. In conclusion, our data demonstrated that PMPs and GMPs promote an increase in cancer cell death. Furthermore, the combination of the drug-loaded MPs indicated a reduction in drug resistance and clear impairment in colony formation. Further studies are in progress to investigate combination MPs injections in a mouse model of pancreatic cancer to confirm in vivo efficacy. The described drug delivery method has the potential to be a more efficient local treatment modality than systemic drug against pancreatic cancer.

#1092

PF562271, a Pyk2 inhibitor, reduces glioma tumor growth and invasion in C57Bl6-Gl261 mouse glioma model.

Jescelica Ortiz-Rivera,1 Kimberleve Rolón-Reyes,2 Alejandro Albors,1 Luis Cubano,1 Lilia Kucheryavykh1. 1 _Universidad Central del Caribe, Bayamon, PR;_ 2 _University of Puerto Rico, Rio Piedras, PR_.

Glioblastoma is an extraordinarily aggressive type of brain cancer due to its invasive and proliferative nature. The tumor microenvironment, with microglia as a critical player, has an important role in tumor progression. Microglia infiltrate majority of gliomas and release factors, which favor tumor growth and invasion. Previously we demonstrated that microglia residing within the tumor stimulate glioma cell invasion through the proline rich tyrosine kinase 2 (Pyk2) signaling cascade. We hypothesize that the use of pharmacological inhibitors of Pyk2 together with currently applied chemotherapy can significantly reduce invasiveness of glioblastoma tumor and improve the outcome of the treatment. Using C57Bl/6-Gl261 mouse glioma implantation model we investigated the effect of the combined treatment for glioblastoma with temozolomide (TMZ, 50 mg/kg, once/day, orally) together with a Pyk2 inhibitor PF562271 (twice/daily, 25 mg/kg, orally) vs. TMZ monotherapy. Treatment was provided during 14 days, beginning the 5th day after glioma implantation. For the assessment of the effectiveness of treatment animals' survival, tumor size, and invasion area were evaluated. Western blot were used for the evaluation of the level of Pyk2 phosphorylation in tumor cells. For the purification of glioma cells from total tumor tissue Percol gradients were used. The study revealed that treatment with PF562271 reduced invasion of glioma cells at the tumor edge, while TMZ reduced the tumor growth. Combined treatment showed significantly more prominent effect on reduction of tumors growth compared to TMZ monotherapy and additionally reduced invasiveness of tumors similar to PF562271 monotherapy. In both PF562271 monotherapy and combined treatments the downregulation of Pyk2 phosphorylation in glioma cells has been recorded. Survival analysis demonstrated a significance increase of survival of animals received combined treatment compare to TMZ monotherapy. In conclusion, we can state that combined treatment with TMZ together with PF562271 reduced Pyk2-related tumor growth and invasiveness and increased animal survival. This research was made possible by NIH grant numbers: 1SC2GM102040, G12MD007583, R25GM110513, Puerto Rico Science, Technology, and Research Trust grant number 2016-00157, and US Department of Education Grant number P031S130068.

#1093

**Synergistic action of sorafenib and carfilzomib against hepatocellular carcinoma** in vitro **and** in vivo **.**

Chao Jiang,1 Rui Xu,2 Xiao-Xing Li,1 Hui-Yun Wang,1 X.F. Steven Zheng1. 1 _Sun Yat-sen University Cancer Center, Guangzhou, China;_ 2 _Cancer Center of Guangzhou Medical University, Guangzhou, China_.

Background: Sorafenib, a multikinase inhibitor, is currently the only approved systemic drug for advanced hepatocellular carcinoma (HCC), but has demonstrated limited survival benefits for patients. Carfilzomib (CFZ) is a second-generation proteasome inhibitor approved for the treatment of multiple myeloma. In this study, we aim to investigate the synergistic antitumor effect of the combined treatment of sorafenib and CFZ in hepatocellular carcinoma and the underlying molecular mechanisms.

Methods: Hep3B and Bel-7402 HCC cell lines were treated with sorafenib and/or CFZ at clinically relevant concentrations, after which effects on cell viability and proliferation were analyzed using MTS method; cell migration and invasion were evaluated in transwell assay; cell apoptosis were monitored by flow cytometry. Western blot assay was used to investigate the potential molecular mechanism of the synergistic antitumor effect of the combined treatment. In vivo efficacy was determined in nude mice with Hep3B xenografts.

Results: MTS results showed that CFZ could improve the drug sensitivities of sorafenib in Hep3B and Bel-7402 cells when combined these two drugs together. Moreover, cell proliferation was more significantly suppressed in combinational treatment compared to single agent (p < 0.01) in the two tested cell lines. The colony formation assay showed similar results with the MTS assay. The results from flow cytometry showed that sorafenib or CFZ alone induced about 11% apoptotic cells in Hep3B, when administered in combination the percentage of apoptotic cells significantly increased to about 20.78% (p < 0.01). Similar results were observed in Bel-7402. Results of transwell assay demonstrated that cell migration and invasion ability were inhibited when treated with sorafenib or CFZ alone; and cells treated with the combinational therapy migrated and invaded even less (p < 0.05). The in vivo xenograft tumor model showed that treatment with sorafenib or CFZ inhibited Hep3B tumor growth (p < 0.05); moreover, treatment with the combination more significantly inhibited tumor growth (p < 0.01). Mechanistically, combined treatment of the two drugs suppressed the epithelial-mesenchymal transition process by increasing E-cadherin and decreasing N-cadherin and β-catenin. Furthermore, the drug combination activated the mitochondria-related apoptosis pathway, showing up-regulation of cleaved caspase 3/7/9. Up-regulations of PERK/eIF2α/ATF4/CHOP pathway implicated the involvement of ER stress-mediated apoptotic pathway following the drug combination treatment.

Conclusion: In conclusion, these findings suggest that the combination of sorafenib and CFZ shows synergistic antitumor activities in HCC, potentially providing a novel therapeutic strategy for patients with advanced hepatocellular carcinoma.

#1094

Combining the pan-FGFR inhibitor AZD4547 with radiation in lung and head and neck squamous cell carcinoma.

Andrew M. Baschnagel, Chunrong Li, Alecia M. Morgan, Sean R. Brennan, Kalley A. Russo, Paul M. Harari. _Univ. of Wisconsin School of Medicine, Madison, WI_.

The fibroblast growth factor receptors (FGFR1, FGR2, FRG3, FGR4) are frequently altered or overexpressed in both lung and head and neck squamous cell carcinomas (SCC). Amplification of FGFR1 has been reported to be as high as 22% in lung SCC and 17% in head and neck SCC (HNSCC). FGFRs have been identified as a potential therapeutic target in lung and head and neck cancers. In addition, FGFRs activate multiple pathways involved in the radiation response including RAS/RAF/MAPK, PI3K/AKT and STAT. AZD4547 is a potent and selective tyrosine kinase inhibitor of the FGFR family and is currently being investigated in clinical trials. Since a substantial portion of lung cancer and HNSCC patients are treated with radiotherapy, we examined the anti-tumor effects of AZD4547 in combination with radiation in both lung and head and neck cancer model systems. FGFR protein and RNA expression and cell proliferation with AZD4547 were evaluated in 8 lung and 8 head and neck cancer cell lines. Clonogenic survival assays were performed on 4 lung and 4 head and neck cancer cell lines and xenograft experiments were performed on one HNSCC cell line. These cell lines demonstrated varying levels of FGFR1, 2 and 3 protein and RNA expression. The half maximal inhibitory concentration (IC50) of AZD4547 in these cell lines ranged from 0.05 to 128 uM. Sensitivity to AZD4547 did not correlate directly with protein or RNA expression. In vitro clonogenic survival assays showed limited effects of combining AZD4547 with radiation. However, significant tumor growth delay was observed with the combination of radiation and AZD4547 compared to radiation or drug alone in the SCC1483 FGFR2 expressing HNSCC xenograft model. These findings suggest that AZD4547 can augment the response of radiation in an in vivo model system. Further studies are underway to test these findings in additional cell lines including FGFR dependent amplified cancer cell lines.

#1095

A combination of suberoylanilide hydroxamic acid and quinacrine is an effective therapeutic approach in preclinical settings of upper gastrointestinal cancers.

Shoumin Zhu, Wael El-Rifai. _Surgery, Nashville, TN_.

Background: Quinacrine (QC), an antimalarial drug, has been shown to possess anticancer effects. Suberoylanilide hydroxamic acid (SAHA) inhibits class I and class II HDACs and is approved for cancer therapy. Developing novel approaches to overcome cancer drug resistance could significantly enhance current therapeutic approaches and improve patient care.

Methods: ATP-GLO, clonogenic survival, Annexin-V apoptosis assay, comet assay and DNA double-strand breaks (DSB) kits were used. The mRNA and protein levels were evaluated by quantitative real-time PCR and Western blot analyses.

Results: A combination of QC/SAHA significantly increased cell death in all cancer cell lines and had no effect on immortalized non-cancer cell lines (HFE145, NIH-3T3 and EPC2) (P<0.01). Clonogenic survival assay indicated that QC/SAHA co-treatment led to significantly lower number of cancer cell colonies, as compared to single agents and controls (P<0.01). Of note, the QC/SAHA combination led to an increase in the the sub-G0 population in AGS (9-fold), MKN-28 (14-fold), FLO1 (5.6-fold), and SNU1 (4-fold) cells (P<0.01). These results were confirmed using the Annexin V apoptosis induced significantly higher levels of apoptosis (10 - 20 fold) as compared to single agent and control (P<0.01). Treatment with QC/SAHA combination induced high levels of DSB (>20 fold, P<0.01). Comet assay data showed increased DNA damage compared with vehicle-treated cells (8-fold, P<0.01). Western blot analysis demonstrated a notable increase in activation of PARP, caspases 3, 9 and γ-H2AX following QC/SAHA co-treatment in all cancer cell lines. Interestingly, the combination of QC/SAHA substantially decreased the protein levels of both wtP53 and mutP53 in these cells. Tumor xenograft data confirmed that a combination of QC/SAHA is more effective than a single agent in abrogating tumor growth (P<0.05).

Conclusion: Our novel findings show that QC and SAHA have a synergistic effect on cancer cell death. The results provide compelling evidence that increased DNA damage mediates the cytotoxic effect of combined QC/SAHA. Such effect is likely related to mutP53 and wtP53 protein degradation induced by QC/SAHA combination. Our findings provide a rationale for a clinical evaluation of combined QC/SAHA therapy in gastro-esophageal cancers. Ongoing studies are currently being undertaken to understand mechanisms that regulate the degradation of wtp53 and mutp53 proteins.

#1096

**Co-targeting of AKT and Pim kinases in mouse** PTEN **-deficient prostate cancer.**

Marco A. De Velasco,1 Koichi Sugimoto,1 Yurie Kura,1 Naomi Ando,1 Noriko Sato,1 Kazuko Sakai,1 Barry R. Davies,2 Dennis Huszar,2 Masahiro Nozawa,1 Kazuhiro Yoshimura,1 Kazuhiro Yoshikawa,3 Kazuto Nishio,1 Hirotsugu Uemura1. 1 _Kindai University Faculty of Medicine, Osaka-Sayama, Japan;_ 2 _AstraZeneca, Macclesfield, United Kingdom;_ 3 _Aichi Medical University, Nagakute, Japan_.

AKT and Pim kinases modulate programmed cell death by the phosphorylation of common substrates that regulate apoptosis and other survival processes. Evidence suggests that the antitumor effects of targeted Akt inhibition can be inhibited or diminished by the JAK/STAT-dependent induction of Pim kinases. In this study we examined the therapeutic potential of co-targeting AKT and Pim in a genetically engineered mouse model of prostate cancer driven by the conditional inactivation of PTEN. The antitumor effects of AZD5363, a pan AKT inhibitor, and AZD1208, a highly potent Pim kinase inhibitor, were investigated as monotherapy or in combination on mice harboring castration-naïve prostate tumors and mice that developed castration-resistant disease. Mice were randomized treated for four weeks. Safety and tolerability was assessed by bodyweight changes. Antitumor activity was determined by differences in tumor burden, proliferation and apoptosis and histology. Molecular activity was assessed by examining the phosphorylation of common substrates by western blot analysis. Treatments were well-tolerated and no significant differences in bodyweight changes were observed. In castration-naïve prostate tumors, treatments with AZD5363, AZD1208 and AZD5363/AZD1208 resulted in 11.9%, 13.5% and 36.9% reductions of tumor burden compared to vehicle treated controls, respectively, P<0.001. The treatment combination of AZD5363/AZD1208 demonstrated significant antitumor activity compared to monotherapy, P<0.001. In the castration-resistant tumors, treatments with AZD5363, AZD1208 and AZD5363/AZD1208 resulted in 21%, 9.5% and 27% reductions of tumor burden compared to vehicle treated controls, respectively, P<0.004. Although the differences in in tumor burden were not statistically between monotherapy and combination therapy, a notable degree of tumor gland regression was observed in tumors treated with AZD5363/AZD1208. Overall, combination therapy showed a synergistic effect by inhibited or impaired phosphorylation of PRAS40, eIF4B and BAD in both castration-naïve and castration-resistant tumor models. Inhibition of both pathways enhanced the reduction of tumor cell proliferation and increased apoptosis. Overall, our findings provide in vivo data to support redundancy between AKT/Pim survival pathways and suggest that a therapeutic approach of combined AKT/Pim kinase inhibition may be possible therapeutic approach for AKT-driven prostate cancer.

#1097

Enhancing the efficacy of Temozolomide with Biochanin A: Mechanistic and metabolic effects.

Vilas Desai,1 Aditi Jain,2 Maryna Perepelyuk,1 Annie Chhun,1 Christopher Farrell,1 James C. K. Lai,3 Alok Bhushan1. 1 _Thomas Jefferson University, Philadelphia, PA;_ 2 _University of Kentucky, Lexington, KY;_ 3 _Idaho State University, Pocatello, ID_.

Enhancing the efficacy of Temozolomide with Biochanin A: Mechanistic and Metabolic effects

Glioblastoma multiforme (GBM) is the most frequent, primary malignant brain tumor prevalent in humans. GBM is characterized by aggressive cell proliferation and rapid invasion of normal brain tissue resulting in poor patient prognosis. Furthermore, the current standard therapy of surgical resection followed by radiotherapy and chemotherapy with Temozolomide is not very effective. The inefficacy of the currently available chemotherapeutic agents for GBM can be attributed to the challenges of drug delivery to the tumor. Our laboratory has previously reported the anticancer potential of isoflavones like Biochanin A against oral, pancreatic, breast and brain cancers. In this study, we tested our hypothesis that, Biochanin A sensitizes the GBM cells to Temozolomide, thereby increasing its efficacy in combination treatment. We used the MTT assay to determine the effects of Biochanin A alone and in combination with Temozolomide on survival of U-87 MG cells. Furthermore, two different nanoformulations of Single Walled Carbon Nanotubes (SWCNTs) functionalized with Biochanin A and Atorvastatin and SWCNTs functionalized with Temozolomide were synthesized and their effects on cell viability of U-87 MG glioblastoma cells tested. The cell viability results indicate a decrease in cell proliferation with the combination treatment as compared to the individual drug treatment. Similarly, the flow cytometry analysis showed an increased induction of apoptosis with combination treatment as compared to the drugs alone. Western-blot analysis and Immunofluorescence imaging methods were used to elucidate some of the cell survival/proliferation mechanisms involving EGFR, p-AKT, MMP-2, MT-MMP1. The effects of the drugs on U-87 MG cell metabolism (parameters like ECAR & OCR) studied using Seahorse XFp analyser, suggest an increase in the mitochondrial respiration. Our results provide support for our hypothesis and the data shows that it may have implications in increasing the efficacy of the conventional treatments for GBM. These results will potentially aid in designing new therapies that can improve the outcome of GBM.

#1098

Delta-tocotrienol chemosensitizes human pancreatic tumor metastasis to gemcitabine targeting cancer stem cells.

Kazim Husain, Said M. Sebti, Mokenge P. Malafa. _Moffitt Cancer Center, Tampa, FL_.

Background: Pancreatic cancer, a lethal malignancy is the fourth leading cause of cancer-related deaths in the United States. The standard chemotherapy with gemcitabine is ineffective in patients with metastatic tumors. Pancreatic cancer stem cells (CSCs) have been implicated in the development of pancreatic cancer metastasis, resistance to chemotherapy and its recurrence following surgical extirpation. We have shown that natural vitamin E δ-tocotrienol is the most bioactive tocotrienols against pancreatic cancer in vitro as well as in vivo models. The purpose of this study was to evaluate the chemosensitization of pancreatic tumor metastasis by δ-tocotrienol to gemcitabine in vitro as well as in vivo.

Methods: In vitro human metastatic pancreatic cancer cells L3.6pl and pancreatic cancer stem cells (PCSC) were treated with gemcitabine (5 μM) and δ-tocotrienol (50 μM) alone and in combination. Treated cells used for epithelial to mesenchymal transition (EMT), migration/invasion, microsphere/spheroid, and signaling markers assays. PCSC expressing luciferase was orthotopically implanted into pancreas of Athymic nude mice (n = 20) and after one week they were randomized into four groups: 1) vehicle control (ethanol extracted olive oil), 2) gemcitabine (100 mg/kg, IP, twice a week), 3) δ-tocotrienol (200 mg/kg, orally twice a day) and 4) gemcitabine + δ-tocotrienol. The treatment was continued for 4 weeks. The tumor volume, tumor weight, metastasis were recorded.

Results: Gemcitabine slightly inhibited the growth, migration/invasion of L3.6pl and PCSC in vitro and PCSC tumor growth (tumor volume and weight) in vivo. δ-tocotrienol significantly inhibited the growth, microsphere/spheroid, migration/invasion, EMT(E-cadherin to vimentin), angiogenesis (VEGF), PCSC transcription factors (Nanog, Oct4, and Sox2), tumor growth and liver/lung metastasis compared to control. The combination of both drugs synergistically inhibited the cancer and stem cell growth, EMT, migration/invasion, microsphere/spheroid, metastasis, angiogenesis, and PCSC transcription factors in vitro as well as in vivo.

Conclusion: Vitamin E δ-tocotrienol chemosensitizes human pancreatic tumor metastasis to gemcitabine through inhibition of EMT, migration, invasion, angiogenesis, cancer stem cell self-renewal, tumor growth and metastasis.

#1099

**Testosterone abolishes cabazitaxel efficacy in an** in vivo **model of prostate cancer and affects intratumoral concentrations.**

Lisanne Mout, Corrina de Ridder, Debra Stuurman, Peter Bruijn, Ron H. Mathijssen, Ronald de Wit, Martijn P. Lolkema, Wytske M. Weerden. _Erasmus MC, Rotterdam, Netherlands_.

Introduction Docetaxel combined with castration provides a major survival benefit in newly diagnosed metastatic PCa. Conversely, docetaxel without castration in high-risk localized PCa is not active. Cabazitaxel is a new taxane retaining its activity in tumors resistant to docetaxel and new hormonal therapies. We aimed at better characterizing in vivo the relationship between cabazitaxel and circulating androgens in a preclinical human-derived xenograft model of castration-resistant prostate cancer (CRPC). Material and methods Athymic nude mice were inoculated with the androgen receptor wt, PSA secreting CRPC cell line PC346C-DCC-K. Mice were surgically castrated when tumors were established (tumor volume (TV) of 150 mm3). After 7 days, mice were randomized to receive testosterone (40 mg) or empty pellet. The following day mice were injected with one bolus injection of cabazitaxel (33 mg/kg) or NaCl intraperitoneally. Mice were sacrificed when tumors exceeded a volume of 1,500 mm3 or the maximum follow-up of 90 days after cabazitaxel treatment. Blood was sampled biweekly for PSA and testosterone and analyzed by an immunoassay. In a second in vivo experiment intratumoral uptake of cabazitaxel was determined in the presence and absence of testosterone using a similar experimental set-up, with the exception that mice were sacrificed 7 days after cabazitaxel treatment. Here, intratumoral cabazitaxel concentrations were determined in snap frozen tumors by LC/MS-MS and corrected for tumor weight. Results Cabazitaxel treatment of castrated male mice bearing PC346C-DCC-K tumors resulted in a complete or near-complete antitumor response. In contrast, in mice supplemented with testosterone the anti-tumor effect of cabazitaxel was not significantly different from placebo (median time till TV 1,500 mm3 48 and 45 days resp.). Importantly, testosterone supplementation alone did not significantly affect tumor growth, confirming the CRPC nature of the (PC346C-DCC-K) xenograft. Interestingly, in tumors of testosterone supplemented castrated mice the intratumoral cabazitaxel concentrations were significantly decreased compared to mice that did not receive testosterone (0.39 ng cabazitaxel per mg tumor tissue vs. 1.36 ng/mg, t-test p=0.0032). Conclusions These findings indicate that circulating testosterone significantly impairs the efficacy of cabazitaxel. Testosterone supplementation may alter the metabolism of cabazitaxel, or may interfere with uptake and/or accumulation of cabazitaxel in PCa cells. We will further investigate the relationship between circulating androgens and intratumoral cabazitaxel accumulation as well as anti-tumor efficacy. The study was supported by a research grant from Sanofi

#1100

Synergistic interaction between gemcitabine and PD-0325901 in C-33A cervical carcinoma cell line.

Macarena Irigoyen, Gonzalo Castillo. _BIOENSIS, LLC., Bellevue, WA_.

Cervical cancer affects over half a million women worldwide, and is one of the most common causes of death in women. Treatment of cervical cancers consists of surgery, radiation, chemotherapy, or combination of therapies. However, about half of patients with advance cancer will show recurrent tumors. Signaling cascades are often dysregulated in human cancer cells making them attractive targets for combination drug therapies. PD-0325901, an inhibitor of the mitogen-activated protein kinase, is a promising target for metastatic cancer. Another drug used to treat cervical carcinoma is gemcitabine, a nucleoside analog inhibitor of the ribonucleotide reductase and DNA damage inducible protein 45a (Gadd45a). This drug is generally used in combination with other drugs, after reoccurrence of the disease state. We decided to evaluate the efficacy of gemcitabine in combination with PD-0325901 in C-33A, a metastatic cervical cancer-derived cell line. We evaluated the potency of gemcitabine and PD-0325901 alone and in combination. Cell viability after incubation with the drugs was determined at 72 hours and at 7 days by measuring ATP content. Synergy was determined assessed utilizing BIOENSIS's suite of drug combination analysis tools including: isobologram, combination index (CI) and Bliss analysis. Isobologram analysis at 72 hours of incubation revealed that the mean values of the observed data were significantly smaller than those of the predicted maximum additive values, indicating synergy. Bliss and combination index analysis confirmed the observation, showing a synergistic effect at concentrations between 2 - 200 nM PD-0325901 and 0.3 - 3 nM gemcitabine. Similar patterns of synergy were observed with 7 day incubations of C-33A with gemcitabine and PD-0325901. These results indicate that gemcitabine and PD-0325901 showed significantly more growth inhibition than either drug alone in the C-33A tumor cell line, suggesting that the combination of gemcitabine - PD-0325901 could be used in cervical cancer therapy.

#1101

**Telomerase inhibitor imetelstat in combination with the BCL-2 inhibitor venetoclax enhances apoptosis** in vitro **and increases survival** in vivo **in acute myeloid leukemia.**

Joshua J. Rusbuldt, Leopoldo Luistro, Diana Chin, Melissa Smith, Amy Wong, Margarita Romero, Aleksandra Rizo, Jacqueline Bussolari, Fei Huang, Amy (Kate) Sasser. _Janssen Research & Development, LLC, Spring House, PA_.

Background/Aims: Acute myeloid leukemia (AML) is an aggressive cancer with limited treatment options outside of chemotherapy. Improved therapies with novel mechanisms of action are desperately needed to fill this need. Both hTERT, the catalytic subunit of telomerase, and BCL-2, an apoptotic regulator, are overexpressed in AML, correlating with disease severity and poor prognosis respectively. Imetelstat is a novel, first-in-class competitive inhibitor of telomerase with clinical activity in hematologic malignancies. Venetoclax, an approved BCL-2 inhibitor for CLL, has shown a promising clinical benefit in AML patients. Preclinical evidence shows that downregulation of hTERT induces apoptosis via disruptions of hTERT and BCL-2 interaction; we hypothesize that inhibiting both targets would yield greater anti-tumor activity in AML compared to treatment with either agent alone.

Methods: AML cell lines and AML patient's PBMC samples were treated with imetelstat or venetoclax alone, or in combination, and viable and apoptotic populations of cells were evaluated by flow cytometry. Telomerase activity, hTERT expression and mitochondrial dysfunction were investigated for mechanism of action. Furthermore, an in vivo study in the MOLM-13 AML disseminated model was conducted to assess efficacy and survival.

Results: A dose-dependent synergistic activity in inducing apoptosis was observed in multiple AML cell lines when combining imetelstat with venetoclax. In the MOLM-13 cell line, single-agent imetelstat and venetoclax had modest apoptotic activity after 48 hours (22% and 30% respectively), but the combination achieved 88% at 48 hours and nearly 100% at 96 hours. Similarly enhanced apoptotic activity was also observed in PBMCs purified from 4 AML patient whole blood samples. Molecular analyses showed combining imetelstat with venetoclax reduced hTERT expression and telomerase activity much more strongly than either agent alone. Furthermore, in vivo studies showed all mice tolerated the combination of imetelstat with ABT-199 well, with increased life span as compared to the vehicle control (68.1%, p=0.0001), to imetelstat (39.6%, p=0.0011) alone, or to venetoclax (23.3%, p=0.0001) alone. In the combination group, 40% of treated mice were alive 77-days after treatment discontinued whereas all mice of the other single agent arms died within two weeks, demonstrating a significant survival benefit.

Conclusions: To our knowledge, this is the first investigation combining imetelstat with venetoclax in AML, and the results demonstrated a synergistic effect on induction of apoptosis in cell lines and patient samples in vitro, which translated into prolonged survival in xenograft models, thus providing a strong rationale for clinical exploration of this combination.

#1102

**Anti-tumor effects of imipridone ONC201 in combination with anti-angiogenic agents significantly impact on colorectal cancer growth** in vivo **.**

Jessica Wagner, Wafik El-Deiry. _Fox Chase Cancer Center, Philadelphia, PA_.

Small molecule imipridone ONC201 is an investigational anti-tumor agent with a wide therapeutic index and broad-spectrum efficacy in vivo. ONC201 upregulates intratumoral TRAIL expression and the integrated stress response pathway. A Phase I clinical trial using ONC201 as therapy in advanced cancer patients has been completed and the drug has progressed into several Phase II trials in multiple cancer types. Colorectal cancer (CRC) remains one of the leading causes of cancer worldwide and metastatic disease continues to have a poor prognosis. Clinical trials in CRC and other tumor types have demonstrated that therapeutics targeting the vascular endothelial growth factor (VEGF) pathway, such as bevacizumab, are effective in combination with certain chemotherapeutic agents. Bevacizumab is a humanized monoclonal antibody that targets VEGF and is an FDA-approved treatment for advanced CRC patients, in addition to other tumor types. We are investigating the potential combination of VEGF inhibitors such as bevacizumab and ONC201 in both CRC xenograft and patient-derived xenograft (PDX) studies. Our results demonstrate significant tumor regression and occasional tumor ablation in human xenografts with the combination of ONC201 with bevacizumab, and in syngeneic MC-38 colorectal cancer xenografts using a murine VEGF-A inhibitor. Non-invasive angiogenesis imaging demonstrated the impact of this combination on decreasing tumor growth and tumor metastasis. With the use of both a murine VEGF inhibitor in syngeneic models, and bevacuzimab in human cell line-derived xenografts, we have demonstrated that combining anti-angiogenic therapies with ONC201 could enhance antitumor efficacy. Thus, ONC201 in combination with anti-angiogenic therapies such as bevacizumab represents a promising combinatorial approach that may be exploited in the clinic for the treatment of CRC.

#1103

Novel theranostic strategy against scirrhous gastric cancer; combination of chemotherapy and fluorescence oncolytic adenovirus.

Wataru Ishikawa, Satoru Kikuchi, Hiroshi Tazawa, Shinji Kuroda, Kazuhiro Noma, Hiroyuki Kishimoto, Takeshi Nagasaka, Masahiko Nishizaki, Shunsuke Kagawa, Toshiyoshi Fujiwara. _Okayama University, Okayama, Japan_.

Background: Gastric cancer is one of the most common cancers worldwide, especially in Eastern Asia. Scirrhous gastric cancer (SGC), which is characterized by poorly differentiated tumor cells that diffusely infiltrate the gastric wall, accounts for 10% of all gastric cancers, and often causes peritoneal metastasis. Peritoneal carcinomatosis shows the worst prognosis among various metastatic patterns. Since it is difficult to detect peritoneal micrometastasis preoperatively or intraoperatively, SGC patients with peritoneal micrometastasis show the recurrence and refractory to conventional systemic chemotherapy after surgery. In recent year, intraperitoneal (i.p.) administration of paclitaxel (PTX) has been shown to have therapeutic effects against ovarian cancer with peritoneal metastasis. However, the effective treatment option to overcome peritoneal metastasis of SGC has not been developed yet, although i.p. administration of some chemotherapeutic agents has been attempted to SGC. Therefore, a novel therapeutic strategy is required for the treatment of peritoneal metastasis of SGC.

Methods: We previously developed a telomerase-dependent replication-selective adenovirus OBP-401 (TelomeScan), which can replicate within the tumor cells selectively and express green fluorescent protein (GFP). Moreover, OBP-401 infection also induces tumor-specific cell death in monotherapy or combination therapy with chemotherapy. In this study, we assessed synergistic effects in combination with novel theranostic agent OBP-401 and chemotherapeutic agent paclitaxel (PTX) using human SGC cell lines (GCIY and KATO III). In vitro experiment, SGC cells and normal human lung fibroblast (NHLF) cells were infected with OBP-401 at various doses. PTX was administrated after 48 hours of virus infection. Twenty-four hours after PTX administration, we evaluated the anti-tumor effect by XTT assay, and analyzed the synergistic effect by CalcuSyn Software.

Results: SGC cells were visualized as GFP-positive cells selectively by the infection of OBP-401, whereas NHLF cells were not visualized with OBP-401. In both SGC cell lines, the suppression of cell viability dose-dependently by administration of OBP-401 or PTX alone was confirmed. OBP-401 synergistically suppressed the viability of SGC cells in combination with PTX as compared to monotherapy.

Conclusions: These results suggest that OBP-401 has a promising potential to detect peritoneal micrometastasis of SGC intraoperatively and combination of OBP-401 and PTX would be a novel theranostic strategy for the treatment of peritoneal metastasis of SGC.

#1104

Novel specific RTK targeting of EGFR/FAK axis in glioblastoma invasion.

Raghupathy Vengoji, Satyanarayana Rachagani, Suprit Gupta, Kavita Mallya, Maneesh Jain, Moorthy Ponnusamy, Surinder Batra, Nicole Shonka. _University of Nebraska Medical Center, Omaha, NE_.

Background: Glioblastoma (GBM) is the most aggressive primary brain tumor with a median survival rate of 14.6 months. Currently, the first-line treatment includes surgical resection, chemoradiation, and adjuvant chemotherapy with temozolomide. However, GBM recurs most often within 6.9 months. Receptor tyrosine kinases are dysregulated in GBM, with epidermal growth factor receptor (EGFR) representing 57.4% of the deleted/mutated GBM. In addition, 30 - 40% of GBM patients with EGFR amplification carry an oncogenic gene rearrangement EGFR variant III (EGFRvIII) which is constitutively active. Yet most EGFR inhibitors have shown very little clinical efficacy in GBM.

Methods: Afatinib, blood-brain barrier penetrant pan-EGFR inhibitor, covalently binds and irreversibly inhibits signaling from EGFR. Afatinib also persistently inhibits ErbB homo and hetero-dimers. Using GBM cell lines U87MG and U87MG transfected with wild type EGFR, EGFRvIII and EGFRvIII with dead kinase domain, we evaluated the efficacy of afatinib alone and in combination with temozolomide.

Results: Afatinib treatment resulted in a dose dependent decrease in the proliferation of U87MG cells transfected with EGFRvIII. The IC50 value for this cell line is 2µM (afatinib). 50µM temozolomide inhibited cell proliferation by 50%. We evaluated the combinational efficacy of IC25 of both. Afatinib effectively blocked EGFR signaling even 72 hours after treatment in the U87MG cell line transfected with either wild type EGFR or EGFRvIII. This was evidenced by decreased phosphorylation of EGFR (Tyr 1068) and its downstream signaling. Focal adhesion kinase (FAK) signaling in EGFRvIII expressing cells, largely responsible for the invasiveness of GBM, was abolished by afatinib. Furthermore, treatment with afatinib and temozolomide significantly decreased the in vitro tumorigenicity (anchorage dependent growth - colony formation assay as well as anchorage independent growth - soft agar assay) of EGFRvIII expressing GBM cells.

Conclusion: Altogether, these results support synergistic efficacy of afatinib and temozolomide in EGFRvIII expressing GBM.

#1105

A chemotherapeutic approach to kill cancer stem cell rich ovarian ascites.

Siddik Sarkar, Obeid M. Malekshah, Arash Hatefi. _Rutgers University, Piscataway, NJ_.

Purpose: More than one third of ovarian cancer patients present with cancer cells in the abdomen (also known as ascites) at diagnosis, and almost all have ascites at recurrence (Ahmed and Stenvers 2013). It is well-documented that subpopulations of the ascites show cancer stem cell-like characteristics which possess enhanced resistance to chemotherapies and the capacity for distal metastatic spread and recurrent disease. Therefore, ascites are a major source of morbidity and mortality for ovarian cancer patients. The major deficiency that currently exists is that there is no effective and safe therapy for ascites since they are rich in cancer stem cells (CSCs) and resistant to chemotherapy. To overcome this deficiency, it is our objective to develop an effective chemotherapy approach that can destroy the CSC-rich ascites and inhibit recurrence.

Methods: Several established human ovarian cancer cells as well as ascites obtained from the abdomen of an ovarian cancer patient (ASCITES) were cultured. Cells were sorted by FACS Cell Sorter to determine the percentage of cancer stem cells (CSCs) in each cell line. Dose response curves of different chemotherapeutic drugs such as Cisplatin (CDDP), 6-Methylpurine (6-MP), 5-Flurouracil (5-FU), SN-38, Monomethyl auristatin E (MMAE) and Etoposide were studied in suspension culture enriched with cancer stem cells. The relative change in total CSC-rich spheroid mass which considers both number and volume of spheroids was used to determine Killing Index for each chemotherapeutic drug. To investigate inhibition of recurrence, the spheroids were monitored for 30 days post treatment and the viability of CSC-rich spheroids were studied by fluorescent microscopy.

Results: Primary human ovarian A2780 and ASCITES (derived from ovarian cancer patient Ascitic fluid) were found to have CSCs with unlimited renewal capacity in suspension culture. While Cisplatin, 5-FU, 6-MP and Etoposide showed efficacy in killing CSC spheroids at effective doses around 1-10 μM, but MMAE and SN-38 showed the ability to either significantly diminish or completely eradicate CSC spheroids with effective doses as low as 10 nM. Importantly, no evidence of recurrence (repopulation of spheroids) was observed in MMAE and SN-38 treated group even after 30 days of post treatment.

Conclusion: These promising results with the two chemotherapeutic drugs that show their ability to kill highly drug-resistant CSC-rich ovarian spheroids open the door to further investigate their use in the clinic. Currently, we are investigating the potential use of these two drugs in combination but at very low concentrations for the treatment of abdominal ovarian metastasis.

#1106

Alvocidib potentiates the activity of venetoclax in preclinical models of multiple myeloma.

Mark Livingston, Wontak Kim, Hillary Haws, Peter Peterson, Clifford J. Whatcott, Adam Siddiqui-Jain, Steven Weitman, David J. Bearss, Steven L. Warner. _Tolero Pharmaceuticals, Inc., Lehi, UT_.

The proteasome inhibitor bortezomib is widely used in the treatment of patients with multiple myeloma (MM). The expression levels of many proteins increase as a result of bortezomib treatment, including the pro-apoptotic protein NOXA. NOXA functions to sequester the anti-apoptotic BCL-2 family member, MCL-1. High levels of MCL-1 and/or low levels of NOXA have been implicated in bortezomib resistance and negative patient outcomes, including short duration of treatment response. The BCL-2-specific BH3 mimetic venetoclax (ABT-199) has also been explored in multiple hematological malignancies, including the treatment of MM. Venetoclax induces apoptosis in a BCL-2 specific manner by directly inhibiting BCL-2 function. However, intrinsic resistance to venetoclax treatment observed in MM patient samples has been attributed to a low BCL-2-to-MCL-1 gene expression ratio, suggesting a central role for MCL-1 in cell survival in this context as well. Increased MCL-1 expression is a known resistance mechanism to venetoclax treatment in a variety of cell types including chronic lymphocytic leukemia and lymphomas. Considering the central role of MCL-1 to treatment efficacy in MM, we investigated the ability of an MCL-1-lowering agent, namely the CDK9 inhibitor alvocidib, to potentiate the activity of venetoclax in MM. Alvocidib suppresses MCL-1 expression via CDK9-mediated regulation of RNA polymerase II. Alvocidib has achieved robust improvements in the clinical response rates of high-risk, newly diagnosed acute myeloid leukemia (AML) patients as part of the time-sequential ACM regimen (alvocidib + cytarabine + mitoxantrone). We therefore hypothesized that alvocidib would potentiate the activity of venetoclax in MM through an MCL-1-dependent mechanism. In this report, we demonstrate that alvocidib inhibits the protein expression of MCL-1 in MM cells in a time-dependent fashion, up to 96 hours. In cell viability assays, the addition of up to 100 nM venetoclax resulted in a 2.8-fold reduction in the IC50 of alvocidib in the cultured OPM-2 cell line. Conversely, the potentiation of venetoclax activity with the addition of alvocidib resulted in a more than 500-fold decrease in IC50 in the relatively venetoclax-resistant OPM-2 cells. Additional studies are currently underway to investigate the efficacy of alvocidib and venetoclax in the context of bortezomib resistance where low NOXA may contribute to enhanced cell survival via MCL-1. Taken together, our data suggest that the combination of alvocidib with venetoclax may constitute a novel therapeutic regimen in the treatment of MM. Further, it suggests that CDK9-mediated targeting of MCL-1 may offer a clinical route to addressing intrinsic resistance in MM patients.

#1107

Co-treatment with a C1B5 domain peptide of protein kinase Cγ and a low dose of gemcitabine effectively inhibited pancreatic cancer growth in mouse peritoneal cavity.

Alejandro Zulbaran, Kelsey Monson, Susumu Ishiguro, Atsushi Kawabata, Deepthi Uppalapati, Naomi Ohta, Masaaki Tamura. _Kansas State University College of Veterinary Medicine, Manhattan, KS_.

Although the gemcitabine is an effective chemotherapeutic agent for pancreatic cancer, unacceptable side effects often accompany. Since we have previously discovered that PKCγ C1B domain peptides effectively control tumor growth without any side effect (Kawabata et. al, Cancer Biol Ther, 2012), we sought to examine the efficacy of co-treatment with this peptide and a low dose of gemcitabine on the growth of pancreatic cancer. Although individual and co-treatment with C1B5 peptide (1µM) and gemcitabine (20 nM) weakly inhibited growth of PAN02 murine pancreatic acinar cell carcinoma in 2D culture, either treatment effectively attenuated spheroid growth on PAN02 cells in 3D culture with 48.2% and 35.8% inhibition, respectively. Combination treatment with the C1B5 peptide and gemcitabine further attenuated the growth of PAN02 cells (69.5% inhibition). In mice bearing peritoneal allograft tumors of PAN02 cells (2.5 x 105 cells/mouse), combination treatment with C1B5 peptide at 20 mg/kg (every other day) and gemcitabine 15 mg/kg (every three days) markedly inhibited tumor growth of PAN02 allografts (94% inhibition) more than individual treatment with gemcitabine (76% inhibition) or C1B5 peptide (39% inhibition). The tumor growth inhibition by the combination treatment was similar to the higher dose (50 mg/kg) of gemcitabine alone treatment. Peritoneal cavity infiltrated neutrophils and granzime B+ lymphocyte numbers were significantly higher in combination treatment group than in control group. In cell culture study, the treatment with C1B5 peptide alone (1µM) significantly increased INF-γ, IL-2, and TNF-α mRNA levels, suggesting that C1B5 peptide directly stimulated Jurkat cell activation. These studies suggest that stimulation of leucocyte migration toward cancer tissues and activation of cytotoxic T cells may play important roles in tumor growth attenuation by the combination treatment of C1B5 peptide and gemcitabine. Taken together, the current study suggests that C1B5 peptide offers an effective combination treatment strategy to reduce side effects associated with gemcitabine without losing tumoricidal effect of this agent. This work is supported in part by Kansas State University Johnson Cancer Research Center, NIH grants P20 GM103418, and Kansas State Bioscience Authority Collaborative Cancer Research grant.

### DNA Repair

#1108

A novel, selective inhibitor of DNA-dependent protein kinase (DNA-PK) potentiates the effects of DNA-damaging therapies in hepatocellular carcinoma.

Catherine E. Willoughby,1 Huw D. Thomas,1 Tommy Rennison,2 Celine Cano,2 Helen L. Reeves,3 Stephen R. Wedge1. 1 _Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, United Kingdom;_ 2 _School of Chemistry and Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, United Kingdom;_ 3 _Liver Unit, Freeman Hospital, Newcastle upon Tyne Hospitals NHS Foundation Trust and Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, United Kingdom_.

DNA-dependent protein kinase (DNA-PK) is a key component in the repair of DNA double-strand breaks via non-homologous end-joining. Studies have found elevated DNA-PK expression and activity in hepatocellular carcinoma (HCC) to be strongly correlated with increased tumor grade, resistance to DNA-damaging therapies and poor survival (1,2).

We have explored the selective inhibition of DNA-PK in combination with DNA-damaging agents as a potential therapeutic approach in HCC using NDD0004 - a novel, orally-bioavailable small molecule inhibitor of DNA-PK (in vitro IC50 = 8 nM).

NDD0004 was evaluated in a panel of DNA-PK overexpressing human HCC cell lines (Hep3B, HepG2, Huh7) in combination with ionizing radiation or the topoisomerase II poison doxorubicin. DNA-PK activity was determined by Ser2056 phosphorylation status, DNA damage quantified by γH2AX levels, cell proliferation determined by SRB assays and cell survival assessed using clonogenic assays. In vivo efficacy was evaluated using a novel murine model of localized and sustained doxorubicin therapy, involving intra-tumoral injection of doxorubicin-loaded DC M1 polymer beads into established Huh7 human HCC xenografts in CD1 nude mice. Oral twice-daily treatment with 30 mg/kg NDD0004 or vehicle control was commenced 1 hour following bead implantation and continued for up to 20 days (6 mice per group).

NDD0004 dose-dependently inhibited activation of DNA-PK in HCC cell lines in vitro in response to ionizing radiation, and significantly increased and sustained DNA damage following treatment. Furthermore, NDD0004 sensitized DNA-PK overexpressing HCC cell lines to doxorubicin and ionizing radiation in proliferation and survival assays by ≥ 5-fold. Combining NDD0004 with doxorubicin-loaded beads in vivo significantly inhibited the rapid growth of HCC tumors when compared to treatment with doxorubicin-loaded bead monotherapy, with the time taken for tumor volumes to quadruple (RTV4) being extended from 11 to 18 days (P<0.01, Mann-Whitney). Individual and combination treatments were well tolerated throughout. Immuno-histochemical analysis revealed γH2AX levels in hepatocytes surrounding doxorubicin-loaded beads to be significantly increased by NDD0004 co-treatment for 72-hours (P<0.02, 2-way ANOVA).

In conclusion, selective inhibition of DNA-PK catalytic activity sensitized DNA-PK overexpressing HCC cell lines to doxorubicin chemotherapy and ionizing radiation in vitro, and augmented the anti-tumor activity of localized chemotherapy in HCC xenografts in vivo. These data support the concept of combining a DNA-PK inhibitor with localized DNA-damaging therapies in the treatment of HCC.

1. Cornell et al. (2015) Clin Cancer Res. 21: 925-33

2. Evert et al. (2013) Br J Cancer. 109: 2654-64

#1109

MutSα and MutSβ mismatch repair complexes differentially regulate temozolomide efficacy & sensitizing effects of veliparib in glioblastoma cells.

Shiv K. Gupta, Emily J. Smith, Ann M. Tuma, Brett L. Carlson, Mark A. Schroeder, Gasper J. Kitange, Jann N. Sarkaria. _Mayo Clinic, Rochester, MN_.

Mismatch repair (MMR) is essential for temozolomide (TMZ) sensitivity, and MMR deficiency causes TMZ resistance and disease recurrence. However, the role MMR in TMZ response has been linked with Msh2/Msh6 (MutSα), while role of Msh3, a component of MutSβ remains unclear. To delineate role of MutSα and MutSβ in TMZ response, we used siRNA to disrupt MSH6 and MSH3 expression. As expected, silencing of MSH6 led to resistance in a TMZ-sensitive U251 cell line. In contrast, silencing of MSH3 enhanced TMZ sensitivity as compared to the control cells. These results support established role of MutSα in TMZ response via replication stress and DNA double strand breaks (DSB), and reveal that MutSβ may potentially influence TMZ response by modulating DSB repair. Consistently, host cell reactivation (HCR) assays in U251 expressing DRGFP reporter showed decreased homologous recombination (HR) after MSH3 knockdown (KD), while Msh6 KD had no effect on HR efficiency. Supporting the concept of synthetic lethality of HR with PARP inhibition, clinically relevant dose of veliparib (1 µM) showed a modest but consistent increase in TMZ sensitization measured in terms of potentiation factor (PF, ratio of relative growth with TMZ alone/ TMZ and veliparib) in MSH3 KD (PF 1.6±0.2) as compared to control cells (PF 1.1±0.2). Sensitizing effect of 1 µM veliparib in MSH6 KD (PF 1.7±0.5) was comparable to that in MSH3 KD cells. Interestingly, supra-therapeutic dose of veliparib (10 µM) induced more pronounced sensitization in MSH6 KD cells (PF 2.6±0.3) as compared to MSH3 KD (PF 2.4±0.3) or control cells (PF 1.6±0.2). Consistently, veliparib (10 µM) alone had no impact on DNA damage signaling, while TMZ induced prominent increase in phosphorylation of KAP1, Chk1 and Chk2 in control and MSH3 KD cells, but not in MSH6 KD cells; veliparib/TMZ co-treatment led a robust signaling in all 3 types of cells. Increased sensitization by veliparib after MSH3 KD was also examined in a TMZ-resistant (GBM22TMZ) PDX model carrying resistance due to a homozygous mutation in MSH6, and a 4-fold increase in HR efficiency. In clonogenic assays silencing of MSH3 or BRCA1 had no effect on TMZ sensitivity in GBM22TMZ cells. However, veliparib alone (10 µM) decreased colony formation with relative colony formation 0.54±0.03 and 0.32±0.06 in MSH3 and BRCA1 KD cells, respectively as compared to 0.82±0.04 in controls. TMZ/veliparib co-treatment in GBM22TMZ led to further decrease in colony formation in MSH3 KD and BRCA1 KD cells with relative colony formation of 0.30±0.04 and 0.19±0.02, respectively versus 0.46±0.03 in control. Taken together, our results suggest that MutSα and MutSβ differentially regulate cytotoxic effects of TMZ. Decreased HR efficiency and enhanced sensitizing effects of veliparib in MSH3 KD cells partially define synthetic lethality of MMR deficiency with PARP, and may help development of PARP inhibitors as sensitizer of TMZ therapy in GBM.

#1110

AsiDNA induce tumor sensitivity to PARP inhibitors in homologous recombination proficient breast cancer AsiDNA induce tumor sensitivity to PARP inhibitors in homologous recombination proficient breast cancer.

marie DUTREIX,1 Wael Jdey,2 sylvain Thierry,1 Inna Kuperstein,3 Graham Dixon4. 1 _Institut Curie, Orsay, France;_ 2 _Institut Curie, Onxeo, Orsay, France;_ 3 _Institut Curie, Paris, France;_ 4 _Onxeo, Paris, France_.

Purpose: PARP inhibitors (PARPi) have shown significant benefits in cancer patients with BRCA mutations. However, their major limitations are the necessity of homologous recombination (HR) deficiency and the rapid emergence of resistance. In the current study, we propose a novel therapeutic strategy, based on drug combination to promote sensitivity to PARPi independently of the tumor genetics.

Experimental design: We used AsiDNA, a DNA repair pathways agonist (Dbait concept), consisting in small molecules mimicking double-strand DNA breaks to activate ectopic signaling of DNA damage and prevent recruitment at damage sites of HR repair enzymes. We characterized the DNA repair inhibition activity of AsiDNA by monitoring repair foci formation and DNA damage and analyzed the cell survival to AsiDNA monotherapy and combination with the PARPi Olaparib of 21 tumor cell lines, and 3 non-tumor cell lines. Efficacy of the combination treatment was analyzed in tumor xenografts derived from the MDA-MB-231 cell line showing reduced sensitivity to AsiDNA and Olaparib in vitro. We screened for the frequency of clones resistant to two weeks treatment with AsiDNA or Olaparib in the 4NQO-mutagenized haploid KBM7 cells.

Results: Molecular analyses of repair foci formed after irradiation demonstrate that AsiDNA inhibits recruitment of RAD51 and 53BP1 whereas Olaparib prevents XRCC1. Combination of both drugs increases the accumulation of unrepaired spontaneous damage resulting in an increase of cell death in all tumor cell lines. The synergy of the association of AsiDNA with PARPi was also confirmed with 6 other PARPi. AsiDNA do not induce any increase in DNA damage or lethality in non-tumor cells when used in monotherapy as well as in association with PARPi. Multi-omics analysis of tumor cells highlighted a correlation between similarity to normal cells profile and AsiDNA resistance. Selection of resistant clones allowed the emergence of resistance to Olaparib (1.45%), Imatinib (0.6%) and 6-Thioguanine (1.6%) but not to AsiDNA (<0.07%), indicating that the drug acts through multiple targets. In the MDA-MB-231 xenograft model, whereas Olaparib failed to prevent tumor growth and AsiDNA provided only a 62% increase in tumor growth delay, the combination of Olaparib and AsiDNA increase 215% days the mean tumor growth delay.

Conclusion: Our results highlight the therapeutic interest of combining AsiDNA and PARPi to recapitulate synthetic lethality in all tumors independently of their HR status. Moreover, the low frequency of appearance of resistant clones to AsiDNA suggests a sustained efficacy during treatment unlike most targeted therapies.

#1111

The chromatin remodelers RUVBL1 and RUVBL2 are prognostic factors and therapeutic targets in non-small cell lung cancer due to their roles in DNA replication, repair, and radiosensitization.

Paul Yenerall,1 Rahul Kollipara,1 Amit Das,1 Pamela Villalobos,2 Long Shan Li,1 Brenda Timmons,1 Luc Girard,1 Jaime Rodriguez-Canales,2 Ignacio Wistuba,2 John Minna,1 Ralf Kittler1. 1 _Univ. of Texas Southwestern Medical, Dallas, TX;_ 2 _UT-MD Anderson Cancer Center, Houston, TX_.

Despite advances in targeted agents and immunotherapy, non-small cell lung cancer (NSCLC) remains the number one cause of cancer-related death. To identify new therapeutic targets in NSCLC, we performed an siRNA screen directed against genes involved in chromatin remodeling. This screen showed that RUVBL1 and RUVBL2 (herein collectively referred to as RUVBL1/2) were universally but differentially required for the viability of 24 NSCLC cell lines, which was an on-target effect. Various independent gene expression datasets/platforms show that NSCLC patient tumors have increased levels of RUVBL1 and RUVBL2 mRNAs, in comparison to normal lung, and that patients with high levels of RUVBL1 or RUVBL2 have a poorer prognosis, suggesting that RUVBL1/2 may play an important role in NSCLC tumors. To confirm this at the protein level, we validated an antibody against RUVBL1 for immunohistochemistry, stained clinically annotated NSCLC tissue microarrays for RUVBL1, and found that patients with higher levels of RUVBL1 protein also have a poorer prognosis. To better understand the role of RUVBL1/2 in NSCLC at a molecular level, we measured the distribution of cells in the cell cycle following RUVBL1/2 KD, determined RUVBL1/2 interacting proteins by immunoprecipitation followed by tandem mass-spec (IP-MS/MS), and measured gene expression changes following RUVBL1/2 KD by RNA-seq. Depletion of RUVBL1/2 arrested cells in S-phase and promoted pan-γH2AX positivity, IP-MS/MS showed an over-representation of proteins involved in DNA repair and replication, and gene set enrichment analysis of the RNA-seq data displayed a downregulation of transcripts involved in DNA replication and repair, strongly implicating RUVBL1/2 in these processes. To further probe the effects of RUVBL1/2 loss, we performed low-level knock down (KD) of RUVBL1/2, such that viability is largely unaffected, and then measured the viability of cells in response to various drugs (n=35). These drugs target a wide variety of biological processes; however, the only drugs with increased efficacy in the presence of RUVBL1/2 KD were those that damage DNA, target ATR or its downstream partner CHEK1, or target some mitotic proteins, further implicating RUVBL1/2 in DNA replication and/or repair. Due to their roles in replication, we reasoned that RUVBL1/2 KD may enhance the effects of ionizing radiation, a treatment frequently given to NSCLC patients. Low level KD of RUVBL1/2 decreased the clonogenic potential of multiple NSCLC cell lines, which can be phenocopied by depleting independent subunits of RUVBL1/2-containing chromatin remodeling complexes. Finally, we show that RUVBL1/2 depend upon their ATPase activity to support NSCLC viability, suggesting that small molecule inhibitors of this protein may be efficacious in the treatment of NSCLC, especially when combined with radiation therapy.

#1112

Forced reduction of DSS1, a member of TREX2 complex, highly sensitizes chemotherapy to breast cancer cells in a BRCA2-independent manner.

Kazuhiko Kuwahara,1 Naomi Gondo,2 Andri Rezano,3 Zhenhuan Zhang,4 Yukari Hato,5 Kiyotaka Kuzushima,2 Hiroji Iwata,6 Tatsuya Toyama,5 Eisaku Kondo1. 1 _Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan;_ 2 _Aichi Cancer Center Research Institute, Nagoya, Japan;_ 3 _Universitas Padjadjaran, Bandung, Indonesia;_ 4 _University of Florida, Gainesville, FL;_ 5 _Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan;_ 6 _Aichi Cancer Center Hospital, Nagoya, Japan_.

[Introduction] DSS1 (deleted in split-hand/split-foot malformation 1) was originally identified as a BRCA2-associated/stabilizing protein. By comparison of DSS1 mRNA level, we reported that the high DSS1 expression groups in breast cancer patients showed worse prognosis in relapse-free survival (RFS); however, DSS1 expression per se was not correlated with other clinical parameters including cellular proliferation or tumor grade, suggesting that the level of DSS1 affected the sensitivity of DNA-damage agents in breast cancer cells (Rezano et al., 2013). It still remains unclear why the expression level of DSS1 is associated with chemosensitivity, because DSS1 has multifunctional properties regulating homologous recombination and RNA-mediated genomic stability. In this study, we investigated if the effect of DSS1 in chemosensitivity is influenced by two representative DSS1-associated molecules, BRCA2 and PCID2.

[Methods] We established MCF7 overexpressing DSS1 (MCF7/DSS1) by retroviral transfection. DSS1, BRCA2, or PCID2 knockdown in MCF7 was performed using siRNA transfection. The susceptibility to the cytotoxic chemotherapy such as doxorubicin and paclitaxel in breast cancer cells was evaluated by flow cytometry to detect apoptosis and colony assay. In addition, we compared patient survival by dividing patients into high or low expression group of BRCA2 or PCID2 using cohort studies.

[Results] BRCA2 depletion in MCF7/DSS1 did not alter the chemoresistance. Although DSS1 knockdown induced the downregulation of BRCA2, direct depletion of BRCA2 did not show marginal effect to chemosensitivity, suggesting that the enhancement of chemosensitivity by DSS1 knockdown was not related to BRCA2 expression. Importantly, over- and under-expression of PCID2 induced similar effect to chemosensitivity compared to those of DSS1. There was a trend that high PCID2 expression group in breast cancer patients showed worse prognosis in RFS.

[Conclusion] DSS1 could be a molecular target to increase chemosensitivity, which is independent of BRCA2 expression. Rather, the transcription-coupled DNA damage induced by impaired TREX2 complex might be associated with induction of chemosensitivity.

#1113

Inhibition of DNA repair by NR1D1 enhances chemosensitivity of breast cancer cells.

Na-Lee Ka, Tae-Young Na, Hyelin Na, Mi-Ock Lee. _Seoul National University, Seoul, Republic of Korea_.

Most chemotherapeutic agents exert cytotoxic effects by inducing excessive DNA lesions in cancer cells. Therefore, regulating DNA repair networks is a critical factor that determines sensitivity of cancer cells to chemotherapeutic drugs. In this study, we identified the role of nuclear receptor NR1D1 in DNA repair, which enhanced chemosensitivity in breast cancer cells. NR1D1 inhibited recruitment of the DNA repair complex to damaged DNA sites, thereby impaired proper DNA repair upon doxorubicin treatment in breast cancer cells. Interaction with Poly(ADP-ribose) polymerase-1 and subsequent PARylation were critical steps that allow NR1D1 to translocate to DNA double-strand breaks. In agreement, depletion of NR1D1 in MCF-7 cells resulted in resistance to a chemotherapeutic drug, doxorubicin, in both in vitro and in vivo experiments. Further, NR1D1 expression level was correlated positively with clinical outcomes in breast cancer patients who received chemotherapy. Thus, NR1D1 and its ligands may provide better therapeutic options that could enhance the outcomes of adjuvant chemotherapy in breast cancer patients.

#1114

Combining anti-androgen therapy and PARP inhibition results in synergistic cytotoxicity of metastatic castration-resistant prostate cancer (mCRPC) cells.

Sahithi Pamarthy, Vinay Sagar, Rajita J. Vatapalli, Zachary Reinstein, Muhammad Zayd Ansari, Benedito A. Carneiro Filho, Francis J. Giles, Sarki A. Abdulkadir. _Northwestern University, Chicago, IL_.

Androgen deprivation therapy is an efficient first line therapy for advanced prostate cancer. However, in most cases resistance inevitably develops resulting in metastatic castration-resistant prostate cancer (mCRPC). Abiraterone and enzalutamide are approved to treat mCRPC and act by inhibiting androgen biosynthesis and androgen receptor (AR) signaling, respectively. Poly (ADP-ribose) polymerase 1 (PARP1) is a key enzyme in the DNA damage repair pathway and has also been implicated in AR signaling cascade. Recent reports suggest significant efficacy combining anti-androgens and inhibitors of Poly (ADP-ribose) polymerase (PARP) particularly against cells harboring mutations in the DNA repair pathway. Here, we provide evidence of synergistic cytotoxicity combining PARP inhibition and androgen signaling ablation in the AR positive human CRPC cell line 22RV1 harboring mutation in the DNA repair gene BRCA2. The PARP inhibitor talazoparib (IC50 - 0.05 μM) was upto 20 times more potent than olaparib (IC50 – 1.0 μM) in inducing cell death in 22RV1 as seen by cell viability and clonogenic assays. When combined with AR antagonist enzalutamide (IC50 – 0.25 μM) or androgen synthesis inhibitor abiraterone (IC50 – 1.25 μM) both PARP inhibitors showed significant synergy, although talazoparib was more prominent owing to its superior potency. Higher concentrations of enzalutamide and abiraterone (>10μM) induced PARP cleavage and increased expression of γ-H2AX, a marker for DNA damage. Our results suggest that AR signaling inhibition involves PARP mediated apoptosis and highlight the crosstalk between PARP and AR pathways. In summary, combining anti-androgen therapy and PARP inhibitors carry significant therapeutic potential for CRPC patients harboring mutations in DNA repair pathway.

#1115

Germline single nucleotide polymorphisms in DNA repair genes in urothelial cancer patients.

Bishoy M. Faltas, Panagiotis J. Vlachostergios, Linda Lam, Tuo Zhang, Olivier Elemento, Mark A. Rubin. _Weill Cornell Medical College, New York, NY_.

Introduction: Germline single nucleotide polymorphisms (SNPs) have been investigated in several cancers. Repair of DNA damage is a key cellular process involved in the development of chemotherapy-resistance. The role of germline SNPs in DNA damage repair genes (DRGs) in determining resistance to DNA-damaging agents in urothelial carcinoma (UC) patients is not completely understood.

Methods: We examined a cohort of 53 UC patients (median age 67, 42 males) enrolled in our IRB-approved Precision Medicine program. Patients had histologically confirmed UC (43 bladder, 10 upper tract UC) and received treatment with platinum-based chemotherapy. We isolated germline DNA from peripheral blood lymphocytes or buccal swabs, and used whole exome sequencing (WES) to examine germline SNPs. As a reference for SNP frequencies we used the Exome Aggregation Consortium (ExAC) database, which represents the largest, randomly selected, germline WES database in the general population, including 60,706 individuals, among which 7,601 patients with multiple cancer types from the Tumor Cancer Genome Atlas (TCGA) cohort.

Results: Twelve different DRG SNPs were identified in germline DNA samples from 53 patients, affecting genes involved in non-homologous end-joining (RECQL4, n = 18, 54.50%; POLQ, n = 2, 6%), nucleotide excision repair (ERCC6, n = 2, 6%; XPA, n = 1, 3%; CCNH, n = 1, 3%; POLK, n = 1, 3%), homologous recombination (RNF168, n = 1, 3%; RAD17, n = 1, 3%; POLE, n = 1, 3%), Fanconi anemia pathway (POLN, n = 1, 3%), mismatch repair (EXO1, n = 1, 3%) and mitochondrial DNA repair (POLG, n = 2, 6%). The frequency of rs11342077 of the DNA helicase RECQL4 was significantly higher in our cohort (18/53, 34%) compared to its frequency in the ExAC database (p<0.01%). There was no significant difference in overall survival (OS) between patients with and without DRG SNPs (log-rank p=0.46). There was no significant association between the most commonly identified SNP, rs11342077, and overall survival among UC patients with DRG SNPs (log-rank p=0.39). Overall, the presence of DRG SNPs in our cohort occurred at a significantly higher frequency (33/53, 62.3%) compared to the ExAC database (0.56%, chi-square p<0.01).

Conclusions: Germline SNPs in DNA repair pathway genes are common in UC. Additional study of the role of these SNPs as potential biomarkers of response DNA damaging chemotherapeutic agents, including platinum-based chemotherapy or/and PARP inhibitors is needed in a larger cohort.

#1116

Targeting replication stress by carbazole blue- A novel strategy to treat triple negative breast cancers.

Subapriya Rajamanickam,1 Kaitlyn Bates,1 Santosh Timilsina,1 JunHyoung Park,2 Benjamin Onyeagucha,1 Panneerdoss Subbarayalu,1 Nourhan Abdelfattah,1 Kwang Hwa Jung,2 Edward Favours,1 Tabrez A. Mohammad,1 Hung-I Harry Chen,1 Benny A. Kaipparettu,2 Yidong Chen,1 Jack L. Arbiser,3 Manjeet K Rao1. 1 _University of Texas Health Science Center San Antonio, San Antonio, TX;_ 2 _Baylor College of Medicine, Houston- 77030, TX;_ 3 _Emory University School of Medicine, Atlanta-30322, GA_.

Background: Triple-negative breast cancers (TNBC) are the most aggressive forms of breast cancer and almost 60% of patients with TNBCs develop chemo-resistance, leading to recurrence, poor prognosis and poor survival. TNBCs have been reported to have high levels of replication stress, which plays pivotal role in genomic instability, and therapy resistance. Targeting replication stress is an emerging approach for better TNBC treatment. Here, we evaluated the anticancer efficacy of carbazole blue (CB), a synthetic analogue of carbazole that we recently synthesized on TNBC cells growth and progression.

Experimental Design: The effect of CB on breast cancer growth was assessed in vitro as well as in orthotopic mouse xenograft and PDX-models of breast cancer. In addition, the therapeutic efficacy and safety of CB was determined in long term toxicity studies in mice and also in ex-vivo explants from breast cancer patients. The mechanism of action of CB was evaluated by performing gene expression, cell cycle, apoptosis and DNA repair studies as well as proteins involved in the above mentioned mechanisms.

Results: Our results demonstrated that CB inhibits short and long term viability of TNBC cells in a dose dependent manner without affecting normal mammary epithelial cells. We show that the systemic delivery of CB using nanoparticle-based delivery approach suppressed breast cancer growth without inducing toxicity in preclinical and PDX mouse models of triple negative breast cancer. Our long term toxicity studies reveled that CB treatment did not induce any toxicity in Balb/c mice. Using ex-vivo explants from breast cancer patients, we demonstrated that CB modulated breast cancer growth. Consistent with that, our results revealed that CB treatment induced G1/S cell cycle arrest and apoptosis in TNBCs. Interestingly, our gene expression analysis revealed that CB modulates expression and activity of several genes known to be involved in DNA replication and DNA repair machinery.

Conclusions: Our results for the first time showed the CB can serve as a novel and potent therapeutic agent for treating breast cancer in general and TNBC in particular. These findings highlight the potential of CB to be applied as a safe regimen for treating breast cancer patients. As exploiting replication stress to treat cancer is gaining major interest, compound/s that may induce replication stress and inhibit DNA repair ability of cancer cells, has immense translational potential.

#1117

Triple negative breast cancer patient-derived xenografts as a translational model for discovery of novel therapeutic targets.

Margarite D. Matossian,1 Hope E. Burks,1 Annie C. Bowles,1 Rachel A. Sabol,1 Van T. Hoang,1 Bruce Bunnell,1 William J. Zuercher,2 David H. Drewry,2 Carrow Wells,2 Krzysztof Moroz,3 Matthew E. Burow,1 Bridgette Collins-Burow1. 1 _Tulane University School of Medicine, New Orleans, LA;_ 2 _University of North Carolina, Chapel Hill, NC;_ 3 _Louisiana Cancer Research Center, New Orleans, LA_.

Triple negative breast cancers (TNBCs) constitute 12% of all breast cancers, and is approximately twice more prevalent in African-American populations. Louisiana has a high proportion of African-American residents (32.5% in 2015), and thus hosts a higher population of TNBC patients. TNBCs have an aggressive phenotype that is elusive to the targeted therapeutics used to treat other breast cancer subtypes. The claudin-low molecular subtype has higher rates of metastases and recurrence. Certain kinase families have been extensively studied as regulators of epithelial-mesenchymal transition (EMT), a process involved in the initiation of cancer metastasis. Discovery of novel kinase targets within the subset of uncharacterized kinases could provide important insight into future targeted therapies. However, current models utilized in target discovery research are limited by the inability to accurately recapitulate the complex stromal architecture and heterogenous genetic and molecular composition of breast cancer. Furthermore, immortalized cell lines are limited to a 2D environment and over time acquire mutations that may not reflect the primary tumor. Recently, our laboratory has successfully established two TNBC patient-derived xenograft (PDX) models derived from African-American patients, and generated cell lines (TU-BCx-2K1, TU-BCx-2O0) and mammospheres. One of these models, 2O0, presents tumor architecture, cellular composition, genomic (qRT-PCR) and protein (western blot) expressions that are concordant with a claudin-low subtype. Furthermore, we show that both TNBC models metastasize to the lungs, and exhibit molecular characteristics consistent with mesenchymal phenotypes. We utilized these translational PDX models to screen a library of small molecule inhibitors that represent a variety of kinase pathways to identify novel therapeutic targets and/or pathways that are specific to TNBC subtypes. We found in a preliminary cell morphology screen using three TNBC cell lines (MDA-MB-231, BT549, MDA-MB-157), two small molecule inhibitors that increased epithelial marker (CDH1) gene expression, suppressed mesenchymal (VIM, c-FOS, SNAI1, ZEB1) expression and/or suppressed cellular motility in transwell migration assays. We observed after ex vivo treatments with our PDX tumors the two compounds increase the epithelial marker CDH1 expression, and suppress mesenchymal markers (VIM, MMP2) expressions. We confirm these findings in the TU-BCx-2K1 cell line. Kinase array data revealed candidate kinases responsible for the observed EMT changes in the two compounds of interest (NEK5, NEK9, NEK1 potentially affect cell motility; SRC-family kinases, TAOK2, STK10 potentially affect EMT gene changes); we plan to utilize the PDX cell lines to characterize these kinases in EMT. We aim to ultimately discover novel therapeutic targets specific to different TNBC molecular subtypes.

#1118

Synthetic lethality of CDK12 inhibition in tumors with EWS/FLI rearrangements.

Amanda L. Balboni,1 Bjorn Stolte,1 Amy Saur Conway,1 Gabriela Alexe,1 Emily Jue Wang,1 Nicholas Kwiatkowski,1 Tinghu Zhang,1 Brian J. Abraham,2 Peter Kalev,1 Dipanjan Chowdhury,1 Cyril H. Benes,3 Richard A. Young,2 Nathanael S. Gray,1 Kimberly Stegmaier1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Whitehead Institute, Cambridge, MA;_ 3 _Massachusetts General Hospital, Boston, MA_.

THZ1 is a potent, covalent inhibitor of the transcriptional CDKs, CDK7/12/13. Chemical genomic profiling of THZ1 across >1,000 diverse cancer cell lines revealed that EWS/FLI- rearranged Ewing sarcoma cells were remarkably sensitive to this molecule. We demonstrated that THZ1 inhibits the phosphorylation of the C-terminal domain of RNA Polymerase II, decreased colony formation capacity, and induced apoptosis in a dose-dependent manner in Ewing sarcoma cell lines. Using selective CDK7 and CDK12/13 inhibitors, we revealed that the primary target of THZ1 in Ewing sarcoma is CDK12/13. Genetic suppression of CDK12, but not CDK13, induced strong anti-viability effects, confirming CDK12 as the primary target. Treatment of Ewing sarcoma cell lines with THZ531, a novel CDK12/13 selective inhibitor, preferentially repressed genes involved in DNA damage repair. Additionally, suppression of EWS/FLI rendered Ewing sarcoma cells resistant to THZ531 and partially rescued the anti-viability effects of CDK12 knockdown. These results suggest that EWS/FLI imparts vulnerability to DNA damage repair inhibition and implicate a synthetic lethal relationship between the tumor-specific expression of EWS/FLI and CDK12 inhibition. Furthermore, we demonstrated that CDK12 and PARP inhibitors are highly synergistic in vitro, inducing widespread yH2AX foci formation. Interestingly, THZ531 impairs the ability of the PARP inhibitor, olaparib, to induce RAD51 foci formation, suggesting that THZ531 specifically causes a defect in homologous recombination repair. Moreover, we observed striking synergy of THZ1 and olaparib in two mouse models of Ewing sarcoma with limited toxicity observed. These findings have important translational significance as clinical trials with PARP inhibitors as single agents in Ewing sarcoma failed to demonstrate efficacy, highlighting the need to identify combination therapies that will enhance the activity of PARP inhibition. We anticipate that CDK12 and PARP inhibitor combinations will be of therapeutic interest in other ETS-rearranged tumors, as well as tumors with defects in DNA repair.

#1119

Targeting tankyrases as a therapeutic strategy for triple-negative breast cancer.

Maya Mathew, Loredana Campo, Jung-Lye Kim, Geun-Hyoung Ha, Eun-Kyoung Breuer. _Loyola University Medical Center, Maywood, IL_.

Tankyrases (TNKS1 and TNKS2) are members of the human poly(ADP-ribose) polymerase (PARP) family and catalyze PARsylation by using NAD+ as a substrate to modify various proteins in order to regulate various cellular processes including Wnt/β-catenin signaling. TNKS1 has been shown to be overexpressed and to be correlated with highly aggressive disease and prognosis in various types of human cancers. A small molecule inhibitor of TNKS1, XAV-939 has been proven to be a potent inhibitor of Wnt/β-catenin signaling by stabilizing Axin. However, the effect of tankyrase inhibitors as potential breast cancer therapy is still under investigation. In this study, we found that TNKS1 is highly expressed in breast cancer cell lines, particularly in triple-negative breast cancer (TNBC) cells and that the expression of TNKS1 correlates with anti-proliferative efficacy of tankyrase inhibitors, XAV-939 and JW55. Also, XAV-939 treatment suppressed cell migration and invasion capabilities and epithelial-mesenchymal transition (EMT) signaling in triple-negative breast cancer cell lines. Moreover, XAV-939 reduced expression of stemness markers. It was shown that polo-like kinase 1 (PLK1) functions as a positive regulator of TNKS1 protein stability. Interestingly, we found that combination treatment of XAV-939 with PLK inhibitor GW843682X significantly suppressed cell growth, clonogenic potential, migratory and invasive capabilities of breast cancer cells. The combination of XAV-939 with ionizing radiation (IR) also showed an additional therapeutic effect. Therefore, our findings highlight the important value of TNKS1 as a therapeutic target and suggest the potential for improving the clinical efficacies of tankyrase inhibitors in combination with either PLK inhibitors or radiotherapy for patients with triple-negative breast cancer.

#1120

Chronopharmacology of cisplatin: role of the circadian rhythm in modulating cisplatin-induced toxicity in melanoma mouse model.

Panshak Dakup, Kenneth Porter, Rajendra Gajula, Shobhan Gaddameedhi. _Washington State Univ. College of Pharmacy, Spokane, WA_.

Purpose: The goal of this research is to highlight novel and divergent molecular mechanisms of the circadian system in modulating the response of cisplatin therapy against melanoma tumors. Cisplatin is one of the most commonly used chemotherapeutic drugs in treating a variety of tumors including cancers of ovaries, testis, lungs, blood and solid tumors of the head and neck, and is more recently under clinical trials for potential application in melanoma tumors. However, the major limitation of cisplatin as a chemotherapeutic drug is its tumor resistance and nephrotoxicity. Hence, improving the effectiveness and reducing the toxicities associated with cisplatin therapy are desirable outcomes. Studies on human models have shown decreased renal and blood toxicity by time-of-the-day. Consequently, our project seeks to study the chronopharmacological effects and understand the mechanisms that have been successfully demonstrated in human models. We hypothesize a mechanistic, circadian rhythm-based cause for these outcomes.

Experimental Design: Studies were done on B16F10 melanoma mouse models of wild-type and circadian disrupted Per1/2 -/- animals treated with 3 doses of 5 mg/kg cisplatin in the morning (7 AM) and evening (5 PM). Animal weights and tumor sizes were measured regularly post-treatment. Upon sacrifice, tissues (skin, kidney, and tumor) were harvested and analyzed for cisplatin-induced toxicities and DNA damage responses using H & E staining, western blot, and KIM-1 and immuno-slot blot assays.

Results: Weight measurements show a clock-regulated response to cisplatin toxicity. AM-treated wild-type animals showed significant weight loss compared to PM-treated wild-type animals. This treatment time differential is lost in the Per1/2 -/-. On a molecular level, kidney tissue DNA samples showed clock-controlled cisplatin-DNA repair activity in wild-type animals compared to Per1/2 -/-. These initial findings strongly suggest that nephrotoxicity and DNA damage response function might be regulated by the circadian rhythm.

Conclusions: These findings indicate a possible mechanism for the chronopharmacology of cisplatin in minimizing the toxicity associated with it and reveal a target for future study of additional mechanistic causes of circadian dosing changes in cisplatin and other genotoxic stress-mediated anti-cancer agents.

#1121

Redox-mediated inactivation of MGMT DNA repair through thiolation or nitrosylation of the active site cysteine145 sensitizes human gliomas to alkylating agents in vitro and in xenograft models.

Debasish Basak, Kalkunte S. Srivenugopal. _Texas Tech University Health Sciences Center, Amarillo, TX_.

O6-Methylguanine-DNA methyltransferase (MGMT) is a simple and unique antimutagenic DNA repair protein that plays a crucial role in conferring tumor resistance to various alkylating agents in brain tumor therapy. MGMT repairs O6-alkylguanines by transferring the alkyl groups to its active site cysteine (Cys145) in a stoichiometric/suicidal reaction thereby restoring the original nucleotide sequence in a single step. Although O6-benzylguanine (BG), a pseudosubstrate inhibitor, currently in clinical trials, potently inhibits MGMT and improves the efficacy of alkylating agents, unacceptable levels of bone marrow toxicity has been a great concern. For developing new MGMT inhibitors, we exploited the reactive nature of the active site Cys145 (pKa 4.5) and its susceptibility for thiolation and nitrosylation, both of which inactivate the MGMT. A new strategy involving inhibition of S-nitosoglutathione reductase (GSNOR) was also explored to increase the steady-state levels of nitrosylated MGMT. First, we designed a redox perturbing glutathione mimetic, a homoglutathione disulfide (hGTX) that has been stabilized with the addition of small amounts of cisplatin (1000:10). Spermine NONOate and S-nitroso penicillamine (SNAP) served as the protein-nitrosylating agents. N6022, a specific and reversible inhibitor of GSNOR was used with nitrosylators in some assays. Both the hGTX and spermine NONOate at 100-400 µM concentrations for 24 h inhibited the repair activity of MGMT >75% in HT29, SF188, and T98G cells. The NONOate strongly curtailed the DNA repair activity of recombinant MGMT showing a direct interaction. The inactivated MGMT protein was promptly eliminated from glioma cells. MGMT disappeared from cells much faster in spermine NONOate +N6022 treated cells, suggesting an extended steady-state of nitrosylated MGMT and its subsequent degradation. MGMT depletion was accompanied by 4-6 fold increases in cell killing by BCNU or temozolomide (TMZ). Binding of the biotinylated-O6-benzylguanine to the MGMT protein was prevented by hGTX and spermine NONOate treatments indicating the Cys145 was indeed the site of modification. In the nude mice bearing T98G or HT29-luc2 xenografts, combinations of hGTX and TMZ or spermine NONOate +TMZ produced markedly synergistic tumor regressions compared with TMZ alone. A reduction of MGMT protein as by immunoblotting and diminished MGMT activity levels in tumor tissues was verified. Apoptosis regulatory proteins were upregulated in excised tumors and H&E staining confirmed no discernible host organ toxicities. Our studies highlight the options for redox-driven therapeutic strategies for MGMT [supported by CPRIT grants RP130266 and RP170207 to KSS].

#1122

ATR inhibitors synergize with PARP inhibitors in killing glioblastoma stem cells and treating glioblastoma.

Jianfang Ning, Hiroaki Wakimoto, Robert L. Martuza, Samuel D. Rabkin. _Massachusetts General Hospital, Boston, MA_.

PARP inhibitors (PARPi) have been used alone or in combination with other agents for the treatment of tumors with homologous repair (HR) deficiencies. However, challenges remain for the treatment of tumors that are PARPi-resistant or HR-proficient. Glioblastoma (GBM) is an invariably lethal tumor that is not associated with HR deficiencies. GBM stem cells (GSCs), thought to be critical for tumor growth and resistance to therapy, can be isolated from GBM specimens and are representative of the patient's tumors. GSCs exhibit variable PARPi sensitivity, with at least half being resistant. In order to enhance the antitumor efficacy of PARPi for GBM, we examined the combination of PARPi with inhibitors of oncogenic or DNA damage pathways, including inhibitors of PTEN, PI3K, ATM, ATR, and temozolomide, some of which have been reported to sensitize cancer cells to PARPi. Only inhibitors of ATR (VE821, VE822, AZ20) synergized with PARPi in killing GSCs in vitro. VE822 inhibited ATR activity both in vitro and in vivo, and modestly, but significantly, extended survival in mice bearing GSC-derived tumors. However, ATR inhibitor (ATRi) in combination with PARPi further prolonged survival, compared to each drug alone, even in mice bearing PARPi-resistant GSC-derived tumors. This is the first report that ATRi alone or in combination with PARPi is effective in treating GBM and provides a rationale for clinical trials for GBM.

#1123

Sphingolipids transfer proteins (GLTP and CPTP) regulate the neoplastic progression of colon and breast cancer cells.

Shrawan K. Mishra, Rhoderick E. Brown. _Hormel Institute, University of Minnesota, Austin, MN_.

Sphingolipids have previously been shown to affect cancer progression by regulating cell death (apoptosis) and survival (autophagy). While apoptosis is promoted by elevations in sphingosine and ceramide, increases in sphingosine-1-phosphate and ceramide-1-phosphate can tip the balance toward survival. Changes in the expression of sphingolipid transfer proteins such as GLTP (glycolipid transfer protein) and CPTP (ceramide-1-phosphate transfer protein) have been shown to modulate cell shape change and regulate pro-inflammatory cytokine release, respectively, in human cancer epithelial cell lines. To gain a better understanding of the situation, we investigated further. We discovered that GLTP overexpression inhibits the growth of human colon carcinoma cell lines (HT-29; HCT-116), but spares normal colonic epithelial cells (HCEC) largely due to growth arrest at the G0/G1 cell cycle checkpoint. Mechanistically, we found that GLTP overexpression modulated cell cycle progression by upregulating Kip1/p27 and Cip1/p21 protein and mRNA levels, while decreasing CDK2, CDK4, cyclin E and cyclin D1 protein levels. The cell cycle arrest and growth inhibition induced by GLTP overexpression led to apoptotic cell death of HT-29 cells. Neither GLTP nor CPTP overexpression induced apoptosis in breast cancer cell lines (T47D and HTB-126). However, depletion of CPTP, but not GLTP, induced autophagy as indicated by increased LC3 transcript and autophagosome levels (GFP-LC3-II puncta). In the breast cancer cell lines, increased transcript levels of cPLA2 and CERK were detected consistent with the earlier pro-inflammatory effect reported for CPTP depletion in A549 lung epithelial carcinoma cells. Our findings shed light on the mechanistic ways by which GLTP and CPTP affect apoptosis and autophagy to impact cancer progression. Future in vivo studies are planned to ascertain the role(s) of these new players in cancer development and therapy.

[Support: NIH GM45928, HL-125353, Paint-the-Town-Pink (PTTP) Cancer Funds, Hormel Fdn]

#1124

Assessment of HRD score as predictor of chemosensitivity of PDAC PDX xenograft models to DNA-damaging chemotherapy.

Vladimir Khazak,1 Natalia Skobeleva,2 Anastasiia Vetkina,2 Ilya Serebriiskii,2 Kirsten M. Timms,3 Angela Davies,4 Igor Astsaturov2. 1 _NexusPharma Inc., Philadelphia, PA;_ 2 _Fox Chase Cancer Center, Philadelphia, PA;_ 3 _Myriad Genetics, Inc., Salt Lake City, UT;_ 4 _Champions Oncology, Inc., Hackensack, NJ_.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating malignancy that affects 44,000 individuals annually in the US, with almost 90% lethality even when diagnosed prior to metastasis. There is an urgent unmet medical need both for new therapies as well as better matching of existing therapies to patients. To address this emergency, we are assessing the feasibility of implementing a strategy of using HRD (Homologous Recombination Deficiency) scores for better therapy matching in PDAC patients. Using a panel of 77 patient-derived xenograft (PDX) models that were developed from fresh surgical PDAC tumor samples, HRD scores were generated based on analysis of three biomarkers (LOH, TAI and LST) and mutational data for 45 genes. All 77 samples met inclusion criteria, 75 FFPE specimens generated mutation data. HRD analysis was successful for 71 specimens (range= 1 - 63 (median=22)), with the primary cause of failure identified as high non-tumor content. 53 PDX models had mutations in KRAS gene and 45 in TP53. We have also identified 4 PDX models with mutations in BRCA2, 3 models with mutations in ATM, 4 models with mutations in RAD51. We have also found frequent mutations in several other DNA repair genes (ATR, PALB2, MLH1, MSH2, MSH3, MSH6, FANCM), but most of these models retained one functional allele and were not associated with a high HRD score. Using this genomic analysis, all 71 PDX models were stratified into three clusters with high, medium and low HRD scores. Three PDX models with the highest and lowest HRD scores each were selected for an in vivo study with DNA-damaging platinum-based chemotherapeutic agents Cisplatin and Carboplatin, as well as with a clinically relevant PARP inhibitor Niraparib. The results of the PDX study will be reported and compared with responses to chemotherapy using RECIST V1.1 in patients.

#1125

Eukaryotic Elongation Factor 2 Kinase (eEF-2K) is a novel therapeutic target in BRCA1+ mutated breast cancer.

Elif Asik,1 Nermin Kahraman,1 Tulin Guray,2 Murvet Volkan,2 Gabriel Lopez-Berestein,1 Bulent Ozpolat1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _METU, Ankara, Turkey_.

BReast CAncer 1 and 2 (BRCA1/2) tumor suppressor genes are frequently mutated in familial breast and ovarian cancers. Recent clinical trials in breast cancers with BRCA1/2 mutations indicate that 50-75% of patients may not have response to poly (ADP-ribose) polymerase inhibitors (PARP) inhibitors. Thus, identification of novel molecular targets and therapeutic interventions are needed to improve poor clical outcome and patient survival. Here we report that Eukaryotic Elongation Factor-2 Kinase (EF2K), an atypical kinase, is highly upregulated in BRCA1+ mutated cell lines and involved in colony formation, migration/ invasion and enhances the effect of PARP inhibitors. Furthermore, in vivo therapeutic targeting of EF2K by once a week systemic injections with magnetic nanoparticles (MNPs) incorporating EF2K siRNA significantly inhibits growth of BRCA1+ tumors in orthotopic xenograft models in mice by inhibition of tumor cell proliferation, angiogenesis and induction of robust apoptosis, which were associated with in vivo inhibition of clinically relevant molecular pathways including, proliferation, survival/drug resistance (PI3K/Akt, c-Myc), cell cycle (Cyclin D1), motility/invasion (Src/FAK/Paxillin), translation (4E-BP1), angiogenesis (VEGF) and stem-cell markers. No toxicity was detected during the study and by postmortem analysis of major organs, indicating that targeting EF2K is safe. The combination of PARP inhibitor (AZD2281, Oleparib) with MNP-EF2K siRNA enhanced significantly growth inhibition in MDA-MB-436 cells indicting EF2K siRNA sensitize BRCA1 + mutation breast cancer cells to PARP inhibitors. Collectively, our results suggest, for the first time, that EF2K is involved in tumorigenesis and progression and may serve as a potential therapeutic target in BRCA1+ mutated breast cancers.

#1125A

β-hydroxybutyrate inhibits histone deacetylase activity and radiosensitizes malignant glioma cells.

Eric C. Woolf,1 Alex P. Rossi,1 Helena B. Silva-Nichols,1 Kara D. Gardner,1 Nelofer Syed,2 Adrienne C. Scheck1. 1 _Barrow Neurological Inst., Phoenix, AZ;_ 2 _Imperial College London, United Kingdom_.

Several preclinical studies support the use of the therapeutic ketogenic diet (KD) and/or caloric restriction (CR) as an adjuvant to cancer therapy. This data not only suggests that this approach alters multiple hallmarks of cancer growth, but that it may also enhance other therapeutic modalities. We have demonstrated that the unrestricted KD greatly increases survival in a mouse model of malignant glioma when administered in combination with radiation, yet the underlying mechanisms are not fully elucidated. Mounting evidence suggests that the abnormal epigenetic landscape in tumors may, among other things, impact radiosensitivity. Specifically, many cancers exhibit increased histone deacetylase (HDAC) activity which contributes to the epigenetic milieu found in tumors and alters DNA damage repair. It was recently demonstrated that the ketone body β-hydroxybutyrate (BHB) inhibits HDAC activity in normal mouse tissue. As the use of HDAC inhibitors as anti-cancer agents has generated great interest, we examined the effect of BHB on HDAC activity and radiosensitivity in the context of malignant glioma. We found that BHB radiosensitizes mouse glioma, human glioma and human glioma stem-like cells in vitro. We also demonstrate that BHB inhibits HDAC activity in a dose dependent manner and alters key components of DNA damage repair. Taken together this data opens up another avenue for understanding the mechanisms underlying the KD and its impact on radiation therapy, ultimately helping us better understand how to implement it for clinical use in oncology. 

### New Targets 1

#1126

Potent inhibition of HCMV by modulating the cellular SNARE syntaxin 5.

Dhimant H. Desai,1 Linda Cruz,1 Nicholas T. Streck,1 Trisha D. Desai,2 Aron Lukacher,1 Shantu G. Amin,1 Nicholas J. Buchkovich1. 1 _Penn State Univ. College of Medicine, Hershey, PA;_ 2 _Cumberland Valley High School, Mechanicsburg, PA_.

Human cytomegalovirus (HCMV), also referred to as human herpesvirus-5 (HHV-5), can cause serious and even fatal disease in immunocompromised individuals and newborns, namely individuals with AIDS, solid organ transplant recipients, chemotherapy patients and recipients of bone marrow and stem cell transplants. HCMV is a ubiquitous virus found throughout all geographic regions and socioeconomic groups, infecting greater than 50% of adults in industrialized countries and as many as 100% in developing countries. Although current therapeutics improves clinical outcomes, they are limited by toxicity, intravenous infusion and the development of resistance by the virus. Thus, there is a pressing need to develop novel therapeutics to prevent HCMV infections with concomitant organ toxicity. Formation of the cytoplasmic viral assembly compartment (cVAC) is an important step for efficient HCMV assembly. To do this, the virus must alter and repurpose the normal cellular balance of membrane and protein flux, a process that is not well understood. We have identified a compound Retro94, which potently inhibits production of infectious HCMV virions in cells by operating against a host cell process, rather than directly targeting the virus. The presence of Retro94 results in the severely impaired production of infectious virions, as great as 5 logs (99-99.99%). Here we discuss in vitro, in vivo, stability, and binding study of Retro94. Overall, our findings have identified Retro94, as novel agent that affects key cellular trafficking factors important for supporting HCMV infection.

#1127

Aberrant nuclear expression of GSK-3beta in human head and neck carcinoma.

Maria Matsangou,1 Andrey Ugolkov,2 Timothy J. Taxter,3 Sandeep Samant,4 Andrew P. Mazar,2 Francis J. Giles1. 1 _Robert H Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL;_ 2 _Chemistry of Life Processes Institutue, Northwestern University, Chicago, IL;_ 3 _Department of Pathology, Northwestern University, Chicago, IL;_ 4 _Department of Otolaryngology, Northwestern University, Chicago, IL_.

Background: Recurrent/metastatic head and neck squamous cell carcinoma (SCCHN) and salivary gland malignancies are difficult to treat with limited standard of care options at the present time. Glycogen Synthase Kinase-3beta (GSK-3beta), a serine/threonine protein kinase, has been implicated as a potential therapeutic target in human cancer. Our in vivo studies demonstrated that our novel GSK-3 inhibitors significantly potentiated the effects of conventional chemotherapy in patient-derived xenograft models of glioblastoma and breast cancer leading to regression of tumors. In order to develop a rationale to test our novel GSK-3 inhibitors in head and neck (H&N) malignancies, we evaluated the expression pattern of GSK-3beta in human H&N benign tissue and malignant tumors.

Methods: We used immunohistochemical staining of H&N tumor tissue Microarray (TMA), 48 total cases (20 benign tissues, 28 malignant), to study the expression pattern of GSK-3beta. GSK-3beta nuclear accumulation was defined as positive staining of more than 50% of cancer cell nuclei throughout the tumor regardless of cytoplasmic staining.

Results: Of the 20 malignant H&N samples (15 SCCHN, 1 nasopharyngeal and 5 other histology), 13 (65%) were found to have aberrant nuclear accumulation of GSK-3beta. Amongst SCCHN, 73% (11 of 15 samples) had aberrant nuclear accumulation of GSK-3beta. In contrast, none (0%) of the 11 benign non-salivary H&N tissue showed detectable expression of GSK-3beta. Of interest, 60% of salivary adenoid cystic carcinoma specimens and 44% of benign salivary gland tissue showed GSK-3beta expression.

Conclusions: Our results demonstrate for the first time, that there is aberrant nuclear expression of GSK-3beta in SCCHN. This finding supports further exploring of our novel GSK-3beta inhibitor as a potential therapeutic target for recurrent/metastatic SCCHN and also, as a potential prognostic and predictive biomarker for risk of recurrent disease and chemo- or radio-resistance. Its role in salivary gland malignancies requires further studying.

#1128

Identification of arginine methyltransferase PRMT5 as a novel therapeutic target in T-cell acute lymphoblastic leukemia.

Yunyue Wang,1 Hui Huang,2 Daniel Diolaiti,3 Marta Sanchez Martin,1 Beata Modzelewski,3 Lianna J. Marks,3 Allison R. Rainey,3 Ervin S. Gaviria,3 Maria L. Sulis,1 Filemon S. Dela Cruz,3 Adolfo A. Ferrando,1 Andrew L. Kung3. 1 _Columbia University, New York, NY;_ 2 _EMD serono, CT;_ 3 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Advances in risk-adapted cytotoxic chemotherapy, hematopoietic stem cell transplantation and supportive care have contributed to significant improvements in the survival of patients with acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML) over the past few decades. However, despite such progress, a significant percentage of both adult and pediatric leukemia patients become refractory to therapy or relapse and eventually die of disease. Hence, there remains an urgent need for the development of effective and targeted therapies for acute leukemia. Recent genetic profiling of solid and hematologic malignancies has identified epigenetic factors as a critical group of genes recurrently mutated in cancer. Additionally, epigenetic dysregulation has been shown to play an important role in the development, progression and maintenance of leukemia. Therefore, pharmacological inhibition of epigenetic factors represents a potential avenue for the development of novel epigenetic-targeted therapies.

In order to identify epigenetic vulnerabilities in leukemia, we developed an epigenetic-focused shRNA screen to search for novel therapeutic targets in human leukemia cell lines both in vitro and in vivo. Specifically, T- and B-ALL cell lines were transduced with a library of shRNAs targeting 449 genes including epigenetic readers, writers and erasers and other chromatin-related factors. Selected cells were subsequently cultured in vitro and concurrently injected into mice. Engraftment of inoculated cells and disease progression were monitored through bioluminescence imaging. Amongst the universe of epigenetic regulatory proteins, the arginine methyl transferase, PRMT5, emerged as the most significantly depleted factor in both in vitro and in vivo screenings.

Chemical inhibition of PRMT5 enzymatic activity effectively reduced protein symmetric dimethyl arginine methylation, altered splicing, inhibited cell growth and promoted apoptosis of both ALL and AML cell lines in vitro. In addition, inhibition of PRMT5 in vivo using patient-derived xenograft (PDX) T-ALL mouse models demonstrated diminished tumor growth and prolonged survival. Notably,

quantification of peripheral blood cell numbers showed that pharmacologic PRMT5 inhibition was well tolerated and did not affect normal hematopoiesis in mice suggesting that a therapeutic window exists for anticancer drugs targeting PRMT5 in acute leukemia. Overall, our data indicates that pre-mRNA processing and in particular RNA splicing modulation may represent novel therapeutic targets in leukemia.

#1129

Sigma receptors: Novel targets for the treatment of highly malignant tumors.

Michela Cortesi,1 Sara Pignatta,1 Simona Collina,2 Andrea Rocca,1 Alice Zamagni,1 Chiara Arienti,1 Michele Zanoni,1 Luigino Tosatto,3 Daniela Bartolini,3 Evandro Nigrisoli,3 Annamaria Marra,2 Marta Rui,2 Anna Tesei1. 1 _Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (I.R.S.T.) IRCCS, Meldola (FC), Italy;_ 2 _University of Pavia, Pavia, Italy, Pavia, Italy;_ 3 _M. Bufalini Hospital, Cesena, Italy_.

Introduction. More than 7 million patients are diagnosed with cancer every year, and approximately 24 million new cases of cancer will probably be registered by 2035. Over the past decades, numerous cancer targets have been identified and, despite promising results in this field, the discovery of novel targeted therapies is still a largely unmet need. Sigma receptors (SRs), more highly expressed in tumor cells than in normal cells, have aroused the interest of the scientific community because of increasing evidence of their involvement in proliferation and survival signaling pathways of cancer cells. Glioblastoma (GBM) and pancreatic cancer (PCa) are two of the most aggressive and lethal solid tumors. GBM patients usually relapse within 7-10 months of the end of first-line treatment with median survival of about 14.6 months while PCa is the fourth most fatal cancer in both men and women, with median survival of 6-12 months. Little or no improvement in prognosis has been obtained over the past 20 years. Thus, new approaches to the treatment of both tumors are urgently needed.

In the present work, 3D GBM primary cultures endowed with stemness features and PCa cell lines were used to evaluate the antitumor activity of a dual S1R and S2R ligand RC-106, a novel sigma receptor modulator.

Methods. RC-106 tissue distribution studies were performed in mice. PCa cell lines were assessed by cytotoxic assay (CellTiter 96® AQueous One Solution Cell Proliferation Assay). Molecular analyses were performed by qRT-PCR and Western blot. Apoptosis and cell cycle were analyzed by flow cytometry. GBM primary cultures were isolated from surgical tumor samples. A rotatory cell culture system (RCCS) was used to obtain 3D cell cultures. Growth and morphology of the colonies were monitored by open-source AnaSP and ReViSP software tools. Molecular analyses were performed by flow cytometry and qRT-PCR. 3D cell viability was measured using CellTiter-Glo® 3D Cell Viability Assay. Apoptosis and cell cycle were analyzed by flow cytometry.

Results. RC-106 preferentially accumulate in mice pancreas and brain after intravenous administration, supporting the use of sigma receptor modulators as a novel therapy for these

tumors. Treatment with RC-106 strongly inhibited cell proliferation and survival regardless of the type of cancer investigated. 25 μM of the drug impaired the in vitro clonogenic ability of tumor cells, an effect lasting for up to 42 days, the longest time tested. RC-106 also led to the induction of substantial apoptosis mediated by caspase 3 and 9 activation, and to a modulation of the Akt pathway. Furthermore, treatment with RC-106 significantly reduced the growth of human GBM and PCa spheroids.

Conclusions. (RC-106) represents the hit compound of a new class of dual-action ligands targeting S1R and S2R potentially useful for the treatment of cancer disease.

#1130

Targeting the tumor-associated autoantigen alpha-enolase in prostate cancer.

Tino Wilson Sanchez,1 Guangyu Zhang,1 Jitian Li,2 Liping Dai,2 Saied Mirshahidi,1 Nathan R. Wall,1 Clayton Yates,3 Colwick Wilson,4 Susanne Montgomery,4 Jian-Ying Zhang,2 Carlos A. Casiano1. 1 _Loma Linda University School of Medicine, Loma Linda, CA;_ 2 _University of Texas, El Paso, El Paso, TX;_ 3 _Tuskegee University, Tuskegee, AL;_ 4 _Loma Linda University School of Behavioral Health, Loma Linda, CA_.

Prostate cancer (PCa) is the most commonly diagnosed cancer and second leading cause of cancer-related deaths in American men. African American (AA) men are more likely to be diagnosed with aggressive PCa at a younger age and twice as likely to die from the disease as European American (EA) men. In order to reduce PCa mortality and its associated racial disparities, there is a critical need to identify and target pathways responsible for PCa aggressiveness. An unexplored target is the plasminogen system, which is key to cancer cell migration and tissue invasion during metastasis. During inflammation, activation of the plasminogen pathway degrades extracellular fibrin networks; however, in the context of cancer, this activation promotes cancer tissue invasion and metastasis. Mounting evidence shows that the glycolytic enzyme alpha-enolase (ENO1) plays a vital role in both increased energy metabolism and plasminogen activation during cancer progression and metastasis. Using immunoseroproteomic profiling of anti-tumor autoantibody responses in AA and EA men with PCa, we identified several tumor-associated autoantigens with functions in glycolysis and plasminogen signaling, including ENO1, annexin A2, fructose bisphosphate aldolase, glucose-regulated protein 78, glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. In a cohort of PCa (N=157) and non-PCa sera (N=183), we found a significantly higher frequency of autoantibodies to ENO1 in patients with PCa (p<0.05). Surprisingly, when we probed a panel of non-PCa and PCa cell lines with anti-ENO1 positive sera from AA and EA patients with PCa, we observed that AA patients showed increased immunoreactivity to this protein in metastatic PCa cells, with decreased reactivity in docetaxel-resistant cell lines. By contrast, the anti-ENO1 EA-PCa sera and a monoclonal mouse anti-ENO1 showed relatively uniform immunoreactivity across the same panel cell lines. This suggested that the AA-PCa patients are producing antibodies to a different ENO1 variant than EA-PCa patients. Proteomic analysis of post-translational modifications (PTM) in ENO1 showed differences in ENO1 PTM profiles between the parental PC3 bone metastatic cell line and the PC3 docetaxel-resistant cell line, which could explain the differential autoantibody reactivity observed in AA men. Given that ENO1 is up-regulated in PCa tissue, plays a role in glucose metabolism and plasminogen activation during cancer progression, and that the immune system of a cohort of AA-PCa patients recognizes an alternately modified variant of ENO1 that is upregulated in metastatic PCa, we elected to target this protein in metastatic PCa cell lines. ENO1 depletion via siRNA knockdown in PC3 cells led to increased cell death, with concomitant decrease in PCa proliferation. Taken together, these results highlighted the value of immunoproteomics as a tool to identify novel therapeutic targets, with ENO1 as a promising target in metastatic PCa.

#1131

O-GlcNAc transferase inhibition in breast cancer cells.

Anna Barkovskaya,1 Lina Prasmickaite,1 Damien Y. Duveau,2 Ian G. Mills,3 Gunhild M. Mælandsmo,1 Siver A. Moestue,4 Harri M. Itkonen5. 1 _Oslo Univ. Hospital Inst. for Cancer Res., Oslo, Norway;_ 2 _Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD;_ 3 _PCUK/Movember Centre of Excellence for Prostate Cancer Research, Centre for Cancer Research and Cell Biology (CCRCB), Queen's University Belfast, Belfast, United Kingdom;_ 4 _NTNU, Department of Circulation and Medical Imaging, Trondheim, Norway;_ 5 _Department of Microbiology and Immunobiology, Harvard Medical School, Harvard Institute of Medicine, Boston, MA_.

O-linked-N-acetylglucosamination (O-GlcNAcylation) is a post-translational modification that occurs on serine and threonine amino acid residues of the intracellular proteins. O-GlcNAcylation of numerous targets is catalyzed by a single enzyme - O-GlcNAc-transferase (OGT). O-GlcNAcylation impacts protein activation and stability thereby regulating several key biological functions, and is heavily involved in cancer development and progression. OGT expression and total protein O-GlcNAcylation are elevated in many cancers compared to non-malignant adjacent tissues. In breast cancer, this increase also has a positive correlation with the tumor grade suggesting a possible link to metastasis and disease progression. Here we investigated the effects of OGT inhibition in two triple-negative (TNBC) and two receptor-positive luminal breast cancer cell lines. Treatment of cells with an OGT inhibitor as well as OGT knockdown led to a strong decrease in viability and proliferation of the TNBC cells, but significantly less so in the luminal receptor-positive cell lines. This differential response was evaluated using analysis of apoptosis, cell cycle, gene expression and a reverse-phase antibody-based protein array (RPPA). We found that cell cycle progression was affected by OGT inhibition. In addition, proteomics data pointed towards the transcription factor hairy and enhancer of split-1 (HES1) as a possible mediator of the cytotoxicity observed in the TNBC cell lines. We currently work to elucidate the molecular mechanisms of HES1 regulation by OGT and its effect on TNBC cell survival and apoptosis. In conclusion, OGT inhibition has a pronounced cytotoxic effect on the TNBC cells and may have some potential for therapeutic use in the future.

#1132

Lactate dehydrogenase expression in African American women with triple-negative breast cancer.

Odalys J. Torres-Luquis, Sulma Mohammed. _Purdue University, West Lafayette, IN_.

The incidence of breast cancer in African American women (AAW) is much lower than those of their Caucasian counterparts. However, the age adjusted mortality rates are much greater for AAW compared to Caucasian American women (CAW). These differences in survival between AAW and CAW were attributed to socioeconomic factors and access to health care. Yet, after adjusting these differences, AAW still had lower survival rates than CAW. Genetic and immunohistochemical studies have revealed that biological variations may account for this disparity. These studies have suggested as a causative: a higher prevalence of basal-like tumors in premenopausal AAW and low prevalence of luminal tumor. These are characterized by being high-grade aggressive tumors that are estrogen receptor negative (ER-), progesterone receptor negative (PR-), human epidermal growth factor receptor-2 (HER-2) +/-; with high rate of lymph node involvement. Despite this understanding, there is little evidence to indicate which genetic or non-genetic factors contribute to causing aggressive breast cancer thereby limiting the development of prevention and treatment option. We proposed that the phenotypic diversity of breast cancer might be accompanied by a corresponding diversity in protein expression patterns that can be reveal using proteomic technologies. We hypothesized that in addition to ER- and PR-, and the absence in expression of HER-2, the protein composition of triple-negative tumors varies among African-American and Caucasian. Two-dimensional gel electrophoresis, mass spectrophotometry and data analysis of protein from breast cancer tissues were used to identify differentially expressed proteins from African American and Caucasian women. Western blot of luminal and TNBC cell lines were used to confirm the expression of identified proteins. We have found that many proteins were differentially expressed in AAW breast cancer tissues compared to CAW. One of these is lactate dehydrogenase which was found to be overexpressed in tissues from AAW. Western blot analysis confirmed expression.

#1133

Discovery of novel small molecule inhibitors of RNA-binding protein Musashi-1.

Lan Lan,1 Amber Smith,1 Xiaoqing Wu,1 Anuradha Roy,1 Ragul Gowthaman,1 John Karanicolas,2 Amber D. Somoza,3 Clay C.C. Wang,3 Berl Oakley,1 Roberto De Guzman,1 Kristi Neufeld,1 Liang Xu1. 1 _University of Kansas, Lawrence, KS;_ 2 _Fox Chase Cancer Center, Philadelphia, PA;_ 3 _University of Southern California, Los Angeles, CA_.

1) Background and Objective: Musashi-1(Msi1) is an RNA-binding protein that negatively regulates translation by binding to the 3ʹ-UTRs of target mRNAs including Numb, APC and p21WAF-1, key regulators of Notch, Wnt signaling and cell cycle progression, respectively. We aim to identify small molecule inhibitors of Msi1's mRNA-binding activity thus blocking the growth of a broad range of cancer cells.

2) Methods: Fluorescence polarization assay was used to screen for small molecules that disrupt the binding of Msi1 to its consensus RNA binding site. Hits were validated by surface plasmon resonance, amplified luminescent proximity homogeneous assay and nuclear magnetic resonance.

3) Results: Among ~2,000 small molecules we screened, we found several clusters of compounds that disrupt the Msi1-numb RNA binding potently, including natural products, herbal extracts and fungal metabolites. One of the clusters is the derivatives of the secondary metabolites from Aspergillus nidulans. The top hit, Aza-9, from this cluster was validated by amplified luminescent proximity homogeneous assay. Further validation by surface plasmon resonance and nuclear magnetic resonance indicate that Aza-9 binds to Msi1 directly, in the RNA binding pocket.

4) Discussion and Conclusions: Our study supports the hypothesis that Msi1 inhibition is a viable and effective anti-cancer strategy. More target validation assays will be carried out for the compounds from other clusters.

#1134

Tissue transglutaminase is a new therapeutic target to overcome steroid resistance in T cell acute lymphoblastic leukemia.

Hyun Joo Jung. _Ajou University Hospital, Suwon, Republic of Korea_.

Background: The treatment response to steroid is the most important predictive factor for treatment outcome of T-cell acute lymphoblastic leukemia (T-ALL). To improve survival for steroid-resistant T-ALL patients, it is necessary to develop innovative therapeutic strategies to overcome steroid resistance. This study attempts to determine new therapeutic strategies to overcome steroid resistance for patients with T-ALL. Tissue transglutaminase (TG2) is a intracellular calcium-dependent protein cross-linking enzyme reported to be highly expressed in various metastatic or drug-resistant cancer cells, and chronic expression of TG2 constitutively activates nuclear factor kappa B (NF-kappaB).

Methods: We used biochemical and molecular methodologies to demonstrate that TG2 modulation leaded to overcome the steroid resistance in T-ALL.

Results: We generated the steroid-adapted subclones of T-ALL cell lines that were extremely less sensitive to steroid than their parent T-ALL cells. We found that steroid-resistant subclones of T-ALL cells expressed much higher levels of TG2 than their parent cells and the modulation of TG2 activities altered NF-kappaB expression in this steroid-resistant cells. Inhibition of TG2 suppressed steroid resistance and improved the cytotoxicity of leukemia cells in steroid-adapted subclones of T-ALL.

Conclusion: This study shows that TG2 expression is elevated with steroid resistance in T-ALL and the modulation of TG2 induces the cytotoxicity of steroid-adapted T-ALL. TG2 could be a new therapeutic target to overcome steroid resistance in T-ALL. Furthermore, change of TG2 expression could serve as markers of induced steroid resistance in T-ALL.

#1135

DRD2 is critical for pancreatic cancer and promises pharmacological therapy by already established antagonists.

Pouria Jandaghi,1 Hamed S. Najafabadi,1 Andrea Bauer,2 Andreas I. Papadakis,1 Matteo Fassan,3 Anita Hall,1 Anie Monast,1 Maryam Safisamghabadi,1 Magnus von Knebel Doeberitz,4 John P. Neoptolemos,5 Eithne Costello,6 William Greenhalf,6 Aldo Scarpa,7 Bence Sipos,8 Daniel Auld,1 Mark Lathrop,1 Morag Park,1 Markus W. Büchler,9 Oliver Strobel,9 Thilo Hackert,9 Nathalia Giese,9 George Zogopoulos,1 Veena Sangwan,1 Sidong Huang,1 Jörg D. Hoheisel,4 Yaser Riazalhosseini1. 1 _McGill University, Montreal, Quebec, Canada;_ 2 _Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany;_ 3 _ARC-NET Center for Applied Research on Cancer, University and Azienda Ospedaliera Universitaria Integrata, Verona, Italy;_ 4 _Deutsches Krebsforschungszentrum (DKFZ), Germany;_ 5 _National Institute for Health Research, Liverpool Pancreas Biomedical Research Unit, Liverpool, United Kingdom;_ 6 _National Institute for Health Research, Liverpool Pancreas Biomedical Research Unit, United Kingdom;_ 7 _ARC-NET Center for Applied Research on Cancer, University and Azienda Ospedaliera Universitaria Integrata, Italy;_ 8 _Institute for Pathology and Neuropathology, Universitätsklinikum Tübinge, Germany;_ 9 _Department of Surgery, University Hospital Heidelberg, Germany_.

Introduction and aims: Although the overall five-year survival of all patients with cancer stands at 63%, for pancreatic cancer patients, it is a disheartening 8% - a number that remains largely unchanged for three decades. Of the patients diagnosed with pancreatic cancer, about 85% exhibit pancreatic ductal adenocarcinoma (PDAC). Most of these patients die within 4 to 6 months after diagnosis. The poor prognosis is caused by the detection at only late stages, and lack of effective options for chemotherapy. The widely used chemotherapeutic agent gemcitabine, confers a median survival advantage of only 6 months, and resistance to therapy develops in the vast majority of patients. Given this poor prognosis of patients with PDAC, there is an urgent need to find more effective therapies.

Experimental procedures: Microarrays were used to perform global gene expression profiling in 195 PDAC and 41 normal pancreatic tissue samples. Using these profiling data, we undertook an extensive analysis of PDAC transcriptome by superimposing the pathway context and interaction networks of aberrantly expressed genes to identify factors with central roles in PDAC pathways. Next, tissue microarray analysis (TMA) were used to verify the expression of the candidate target in independent set of 152 samples comprising 40 normal pancreatic tissues, 49 chronic pancreatitis sections (CP) and 63 PDAC samples. We further validated the functional relevance of the candidate molecule through RNA interference (RNAi) and pharmacological inhibition in vitro and in vivo.

Results: We identified dopamine receptor D2 (DRD2) as a key modulator of cancer pathways in PDAC. DRD2 up-regulation at the protein level was validated in a large independent sample cohort. Most importantly, we found that blockade of DRD2, through RNAi or pharmacological inhibition using FDA-approved antagonists hampers the proliferative and invasive capacities of pancreatic cancer cells while modulating cAMP and endoplasmic reticulum stress pathways. Also, we observed a potent effect of DRD2 antagonists on inhibition of cancer cell proliferation using different model of primary and metastatic tumor cells derived from spontaneous pancreatic cancer mouse models and patient-derived pancreatic adenocarcinoma mouse xenograft (PDX) models.

Conclusions: Our findings demonstrate that inhibiting DRD2 represents a novel therapeutic approach for PDAC. Since DRD2 inhibitors are already in the clinic for the management of schizophrenia, our results from this study could support a drug repurposing strategy to expedite clinical evaluation of these agents as novel therapy against pancreatic cancer.

#1136

Heat shock protein 90 inhibitors suppress PDGFRA reactivation and other receptor tyrosine kinase activation important in drug resistant gastrointestinal stromal tumors.

Masahiro Yamamura, Akira Yamauchi, Naoki Katase, Makoto Okawaki, Yousuke Katata, Futoshi Kuribayashi, Junichi Kurebayashi, Yoshiyuki Yamaguchi. _Kawasaki Medical School, Kurashiki, Japan_.

Background: Inhibition of KIT oncoproteins by imatinib induces clinical responses in most gastrointestinal stromal tumor (GIST) patients. However, many patients develop imatinib resistance due to secondary KIT mutations. The drug resistance mechanism has only been partially elucidated, and the sunitinib resistance mechanism is also unknown. In this study, we elucidate the new imatinib resistant mechanism and find effective inhibitors.

Methods: We have established imatinib-resistant cells GIST-T1R by culturing cells with increasing concentrations of imatinib. We analyzed receptor tyrosine kinase and intracellular signal protein strongly expressed in resistant cells using Western blotting and Phosphorylation Array. Then, the antitumor effect was examined using an agent that inhibits strongly expressed molecules of the resistant cells.

Results: The IC50 of imatinib in the sensitive cells(GIST-T1) and the resistant cells(GIST-T1R) was 10 nM and 20 μM, respectively, which demonstrated 2000-fold difference. These imatinib resistant cells were also cross-resistant to sunitinib. In western blotting, PDGFRA phosphorylation and protein expression were enhanced in GIST-T1R compared to GIST-T1. When GIST-T1 was treated with imatinib, KIT phosphorylation was suppressed but PDGFRA phosphorylation was enhanced. Imatinib treatment of GIST-T1R enhanced PDGFRA phosphorylation and protein expression as compared to before imatinib treatment. Phosphorylation of PDGFRA in these resistant cells could not be suppressed by regorafenib. Furthermore, in the protein phosphorylated array, in addition to phosphorylation of PDGFRA in resistant cells, phosphorylation of multiple RTKs was also enhanced. An HSP90 inhibitor AUY922 capable of simultaneously suppressing a plurality of proteins was used. AUY922 suppressed GIST-T1 and GIST-T1R cell proliferation at low concentrations and was more effective than regorafenib. Furthermore, AUY922 inhibited phosphorylation and protein expression of PDGFRA in drug-resistant cells, as well as phosphorylation of other several receptor tyrosine kinases.

Conclusion: Activation of PDGFRA was considered to be important for the mechanism of drug resistance of GIST. Furthermore, since activation of multiple receptor tyrosine kinases can be observed simultaneously in drug resistance, it is considered that inhibiting a single target is not effective. Since HSP90 inhibition suppresses PDGFRA protein expression and possibly inhibits multiple target proteins at the same time, it is the most effective treatment for drug resistant GIST.

#1137

ADAM8 drives aggressive phenotype of triple-negative inflammatory breast cancer & constitutes a novel therapeutic target.

Mathilde Romagnoli,1 Stefania Pianetti,1 Sonia G. Das,1 Delphine Loussouarn,2 Carole Gourmelon,2 Mario Campone,2 Sophie Barillé-Nion,3 Giang T. Nguyen,1 Srimathi Srinivasan,1 Gail E. Sonenshein,1 Nora D. Mineva1. 1 _Tufts Univ. School of Medicine, Boston, MA;_ 2 _Institut de Cancérologie de l'Ouest, Saint-Herblain, France;_ 3 _UMR 892 INSERM, Nantes, France_.

Inflammatory Breast Cancer (IBC) is a rare, highly aggressive form of cancer that is frequently locally advanced or metastasized at the time of diagnosis. The Triple-Negative subtype of IBC (TN-IBC), in particular, is characterized by very poor overall survival. TN-IBC lacks targeted therapies and is primarily treated with radiation or chemotherapy, which are inefficient. Recently, we identified the cell surface transmembrane ADAM8 (A Disintegrin and Metalloproteinase) protein as a driver of Triple-Negative Breast Cancer (TNBC) growth and metastasis via its Metalloproteinase (MP) and Disintegrin (DI) domains, respectively. In in vivo proof-of-concept experiments with a prototype reagent, we demonstrated that simultaneous, antibody-based, targeting of the ADAM8 MP and DI domains is an effective therapeutic approach for TNBC (Romagnoli et al., EMBO Mol. Med. 6:278, 2014).

The aggressive behavior of IBC cells has been attributed to a stem-like cancer cell compartment with high ALDH activity (ALDH+). Our studies of the green tea compound epigallocatechin-3 gallate (EGCG) revealed that it reduced growth of tumors derived from ALDH+ SUM-149 TN-IBC cells. We now report that EGCG concurrently decreases tumor levels of ADAM8 mRNA. This led us to assess the role of ADAM8 in TN-IBC. Tumor biopsies from 15 patients taken at the time of diagnosis and/or after neo-adjuvant treatment with chemotherapy, as well as paired lymph node and skin samples (when available) were analyzed by immunohistochemistry for ADAM8 expression. We report that 45.5% of primary TN-IBC patient tumors and 50.0% of metastases within the axillary lymph nodes express high levels of ADAM8 and its expression is largely unaffected by chemotherapy. In addition, ADAM8 expression was higher in the aggressive ALDH+ stem-like SUM-149 cell compartment. Consistently, knockdown of ADAM8 dramatically reduced the ability of SUM-149 cells to grow in an anchorage independent fashion and to migrate through Matrigel. A newly prepared anti-human ADAM8 mouse monoclonal antibody (ADP13) inhibited the MP and DI domains of ADAM8 on SUM-149 cells. ADP13 reduced orthotopic growth of tumors derived from SUM-149 cells by 40% in mice treated by i.p. injection 2 days a week with a dose of 4.5 mg/kg (n=7). Dose-response curves and survival experiments are in progress.

Conclusions: ADAM8 expression is present in almost half of TN-IBC patient tumors and their metastases, and promotes aggressive phenotype of TN-IBC cells in in vitro 3D-assays. A pre-clinical mouse model of TN-IBC validated ADAM8 as an accessible and promising new target for therapeutic intervention against this highly aggressive disease.

#1138

**The protein disulfide isomerase inhibitor XCE853 inhibits** in vitro **,** ex-vivo **and** in vivo **growth of human tumors.**

Gregoire Pierre Prevost,1 Anne Chauchereau,2 Patrick Ladam,3 Renaud Seigneuric,4 Denis Carniato,5 Marc-Henry Pitty,6 Paul Foster7. 1 _CIPREVO, Antony, France;_ 2 _Gustave Roussy, Villejuif, France;_ 3 _Université Paris XIII, Bobigny, France;_ 4 _Université de Bourgogne, Dijon, France;_ 5 _DC2, Marcoussis, France;_ 6 _Oregon Therapeutics, Paris, France;_ 7 _University of Birmingham, Birmingham, United Kingdom_.

Protein disulfide isomerase (PDI) is a chaperone protein that regulates oxidative protein folding as well as cell viability. Increased PDI levels have been documented in a variety of human cancers associated with a poor overall survival, including ovarian, prostate, brain and lung cancers. Inhibition of PDI activity leads to apoptosis in cancer, suggesting that PDI is a promising druggable target. XCE853 is a synthetic small molecule displaying an excellent docking with the catalytic domain of the human PDI. XCE853 inhibits in vitro recombinant PDI enzymatic activity. In addition, the proliferation of a large panel of human tumor cells is blocked by XCE853 with IC50s in the nanomolar range through an irreversible cytolysis leading to a tumor cell death by autophagy and particles release (vesicles or protein aggregates). XCE853 is also active on a large panel of drug resistant human cancer cells. XCE853 induced an irreversible cytolysis of human tumor cells after a short in vitro exposure independently of efflux pumps. In addition, the ex-vivo approach using fresh human tumor explants cultivated in 3 dimensions with low concentrations of XCE853 has shown a strong decrease of the proliferation (KI-67 labeling) in several tumor types. Finally, XCE853 displayed excellent oral bioavailability in mice and was able to block the growth of several human cancers using in vivo xenograft models leading to a complete tumor growth arrest even after the cessation of the treatment. Altogether, these data strongly support further efforts to move this drug candidate to the preclinical studies to access advanced cancer patients.

#1139

G protein-coupled receptors (GPCRs): unrecognized potential therapeutic targets in cancer.

Paul A. Insel, Krishna Sriram, Shu Z. Wiley, Thalia McCann, Randall P. French, Andrew M. Lowy. _UCSD, La Jolla, CA_.

G protein-coupled receptors (GPCRs) are the largest class of signaling receptors in humans and other species and in addition, the most widely targeted class for FDA-approved therapeutics. However, GPCRs are not commonly targeted in cancers other than endocrine tumors. We hypothesize that GPCRs may have been "missed" as targets in cancer, perhaps in part because they have not been frequently identified as driver mutations; however, GPCRs may have unappreciated utility as therapeutic targets in a variety of tumors. To test this hypothesis, we have used unbiased approaches (Taqman GPCR arrays and RNA-seq), mining of publicly available datasets (e.g., The Cancer Genome Atlas, TCGA), and validation studies (e.g., protein and functional analyses) to assess GPCRs in a variety of human tumors, cancer cells and stromal cells (cancer-associated fibroblasts, CAFs). A major focus of our efforts has been on pancreatic ductal adenocarcinoma (PDAC) cells/tumors and pancreatic CAFs (PCAFs). We find that PDAC cells and PCAFs express >70 different GPCRs, including many orphan GPCRs (receptors without known physiologic agonists) and that numerous GPCRs are expressed at much higher levels than in normal precursor cells (pancreatic ductal epithelial cells for PDAC cells and pancreatic stellate cells and pancreatic fibroblasts for PCAFs). We find that a cluster of GPCRs is overexpressed in PDAC tumors. Two such receptors are orphan GPCRs, Orphan 1 and Orphan A, neither of which is mutated but each has high expression, respectively, in PCAFs and PDAC cells. Orphan 1 and Orphan A have at least 2-fold higher expression in >90% of PDAC tumors in TCGA compared to that in normal pancreatic tissue (compiled from the GTEX database). Higher expression of orphan A is associated with decreased survival and remission, while Orphan 1 expression correlates with that of numerous fibrotic genes. Importantly, both Orphan 1 and Orphan A are functional and appear to contribute to the malignant phenotype. For example, siRNA knockdown of Orphan 1 in PCAFs blunts production of profibrotic markers and decreases ability of conditioned media from PCAFs to enhance proliferation of PDAC cells while transfection of Orphan A increases DNA synthesis of normal pancreatic ductal epithelial cells. Taken together, these and other results suggest that in addition to their role in endocrine tumors, GPCRs represent previously unrecognized contributors to cancer through actions on tumor cells and stromal cells. These data suggest the possibility that highly expressed GPCRs (such as Orphan A) may function as oncogenes. Thus, GPCRs may be druggable, novel targets for the treatment of cancer, including pancreatic cancer, a tumor for which new therapeutics are an important, unmet need.

#1140

Characterization of a novel PDE10 inhibitor in lung tumor cells and an orthotopic mouse model of lung cancer.

Veronica Ramirez-Alcantara,1 Bing Zhu,1 Xi Chen,1 Rajkumar Savai,2 Prema Subbarayal,2 Michele A. Schuler,3 Kevin J. Lee,1 Ashley S. Lindsey,1 Kristy L. Berry,1 Dennis Otali,4 Joshua Canzoneri,5 Jacob Valiyaveettil,1 Adam Keeton,1 Lori Coward,6 Gregory Gorman,6 William Grizzle,4 Michael Boyd,5 Gary A. Piazza1. 1 _University of South Alabama Mitchell Cancer Institute, Drug Discovery Research Center, Mobile, AL;_ 2 _Max Planck Institute for Heart and Lung Research, Molecular Mechanism in Lung Cancer, Bad Nauheim, Germany;_ 3 _University of South Alabama, Department of Comparative Medicine, Mobile, AL;_ 4 _University of Alabama at Birmingham, Department of Pathology, Birmingham, AL;_ 5 _ADT Pharmaceuticals, Inc., Orange Beach, AL;_ 6 _Samford University, Pharmaceutical Sciences Research Institute, McWhorter School of Pharmacy, Birmingham, AL_.

BACKGROUND: Screening a focused library of indene derivatives for PDE10 inhibitory activity identified novel leads with potent and selective tumor cell growth inhibitory activity. ADT-030 emerged from lead optimization chemistry with excellent drug-like properties and oral bioavailability. Here we characterize the anti-tumor activity of ADT-030 in human lung tumor cells and an orthotopic mouse model of lung cancer.

METHODS: Growth inhibitory activity of ADT-030 was measured in a panel of human lung tumor cell lines by ATP quantification following 72 h of treatment. The effect of ADT-030 on intracellular cGMP/cAMP was measured in whole cell lysates using a competitive ELISA assay. PDE inhibitory activity of ADT-030 was evaluated in lysates of human lung tumor cells and by recombinant PDE isozymes using the IMAP fluorescence polarization PDE assay. Activation of PKG signaling and suppression of β-catenin levels in response to ADT-030 treatment was evaluated by Western blot using whole cell lysates of human lung tumor cells. ADT-030 was orally administrated to C57BL/6 mice and free levels quantified in plasma and tissues by LC-MS. Anti-tumor activity of ADT-030 was evaluated in athymic nude-Foxn1nu mice after inoculating the left lung with 1x106 A549 lung tumor cells and treating once daily by oral administration at dosages ranging from 25 - 125 mg/kg. Tumor growth was monitored by in situ bioluminescence using IVIS as well as necropsy and pathological grading after 4 weeks of treatment.

RESULTS: ADT-030 inhibited the growth of human lung tumor cell lines with IC50 values in the low micromolar range by inducing apoptosis, while appreciably higher concentrations were required to affect the growth of normal human airway epithelial cells. ADT-030 treatment of human lung tumor cells increased both intracellular cGMP and cAMP levels, activated PKG and suppressed β-catenin within the same concentration range as required for tumor cell growth inhibition. Pharmacokinetic studies in mice demonstrated a half-life suitable for once a day dosing. Tissue distribution studies revealed appreciably higher concentrations of ADT-030 in lungs relative to plasma and other tissues, with the highest accumulation measured in the parenchyma. ADT-030 was well tolerated in mice implanted with A549 tumor cells and displayed strong anti-tumor activity as evident by reduced luminescence, tumor grading, and double-blinded pathological evaluation.

CONCLUSIONS: ADT-030 represents a prospective drug development candidate with favorable drug-like properties that concentrates in lung after oral administration exhibiting a strong anti-tumor activity in a pre-clinical mouse model. The mechanism of lung tumor cell growth inhibition involves PDE10 inhibition, elevation of cGMP, activation of PKG, and attenuation of β-catenin.

#1141

Evaluation of GABRP as a novel therapeutic target in triple negative breast cancer.

Vikram B. Wali, Gauri A. Patwardhan, Christos Hatzis, Lajos Pusztai. _Yale Univ. School of Medicine, New Haven, CT_.

Triple Negative breast cancers (TNBC) represent nearly 20% of all breast cancers but do not have any targeted therapies available due to lack of amplification of HER2 and expression of estrogen and progesterone receptors. To identify potentially druggable targets for TNBC, we compared TNBC to non-TNBC tumors in large independent gene expression clinical datasets to identify highly expressed genes in TNBC that encode for proteins expressed on the cell surface. We found gamma-aminobutyric acid type A receptor pi (GABRP) to be the most overexpressed receptor protein gene in TNBC, while its expression in normal tissues was low and comparable to that of proteins targeted by drugs or therapeutic antibodies already in clinical testing. We compared GABRP mRNA and protein expression across panel of breast cancer cell lines and found it to be particularly elevated in HCC1143 and MDA-MB-468 TNBC cells. Knockdown of GABRP using siRNA suppressed TNBC cell growth and stable GABRP knock-down by shRNA suppressed growth of MDA-MB-468 xenografts in nude mice. Sub-cellular fractionation of TNBC cells and subsequent Western blot analysis confirmed that GABRP is located predominantly in the cell membrane. GABRP protein expression on cell surface was also detected by flowcytometry. Interestingly, treatment with 5-100μg/ml of anti-GABRP antibody that binds to the extracellular domain (ECD) itself inhibited growth of TNBC cell lines expressing higher GABRP, while the same doses of a control antibody had no effect in these cells in culture. Our goal is to generate an antibody drug conjugate (ADC) for TNBC by conjugating anti-GABRP antibody with a maytansinoid such as DM1, which has proven cytotoxicity in breast cancer cells. Together, our results suggest that GABRP is a potential therapeutic target for triple-negative breast cancers.

#1142

Novel target discovery for glioblastoma using chemical biology fingerprinting.

Darren Finlay,1 Pedro Aza-Blanc,1 Harshil Dhruv,2 Alexey Eroshkin,1 Craig Hauser,1 Jeff Kiefer,2 Seungchan Kim,2 Tao Long,1 Robert G. Oshima,1 Sen Peng,2 Gil Speyer,2 Michael Berens,2 Kristiina Vuori1. 1 _Sanford Burnham Prebys Med Discovery Institute, La Jolla, CA;_ 2 _TGen, Phoenix, AZ_.

The most common adult brain tumor is Glioblastoma Multiforme (GBM), an extremely aggressive cancer with only scant treatment options. Even with standard of care most patients present with a recurrence and the median survival is only circa 15 months. The need, therefore, for new therapeutic targets and treatment options is pressing. Here we describe here a multipronged approach to identifying said targets. We present an established methodology for the isolation and culture of patient derived GBM samples that retain the "stem-like" fraction thought to underlie resistance and recurrence. Furthermore we show genomically that these samples represent specific subtypes of the disease yet still form distinct groups in unbiased clustering analysis. Thus we have multiple representative patient derived cultures that are suitable for our drug discovery and chemical biology analyses. Using a process we term Chemical Biology Fingerprinting (CBF) we utilize small focused, and clinically relevant, chemical collections in order to identify patterns of chemovulnerabilities across multiple samples. This allows an unbiased yet cancer relevant sub-stratification and the identification of agents, and therefore targets, which may be relevant for GBM patient subtypes. Indeed our use of the highly annotated NCI CTD2 Informer Set of chemicals allows ready drug-to-target mapping and facilitates data sharing across the CTD2 network. Moreover, already defined subgroups can be clustered to find agents, or groups of agents, that show selective activity against traditional classifications (e.g. proneural, mesenchymal etc.). Finally our strategy is permissive for the identification of "exceptional responders". That is, individual patient samples that respond to a specific drug whilst most samples are refractory. In sum we demonstrate generation of patient derived models and identify specific, and novel, drugs that may be relevant for specific GBM subtypes. Supported by NIH U01CA168397

#1143

Targeting the transcriptional kinases CDK12 and CDK13 in breast and ovarian cancer.

Michael Bradley, Jason Marineau, Yoon Choi, Kristin Hamman, Goran Malojcic, David Orlando, Yixuan Ren, Nan Ke, Shanhu Hu, Eric Olson, Christian Fritz, Christopher Roberts. _Syros Pharmaceuticals, Cambridge, MA_.

CDK12 and CDK13 regulate expression of large transcripts requiring substantial processing to produce mature mRNA. This transcriptional regulation includes coordinated phosphorylation of specific repeats within the C-terminal domain of RNA polymerase II and association with RNA processing factors (Chila, 2016). RNAi knockdown of CDK12 in cell culture decreases expression of DNA damage response genes, including BRCA1 and ATR, while enhancing sensitivity to DNA damaging agents (Blazek, 2011; Liang, 2015). Recently THZ531, a selective covalent inhibitor of CDK12 and CDK13, was shown to decrease expression of DNA damage response genes in cell culture (Zhang, 2016). Here we present further studies with THZ531 to guide our discovery program toward molecules suitable for clinical development and to explore mechanistic rationales for combining a CDK12/13 inhibitor with PARP inhibitors or DNA damaging agents for difficult-to-treat cancers such as high-grade serous ovarian cancer and triple-negative breast cancer. Using THZ531 as a benchmark, we developed assays capable of discriminating sub-nM inhibitors, including quantifying time-dependent covalent inhibition and cell-based CDK occupancy. Since CDK7, like CDK12 and CDK13, contains a cysteine residue proximal to the kinase active site, these approaches are critical to understand covalent inhibitor selectivity. Furthermore, we performed kinome paneling studies to better understand selectivity of this scaffold in support of our ongoing efforts to optimize CDK12/13 potency and selectivity. To pharmacologically investigate the previously reported effects of CDK12 RNAi, growth inhibition of a panel of ovarian and breast cancer cell lines was assessed following treatment with THZ531 (OVA EC50 = 50-200 nM (n=6); BRCA EC50 <50 nM (n=4)). Expression profiling revealed that THZ531 treatment resulted in different sets of genes being affected than was observed following treatment with inhibitors targeting CDK7, CDK9 or BET-bromodomain proteins. Additionally, CDK12/13, CDK7 and CDK9 inhibitors were profiled in a broad cell line panel (n>400) to reveal relationships between inhibitor sensitivity, mutation status, gene expression, and potential oncology indications that may be addressed by these different mechanisms. Finally THZ531 was synergistic with both PARP inhibitors and DNA damaging agents in ovarian and breast cancer cell lines. These data highlight cancer indications and combinations that may be particularly amenable to treatment with CDK12/13 inhibitors. While the pharmacokinetic properties of THZ531 preclude adequate target engagement in tumor tissue at tolerated doses in mouse model systems, our ongoing medicinal chemistry program is progressing to identify and optimize CDK12/13 inhibitors suitable for clinical evaluation.

#1144

Dual inhibition of MEK1/2 and PLK1 specifically targets aggressive breast cancer cell population with CEP55 elevated expression.

Debottam Sinha, Murugan Kalimutho, J. Alejandro Lopez, Kum Kum Khanna. _QIMR Berghofer Medical Research Institute, Brisbane, Australia_.

Triple negative breast cancers (TNBCs), lacking the expression of ER, PR and HER2 amplification, are the most aggressive form of breast cancer (BC). Due to their heterogeneity influenced by high level of genomic instability and aneuploidy, treatment of TNBC patients is one of the biggest challenges faced in the clinics. CEP55, discovered first by our laboratory, is a key regulator of cytokinesis malfunction of which has been shown to cause multinucleation. Furthermore, ERK2/PLK1 critically regulates the functional role of CEP55 via phosphorylation of CEP55 at particular stages of mitosis, allowing it to localize to the midbody for accurate cytokinesis. Research has demonstrated association of CEP55 with various cancers especially BC as over-expression of CEP55 mRNA is associated with worse BC prognosis and poor survival. We hypothesized that, CEP55 regulates the fate of aneuploid cell population, which are highly dependent on mitotic genes for tumor progression among aggressive BC, thus can be targeted for therapy development. We have performed a series of in vitro studies demonstrating that depletion of CEP55 sensitizes cells to anti-mitotic drugs like PLK1 inhibitor (BI2536) and leads to unscheduled CDK1/Cyclin B activation and favor CDK1-Caspase 3-dependent mitotic catastrophe. We also demonstrate that ERK1/2 transcriptionally controls CEP55 mRNA and due to lack of a specific small molecule inhibitor against CEP55, inhibition of MEK1/2 using the small molecule inhibitor Selumetinib, can mimic depletion of CEP55 in vivo. Thus, we rationalized the usage of a MEK1/2 inhibitor in combination with a PLK1 inhibitor across a series of BC cell lines. We observed synthetic lethality among the aggressive hormone receptor negative cell lines, which expressed higher expression of CEP55 compared to normal like and hormone receptor positive BC cell lines with lower CEP55 level. The combination synergistically increased apoptosis of aneuploid population via premature entry of these cells into mitosis in the presence of antimitotic drugs due to exhaustion of CEP55. We have also validated this synergistic effect of MEK1/2 and PLK1 inhibition using xenograft models, results of which imitated the in vitro findings. Therefore, we propose a novel treatment strategy of MEK1/2-PLK1 dual combination for selectively targeting CEP55 over-expressing BC in the clinics.

#1145

Overcoming chemoresistance and blocking metastasis in breast cancer by targeting HuR.

Xiaoqing Wu,1 Rebecca Marquez,1 Shuang Han,1 Lan Lan,1 Dan A. Dixon,2 Jeffrey Aubé,3 Danny R. Welch,2 Liang Xu1. 1 _University of Kansas, Lawrence, KS;_ 2 _University of Kansas Medical Center, Kansas City, KS;_ 3 _University of North Carolina, Chapel Hill, NC_.

Patients with metastatic breast cancer have a dismal 5-year survival rate of only 24%. Chemoresistance is another contributor to the high mortality of advanced breast cancer. The RNA-binding protein, Hu antigen R (HuR), is overexpressed at all stages of breast cancer development. Cytoplasmic HuR accumulation correlates with high-grade malignancy, poor distant disease-free survival and serves as a prognostic factor for poor clinical outcome in breast cancer. HuR promotes tumorigenesis by promoting mRNA stability and translation of proteins implicated in proliferation, survival, angiogenesis, invasion, and metastasis. HuR also modulates sensitivity of breast cancer cells to chemotherapy. Taken together, HuR is an emerging target for breast cancer therapy, especially metastatic breast cancer. Using shRNA and CRISPR technologies to modulate HuR expression in breast cancer cells, we found that cells with HuR KD/KO were less invasive and more sensitive to chemotherapy. In an effort to discover small molecules that disrupt the HuR-mRNA interaction and block HuR functions in breast cancer progression, metastasis and chemoresistance, high throughput screening (HTS) was carried out in several chemical libraries (~23,000 compounds) using fluorescence polarization (FP) assay, which identified several initial hits with sub-micromolar inhibitory constants (Ki). Those potential inhbitors were then validated by ALPHA assay (Amplified Luminescent Proximity Homogeneous Assay), confirmed by Surface Plasmon Resonance (SPR). In cell-based assays, top hit KH-3 and its optimized analogs, specifically shortened HuR target mRNA half-lifes and decreased the level of the encoded proteins. Moreover, those compounds inhibited invasion and restored chemosensitivity. qPCR arrays focusing on specific pathways revealed that HuR inhibitors potently upregulated metastatic suppressors and downregulated genes frequently overexpressed in lung metastases. In animal studies, KH-3 not only exhibited potent antitumor efficacy in orthotopic xenografts of breast cancer, but also efficiently blocked experimental metastasis. In conclusion, we identified potent and specific small molecule inhibitors of HuR-mRNA interaction for potential anti-metastatic therapy for breast cancer with HuR overexpression.

#1146

Biomarker-driven targeted oral treatment strategy for bladder cancer.

Daley S. Morera, Andre Jordan, Vinata B. Lokeshwar. _Augusta University, Augusta, GA_.

INTRODUCTION AND OBJECTIVE: Hyaluronic acid (HA) family of molecules, HA-synthases (HAS-1,2,3), HA-receptors (CD44, RHAMM) and hyaluronidase (HYAL-1) are markers for bladder cancer (BCa) diagnosis and predicting prognosis. HA-family promotes tumor growth and metastasis by inducing epithelial mesenchymal transition (EMT). 4-Methylumbelliferone (4-MU) is an orally bioavailable dietary supplement that inhibits HA synthesis. We evaluated the expression of HA family and EMT markers in bladder tissues as well as the antitumor effects of 4-MU as a potential targeted therapeutic agent in preclinical models of BCa.

METHODS: Quantitative PCR was used to measure mRNA expression of HA-family and EMT genes (β-catenin, Twist, and Snail) in 72 bladder tissue specimens (28 normal; 44 tumor); follow-up: 20.3±2.5 months; median 17 months. The effect of 4-MU (0-0.6 mM) on cell proliferation, apoptosis, intracellular signaling, and the expression of HA receptors, and EMT genes were examined in BCa cell lines by Q-PCR, immunoblotting, proximal ligation and PI-3K activity assays. Mechanism of action was analyzed by HA addition and mAkt overexpression. Effect of oral administration of 4-MU (100, 200-mg/kg) on tumor growth was analyzed in subcutaneous xenografts.

RESULTS: HAS-1, -2 -3, HYAL-1 and Snail levels were 10-20-fold elevated in BCa tissues as compared to normal bladder (P<0.001). In univariate analysis, HAS-1, -2, HYAL-1 and Twist levels correlated with metastasis (P<0.001); HYAL-1 was an independent predictor of metastasis. 4-MU inhibited cell proliferation, chemotactic motility and invasion in a dose-dependent manner; 50-70% inhibition at IC50 (0.4 mM) for HA-synthesis inhibition. 4-MU induced apoptosis (>3-fold) via the death receptor pathway. 4-MU downregulated HA-signaling, specifically transcript and/or protein levels of CD44, RHAMM, p-Akt, β-catenin, pβ-catenin(S552). Snail and twist were downregulated by 2-5-fold, but pβcatenin((T41/S45), pGSK-3α/β and E-cadherin levels were increased. 4-MU also inhibited CD44/PI-3K complex formation and PI-3K activity. HA addition or myristoylated Akt expression attenuated 4-MU effects. In xenograft studies, 4-MU oral treatment abrogated tumor growth of established tumors (vehicle, day 50: 766±221 mm3; 4-MU: 128±61, day 50) by abrogating HA-synthesis. No weight loss or serum or organ toxicity was observed in treated mice.

CONCULUSION: This study demonstrates that HA-family signaling is upregulated in BCa and can be specifically targeted for treatment by a non-toxic dietary supplement. Support: 7R01 CA072821-16; 7R01CA176691-03

#1147

Probing the roles of SUMOylation in cancer cell biology using a selective SUMO activating enzyme inhibitor.

Sai M Pulukuri, Xingyue He, Stephen Grossman, Jessica Riceberg, Erik Koenig, Teresa Soucy, Keli Song, Anna Kreshock, Dylan England, Hirotake Mizutani, Larry Dick, James Brownell, John Newcomb, Steve Langston, Eric Lightcap, Katherine Galvin. _Takeda Pharmaceuticals, Inc, Cambridge, MA_.

SUMOylation has been implicated in many cellular processes that are important for cancer cell survival, including cell cycle, chromosome structure and segregation, nuclear and subnuclear organization, transcription and DNA damage repair. However, a potent and selective inhibitor to target the SUMO pathway has been lacking. The SUMO-activating enzyme (SAE) is an essential enzyme in the pathway that initiates the SUMOylation process. Here we report the identification of the first mechanism-based SAE inhibitors with nanomolar potency in cellular assays. These inhibitors selectively block SAE enzyme activity and total SUMOylation in cells, which leads to reduced cancer cell proliferation. Moreover, SAE inhibition resulted in disruption of PML nuclear bodies and redistribution of DAXX. In vivo administration of SAE inhibitor into tumor bearing mice results in modulation of several biomarkers including a significant reduction in SUMO-conjugates and E2 Ubc9 thioesters, demonstrating SUMO pathway inhibition. Our results demonstrate the feasibility of inhibiting the SUMO pathway with small molecule inhibitors and provide tools to study the SUMO biology in cancer.

#1148

Bazedoxifene as a novel GP130 inhibitor for the treatment of triple-negative breast cancer.

Jilai Tian,1 Yang Cao,2 Xiang Chen,1 Hui Xiao,3 Chenglong Li,4 Jiayuh Lin1. 1 _University of Maryland, Baltimore, MD;_ 2 _Huazhong University of Science and Technology, Wuhan, China;_ 3 _The Ohio State University, Columbus, OH;_ 4 _University of Florida, Gainesville, FL_.

Triple negative breast cancer (TNBC) is the only subtype of breast cancer still lacking effective therapeutic options. Thus, finding novel targets and therapies to treat TNBC is an urgent clinical issue. IL-6 is one of the principal oncogenic mediators in breast cancer and systemic IL-6 levels correlates with poor prognosis, advanced disease, and metastases. Importantly, growth of TNBC cells are replied on autocrine cytokines including IL-6. Therefore, IL-6 signaling represents a novel approach with a potential to improve the therapeutic efficacy. To date, however, no small molecules that target IL-6 signaling are available for clinical cancer therapy. IL-6 binds to IL-6 Rα, then recruits GP130 to form the IL-6/IL-6 Rα/GP130 heterotrimer and triggers a signaling cascade downstream including JAK/STAT3, PI3-K/AKT/mTOR, and MEK/ERK. Therefore, it is possible to target IL-6 signaling by blocking IL-6 binds to GP130 or IL-6 Rα and thus inhibiting its signaling cascade downstream.To effectively target IL-6 signaling, we have utilized a novel drug discovery approach combining Multiple Ligand Simultaneous Docking and drug repurposing to target GP130, since its structure is more druggable than IL-6 Rα. We have identified a FDA-approved drug Bazedoxifene (for the prevention of the postmenopausal osteoporosis in postmenopausal women) with a novel function to inhibit IL-6 and GP130 protein-protein interactions and thus blocking IL-6 signaling cascade downstream. In this study, we examined the therapeutic effect of Bazedoxifene on TNBC, aiming to verify Bazedoxifene as a Novel IL-6/GP130 Inhibitor for TNBC treatments.Our data of western blot showed Bazedoxifene inhibited increased STAT3 phosphorylation induced by IL-6. In addition, Bazedoxifene inhibited phosphorylation of AKT and ERK in TNBC cells that were also produced by IL-6. Furthermore, combination of Bazedoxifene with paclitaxel exhibited more significant inhibition than single agent alone on cell viability in TNBC cells either in 2D or 3D culture model in vitro. Results of colony formation showed that Bazedoxifene could significantly inhibit cell survive and proliferation at 20 μM, which were more effective than Evista and SC144. Moreover, Bazedoxifene could inhibit cell migration. We tested the increased proliferation of TNBC cells using BrdU with the added IL-6, which were then inhibited by Bazedoxifene, demonstrating the Bazedoxifene could achieve a competed inhibition of IL6. The result of significant inhibition of tumor growth in vivo furtherly verified the therapeutic effects of Bazedoxifene. All results showed the significant therapeutic effects of Bazedoxifene in TNBC cells by competed blocking IL6 signaling. Thus, Bazedoxifene holds a great potential for TNBC therapy.

#1149

Targeting Rad6B suppresses melanomagenesis.

Ashapurna Sarma,1 Sarah Petit,1 Fangchao Liu,2 Pratima Nangia-Makker,1 Guangzhao Mao,2 Malathy Shekhar1. 1 _Wayne State University and Karmanos Cancer Institute, Detroit, MI;_ 2 _Wayne State University, Detroit, MI_.

Active β-catenin signaling in melanoma is associated with aggressive disease. We have previously shown that Rad6B, an ubiquitin conjugating enzyme, is a positive regulator of β-catenin stability and transcriptional activity, and is itself a transcriptional target of β-catenin. This stabilization is mediated by Rad6B-induced K63-linked polyubiquitination of β-catenin that renders it insensitive to 26S proteasomal degradation. Immunohistochemical analysis of human cutaneous melanoma samples showed the first detectable expression of Rad6B in melanocyte hyperplasia and continued Rad6B and β-catenin overexpression in primary and metastatic melanomas. These data implicate a critical role for Rad6B in melanocyte transformation and a continuing role in melanoma progression. To analyze the role of Rad6B in melanomagenesis, we targeted Rad6B expression/activity in human M14 metastatic melanoma cells by using a Rad6-selective small molecule inhibitor SMI#9 and the CrispR/Cas9 gene editing system. Immunoblot analysis showed that compared to controls, M14 cells treated with SMI#9 showed decreased levels of β-catenin, and β-catenin driven transcriptional targets Rad6, vimentin, Mitf-M, and Melan A. Although Snail levels were not altered, SMI#9 treated cells showed increase in phosphorylated Snail. Immunocytochemical staining verified the western blot data and showed cytoplasmic relocalization of PCNA and Snail in SMI#9 treated cells which is consistent with increase in phosphorylated Snail. Concordant with the decrease in vimentin and unphosphorylated Snail levels, Rad6B-inhibited cells display poor capacity to migrate as compared to controls. Whereas Rad6B knockout cells were severely growth impaired, Rad6B edited clones showing partial expression (potentially from only one allele) similarly showed decreased levels of β-catenin, vimentin, and Mitf-M. Similar to Rad6B-inhibited cells, Rad6B edited cells were migration- and invasion-impaired. Whereas control cells produced robust tumors and overt lung metastases, the Rad6B gene edited cells were poorly tumorigenic. Rad6B gene edited clones showed complete loss of Melan A. Since Mitf-M is a transcriptional regulator of Melan A and a key player in melanocyte lineage specification, our data suggest that targeting Rad6B could potentially inhibit melanomagenesis by reprogramming melanoma cells. Supported by NIH CA178117 and 3Balls Racing.

#1150

Downregulation of Hedgehog signaling via Sirt6 activation is important to docosahexaenoic acid-induced cell death in human EGFR mutant non-small cell lung cancer.

Soyeon Jeong,1 Kaipeng Jing,1 Soyeon Shin,2 Seung-Hyeon Han,3 Yoon-Seon Yoo,3 Prashanta Silwal,3 Young-Joo Jeon,3 Jun-Young Heo,4 Gi-Ryang Kweon,4 Seung-Kiel Park,5 Jong-Il Park,5 Tong Wu,6 Kyu Lim7. 1 _Dept. of Biochemistry, Infection Signaling Network Research Center, Chungnam National University, Daejeon, Republic of Korea;_ 2 _Dept. of Biochemistry, Cancer Research Institute, Chungnam National University, Daejeon, Republic of Korea;_ 3 _Dept. of Biochemistry, Dept. of Medical Science, School of Medicine, Chungnam National University, Daejeon, Republic of Korea;_ 4 _Dept. of Biochemistry, Dept. of Medical Science, School of Medicine, Infection Signaling Network Research Center, Chungnam National University, Daejeon, Republic of Korea;_ 5 _Dept. of Biochemistry, Chungnam National University, Daejeon, Republic of Korea;_ 6 _Department of Pathology and Laboratory medicine, Tulane University School of Medicine, New Orleans, LA;_ 7 _Dept. of Biochemistry, Dept. of Medical Science, School of Medicine, Cancer Research Institute, Infection Signaling Network Research Center, Chungnam National University, Daejeon, Republic of Korea_.

Docosahexaenoic acid (DHA) induces apoptotic cell death through several mechanisms in cancer cells. Here we report that DHA-induced apoptotic cell death is related to the inhibition of Hedgehog signaling via SIRT6 activation in human PC9 and H1975 nonsmall cell lung cancer (NSCLC). DHA-induced cell death was confirmed increase of the protein levels of cleaved PARP and caspase-3 Bax as well as the number of TUNEL positive cells and the Sub-G1 proportion in PC9 and H1975. DHA significantly increased the SIRT6 levels. Knockdown of SIRT6 by siRNAs inhibited apoptosis induced by DHA, while SIRT6 overexpression increased apoptotic cell death, indicating that DHA-induced SIRT6 activation results in apoptosis. In addition, SIRT6 activation by DHA treatment was associated with downregulation of Hh signaling and knockdown of SIRT6 resulted in augmentation of Hh signaling, suggesting elevation of SIRT6 levels in DHA-treated NSCLC cells leads to downregulate the Hh signaling. Smo activator SAG increased the protein levels of Hh signaling molecules by DHA, and SAG plus DHA treatment decreased cleaved PARP levels, implying that DHA-inhibited Hh signaling is related to apoptotic cell death. To unveil the effects of endogenous DHA on apoptosis via SIRT6-mediated Hh signaling downregulation, we used PC9 stable cell lines of fat-1 (ω3-desaturase) gene. The SIRT6 levels were significantly increased and Hh signaling molecules were diminished in fat-1 stable PC9 (f-PC9) cells, compared with the cells transfected with the control vector (c-PC9). Moreover, SIRT6 expression was elevated and Gli and smo levels were inhibited in tumor tissues from f-PC9 cells-injected mice, demonstrating that DHA regulates SIRT6 and Hh signaling in vivo. Taken together, these results suggest that DHA may induce apoptotic cell death through SIRT6-modulated Hh signaling suppression in human NSCLC cells. These findings implicate that ω3-PUFAs may represent a potential effective therapy for the chemoprevention and treatment of human NSCLC. [This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (2007-0054932, NRF-2015R1D1A1A01056887) and by the framework of international cooperation program managed by National Research Foundation of Korea (2015K2A2A6002008)].

#1151

SY-1365, a potent and selective CDK7 inhibitor, exhibits promising anti-tumor activity in multiple preclinical models of aggressive solid tumors.

Shanhu Hu, Nan Ke, Yixuan Ren, Sofija Miljovska, Nisha Rajagopal, Michael McKeown, David Orlando, Kevin Sprott, Yoon J. Choi, Eric Olson, Christian C. Fritz. _Syros Pharmaceuticals, Cambridge, MA_.

CDK7 has recently emerged as an attractive target in cancer since its inhibition decreases the transcript levels of oncogenic transcription factors, especially those driven by super-enhancers (SEs). Cancers have been hypothesized to be addicted to SE regulated genes and simultaneous suppression of multiple SE associated genes through CDK7 inhibition might represent a novel, powerful way to selectively kill cancer cells. Previously, we reported that SY-1365, a highly selective covalent CDK7 inhibitor, induces apoptosis in leukemia cells, but not in non-malignant cells, and demonstrates anti-tumor activity in in vivo models of leukemia. In this study, we extend these findings to the identification of multiple solid tumors that are susceptible to SY-1365 and compare the effects of SY-1365 on gene expression to other gene control agents. SY-1365 was screened in a panel of solid tumor cell lines, revealing activity in breast, ovarian, colorectal and lung cancer cells with low nM EC50 and rapid induction of apoptosis. In breast cancer, a subset of triple negative breast cancer (TNBC) cell lines were found to be more sensitive than luminal breast cancer cell lines, so we extended our studies to in vivo models and showed substantial tumor growth inhibition in multiple patient-derived xenograft (PDX) models of TNBC. Since other compounds have been reported to modulate the expression of SE regulated genes, we compared the transcriptional effects of SY-1365 treatment with those of a pan-CDK inhibitor (flavopiridol), a CDK9 inhibitor (NVP2) and a BRD4 inhibitor (JQ1) using microarray analysis. Applying principal component analysis, we observed a unique transcriptional response elicited by SY-1365 compared to the other inhibitors. NVP2 and flavopiridol inhibited an overlapping and much larger gene set than either JQ1 or SY-1365. Interestingly, SY-1365 treatment decreased the expression of oncogenic transcription factors, cell cycle checkpoint regulators and DNA damage response genes. The downregulation of transcripts involved in apoptosis and DNA damage response by SY-1365 suggests that CDK7 inhibition might synergize with targeted agents that affect these processes. Indeed, we observed that SY-1365 was synergistic with the PARP inhibitor niraparib and the Bcl-2 inhibitor venetoclax in triple negative breast cancer and AML cell lines, respectively. In summary, we have identified TNBC, ovarian and small cell lung cancer cell lines as highly sensitive to SY-1365 in vitro and have observed substantial tumor growth inhibition in PDX models of TNBC. SY-1365 induced a distinct transcriptional response compared with other transcriptional inhibitors, with apoptotic and DNA damage pathways being central. Finally, these mechanism of action studies support a rationale for investigating combinations of SY-1365 with inhibitors of PARP and Bcl-2.

#1152

Lipid elongation: an unexplored therapeutic target in prostate cancer.

Zeyad D. Nassar,1 Margaret M. Centenera,1 Jelle Machiels,2 Samuel J. Polacek,1 Katarzyna Bloch,2 Wayne D. Tilley,1 Luke A. Selth,1 Johannes V. Swinnen,2 Lisa Butler1. 1 _University of Adelaide, Adelaide, Australia;_ 2 _KU Leuven - University of Leuven, Leuven, Belgium_.

Dysregulated lipid metabolism is one of the hallmarks of cancer, particularly for prostate cancer (PCa). PCa cells exhibit distinctive metabolic features such as upregulation of enzymes involved in de novo synthesis, uptake and beta-oxidation of lipids, which promote prostate cancer growth, metastasis, and drug resistance. Androgen signalling is a major driver of both PCa growth and lipid metabolism in PCa cells, however the precise effects of androgens on cellular lipid composition and the molecular pathways by which androgens regulate lipid metabolism in PCa cells are yet to be elucidated. In this study we investigated the effect of androgens on the lipid composition of PCa cell membranes and the enzymes involved in lipid metabolism, and explored the influence of these enzymes on tumour cell behaviour such as cell migration, proliferation and attachment. PCa cell lines (AR positive and negative) were cultured in the absence or presence of androgens or the anti-androgen enzalutamide, and changes in intact phospholipid species were assessed by ESI-MS/MS-based lipidomics. This analysis revealed a complexity of changes in phospholipid profiles in response to androgen treatment. Strikingly, elongation of the fatty acyl chains was consistently observed for multiple phospholipid classes in response to the androgens mibolerone or 5α-dihydotestosterone, whereas inhibition of elongation was observed in the presence of enzalutamide. Transcriptional analysis of critical lipid metabolism pathways revealed that the enzymes that catalyse lipid elongation (ELOVLs) were markedly induced by androgens in multiple PCa cell lines, and siRNA depletion of these enzymes, either alone or in combination, reversed the androgen-induced fatty acyl elongation phenotype. The androgenic regulation of ELOVL enzymes was confirmed in clinical PCa cohorts and in primary tumours cultured as explants. Targeting ELOVL gene expression also significantly attenuated the tumorigenic properties of PCa cells. ELOVL downregulation decreased LNCaP cell migration, and adhesion to fibronectin. Furthermore, ELOVL knock down significantly decreased three-dimensional spheroid growth of LNCaP cells using a hang drop assay. The impact of these enzymes on the lipid profile of PCa cell membrane and cell viability, adhesion and migration suggests that they may represent promising and previously unexplored therapeutic targets.

#1153

Heparan sulfate proteoglycans as novel target in cancer precise therapy.

Chiara Falciani, Jlenia Brunetti, Lorenzo Depau, Alessandro Pini, Giulia Riolo, Elisabetta Mandarini, Luisa Bracci. _University of Siena, Siena, Italy_.

Heparan sulfate proteoglycans (HSPGs) have crucial regulatory roles in tumor onset and progression. HSPGs

are composed of a core protein and glycan chains characterized by repeated disaccharide units which can

be sulfated at different amount and position. HSPG have enormous structural diversity due to the different

possible modifications of the single saccharide units within the polysaccharide, such as position, sulphation

and acetylation. As a result HSPG can bind and modulate their binding to signaling molecules such as

growth factors, morphogens and chemokines1 .

HSPG proved to be important in mediating cancer development and progression by enhancing the binding

of growth factors, morphogens and cytokines to their cognate receptors, thus activating signaling pathways

that give rise to angiogensis, cell growth and proliferation, together with invasion and metastasis 2-3.

NT4 is a branched peptides that targets HSPGs. NT4 specifically binds to sulfated glycosaminoglycans on

cancer cells and tissues. NT4 can be conjugated to many different cytotoxic units and tracers. NT4

conjugated to paclitaxel produced tumor regression in a breast cancer orthotopic mouse model 4. NT4

conjugated to tracers can discriminate between tumor and healthy tissue in different human cancer

specimen5.

We will show the ability of NT4 to drive tracers onto tumor lesions by means of Qdots and in vivo imaging,

proving their promising features as theranostics. We will also show NT4 ability to interfere with HSPG-

modulated activities such as: tumor cell proliferation, migration and invasion of matrix; as well as

endothelial cells proliferation, migration and tube formation. NT4-HSPG interactions and consequent

modulation of signaling pathways will prove the importance of this versatile tool, NT4, in addressing tumor

cells and interfering in their cell-cell and cell-matrix communications.

Bibliography

1. Gharbaran R. et al. Tumour Biol. 2016; 37:11573-11588.

2. Purushothaman A et al. Blood. 2010; 115:2449-57.

3. Lee JH et al. J Biol Chem. 2009; 284:27167-75.

4. Brunetti, J. et al. Scientific Reports. DOI:10.1038/srep17736

5. Falciani C. et al. J Med Chem. 2013; 56:5009-18.

#1154

Targeted silencing of elongation factor 2 kinase (EF2K) suppresses invasion, cell proliferation and growth in an orthotopic model of ovarian cancer.

Mumin Alper Erdogan, Bulent Ozpolat. _UT MD Anderson Cancer Center, Houston, TX_.

Ovarian cancer (OCa) is one of the most aggressive and deadliest cancer and is currently incurable disease with the poorest prognosis and survival rates (4%, 5year survival rates). One of the hallmarks of OCa is its early invasion and metastasis, resistance to chemotherapeutics and radiotherapy and aggressive tumor progression. These features contribute to a poor prognosis of this highly complex malignancy with many molecular alterations and mutations involving multiple genes. The major reason for patient death is due to metastasis and resistance to current therapies that include chemotherapy, hormonal therapy and radiation. Therefore, novel molecular targets and therapeutics strategies are urgently needed to enhance the efficacy of current therapies and prolong patient survival. Recently, we discovered that Eukaryotic elongation factor-2 kinase (EF2K), an atypical kinase, is dramatically up-regulated in cells and promotes cell survival and proliferation, its inhibition significantly reduces cell growth and colony formation. However, the role that EF2K in OCa and pathways regulating cell proliferation, colony formation, survival, invasion/metastasis and resistance remains is largely unknown. Here, we show that specific inhibition of EF2K by siRNA-mediated knockdown markedly inhibits OCa (HeyA8 and SKOV3ip1) cell proliferation, colony formation and invasion/migration in vitro. This targeting resulted in a substantial decrease in eEF2 phosphorylation in the tumors, and led to the inhibition of tumor growth and metastasis. Lack of function demonstrated that EF2K regulates Beta1-integrin, p-SRC, p-FAK, p-AKT, p-IF2alpha, p-ERK expression. Our western blot data suggested that EF2K may enhance tumorigenesis and metastasis through the upregulation of pro-tumorigenic, metastatic proteins and pathways in OCa. Furthermore, we found that therapeutic inhibition of EF2K by systemically administered (i.v) nanoliposomal EF2K siRNA inhibited growth SKOV3 tumor xenografts and intraperitoneal tumor nodules (intraperitoneal metastatic model) in nude mice (p<0.05), demonstrating the role of EF2K in tumorigenesis and metastatic phenotype. We also found that treatment with EF2K siRNA resulted in significant decrease in mean tumor weight, induced apoptosis (as judged by a positive TUNEL assay) and the downregulation of the proliferative marker Ki-67. Collectively, our data suggest, for the first time, the pivotal role of eEF2K in OCa cell proliferation, invasion and tumor progression and establish eEF2K as a novel potential therapeutic target in highly aggressive and lethal ovarian cancer.

#1155

Potent anti-cancer effects of selective GPR132/G2A agonist imipridone ONC212 in leukemia and lymphoma.

Varun Vijay Prabhu,1 Neel Madhukar,2 Rohinton Tarapore,1 Mathew Garnett,3 Ultan McDermott,3 Cyril Benes,4 Neil Charter,5 Sean Deacon,5 Wolfgang Oster,1 Michael Andreeff,6 Olivier Elemento,2 Martin Stogniew,1 Joshua Allen1. 1 _Oncoceutics, Inc, Philadelphia, PA;_ 2 _Weill Cornell Medicine, New York, NY;_ 3 _Wellcome Trust Sanger Institute, Hinxton, United Kingdom;_ 4 _Massachusetts General Hospital, Boston, MA;_ 5 _DiscoverX Corporation, Fremont, CA;_ 6 _The University of Texas M.D. Anderson Cancer Center, Houston, TX_.

ONC201, founding member of the imipridone class of compounds, is a highly selective small molecule G protein-coupled receptor (GPCR) antagonist that is in Phase I/II advanced cancer clinical trials. In this study, we evaluated GPCR engagement and antitumor activity of ONC212, an imipridone that is a chemical analogue of ONC201.

Experimental GPCR profiling using the PathHunter® β-Arrestin assay, determined that ONC212 is a selective agonist of the orphan GPCR GPR132/G2A. Multidose validation confirmed nanomolar GPR132 agonist activity (EC50 ~400nM). GPR132 is a stress-inducible orphan GPCR that causes G2/M arrest. The tumor suppressive role of GPR132 has been demonstrated most notably in lymphoid leukemia. Gene expression analysis of samples in the Cancer Genome Atlas (TCGA) revealed GPR132 is expressed in a range of tumor types with highest expression in leukemia and lymphoma.

The in vitro efficacy of ONC212 was assessed in the Genomic of Drug Sensitivity in Cancer (GDSC) collection of cell lines (>1,000 cell lines). Cell viability assays were performed at 72 hours post-treatment to generate dose responses curves at concentrations from 78nM up to 20μM. ONC212 was broadly efficacious across most solid tumors and hematological malignancies in the low nanomolar range. Ranking the sensitivity of cancer lines to ONC212 by tumor type revealed that leukemia and lymphoma are the most responsive tumor types based on completeness of response (area under the dose-response curve). Interestingly, we also observed that high expression of GPR132 significantly correlated with ONC212 efficacy in GDSC cell lines, suggesting the importance of GPR132 agonist activity for ONC212 efficacy.

ONC212 was tested in 62 human leukemia cell lines that included acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML) and hairy cell leukemia. ONC212 demonstrated broad spectrum anti-leukemic activity and was equally efficacious across all leukemia subtypes tested. Most cell lines (60/62) were responsive to ONC212 with GI50 ranging from <78nM to 456nM. Within ALL, both B-cell and T-cell ALL were highly sensitive to ONC212. ONC212 reduced cell viability in AML independent of complex karyotypes and p53 mutations that are associated with poor clinical prognoses.

ONC212 was also tested in 58 lymphoma cell lines comprised of anaplastic large cell lymphoma, Burkitt's lymphoma, cutaneous T-cell lymphoma (CTCL), diffuse large B-cell lymphoma (DLBCL), Mantle cell lymphoma (MCL), Follicular Lymphoma and Hodgkin's lymphoma. ONC212 reduced cell viability in most cell lines (56/58) and was equally efficacious regardless of subtypes with GI50 ranging from <78nM to 261nM.

Thus, GPR132 agonist ONC212 possesses robust anti-cancer activity in hematological malignancies irrespective of leukemia and lymphoma subtype and provides further validation of the anti-cancer efficacy of the novel imipridone class of small molecules.

### Novel Molecular Targets 1

#1156

Epigenetic re-wiring of breast cancer by pharmacological targeting of C-terminal binding protein.

Jung S. Byun, Samson Park, Dae IK Yi, Mohamed Kabbout, Genqing Liang, Kevin L. Gardner. _National Institutes of Health, Bethesda, MD_.

The C-terminal binding protein (CtBP) is a family of dimeric nuclear proteins whose levels are increased in cancers of the colon, ovaries, prostate and breast. Elevated CtBP expression is associated with poor cancer survival and can also distinguish those node negative breast cancer patients who will show worse survival. This implicates CtBP as both a biomarker and a promising candidate for therapeutic intervention. As a dimer, CtBP provides a scaffold that couples multiple different DNA-binding transcriptional regulators with a variety of chromatin modifying protein complexes to alter the epigenetic landscape throughout the nucleus. These properties provide the rationale for pharmacological targeting of CtBP to change epigenetically regulated gene expression in cancer cells. In this study, we employ computer assisted drug design to screen for optimal quantitative structure-activity relationships (QSARs) between small molecules and CtBP to identify 24 potential CtBP inhibitors. Functional screening of these compounds identifies 4 lead compounds with low toxicity and high water solubility. Treatment of breast cancer cells at micro-molar concentrations of these small molecular inhibitors induces significant de-repression of epigenetically silenced pro-epithelial genes in the mesenchymal, triple negative breast cancer cell line, MDA-MB-231. This re-activation is associated with eviction of CtBP from the respective gene promoters, disrupted recruitment of CtBP-chromatin modifying protein complexes, increased deposition of activating epigenetic histone marks, and upregulation of both pro-epithelial gene mRNA and protein expression. In functional assays, CtBP inhibition by these small molecular inhibitors decreases cellular invasion, and improves DNA repair. FRET (Förster resonance energy transfer) analysis demonstrates that CTBP inhibition results in decreased FRET intensity, suggesting that CTBP dimerization is repressed by CTBP inhibition. In addition, pharmacological inhibition of CtBP combines with established epigenetically targeted drugs to synergistically decrease cell migration and potentiate the reactivation of silenced pro-epithelial gene expression in triple negative cancer cells. Finally, CTBP inhibition results in transcriptional repression of MDR1 expression and reduces the population of Doxorubicin resistant cells in the triple negative breast cancer cell lines. These findings implicate the possible use of this class of compounds in strategies for therapeutic intervention that may increase the efficacy and decrease the acquired resistance to targeted therapeutic intervention in breast cancer.

#1157

Screening and identification of new generation glucose transporter inhibitors as anticancer therapeutics.

Yanrong Qian, Pratik Shriwas, Xuan Wang, Dennis Roberts, Emma Kessler, Stephen Bergmeier, Xiaozhuo Chen. _Ohio Univ., Athens, OH_.

Cancer is a top killer second only to cardiovascular diseases. Cellular metabolic reprogramming and altered energetics are key features of cancer. Opportunistic uptake of extracellular nutrients has recently been named as an emerging hallmark of cancer metabolism. Glucose is a key nutrient taken in by cancer cells for satisfying their high energy and biosynthesis needs. Drastic upregulation of glucose uptake and glycolytic activity, a phenomenon called the Warburg effect, is prevalent in cancers. Cancer cells overexpress specific glucose transporters (Gluts), particularly Glut1, on cell membranes for taking in glucose via facilitated diffusion. Considering the addiction of cancer cells for glucose and their sensitivity and vulnerability to changes in glucose supply, Gluts, especially Glut1, have been recognized as an attractive anticancer target. We previously reported the identification of first generation lead Glut1 inhibitor WZB117 (1). WZB117 treatment of human nonsmall cell lung cancer (NSCLC) A549 cells resulted in inhibition of glucose uptake, glycolysis and induction of apoptosis and necrosis. In addition, WZB-117 reduced tumor growth by ~70% in an A549 tumor mouse model. WZB117 inhibit cell growth of 7 cancer cell lines in 4 cancer types with IC50 values lower than 10 μM. Through a structure activity relationship (SAR) study of WZB117, a second generation lead, DRB18, has been identified. DRB-18 is much more stable and 5 to 10 times more potent than WZB117 in inhibiting growth and proliferation in about 90% of the 60 different cancer cell lines in 9 major cancer types as revealed in NCI-60 screenings. And its IC50s in many cancer cell lines are in the high nM range. In this study, we also report new compounds screening results for the objective of identifying novel compounds with higher glucose transport-inhibitory and cancer cell growth-inhibitory activities using nonsmall cell lung cancer cell lines as models. References: 1. Liu, Y., Cao, Y., Zhang, W., Bergmeier, S., Qian, Y., Akbar, H., Colvin, R., Ding, J., Tong, L., Wu, S. and Hines, J., 2012. A small-molecule inhibitor of glucose transporter 1 downregulates glycolysis, induces cell-cycle arrest, and inhibits cancer cell growth in vitro and in vivo. Molecular cancer therapeutics, 11(8):1672-1682.

#1158

Modulation of splicing by inhibiting the kinase SRPK1 as a novel therapeutic strategy in myeloid leukemia.

Konstantinos Tzelepis,1 Etienne De Braekeleer,1 Michael Seiler,2 Isaia Barbieri,3 Sam Robson,3 Yu Hsuen Yang,4 Malgorzata Gozdecka,1 Monika Dudek,1 Grace Collord,1 Oliver M. Dovey,1 Emmanouil Metzakopian,1 Dimitrios Garyfallos,1 Jonathan L. Cooper,1 Silvia Buonamici,2 Hannes Ponstingl,1 Michael R. Stratton,1 Allan Bradley,1 Brian J. Huntly,5 Cristina Pina,6 Tony Kouzarides,3 Kosuke Yusa,1 George S. Vassiliou1. 1 _Sanger Institute, Cambridge, United Kingdom;_ 2 _H3 Biomedicine, Boston, MA;_ 3 _Gurdon Institute, Cambridge, United Kingdom;_ 4 _University College of London, London, United Kingdom;_ 5 _Cambridge University Hospitals NHS Trust, Cambridge, United Kingdom;_ 6 _NHS Blood and Transplant, Cambridge Biomedical Campus, Cambridge, United Kingdom_.

Acute myeloid leukemia (AML) is an aggressive cancer with a poor prognosis, for which the therapeutic landscape has changed little for decades. Aberrant mRNA splicing plays a key role in cancer development and genes coding for several of the major components of the spliceosome are targeted by somatic mutations in numerous cancers including myelodysplastic syndromes and AML. Recently, myeloid neoplasms bearing spliceosome gene mutations were shown to be preferentially susceptible to pharmacological disruption of the spliceosome. Here we report that targeting the spliceosome can also be an effective therapeutic strategy in other types of AML.

Recently, we generated a comprehensive catalogue of genetic vulnerabilities in AML using CRISPR-Cas9 genome-wide recessive screens and reported several novel intuitive and non-intuitive therapeutic candidates. Amongst these we identify SRPK1, the gene coding for a serine-threonine kinase that phosphorylates the major spliceosome protein SRSF1. Here, we demonstrate that targeted genetic disruption of SRPK1 in AML driven by MLL-fusion genes, led to differentiation and apoptosis. Additionally, mice transplanted with human AML cell lines carrying the MLL-AF9 fusion gene, namely MOLM-13 and THP-1, presented a significant prolongation of survival when SRPK1 was genetically disrupted by CRISPR-Cas9 editing. Similar effects were seen with pharmacological inhibition of SRPK1 in vitro and in vivo. At the molecular level we show that genetic or pharmacological inhibition of SRPK1 was associated with profound changes in the splicing of multiple genes involved in the MLL leukemogenic program in association with significant changes in enzymatic modifications of core histone tails.

We proceeded to perform a genome-wide CRISPR drop-out screen for sensitizers of MOLM13 cells to pharmacological inhibition of SRPK1 and identified, amongst other genes, BRD4 as a sensitizer. We go on to show that the BRD inhibitor iBET-151 synergizes with SRPK1 inhibition to kill MOLM-13 both in vitro and in vivo. Preliminary data indicates that SRPK1 inhibition has overlapping molecular effects to BRD inhibition. We are currently investigating the molecular bases of this observation.

Our work identifies SRPK1 as a novel therapeutic target in AML that can be used alone or in conjunctions with drugs targeting epigenetic modifications to improve their anti-leukemic effects.

#1159

Targeting novel mucin synthesis pathway for pancreatic cancer treatment.

Altaf Mohammed, Naveena B. Janakiram, Venkateshwar Madka, Gaurav Kumar, Scott Edgar, Gopal Pathuri, Taylor Bryant, Yuting Zhang, Hariprasad Gali, Yan Daniel Zhao, Stan Lightfoot, Chinthalapally V. Rao. _Univ. of Oklahoma Health Sciences Ctr., Oklahoma City, OK_.

Pancreatic cancer (PC) is most fatal and is the fourth leading cause of cancer related deaths in USA with a least five year survival of <7%. Although several overexpressed mucin subtypes (MUC1, MUC4, MUC5AC, and MUC16) that impede drug delivery to PC have been targeted with limited results, core enzymes involved in mucin biosynthesis have yet to be evaluated as potential targets. The core 2 β 1,6 N-acetylglucosaminyltransferase (GCNT3/C2GNT) plays a significant role in mucin glycan biosynthesis. We analyzed GCNT3 expression on 180 specimens from individuals with PC (90) or matched controls (90). Patients with low GCNT3 expression survived longer than patients with high GCNT3 expression (median survival: 17.5 vs. 10.5 months, p=0.036). We observed a significant increase in GCNT3 mRNA expression (103.16 folds; p<0.0001) in correlation with increased mucins subtypes (p<0.05) in the pancreatic tumors from genetically engineered mouse (GEM) compared with pancreata from wild-type mice, as determined by Next-generation Sequencing/RNAseq analysis. In in silico studies, blind docking simulations revealed that a novel drug talniflumate binds to GCNT3 with a better docking affinity of -8.3 kcal/mol. We synthesized talniflumate and determined quality/purity using NMR, HRMS, and HPLC for in vitro and in vivo studies. Pancreata from 6-week-old Kras GEM (N=6/group) treated with talniflumate showed a significant decrease in GCNT3 and mucin expression in PanIN lesions. In human PC MiaPaCa cell line, talniflumate reduced GCNT3 expression and the number of mucin-positive cells (38% p<0.0001). Further, PC cells (BxPc3 and Panc1) were treated with talniflumate for 24 hrs for mucin disruption followed by radioactive 3H gemcitabine treatment for 2 hrs. The cells treated with talniflumate showed 40% (p<0.03) and 50% (p<0.025) increased 3H gemcitabine uptake in BxPc3 and Panc1 cells compared to respective controls.

GCNT3 CRISPR knock out PC cells and talniflumate treated PC cells showed a reduction in cell proliferation and significantly reduced spheroid formation (p<0.05). In in vivo, we did not observe any overt toxicity upon talniflumate treatment as determined by liver enzyme toxicity and body weight gain (N=5 mice/group). BxPC3 cells implanted nude mice (N=5 mice/group) treated with talniflumate or GCNT3 siRNA showed a marked reduction in xenograft tumors compared with untreated controls. Both talniflumate and GCNT3 siRNA significantly inhibited xenograft tumor weight (48% p<0.002 with siRNA, 63% p<0.0001 with talniflumate) and tumor volume (75% p<0.0001 with both siRNA and talniflumate) and GCNT3 enzyme activity (28% p<0.03). Transcriptome analysis of xenograft tumors (N=3/group) treated with GCNT3 SiRNA or talniflumate showed reduced GCNT3 and top hits related to mucin expression. These results justify the use of talniflumate for mucin synthesis disruption, thereby enhancing the uptake of gemcitabine and thereby efficacy for PC treatment.

#1161

Dimethylaminoparthenolide (DMAPT), an oral nuclear factor kappa B inhibitor (NFκB), enhances radiation therapy and enhances epidermal growth factor tyrosine kinase inhibitor (EGFR TKI) activity.

Katherine L. Morel,1 Pamela J. Sykes,1 Prafulla C. Gokhale,2 Gwo-Shu Mary Lee,2 Hong Tiv,2 Christopher J. Sweeney2. 1 _Flinders University, Bedford Park, Australia;_ 2 _Dana-Farber Cancer Institute, Boston, MA_.

Introduction: NFκB can promote cancer and resistance to therapy as well as mediate tissue injury in response to radiation (XRT). We assessed DMAPT efficacy alone and in combination with XRT, and also with an EGFR TKI, and assessed whether it can mitigate XRT side effects.

Methods: Six-week old male TRAMP (Transgenic Adenocarcinoma of the Mouse Prostate) mice received DMAPT (100 mg/kg) orally or vehicle control 3x/week until palpable tumors formed and lung metastases were analyzed histologically. In separate studies, 16 week old TRAMP mice were treated with 100 mg/kg DMAPT 3x over 1 week before 6 Gy whole-body XRT. Cancer and normal tissues were analyzed for apoptosis for up to 72 hours post-XRT. NCr nude mice were inoculated with H1975 (T790M mutation non small cell lung cancer, NSCLC) subcutaneously. When tumors reached 200 mm3, treatment (Rx) commenced: vehicle, DMAPT 100mg/kg/day, EGF TKI-AZD9291 25mg/kg/day, combination.

Results: Long-term Rx with DMAPT extended the median time to palpable prostate tumor by 41.3 (p = 0.0013) days. Chronic DMAPT Rx reduced the number of metastatic lesions/mm2 in TRAMP lungs 20-fold (0.077 ± 0.12 SD) compared to a vehicle control (1.47 ± 1.28 SD) (p = 0.0004). XRT-induced apoptosis doubled in TRAMP prostates (with moderate to high grade PIN lesions) treated with DMAPT prior to 6 Gy XRT (101.3 % increase, p = 0.039). DMAPT induced the greatest radiosensitivity in TRAMP prostates with higher grade PIN (R2 = 0.79, p = 0.0001), while apoptosis frequency in tissues with lower grades of PIN was the same as vehicle control TRAMP mice (R2 = 0.024, p = 0.3). DMAPT also reduced XRT-induced apoptosis in healthy TRAMP spleen (32.9 % reduction, p = 0.003) and rectum (28.7 % reduction, p = 0.0001). In the H1975 experiment, DMAPT monotherapy did not differ from the vehicle controls. In the single agent AZD9291 group, 2 of the 8 mice had resistance emerge during Rx and adding DMAPT to AZD9291 reversed resistance in one of these. Rx was held at Day 220 in 3 remaining AZD9291 treated mice with no evidence of tumor and only 1 mouse was alive with no tumor at day 260. For mice receiving Rx with AZD9291 and DMAPT from D1, resistance emerged in 1 mouse, and at D140 all remaining mice with no evidence of tumor had Rx stopped, and 3 mice were still tumor free at D260. Two mice had regrowth, were retreated, and at D200 without evidence of tumors had Rx held, and one had no tumor at D260 at the end of the experiment. In total, 4 of 8 mice in the AZD9291 and DMAPT had no tumor off therapy at D260.

Conclusion: Radiation and EGFR TKI resistance has been linked to NFκB activation. DMAPT slows down cancer progression and decreases metastatic lesions in a prostate cancer mouse model, protects normal tissue from radiation induced apoptosis, augments radiation induced apoptosis in prostate cancer and augments EGFR TKI efficacy in T790M mutant NSCLC.

#1162

The evaluation of INCB059872, an FAD-directed covalent inhibitor of LSD1, in preclinical models of Ewing sarcoma.

Valerie Dostalik Roman, Min Ye, Huiqing Liu, Melody Diamond, Antony Chadderton, Yvonne Lo, Xuesong M. Liu, Jin Lu, Chunhong He, Liangxing Wu, Timothy Burn, Richard Wynn, Wenqing Yao, Gregory Hollis, Gregory Hollis, Peggy Scherle, Bruce Ruggeri, Sang Hyun Lee. _Incyte Corporation, Wilmington, DE_.

Ewing sarcoma is a rare bone cancer affecting predominantly children. The chromosomal translocation of chromosomes 11 and 22 results in the EWS/FLI gene fusion oncoprotein that is associated with ~85% of Ewing sarcoma cases. The EWS/FLI fusion protein is involved in deregulating gene expression and consequently causing cellular transformation. It was previously reported that lysine specific demethylase 1 (LSD1) regulates EWS/FLI transcriptional activity via its functional interaction through the NuRD co-repressor complex. We therefore evaluated whether inhibition of LSD1 could have anti-tumor effects in Ewing sarcomas that express the EWS/FLI fusion oncoprotein. INCB059872 is a potent, selective, and orally available FAD-directed covalent inhibitor of LSD1. To investigate the potential utility of INCB059872 in Ewing sarcoma, the A673 cell line having the characteristic chromosomal translocation was chosen as the experimental model system. INCB059872 inhibition of LSD1 did not significantly alter A673 proliferation in vitro. However, INCB059872 inhibited oncogenic transformation as determined by colony formation clonogenicity assays. NKX.2.2 was previously identified as a critical downstream target molecule of the EWS-FLI fusion oncoprotein that is required for transformation. A significant downregulation of NKX2.2 was observed in A673 cells treated with INCB059872, suggesting that INCB059872 mediates its effects through modulation of the EWS/FLI -NKX2.2 axis. Oral administration of INCB059872 significantly suppressed the growth of both A673 and SK-ES Ewing sarcoma xenografts in vivo. In addition, in vivo efficacy was evaluated in patient derived xenograft (PDX) models that were developed from relapsed tumor tissues of Ewing sarcoma patients. Notably, a subset of PDX models having EWS/FLI translocations (3/6) exhibited significant tumor growth inhibition at well-tolerated doses of INCB059872. Molecular signatures obtained from RNA-Seq data with these PDX models exhibited intrinsic differences between responders and non-responders, suggesting additional molecular or genetic variations may contribute to their sensitivity to INCB059872. Studies identifying potential candidate molecular mechanisms are underway. Together, these data suggest that INCB059872 may be therapeutically efficacious in a subset of Ewing sarcoma patients.

#1163

Non clinical pharmacology, pharmacokinetics and safety profiling of CB-103: A novel first-in-class small molecule inhibitor of the NOTCH pathway.

Rajwinder Lehal,1 Viktoras Frismantas,2 Charlotte Urech,1 Maximilien Murone,1 Dirk Weber,1 Michael Bauer,1 Jean-Pierre Bourquin,2 Freddy Radtke3. 1 _Cellestia Biotech AG, Basel, Switzerland;_ 2 _Univeristy Children's Hospital, Zurich, Switzerland;_ 3 _Ecole Polytechnique Federale de Lausanne, Lausanne, Switzerland_.

NOTCH signaling is a developmental pathway known to play critical roles during embryonic development as well as for the regulation of self-renewing tissues. Aberrant activation of NOTCH signaling leads to deregulation of the self-renewal process resulting in sustained proliferation, evasion of cell death, loss of differentiation capacity, invasion and metastasis, all of which are hallmarks of cancer. Over activation of NOTCH in human cancers can be a consequence of over expression of NOTCH ligands/receptors, GOF mutations in NOTCH receptors as well as chromosomal translocations leading to constitutive activation of the pathway by producing truncated version of NOTCH1 or NOTCH2. Over activation of NOTCH and its oncogenic role in various cancers is prognostically relevant with shorter survival seen in patients harbouring these genetic alterations. Given the importance of Notch signaling in human cancers, several therapeutic approaches have been utilized to block NOTCH signaling. Two of these strategies are; a) the use of monoclonal blocking antibodies (mAbs) against NOTCH ligands and receptors and b) the use of small molecule γ-secretase inhibitors (GSIs). However, these approaches can only be effective if tumor cells express full-length ligand or receptor molecules. On the contrary, in human cancers harbouring NOTCH gene fusion due to chromosomal translocations, the use of mAbs and GSIs will have very limited clinical benefits. A third, yet not fully explored approach would be the blockage of NOTCH signalling by targeting the most downstream complex in the NOTCH signal transduction cascade, the NOTCH transcriptional activation complex, using small molecule inhibitors.

Previously we have reported the discovery and characterization of CB-103, a novel first-in-class orally-active small molecule inhibitor of the NOTCH pathway. CB-103 inhibits NOTCH signaling by targeting the NOTCH transcriptional activation complex in the nucleus. CB-103 has shown the ability to block NOTCH signalling in various human cancer cell lines with active NOTCH pathway. CB-103 has also been tested in in vivo models. In a GSI/mAb resistant model of human triple negative breast cancer, CB-103 has demonstrated excellent anti-tumor efficacy. In this study we present additional pharmacology, pharmacokinetics data and provide an overview of the good safety and tolerability seen with CB-103 in the non-clinical studies. Based on these findings CB-103 has been selected as clinical candidate to be investigated in a FIM, phase I/IIA clinical study in advanced cancer patients.

#1164

A novel hypoxia-inducible factor-1 inhibitor IDF-11774 regulates cancer metabolism, thereby suppressing tumor growth.

Misun Won,1 Hyun Seung Ban,1 Kyeong Lee,2 Hongsub Lee,3 Bo-Kyung Kim,1 Hwan Mook Kim,4 Ravi Naik,2 Song-Kyu Park,5 Joon-Tae Park,3 Inhyub Kim,1 Miso Nam,6 Geum-Sook Hwang6. 1 _Korea Research Inst. of Biosci. & Biotech., Daejeon, Republic of Korea; _2 _Dongguk University-Seoul, Republic of Korea;_ 3 _ILDONG Pharmaceutical Co. Ltd, Republic of Korea;_ 4 _Gachon University, Republic of Korea;_ 5 _Korea University, Republic of Korea;_ 6 _Korea Basic Science Institute, Daejeon, Republic of Korea_.

HIF-1 is associated with poor patient prognosis and therapeutic resistance of cancer. We have developed a novel hypoxia-inducible factor (HIF)-1 inhibitor, IDF-11774, as a clinical candidate for cancer therapy. Under hypoxic condition, IDF-11774 inhibited the accumulation of HIF-1α in vitro and in vivo in colorectal carcinoma HCT116 cells. IDF-11774 suppressed the angiogenesis of cancer cells by reducing the expression of HIF-1 target genes. Moreover, IDF-11774 reduced glucose uptake, leading sensitizing cell growth on low glucose condition. IDF-11774 also decreased the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). Metabolic profile of IDF-11774-treated cells revealed low levels of NAD+, lactate, and intermediates in glycolysis and tricarboxylic acid (TCA) cycle. More importantly, we observed elevated AMP and diminished ATP level, leading high AMP/ATP ratio. Apparently, phosphorylation of AMPK increased, leading inhibition of mTOR signaling in cells. In vivo xenograft assays demonstrated that IDF11774 has significant anti-cancer efficacy by targeting cancer metabolism in mouse models containing the KRAS, PTEN or VHL mutation, which often occurs in many malignant cancers. Collectively, IDF-11774 suppresses the hypoxia-induced HIF-1α accumulation, thereby repressing tumor growth by regulating cancer metabolism.

#1165

Plitidepsin targets the moonlighting functions of eEF1A2 in cancer cells.

Alejandro Losada, Maria Jose Muñoz, Juan F. Martínez-Leal, Juan M. Domínguez, Carlos M. Galmarini. _PharmaMar S.A., Colmenar Viejo, Spain_.

Plitidepsin, a cyclic depsipeptide of marine origin, has shown potent anticancer activity in preclinical assays and recently finished with positive results a pivotal phase III trial (clinicaltrials.gov identifier: NCT01102426) for the treatment of multiple myeloma patients. We have recently found that eukaryotic elongation factor 1A2 (eEF1A2), one of the two isoforms of the alpha subunit of eEF1, is the pharmacological target of plitidepsin. Although it shares 96% homology with eEF1A1 (the other isoform), they display an exclusive pattern of expression, being eEF1A2 solely expressed in brain and muscle in healthy individuals. However, it has been found that many tumors abnormally overexpress this protein, including multiple myeloma, prostate, pancreatic, ovarian, breast, lung and liver cancers. Furthermore, although eEF1A2 canonical function consists in the delivery of aminoacyl-tRNAs to the A site in the ribosome, it has been shown to have pro-oncogenic moonlighting activities, including inhibition of apoptosis, protein degradation by the proteasome, heat shock response, cytoskeleton organization and regulation of oxidative stress. We now investigated several of the pro-oncogenic activities of eEF1A2 to analyze the impact that plitidepsin could have preventing them. Indeed, we observed that plitidepsin interfered with the interaction between eEF1A2 and Peroxiredoxin 1 (PRDX1), a complex that allosterically enhances the enzymatic activity of PRDX1. This way, plitidepsin would diminish PRDX1 antioxidant activity, possibly originating the oxidative stress that has been described in the bibliography as one of the first effects triggered by the drug in cancer cells. PRDX1 only interacts with the GDP-bound form of eEF1A2, while plitidepsin exclusively binds to the GTP-bound form, most probably sequestering this protein from the pool that could interact with and activate PRDX1. Furthermore, we have confirmed that eEF1A2 interacts with Sphingosine kinase 1 (SPHK1), a complex that has been described in the bibliography as having enhanced SPHK1 activity. SPHK1 phosphorylates sphingosine producing sphingosine-1-P, second messenger that binds to its receptors in the cell membrane and conveys growth and survival signals to the cell. We could see that plitidepsin treatment reduced the production of sphingosine-1-P in HeLa cells, destabilizing the equilibrium towards the pro-apoptotic ceramide/sphingosine side and promoting cell death. Thus, through its binding to eEF1A2, plitidepsin derails a series of its moonlighting functions that are essential for the survival of tumor cells, driving them into apoptosis.

#1166

Identification of a novel quinoxaline-isoselenourea targeting STAT3 pathway as a potential melanoma therapeutic.

Verónica Alcolea,1 Deepkamal Karelia,2 Manoj K. Pandey,3 Daniel Plano,1 Parvesh Singh,4 Rosalyn B. Irby,2 Juan Palop,1 Shantu Amin,2 Carmen Sanmartín,1 Arun K. Sharma2. 1 _Univesrsity of Navarra, Pamplona, Spain;_ 2 _Penn State Univ. College of Medicine, Hershey, PA;_ 3 _Cooper Medical College of Rowan University, Camden, NJ;_ 4 _University of Kwa-Zulu Natal, Durban, South Africa_.

The prognosis for patients with metastatic melanoma remains very poor. Constitutive STAT3 activation has been correlated to larger tumor size, metastasis, acquired resistance against vemurafenib (PLX-4032), and poor patient survival suggesting its potential as a molecular target. We recently designed a series of isoseleno- and isothio-urea derivatives of several biologically active heterocyclic scaffolds. The cytotoxic effects of lead isoseleno-/isothio-urea derivatives (compounds 1/3) were studied in a panel of five melanoma cell lines, including B-RAFV600E mutant and wild type (WT) cells. Compound 1 (IC50 range 0.8-3.8 µM) showed lower IC50 values than 3 (IC50 range 8.1-38.7 µM) and the mutant B-RAF specific inhibitor PLX-4032 (IC50 range 0.4->50 µM), especially at shorter treatment time (24 h). These effects are long-lasting since melanoma cells did not recover their proliferative potential after 14 days of the treatment. In addition, we confirmed that compound 1 induces cell death by apoptosis using Live and Dead, Annexin V and Caspase3/7 apoptosis assays. Furthermore, compound 1 reduces protein levels of STAT3 and its phosphorylation, as well as decreases the expression of STAT3 regulated genes involved in survival and metastasis such as survivin and c-myc. Compound 1 also upregulates the cell cycle inhibitor p21. Docking studies further revealed the favorable binding of 1 with SH2 domain of STAT3 suggesting its activity through STAT3 inhibition. Taken together, our results suggest that compound 1 induces apoptosis by means of the inhibition of STAT3 pathway to non-specifically target both B-RAF mutant and WT melanoma cells with much better cytotoxicity than the current therapy PLX-4032.

#1167

Development of BT1718, a novel Bicycle Drug Conjugate for the treatment of lung cancer.

Gavin Bennett, Robert Lutz, Peter Park, Helen Harrison, Kevin Lee. _Bicycle Therapeutics Ltd, Cambridge, United Kingdom_.

BT1718 is a Bicycle Drug Conjugate (BDC®) comprising a constrained bicyclic peptide (Bicycle®) that binds with high affinity and specificity to membrane type 1-matrix metalloprotease (MT1-MMP; MMP14) covalently linked through a hindered disulfide linker to the potent anti-tubulin agent DM1. MT1-MMP is involved in normal tissue remodeling and is also expressed in tumor associated stromal cells. However, overexpression is linked to increased tumor aggression and metastasis. Specifically, over-expression of MT1-MMP is also associated with poor clinical prognosis and shorter survival times in patients with NSCLC and a range of other solid tumors. The Bicycle MT1-MMP binding element within BT1718 was identified using a proprietary phage display peptide technology consisting of highly diverse phage libraries of linear amino acid sequences constrained into two loops by a central tri-functional chemical scaffold. These MT1-MMP binding Bicycles exhibit a profound affinity and specificity, more often associated with monoclonal antibodies, whilst their low molecular weight (1.5-3 kDa), akin to that of a small molecule, aids in rapid extravasation and tumor penetration. Together these attributes make Bicycles an ideal format for the targeted delivery of cytotoxic payloads. We evaluated the ability of BT1718 to bind to and kill tumor cells in vitro and in vivo in a panel of tumor cells. BT1718 demonstrated MT1-MMP target-specific binding and MT1-MMP-dependent cell killing of lung tumor cells in vitro as well as efficacy across a panel of lung tumor xenograft mouse models. For example, in the Met-amplified squamous NSCLC lung EBC-1 model, complete regressions were observed in all mice at doses as low as 5 mg/kg (iv) twice weekly and across a range of other dosing schedules, from daily to weekly. MT1-MMP-dependent activity was demonstrated by blocking target specific interactions through co-administration of an excess of unconjugated Bicycle binder, which inhibited tumour regression, or a non-binding Bicycle, which had no effect. Further evaluation in patient-derived lung xenograft (PDX) models indicates a similar activity to that seen in cell-line derived xenografts, with efficacy seen from 3mg/kg twice weekly and rapid full regression of tumors at higher doses. The molecular attributes of these Bicycles: rapid tumor penetration and specific binding, makes them ideal therapeutics for targeted delivery of toxins as Bicycle drug conjugates (BDCs). The small size of the BDC may offer a significant advantage to other targeted cytotoxic approaches such as antibody-drug conjugates due to rapid extravasation and improved tumor penetration. BT1718, a Bicycle Drug Conjugate, shows potent anti-tumor activity in human lung tumor xenograft models and IND-enabling studies are underway.

#1168

Efficacy of the IRAK4 inhibitor CA-4948 in patient-derived xenograft models of diffuse large B cell lymphoma.

Robert N. Booher, Maria Elena Samson, Guang-Xin Xu, Hongsheng Cheng, David P. Tuck. _Curis, Inc, Lexington, MA_.

IRAK4 kinase activity is required for toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling in a variety of myeloid and lymphoid cell types. Recruitment of IRAK4 to these receptors and its subsequent activation is facilitated by the MYD88 adaptor protein, which is mutated in ~22% of DLBCL cases. The MYD88 L265P activating mutation is found in ~30% of the activated B-cell (ABC) and ~6% of germinal center B-cell (GCB) subtypes of DLBCL and leads to constitutive activation of NF-κB signaling that is associated with worse prognosis. Thus, the development of small molecule inhibitors targeting IRAK4 is an attractive anticancer strategy for MYD88 mutation-containing cancers such as DLBCL.

We are developing an IRAK4 inhibitor, CA-4948, as a therapeutic agent for hematological cancers with dysregulated TLR/MYD88/IRAK4 signaling. CA-4948 (previously AU-4948) is a selective and potent IRAK4 kinase inhibitor with in vivo activity in a TLR4-induced cytokine release model. CA-4948 exhibits favorable DMPK properties, oral bioavailability, and is well tolerated in mice. Furthermore, CA-4948 was previously shown to exhibit dose-dependent efficacy in ABC-DLBCL MYD88-L265P xenograft tumor models using cell lines OCI-LY3 and OCI-LY10.

Here, we report the efficacy results from testing CA-4948 in a panel of well characterized, patient-derived DLBCL tumor xenograft (PDX) mouse models. CA-4948 exhibited the greatest efficacy in four of the five ABC-DLBCL PDX models tested as compared to GBC-DLBCL and ABC/GCB DLBCL PDX models. Furthermore, CA-4948 was efficacious in ABC-DLBCL PDX tumors containing activating mutations in both TLR/IL-1R and BCR signaling pathways (MYD88 and CD79B double mutants). Interestingly, the one ABC-DLBCL PDX model that failed to respond to CA-4948 treatment contained a MYD88 L265P mutation as well as a BCL6 translocation. While this particular PDX model was resistant to CA-4948, and showed a weak anti-tumor response to single-agent ibrutinib, the combination treatment of ibrutinib and CA-4948 exhibited a synergistic tumor growth inhibition effect. In summary, CA-4948 exhibited anti-tumor activity in ABC-type DLCBL PDX tumor models including those containing combinations of activating mutations in the TLR/IL-1R and BCR signaling pathways. These results underscore the therapeutic potential of IRAK4 kinase inhibition by CA-4948, as a single-agent or in combination with BCR inhibitors, for the treatment of DLBCL.

#1169

mtTERT promoter as a target for treatment of glioblastoma.

Saumya R. Bollam,1 Harshil D. Dhruv,1 Hyun-Jin Kang,2 Sen Peng,1 Vijay Gokhale,2 Laurence Hurley,2 Michael Berens1. 1 _Translational Genomics Research Institute, Phoenix, AZ;_ 2 _University of Arizona, Tuscon, AZ_.

Approximately 86% of GBM tumors exhibit mutation at -124 or -146 bases upstream of the ATG start site in the transcription activating promoter region of Human telomerase reverse transcriptase (hTERT). Mutation in the promoter region of hTERT impairs repression, leading to overexpression of hTERT; inappropriate hTERT is associated with oncogenesis, tumor maintenance, and resistance to apoptosis. We surveyed long-term glioma cell lines and glioma PDX models for mt-hTERT; mRNA and protein expression of hTERT were assessed by qPCR and western blot. The -124 and -146 mutations are located in the major 5-12 G-quadruplex and result in misfolding of the silencer element and consequent over-expression of hTERT. Using a diverse small molecule library, we identified a small drug-like pharmacological chaperone (pharmacoperone) molecule, TG-4260, which binds to the 26 mer base-pair heteroduplex loop, which is the nucleation site for cooperative folding of the major 5-12 G-quadruplex. The chaperone effect of TG-4260 corrects DNA hTERT G-quadruplex misfolding resulting from the somatic mutations and restores the silencer function of the G-quadruplex thereby reducing hTERT activity. TG-4260 directly decreases the transcription activity of the WT and the −124, −124/125, −138/139, and −146 mutants to a similar extent and suppresses the downstream gene BCL2, which activates caspase-3 and produces cell-cycle arrest, leading to cell death. Finally, TG-4260 significantly inhibits telomerase and shortens telomere length after five days of treatment and induces a senescence-like phenotype. This is the first example of the use of a pharmacoperone molecule to correct the misfolding of a DNA G-quadruplex element resulting from mutations in an early folding intermediate. Finally, we screened GBM cell models against a novel small molecule inhibitor that interferes with mutated hTERT promoter and demonstrated that TG-4260 selectively suppresses glioma cell viability without affecting non-transformed normal human astrocytes.

#1170

Notch pathway is overexpressed and is a therapeutic target in clear cell renal cancer.

Tushar D. Bhagat,1 Yiyu Zou,1 Shizeng Huang,2 Jihwan Park,2 Matthew B. Palmer,2 Caroline Hu,1 Weijuan Li,1 Niraj Shenoy,1 Orsolya Giricz,1 Yiting Yu,1 Yi-An Ko,2 María Concepción Izquierdo,2 Esther Park,2 Nishanth Vallumsetla,1 Remi Laurence,1 Robert Lopez,1 Masako Suzuki,1 James Pullman,3 Justin Kaner,1 Benjamin Gartrell,1 A. Ari Hakimi,4 John Greally,1 Bharvin Patel,5 Karim Benhadji,5 Kith Pradhan,1 Amit Verma,1 Katalin Susztak2. 1 _Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, NY;_ 2 _University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA;_ 3 _Montefiore Medical Center, Bronx, NY;_ 4 _Sloan Kettering Cancer Center, New York, NY;_ 5 _Eli Lilly, Indianapolis, IN_.

Clear cell renal cell carcinoma (CCRCC) is an incurable malignancy in advanced stages and needs newer therapeutic targets. We conducted a transcriptomic analysis of CCRCCs and matched microdissected renal tubular controls and observed an overexpression of NOTCH ligands [JAGGED1, JAGGED2) and Delta like (DLL3) family of ligands] and receptors (NOTCH1, NOTCH2, NOTCH3 and NOTCH4) in tumor tissues. Examination of the TCGA RNA-Seq dataset also revealed widespread activation of NOTCH pathway in a large cohort of CCRCC samples. Samples with NOTCH pathway activation were also clinically distinct and were associated with better overall survival.

Parallel high resolution DNA methylation and copy number analysis with the HELP assay demonstrated that both genetic and epigenetic alterations led to NOTCH pathway activation in CCRCC. NOTCH ligands, JAGGED1 was overexpressed and associated with loss of methylation of its intronic enhancer. The other NOTCH ligand, JAGGED2 was overexpressed in and associated with gene amplification in distinct CCRCC samples.

To test the causality, we transgenically expressed the intracellular domain of NOTCH1 in mice renal tubules with tubule specific deletion of VHL. The Kspcre VHLf/f ICNotch1 mice exhibited dysplastic hyperproliferation of tubular epithelial cells confirming the procarcinogenic role of NOTCH in vivo. Alteration of cell cycle pathways was seen in murine renal tubular cells with NOTCH overexpression and molecular similarity to human tumors was observed, demonstrating that human CCRCC recapitulates features and gene expression changes observed in mice with transgenic overexpression of the Notch intracellular domain.

Finally, treatment with clinical, gamma secretase inhibitor, LY3039478, led to inhibition of CCRCC cells in vitro and in vivo in CCRCC xenografts. In summary, these data reveal the mechanistic basis of NOTCH pathway activation in CCRCC and demonstrate this pathway to a potential therapeutic target.

#1171

Therapeutic applications of the selective high affinity ligand SH7139 may extend beyond NHL to many other types of solid tumors.

Monique Cosman Balhorn, Rod Balhorn. _SHAL Technologies, Inc., Livermore, CA_.

SH7139, the first of a new class of cancer therapeutics developed for treating non-Hodgkin's lymphoma, is unusual in that both targeting and pro-drug functionalities have been incorporated into the same small molecule. Functioning similar to an antibody-drug conjugate, SH7139 targets a unique structural epitope within the antigen-binding pocket of HLA-DR10. HLA-DRs containing this epitope within the β-subunit are reported to be over-expressed in approximately 85% of B-cell lymphomas. Upon binding to HLA-DR molecules located on the tumor cell's surface, SH7139 is transported into the cytoplasm where it is concentrated and subsequently metabolized. A series of metabolic products derived from the SHAL's recognition elements (three small molecules that are linked together to create SH7139 and to provide targeting selectivity) are generated as the drug is broken down, each of which inhibits one or more activities required for tumor cell survival. Preclinical studies with SH739 have demonstrated remarkable efficacy in treating B-cell lymphoma xenografts in mice, providing permanent cures for up to two-thirds of the animals at a human equivalent dose as low as 0.41 μg/kg. Biopsy tissue binding studies conducted with SH7129, a biotinylated form of SH7139, and streptavidin-horse radish peroxidase detection have shown the drug binds to a significant fraction of tumors obtained from patients diagnosed with multiple myeloma and each of the B-cell lymphoma subtypes tested to date (DLBC, Burkitt's, Mantle Cell, Follicular, MALT, and CLL). SH7129 was also observed to bind to tumor biopsies obtained from of a number of patients diagnosed with peripheral T-cell and nodular sclerosis Hodgkin's lymphomas, a result consistent with observations reported by others that HLA-DRs are expressed in a subset of these lymphomas. HLA-DR expression has also been reported to occur in or be linked to a number of other types of cancer, including melanoma, cervical, ovarian, pancreatic and lung cancers. SH7129 staining of tumor microarrays have shown biopsy cores from a subset of patients diagnosed with each of these cancers also bind SH7139. While in vivo efficacy has only been tested in Burkitt's (Raji), Mantle cell (Granta-519), and T-cell (Jurkat, a cell line control lacking HLA-DR and showing no efficacy) lymphoma xenografts, these tissue binding results suggest that in addition to the majority of the NHL subtypes, nodular sclerosis Hodgkin's lymphoma, multiple myelomas, as well as a subset of melanomas, ovarian, cervical, pancreatic, and lung cancers may also respond to SH7139 therapy. This research was supported by the National Cancer Institute Phase II SBIR Award R44CA159843.

#1172

In vivo **pharmacokinetic properties and antitumor efficacy of porcupine lead inhibitors in the orthotopic murine MMTV-Wnt1 breast tumor model and the human HPAF-II pancreatic xenograft mouse model.**

Vishal Pendharkar,1 Yun Shan Chew,1 Vithya Manoharan,1 Choon Bing Low,1 Hongqian Esther Ong,1 Soo Yei Ho,1 Wei Ling Wang,1 Jeyaraj Duraiswamy Athisayamani,1 Babita Madan,2 David Virshup,2 Thomas Hugo Keller,1 May Ann Lee,1 Alex Matter,1 Jeffrey Hill,1 Kanda Sangthongpitag1. 1 _Experiemental Therapeutic Centre, Singapore, Singapore;_ 2 _Duke-NUS Medical School, Singapore, Singapore_.

Porcupine (PORCN), a muti-pass integral membrane-bound-O-Acyl acyltransferase (MBOAT), resides in the in the endoplasmic reticulum (ER) and is required for biogenesis of Wnt ligands. The secreted mature Wnt ligands bind to their cognate receptors (Frizzled, LRP5/6 and transmembrane receptor ROR) to form the ligand - receptor complex which is capable to activate the Wnt-β-catenin signalling cascade and downstream signalling pathways such as mTOR, GSK3, Akt, and PKC. The deregulation of and aberrant activation of the various components of Wnt-β-catenin signalling pathway have been implicated in tumorigenesis, cell proliferation, survival and differentiation. Hyperactivity of PORCN is found to be associated with cancerous cell growth. Knockdown of Porcn mRNA significantly reduced the proliferation of breast cancer cells and resulted in the delay of MDA-MB-231 tumor formation in mouse xenograft models (Covey et al 2012). Loss of function mutations of RNF43, a negative regulation of Wnt-signalling via Frizzled receptor, is recently reported to be involved with pancreatic ductal adenocarcinoma. Inhibition of the PDAC cell lines bearing RNF43 mutations enhanced Wnt-β-catenin signalling and resulted in suppression of proliferation and differentiation of PDAC tumor cells (Jiang et al 2013). Taken together porcupine could be an attractive therapeutic approach for a particular Wnt-driven cancer population. We have identified the porcupine lead compounds (ETC-159, ETC-535, ETC-611 and ETC-539) from different novel chemical scaffolds. The aim of this study was to evaluate their pharmacokinetic properties and antitumor efficacy in different cancer mouse models, the murine MMTV-Wnt1 breast cancer and the human pancreatic HPAF-II cancer. All porcupine lead compounds had good oral pharmacokinetic properties with the absolute oral bioavailability greater than 42%. They had the maximum tolerated dose (MTD-7d) up to 200 mg/kg. They produced antitumor efficacy ranging from 24% to 79% at 1 mg/kg, 38% to 89% at 3 mg/kg, and 58 to 97% at 10 mg/kg in MMTV-Wnt1 tumor mouse model. In vivo inhibition of PORCN led to reduce the expression level of Axin2 in MMTV-Wnt tumors upto 8h. At 100 mg/kg, they produced antitumor efficacy ranging from 34% to 91% in human HPAF-II pancreatic xenograft mouse model. Of 4 porcupine lead compounds, ETC-159 demonstrated great oral pharmacokinetic properties and produced significantly antitumor efficacy (p value < 0.0001) in both cancer mouse models. ETC-1922159 was selected as the preclinical development candidate and currently is under investigation in Phase 1 clinical trial.

#1173

**A novel selective Mcl-1 inhibitor exhibits** in vitro **and** in vivo **efficacy in melanoma.**

Karson J. Kump, Lei Miao, Ahmed S. Mady, Katherine Lev, William Giblin, Mary E. Skinner, David B. Lombard, Zaneta Nikolovska-Coleska. _University of Michigan, Ann Arbor, MI_.

Metastatic melanoma is the deadliest form of skin cancer that still has limited treatment options and dismal 5-year year survival rates as low as 15%. Only recently has there been significant progress in the treatment of metastatic melanoma, advent by molecular targeted drugs and immunotherapy, but their limitations are exposed as these cancers are quickly able to develop resistance. Dysregulation of apoptotic machinery in melanoma allows the cancer cells to evade cell death and contributes to treatment resistance. Up-regulation of Mcl-1, a member of the Bcl-2 family of anti-apoptotic proteins, has been correlated with melanoma progression and metastasis. Mcl-1 amplification is one of the most common genetic aberrations found in human cancers and has been labeled as a marker of aggressive oncogenesis and poor patient prognosis. Previous studies have pointed to Mcl-1 as a viable therapeutic target for the treatment of melanoma and conclude that small molecule Mcl-1 inhibitors may appeal this unmet medical need. We have discovered and characterized a new class of selective small molecule Mcl-1 inhibitors using various biochemical, functional, and cell based assays; further development of these compounds allowed us to achieve potent low-nanomolar binding affinity for Mcl-1 and more than 300-fold selectivity over Bcl-2/Bcl-xL. Our most potent inhibitor, 483-LM, was screened across a panel of human melanoma cell lines using a cell proliferation assay and revealed varying levels of sensitivity. A BH3 profiling assay demonstrates that C8161, the cell line most sensitive to our inhibitor, solely relies on Mcl-1 for survival. The Mcl-1 dependence of C8161 might contribute to the known metastatic nature of this cell line. Mechanistic studies revealed that 483-LM effectively engaged the endogenous Mcl-1 protein after treatment of C8161, as determined by a CETSA assay, and prompted disruption of protein-protein interactions between Mcl-1 and several pro-apoptotic proteins, including Bax, Bak and Bim. This was followed by induction of Bax/Bak dependent apoptosis and activation of hallmarks of the intrinsic apoptotic pathway, including mitochondrial outer membrane depolarization, caspase activation, and PARP cleavage. Importantly, treatment with 483-LM caused massive up-regulation of the pro-apoptotic BH3-only protein Noxa, an effect apparent after an 8 hour treatment, which contributes to the induction of cell death. The in vivo efficacy studies with 483-LM showed significant effect on tumor growth and caspase-3 activation in tumor samples. Overall, our data indicate that Mcl-1 inhibitors are a promising treatment option for aggressive metastatic melanoma and warrant further preclinical investigation of 483-LM as a promising selective Mcl-1 inhibitor to be used as a single agent and in combination with chemotherapy and immunotherapy.

#1174

CT179 degrades the olig2 transcription factor in glioblastoma stem-like cells and prolongs survival.

Gordon R. Alton,1 Graham Beaton,1 Susan Knowles,1 Gregory Stein,1 Santosh Kesari2. 1 _Curtana Pharmaceuticals, Austin, TX;_ 2 _Pacific Neuroscience Institute, Santa Monica, CA_.

Olig2 is a bHLH transcription factor that has been shown to be a key driver of glioblastoma (GBM) tumorigenesis. Olig2 is expressed in human tumor-derived glioma stem-like cells. A growing body of evidence suggests that glioma stem-like cells (GSCs) are more representative of their parent tumors when cultured under defined serum-free conditions with the mitogens epidermal growth factor (EGF) and fibroblast growth factor (FGF). Furthermore, recent research has indicated that human Proneural GSCs grown in vitro in PDGF are more tumorigenic in vivo. We have designed and synthesized a potent inhibitor of Olig2 that possesses all the necessary properties for clinical development as an important new GBM therapeutic. CT179 dose dependently causes the degradation of Olig2 in multiple GBM cell lines and this correlates to G2/M arrest and increased apoptosis. CT179 also induces the loss of expression of EGFR and PDGFR in human GSCs. CT179 achieves high concentrations in the CNS with a long duration of pharmacologic action. Furthermore, CT179 significantly extends survival of mice implanted orthotopically with patient-derived human tumor GSCs. Immunohistochemistry demonstrates a marked reduction of Olig2 positive cells in the tumor bearing animals. Together, these results indicate that CT179 has significant promise as a new therapeutic for the treatment of human GBM.

#1175

Inhibition of NAMPT as a novel therapeutic strategy for infant leukemia.

Klaartje Somers,1 Shiloh Middlemiss,1 Asel Biktasova,1 Mawar Karsa,1 Leanna Cheung,1 Angelika Kosciolek,1 Kathryn Evans,1 Chelsea Mayoh,1 Ursula R. Kees,2 Lioubov Korotchkina,3 Olga B. Chernova,3 Richard B. Lock,1 Andrei V. Gudkov,4 Michelle Haber,1 Murray D. Norris,1 Michelle J. Henderson1. 1 _Children's Cancer Institute, University of New South Wales, Randwick, Australia;_ 2 _Telethon Kids Institute, University of Western Australia, Perth, Australia;_ 3 _Tartis, Inc., Buffalo, NY;_ 4 _Roswell Park Cancer Institute, Buffalo, NY_.

Novel targeted therapies are urgently needed for infant leukemia as this disease is highly aggressive and refractory to treatment resulting in poor survival rates. The NAD (Nicotinamide Adenine Dinucleotide) producing enzyme NAMPT (Nicotinamide Phosphoribosyltransferase) has been considered an attractive selective target for anti-cancer therapy due to the high dependency of tumor cells on NAD for energy metabolism and activity of NAD-dependent enzymes such as poly-ADP-ribose polymerases (PARPs) and sirtuins that play key roles in cancer cell survival. A newly developed NAMPT inhibitor, OT-82, was initially isolated for its high selective toxicity against a panel of adult leukemia cell lines. Here we investigated NAMPT inhibition as a therapeutic strategy for infant leukemias characterized by rearrangement of the MLL gene (MLL-r), by testing the potency of OT-82 in a panel of preclinical in vitro and in vivo models of MLL-r leukemia that are based on the use of patient-derived xenograft (PDX) cells.

OT-82, as a single agent, dramatically reduced the viability of all tested MLL-r leukemia cell lines (n=9) and MLL-r leukemia PDX (n=6) with IC50s ranging from 0.15 to 3.82nM. While the IC50 for OT-82 correlated significantly with the IC50 of other NAMPT inhibitors STF-118804 and FK866, OT-82 was the most potent compound. When combining OT-82 with chemotherapeutic agents currently used to treat infants with leukemia, we observed significant synergy between OT-82 and cytarabine indicating the potential of OT-82 for chemosensitization. Consistent with NAMPT inhibition, OT-82 reduced cytosolic NAD+ levels in MLL-r leukemia cells and inhibited the activity of the NAD-requiring enzymes PARP-1 and SIRT-1, as exemplified by a decrease in PARylated PARP-1 levels and a p53-mediated increase in p21 levels, leading to apoptosis induction. Interestingly, despite the remarkable potency of OT-82 in killing MLL-r leukemia cells, a 25-fold difference in IC50 levels was noted across the cell line panel, with those lines harboring the MLL translocations most prevalent in infants, namely t(4;11) and t(11;19) translocations, being the most sensitive to the compound. A positive correlation was noted between baseline NAMPT mRNA levels and OT-82 IC50 (r=0.7712, P=0.015).

Further in vivo testing of OT-82 showed impressive efficacy of the compound in MLL-r leukemia PDX-based animal models (n=6). OT-82 (p.o. 3x week, 40-50 mg/kg, for 3 or 6 weeks) was well tolerated and significantly delayed leukemia progression in 6/6 MLL-r leukemia xenografts with 5/6 achieving objective responses. OT-82 given as monotherapy was as effective as the routinely used triple combination treatment of vincristine, L-asparaginase and dexamethasone.

Overall, these results demonstrate that NAMPT inhibition using OT-82 is highly effective against MLL-r leukemia and when combined with current chemotherapies may offer a more selective and potent therapeutic option for infants suffering from this disease.

#1176

Preclinical characterization of the pharmacokinetic-pharmacodynamics-efficacy relationship of novel allosteric SHP2 inhibitors.

Minying Pu, Laura R. La Bonte, Stan Spence, Kathy Hsiao, Shumei Liu, Brant Firestone, Ping Wang, Pascal D. Fortin, Ying-Nan P. Chen, Matthew J. LaMarche, Matthew J. Meyer. _Novartis Insts. for BioMedical Research, Cambridge, MA_.

SHP2 is a non-receptor protein tyrosine phosphatase downstream of receptor tyrosine kinases (RTK). Mutations yielding constitutive activation of SHP2 primarily lead to activation of the MAPK pathway and have been found in multiple tumor types. These observations make SHP2 a potentially promising therapeutic target for the treatment of cancers with RTK dependence. Recently, a novel allosteric mechanism of SHP2 inhibition was identified where the autoinhibited form of SHP2 is stabilized via small molecule binding. Herein we describe efforts to characterize the pharmacokinetic (PK)/pharmacodynamic (PD)/efficacy relationship of orally bioavailable novel allosteric SHP2 inhibitors.

Single dose PK/PD studies were conducted in nude mice engrafted with the EGFR amplified esophageal squamous cell carcinoma cell line, KYSE520. The allosteric SHP2 inhibitor SHP099 achieved time and dose dependent increases in plasma concentrations and concomitant reductions in tumor pERK that could be described by an Emax model. Tumor pERK levels were reduced by 50 to 70% when SHP099 unbound plasma concentrations exceeded the in vitro cellular PD IC50, suggesting that exposure above this threshold was required for pathway inhibition in vivo. A second allosteric SHP2 inhibitor, SHP065, was also profiled in vivo and yielded data consistent with this hypothesis. Two additional allosteric SHP2 inhibitors (SHP156, SHP393) that achieved unbound plasma concentrations approximating their cellular IC50s failed to modulate tumor pERK. The totality of these data support the hypothesis that unbound plasma concentrations in excess of the cellular IC50 is required for allosteric SHP2 inhibitors to inhibit the MAPK pathway in vivo.

We further demonstrated that SHP099 achieves dose dependent inhibition of KYSE520 tumor xenograft growth in nude mice. Integration of the antitumor efficacy data and pERK inhibition data revealed a direct linear relationship between tumor growth inhibition and the fraction of time between dosing intervals in which pERK is inhibited by at least 50%. To test this model, SHP099 PK data from nude rats was applied to the Emax model and the resulting predicted PD responses were applied to the PD/efficacy model to predict SHP099 anti-tumor efficacy in nude rats. Data generated from a SHP099 KYSE520 efficacy study in nude rats demonstrated that the exposure/response model was remarkably robust. Doses of 8 mg/kg qd, 25 mg/kg qd, or 75 mg/kg q2d yielded observed T/C of 70, 14, and 18%, respectively; versus a model predicted T/C of 75, 6, and 6%, respectively. In summary, we describe for the first time successful efforts to characterize the PK/PD/efficacy relationship of novel allosteric SHP2 inhibitors. These exposure/response models served as a basis for further allosteric SHP2 inhibitor drug discovery efforts and begin to inform rational approaches to dose and schedule selection in clinic.

#1177

Targeting NEDD8 to uncover an exceptional responder molecular subtype in glioblastoma.

Lauren K. Hartman,1 Darren Finlay,2 Peiwen Pan,2 Seungchan Kim,1 Gil Speyer,1 Jeff Kiefer,1 Harshil Dhruv,1 Kristiina Vuori,2 Michael Berens1. 1 _Translational Genomics, Phoenix, AZ;_ 2 _Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA_.

Neddylation is a post-translational mechanism that marks proteins for degradation through activity of NEDD8 Activating Enzyme (NAE). NAE blocks cullin-RING ligases from initiating proteosomal degradation of select substrates including cell cycle regulators and apoptosis modulators. MLN4924, or Pevonedistat, targets NAE and inhibits Neddylation and induces apoptosis in sensitive cells. We have discovered, in a cohort of glioblastoma PDX models, an exceptional responder to MLN4924 (GBM102). Most pertinently the effects we observe are in a PDX cultured as 3D neurospheres that more closely resemble the true tumor architecture, heterogeneity, and "stem-like" phenotype characteristic of tumor growth. We have leveraged RNAseq expression data from Cancer Cell Line Encyclopedia (CCLE) and Pevonedistat response data from The Cancer Therapeutics Response Portal (CTRP) to apply a network-based analysis to identify pathways enriched with differential dependencies between cell lines sensitive and non-sensitive to MLN4924. The analysis also identifies potential mediating genes that appear to play critical roles in such differential dependency networks. Identified differential networks and mediators provide insight for cellular mechanisms underlying drug response. Additionally, we also investigated the efficacy of MLN4924 against orthotopic glioma PDX models (GBM102 and GBM116) in vivo to validate our findings in vitro. Thus genomic characterization of patient samples may lead to the identification of a molecular signature which is associated with a subset of GBMs vulnerable to MLN4924. As the treatment options for GBM are extremely limited, this may highlight a novel alternative opportunity to treat a select fraction of patients with this aggressive disease.

#1178

Unbiased efficacy-based screening of small molecules highly selective against hematological malignancies revealed inhibitor of NAD synthesis.

Lioubov Korotchkina,1 Sangeeta Joshi,1 Slavoljub Vujcic,1 Ilya Toshkov,1 Mikhail Chernov,2 Denis Kazyulkin,1 Katerina Andrianova,1 Alexander Polinsky,1 Olga Chernova,1 Andrei V. Gudkov2. 1 _Oncotartis Inc., Buffalo, NY;_ 2 _Roswell Park Cancer Inst., Buffalo, NY_.

Effective anticancer drugs can be directed against cell lineage-specific, rather than tumor-specific, targets (i.e., androgen receptor, CD20, etc.). We sought to conduct systematic search for novel agents that are selectively cytotoxic to hematopoietic (HP) malignancies presumably targeting HP lineage-specific survival factors. Cell-based screening and subsequent validation of small-molecules in a broad panel of HP and non-HP cancer cell lines followed by a hit-to lead optimization of the selected class of compounds has led to a molecule named OT-82 characterized by highly selective cytotoxicity against all HP-derived cancers tested (14 cell lines, representing human AML, ALL, CML and lymphoma cells) active within single nanomolar range of concentrations. OT-82 killed sensitive cells by induction of apoptosis and was up-to 20-fold more toxic to HP-derived than to solid tumor-derived cell lines and normal primary cells of HP and non-HP origin. OT-82-based affinity chromatography of cell lysates followed by mass spectrometry led to the identification of nicotinamide phosphorybosyltransferase (NAMPT) as the OT-82 target. NAMPT catalyzes the rate limiting step in a major pathway of synthesis of NAD, an energy metabolism mediator and essential cofactor of PARP and sirtuins. NAMPT treatment caused dramatic reductions in cellular NAD levels. Nicotinic acid (NA, vitamin B3), a substrate for the alternative NAD synthesis pathway, protected cells from the cytotoxic effects of OT-82. Oral administration of OT-82 was efficacious in subcutaneous and systemic xenograft models of human AML, ALL, erythroleukemia and multiple myeloma including complete response in several models. Therapeutic doses of OT-82 in mice were significantly lower than maximal tolerated dose (MTD); toxicities observed at doses above MTD were largely limited to HP and lymphoid organs. Toxicity profile of OT-82 determined in GLP in mice and non-human primates was favorably different from that reported for other reported clinical stage NAMPT inhibitors and did not involve any detectable retinal or cardiac toxicity. These results indicate high lineage specific and malignant transformation-specific dependence of HP neoplastic cells on NAMPT, presumably due to the strong addiction of this cell type on the NAD levels, and highlight OT-82 as a prospective clinical candidate for the treatment of hematological malignancies.

#1179

EP4 receptor antagonism in paclitaxel-resistant ovarian clear cell carcinomas that overexpress class III β-tubulin.

Dana M. Roque,1 Danielle Meir-Levi,1 Gautam G. Rao,1 Paul Staats,2 Amy Fulton,2 Jocelyn Reader1. 1 _Division of Gynecologic Oncology, University of Maryland School of Medicine-Greenebaum Cancer Center, Baltimore, MD;_ 2 _Department of Pathology, University of Maryland School of Medicine, Baltimore, MD_.

OBJECTIVES: Advanced ovarian clear cell carcinoma (OCCC) is associated with a survival disadvantage relative to ovarian serous carcinoma following platinum/taxane-based chemotherapy and optimal cytoreduction. Prostaglandin E2 (PGE2) contributes to disease progression through modulation of several G-protein coupled receptors (EP1-4) [Fig.1a]. The addition of upstream COX inhibition to platinum/taxane-based chemotherapy in the first-line phase II setting in ovarian cancer has been disappointing,a possibly due to compensatory upregulation of COX isoenzymes and negation of protective effects of EP1. Selective antagonism of EP4 may therefore pose a more rational strategy than global COX inhibition. Paclitaxel administration has been shown to upregulate components of the COX pathway.b Class III β-tubulin is a marker for paclitaxel resistance and is widely overexpressed in OCCC. The purpose of this study is to demonstrate that EP4 inhibition may overcome paclitaxel resistance in OCCC that overexpress class III β-tubulin.

METHODS: Expression of EP4 receptor and class III β-tubulin was quantified using immunohistochemistry and Western blot in solid tissues and cell lines. Standard metabolic growth and migration assays were employed to test the effects of drug treatment (paclitaxel and EP4 inhibitors RQ-15986/AH-23848) with and without EP4 silencing using siRNA.

RESULTS: OCCC overexpress class III β-tubulin/EP4 relative to normal ovary [Fig. 1b/c]. EP4 staining intensity was 2+ in 100% OCCC using an ovarian cancer tissue microarray (62 cores, 13% OCCC); this rate was only 26-56% among other histologies. EP4 inhibition reduces growth of paclitaxel-resistant cells [Fig. 1d]. Likewise, treatment with EP inhibitors [Fig.1 e-top] and silencing of EP4 resulted in reduced migration [Fig.1 e-bottom].

CONCLUSIONS: Selective antagonism of PGE2 through EP4 receptor inhibition may represent a powerful targeted therapy for paclitaxel-resistant OCCC. Further study including simultaneous treatment (EP4 inhibitor+paclitaxel) and larger samples sizes is required.

REFERENCES: a Reyners et al. A randomized phase II study investigating the addition of the specific COX-2 inhibitor celecoxib to docetaxel plus carboplatin as first-line chemotherapy for stage IC to IV epithelial ovarian cancer, fallopian tube or primary peritoneal carcinomas: the DoCaCel study. Ann Oncol 2012; 23:2986-902. b Moos et al. Effects of taxol/taxotere on gene expression in macrophages: induction of the PGH synthetase-2 isoenzyme. J Immunol 1999;162:476-73.

#1180

Targeting the HSP40/HSP70 chaperone axis as a novel strategy to treat castration-resistant prostate cancer.

Michael A. Moses,1 Yeong Sang Kim,1 Genesis Rivera-Marquez,1 Matthew J. Watson,1 Sunmin Lee,1 Andrea Kravats,1 Sue Wickner,1 Jason Gestwicki,2 Jane Trepel,1 Len Neckers1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _University of California, San Francisco, San Francisco, CA_.

Castration-resistant prostate cancer (CRPC) is frequently characterized by elevated expression of nuclear receptors able to at least partially maintain the androgen receptor (AR) transcriptional program. Elevated expression of a number of constitutively active AR splice variants lacking the ligand binding domain (LBD) (e.g., ARv7, which is ligand-independent and correlates with poor prognosis, reduced survival, and resistance to existing LBD-targeted standard of care therapy) is a frequent occurrence in CRPC. Thus, alternative approaches to disrupt AR signaling in CRPC are of great clinical importance, and a single strategy able to target AR and ARv7 remains a critical unmet need. As a steroid hormone nuclear receptor, the AR exists in an interactive and dynamic cycle with the molecular chaperones (heat shock proteins, HSPs) HSP40/HSP70/HSP90 for proper folding and remodeling of the AR LBD to bind ligand. Notably, HSP90 inhibitors promote AR degradation and display efficacy in prostate cancer xenograft models. Although it has been shown that ARv7 functions independently of HSP90, additional chaperone requirements of LBD-deficient ARv7 are not known. Thus, we tested the hypothesis that both AR and ARv7 are dependent on HSP40/HSP70 and that targeting these chaperones with specific inhibitors (C86 and JG98, respectively) will lead to AR/ARv7 destabilization and loss of transcriptional activity in models of CRPC. To determine if AR proteins associate with HSP40/HSP70, 22Rv1 CRPC cells (expressing endogenous AR and ARv7) were first transfected with FLAG-HSP40 or FLAG-HSP70. Immunoprecipitation with FLAG beads revealed AR and ARv7 associated with both chaperones, indicating potential functional dependence of these nuclear receptors on HSP40/HSP70. To further characterize these interactions, 22Rv1 lysate was probed with biotinylated-C86 and subjected to IP with streptavidin beads. C86 bound a significant fraction of HSP40 complexed with HSP70, AR, and ARv7. Excess unlabeled C86 or JG98 effectively competed away binding of HSP40/HSP70 to biotinylated-C86 with concomitant loss of associated AR and ARv7. Treatment of 22Rv1 cells with C86 or JG98 led to a time and dose-dependent decrease in AR and ARv7 protein, concomitant with a significant loss of viability. We also observed that HSP40/HSP70 inhibition markedly reduced AR and ARv7 transcriptional activity, as indicated by decreased AR (KLK3, TMPRSS2) and ARv7 (UBE2C) target gene expression. Finally, treatment of mice bearing 22Rv1 xenografts with JG231 (an analog of JG98 with enhanced PK properties) led to significantly smaller tumors relative to vehicle treated mice. Together, these data confirm the continued dependence of AR and ARv7 on HSP40/HSP70 molecular chaperones and they demonstrate the feasibility of targeting the HSP40/HSP70 axis to abrogate sustained AR-mediated signaling in CRPC.

#1181

Discovery and development of novel highly potent and selective inhibitors of USP19 using UbiPlex™.

Gerald Gavory, Colin O'Dowd, Ewelina Rozycka, Anthony Dossang, Ashling Henderson, Caroline Hughes, Hugues Miel, Oliver Barker, Joana Costa, Peter Hewitt, Mary McFarland, Lauren Proctor, Tim Harrison. _Almac Discovery, Belfast, United Kingdom_.

Over the past decade, protein ubiquitination has emerged as an important post-translational modification with regulatory functions in all important cellular processes. Deubiquitinating enzymes (DUBs) including ubiquitin specific proteases (USPs) are cysteine proteases that catalyse the de-ubiquitination of protein substrates including tumor suppressors and oncogenes, hence regulating their levels and/or function. As a result of their increasing implications in the etiology of numerous pathological conditions including cancer, DUBs are emerging as an attractive and promising target class for the development of 1st in class medicines with high therapeutic impact. However, despite 15 years of intense research DUBs have proved largely refractory to drug discovery efforts.

Herein, we further describe the application of Ubi-Plex™, our drug discovery platform for the identification and optimisation of DUB inhibitors. In particular, we will highlight the versatility and robustness of Ubi-Plex™ by describing the outcome of our focussed library screening, hit identification, hit validation and elaboration activities on USP19.

A series of novel, highly potent (e.g. IC50 < 10 nM) and reversible USP19 inhibitors have been identified. Further profiling has also demonstrated excellent selectivity against a large panel of DUBs and other non-related enzymes (e.g. kinases, proteases). These inhibitors are cell-permeable and exhibit potent target engagement in cells with EC50 values < 30 nM. Finally, we will describe our progress towards the development of lead molecules with drug-like properties with the aim to rapidly establish in vivo proof-of-concept studies.

In summary, this work further exemplifies the tractability of the DUB target family and reports the discovery and detailed profiling of the first highly potent and selective inhibitors of USP19. These molecules may provide opportunities for the development of new anticancer therapeutics as well as for the treatment of muscle wasting disorders including cachexia.

#1182

A novel curcumin derivative inhibits active ras and its downstream pathway in pancreatic cancer.

Naveen Mallangada,1 Gerardo G. Mackenzie2. 1 _Stony Brook University, Stony Brook, NY;_ 2 _University of California, Davis, Davis, CA_.

Pancreatic Cancer's (PC's) 5-year survival rate of only 6.7% indicate the need to improve treatment modalities. Despite decades of research, current chemotherapy and radiation therapy regimens offer minimal or no help. It is critical to develop new agents for the effective management of PC. Given that Kras mutations initiate and maintain PC, inhibition of this pathway is widely considered a therapeutic target of exceptional importance. Our laboratory is currently exploring the chemotherapeutic efficacy of a novel Chemically-Modified Curcumin (CMC2.24) as a potential chemotherapeutic agent for PC. Preliminary studies have shown that CMC2.24 has higher bioavailability than curcumin, as shown in pharmacokinetic studies in rats, and inhibits PC growth in vitro and in vivo. However, the exact mechanism on how CMC2.24 reduces cell growth remains unidentified. The objective of this work was to determine the mechanism of CMC2.24 in PC. Using human PC MIA PaCa-2 and Panc-1 cell lines and pancreatic acinar explants from Kras mutant mice, we explored the effects of CMC2.24 on Ras activation, ERK phosphorylation, mitochondrial reactive oxygen species, mitochondrial ATP production, and intrinsic apoptosis. In human PC MIA PaCa-2 cells CMC2.24 inhibited Ras activation by 90% (p<0.05). This was confirmed in primary acinar explants isolated from Kras mutant mice in which CMC treatment reduced Ras activation by 70%, compared to control. Furthermore, CMC 2.24 treatment reduced the phosphorylation MEK, ERK and c-Raf, down-stream pathway of Ras, both in vitro and in vivo. The effect of CMC2.24 on ERK phosphorylation was confirmed by Immunofluorescence, showing a significant decrease in ERK phosphorylation (47.8%; p<0.05). Moreover, CMC2.24 treatment enhanced the levels of superoxide anion in mitochondria by 200% (p<0.02), decreased ATP levels in a concentration-dependent manner (p<0.05), and induced intrinsic apoptosis, as shown by the increase in caspase 9 and Parp cleavage downstream of cytochrome C cytosolic release. In conclusion, our results indicate that the Ras pathway is a key molecular target for CMC2.24 and that CMC induces apoptosis in PC cells through the intrinsic pathway.

This research was supported by the Stony Brook Cancer Center and a URECA Summer Grant.

#1183

Structure-activity studies and biological evaluations of ERGi-USU, a highly selective inhibitor for ERG-positive prostate cancer cells.

Ahmed A. Mohamed,1 Charles P. Xavier,1 Gauthaman Sukumar,2 Samuel D. Banister,3 Vineet Kumar,3 Shyh-Han Tan,1 Shilpa Katta,1 Lakshmi Ravindranath,1 Muhammad Jamal,1 Taduru Sreenath,1 David G. McLeod,1 Gyorgy Petrovics,1 Albert Dobi,1 Meera Srivastava,2 Sanjay Malhotra,3 Clifton Dalgard,2 Shiv Srivastava1. 1 _Uniformed Services Univ. of the Health Sci., Rockville, MD;_ 2 _Uniformed Services Univ. of the Health Sci., Bethesda, MD;_ 3 _Stanford University, Standford, CA_.

Introduction and objectives: While new prostate cancer (CaP) treatments (Abiraterone and Enzalutamide) have improved survival in castration-resistant prostate cancer (CRPC), their benefits are short-lived and drug resistance develops likely due to numerous adaptive mutations. Therefore it is a critical to develop effective novel inhibitors to target other signaling pathways that promote or contribute to prostate tumorigenesis. Accumulating evidence has established the androgen regulated TMPRSS2-ERG fusion as a common oncogenic driver that contributes to the early development and progression of over half of CaP. Therefore, ERG oncoprotein and ERG dependent pathways are promising targets for CaP therapy in early stages when cancer is most responsive to treatment. We previously identified a small molecule inhibitor, ERGi-USU, which selectively inhibits ERG protein and cell growth in ERG positive tumor cell lines and mouse xenograft models. In an effort to further develop ERGi-USU with enhanced efficacy we performed detailed structure-activity relationship (SAR) evaluation of ERGi-USU core structure and developed new derivatives.

Methods: Based on SAR of the core structure of ERGi-USU, 48 new derivatives were designed and synthesized by substitutions with alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, heteroaryl or hydroxyl groups. The new ERGi-USU derivatives were evaluated for inhibition of cell growth and ERG protein levels in the TMPRSS2-ERG fusion harboring CaP cell line, VCaP. Four of these compounds have been selected for evaluation of ERG selectivity by defining IC50 in ERG positive malignant cells (VCaP, KG1, MOLT-4 and COLO320), ERG negative CaP cell line (LNCaP) or ERG positive normal primary endothelium-derived cells (HUVEC).

Result: Like parental compound, four new ERGi-USU derivatives exhibited inhibition of cell growth and ERG protein levels in ERG positive VCaP, KG1, MOLT-4 and COLO320 cell lines, with no or minimal effects on LNCaP and HUVEC cells. One of the new derivatives (ERGi-USU#6) showed increased efficacy for cell growth inhibition (IC50=0.074µM) compared to the parental ERGi-USU (IC50=0.200µM). Other three new compounds showed similar IC50 as the ERGi-USU.

Conclusion: Comprehensive evaluation of ERGi-USU derivatives along with parental compound has continued to underscore selective inhibition of ERG positive tumor cells by these small molecules.

#1184

Characterization of novel STAT5 inhibitors to interfere with the oncogenic activities of STAT5 in hematopoietic diseases.

Bettina Wingelhofer,1 Barbara Maurer,1 Elizabeth C. Heyes,1 Patricia Freund,1 Abbarna A. Cumaraswamy,2 Jisung Park,2 Stefan Kubicek,3 Peter Valent,4 Patrick T. Gunning,2 Richard Moriggl1. 1 _Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria;_ 2 _University of Toronto Mississauga, Mississauga, Ontario, Canada;_ 3 _CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria;_ 4 _Medical University of Vienna, Vienna, Austria_.

Activation of the transcription factor STAT5 is essential for the pathogenesis of acute myeloid leukemia (AML) containing the FLT3 internal tandem duplication (ITD). FLT3 ITD is a constitutively active tyrosine kinase (TK) that drives the activation of STAT5 leading to the growth and survival of AML cells. Although there has been some success in identifying TK inhibitors that block the function of FLT3 ITD, treatment options are still limited. This is mainly due to drug resistance development by mutations that allow for the continued activation of STATs. Since STAT5 represents a critical mediator of malignant cellular behavior and sits at the convergence point of many kinase pathways, the direct targeting of STAT5 may be an effective means of overcoming this resistance to TK inhibitors. First, we screened a library of potential STAT5 inhibitors for specific SH2 domain binders using a fluorescent polarization assay. Thereby, we identified small inhibitory molecules, called AC-3-019 and AC-4-130, that bind to the SH2 domain of STAT5, subsequently resulting in the disruption of the reciprocal STAT5-phosphopeptide interactions. They efficiently blocked kinase-mediated phosphorylation, dimer formation, nuclear translocation, DNA binding and STAT5 mediated target gene expression. Further, we observed a time- and dose-dependent impairment of proliferation, blocked cell cycle progression and increased apoptosis. Studies with human AML patient-derived samples similarly showed an induction of apoptotic cell death and decreased colony forming capabilities. A combinatorial drug screen revealed synergistic effects with TK inhibitors, as well as with drugs standardly used in the treatment of AML patients, e.g. Cytarabine. Finally, AC-3-019 and AC-4-130 significantly suppressed tumor growth in vivo without general toxicity in healthy organs. Overall, our findings indicate that AC-3-019 and AC-4-130 are potent and selective inhibitors of STAT5. These compounds provide lead structures for further chemical modifications and clinical development for the identification of compounds to improve existing therapies.

#1185

H3B-8800, a novel orally available SF3b modulator, shows preclinical efficacy across spliceosome mutant cancers.

Silvia Buonamici,1 Akihide Yoshimi,2 Michael Thomas,1 Michael Seiler,1 Betty Chan,1 Benjamin Caleb,1 Fred Csibi,1 Rachel Darman,1 Peter Fekkes,1 Craig Karr,1 Gregg Keaney,1 Amy Kim,1 Virginia Klimek,3 Pavan Kumar,1 Kaiko Kunii,1 Stanley Chun-Wei Lee,2 Xiang Liu,1 Crystal MacKenzie,1 Carol Meeske,1 Yoshiharu Mizui,1 Eric Padron,3 Eunice Park,1 Ermira Pazolli,1 Sudeep Prajapati,1 Nathalie Rioux,1 Justin Taylor,2 John Wang,1 Markus Warmuth,1 Huilan Yao,1 Lihua Yu,1 Ping Zhu,1 Omar Abdel-Wahab,2 Peter Smith1. 1 _H3 Biomedicine, Cambridge, MA;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 3 _Moffitt Cancer Center and Research Institute, Tampa, FL_.

Genomic characterization of hematologic and solid cancers has revealed recurrent somatic mutations affecting genes encoding the RNA splicing factors SF3B1, U2AF1, SRSF2 and ZRSR2. Recent data reveal that these mutations confer an alteration of function inducing aberrant splicing and rendering spliceosome mutant cells preferentially sensitive to splicing modulation compared with wildtype (WT) cells.

Here we describe a novel orally bioavailable small molecule SF3B1 modulator identified through a medicinal chemistry effort aimed at optimizing compounds for preferential lethality in spliceosome mutant cells. H3B-8800 potently binds to WT or mutant SF3b complexes and modulates splicing in in vitro biochemical splicing assays and cellular pharmacodynamic assays. The selectivity of H3B-8800 was confirmed by observing lack of activity in cells expressing SF3B1R1074H, the SF3B1 mutation previously shown to confer resistance to other splicing modulators.

Although H3B-8800 binds both WT and mutant SF3B1, it results in preferential lethality of cancer cells expressing SF3B1K700E, SRSF2P95H, or U2AF1S34F mutations compared to WT cells. In animals xenografted with SF3B1K700E knock-in leukemia K562 cells or mice transplanted with Srsf2P95H/MLL-AF9 mouse AML cells, oral H3B-8800 treatment demonstrated splicing modulation and inhibited tumor growth, while no therapeutic impact was seen in WT controls. These data were also evident in patient-derived xenografts (PDX) from patients with CMML where H3B-8800 resulted in a substantial reduction of leukemic burden only in SRSF2-mutant but not in WT CMML PDX models. Additionally, due to the high frequency of U2AF1 mutations in non-small cell lung cancer, H3B-8800 was tested in U2AF1S34F-mutant H441 lung cancer cells. Similar to the results from leukemia models, H3B-8800 demonstrated preferential lethality of U2AF1-mutant cells in vitro and in in vivo orthotopic xenografts at well tolerated doses.

RNA-seq of isogenic K562 cells treated with H3B-8800 revealed dose-dependent inhibition of splicing. Although global inhibition of RNA splicing was not observed; H3B-8800 treatment led to preferential intron retention of transcripts with shorter and more GC-rich regions compared to those unaffected by drug. Interestingly, H3B-8800-retained introns commonly disrupted the expression of spliceosomal genes, suggesting that the preferential effect of H3B-8800 on spliceosome mutant cells is due to the dependency of these cells on expression of WT spliceosomal genes.

These data identify a novel therapeutic approach with selective lethality in leukemias and lung cancers bearing a spliceosome mutation. Despite the essential nature of splicing, cancer cells without a spliceosome mutation were less sensitive to H3B-8800 compared with potent eradication of mutant counterparts. H3B-8800 is currently undergoing clinical evaluation in patients with MDS, AML, and CMML. 

### Reversal of Drug Resistance

#1186

Propranolol could overcome BRAF inhibitors resistance by multiple mechanisms in melanoma.

Chengfang Zhou,1 DongYa Shen,1 WeiLi Wang,1 Shangchen Xie,1 Ping Liao,1 Xiang Chen,2 Howard L McLeod,3 Yijing He2. 1 _Department of Clinical Pharmacology, XiangYa Hospital, Central South University, Institute of Clinical Pharmacology, Central South University, Hunan Key Laboratory of Pharmacogenetics, Changsha, China;_ 2 _Department of Dermatology, XiangYa Hospital, Central South University, Changsha; China Hunan Key Laboratory of Skin Cancer and Psoriasis, Changsha, China;_ 3 _Moffitt Cancer Center, Tampa, FL_.

BRAF(V600E) is the most common oncogenic mutation in melanoma and leads to constitutive activation of the MAPK pathway, which results in uncontrolled cell growth. Selective BRAF inhibitors such as vemurafenib have been observed to neutralize oncogenic the signaling, inhibit cellular growth, and improve patient outcome. Although these mechanisms of vemurafenib resistance have been reported, few studies focused on how to overcome the resistance. Propranolol, a non-selective β-blocker, was confirmed to involve in multiple anticancer effects. Our previous study also showed propranolol inhibited melanoma by suppressing MAPK and AKT pathways in vitro an in vivo. But its efficacy and mechanism of overcoming vemurafenib still remain unknown in melanoma. Here, we explored the effect of propranolol on the A375, P-8 (patient-derived melanoma cell line) vemurafenib resistance cell line and resistance mice xenografts. Cell viability assay demonstrated that 2μM -20μM vemurafenib couldn't decrease the proliferation but 24h-120h of incubation of 2μM-200μM propranolol inhibited viability with a concentration and time dependent manner in the two resistance cell line. TUNEL staining showed 24h incubation of 20μM propranolol alone or plus 4μM vemurafeinb obviously increased cell apoptosis. Mice received daily ig. administration of propranolol at the dose of 2 mg/kg alone or plus 10 mg/kg for 21days. The mean tumor volume at day 21 in resistance A375 xenografts was 221.13 ± 7.65mm3vs. 904.12 ± 70.57mm3 vs. 2021 ± 316.24mm3for the propranolol plus vemurafenib, propranolol alone, vemurafenib alone, respectively. Propranolol improved mice survival, 28.6% animal dead in plus group, 57.1% mice dead in the propranolol group, and 71.5% animal dead in vemurafenib at end of treatment. IHC showed propranolol also reduced Ki67 index both in propranolol and plus group when compared with vemurafenib treated mice. Furthermore, RNA sequencing was performed to explore the mechanism of propranolol overcoming the resistance, the data showed propranolol largely reduced mRNA levels of IGF family (IGFBP3, IGFLR1, IGFBP6, IGF2, IGF1R.etc.) but elevated the expressions of innate immune related genes (NKG7, TLR9, NCR3.etc.) in A375-vemr cell line. These results provide a strategy of therapeutic resistance for the clinic, importantly, this study also provide a clue targeting IGF family and regulating innate immune might be a potential strategy to suppress resistance in BRAF inhibitor therapies in melanoma.

#1187

Synthetic lethality screening reveals ATR as responsible for oxaliplatin resistance in colorectal cancer cells.

Eve Combes,1 Augusto Faria-Andrade,1 Diego Tosi,1 Pierre Martineau,1 Maguy Del Rio,1 Roderick Beijersbergen,2 Nadia Vie,1 Céline Gongora1. 1 _IRCM, INSERM U1194, Montpellier, France;_ 2 _The Netherlands Cancer Institute, Amsterdam, Netherlands_.

Despite the recent advances achieved in the treatment of colon cancer, tumor resistance is a frequent cause of chemotherapy failure. Our work was aimed to determine the molecular mechanisms involved in the resistance to oxaliplatin, an anticancer agent widely used in colorectal cancer treatment. To this end, we establish an oxaliplatin-resistant cellular model from the colon adenocarcinoma cell line HCT-116. Among cellular clones obtained, we used one displaying mild resistance (10 fold called HCT116-R1) to perform short hairpin RNA-based loss of function genetic screen in order to identify genes that can modulate the cellular response to oxaliplatin by revealing genes the silencing of which causes drug sensitivity (synthetic lethal interactions with the drug). Using this screen, we have identified ATR (Ataxia-telangiectasia mutated and rad3 related), a protein that plays a key role in the repair of DNA double-strand breaks induced by various DNA damaging agents, including platinum derivatives. We further validated ATR implication in oxaliplatin resistance by showing that (i) shRNA-mediated repression of ATR in HCT116-R1 oxaliplatin-resistant CRC cells sensitizes such cells to the drug; (ii) co-incubation with the ATR inhibitor VE-822 (or VX-970) and oxaliplatin led to a dramatic synergistic effect in six different CRC cell lines (two oxaliplatin-sensitives: HCT116 and SW48; and four oxaliplatin-resistant: HCT116-R1, HCT116-R2, SW48-R1 and SW48-R2) using 2D and 3D cell growth inhibition assays and in vivo. The synergistic effect was evaluated using dose matrix data (an algorithm that was implemented in our group); (iii) the synergistic effect of oxaliplatin and VE-822 was accompanied by an increase of ssDNA, DNA double-strand breaks, growth arrest and apoptosis induction. In conclusion, our preliminary data confirm the results of our screen by demonstrating for the first time the functional role of ATR in the sensitivity to oxaliplatin.

#1188

Sodium phenylbutyrate has an antineoplastic effect and enhances the cytotoxicity of 5-fluorouracil and irinotecan in colorectal cancer cell lines.

Maha Al-Keilani, Ruba Darweesh. _Jordan University of Science and Technology, Irbid, Jordan_.

Colorectal cancer is the third most common cancer in the world and it is the second leading cause of cancer-related deaths in the United States. About 25% of colorectal cancer cases present with metastases at time of diagnosis and 50% of patients who undergo surgery will ultimately develop metastatic disease. Consequently, adjuvant chemotherapy is required in many cases and it aims to prevent tumor progression and metastasis.

Resistance to chemotherapy is the biggest obstacle in the treatment path of advanced colorectal cancer. Thus, highlighting the urgent need for the identification of new therapeutic agents that can enhance the cytotoxicity of the currently approved anticancer drugs.

Sodium phenylbutyrate (NaPB), a salt of a short chain fatty acid that is used for treatment of urea cycle disorders, is under investigation for its antineoplastic potential. NaPB acts as a histone deacetylase inhibitor and it has been shown to be associated with reduced ER stress and enhanced JNK signaling pathway. This indicates that NaPB may enhance the cytotoxicity of the currently approved agents for colorectal cancer including oxaliplatin, 5-fluorouracil, and irinotecan.

The aims of this study were to identify the antineoplastic potential of NaPB in colorectal cell lines, and to identify its effect on the cytotoxicity of oxaliplatin, 5-fluorouracil, and irinotecan.

MTT proliferation assay showed that NaPB possessed a concentration dependent killing effect against HCT-116 and HT-29 colorectal cancer cell line (IC50 values were 5 and 10 mM respectively). Moreover, at clinically achievable and nontoxic concentration (2.5 mM), NaPB showed a synergistic effect in HCT-116 on 5-fluorouracil (R=14.176, p=0.006), on irinotecan (R=53.86, p=0.0117), and on oxaliplatin (R=20, p=0.004), but resulted in extensive cytotoxicity when used at 5 mM and 10 mM.

In HT-29 cells, NaPB at concentrations 5 mM and 10 mM, showed a synergistic effect on 5-fluorouracil (R=5.5, p<0.001; and R=125, p<0.001 respectively), on irinotecan (R=5.7, p=0.016; and R=21.3, p=0.018 respectively), and on oxaliplatin (R=2.4, p=0.003; and R=3, p=0.002 respectively). The addition of NaPB at concentration of 20 mM to the previous drugs resulted in profound cytotoxic effect toward HT-29 cells.

As a conclusion, NaPB is a promising new adjuvant antineoplastic agent that can be used to enhance the cytotoxicity of 5-fluorouracil, irinotecan, and oxaliplatin in colorectal cancer.

#1189

Enhanced YAP expression leads to EGFR TKI resistance in lung adenocarcinomas.

Ting-Fang Lee, Yu-Chi Tseng, Cheng-Wen Wu. _National Yang-Ming University, Taipei, Taiwan_.

Epidermal growth factor receptor (EGFR) mutation is prevalently expressed in lung adenocarcinoma cases and acts as one of the major driving oncogenes. EGFR tyrosine kinase inhibitors (TKIs) have been used in patients with EGFR-mutant as an effective targeted therapy in lung adenocarcinoma, but drug resistance and tumor recurrence inevitably occurs. Recently, Yes-associate protein (YAP) has been reported to promote multiple cancer cell properties, such as promoting cell proliferation, EMT and drug resistance. This study investigated the roles of YAP in TKI-resistant lung adenocarcinoma. In TKI-sensitive cells, enhanced YAP expression leads to TKI resistant. Also, upregulated YAP expression and activation were detected in long term TKI-induced resistant cells. With reduced YAP expression using shRNA or YAP inhibitors, TKI-resistant cells become TKI-sensitive. Moreover, combined EGFR TKI and an YAP inhibitor statin, prolonged survival among lung cancer patients analyzed by Taiwan National Health Insurance Research database. These observations revealed the importance of YAP in promoting TKI-resistance and combined YAP inhibition can be a potential therapy delaying the occurrence of TKI-resistance in lung adenocarcinoma.

#1190

Comprehensive high-throughput screen for combination therapies to block acquired resistance to targeted drugs.

Izhak Haviv. _Bar Ilan Univ., Zfat, Israel_.

Background - Genetic and epigenetic alterations provide the selective advantage for cancer cell acquired drug resistant (ADR). A bottleneck in implementing drug combination is the challenging task of identifying combinations with acceptable risk benefit ratio. High throughput functional genomic (RNAi) screen is an ideal tool to comprehensively identify double and triple inhibitor combinations to prevent ADR. Unfortunately, most anticancer agents are antagonists (antagonists operate in the same direction as the RNAi), therefore RNAi hits with possible clinical value are the ones that disappear during the screen.

Materials and Methods - We use lentiviral-borne shRNA libraries (mainly kinome collection) to transduce the different RNAis into patient-derived cancer cells. The resulting mixed cancer cell population (each lost one gene) is subject to negative screen, to identify mediators of ADR. This process was performed with approved cancer drugs, such as erlotinib, olaparib, pazopanib, etc. We developed a proprietary unique way to identify the RNAi clones that disappear/extinct, specifically in the presence of the investigated drug. RNAi clones are tracked using next generation sequencing, and quantified in treated and untreated cancer cell cultures or xenografts. The resulting drug combinations identified through this screen are tested directly on a platform of patient-derived xenografts.

Results - We identified known boosters of the EGFR activity, such as DYRK1 (maintains level of EGFR protein), IKBKE (downstream survival mediator), which when reduced by their RNAi, enhance the response to erlotinib. Consequently, known inhibitors of JAK1, CDK4/6, and PI3K combined to egfr inhibitors generated more durable remissions in egfr positive cells. Remarkably, even tumors without egfr mutations were sensitive to the combinations. A similar discovery of novel combinations were found for olaparib and pazopanib. Mouse harboring patient derived xenografts tolerated combination therapy fairly well.

Conclusion - Our findings demonstrate the value of clinically testing the efficacy of drug combinations empirically designed to block ADR. Animal model support our findings with acceptable toxicity.

#1191

**The dopamine (DA) D** 3 **receptor antagonists (PG01037, NGB2904, SB-277011A, and U99194) significantly attenuate ABCG2-mediated multidrug resistance .**

Noor Hussein,1 Haneen Amawi,1 Charles R. Ashby,2 Karthikeyan Chandrabose,3 Roopali Mittal,4 Ryann Christman,1 Piyush Trivedi,3 Amit Tiwari1. 1 _University of Toledo, Toledo, OH;_ 2 _St. John's University, Queens, NY;_ 3 _Rajiv Gandhi Proudyogiki Vishwavidyalya, Bhopal, India;_ 4 _OU Medical Center, Oklahoma City, OK_.

ATP-binding family G2 (ABCG2) transporters are known to produce multidrug resistance (MDR) and limit successful cancer chemotherapy. Using molecular modeling, we obtained data indicating that highly potent and selective dopamine (DA) D3 receptor antagonists had significant docking scores for the active site of the ABCG2 transporter. In this in vitro study, we determined the effect of the D3 receptor antagonists (PG01037, NGB2904, SB-277011A, and U99194) on MDR resulting from the overexpression of ABCG2 transporters. The D3 receptor antagonists alone did not significantly affect the viability of HEK293/ABCG2, H460/MX-20, S1-M1-80 or A549-MX-10 cells, which overexpress ABCG2 transporters. However, the D3 receptor antagonists (PG01037, NGB2904, SB-277011A, and U99194) significantly increased the efficacy of the anticancer drugs mitoxantrone and doxorubicin in the above mentioned cell lines. Efflux studies indicated that both PG01037 and NGB2904 significantly decreased the efflux of rhodamine 123 from H460-MX20 cells. Interestingly, PG01037 and NGB2904 significantly decreased the expression levels of the ABCG2 protein levels as shown by immunocytochemical and Western blot analysis. This suggests that D3 antagonists inhibit both function and expression of ABCG2 transporters at relevant non-toxic concentrations. In conclusion, our in vitro results indicate that PG01037, NGB2904, SB-277011A, and U99194 reverse resistance to mitoxantrone and doxorubicin mediated by overexpression of ABCG2 transporters. Further mechanistic and animal studies are warranted to establish the clinical use of D3 receptor antagonists in reversing ABCG2-mediated MDR.

#1192

**Regorafenib (Stivarga®) reverses BCRP-mediated multidrug resistance** in vitro **and** in vivo **.**

Yun-Kai Zhang,1 Yi-Jun Wang,1 Guan-Nan Zhang,1 Wen-Ji Zhang,2 Meng-Ning Wei,2 Zhi Shi,2 Zhe-Sheng Chen1. 1 _St. John's University, Jamaica, NY;_ 2 _Jinan University, Guangzhou, China_.

Overexpression of breast cancer resistance protein (BCRP) has been shown to produce multidrug resistance (MDR) in various kinds of cancers, such as breast, colon, lung and ovarian cancers. Regorafenib, an oral multi-kinase inhibitor, was found to have inhibitory effects on BCRP-mediated MDR both in vitro and in vivo. Regorafenib significantly sensitized BCRP-overexpressing cancerous cells to BCRP substrates by increasing their intracellular accumulation. There are no significant changes in the expression level or the subcellular distribution of BCRP in the cells exposed to regorafenib. Our mechanism studies revealed that regorafenib inhibited the ATP-driven efflux function of BCRP. Our induced-fit docking and molecular dynamics simulations suggested the existence of strong and stable interactions between regorafenib and BCRP protein. Animal study revealed that the combination of regorafenib and topotecan resulted in great inhibitory effects on the growth of S1-M1-80 xenograft tumors. Regorafenib significantly increased the intratumoral concentration of topotecan but had no significant effects on topotecan plasma concentration. In conclusion, our study indicated that regorafenib could be useful in combating multidrug resistance in cancer treatments.

#1193

DS-8201a, a novel HER2-targeting ADC with a novel DNA topoisomerase I inhibitor, abrogates the resistance to T-DM1 in HER2-positive gastric cancer: a preclinical study.

Yoshikane Nonagase,1 Naoki Takegawa,1 Kimio Yonesaka,1 Kazuko Sakai,1 Yusuke Ogitani,2 Junji Tsurutani,1 Kazuto Nishio,1 Kazuhiko Nakagawa1. 1 _Kindai University Faculty Of Medicine, Osakasayama, Osaka, Japan;_ 2 _Daiichi-Sankyo Co., Ltd., Tokyo, Japan_.

Background Anti-HER2 therapies are beneficial for patients with HER2-positive breast and gastric cancer. T-DM1 is an HER2-targeting antibody-drug conjugate (ADC), which is structurally composed of the anti-HER2 antibody trastuzumab and the tubulin inhibitor DM1. T-DM1 has shown efficacy in patients with advanced breast cancer, but all patients eventually develop resistance to T-DM1. DS-8201a is a novel ADC composed of an anti-HER2 antibody and a novel potent topoisomerase I inhibitor DX-8951 derivative. DS-8201a achieved a high drug-to-antibody-ratio (DAR: 7-8) and homogeneous drug conjugation. The aim of this study was to elucidate the mechanisms of T-DM1 resistance, and evaluate the efficacy of DS-8201a in a T-DM1-resistant HER2-positive gastric cancer cell line.

Materials and methods The T-DM1-resistant NCI-N87 cell line (N87-TDMR) was established by a step-wise method—the parent HER2-positive N87 cell line was exposed to up to 4 µg/ml of T-DM1. The profile of N87-TDMR was assessed by immunoblotting, DNA microarray, and quantitative reverse transcription-PCR (qRT-PCR). The sensitivity of parent N87 and N87-TDMR cells to T-DM1 or DS8201a was assessed by an in vitro growth inhibition assay as well as through the mouse xenograft model study.

Results N87-TDMR cells were found to be resistant to T-DM1 in the in vitro growth inhibition assay (50% growth inhibitory concentration; >10 µg/ml in N87-TDMR, 0.055 µg/ml in N87) as well as in the in vivo xenograft model study, but HER2 expression was maintained in the N87-TDMR cells. A comprehensive expression analysis revealed that N87-TDMR cells showed increased ATP-binding cassette (ABC) transporter expression such as ABCC2 and ABCG2 compared with N87 cells (50.2 times; 8.0 times higher than that measured by qRT-PCR relatively). Inhibition of ABCC2 and ABCG2 by MK571 recovered the sensitivity to T-DM1 in N87-TDMR cells. Since DS-8201a has higher DAR than T-DM1 and the unique topoisomerase I inhibitor, this resistance was expected to be abrogated. DS-8201a showed anti-tumor efficacy against N87-TDMR xenograft cells in the mouse model (relative tumor growth rate: 244.2% with T-DM1, -39.2% with DS-8201a; n=10 for each treatment).

Conclusions The HER2-positive gastric cancer cell line used in this preclinical study acquired resistance to T-DM1 depending on the up-regulation of ABCC2 and ABCG2 expression. However, DS-8201a could abrogate this T-DM1 resistance. These results provide a rationale for DS-8201a to be investigated for its efficacy in patients with T-DM1-resistant breast or gastric cancer.

#1194

Neutralization of IGF-I and -II ligands with the fully humanized bispecific monoclonal antibody xentuzumab inhibits AR-V7-induced enzalutamide resistance in castration-resistant prostate cancer.

Gang Liu,1 Holly M. Nguyen,1 Kristine van Maltzan,1 Shihua Sun,1 Cynthia Sprenger,1 Ulrike Weyer-Czernilofsky,2 Eva Corey,1 Stephen R. Plymate1. 1 _Univ. of Washington, Seattle, WA;_ 2 _Boehringer Ingelheim, Ingelheim, Germany_.

Background: Recent data from multiple investigators have shown that an increase in constitutively active androgen receptor (AR) variants such as AR-V7 is associated with more aggressive prostate cancer and a significantly shortened overall survival. Additionally, expression of AR-V7 in circulating tumor cells or increased expression in metastatic tissue is a biomarker for resistance to currently available anti-androgen therapies, including enzalutamide (ENZA) and abiraterone, resulting in castration resistant prostate cancer (CRPC). Insulin-like growth factor (IGF) signaling pathways have been incriminated as mechanisms of resistance but little is known about the potential role of IGFs in CRPC growth in relation to AR variants. In this preclinical study we examined the effects of IGF-I and -II inhibition in LuCaP 96CR, an ENZA resistant prostate cancer patient-derived xenograft.

Hypothesis: IGF-I and -II neutralization will block AR-V7 activity and inhibit castration-resistant prostate cancer growth.

Study procedures: LuCaP 96CR was implanted s.c. into castrate SCID mice (15 mice per group). When tumors exceeded 150mm3 animals were randomized into groups: 1) Control; 2) ENZA (50 mg/kg, QD, po), 3) xentuzumab (BI 836845[1], 200 mg/kg QW IP) in combination with ENZA. At the end of the study, tumors were collected for preparation of RNA and protein lysates and histology.

Results: ENZA did not show significant inhibitory effects on LuCaP 96CR, but the combination of xentuzumab and ENZA resulted in significant tumor inhibition (p<0.001) vs. ENZA alone. AR full length (AR-FL) mRNA and protein increased after ENZA treatment (p<0.001) but did not further rise significantly when xentuzumab was co-administered. In contrast, significantly elevated AR-V7 mRNA and protein levels were detected in tumors from mice treated with the xentuzumab and ENZA combination (p<0.001). Downstream markers of AR-V7 activity, UBE2C and UGT2B17 mRNA, increased after ENZA treatment when compared to vehicle (UGT2B17, p<0.01); no further significant increase of UGT2B17 was detected after combination treatment and UBCE2C significantly decreased (p<0.001).

Summary: These data show that addition of xentuzumab provides tumor inhibition of ENZA-resistant CRPC. Interestingly, associated with a significant decrease in tumor growth, this treatment resulted in increased AR-V7 mRNA and protein expression but no significant increases in downstream markers of AR-V7 activity, UBE2C and UGT2B17.

Conclusion: Our results suggest that the IGF-I and -II neutralizing antibody xentuzumab may reverse AR-V7-mediated ENZA resistance.

#1195

TNFα induces multiresistance to HER2-targeted TNFα induces multiresistance to HER2-targeted therapies in HER2-positive breast cancer.

María F. Mercogliano,1 Mara De Martino,1 Sofia Bruni,1 Leandro Venturutti,1 Martín Rivas,2 Matías Amasino,1 Cecilia J. Proietti,1 Patricia V. Elizalde,1 Roxana Schillaci1. 1 _IBYME-CONICET, CABA, Buenos Aires, Argentina;_ 2 _Weill Cornell Medical College, Manhattan, NY_.

HER2 positive (HER2+) is a breast cancer (BC) subtype characterized by HER2 overexpression/amplification that affects nearly 15% of BC patients and correlates with poor prognosis. These patients receive trastuzumab (T), an anti-HER2 monoclonal antibody, but resistance events (40-60%) hamper its clinical benefit. Previously we have demonstrated that TNFα (TNF) induced mucin 4 (MUC4) expression and turned T-sensitive cell lines and tumors into resistant ones.

Nowadays, new anti-HER2 therapies are being used in the clinical setting, such as lapatinib (a dual inhibitor of EGFR and HER2), and antibodies like T-DM1 (combines TZ with the anti-microtubule agent

emtansine), and pertuzumab (P) that impeds HER2 dimerization.

The aim of this work was to study the role of TNF in resistance to the new HER2-targeted therapies.

We used BT-474-C (control cells) and BT-474-T2, engineered in our lab to stably overexpress TNF, and were proven to be sensitive and resistant to T, respectively.

We performed dose-response curves for T-DM1, they show that inhibits proliferation of BT-474- C cells at 0.01 μg/ml. On the other hand, BT-474-T2 cells were resistant in the same experimental conditions and they exhibited reduced T-DM1 binding with respect to BT-474-C. BT-474-C cells were sensitive to low concentrations of T-DM1 with 0.51 nmol/L, but in BT-474-T2 cells T-DM1 was ~10 times less potent than control cells (IC 50 3.34 nmol/L). When we abrogated MUC4 expression, BT-474-T2 cells were sensitized to T-DM1, showing that TNF-induced MUC4 expression is responsible for T-DM1 resistance in this cell line.

We assessed the effect of lapatinib performing a dose-response curve. Results shown a similar IC50 for BT-474 C and T2 cells (0.26 μM and 0.28 μM, respectively).

When we studied P effect, we observed that the combination of T+P was more effective inhibiting proliferation in BT-474-C cells than T alone, despite these results binding of the antibody showed no change between the cell lines. In BT-474-T2 cells proliferation was slightly inhibited by the combined treatment. In vivo experiments showed that BT-474-C tumors were sensitive to T and the combination of T+P, but BT-474-T2 tumors did not respond to any of these treatments.

These results suggest that TNF plays an important role in multiresistance to HER2-targeted therapies, specifically T-DM1 and P, but not in lapatinib resistance. We propose TNF as an attractive target and we suggest that HER2+ patients resistant to T could be eligible for a combination of HER2-targeted therapies and a TNF-blocking treatment to overcome resistance.

#1196

Epigenetic drugs modulate long noncoding RNAs expression in BRAF inhibitor-resistant melanoma.

Barbara Montico,1 Giorgio Giurato,2 Katy Mastorci,1 Aurora Rizzo,1 Maria Ravo,2 Francesca Rizzo,2 Alessandro Weisz,2 Riccardo Dolcetti,3 Francesca Colizzi,1 Luca Sigalotti,4 Elisabetta Fratta1. 1 _Centro di Riferimento Oncologico, Aviano, Italy;_ 2 _University of Salerno, Salerno, Italy;_ 3 _University of Queensland Diamantina Institute, Brisbane, Australia;_ 4 _University Hospital of Udine, Udine, Italy_.

Emergence of drug resistance is the major cause of failure of BRAF inhibitors (BRAFi) treatment in cutaneous melanoma (CM). Epigenetic modifications are known to physiologically trigger massive modifications in cellular commitment and several studies report a correlation between the drug-resistant phenotype and epigenetic alterations of tumor cells. In this setting, long noncoding RNAs (lncRNAs) represent a class of gene regulators acting at epigenetic, transcriptional and post-transcriptional level. Several studies have implicated lncRNAs in chemoresistance through their ability to impair cell cycle arrest and apoptosis, but also to induce and modulate epithelial-mesenchymal transition and cell adhesion-associated signaling pathways. LncRNAs interact with histone modifying complexes and/or DNA methyltransferases, being also targets of these epigenetic mediators. Furthermore, epigenetic drugs have been recently identified as modulators for lncRNAs function as well as their related targeting signals. Starting from these evidences, we asked the question whether epigenetic drugs could differentially affect the survival of BRAFi-resistant (VR) and -sensitive CM cells, investigating the mechanistic network involved, with a specific focus on the role of lncRNA. A panel of BRAFi-sensitive and VR CM cell lines was treated with the FDA-approved HDAC inhibitor vorinostat (SAHA). FACS analysis of annexin V-FITC/propidium iodide stained cells showed that SAHA cytotoxic activity was more pronounced on VR CM cells than on their parental counterparts. RNA-Seq analysis revealed that a large number of differentially expressed lncRNAs was modulated in VR CM cells treated with SAHA. Intriguingly, the expression of several VR up-regulated lncRNAs was decreased to levels similar to those observed in the matched parental cells. Functional analysis indicated these lncRNAs were statistically enriched in pathways involving cellular growth and proliferation, but also cellular assembly and organization. Though additional studies are required, epigenetic modulation of VR-associated lncRNAs promises to have significant therapeutic potential to restore BRAFi sensivity in CM, being concomitantly effective in killing VR cells as monotherapy. Based on our preliminary data, we could anticipate that the combined use of epigenetic and targeted drugs would increase therapeutic efficacy in CM patients relapsing to BRAFi.

#1197

Endothelin-1 receptor/β-arrestin1 is an actionable node that regulates YAP/TAZ signaling and chemoresistance in high-grade ovarian cancer.

Piera Tocci,1 Roberta Cianfrocca,1 Laura Rosanò,1 Rosanna Sestito,1 Valeriana Di Castro,1 Giovanni Blandino,2 Anna Bagnato1. 1 _Preclinical Models and New Therapeutic Agents Unit - Regina Elena National Cancer Institute, Rome, Italy;_ 2 _Oncogenomic and Epigenetic Unit - Regina Elena National Cancer Institute, Rome, Italy_.

The widespread activation of YAP and TAZ transcriptional regulators has been shown to promote tumor progression and resistance to anti-cancer therapies. Extensive studies have identified different upstream regulators of YAP/TAZ, including G-protein coupled receptor (GPCR). However how GPCR signals regulate YAP/TAZ puzzles have remained elusive. Among the GPCR, we reveal that the endothelin-1 (ET-1) receptor activates YAP/TAZ pathway in high-grade serous ovarian cancer (HG-SOC) sensitive and resistant cell lines and primary cultures. In HG-SOC cells, the aberrant activation of ET-1 receptor (ET-1R), promotes an enhanced nuclear shuttling of dephosphorylated YAP and TAZ in a time-dependent manner, through the scaffold protein β-arrestin1 (β-arr1). Interestingly, ET-1-induced YAP/TAZ nuclear accumulation is further improved in platinum-resistant cells and is prevented by expressing a mutant β-arr1 incapable of nuclear distribution. Moreover, the ET-1/ETAR axis activation induces the co-localization of β-arr1, YAP and TAZ in the nucleus. Mechanistically, in sensitive and to a greater extent in resistant cells, ET-1 induces the formation of a complex β-arr1/Trio, a Rho-Guanine nucleotide exchange factor family member, which dephosphorylates YAP, inducing its nuclear accumulation and the transcription of YAP/TAZ target genes. β-arr1, RhoA or Trio depletion, as well as the treatment with the dual ETAR/ETBR antagonist macitentan, significantly reduces RhoA GTPase activity and YAP nuclear translocation, suggesting that ET-1/ETAR axis regulates YAP/TAZ activity by driving β-arr1/Trio/RhoA pathway. According with these results, the ET-1-induced upregulation of YAP/TEAD target genes, such as CYR61, CTGF, ANKRD1 and EDN1, as well as TEAD transcriptional activity and cell invasion, are inhibited upon β-arr1, Trio, or YAP silencing, or macitentan treatment, demonstrating that β-arr1-mediated intersections are required for ET-1-induced YAP signaling. At the chromatin level, the activation of ET-1 axis promotes the recruitment of YAP, TEAD and β-arr1 on TEAD binding sites of CTGF, ANKRD1 and EDN1 promoters, an effect inhibited by macitentan treatment. These results indicate that β-arr1 can act as a transcription co-activator that bind TEAD thereby activating transcription of EDN1, which in turn, can sustain persistent YAP/TAZ activity through an autocrine loop. In murine orthotopic model of patient-derived xenograft (PDX) of HG-SOC, ET-1R blockade by macitentan, inhibits tumor growth, enhances the sensitivity to chemotherapy and reduces YAP and TEAD transcriptional activity. Altogether our results establish for the first time YAP/TAZ as critical downstream effectors of ET-1R/β-arr1 signaling, providing mechanistic insights that targeting ET-1R signaling overcomes YAP/TAZ driven platinum-based therapy failure in HG-SOC.

#1198

Neuropilin-1 blockade chemosensitizes pancreatic cancer cells via dual inhibition of epithelial-mesenchymal transition and autophagy.

Pratiek N. Matkar, Krishna K. Singh, Gerald Prud'homme, David Hedley, Howard Leong-Poi. _Univ. of Toronto, Toronto, Ontario, Canada_.

Introduction: Fibrotic and malignant pancreatic ductal adenocarcinoma (PDAC) tumors are often resistant to chemotherapy, and remain a leading cause of morbidity and mortality. Epithelial–mesenchymal transition (EMT), a process by which epithelial cells lose their cell polarity and adhesion to become migratory and invasive mesenchymal stem cells, contributes to chemoresistance in PDAC. Likewise, PDAC tumors benefit from autophagy, a self-degradation pathway that confers a survival benefit against metabolic stress. Over-activation of EMT and autophagy has been linked to increased drug resistance in PDAC. Both these processes are largely mediated by TGFβ1. Recently, we demonstrated the role of neuropilin-1 (NRP-1) in TGFβ1-dependent endothelial-mesenchymal transition and fibrosis in PDAC. However, the potential mechanisms linking NRP-1 with EMT and autophagy-mediated chemoresistance remain unexplored. We hypothesized that NRP-1 blockade will enhance chemotherapeutic effect, by suppressing EMT and autophagy in PDAC.

Methods: Human PDAC cells (BxPC-3) or serially gemcitabine exposed chemoresistant (CR-BxPC-3) cells were transfected with siNRP-1 or scramble using DharmaFECT-2. For EMT and chemotherapy studies, transfected cells were treated with TGFβ1 (5ng/mL, 24h), followed by gemcitabine (1μM) for 48h. RNA (24h) and protein (48h) was isolated using TRIzolTM and RIPA, respectively. EMT markers were evaluated by RT-PCR and immunoblotting/immunostaining. For autophagy studies, cells were serum starved post NRP-1 silencing and protein was isolated. For autophagy inhibition, cells were treated with bafilomycin-A1 (100nM) for 24h post NRP-1 silencing and protein was isolated. Autophagy flux (LC3-II/I) and P62 were measured by immunoblotting/immunostaining. Cell viability was evaluated by MTT assay.

Results: SiNRP-1 treatment in BxPC-3 and CR-BxPC-3 cells resulted in NRP-1 knockdown and promoted cell-death after chemotherapy. TGFβ1-stimulated BxPC-3 cells demonstrated a morphological change consistent with EMT that was revoked by siNRP-1. Also, siNRP-1 maintained epithelial cellular morphology via gain of epithelial and loss of mesenchymal markers. Remarkably, CR-BxPC-3 cells exhibited enhanced EMT features. SiNRP-1 inhibited EMT and promoted cell-death by overcoming gemcitabine resistance in CR-BxPC-3 cells, possibly through reduced ERK signaling. Moreover, siNRP-1 inhibited autophagy (decreased LC3-II/I ratio and increased P62 levels) following starvation or chemotherapy stress, and enhanced cell-death after chemotherapy and autophagy inhibition in BxPC-3 cells.

Conclusions: Our findings define a previously undetermined role of NRP-1 in regulating autophagy and TGFβ1-induced EMT in PDAC, suggesting that NRP-1 may represent a novel therapeutic target to overcome chemoresistance through reduced EMT and autophagy.

#1199

**Overcomes AXL and Met mediated erlotinib/gefitinib cross resistance in non-small cell lung cancer cells by** Marsdenia tenacissima **extract.**

Shu-Yan Han, Hong Sun, Dong Xue, Wei Zhao, Yan-Na Jiao, Ping-Ping Li. _Peking University Cancer Hospital & Institute, Beijing, China_.

Apart from EGFR T790M mutation, the bypass activation of c-Met and Axl kinase also can lead to the resistance to tyrosine kinase inhibitors (TKIs) in NSCLC. Axl and c-Met share same downstream pathways with EGFR, thus combined treatments of EGFR inhibitors with Axl or Met inhibitor are promising to overcome acquired resistance of TKIs.

Our previous work showed that the water extract of Marsdenia tenacissima (MTE), which used to treat cancer in clinics for decades, restored gefitinib sensitivity in resistant NSCLC cells with EGFR T790M mutation or K-ras mutations in vitro and in vivo. However, the potential effeicacy of MTE on Axl and c-Met mediated resistance has not yet been fully understood, and the related molecular mechanisms also need to be elucidated.

The present study was performed on HCC827/ER cells, which was established by exposing parental HCC827 cells to erlotinib. HCC827/ER cells are with Axl activiation and c-Met amplification, and show dual-resistance to erlotinib and gefitinib. We evaluated the effects of MTE to restore erlotinib/gefitinib sensitivity with three different combinations. Cell viability and cell apoptosis were determined by MTT and flow cytometry, respectively. The c-Met amplification was assessed with TaqMan real-time PCR. Signaling pathways were examined by Western blotting to reveal the possible mechanisms. The in vivo effects of MTE combined with erlotinib/gefitinib were tested on HCC827/ER xenograft mice, and tumor tissues were subjected to immunohistochemistry analysis and Western blotting.

Our results indicated the MTE→MTE+Erlotinib/Geftinib (M→M+E/G) treatment was the most potent combinations. Compared with control group and each single, M→M+E/G treatment induced significant apoptosis, obviously inhibited EGF-induced phosphorylation of PI3K/Akt/mTOR and ERK1/2, down-regulated HGF/c-Met activation in HCC827/ER cells. Axl is a receptor tyrosine kinase which strongly associated with EMT phenotype. The M→M+E/G treatments could not restrain Axl expressions but remarkably reduced phospho-Axl levels and inhibited EMT phenotype in HCC827/ER cells. Surprisingly, MTE alone caused prominent p-Axl inhibition, along with up-regulated E-cadherin and decreased

mesenchymal markers. The mouse tumors were remarkably restrained by the M→M+E/G combinations, and it was significant compared with each drug alone (P<0.05). Accordingly, cell apoptosis was extended, PCNA expression and tumor angiogenesis (VEGF and CD105) were reduced and EMT phenotype were regulated in tumor tissues. The p-Met and p-AXL were also considerably suppressed by the combined treatments.

The present data revealed that MTE restored TKIs such as erlotinib and gefitinib efficacy in resistant NSCLC cells with Axl activation and c-Met amplification in vitro and in vivo. It suggests that the addition of MTE may be a promising therapeutic strategy to overcome TKIs resistance in NSCLC.

#1200

Novel strategy to overcome platinum resistance in uterine leiomyosarcoma; blocking ATP7B by copper ion.

Mamoru Kakuda, Shinya Matsuzaki, Ruriko Nakae, Yusuke Tanaka, Eiji Kobayashi, Yutaka Ueda, Kiyoshi Yoshino, Tadashi Kimura. _Osaka University, Suita-City, Japan_.

Objective: Resistance to platinum drugs remains a significant problem. Our objective is to elucidate the role of ATP7B, a copper transporter which excretes intracellular platinum giving drug resistance, in gynecologic malignancies and to develop a method which can overcome the platinum resistance.

Methods: The expression of ATP7B was evaluated in 3 uterine leiomyosarcoma (LMS) cell lines (SK-LMS, SK-UT1 and SKN) and 26 clinical samples of LMS by immunohistochemical (IHC) staining, respectively. The IC50 values of cisplatin were measured by modified MTT assay. Intracellular platinum accumulation was determined by ICP-MS analysis in SK-LMS cells and ATP7B-stably suppressed cells (SK-LMS-7B cells), which transfected the pRS ATP7B shRNA vector. To analyze in vivo platinum sensitivity, SK-LMS and SK-LMS-7B cells were subcutaneously injected into nude mice. Cisplatin (3 mg/kg) or PBS was administered i.p. to xenografted mice twice weekly for 28 days. Further, we attempted to show the improvement of platinum sensitivity by using premedication for CuSO4 before cisplatin treatment in vitro and in vivo model.

Results: Expression of ATP7B was observed in all 3 LMS cell lines. In clinical samples, ATP7B was over expressed in 14 of 26 (53.8%) cases. In drug resistance analysis, significantly lower IC50 values of cisplatin were observed in SK-LMS-7B cells (4.3μM, p < 0.01) compared with SK-LMS cells (17μM) indicating knockdown of ATP7B improved platinum sensitivity. Also, significantly higher platinum accumulation was detected in SK-LMS-7B cells (7.2ng/dish, p < 0.01) compared with SK-LMS cells (52ng/dish). In in vivo analyses, a significantly higher anti-tumor effect of cisplatin was observed in SK-LMS-7B-xenografted mice compared with SK-LMS-xenografted mice (56±27 μg vs 214±68 μg, p<0.05). Pretreatment with CuSO4 (15μM) before cisplatin treatment for SK-LMS cells significantly reduced IC50-values (17 μM to 3.9 μM, p<0.05). In addition, an elevated intracellular platinum accumulation was observed in cells from pretreatment group (15.0 to 68.4 ng/dish, p<0.05). A significant improvement of platinum sensitivity was observed in the premedication group (CuSO4 premedication group; 65±40 μg vs no pretreatment group; 230±60 μg, p<0.05).

Conclusion: ATP7B contributes platinum resistance in LMS. Presumably, ATB7B preferentially excrete copper ion thereby platinum can be accumulate in cells. Thus, pretreatment with CuO4 effectively improved platinum sensitivity in vitro and in vivo by blocking ATP7B.

#1201

Disrupted endoplasmic reticulum-mitochondria contacts promote multidrug resistance.

Jorida Coku,1 Madison C. Pedrotty,1 David M. Booth,2 Sharon Kim,1 Annette Vu,1 C. Patrick Reynolds,3 György Hajnóczky,2 Michael D. Hogarty1. 1 _University of Pennsylvania, Philadelphia, PA;_ 2 _Thomas Jefferson University, Philadelphia, PA;_ 3 _Texas Tech University Health Sciences Center, Lubbock, TX_.

Background: Most high-risk neuroblastoma patients succumb to lethal therapy resistant disease acquired during the course of intensive multimodality treatment. This acquired therapy resistance is largely attributed to insensitivity to drug-induced apoptosis, however the exact mechanisms remain unknown. Apart from integrating death signals, mitochondria (mito) interact with the endoplasmic reticulum (ER) at close contact sites known as mitochondria associated membranes (MAM) of the ER to regulate calcium and lipid transfer and apoptotic sensitivity, a process often derailed in therapy resistant cancers. ER-mito contact sites are juxtaposed by various tethering protein complexes that include MFN2 and PACS2. Pathologic deregulation of these contact sites has been implicated in the genesis of neurodegenerative and metabolic disorders. Here, we show that disruption of bona fide ER-mito tethering proteins in therapy sensitive neuroblastomas induces apoptotic insensitivity and a drastic shift towards a resistance-like phenotype.

Methods and Results: Previously we showed that isolated mitochondria from tumors at relapse resist induction of mitochondrial apoptosis when primed with the terminal death effectors that are downstream of therapeutic stress (tBid and Bim-BH3 peptide). We isolated mitochondria from seven matched isogenic tumor pairs obtained at diagnosis (DX) and relapse (REL) and quantified their apoptotic response to tBid and Bim by measuring cytochrome C release by ELISA. Electron microscopy (EM) image analyses of ER-mito contact sites revealed that REL tumors contain up to 70% fewer ER-mito interactions than their matched DX tumors, as confirmed by IB for organelle-specific proteins. Here, we recapitulate the post-therapy resistance phenotype in DX therapy sensitive cells by shRNA silencing of MFN2 or PACS2 and confirm their apoptotic resistance by BH3 profiling and drug response in vitro. A 60% decrease in MFN2 protein in DX cells partially phenocopied the resistance profile of isogenic REL cells. The degree of apoptotic resistance correlated with the extent of protein knockdown for MFN2. Treatment of shMFN2 and shPACS2 DX cells with ABT-737, a BH3 mimetic, or carboplatin, increased their IC50s multiple-fold compared to control cells, paralleling their blunted mitochondrial apoptotic response.

Conclusions: Our data implicate ER-mito contact sites as positive regulators of apoptosis, whose disruption may be necessary for apoptotic attenuation in therapy resistant cancer cells to enable apoptosis evasion and survival. We present a potential mechanism for broad therapy resistance arising under therapeutic stress that selects for reduced communication of ER with mitochondria.

#1202

Targeting PIK3C3 to overcome drug resistance resulted from prosurvival autophagy in cancer cells.

CHUN-HAN CHEN. _Taipei Medical University, Taipei, Taiwan_.

Autophagy is an important cellular recycling mechanism in which portions of cytosol or organelles are sequestered into a double-membrane structure and delivered to lysosome for degradation. PIK3C3 is the only class III PI3K, which specifically generates phosphatidylinositol 3-phosphate (PtdIns3P). It in turn recruits proteins containing FYVE or PX domains, thereby initiating various complexes at the membranes of endosomes, phagosomes and autophagosomes. Recently, several studies have shown that autophagy is upregulated in cancer cells when exposing to chemotherapy and radiotherapy. Therefore, the rationale of targeting autophagy addiction in cancer was proposed by combining autophagy inhibition with agents that induce autophagy as a pro-survival response to increase their therapeutic efficacy. We stably knocked down PIK3C3 in cancer cell lines and combined with clinical therapeutic agents reportedly induce pro-survival autophagy. Cell viability was examined by MTT assay and combination index was determined by Compusyn software. The autophagy flux was evaluated by detecting protein levels of LC-3II and p62 through western blot analysis. The results showed PIK3C3 knockdown synergistically sensitize the killing effects of gefitinib and gemcitabine in A549 cells and Panc1 cells, respectively. Both of gefitinib and gemcitabine increased autophagy flux, as evidence by the increase of LC3-II and decrease of autophagy substrate, p62. However, the combination of PIK3C3 knockdown and therapeutic agents increased both of the protein levels of LC3-II and p62. These data suggest incomplete autophagy when the function of PIK3C3 was inhibited. The phenomenon was confirmed by bombing autophagy inducer and PIK3C3 inhibitors. Moreover, no appreciable apoptotic cell death was observed. Taken together, our study revealed a potential strategy by targeting PIK3C3 to overcome drug resistance associated with the pro-survival autophagy in cancer.

#1203

MiR-25 can sensitize cisplatin-resistant ovarian cancer cells to cisplatin through enhancing cisplatin-mediated G2/M arrest.

Huaping Chen, Adam Steg, Zachary C. Dobbin, Ashwini A. Katre, Ronald D. Alvarez, Charles N. Landen. _University of Alabama at Birmingham, Birmingham, AL_.

Recurrent chemoresistant ovarian cancer is responsible for the high mortality rate in ovarian cancer patients. The molecular mechanisms underlying the chemoresistant ovarian cancer are not fully understood. MicroRNAs (miRs) expression has been recognized as one of the core regulators of gene expression in recent years. However, whether miRs are involved in chemoresistance of ovarian cancer has not been fully elucidated. Current study was performed to interrogate the role of a number of critical miRs in chemoresistance of ovarian cancer. These miRs include miR-21, miR-25, miR-128a, miR-141, miR-182, miR-214, miR-29c, miR-200a, miR-506 which have been identified to play key roles in ovarian cancer by previous study. The association between expression pattern of these miRs and chemoresistance of ovarian cancer was revealed through comparing their expression in chemosensitive versus -resistant ovarian cancer cell lines or chemosensitive and treated recurrent chemoresistant patient derived xerograph (PDX) samples via Taqman real-time quantitative PCR. Involvement of aberrant expressied miRs in chemoresistant ovarian cancer was further validated through treating cells with antagomers or mimics for corresponding miRs. Cell cycle analysis was performed to study the role of miRs in cell cycle progression. We demonstrate that miR-21, miR-25, miR-128a, miR-141, miR-182, miR-214 and miR-506 are upregulated, while miR-29c and miR-200a are downregulated in chemoresistant ovarian cancer cells or PDX samples. Inhibiting miR-21, miR-128a, miR-182 or overexpression of miR-101 does not lead to significant sensitization, suggesting the differential expression of these miRs in chemoresistant cells does not contribute directly to ovarian cancer cell-mediated chemoresistance. However, inhibiting miR-25 leads to a 3-fold decrease in the IC50 to cisplatin in A2780CP20 cells (from 1 μM to 0.333 μM), indicating sensitization to chemotherapy. Furthermore, inhibiting miR-25 can arrest more cells in G2/M phase by itself or in combination with cisplatin. Collectively, these data indicate that miR-25 could be a potential therapeutic target for treating cisplatin resistant ovarian cancer patients.

#1204

Enhanced efficacy of selumetinib by pretreatment of 5-FU in preclinical Ras/Raf-mutant colorectal cancer models.

Hye Yeon Jang,1 Haeng Jung Lee,2 Yeo Jin Nam,1 Won Dong Kim,3 Yong Ju Bae,3 Jin Hwang Jung,4 Seung Jin Lee,1 Dae Hyuk Moon5. 1 _Chungnam National University, Daejeon, Republic of Korea;_ 2 _Asan Institute for Life Sciences, Asan Medical Center, Seoul, Republic of Korea;_ 3 _Gachon University, Incheon, Republic of Korea;_ 4 _Asan Medical Center, University of Ulsan, College of Medicine, Seoul, Republic of Korea;_ 5 _Asan Medical Center, University of Ulsan, Seoul, Republic of Korea_.

Optimal strategy for the application of MEK inhibitor as combination therapy has not been established in Ras/Raf-mutant colorectal cancer (CRC). As we preliminary observed that 5-FU-induced pharmacodynamic changes were restored by ERK inhibition, we hypothesized that post-treatment of MEK inhibitor would enhance antitumor activity of 5-FU. Treatment of 5-FU for 2 days followed by selumetinib for another 2 days (FS schedule) exhibited synergism for cell viability, whereas reverse or concomitant combination showed antagonism or inconsistent response, respectively, in 8 kinds of CRC cell lines including BRAFV600E colo205, BRAFV600E RKO, or KRASG13D HCT8 cells. Combination as FS schedule persistently down-regulated the phosphorylation of ERK and pS6 throughout treatment period, whereas selumininib alone or reverse combination transiently decreased within 24 hr. The cells treated as FS schedule exhibited higher apoptosis and lower capacity of anchorage-independent cell growth than the cells under single or reverse combination. Microarray analysis revealed the distinct groups of genes underlying different efficacy by schedule-dependent treatment. Tumor growth in mice administered 5-FU at 10 mg/kg/day for colo205 and at 30 mg/kg/day for HCT8 for 7 days followed by selumetinib at 10 mg/kg/day for colo205 and 25 mg/kg/day for HCT8 for another 7 days was significantly retarded than that in mice treated with single agents. But the combination in reverse sequence induced comparable tumor growth to single agents. Decreased expression of Ki67 was observed in tumors from mice treated as FS scheduling. Our results suggest that the schedule-dependent combination of 5-FU and selumetinib may be benefit in patients having CRC with hyper-activation of Ras/RAF pathway.

#1205

**Rubrofusarin and toralactone sensitize resistant MCF-7** adr **cell line to paclitaxel via inhibiting P-glycoprotein efflux activity.**

Salwa D. Alqahtani,1 Hanan A. Assiri,1 Fahad A. Al-Abbasi,1 Ali M. El-Halawany,2 Ahmed M. Al-Abd3. 1 _King Abdulaziz University, Jeddah, Saudi Arabia;_ 2 _Cairo University, Cairo, Egypt;_ 3 _National Research Centre of Egypt, Giza, Egypt_.

The use of anti-cancer adjuvant therapy is rationalized by potentiating the efficacy and/or protecting from major side effects of chemotherapeutics. Paclitaxel is an effective spindle toxin anticancer agent used for the treatment of breast cancer. Rubrofusarin and toralactone are naturally occurring structurally related naphthopyrones with potent antioxidant and hence chemoprotective activity. The purpose of this study is to investigate the influence of rubrofusarin and toralactone on the cytotoxic profile of paclitaxel (PTX) against chemo-resistant breast cancer cell line (MCF-7adr) compared to parent MCF-7 cells. MCF-7adr was prepared by continuous culturing of MCF-7 cells in media containing subcytotoxic concentration of doxorubicin (IC10%) for two months duration. Toralactone and rubrofusarin showed moderate cytotoxic effects against MCF-7 cells with IC50's of 32.4±4.2 µM and 13.9±6.1 µM, respectively. Interestingly, toralactone and rubrofusarin showed stronger cytotoxicity against MCF-7adr cells with IC50's of 7.3±0.7 µM and 7.8±0.6 µM, respectively. PTX treatment was opposed by significant resistance from MCF-7adr cells compared to parent MCF-7 cells having IC50's of 4.9±0.9 µM and 57.7±4.5 nM, respectively. Combination of torolactone or rubrofusarin with PTX did not significantly improve its cytotoxicity against MCF-7; IC50's were 24.2±2.2 nM and 69.7±6.6 nM, respectively compared to 57.7±4.5 nM for PTX alone. Surprisingly, combination with either torolactone or rubrofusarin abolished the resistance of MCF-7adr to PTX resulting in IC50's of 3.4±0.2 nM and 4.9±0.5 6 nM, respectively compared to 4.9±0.9 µM for PTX alone. Using flowcytometric analysis, rubrofusarin and toralactone increased median intracellular fluorescence signal count of rhodamine dye (non-cytotoxic P-gp substrate) within MCF-7adr from 1794.7±21.5 to 2003.3±12.5 and 2113±10.5 cellular events, respectively. Using purified membrane bound humanized recombinant P-gp molecules; it was found that both rubrofusarin and toralactone might be inhibiting P-gp efflux activity via dual interaction with both P-gp ATPase enzyme subunit and P-gp active binding site. In both MCF-7 and MCF-7adr cells, PTX exerted significant cell cycle block at G2/M-phase with significant increase in the pre-G dead cell fraction from 3.4±0.9% to 23.6±2.1% and from 1.4±0.6% to 17.2±3.2%, respectively. Neither rubrofusarin nor toralactone exerted any significant influence for PTX induced cell cycle interruption in both normal (MCF-7) and resistant (MCF-7adr) cell lines. In conclusion, rubrofusarin and toralactone can enhance the cytotoxic profile of P-gp substrates, such as PTX, via inhibiting P-gp efflux activity and enhancing the intracellular entrapment of P-gp substrates.

#1206

miR-205 replenishment in prostate cancer cells: A novel nanoparticle approach.

Prashanth K. Nagesh, Pallabita Chowdhury, Vijayakumar N. Boya, Vivek K. Kashyap, Sheema Khan, Bilal B. Hafeez, Nadeem Zafar, Stephen W. Behrman, Subhash C. Chauhan, Meena Jaggi, Murali M. Yallapu. _University of Tennessee Health Science Center, Memphis, TN_.

Introduction

Prostate cancer (PrCa) is the most common male malignancy among men in the United States. Recent studies suggest that low expression of miR-205 is seen in PrCa cell lines and tumors in comparison to normal prostatic epithelial cells. A numer of studies have shown that restoration of miR-205 in PrCa cells resulted in supression of cell growth, epithelial-to-mesenchymal transition, and chemosensitization. However, due to the poor pharmacological kinetics and low in vivo stability of miR-205, serious limitations are being experienced at the clinical level. Therefore, we have chosen a novel nanoparticle-based approach to deliver miR-205, for improved therapeutic benefits in PrCa.

Methods

A novel miR-205 nanoparticle formulation (named miR-MPG) was generated which is composed of an iron oxide core layered with polyethylene imine (PEI), and NHS-PEG-NHS (PEG) polymer. The miR-205 withholding and release characteristic of miR-PEG were examined through fluorescence quenching and agarose gel electrophoresis. Hemocompatibility of this formulation was examined using a hemolysis assay. Cellular uptake of miR-MPG formulation was evaluated using flow cytometry and confocal studies. Further, therapeutic and chemosensitization activity of miR-205 were assessed using cell-culture based assays. Molecular effects associated with the PrCa cells growth inhibiton were evaluated through protein profiling and qRT-PCR analyses.

Results and Dissusion

miR-MPG formulation exhibited optimal particle size and zeta potential, which are suitable for cancer therapeutics. Agarose gel electrophoresis binding studies suggested 5 µg of nanoparticle formulation is optimum to hold 1 µg of miR-205 mimic. Release of miR-205 from miR-MPG was determined with respect to concentration of anionic molecules and in a time-dependent manner. We observed no hemolysis during miR-MPG interaction with the red blood cells indicating its hemocompatibility. In addition, miR-MPG particles exhibited superior internalization and endosmal escape in PrCa cells. This formulation displayed enhanced sensitization of PrCa cells to docetaxel. Additionally, it induced the expression of apoptotic proteins (Bax, Bim, cleaved PARP, and caspase 3), and downregulated the anti-apoptotic proteins (Bcl-2 and survivin). Moreover, the expression of the chemoresistance-associated proteins (PSMA and MDR1) was profoundly inhibited in cells treated with miR-MPG in the presence of docetaxel. Further dataset of qRT-PCR studies showed induced expression of the miR-205 and affected the expression of its downstream genes. These results suggest that miR-MPG formulation may serve as an ideal delivery vehicle to deliver miR-205.

Conclusion

Results from this study suggests that successful delivery of miR-205 through miR-MPG nanoparticles can induce sensitization potential for docetaxel treatment. This novel therapeutic modality might be effective for PrCa patients undergoing chemotherapy.

#1207

**SNS-062 demonstrates efficacy in chronic lymphocytic leukemia** in vitro **and inhibits C481S mutated Bruton tyrosine kinase.**

Catherine A. Fabian,1 Sean D. Reiff,1 Daphne Guinn,2 Linda Neuman,3 Judith A. Fox,3 Wendy Wilson,3 John C. Byrd,1 Jennifer A. Woyach,1 Amy J. Johnson1. 1 _The Ohio State University, Columbus, OH;_ 2 _Georgetown University, Washington, DC;_ 3 _Sunesis Pharmaceuticals, South San Francisco, CA_.

Introduction: In order to address the issue of acquired resistance to ibrutinib, we sought to characterize the Bruton agammaglobulinemia tyrosine kinase (BTK) inhibitor SNS-062 in preclinical models of chronic lymphocytic leukemia (CLL). Methods: Primary CLL B cells were isolated from the whole blood of consented patients by ficoll density centrifugation and Rosette-Sep negative selection. Annexin V and propidium iodide flow cytometry was used to measure patient CLL cell viability and 7-AAD was used to measure viability in stromal co-culture. CD40 and CD86 expression was evaluated via flow cytometry subsequent to sustained 3.2uM CpG stimulation. BCR signaling in primary CLL cells was investigated by immunoblot following 1 hour treatment and following 1 hour or 24 hours of incubation with SNS-062 in XLA cell lines. ITK inhibition was investigated via immunoblot after stimulation with anti-CD3 and anti-CD28 and incubation with SNS-062 for 1 hour. SNS-062 was used at a concentration of 1uM in preclinical studies unless otherwise noted. Measurement of kinase activity in human recombinant WT BTK or C481S BTK was performed in a FRET kinase assay. Results: Immunoblots of BTK and ERK phosphorylation of XLA cells transfected with WT or C481S BTK demonstrated that SNS-062 inhibition is comparable to that of ibrutinib in WT BTK and greater than that of ibrutinib in C481S BTK. Using a recombinant kinase assay, we found the IC50 of SNS-062 against WT BTK to be 4.6nM and C481S BTK to be 1.1nM, suggesting that SNS-062 retains activity against the mutated BTK variant. Additionally, SNS-062 was found to be six times more potent than ibrutinib and greater than 640 times more potent than acalabrutinib against C481S BTK. SNS-062 demonstrates dose-dependent inhibition of BTK in primary patient CLL cells comparable to ibrutinib via immunoblot for BTK phosphorylation. The viability of primary patient cells treated with 0.1uM, 1.0uM, and 10.0uM SNS-062 for 48 hours was measured to be 96.7%, 96.1%, and 88.1%, respectively, that of the untreated condition. At 48 hours, SNS-062 decreased viability of primary CLL cells in the presence of HS5 stromal protection by 5.5%. SNS-062 was found to decrease CpG induced CD40 and CD86 expression by 8.7% and 15.7%, respectively. Using an in vitro kinase assay, SNS-062 inhibited ITK with an IC50 value of 24nM. An immunoblot of anti-CD3/CD28 stimulated Jurkat cells revealed that SNS-062 decreased the phosphorylation of ERK, implying inhibition of ITK. Conclusion: Unlike ibrutinib, SNS-062 inhibits BTK signaling in the presence of the C481S mutation and may address acquired resistance to covalent BTK inhibitors. SNS-062 decreases B cell activation markers, viability, and stromal cell protection in primary patient CLL cells and was shown to inhibit ITK, suggesting support of T cell mediated antitumor activities. These data support further investigation of this molecule and advancement into clinical trials.

#1208

Analysis of the combinatorial antiproliferative effect of pan RAF inhibitor AZ-628 and MEK1-inhibitor AZD-6244 on a large panel of tumor cell lines.

Alokta Chakrabarti, Daniel Feger, Sarah Umber, Orysya Stus, Marianne Birkle, Oliver Siedentopf, Jan E. Ehlert. _ProQinase GmbH, Freiburg, Germany_.

Cancer is a highly complex, multigenic disease with tumor cells underlying constant transition. Single drug treatments against specific targets frequently result in only partial success because mutations and redundant pathways cause drug resistance. Therefore, drug combinations that effect different synergistically acting targets in the cancer cell in parallel have become a promising strategy to improve the success in many fields of cancer therapy. One example for such an approach is the co-treatment of B-Raf driven tumors with Raf and MEK1 inhibitors. In our study, we determined the combinatorial effect of the pan-Raf inhibitor AZ-628 and the MEK1 inhibitor AZD-6244 on the viability of a large panel of more than 100 tumor cell lines. Whittaker et al. (Mol.CancerTher, 2015) could already show that this drug combination has a significant synergistic effect in several melanoma and colon cancer cell lines. The authors showed evidence that synergistic activity was correlated with enhanced mutation-triggered RAS/RAF/MAPK signaling with a special role for C-Raf in resistance due to a negative feedback mechanism. We here applied the combination of AZD-6244 and AZ-628 in a broad checkerboard pattern to a multitude of cell lines from other entities beyond melanoma and colorectal cancer. Based on these results we correlated the observed synergistic and non-synergistic effects with gene expression profiles of these cell lines and analyzed the requirement of a MAPK activating signaling. Our approach revealed that synergistic activity is not confined to melanoma and colon cancer but is observed in tumors from other entities as well. This observation may expand the usefulness of MEK/Raf inhibitor co-treatment to a larger panel of cancer types.

#1209

FGFR inhibition re-sensitizes BRAF/MEK dual resistant cells to the BRAF/MEK inhibitor combination.

Victoria E. Wang,1 Jeffrey Settleman,2 Frank McCormic1. 1 _UCSF, San Francisco, CA;_ 2 _Genentech, South San Francisco, CA_.

The BRAF-MEK pathway is frequently mutated in human melanoma. Inhibitors of these kinases have proven to prolong survival in melanoma patients. However, clinical benefit is relatively short-lived due to acquired drug resistance. To explore mechanisms of resistance in melanoma cells treated with dual BRAF and MEK inhibitors, A375, a cell line harboring the BRAF V600E mutation, was treated with escalating doses of vemurafenib and cobimetinib to generate a cellular population resistant to combination therapy. These cells display a reversible resistance phenotype. Upon initial treatment of the drug combination, upregulation of pSTAT3 is observed but the cells lose dependency on the pSTAT3 pathway upon prolonged drug treatment. A pharmacological synthetic lethal screen was performed and the FGFR inhibitor class re-sensitizes the cells to BRAF and MEK inhibition through inhibitions of pERK. The dual resistant cells also displayed hyperactivation of the PI3K-AKT pathway. Interrogation of patient samples confirmed these findings. These findings provides a biological rationale for a polytherapy strategy using a FGFR inhibitor in patients with resistance to dual BRAF and MEK inhibition. <!--EndFragment-->

#1210

Hederagenin overcomes the cisplatin resistance of head and neck cancer by targeting the antioxidant defense mechanisms.

Daiha Shin,1 Eun Hye Kim,1 Hyejin Jang,1 Jaewang Lee,1 Ji Won Kim,1 Jae Ryung Lee,1 Minsu Kwon,2 Seungho Baek,3 Jong-Lyel Roh1. 1 _Asan Medical Center, Univ. of Ulsan College of Medicine, Seoul, Republic of Korea;_ 2 _Gyeongsang National University School of Medicine and Gyeongsang National University Hospital, Jinju, Republic of Korea;_ 3 _College of Korean Medicine, Woosuk University, Wanju, Jeonbuk, Republic of Korea_.

Background: Cisplatin is a first-line chemotherapeutic agent for head and neck cancer (HNC). Acquired resistance to cisplatin is the most common reason for the failure of cisplatin chemotherapy. Hederagenin is extracted from the leaves of Cyclocarya paliurus, also known as sweet tea, which is one of the most popular teas utilized in traditional Chinese medicine. There is accumulating evidence it exhibited significant cytotoxicity against several types of cancers, however, which has been rarely tested in HNC cells. Herein, we examined the potential utility of hederagenin for overcoming cisplatin resistance in HNC cells and further clarified its molecular mechanisms of action.

Methods: Parental and cisplatin-resistant HNC cells, and Nrf2 or SLC7A11 gene overexpressed HNC cell lines and other human HNC cells were used. The cells were used to examine the effects of hederagenin treatment in HNC cell lines by measuring cell viability, cell cycle, cell death, reactive oxygen species (ROS) production, glutathione level, mitochondrial membrane potential (ΔΨm), and protein expression. The anti-tumor effect of hederagenin in mouse tumor xenograft models was also tested.

Results: Hederagenin induced cell death in both cisplatin-sensitive and -resistant HNC cells. Hederagenin treatment resulted in effective death of cisplatin-resistant HNC cells in a form of apoptotic cell death with the ΔΨm change. Hederagenin inhibited Nrf2 and SLC7A11 resulting in an increased ROS accumulation in HNC cells, of which effects were reversed by the pretreatment of antioxidant trolox. In addition, hederagenin caused the apoptosis effectively induced by targeting the antioxidant defense mechanisms in HNC cells. Subsequently, hederagenin activated signaling pathways of cell death involving caspase-3, PUMA, and PARP in the cells. The growth inhibitory effects of hedragenin were also confirmed in the tumor xenograft model implanted with cisplatin-resistant HNC cells.

Conclusions: Our data suggests that hederagenin can overcome the resistance of cisplatin in resistant HNC cells via targeting antioxidant systems.

#1211

Lurbinectedin reverses platinum dependent IRF1 overexpression and nuclear localization, partially responsible for resistance to platinum drugs in ovarian cancer.

Gema Santamaria Nuñez, Maria Jose Guillén, Juan F. Martínez-Leal, Pablo Avilés, Carlos M. Galmarini. _PharmaMar S.A., Colmenar Viejo, Spain_.

Lurbinectedin (PM1183) is a new synthetic compound from the tetrahydroisoquinoline family, which has demonstrated a strong antiproliferative activity against a panel of human tumor models in preclinical assays and is currently being evaluated in phase III clinical trials in platinum-resistant ovarian cancer and small cell lung cancer. Lurbinectedin binds to DNA, inhibits trans-activated transcription, induces the degradation of elongating RNA Pol II and fools nucleotide excision repair to produce dsDNA breaks that need to be repaired mainly by homologous recombination (HR)1,2. Nearly 70% of patients diagnosed with ovarian cancer are in advanced stage, and the vast majority of them will eventually relapse after a primary cytoreductive surgery and several cycles of standard adjuvant chemotherapy including a platinum drug and a taxane. After a period of treatment with platinum drugs, patients will finally develop resistance, usually mediated by mechanisms such as drug detoxification or efflux and enhanced DNA repair. IRF-1 transcription factor expression has been shown to be up-regulated by cisplatin (CDDP) in ovarian cancer cells and might be limiting the response to the drug, likely by inhibiting cell proliferation3. Here we took advantage of the A2780/A2780cis human ovarian cancer cell lines, the second being a cisplatin resistant derivative, to investigate the role of IRF1 in the response of human ovarian cancer cells to cisplatin and lurbinectedin. A2780cis cells are, indeed, more resistant to cisplatin that their parental cell line but they do not differ in their resistance to lurbinectedin. Basal IRF-1 protein levels were actually higher in A2780cis cells than in their parental cell line, contributing to their resistance to cisplatin. Furthermore, cisplatin treatment induced the overexpression and nuclear localization of IRF-1 both, in A2780 and A2780cis cell lines. Contrarily, lurbinectedin did not induce the overexpression of IRF-1 neither in A2780 nor in A2780cis, explaining why this latter cell line is not resistant to the compound. Furthermore, lurbinectedin co-treatment with cisplatin reduced the expression of IRF-1 in A2780 and, more importantly, in A2780cis cells, explaining the synergism the combination has on these tumor cell lines. Thus, lurbinectedin not only did not activate the same mechanisms of resistance as cisplatin in ovarian cancer cells, but even reversed the resistance of these resistant cells to platinum drugs. 1 Santamaría Nuñez et al, 2016. Mol Cancer Ther 15(10):2399-2412 2 Romano et al, 2013. Int J Cancer. 2013 Nov;133(9):2024-33 3 Pavan et al, 2013. Eur J Cancer 49(4):964-973

#1212

Co-targeting epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase overcomes EGFR inhibitor resistance in head and neck squamous cell carcinoma patient-derived models.

Xiaoming Ouyang, Ashley Barling, Aletha Lesch, Jeffrey Tyner, Sophia Jeng, Christina Zheng, Sara A. Courtneidge, Shannon McWeeney, Molly Kulesz-Martin. _Oregon Health & Science University, Portland, OR_.

Background: HNSCC is the sixth most common cancer worldwide. EGFR is overexpressed in up to 90% of HNSCC and associated with poor outcome. An EGFR monoclonal antibody is the only approved molecular targeted therapy for HNSCC, however, resistance eventually occurs in all patients.

Methods: Functional screens including a small-molecule kinase inhibitor panel and an RNAi screen panel were used to identify agents that synergized with EGFR inhibitors in reducing cell viability in HNSCC patient-derived tumor cells. Effective combination therapies were validated in scale-up experiments in 2D and 3D, and their true targets were evaluated using siRNAs to rule out off-target effects of the drugs. Cell viability, cell number and cell colony formation ability were tested upon siRNA treatment. qRT-PCR and immunofluorescent staining was used to test ALK expression in tumor cells before and after anti-EGFR treatment.

Results: Two ALK inhibitors on the drug screen panel showed synergistic effects with EGFR inhibitors in 6/8 HNSCC patients' tumor cells, despite ineffectiveness of single drug. Scale-up dose-response experiments confirmed patient cell sensitivity to 4 different ALK inhibitors in combination with the EGFR inhibitor Gefitinib. siRNA targeting ALK synergized with Gefitinib in reducing cell viability in the patient cells that responded to EGFR/ALK inhibitor combinations, indicating specificity to ALK. Scale-up RNAi experiments confirmed synergy between siRNAs targeting ALK and EGFR in reducing cell viability, cell number, and colony formation ability. Inhibition of EGFR by siRNA or Gefitinib each increased ALK expression at the mRNA level and protein level, suggesting induction of ALK as a novel mechanism of EGFR inhibitor resistance in HNSCC.

Conclusion: We identified EGFR/ALK inhibitor combinations as a potential strategy for treating EGFR inhibitor resistant HNSCC.

#1213

Nuclear export of E2F7 in squamous cell carcinoma in an actionable event that reverses resistance to anthracyclines.

Alba Natalia Saenz Ponce,1 Yosef Landesman,2 Trinayan Kashyap,2 Alexander Guminski,3 Orla Gannon,1 Nicholas Saunders1. 1 _University of Queensland, Brisbane, Australia;_ 2 _Karyopharm Therapeutics, Newton, MA;_ 3 _Royal North Shore Hospital, NSW, Australia_.

Objectives and Background: Squamous cell carcinoma (SCC) of the skin or head and neck region is a major cause of cancer death in the western world. Patient with advanced SCC have few therapeutic options and SCC remains resistant to current treatments. Overcoming drug resistance in SCC would significantly change patient outcomes and treatment strategies. We previously showed that SCCs express the activating transcription factor E2F1 and the inhibitory transcription factor E2F7, which are mutually antagonistic. We showed that E2F1/7 controlled the sensitivity to anthracyclines in SCC cells via E2F-dependent regulation of sphingosine kinase 1 (Sphk1) and its product sphingosine-1-phosphate (S1P). In addition, we showed that S1P treatment of cells induces profound anthracycline resistance. However, it was consistently difficult to reconcile the mutual overexpression of E2F1 and E2F7 with a seemingly pro-E2F1 environment within the SCC cells (i.e. pro-proliferation, differentiation-suppressive and pro-survival). In this study we have examined the role of nuclear export pathways in determining nuclear E2F activation in SCC.

Methods: We have interrogated human tissue microarrays of normal stratified epithelia and SCC of the skin or head and neck tumours from patient samples. To validate our in vivo findings we used a suite of SCC cell lines and examine the nuclear and cytoplasmic localization of E2F1 and E2F7 by immunofluorescence and western blotting. We deployed inhibitors of the Exportin 1 (XPO1) pathway and have used siRNA against XPO1 to confirm a functional role of XPO1 in the nuclear export of E2F7. We have used inhibitors of XPO1 in vitro and in vivo to examine their ability to enhance the cytotoxic responses to anthracyclines.

Results & Conclusions: We report that E2F7 is selectively localized to the cytoplasm of SCC cells and tumors but is exclusively nuclear in normal cells and tissues. In contrast, E2F1 is almost exclusively localized to the nucleus in normal and SCC cells and tissues. The localization of E2F7 to the cytoplasm could be reversed using inhibitors of the nuclear export protein XPO1 and siRNA against XPO1. XPO1-dependent nuclear export of E2F7 shows that this pathway is selectively activated in SCC. The inactivation of XPO1 is now possible using selinexor, a Selective Inhibitor of Nuclear Export (SINE) compound currently in advanced clinical trials to treat solid and hematological malignancies. We show that treatment of SCC cells with selinexor reverses anthracycline resistance. Significantly, we show that a combination of selinexor + doxorubicin in vivo induces profound anticancer activity.

Significance: Our studies show that i) E2F7 is selectively mislocalized in SCC, ii) E2F7 nuclear export is XPO1-dependent, iii) dysregulation of XPO1 causes derepression of S1P-mediated anthracycline resistance and iv) anthracycline resistance is reversed with inhibitors of XPO1 in vivo.

#1214

The polymeric fluoropyrimidine F10 is effective towards cell and organoid models of 5-FU-resistant colorectal cancer.

William H. Gmeiner,1 Anthony Dominijanni,1 Olcay Boyacioglu,2 Steven Forsyth,1 Aleks Skardal,1 Boris Pasche1. 1 _Wake Forest Univ. School of Medicine, Winston-Salem, NC;_ 2 _Adnan Menderes University, Aydin, Turkey_.

Background: Fluoropyrimidine drugs (FPs, e.g. 5-fluorouracil (5-FU)) constitute the backbone of most chemotherapy regimens for treating locally-advanced and metastatic colon cancer (e.g. FOLFOX, FOLFIRI, FOLFOXIRI). While current FPs improve survival and are the preferred drugs for most colorectal (CRC) cases, drug resistance is a serious problem. One of the most prevalent causes of 5-FU resistance is mutation in TP53 which occur in more than one-third of stage III CRC tumors. 5-FU requires wtp53 for initiation of apoptosis and CRC cells with mtp53 are markedly less sensitive to 5-FU.

Methods: To test if F10 induced p53-independent apoptosis in CRC cells, we obtained p53-/- and p53+/+ HCT-116 cells from Professor Vogelstein (Johns Hopkins) and evaluated drug-response in cell-based, organoid, and xenograft models. Cell viability and apoptosis were assessed using Cell-Titer Glo and Caspase Glo assays (Promega). Cell-cycle distribution was evaluated by flow cytometry analysis of propidium iodide stained cells. Thymidylate synthase (TS) activity was assessed using a 3H-release assay and Topoisomerase 1 cleavage complex (Top1cc) formation was assessed using an in vivo complex of enzyme (ICE) bioassay. Cell division was quantified by time-lapse video microscopy. Organoids were developed by bioprinting cells on a biogel matrix to produce a center comprised of tumor cells encompassed by stromal cells. Drug effects in each cell type were evaluated using a "Live-Dead" assay. Drug effects in vivo were evaluated using xenograft models.

Results: p53-/- HCT-116 cells were resistant to 5-FU, but sensitive to F10 and underwent p53-independent apoptosis. While F10 is more efficient than 5-FU at inhibiting TS, it uniquely supported continued DNA synthesis, mitosis, and cell division in p53-null cells while 5-FU-treated cells underwent S-phase arrest under thymineless conditions. Following cell division, F10 induced high levels of Top1cc in daughter cells causing massive DNA damage. Using a DT40 knockout system we determined that Tdp1 and PARP-1 were important for F10-induced DNA damage. Organoid models demonstrated selective killing of malignant cells relative to stroma by F10, but not 5-FU. Xenograft models are in progress.

Conclusions: F10 is much more potent than 5-FU and induces p53-independent apoptosis. F10 differs from 5-FU in supporting continued DNA synthesis under thymineless conditions leading to Top1-mediated DNA damage, Top1 depletion, genomic instability and activation of the extrinsic apoptotic pathway.

#1215

Apigenin overcomes drug resistance by blocking signal transducer and activator of transcription 3 (STAT3) signaling in breast cancer.

Hye-Sook Seo,1 Jin Mo Ku,1 Se Hyang Hong,1 Hyeong Sim Choi,1 Jong-Kyu Woo,2 Bo-Hyoung Jang,1 Yong Cheol Shin,1 Seong-Gyu Ko1. 1 _Kyung Hee University, Seoul, Republic of Korea;_ 2 _Seoul National University, Seoul, Republic of Korea_.

Drug resistance in chemotherapy is a major obstacle for successful treatment of cancer. Drug resistance is caused by various reasons including the overexpression of P-glycoprotein (P-gp, MDR1). Development of new useful compound which overcomes drug resistance is urgent. Apigenin, a dietary flavonoid, is reported as an anti-cancer drug in vivo and in vitro. In the present study, we investigated whether apigenin is able to reverse drug resistance using adriamycin-resistant breast cancer cells (MCF-7/ADR) and xenograft mouse model. As a result, apigenin significantly decreased cell growth and colony formation in MCF-7/ADR and its parental MCF-7 cells. This growth inhibition was related with accumulation of subG0/G1 apoptotic population and increase of apoptosis cell number. Apigenin reduced the mRNA expressions of multi-drug resistance 1 (MDR1) and multi-drug resistance associated proteins (MRPs) in MCF-7/ADR cells. Apigenin also down-regulated the expression of P-gp. Apigenin reversed drug efflux from MCF-7/ADR cells resulting in Rho123 accumulation. Inhibition of drug resistance by apigenin is related with suppression of STAT3 signaling pathway. Apigenin decreased STAT-3 activation (p-STAT3) and its nuclear translocation, and inhibited the secretion of VEGF and MMP-9 which are STAT3 target genes. STAT3 inhibitor, JAK inhibitor I and HIF-1α inhibitor decreased cell growth in MCF-7 and MCF-7/ADR cells. In conclusion, apigenin overcomes drug resistance, and this study advances human health.

### Targeting Growth Factor and Intracellular Signaling

#1216

Overcoming cisplatin resistance through the combination treatment with CK2 inhibitor, CX-4945, in gastric cancer.

Hyun Myong Kim,1 Inhye Jeong,1 Kyu Hyun Park,2 Tae Soo Kim,2 Woo Sun Kwon,2 Hei-Cheul Jeung,2 Minkyu Jung,3 Sun Young Rha4. 1 _Song-dang Institute for Cancer Research, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Republic of Korea;_ 2 _Song-dang Institute for Cancer Research, Yonsei University College of Medicine, Seoul, Republic of Korea;_ 3 _Song-dang Institute for Cancer Research, Division of Medical Oncology, Department of Internal Medicine, Yonsei Cancer Center, Yonsei University Health System, Seoul, Republic of Korea;_ 4 _Song-dang Institute for Cancer Research, Brain Korea 21 PLUS Project for Medical Science, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea_.

Platinum-based antineoplastic drugs are chemotherapeutic agents to usually treat gastric cancer (GC) include cisplatin. However, the majority of cancer patients will eventually relapse with cisplatin-resistant disease. Especially, increased DNA repair is drug targetable mechanism and useful in the treatment strategy of cisplatin-resistant cancer. Casein kinase 2 (CK2) has critical role of multiple cellular processes with DNA repair. For this reason, research for CK2 expression correlated with DNA repair mechanism is important in gastric cancer. Combination of cisplatin and CK2 inhibitor (CX-4945, also known as Silmitasertib, Senhwa Biosciences, USA) may improve cisplatin-induced DNA damage for GC treatment. In this study, we screened sensitivity of cisplatin and CX-4945 in 49 GC cell lines by MTT assay. For molecular profiling, we analyzed variants and gene expression using whole exome sequencing and RNA sequencing. RNA and protein expression of CK2 subunits (α/α') using real-time RT-PCR and Western blot. Also, activity of CK2α was measured by ELISA. Combination treatment performed different schedules including concurrent and sequential. Synergistic effect was analyzed by Bliss Independence model. As CK2 profiling, CK2α' mRNA expression was a tendency to correlated with CX-4945 sensitivity(p=0.0504). Moreover, CK2α' protein expression was a correlated with CX-4945 sensitivity(p=0.0252). Other molecular profiling did not reveal any clear correlations. Twenty one (Group1: cisplatin extremely resistant and CK2 high, Group2: cisplatin intermediate resistant regardless of CK2 expression) cell lines were performed combination treatment. Both YCC-21 and YCC-28 cell lines show synergistic effect (+20% and +22%, respectively) in concurrent schedule. Only MKN-74 cell line show synergistic effect (+11%) in Pre-addition. The 4 cell lines (YCC-18, YCC-38, SNU-1 and -216) were show synergistic effect (+10%, +37%, +11% and +19%, respectively) in Post-addition. In conclusion, early inhibition of CK2 shows synergistic effect in group 1, because CK2 is high expression. In group 2, late inhibition of CK2 demonstrated synergistic effect. It means CK2-related DNA repair is up-regulating for repair to cisplatin-induced DNA damage. Personalized the treatment schedule for inhibit CK2-induced DNA repair is a new strategy to restore cisplatin resistant in GC.

#1217

Blockade of Aurora kinase A synergizes with platinum and radiation in non-small cell lung cancer cells.

Huijie Liu,1 Dongwei Zhang,2 Alain Borczuk,3 Vincent Chau,4 Roman Perez-Soler,5 Balazs Halmos,5 Haiying Cheng5. 1 _Albert Einstein College of Medicine, Bronx, NY;_ 2 _Columbia University Medical Center, New York, NY;_ 3 _Weill Cornell Medical College, New York, NY;_ 4 _Montefiore Medical Center, Bronx, NY;_ 5 _Albert Einstein College of Medicine /Montefiore Medical Center, Bronx, NY_.

Background: Most patients with advanced lung cancer will receive platinum-based therapy during their treatment course despite limited clinical benefits. Thus, strategies to improve platinum-based therapy continue to be needed. Through a functional genetic screen, we have identified Aurora kinase A as a potentially actionable candidate that can modulate the activity of platinum compounds. Aurora A is a serine/threonine kinase and a key regulator of the mitotic process. Through its mitotic and non-mitotic roles, the aberrant activation of Aurora A can lead to oncogenic transformation and has emerged as a promising therapeutic target in cancer.

METHODS: High-throughput RNAi screen was used to identify potential candidates for cisplatin resistance. Cell proliferation/viability was determined by MTS assays and colony formation assays. Immunohistochemistry was performed to determine the overexpression of Aurora A in specimens from lung cancer patients. Western blot was used to explore the cell signaling mechanisms in cisplatin, radiation, and/or MLN8237 (alisertib) treated NSCLC cell lines. β-Galactosidase staining was performed to determine the senescence following cisplatin and/or MLN8237 treatment in NSCLC cell lines.

RESULTS: Aurora Kinase A is widely expressed in lung cancer specimens. The expression of Aurora A is upregulated following treatment with Cisplatin in NSCLC cells, such as PC9, A549, H1703, and H460. Inhibition of Aurora A activity with either siRNA-mediated knockdown or pharmacological blockade (MLN8237) significantly improved sensitivities to cisplatin and ionizing radiation, both of which cause DNA damage and cell death. On the other hand, inhibition of Aurora A activity by itself was associated with reduced cell proliferation and survival of NSCLC cell lines. Moreover, co-administration with Cisplatin and AURKA siRNA resulted in sustained DNA double strand breaks and more pronounced apoptosis. The combination of AURKA inhibitor (MLN8237) with Cisplatin similarly led to enhanced DNA damage. Similar effects were observed with radiation concomitant with inhibition of Aurora A. Furthermore, MLN8237 induced marked apoptosis in the p53 mutant cell line, PC9, whereas more senescence was noted in p53 wild type A549 cells.

CONCLUSIONS: Our study indicates a significant role for Aurora Kinase A as a common modulator of p53-dependent resistance in the context of DNA-damaging platinum and radiation therapy. Aurora A inhibition can potentially improve the efficacy of these common treatment modalities in lung cancer management.

#1218

Synthetic lethal targeting of p53 mutant cells with prenylation inhibitors.

Shay R. Ferdosi,1 Benjamin Katchman,2 Jia Loo,1 Harneet Grewal,1 Seron Eaton,1 Shanshan Yang,1 Jin Park,1 Joshua Labaer,1 Karen S. Anderson1. 1 _Arizona State University, Tempe, AZ;_ 2 _Eccrine System Inc, Cincinnati, OH_.

Introduction: TP53 is mutated in 30-40% of breast cancers and is associated with poor prognosis. Mutation of TP53 causes increased cellular proliferation, migration and invasion, and downstream activation of multiple pathways. HMG CoA reductase inhibitors, such as simvastatin, inhibit tumorigenic properties induced by TP53 mutation. Mechanism of this response is an important question in targeted cancer therapy. It is well known that HMG CoA reductase inhibitors block cholesterol production and prenylation. Therefore, we hypothesized that prenylation inhibitors would target p53 mutant cells primarily by inhibiting activation of the ras family induced by TP53 mutation.

Method: We generated 5 MCF10A stably transduced cell lines over-expressing each of the common TP53 point mutations, R273H, G245S, R248Q, Y234C, or wild type p53. Total Ras, Ras-GTP, total RhoA and RhoA-GTP was measured by immunoprecipitation and immunoblot. Proliferation was measured using CellTiterGlo. The functional effects of a panel of prenylation inhibitors was measured using a caspase3 reporter assay, and migration was measured with a scratch assay. Illumina mRNA sequencing was performed to measure gene expression of mutant and wild type cells before and after simvastatin treatment (2.5µl, 48 h). The RNAseq was analyzed with the DAVID Enrichment tool to evaluate ras pathway activation induced by TP53 mutation.

Results: Mutation in TP53 was associated with significant activation of both wild-type Ras and RhoA. Proliferation of cells expressing mutant TP53 was sensitive to a panel of prenylation inhibitors, including simvastatin. Growth inhibition by simvastatin correlated with induction of apoptosis, and could be fully rescued by addition of farnesylpyrophosphate or geranylgeranylpyrophosphate, suggesting that simvastatin functionally blocks both prenylation pathways which are activated in the presence of mutant p53 (IC50(wt/mut) = 4). RNAseq analysis confirms that Ras signaling pathways, including GAPs and GEFs are enriched in the presence of mutant TP53, and FOXO signaling is significantly targeted post statin treatment (P<0.01).

Conclusion: p53 mutant breast cancer cells are highly sensitive to prenylation inhibition due to activation of both ras and rhoA. Simvastatin inhibits both farnesylation and geranylgeranylation, effectively blocking ras pathway activation and induction of proliferation in p53 mutant cell lines. A gene expression panel associated with ras pathway activation was identified and may be predictive biomarkers for sensitivity to statin therapy.

#1219

Integrative kinome exome sequencing and copy number profiling of ovarian clear cell carcinoma.

J.j. Caumanns,1 G.b.a Wisman,1 K Berns,2 T. Tomar,1 R.s.n. Fehrmann,1 R Bernards,2 A.g.j. Van der Zee,1 S De Jong1. 1 _University Medical Centre Groningen, Groningen, Netherlands;_ 2 _Netherlands Cancer Institute, Amsterdam, Netherlands_.

Introduction: High stage ovarian clear cell carcinoma (OCCC) is less responsive to chemotherapy and has a worse prognosis than other ovarian cancer subtypes. The most frequently mutated genes in OCCC have been characterized in recent years. These include deleterious mutations in the tumor suppressor gene ARID1A, epigenetic silencing and deleterious mutations in the PI3K antagonist PTEN as well as activating mutations in the PI3K catalytic subunit PIK3CA. Yet, therapeutic strategies that utilize these genetic aberrations are lacking. In the present study, we aimed to identify and validate new kinase targets in OCCC.

Methods: To determine new mutations and copy number gains and losses, kinome sequencing was performed on DNA isolated from tumor samples (n=124) and matched controls (n=47) and high-coverage SNP analysis was implemented for 109 of the collected OCCC tumor samples. Inhibitor sensitivity screening was performed on 17 OCCC cell lines to validate potential kinase targets, following in vivo validation in OCCC patient-derived xenografts (PDX).

Results: Mutations in ARID1A, PIK3CA, PTEN, KRAS and TP53 corresponded to frequencies found in literature. Most identified mutations could be designated to the PI3K/Akt/mTOR and MAPK signal transduction pathway, and HER family of receptor tyrosine kinases. The PI3K pathway related genes PIK3R1, ERBB3 and AKT1 were significantly mutated in our dataset and not described in OCCC before. EGFR, ERBB2 and PIK3CA emerged as most frequently amplified kinases. Combining mutations and copy number alterations in these pathways, 91% of all tumors were affected. The highly positive staining of p-S6 (90% of OCCC tumors) indeed suggests high mTORC1/2 activity in these tumors. Inhibitor screening demonstrated subsets of cell lines to be sensitive to EGFR or ERBB2 inhibition. Furthermore, the vast majority of OCCC cell lines was susceptible to inhibition of PI3K and MAPK downstream target mTORC1/2 using AZD8055, which efficacy was further demonstrated in several OCCC PDX models. In contrast, mTORC1 inhibition alone was less effective.

Conclusion: Most kinase mutations and copy number alterations in OCCC can be related to PI3K and MAPK pathway activation. In vitro and in vivo data suggest mTORC1/2 inhibition as a novel treatment strategy in OCCC. Combinations of EGFR or ERBB2 inhibition with mTORC1/2 targeted therapy can be envisioned for OCCC tumors with EGFR or ERBB2 alterations. Supported by a grant from the Dutch Cancer Foundation: RUG 2012-5477

#1220

Coordinated chemical-genetics approach identifies PTP4A3-mediated regulation of colon cancer cell migration and extracellular matrix interactions.

Kelley E. McQueeney,1 Joseph M. Salamoun,2 Isabella K. Blanco,1 Paula Pekic,1 Jennifer Ahn,1 Elizabeth R. Sharlow,1 Peter Wipf,2 John S. Lazo1. 1 _University of Virginia, Charlottesville, VA;_ 2 _University of Pittsburgh, Pittsburgh, PA_.

Aberrant regulation of protein phosphorylation is an exceedingly common driver of human cancers. It is notable that we understand much less about the role of protein tyrosine phosphatases in human malignancies compared to tyrosine kinases. The membrane-associated, intracellular, protein tyrosine PTP4A3 is highly overexpressed in multiple tumor types including colorectal cancer and has been associated with tumor metastases. We have, therefore, investigated the role of PTP4A3 in colon cancer migration and invasion. The Ptp4a3 gene was expunged from colon tumor cells derived from Ptp4a3flox/flox mice and the resulting cells exhibited impaired migration, invasion, and colony formation compared to the wildtype isogenic cells. We characterized a potent, selective, and noncompetitive small molecule inhibitor of PTP4A3, JMS-631-053, which also disrupted colon cancer cell migration, invasion, and colony formation. PTP4A3 deletion increased the expression of extracellular matrix and adhesion genes, including the tumor suppressor Emilin 1. Expression of these extracellular matrix genes is mutually exclusive with PTP4A3 expression in tumors derived from patients with colorectal cancer. These chemical and biological reagents reveal a previously unknown communication between the intracellular PTP4A3 phosphatase and the extracellular matrix and support continued efforts to pharmacologically target PTP4A3 for cancer therapy.

#1221

Triple-negative breast cancer (TNBC) phosphoproteomics.

Sara Fernandez Gaitero,1 Ivana Zagorac,1 Jose Francisco Lopez-Acosta,1 Gonzalo Gomez-Lopez,1 David González Pisano,1 Javier Muñoz Peralta,1 Luis Manso,2 Soledad Alonso,3 Renske Penning,4 Maarten Altelaar,4 Albert JR Heck,4 Miguel Quintela-Fandino1. 1 _Spanish National Cancer Research Center (CNIO), Madrid, Spain;_ 2 _12 de Octubre University Hospital, Madrid, Spain;_ 3 _Hospital Universitario de Guadalajara, Guadalajara, Spain;_ 4 _Netherlands Proteomics Centre, Utrecht, Netherlands_.

Background: We hypothesized that the biphasic relapse pattern of TNBC could be explained by a limited number of activation patterns of signaling nodes. In addition, we sought to determine whether the hyperactive signaling nodes, distinguishing the cases with favorable vs adverse outcome, could be potential targets.

Methods: Training set of 34 frozen tumor samples divided in two sets, (A) 13 patients, relapsed in <4 years; (B) 21 patients relapse-free >12 years, (mimicking the percentage and relapse patterns of unselected TNBC, but paired for T, N, G and Ki67). TNBC cell lines: 7 indolent (no metastases in 60 weeks) and 3 aggressive, develop metastases and kill recipient mice in <4 months. Shotgun phosphoproteomics and TiO2-IMAC phosphopeptide enrichment coupled with mass spectrometry runs in a Orbitrap Elite Mass Spectrometer was performed. Spectra were processed with MaxQuant software. Differentially expressed phosphopeptides were obtained by applying linear models R limma package. Differential kinase activation driving the profiles segregating cured vs. relapsing cases was done using linear sequence motif analysis. The hyperactivated kinases were validated in an independent set of 113 consecutive TNBC cases with 12+ years of follow-up spotted in TMAs by using an in-house algorithm for immunohistochemistry coupled with computer-aided quantitation using an Ariol scanning, we took the kinases in the upper quartile (high activity). Survival analysis was performed with KM curves and log rank test; and Cox proportionate hazards model was used for multivariate models.

Results: 11405 phosphopeptides were identified and quantified in the training set. Supervised clustering of relapsed vs. cured cases showed that 161 and 541 peptides were significantly up-regulated in the A and B groups, respectively (FDR<0.15). After kinase-to-kinase co-linearity was ruled-out , gathering the high activity (upper quartile) of six kinases (a combined variable, herein Var1) showed statistically significant association with relapse, being these: PRKCE, pERK, c-KIT, CDK6, pP70S6K and pPNKP. Cox proportional hazards model of any of the six probes high (var1) vs rest: 9.9 vs. not reached years (P<0.001). Patients that had any of the 6 kinases high have 47% of chance to relapse (only 2 out of 42 relapsed patients have 0/6 active kinases) vs patients with Var1 negative, 7% of chance (29 patients out of 72 have 0/6 active kinases) we also observed constants patterns of activations in the different sets expressions of kinases. We considered the kinases at Var1 as a potentially targets and we developed a pharmacological in vitro assay, testing pairs inhibitors on 10 TNBC cell lines; 99.3% of the combinations were supra-additive.

Conclusion: High throughput p-proteomics allows a parsimonious segregation of early TNBC cases, easily detecting the cases with long-term cure vs the remaining while identifying potential therapeutic solutions for the patients falling in the adverse prognostic subgroups.

#1222

Developing kinase inhibitors with polypharmacological profiles for the treatment of resistant cancers.

Suman Rao,1 Tan Li,1 Robert Everley,2 Guangyan Du,1 Peter Sorger,2 Nathanael Gray1. 1 _Dana Farber Cancer Institute, Harvard Medical School, Boston, MA;_ 2 _Harvard Medical School, Boston, MA_.

Small molecule kinase inhibitors targeting a specific oncoprotein driving cancer cells have shown great clinical success in the past (e.g. imatinib against leukemia). However, resistance to targeted therapies quickly arises due to plasticity associated with cancer cell signaling and tumor heterogeneity. One way to overcome this problem is to rationally design drugs with polypharmacology that can target multiple signaling pathways engaging in cancer cell proliferation and survival. To this end, we have developed a strategy based on combining phenotypic screens of small molecule inhibitors and affinity-based mass spectrometry to identify drugs with multi-targeted properties and piloted this approach using the highly resistant KRAS mutant non-small cell lung cancer cell lines. We began this study by screening kinase inhibitors targeting MEK, ERK, PI3K, Akt and mTOR and found that the lung cancer cells were largely resistant to single drugs but showed enhanced sensitivity towards triple inhibition of MEK, ERK and PI3K. These cell lines were then screened with a library of covalent and non-covalent kinase inhibitors in an attempt to identify a small molecule inhibitor that could mimic the activity of the triple drug combination. This led to the discovery of SM1-71 that not only recapitulated the growth inhibitory activity of MEK+ERK+PI3K inhibition, but also blocked phosphorylation of ERK, Akt, mTOR and S6K in cells. Target profiling of the drug was carried out using chemoproteomics, which revealed that SM1-71 inhibited 55 kinases at a 1μM concentration. Dose-dependent evaluation further determined the IC50 of inhibition across the 55 kinases and rank ordered them based on binding affinity. Top hits including MEK1, MEK2, ERK1, ERK2, AURKA, SRC and RPS6KA3 were confirmed using alternate kinase screens and cellular assays. Growth factor receptors such as IGF1R, MET and EGFR were also identified and further validated using cell-based assays. Taken together, these results elucidated the polypharmacology associated with SM1-71 and uncovered key targets responsible for inducing strong cytotoxicity in KRAS mutant lung cancer cells. Through the combination of phenotypic screens and mass spectrometry-based target identification, we have demonstrated the proof of principle for developing kinase inhibitors with polypharmacology targeting resistant cancers.

#1223

Evaluation of the dual p38/NLK kinase inhibitor as potential new therapeutic agent for tamoxifen-resistant breast cancer.

XIAN WANG,1 Xixi Cao,2 Jamunarani Veeraraghavan,2 Lanfang Qin,2 Jin-Ah Kim,2 Ying Tan,2 Susan G. Hilsenbeck,2 Rachel Schiff,2 Xiaosong Wang1. 1 _University of Pittsburgh, Pittsburgh, PA;_ 2 _Baylor College of Medicine, Houston, TX_.

Background: Endocrine therapy has been considered an effective initial treatment for ER positive breast cancer and tamoxifen is the most commonly used endocrine agent. However, about half of the patients develop resistance or relapse eventually. No effective targeted therapy exists to overcome it. In our previous study, we have identified the role of nemo-like kinase (NLK) in breast cancer endocrine resistance -- a serine-threonine kinase that functions in stress response and neurite outgrowth. In addition to NLK, activation of the other stress kinases such as p38 MAPK has been reported to modulate ER signaling and promote endocrine resistance. In this study, we have identified a highly selective dual p38 and NLK kinase inhibitor (PNKI). This study aimed to evaluate the therapeutic effect of the PNKI inhibitor in tamoxifen-resistant breast cancers using in vitro and preclinical mouse models.

Experimental design and methods: To determine the effect of PNKI on tamoxifen-resistant breast cancer cells, we treated a primary tamoxifen-resistant breast cancer cell line MDAMB415 and an acquired-resistant line MCF7 TamR, with 0.5 uM PNKI in the presence of different doses of Tamoxifen. To evaluate the therapeutic effect of PNKI in a T47D-derived xenograft tumor model with acquired tamoxifen resistance, we administered PNKI alone or in combination with Fulvestrant, or with the mTOR inhibitor Everolimus. Mice bearing T47D-TamR xenografts were randomized into six treatment groups (Vehicle, PNKI, Fulvestrant, Fulvestrant + PNKI, Everolimus, Everolimus + PNKI). Tumor growth was tracked closely. The tumors harvested 2 weeks following treatments were profiled with Reverse Phase Protein Array (RPPA) to assess the early signaling changes after treatments. Furthermore, the therapeutic effect of PNKI were also evaluated in a patient-derived xenograft (PDX) model of de novo endocrine resistant breast cancer. Mice bearing the PDX tumors were randomized to one of four treatment groups (Vehicle, PNKI, Everolimus, Everolimus + PNKI) and tumor growth curve was measured.

Summary of the Results: Breast cancer cell lines with either de novo or acquired Tamoxifen resistance became more sensitive to tamoxifen when treated with 0.5 uM PNKI. The concomitant treatment of PNKI and Everolimus results in significant decrease of tumor growth in the T47D-TamR xenograft tumor compared to Fulvestrant, Fulvestrant + PNKI, PNKI, or Everolimus treatments. RPPA data revealed that a majority of key survival signaling in breast cancer are repressed only when PNKI are combined with Everolimus. The de novo endocrine-resistant PDX tumors showed diverse response to PNKI mono-treatment, whereas the combination of PNKI and Everolimus resulted in significantly decreased tumor growth.

Conclusion: The PNKI exhibited potential therapeutic value as adjuvant agent to the mTOR inhibitor everolimus for acquired or de novo tamoxifen-resistant breast cancer.

#1224

Combination of rapamycin and crizotinib induces partial remission of pleural mesothelioma in a patient-derived xenograft model.

Dina Mönch,1 Sabine Bode-Erdmann,1 Jörg Kalla,2 Jörn Sträter,3 Carsten Schwänen,3 Roger-Fei Falkenstern-Ge,4 Martin Kimmich,4 Martin Kohlhäufl,4 Godehard Friedel,4 German Ott,1 Claudia Kalla1. 1 _Department of Clinical Pathology, Robert-Bosch-Krankenhaus, Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, University of Tübingen, Stuttgart, Germany;_ 2 _Institute of Pathology, Schwarzwald-Baar-Klinikum, Villingen-Schwenningen, Germany;_ 3 _Ambulantes Onkologiezentrum, Klinikum Esslingen and Institute of Pathology, Esslingen, Germany;_ 4 _Center for Pulmonology and Thoracic Surgery, Klinik Schillerhöhe, Stuttgart, Germany_.

Introduction: Malignant pleural mesothelioma (MPM) is a neoplasm with inferior prognosis and notorious chemotherapeutic resistance. New treatment approaches may be based on specific inhibitors against kinases that are overexpressed in MPM, such as mTOR and MET. Here we examined the tyrosine kinase ALK as a potential therapeutic target and the combinatorial efficacy of the ALK/MET inhibitor crizotinib and the mTOR inhibitor rapamycin.

Methods: We investigated the ALK status and the expression of mTOR and MET in 145 primary MPM and 8 murine patient-derived xenograft models. ALK overexpression, rearrangement and mutation were studied by qRT-PCR, FISH, immunohistochemistry and sequence analysis. Expression of mTOR and MET was analyzed by qRT-PCR and immunohistochemistry. The combined anti-tumor effect of crizotinib and rapamycin was evaluated in a patient derived xenograft model. Effects of single drug vs. combination treatment on proliferation, apoptosis and autophagy were assessed using Ki-67 immunohistochemistry, TUNEL assay and LC3B immunofluorescence, respectively.

Results: Overexpression of ALK transcripts was detected in 25 (19.5%) of 128 interpretable primary MPM and two xenograft tumors and was neither associated with ALK rearrangement nor with mutation of the kinase domain. ALK protein was expressed in 8.3% MPM and the two xenograft tumors expressing ALK transcript. mTOR protein expression was detected in 28.7% MPM, co-expressed with ALK and/or MET in 17.8% MPM. Applied to a patient derived MPM xenograft model that co-expressed ALK, MET and mTOR, crizotinib alone did not exert anti-tumor growth activity, but enhanced the anti-tumor effect of rapamycin: Rapamycin was effective in 3 of 5 tumors (pathological partial remission in 1, stable tumor in 2 cases). Combination treatment was active in all 5 tumors (partial remission in 4, stable tumor in 1 case). Simultaneous treatment with rapamycin and crizotinib, however, significantly suppressed tumor proliferation compared to rapamycin-single treatment. Autophagy was induced by both single drug treatments and distinctly enhanced by combination treatment, while apoptosis was not promoted.

Conclusion: Dual combinatory treatment of rapamycin and crizotinib is more effective than rapamycin as single agent in suppressing MPM tumor growth and therefore merits further investigation.

#1225

A unified model of RAF inhibitor action determines inhibitor activity in BRAF-dependent tumors.

Zoi Karoulia,1 Yang Wu,2 Tamer A. Ahmed,1 Qisheng Xin,2 Julien Bollard,1 Clemens Krepler,3 Xuewei Wu,1 Chao Zhang,4 Gideon Bollag,4 Meenhard Herlyn,3 James A. Fagin,5 Amaia Lujambio,1 Evripidis Gavathiotis,2 Poulikos I. Poulikakos1. 1 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 2 _Albert Einstein College of Medicine, Bronx, NY;_ 3 _The Wistar Institute, Philadelphia, PA;_ 4 _Plexxikon, California, CA;_ 5 _Memorial Sloan Kettering Cancer Center, New York, NY_.

The FDA approved RAF inhibitors vemurafenib and dabrafenib have elicited profound responses in melanoma patients with tumors harboring BRAFV600E mutation, but resistance limits their effectiveness. Furthermore, RAF inhibitors exhibit only modest efficacy in colorectal and thyroid BRAFV600E tumors and they are not effective in tumors harboring non-V600 BRAF mutations. While RAF/MEK inhibitor combinations demonstrate increased clinical efficacy, drug resistance develops as well, through similar mechanisms to those identified in RAF inhibitor monotherapy. This suggests that insufficient RAF inhibition limits treatment efficacy and indicates the need for more effective therapeutic approaches to potently and durably target RAF/ERK signaling.

Although the unique biochemical properties of RAF inhibitors have been a topic of intensive investigation, a unified model of RAF inhibitor action has been lacking. Our biochemical and structural analysis in a panel of RAF inhibitors with different structural properties revealed that they exert their effects via the combined outcome of two distinct allosteric mechanisms. RAF priming and dimerization are the result of RAF/RAS-GTP interaction upon inhibitor binding to RAF and stabilization of the αC-helix in the IN (active) position. Resistance of the dimeric RAF to vemurafenib and dabrafenib is the result of stabilization of the αC-helix of the drug-bound protomer towards the OUT (inactive) position, a conformation that sterically prevents occupancy of both RAF protomers. Next generation RAF inhibitors that stabilize the αC-helix of RAF towards the IN position (αC-IN RAF inhibitors) allow occupancy of both RAF protomers and thus accomplish potent inhibition of the dimeric form of RAF.

We further provide structural, biochemical and cell-based evidence that next generation αC-IN RAF inhibitors (such as MLN480, TAK632, LY3009120, AZ628 and others) will be more effective for the treatment of patients with tumors expressing non-V600 BRAF mutants and in BRAFV600E colorectal

and thyroid tumors. However, αC-IN RAF inhibitors are predicted to have a low therapeutic window, since they inhibit dimeric wild-type BRAF also in normal cells.

Together our findings provide a blueprint for the development of RAF inhibitors with defined biochemical properties and support therapeutic strategies in which relatively lower, non-toxic concentrations of αC-IN inhibitors will be administered in combination with currently approved RAF and MEK inhibitor regimens for the treatment of BRAFV600E melanoma, colorectal and thyroid cancers, as well as for tumors with BRAF mutations other than V600.

#1226

Targeting BTK family in ER+ breast cancer with Ibrutinib for personalized precision medicine: a double-edged sword.

Yesim Gökmen-Polar, Xiaoping Gu, Edyta Vieth, Sunil Badve. _Indiana Univ. School of Medicine, Indianapolis, IN_.

Background: Metastasis is the principal cause of morbidity and mortality from breast cancer. Although current therapies are effective in early-stage of breast cancer, resistance to therapy is common leading to the development of metastasis. The commonest site of metastases is the regional lymph nodes. The presence and the number of involved lymph nodes remains the single best predictor of metastases and an important guide for the use of systemic adjuvant therapy. Identification of the underlying molecular targets of lymph node metastasis and a better understanding how to therapeutically target these will be a significant step in the goal of prevention of metastases.

Experimental Design: To determine the key players associated with the development of metastasis in breast cancer, we performed RNA sequencing (Illumina Paired-end RNA-seq) analysis in a cohort of matched pairs of primary and (nodal) metastatic tumors (n=18). Gene expression profiles were analyzed using DESeq2. For targeted therapeutics, we performed orthotopic tumor implantation into mammary fad pads of athymic nude mice of breast cancer models.

Results: Our results identified significant differential expression of the Bruton's tyrosine kinase (BTK) family members in nodal metastasis. Among the family members, BTK, BLK, RLK/TXK and JAK3 are expressed at very high levels in the metastatic lesions as compared to matched primary tumors. Among the 51 breast cancer cell lines analyzed, the BTK genes were significantly upregulated in luminal, but not basal cell lines. Categorized by the molecular subtypes of breast cancer, hormone receptor- positive (HR) and /or HER2 overexpressing breast cancer cell lines present higher BTK expression. Among the 51 breast cancer cell lines analyzed, the BTK genes were significantly regulated in both ER+ HER2- (T47D and ZR75.1) and ER+HER2+ (BT-474 and ZR75.30) breast cancer cell lines. Ibrutinib, a selective and irreversible inhibitor of BTK targeting the cysteine-481 residue in the active site, exhibited potent activity in preclinical models of BTK-expressing targets. Ibrutinib decreased primary tumor growth significantly in ER+HER2+ (BT-474) mouse xenograft model in a dose dependent manner (10 mg/kg vs 25 mg/kg; P< 0.05), whereas it promoted the tumor growth in a significant manner in ER+HER2- xenograft models (T47D and ZR75.1; P<0.05). The differences might be due to the differences in model systems due to the HER2 presence.

Conclusion: Our data suggests that differential expression of target genes can be used to select agents as treating breast cancer. However, caution is necessary, as, in some situations, this might lead to enhancement of the growth in in vivo in subsets of breast cancer.

#1227

Angiotensin-(1-7) reduces triple negative breast cancer brain metastatic growth in association with decreased c-Met signaling.

E. Ann Tallant, Guorui Deng, Wenhong Chen, Linda J. Metheny-Barlow, Patricia E. Gallagher. _Wake Forest School of Medicine, Winston-Salem, NC_.

Patients with triple negative breast cancer often develop metastatic brain disease and have limited treatment options due to the lack of targeted therapeutics that efficiently cross the blood brain barrier. In the current study, we investigated the effect of angiotensin-(1-7) [Ang-(1-7)], a heptapeptide hormone with anti-proliferative and anti-metastatic activities, on the growth of murine brain tumors using brain trophic BR.5-4T1 cells in a model of metastatic triple negative breast tumor growth. BR.5-4T1 cells (1.25 x 104) were injected into the internal carotid artery of female BALB/c mice; after two days, mice were implanted with osmotic mini-pumps to deliver 24 µg/kg/h Ang-(1-7) and all mice were sacrificed after 20 days of treatment. Ang-(1-7) treatment significantly reduced metastatic tumor number in the brain; an average of 20 metastatic lesions per mouse was observed in the brains of an untreated control cohort while an average of 3 metastatic tumors per mouse was observed in the Ang-(1-7)-treated cohort. Tumor size was also reduced 17-fold by treatment with Ang-(1-7). Ki67, a marker of proliferation, was decreased from 29.1±6.7% to 6.4±2.2% in tumors from mice treated with Ang-(1-7), indicating that the heptapeptide hormone decreased metastatic tumor burden in the brain. Aberrant activation of the c-Met receptor tyrosine kinase signaling pathway facilitates tumor cell invasion and metastasis. Treatment with Ang-(1-7) significantly reduced the percentage of metastatic tumor cells exhibiting phospho-c-Met immunoreactivity, from 20.8±3.4% in tumors from untreated mice to 6.7±2.6% in mice treated with Ang-(1-7). Protein phosphatase 1b (PTB1b), which dephosphorylates and negatively regulates phospho-c-Met, was increased in the tumor cells of mice treated with Ang-(1-7) compared to tumor cells from untreated animals, from 4.1±1.4% in sections of tumors from control mice compared to 8.8±0.9% in sections of tumors from mice treated with Ang-(1-7). These results suggest that Ang-(1-7) increases PTP1b to antagonize c-Met signaling and decrease triple negative breast cancer brain metastasis. In support of a role for c-Met signaling in the regulation of metastatic brain disease in breast cancer patients, analysis of publicly available breast cancer cohort databases demonstrated a significant correlation between low c-Met/high PTP1b expression and improved survival outcome, including an increase in brain metastasis-free survival and distant-metastasis free survival. This is the first report to demonstrate that Ang-(1-7) reduces metastatic triple negative breast cancer localized to the brain in association with increased protein phosphatase PTP1b and decreased phospho-c-Met, suggesting that the heptapeptide hormone may serve as a novel targeted therapy to reduce metastatic brain tumor burden in triple negative breast cancer patients.

#1228

Nintedanib selectively inhibits the activation and tumor-promoting effects of fibroblasts from lung adenocarcinoma patients.

Jordi Alcaraz,1 Marta Gabasa,2 Rafael Ikemori,1 Noemí Reguart3. 1 _University. of Barcelona, Barcelona, Spain;_ 2 _University of Barcelona, Barcelona, Spain;_ 3 _Hospital Clínic de Barcelona, Barcelona, Spain_.

Nintedanib (BIBF 1120) is a multikinase inhibitor recently approved to treat lung adenocarcinoma (ADC) patients based on the therapeutic benefits reported in clinical trials on ADC but not on squamous cell carcinoma (SCC), which are the two most common lung cancer subtypes. However, the biological processes underlying the selective anticancer function of nintedanib in ADC remain obscure. To shed light on this gap of knowledge, we examined the role of tumor-associated fibroblasts (TAFs) in the differential effects of nintedanib in ADC and SCC. Because TAFs are largely quiescent and activated in vivo, we focused on the antifibrotic effects of nintedanib on TAFs stimulated with TGF-β1, which is a potent fibroblast activator frequently upregulated in lung cancer. Nintedanib dose-dependently inhibited the TGF-β1-induced expression of activation markers in both ADC-TAFs and control fibroblasts derived from uninvolved parenchyma, whereas such inhibiton was very modest in SCC-TAFs, thereby suggesting that TAF activation is regulated by different mechanisms in ADC and SCC. Remarkably, nintedanib abrogated the stimulation of growth and invasion in a panel of carcinoma cell lines induced by secreted factors from activated TAFs in ADC but not SCC. These results reveal that nintedanib is a potent inhibitor of fibrosis and its associated tumor-promoting effects in ADC, and strongly suggest that the subnormal antifibrotic response of SCC-TAFs to nintedanib may underlie the lack of therapeutic benefits of this drug in SCC. Our findings also support that preclinical models based on carcinoma-TAF interactions may help unraveling the resistance mechanisms of SCC-TAFs to nintedanib and testing new combined therapies to further expand the therapeutic effects of this drug.

#1229

RNAi-based β-catenin inhibition in combination with MEK inhibition promotes synergistic anti-tumor efficacy and overcomes resistance with MEK inhibition promotes synergistic anti-tumor efficacy and overcomes resistance.

Shanthi Ganesh, Serena Shui, Girish Chopda, Martin Koser, Kevin Craig, Chaitali Dutta, Cheng Lai, Hank Dudek, Weimin Wang, Bob D. Brown, Marc Abrams. _Dicerna Pharmaceuticals, Inc, Cambridge, MA_.

Despite recent advances in early detection and therapeutic intervention, colorectal cancer (CRC) remains one of the most deadly cancers in the United States. The Wnt/β-catenin and KRAS/BRAF-driven MAPK pathways were reported to cooperate in inducing CRC initiation and progression. Several studies have also identified Wnt signaling as a resistance mechanism in MAPK pathway-activated CRCs. Thus far, independent targeting of these single oncogenic events in CRC has yielded limited therapeutic success. RNA interference (RNAi) triggers are capable of silencing any expressed mRNA with high potency and specificity, including previously un-druggable targets such as β-catenin, an oncogene frequently dysregulated in CRC. DCR-BCAT is a chemically-optimized DsiRNA targeting CTNNB1, the gene which encodes β-catenin, formulated in a second-generation EnCore lipid nanoparticle. We have previously reported extensive preclinical pharmacology for DCR-BCAT in mouse tumor models of diverse origin. (Ganesh et al, Mol Cancer Ther 2016). Here we demonstrate that the dual inhibition of β-catenin and MEK demonstrates synergistic efficacy in not only primary CRC, but also in aggressive liver metastases of CRC in preclinical models. This combination of DCR-BCAT and the MEK inhibitor trametinib was also efficacious in tumors which are known to be resistant to trametinib, as well as tumors which acquire resistance due to drug exposure. These data support clinical development of these combination approaches for this first-in-class RNAi therapeutic.

#1230

Talazoparib, a second generation PARP inhibitor, is a novel therapy for PTEN mutants in prostate cancer.

Zachary Z. Reinstein, Rajita Vatapalli, Jonathan Anker, Sahithi Pamarthy, Benedito Carneiro, Sarki Abdulkadir. _Northwestern University, Chicago, IL_.

Prostate cancer is the most common cancer among men, other than skin cancer, in the United States. When the cancer enters into the metastatic castration resistant stage (mCRPC), few treatment options are currently available and the disease is considered incurable. The purpose of this study is to investigate a new, targeted therapy against prostate cancers, including mCRPC. Talazoparib is a novel PARP inhibitor with up to 100-fold higher potency and PARP affinity when compared to PARP inhibitors such as olaparib, veliparib, and rucaparib. Current literature suggests that PTEN mutants have high sensitivity to talazoparib. PTEN is a tumor suppressor gene that is commonly lost in prostate cancers. A murine prostate cancer cell line, Myc-CaP, which has been modified to overexpress the oncogene Myc, was edited by the CRISPR-Cas9 system to knock out the PTEN gene. Myc-CaP and Myc-CaP PTEN KO cells were treated with olaparib, an FDA approved, first generation PARP inhibitor, and talazoparib. Talazoparib decreased cell viability significantly more in PTEN KO cells versus PTEN wild-type (WT) cells. No such trend was observed when these cells were treated with olaparib. In addition, IC50 values in prostate cancer cell lines with heterozygous or homozygous deletion of PTEN were up to 20 times lower with the talazoparib treatment when compared to olaparib. Not only does this indicate that talazoparib is much more potent than olaparib, but also preferentially impairs cell growth in PTEN mutants. PTEN's signal transduction prevents the phosphorylation of AKT, which is necessary for cell proliferation. When cells were treated with an AKT inhibitor, MK2206, alone or in combination with talazoparib, pAKT protein levels decreased, but the greatest effect was seen when MK2206 was used in combination with a low dose of talazoparib in the Myc-Cap cell line with wt PTEN. Western blot analysis showed that PTEN was upregulated following talazoparib treatment. These results indicate synergy of talazoparib and MK2206 in reducing the phosphorylation of AKT in a dose-dependent manner. Talazoparib is by far the most potent PARP inhibitor developed to date. With talazoparib potentially acting on prostate cancer with PTEN mutations, talazoparib treatment may expand current PARP inhibitor treatment in advanced prostate cancers and thereby elicit a response in as many as 80% of patients. Thus, talazoparib has vast therapeutic potential in prostate cancer.

#1231

**Restoration of tumor suppression** in vivo **by systemic delivery of PTEN mRNA nanoparticles.**

Yingjie Xu,1 Mohammad Ariful Islam,2 Harshal Zope,2 Morteza Mahmoudi,2 Robert S. Langer,3 Jinjun Shi,2 Bruce R. Zetter,1 Omid C. Farokhzad2. 1 _Boston Children's Hospital, Harvard Medical School, Boston, MA;_ 2 _Brigham and Women's Hospital, Harvard Medical School, Boston, MA;_ 3 _Massachusetts Institute of Technology, Cambridge, MA_.

PTEN (phosphatase and tensin homolog on chromosome ten) is one of the most common lost or mutated tumor suppressor genes in human cancers, including ~50% of metastatic castration-resistant prostate cancer (mCRPC). By catalyzing PIP3 dephosphorylation, PTEN negatively regulates the PI3K-AKT-mTOR pathway, which is frequently altered in mCRPC. Reintroduction of functional PTEN for mCRPC treatment has proven difficult. By employing self-assembled lipid-polymer hybrid nanoparticle platforms, we successfully reintroduced PTEN mRNA to PTEN-null prostate cancer cells both in vitro and in vivo. These mRNA-loaded nanoparticles demonstrate high protein expression efficiency, low toxicity and good stability in serum and tumor accumulation. We confirmed that restoration of PTEN in PTEN-null prostate cancer cells inhibits the PI3k-AKT pathway, reduces cell viability and enhances apoptosis in vitro. Systemic delivery of PTEN mRNA-loaded nanoparticles in prostate xenograft tumors results in ~85% inhibition of tumor growth and leads to tumor cell death without toxic side effects in vivo. In summary, this work provides proof of concept of mRNA-based gene therapy for systemic restoration of functional PTEN for tumor suppression in vivo. It represents a novel approach to PI3K-AKT pathway inhibition, with the potential to specifically target cancers with loss of PTEN function.

#1232

Development of novel therapeutic splice-switching oligonucleotides targeting oncogenic RNA isoforms driving race-related aggressive prostate cancer.

Jennifer A. Freedman, Timothy Robinson, Bonnie LaCroix, Brendon Patierno, Daniel George, Bruce Sullenger, Steven Patierno. _Duke Cancer Inst., Durham, NC_.

Background: Age-adjusted incidence and mortality rates for prostate cancer (PCa) among African American (AA) men are 1.6- and 2.4-fold greater, respectively, than among white men. More aggressive characteristics of AA PCa account for a significant component of the PCa disparity, in addition to social determinants of health. Previous work from our laboratory and others have identified deregulation of the androgen receptor (AR) and epidermal growth factor receptor (EGFR) pathways in AA versus white PCa. Thus, there is an urgent need to develop a novel therapeutic strategy capable of inhibiting oncogenic AR and EGFR RNA isoforms enriched for in AA PCa.

Methods: We have designed and synthesized novel chemically modified splice switching oligonucleotides (SSOs) to: 1) correct aberrant splicing leading to production of AR-V7 (AR-V7 SSO), an AR variant that lacks the ligand binding domain, is constitutively active and associates with castration-resistant PCa, poorer clinical outcomes and resistance to androgen ablation/AR inhibition therapies, 2) drive production of AR45 (AR45 SSO), an AR variant that has a unique, non-functional transactivation domain and acts in a dominant-negative manner and 3) drive production of inhibitory or dominant-negative EGFR isoforms (EGFR-TM or EGFR-TK SSOs), lacking the transmembrane or tyrosine kinase domain, respectively.

Results: Transfection of a panel of PCa cell lines derived from AA and white patients with AR-V7 SSO decreases AR-V7 RNA and protein in a dose-dependent manner. Preliminary data suggests this biochemical response correlates with a biologically significant phenotype, as AR-V7 SSO also decreases proliferation, proliferation in the presence of enzalutamide and colony forming ability of PCa cells. Following transfection of PCa cell lines with AR45 SSO, an increase in AR45 RNA is seen and simultaneous decreases in wild type AR RNA and AR-V7 RNA are seen. In addition, modulation of AR signaling is detected, with decreases in RNAs corresponding to AR-induced target genes and increases in RNAs corresponding to AR-repressed target genes. Transfection of PCa cell lines with EGFR-TM or EGFR-TK SSOs increase RNA corresponding to these isoforms in a dose-dependent manner. Furthermore, a decrease in phosphorylated EGFR protein is detected. Further studies to examine the effects of these SSOs on transactivation activity, signaling and PCa cell biology and the therapeutic efficacy of these SSOs in AA and white PCa patient-derived explants are underway.

Conclusions: These studies suggest that SSOs can be developed to modulate AR and EGFR signaling. Such SSOs could represent a novel therapeutic strategy with the potential to reduce PCa disparities for AA men and improve outcomes for men of all races with aggressive disease driven by these mechanisms.

#1233

WNT inhibitor ICG-001 prevents visceral metastatic triple negative breast cancer in a chemo-resistant patient derived xenograft -PDX-model.

Iram Fatima, Ikbale El Ayachi, Chidi Zacheaus, Joseph Kerby Gray, Raya Krutilina, Tiffany N. Seagroves, Susan A. Krum, Gustavo A. Miranda-Carboni. _University of Tennessee Health Science Center, Memphis, TN_.

Triple negative breast cancer (TNBC) is characterized by absence of the estrogen receptor (ER) and progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) amplification. Such cancers are highly aggressive and frequently metastasize to the lung and brain. Unlike other breast cancer subtypes such as, ER+, PR+ and/or HER2+, TNBCs have no specific targeted-therapeutics; therefore, studies should be directed for development of targeted therapies to treat this condition. Recent studies indicate that Wnt/β-catenin signaling is particularly activated in TNBC and is associated with reduced overall survival in all patients. Therefore, pharmacological targeting of Wnt signaling pathway constitutes an ideal approach for treating TNBC and various Wnt inhibitors are currently in use in clinical trials, such as; ICG-001 in metastatic colon cancer. The inhibitory effects of ICG-001 have not been tested in chemoresistant TNBC PDX tumors. Herein, we report for the first time that ICG-001 compound selectively inhibited the growth of several European American (EA) and African American (AA) triple negative breast cancer subtypes MSL and BSL-1 cell lines both in vitro and in vivo models. To further investigate the precise mechanisms of action in the regulation of Wnt/β-catenin signaling by ICG-001, we performed Western blot analysis, apoptosis assays, cell cycle assays and quantitative real-time reverse transcriptase- polymerase chain reactions in human triple negative breast cancer cells. This compound significantly interfered with Wnt/β-catenin signaling, and its inhibition led to downregulation of important downstream targets such as Axin2, HMGA2, PCNA, c-myc and Cyclin D1, which in turn led to inhibition of proliferation, cell cycle progression and metastasis confirming our previous results too. In addition ICG-001 inhibited the invasion and motility of tumor cells and showed inhibition and prevention of visceral metastatic PDX tumors from both chemoresistant EA and AA women. These results indicate that the Wnt inhibitor ICG-001 could constitute a powerful new chemotherapeutic agent against triple negative breast cancer.

#1234

The novel FGFR4-selective inhibitor INCB062079 is efficacious in models of hepatocellular carcinoma harboring FGF19 amplification.

Bruce Ruggeri, Matthew Stubbs, Yan-ou Yang, Ashish Juvekar, Liang Lu, Sindy Condon, Darlise DiMatteo, Xiaoming Wen, Paul Collier, Timothy Burn, Liangxing Wu, Daniel Wilson, Swamy Yeleswaram, Alan Roberts, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Phillip CC Liu. _Incyte Corporation, Wilmington, DE_.

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer with limited treatment options for advanced stage disease. Thus, there is a critical medical need for improved therapies. In approximately 10% of HCC, a focal amplicon at 11q13 harboring FGF19 has been reported. High levels of FGF19 have been shown to drive HCC tumor development and progression in preclinical models, suggesting that selective targeting of FGFR4, a high affinity receptor for FGF19, may be efficacious in these HCC tumors. INCB062079 a potent and selective irreversible inhibitor of FGFR4 (>250-fold vs FGFR1/2/3) suppresses the growth of HCC cell lines driven by amplification and overexpression of FGF19. In subcutaneous xenograft models of HCC, oral dosing of INCB062079 at tolerated doses resulted in dose-dependent inhibition of tumor growth with regressions observed at higher doses consistent with inhibition of FGFR4 signaling in the tumors. In combination with sorafenib, the only approved targeted therapy for HCC, FGFR4 inhibition exhibited additive tumor growth inhibition in the Huh7 model. To assess exposure of INCB062079 in orthotopic tumors after oral dosing, Hep3B tumors were implanted surgically into the liver and their development monitored by analysis of plasma alpha-fetoprotein (AFP). At efficacious doses, INCB062079 strongly suppressed the levels of AFP and FGF19 secreted by the tumors, and their levels correlated well with the reduction in terminal liver tumor mass, suggesting that these may be surrogate markers for response of HCC tumors to INCB062079. In two PDX models of HCC with amplification of FGF19 (4-6 CNV), INCB062079 administration reduced tumor growth by 50-66% at doses that were well tolerated. Additional surrogate markers for FGFR4 inhibition were explored including several parameters related to FGF19 regulation of bile acid metabolism. The mRNA levels of CYP7A1, encoding cholesterol 7a-hydroxlyase, the rate limiting enzyme in bile acid synthesis, were induced in the livers of cynomolgus monkeys upon dosing with INCB062079. Correspondingly there was a dose-dependent increase in fecal bile acids. In summary these data demonstrate that INCB062079 is highly and selectively efficacious in models of HCC with FGF19-FGFR4 oncogene addiction and elicits pharmacodynamic responses in primates providing support for the clinical evaluation of INCB062079 in genetically selected liver cancer patients.

#1235

EPHA2-targeted therapy enhances the cytotoxicity of eicosapentaenoic acid against triple-negative inflammatory breast cancer.

Angie M. Torres-Adorno,1 Heidi Vitrac,2 Yuan Qi,3 Yiwen Yang,4 Peiying Yang,5 Bedrich L. Eckhardt,6 Naoto T. Ueno6. 1 _The University of Texas Graduate School of Biomedical Sciences at Houston, Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Center for Membrane Biology, University of Texas Health Science Center, Houston, TX;_ 3 _Bioinformatics & Computer Biology, The University of Texas MD Anderson Cancer Center, Houston, TX; _4 _Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, TX;_ 5 _General Oncology Research, The University of Texas MD Anderson Cancer Center, Houston, TX;_ 6 _Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX_.

Background: Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer. We have previously reported that mediators of inflammation, such as COX-2, promote the growth of Triple-Negative receptor (TN) IBC xenografts; therefore, inflammation in TN-IBC has a unique opportunity as a therapeutic strategy. Eicosapentaenoic acid (EPA), a non-toxic omega-3 fatty acid with anti-inflammatory properties, has partially reduced tumor growth in pre-clinical models of TN-IBC. Therefore, our goal is to develop a novel non-toxic approach that enhances EPA efficacy against TN-IBC in combination with targeted therapy.

Methods and Results: Using a high-throughput, siRNA screen (939 genes) in the TN-IBC cell line SUM149PT, we identified Ephrin type-A receptor 2 (EPHA2), an oncogenic cell-surface receptor tyrosine kinase, as a target that modulates the sensitivity of TN-IBC cells to EPA treatment. To determine the clinical relevance of EPHA2, we interrogated a meta-analysis of breast cancer mRNA expression data sets, and found that high EPHA2 tumor expression was significantly correlated with poor overall survival in TN-IBC patients, compared to low EPHA2 expressing tumors (P = 0.01). We observed no significant correlations to other breast cancer subtypes. Similar findings were observed in vitro were EPHA2 expression predominantly occurred in the TN-IBC subtypes (19 of 30) among 49 breast cancer cell lines. Gain/loss-of-expression studies were performed to functionally validate EPHA2 as a synergistic combinational target with EPA in two EPHA2-expressing TN-IBC models, SUM149PT and BCX010, using proliferation and apoptosis assays in vitro and established tumor xenografts in vivo. EPHA2 gene silencing significantly reduced cell growth and induced apoptosis in combination with EPA when compared with untreated control and monotherapy in vitro (P < 0.05) and in vivo (P < 0.001), while vector-induced EPHA2 expression reversed cell growth reduction and apoptosis induction following combination treatment with EPA in vitro (P < 0.05). To translate our findings to the clinic, we validated that dasatinib, a small molecule inhibitor of EPHA2, in combination with EPA significantly enhanced cell death of SUM149PT and BCX010 cells in vitro when compared to non-treated and monotherapy (P < 0.05). Finally, using membrane fluidity assessment and reverse-phase protein array (300 antibodies), we determined that combination treatment efficacy depended on EPA/EPHA2 inhibition-mediated increase in cell membrane rigidity (P < 0.001, compared to monotherapy), which subsequently inhibited receptor tyrosine kinase signaling activity, potentially resulting in induction of apoptosis.

Conclusions: Our preclinical findings provide a rationale for the development of a phase 1 clinical trial investigating combination EPA and EPHA2-inhibitors in patients with EPHA2-positive TN-IBC.

#1236

Novel androgen receptor inhibitors in triple negative breast cancer.

Saswati Bhattacharya, Yuanqi Cai, Steven Kurina, Pamela Westmark, Benjamin Hokanson, Matthew Anderson, Ruth O'Regan. _UW Madison, Madison, WI_.

Triple negative breast cancer (TNBC), which lacks expression of hormone receptors and human epidermal growth factor receptor 2, has the worst prognosis of all breast cancer subtypes, due to inherent chemo-resistance and a lack of therapeutic targets. A subset of TNBC has been shown to express androgen receptor (AR) but despite promising pre-clinical data, clinical trials evaluating the use of approved anti-androgens (bicalutamide, enzalutamide) in AR-positive TNBC has shown modest efficacy to date. In prostate cancer, bicalutamide binds to AR with low affinity and in the presence of AR gene amplification or mutation, can exhibit partial agonist activity leading to resistance. In order to increase affinity for AR and to avoid AR antagonist to agonist conversion synthesized 7-substituted umbelliferones (UMB) derivatives with a distinct scaffold modified from available AR antagonists (Kandil et al Bioorganic & Medicinal Chemistry Letters, 2016). These agents exhibited inhibitory activity in human prostate cancer cells lines at sub-micro molar level, a 50-fold and 30-fold improvement over bicalutamide and enzalutamide, respectively. We hypothesized that these novel AR antagonists would be effective in inhibiting the growth of AR-positive TNBC cells. Methods: TNBC AR negative mesenchymal MDA-MB-231 (as control) and AR positive MDA-MB-231 cells were utilized for determination of the efficacy of the UMB derivatives. For proliferation assays, viability of the cells was monitored over a period of 72 hours with redox dye Cell Titer-Blue. Standard Western blotting techniques were used for protein detection. For apoptosis analysis, standard flow cytometry reagents, FITC labelled Annexin V and Propidium Iodine(PI) mix were utilized. Results: UMB derivatives decreased the expression of AR in AR-positive MDA-MB-453 in a dose dependent manner. UMB derivatives, at micro Molar concentration decreased cell proliferation of AR-positive and interestingly AR-negative cell lines by 20 to 50%, also in a dose dependent manner. UMB derivatives induced apoptosis in both cell lines. Interestingly, UMB derivatives induced a change from a mesenchymal to epithelial phenotype in AR-negative MDA-MB-231 cells, which was confirmed by decreased expression of vimentin and other mesenchymal markers. Conclusions: Novel UMB derivatives inhibit growth and cause apoptosis in TNBC cells and induce mesenchymal-to-epithelial transition in an AR-negative mesenchymal TNBC cell line. Targeting AR with novel AR antagonists seem to be a promising therapeutic approach for AR-positive TNBC and further evaluation of AR antagonists in AR-negative mesenchymal TNBC is warranted.

#1237

PDGF induces cell growth and changes in glucose metabolism in colon cancer in the absence of PDGF receptors.

Romana Moench, Tanja Grimmig, Christoph-Thomas Germer, Ana Maria Waaga-Gasser, Martin Gasser. _Univ. of Würzburg, Wuerzburg, Germany_.

Background: The Platelet derived growth factor (PDGF) and its receptors play a major role in inducing proliferation, migration, and angiogenesis by activating intracellular PI3K/Akt/mTOR signaling events in different solid tumors and therefore represent attractive targets in tumor therapy. Recently we showed a PDGF-induced activation of metabolism and proliferation in HT29 colon cancer cells in the absence of specific PDGF receptors. The aim of this study was to analyze PDGFR and VEGFR expression and possible alternative PDGF binding partners in colorectal cancer (CRC).

Methods: Human HT29 colon cancer cells were cultured and stimulated with PDGF in a time-dependent manner. Additionally, VEGFR2 and EGFR inhibition was performed to analyze alternative PDGF signaling events. Whole cell or RNA extracts were analyzed by Western Blot and RT-q-PCR for receptors and PI3K/Akt/mTOR signaling. To investigate the effects of specific receptor inhibition on proliferation, MTS proliferation assays were performed. Moreover, mRNA levels of PDGFR and VEGFR in tumors from patients with CRC were analyzed by RT-qPCR.

Results: Human UICC stage I-IV tumors exhibited a significantly increased PDGFRβ and VEGFR1,2 gene expression. As observed previously, HT29 colon cancer cells showed only positivity for VEGFR1,2, but no PDGFR protein expression. Despite that, stimulation with PDGF resulted in increased proliferation and metabolic changes. In contrast, Caco-2 and SW480 colon cancer cells showed PDGFR and VEGFR expression on protein level. Interestingly, PDGF stimulation increased VEGFR1 and 2 gene expression in HT29 colon cancer cells and secondly inhibition of VEGFR2 and EGFR showed alterations in proliferation and Akt signaling in PDGF stimulated cancer cells.

Conclusion: Despite the absence of PDGF receptors in HT29 colon cancer cells, PDGF induced proliferating and metabolic effects in the tumor cells, suggesting an alternative binding partner on the tumor cell surface. Further investigation of the alternative PDGF binding partners could be of clinical relevance in CRC.

#1238

Targeting the platelet derived growth factor receptor (PDGFR) with the receptor tyrosine kinase inhibitor ponatinib in small cell carcinoma of the ovary, hypercalcemic type.

Jessica Diane Lang,1 William Hendricks,1 Pilar Ramos,1 Holly Yin,1 Chris Sereduk,1 Jeffrey Kiefer,1 Yemin Wang,2 Anthony N. Karnezis,2 Bernard Weissman,3 David Huntsman,2 Jeffrey Trent1. 1 _Translational Genomics Research Institute, Phoenix, AZ;_ 2 _British Columbia Cancer Agency, Vancouver, British Columbia, Canada;_ 3 _University of North Carolina at Chapel Hill, Chapel Hill, NC_.

Subunits of the SWI/SNF chromatin-remodeling complex are tumor suppressors that are inactivated in ~20% of all cancers, yet few targeted treatments have shown selective activity in SWI/SNF-mutant cancers. Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare, aggressive ovarian cancer in young women that is universally driven by SWI/SNF dysregulation. Given that two-year survival following standard high-dose chemotherapy and radiation in SCCOHT is less than 35%, a great need exists for effective targeted therapies to improve outcomes for these women. We previously demonstrated that SCCOHT tumors are driven by inactivating mutations in SMARCA4, one of two mutually exclusive SWI/SNF ATPases. In addition, we have shown that SCCOHT lacks expression of the alternative SWI/SNF ATPase, SMARCA2. We have now found through integrated genomic and functional analyses in SCCOHT tumors and cell lines that SMARCA4 loss correlates with increased expression of receptor tyrosine kinases (RTKs) including the platelet derived growth factor receptors (PDGFRs). Through integration of high-throughput RNA interference and drug screens in SCCOHT cells we have identified sensitivity to RTK knockdown and RTK inhibitors including the FDA-approved oncology drug, ponatinib. These data corroborate prior studies showing RTK dependence in rhabdoid tumors, rare cancers that are also driven by mutations in the SWI/SNF complex. Of the known ponatinib targets, PDGFR-alpha and FGFR1 were highly expressed in SCCOHT tumors, as confirmed in RNA-Seq data (four tumors) and a SCCOHT tissue microarray (TMA; ten tumors). Furthermore, PDGFR-alpha and -beta phosphorylation and downstream signaling are inhibited by ponatinib in SCCOHT cells, suggesting that these tumors are sensitive to ponatinib due to dependence on signaling through these RTKs. Finally, given ponatinib's potency in vitro and the proposed mechanism of action, we tested this agent in xenograft models of SCCOHT. In addition to confirming efficacy in a SCCOHT cell line xenograft model, superior efficacy was demonstrated in two patient-derived xenograft (PDX) models of SCCOHT with ponatinib. Thus, ponatinib effectively targets SWI/SNF-mutant SCCOHT tumors through inhibition of PDGFR signaling and may have clinical utility for the treatment of these cancers.

#1239

The expression of salvage pathway enzymes in non-small cell lung cancer cells.

Michelle H. Townsend, Evita G. Weagel, Michael D. Anderson, Edwin Velazquez, Richard A. Robison, Kim L. O'Neill. _Brigham Young University, Provo, UT_.

The aim of this study is to evaluate the expression of the salvage pathway enzymes DCK, APRT, and HPRT in lung cancer cells to determine if they could serve as biomarkers for lung cancer diagnosis and potential treatment. In both men and women, lung cancer is the most lethal cancer in the world, and accounts for more than 30% of cancer-related deaths. We chose to evaluate the salvage pathway enzymes due to an established relationship between the serum biomarker Thymidine Kinase 1 (TK1) and lung cancer. Two non-small cell lung carcinoma cell lines were utilized for this analysis (NCI-H460 and A549) along with cancer tissue and healthy tissue from 27 lung squamous carcinoma patients. The surface localization of these enzymes was determined utilizing flow cytometry, confocal microscopy, and scanning electron microscopy, while upregulation within tissue was assessed using immunohistochemistry (IHC). Throughout our investigation, we found no significant expression of DCK or APRT on the surface of non-small cell lung cancer cells, but found a significant presence of HPRT on the plasma membrane of both NCI-H460 and A549 cells. The average population florescence of cells treated with HPRT antibodies increased by 24.3% and 12.9% in NCI-H460 and A549 cells, respectively, in comparison to controls. To ensure expression was not attributed to cytoplasmic HPRT, confocal microscopy was performed to visualize HPRT binding on the plasma membrane. After staining NCI-H460 cells treated with both fluorescent antibodies and a membrane-specific dye, we observed direct overlap between HPRT and the membrane of the cancer cells. Additionally, gold conjugated antibodies were used to label and quantify the amount of HPRT on the cell surface using scanning electron microscopy and EDAX. Further confirming HPRT presence, the gold weight percentage of the sample increased significantly when NCI-H460 cells were exposed to HPRT antibody (p-value 0.012) in comparison to isotype controls. Finally, the general upregulation of the protein was observed in patient tissue samples, with approximately 44% of lung cancer tissues showing significant HPRT upregulation when compared to healthy tissue samples. This differential upregulation shows an altered HPRT expression within some patients, which may help explain the presence of this presumed cytosolic enzyme on the surface of lung cancer cells. These results strongly indicate a unique relationship between cancer cells and HPRT and suggest HPRT as a possible biomarker for the detection and treatment of non-small cell lung cancers.

#1240

Evaluating clinically relevant pharmacological agents in a rat ambulation model to ameliorate PEGylated recombinant hyaluronidase PH20 (PEGPH20)-mediated musculoskeletal adverse events.

Li Ma,1 Marc R. Azar,2 Michael Shepard,1 Ya Huang,1 Piet A. Vervaet,1 Daniel C. Maneval,1 Curtis B. Thompson,1 Rudy Paladini1. 1 _Halozyme Therapeutics Inc., San Diego, CA;_ 2 _Behavioral Pharma, La Jolla, CA_.

Some cancers accumulate high levels of the glycosaminoglycan hyaluronan (HA) within the tumor microenvironment (TME), and this HA accumulation is associated with rapid tumor progression and poor clinical outcomes. As preclinical data have demonstrated that enzymatic degradation of HA, via intravenous (IV) administration of PEGylated recombinant human hyaluronidase PH20 (PEGPH20), increases therapeutic efficacy in HA-accumulating tumors, several clinical trials evaluating PEGPH20 in combination with anti-cancer therapies are ongoing. The most advanced clinical trial (Phase 3) is evaluating PEGPH20 plus Abraxane® (nab-paclitaxel)/gemcitabine (AG) in patients with stage IV untreated pancreatic ductal adenocarcinoma (PDA)(NCT02715804). The safety profile of PEGPH20/Abraxane/gemcitabine (PAG) was generally similar to AG alone in an interim analysis of the Phase 2 data, with the exception of a potential imbalance of thromboembolic events (TEs), and an increased incidence of musculoskeletal events (MSEs) in the PAG arm, with the predominant MSE being muscle spasms (55.4% PAG vs. 1.6% AG, all grades)(Bullock, ASCO 2016). In an effort to find pharmacological agent(s) that might decrease MSEs in PAG-treated patients, a rat locomotor activity assay was developed. In brief, locomotor activity of male Sprague Dawley rats was measured with an automated infrared photobeam monitoring apparatus (Hamilton/Kinder®, San Diego) during the 12h dark cycle when rats are normally active. Any PEGPH20-mediated decrease in rat locomotor activity was interpreted as MSE-like effects. In response to PEGPH20 (1 mg/kg) the locomotor activity of male rats was reduced by ~75% for the first 2 dark cycles post-treatment (48h), and then gradually returned to vehicle baseline levels by days 4-5. FDA approved drugs used clinically to alleviate pain were subsequently tested for their ability to ameliorate the effects of PEGPH20 on locomotor activity. Agents included anti-inflammatories, anti-spasmodics, neuropathic pain medications, neuroleptics, diuretics, anti-hypertensives, anti-fibromyalgics and opioids. The steroid dexamethasone, currently being used in the Phase 2 and Phase 3 PDA clinical trials to manage MSEs, was the positive control. As a class, nonsteroidal anti-inflammatory drugs (NSAIDs), including ketorolac, ketoprofen, diclofenac, and piroxicam, increased rat ambulation by ~40% on the first night following PEGPH20 treatment compared to PEGPH20 alone. Mobility in rats treated with NSAIDs/PEGPH20 increased ~60% on the second night compared to PEGPH20 alone. No other drug classes increased rodent ambulation. These data suggest that NSAIDs may be useful in partially alleviating the MSEs experienced by patients receiving PEGPH20.

#1241

A preclinical study of the combined treatment of arginase and canavanine in pancreatic cancer.

Tsz Tung Kwong, Chi Hang Wong, Herbert Ho Fung Loong, Stephen Lam Chan. _The Chinese University of Hong Kong, Hong Kong, Hong Kong_.

Background: Arginine deprivation is a novel approach for cancer treatment as some cancers were found to be arginine auxotrophic. PEG-BCT-100, a pegylated form of recombinant human arginase, was used to investigate the effect of arginine deprivation on pancreatic cell lines. On the other hand, Canavanine (CAV), a natural toxic analog of arginine isolated from leguminous plant, has also demonstrated its ability in inhibiting cancer cell proliferation. It alters the protein structure and causes critical metabolic defects in the cells by replacing arginine into newly synthesized proteins during translation. In this study, we have also tested the efficacy of combined arginase and CAV treatment on pancreatic cell lines.

Methods: Pancreatic cell lines (MIA PaCa-2, CFPAC-1) and normal fibroblast cell line (WI-38), were either incubated in normal or arginine free condition. The corresponding IC50 of CAV was determined by cell viability assay. To evaluate the responsiveness to the combined treatment, cells were monitored by live cell imaging under the conditions of PEG-BCT-100 treatment alone (0.3IU/ml) or in combination with CAV (10μM or 50μM). The apoptotic effect of the combined treatment was examined by Western blotting and Annexin V assay.

Results: Either arginase or CAV treatment could inhibit pancreatic cancer cell growth. The cells treated with CAV in arginine free condition demonstrated an inhibition of cell proliferation and its corresponding IC50 was significantly lowered from millimolar to micromolar concentration when arginine was withdrawn. Strong synergism of CAV and PEG-BCT-100 was observed after 48-hour treatment with enhanced apoptosis in pancreatic cell lines, but not in normal fibroblast cells. Both early and late apoptosis were observed in co-treated pancreatic cancer cells as indicated by Annexin V assay and the detection of polyADP ribosyl polymerase (PARP) in Western blot.

Conclusion: Arginine deprivation by PEG-BCT-100 combined with CAV treatment showed a strong synergetic effect on inducing cell death in pancreatic cell lines, suggesting that the supplement of CAV could facilitate the treatment outcome of arginine deprivation by arginase. This combined treatment may serve as a treatment strategy for pancreatic cancer.

#1242

Ornithine decarboxylase as a therapeutic target in endometrial cancer.

Hong Im Kim,1 Chad R. Schultz,1 Andrea L. Buras,1 Elizabeth Friedman,2 Alyssa M. Fedorko,1 Leigh G. Seamon,3 Gadisetti Chandramouli,4 André S. Bachmann,1 John I. Risinger1. 1 _Michigan State University, Grand Rapids, MI;_ 2 _Grand Rapids Medical Education Partners, Grand Rapids, MI;_ 3 _Spectrum Health, Grand Rapids, MI;_ 4 _GenEpria, Columbia, MD_.

Endometrial cancer is the 4th most common cancer and the 6th deadliest cancer in US women. The American Cancer Society estimates there will be 60,050 new endometrial cancers in 2016, an increase of more than 10% from the previous year highlighting the need for more effective treatments and prevention. Ornithine Decarboxylase (ODC) a key enzyme in polyamine synthesis is often overexpressed in cancers and contributes to cell proliferation and tumor growth. Therefore, ODC and the polyamine pathway are considered rational targets for cancer treatment or prevention. We noted ubiquitous expression of ODC1 in our previously published endometrial cancer gene array data and confirmed this in the cancer genome atlas (TCGA), finding expression in all four molecular sub-types with highest expression in copy number high cancers which have the worst clinical outcomes. Therefore, we explored the association of ODC1 gene expression with clinical outcomes of overall survival (OS) and recurrence in the TCGA cohort and noted that elevated ODC1 was significantly related to OS (Wald test p=0.001) and recurrence (p=0.01). Importantly, we confirmed these observations using QRT-PCR in a validation cohort of 60 endometrial cancers and found that endometrial cancers with elevated ODC1 had significantly shorter recurrence-free intervals (p=5.59x10-5) and elevated hazard ratio=3.72. Similar to TCGA data we also noted a strong trend to worse OS ( p=0.00014) with elevated hazard ratio 3.81. Numerous studies including clinical trials have examined the chemopreventive and anti-tumor effects of difluoromethylornithine (DFMO), a specific inhibitor of ODC. We found that DFMO treatment significantly reduced cell proliferation, cell viability, and colony formation in human cell line models derived from undifferentiated, endometrioid, serous, MMT and clear cell endometrial cancers. In contrast, immortalized uterine endometrial epithelial cells (EM E6/E7 TERT1) were less sensitive to DFMO. To confirm the significant effects of DFMO in vitro we performed an in vivo study with human endometrial cancer (ACI-98) tumor-bearing athymic nude mice. Xenografted mice were either treated with 2% (w/v) DFMO supplied in drinking water or water only (n=10/group). DFMO significantly reduced the tumor burden in mice compared to controls (p=0.0023). ODC-regulated polyamines (putrescine [Put] and spermidine [Spd]) known activators of cell proliferation were strongly decreased in response to DFMO, in both tumor tissue ([Put] (p=0.0006), [Spd] (p<0.0001)) and blood plasma ([Put] (p<0.0001), [Spd] (p=0.0049)) of treated mice. Results of these studies indicate that some endometrial cancers appear particularly sensitive to DFMO. Our findings indicate that the polyamine pathway in endometrial cancers in general and specifically those most clinically relevant endometrial cancers could be targeted for effective treatment, chemoprevention or chemoprevention of recurrence.

## CANCER CHEMISTRY:

### Cheminformatics and Computational Strategies for Drug Discovery

#1243

FAM134B mutation in esophageal squamous cell carcinoma: Its clinical significance and quantification by electrochemical methods.

Md. Hakimul Haque,1 Vinod Gopalan,1 Muhammad J. A. Shiddiky,2 Alfred K. Lam1. 1 _Griffith University, Southport, Australia;_ 2 _Griffith University, Nathan, Australia_.

Aim: The aim of this research was to detect novel sites of FAM134B mutations, copy number variations and their clinicopathological significance in esophageal squamous cell carcinoma (ESCC) patients. Also, this study was intended to develop a simple and inexpensive electrochemical detection method for the analysis of FAM134B mutations using a single-use and disposable screen-printed electrode.

Method: Approximately 102 fresh tissue samples of ESCC and matched non-cancer adjacent tissues were recruited. The DNA copy numbers of FAM134B were initially studied by qRT-PCR. The FAM134B mutations were then quantified via high resolution melt curve (HRM) and Sanger sequencing analysis. In order to quantify the level of point mutation or SNPs in FAM134B gene, a new electrochemical method was also developed. The underlying working principle of the method is relied on the base dependent affinity interactions towards gold electrode. Since two DNA sequences with different DNA base compositions (i.e., amplified mutated sequences will be distinctly different than its wild type sequence) will have different adsorption affinity towards an unmodified gold electrode, accurate measurement of adsorbed DNA on the electrode surface will give the measure of point mutation or SNPs present in the DNA sequences. Target DNA sequences were first extracted from clinical samples and then PCR amplified and purified prior to adsorption on a single-use screen-printed gold electrode. The amount of mutation sites on a DNA sequence is quantified by monitoring the Faradaic current generated by the [Fe(CN)6]3-/4- system present in the electrolyte solution.

Result: Amplification of FAM134B DNA was noted in 37% of ESCC tissues whereas 35% cases showed loss of FAM134B copies compared to matched non-tumor tissues. Overall, thirty-seven FAM134B mutations were documented in exons 4, 5, 7, 9 as well as introns 2, 4-8 of FAM134B. Also, FAM134B mutations were detected in all the metastatic ESCC cases and in 14% (8/57) of the primary ESCC. Using the new electrochemical method, we were able to detect mutations in 50 ng of target PCR-amplified product within 1 h with high reproducibility (% RSD= <2) and specificity.

Conclusion: DNA copy number variations and frequent mutations of FAM134B in metastatic lymph node tissues in ESCC patients indicate its critical role in the pathogenesis of ESCCs. Also the mutation detection via electrochemical methods was successful distinguishing single point mutation in DNA from oesophageal cancer implying its potential application in point mutation detection in clinical diagnostics.

#1244

**An** in vitro **biological assay to identify small molecule upregulators of let-7 miRNA in LIN28- positive ovarian cancer cells.**

Miriam-Rose Menezes,1 Goeun Bae,2 Yong Sung Park,2 Julien Balzeau,1 Clifford C. Stephan,2 John P. Hagan1. 1 _University of Texas Health Science Center Houston, Houston, TX;_ 2 _TAMHSC Institute of Bioscience and Technology, Houston, TX_.

Onco-fetal LIN28, present in two isoforms A and B and expressed in cancer cells, promotes tumorigenesis by suppressing the biogenesis of the tumor suppressor microRNA, let-7, thereby activating an array of oncogenes including RAS, MYC, HMGA2 and Cyclin D1. Studies have demonstrated a positive correlation between LIN28 A/B expression and advanced epithelial ovarian cancers (Histological grade 2 or 3). Correlation of LIN28 expression to a poor progression-free survival and resistance to platinum-based therapies makes LIN28-let-7 pathway an attractive target for ovarian cancer chemotherapy. The overall goal of this study was to identify small molecules that upregulate let-7 levels in LIN28A or LIN28B positive ovarian cancer cells. We hypothesize that induction of let-7 levels would potentially improve prognosis for ovarian cancer patients bearing LIN28 positive tumors. To test this hypothesis, we developed a dual luciferase let-7 reporter assay in which let-7 binding sites found in the 3' UTR of the HMGA2 gene were cloned downstream of the nanoluciferase reporter gene. In this assay, elevated let-7 levels would result in repression of the nanoluciferase signal. Data obtained was normalized to Firefly luciferase activity. We tested efficacy of the assay system using let-7 mimics and inhibitors in LIN28A (OVK-18) and LIN28B (TOV-112D) positive ovarian cancer cell lines stably expressing the reporter cassette that were generated in house. Transfection of the let-7 mimic resulted in a 0.5 fold decrease in both cell lines relative to the negative controls, whereas transfection of the let-7 inhibitor resulted in a two-fold increase in relative luminescence. We miniaturized the assay to 384-well format for small molecule screening. The Z prime (Z') scores for both cell lines was 0.58 and 0.84 respectively, indicating that the assay was robust to carry out the screens. We performed a primary screen using the Prestwick and LOPAC1280 libraries for each cell line in duplicate as well as a counter screen using a reporter cassette that lacked let-7 binding sites. A compound was identified as a hit if it ranked within the top 100 compounds in the primary screen and outside the top 250 compounds in the counter screen. The hit rate for LIN28A positive cell line was 80 % for the Prestwick and 74 % for the LOPAC1280 libraries. For the LIN28 B positive cell line, the hit rate was 73 % and 53 % for the Prestwick and LOPAC libraries. The hits were validated by assaying for let-7 levels using qRTPCR. The hits identified included compounds that inhibited PI3K-mTOR, nFkB, c-MYC, Cyclin D1, BET1 as well as aurora kinase. These targets have been reported in the literature to downregulate let-7 levels. In conclusion, our assay-system is the first to utilize a biological assay of let-7 levels for small molecule library screening thus serving as a valuable tool for cancer drug discovery.

#1245

Screening methodologies for the discovery of small molecule melanoma therapeutics targeted at the ErbB4 receptor tyrosine kinase.

Richard L. Cullum,1 John T. Piazza,1 Jared I. Senfeld,1 Logan T. Neel,1 Ram B. Gupta,2 Allan E. David,1 David J. Riese1. 1 _Auburn University, Auburn, AL;_ 2 _Virginia Commonwealth University, Richmond, VA_.

Introduction: Gain-of-function mutations in the ErbB4 receptor tyrosine kinase have been found in a significant fraction of melanoma cell lines that are dependent on ErbB4 for proliferation. However, there is a scarcity of therapeutics for treating these ErbB4-dependent tumors. Consequently, we have developed high-throughput screening assays to identify small molecule ErbB4 antagonists that may hold promise as targeted melanoma therapeutics. Our approach is based on the observation that the Q43L mutant of the ErbB4 agonist Neuregulin 2beta (NRG2b) functions as a partial agonist at ErbB4. NRG2b/Q43L stimulates ErbB4 tyrosine phosphorylation, fails to stimulate ErbB4-dependent cell proliferation, and competitively antagonizes agonist stimulation of ErbB4-dependent cell proliferation.

Experimental procedures: Therefore, we have developed three high-throughput screening assays to identify ErbB4 partial agonists that function as antagonists. The primary screen identifies molecules that stimulate ErbB4 tyrosine phosphorylation. The secondary screen identifies molecules that stimulate or fail to stimulate ErbB4-dependent cell proliferation. The tertiary screen identifies molecules that antagonize agonist stimulation of ErbB4-dependent cell proliferation.

Results: A phospho-ErbB4 sandwich ELISA assay identifies molecules that stimulate ErbB4 tyrosine phosphorylation with high sensitivity and fidelity (Z' >0.5). IL3-independence assays in conjunction with MTT assays using a cell line that displays ErbB4-dependent cell proliferation distinguish between molecules that stimulate and fail to stimulate ErbB4-dependent proliferation (Z'>0.5) and identify molecules that antagonize agonist stimulation of ErbB4 dependent proliferation. These assays have been used to identify small molecules that stimulate ErbB4 tyrosine phosphorylation. Efforts to determine whether these hits function as ErbB4 full agonists or partial agonists (antagonists) are underway and will be reported. Structures of these small molecule ErbB4 full and partial agonists may be reported, pending submission of a provisional patent application.

Conclusions: We have validated an HTS strategy for identifying ErbB4 partial agonists that function as ErbB4 antagonists and deployment of that strategy has led to the identification of several hits (~20 from the primary screen). Such molecules may hold promise as targeted therapeutics for melanoma and other ErbB4-dependent tumors.

#1246

**Development of a novel KRAS-targeting agent: systematic validation using** in silico **, in solution, cell models, PDX and transgenic mouse models.**

Arsheed Ahmad Ganaie,1 Hifzur R. Siddique,1 Ishfaq Sheikh,1 Lei Wang,1 Aijaz Parray,1 Jayanth panyam,2 Peter Villalta,1 Joshua Liao,1 Yibin deng,1 Mohammad saleem1. 1 _Univ. of Minnesota Hormel Inst., Austin, MN;_ 2 _Univ. of Minnesota Hormel Inst., Minneapolis, MN_.

Aberrant KRAS signaling plays an important role in the pathogenesis of malignancies including pancreatic (PDAC), colon and lung cancer. Because therapies targeting either downstream or associated factors of KRAS pathway have shown dismal results in clinics, there is a need to identify effective agents targeting active KRAS (GTP-KRAS) protein. In this study, we screened a chemical library for their efficacy to inhibit KRAS activity using in silico methods. Based on their binding energy, we selected a candidate agent (LP1) that exhibited high potential of binding to KRAS protein at a region where KRAS-activator protein "SOS1" binds to, and GDP-GTP exchange takes place. We next investigated if LP1 exhibits the efficacy of KRAS binding in biological solution using recombinant proteins and employing drug development techniques i.e., florescence (FL)-based competition, Isothermal Titration Calorimetry (ITC) and Surface Plasmon Resonance (SPR) assays. The FL-based study show that LP1 inhibits the binding of SOS1 protein and GTP molecules to KRAS protein. The ITC and SPR analysis showed that LP1 binds to the KRAS protein (at 10 μM). We next determined the KRAS-inhibitory potential of LP1 in activated-KRAS cells representing premalignant and malignant models of PDAC, colon and lung cancer. Dissociation study involving incubation of KRAS/SOS1 proteins showed the LP1 inhibits the binding of SOS1 to KRAS. Notably, LP1 therapy significantly decreased the (i) KRAS-GTP protein levels (ii) growth and (iii) proliferation of activated-KRAS single cell and 3D Organoid cultures. We next tested KRAS-inhibitory efficacy of LP1 in PDAC patient-derived (PD) tumor explants and found that LP1 therapy (10 days) significantly decreased growth of, and KRAS-GTP levels in PD explants. Based on the in silico, in solution, cell model and PD-explant data, we determined the efficacy of LP1 as a KRAS inhibitor in in vivo conditions. We first performed the pharmacokinetic study of LP1 in mice and observed that LP1 is physiologically available in the blood (peak of 15-20 μM) after oral and intraperitoneal administrations and detectable up to 24h post-administration. We next tested LP1 therapy in tumor xenograft models and show that LP1 therapy significantly reduces the tumorigenicity of KRAS-activated PDAC and colon cell-derived tumors implanted in athymic mice. We next tested efficacy of LP1 administration on the developmental phases of PDAC disease (from normal to high grade neoplasia/early stage carcinoma) and show that LP1 feeding for six months caused a significant inhibition of PanIN development in KPCG12D transgenic mouse model. Notably the analysis of tumors xenograft models and pancreatic tissues of KPCG12D mice receiving LP1 therapy exhibited reduced KRAS-GTP levels. Taken together, these data show that LP1 is a potent KRAS activity inhibitor and a potential candidate for clinical use against KRAS-driven cancers.

#1247

Identifying novel regulators of the Unfolded Protein Response (UPR) by genome-scale CRISPR-Cas9 knockout screens.

Nektaria Maria Leli, Souvik Dey, Lauren Brady, Constantinos Koumenis. _University of Pennsylvania, Philadelphia, PA_.

Development and growth of a tumor as well as its ability to metastasize involves a complex relationship with its tissue microenvironment. A proliferating tumor encounters several microenvironmental stress conditions such as hypoxia, lack of nutrients and acidosis. To cope with these conditions, cancer cells have developed elaborate cytoprotective mechanisms which provide them with distinct advantages to thrive. Thus, deciphering the signaling pathways which get activated in the tumor microenvironment has been paramount to develop new therapeutic strategies for treatment. The Unfolded Protein Response (UPR) is an adaptive prosurvival pathway elicited by stresses in the tumor microenvironment (e.g., hypoxia, low glucose) and involves translational and transcriptional activation of effector genes which act to relieve cellular stress and block cancer cell death. We developed a strategy to comprehensively analyze critical mediators of cell fate in response to UPR activation. We have delivered a lentiviral genome-scale CRISPR Cas9 knockout (GeCKOv2) library to Sq20B cells (human squamous head and neck carcinoma) and A375 (human melanoma) cells. The library is targeting 19,050 genes with 123,411 unique guide sequences and enables both negative and positive selection screening. We used the GeCKO v2 library to identify genes essential for triggering the UPR in response to thapsigargin and tunicamycin, known specific activators of ER stress. Our highest-ranking candidates include BIRC5/Survivin, a well-studied molecule that acts as an inhibitor of apoptosis and is highly expressed in cancer cells and eukaryotic translation initiation factor eIF6, whose overexpression increases motility and invasiveness of cancer cells. Our preliminary results indicate that loss of Survivin and eIF6 dramatically enhance sensitization of cells to various ER stress conditions. Moreover, this synergistic outcome is observed when cells are treated with YM155, a small molecule that selectively suppresses Survivin and is used in phase I/II clinical trials. Lastly, morphological changes like endoreduplication are observed after long term absence of Survivin indicating its endogenous ER stress inducing role. Taken together, Survivin and eIF6 are important mediators of survival following ER stress and characterizing the pathways involved can lead to the development of novel targeted agents and therapeutic approaches. 

## PREVENTION RESEARCH:

### Biologic and Biochemical Mechanisms in Cancer Prevention

#1248

Adult calorie restriction reduces weight gain and mammary tumor incidence in obesity prone MMTV-TFG α mice -.

Michael E. Grossmann, Nancy K. Mizuno, Da-Qing Yang, D. Joshua Liao, Margot P. Cleary. _Univ. of Minnesota Hormel Inst., Austin, MN_.

Obesity is associated with increased risk of postmenopausal breast cancer resulting in recommendations for weight gain prevention and/or weight loss. However, there is little experimental data indicating if calorie restriction (CR) initiated in adulthood impacts mammary tumorigenesis. We have used female MMTV-TGF-α mice which develop mammary tumors in the 2nd yr of life as a model of postmenopausal breast cancer. From 10-30 weeks of age (woa) mice were fed a diet with 33% fat calories. At 30 woa based on body weight mice were classified as Obesity-Prone (Ob-Pr), Overweight (Ow) or Obesity-Resistant (Ob-R). Half in each weight category were then fed a 25% CR diet with reduced carbohydrate content (43% fat calories). The remaining mice continued on ad libitum (AL) feeding of the 33% fat diet. This resulted in six experimental groups (n = 26-31/group). Food intakes were monitored and body weights and mammary tumor status were determined weekly. Mice were sacrificed either at 90 woa or earlier if tumor size dictated. Mice in the CR-Ob-Pr group lost an average of 1.12 grams while mice in the AL-Ob-Pr group gained 16.03 grams from 30 woa until euthanasia. CR-Ob-Pr mice had a reduced palpable tumor incidence as compared to AL-Ob-Pr mice prior to euthanasia (12.90% versus 61.54% P = 0.0002). Indolent tumors were discovered at euthanasia with the CR-Ob-Pr mice having a larger number than AL-Ob-Pr mice resulting in final tumor positive mice in these groups of 54.84% vs 65.38%, respectively. Interestingly, the Ob-R mice had final tumor incidence of 38.71% and 73.08% for the CR-Ob-R vs the AL-Ob-R (P<0.01). Average tumor weight was highest in the AL-Ob-Pr group with CR resulting in a significantly lower tumor weight (P< 0.01). The AL-Ob-Pr group had the shortest average survival time of 82.96 woa while CR of the Ob-Pr mice resulted in increased survival to 89.77 woa (P < 0.001). A comparison of the survival curves of these two groups showed that CR resulted in significantly increased survival (P < 0.0001). Results of this study indicate that initiation of moderate 25% CR during adulthood reduces weight gain, mammary tumor incidence, and tumor weight as well as increases lifespan even when a moderately high fat diet was consumed. This information supports further efforts to be made to prevent weight gain as women approach menopause to delay or prevent breast cancer development. Support: NIH-CA157012, The Hormel Foundation and Paint the Town Pink.

#1249

Procyanidin B2-3,3′-di-O-gallate targets cancer cell metabolism in its efficacy against human prostate carcinoma cells.

Chapla Agarwal, Dileep Kumar, Alpna Tyagi, Natalie Serkova, Michael Wempe, Komal Raina, Rajesh Agarwal. _Univ. of Colorado Denver, Aurora, CO_.

The metabolic profile of various cancerous tissues can be correlated with cell growth and death, specific tumor type as well as the pathological stage of tumor. In recent times, prostatic tissue metabolomics using spectroscopy methods has helped to identify and establish the metabolic profiles specific to prostate cancer (PCa) malignancy. Thus, the evaluation of the anticancer efficacy of an agent can be considered incomplete without assessing its effect on the metabolic profile of the tumor tissue. With this rationale, herein, we carried out a metabolomics study on human PCa cell lines after procyanidin B2-3,3′-di-O-gallate (B2G2) exposure, which was recently identified by us as most effective agent in grape seed extract for growth inhibition and apoptotic death of human PCa cells. Specifically, we employed quantitative high-resolution nuclear magnetic resonance spectroscopy (1H-, 13C- and 31P-NMR) to assess the metabolic profile and energy state of the B2G2-treated human PCa cell lines PC3 and C42B. This approach helped us assess global metabolic profile, including glucose metabolism, energy state, and lipid metabolism in these cells after B2G2 treatment. We also studied the time-course (4-72 hrs) of B2G2 effect on glucose uptake and lactate release in the media of B2G2-treated PCa cells. Our results indicated that there was differential effect of B2G2 on mitochondrial glucose metabolism in these cells. While glucose uptake was markedly reduced as a function of time, there was also a significant decrease in extracellular lactate export with increased B2G2 exposure-time. Importantly, B2G2 preserved citrate concentration in the PCa cells as indicated by an increase in citrate levels after treatments; citrate generation is indicative of normal secretion functions of the prostate epithelial cells. The fact that the citrate was not further utilized for cholesterol synthesis was also confirmed by a decrease in cholesterol levels in these cells, indicating decreased cholesterogenesis by B2G2. Together, these results suggest that B2G2 differentially induces metabolic alterations in various PCa cell lines, which could be associated with its effects on cell growth, proliferation and death, as well as androgen dependency (or lack of it). In in vivo study examining B2G2 effect on PCa PC3 tumor xenograft growth in athymic nu/nu mice, B2G2 treatment (dose: 5mg/kg body weight of mice; given daily as intraperitoneal injections) significantly decreased tumor volume and tumor weight by 61% and 52% (both P≤ 0.01), respectively, after 5 weeks of treatments. Taken together, these studies indicate anti-PCa efficacy of B2G2 under both in vitro and in vivo scenario and warrant further extensive studies to establish its efficacy in other PCa models.

#1250

Curcumin inhibits migration and growth of human colon cancer cells through covalent modification of oncogenic SIRT1: Cysteine 67 as a potential binding site.

Yeon-Hwa Lee,1 Na-Young Song,1 Do-Hee Kim,1 Hye-Kyung Na,2 Young-Joon Surh1. 1 _Tumor Microenvironment Global Core Research Center, College of Pharmacy, Seoul National University, Seoul, Republic of Korea;_ 2 _College of Human Ecology, Sungshin Women's University, Seoul, Republic of Korea_.

Silent mating type information regulator 2 homolog 1 (SIRT1), an NAD+-dependent histone/protein deacetylase, has diverse physiological functions, including metabolic regulation and stress response. Despite extensive research, however, the role of SIRT1 in tumorigenesis remains controversial. Recent studies have demonstrated that SIRT1 is abnormally overexpressed in several human malignancies, and elevated levels of SIRT1 are correlated with the tumor invasion and metastasis. Curcumin (diferuloymethane), a major component of the spice turmeric (Curcuma longa L.), has been reported to possess anti-inflammatory and anti-carcinogenic properties. In the present study, we found that SIRT1 is predominantly overexpressed in the cytoplasm of several colorectal cancer cells as well as colon tumors. Curcumin abrogated migration and colony forming capability of human colon cancer (HCT-116) cells. This prompted us to investigate the effect of curcumin on the expression of SIRT1 and underlying molecular mechanisms in the context of its inhibition of the migration and growth of these cells. When HCT-116 cells were treated with curcumin, the protein expression of SIRT1 was significantly reduced, but the level of its mRNA transcript remained unchanged. The curcumin-induced suppression of SIRT1 protein expression was abrogated by the proteasomal inhibitor, MG-132. When HCT-116 cells were treated with curcumin, ubiquitination of SIRT1 was elevated. Notably, tetrahydrocurcumin, a non-electrophilic analogue of curcumin that lacks the α,β-unsaturated carbonyl moiety, failed to ubiquitinate and degrade SIRT1. Nano-LC-ESI-MS/MS analysis revealed the modification of the SIRT1 cysteine 67 residue. In line with this observation, the protein stability of a mutant SIRT1 in which cysteine 67 was replaced by alanine (SIRT1-C67A) was unaffected by curcumin treatment. Furthermore, migration and anchorage-independent growth of cells expressing SIRT1-C67A were barely inhibited by curcumin compared with those in cells harbouring wild-type SIRT1. Lysates of HCT-116 cells incubated with curcumin-conjugated Sepharose 4B beads exhibited covalent binding of curcumin to SIRT1. However, such direct interaction was markedly reduced in cells with SIRT1 cysteine 67 mutation. Taken together, these findings suggest that curcumin exerts inhibitory effects on progression of colon cancer through destabilization of oncogenic SIRT1. The electrophilic α,β-unsaturated carbonyl group present in curcumin can covalently modify SIRT1, preferentially at the cysteine 67 residue, facilitating its degradation via the ubiquitin-proteasome pathway in HCT-116 cells.

#1251

Pioglitazone inhibits periprostatic white adipose tissue inflammation in obese mice.

Miki Miyazawa,1 Kotha Subbaramaiah,1 Priya Bhardwaj,1 Xi Kathy Zhou,1 Hanhan Wang,1 Domenick J. Falcone,1 Dilip D. Giri,2 Andrew J. Dannenberg1. 1 _Weill Medical College of Cornell University, New York, NY;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Obesity is associated with an increased incidence of high-grade prostate cancer (PC) and poor prognosis for PC patients. Recently, we showed that obesity-related periprostatic white adipose tissue (WAT) inflammation, characterized by crown-like structures (CLS) consisting of dead or dying adipocytes surrounded by macrophages, was associated with high-grade PC in men. It's possible, therefore, that agents that suppress periprostatic WAT inflammation will alter the natural history of PC. Pioglitazone, a ligand of PPARγ, is used to treat diabetes and possesses anti-inflammatory properties. Here our main objectives were to determine if pioglitazone inhibited obesity-related periprostatic WAT inflammation in mice and then to elucidate the underlying mechanism. Mice were fed either a high fat (HF) diet or low fat (LF) diet to determine if obesity caused periprostatic WAT inflammation as defined by the presence of CLS. To determine if pioglitazone inhibited obesity-associated periprostatic WAT inflammation, obese HF diet fed mice were either continued on HF diet alone or treated with HF diet containing two doses (0.005% or 0.05% w/w) of pioglitazone. Treatment with pioglitazone reduced the density of CLS in periprostatic WAT, and suppressed levels of TNF-α, TGF-β and the chemokine monocyte chemoattractant protein-1 (MCP-1). Importantly, the ability of pioglitazone to suppress periprostatic WAT inflammation was abrogated in MCP-1 knock out mice. Pioglitazone caused dose-dependent induction of both adiponectin, an anti-inflammatory adipokine, and its receptor AdipoR2 in cultured 3T3-L1 cells and in periprostatic WAT of obese mice. Pioglitazone blocked TNF-α-mediated induction of MCP-1 in 3T3-L1 cells, an effect that was attenuated when either adiponectin or AdipoR2 were silenced. Taken together, pioglitazone-mediated induction of adiponectin suppressed the elevation in MCP-1 levels thereby attenuating obesity-related periprostatic WAT inflammation. These findings strengthen the rationale for future efforts to determine whether targeting the PPARγ-adiponectin-MCP-1 axis will decrease periprostatic adipose inflammation and thereby reduce the risk of high-grade PC or improve outcomes for men with PC.

#1252

A pre-clinical model for lung cancer interception: gene expression similarities between human and mouse bronchial premalignant lesions.

Sarah A. Mazzilli,1 Anna Tassinari,1 Xiaohui Xiao,1 Gang Liu,1 Samjot Dhillon,,2 Candace Johnson,2 Mary Reid,2 Marc Lenburg,1 Avrum Spira,1 Jennifer Beane1. 1 _Boston University, Boston, MA;_ 2 _Roswell Park Cancer Institute, Buffalo, NY_.

The molecular events that drive the development of the premalignant lesions (PMLs) that precede lung squamous cell carcinoma (SCC) are poorly understood. A major limitation to understanding PMLs and developing interventions is the lack of preclinical models to test candidates derived from human studies. Previous work by our group and others suggests that the N-nitroso-tris-chloroethylurea (NTCU) mouse model of lung SCC may be a candidate for modeling human PMLs based on histologic similarities, but its molecular relationship to human disease is limited. In this study, we investigate the transcriptomic similarities between PMLs from the NTCU treated mice and humans. RNA from 40 whole lung sections (curls) and laser capture microdissected (LCM) of PMLs of varying histology from SWR/J and A/J mice treated with NTCU were profiled by RNA-Seq. RNA from 131 human endobronchial biopsies representing analogous histological grades were also profiled by RNA-Seq as part of the Pre-Cancer Genome Atlas (PCGA). Linear modeling was used to identify gene expression differences associated with increasing histological severity, mouse strain and sample type. Concordance of gene expression associated with histology between species was investigated using GSEA and GSVA. Biological pathways were inferred using EnrichR and GSEA. A gene expression signature associated with increasing histological severity across all mouse samples was identified (1195 genes, FDR<0.05), which was concordant with gene expression differences associated with increasing histological severity of human PMLs (FDR<0.05). We identified a 51-gene (p<0.001) and a 178-gene (p<0.001) signatures associated with increasing histology in the all SWR/J and A/J samples respectively. These gene expression signatures are enriched for biological pathways involved in immune modulation, occurring with progressing lesions. Conversely, genes whose expression changed with increasing histology in a model that included only LCM samples (153 genes at p<0.001) revealed epithelial-specific processes including the up-regulation of KRT5 and p63 and loss of TTF1. Additionally, in the LCM samples, we identified up-regulation of genes involved in proliferation including a number of genes involved in cell structure, metabolism, transcription and translation, anti-apoptosis as well as several immunosuppressive genes. The major finding of these studies is that there are molecular similarities between PMLs from human and NTCU treated mice, which suggest that this mouse model may be useful to investigate targeted early intervention to halt or reverse PML progression towards lung cancer.

#1254

Mechanistic interrogation of pre-treatment low dose aspirin effects in HER 2 positive breast cancer.

Ian S. Miller,1 Sonja Khan,2 Liam P. Shiels,3 Sudipto Das,1 Bruce Moran,3 Finbarr P. Leacy,1 Paul M. Loadman,4 Robert S. Kerbel,5 Darran O' Connor,1 Kathleen Bennett,1 Róisín M. Dwyer,2 Annette T. Byrne1. 1 _Royal College of Surgeons in Ireland, Dublin, Ireland;_ 2 _National University of Ireland, Galway, Galway, Ireland;_ 3 _University College Dublin, Dublin, Ireland;_ 4 _University of Bradford, Bradford, United Kingdom;_ 5 _University of Toronto, Toronto, Ontario, Canada_.

Background: Prior data (Barron et al. Cancer Res. 2014 74:4065-77) suggests that pre-diagnostic exposure to aspirin can have significant effects on breast tumor biology and patient outcome. It has been proposed that aspirin inhibition of COX-2 may suppress lymphangiogenesis and metastasis (Karnezis et al Cancer Cell. 2014. 21:181-95). Here, we sought to recapitulate pre-diagnostic aspirin exposure in rodent models of Her2+ breast cancer and elucidate mechanisms of action. We also determined the effect of aspirin on tumor stroma, using a co-culture system of human tumor and mesenchymal stem cells (MSC).

Methods: NOD/SCID mice were orthotopically implanted with Her2+ MDA-MB-231 or HCC1954 cells. 48hr later, animals began a daily low dose [30mg/kg or 120mg/kg] of aspirin, until tumors reached 250mm3. They were then resected. 3 weeks later, HCC1954 implanted animals were treated with trastuzumab (15mg/kg) and paclitaxel (5mg/kg) for 6 weeks. Primary tissues were analysed by immunohistochemistry to assess VEGF-C, -D, COX-2, LYVE1 and CD31. RNAseq was performed on tumours to identify aspirin perturbed molecular pathways. To determine the stromal response to aspirin, patient derived MSCs were cultured either alone or with HCC1954 cells and exposed to aspirin (2.5 or 7.5mM). Secreted VEGF-C was quantified. A tubule formation assay was performed to determine the impact of aspirin on angiogenesis. Pro-angiogenic protein expression was investigated using a human angiogenesis array platform.

Results: A significant delay in tumor growth was observed in both tumor models following aspirin treatment (p<.01). Assessment of metastatic progression revealed that 120 mg/kg aspirin significantly (p<.05) increased time to metastasis and reduced primary regrowth in the MDA MB 231 model (p<.01). Immunohistochemical analysis of VEGF C, D and LYVE1 showed a significant dose dependant reduction (p<.01) in both models. RNAseq pathway analysis revealed a significant over-representation of mitochondrial electron transport chain genes. Downstream factors of AMPK showed significant (p<.01) upregulation suggesting alterations in metabolism. Aspirin (7.5mM) exposure resulted in loss of VEGF-C secretion from co-cultured tumor / MSC cell populations. Conditioned media harvested following aspirin treatment limited support tubule formation. Expression of pro-angiogenic factors in HCC1954 cells showed alterations following treatment, with the greatest decrease seen in Urokinase Plasminogen Activator (-42%) and its inhibitor Serpine1 (+55%).

Conclusion: We have successfully recapitulated pre-treatment aspirin response in surgical resection models of Her2+ breast cancer, with IHC analysis confirming the impact of treatment on angiogenic and lymphangiogenic factors. RNAseq analysis implicates aspirin mediated alterations in cellular metabolism. Our data further reveals increased response to aspirin in stromal cell populations.

#1255

PhIP/DSS-induced colon carcinogenesis in CYP1A-humanized mice and its prevention by tocopherols.

Chung S. Yang,1 Jayson Chen,1 Anna B. Liu,1 Mao-Jung Lee,1 Hong Wang,1 Guangxun Li,1 Lanjing Zhang,2 Kenneth Reuhl,1 Nanjoo Suh1. 1 _Rutgers University, Piscataway, NJ;_ 2 _University Medical Center of Princeton, Princeton, NJ_.

In order to establish a more physiologically relevant colorectal cancer model, we recently developed a colon-carcinogenesis model induced by the meat-derived dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and promoted by dextran sodium sulfate (DSS)-induced colitis in the cytochrome P450 1A-humanized (hCYP1A) mice. PhIP/DSS treatments caused rapid destruction of the colonic mucosa with severe inflammation, followed by the presence of reactive changes and low-grade dysplastic lesions, and then manifestation of high-grade dysplastic lesions and finally adenocarcinomas in the middle to distal colon of the hCYP1A mice after 10 weeks. Molecular analysis of the high-grade dysplastic lesions present at early time-points indicates Ctnnb1/β-catenin mutations and β-catenin nuclear accumulation in high-grade dysplastic lesions, but not in low-grade dysplastic lesions or adjacent normal tissues. Using hCYP1A/Lgr5-EGFP mouse, which harbors EGFP-tagged Lgr5 allele, we also investigated the role of Lgr5+ colon stem cells in the PhIP/DSS-induced colon carcinogenesis and found the presence of Lgr5-EGFP-expressing cells amidst ulcerated mucosa, high-grade dysplastic lesions and adenocarcinomas, suggesting a possible role of Lgr5+ stem cells in this colon-carcinogenesis model. In addition, our study demonstrated strong cancer preventive effects of tocopherols (T), the major forms of vitamin E, in PhIP/DSS-induced colon carcinogenesis. Dietary supplementation with δ-T and γ-T significantly reduced colon tumor formation and suppressed the expression of markers of oxidative and nitrosative stress as well as pro-inflammatory mediators in tumors and adjacent tissues. By administering δ-T at different time points, we found that the inhibitory effect of δ-T against colon carcinogenesis was mainly due to protection against early cellular and DNA damages caused by PhIP. α-T was found to be ineffective in inhibiting colon tumorigenesis and less effective in attenuating the molecular changes. Altogether, PhIP/DSS-induced colon carcinogenesis is likely initiated from residual epithelial cells (possibly Lgr5+ colon stem cells) and promoted by colitis, and subsequently developed into high-grade dysplasia and adenocarcinoma. These events are effectively inhibited by δ-T and γ-T, but not α-T. (This work was supported by the US NIH grants RO1 CA133021, RO1 AT007036, and F31 CA168333 as well as shared facilities funded by center grant P30 CA72720 and P30 ES005022).

#1256

Non-steroidal anti-inflammatory drugs induce ER stress and have an immunomodulatory role in the suppression of colorectal tumorigenesis.

Rochelle E. Fletcher,1 Lin Zhang,2 Brian Leibowitz,3 Jian Yu4. 1 _University of Pittsburgh Cancer Institute, Pittsburgh, PA;_ 2 _University of Pittsburgh, Department of Pharmacology, Pittsburgh, PA;_ 3 _University of Pittsburgh, Department of Pathology, Pittsburgh, PA;_ 4 _University of Pittsburgh, School of Medicine, Pittsburgh, PA_.

Colorectal cancer (CRC) is the second-leading cause of cancer-related deaths in the United States. A number of epidemiological studies, clinical trials, and animal studies have demonstrated the chemopreventive effect of non-steroidal anti-inflammatory drugs (NSAIDs) on CRC. However, the exact mechanism by which NSAIDs suppress colorectal tumorigenesis has remained unclear. Our previous study showed that NSAID-induced death receptor signaling, along with loss of the gatekeeper APC tumor suppressor, triggers a synthetic lethal interaction and apoptotic death in an otherwise normal cell. In this study, we determined how NSAIDs induce death receptor signaling, and found a critical role of the endoplasmic reticulum (ER) stress response. Treating CRC cells with the NSAIDs sulindac or indomethacin upregulated C/EBP homologous transcription factor (CHOP), a key mediator of the ER stress response that is required for the induction of death receptor 5 (DR5) by NSAIDs. Blocking ER stress abolished the apoptotic effect of NSAIDs in CRC cells and in normal colonic epithelial cells with knockdown of APC, and also suppressed the chemopreventive activity of NSAIDs in APCMin/+ mice. Furthermore, we identified plasma membrane translocation of calreticulin in cells undergoing NSAID-induced apoptosis, suggesting the involvement of immunomodulation in this tumor suppression. Our results provide novel insight into the chemopreventive mechanism of NSAIDs, which may help design more effective chemopreventive strategies and agents.

#1257

Paternal sub-optimal nutrition leads to programming of daughters' breast cancer risk in a mouse model.

Raquel Santana,1 Elissa Carney,1 Hong Cao,1 Johan Clarke,1 M. Idalia Cruz,1 Lu Jin,1 Yi Fu,2 Zuolin Cheng,2 Joseph (Yue) Wang,2 Sonia de Assis1. 1 _Georgetown Lombardi Comp. Cancer Ctr., Washington, DC;_ 2 _Virginia Tech University, VA_.

Background: Dietary patterns are known to induce epigenetic changes in paternal sperm. Acquired epigenetic traits have been shown to be transmitted from parents to offspring via the germ-line (eggs and sperm) and modulate disease risk in offspring. We have shown before that paternal overweight around the time of conception leads to reprogramming of the mammary gland tissue and breast cancer risk in the offspring. Here, we investigated whether paternal sub-optimal nutrition (low protein diet) could epigenetically reprogram the father's germ-line and alter daughters' likelihood of developing breast cancer, using a mouse model.

Methods: Male mice were fed control (Con) or low-protein (LP) diets from weaning until sexual maturity; at this point, males were housed together with female mice reared on control diet. Once a vaginal plug was detected, males were euthanized for sperm collection. Pregnant dams were fed the control diet throughout pregnancy and after giving birth. Pups were fed the control diet throughout the experiment. A subset of female offspring was euthanized for mammary tissue harvesting on post-natal day (PND) 50, which was used for morphologic and molecular analyses. Another sub-set of female offspring was treated with 9,12-dimethylbenz[a]anthracene (DMBA) to induce mammary tumors.

Results: We found that male LP consumption alters the non-coding RNA content and DNA methylation patterns of the sperm in agreement with previous findings. We also observed that, compared to Con offspring, the female offspring of LP fathers had lower birth weight (p=0.035). This decrease in body weight persisted through sexual maturity when females begun to gain more weight than their control counterparts (p>0.001). In addition, mammary glands of LP offspring were larger(p=0.02) and had increased epithelial density (p=0.001) when compared to Con on PND50. In addition, we found that LP mammary glands had epigenetic alterations, including in DNA methylation and the miRNA expression profiles. These changes in normal mammary tissue were associated with higher rates of DMBA-induced mammary tumor (p=0.04), increased tumor growth (p=0.03) and reduced tumor latency in LP offspring (p=0.03). Mammary tumors of LP offspring had lower rates of apoptosis, which may explain the increased tumor growth in this group. However, the mechanisms mediating this effect still needs to be elucidated.

Conclusion: Paternal sub-optimal nutrition programs the female offspring mammary gland development and breast cancer risk. Whether this increase in cancer risk is due to local mammary tissue changes or whether systemic changes play a role remains to be investigated. It also remains to be determined whether epigenetic changes in paternal sperm are functionally linked to changes in mammary gland development and cancer risk.

#1258

Lifestyle improvements delay cancer onset and progression by adapting endocrine and immune signaling in mouse models.

Grant D. Foglesong, Stephen M. Bergin, Wei Huang, Michael A. Caligiuri, Lei Cao. _The Ohio State University, Columbus, OH_.

Metabolic syndromes instigate substantial morbidity and mortality worldwide via its negative effects of many diseases including cancer. Obesity in particular has been deemed a causative factor for breast cancer in post-menopausal women, while worsening prognosis regardless of menopausal status. Importantly, progression of many metabolic syndromes can largely be slowed or reversed with aggressive lifestyle changes, thus also having the potential to mitigate cancer onset. A mouse model of environmental enrichment (EE) to improve motosensory, cognitive, and social stimulation by increasing physical engagement and social interaction triggers vast improvements in overall health. These include boosting mental health, reducing adiposity, prevention of diet-induced obesity (DIO), promoting the white to brown fat transition, enhancing insulin sensitivity, improving immune function, limiting inflammation, and inhibition of cancer growth. We have elucidated the central mechanism of EE to be the activation of the hypothalamic-sympathoneural-adipocyte (HSA) axis, a specific neuroendocrine route in which the brain communicates with adipose tissue via the sympathetic nervous system. Once activated, norepinephrine (NE) is released directly onto adipose tissue which induces the observed metabolic improvements. Breast cancer is uniquely exposed to this adipose-NE so we sought to explore this distinctive microenvironment in the context of both local and systemic endocrine and immune signaling in varied body mass states and menopausal statuses. Our data showed that EE diminished leptin levels and delayed cancer onset in the MMTV-PyMT spontaneous mouse model of breast cancer and slowed growth in a DIO allograft model. In contrast, EE failed to attenuate tumor progression in ob/ob mice in the absence of leptin, but rather enhanced tumor growth, suggesting that the leptin-NE relationship is one of the key peripheral mediators of the EE anti-breast cancer effects. We also observed significant immune cell modulation in the adipose tissue following EE. Initial characterization revealed a decrease in the proportion of tumor-associated macrophages (TAMs) and an overall anti-inflammatory response which together with the drop in leptin may contribute to the observed anti-cancer effects. We plan to further elucidate this EE-induced adipose and immune cell modulation with the ultimate goal of developing therapeutic strategies to treat both metabolic syndromes as well as associated and possibly independent cancer types.

#1259

Stem cell property-suppressing microRNAs are stimulated and their target proteins inhibited by vitamin D and progesterone in ovarian cancer cells.

Rebecca Rosales, Jane Turbov, Jennifer Yoo, Gustavo C. Rodriguez, Larry G. Thaete. _NorthShore University HealthSystem, Evanston, IL_.

Vitamin D (1,25(OH)2D3) and progesterone (P4) have both been shown to reduce the incidence of ovarian cancer. Mechanisms involved in their actions are not fully known but each one can enhance the effect of the other. Six microRNAs (miRNAs), associated with suppression of cancer stem cell properties, were investigated first in a screening array and then with a more definitive RT-PCR investigation of the response of these miRNAs to 100 nM 1,25(OH)2D3 and 100 nM P4. These experiments were conducted in cells that do (OVCAR-3-PGR) and do not (SKOV3) express nuclear progesterone receptors (PGR).

Let-7a and Let-7b are known to decrease expression of Ras and c-Myc and reduce stem cell self-renewal. Let-7b expression was increased (p<0.001) at 48 h in response to combined 1,25(OH)2D3 and P4 when PGR were present, and in response to individual and combined treatments at 72 h (p<0.01). Correspondingly, c-Myc expression was decreased (p<0.001) by both P4 and combined P4 and 1,25(OH)2D3. Let-7a expression was not affected by these treatments. No treatment-related change in the production of ras protein was observed compared to controls. In the absence of PGR (SKOV3 cells), c-Myc was suppressed by combined P4 and 1,25(OH)2D3 treatment at 24 h and 48 h.

miR-26a and miR-101 are known to downregulate EpCAM and EZH2, leading to decreased invasion and metastasis and to reduced proliferation. miR-101 increased (p<0.001) at 48 h in response to combined 1,25(OH)2D3 and P4 in cells expressing PGR. It was increased (p<0.05) at 36 h and showed synergy in the screening array in the absence of PGR. Target proteins EZH2 and EpCAM decreased moderately (p<0.05) at 72 h combined treatment, both with and without PGR present. miR-26a expression was not affected by these treatments.

miR-200b and miR-200c are known to suppress expression of ZEB1 which in turn decreases Bmi1 and Sox2 to increase E-cadherin (epithelial marker) and decrease vimentin (mesenchymal marker), thus inhibiting the epithelial-to-mesenchymal transition. Expression of miR-200b was increased (p<0.05) at 24 h and 72 h in response to P4 when PGR were present. miR-200c expression was not responsive to these treatments. ZEB1 was decreased (p<0.01) at 48 h in response to combined 1,25(OH)2D3 and P4.

These results point not only to differences among cancer cell lines but they also emphasize a possible mechanism by which P4 and 1,25(OH)2D3 may work together to prevent the occurrence of ovarian cancer, namely by suppressing stem cell renewal and the formation of neoplastic lesions. The results also reveal that P4 may act via other signaling pathways than its primary nuclear receptors to effect suppression of stem cell properties. Increased expression of cancer stem cell-suppressing miRNAs may be an important mechanism mediating the efficacy of a progestin-based ovarian cancer prevention strategy.

#1260

Diet and exercise-induced weight maintenance may be preventing mammary tumor growth and metastatic burden by enhancing antitumor immunity and/or reducing protumorigenic factors.

William J. Turbitt, Yitong Xu, Andrea M. Mastro, Connie J. Rogers. _The Pennsylvania State University, University Park, PA_.

Two lifestyle factors that increase cancer risk and progression are weight gain and sedentary behavior. Possible mechanisms underlying this relation include changes in metabolic, inflammatory, and immune mediators. Few studies have examined the effect of body weight and exercise on the efficacy of immunotherapeutic strategies. An emerging immunotherapeutic strategy is PD-1 checkpoint blockade, which selectively targets the membrane protein programmed cell death-1 (PD-1) on T cells to promote a sustained, antitumor effector response. Thus, the goal of the current study was to determine if preventing weight gain through diet (10% reduction in calories) and exercise (voluntary running wheel activity) will improve the response to the dual administration of a whole tumor cell vaccine and PD-1 checkpoint blockade. Female BALB/c mice were randomized to sedentary, weight gain (WG) or exercising, weight maintenance (WM) groups (n=32/group). After 8 weeks, all mice were orthotopically injected with 5x104 luciferase-transfected 4T1.2 cells into the fourth mammary fat pad and continued on their intervention for 35 days. After injection, WG and WM mice were randomized into vaccination (VAX) or vehicle (VEH) groups, and 1x106 irradiated 4T1.2 cells or HBSS vehicle control, respectively, was administered at day 7 post-tumor injection. Mice were further randomized (n=8/group) to receive anti-PD-1 (10 mg/kg/mouse) or isotype control at day 9 and 12 post-tumor injection. All WM groups, regardless of immunotherapy intervention, weighed significantly less than WG groups over the course of the study (p<0.001). Mean tumor volume (p<0.001) and tumor weight at sacrifice (p=0.076) were significantly lower with PD-1 treatment in the WG but not WM groups. Furthermore, metastatic burden in the lung (p=0.032) and the number of splenic myeloid-derived suppressor cells (p=0.058) was reduced with VAX+PD-1 treatment in the WG but not WM groups. Thus, the combination of VAX+PD1 was effective at reducing primary tumor growth and metastatic burden and improving immune outcomes only in mice that gained weight over the course of the study. However, diet and exercise alone was effective in reducing tumor growth and metastatic burden. The lack of responsiveness to vaccination + anti-PD-1 treatment in WM mice suggests that WM achieved through diet and exercise may be enhancing antitumor immunity and/or reducing protumorigenic factors (i.e. similar mechanisms mediated by vaccination + anti-PD-1 therapy). These data demonstrate that preventing weight gain through diet and exercise may be an important recommendation to maintain prolonged antitumor effector responses and improve clinical outcomes.

#1261

Lipid-peroxidation derived DNA damage is prevented in obesity-related hepatocarcinogenesis through CD4+ mediated apoptosis in the livers of mice on a green-tea diet.

Heidi Coia, Ning Ma, Marcin Dyba, Fung-Lung Chung. _Georgetown UNIV., Washington, DC_.

Using a C57BL/6J bioassay, we assessed whether formation of a lipid-peroxidation (LPO)-derived DNA adduct would induce mutations during the progression of obesity-induced hepatocarcinogenesis and if its formation could be prevented using a diet infused with Theaphenon E, a mixture of polyphenols extracted from green tea. Obesity is now the leading risk factor for the development of hepatocellular carcinoma in the United States. We have conducted a life-time C57BL/6J tumor bioassay to assess the role of obesity in the formation of γ-hydroxy-1,N2-propanodeoxyguanosine (γ-OHPdG), a promutagenic DNA adduct formed endogenously by LPO. Previously, we have shown γ-OHPdG was detected in FFPE liver tissues of patients with different stages of liver disease, demonstrating that its formation is consistent with LPO in the early stages of liver disease and suggesting that it may be a source of endogenous DNA damage in hepatocarcinogenesis. Tea polyphenols, predominately EGCG, are antioxidants that have been shown to prevent obesity and tumorigenesis. For the bioassay, mice were fed either a high fat, low fat, or high fat 2% TE diet starting at 4 weeks of age. Tissue and blood were collected over the course of 80 weeks at 11 separate time points. Health of the mice was assessed using serum concentrations of ALT and AST, gross morphology and immunohistochemistry of TUNEL, Ki67 and CD4+. In contrast to the high-fat and low-fat diet mice, the TE treated mice maintained a healthy body weight, liver to body weight ratio and low levels of AST and ALT enzymes which are elevated in liver disease. Additionally, the health of the liver appears to be maintained through increased CD4+ mediated apoptosis in TE treated mice as demonstrated through decreased Ki67 expression and TUNEL staining showing increased apoptosis in TE treated mice livers in conjunction with increased CD4+ cells. The CD4+ immune response may, therefore, be targeting cells with mutagenic potential. Similar to the trend indicated by the clinical samples, during hepatocarcinogenesis in the high fat diet mice, adduct levels were increased initially and likely fixed into mutation at later time points, where their levels decreased, as quantified by LC-MS/MS. In contrast, in the TE treated mice, the average γ-OHPdG levels remained consistently low. Further studies incorporating whole exome sequencing of the tumors that were formed during the life-time bioassay will indicate whether γ-OHPdG may contribute to the mutations that occured. In conclusion, our study has shown that a robust immune system response mediated by TE may be an underlying mechanism to combat the DNA adduct-induced DNA damage that may lead to HCC.

#1262

Aspirin inhibits the carcinogenesis of esophageal squamous cell carcinoma and enhances its responses to cisplatin.

Zhigeng Zou, Hongjun Fan, Xiying Yu, Shuming Zhang, Liping Guo, Wei Jiang, Shih-Hsin Lu. _Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China_.

Esophageal squamous cell carcinoma (ESCC) is one of the most lethal malignancies. Over 70% of ESCC cases occur in China. Unfortunately, the treatment of ESCC has hardly been improved all these years. Several studies suggested that aspirin (ASA) might decrease the risk of ESCC and prolonged the survival of patients with ESCC. However, it is unclear if ASA could prevent ESCC and/or enhance ESCC treatment by chemotherapy. In this study, a N-nitroso-N-methylbenzylamine (NMBzA) - induced ESCC model was employed to prove that aspirin could prevent the growth of esophageal tumor. F344 rats were treated with NMBzA by subcutaneous injection with or without ASA in drinking water (2mg/ml). After 35 week ASA-NMBzA or NMBzA alone treatment, rats were killed and esophageal tumor development were examined. The results showed that F344 rats treated with NMBzA and receiving a daily intake of ASA developed much less tumors than F344 rats treated with NMBA alone both in amount and in size (Tumor count: 2.40 ± 1.57 vs 10.85 ± 3.86 , P < 0.001; Tumor volume: 11.10 ± 13.38 mm3 vs 70.79 ± 41.65 mm3, P < 0.001). Immunohistochemical analysis indicated that a higher rate of apoptosis was observed in the basal layer of esophageal epithelium in ASA-NMBzA treated rats than in NMBzA alone treated rats. These results indicated that ASA prevented development of esophageal tumors in rats induced by NMBzA. Moreover, using in vitro human ESCC cell culture and in vivo xenograft models, we showed that ASA has strong beneficial effects on inhibition of ESCC cell proliferation and colony formation, reduction of ESCC cancer stem cells and enhance of ESCC cell cytotoxicity induced by cisplatin treatment. Biochemical analysis revealed that these effects of ASA on human ESCC cells in vitro and in vivo were due to inhibiting the repairing of DNA damage, decreasing the efflux activity and ALDH1 activity of the tumor cells, and blockades of PI3K/Akt pathway. Thus, our results demonstrated a positive role of aspirin in the prevention and treatment of ESCC.

#1263

Andrographolide inhibits prostate cancer by suppressing cytokine involved in promoting epithelial to mesenchymal transition.

Hina Mir,1 Neeraj Kapur,1 Guru Sonpavde,2 Shailesh Singh1. 1 _Morehouse School of Medicine, Atlanta, GA;_ 2 _UAB Comprehensive Cancer Center, Birmingham, AL_.

Mesenchymal phenotypes are responsible for cancer progression and poor therapeutic response. Hence, approaches targeting mesenchymal phenotype or inhibiting epithelial to mesenchymal transition will significantly prevent cancer progression or improve therapeutic outcome. Using in vitro models and molecular approaches we have shown that Andrographolide (AG), a compound isolated from Andrographis paniculata, shows promising effect on mesenchymal cells. It disposes PCa cells towards epithelial phenotype by modulating E-cadherin, ZEB-1, SNAIL and TWIST. In addition to these, significant inhibition in IL-6, which is known to be a poor prognostic maker, was observed in PCa cells treated with AG compared to untreated cells. This reduction in IL-6 could be due to AG induced suppression of SOCS (Suppressor of cytokine signaling) molecules. Therefore, our data underscores the ability of AG to impede cancer growth by impacting EMT and rationalizes its application as a potent therapeutic agent.

#1264

Studying senescence in prostate of selenium treated rats undergoing carcinogen-induced, androgen-promoted prostate carcinogenesis.

Kartick C. Pramanik,1 Michael Schlicht,2 Maarten Bosland,2 Chang Jiang,1 Yibin Deng,3 Junxuan Lu1. 1 _Penn State University, College of Medicine, Hershey, PA;_ 2 _University of Illinois at Chicago, IL;_ 3 _The University of Minnesota Hormel Institute, MN_.

Preclinical studies from us and others have shown that oral intake of next-generation selenium (Se) forms, especially methylseleninic acid (MSeA) and Se-methylselenocysteine (MSeC) inhibits mouse prostate carcinogenesis. Our recent study has shown that MSeA suppresses pten-deficient mouse high grade prostatic intraepithelial neoplasia progression to adenocarcinoma in association with superactivating p53-p21 mediated cellular senescence. To address the question whether the activation of senescence is induced in prostate carcinogenesis driven by different etiology, we sought to test the in vivo effect of different selenium forms fed to rats undergoing chemically induced, androgen promoted prostate carcinogenesis. Wistar-Unilever (WU) rats (10-12 wks) were sequentially treated with androgen receptor blocker flutamide for 21 days (10 mg/kg/day by gavage), followed by testosterone propionate (TP, 10 mg/kg, s.c) on day 22. Three days after TP, chemical carcinogen methylnitrosourea (MNU, 30 mg/kg/bw) was administered by i.p. injection. One week later, the rats received testosterone-containing silastic implants. Rats were randomized to groups fed the AIN-93M diet or supplemented with 3 ppm of Se as MSeA, MSeC, or selenomethionine (SeMet). Separate groups of rats were fed these diets, but did not receive the carcinogen treatment. After 16 weeks of this dietary treatment, rats were euthanized. Blood, major organs and different prostate lobes were dissected and weighed. The body weight of carcinogen-exposed rats was significantly lower than their non-carcinogen-exposed counterparts. However, total prostate complex weight was higher in carcinogen-treated groups than the non-carcinogen groups, consistent with MNU and extra-testicular androgen promoting prostate epithelial cell proliferation. There were no significant organ-to-body weight ratio differences of various prostate lobes in the different dietary groups, regardless of the carcinogen exposure status. In order to test the involvement of senescence in prostate of rats fed different Se diets, we have undertaken steps to optimize staining method for senescence-associated β-galactosidase (SA-βgal) activity in different lobes of prostate frozen sections. Together, with whole slide imaging using an Aperio Digital Pathology Scanner for quantitation, we are afforded an approach for more objective interrogation of the role of senescence in this carcinogenesis model and chemoprevention by selenium compounds.

#1265

Mechanisms underlying genomic integrity in breast cancer-derived dormant circulating tumor cells.

Debasish Boral,1 Haowen N. Liu,1 Wei Yin,1 Monika Vishnoi,1 Marc L. Sprouse,1 Jenny C. Chang,2 Jean Paul Thiery,3 Scamardo Antonio,4 David S. Hong,4 Dario Marchetti1. 1 _Houston Methodist Research Institute, Houston, TX;_ 2 _Houston Methodist hospital, Houston, TX;_ 3 _National University of Singapore, Singapore, Singapore;_ 4 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Patients with metastatic breast cancer have a five-year survival rate of 22% compared to 99% in patients with cancer localized to the breast. This disparity in clinical outcome is at least in part caused by the lack of diagnostic tools for early detection of metastatic recurrence. This can be especially valid for breast cancer patients having brain metastases (BCBM), 10-20% of whom are diagnosed only at autopsy. The early detection of circulating tumor cells (CTCs) in peripheral blood during the metastatic cascade provides a window of opportunity to prevent BCBM onset. However, to target CTCs causing BCBM, one must dissect mechanisms that allow CTCs to retain their brain metastatic potency while remaining dormant in circulation. We hypothesized that hyperactive mechanisms of DNA repair preserve the genomic make-up of dormant CTCs allowing them to maintain over time the potential for BCBM competence. Comprehensive analyses of breast cancer patient-derived CTC transcriptomes showed that CTC gene signatures closely relate to basal-like breast cancer cell lines rather than the molecular subtype of the primary tumor. However, unlike actively cycling cell lines, a large proportion of CTCs were mitotically dormant (Ki67-/PCNA-) with low overall mRNA expression and transcriptional activity. Upon systemic injection of patient-derived common lineage-depleted cells into immuno-compromised mice, the majority of CTCs were found at metastatic sites in a state of mitotic/metabolic dormancy. Second, dormant CTCs had a lower incidence of double-strand DNA breaks (DSB) than proliferating cells as assessed by Serine139 phosphorylation status of gamma H2AX. Third, we discovered that the human telomere-associated protein RIF1, a mediator of alternative non-homologous end joining repair of DNA DSB interplaying with BRCA1/2, was significantly up-regulated in the Ki67-/PCNA- CTC population. RIF1/BRCA1 foci formation was confirmed in areas of DNA damage in these cells. Lastly, shRNA-mediated RIF1 knockdown promoted dormant cell populations to become more susceptible to UV and bleomycin-induced DNA damage while activating p38 and ERK stress-response pathways as well as their phosphorylation ratio. Moreover, RIF1 knockdown in MDA-MB231BR clone attenuated its metastatic competence to the brain. Collectively, these findings suggest that RIF1 regulates the genomic integrity of dormant CTCs allowing them to survive over time while in circulation. Further elucidation of RIF1-mediated CTC pathways and its dynamics with BRCA1/2 may provide novel strategies for therapeutic intervention. This can be clinically useful in breast cancer patients that are yet to develop overt BCBM.

#1266

Metformin represses esophageal carcinogenesis in NMBzA-treated rat model through inhibiting AMPK/mTOR and Stat3 signaling pathways.

Hongjun Fan, Zhigeng Zou, Xiying Yu, Liping Guo, Wei Jiang, Shih-Hsin Lu. _National Cancer Center/ Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China_.

Esophageal cancer is one of the most aggressive tumor types because of its invasiveness and metastatic potential. Metformin is one of the most used diabetic drugs for the management of type 2 diabetes mellitus in the world. The role of metformin in prevention of the development and progression of a variety of human tumors has been studied. However, the detailed mechanisms have not yet been fully understood. In the present study, we investigated the effects of metformin on the suppression of esophageal carcinogenesis in a rat model, in which F344 rats were treated with N-nitroso-N-methylbenzylamine (NMBzA 0.30 mg/kg s.c.) three times per week for 35 weeks to induce esophageal tumors. To monitor the effects of metformin in this model, one group of rats were administered with metformin (3 g/L) in the drinking water at the first NMBzA injection. Our results showed that although there was no significant difference in body weight in rats of different groups, rats treated with NMBzA and metformin together significantly reduced the tumor formation and tumor volume when compared with rats treated with NMBzA alone. Statistic analyses demonstrated that the tumor numbers was reduced in NMBzA-treated rats received metformin to an average of 1.85 ± 1.09 tumors per rat when compared with 10.85 ± 3.86 (P < 0.001) in rats without metformin, while the tumor volume was decreased from 70.79 ± 41.65 mm3 per rat without metformin administration to 8.64 ± 13.45 mm3 (P < 0.001) with metformin administration. In addition, 7 out of 24 rats in the NMBzA-treated group died before week 35 but no rats died in the other groups. Furthermore, immunoblotting analysis indicated that p-mTORSer2448, p-Stat3Tyr705, and Cyclin D1 protein levels significantly decreased, while p-AMPKThr172 significantly increased in tumors obtained from rats treated with NMBzA and metformin when compared with tumors obtained from rats treated with NMBzA alone. Thus, our results indicated that metformin suppressed NMBzA-induced esophageal carcinogenesis via inhibition of the AMPK/mTOR and Stat3 signaling pathways. Together, our study suggested that metformin might have a potential use for treatment and prevention of esophageal cancer.

#1267

Emodin exerts its anticancer effect on colon cancer cells by inhibiting proliferation and inducing apoptosis.

Ian T. Saunders, Hina Mir, Neeraj Kapur, Shailesh Singh. _Morehouse School of Medicine, Atlanta, GA_.

Despite state of the art diagnostic and therapeutic options offered in the clinics, colon cancer is still a major health care concern. Therapeutic outcome is often compromised due to late diagnosis and development of resistance against treatment options used to treat advanced disease. Hence, the need to explore new avenue to treat/prevent colon cancer more effectively still exist. In this study we have investigated potential impact of Emodin, an anthraquinone derived from plant (Rheum rhabarbarum) on colon cancer cell proliferation, survival and apoptosis. Our data show that emodin reduces the cell viability and inhibits proliferation of human colon cancer cell (COLO-201 and DLD-1) in a time-dependent manner. Furthermore, FACS analysis show higher percentage of apoptotic cells with Emodin treatment compared to untreated cells. Interestingly, pro-inflammatory and anti-inflammatory cytokines produced by colon cancer cells were modulated with Emodin treatment. Cancer cells often use these cytokines to achieve their goal to progress and escape the treatment. Our data implies Emodin could be a promising agent that could be used for chemoprevention as well as in therapeutic combination to improve efficacy of conventional therapy.

#1268

Bitter melon efficacy against human pancreatic cancer cells: possible involvement of cellular stemness and metabolome targets.

Deepanshi Dhar, Gagan Deep, Sushil Kumar, Chapla Agarwal, Natalie Serkova, Michael Wempe, Komal Raina, Rajesh Agarwal. _Univ. of Colorado Denver School of Pharmacy, Aurora, CO_.

Pancreatic cancer (PanC) has a dismal 5-year survival rate of < 5%. Symptomless progression, late diagnosis and rising drug resistance to leading chemotherapeutic agents like gemcitabine, add to PanC severity. Hence, there is a critical requirement for identifying alternative novel non-toxic agents (dietary/non-dietary) aimed at effective management of PanC with minimal patient distress. Bitter melon (Momordica charantia), a dietary agent, is actively being investigated for its anti-cancer potential against a variety of malignancies, including our work in PanC. Recent studies by us have shown bitter melon juice (BMJ) efficacy against a panel of human PanC cell lines in culture and tumor xenografts. For mechanistic studies, here we evaluated the efficacy of BMJ against cancer stem cells (CSCs) pool in PanC cells. Results indicated that BMJ treatment (0.25-2% v/v) significantly decreased CSC enriched (CD44+CD24+EpCAMhigh) PanC cell population. Successive immunofluorescence/ immunohistochemical analysis of BMJ exposed cells, spheroids and MiaPaCa2 xenograft tumors showed a significant reduction in CSC markers/ regulatory molecules. Since cancer cell metabolism is an important component to be targeted to control cancer growth and progression, we next assessed BMJ efficacy in that direction. Indeed, BMJ mediated its anti-PanC effect via activating the key metabolic regulator AMPK. Concomitantly, to determine whether BMJ-effects were nutrient/energy stress dependent, we next sought to determine the cellular energy profile and major metabolic changes induced by BMJ. PanC cell metabolome analysis was done, as a function of time, to determine the shift in PanC cell metabolism on BMJ exposure. 1H-, 13C- and 31P-NMR spectra of PanC cell monolayers treated with BMJ (2% v/v) at early ( 4h) and late (72h) time points showed significantly altered energy status and modulation of major metabolic pathways involved in PanC cell growth and proliferation. A significant reduction in glucose uptake and cellular energy levels (ATP/ADP) ratio was observed, but lactate export was increased. BMJ also modulated nucleotides and aromatic amino acids involved in protein precursors/synthesis for PanC growth and proliferation. Total fatty acids and phosphatidylcholine levels (a precursor of membrane lipids) were also downregulated by BMJ. Glutamine and glutamate levels were constrained by BMJ, indicating that it could potentially limit PanC cell ability for targeting an alternative energy source. Furthermore, the cellular levels and membrane localization of GLUT1 (major glucose transporter overexpressed in PanC) were also suppressed after BMJ treatment. Collectively, these results suggest that BMJ targets PanC CSCs and cellular metabolism in its efficacy against this deadly malignancy (supported by CA195708).

#1269

**Preventive potentials of the fruits of** Detarium microcarpum **on** N **-methyl nitrosourea (MNU) induced colon carcinogenesis in rats.**

Mubarak L. Liman,1 Sunday E. Atawodi2. 1 _Nuhu Bamalli Polytechnic, Zaria- Kaduna, Nigeria;_ 2 _Ahmadu Bello University, Zaria- Kaduna, Nigeria_.

Detaruim microcarpum Guill. & Perr is a leguminous African plant with significant nutritional and medicinal uses. In an experimental N-methyl nitroso urea (MNU) induced colon carcinogenesis in rats, various concentrations of pulverized D. microcarpum fruits were included in diets of rats and the chemopreventive effects of these supplemented diets were investigated. The rats were divided into 10 groups (n=7) and fed for 10 weeks with diets containing 2.5%, 5.0% and 10.0% D. microcarpum. Subsequently, the feeding continues for another 12 weeks but with concomitant intra-rectal administration of MNU at every 72 hour interval. Respective control groups fed similar concentrations of supplemented diets, normal diet with or without MNU intoxication were also included. All rats were sacrificed at the end of the 22 weeks experiment; blood collected for hematological analysis and serum was separated for carcinoembryogenic antigen (CEA) assay and other biochemical tests. Liver, kidney and colon were also collected for antioxidant and tissue peroxidation assays. Sections of the colon were subjected to histopathological studies and immunohistochemical (IHC) staining using mutL homolog 1 (MLH1) antibody. A significant (p<0.05) elevation was observed in levels of CEA (210 ± 18 pg/ml) in the MNU positive control group when compared with the test groups and the negative control group (70 ± 6 to 140 ±11 pg/ml). There were significant decreases (p<0.05) in the major hematological parameters (PCV, hemoglobin, WBC etc) levels, catalase and superoxide dismutase (SOD) activities in the MNU control group when compared to other groups. A concomitant significant increases (p<0.05) was also observed in the levels of liver function enzymes and kidney function biomarkers as well as thiobarbituric reactive substance (maliondealdehyde) in the MNU control group when compared to the test groups and other controls . It was further observed that these variations in the CEA and antioxidant status markers were dose dependent in many instances. Histopathological and IHC staining of the colons in the treated groups showed mild changes in tissues with no expression of the MLH1 antibody when compared with the MNU control group having moderate damage and expression of the antibody. Hence it was concluded that the inclusion of fruits of D. microcarpum in the diet protected the organs and tissues of the rats from induced MNU toxicity; ameliorated oxidative stress in colon carcinogenesis model and possibly prevented the initiation of the process of carcinogenesis implying that the fruits possess significant health benefits which may have warranted their usage in foods and also in traditional medicine in Africa.

## EPIDEMIOLOGY:

### Genetic Variation (Non-GWAS) and Cancer Risk, Prognosis, or Mechanisms

#1270

Vitamin E related genetic variants and prostate cancer risk: A Mendelian randomization analysis.

Tracy M. Layne, Shakira M. Nelson, Stephanie J. Weinstein, Eric Karlins, Stephen J. Chanock, Demetrius Albanes, The PRACTICAL Consortium. _National Cancer Institute, Bethesda, MD_.

Alpha-tocopherol is a highly biologically active form of vitamin E that has been inconsistently associated with prostate cancer risk in cohorts and supplementation trials. In order to further elucidate the association using a Mendelian randomization approach that avoids sources of bias common in observational research, we examined genetic variants related to vitamin E status, including those from GWAS analyses of circulating vitamin E, in relation to prostate cancer risk in the Prostate Cancer Association Group to Investigate Cancer Associated Alterations in the Genome (PRACTICAL) Consortium. The analysis included 38,868 cases and 25,210 controls from 35 participating cohorts and examined the association between 847 vitamin E-related single nucleotide polymorphisms (SNPs) in (or near) six genes and risk of prostate cancer. Logistical regression was used to estimate meta-analyzed case-control odds ratios (OR) and 95% confidence intervals (CI) for the per allele risk associations. Both genotyped and imputed data were examined, and results for the latter are presented based on the consistency between the two sets of findings. In addition to examining SNPs demonstrating nominal significance (alpha-error p<0.05), a Bonferroni threshold of <0.00006 was applied to adjust for multiple comparisons. We found 89 SNPs were nominally significantly associated with prostate cancer risk. This included SNPs involved in vitamin E transport (i.e., SEC14L2 and SCARB1) and metabolism (i.e., BUD13/ZNF259). The top three risk-associated SNPs (P <0.001) included rs141696823 (OR = 0.59, P=6.0x10-5) in CYP4F8 (cytochrome P450 family 4 subfamily F member 8) on 19p13.12 which functions as a prostaglandin hydroxylase in the seminal vesicles, and rs1915379 (OR = 1.04, P=0.0002) in CYP4F3 (cytochrome P450 family 4 subfamily F member 3) on 19p13.12 which oxidizes arachidonic acid and omega-hydroxylates tocopherol phytyl side chains, and has been directly associated with higher circulating alpha-tocopherol. The final SNP (r185614299, OR = 1.19, P=0.0004) is in NKAIN3 on 8q12.3, which encodes the Na+/K+ transporting ATPase Interacting Protein 3, a plasma membrane enzyme responsible for maintenance of intracellular electrolyte gradients. Vitamin E has been shown to reduce cell membrane lipid peroxidation, thereby promoting Na/K-ATPase activity. Other variants on this gene have previously been associated with serum alpha-tocopherol concentration in response to vitamin E supplementation in the ATBC Study. The present Mendelian randomization analysis suggests genetic variants related to vitamin E metabolism, its role as an antioxidant, and its function in inflammatory processes, may be associated with prostate cancer risk.

#1271

**Genetic variants in the 16q24.3 region of melanocortin-1-receptor (** MC1R) **and prostate cancer risk: A Mendelian randomization analysis of 39,000 cases and 25,000 controls.**

Stephanie J. Weinstein, Tracy M. Layne, Jiaqi Huang, Eric Karlins, Stephen J. Chanock, Demetrius Albanes, the PRACTICAL Consortium. _National Cancer Inst., Bethesda, MD_.

We previously reported that men with naturally red hair had a significantly lower risk of prostate cancer (HR=0.46, 95% CI 0.24-0.89) compared with the most prevalent hair color, light brown (Br J Cancer 2013;109(3):747-750). The red hair phenotype is encoded by variants in the melanocortin-1-receptor (MC1R) gene that controls expression of the eumelanin and pheomelanin pigments. In order to re-examine this association through the relatively unbiased Mendelian randomization approach, we examined single nucleotide polymorphisms (SNPs) in and near MC1R in relation to prostate cancer risk using data from the Prostate Cancer Association Group to Investigate Cancer Associated Alterations in the Genome (PRACTICAL) consortium. The analysis includes 38,868 men diagnosed with prostate cancer and 25,210 controls from 35 studies participating in PRACTICAL Consortium (Nat Gen 2014;46(10)1103-1109). Logistic regression modeling was used to estimate the odds ratio (OR) and confidence interval (CI) for the per allele prostate risk associations with 148 genotyped SNPs and 164 imputed SNPs. Variants demonstrating nominal significance (alpha-error p<0.05) as well as those reaching a stringent Bonferroni threshold of p<0.0003 adjustment for multiple comparisons were examined. We identified prostate cancer risk associations with 30 genotyped and 36 imputed polymorphisms that were nominally significant, three of which achieved Bonferroni significance and were in a region of 16q24.3 that includes MC1R, GAS8 (growth arrest specific-8, an alleged tumor suppressor that is deleted in some prostate cancers), and transcription factor-25 (TCF25). These were rs4628973 and rs3743824 that were in high LD (r2=0.931; prostate cancer risk associations p=0.00029 and 0.00023, respectively), as well as rs6500461 that was independently associated with risk (r2=0.20 with rs4628973; p=0.00020). This large-scale Mendelian randomization analysis suggests genetic variants near MC1R, which directs melanocyte melanin synthesis, secretion and human pigmentation, may be associated with risk of prostate cancer.

#1272

Prostate cancer risk and vitamin a related genetic variants in the PRACTICAL Consortium.

Shakira M. Nelson,1 Tracy M. Layne,1 Stephanie J. Weinstein,1 Eric Karlins,2 Stephen J. Chanock,1 Demetrius Albanes,1 PRACTICAL Consortium. 1 _NCI, Rockville, MD;_ 2 _Cancer Genomics Research Laboratory, Rockville, MD_.

Vitamin A compounds including retinol are thought to play a protective role in human carcinogenesis. However, recent studies have shown that men with higher serum retinol concentrations are at increased risk of both overall and aggressive prostate cancer, the most common cancer in men within developed populations. The biological mechanism underlying this association remains unclear. In the present investigation, we examined the associations between common genetic variants related to serum retinol status and prostate cancer risk in the Prostate Cancer Association Group to Investigate Cancer Associated Alterations in the Genome (PRACTICAL) Consortium. The analysis includes 38,868 cases and 25,210 controls from 35 participating PRACTICAL studies. A Mendelian randomization approach was used to examine the individual association between 135 vitamin A-related single nucleotide polymorphisms (SNPs) and risk of overall prostate cancer. Logistical regression was used to estimate the odds ratio (OR) and confidence interval (CI) for the per allele association between each SNP and risk. Associations were examined using both genotyped and imputed data, and results for the latter are presented here based on their substantial similarity. In addition to examining SNPs demonstrating nominal significance (alpha < 0.05), a Bonferroni cut-point of < 0.0003 was used to adjust for multiple comparisons. Seven SNPs were found to be nominally significant at the p < 0.05 level; however, none remained significant after Bonferroni correction. Of these, five SNPs (rs4799751, OR = 0.97; rs9963553, OR= 1.03; rs9967180, OR=1.03; rs2167689, OR= 0.98; rs1941356, OR= 0.98) are located in or near three genes: transthyretin gene (TTR), trafficking protein particle complex 8 (TRAPPC8), and beta-1,4-galactosyltransferase 6 (B4GALT6). The carrier protein transthyretin is the only gene known to be related to retinol status based on prior GWAS analyses, while B4GALT6 encodes type-II membrane-bound glycoproteins which may be used as a potential cancer biomarker. The two other SNPs, rs1377189 (OR=0.96) and rs117053512 (OR=0.97), are located in desmoglein 3 (DSG3), whose encoded protein plays a role in maintenance of tissue architecture which may be upregulated in malignancies. Our large-scale Mendelian randomization analysis reveals some evidence of a relatively weak association between genetic variants related to vitamin A status and prostate cancer risk. These associations should be further examined in aggressive and fatal cancers.

#1273

Pathway analysis of insulin-like growth factor candidate genes and risk of pediatric rhabdomyosarcoma.

Libby Morimoto,1 XIaorong Shao,1 Anand Chokkalingam,1 Joseph Wiemels,2 Xiaomei Ma,3 Catherine Metayer1. 1 _University of California, Berkeley, Berkeley, CA;_ 2 _UCSF, San Francisco, CA;_ 3 _Yale University, New Haven, CT_.

Pediatric rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children under 15 years of age. Due largely to its rarity, the etiology of pediatric RMS is poorly understood. Patterns of fetal growth were previously shown to be independent risk factors for RMS among non-Hispanic white (NHW) children; however, the underlying biologic mechanisms remain unclear. Alterations in the insulin-like growth factor (IGF) system, the primary regulator of growth during the fetal and early life period, may provide clues. Using an innovative linkage between the California birth records and cancer registry information, we conducted a population-based case-control study (1982-2009) of candidate genes involved in IGF signaling and risk of pediatric RMS. Fifteen genes involved in the IGF pathway were selected: IGF1, IGF2, IGF1R, IGF2R, IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP6, INS, INSR, IRS1, GH1, and GHRH. DNA extracted from the archived newborn blood spots of all available RMS cases (n=644) was genotyped on the Illumina OmniExpress Exome array (n=964,043 SNPs); after stringent quality control (QC), a total of 633 RMS cases (360 embryonal RMS [eRMS] and 197 alveolar RMS [aRMS]) were available for analysis. A total of 815 controls genotyped on the Illumina OmniExpress Exome array and 3,922 controls genotyped on the Affymetrix Axion LAT array (n=801,830 SNPs) from the same base population of California children, with no evidence of RMS, were included in this analysis. After imputation to increase coverage of the genome and post-imputation QC to identify and remove platform effects, a total of 5,556,335 SNPs were available for analysis. Among NHW children, SNPs in IGF1 (rs10860869, p=0.0031) and IGF1R (rs62023616, p=0.00074; rs62023648, p=0.00076) were significantly associated with RMS risk after adjustment for multiple comparisons. Analyses stratified by histologic subtype showed that these associations were limited to eRMS, and of stronger magnitude. Among Hispanic children, 12 SNPs in IGF1R (p-value range: 0.0007-0.0027) and 3 SNPs in IGFBP1 (p-values: 0.0016-0.0023) were significantly associated with eRMS risk. The SNPs found to be associated with RMS were located in intergenic and intronic areas of the genes, areas that may influence gene regulation. Our results support the hypothesis that genetic variation in IGF1 signaling pathway modulate the risk of RMS, especially eRMS, among NHW and Hispanic children.

#1274

Associations between genetic polymorphisms in genes related to estrogen metabolism and function and prostate cancer risk: results from the Prostate Cancer Prevention Trial.

Li Tang,1 Mary Platek,1 Song Yao,1 Cathee Till,2 Phyllis Goodman,2 Catherine M. Tangen,2 Yue Wu,1 Elizabeth A. Platz,3 Marian L. Neuhouse,2 Frank Z. Stanczyk,4 Juergen K. Reichardt,5 Regina M. Santella,6 Ann Hsing,7 William D. Figg,8 Scott M. Lippman,9 Ian M. Thompson,10 Christine B. Ambrosone1. 1 _Roswell Park Cancer Inst., Buffalo, NY;_ 2 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 3 _Johns Hopkins Bloomberg School of Public Health, Baltimore, MD;_ 4 _University of Southern California, Los Angeles, CA;_ 5 _University of Sydney, Sydney, Australia;_ 6 _Columbia University, New York, NY;_ 7 _Stanford University, Stanford, CA;_ 8 _National Cancer Institute, Baltimore, MD;_ 9 _UC San Diego Moores Cancer Center, San Diego, CA;_ 10 _University of Texas Health Science Center at San Antonio, San Antonio, TX_.

Background: Substantial preclinical data corroborate the critical role of estrogen in prostate cancer development; however, epidemiological studies found no associations between circulating estrogen levels and prostate cancer risk. It was hypothesized that intraprostatic estrogen milieu may play a more important role than circulating estrogen in prostate carcinogenesis. Since it is difficult to obtain data on prostatic estrogen levels, we tested the hypothesis indirectly by investigating associations of prostate cancer risk with genetic variations of enzymes that are involved in estrogen synthesis, metabolism and function, and may affect intraprostatic estrogen milieu.

Methods: A panel of 36 potentially functional single nucleotide polymorphisms (SNPs) in estrogen-related genes was assembled based on information obtained in the literature. After removing SNPs with call rate <95% (1 SNP) or minor allele frequency <3% (10 SNPs), a total of 25 SNPs in 13 genes (PGR, ESR1, ESR2, CYP17A1, HSD17B1, CYP19A1, CYP1A1, CYP1B1, COMT, UGT1A6, UGT1A10, UGT2B7, UGT2B15) were examined for associations with prostate cancer risk using data and DNA samples from 1617 cases and 1731 controls in the Prostate Cancer Prevention Trial (PCPT). Cases and controls were frequency-matched on age, treatment arm and family history. Logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) separately in the placebo and finasteride arms, adjusting for age, race and family history. Results were similar when restricting analysis to white men only.

Results: Panels of SNPs that were significantly associated with prostate cancer risk were different according to treatment arm, showing rs1801132 in ESR1, rs700518 in CYP19A1, and rs4124874 in UGT1A6 in the placebo arm and rs2445765 in CYP19A1 and rs4680 in COMT in the finasteride arm. When stratified by circulating estrogen and androgen levels, significant associations were only observed in either the high or low category of serum hormone levels; no SNPs were significantly associated with prostate cancer risk in both categories. CYP19A1 was the only gene harboring SNPs that were significantly associated with prostate cancer risk in both the placebo (rs700518) and finasteride arms (rs2445765). In haplotype analysis using all three CYP19A1 SNPs genotyped in the study (rs700518, rs2445765 and rs700519), compared with non-risk haplotype (GCC), certain CYP19A1 haplotypes were significantly associated with increased prostate cancer risk in both arms.

Conclusion: Associations between prostate cancer risk and SNPs in genes involved in estrogen metabolism and function are complicated, and markedly modified by other factors such as finasteride treatment or circulating hormone levels. Supported by grant U10CA37429, 5UM1CA182883 and P01CA108964 from the NCI.

#1275

Genetic variations in cancer-related significantly mutated genes and lung cancer susceptibility.

Liren Zhang,1 Yanwei Zhang,1 Rong Li,1 David W. Chang,1 Yuanqing Ye,1 John D. Minna,2 Jack A. Roth,1 Baohui Han,3 Xifeng Wu1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _University of Texas Southwestern Medical Center, Dallas, TX;_ 3 _Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai, China_.

Cancer initiation and development are driven by key mutations in driver genes. Applying high-throughput sequencing technologies and bioinformatics analyses, The Cancer Genome Atlas (TCGA) project has recently identified panels of genetic mutations that contributed to or associated with the etiology of various cancers. However, there are few studies investigating the germline genetic variations in these highly mutated genes and lung cancer susceptibility. In this multi-phase study, we aimed to comprehensively evaluate the 1655 tagSNPs located in the 127 significantly mutated genes (SMGs) identified by TCGA, and test their association with risk of lung cancer in a total of 7355 patients and 7301 healthy controls. We found that 11 SNPs in 8 genes showed consistent association (P<0.1) and 8 SNPs significantly associated with lung cancer risk (P<0.05) in both discovery and validation phases. The most significant SNP A was associated with a decreased risk of lung cancer (OR=0.91, 95%CI: 0.87-0.96, P=2.3×10-4). Cumulative analysis of the 11 SNPs showed consistently elevated risk with increasing number of unfavorable genotypes (P for trend=1.2×10-10). In stratified analyses, the association of SNP A and lung cancer risk appeared stronger among population of younger age at diagnosis and never smokers. The eQTL-analysis indicated that genotypes of four SNPs significantly correlated with the expression of their genes respectively. From TCGA data, expression of the identified genes were significantly different in lung tumors compared with normal tissues, and the genes' highest mutation frequency was found in lung cancers. Integrative pathway analysis (IPA) indicated the identified genes were mainly involved in major cell cycle regulatory pathway suggesting the underlying biological processes. In summary, this study revealed novel genetic variants in SMGs associated with lung cancer risk, which might contribute to elucidating the biological network involved in lung cancer development.

#1276

Gene-environment interaction relevant to estrogen and risk of breast cancer.

JooYong Park,1 Ji-Yeob Choi,1 Seokang Chung,1 Nan Song,2 Sue K Park,3 Wonshik Han,4 Dong-Young Noh,4 Sei-Hyun Ahn,5 Mi-Kyung Kim,6 Keun-Young Yoo,3 Wei Zheng,7 Daehee Kang3. 1 _Department of Biomedical Sciences, Seoul National University College of Medicine, SEOUL, Republic of Korea;_ 2 _Cancer Research Institution, Seoul National University College of Medicine, SEOUL, Republic of Korea;_ 3 _Department of Preventive Medicine, Seoul National University College of Medicine, SEOUL, Republic of Korea;_ 4 _Department of Surgery, Seoul National University College of Medicine, SEOUL, Republic of Korea;_ 5 _Department of Surgery, University of Ulsan College of Medicine and ASAN Medical Center, SEOUL, Republic of Korea;_ 6 _Division of Cancer Epidemiology and Management, National Cancer Center, Goyang-si, Gyeonggi-do, Republic of Korea;_ 7 _Department of Medicine, Division of Epidemiology, Vanderbilt Epidemiology Center, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashvile, TN_.

Introduction:

Many established reproductive risk factors for breast cancer are mediated by hormonal mechanisms, and most parts are involved in estrogens. In addition, the estrogen level controlled by genetic factors might be a sort of determinants to breast cancer risk involving estrogen metabolism. This study aims to examine gene and environment interaction (GxE) between candidate genes which are involved in estrogen metabolism and environmental factors which are related to estrogen exposure.

Methods:

GxE analyses were conducted in the Korean 1,970 breast cancer cases and 2,052 controls which were recruited in the Seoul Breast Cancer Study (SEBCS), a multicenter case control study. 137 candidate genes involved in estrogen metabolism were searched from couple of databases and 11,555 SNPs (2,472 typed SNPs and 9,083 imputed SNPs) from the range of candidate genes were included in GxE analyses with 8 established environmental factors. Statistical analyses were performed by using GxEScan (ver. Beta 0.4.0; http://biostats.usc.edu/software) for GxE test.

Results:

There were three significant interaction between 11,555 SNPs and 8 environmental factors. Firstly, interaction rs851998 nearby ESR1 with height was shown in the GE|2df model (p-2df = 1.1x10-4) This SNP had protective effect marginally (ORG = 0.82, 95% CI: 0.75-0.90) and further protective effect for women whose height is smaller than 160cm (OR G | height ≥ 160cm = 0.78, 95% CI: 0.70-0.87, p = 1.09x10-5). Second interaction was shown between rs13035764 of NCOA1 and age at menarche in the GE|2df model (p-2df = 1.2x10-3). This SNP also had protective effect marginally (ORG = 0.85, 95% CI: 0.77-0.97) and more protective effect when age at menarche is 14 years and over (OR G | age at menarche ≥ 14years = 0.82, 95% CI: 0.73-0.97, p=3.6x10-4). Lastly, there was marginally significant interaction between rs4140979 of FSHR and age at first full term pregnancy (FFTP) in the EDGxE model. Although this SNP had no marginal effect, qualitative interaction was shown with FFTP (OR G | FFTP < 27years = 0.88, 95% CI: 0.78-1.00, p=0.0482; OR G | Nulliparity or FFTP ≥ 27 years = 1.30, 95% CI: 1.10-1.53, p=0.002).

Conclusion:

Three significant interactions were identified by focusing two step methods. Replication should be performed in the independent and larger population due to statistical limitation.

#1277

Interaction of insulin-like growth factor-I and insulin resistance-related genetic variants with obesity and lifestyle factors on postmenopausal breast cancer risk.

Su Yon Jung,1 Gloria Ho,2 Thomas Rohan,3 Howard Strickler,3 Jennifer Bea,4 Jeanette Papp,5 Eric Sobel,6 Zuo-Feng Zhang,7 Carolyn Crandall6. 1 _University of California, Los Angeles, Los Angeles, CA;_ 2 _Hofstra Northwell School of Medicine, Feinstein Institute for Medical Research, Great Neck, NY;_ 3 _Albert Einstein College of Medicine, Bronx, NY;_ 4 _University of Arizona Cancer Center, Tucson, AZ;_ 5 _David Geffen School of Medicine, University of California, Los Angeles, CA;_ 6 _David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA;_ 7 _Fielding School of Public Health, University of California, Los Angeles, Los Angeles, CA_.

Objectives: Genetic variants and traits in metabolic signaling pathways may interact with obesity, physical activity, and exogenous estrogen (E), influencing postmenopausal breast cancer risk, but these inter-related pathways are incompletely understood. Stratifying via obesity and lifestyle modifiers, we evaluated the effects of insulin-like growth factor-I (IGF-I)/insulin resistance (IR)–related traits on breast cancer risk as a mediator or influencing factor in this case-cohort study.

Method: Using 75 single-nucleotide polymorphisms (SNPs) in genes related to IGF-I/IR traits and signaling pathways, and data from 1,003 postmenopausal women in Women's Health Initiative Observation ancillary studies, we assessed the effects of IGF-I/IR-traits (fasting total and free IGF-I, IGF binding protein-3, glucose, insulin, and homeostatic model assessment–insulin resistance) by proportional change estimation. Using traits as mediators of the IGF-I/IR–traits genetic variants-cancer relationship, we partitioned total effect of these genetic variants on cancer risk into two putative mechanisms: 1) indirect (mediated by traits) and 2) direct (through pathways other than traits). For the effects of IGF-I/IR traits on IGF-I/IR signaling pathways–relevant genetic variants–cancer risk, we used the same algorithm as that for mediator, but interpreted as an influential factor.

Results: Seven SNPs in IGF-I and INS genes were associated with breast cancer risk. These associations differed between non-obese/active and obese/inactive women and between exogenous E nonusers and users. The mediation effects of IGF-I/IR traits on these SNPs–cancer relationship differed between strata, but only roughly 35% of the cancer risk due to the SNPs was mediated by IGF-I/IR traits. Similarly, carriers of 20 SNPs in PIK3R1, AKT1/2, and MAPK1 genes (signaling pathways–related genetic variants) had different associations with breast cancer between strata, and the proportion of the SNP–cancer relationship explained by IGF-I/IR traits varied 45-50% between the strata.

Conclusions: Our findings suggest that IGF-I/IR genetic variants interact with obesity and lifestyle factors, altering cancer risk partially through pathways other than IGF-I/IR traits.

#1278

Effect modifications of vitamin D receptor common polymorphisms association with prostate cancer by serum vitamin D related behavioral and biological factors.

Ken Batai,1 Adam B. Murphy,2 Ebony Shah,1 Chiledum Ahaghotu,3 Rick A. Kittles1. 1 _University of Arizona, Tucson, AZ;_ 2 _Northwestern University, Chicago, IL;_ 3 _Carney Hospital-Steward Health System, Dorchester, MA_.

Background: Several roles have been proposed for vitamin D and the vitamin D receptor (VDR) in prostate cancer (PCa) pathogenesis and progression. However, previous genetic epidemiologic studies have provided limited support linking VDR polymorphisms to PCa due to complexity of vitamin D metabolism and potential gene and environmental interactions. In this study, we investigated the association of VDR genotypes with PCa incidence and aggressiveness in African Americans (AAs) and European Americans (EAs) and if the associations were modified by behavioral and biological factors that influence serum vitamin D levels.

Methods: The total of 810 AAs and 487 EAs from Chicago, IL and Washington, D.C. were included in this study. Seven single-nucleotide polymorphisms (SNPs) in and around the VDR gene and 105 ancestry informative markers were genotyped. We performed logistic regression analyses adjusting for relevant variables.

Results: In EAs, TaqI (rs73136) and BsmI (rs1544410) minor alleles revealed a protective effect against PCa, while in AAs, FokI (rs2228570) was associated with overall PCa risk (OR=0.74, 95% C.I.: 0.57-0.96). The heterogeneous associations observed in our study could be due to difference in prevalence of vitamin D deficiency between AAs and EAS and effect modifications by behavioral and biological factors that affect serum vitamin D levels. Therefore, we investigated if behavioral and biological factors that influence serum vitamin D modified the associations between VDR polymorphisms and PCa, and we found evidence suggesting that these behavioral and biological factors modify the effect of VDR on PCa. In AAs, we observed statistically significant interaction between two VDR polymorphisms, FokI and TaqI, and vitamin D intake (PInteraction=0.01 and PInteraction=0.03 respectively). We also observed evidence of interactions between VDR gene variants and other behavioral and biological factors that lower serum vitamin D levels in both AAs and EAs. In AAs, calcium intake and skin pigmentation may modify the effects of VDR on PCa. In EAs, BMI may modify the effect of VDR.

Conclusions: Although a larger sample size is necessary to confirm the observation, we demonstrated that vitamin D related behavioral and biological factors modify the effect of VDR genotypes on PCa. Impact: The VDR gene is involved in PCa pathogenesis and progression, and polymorphisms in VDR gene and vitamin D are likely to alter the function of the gene.

#1279

A polymorphism of VEGF -2489C/T is associated with prostate cancer susceptibility in Mexicans.

Abril Martínez-Rizo,1 Xavi Casillas-Rangel,1 Cuerpo Académico Investigación Bioquímica (UAN-CA-266), Hilda Andrade-Madrid,2 Veronica Benites-Godinez,2 Nallhely Rodríguez-Ocampo1. 1 _Autonomous University of Nayarit, Tepic, Mexico;_ 2 _Mexican Social Security Institute, Delegation Nayarit, Tepic, Mexico_.

Introduction: Prostate cancer (CaP) encompasses a range of malignant prostate tissue dysplasias that primarily affects men over 50 years, is the second leading cause of cancer mortality in males. VEGF expression is associated with tumor progression and metastasis in patients with CaP. The SNP -1154 G/A is associated with the severity of CaP populations in Tunisia and the United Kingdom while -634 G/C alone has been associated in Tunisian population. It is unknown whether these polymorphisms could serve as biomarkers in the Mexican population.

Aim: To associate VEGF polymorphisms -1154 G/A and - 634 G/C with the severity of prostate cancer in patients of western Mexico.

Materials and methods: The gDNA was analyzed a total of 525 men of whom 301 are presented HBP and 224 patients with clinical diagnosis of prostate cancer. For association with severity they were subclassified into high (≥7) and low grade (≤6) following the Gleason scale. Genotyping was performed by Real Time PCR using TaqMan probes system.

Results. When we compare the allelic frequencies in the control group, they are in Hardy-Weinberg equilibrium. However, no significant association of the disease with polymorphisms or severity or progression was found.

Conclusions: There is no association between prostate cancer and genetic polymorphisms at -634 and -1154 position of the promoter region of VEGF.

#1280

Functional characterization of prostate cancer risk loci by SNPs-seq and STARR-seq.

Peng Zhang,1 Jing Zhu,2 Sufyan Suleman,3 Yong-Chen Guo,4 Mei-Jun Du,5 Li-Dong Wang,1 Gong-Hong Wei,3 Liang Wang5. 1 _the First Affiliated Hospital of Zhengzhou University, ZhengZhou, China;_ 2 _Harbin Medical University, Harbin, China;_ 3 _University of Oulu, Oulu, Finland;_ 4 _Jining Medical University, Jining, China;_ 5 _Medical College of Wisconsin, Milwaukee, WI_.

Background. By SNP genotyping and RNA sequencing of 471 normal prostate samples, we recently created a prostate tissue-based eQTL dataset and identified significant eQTL signals at 51 prostate cancer risk loci. To functionally characterize these risk SNPs, we developed a massively parallel sequencing technology to screen SNPs for their allele-dependent protein binding differences. We combined this technology (called SNPs-seq) with another high throughput assay (called STARR-seq) to screen the risk loci with significant prostate-specific eQTL signals.

Methods. To select candidate functional SNPs in eQTL regions, we took advantage of existing epigenomic datasets and available tools including ENCODE, HaploReg, and Regulome. For all selected SNPs, we first made allele-specific double-strand oligos and performed DNA-protein binding assays. We then performed sequencing analysis on the protein-bound DNA oligos and determined allele-specific protein binding differences. To evaluate reproducibility of SNPs-seq, we performed each assay in duplicates. We cloned SNPs-seq screened SNP regions showing allele-specific protein binding differences into the STARR-seq vector to further determine allele-specific enhancer activities. Finally, we performed EMSA and luciferase reporter assays to validate a set of promising candidate SNPs.

Results. From 51 risk loci with strong eQTL signals, we selected 374 SNPs with strong indication of regulatory potential, as evidenced by overlapping with epigenomic marks. When comparing technical duplicates, sequence read counts from the SNPs-seq showed significant correlation with r2>=0.99. By normalizing input controls, we found 101 of the 374 SNPs showing significant allelic protein binding differences (>=1.5-fold binding difference between variant and reference alleles). Interestingly, three published functional SNPs (rs12769019, rs10993994, and rs4907792) were also among the significant SNPs, validating SNPs-seq as functional SNP screening tool. To further validate the candidate SNPs from SNPs-seq, we applied STARR-seq and tested the 101 SNPs-containing sequences (371-686bp) in LNCaP cell line under androgen treatment. This analysis revealed 11 SNPs that not only demonstrated enhancer/repressor activity but also functioned with allelic differences. EMSA and luciferase reporter assays confirmed 6 SNPs with allele-dependent enhancer/repressor activity.

Conclusions. We developed a high throughput sequencing-based technology to screen large number candidate SNPs for their allelic protein binding differences. The SNPs-seq coupled with STARR-seq will provide a powerful strategy for functionally characterizing risk loci in prostate cancer and other common diseases. Further understanding genetic role of prostate cancer etiology may facilitate the translation of population-based discovery into biological mechanisms and eventually benefit clinical practice.

#1281

**Germline mutations in** NBN **conferring DNA damage response defects are found in patients with multiple cancer types.**

Sabine Topka, Michael F. Walsh, Ann Maria, Annie Lincoln, Diana Mandelker, Liying Zhang, Marc Ladanyi, Michael F. Berger, Mark E. Robson, Joseph Vijai, Kenneth Offit. _Memorial Sloan Kettering Cancer Ctr., New York, NY_.

Nibrin, the protein encoded by the NBN gene forms a complex with Mre11 and Rad51 (MRN complex) that is crucial for DNA damage repair. Mutations in NBN are found in >90% of patients with Nimjegen breakage syndrome (NBS), an autosomal recessive disorder characterized by growth retardation, microcephaly, radiosensitivity, immunodeficiency and increased cancer risk. Most of NBS patients harbor the common founder mutation c.657del5 that leads to expression of a hypomorphic 70kDa C-terminal fragment produced by alternative translation initiation. Cell lines derived from these patients show increased sensitivity to DNA-damaging agents, chromosome instability, and abnormal cell cycle checkpoint function. Several studies have addressed cancer incidence in individuals with germline NBN mutations, showing increased cancer risk for individuals harboring the c.657del5 founder mutation and for carriers of the R215W missense mutation across multiple cancer types. For other NBN mutations conflicting reports exist as to their association with cancer risk. Here, we report newly identified NBN germline frameshift and truncating mutations in patients with multiple cancer types, including prostate, lung, breast cancer, acute lymphocytic leukemia, and chronic lymphocytic leukemia. Modeling these mutations in an NBN-deficient cellular background showed expression of a novel C-terminal truncated fragment that can bind to Mre11. Cells expressing these mutant proteins display attenuated DNA damage repair function and decreased overall survival following induction of DNA damage. Impaired Chk2 phosphorylation was also observed, indicating cell cycle checkpoint deficiencies. Thus, the NBN germline mutations identified here could contribute to genomic instability predisposing to tumorigenesis. Further in vitro studies with these and additional germline mutations occurring in cancer patients are ongoing, in order to better understand the role of this pathway of DNA damage repair in susceptibility to malignancies.

#1282

**Analysis of missense variants in** BRCA1 **BRCT domains.**

Giuliano Di Pietro,1 Vanessa Camara Fernandes,2 Kwabena Amankwah,3 Cara Shields,3 Volha A. Golubeva,3 Carly Harro,3 Marcelo Alex Carvalho,4 Alvaro N. Monteiro3. 1 _Federal University of Sergipe, Sao Cristovao, Brazil;_ 2 _Instituto Nacional de Câncer, Rio de Janeiro, Rio de Janeiro, Brazil;_ 3 _Moffitt Cancer Center, Tampa, FL;_ 4 _Instituto Federal do Rio de Janeiro, Rio de Janeiro, Brazil_.

Currently, only ~¼ (133/600) of documented single nucleotide variation leading to missense changes in the BRCA1 gene have been characterized as pathogenic or non-pathogenic (IARC Classes 4 & 5 and 1 & 2, respectively) posing a challenge for carriers of these variants. A detailed understanding of structure-function relationships is critical for the assessment of these variants in the interpretation of data from functional assay and for the development of functional impact predictors. Thus, we have systematically probed the C-terminus of BRCA1 including the N-terminal border of the BRCT domains (M1650, S1651), the BRCT linker region (G1738, D1739, V1740, G1743, H1746, G1748, P1749, R1753, and D1757), and the alpha 1 and alpha'1 helices in BRCT[N] and [C], respectively. We assessed the functional impact of 47 missense variants using a validated transcriptional assay by a protein fusion of the GAL4 DNA-binding domain to BRCA1 C-terminus (aa 1,396-1,863) in HEK 293FT cells. Analysis of functional data redefined the BRCT border (previously thought to be at S1651) at residue M1650. Overall, mutations in the BRCT linker had severe impact on the function. Mutations in the alpha1 and alpha'1 helices also tended to display impaired function despite the lack of functional impact of 16 out of the 17 variants previously analyzed. In summary, our study provides increased granularity in our understanding of BRCA1 regions less tolerant to changes and therefore more likely to contain pathogenic variants. This knowledge will be critical for the assignment of different prior probabilities of pathogenicity given the variant location, improving risk assessment and clinical management of carriers of BRCA1 missense alleles

#1283

**Mutations in the** Duffy Antigen Receptor for Chemokines (DARC/ACKR1) **gene result in population-private variants.**

Rachel Martini, Brittany Jenkins, Melissa Davis. _University of Georgia, Athens, GA_.

By studying the breast cancer microenvironment, we can specifically address novel aspects of cancer development and progression at the molecular level. Chemokines are small signaling molecules that are an important component of this microenvironment, as they shape cellular composition of this space through recruitment of immune cells, among other functions. DARC, the Duffy Antigen Receptor for Chemokines, is an atypical chemokine receptor (ACKR1) that can bind different classes of chemokines. It is a non-signaling receptor that mainly acts as a regulator of chemokine homeostasis by removing them from circulation to recruit appropriate immune cell types. The purpose of this study is to determine how population-private variants of the DARC/ACKR1 gene across global population render distinct function and define aspects of human diversity in immune responses. A well-studied example of this would be the Duffy-null allele. This allele carries a mutation that is population-specific to recent decedents of Sub-Saharan Africans, and removes expression of DARC on red blood cells. Loss of expression on red blood cells presumably causes these "Duffy-Null" individuals to lose the ability to sequester chemokines, therefore losing homeostatic balance of these molecules in circulation. To identify additional mutations, we completed in silico analysis of minor allele frequencies across the gene in the 1000 Genomes Project data, and identified several other putative functional mutations that likely result in additional gene variants, specific to geographic ancestry groups and likely under similar selection as the Duffy null allele. We have prioritized our initial functional study to include mutations in the regulatory regions of the gene, as these mutations can change how and where the gene is expressed. Future work is focused on investigating these functional variants in situ, using human tissue and human breast cell lines, by isolating and re-engineering ancestry-specific variants in these cell lines. By identifying and determining functional outcomes of these population-specific mutation, we can observe differences in immune responses across these global populations, further defining the role that DARC/ACKR1 plays in the breast tumor microenvironment.

#1284

Identification of potential cancer regulatory germline single-nucleotide polymorphisms in the non-coding genome.

Diptee A. Kulkarni, Kijoung Song, Karl Guo. _GlaxoSmithKline, King of Prussia, PA_.

Tumorigenesis in sporadic cancers is mainly driven by somatic genetic alterations such as driver mutations in protein coding genes or chromosomal changes comprising deletions, amplifications or translocations resulting in loss of tumor suppressor proteins, gain of oncogenic proteins or expression of aberrant fusion proteins, respectively. Some cancers lack such somatic changes, but are addicted to expression of certain genes for their sustained proliferation and survival. There is evidence of such oncogenic addiction to LIM domain only 1 (LMO1) expression in neuroblastoma (NB). Genome-wide association studies (GWAS) have identified robust associations between germline single-nucleotide polymorphisms (SNPs) within LMO1 and NB susceptibility with the causal SNP being rs2168101. Investigation of the mechanism of NB dependency on LMO1 showed that LMO1 expression in NB cells is regulated by rs2168101, which resides within a highly conserved tissue-specific super enhancer in LMO1 intron 1 and drives LMO1 expression through GATA3 transcription factor binding. This makes LMO1-dependent NB a unique example of sporadic cancer driven by germline genetic variation. Numerous GWAS have identified significant associations between germline SNPs in the non-coding genome and cancer risk or outcomes. To identify additional examples of regulatory SNPs as cancer drivers, we merged published genome-wide significant associations from cancer GWAS with genome regulatory data from ENCODE (Encyclopedia of DNA Elements; Nature. 2012 Sep 6; 489 (7414): 57-74)) and searched for clusters of cancer associated SNPs that resided within gene regulatory elements. Gene regulatory elements were defined as those marked by active epigenetic features and chromatin accessibility in cancer cell lines. Of the ~1,600 unique, genome-wide significant SNPs from cancer GWAS with regulatory evidence, we identified 46 clusters of 3 or more putative regulatory SNPs near 28 genes. These clusters were particularly enriched within ovarian cancer associated loci. Mechanistic studies such as reporter assays and genome editing in relevant cell types are being considered to identify the causal SNPs from these clusters regulating gene expression and driving tumorigenesis, which in turn may lead us to new cancer targets.

#1285

Analysis of esophageal cancer SNPs and gene expression to predict ESCC risk.

Lilly A. Ding,1 Molly P. Ding,1 Xin Song,2 Lidong Wang,2 Liang Wang3. 1 _None, Bethesda, MD;_ 2 _First Affiliated Hospital, Zhengzhou, China;_ 3 _Medical College of Wisconsin, Milwaukee, WI_.

Esophageal cancer is one of most common cancers worldwide and its incidence has increased rapidly over the past decades. The majority of esophageal cancers is the esophageal squamous cell carcinoma (ESCC), which is particularly prevalent in Asian and South African areas. Most cases of ESCC are diagnosed at advanced stages, with an overall 5‐year survival rate of 10–20%. Currently, genome-wide association studies (GWAS) are used to identify high risk individuals for the development of ESCC. Many SNPs have been identified to associate with a risk of ESCC. However, the effect size attributable to individual SNPs was typically modest, suggesting that genetic variants may only account for a very small amount of genetic risk and heritability of ESCC. The combined effect of multi genetic variants with altered mRNA expression play critical roles in ESCC. In this study, we used several existing bioinformatics databases (HaploReg, GTEx Portal and cBio Portal). We first selected 450 potential ESCC risk SNPs from HaploReg and then defined which SNPs caused gene expression alterations in the esophagus through expression quantitative trait loci (eQTL). Nine SNPs in eight genes (MUC1, ALS2CR12, ADHB1, TMEM173, XBP1, PLCE1, HEATR3 and SMG6) were identified as having altered mRNA expression. The altered mRNA expression of these seventeen genes are: MUC1, THBS3, GBAP1, EFNA1, SCAMP3, CASP8, CASP10, ALS2CR12, STRADB, ADH4, TMEM173, CYCTM1, DNAJC18, DNAJC20, NOC3L, HEATR3 and SRR. These altered gene expressions in the mucosal layer of the esophagus metabolize enzymes, repair DNA, and prevent cell death and inflammation, all of which may indicate that the existing altered esophageal mRNA levels plus the predisposition of normal esophageal epithelial cells can lead to harmful environmental carcinogens, thus attributing to ESCC development. In order to validate these gene alterations related to ESCC SNPs in screening and early detection of high risk individuals, DNA genotyping of this group of SNPs with a prospective cohort (n=5451) in a high risk area of ESCC will be tested in future studies.

#1286

Targeted deep sequencing of colorectal tumor tissues to study associations of tumor subtypes with germline genetic, lifestyle, and environmental risk factors.

Syed H. Zaidi,1 Wei Sun,2 Jeroen Huyghe,2 Catherine S. Grasso,2 Quang Trinh,1 Charles Connolly,2 Amy French,3 Jasmine Mu,4 Marios Giannakis,5 Eve Shinbrot,6 Ivan Borozan,1 Michael J. Quist,2 Hermann Brenner,7 Daniel Buchanan,8 Peter Campbell,9 Andrew Chan,10 Jenny Chang-Claude,7 Vincent Ferretti,1 Charles Fuchs,5 Andrea Gsur,11 Marc Gunter,12 Tabitha Harrison,2 Michael Hoffmeister,7 Wen-Yi Huang,13 Paul Krzyzanowski,1 Stephen Lee,1 Mathieu Lemire,1 Jessica Miller,1 Danielle Pasternack,1 Cherie Teney,1 Elaine Mardis,14 Polly Newcomb,2 Lincoln Stein,1 Lee Timms,1 David Wheeler,6 Christina Yung,1 Niha Zubair,2 Levi Garraway,5 Shuji Ogino,15 Li Hsu,2 Steven Gallinger,1 Stephen Thibodeau,3 Thomas Hudson,1 Ulrike Peters2. 1 _Ontario Institute for Cancer Research, Toronto, Ontario, Canada;_ 2 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 3 _Mayo Clinic, Rochester, MN;_ 4 _Broad Institute, Cambridge, MA;_ 5 _Dana-Farber Cancer Institute, Boston, MA;_ 6 _Baylor College of Medicine, Houston, TX;_ 7 _German Cancer Research Center (DKFZ), Germany;_ 8 _The University of Melbourne, Australia;_ 9 _American Cancer Society, Atlanta, GA;_ 10 _Massachusetts General Hospital, Boston, MA;_ 11 _Medical University of Vienna, Austria;_ 12 _International Agency for Research on Cancer, France;_ 13 _National Cancer Institute, NIH, DHHS, Bethesda, MD;_ 14 _Washington University School of Medicine, St. Louis, MO;_ 15 _Harvard Medical School, Boston, MA_.

The Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO) in collaboration with the Colorectal Cancer Family Registry (CCFR) aims to identify genetic variants and environmental risk factors that impact colorectal cancer (CRC). Over 30 studies from North America, Europe, and Australia participate in GECCO. These studies have collected clinical, epidemiological, and survival data, as well as blood and tumor biospecimens, for over 80,000 CRC cases and controls.

The current study aims to conduct targeted deep sequencing of tumors and matching normal DNA to identify recurrent and novel somatic and germline variants in 4,200 CRC cases. To achieve this goal, an AmpliSeq targeted sequencing panel of 1.12 Mbp was constructed to sequence the coding regions of 190 significantly mutated genes identified from whole exome sequencing datasets generated by the Nurses' Health Study and Health Professional's Follow-up Study, and The Cancer Genome Atlas. The panel also covers coding regions of 15 genes with germline high penetrance mutations in CRC, 54 regions to detect CRC-related copy number alterations (CNAs), and microsatellite and homopolymer repeat regions to identify defective DNA mismatch repair. Primers were also included to detect Fusobacterium nucleatum DNA in tumor biopsies, as F. nucleatum is thought to promote CRC carcinogenesis.

Sequencing of the DNA libraries on Illumina HiSeq 2500 produced a mean coverage of greater than 500X for tumor DNA and 100X for normal DNA, with >85% of the bases covered at the target at 50x. So far, targeted sequencing of >1,500 DNA samples from CRC tumors and normal tissues has identified recurrent and novel somatic mutations, germline genetic variants, and hypermutation status of the tumors due to defective DNA mismatch repair or pathogenic mutations in the POLE gene. Targeted sequencing has also allowed quantification of the F. nucleatum DNA in tumor biopsies; the results were validated by a multiplex QPCR assay.

At the AACR annual meeting, we will present targeted sequencing results generated from the first two GECCO-participating studies (n=1,300 cases). These data will be valuable for future association testing of somatic mutations, CNAs, hypermutation status, and F. nucleatum with germline genetic variants, lifestyle, and environmental risk factors and survival. This large study will allow development of better strategies for diagnosis, treatment, and prevention of CRC.

#1287

Multigene panel testing and risk estimates in 10,233 ovarian cancer cases.

Jenna Lilyquist,1 Holly LaDuca,2 Eric Polley,1 Hermela Shimelis,1 Chunling Hu,1 Raymond Moore,1 Steven N. Hart,1 Fergus J. Couch,1 Jill Dolinsky,2 David E. Goldgar3. 1 _Mayo Clinic, Rochester, MN;_ 2 _Ambry Genetics, Aliso Viejo, CA;_ 3 _University of Utah School of Medicine; Huntsman Cancer Institute, Salt Lake City, UT_.

Germline pathogenic variants in BRCA1 and BRCA2 account for 10% of ovarian cancer (OC), including ovarian, fallopian tube, and primary peritoneal carcinomas. Pathogenic variants in BRIP1, RAD51C, RAD51D, and other cancer predisposition genes have been observed in another 2% to 5% of OCs. However, the specific genes associated with OC and estimates of risk associated with pathogenic variants in the individual genes are not well defined. We sought to determine the relevance of multigene panel testing results for OC cases. The study was focused on 140,449 individuals, including 10,233 OC cases, receiving clinical panel testing of cancer predisposition genes. Standardized relative risks (SRR) for pathogenic variants in 18 cancer predisposition genes were estimated using reference controls from the Exome Aggregation Consortium (ExAC). The median (range) age at diagnosis of OC was 57 (21-90) years. OC cases were 76.2% Caucasian, 3.6% African American, 4.6% Asian, 4.7% Hispanic, and 10.9% unknown/other. Among the 10,233 OC cases, 1391 (13.6%) had pathogenic mutations, including 1032 pathogenic mutations among the 7793 Caucasian OC cases (13.2%). Using the non-Finnish European ExAC (excluding TCGA samples) reference controls, the allele frequency for all pathogenic variants in each gene was summed and compared to the frequency of mutations in the Caucasian OC cases. Pathogenic variants in the known OC predisposition genes: BRCA1, BRCA2, BRIP1, MSH2, MSH6, RAD51C, and RAD51D were associated with a high risk of OC (SRR>4.0). Additionally, significant associations were observed for pathogenic variants in suspected OC risk genes ATM and PALB2 (SRR=2.06 and 2.78, respectively). This study identified several genes routinely screened on multigene panel testing that confer high or moderate risks of OC. Associations with known OC predisposition genes were confirmed. In addition, this study provides evidence that PALB2 is a moderate risk OC gene, and that ATM may confer lower to moderate risks of OC. If confirmed in future studies, these risks should be carefully considered in future screening and management of OC patients. In contrast, BARD1 and genes in the MRN complex were not associated with clinically relevant risks of OC.

#1288

Telomere length genetic risk score is associated with breast cancer risk.

Laurie Grieshober,1 Jean Wactawski-Wende,1 Rachael Hageman Blair,1 Lina Mu,1 Leah Preus,1 Jing Nie,1 Jiali Han,2 Jaymie R. Meliker,3 Thomas Rohan,4 Heather M. Ochs-Balcom1. 1 _University at Buffalo, Buffalo, NY;_ 2 _Indiana University, Indianapolis, IN;_ 3 _Stony Brook University, Stony Brook, NY;_ 4 _Albert Einstein College of Medicine, Bronx, NY_.

Background: Genome-wide association studies (GWAS) have identified associations of telomere maintenance genes with breast cancer risk. This, coupled with the recognized role of telomere dysfunction as a cancer hallmark, has motivated the need for further study of telomeres and breast cancer. Further, the meta-GWAS discovery of seven single nucleotide polymorphisms (SNPs) associated with telomere length (TL) enables the use of an aggregated genetic risk score (GRS) for TL. We hypothesized that a higher GRS (representing shorter TL) would be associated with increased risk of breast cancer, and that a strengthened association may exist between higher TL GRS and aggressive breast cancer risk.

Methods: In a race/ethnically diverse sample of invasive breast cancer cases (N=1,108) and non-cases (N=20,023) from the Women's Health Initiative, we derived an unweighted TL GRS using seven previously identified TL-associated SNPs. Women were postmenopausal and the average age at enrollment was 63.9 years. We tested TL GRS associations with overall breast cancer risk and for estrogen receptor (ER), progesterone receptor (PR), and her2/neu (HER2) status-specific breast cancer risk using Cox proportional hazards models adjusted for age and race/ethnicity (in the entire sample) and age in strata of European American (EA; N=9,796), African American (AA; N=7,504), and Hispanic American (HA; N=3,229) women. We also considered reproductive risk factors, family history of breast cancer, hormone therapy, tumor characteristics, BMI, physical activity, smoking, alcohol, and US region as potential confounders.

Results: We observed a small but statistically significant association between higher TL GRS (shorter TL) and reduced risk of overall breast cancer in the entire sample (HR=0.96, 95% CI: 0.93-1.00). Results of race/ethnicity-stratified analyses for TL GRS and overall breast cancer risk were not significant. The TL GRS was independently associated with decreased risk of ER-negative, PR-positive, and HER2-positive breast cancer risk in the entire sample (HR=0.82, 95% CI: 0.71-0.94, HR=0.94, 95% CI: 0.89-0.99, and HR=0.88, 95% CI: 0.79-0.99, respectively). Similarly, the TL GRS was associated with a reduced risk of ER-negative breast cancer in AA (HR=0.79, 95% CI: 0.64-0.97) and independently with PR-positive and HER2-positive breast cancer risk in HA (HR=0.85, 95% CI: 0.75-0.97 and HR=0.79, 95% CI: 0.58-0.99, respectively). Adjustment for other covariates did not influence hazards.

Conclusions: Our results suggest that higher TL GRS is associated with reduced risk of overall breast cancer and with the development of specific breast cancer subtypes, such as ER negativity and/or PR and HER2 positivity. While our focus on GWAS-implicated SNPs may not encompass the full spectrum of genes involved in TL, further studies into the potential mechanism for shorter TL and reduced breast cancer risk are needed.

#1289

Evaluation of vitamin D receptor regulated genes reveals EGFR polymorphism is associated with high-grade serous ovarian cancer in African American women.

Delores J. Grant,1 Ani Manichaikul,2 Lauren C. Peres,2 Xin-Qun Wang,2 Ann G. Schwartz,3 Anna Wu,4 Edward Peters,5 Patricia Moorman,6 Michele L. Cote,7 Melissa Bondy,8 Linda E. Kelemen,9 Jill Barnholtz-Sloan,10 Temitope O. Keku,11 Cathrine Hoyo,12 Andrew Berchuck,6 Paul Pharoah,13 Joellen M. Schildkraut,2 African American Cancer Epidemiology Study (AACES) and the Ovarian Cancer Assoc Consortium (OCAC)2. 1 _North Carolina Central Univ., Durham, NC;_ 2 _University of Virginia, Charlottesville, VA;_ 3 _Wayne State University, Detroit, MI;_ 4 _Keck School of Medicine of USC, Los Angeles, CA;_ 5 _Louisiana State University Health Sciences, New Orleans, LA;_ 6 _Duke University Medical Center, Durham, NC;_ 7 _Wayne State University School of Medicine, Detroit, MI;_ 8 _Baylor College of Medicine, Houston, TX;_ 9 _Medical University of South Carolina, Charleston, SC;_ 10 _Case Western Reserve University School of Medicine, Cleveland, OH;_ 11 _University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 12 _North Carolina State University, Raleigh, NC;_ 13 _University of Cambridge, Cambridge, United Kingdom_.

Few studies have evaluated genetic risk factors for ovarian cancer in African American women. In a previous study with small sample sizes, Vitamin D Receptor (VDR) variants were shown to increase risk of epithelial ovarian cancer (EOC) in that population. This study sought to replicate those findings and assess gene variants from the VDR regulatory pathway. We assessed SNPs (genotyped+imputed) from 5 gene regions which included VDR and genes regulated by VDR, including UGT1A, UGT2B, CYP3A4/5 and EGFR in 755 EOC cases and 1,235 controls among women of African ancestry from the Ovarian Cancer Association Consortium (OCAC). We also performed analyses restricted to 537 patients with HGSOC represented in the overall analysis of EOC. SNPs were genotyped using the custom-designed 533,631 SNP Illumina OncoArray with imputation to the 1,000 Genomes Phase 3 v5 reference set for genotyped and imputed SNPs evaluated at the 5 gene regions (918 SNPs in UGT1A, 6302 SNPs in UGT2B, 410 SNPs in CYP3A4/5, and 824 SNPs in EGFR). Logistic regression was performed using an additive 1 degree of freedom model for genetic inheritance with adjustment for two principal components of ancestry to estimate odds ratios (OR) and 95 % confidence intervals (CI). For each gene region, we applied a gene-specific Bonferroni-threshold for statistical significance defined as 0.05 / number of SNPs examined for that gene. Based on this significance threshold, a statistically significant association with HGSOC was identified in the EGFR region for the imputed SNP, rs114972508 (per allele OR = 2.32, 95% CI = 1.58, 3.40 p=1.6e-05, imputation R-squared=0.89). We further identified suggestive associations (P < 1.0e-06) with EOC for 10 imputed SNPs located within the UGT2B4 gene. We did not replicate previous associations in the 282 SNPs examined in the VDR gene for EOC overall or among HGSOC. In summary, we identified statistically significant association for variants from the EGFR region, and suggestive evidence of association for variants within the UGT2B region in genetic association analyses of ovarian cancer in women of African Ancestry. Data from in vitro experiments suggest that EGFR transcription and proliferative function is suppressed via VDR binding. Thus EGFR association with HGSOC may be a marker of VDR activity. UGT2B4 variants have not been previously explored in ovarian cancer but shown to be nominally associated with breast cancer in women of African ancestry. UGT2B4 enzymes, part of the phase II liver detoxification pathway, are important in the clearance of steroid hormones, bile acid and drug metabolism. Alterations in EGFR and UGT2B4 could perturb enzyme efficacy and proliferation in ovaries and impact susceptibility to ovarian cancer. Future studies will be needed to validate the associations of the imputed SNPs and to determine the impact of EGFR and UGT variants on cancer development.

#1290

**Association of common genetic variants with** TMPRSS2 ERG **fusion status in prostate cancer.**

Indu Kohaar,1 Lakshmi Ravindranath,1 Denise Young,1 Amina Ali,2 Qiyuan Li,3 Albert Dobi,1 David McLeod,1 Inger L. Rosner,2 Isabell Sesterhenn,1 Matthew Freedman,4 Shiv Srivastava,1 Gyorgy Petrovics1. 1 _CPDR/USUHS, Rockville, MD;_ 2 _WRNMMC/USUHS, Bethesda, MD;_ 3 _Xiamen University, China;_ 4 _Dana-Farber Cancer Institute, Boston, MA_.

Introduction and Objectives: Oncogenic activation of ERG resulting from prevalent gene fusions is present in two thirds of prostate cancer (CaP) patients of European Ancestry including Caucasian Americans (CA). Our laboratory and others have recently reported that major cancer driver genes, including ERG, show significant racial/ethnic differences in CaP with lower frequencies in African Americans (AA), Africans and Asians. Racial differences of CaP associated SNPs have also been extensively described. However, there is limited data on germline association with ERG fusion status. The goal of this study is to identify germline molecular determinants associating with ERG status of CaP.

Methods: Blood derived genomic DNA samples were prepared from 270 AA men and 129 CA men treated by radical prostatectomy at Walter Reed National Military Medical Center (WRNMMC). ERG status was determined in whole mounted prostate specimens by immuno-histochemistry (IHC) for ERG protein expression as a surrogate for the TMPRSS2-ERG fusion. Blinded blood samples were genotyped for SNPs on the Illumina Golden Gate platform using Infinium Oncoarray, a 500K genome wide BeadChip kit from Illumina. Data analysis approaches included association analyses based on logistic regression, Principal Component Analysis (PCA) and Efficient Mixed-Model Association eXpedited (EMMAX) analysis. Genotype imputation analysis is being performed by IMPUTE2 program.

Results: After applying rigorous sample and SNP QC steps on the datasets, SNP genotyping analysis was performed in 321 patients with 478,299 SNPs. Logistic regression, principal component analysis by EIGENSTRAT and a variance component approach, EMMAX analysis were performed to account for population structure. By EMMAX we identified SNPs associated with ERG status. The SNPs most significantly (<10-5) associated with ERG fusion status included rs6698333, an intron variant of Kruppel-like factor 17 (KLF17) and two SNPs (rs1889877, rs3798999) in the intron of adhesion G protein-coupled receptor B3 (ADGRB3). Fine-mapping of SNPs is underway by genotype imputation analysis (IMPUTE2) using the 1000 Genomes reference dataset, followed by independent validation.

Conclusions: This study identified SNPs differentially associated with ERG status of CaP, a major driver oncogene in CaP. Although the biological significance as it relates to ERG status of CaP still needs to be determined, these SNPs, with independent validation, may help as markers in stratifying patients early (even before CaP is detected) for targeted prevention and treatment options.

#1291

Mutation analysis of endometrial cancer in a population-based study by targeted next-generation sequencing.

Maxine Chen, Marta Crous-Bou, Michael J. Downing, Evan L. Busch, Kimberley Glass, Jennifer Prescott, George L. Mutter, Immaculata De Vivo. _Brigham and Women's Hospital and Harvard Medical School, Boston, MA_.

Endometrial carcinoma (EC), a malignancy that arises from the epithelial lining of the uterus, is heterogeneous at histologic and molecular levels. Risk factors and outcomes also differ by type. Though prior studies characterized the genomic landscape of endometrial carcinoma, few integrated histologic, clinical, and prospectively collected epidemiologic data into the analysis. We collected formalin-fixed paraffin embedded tumor tissue from women enrolled in the Nurses' Health Study who were diagnosed with EC between 1976 and 2012. We targeted 50 cancer related genes for high-throughput sequencing to identify genetic variants in 37 ECs and correlate findings with immunohistochemical, histologic, and epidemiologic data. Case selection was designed to include maximal power to discover genetic changes associated with p53 immunohistochemical status and clinical stage of disease at diagnosis. Mutations most frequently occurred in TP53 (57%), PTEN (46%), and PIK3CA (38%). TP53 mutations were seen in 83% of ECs that immunostained positive for mutant p53, with the most frequent TP53 mutations occurring in R248. Well-differentiated endometrioid tumors had elevated frequencies of PTEN and PIK3CA mutations compared to less differentiated tumors (p < 0.05). The mutation profiles of these samples are consistent with previous studies, supporting the viability of archival paraffin-embedded tissue in mutation detection. This study's interdisciplinary approach to tumor characterization may help inform future development of personalized models for EC.

#1292

Germline genetic, molecular and environmental factors modulate APOBEC mutagenesis in human tumors.

A. Rouf Banday, Ariunaa Bayanjargal, Krizia-Ivana Udquim, Olusegun O Onabajo, Ludmila Prokunina-Olsson. _National Cancer Institute, National Institutes of Health, Bethesda, MD_.

Background: Activity of APOBEC3 enzymes has been identified as a main source of somatic mutations of a specific type (C to T or G substitutions in the TCA or TCT motifs). This mutation pattern, known as the APOBEC signature, has been described in 16 of 30 tumor types explored by TCGA, with bladder cancer being one of top cancers enriched with APOBEC-signature mutations. Recently, we demonstrated that a risk allele of a bladder cancer GWAS SNP rs1014971 was significantly associated with increased APOBEC3B (A3B) mRNA expression and APOBEC mutagenesis in bladder tumors. Here, we aimed to further explore factors that can modulate APOBEC mutagenesis in human tumors.

Methods: Isoform-specific expression analysis of all APOBEC3 genes (A3A, A3B, A3C, A3D, A3G, A3F, and A3H) was performed in TCGA datasets for seven tumor types enriched for APOBEC mutations (> 25% of the samples) - bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cervical and endocervical cancers (CESC), head and neck squamous cell carcinoma (HNSC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), and ovarian serous cystadenocarcinoma (OV). Expression and APOBEC mutagenesis pattern were analyzed by multivariate linear regression in relation to rs1014971 genotypes and adjusting for age, sex, race, DNA CpG methylation, copy number alterations (CNA), and human papilloma virus (HPV) infection status (where relevant). Survival analysis was performed using multivariate Cox regression models that included age, sex, tumor stage, rs1014971, and load of APOBEC mutations or mRNA expression of APOBEC3 splicing forms.

Results: Analysis of APOBEC mutagenesis identified significant interplay between SNP rs1014971, expression of specific APOBEC3 splicing isoforms, and environmental factors (such as HPV infection). For example, SNP was important in BLCA and sub-types of HNSC, while HPV infection status was the main factor associated with APOBEC mutagenesis in CESC and HNSC. Importantly, variable contribution of mutagenic and non-mutagenic isoforms of A3A and A3B, which are generated by alternative splicing, emerged as significant factors contributing to APOBEC mutagenesis across human tumors. Survival of the cancer patients was also differentially influenced by these factors.

Conclusions: These findings suggest that APOBEC mutagenesis pattern is tissue-specifically affected by germline genetic, molecular and environmental factors. Deeper understanding of these factors and their interplay may pave the way for preventive and treatment measures of human cancers towards precision medicine.

#1293

ABO blood type and cancer risk: preliminary findings from a phenome analysis.

Alicia Beeghly-Fadiel, Ayush Giri, Lisa Bastarache, Jill Pulley, Jeremy Warner, Josh Denny. _Vanderbilt University Medical Center, Nashville, TN_.

Introduction: ABO blood type has long been implicated in disease susceptibility, including cancer. However, evidence for associations with many malignancies is mixed. We applied a novel phenome approach to test to cancer codes from electronic medical records (EMR) in relation to ABO blood type in a large predominantly Caucasian study population.

Approach: Among adults aged 18-100, cancer case and control status were assigned using 58 general neoplasm related phenome codes to de-identified EMR at the Vanderbilt University Medical Center. Blood type from serologic assays was ascertained from EMR-linked laboratory reports. Associations between blood type and cancer phenomes were quantified with Odds Ratios (OR) and corresponding 95% Confidence Intervals (CI) from logistic regression in models adjusted for sex and stratified by race/ethnicity. Only analyses with at least 100 cases per strata were conducted.

Results: Among 221,015 Non-Hispanic Caucasians, 37,841 Blacks, 7,714 Hispanic Caucasians, and 3,616 Asian subjects with ABO blood type available in linked EMR, we evaluated 56, 37, 4, and 3 general cancer phenome codes, respectively. After employing Bonferroni corrections, ABO blood type was significantly associated with cancers of the pancreas, ovary, cervix, skin, and hematopoietic system. Caucasians with blood type O were less likely to have ovarian cancer (OR: 0.82, 95% CI 0.73-0.91) and pancreatic cancer (OR: 0.83, 95% CI: 0.74-0.92), and more likely to have squamous cell or other skin cancer (OR: 1.08, 95% CI: 1.04-1.13) and myeloid leukemia (OR: 1.15, 95% CI: 1.06-1.25) than those with other blood types (A, B, or AB). Hispanic Caucasians with blood type O were less likely to have cervical cancer (OR: 0.56, 95% CI: 0.38-0.82) than those with other blood types. No associations surpassed correction for multiple comparisons among Blacks or Asians.

Conclusions: Our phenome approach confirmed known associations between blood type and risk of pancreatic and ovarian cancer, and adds to accumulating evidence supporting associations with skin cancer and leukemia. Our novel cervical cancer association among Hispanic Caucasians and other nominally significant findings, especially in understudied non-Caucasians, should be further evaluated in large and diverse populations. In addition, research to determine how ABO blood type may influence cancer development and progression, and if such associations can be exploited for risk prediction or cancer prevention is warranted.

#1294

**Prevalence and spectrum of germline rare variants in** BRCA1/2 **and** PALB2 **among breast cancer cases in Sarawak, Malaysia.**

Xiaohong (Rose) Yang,1 Beena Devi,2 Hyuna Sung,1 Jennifer Guida,1 Yanzi Xiao,1 Lisa Garland,3 Nan Hu,1 Maria Rodriguez-Herrera,1 Chaoyu Wang,1 Kristine Jones,3 Wen Luo,3 Belynda Hicks,3 Tieng Swee Tang,2 Karobi Moitra,1 Mike Dean1. 1 _NCI/NIH/DHHS, Bethesda, MD;_ 2 _Sarawak General Hospital, Kuching, Malaysia;_ 3 _Leidos Biomedical Research, MD_.

Germline mutations in the BRCA1 and BRCA2 genes result in predisposition to breast and ovarian cancer. Detection of BRCA mutation carriers can lead to improved prevention and therapeutic interventions such as the targeted therapy using poly ADP ribose polymerase (PARP) inhibitors, which are particularly effective in BRCA mutation carriers. The spectrum of BRCA mutations varies depending on geographic origin, population, and ethnic group; however, the prevalence of BRCA mutations in non-Caucasian populations has been poorly characterized, particularly in a population-based setting. The goal of this study was to characterize the spectrum of germline mutations in BRCA1/2 and PALB2 in unselected breast cancer cases who were seen in Sarawak General Hospital, Malaysia, where 93% of the breast cancer patients in Sarawak are treated. We performed targeted sequencing using a validated AmpliSeq panel on 467 cases with available risk factor questionnaire and clinical follow-up data. Breast cancer subtypes were defined by the joint expression of ER, PR, and HER2. Common variants with frequency >1% in any public database (1000 Genome, Exome Sequencing Project, The Exome Aggregation Consortium) were excluded. Pathogenic variants included known pathogenic variants in ClinVar, loss of function variants (frameshift and stop-gain), and variants that alter the first or second base of the splice site. Variants of unknown significance (VUS) were also defined using the ClinVar classification. We found 10 BRCA1 pathogenic variants in 12 patients, 10 BRCA2 pathogenic variants in 15 patients, and 4 PALB2 pathogenic variants in 4 patients, which gave a BRCA mutation prevalence of 5.78% among the unselected breast cancer cases in this population. All these variants were extremely rare in the general population (<0.05%). Only 4 of them were recurrent variants (2 in BRCA1 and 2 in BRCA2) and all four are known variants that were reported previously in other populations. In addition, we identified a novel deleterious mutation (stop-gain) that has never been reported and a number of VUS variants in this population. Patients with pathogenic BRCA and PALB2 variants were associated with an earlier age at onset (7 years younger, p=0.0005), a positive family history (20% higher, p=0.01), and the triple-negative (TN) subtype (56% vs. 18%, p<0.0001), compared to patients without mutations. Mutation carrier cases had worse survival compared to non-carriers, however, the association was mostly driven by the higher frequency of TN in mutation carriers. Our study for the first time reported the prevalence of germline mutations in BRCA and PALB2 in a quasi-population-based case series unselected for age and family history in an under-studied Asian population. Our results may have important clinical implications for performing genetic testing on selected patients in a low-resource setting.

#1295

Genomewide gene-diabetes and gene-obesity interaction scan in 7095 cases and 10710 controls from Pancreatic Cancer Cohort Consortium and Pancreatic Cancer Case Control Consortium.

Hongwei Tang,1 Lai Jiang,2 Laufey T. Amundadottir,3 Harvey A. Risch,4 Rachael S. Stolzenberg-Solomon,3 Alison P. Klein,5 Brian M. Wolpin,6 Gloria Petersen,7 Nilanjan Chatterjee,8 Donghui Li,1 Peter Kraft,2 Peng Wei1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Harvard T.H. Chan School of Public Health, Boston, MA;_ 3 _National Cancer Institute, National Institutes of Health, Bethesda, MD;_ 4 _Yale School of Public Health, New Haven, CT;_ 5 _Johns Hopkins School of Medicine, Baltimore, MD;_ 6 _Dana-Farber Cancer Institute, Boston, MA;_ 7 _Mayo Clinic, Rochester, MN;_ 8 _The Bloomberg School of Public Health, Baltimore, MD_.

Introduction: Obesity and diabetes are two major modifiable risk factors for pancreatic cancer. Genetic variation affecting the risk of obesity- and diabetes-related pancreatic cancer have yet to be comprehensively investigated at the genome-wide level.

Aims: To identify novel gene-environment interactions (GxE) of obesity/diabetes on pancreatic cancer risk.

Methods: We conducted a multi-stage genome-wide gene-diabetes and gene-obesity interaction study using GWAS data of 7095 cases and 10710 controls from Pancreatic Cancer Consortium (PanScan I, PanScan II and PanScan III) and Pancreatic Cancer Case Control Consortium (PanC4). About 870,000 SNPs, having MAF ≥ 0.005 and having been genotyped in at least one study, were analyzed. Missing genotypes were imputed using IMPUTE2 and the Haplotype Reference Consortium (HRC) reference panel. Case-control (CC) and case-only (CO) methods were used for SNP-level GxE analysis. Fixed-, random- and joint-effect models built in "rareGE" R package were used for gene-based GxE analysis. About 20,000 genes that have at least one SNP genotyped were included in the analysis. Age, gender and principal components accounting for subpopulation structure were adjusted in both SNP-level and gene-level analyses. Obesity was defined as body mass index ≥30 mg/m2. Diabetics with <3-year duration were excluded for gene-diabetes interaction analysis. The PanScan I, II, III and PanC4 datasets were analyzed separately followed by meta-analysis of the summary statistics. Fixed-effect and Fisher's p-value based meta-analysis was applied for SNP-level and gene-level GxE analysis, respectively.

Results: No genome-wide significant (P<5 x 10-8 ) interaction at the SNP level was detected in any of the PanScan or PanC4 datasets or in the meta-analysis. A significant interaction of diabetes and FAM63A (family with sequence similarity 63 member A) gene (P<2.5 x 10-6) was observed in the meta-analysis (PFixed= 3.4 x 10-5, PRandom= 1.9 x 10-6, PJoint= 6.8 x 10-7) . This interaction showed nominal significance in all individual dataset analyses. The corresponding P values for this interaction were 4.2 x 10-2, 3.8 x 10-2 and 6.8 x 10-2 in PanScan I, 2.8 x 10-2, 3.9 x 10-2 and 6.5 x 10-2 9.1 x 10-3, 2.2 x 10-4 and 8.8 x 10-6 in PanScan III, and 3.1 x 10-3, 3.4 x 10-3 and 8.8 x 10-3 in PanC4, respectively.

Conclusions: In the largest genome-wide GxE analysis for pancreatic cancer to date, we identified a significant diabetes interacting gene in pancreatic cancer, which was consistently replicated in 4 individual GWAS studies. This observation identifies FAM63A as a candidate for future functional studies to understand its role in pancreatic cancer etiology. Further increasing the sample size may unveil additional genetic loci that contribute to the susceptibility to pancreatic cancer via G x E.

#1296

Bayesian fine-mapping using summary data of 145,000 subjects refines common risk associations, discovers secondary signals and novel candidate genes for prostate cancer.

Zsofia Kote-Jarai,1 Tokhir Dadaev,1 Ed Saunders,1 Paul Newcombe,2 Ezequiel Anokian,1 Daniel Leongamornlert,1 Ali Amin Al Olama,2 Christopher Haiman,3 Ros Eeles,1 David Conti,3 The PRACTICAL/ELLIPSE Consortium. 1 _Inst. of Cancer Research, Sutton, United Kingdom;_ 2 _University of Cambridge, Cambridge, United Kingdom;_ 3 _University of Souther California, Los Angeles, CA_.

Genome-wide association studies (GWAS) have identified more than 160 prostate cancer (PrCa) genetic risk loci, however these variants rarely point directly to the true underlying functional variant driving the association. In this fine-mapping study to narrow the credible causal variant set for 80 PrCa regions representing 89 original independent GWAS signals, we performed Bayesian variable selection in combination with functional annotation and quantile regression. We used imputed data for 83,511 PrCa cases and 62,283 controls investigated with high-density genotyping arrays from the OncoArray, iCOGS and 5 previous GWAS studies from the PRACTICAL/ELLIPSE consortia. To facilitate fine-mapping from one-at-a-time SNP associations meta-analyzed over the consortia we first applied JAM, a novel Bayesian algorithm which searches multi-SNP models in summary data by imputing the correlation structure according to a reference panel. JAM provides inference on the number of independent signals, as well as the set of potential SNPs driving those signals. We utilized functional annotation and eQTL analysis (TCGA prostate tumor data) in combination with quantile regression to further prioritize the most likely causal variants within the credible set of SNPs and identify potential candidate genes and functional mechanisms. The median credible set size from JAM was 17 SNPs per region, shrinking the post-QC input set of variants by about 98%. In 13 regions evidence was found for multiple independent signals, up to a maximum of 5 SNPs. Within the single hit regions, almost half had less than 10 variants selected. In 34 regions the credible set included at least one SNP that was co-localized with a significant eQTL. Quantile regression highlighted enrichment for variants in promoters, DNase hypersensitivity site and eQTLs - representing candidate biological mechanisms underpinning disease development. This study has substantially reduced and prioritized the candidate causal PrCa risk variants within previously known GWAS regions, identifying a small subset of variants for further functional investigation and novel candidate genes at a number of loci.

#1297

Genetic reclassification of prostate-specific antigen levels for personalized prostate cancer screening.

Rebecca E. Graff,1 Thomas J. Hoffmann,1 Michael N. Passarelli,2 Nima C. Emami,1 Lori C. Sakoda,3 Eric Jorgenson,3 Laurel A. Habel,3 Jun Shan,3 Dilrini K. Ranatunga,3 Charles P. Quesenberry,3 Chun R. Chao,4 Nirupa R. Ghai,4 David Aaronson,5 Joseph Presti,5 Tobias Nordström,6 Zhaoming Wang,7 Sonja I. Berndt,7 Stephen J. Chanock,7 Jonathan D. Mosley,8 Robert J. Klein,9 Mridu Middha,9 Hans Lilja,9 Olle Melander,10 Mark N. Kvale,1 Pui-Yan Kwok,1 Catherine Schaefer,3 Neil Risch,1 Neil Risch,1 Stephen K. Van Den Eeden,3 John S. Witte1. 1 _University of California, San Francisco, San Francisco, CA;_ 2 _Geisel School of Medicine at Dartmouth, Hanover, NH;_ 3 _Kaiser Permanente Northern California, Oakland, CA;_ 4 _Kaiser Permanente Southern California, Pasadena, CA;_ 5 _Kaiser Oakland Medical Center Northern California, Oakland, CA;_ 6 _Karolinska Institutet, Stockholm, Sweden;_ 7 _National Cancer Institute, National Institutes of Health, Bethesda, MD;_ 8 _Vanderbilt University, Nashville, TN;_ 9 _Memorial Sloan-Kettering Cancer Center, New York, NY;_ 10 _Lund University, Malmö, Sweden_.

Background: Prostate-specific antigen (PSA) screening for prostate cancer results missed diagnoses as well as many unnecessary biopsies and their associated morbidities. Because PSA levels are heritable, traditional PSA screening may be improved by adjustment for constitutive germline genetics that influence PSA independently of prostate cancer.

Methods: In a previous genome-wide association study in non-Hispanic white men without prostate cancer in the Kaiser Permanente Research Program on Genes, Environment and Health cohort, we identified 40 single nucleotide polymorphisms (SNP) independently and significantly associated with PSA levels. Among them, 24 were not associated with prostate cancer risk. To calculate genetically normalized PSA (i.e., PSA'), we multiplied each man's most recent PSA measurement by a factor that adjusted for the amount by which his 24 PSA-specific SNPs may have increased his PSA level. We then compared how men without prostate cancer were classified based on their PSA and PSA' levels with regard to decisions for diagnostic testing.

Results: PSA and PSA' were highly correlated among non-Hispanic white men without prostate cancer (r2: 0.959; 95% CI: 0.958-0.960). Still, 4.6% were reclassified from above to below a cutpoint of 2.5 ng/ml when using the PSA' measure, and 2.7% were reclassified from below to above this cutpoint (4.6% - 2.7% = 1.9% net reclassified to below the cutoff). The reclassification was even more pronounced when restricting to controls with a negative prostate biopsy: 5.0% downward and 1.5% upward (3.5% net reclassified to below the cutoff).

Conclusions: Normalizing PSA levels using germline PSA variants may reduce the number of subjects without prostate cancer receiving unnecessary biopsies.

#1298

Genetic variants related to longer telomere length are associated with increased risk of renal cell carcinoma.

Mitchell J. Machiela,1 Jonathan N. Hofmann,1 Robert Carreras-Torres,2 Nathaniel Rothman,1 Paul Brennan,2 Mattias Johansson,2 Stephen J. Chanock,1 Kevin M. Brown,1 Ghislaine Scelo,2 Mark P. Purdue1. 1 _National Cancer Institute, Rockville, MD;_ 2 _International Agency for Research on Cancer, Lyon, France_.

Telomere length in peripheral blood leukocytes has been evaluated as a potential biomarker for renal cell carcinoma (RCC) risk in numerous observational studies, but association results have been inconsistent. These findings may have been affected by several limitations, including bias from reverse causation, reliance on a single blood specimen, residual confounding or measurement outside of the etiologically relevant time period. Germline genetic variations associated with leukocyte telomere length are not affected by an individual's exposure to confounders and may act as unconfounded markers of the relationship between telomere length and RCC risk. We performed an analysis of genetic variants associated with leukocyte telomere length to assess the relationship between telomere length and RCC risk. Genotypes from nine telomere length associated variants were aggregated for 10,785 RCC cases and 21,579 cancer-free controls. We found that the number of telomere length variants associated with RCC risk (P-value<0.05) was significantly higher than what would be expected by chance (5/9 variants, binomial P-value=3.32×10−5). We aggregated the telomere length associated variants into a weighted genetic risk score (GRS), where one GRS unit equals to an inferred one Kb change in telomere length. Genetically inferred telomere length was associated with an increased risk of RCC (OR=2.07, 95% CI=1.70-2.52, P-value=7.14×10−13). This association was consistent across increasing deciles of telomere length GRS (Trend P-value=5.58×10−12). As a sensitivity analysis, we removed two telomere length variants in linkage disequilibrium with RCC genome-wide associated regions (rs10936599 and rs9420907) from the telomere length GRS, the association with RCC risk remained statistically significant (OR=1.73, 95% CI=1.35-2.20, P-value=1.07×10−5). When performing analyses by RCC subtype (clear cell=5,574 cases, papillary=573 cases and chromophobe=203 cases), all subtypes investigated showed positive GRS effect estimates, suggesting higher risk for longer telomeres, however, due to limited sample sizes the estimates were only significant for clear cell (OR=1.93, 95% CI=1.50-2.49, P-value=3.79×10−7) and papillary subtypes (OR=1.96, 95% CI=1.01-3.81, P-value=0.046). In conclusion, our results suggest a genetic background that favors longer telomeres is associated with increased RCC risk.

#1299

Common germline risk loci and the tumor anticancer immune response in breast cancer.

Siddhartha Kar, Jonathan Tyrer, Paul Pharoah, Breast Cancer Association Consortium (BCAC). _University of Cambridge, United Kingdom_.

The relationship between common germline SNPs that predispose to cancer and the anticancer immune response as measured in tumors is uncharacterized. We investigated this relationship in breast cancer, leveraging availability of four large-scale resources for this cancer type: (1) 159 independent SNP loci associated with overall breast cancer risk at P < 5 x 10-8, including 65 unpublished SNPs, identified by a recent meta-analysis of over 220,000 individuals of European ancestry (EUR) from existing GWAS and new studies from the OncoArray Network (2) Matched germline genotype and tumor transcriptomic profiles from 701 (EUR) breast cancer cases from TCGA (3) a general measure of anticancer immunity - fraction of total immune cells in these TCGA tumors inferred from gene expression signatures by ESTIMATE scoring (Yoshihara et al, Nat Commun 2013) (4) specific measures of anticancer immunity - levels of 15 immune cell types and functions in these TCGA tumors inferred from gene expression signatures by ssGSEA (Rooney et al, Cell 2015). Linear regression with and without adjustment for covariates (age at diagnosis, stage, tumor neoantigen load, tumor ESR1 expression) was used to test association between germline SNP genotype and the tumor anticancer immune response measures. SNPs were evaluated individually and collectively as a polygenic risk score (PRS) weighted by the per-allele log OR derived from the OncoArray analysis. The breast cancer risk-conferring allele for 9 of the 159 SNPs was associated with reduced tumor general immune infiltration (ESTIMATE score) at P < 0.05 both before and after adjustment for covariates. The most significant of these associations with ESTIMATE score (Punadjusted = 0.0003; Padjusted = 0.001) was for rs71801447, the lead SNP at 2q13, a breast cancer risk locus identified by the OncoArray. This SNP was associated at P < 0.05 with 12 of the 15 specific immune cell types and functions both before and after adjustment. The risk allele correlated with reduction in each of these 12 tumor immune phenotypes, with the strongest associations identified for CD4+ regulatory T cell (Padj = 0.003) and CD8+ T cell infiltrates (Padj = 0.007). SNP rs71801447 lies in the 3'-UTR of BCL2L11 and its risk allele was associated with reduced expression of BCL2L11 in the same set of TCGA breast tumors (P = 0.002). Moreover, SNPs in strong LD (r2 > 0.98) with rs71801447 overlapped enhancers in CD4+ and CD8+ T cells and in HMEC and MCF7 breast cells that were predicted to interact with the promoter of BCL2L11 in each of these cell types. Finally, as a PRS, breast cancer risk was significantly associated with reduced tumor type I interferon activity (Punadj = 0.006; Padj = 0.02). Our findings implicate BCL2L11, a well-known regulator of T and breast cancer cell apoptosis, as a potential link between germline risk SNPs and immune response to tumors in breast cancer and suggest that modulating antitumor immunity may be a mechanism of action for some established risk loci.

### Genome-Wide Association Studies/Post-GWAS Studies

#1300

Genetic predictors of gene expression associated with risk of colorectal cancer.

Stephanie A. Bien,1 Xingyi Guo,2 Yu-Ru Su,1 Tabitha A. Harrison,1 Conghui Qu,1 Yingchang Lu,2 Jiron Long,2 Sai Chen,3 Andrew T. Chan,4 David V. Conti,5 Hyun M. Kang,3 Michael Hoffmeister,6 Thomas J. Hudson,7 Mark A. Jenkins,8 Loic Le Marchand,9 Polly A. Newcomb,1 Martha L. Slattery,10 Emily White,1 Goncalo R. Abeçasis,3 Stephen B. Gruber,5 Deborah A. Nickerson,11 Stephanie L. Schmit,12 Graham Casey,13 Li Hsu,1 Wei Zheng,2 Ulrike Peters,1 GECCO-CCFR-AAAC-CORECT. 1 _Fred Hutchinson Cancer Research Ctr., Seattle, WA;_ 2 _Vanderbilt University, TN;_ 3 _University of Michigan, MI;_ 4 _Harvard Medical School, MA;_ 5 _University of Southern California, CA;_ 6 _German Cancer Research Center (DKFZ), Germany;_ 7 _Ontario Institute for Cancer Research, Ontario, Canada;_ 8 _The University of Melbourne, Australia;_ 9 _University of Hawaii Cancer Center, HI;_ 10 _University of Utah Health Sciences Center, UT;_ 11 _University of Washington, WA;_ 12 _Moffitt Cancer Center, FL;_ 13 _University of Virginia, VA_.

To date, genome-wide association studies (GWAS) have reported common variants in over 50 loci

with weak to moderate effects on CRC risk. These genetic factors in aggregate explain only a small

fraction of familial risk of CRC. To aid in the discovery of novel CRC loci, we integrated large

transcriptome data, including those generated in the Genotype-Tissue Expression (GTEx) Project in

genetic association analyses of CRC. The computational method, PrediXcan, was used to predict

transcript levels in relevant tissues and perform gene-level association tests with CRC. Prediction

models were developed using whole blood transcriptomes (n=922) from the depression genes and

networks (DGN), as well as colon transcriptomes (transverse n=169 and sigmoid n=124) from

GTEx datasets, along with high-density genotyping data from the same subjects. Genetically

determined expression levels were tested for association with CRC in 12,186 cases and 14,718

controls from GECCO-CCFR and suggestive associations (false discovery rate = 0.2) were evaluated

in 7,481 cases and 17,796 controls from the Asia Colorectal Cancer Consortium (ACCC) and 22,974

cases and 14,392 controls from the Colorectal Transdisciplinary (CORECT) study. We attempted to

replicate novel associations for eight genes and found statistically significant associations with

CXCR1 (OR=1.21 (1.10-1.33), p-value=7.8x10-5) and CXCR2 (OR=1.24 (1.11-1.38), p-value=9.9x10-

5). We also recovered previous associations at six known GWAS loci, thereby providing additional

support for putative target genes. CXCR1 and CXCR2 are therapeutic targets for the anticancer agent

Reparixin, which is currently being investigated in a stage II clinical trial for triple negative breast

cancer. As such, these findings provide preliminary support for new molecular targets that could

potentially repurpose a putative cancer therapeutic. These findings highlight the utility integrating

transcriptome data for novel discovery and biological insight of risk loci.

#1301

Identification of novel susceptibility loci and genes for prostate cancer risk: A large transcriptome-wide association study in over 143,000 subjects.

Lang Wu,1 Jirong Long,1 Yingchang Lu,1 Xingyi Guo,1 Bogdan Pasaniuc,2 Kathryn L. Penney,3 Zsofia Kote-Jarai,4 Christopher A. Haiman,5 Rosalind A. Eeles,4 Wei Zheng,1 the PRACTICAL consortium. 1 _Vanderbilt University Medical Center, Nashville, TN;_ 2 _University of California, Los Angeles, Los Angeles, CA;_ 3 _Harvard T.H. Chan School of Public Health, Boston, MA;_ 4 _The Institute of Cancer Research, London, United Kingdom;_ 5 _University of Southern California, Los Angeles, CA_.

Common genetic variants in over 150 loci have been found to be associated with prostate cancer (PrCa) risk through GWAS. These variants, however, explain only a small fraction of PrCa heritability, and the genes responsible for the detected associations remain largely unknown. It has been suggested that many GWAS-identified associations may be driven by the regulation of risk variants on the expression of disease causal genes. To identify novel PrCa risk loci and possible causal genes at known risk loci, we performed a transcriptome-wide association study (TWAS) to evaluate associations of genetically predicted gene expressions with PrCa risk.

We used RNA sequencing data from normal prostate tissues and high-density genotyping from 73 European descendants included in the Genotype-Tissue Expression Project and established genetic models to predict gene expression level. Given that the regulatory mechanisms for most genes are similar across most human tissues, we also built cross-tissue models using gene expression data generated in all tissues from 369 European descendants to increase the statistical power. Based on prediction performance, we selected 22,126 genes and conducted association analyses of their predicted expression with PrCa risk using GWAS data obtained from more than 143,000 subjects included in PRACTICAL/ELLIPSE consortia.

We identified 140 genes showing an association of their predicted expression levels with PrCa risk at P < 2.26×10-6, a Bonferroni-corrected significance threshold, including 105 protein-coding genes, 33 long non-coding RNAs, and 2 processed transcripts. Seven of these associated genes are located more than 1Mb away from any of the risk variants identified in PrCa GWAS, representing potential novel risk loci. Of the remaining 133 genes located in known risk loci, 100 have not been reported from previous eQTL analyses. The associations for 25 of these genes remained significant at P < 3.76×10-4 (0.05/133) after adjusting for the risk variants reported in the initial GWAS. Our study also identified 33 genes that were previously reported based on eQTL and fine-mapping analyses. For many of the identified genes, somatic changes of indels, nonsense mutations, splice site variants, translation start site variants, or missense mutations were detected in PrCa patients in the TCGA, including known PrCa driver genes NKX3-1 and PLXNA1. Pathway enrichment analysis showed that cancer related functions were significantly enriched for the identified genes. The top canonical pathways identified included prostate cancer signaling, ATM signaling, AMPK signaling, protein ubiquitination pathway, and antigen presentation pathway.

In summary, we conducted the first large PrCa TWAS and identified multiple novel susceptibility loci and genes for PrCa risk. Our study provided substantial new information towards the understanding of PrCa genetics and biology.

#1302

Genome-wide association study of glioma reveals specific differences in genetic susceptibility to glioblastoma and non-glioblastoma.

Ben Kinnersley,1 Beatrice S. Melin,2 Jill S. Barnholtz-Sloan,3 Margaret R. Wrensch,4 Christoffer Johansen,5 Dora Il'yasova,6 Quinn Ostrom,3 and members of GICC, Karim Labreche,7 Jeanette E. Eckel-Passow,8 Paul A. Decker,8 Marianne Labussière,7 Ahmed Idbaih,7 Khe Hoang-Xuan,7 Anna-Luisa Di Stefano,7 Karima Mokhtari,7 Jean-Yves Delattre,7 Pilar Galan,9 Konstantinos Gousias,10 Johannes Schramm,10 Minouk J. Schoemaker,1 Sarah J. Fleming,11 Stefan Herms,12 Stefanie Heilmann,13 Marcus M. Nöthen,13 Heinz-Erich Wichmann,14 Stefan Schreiber,15 Anthony Swerdlow,1 Mark Lathrop,16 Matthias Simon,10 Marc Sanson,7 Preetha Rajaraman,17 Stephen Chanock,17 Martha Linet,17 Zhaoming Wang,17 Meredith Yeager,17 Rose K. Lai,18 Elizabeth B. Claus,19 Sara H. Olson,20 Robert B. Jenkins,21 Richard S. Houlston,1 Melissa L. Bondy22. 1 _Institute of Cancer Research, Sutton, United Kingdom;_ 2 _Umeå University, Umeå, Sweden;_ 3 _Case Western Reserve University, Cleveland, OH;_ 4 _University of California, San Francisco, CA;_ 5 _Institute of Cancer Epidemiology, Copenhagen, Denmark;_ 6 _Georgia State University, Atlanta, GA;_ 7 _Institut du cerveau et de la moelle épinière (ICM), Paris, France;_ 8 _Mayo Clinic College of Medicine, Rochester, MN;_ 9 _Université Paris 13 Sorbonne Paris Cité, Paris, France;_ 10 _University of Bonn Medical Center, Bonn, Germany;_ 11 _Centre for Epidemiology and Biostatistics, University of Leeds, Leeds, United Kingdom;_ 12 _University of Basel, Basel, Switzerland;_ 13 _Institute of Human Genetics, University of Bonn, Bonn, Germany;_ 14 _Institute of Medical Informatics, Ludwig Maximilians University, Munich, Germany;_ 15 _University Clinic Schleswig-Holstein, Kiel, Germany;_ 16 _Génome Québec, McGill University, Montreal, Quebec, Canada;_ 17 _National Cancer Institute, Bethesda, MD;_ 18 _Keck School of Medicine, University of Southern California, Los Angeles, CA;_ 19 _Brigham and Women's Hospital, Boston, MA;_ 20 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 21 _Mayo Clinic Comprehensive Cancer Center, Rochester, MN;_ 22 _Dan L. Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX_.

BACKGROUND: Glioma accounts for ~27% of all primary brain tumors and is responsible for ~13,000 cancer-related deaths in the US each year. Glioma tumors can be broadly classified into glioblastoma (GBM) and lower-grade non-GBM. Typically gliomas have a poor prognosis irrespective of medical care, with the most common form, GBM, having a five-year survival rate of only 5%. While genome-wide association studies (GWAS) have transformed our understanding of glioma susceptibility, individual studies have had limited power to identify risk loci.

METHODS: We performed the largest glioma GWAS to date, comprising a meta-analysis of six existing GWAS (6,405 cases, 14,100 controls) as well as new GWAS from the Glioma International Case Control Consortium (GICC; 4,572 cases and 3,286 controls) and University of California, San Francisco (UCSF)-Mayo (1,519 cases, 804 controls), totaling 12,496 cases (6,191 classified as GBM, 5,819 as non-GBM) and 18,190 controls.

RESULTS: We identified five new risk loci for GBM at 1p31.3 (rs12752552; near JAK1, P=2.04×10-9, odds ratio (OR)=1.22), 11q14.1 (rs11233250; P=9.95×10-10, OR=1.24), 16p13.3 (rs2562152; near MPG, P=1.93x10-8, OR=1.21), 16q12.1 (rs10852606; HEATR3, P=1.29×10-11, OR=1.18), 22q13.1 (rs2235573; P=1.76×10-10, OR=1.15) and eight for non-GBM at 1q32.1 (rs4252707; MDM4, P=3.34×10-9, OR=1.19), 1q44 (rs12076373; AKT3, P=2.63×10-10, OR=1.23), 2q33.3 (rs7572263; near IDH1, P=2.18×10-10, OR=1.20), 3p14.1 (rs11706832; LRIG1, P=7.66×10-9, OR=1.15), 10q24.33 (rs11598018; OBFC1, P=3.39×10-8, OR=1.14), 11q21 (rs7107785; P=3.87×10-10, OR=1.16), 14q12 (rs10131032; P=5.07x10-11, OR=1.33) and 16p13.3 (rs3751667; P=2.61×10-9, OR=1.18). Case-only analyses confirmed the specificity of 11q14.1, 16p13.3 and 22q13.1 associations for GBM and 1q44, 2q33.3, 3p14.1, 11q21 and 14q12 for non-GBM tumors. In the combined meta-analysis, among previously published glioma risk SNPs, those for all glioma at 17p13.1 (TP53), GBM at 5p15.33 (TERT), 7p11.2 (EGFR), 9p21.3 (CDKN2B-AS1) and 20q13.33 (RTEL1) and for non-GBM at 8q24.21 (CCDC26), 11q23.2, 11q23.3 (PHLDB1) and 15q24.2 (ETFA) showed even greater evidence for association. SNPs at 10q25.2 and 12q12.1 for non-GBM tumors retained genome-wide significance (i.e. P<5.0x10-8). Associations at the previously reported loci for GBM at 3q26.2 (near TERC) and 12q23.33 (POLR3B) did not retain statistical significance.

CONCLUSIONS: Our findings substantiate genetic susceptibility to GBM and non-GBM glioma being highly distinct, consistent with their distinctive molecular profiles presumably resulting from different etiological pathways. Functional analyses should lead to further insights into the biological basis of the different glioma histologies. Such information can inform gene discovery initiatives and therefore have a measurable impact on the successful development of new therapeutic agents.

#1303

A genome-wide association study of prostate cancer in Latinos.

Hannah Hopp,1 Sue Ingles,1 Chad Huff,2 Xin Sheng,1 Brandi Weaver,3 Mariana Stern,1 Sara Strom,2 Ian Thompson,4 David Conti,1 Christopher A. Haiman1. 1 _Department of Preventive Medicine, Keck School of Medicine, University of Southern California Norris Comprehensive Cancer Ctr., Los Angeles, CA;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _University of Texas Health Science Center, San Antonio, TX;_ 4 _University of Texas Health Science Center, San Antonio, TX_.

Recent studies estimate that individuals of African or Latin American ancestry represent less than 4% of samples analyzed to date in genome-wide association studies. The clinical value of genetic information in guiding personalized medicine in populations of non-European ancestry will require additional discovery and risk locus characterization efforts in these populations. Here, we performed a genome-wide association study (GWAS) meta-analysis of prostate cancer in Latino men to search for risk loci that may be important in this population. We combined GWAS data for 1034 cases and 1046 controls genotyped with the Illumina 660 Beadarray with GWAS data for 1235 cases and 1053 controls genotyped with the Illumina Oncoarray as part of the ELLIPSE U19 GAME-ON Consortium. A total of ~11 million genotyped and imputed SNPs of ≥1% frequency were tested for association with prostate cancer risk in logistic regression models controlling for age, study and genetic ancestry. Genome-wide significant associations were observed with 24 variants all located at 8q24 (128.484-128.548), and which capture the first reported prostate cancer susceptibility locus in 'region 1' of 8q24. The most significant association genome-wide was with SNP rs7824776 (risk allele frequency, 0.35; OR=1.69, p=3.4x10-11). No novel genome-wide significant associations were noted outside of 8q24. We observed a high degree of generalizability of known prostate cancer risk loci, with 78 (76%) of the 103 known risk variants having effects that were directionally consistent in their association with prostate cancer risk as previously reported, of which 31 (30%) were statistically significant with p < 0.05. In addition to these findings from the largest GWAS of prostate cancer in Latinos conducted to date, we will also present the results investigating effect heterogeneity by local ancestry (i.e. proportion Native American vs. European). In addition, we will present a comparison of polygenic risk models between Latinos, African Africans and men of European ancestry that incorporate the known risk loci to better understand how genetic risk tracks with population differences in prostate cancer incidence.

#1304

The contribution of rare and low-frequency variants to colorectal cancer heritability.

Jeroen R. Huyghe,1 Sai Chen,2 Hyun M. Kang,2 Tabitha Harrison,1 Sonja I. Berndt,3 Stephane Bézieau,4 Hermann Brenner,5 Graham Casey,6 Andrew T. Chan,7 Jenny Chang-Claude,5 Steven J. Gallinger,8 Stephen B. Gruber,9 Andrea Gsur,10 Michael Hoffmeister,5 Thomas Hudson,11 Mark A. Jenkins,12 Loic Le Marchand,13 Polly A. Newcomb,1 John D. Potter,1 Conghui Qu,1 Martha L. Slattery,14 Joshua D. Smith,15 Emily White,1 Goncalo R. Abecasis,2 Li Hsu,1 Deborah A. Nickerson,15 Ulrike Peters,1 on behalf of CCFR and GECCO. 1 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 2 _University of Michigan, Ann Arbor, MI;_ 3 _National Cancer Institute, Bethesda, MD;_ 4 _Centre Hospitalier Universitaire Nantes, Nantes, France;_ 5 _German Cancer Research Center, Heidelberg, Germany;_ 6 _University of Virginia, Charlottesville, VA;_ 7 _Massachusetts General Hospital, Boston, MA;_ 8 _Mount Sinai Hospital, Toronto, Ontario, Canada;_ 9 _USC Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA;_ 10 _Medical University of Vienna, Vienna, Austria;_ 11 _Ontario Institute for Cancer Research, Toronto, Ontario, Canada;_ 12 _The University of Melbourne, Melbourne, Australia;_ 13 _University of Hawaii Cancer Center, Honolulu, HI;_ 14 _University of Utah Health Sciences Center, Salt Lake City, UT;_ 15 _University of Washington, Seattle, WA_.

Studies that estimate complex disease heritability based on genome-wide common SNP array data have shown that a large fraction of heritability is contributed from variants that do not reach genome-wide significance at current genome-wide association study (GWAS) sample sizes. The contribution of rare variants to heritability has yet to be explored for many complex diseases. Despite the decreasing cost of sequencing, it still remains prohibitively expensive to sequence sufficient samples for well-powered genetic association studies of rare variants. However, with increasingly large and denser imputation reference panels it has become feasible to accurately impute variants with minor allele frequencies (MAFs) as low as 0.1%, enabling study of a subset of rare risk variants. In previous work, we used restricted maximum likelihood (REML) to estimate the total additive heritability of colorectal cancer (CRC) based on common SNP array genotypes. Here, we expand this work using imputed genotype data and a larger sample size. We performed whole-genome sequencing of 1,961 CRC cases and 981 controls, and subsequently imputed these haplotypes into 11,895 unrelated CRC cases and 14,659 unrelated controls that are part of the Colorectal Cancer Family Registry (CCFR) and the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO). We estimated heritability from individual-level imputed genotype data using LD- and MAF-stratified GREML, as implemented in GCTA. In total, we analyzed 17,649,167 imputed genetic variants with minor allele count >3. Published heritability estimates for CRC from family-based studies vary from 12% to 35%. Based on common genotyped SNPs, we previously estimated heritability to be 7.42% (95% CI: 4.71-10.12%) on the underlying liability scale, assuming a population prevalence of 0.004. For imputed genotypes, we estimate the total heritability to be 12.0% (95% CI: 9.65-14.35). Using a likelihood ratio test, we demonstrate a significant contribution of variants with MAF ≤1% to CRC genetic risk (P=0.003). Because of the imperfect imputation accuracy for very rare variants, their contribution is likely higher. These results suggest that with additional sequencing, improved imputation accuracy, and larger GWAS, we should expect to start discovering rare variant associations for CRC risk.

#1305

A genome-wide association study of prostate cancer in Uganda.

Zhaohui Du,1 Alexander Lubmawa,1 Susan Gundell,2 Peggy Wan,2 Nalukenge Cissy,1 Muwanga Proscovia,1 Lutalo Moses,1 Nansereko Deborah,1 Ndaruhutse Olivia,1 Katuku Molly,1 Lubwama Alexander,1 Rosemary Nassanga,1 Benson Masaba,1 Sam Kaggwa,1 Dan Namuguzi,1 Vicky Kiddu,1 Asiimwe Luke,1 Kuteesa J,1 Dabanja M. Henry,1 David Conti,2 Christopher A. Haiman,2 Stephen Watya1. 1 _Mulago Hospital Department of Surgery, Urology Unit, Uganda;_ 2 _Department of Preventive Medicine, Keck School of Medicine, University of Southern California USC /Norris Comprehensive Cancer Ctr., Los Angeles, CA_.

The greater incidence of prostate cancer in men of African ancestry remains one of the most important unanswered health disparities globally. No established environmental/lifestyle risk factors have been identified, with the only established risk factors being age, race/ethnicity and family history, all of which implicate genetic susceptibility. GWAS have clearly validated the importance of genetic susceptibility in prostate cancer, with ~100 common risk loci identified to date which in aggregate explain 33% of the familial risk. Genetic studies in African ancestry populations have provided strong evidence for genetic factors in contributing to the greater incidence of prostate cancer in men of African ancestry. To further explore this hypothesis, we conducted a genome-wide association study (GWAS) of prostate cancer among Ugandan men. Specifically, we genotyped the Illumina OncoArray, which includes a 260K GWAS backbone, in 560 prostate cancer cases (119 with Gleason score ≥8) and 480 controls and tested the associations of 448,939 genotyped and 16,396,662 imputed variants with >1% frequency. The most statistically significant variants were observed at the 8q24 risk locus (rs72725854, OR=3.37, P=2.14x10-13). We also observed suggestive signals with 106 variants outside of known risk regions with p-values <10-5 and >10-7. Of the 104 known risk variants, 100 are polymorphic in Uganda men, of which, 66 (66%) had effects that were directionally consistent in their association with prostate cancer risk as previously reported and 8 (8%) were significantly associated with risk at p < 0.05, with the most statistically significant variants being rs16901979 at 8q24 (OR=1.45, p=0.0001) and rs1512268 at 8p21.2 (OR=1.31, p=0.0087). In addition to these findings, we will also present the results from replication testing of the most significant associations from the GWAS in the Ghana Prostate GWAS Study and the African Ancestry Prostate Cancer Consortium, as well as provide a detailed comparison of polygenic risk models of the known prostate cancer variants between these two African populations, African Africans and men of European ancestry.

#1306

Two distinct regulatory mechanisms underlie estrogen receptor negative breast cancer susceptibility at the 2p23.2 locus.

Carly M. Harro,1 Gustavo Mendoza-Fandino,1 Nicholas T. Woods,2 Xueli Li,1 Fergus J. Couch,3 Alvaro N. Monteiro1. 1 _H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL;_ 2 _Eppley Institute for Research in Cancer, Omaha, NE;_ 3 _Mayo Clinic, Rochester, MN_.

Genome Wide Association Studies (GWAS) are designed to identify single nucleotide polymorphisms (SNPs) mapping to genomic regions associated with a particular trait or disease. Currently, 19 genomic loci associated with risk for estrogen receptor negative breast cancer have been identified. However, for most of these loci the mechanisms driving susceptibility have remained elusive. The 2p23.2 locus, defined by SNP rs67073037 (p = 4.76 x 10-9), contains 79 SNPs exhibiting genome-wide significant associations as well as four candidate genes: SPDYA, TRMT61B, WDR43, and FAM179A. Post-GWAS functional analysis revealed two candidate causal regulatory elements: an enhancer-like element defined by SNP rs44017214 located in the first intron of the WDR43 gene; and a segment in the 3'-UTR region of WDR43 defined by SNPs rs11680458 and rs11331880 which are predicted to have allele specific binding of non-coding microRNAs miR-548 and miR-376, respectively. We validated allele specific transcription factor binding to rs44017214 using competitive binding of USF1/USF2 to the risk allele in both MCF10A and CAL51 cell lines in Electrophoretic Mobility Shift Assays (EMSA). Additionally, rs44017214 was removed using CRISPR-cas9, and expression of neighboring genes was evaluated by qRT-PCR. No change in expression was found for any gene in a 2 Mb region surrounding the SNP, with the exception of PLB1. This suggests that the enhancer element in the intron of WDR43 has PLB1 as its target. To assess the miRNA binding site impact on WDR43 expression the pMIR-REPORT Luciferase allele-specific vector was co-transfected with miR-141/200, miR548, or miR-376 in MCF10A cells. The rs1131880 allele C in the WDR43 3'-UTR lead to the suppression of luciferase expression by miR-376 compared to the G allele. This study reveals two independent regulatory mechanisms within the 2p23.2 locus; (i) modulation of enhancer activity by allele specific binding of transcription factors USF1/USF2 to rs4401714, and (ii) decreasing the expression of WDR43 through the formation of a miR-376 binding site at the 3'-UTR of the WDR43 gene. Elucidating the functional significance of these common variants in the novel susceptibility loci 2p23.2 furthers our knowledge of the etiology of estrogen receptor negative breast cancer.

#1307

Identification of novel epithelial ovarian cancer loci in women of African ancestry from the Ovarian Cancer Association Consortium.

Ani Manichaikul,1 Lauren C. Peres,1 Xin-Qun Wang,1 Kate Lawrenson,2 Sarah Abbott,1 Ann G. Schwartz,3 Anna H. Wu,4 Edward Peters,5 Patricia G. Moorman,6 Michele L. Cote,3 Melissa Bondy,7 Linda Kelemen,8 Ellen L. Goode,9 Jill Barnholtz-Sloan,10 Simon A. Gather,2 Andrew Berchuck,6 Jennifer A. Doherty,11 Paul Pharoah,12 Joellen Schildkraut1. 1 _University of Virginia, Charlottesville, VA;_ 2 _Cedars-Sinai Medical Center, Los Angeles, CA;_ 3 _Wayne State University School of Medicine, Detroit, MI;_ 4 _Keck School of Medicine of USC, Los Angeles, CA;_ 5 _Louisiana State University Health Sciences Center School of Public Health, New Orleans, LA;_ 6 _Duke University Medical Center, Durham, NC;_ 7 _Baylor College of Medicine, Houston, TX;_ 8 _College of Medicine, Medical University of South Carolina, Charleston, SC;_ 9 _Mayo Clinic, Rochester, MN;_ 10 _Case Western Reserve University School of Medicine, Cleveland, OH;_ 11 _University of Utah, Salt Lake City, UT;_ 12 _University of Cambridge, Cambridge, United Kingdom_.

Epithelial ovarian cancer (EOC) is a rare but deadly disease for which there is notably poorer survival in women of African Ancestry (AA) compared to women of European Ancestry (EA). Previous EA-based genome-wide association studies (GWAS) have identified 30 common, low penetrant EOC susceptibility alleles. Using the custom-designed 533,631 SNP Illumina OncoArray and imputation to ~12 million genetic variants in the 1000 Genomes Phase 3, we conducted a GWAS in 755 AA EOC cases, including 537 high-grade serous ovarian cancer (HGSOC) cases, and 1,235 AA controls from the Ovarian Cancer Association Consortium (OCAC). We identified novel susceptibility loci with suggestive evidence of association with EOC, based on a threshold of P<1x10-6, at three distinct loci including 10p15.1 (lead SNP rs4525119, intronic to AKR1C3, P=4.9 x 10-7, minor allele frequency [MAF]=0.33), 3p25.3 (lead SNP rs7643459, intronic to LOC101927394, P=8.4 x 10-7, MAF=0.36), and 4q13.3 (lead SNP rs4286604, 15 kb 3' of UGT2A1, P=8.5x10-7, MAF=0.27). In analysis of HGSOC, we identified suggestive evidence of association at six distinct loci including 5q11.2 (lead SNP rs37792, 132 kb 5' of FST, P=6.0x10-8, MAF=0.34), Xq27.2 (lead SNP rs57403204, 81 kb 3' of MAGEC1, P=1.7x10-7, MAF=0.06), 10p15.1 (lead SNP rs79079890, LOC105376360 intronic, P=3.0x10-7, MAF=0.03), 17p25.1 (lead SNP rs66459581, 5 kb 5' of PRPSAP1, P=5.1x10-7, MAF=0.23), 15p12 (lead SNP rs116046250, GABRG3 intronic, P=8.7x10-7, MAF=0.05), and 4q21.21 (lead SNP rs192876988, 30 kb 3' of GK2, P=9.2x10-7, MAF=0.05). These SNPs showed no evidence of association with risk in the OCAC EA GWAS including up to 23,543 EOC cases and 29,444 controls. Of the SNPs identified with suggestive evidence of association in our GWAS, two are located near genes known to regulate hormones and previously reported in relation to diseases of the ovary (AKR1C3 and FST), two have been linked to cancer (AKR1C3 and MAGEC1) and one represents testis-specific expression (GK2). Of the 30 SNPs identified previously in a EA EOC GWAS, we observed nominally significant associations (P<0.05) with consistent direction of effect for eight SNPs in AA women. Our investigation presents evidence of (1) variants for EOC shared among EA and AA women and (2) novel EOC risk loci of importance for AA women.

#1308

Transcriptome-wide association study among 66,450 women to identify candidate susceptible genes for ovarian cancer risk.

Yingchang Lu,1 Joellen M. Schildkraut,2 Thomas A. Sellers,3 Lang Wu,1 Xingyi Guo,1 Bingshan Li,1 Y. Ann Chen,3 Jennifer B. Doherty,4 Simon Gayther,5 Ellen L. Goode,6 Hae Kyung Im,7 Siddhartha Kar,8 Kate Lawrenson,9 Ani W. Manichaikul,2 Jennifer B. Permuth,3 Brett M. Reid,3 Jamie K. Teer,3 Paul Pharoah,8 Wei Zheng,1 Jirong Long1. 1 _Vanderbilt University Medical Center, Nashville, TN;_ 2 _University of Virginia, Charlottesville, VA;_ 3 _Moffitt Cancer Center, Tampa, FL;_ 4 _University of Utah, Salt Lake City, UT;_ 5 _Cedars-Sinai Medical Center, Los Angeles, CA;_ 6 _Mayo Clinic, Rochester, MN;_ 7 _University of Chicago, Chicago, IL;_ 8 _University of Cambridge, Cambridge, United Kingdom;_ 9 _Cedars -Sinai Medical Center, Los Angeles, CA_.

Background: Large-scale genome-wide association studies (GWAS) have identified ~35 loci associated with epithelial ovarian cancer risk. However, these genetic loci together explain only a small portion of the heritability of this malignancy. The large majority of the GWAS-identified variants are located in non-coding regions, thus possible causal genes in these loci remain largely unknown. We performed a transcriptome-wide association study (TWAS) to search for novel genetic loci for ovarian cancer risk and plausible causal genes at GWAS-identified loci.

Method: Transcriptome data from normal ovarian tissue samples (n=68) and all tissue samples (n = 369), along with their high-density genotyping data, obtained from only European descendants included in the Genotype-Tissue Expression Project (GTEx), were used to build ovarian linear prediction models and cross-tissue models (to increase statistical power) using the elastic net method. Based on model performance, we evaluated 17,121 genes for their cis-predicted gene expressions in relation to ovarian cancer risk using summary statistics data generated in GWAS of ovarian cancer from 25,509 cases and 40,941 controls. MetaXcan was used to integrate gene expression prediction model with summary statistics.

Results: We identified 35 genes with predicted expression levels associated with ovarian cancer risk at P value < 2.2 × 10-6, the Bonferroni corrected significance level for multiple comparisons. Of these, 12 genes at 4 genetic loci are located ≥500kb away from risk SNPs previously reported in GWAS, representing potential novel genetic loci for ovarian cancer risk. The remaining 23 genes at 12 loci are located within known ovarian cancer risk loci. Fifteen of these genes at the 12 loci have not been reported in previous studies. Analyses by ovarian cancer histological subtypes showed that the majority of these 35 genes are associated with serous invasive carcinoma. Several new associations were identified in histological subtype analyses.

Conclusion: In this TWAS we identified multiple genes with predicted expressions related to ovarian cancer risk and provide substantial new information to enhance the understanding of ovarian cancer biology and genetics.

#1309

Association between insulin resistance and breast cancer risk: A Mendelian randomization analysis of data from 228,000 women of European descent.

Xiang Shu,1 Lang Wu,1 Nikhil K. Khankari,1 Kyriaki Michailidou,2 Manjeet K. Bolla,2 Jean Wang,2 Joe Dennis,2 Xiao-ou Shu,1 Jacques Simard,3 Douglas F. Easton,2 Wei Zheng1. 1 _Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN;_ 2 _University of Cambridge, Cambridge, United Kingdom;_ 3 _Centre Hospitalier Universitaire de Québec Research Center, Laval University, Québec City, Quebec, Canada_.

Background: Epidemiologic studies suggest that insulin resistance may be associated with breast cancer risk. We conducted Mendelian randomization (MR) analyses to reduce the biases associated with previous studies and provide evidence for causal inference.

Materials and Methods: We used genetic variants identified in genome-wide association studies for circulating fasting insulin (15 variants), early insulin secretion (16 variants), fasting proinsulin (8 variants), fasting glucose (35 variants), and 2-hour glucose (8 variants) as instruments in MR analyses. To reduce possible pleiotropic effects, variants associated with obesity were removed from the instruments. We first evaluated the association of these instruments with type 2 diabetes risk in 110,452 subjects to assess instrument validity. We then investigated the association of these instruments with breast cancer risk using data obtained from 122,977 cases and 105,974 controls of European descent included in the Breast Cancer Association Consortium (BCAC). Odds ratios (OR) were calculated to measure the associations of instrumental variables with risk of overall breast cancer and its subtypes defined by estrogen-receptor [ER] status.

Results: All instrumental variables constructed for this study were strongly associated with type 2 diabetes risk with ORs of 3.01 (p=7.86x10-5), 0.22 (p=3.54x10-14), 1.90 (p=8.28x10-4), 6.11 (p=3.59x10-19), and 1.91 (p=6.8x10-16) for per unit increase of fasting insulin, early insulin secretion, fasting proinsulin, fasting glucose, and 2-hour glucose levels, respectively. Statistically significant associations with overall breast cancer risk were found for fasting insulin (OR=1.36 for per unit increase, 95% CI=1.09-1.70, p=0.011) and fasting proinsulin (OR=1.21, 95% CI=1.06-

1.38, p=0.011). These associations were observed only for ER-positive breast cancer. No statistically significant association at p<0.05 was found for early insulin secretion, fasting glucose, or 2-hour glucose levels.

Conclusions: Our study provides strong support that certain insulin resistance traits may be causally associated with risk of breast cancer, particularly ER-positive breast cancer.

#1310

Identification of pleiotropic cancer susceptibility variants from genome-wide association studies reveals functional characteristics.

Yi-Hsuan Wu,1 Rebecca E. Graff,1 Michael N. Passarelli,2 Thomas J. Hoffmann,1 Elad Ziv,1 John S. Witte1. 1 _University of California, San Francisco, San Francisco, CA;_ 2 _Geisel School of Medicine, Dartmouth College, Hanover, NH_.

Background: There exists compelling evidence that some genetic variants are associated with the risk of multiple cancers (i.e., pleiotropy). However, the biological mechanisms of the pleiotropic effects are unclear. Thus, we investigated the functional effects for genetic variants associated with the risk of multiple cancers.

Methods: The National Human Genome Research Institute-European Bioinformatics Institute (NHGRI-EBI) GWAS Catalog contains 28,643 variant-trait associations with p < 10-5. We utilized the Experimental Factor Ontology (EFO) to classify cancer traits and obtained all associations between variants and cancer risk. Based on pairwise linkage disequilibrium (LD) determined from the European (EUR) population of Phase 3 of the 1000 Genomes Project in LDlink, correlated variants (R-squared ≥ 0.8) were clustered into groups. Variant groups associated with the risk of multiple cancers were annotated using the Ensembl Variant Effect Predictor (VEP), and tested for functional enrichment using the DAVID Functional Annotation Tool.

Results: We identified 1,456 variant-cancer risk associations. The majority (57.1%) of the associations were discovered in European ancestry populations, 19.0% in East Asians, 7.9% in an African Americans or Afro-Caribbeans, and 7.1% in Hispanics or Latin Americans. Removing duplicates, we found 1,034 unique variant-cancer risk associations for 1,005 unique variants and 27 unique cancer sites. After clustering correlated variants, we identified 29 pleiotropic variant groups, of which 2 were associated with risk of five different cancer sites. Variant group rs10936599 and rs12696304 within the MYNN gene (7.8 kb from the TERC gene) was associated with leukemia, multiple myeloma, and colorectal, skin, and bladder cancer. Variant group rs31489, rs31490, rs401681, and rs4975616 within the CLPTM1L gene (22.8 kb from the TERT gene) was associated with leukemia, and lung, pancreatic, skin, and bladder cancer. Among the 42 unique variants that composed the 29 pleiotropic variant groups, 41 variants were within a known gene, and only 1 variant was in an intergenic region. The 41 variants mapped to 26 genes, which were enriched in cellular response to hypoxia (p= 0.0071), establishment of protein localization to telomere (p= 0.0071), and ubl conjugation (p= 0.0072).

Conclusion: This study identified and functionally characterized genetic variants showing pleiotropic effects on cancer risk. Our findings improve the understanding of shared biological mechanisms common to different cancers. Clinical implications could include the classification of cancers based on etiology, genetic testing for multiple cancers, and repurposing cancer treatments.

#1311

Germline variation at 8q24 and prostate cancer risk.

Kan Wang,1 Ali Amin Al Olama,2 Rosalind Eeles,3 David Conti,1 Zsofia Kote-Jarai,3 Christopher A. Haiman1. 1 _Department of Preventive Medicine, Keck School of Medicine, USC/Norris Comprehensive Cancer Ctr., Los Angeles, CA;_ 2 _Centre for Cancer Genetic Epidemiology, Department of Public Health and Primary Care, University of Cambridge, Cambridge,, United Kingdom;_ 3 _The Institute of Cancer Research, London, United Kingdom_.

The 8q24 region harbors multiple risk variants for distinct cancers including 7 for prostate cancer, the majority of which lie in separate linkage disequilbrium blocks. It is not known whether a common biological mechanism underlies the association of genetic variation with cancer risk at 8q24, or whether there are site-specific functions of regulatory elements that are affected in this region. Given the proximity, the MYC oncogene is a likely candidate as are multiple long non-coding RNAs in the region. To further understand the contribution of germline variation to prostate cancer risk we performed a comprehensive fine-mapping analysis of the region in men of European ancestry from the PRACTICAL/ELLIPSE Consortium. More specifically, we tested 1,731 genotype tag SNPs and 12,221 imputed variants spanning the risk region (127.3-129.0Mb) in 56,363 prostate cancer cases and 37,386 controls of European ancestry that were genotyped with the Illumina OncoArray. We performed stepwise logistic regression and identified 13 variants with risk allele frequencies between 0.006 and 0.998 that surpassed genome-wide statistical significance (p-values between 3.2x10-8 and 8.0 x10-78) and with per allele odds ratios ranging from 1.11(rs5013678) to 2.68(rs183373024). Ongoing analyses that will be presented include incorporating existing GWAS and fine-mapping data (iCOGs) for men of European and African ancestry (35,000 cases and 35,000 controls) using JAM, a Bayesian approach that investigates multi-SNP models using marginal meta-analysis statistics. Leveraging the power from the overall multiethnic meta-analysis (>93,000 cases and >72,000 controls) will provide further insight into the number of independent signals in the region and their contribution to prostate cancer risk in these populations.

#1312

Genetic variation in the Hippo pathway and breast cancer risk in women of African ancestry in the ROOT Consortium.

Shengfeng Wang,1 Yonglan Zheng,1 Temidayo O. Ogundiran,2 Oladosu Ojengbede,2 Wei Zheng,3 Katherine L. Nathanson,4 Barbara Nemesure,5 Stefan Ambs,6 Dezheng Huo,1 Olufunmilayo I. Olopade1. 1 _University of Chicago, Chicago, IL;_ 2 _University of Ibadan, Ibadan, Nigeria;_ 3 _Vanderbilt University, Nashville, TN;_ 4 _University of Pennsylvania, Philadelphia, PA;_ 5 _State University of New York at Stony Brook, Stony Brook, NY;_ 6 _National Cancer Institute, Bethesda, MD_.

Background: The Hippo pathway controls organ growth by regulating cell proliferation and apoptosis. To our knowledge, it is still unclear about its role in the development of breast cancer. Identifying the relevant genes and single-nucleotide polymorphisms (SNPs) should shed light on the pathway's mechanism in carcinogenesis. Methods: We examined 47,419 SNPs in 37 Hippo pathway genes in the genome wide association study of breast cancer conducted in the African Diaspora (ROOT consortium), which included 3,686 participants of African ancestry from Nigeria, USA, and Barbados (1,657 cases and 2,029 controls). Gene-level analyses were conducted using the adaptive rank truncated product (ARTP) test for 10,771 SNPs that weren't highly correlated (r2 < 0.8), and SNP-level analyses were conducted with logistic regression. Results: The Hippo pathway was significantly associated with risk of estrogen receptor positive (ER+) breast cancer (pathway-level P=0.028). Gene-based analyses revealed that WWC1 (gene-level P=0.001) was responsible for this association, with rs116516633 in this gene being statistically significant after gene-level adjustment for multiple comparisons [odds ratio (OR) =0.53 for each G allele, 95% confidence interval (CI) =0.41-0.70, Padj=0.002]. In addition, two SNPs in LATS2 (rs142900440, OR=0.42, 95% CI: 0.28-0.64; rs58674288, OR=0.38, 95% CI =0.24-0.61) were associated with risk of ER+ breast cancer. In the analysis of ER- breast cancer risk, rs2579161 in DLG5 (OR=1.38, 95% CI: 1.17-1.63) and rs11062429 in TEAD4 (OR=1.39, 95% CI: 1.17-1.64) were statistically significant. These associations remained significant after Bonferroni-correction at the gene-wide level (all P< 0.05). Conclusions: We found evidence of associations of the Hippo pathway with ER+ breast cancer risk in women of African ancestry. Our findings supported the potential mechanism that WWC1 functions as an upstream member of the pathway, and highlighted the importance of further studies.

Table 1: Gene-wide significant tested SNPs for all breast cancer, ER+ tumors or ER- tumors

---

Gene-SNP | Function | Major/minor alleles | Minor allele frequency among controls | All cases vs controls | ER+ cases vs controls | ER- cases vs controls

OR (95% CI) | Padj | OR (95% CI) | Padj | OR (95% CI) | Padj

Overall | |  | |  | |  | |

|

TEAD1-rs16911695 | Intron | G/A | 0.24 | 1.24 (1.11-1.39) | 0.10 | 1.30 (1.07-1.58) | 1.00 | 1.34 (1.10-1.63) | 1.00

ER+ | |  | |  | |  | |

|

LATS2-rs142900440 | Intron | T/C | 0.04 | 0.78 (0.61-0.98) | 1.00 | 0.42 (0.28-0.64) | 0.01 | 0.80 (0.54-1.19) | 1.00

LATS2-rs58674288 | Intron | C/T | 0.03 | 0.73 (0.56-0.95) | 1.00 | 0.38 (0.24-0.61) | 0.01 | 0.70 (0.45-1.08) | 1.00

WWC1-rs116516633 | Non-coding transcript variant | A/G | 0.10 | 0.82 (0.71-0.96) | 1.00 | 0.53 (0.41-0.70) | 0.002 | 0.81 (0.62-1.06) | 1.00

ER- | |  | |  | |  | |

|

DLG5-rs2579161 | Upstream gene variant | G/A | 0.49 | 1.08 (0.98-1.19) | 1.00 | 0.99 (0.85-1.17) | 1.00 | 1.38 (1.17-1.63) | 0.03

TEAD4-rs11062429 | Downstream gene variant | C/G | 0.43 | 1.01 (0.92-1.11) | 1.00 | 0.94 (0.79-1.00) | 1.00 | 1.39 (1.17-1.64) | 0.047

#1313

Characterization of germline susceptibility to male breast cancer.

Eleni Perrakis, Sarah Maguire, Katarzyna Tomczyk, Anthony Swerdlow, Nick Orr. _ICR, London, United Kingdom_.

Male breast cancer (MBC) accounts for approximately 1% of all breast cancer cases. Family history is an established risk factor for MBC and 10% of cases have germline BRCA2 mutations. We recently demonstrated that common germline polymorphisms contribute to MBC predisposition and that there is overlap between genetic susceptibility loci for MBC and female breast cancer (FBC).

Here we report a comprehensive evaluation of all 107 known FBC predisposition loci in an association analysis of 1,032 MBC cases and 2,795 population matched controls. A total of 21 FBC predisposition single nucleotide polymorphisms (SNPs) showed evidence of association with risk of MBC. For approximately a third of these SNPs, the risk estimates were significantly larger in MBC than FBC. The magnitude of effects observed suggest that common susceptibility variants may be of value for risk stratification in men who have a high absolute risk of breast cancer such as BRCA2 mutation carriers. Although our data suggests that MBC predisposition shares many features in common with FBC, there are striking differences. Surprisingly, given the predominance of hormone receptor positive tumors in MBC, we observed no evidence of association between SNPs at FGFR2 and MBC risk suggesting that the estrogen receptor status of MBC tumors does not explain the observed SNP associations.

In summary, the data presented in this abstract significantly advance our understanding of genetic factors that contribute to MBC predisposition and further underscore the growing consensus that male breast cancer is not simply analogous to hormone receptor positive female breast cancer.

#1314

Trans-ethnic HLA fine-mapping of the MHC region identified several independent variants influencing susceptibility to lung cancer.

Aida Ferreiro-Iglesias,1 Corina Lesseur,1 James McKay,1 Rayjean J. Hung,2 Christopher I. Amos,3 Paul Brennan,1 on behalf of OncoArray consortium1. 1 _International Agency for Research on Cancer (IARC-WHO), Lyon, France;_ 2 _Lunenfeld-Tanenbaum Research Institute of Sinai Health System, University of Toronto, Toronto, Ontario, Canada;_ 3 _Geisel School of medicine, Dartmouth College, Lebanon, PA_.

Background: Genetic variants within the major histocompatibility complex (MHC) are associated with lung cancer. However, debate persists about the identity of the true causal variants, in part due to the broad linkage disequilibrium (LD) characteristic of the MHC and to the complexity and cost of complete HLA genotyping. Here, we assessed the role of the MHC in modulating lung cancer risk in Asians and Europeans using a broad set of HLA variants to detect the main alleles and the presence of independent effects elsewhere in this genomic region.

Material and methods: Association between the HLA variants and risk of lung cancer was examined in two collections of samples of different ethnicity (18,686 cases / 15,190 controls and 2,324 cases / 1,646 controls of European and Asian ancestry, respectively). Using existing genome-wide SNP data from the lung cancer OncoArray study, we imputed and tested classical alleles and amino acid polymorphisms in HLA-A, HLA-B, HLA-C, HLA-DQA1, HLA-DQB1, HLA-DRB1, HLA-DPA1 and HLA-DPB1, as well as 7,258 SNPs across the MHC. All the analyses were performed assuming an additive genetic model adjusted for sex and principal components as covariates. The study-wide significant threshold was p=6.03 x 10-6.

Results: Conditional, haplotype and stratified analyses identified two independent HLA allele effects associated with lung cancer squamous cell carcinoma in Europeans. One is HLA-DQB1*06 that showed a significant protective effect (OR=0.85, 95%CI 0.80-0.90, p=3.05 x10-8). The other effect is attributed to the long ancestral 8.1 haplotype (OR=1.30, 95%CI 1.18-1.42, p=4.78 x10-8), which contains class I and class II HLA alleles (A*0101 - B*0801 - C*0701 - DRB1*0301 - DQB1*0201 - DQA1*0501). In addition, single-amino-acid polymorphisms in HLA-B (at positions 9, 156, 163 and 178) and HLA-DRB1 (at positions 26, 71 and 74) located in peptide-binding grooves, suggest these proteins as possible functional contributors within the haplotype. In Asians, we observed two independent HLA allele effects associated with lung adenocarcinoma : HLA-DQB1*0401 (OR=1.67, 95% CI 1.35-2.05, p=1.59 x10-6), and HLA-DRB1*0701(OR=1.62, 95% CI 1.31-2.01, p=5.48 x10-6) as well as an intronic SNP in HLA-A (rs2256919) (OR=0.75, 95% CI 0.67-0.83, p=1.75 x10-7).

Conclusions: We established an association between HLA haplotype 8.1 and squamous lung cancer risk in Europeans. In contrast, in Asians we observed associations only with class II HLA alleles and risk of lung adenocarcinoma. We did not observed shared HLA alleles effects between populations. This ethnic heterogeneity in classical HLA allelic associations with lung cancer may be explained by allele frequency differences between populations and also different exposures that interact with HLA. Furthermore, our study illustrates the value of high-resolution imputation for fine-mapping causal variants in the MHC.

#1315

Estimating sex-specific effects of genetic loci associated with glioma risk.

Quinn T. Ostrom,1 Ben Kinnersley,2 Margaret Wrensch,3 Jeanette E. Eckel-Passow,4 Georgina Armstrong,5 Terri Rice,3 Yanwen Chen,1 John Wiencke,3 Lucie McCoy,3 Helen Hansen,3 Christopher Amos,6 Jonine L. Bernstein,7 Elizabeth B. Claus,8 Dora Il'yasova,9 Christoffer Johansen,10 Daniel Lachance,11 Rose Lai,12 Ching C. Lau,5 Ryan T. Merrell,13 Sara H. Olson,7 Siegal Sadetzki,14 Joellen Schildkraut,15 Sanjay Shete,16 Richard S. Houlston,2 Robert B. Jenkins,11 Beatrice Melin,17 Melissa Bondy,5 Jill S. Barnholtz-Sloan1. 1 _Case Western Reserve University School of Medicine, Cleveland, OH;_ 2 _The Institute of Cancer Research, Sutton, United Kingdom;_ 3 _University of California, San Francisco, San Francisco, CA;_ 4 _Mayo Clinic College of Medicine, Rochester, MN;_ 5 _Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX;_ 6 _Geisel School of Medicine at Dartmouth, Hanover, NH;_ 7 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 8 _Yale University, New Haven, CT;_ 9 _Georgia State University, Atlanta, GA;_ 10 _Danish Cancer Society, Copenhagen, Denmark;_ 11 _Mayo Clinic Comprehensive Cancer Center, Mayo Clinic, Rochester, MN;_ 12 _Keck School of Medicine, University of Southern California, Los Angeles, CA;_ 13 _NorthShore University HealthSystem, Evanston, IL;_ 14 _School of Public Health, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel;_ 15 _University of Virginia School of Medicine, Charlottesville, VA;_ 16 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 17 _Faculty of Medicine, Umeå University, Umeå, Sweden_.

BACKGROUND: Incidence of glioma varies significantly by sex, and most glioma histologies occur with greater incidence in males. Previous analyses have examined the impact of estrogen exposure as a risk factor for these tumors, but have found results of varying significance and low effect size. There may be differences in effect of previously discovered risk alleles that contribute to sex differences.

METHODS: Using data collected for three previous glioma GWAS in European-ancestry populations (MD Anderson Cancer Center, the San Francisco Adult Glioma Study, and the Glioma International Case Control Study) we assessed sex-specific effects for 14 previously identified and 13 newly identified glioma risk SNPs (27 total) overall and for glioblastoma (GBM) and non-GBM tumors separately. There were 3,892 male cases (59% GBM), 4,522 male controls, 2,500 female cases (52% GBM) and 4,940 female controls. Sex-specific odds ratios (ORM and ORF), 95% confidence intervals (95% CI) and p values (pM and pF) were generated using stratified logistic regression models. Data from each study were analyzed separately and combined using inverse variance weighted meta-analysis. Results were considered statistically significant at p<6.2x10-4.

RESULTS: In GBM, rs11979158 (7p11.2, pM=1.01x10-10, ORM=1.43 [95% CI: 1.28-1.59]; pF=3.43x10-3, ORF=1.22 [95% CI: 1.07-1.39]) and rs2562152 (16p13.3, pM=5.59x10-4, ORM=1.23 [95% CI: 1.09-1.39]; pF=1.22x10-1, ORF=1.12 [95% CI: 0.97-1.29]) had significant effect in males only. In non-GBM gliomas, rs12076373 (1q44, pM=3.56x10-7, ORM=1.41, 95% CI: 1.23-1.61; pF=1.76x10-2, ORF=1.20, 95% CI: 1.03-1.38), rs11979158 (7p11.2, pM=1.77x10-5, ORM=1.32 [95% CI: 1.16-1.49]; pF=2.73x10-1, ORF=1.08 [95% CI: 0.94-1.24]), and rs3751667 (16p13.3, pM=9.44x10-7, ORM=1.30 [95% CI: 1.17-1.44]; pF=5.18x10-2, ORF=1.13 [95% CI: 1.00-1.28]) had significant effect in males only. Effect size for rs55705857 (8q24.21) varied significantly by sex, with ORM=2.63 (95% CI: 2.24-3.09, pM=4.42x10-32), as compared to ORF=3.95 (95% CI: 3.28-4.76, pF=1.82x10-47). A sensitivity analysis was performed due to allele frequency heterogeneity by study and results did not change.

CONCLUSIONS: Sex differences and other demographic differences in cancer susceptibility can provide important clues to etiology, and these differences can be leveraged for discovery in genetic association studies. Significant differences in effect size may suggest variation in genetic effect of risk alleles or in unidentified risk factors that vary in prevalence or effect by sex. There may also be differences in the distribution of molecular subtypes within each histology by sex. Further investigation using an agnostic approach may further elucidate the relationship between effect of risk alleles and sex.

#1316

Germline genetic signals across multiple aggressive prostate cancer phenotypes.

Caroline G. Tai,1 Nima C. Emami,1 Thomas J. Hoffmann,1 Lori C. Sakoda,2 Eric Jorgenson,2 Laurel A. Habel,2 Jun Shan,2 Dilrini K. Ranatunga,2 Chun R. Chao,3 Nirupa R. Ghai,3 David Aaronson,4 Joseph Presti,4 Catherine Schaefer,2 Neil Risch,1 Stephen K. Van Den Eeden,2 John S. Witte1. 1 _University of California, San Francisco, San Francisco, CA;_ 2 _Kaiser Permanente Northern California, Oakland, CA;_ 3 _Kaiser Permanente Southern California, Pasadena, CA;_ 4 _Kaiser Oakland Medical Center, Oakland, CA_.

Prostate cancer (PrCa) is documented to be highly heritable. But many known prostate cancer loci are not associated with aggressive prostate cancer phenotypes such as high-grade cancer or biochemical failure. Previous efforts have been largely underpowered or not replicated.

We conducted a study of 6,321 men with PrCa and 5,663 controls of European ancestry recruited from Kaiser Permanente (KP) matched by age and self-reported race. Phenotype data combined patient-level demographic, clinical, and tumor data from KP databases with data from 1994 to 2016. High-grade PrCa was defined using Gleason score: low-grade defined as ≤ 7 (3+4), high-grade defined as ≥ 7 (4+3). Biochemical failure was defined for cases using post-treatment prostate specific antigen (PSA) values: post-surgery failure if PSA ≥ 0.4 ng/mL; post-radiation failure if PSA value ≥ 2 ng/mL + nadir; and no failure if PSA did not reach specified threshold. Early-onset disease was defined as age < 55 years at diagnosis. Men were genotyped for over 650,000 SNPs using an ethnic-specific Affymetrix Axiom array and imputed to 1000 Genomes. We tested genetic associations for multiple aggressive phenotypes (410 early-onset cases, 1324 biochemical failure cases, and 886 high grade cases) in both a case-only (e.g., high vs. low grade) and case-control (e.g., high grade vs. no prostate cancer controls) analysis using logistic regression adjusting for ancestry principal components, age, BMI, and kit for batch effects. PrCa specific mortality was too rare to be evaluated.

We identified several known and novel loci. Case-control analyses highlighted known prostate cancer loci at 8q24, 10q11, 11q13, 16q22, and 19q13. Previous reports of case-only analyses also identified 8q24, 20p13 and the gene, KLK3 in 19q13, to be associated with high-grade PrCa; we confirmed these signals in our study. Previous family-based studies identified 15q11 as a familial or early-onset prostate cancer locus.

These aggressive-specific variants could perform better in predicting clinically actionable prostate cancers than variants for overall prostate cancer and can improve the predictive ability of pre-diagnostic PSA.

#1317

Simultaneous identification of candidate melanoma risk variants using massively parallel reporter assay.

Jiyeon Choi, Tongwu Zhang, Michael Kovacs, Mai Xu, Nghi Lam, Leandro Colli, Kevin Brown. _NCI, Bethesda, MD_.

Recent melanoma Genome-Wide Association Study (GWAS) has identified 20 common susceptibility loci via meta-analysis of ~15,000 cutaneous malignant melanoma cases. While GWAS is extremely powerful in identifying genomic regions contributing to melanoma risk, teasing out functional variants and unraveling molecular mechanisms by which the risk is conferred presents a tremendous challenge due to limited resolution of genetic structure and lack of high-throughput assay system. To simultaneously identify functional risk variants from multiple GWAS loci, we employed recently developed Massively-Parallel Reporter Assays (MPRA). Based on the hypothesis that many GWAS functional variants regulate gene expression through transcriptional mechanism, we systematically examined allele-specific transcriptional activities of genetically indistinguishable candidate variants. Out of ~2,800 melanoma-associated variants (r2>0.4, EUR) from 16 melanoma loci for which transcriptional mechanism could be applied, 835 variants were prioritized based on their relevance as melanocyte-/melanoma-specific cis-element represented by ENCODE annotation as open chromatin or putative promoter/enhancer histone marks in melanocytes and melanoma cell lines. Pooled oligoes of 145bp encompassing each variant with either melanoma risk or protective allele coupled with 10 different unique sequence tags were cloned into luciferase vectors and transfected to melanoma cell lines. Resulting expressed tag counts were subsequently determined using massively parallel sequencing. We were able to identify ~200 potentially functional variants displaying allelic transcriptional activity by combining transfections in a melanoma cell line and HEK293FT cell line. Top functional SNPs from melanoma GWAS loci were also shown as significant eQTL SNPs in human melanocytes including MX2 on chromosome 21. Overlaying MPRA functional SNPs with melanocyte eQTL SNPs further enabled isolation of a functional risk variant among several highly linked MX2 eQTL SNPs. The results from this analysis will greatly accelerate the identification of functional melanoma risk variants and further shed light on molecular mechanisms of genetic susceptibility of melanoma in population.

#1318

A genome-wide association study of Waldenström macroglobulinemia/lymphoplasmacytic lymphoma demonstrates association with chromosome 6.

Mary L. McMaster,1 Sonja I. Berndt,1 Shengchao A. Li,1 Susan Slager,2 Joseph Vijai,3 Charles C. Chung,1 Bin Zhu,1 Laurie Burdette,1 Brenda Birmann,4 Elizabeth E. Brown,5 James R. Cerhan,2 Karin Ekstrom-Smedby,6 Henrik Hjalgrim,7 Geffen Kleinstern,2 Brian K. Link,8 James McKay,9 Alain Monnereau,10 Lindsay M. Morton,1 Alexandra Nieters,11 Nathaniel Rothman,1 Christine F. Skibola,12 Alex Smith,13 Lauren R. Teras,14 Claire M. Vajdic,15 Roel Vermeulen,16 Belynda Hicks,1 Lynn R. Goldin,1 Neil E. Caporaso1. 1 _NCI-DCEG, Bethesda, MD;_ 2 _Mayo Clinic, Rochester, MN;_ 3 _Memorial Sloan-Kettering Cancer Center, New York, NY;_ 4 _Brigham and Women's Hospital and Harvard Medical School, Boston, MA;_ 5 _University of Alabama at Birmingham, Birmingham, AL;_ 6 _Karolinska Institutet, Stockholm, Sweden;_ 7 _Statens Serum Institut, Copenhagen, Denmark;_ 8 _The University of Iowa, Iowa City, IA;_ 9 _International Agency for Research on Cancer, Lyon, France;_ 10 _Center of Research in Epidemiology and Statistics Sorbonne Paris Cite, Paris, France;_ 11 _University Medical Center Freiburg, Freiburg, Germany;_ 12 _Emory University, Atlanta, GA;_ 13 _University of York, York, United Kingdom;_ 14 _American Cancer Society, Atlanta, GA;_ 15 _University of New South Wales, Sydney, Australia;_ 16 _Utrecht University, Utrecht, Netherlands_.

Waldenström macroglobulinemia (WM) is a unique subset of lymphoplasmacytic lymphoma (LPL) that is defined by the presence of an LPL infiltrate in the bone marrow together with a monoclonal IgM protein in the serum. A somatic activating mutation, MYD88 L265P, occurs in 85+% of WM and in 25%-50% of patients with the precursor condition, IgM monoclonal gammopathy of undetermined significance (MGUS); however, germline MYD88 mutations have not been observed in WM patients, and the genetic basis for WM predisposition remains undefined. To identify novel WM susceptibility loci we conducted a two-stage genome-wide association study (GWAS) in over 450 WM cases and 4300 controls of European ancestry. Discovery (stage 1) included 217 WM cases (40% familial) and 3798 controls genotyped on the Illumina Omni Express or Illumina Omni2.5 platforms following standard quality control procedures. The genotyped data were imputed using the Haplotype Reference Consortium panel as a reference and analyzed using logistic regression. In stage 1, we identified three loci on chromosomes 6, 14 and 3 significantly associated (P<5.0x10-8) with risk of WM. Eleven promising SNPs in these and other suggestive loci (P<5.0x10-7) were selected for replication (stage 2) in 269 WM or LPL cases (4% familial) and 571 controls, and genotyping was conducted using standard methods on Taqman and Sequenom platforms or Sanger sequencing (1 SNP). Preliminary results confirm replication of the chromosome 6 locus. Stratification on familial status will illuminate the contribution of familial disease. These results will provide insight into the underlying genetic basis of WM susceptibility.

#1319

Epigenome-wide association study reveals differential DNA methylation consistent with progression of multiple myeloma.

Stephen D. Gragg,1 Devin Absher,2 Xiangqin Cui,3 Christina Pillion,1 Richard Myers,2 Shaji Kumar,4 Luciano Costa,1 Brian Chiu,5 Celine Vachon,4 Elizabeth Brown1. 1 _University of Alabama at Birmingham School of Medicine, Birmingham, AL;_ 2 _HudsonAlpha Institute for Biotechnology, Huntsville, AL;_ 3 _University of Alabama at Birmingham School of Public Health, Birmingham, AL;_ 4 _Mayo Clinic, Rochester, MN;_ 5 _University of Chicago, Chicago, IL_.

Purpose: Multiple myeloma (MM) is the second most common hematological malignancy in the US. It is characterized by a clonal expansion of plasma cells in the bone marrow and extramedullary sites and is preceded by two precursor conditions including monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM). Strong evidence suggests a germline and environmental etiology. However, efforts to characterize heritable changes in gene activity, such as DNA methylation, have not been widely reported.

Methods: We examined epigenome-wide DNA methylation as markers of MGUS, SMM and MM in peripheral blood obtained from treatment-naïve European American cases with heavy-chain IgG or IgA MGUS (n=60), SMM (n=31) and MM (n=54) and age- and sex-matched controls (n=79) included from the University of Alabama at Birmingham, University of Chicago and the Mayo Clinic, Rochester [54.5% males; mean age, 64 years (range, 36 to 86)]. We quantified DNA methylation of over 450,000 CpG and non-CpG loci using the Infinium HumanMethylation450 array (Illumina). Differentially methylated positions were calculated using a general linear model framework adjusted for confounders and cellular heterogeneity.

Results: A total of 6 CpGs were differentially methylated in MM cases compared to controls at a level of genome-wide statistical significance. MM was associated with hypomethylation at differentially methylated positions inside SBNO2 (P=3.37x10-10), WIZ (P=1.12x10-8), CA6 (P=4.29x10-8) and ADORA1 (P=3.68x10-8) as well as intergenic positions proximal to TNFRSF8 (Chr 1p36.22; P=2.24x10-9) and ENDOV (Chr 17q25.3; P=2.74x10-8). Each of these loci, with the exception of CA6 and ADORA1, were hypomethylated in each of the 3 plasma cell dyscrasia phenotypes including MGUS and SMM and MM cases compared to controls (P<0.03), albeit not at a level of genome-wide statistical significance.

Conclusions: These preliminary findings suggest that differences in DNA methylation may contribute to altered risk of MM, as well as its precursor conditions, and may play a role in plasma cell dyscrasia progression as a consequence of heritable changes in gene activity due to past exposures. Replication in a large yet similarly well-characterized population is warranted.

#1320

Non-additive and interaction effects of HLA class 2 polymorphism contributing to risk of glioma.

Chenan Zhang, Kyle Walsh. _University of California, San Francisco, San Francisco, CA_.

Although genome-wide association studies have identified a number of susceptibility loci for adult glioma, little is still known regarding whether polymorphisms in the human leukocyte antigen (HLA) region contribute risk. HLA associations have previously been reported for a number of malignancies, with selected HLA polymorphisms investigated in a subset of glioma studies. However, no systematic analysis has been conducted to date, and no investigation into potential non-additive effects of such associations has been described. In this study, we conducted comprehensive genetic analyses of HLA variants in the major histocompatibility complex (MHC) region among 1,746 adult glioma patients and 2,312 controls from the GliomaScan Consortium. Genotype data were generated with the Illumina 660-Quad array, containing 1,822 SNP probes across the MHC region. We imputed HLA alleles using a reference panel of 5,225 individuals in the Type 1 Diabetes Genetics Consortium who underwent high-resolution HLA typing via next-generation sequencing. Subjects were of European-ancestry, and case-control comparisons were adjusted for population stratification using ancestry-informative principal components. Because alleles in different loci across the MHC region are linked, we created multi-locus haplotypes consisting of genes DRB1, DQA1, and DQB1. Although none of the haplotypes were associated with glioma in additive models, DRB1*15:01-DQA1*01:02-DQB1*06:02 showed an improvement in model fit after the inclusion of a non-additive term, which was significant after Bonferroni correction (P = 2.5x10-3). Furthermore, the interaction of DRB1*15:01-DQA1*01:02-DQB1*06:02 with haplotype DRB1*04:01-DQA1*03:01- DQB1*03:01 resulted in a 3.80-fold increase in the odds of glioma (P = 8.8x10-3). Our results indicate that the DRB1*15:01-DQA1*01:02-DQB1*06:02 haplotype contributes to the risk of glioma via a dominant effect, with heterozygosity conferring greater risk of glioma than expected from homozygote disease risk in an additive model.

#1321

Transcriptome-wide association study of prostate cancer risk.

Maxine Chen,1 Alexander Gusev,2 The PRACTICAL Consortium, Massimo Loda,3 Lorelei A. Mucci,2 Meir J. Stampfer,1 Peter Kraft,2 Kathryn L. Penney1. 1 _Brigham and Women's Hospital and Harvard Medical School, Boston, MA;_ 2 _Harvard T.H. Chan School of Public Health, Boston, MA;_ 3 _Dana-Farber Cancer Institute, Boston, MA_.

Though more than 150 single nucleotide polymorphisms (SNPs) associated with prostate cancer have been identified through GWAS, we still do not understand the functional consequences of common genetic variation in this disease. Genetic variants may influence complex diseases like prostate cancer through gene expression. However, very large studies evaluating gene expression in prostate cancer are currently infeasible due to the limited availability of prostate cancer tissue and expense of collecting expression data. Alternative approaches have been developed to identify expression-trait associations in study populations without directly measured expression data. One such approach (Gusev et al. Nat Genet. 2016) uses a set of reference individuals with both gene expression and SNP data to develop genetic prediction models of gene expression values. These models are then used to impute cis genetic components of gene expression for a much a larger set of individuals with only genotype and phenotype information. We used Affymetrix gene expression microarray data from normal prostate and prostate tumor tissue and genotype data from prostate cancer patients in the Health Professionals' Follow-up Study and the Physicians' Health Study (N=187) as a reference panel for creating the predicted models of the genetic components of expression. We imputed gene expression values into Oncoarray GWAS summary statistics from the PRACTICAL consortium (N ~ 75,000) to assess the association between predicted expression and prostate cancer risk. We present the results of our transcriptome-wide association study in normal prostate and prostate tumor tissue, identifying genes significantly associated with prostate cancer risk after correcting for multiple testing. These genes may provide insight into how genetic variants influence prostate cancer through their effect on gene expression.

#1322

A meta-analysis of genome-wide association studies identifies novel loci that influence breast cancer prognosis.

Latha Kadalayil,1 Sofia Khan,2 Heli Nevanlinna,2 Peter A. Fasching,3 Fergus J. Couch,4 John Hopper,5 Jianjun Liu,6 Tom Maishman,1 Lorraine Durcan,1 Carl Bloomqvist,2 Andy Collins,1 Dianna Eccles,1 William Tapper1. 1 _University of Southampton, Southampton, United Kingdom;_ 2 _University of Helsinki, Helsinki, Finland;_ 3 _Friedrich-Alexander University Erlangen-Nuremberg, Erlangen, Germany;_ 4 _Mayo Clinic, Rochester, MN;_ 5 _University of Melbourne, Melbourne, Australia;_ 6 _Genome Institute of Singapore, Singapore, Singapore_.

Familial studies were among the first to indicate that breast cancer prognosis has a heritable component. Subsequently many variants associated with prognosis have been identified using a range of techniques including genome-wide association studies (GWAs). Despite these advances, much of the heritability remains unexplained. In young women, breast cancer is characterised by a higher incidence of adverse pathological features, unique tumour gene expression profiles and worse survival. In addition, the association of risk with conventional epidemiological exposures is less clear in women with early onset. We hypothesise that some of these difference between early and late onset could be influenced by germline variation.

To identify additional variants that influence breast cancer prognosis we conducted a two stage meta-analysis of four GWAs consisting of 6,042 patients from the UK (POSH), Finland (HEBCS), Germany (SUCCESS-A) and Australia (ABCFS). Cox-regression analyses were used to investigate overall survival (OS, n=1,101 events) and disease-free survival (DFS, n=1,316 events) with correction for oestrogen status (ER). These survival analyses were repeated in a subset of patients with early onset (aged ≤40 at diagnosis, n=2,315 patients, OS n=604 events, DFS n=716 events). Meta-analysis identified two intronic SNPs in ADAMTSL1 that were associated exclusively with early onset DFS, rs715212 (Pmeta=3.54x10-5) and rs10963755 (Pmeta=3.91x10-4) without heterogeneity between cohorts. Multivariable Cox-regression demonstrated that the effect of these SNPs were independent of the classical prognostic factors. Most importantly, rs715212 reached genome-wide significance (Pmultivariate=5.37x10-8) in the multivariate model.

ADAMTSL1 encodes a glycoprotein that forms part of the extracellular matrix (ECM) and may function in cell-cell or cell-matrix interactions or may regulate other ADAMTS proteases. Previous studies have shown that ADAMTSL1 is hypermethylated in ER positive breast cancer tumours. Using GTEx to perform eQTL analysis, we found that rs715212 is associated with the expression of several biologically relevant genes AREG (P=0.035), TNF (P=0.015), FASLG (P=0.0031) and EGF (P=0.0018) in breast mammary tissue. Interestingly, separate studies have shown that AREG is overexpressed in ER-positive breast tumours from pre-menopausal women versus post-menopausal women. Furthermore, AREG is differentially expressed between parous and non-parous mammary glands and is persistently downregulated by parity, which suggests it may contribute to the susceptibility of the nulliparous gland to breast cancer.

We conclude that rs715212 is associated with an increased risk of disease progression in patients with early onset and speculate that this could be due to an interaction with the expression of AREG.

#1323

Genome-wide association study of energy intake and its relationship to prostate cancer.

Lai Jiang, Peter Kraft, Kathryn M. Wilson. _Harvard T.H. Chan School of Public Health, Boston, MA_.

Background/Objectives: Previous studies suggested that high energy intake may increase the risk of prostate cancer. However, the mechanisms remain unclear. No genome-wide association studies (GWAS) have been conducted to detect the genetic variations in total energy intake. We aimed to identify genetic variants associated with total energy intake in men and women, and to determine the association between previously identified prostate cancer susceptibility loci and total energy intake.

Methods: We conducted a genome-wide study using combined GWAS data from 12,031 European-ancestry women and 6,743 European-ancestry men from the Nurses' Health Study, Nurses' Health Study II, Health Professionals Follow-up Study, and Physician's Health Study. Total energy intake was measured through validated food-frequency questionnaires. Missing genotypes were imputed using MACH with 1000 Genomes Project ALL Phase I Integrated Release Version 3 Haplotypes as the reference panel. We stratified by sex and adjusted for age, weight, height, physical activity and three principal components accounting for subpopulation structure. Meta-analysis based on p-values across sex was implemented. SNP set analyses, including a weighted fixed-effects (Mendelian randomization, MR) and an unweighted random-effects approach (METASOFT), were used to test the association between prostate cancer risk loci and total energy intake using summary statistics of total energy intake from the combined GWAS and the summary statistics for genome-wide significant SNPs (NSNPs = 104) from a GWAS of prostate cancer in 43,303 cases and 43,737 controls [Al Olama (2014) Nat Genet].

Results: Three SNPs near gene ANKRD33 were genome-wide significantly (P < 5.0×10-8) associated with the increase in total energy intake among men (rs10876214 [P = 5.81×10-10], rs9669605 [P = 1.46×10-8], and rs10783478 [P = 2.42×10-8]). No significant genome-wide association was found in women or the meta-analysis combining men and women. SNP set analyses did not find a statistically significant association between established prostate cancer SNPs and energy intake (PMR = 0.11 and PMETASOFT = 0.09, respectively).

Conclusions: We identified three significant SNPs near gene ANKRD33 associated with increased total energy intake among men. Genetic alleles related to risk of prostate cancer were not associated with total energy intake. Larger GWAS studies of total energy intake are warranted to explore the genetic basis of energy intake, including possible differences between men and women, and the association between total energy intake, prostate cancer, and other cancers and chronic diseases.

#1324

Profile of common prostate cancer risk variants in an unscreened Romanian population.

Paul D. Iordache,1 Bjarni Halldórsson,1 Andrei Manolescu,1 Dana Mates,2 Radu Ursu,3 Viorel Jinga4. 1 _Rekjavik University, Rekjavik, Iceland;_ 2 _National Institute of Public Health, Bucharest, Romania;_ 3 _Genetics Department University of Medicine and Pharmacy Carol Davila, Bucharest, Romania;_ 4 _4Prof. Dr. Th. Burghele Clinical Hospital, Urology Department, University of Medicine and Pharmacy Carol Davila, Bucharest, Romania_.

In the present study we investigated for the first time the profile of common prostate cancer risk variants in an unscreened Romanian population. The study population consisted of 990 unrelated histopathologically confirmed prostate cancer (PCa) cases and 1,034 male controls consisting of patients admitted for urological and surgical conditions, excluding cancer. DNA was extracted from whole blood at deCODE Genetics (Reykjavik, Iceland) and genotyped using Illumina SNP arrays, 24.295.558 variants were imputed using the 1000 Genomes dataset in the 2,024 Romanian subjects. A systematic literature review for variants associated with prostate cancer identified in previous GWAS' was done using the NHGRI catalog as a starting point, identifying 238 unique variants from 28 studies. None of the tested variants in a Romanian only GWAS reached a genome wide significance (p-value lower than 5x10-8) but 807 markers reached p-values of 1x10-6. Thirty-one of the previously-reported SNPs replicated in the Romanian cohort, with the strongest associations seen at: 8q24.21, 11q13.3, 6q25.3, 5p15.33, 22q13.2, 17q12 and 3q13.2. The most signficantly replicated variants in Romania are rs1016343 at 8q24.21 (P = 2.2x10-4), rs7929962 at 11q13.3 (P = 2.7x10-4) and rs9364554 at 6q25.2 (P = 4.7x10-4). Our present study is the first GWAS on prostate cancer performed on a Romanian cohort. The high proportion of clinically significant disease in the Romanian prostate cancer cases will allow the dissection of the association between genetic variation and severe disease. Acknowledgements: This study was funded in part by the European Union FP7 Program (ProMark project 202059) and by the EEA grant (ROMCAN project RO14-0017; EEA-JRP-RO-NO-20131-10191).

#1325

Genome-wide association study of thyroid nodule in Korean population.

Eun Kyung Lee,1 Yul Hwangbo,1 Young Joo Park,2 Jong-Il Kim,3 Yuh-Seog Jung,1 Jun Sun Ryu1. 1 _National Cancer Center, Korea, Goyang, Republic of Korea;_ 2 _Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea;_ 3 _Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, Republic of Korea_.

Thyroid nodule is very common. Several autopsy surveys reported that 30% or more of patients had thyroid nodule. However, inherited genetic risk factors for thyroid nodule remain unknown. Genome-wide association studies (GWAS) are widely used in human genetics to identify genes associated with various diseases. We aimed to find genetic factors for thyroid nodule and conducted case-control GWAS.

Objective: To identify genetic variants associated with susceptibility to thyroid nodules

Methods: We performed a two-stage GWAS for thyroid nodule. The discovery stage involved a genome-wide scan of 713 subjects with thyroid nodule and 591 subjects with normal thyroid from a population-based cohort. replication testing was conducted in an additional 600 cases and 600 controls from the participants of a health check-up.

Results In discovery stage, 21 candidate single nucleotide polymorphisms (SNP) in 20 loci including NKX2-1, ITGB1 and ZBTB25 were selected for replication. The strongest signal located in NKX2-1 (P = 9x10-7), which is known susceptibility locus for differentiated thyroid cancer. However, no SNP showed significant association in replication test. In joint analysis, no association signals reaching genome-wide significance (P <5x10-8) were detected.

Conclusion GWAS for thyroid nodule have failed to identify susceptibility locus. Therefore, genetic effect for development of thyroid nodule could be negligible.

#1326

A pooled genome-wide association study of pancreatic cancer susceptibility loci in American Jews.

Samantha A. Streicher,1 Alison P. Klein,2 Sara H. Olson,3 Robert C. Kurtz,3 Andrew T. DeWan,1 Hongyu Zhao,1 Harvey A. Risch1. 1 _Yale School of Public Health, New Haven, CT;_ 2 _Johns Hopkins School of Medicine, Baltimore, MD;_ 3 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Jews are estimated to be at increased risk of pancreatic cancer compared to non-Jews. The higher risk of pancreatic cancer in Jews can be partially explained by the increased frequency of BRCA1 and BRCA2 mutations in the Ashkenazi Jewish population; however, the remaining 40-70% excess risk of pancreatic cancer in Jews is not explained by other established non-genetic and genetic risk factors. The genetic origins of modern Ashkenazi Jews can be traced back to a founder population bottleneck between the 11th and 15th centuries follow by a rapid endogamous population expansion, making the Ashkenazi Jewish population a genetic isolate ideal for exploring genetic contributions to pancreatic cancer. We conducted a genome-wide association study (GWAS) in a case-control sample of American Jews, largely Ashkenazi, including 406 pancreatic cancer patients and 2,332 controls, identified in the database of Genotypes and Phenotypes (dbGaP) Pancreatic Cancer Cohort and Case-Control Consortium I/II (PanScan I/II), Pancreatic Cancer Case-Control Consortium (PanC4), and Genetic Epidemiology Research on Adult Health and Aging (GERA) data sets. We then examined those single nucleotide polymorphisms (SNPs) with P<10-7 in an expanded sample set, of 539 full- plus part-Jewish pancreatic cancer patients and 4,117 full- plus part-Jewish controls from the same data sets. Jewish ancestry was genetically determined using seeded fast principal component analysis. In our data set, the part-Jewish subjects had mixed genetic ancestry both from Jews and from non-Jewish white-Europeans. Among the full Jews, a novel genome-wide significant association was detected on chromosome 19p12 (rs66562280, OR=1.55, 95% CI=1.33-1.81, P=2.30x10-8). A suggestive association was detected on chromosome 19p13.3 (rs2656937, OR=1.53, 95% CI=1.31-1.78, P=9.96x10-8). Similar associations were seen for these SNPs among the full- plus part-Jews. No SNPs meeting the quality control filter inclusion criteria were found in the full- plus part-Jewish subjects to be significant at the P=10-7 level. Overall, we identified in Jews one novel susceptibility locus and one suggestive novel susceptibility locus for pancreatic cancer that warrant follow-up work. This is the first GWAS conducted for pancreatic cancer in the increased-risk Jewish population. The novel susceptibility locus discovered on chromosome 19p12 could explain up to ~15%, and the novel suggestive susceptibility locus on chromosome 19p13.3 could explain up to an additional ~15% of the increased risk for pancreatic cancer in the Jewish population. 

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

### Cell Growth Signaling Pathways 3

#1327

CD147 contributes to extracellular matrix remodeling and degradation.

Tiffany Tan, Beata Kosmider, Nathaniel Marchetti, Sudhir Bolla, Chenna Mandapati, Gerard Criner, Karim Bahmed. _Temple University, Philadelphia, PA_.

Chronic obstructive pulmonary disease (COPD) is an incurable, progressive lung disease characterized by shortness and difficulty in breathing. Patients with COPD are at higher risk of developing lung cancer and often are associated with poor outcome of lung cancer diagnosis and treatment. Extracellular matrix (ECM) remodeling and degradation is one of the common hallmarks between cancer and COPD. It contributes to degradation of air sacs (emphysema) or small airways (obstructive bronchiolitis). It is also involved in cancer initiation and progression to assist tumor expansion and cancer cell migration. In this study, we analyzed lung tissue and primary alveolar type II (ATII) cells isolated from patients with emphysema and control organ donors. ATII cells secrete and store pulmonary surfactant and restore the epithelium after damage. We performed RNA sequencing in ATII cells to identify dysregulated proteins, which are involved in ECM degradation in emphysema. To further study the mechanism involved in the ECM degradation in this disease, we analyzed the expression of selected proteins and we focused on the role of extracellular matrix metalloproteinase inducer (CD147). CD147 is a cell membrane protein involved in inducing the production of extracellular matrix metalloproteinases (MMPs). First, we found that cells isolated from patients with emphysema have higher expression of CD147 in ATII cells in comparison with controls as detected by western blotting and RT-PCR. This may explain high ECM degradation in this disease. Second, we observed higher MMPs levels in plasma obtained from individuals with this disease. Third, we found significantly higher expression of cathepsin B in ATII cells isolated from emphysema in comparison with controls. Cathepsin B is an enzyme that facilitates a direct degradation of ECM proteins and activate other proteases capable of degrading ECM. Fourth, our data indicate that CD147 interacts with cathepsin B, which may lead to the activation of MMPs and emphysema progression. In summary, our results show for the first time the important role of CD147 in emphysema development. Moreover, we found that cathepsin B may contribute to CD147-mediated disease progression. Further studies will elucidate its role in lung cancer development in patients with emphysema.

#1328

Mechanism of transcriptional regulation of metabotropic glutamate recepter-5 induced by the CXCR4 signaling pathway.

Nobuyuki Kuribayashi, Daisuke Uchida, Makoto Kinouchi, Sayaka Izumi, Kyoko Kuribayashi, Hitoshi Kawamata. _Dokkyo University School of Medicine, Tochigi, Japan_.

We have previously demonstrated that stromal cell-derived factor (SDF)-1/CXCR4 system enhances the metastases of oral cancer cells via induction of mGluR5. In this study, we examined the transcriptional regulation of mGluR5 induced by the SDF-1/CXCR4 system, using oral cancer cells, B88, which express functional CXCR4 and have highly metastatic potentials. mGluR5 was markedly induced both by the exogenous SDF-1 addition and overexpression of SDF-1 in B88 cells. These induction was completely inhibited by a MEK inhibitor, U0126, and partially inhibited by a PI3K inhibitor, wortmannin. Furthermore, these inhibitors significantly inhibited the SDF-1/CXCR4 dependent cell-migration in the presence or absence of mGluR5 agonist, DHPG. Recently, several investigators demonstrated that activation of the ERK1/2 pathway is responsible for the transcription of several cancer-associated microRNAs (miRNAs). Thus, we performed miRNA microarray in SDF-1 stimulated B88 cells. we isolated miR-30 family which has predictive binding sites in 3'-UTR of mGluR5 mRNA in silico analysis. We now analyze the regulation of these miRNA by MEK/ERK1/2 and PI3K/Akt pathway.

#1329

**Inhibition of papillary thyroid cancer cells progression by targeting skp2 via** ros **-** erk **-** chop **-** dr5 **pathways.**

Pratheeshkumar Poyil, Rong Bu, Abdul K. Siraj, Sandeep Kumar P, Khawla S. Al-Kuraya. _King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia_.

S-phase kinase protein 2 (SKP2), is an F-box protein with proteasomal properties and has been found to be overexpressed in a variety of cancers. However its role in papillary thyroid cancer (PTC) has not been elucidated. Therefore, inactivation of SKP2 could be a viable strategy for the treatment of PTC. In the present study, we investigated the role of SKP2 and its ubiquitin-proteasome pathway in PTC using a tissue microarray cohort of 1022 PTC samples, PTC cell lines and Nude mouse model. Our immunohistochemistry data showed that SKP2 was over-expressed in 75.1% of PTC cases and was clinically, significantly associated with extra thyroidal extension (p=0.0331), Tall cell variant (p=0.0070), and presence in surgical margins (p=0.0347). Bortezomib as well as SKP2 specific siRNA caused downregulation of SKP2 leading to dose-dependent growth inhibition and induction of apoptosis via mitochondrial apoptotic pathway in PTC cell lines. Furthermore, we found that treatment of PTC cells with Bortezomib caused up-regulation of DR5 via generation of reactive oxygen species (ROS). Finally, Bortezomib treatment augmented TRAIL mediated anti-cancer effect on PTC xenograft tumor growth in nude mice. These data suggest that Bortezomib is a viable therapeutic option for the treatment of PTC either alone or in combination with other apoptotic agents such as TRAIL.

#1330

HSP90 inhibitor has a possibility to overcome imatinib resistance in gastrointestinal stromal tumors.

Yurina Saito,1 Tsuyoshi Takahashi,1 Satoshi Serada,2 Minoru Fujimoto,2 Koji Tanaka,1 Yasuhiro Miyazaki,1 Tomoki Makino,1 Yukinori Kurokawa,1 Makoto Yamasaki,1 Kiyokazu Nakajima,1 Shuji Takiguchi,1 Masaki Mori,1 Yuichiro Doki,1 Tetsuji Naka2. 1 _Osaka University, Graduate School of Medicine, Suita, Osaka, Japan;_ 2 _National Institutes of Biomedical Innovation, Health and Nutrition, Ibaraki, Osaka, Japan_.

[Background] Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the digestive tract and are reported to harbor gain-of-function mutations in the KIT gene, which contribute to the development of sporadic GISTs. This knowledge has facilitated the development of targeted therapies with tyrosine kinase inhibitors and the revolutionary chemotherapeutic drug imatinib mesylate (IM). In clinical trials, the disease control rate was nearly 85%, and corresponding 2-year overall survival rates ranged from 70-80%, indicating markedly improved patient outcomes compared with anecdotal data in the pre-IM era. Despite its effectiveness, half of GISTs treated with IM develop resistance within 2 years, largely due to the accumulation of additional kinase domain mutations accompanied by concomitant re-activation of the KIT tyrosine kinase, even in the presence of IM. Heat shock protein 90 (HSP90) is one of chaperon molecules required for the proper folding, function, and stability of various client proteins such as KIT. The aim of this study is to clarify the efficacy of HSP90 inhibitor against IM resistant GIST.

[Material and Method] We used the established human GIST cell line GIST-T1, and two IM-resistant cell lines (GIST-T1R8, GIST-T1R9), which had additional kinase domain mutations accompanied by concomitant re-activation of the KIT tyrosine kinase same as clinical samples, by exposure to IM. These resistant cell lines exhibited imatinib IC50 values (>10 μM) that were >1000-fold higher than the parental cell line. We investigated the cytotoxicity and signaling inhibition by HSP90 inhibitor using by the WST-8 assay, caspase3/7 apoptosis assay and western blotting. Immunobolts for KIT and for KIT-depending signaling pathways were observed at 12 hours of treatment. Caspase 3/7 apoptosis assay was measured after 24h.

[Result] HSP90 inhibitor showed growth inhibition not only for GIST T1 but also GIST-T1R8 and GIST-T1R9. Apoptosis induction of these cell lines were also confirmed by the exposure to HSP90 inhibitor in a dose-dependent manner. To find the mechanism of apoptosis induced by HSP90 inhibitor, we investigated the signaling pathway depending on KIT. HSP90 inhibitor showed inhibition of phosphorylated-KIT, a client for HSP90, in both IM sensitive and resistant GIST cell lines. And, it also inhibited downstream of KIT signaling, e.g. p-ERK and p-AKT.

[Conclusions]HSP90 inhibitor showed the anti-tumor efficacy for imatinib resistant GIST in vitro. It might have a possibility to apply for clinical use.

#1331

TGFβ1 induces breast tumor kinase overexpression in triple negative breast cancer via p38 MAPK signaling to glucocorticoid receptors.

Carlos J. Santos Perez, Tarah Regan Anderson, Carol A. Lange. _University of Minnesota Twin Cities, Minneapolis, MN_.

Triple negative breast cancer (TNBC) is the deadliest breast cancer (BC) subtype, accounting for 20-30% of all BCs. It has a heterogeneous pathology and pathogenicity, but it is defined by the lack of estrogen receptor, progesterone receptor, and Her2 epidermal growth factor receptor expression. Because targeted therapy through these receptors is not possible, treatment relies on chemotherapy and surgery, which are often inadequate. Thus, the identification of possible molecular targets is critically important in TNBC. Breast tumor kinase (Brk) is a soluble tyrosine kinase that is overexpressed in 85% of BCs and a driver of aggressive and metastatic phenotypes. Overexpression of Brk mRNA and protein occurs in TNBC by unknown mechanisms. The glucocorticoid receptor (GR), a very potent modulator of cytokine mediated actions of the immune system, is emerging as a mediator of chemoresistance and recurrence in TNBC. We previously demonstrated that GR signaling cooperates with physiologic stress signaling mediated by hypoxia inducible factors HIF-1a and HIF-2 to modulate the expression of Brk mRNA and protein in TNBC cells. Moreover, phosphorylation of GR at ser134 by p38 MAPK is essential for GR regulation of Brk expression. P38 is an essential Ser/Thr kinase that regulates cellular transduction of growth factors, such as Hepatocyte Growth Factor (HGF), and cytokines (e.g. TGFβ1) in TNBC cells, and was previously shown to be co-expressed with Brk in human breast tumors. Herein, we further probed mechanisms of crosstalk between key cytokines, GR, and p38 signaling in the regulation of Brk overexpression. We hypothesize that TGFβ1 signaling modulates EMT and metastasis in part by increasing the expression of Brk in TNBC. Treatment of MDA-MB-231 cells with TGFβ1 for 1, 2, 24 and 48 hours increased Brk protein expression relative to vehicle controls. Additionally, TGFβ1 increased both HIF1 and HIF2 protein levels (at 24 and 48 hours respectively). TGFβ1 regulated Brk expression at the level of mRNA, as measured using RT-PCR. Moreover, TGFβ1 synergized with activated GR to further increase Brk mRNA levels. In contrast, mRNA levels of HIF1 and HIF2 were not modulated by TGFβ1, suggesting that the observed protein increases are due to stabilization of HIFs. Finally, TGFβ1 robustly induced p38-dependent phosphorylation of GR at serine 134. This phosphorylation event promoted ligand-independent GR transcriptional activity at the Brk promoter. Human breast tumors significantly co-express active p38 MAPK and Brk. Our molecular model implicates TGFβ1 signaling (via p38 MAPKs) to phospho-GR in the aberrant overexpression of Brk in TNBC. We conclude that blocking of the TGFβ1 pathway may provide a strategy to inhibit Brk mediated TNBC tumor progression. This work was supported by NIH/NCI R01 CA192178 (to CAL) and T32 GM008244-24.

#1332

NOTCH - HEY1 pathway induces EMT in head and neck squamous cell carcinoma.

Takahito Fukusumi,1 Theresa W. Guo,2 Shuling Ren,1 Akihiro Sakai,1 Mizuo Ando,1 Sunny Haft,1 Chao Liu,1 Joseph A. Califano1. 1 _UC SanDiego, San Diego, CA;_ 2 _Johns Hopkins Medical Institutions, Baltimore, MD_.

Background

Head and neck squamous cell carcinoma (HNSCC) is noted to have a NOTCH mutation rate of 20% within the Cancer Genome Atlas (TCGA) dataset consistent with tumor suppressor gene status. However, NOTCH genes are also upregulated in wild type HNSCC and show signs of downstream activation in a substantial proportion of HNSCC. In this study, we aimed to further explore the potential for NOTCH pathways to be activated in HNSCC.

Material and Methods

mRNA sequence of TCGA data includes 520 HNSCC tumors and 46 normal tissues. We used 447 HNSCC excluding 73 tumors with NOTCH mutations. The activated groups of NOTCH downstream genes were defined as tumors with expression 1 standard deviation above the mean of normal tissue. We used four HNSCC cell lines (SKN3, Cal27, SCC61, SCC090) to ascertain the results of the TCGA analysis in vitro.

Result

In TCGA analysis, HEY1 activated HNSCC had significant more expression of NOTCH pathway genes. In particular NOTCH4 had the most significant correlation with HEY1 activation among NOTCH receptors. To confirm this result in vitro, HEY1 expression was compared between si-control and si-NOTCH4 of SKN3, Cal27, SCC61 and SCC090. As expected, HEY1 expression in all si-NOTCH4 treated cell lines were significant decreased. Next, we examined the relationship about NOTCH4 and an epithelial mesenchymal transition phenotype (EMT). Using TCGA data, we found that mesenchymal marker genes were significantly increased in high NOTCH4 group. In vitro experiments, si-NOTCH4 cells decreased mesenchymal marker gene expression such as Fibronectin and TWIST1. We also examined HEY1 function. Similar to NOTCH4, it was found that HEY1 is significantly related to EMT in silico and in vitro.

Conclusion

The NOTCH4-HEY1 pathway is significantly upregulated in HNSCC and induces EMT.

#1333

Cross talk between mitogen-activated protein kinase signaling cascades and Wnt/β-catenin in HER2+ overexpressing breast cancer cells.

Miguel Nava,1 Nwamaka A. Amobi,1 Yanyuan Wu,1 Robin Farias-Eisner,2 Jay Vadgama1. 1 _Charles Drew University, Los Angeles, CA;_ 2 _University of California, Los Angeles, Los Angeles, CA_.

Introduction: The HER2-positive breast cancer is characterized by an amplification of the HER2 gene, resulting in an increase in HER2 presence on the surface of cells and magnification of downstream intracellular signaling. Treatment of HER2+ breast cancer with trastuzumab decreases tumor volume, reduces metastasis and increases overall survival. Unfortunately, HER2-positive breast cancers develop resistance to trastuzumab. The mechanism of resistance may involve the activation of compensatory networks of different cell signaling pathways. The purpose of the study is to examine the crosstalk between HER2/EGFR and Wnt signaling in HER2-positive breast cancer cells.

Methods: In order to address this, a HER2-positive breast cancer cell line (SKBR3) was used and induced with EGF. The phosphorylation of p38 and ERK1/2 was determined by Western blot analysis. The cellular localization of proteins of interest was examined using biochemically fractionated lysates followed by western blot analysis.

Results: Our data demonstrated an early and transient peak of phospho-p38 at 30min post EGF treatment. Interestingly, both phospho-p38 and phospo-ERK1/2 were detected in the nuclear compartment at this time. β-catenin, the major effector molecule in Wnt signaling, was stabilized 30min post EGF treatment. β-catenin protein levels remained higher in EGF treated cells than untreated cells up to 4h post EGF treatment. It is likely that a stabilized β-catenin following EGF treatment potently modulates a subset of Wnt targets. We are assessing this possibility using chromatin immunoprecipitation based experiments for H3K18ac, H3K27ac, pol2 and β-catenin. Taken together, our data suggests that EGFR signaling promotes Wnt signaling in HER2-positive breast cancer cells. We hypothesize that this promotes cancer stem cell niche maintenance and an EMT phenotype.

#1334

Mesenchymal cell sub-populations selectively modulate paracrine hedgehog signaling in triple negative breast cancer.

Karla P. Ramos, Maribella Domenech. _Univ. of Puerto Rico-Mayaguez, Mayaguez, PR_.

Recent studies correlate Hedgehog (Hh) signaling with reduced survival rates in triple negative breast cancer (TNBC) patients. Activation of hedgehog signaling in the adjacent mesenchyme has been shown to promote tumor growth and it is a poor prognosis factor for TNBC. We developed a novel tumor-mesenchymal in vitro model of hedgehog signaling in TNBC to evaluate the role of mesenchymal cell sub-populations in the proliferative potential and stem cell markers of breast cancer cells using a custom designed multiwell array. As a source of mesenchymal cell sub-populations we evaluated myofibroblasts, mesenchymal stem cells derived from bone marrow or adipose tissue and tumor cells that undergo epithelial-mesenchymal transition (EMT). Tumor cells were culture with 1 or 2 mesenchymal cell sub-populations in adjacent compartments +/- SHH ligand for 72-96hrs. Active Hh signaling was confirmed by up-regulation of main Hh target genes (e.g. Gli1, Patch1). Paracrine Hh signaling only in bone marrow-derived mesenchymal stem cell and EMT significantly increased proliferation (10-15%) of TNBC and normal breast cancer cell lines. Addition of adipose-derived mesenchymal stem cells or pharmacological Hh inhibitors (Cyclopamine and Gant61) partially abolished tumor growth indicating that other non-canonical signals are involve in paracrine Hh-driven tumors. Cancer stem cell markers were selectively modulated in co-cultures. Our results suggest that paracrine Hh signaling-driven by bone marrow and EMT mesenchymal are potential therapeutic targets to treating TNBC.

#1335

Deubiquitinase OTUD6B isoforms are important regulators of growth and proliferation.

Maurizio Bocchetta, Clodia Osipo, Anna Sobol. _Loyola University Chicago, Maywood, IL_.

Deubiquitinases (DUBs) are increasingly linked to the regulation of fundamental processes in normal and cancer cells, including DNA replication and repair, programmed cell death, and oncogenes and tumor suppressors signaling. Here evidence is presented that the deubiquitinase OTUD6B regulates protein synthesis in non-small cell lung cancer (NSCLC) cells, operating downstream from mTORC1. OTUD6B associates with the protein synthesis initiation complex and modifies components of the 48S preinitiation complex. The two main OTUD6B splicing isoforms seem to regulate protein synthesis in opposing fashions: the long OTUD6B-1 isoform is inhibitory, while the short OTUD6B-2 isoform stimulates protein synthesis. These properties affect NSCLC cell proliferation, since OTUD6B-1 represses DNA synthesis while OTUD6B-2 promotes it. Mutational analysis and downstream mediators suggest that the two OTUD6B isoforms modify different cellular targets. OTUD6B-2 influences the expression of cyclin D1 by promoting its translation while regulating (directly or indirectly) c-Myc protein stability. This phenomenon appears to have clinical relevance as NSCLC cells and human tumor specimens have a reduced OTUD6B-1/OTUD6B-2 mRNA ratio compared to normal samples. The global OTUD6B expression level does not change significantly between non-neoplastic and malignant tissues, suggesting that modifications of splicing factors during the process of transformation are responsible for this isoform switch. Because protein synthesis inhibition is a viable treatment strategy for NSCLC, these data indicate that OTUD6B isoform 2, being specifically linked to NSCLC growth, represents an attractive, novel therapeutic target and potential biomarker for early diagnosis of malignant NSCLC.

#1336

Significance of SERPINE2 expression in peritoneal metastasis in gastric carcinoma.

Daisuke Kuroda,1 Junji Kurashige,2 Hiroshi Sawayama,1 Masaaki Iwatsuki,1 Tomoyuki Uchihara,1 Tasuku Toihata,1 Eri Oda,1 Taisuke Yagi,1 Tsugio Eto,1 Keisuke Miyake,1 Mayuko Ouchi,1 Kenichi Nakamura,1 Koichi Kinoshita,1 Kousuke Mima,1 Takatsugu Ishimoto,1 Yasuo Sakamoto,1 Yoshifumi Baba,1 Naoya Yoshida,1 Koshi Mimori,3 Hideo Baba1. 1 _Kumamoto Univ. Graduate School of Medical Sci., Kumamoto, Japan;_ 2 _Kyushu Univ. Graduate School of Medical Sci., Fukuoka, Japan;_ 3 _Kyushu University Beppu Hospital, Kumamoto, Japan_.

Background: Peritoneal metastasis is one of the most frequent causes of death in patients with advanced gastric carcinoma (GC). However, molecular mechanisms driving peritoneal dissemination still remain poorly understood. Here, we aimed to give new insights to the molecular mechanisms that drive the peritoneal dissemination of GC. This study identified candidates of potential regulators of peritoneal dissemination and focused on the significance of one of them in vitro experiments and clinical samples.

Methods: We utilized previously reported combined expression analysis with two cell lines and samples and 200 GC patients to identify driver genes of peritoneal dissemination. The cell lines consist of HSC58, an established cell lines from scirrhous type primary GC and its progeny cell line, 58As9, with in vivo-selected highly peritoneal metastatic potential. In vitro experiments, we utilized the two gastric cancer cell lines, 58As9 and NUGC4 and evaluated the effect of SERPINE2 knockdown on proliferation, spheroid formation, invasion, and motility. As for evaluation of clinical samples, 176 primary gastric carcinomas were included, which were obtained from patients who underwent curative gastrectomy in Kumamoto university between 2005 and 2015. We evaluated expression of SERPINE2 in immunohistochemistry and divided the cohort into two groups by SERPINE2 expression and analyzed relationship with clinicopathological factors and survival.

Results: Enrichment analyses of the profile of the peritoneal dissemination-associated expression signature revealed that poor prognosis and peritoneal metastasis was related with the two pathways, focal adhesion (FDR q=0.00195) and extracellular matrix (FDR q=0.00195). Among the genes related with extracellular matrix, Serine Proteinase Inhibitor, Clade E, Member 2 (SERPINE2) was found to be highly expressed in 58As9 compared with HSC58, and we focused on SERPINE2 as a candidate of potential regulators of peritoneal dissemination. In vitro experiments, the SERPINE2 knockdown was related with decrease in cell motility and invasion, but not with decrease in proliferation, spheroid formation. Immunohistochemistry analysis of the clinical samples revealed higher SERPINE2 expression was significantly related with invasion of serosa and lymphatic vessels, and lymph node matastasis, and peritoneal metastatic metastasis. Survival analysis also revealed SERPINE2 expression was related with poor peritoneal recurrence-free survival (p=0.038), and peritoneal metastasis-specific survival (p=0.036).

Conclusion: We found SERPINE2 as a candidate of driving molecules of peritoneal metastasis partly because it promotes invasive potential. Further study including in vivo study is required to reveal relationship of SERPINE2 with the other molecules and pathways.

#1337

The distribution of the phosphorylated Parkin in lung carcinoma.

Hongwei Wang,1 Songlin Zhang,2 Catherine Ma,1 Yuan Zhou1. 1 _AbboMax, Inc., San Jose, CA;_ 2 _UT Health Science Center at Houston, Houston, TX_.

Parkin, also known as Parkinson Juvenile Disease Protein 2, together with PINK1 and Ubiquitin, form a simple cascade in the clearance of damaged mitochondria. This cascade plays an important role in maintain mitochondral quality controls and is implicated in protesomal degradation of toxic substrates. Recent studies have demonstrated that Parkin to be genetically altered, and aberrantly expressed in a variety of human malignancies including lung, breast and ovarian cancers, these may indicate that Parkin is a tumor suppressor gene whose inactivation may play an important role in tumorgenesis. PINK1-dependent phosphorylation of Parkin(pS65) is a required step in Parkin activation and localization. The aims of this study were to investigate the expression of Parkin and its phophorylated form in various type of lung carcinoma tissues and lung cancer cell lines; the correlation of the protein expression level and the type of the cancer. We choose two forms of the cancer materials. 1) A tissue microarray (TMA) which contains 16 cases of lung carcinoma tissues consisting of 8x adenocarcinoma (AC), 6x squamous cell carcinoma (SCC), and 2x small cell lung carcinoma (SCLC); 2) FFPE slides which contains 9x lung cancer cell lines (A549, H1703, H1975, H2228, H23, H838, HOP62, HOP92, and SKMES1). All the tissues and cells were immunohistostained by Parkin and phospho-Parkin polyclonal antibodies. The reference staining was performed by monoclonal anti-Cytokaratin 7 (CK7) antibodies. From this study, we found Parkin expression level varies from TMA tissues and FFPE cell section which were not correlated with tumor grade. Among the 8 cases of andenocarcinoma, only 3/8 showed marked Parkin expression, 3/8 moderate, and 2/8 mild; for Parkin(pS65), only 1/8 demonstrated marked expression, 2/8 moderate, 2/8 mild, and no staining in 3/8 cases. In SCC cases, 2/6 demonstrated marked Parkin expression, 2/6 moderate, 2/6 mild. The phosphorylated Parkin(pS65) only expressed in 3/6 showed mild expression, not the others. Among 2 cases of SCLC, only one showed marked Parkin expression and less extent of Parkin (pS65), the other sample had no staining. In all FFPE cell sections, the strong expression of Parkin and Parkin (pS65) was found in the lung cancer cell lines. An interesting finding was that Parkin(pS65) strongly presented in cells of mitotic phase in these cell lines except H1703 cell line. All the TMA and FFPE slides were confirmed by CK7 monoclonal antibody staining. In summary, Parkin and its phophorylated Parkin (pS65) were found in adenocarcinoma, squamouse cell carcinoma and small cell lung cancers with differentially expressed levels, the degree of expression level was not correlated with tumor grade in these 16 cases. Phosphorlated Parkin (pS65) was strongly expressed in FFPE cancer cell lines at mitotic phase. It is concluded that the function of Parkin in tumor development and the prognosis remain to be further evaluated; it may provide a potential therapeutic approach for lung cancer.

#1338

Exosome secretion promotes proliferation of African American prostate cancer cells under hypoxia: Role of HIF2Α and RAB signaling.

Gatikrushna Panigrahi, Taylor C. Peak, Sierra L. Patterson, Nicole Kasica, Ravi Singh, Ashok Hemal, Gagan Deep. _Wake Forest Univ Bapt Med Ctr, Winston Salem, NC_.

Background and Objectives: African American men experience worse prostate cancer (PCa) outcomes compared with those of Caucasian men not only in incidence and mortality rates but also harbor higher grade tumors compared to Caucasians. While the disparity is likely multifactorial, there may be biological basis for such phenomenon. Hypoxia in the tumor microenvironment is the key determinant of disease aggressiveness regulating primary growth, angiogenesis, metastasis and treatment outcomes. But role of hypoxia in the aggressiveness of PCa in African-American men has not been characterized yet. Here, we focused on hypoxia signaling, exosome biogenesis and characterization in African-American PCa cells compared to Caucasian PCa cells.

Methods: We employed several techniques (cell viability assays, ultracentrifugation, nanoparticle tracking analyses [NTA], electron microscopy, and western blotting) to compare exosomes biogenesis and underlying molecular mechanisms in African-American and Caucasian PCa cells under normoxic and hypoxic conditions.

Results: Compared to normoxic condition, African-American PCa cells (E006AA-hT and RC77T) either survived better or proliferated under hypoxia (1% O2), while growth of Caucasian PCa cells (PC3 and LNCaP) was strongly inhibited under hypoxia. NTA analyses showed that all PCa cells secreted more exosomes under hypoxia compared to normoxia, but more prominently in African-American PCa cells (E006AA-hT: 4.4 fold, RC77T: 35 fold, PC3: 3 fold and LNCaP: 2 fold). Exosomes secreted by E006AA-hT cells under hypoxia were loaded with higher amount of HSP70, HSP90, IL6 and Annexin II, and lower TGFβ. These exosomes also showed significantly higher MMP9 loading compared to E006AA-hT cell lysates. More importantly, under hypoxia, exosome biogenesis inhibition by GW4869 (20 µM) and 5-(N,N-dimethyl) amiloride (0.1-5 µg/ml) significantly reduced the E006AA-hT cell viability by 30% (p=0.0001) and 20-60% (p< 0.005 - 0.0001), respectively in 48 hrs. Further studies on hypoxia signaling revealed that E006AA-hT cells have significantly higher HIF2α and HIF1β but low HIF1α expression compared to LNCaP cells. Interestingly, HIF2α was present largely in nuclear fraction and its expression was similar under both normoxic and hypoxic conditions in E006AA-hT cells. Studies on the RAB and its effector proteins indicated that RabGAP5 expression was strongly increased under hypoxia in both E006AA-hT and LNCaP cells. Further, compared to LNCaP cells, E006AA-hT cells showed higher expression of Rab5 and Rabex5 but lower Rab4, and these molecules did not change significantly under hypoxia.

Conclusion: African-American PCa cells have growth advantage under hypoxia mainly through higher exosome secretion; and higher expression/activity of HIF2α and RAB-signaling molecules could be associated with PCa aggressiveness in African-Americans.

#1339

Role of PTEN loss in basal-like 2 triple negative breast cancer.

Ericka L. Smith, Christiana S. Kappler, Stephen P. Ethier. _Medical University of South Carolina, Charleston, SC_.

Basal-like 2 (BL2) triple negative breast cancer (TNBC) is an aggressive molecular subtype of breast cancer with a poor clinical outcome. These cancers are characterized by overexpression of epidermal growth factor receptor (EGFR), loss of PTEN protein expression, and presence of mutated TP53. Despite overexpression of EGFR, EGFR-targeted therapies have performed poorly in clinical settings. Therefore, understanding the mechanisms of resistance in these cells is critical for improved patient care. To understand the oncogenic signaling landscape in BL2 TNBC, our lab utilizes the BL2 cell lines SUM-149 and SUM-229, which overexpress EGFR activated by an amphiregulin-mediated autocrine loop, and are PTEN null. In addition, we utilize the BL1 subtype cell line MDA-MB-468, which also exhibits overexpression of EGFR and loss of PTEN. Both the BL1 and BL2 cell lines exhibit elevated levels of phosphorylated AKT (p-AKT) compared to control MCF-10A cells. However, while treatment of SUM-149 and SUM-229 cells with inhibitors targeting EGFR (Gefitinib), type-1 PI3K (BKM120, BKM), mTOR (KU-0063794, KU) and pan-AKT (MK2206, MK) did not reduce p-AKT levels, MCF-10A and MDA-MB-468 cell lines treated with the same panel of targeted inhibitors showed marked inhibition of p-AKT. In addition, re-expression of PTEN in SUM-149 and SUM-229 cells had little effect on p-AKT but dramatically reduced p-AKT in MDA-MB-468 cells. Although PTEN re-expression alone had little effect on p-AKT in SUM-149 and SUM-229 cells, these cells did display decreased AKT phosphorylation following treatment with Gefitinib, BKM, KU, and MK. There are three isoforms of AKT (AKT1, AKT2 and AKT3) of which, AKT3 has been implicated in the aggressive nature of some cancers. Previous studies in our lab showed that SUM-149 and SUM-229 cell lines have higher levels of AKT3 message and protein compared to the other AKT isoforms. AKT3 expression was also higher in the BL2 cell lines compared to BL1 and control cells. This differential expression of AKT isoforms led us to examine the impact of AKT isoform-specific signaling. Using PTEN re-expressing SUM-149 cells, we showed that PTEN re-expression resulted in decreased phosphorylation of AKT1 but not of AKT3. However, PTEN re-expression combined with Gefitinib treatment dramatically reduced phosphorylation of both AKT1 and AKT3. Interestingly, while neither re-expression of PTEN nor Gefitinib expression alone had an effect on colony-forming efficiency, PTEN re-expression coupled with Gefitinib treatment was able to reduce colony formation efficiency in both SUM-149 and SUM-229 cell lines. Taken together, these data indicate that BL2 breast cancers, with overexpression of EGFR and loss of PTEN expression, have an oncogenic signaling network involving AKT3 that contributes to the aggressive and drug-resistant phenotype observed in this subset of TNBC.

#1340

RAS pathway activation and sensitivity to therapeutic agents is correlated with NF1 residual activity in malignant peripheral nerve sheath tumors.

Elliot Kahen, Darcy Welch, Diana Yu, Christopher Cubitt, Jae Lee, Andrew Brohl, Damon R. Reed. _Moffitt Cancer Center, Tampa, FL_.

Background: Malignant Peripheral Nerve Sheath Tumor (MPNST) is a malignant sarcoma that derives from a peripheral nerve or plexiform neurofibroma. Neurofibromatosis type 1 (NF-1) patients are particularly susceptible, with a higher risk, earlier onset, and worse prognosis. The major factor associated with MPNST and NF-1 is Neurofibromin 1, coded by the NF1 gene. NF1 mutation results in RAS hyperactivation. Chemotherapy for MPNST is currently limited, with poor prognosis for metastatic or unresectable tumors. Thus, the development of promising treatment solutions for MPNST to translate to clinical trials is required.

Methods: Here, we seek to identify efficacious chemotherapeutic treatments for MPNST with a combination of drug screening and biological pathway analysis. We used our previously established preclinical system to test FDA approved or promising developmental agents against five cell line models for MPNST. We screened sixty agents with diverse mechanisms of action below published maximum plasma concentrations, and measured effects with a CellTiter-Glo viability assay. Promising agents were then tested in two-drug combinations, allowing for determination of synergism. We then examined the molecular effects of the top candidates with use of antibody arrays that permit detection of a series of phosphorylated proteins.

Results: The group of most efficacious drugs was enriched with agents that target factors downstream of RAS, including MEK, mTOR, and PI3K inhibitors, with microtubule inhibitors, genotoxics, and HDAC inhibitors also demonstrating good results. Strong synergism was observed across our cell line models particularly in combinations containing the dual mTORC1/2 inhibitor INK128. Interestingly, drug sensitivity varied greatly between cell lines, correlating with relative NF1 protein and RAS-GTP levels. We analyzed the activation of the RAS pathway in response to drug treatment with antibody arrays and found that, following treatment, relative phosphorylation signal was more decreased compared to controls in cell lines with lower relative NF1 protein levels. Doxorubicin was able to reduce phosphorylation signal compared to controls to a level near comparable to targeted inhibitors, which could contribute to doxorubicin's current usefulness against MPNSTs. Importantly, we identified combination treatments that were able to greatly reduce the relative phosphorylation signal of RAS pathway members versus control. Combinations containing INK128 resulted in the most pathway shutdown. These findings suggest that MPNSTs may be susceptible to combination treatments targeting RAS pathway members. Moreover, it may be possible to use pathway analysis as a diagnostic tool to predict drug tolerance.

#1341

Talin plays an important role in cell-cell interactions.

Devang M. Patel, Akhilesh Kumar, William Morgan, Michael D. Birnbaum, Chaunhua Jiang, Fangliang Zhang. _Miller School of Medicine, Miami, FL_.

Appropriate cell adhesion is necessary for numerous physiological processes and can be deranged in many diseases, including cancer. It has long been recognized that the cadherins mediated cell-cell adhesion receptor is an important determinant of tumor progression, serving as a suppressor of invasion and metastasis. Recently a role for the C-terminal region of talin (the VAD fragment) in cadherin mediated cell-cell attachment has been suggested, but its molecular mechanism remained unclear. Here, we identified a small region of the VAD fragment required for its localization to cadherin-mediated cell-cell junctions and also found that its localization to cell-cell junctions is independent of its actin binding properties. To identify the binding partner of the VAD fragment at cell-cell junctions we utilized immune-pulldown and found that it interacts with catenins, an important component of cadherin-mediated cell-cell junction. Based on our data, we proposed that the VAD-fragment of talin stabilized and/or stimulated the cell-cell junction by interacting with catenins.

#1342

Loss of claudin-3 expression induces IL6/gp130/Stat3 signaling to promote colon cancer malignancy.

Rizwan Ahmad,1 Balawant kumar,1 Zhimin Chen,2 Steven(Xi) Chen,3 Dominik Muller,4 Subodh M. Lele,1 Mary Kay Washington,5 Surinder K. Batra,1 Punita Dhawan,1 Amar B. Singh1. 1 _UNMC, Omaha, NE;_ 2 _The First Affiliated Hospital of Zhengzhou University, China;_ 3 _University of Miami, Miami, FL;_ 4 _Department of Pediatric Nephrology, Charité, and Berlin Institute of Health,, Berlin, Germany;_ 5 _Vanderbilt University, Nashville, TN_.

Genetic and epigenetic aberrations drive the formation of a benign colorectal adenoma and its progression to the full-blown carcinoma. Hyperactivated Wnt/β-catenin signaling acts as a switch to induce EMT and promote colorectal cancer. Additionally, IL-6/Stat-3 signaling, activated by microbial translocation through the dysregulated mucosal barrier in colon adenomas, facilitates the adenoma to adenocarcinomas transition. However, inter-dependence between these signaling pathways in their capacity to communicate with mucosal barrier to promote colon cancer remains unclear. In current study, we have discovered, using a comprehensive investigative regimen, a novel and tissue specific role of claudin-3, tight junction integral protein, in inhibiting colon cancer progression by serving as the common rheostat of Stat-3 and Wnt-signaling activation. Notably, in our analysis, using mRNA and protein expression, and utilizing samples from a large patient cohort (<250 CRC specimen), we found claudin-3 expression to be significantly suppressed (p<0.001 versus normal) in cancer tissues versus normal mucosa. The colon tissues from the established mouse models of colon cancer (APCmin mice and Azoxymethane (AOM)-DSS-induced colon cancer) demonstrated similar tumor specific decrease in claudin-3 expression. Interestingly, claudin-3 negative tumors retained E-cadherin expression. Most notably, we found a significant and positive correlation (p<0.05) of the greater levels of claudin-3 expression with patient survival. These findings however contrasted an upregulated claudin-3 expression in other cancer types and implicated differential epigenetic regulation. In further studies, naïve claudin-3-/- mice revealed dedifferentiated {significant down regulation of P-27 (p<0.05) and vimentin expression versus WT mice} and leaky colonic epithelium. Moreover, claudin-3-/- mice demonstrated increased colon tumor burden and invasive adenocarcinoma when subjected to colon cancer. Wnt-signaling hyperactivation, albeit in GSK-3β independent manner, characterized colon cancer in claudin-3-/- mice. Claudin-3 loss also upregulated the gp130/IL6/Stat3 signaling in colonic epithelium potentially assisted by infiltrating immune organization. Genetic and pharmacological studies confirmed that claudin-3 loss induces Wnt/β-catenin activation in Stat-3-dependent manner to promote colon cancer. Overall, these novel findings identify claudin-3 as a therapeutic target for inhibiting overactivation of Wnt-signaling to prevent CRC malignancy.

#1343

DUOX1 silencing in lung cancer is associated with enhanced nuclear EGFR localization.

Andrew C. Little, Karamathullah Danyal, David Heppner, Milena Hristova, Albert van der Vliet. _University of Vermont, Burlington, VT_.

Non-small cell lung cancer (NSCLC) remains to be one of the leading causes of cancer-related mortalities worldwide. The NADPH oxidase homolog, Dual Oxidase 1 (DUOX1), is an H2O2 producing enzyme located in the airway epithelium with key roles in mucosal host defense and wound repair mechanisms. Recent studies indicate that DUOX1 is epigenetically silenced in many forms of NSCLC via hypermethylation of its promoter. We previously demonstrated that DUOX1 silencing in lung cancer cells is closely associated with epithelial-to-mesenchymal transition (EMT) and enhanced tumor invasiveness and metastasis. However, the mechanism(s) by which DUOX1 silencing promotes these outcomes are not understood. Previous findings indicate that DUOX1-dependent epithelial host defense pathways are mediated by redox-dependent activation of epithelial signaling via the non-receptor tyrosine kinase, Src, and the receptor tyrosine kinase, EGFR. We therefore hypothesized that loss of DUOX1 in lung cancer may be associated with aberrant regulation of Src and/or EGFR, tyrosine kinases that are frequently overexpressed and activated in lung cancer and strongly contribute to tumor growth and survival. In fact, recent studies have indicated that nuclear localization of EGFR in cancer cells is associated with metastatic cell behavior and poor clinical outcome, and the nuclear EGFR localization depends on Src-dependent phosphorylation of EGFR at Y1101. We observed that overexpression of DUOX1 in alveolar lung cancer A549 cells, which possess EMT-like features and in which DUOX1 is normally silenced, results in redistribution Src to the plasma membrane and decreased nuclear accumulation. DUOX1 overexpression in A549 cells also suppressed EGF-stimulated nuclear translocation of EGFR, which was associated with reduced EGFR phosphorylation at Y1101. Conversely, RNAi-mediated silencing of DUOX1 in the epithelial cancer cell line H292, which normally expresses DUOX1 expression, was found to promote EGF-mediated EGFR nuclear translocation and Y1101 phosphorylation. Since nuclear EGFR is thought to enhance the transcription of target genes related to cell cycle progression and proliferation (e.g. CDK1, Myc, others), we evaluated gene expression of these target genes in our cell models. Indeed, in cells lacking DUOX1, EGF stimulation significantly enhanced mRNA levels of CDK1, Myc, and other target genes for nuclear EGFR, whereas no such induction was seen in cells that express DUOX1. Our findings indicate that DUOX1 silencing in lung cancer may be associated with worse prognosis, partly due to altered spatiotemporal regulation of EGFR and Src and increased nuclear targeting. Since both EGFR and Src are subject to redox regulation by cysteine oxidation, we are currently aiming to elucidate the molecular mechanisms by which these mechanism are affected by altered DUOX1 status.

#1344

Progranulin promotes ubiquitination, sorting and lysosomal degradation of sortilin in castration-resistant prostate cancer cells.

Ryuta Tanimoto,1 Chiara Palladino,1 Simone Buraschi,1 Shi-Qiong Xu,1 Leonard G. Gomella,1 Renato V. Iozzo,1 Antonino Belfiore,2 Andrea Morrione1. 1 _Thomas Jefferson University, Philadelphia, PA;_ 2 _Universita della Magna Graecia, Catanzaro, Italy_.

Introduction and Objective: Despite extensive clinical and experimental studies over the past decades, the pathogenesis of prostate cancer remains largely unknown. Furthermore, the mechanisms promoting the castration-resistant stage of prostate cancer are still very poorly understood. We recently demonstrated that progranulin acts as an autocrine growth factor and promotes castration-resistant prostate cancer cell motility, invasion and anchorage-independent growth. Progranulin was also overexpressed in prostate cancer tissues vis-à-vis non-neoplastic controls. Despite the strong connections with cancer, progranulin's mode of action is not well understood. Furthermore, proteins that regulate early stages of progranulin signaling have not been identified. Sortilin, a transmembrane protein of the Vps10 family, binds progranulin and negatively regulates progranulin action by promoting progranulin uptake and degradation. Significantly, castration-resistant DU145 and PC3 cells express very low sortilin levels, which were associated with high progranulin production and enhanced motility, suggesting that mechanisms regulating sortilin/progranulin levels may contribute to prostate cancer progression.

Methods: Progranulin-mediated sortilin ubiquitination was assessed by transient ubiquitination assays in PC3 and DU145 cells either expressing or depleted of endogenous progranulin. Sortilin stability was assessed by immunoblotting with or without protein synthesis inhibitors and immunofluorescence microscopy with GFP-tagged sortilin. Progranulin-dependent sortilin endocytosis and sorting for degradation were evaluated by confocal microscopy and colocalization with specific intracellular markers.

Results: Prolonged progranulin stimulation induced sortilin ubiquitination in PC3 and DU145 cells, which was associated with enhanced sortilin internalization through a clathrin-dependent pathway. Upon progranulin stimulation, sortilin was then sorted into early endosomes and subsequently targeted for degradation in the lysosomal compartment as assessed by immunoblot and IF experiments in the presence or absence of lysosomal inhibitors. Significantly, sortilin ubiquitination was significantly inhibited in progranulin-depleted PC3 and DU145 cells, which showed enhanced sortilin stability when compared to progranulin-expressing control cells.

Conclusions: Our data demonstrate that progranulin activates a feedback loop by inducing sortilin downregulation via the lysosomal pathway. This novel regulatory mechanism would ensure the maintenance of progranulin signaling in castration-resistant prostate cancer cells by limiting sortilin-mediated progranulin uptake and degradation. Deciphering the cross-talk between sortilin and progranulin signaling could provide avenues to design novel therapeutic strategies in prostate tumors.

#1345

Mutant IDH1 and tissue factor in gliomas.

Dusten Unruh, Snezana Mirkov, Charles D. James, Craig Horbinski. _Northwestern University, Chicago, IL_.

Background: IDH1 mutations (IDH1mut) occur in 20-30% of gliomas, induce DNA hypermethylation, and are associated with a better prognosis than IDH1 wild-type (IDH1wt) gliomas, yet the basis for this remains unclear. Furthermore, venous thromboemboli (VTE) are a serious complication in glioma patients, and the mechanism for this is also unclear. We recently discovered that IDH1mut gliomas are much less likely to develop VTE compared to IDH1wt gliomas. Our data suggests that suppression of Tissue Factor (TF), the primary initiator of coagulation, is a key reason for this. TF also directly enhances tumor malignancy via protease-activated receptor 2 (PAR2). In this study, we further explored the significance of TF on malignancy and thrombosis in IDH1wt and IDH1mut gliomas.

Methods: Assays were performed using 6 patient derived glioma cells, 3 IDH1wt (GBM6, GBM12, GBM43) and 3 IDH1mut (TB09, BT142, GBM164). DNA methylation was analyzed by Illumina Human 850K. TF procoagulant activity (PCA) was determined by FXa generation, using preoperative glioma patient arterial plasma, conditioned medium, or cells. In vivo models of cancer induced thrombosis were created via surgical stenosis of the inferior vena cava of mice engrafted with IDH1wt or IDH1mut gliomas. In vitro markers of malignancy were measured by BrdU incorporation for cell proliferation, serum gradient Matrigel-coated transwell inserts for invasion, soft agar colony formation for clonogenicity. Nude mice (N≥6/group) were orthotopically transplanted with IDH1wt glioma cells and monitored for growth using bioluminescence imaging.

Results: The TF gene, F3, was significantly hypermethylated in IDH1mut cells compared to IDH1wt cells, and IDH1mut glioma cells had lower levels of TF protein and TF PCA compared to IDH1wt cells. Exogenous treatment with the product of IDH1mut enzyme, D-2-hydroxyglutarate, directly suppressed TF PCA by 38% in IDH1wt cells (P=0.03), as did transgene IDH1mut expression. Mice with IDH1mut glioma xenografts produced fewer and smaller IVC thrombi than mice with IDH1wt xenografts (0.9±0.9 vs 10.3±3.6 mg, P=0.02). Patients whose gliomas contained IDH1mut had lower TF PCA compared to IDH1wt (0.5±0.2 vs 1.2±0.2 pg/mL, P=0.04), and there was a strong link between levels of TF PCA and subsequent development of VTE (P=0.03). TF knockdown greatly reduced cell proliferation, invasion, and colony formation of IDH1wt cells. Likewise, the PAR2 antagonist, GB83, inhibited IDH1wt cell proliferation by 54% (P<0.001) and reduced cell invasion by 25% (P<0.05). TF knockdown also reduced in vivo growth of IDH1wt gliomas by 96% at 28 days post engraftment (P=0.03), and extended median survival of engrafted mice by 86% (P=0.001).

Conclusions: IDH1mut mediated suppression of TF expression and PCA may be a critical component of the less thrombogenic, and less malignant, IDH1mut phenotype. Targeting TF-PAR2 signaling may therefore represent a novel therapeutic strategy to reduce IDH1wt glioma malignancy.

#1346

Pan-ERBB inhibitor blocks tumor growth and metastasis.

Joyce A. Schroeder,1 Sabrina Maisel,1 Derrick Broka2. 1 _University of Arizona, Tucson, AZ;_ 2 _Arizona Cancer Therapeutics, Tucson, AZ_.

The Epidermal Growth Factor Receptor family of transmembrane tyrosine kinases (ErbB) is over-expressed, correlates with poor prognosis and negatively correlates with disease free survival in many cancer types. EGFR, HER2 and ErbB3 are known to be highly expressed and active, and have been targeted by both kinase inhibitors and monoclonal antibodies, both with disappointing results. Importantly, though, these therapeutics only target either a single receptor or the kinase domain alone. It is well known that these receptors function as heterodimers, and blocking of a single receptor will only promote the hetero-dimerization of the remaining family members. Furthermore, while the tyrosine kinase activity of these receptors is well known, less appreciated are the kinase-independent mechanisms by which they drive cancer progression. These include the modulation of calcium signaling and mitochondrial and nuclear translocation and activity as transcriptional co-factors. The highly conserved juxta-membrane domain (JD) of the ErbB receptors regulates these kinase-independent functions, as well as receptor homo- and hetero-dimerization. Upon ligand binding, the JD forms anti-parallel dimers between the receptors, resulting in receptor dimerization and intracellular trafficking. These protein-protein interactions result in cell growth, survival, migration and invasion.

We have previously shown that peptides mimicking this JD can act in a dominant-negative fashion, promoting the formation of non-functional ErbB dimers (consisting of EGFR, HER2 and ErbB3) that induce rapid, ErbB-dependent cell death. Using Cell Penetrating Peptides synthesized in tandem with the JD, these peptides (called EJ1) rapidly cross the plasma membrane, bind the ErbB receptors, and induce cell death in cell lines and PDX lines grown in culture. Death results from a combination of apoptotic and necrotic mechanisms, due to inactivation of mitochondrial function and calcium signaling (as well as kinase inhibition). Although EJ1 peptides display rapid ErbB-dependent cell death in vitro, they are rapidly cleared in vivo, limiting their efficacy. To stabilize in vivo activity, the active peptide (which is an alpha-helix) was stabilized with hydrocarbon staples. Based on this, we hypothesized that SAH-EJ1 would be an effective targeted therapeutic for the treatment of ErbB-dependent cancer. We therefore tested SAH-EJ1 in 3 different ErbB-dependent models of cancer, including EGFRvIII-driven glioblastoma, a PDX model of basal-like breast cancer with EGFR and ErbB3 amplification, and a PDX model of lung cancer with EGFR kinase domain mutations driven by Erlotinib treatment. In each of these models, substantial inhibition of tumor growth was observed, as well as extended survival.

#1347

In vivo **efficacy of eflapegrastim in rats with chemotherapy-induced neutropenia.**

Young Hoon Kim,1 InYoung Choi,1 Prasad Kolli,2 Guru Reddy2. 1 _Hanmi Pharmaceuticals, Seoul, Republic of Korea;_ 2 _Spectrum Pharmaceuticals, Inc., Irvine, CA_.

Introduction: Eflapegrastim (SPI-2012, HM10460A) is a novel, long-acting recombinant human granulocyte colony-stimulating factor (rhG-CSF). Eflapegrastim consists of an rhG-CSF conjugated to a recombinant E. coli derived Fc fragment of IgG4 via a polyethylene glycol linker. Eflapegrastim is in clinical development for the treatment of chemotherapy-induced neutropenia in cancer patients. The purpose of this study was to evaluate and compare the efficacy of eflapegrastim, pegfilgrastim and filgrastim in rats with chemotherapy-induced neutropenia.

Methods: Rats were treated with 50 mg/kg of cyclophosphamide (CPA) intraperitoneally to induce neutropenia. Pegfilgrastim was administered subcutaneously as a single dose of 100 µg/kg on Day 1 and filgrastim was administered subcutaneously at a dose of 20 µg/kg daily for five days on days 1 to 5. Eflapegrastim was administered subcutaneously as a single dose on day 1, at doses ranging from 32 µg /kg to 322 µg/kg (or 8.8 µg/kg to 88 µg/kg as G-CSF equivalent). Blood samples were collected for 8 days after drug administration for the measurement of neutrophil counts and the Duration of Severe Neutropenia (DSN).

Results: The DSN in neutropenic rats treated with eflapegrastim was compared with the DSN in neutropenic rats treated with pegfilgrastim or filgrastim. The DSN was 0.2 days when eflapegrastim was administered as a single dose at 88 µg/kg (as G-CSF equivalent) 24 hours after administering CPA. In contrast, the DSN was 3.04 days with filgrastim administered at a dose of 20 µg/kg for 5 days from Day 1 to Day 5 and 2.8 days with pegfilgrastim administered as a single dose of 100 µg/kg 24 hours after administering CPA. At the lowest eflapegrastim dose of 8.8 µg/kg that was about 1/10 of G-CSF equivalent dose for pegfilgrastim, the DSN in eflapegrastim treated rats was 2.94 days. Thus, eflapegrastim was found to be more potent in shortening the DSN with a lower G-CSF equivalent dose when compared to either pegfilgrastim or filgrastim.

Conclusion: Eflapegrastim was about 10 times more potent than pegfilgrastim at G-CSF equivalent doses in this neutropenic rat model

#1348

**GDC-0032, a p110 beta sparing PI3K inhibitor is highly efficient on** PIK3CA **-mutated and** HER2- **amplified breast cancer model.**

Pradip De,1 Yuliang Sun,1 Jennifer Carlson,1 Lori Friedman,2 Casey Williams,1 Nandini Dey,1 Brian Leyland-Jones1. 1 _Avera Research Inst., Sioux Falls, SD;_ 2 _Genentech, San Francisco, CA_.

Background: HER2 amplification/overexpression frequently co-occurs with PI3K pathway activation in breast tumors. Mutations in PIK3CA are known to be involved in a wide range of human cancers including HER2+ breast cancer, and mutant PIK3CA is thought to act as an oncogene. Here, we evaluate the efficacy of GDC0032, a p110 beta-sparing PI3K inhibitor against HER2+ or HER2+/PIK3CA mutated breast cancer cells. MATERIALS AND METHODS: Four HER2+ breast cancer (BC) cell lines consisting of three activating (either helical or kinase domain) PIK3CA mutation (HER2+/ER+ and HER2+/ER-) were analyzed for proliferation, apoptosis, cell cycle and signaling pathway activation assays. Preclinical efficacy of GDC-0032 was also evaluated in vivo in a mouse model. RESULTS: 1) All HER2+ and HER2+/PIK3CA mutant cell lines exhibited low IC50 values (ranging from 0.1 µM-1.5 µM) irrespective of ER-positive or not. 2) GDC-0032 caused a strong differential growth inhibition in both HER2+ and HER2+/ /PIK3CA mutated breast cancer cell lines when compared to lines that were HER2- and PIK3CA wild type by 3D-ON-TOP clonogenic assay. 3) Administration of GDC-0032 induced cell cycle G0/G1 arrest and resulted in increased apoptosis in a dose-dependent manner. Furthermore, induction of apoptosis was more with GDC-0032 when combined with RAD001. 4) GDC0032 also blocked expression of CYCLIN D1. 5) Investigation of the signal transduction revealed that the treatment of GDC-0032 reduced the level of p-AKT (Ser473 and Thr308), and p-S6 expression is indicating the down-regulation of downstream signaling of PI3K and mTOR pathway. 6) GDC-0032 was highly active at reducing established tumor growth in vivo in BC mouse xenografts harboring PIK3CA mutation and HER2 amplification. CONCLUSION: Our in vitro or in vivo data showed that GDC-0032 was highly efficient either as a single agent as well as with RAD001 in HER2+/PIK3CA mutated breast cancer cell lines. Our data suggest that GDC-0032 represents a novel therapeutic option in patients harboring PIK3CA mutations and/or HER2/neu gene amplification in breast cancer.

#1349

NSAIDs: Multiple roles in multiple environments.

Gloria M. Calaf,1 Debasish Roy2. 1 _Instituto de Alta Investigación, Universidad de Tarapacá, Arica, Chile;_ 2 _City University of New York Hostos Community College, Bronx, NY_.

Non steroidal drugs are mainly known for their activities and use as anti-inflammatory, antipyretic compounds. But, in recent observations, they are exposed to use as an effective alternative compounds to prevent or stimulate different metabolic activities and help in preventing various neoplastic progression. They have specific role in controlling estrogen metabolism during breast cancer progression. Sometimes, they are used as an apoptotic induction in various cancers. Their role in hypoxia induced proliferation is also showing promising results. Different NSAIDs will help to induce the activities of various tumor suppressor genes. As chronic inflammation increases the risk for various cancers, therefore, it is important to eliminate inflammation through anti-Inflammatory compounds where NSAIDs are playing a vital role. Most of them are acting to prevent inflammation either via selective or non-selective COX based mechanism. The non-selective COX inhibitor sulindac and the COX-2 selective inhibitor etodolac etc. have been shown to prevent the formation and cause regression of adenomas in patients with familial polyposis. Unfortunately, COX-1 and/or COX-2 inhibition and depletion of physiologically important prostaglandins is associated with gastrointestinal, renal and cardiovascular toxicities that limit the use of NSAIDs and COX-2 inhibitors for cancer chemoprevention In this particular study, we are using various NSAIDs in a radiation-induced estogren treated breast cancer model to establish their role in differential expression of various cancer related genes selected from different cell cycle pathways microarray. Selection of biomarkers from these altered genes will help to develop a valid strategy for cancer prevention through precision based molecular targeted therapy.

Supported by Office of Academic Affairs, Honors Program, COBI, Hostos-CUNY, NY, USA (DR) and Tarapacá University, Arica, Chile (GMC). 

### Cell Growth Signaling Pathways 4

#1350

Direct pharmacological targeting of Gq/11 in uveal melanoma.

Michael D. Onken, Carol M. Makepeace, Shiqi Wang, Kevin M. Kaltenbronn, S. Michinobu Kanai, Tom J. Broekelmann, John A. Cooper, Kendall J. Blumer. _Washington Univ. School of Medicine, St. Louis, MO_.

Uveal (eye) melanoma is a highly aggressive cancer, in which almost half of patients develop distant metastases that are refractory to therapy. In particular, metastatic uveal melanoma has been clinically unresponsive to the immunotherapeutic agents that have shown success in skin tumors, making the need for novel therapeutic approaches to uveal melanoma all the more urgent. Unlike skin melanomas, which are driven by BRAF and NRAS mutations, uveal melanomas arise typically from mutations that result in constitutive activity of the alpha subunit of the heterotrimeric G-protein, Gq, or its paralog G11. The prevalence of constitutively active Gq/11 in uveal melanoma suggests a dependence of these tumors on Gq/11 activity that could be exploited therapeutically. To address this hypothesis, we are using a potent, bioavailable small molecule that binds to and inhibits Gq/11 to target constitutively active Gq/11 in uveal melanoma cells. This inhibitor functions by sequestering wild type or constitutively active Gq/11 in an inactive state. We first used inositol phosphate accumulation assays and confirmed inhibition of both wildtype and constitutively active Gq/11 by the inhibitor in all uveal melanoma cells. We then assayed the affect of Gq/11 inhibition on overall viability of uveal melanoma cells, and found that uveal melanoma cells with mutant Gq/11 were highly sensitive to the small molecule; whereas, uveal melanoma cells with wildtype Gq/11 showed no loss of viability, even at 1000-fold higher concentrations of inhibitor. In Gq-mutant uveal melanoma cells, Gq/11 inhibition caused cell cycle arrest in G1, and dysregulation of several cell cycle regulatory pathways. Inhibitor treatment also caused Gq/11-driven uveal melanoma cells to become more differentiated, as indicated by increased pigmentation, elevated expression of melanin synthesis and melanosome markers, changes in cell morphology and changes in melanocytic versus melanoma gene programs. None of these phenotypic changes were seen in BRAF-driven uveal melanoma cells treated with the Gq/11 inhibitor, demonstrating that the effects of this inhibitor were exquisitely dependent on the constitutively active Gq/11 oncogene. These results establish that Gq/11 is a druggable target in uveal melanoma cells, and show that Gq/11-mutant uveal melanoma cells are exquisitely sensitive to inhibition by small molecule inhibitors. We are currently transitioning these studies to animal models to establish drug efficacy and toxicity and explore treatment and delivery options.

#1351

Alternative splicing of neurofibromin 1 is associated with elevated MAPK activity and poor prognosis in glioma.

Robert Siddaway, Arun Ramani, Man Yu, Michael Brudno, Cynthia Hawkins. _The Hospital for Sick Children, Toronto, Ontario, Canada_.

High-grade gliomas (HGG) are invasive with poor prognosis regardless of age: diffuse intrinsic pontine gliomas (DIPG), arising in the brainstem, are almost universally fatal and the leading cause of brain-tumor death in children; while adult anaplastic astrocytoma and glioblastoma multiforme (GBM) have median survivals of 1-3 years. The mutational spectra of adult and pediatric HGG differ, with pediatric tumours containing recurrent mutations of H3F3A and HIST1H3B. However, alterations leading to RAS/MAPK/PI3K pathway activation, including PDGFRA amplification, EGFRvIII, BRAF-V600E, NF1 deletion, are frequently found, although not all tumours will have mutations in this pathway. The neurofibromin 1 (NF1) gene negatively regulates RAS signalling by stimulating RAS-GTP turnover, thereby leading to RAS-inactivation. The two major isoforms, NF1-I and NF1-II, differ only by inclusion of the 21 aa exon23a in the GAP-related domain of NF1-II. Exon23a-inclusion has been shown to render NF1 10 times less active towards RAS, leading to elevated MAPK signalling. The brain expresses predominantly NF1-I, while the major isoform elsewhere is NF1-II. Here we used RNA-Seq to identify genes alternatively spliced between DIPG and normal brain, identifying an isoform switch from NF1-I in normal brain to NF1-II; we additionally found the same isoform switch in the TCGA adult GBM and LGG cohorts. For both GBM and LGG, RAS/MAPK/PI3K wild-type tumors with elevated NF1-II conferred significantly reduced patient survival compared to RAS/MAPK/PI3K mutant tumors. NF1-exon23a inclusion is known to be repressed in cell model systems by the CELF and ELAV-like families of splice regulators. We further show that members of these gene families are downregulated in HGG. Together, our results indicate a novel mechanism by which gliomas can activate signaling downstream from RAS independent of mutations and tumor grade, which promotes tumorigenesis by regulating pathways such as proliferation and invasion.

#1352

The importance of the RASA1/R-Ras/Ral-A signaling axis in melanoma tumorigenesis.

Kristen Suzanne Hill, Xue Wang, Youngchul Kim, Minjung Kim. _Moffitt Cancer Ctr., Tampa, FL_.

The Ras family of small GTP binding proteins are frequently activated by mutations in melanoma, as shown for NRAS (20%), KRAS (2%) and HRAS (1%). Ras isoforms can also be activated by inactivation of Ras GTPase activating proteins (RasGAPs), such as NF1, RASA1, and RASA2. In our recent study, we observed that inactivation of RASA1 (RAS p21 protein activator 1, also called p120RasGAP) suppressed melanoma via its RasGAP activity toward the R-Ras (related RAS viral (r-ras) oncogene homolog) isoform and that R-Ras was required to promote anchorage-independent growth driven by RASA1 inactivation. Moreover, a low level of RASA1 mRNA expression is associated with decreased overall survival in melanoma patients with BRAF mutations. Based on these observations, we hypothesized that, although not mutated, R-Ras is activated in melanoma by inactivation of RasGAPs and that BRAF activation cooperates with this RasGAP/R-Ras pathway activation in melanoma tumorigenesis. In this study, we addressed the importance of R-Ras, a previously less appreciated member of the Ras small GTPases family, in melanoma tumorigenesis. We observed frequent activation of R-Ras in BRAF mutant human melanoma cell lines. In addition, RNAi-mediated reduced expression of R-Ras suppressed anchorage-independent colony growth and tumor growth. Moreover, among the 3 major RAS effector pathways, reduced R-Ras expression suppressed Ral-A activation, which may explain the mechanism of Ral-A activation in BRAF mutant melanoma. Interestingly, anchorage-independent growth driven by R-Ras activation downstream of RASA1 inactivation was suppressed by both genetic (siRNA targeting Ral-A) and pharmacological (Ral inhibitor BQU57) inhibition of Ral-A. To further investigate the impact of RASA1 loss, and thus R-Ras activation, on BRAF mutant melanoma development in vivo, we generated a RASA1L/L; BRAFCA/CA; Tyr-CreERT2 mouse model in which treatment with 4OHT results in expression of constitutively activated mutant BRAF and deletion of RASA1 in melanocytic lineage cells. Preliminary analysis shows hyperpigmentation of the ear, tail, and foot pad in RASA1L/L BRAFCA/CA mice compared to RASA1+/+ BRAFCA/CA littermates. Tumors generated in this animal model will be analyzed to determine the extent of R-Ras and Ral-A activity in vivo. This study demonstrates the importance of the RASA1/R-Ras/Ral-A signaling pathway in BRAF mutant melanoma and supports the possible combinatorial treatment strategy targeting both the BRAF/MAPK and Ral signaling pathways.

#1353

Polyisoprenylated cysteinyl amide inhibitors disrupt actin cytoskeleton organization, induce cell rounding, and block invasion of NSCLC.

Elizabeth Ntantie, Jerrine Fletcher, Felix Amissah, Olufisayo O. Salako, Augustine T. Nkembo, Rosemary A. Poku, Nazarius S. Lamango. _Florida A &M University, Tallahassee, FL_.

Non-small Cell Lung Cancer (NSCLC) malignancy is dependent on cellular processes that promote metastasis. F-actin organization is central to cell migration, invasion, adhesion and angiogenesis which are all processes involved in metastasis. F-actin remodeling is enhanced by the overexpression and/or hyper-activation of some members of the Rho family of small GTPases. Therefore agents that mitigate hyperactive Rho proteins may be clinically relevant for controlling metastasis. We previously reported the synthesis and characterization of polyisoprenylated cysteinyl amide inhibitors (PCAIs) as potential inhibitors of the cancer phenotype. In this report, we investigate the potential role of PCAIs against NSCLC malignancy and show that as low as 0.5 µM PCAIs significantly inhibit 2D and 3D NCI-H1299 cell migration by 48% and 45%, respectively. PCAIs at 1 µM inhibited 2D and 3D NCI-H1299 cell invasion through Matrigel by 50% and 85%, respectively. Additionally, exposure to 5 µM of the PCAIs, NSL-BA-040 caused a 38% drop in F-actin intensity at the cell membrane 24 h post treatment. Importantly, this drop in F-actin was accompanied by a 73% reduction in the number of filopodia per cell. Interestingly, the polyisoprenyl group of the PCAIs is essential for these effects, as NSL-100, an analog that lacks the farnesyl moiety, does not elicit similar effects on F-actin assembly and organization. Put together, our findings indicate that PCAIs disrupt F-actin assembly and organization to suppress cell motility and invasion and point towards a potential role of PCAIs as effective therapies for NSCLC metastasis and invasion.

#1354

The role of RALA in soft tissue sarcoma tumor growth and metastasis.

Steven T. Sizemore,1 Gina M. Sizemore,1 Reena Shakya,1 Peter Amaya,1 Anisha M. Hammer,1 Alexander H. Rice,2 Jeffrey J. Chalmers,1 Michael C. Ostrowski,1 Arnab Chakravarti1. 1 _The Ohio State University, Columbus, OH;_ 2 _Denison University, Granville, OH_.

Soft tissue sarcomas (STS) are a diverse collection of cancers of mesenchymal origin arising from the connective and supportive tissues of the body. While localized STS are well managed by surgery and radiation; metastasis, particularly to the lung, is frequent. More than 30% of adult STS patients develop lung metastases and the 5-year survival for these patients is a dismal 16%. Treatment options for metastatic STS are limited, thus there is an urgent unmet need for a better understanding of the key molecular pathways that drive metastatic spread in STS and identification of inhibitors of these pathways for clinical application. Through analysis of gene expression data from metastatic STS patient samples, we identified decreased expression of PPP2R1B as a hallmark of metastatic STS. To directly test its function as a suppressor of tumor growth and metastasis in STS, PPP2R1B was stably over-expressed in HT1080 cells, a model of metastatic STS. PPP2R1B expression almost completely abolished HT1080 tumor growth in nude mice. PPP2R1B is a subunit of the PP2A protein phosphatase complex that negatively regulates numerous cancer signaling pathways. However, the functional consequences of decreased PPP2R1B expression in STS are unknown. A combination of high-throughput and targeted approaches were utilized to identify 37 phosphoproteins that are significantly dephosphorylated following PPP2R1B expression in HT1080 cells. One of these phosphoproteins, the small GTPase RALA, exhibited decreased phosphorylation on Ser194 following PPP2R1B expression. RALA is significantly prognostic of STS metastasis and is elevated in more aggressive STS subtypes relative to less aggressive subtypes and normal tissue. RALA knockdown in HT1080 significantly slowed tumor growth and decreased the incidence of pulmonary metastasis, mirroring PPP2R1B overexpression. Importantly, RALA is an actionable therapeutic target for improved treatment of STS. Aurora A inhibitors indirectly inhibit RALA function by preventing RALA Ser194 phosphorylation by aurora A. We found that RALA expression and activity predicted response of STS cell lines to aurora A inhibition. Excitingly, the aurora A inhibitor alisertib nearly eradicated growth of HT1080 tumors in vivo. Exploration of the biological mechanisms through which RALA regulates STS metastasis identified regulation of vesicular traffic as a likely critical function of RALA in this process. These findings identify PPP2R1B, RALA, and aurora A as members of a key molecular pathway that drives STS progression and advocate the use of treatments targeting this pathway to improve outcome for STS patients with advanced disease.

#1355

Linking AP/MS-derived protein-protein interaction networks and pairwise sgRNA screens defines new KRAS effectors in non-small cell lung cancer.

Marcus R. Kelly. _Stanford University, Stanford, CA_.

The KRAS oncogene is mutated in over one fourth of human cancers. Despite decades of work studying known effector pathways, no drugs in clinical use specifically target KRAS-mutant tumors. Uncharacterized feedback mechanisms and parallel pathways have stymied the treatment of KRAS-mutant tumors in patients, and the KRas protein does not easily accommodate the binding of inhibitory small molecules. These challenges demand better characterization of the physical and genetic relationships between Ras regulators and effectors. To that end, we used tandem affinity purification of Kras, Hras and Nras and twelve proteins with known regulatory or effector roles to generate a high-confidence protein-protein interaction (PPI). Interactions were cross-referenced with public PPI, genetic susceptibility, and patient data to assemble a physical PPI map annotated with important functional determinants. The final map contains 287 genes and 990 physical interactions among them. This map was used to construct a library of >1000 sgRNAs covering >100 genes of probable relevance to the Ras pathway. This library was screened for pairwise genetic effects in a dual sgRNA vector system to identify quantitative changes in the growth of the A549 NSCLC line. We will present the quantitative results of this screen and additional validation studies on several previously undescribed regulators and effectors of the Ras proteins. In particular, we highlight unknown interactions between KRAS and the cell adhesion regulator RADIL, and the specificity of Ras-family protein interaction with the Raf and Ral GEF family proteins. These data underscore the limitations of our current understanding of Kras-driven cancers and reveal their genetic vulnerabilities.

#1356

Variable drug responses characterize the functional heterogeneity of Nf1 null tumors.

Daniela Pucciarelli, Ganesh Krishnamurthi, Steve Braunstein, Jean L. Nakamura. _UCSF, San Francisco, CA_.

Introduction: Neurofibromatosis type 1 (NF1) is a autosomal dominant disease with a predisposition to cancer. Biallelic inactivation of the NF1 gene increases risk of developing brain tumors, leukemia, neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs). Somatic mutations in the NF1 gene are also associated with sporadic malignancies including glioblastoma, neuroblastoma, and melanoma. The NF1 tumor suppressor gene encodes the RAS GTPase-activating protein (GAP) neurofibromin. Loss of NF1 results hyperactivation of Ras, MAPK and PI3K signaling pathways components, representing potential therapeutic targets. However, the variable response of NF1 mutant tumors to MAPK and mTOR inhibitors suggests the intrinsic heterogeneity of NF1 mutant tumors. We hypothesized that defining alternative mechanisms and functional sub-classes of Nf1-mutant tumors on the basis of their variable drug sensitivity will produce pre-clinical therapeutics data that will inform clinical trials.

Methods: We previously generated mouse models in which we mutagenized Nf1 heterozygous mice with radiation, recapitulating the susceptibility of patients with NF1 to radiation-induced cancers. These models produced solid tumors such as mammary carcinomas, squamous cell carcinomas, and soft tissue sarcomas, which we determined to be Nf1 null. Cell lines established from these tumors were characterized by a drug sensitivity screen using a custom 94 compound drug library. Drug responses indicate that Nf1 mutant tumor cell lines organize into functional groups sharing similar drug sensitivities. Six candidate drugs each targeting distinct components of MAPK and PI3K signaling pathways were selected from the drug library. After 24 hours of exposure to each inhibitor or control, cells were analyzed for cell proliferation, cell cycle changes, and cell death. Western blotting were performed to determine whether drug exposures produce alterations in their predicted biochemical pathways such as PI3K/Akt, and MAPK pathways.

Results: Nf1 loss did not predict uniform sensitivity of cell lines to treatment with MAPK and mTOR inhibitors. Each compound induced differential effects on viability of Nf1 null tumors cell lines. High variability and cell line-dependent cytotoxic and cytostatic effects were also observed. Although phosphorylated Akt(S473), S6, and p44/42 MAPK varied widely among all untreated Nf1 null cell lines, this did not predict their drug sensitivity. In vitro drug sensitivity data indicates heterogeneous differential sensitivities of Nf1 mutant tumor cells to different drug classes, independent of tumor histology, permitting segregation into functional groups.

Conclusion: Tumor cell lines driven by Nf1 loss demonstrate heterogeneous responses to Ras pathway inhibition, which may be explained by mechanisms of tumor formation after Nf1 loss involve multiple alternative pathways.

#1357

The small GTPase Arf6 potentiates melanoma metastasis by activating Akt.

Lehi Acosta,1 Aaron Rogers,1 Jae H. Yoo,1 Shannon J. Odelberg,1 Dean Y. Li,1 Sheri Holmen,2 Allie H. Grossmann1. 1 _University of Utah, Salt Lake City, UT;_ 2 _Hunstman Cancer Institute, Salt Lake City, UT_.

Arf6 is a member of the Ras-superfamily of small GTPases and controls membrane trafficking and cytoskeletal remodeling, functioning mainly in endocytosis pathways at the cell periphery. Arf6 is activated by various extracellular signals and oncogenic events and has been shown to promote cell migration and pro-invasive phenotype in human cancer cells. Small molecule inhibition of ARF6 reduces spontaneous metastasis in xenograft models of human cutaneous melanoma, suggesting that ARF6 is necessary for disease progression. Using a genetically-engineered mouse model of BRAF-mutant melanoma, we determined whether activation of Arf6 is sufficient to induce spontaneous metastasis in vivo. For melanocyte-specific primary tumor induction, Cre recombinase was delivered via local injection of RCAS virus into DCT-TVA;Cdkn2alox/lox;BRAFV600E mice. In the experimental mice, a constitutively active mutant form of Arf6 (Q67L) was virally delivered with Cre. In this study, we observed a significant increase in spontaneous metastatic disease burden in Arf6 Q67L mice. Likewise, tail vein injection of melanoma cell lines derived from Arf6 Q67L tumors consistently show a diffuse pattern of pulmonary metastasis compared to controls that show rare, microscopic metastasis. Recently, it has been demonstrated that activation of Akt, but not Pten loss, leads to an aggressive phenotype in melanoma that includes the acquisition of brain metastases. Immunohistochemical analysis of phospho-Akt revealed that Arf6 Q67L is sufficient to induce Akt activation in tumors. In addition, ARF6 is necessary for AKT activation in human melanoma lines. We did not observe brain metastasis in DCT-TVA;Cdkn2alox/lox;BRAFV600E \+ Arf6 Q67L mice. When we added Ptenlox/lox allele to this genetic background, however, we observed microscopic brain metastases at a low frequency, suggesting that the combination of Pten loss and Arf6 activation reaches a threshold level of Akt activation that is sufficient to cause brain metastasis. Taken together our data indicate that activation of Arf6 is sufficient to potentiate melanoma metastasis, consistent with the proinvasive cellular phenotype attributed to Arf6. In addition, our data suggests a novel signaling mechanism by which Arf6, a small GTPase involved in trafficking, is somehow involved in Akt activation and that this step may be important for the acquisition of metastatic capacity.

#1358

p21 activated kinase 4 (pak4) as a novel therapeutic target for non-hodgkin's lymphoma.

Asfar S. Azmi,1 Amro Aboukameel,1 Irfana Muqbil,1 Yiwei Li,1 William Senapedis,2 Erkan Baloglu,2 Yosef Landesman,2 Michael Kauffman,2 Sharon Shacham,2 Ayad Al-Katib,1 Ramzi M. Mohammad1. 1 _Wayne State Univ., Detroit, MI;_ 2 _Karyopharm Therapeutics, Newton, MA_.

Objective: The p21-activated kinase 4 (PAK4) is a key downstream effector of the Rho GTPase family and is over-expressed in many different cancer types. PAK4 protein, by virtue of its ability to engage multiple ligands, regulates a repertoire of signaling pathways. A survey of non-Hodgkin's lymphoma (NHL) cell lines shows that there is increase in PAK4 mRNA and/or protein expression when compared to normal peripheral lymphocytes (PBL). Considering PAK4 RNA interference suppresses lymphoma cell proliferation, these findings point to a novel role for PAK4 in promoting NHL cell growth. To this end we examined the impact of the newly developed PAK4 allosteric modulators (PAMs) on NHL proliferation both in vitro and in vivo.

Methods: WSU-FSCCL (representing follicular small cell cleaved lymphoma) and WSU-DLCL2 (diffused large B-cell lymphoma) were exposed to increasing concentrations of different PAM analogs (KPT-7523, KPT-7189, KPT-9037, KPT-9274, or KPT-7010 [inactive]) or the Pan-PAK inhibitor, PF-3758309, in the presence or absence of CHOP (used at IC25) for 72 hrs. Following combination treatment viability was evaluated using Trypan Blue, apoptosis was analyzed using 7AAD, tetrachrome staining, Annexin V FITC and cell cycle arrest was accessed by flow cytometry. Protein and mRNA expression changes were evaluated using immunoblotting and RT-PCR. The toxicity and efficacy of PAMs were evaluated in sub-cutaneous and disseminated xenograft models of NHL.

Results: As single agents, PAMs show anti-proliferative activity in vitro against NHL cell lines (IC50s for: WSU-FSCCL = 50 nM and WSU-DLCL2 = 250 nM) while sparing normal PBL (IC50s in μM range). There was a statistically significant dose-dependent difference in apoptosis induction in NHL cell lines treated with PAMs when compared to vehicle control. PAMs reduced total p-PAK4 and downstream signaling proteins involved in proliferation and apoptosis. In R-CHOP combination studies we observed enhanced viability suppression, increased apoptosis, and concurrent down-regulation of PAK4 signaling pathway proteins when compared to any single agent alone. The clinical compound, KPT-9274, is well tolerated and showed remarkable anti-tumor activity in WSU-DLCL2 sub-cutaneous xenograft in mice (p < 0.01 at 140 mg/kg/bid for 4 weeks with no loss in body weight). Residual tumors analysis showed suppression of PAK4 signaling pathways. Single agent and R-CHOP combination efficacy is currently being evaluated in subcutaneous and systemic WSU-FSCCL and in primary patient derived xenografts in mice.

Conclusions: This is the first study demonstrating a role for PAK4 in diffused large B-cell and follicular small cell cleaved NHL. Our data shows that inhibition of PAK4 could become a viable therapy for NHL either alone or in combination with R-CHOP. Our data is directly applicable to the current Phase 1 trial of KPT-9274 in patients with advanced solid malignancies or NHL.

#1359

How does RAC1 GTP-ase signal Wnt-beta-catenin pathway-mediated integrin-directed metastasis-associated tumor cell phenotypes in TNBC.

Nandini Dey, Jennifer H. Carlson, Casey Williams, Tyler Jepperson, Pradip De, Brian Leyland-Jones. _Avera Research Inst., Sioux Falls, SD_.

Introduction: We reported that Wnt Pathway (WP) upregulation, one of the salient genetic features of metastatic TNBC, controls the acquisition of integrin-directed metastasis-associated (ID-MA) phenotypes of TNBC cells (Dey et al., 2013). RAC-GTPases, small G-proteins which transduce signals from cell surface proteins including integrins, have been implicated in tumorigenesis and metastasis by their role in essential cellular functions like motility. Aim: Here we tested the role of the mechanism of involvement of RAC1 in the regulation of WP-mediated ID-MA phenotypes in TNBC cells and examined how WP signals are transduced via RAC1 in the context of ID-MA phenotypes in TNBC.

Method: Oncoprints showed that the major alterations of RAC1 gene in TNBC were constituted by the amplification/gain of the gene. The collective percentage of alteration(s) in RAC1 in ER+ve BC was 35% as compared to 57% in ER-ve BC (brca/tcga/pub2015). Using pharmacological agents (sulindac sulfide), genetic tools (siRNA), WP modulators (Wnt-C59, XAV939), RAC1 inhibitors (NSC23766, W56) and WP stimulations (LWnt3ACM, Wnt3A recombinant) in a panel of 6-7 TNBC cell lines, we studied fibronectin-directed migration and various motility parameters (actin dynamics, filopodia and lamellipodia by confocal microscopy) in the context of fibronectin-directed RAC1 and Cdc42 activation.

Result: Breaking down by molecular subtypes of BC, RAC1 gene alterations are observed in 20% in PAM50 Luminal A, 35% in PAM50 Luminal B, 37% in PAM50 HER2 enriched and 40% in PAM50 Basal-like. For the samples set of TCGA, Cell 2015, RAC1 gene was altered in 42% total while it was altered 37% in PAM50 Luminal A, 57% in PAM50 Luminal B, 55% in PAM50 HER2 enriched and 60% in PAM50 basal-like. An attenuation of WP, which (a) decreased cellular levels of beta-catenin, as well as its nuclear active-form, (b) decreased fibronectin-induced migration, (c) altered actin dynamics and (d) decreased podia-parameters was successful in blocking fibronectin-mediated RAC1/Cdc42 activity. Both Wnt-antagonists and RAC1 inhibitors blocked fibronectin-induced RAC1 activation and inhibited the fibronectin-induced ID-MA phenotypes following specific WP stimulation. To test a direct involvement of RAC1-activation in WP-mediated ID-MA phenotypes, we stimulated brain-metastasis specific MDA-MB231BR cells with LWnt3ACM. LWnt3ACM-stimulated fibronectin-directed migration was blocked by RAC1 inhibition in MDA-MB231BR cells. In the light of our previous report (De et al., 2016) that WP upregulation stimulates ID-MA phenotypes in TNBC tumor cells, here we provide the first mechanism based evidence to demonstrate that WP upregulation signals ID-MA tumor cell phenotypes in an RAC1-GTPase dependent manner involving exchange-factors like TIAM1 and VAV2.

#1360

Novel function of p21-activated kinase 3 (PAK3) in regulating Akt phosphorylation and pancreatic cancer stem cell phenotypes.

Hsing-Yu Wu,1 Ming-Chen Yang,2 Po-Chen Chu,1 Samuel K. Kulp,2 Ching-Shih Chen1. 1 _Academia Sinica, Taipei, Taiwan;_ 2 _The Ohio State University, Columbus, OH_.

p21-activated kinases (PAKs) are important effectors of the Rho family GTPases and has been implicated in cytoskelestal remodeling, cell proliferation, apoptosis, and transformation. Based on the sequence, structure homology, and activation mechanism, six PAKs are classified into two groups, PAK 1-3 (group I) and PAK 4-6 (group II). PAK kinases are frequently overexpressed in various human tumors and represent therapeutically relevant targets for cancer treatments. Previous studies have shown that PAK1 and PAK4 are upregulated and/or hyperactivated in pancreatic cancer, and promotes pancreatic cancer cell motility and invasion. In our study, we showed that knockdown of PAK3, but not that of PAK1 or PAK2, inhibited pancreatic cancer cell proliferation in vitro, and tumor growth in vivo. In addition, our data showed that PAK3 regulated the protein stability of β-catenin via Akt/GSK-3β signaling pathway in pancreatic cancer cells. The role of PAK3 in regulating Akt/GSK-3β phosphorylation was further confirmed by the ectopic expression of wild-type versus kinase-dead (K297L) PAK3. Equally important, the mammosphere formation, aldehyde dehydrogenase (ALDH) activity and cancer stem cell-associated markers, were also down-regulated in PAK3 knockdown cells, suggesting the involvement of PAK3 in regulating cancer stem cell-like properties in pancreatic cancer cells. Together, these findings suggested that PAK3 as a primary regulator of Akt/GSK-3β/β-catenin signaling for maintaining cancer stem cell phenotypes and promoting tumor growth, which underlies the potential of targeting PAK3 in fostering new therapeutic strategies for pancreatic cancer.

#1361

Characterization of TpeL, a RAS specific clostridial toxin.

Maria T. Abreu-Blanco. _Cancer Research Technology Program, Leidos Biomedical Research, Inc, Frederick, MD_.

Clostridium perfringes lethal toxin (TpeL) belongs to the family of large clostridial glycosylating cytotoxins. Clostridial toxins are glycosyltransferases that modify and deactivate small GTPases of the RHO and RAS subfamily. TpeL mono-glycosylates in the switch I domain (Thr35) of RAC and RAS small GTPases. Glycosylation of RAS at Thr35 prevents binding to the primary effector, RAF kinase and results in a blockade RAS signal transduction. RAS family proteins function as key regulators of cell proliferation, differentiation, survival and gene expression. Moreover, mutations in RAS proteins are highly prevalent in human cancers. Considering the specificity of TpeL for RAS, we decided to investigate the biochemical interaction between TpeL and RAS, and potentially develop tools to disrupt the RAS signaling pathway. In order to assess the effect of TpeL in vivo, we use RAS-dependent MEFs expressing different KRAS mutations and BRAF V600E. We expect that proliferation should be inhibited in MEF cells expressing RAS isoforms, but MEF cells expressing BRAF V600E should not be affected by any toxin. We found that TpeL treatment did not affect the viability of the BRAF V600E cells and only induced toxicity in RAS expressing cells. Consistent with this result, TpeL treatment inhibited MAPK signal transduction in the RAS expressing cells, but not in the BRAF V600E cells. Surprisingly, both cell viability as well as pERK levels remained unaffected in the KRAS Q61R MEF cells treated with TpeL, suggesting that KRAS Q61R is resistant to glycosylation. This may be due in part to the relatively slow intrinsic rate of GTP hydrolysis and very high levels of KRAS-GTP in KRAS Q61R cells. We also developed and adapted biochemical assays to study TpeL-KRAS interaction in vitro. Based on Alpha Assay and UDP Glow, TpeL activity does not appear to be nucleotide specific, in contrast to previously reported findings. We are currently testing if TpeL RAS glycosylation is competitive with or is inhibited by other molecules that bind to switch I, such as the RBD domain of RAF1 or GEF/GAP proteins. These data may indicate that the lack of activity in KRAS Q61R expressing MEF cells is due not to the high levels of RAS-GTP, but to the constitutive binding to effector molecules, which prevent TpeL from accessing KRAS. We also hope to identify the critical residues required for TpeL binding and catalysis. By elucidating the mechanism of substrate binding and substrate specificity we may gain insight into novel binding pockets that could be exploited therapeutically.

#1362

Argonaute 2 controls RAS activation in mouse embryonic fibroblasts.

Ronald F. Siebenaler, Sunita Shankar, Vijaya L. Dommeti, Malay Mody, Arul Chinnaiyan. _University of Michigan Medical School, Ann Arbor, MI_.

The RAS gene family is among the most commonly mutated genes within cancer, and while much research has elucidated the major downstream pathways, including MAPK and PI3K, little progress has been made in successfully targeting mutant RAS in cancer. We recently identified an interaction between the N terminal domain of Argonaute 2 (AGO2), a core component of RNA-induced silencing complex (RISC), and the Switch II domain of KRAS. Furthermore, this interaction was found in all cell lines tested, expressing either wild-type (WT) or mutant KRAS. We found that stable knockdown of AGO2 in KRAS dependent cell lines lead to a decrease in KRAS protein expression with a subsequent decrease in cellular proliferation. Conversely, the overexpression of AGO2 in these cells lead to both an increase in KRAS expression and oncogenesis. In addition, this interaction inhibits the RNAi function of AGO2 by preventing microRNA unwinding in the presence of oncogenic KRAS compared to WT-KRAS. Despite a clear association between mutant KRAS and AGO2 mediating increased KRAS mediated oncogenesis, the precise function of this interaction remains unclear in normal physiology. In order to better assess the endogenous function of the KRAS-AGO2 interaction, we analyzed two mouse embryonic fibroblast cell lines (NIH 3T3 and MEF) with complete knockout of AGO2. Utilizing a Raf-1 RAS binding domain (RBD) pulldown method, we assessed activated WT-RAS levels in AGO2 null NIH 3T3 and MEF cells. We found that knockout of AGO2 lead to an increase in WT RAS-GTP activation compared to normal control cells. Immunoblot analysis also indicates that AGO2 null fibroblasts lead to increase in RAS downstream signaling through the MAPK/ERK and PI3K/AKT pathways. Furthermore, rescue of AGO2 knockout using full length mouse AGO2 decreased wild type RAS activation and its downstream signaling. Taken together, these observations suggest that the AGO2 interaction may suppress WT-KRAS activation, leading to maintenance of RAS-GDP levels. Using RNA-seq, proteome and microRNA analysis, we have begun to identify the pathways that may be involved in RAS activation in AGO2 null cells. Early analyses indicate that AGO2 controls WT-KRAS levels and activity through multiple mechanisms, laying the foundation for a better understanding of the RAS-AGO2 interaction in normal physiology.

#1363

RSK2 provokes invasive signaling in glioblastoma through LARG-dependent activation of Rho GTPases.

Geng-Xian Shi,1 Ling Jin,1 Michelle L. Matter,1 Santosh Kesari,2 Joe W. Ramos1. 1 _Univ. of Hawaii Cancer Ctr., Honolulu, HI;_ 2 _John Wayne Cancer INstitute, Honolulu, HI_.

Malignant gliomas are one of the most aggressive and deadly forms of cancer and can affect any age group. In glioblastoma multiforme (GBM), infiltration of primary tumor cells into the normal tissue and dispersal throughout the brain is a central challenge to successful treatment that remains unmet. Patients with malignant gliomas respond poorly to the standard therapeutic regimen of radiotherapy and chemotherapy that follow tumor resection and have only a 16-month median survival. It is therefore imperative to identify new approaches to specifically attack GBM cell survival, proliferation and invasion. The cellular mechanisms driving GBM-mediated migration and invasion are not fully understood. RSK2 (p90 ribosomal S6 kinase 2) is a kinase that regulates proliferation and adhesion and can promote metastasis. We find that RSK2 is significantly upregulated in vivo in human GBM patient tumors, and that high RSK2 expression significantly correlates with advanced tumor stage and poor patient survival. We demonstrate that active RSK2 regulates GBM adhesion and is essential for cell motility and invasion of patient-derived GBM neurospheres. Importantly, inhibition of RSK2 by either RSK inhibitors or shRNA silencing impairs invasion and combining RSK2 inhibitors with temozolomide improves efficacy in vitro. We further show that the effects of RSK2 on GBM invasion are mediated in part through activation of Rho GTPases. Rho family of GTPases are key regulators of cell polarity, migration and invasion. Here, we identify Rho A, B and C as downstream effectors of (RSK2) in GBM cell migration and invasion. Here, we identified a unique signaling pathway in which RSK2 confers invasiveness through leukemia-associated RhoGEF (LARG)-dependent Rho GTPase activation. RSK2 directly interacts with LARG and nucleotide-bound Rho isoforms, but not Rac1 and Cdc42. Our data support a fundamental importance of residue of Thr577 to the invasive signaling of RSK2. The invasive signaling of RSK2 appears to depend on RhoA and B, but not RhoC. RSK2 activates RhoA through activation of LARG by phosphorylation at Ser1288. Intriguingly, LARG activity is only required for RSK2-T577E, but not RSK2-Y707A-mediated RhoA activation and cell invasion. This further supports a pivotal role of Thr577 in invasive RSK2 signaling. These results establish a unique RSK2-dependent LARG-Rho signaling cascade as an uncompensated factor key to GBM cell migration and invasion. Together, our data provide strong evidence that RSK inhibitors could enhance the effectiveness of existing GBM treatment, and support RSK2 targeting as a promising approach for novel GBM therapy.

#1364

Direct phosphorylation and attenuation of the DLC1 tumor suppressor by SRC kinase: a new mechanism of SRC-dependent activation of Rho with translational implications.

Brajendra K. Tripathi,1 Xiaolan Qian,1 Ming Zhou,2 Dunrui Wang,1 Marian Durkin,1 Alex G. Papageorge,1 Douglas R. Lowy1. 1 _National Institutes of Health, Bethesda, MD;_ 2 _National Cancer Institute, Frederick National Laboratory for Cancer Research, Frederick, MD_.

Many tumors have high SRC activity and constitutive up-regulation of Rho-GTP, which is frequently implicated in the neoplastic process and tumor progression to metastasis, but the mechanistic link is unclear. Here we identify the DLC1 tumor suppressor, which encodes a RhoGAP, as a previously unrecognized direct target of the SRC kinase, in untransformed cells and in tumor-derived cell lines, which upregulates Rho-GTP. SRC kinase directly phosphorylates two Tyrosines in DLC1: Y451 and Y701. SRC phosphorylation of DLC1-Y701, which is located in the DLC1 RhoGAP domain, attenuates its RhoGAP activity and tumor suppressor function by inhibiting the binding of active Rho-GTP to the RhoGAP domain, reducing the hydrolysis of active Rho-GTP to inactive Rho-GDP and resulting in an increase in Rho-GTP. The role of SRC phosphorylation of DLC1-Y451 is to diminish DLC1 binding to tensin, which further reduces the tumor suppressor functions, as measured by cell migration rate, anchorage-independent growth, and tumor formation in nude mice, independently of Rho-GTP. We also found that ERK1 cooperates with SRC by phosphorylating DLC1-S129, which enhances the binding of the SRC SH3 domain to this region of DLC1, and increases SRC-dependent phosphorylation of Y451 and Y701. The cooperative effects reduce RhoGAP activity, increase Rho-GTP and Rho/ROCK/MRLC signaling, and attenuate DLC1 tumor suppressor functions. DLC1 preferentially binds active SRC, which reduces overall SRC activity. Therefore, SRC activity tends to be lower in cells that express high levels of endogenous DLC1, compared with cells in which DLC1 expression has been down-regulated. Although SRC targets two DLC1 functions that contribute to its tumor suppressor activity, DLC1 binds several ligands in addition to tensin that also contribute to its tumor suppressor functions. Therefore, it would not be surprising if high SRC activity were associated with selective pressure for down-regulation of DLC1 expression. Consistent with this hypothesis, the combination high SRC expression and low DLC1 expression was associated with a poor prognosis in lung adenocarcinomas (p=0.005) in TCGA. The potentially reversible nature of the SRC-induced attenuation of DLC1 function can be exploited therapeutically. In two systems that have high SRC activity and express DLC1 - the mouse MMTV-PyMT cancer model and a human lung cancer cell line - the Src inhibitor PP1 has potent antitumor activity, as it reduces DLC1 Tyrosine phosphorylation, reactivates DLC1, and reduces Rho-GTP. However, PP1 has marginal antitumor activity an isogenic DLC1-negative strain of the lung cancer line, as the inhibitor does not reactivate DLC1 and does not alter Rho-GTP levels. The results highlight the potential importance of tumor suppressor reactivation as a biomarker for predicting and monitoring the response to SRC inhibitors.

#1365

Sensitivity of oncogenic KRAS to adenosine triphosphate suppression.

Matthew Holderfield, Kanika Sharma. _Frederick National Laboratory for Cancer Research, Frederick, MD_.

KRAS proteins regulate many cellular processes including initiation of cell division in response to mitogens. Gain of function KRAS mutations appear in approximately 30% of all human cancers and activate effector signal transduction pathways independent of mitogenic stimuli. KRAS driven cancers also frequently have high metabolic rates and may have increased sensitivity to metabolic inhibitors but the underlying mechanism is not fully understood. In this study, we characterized the cellular mechanism of a small molecule inhibitor identified using a transgenic Drosophila melanogaster model that expresses human KRAS-G12V in the fly wing. The compound partially restored wing development in the KRAS-G12V flies and inhibited growth and signal transduction in multiple KRAS driven cancer cells and KRAS-G12V dependent MEF cells but not in MEF cells expressing BRAF-V600E. The compound also had a profound impact on mitochondrial function by inhibiting electron transport (ETC) complex I. Multiple other ETC or ATP synthase inhibitors similarly impaired oncogenic KRAS signal transduction. ATP production through glucose metabolism was sufficient to rescue KRAS function and cellular growth after ETC inhibition, suggesting that inhibition of MAPK signaling is due to low ATP levels rather than a direct consequence of ETC or complex I inhibition. These data indicate that oncogenic KRAS signal transduction requires high cellular ATP levels and suggests a synthetic lethal interaction between KRAS and multiple metabolic targets that could be exploited to target KRAS driven cancers.

#1366

Structural basis of impaired GTP hydrolysis in oncogenic mutants of KRAS.

Timothy Tran,1 Sathiya Dharmaiah,1 Oleg Chertov,1 Timothy Waybright,1 William Gillette,1 Dominic Esposito,1 Dwight Nissley,1 Frank McCormick,2 Andrew Stephen,1 Dhirendra Simanshu1. 1 _NCI RAS Initiative, Frederick National Laboratory for Cancer Research, Frederick, MD;_ 2 _Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA_.

RAS mutations are found in one-third of all human cancers. Among the three RAS isoforms - HRAS, KRAS and NRAS, KRAS is the most commonly mutated gene (in 86% of RAS-driven cancers) and the mutations are often detected in pancreatic, colorectal and lung cancers. RAS proteins function as molecular switches by alternating between inactive GDP-bound and active GTP-bound states. The active or inactive state of RAS proteins is regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). In the GTP-bound state, RAS proteins interact with a variety of effector proteins, such as Raf, PI3K, and RalGDS, leading to activation of several signaling cascades within the cell. In 98% of the cases, oncogenic RAS mutations are found at amino acid positions G12, G13, and Q61 which impair intrinsic and GAP-mediated GTPase function resulting in accumulation of constitutively GTP-bound RAS in cells. To gain insights into the effect of oncogenic mutations on overall structure and GTP hydrolysis, we solved high-resolution crystal structures of wild type and six oncogenic mutants (G12C, G12D, G12V, G13D, Q61L and Q61R) of KRAS4b in complex with GMPPNP (a non-hydrolysable GTP analog) and magnesium. Comparison of GDP and GMPPNP bound structures of wild type (WT) KRAS4b suggests conformational changes that occur when KRAS4b transitions from inactive to active state. Comparison of mutant and wild-type KRAS structures has shown the appearance of new pockets in some cases that could be exploited for structure-based drug design. Structural superposition of mutants vs. wild type KRAS4b in complex with GMPPNP/Mg2+, and KRAS4b mutants vs. WT-HRAS bound to RASA1-GAP provides a rationale for impaired intrinsic and GAP-mediated GTP hydrolysis in the KRAS mutants.

#1367

The dynamic mechanism of SARAH domain in RASSF5 activation by K-Ras4B.

Tsung-Jen Liao,1 Hyunbum Jang,2 Chung-Jung Tsai,2 David Fushman,1 Ruth Nussinov2. 1 _University of Maryland, College Park, MD;_ 2 _National Cancer Institute at Frederick, NIH, Frederick, MD_.

RASSF5 is a tumor suppressor, which acts as an adaptor linking Ras and the Hippo pathways. RASSF5 activates MST1/2, a key upstream protein of the Hippo pathway, through SARAH domain heterodimerization, resulting in Hippo signaling. Signaling stimulates YAP1 phosphorylation, leading to its degradation and thereby cell apoptosis. Association with K-Ras4B promotes RASSF5 to release its SARAH which heterodimerizes with MST1/2. We aim to reveal how K-Ras4B regulates the Hippo pathway through RASSF5 by resolving the structure and conformational dynamics of RASSF5 complexes. Our modeling and simulations help elucidate the intrinsic dynamic interactions of RASSF5 RA (Ras binding) and SARAH domains. Surprisingly, we found that the residue E388 on RASSF5 SARAH plays a significant role in the dynamic mechanism of SARAH homo-/hetero- dimerization and the RA-SARAH association. The mutants E388A and E388K dramatically decrease the strength of RASSF5 homo-SARAH and RASSF5-MST2 hetero-SARAH, suggesting that the E388 mutation may be oncogenic. Simulations of selected RASSF5 configurations with GTP-bound K-Ras4B indicate that the interactions between RA and SARAH are reduced. We conclude that Ras binding weakens the RA-SARAH association, increasing the SARAH dynamic fluctuation. The released RASSF5 SARAH has high propensity to dimerize with MST1/2 SARAH, activating Hippo signaling thus cancer suppression. Funded in whole or in part with Federal funds from the Frederick National Laboratory for Cancer Research, NIH under contract HHSN261200800001E.

#1368

RABL6A, a novel critical regulator of Akt-mTOR signaling in pancreatic neuroendocrine tumor cells.

Shaik Amjad Ume Salma, Jussara Hagen, Jacki Reilly, Ryan Sheehy, Nitija Tiwari, Jackson Nteeba, Scott K. Sherman, Thomas M. O'Dorisio, James R. Howe, Andrew M. Bellizzi, Benjamin W. Darbro, Dawn E. Quelle. _Univ. of Iowa College of Medicine, Iowa City, IA_.

Introduction: A better molecular understanding of pancreatic neuroendocrine tumors (PNETs) is needed to improve patient diagnosis and treatment. Everolimus (mTOR inhibitor) is a standard-of-care therapy for PNET patients based on aberrant activation of the PI3K/Akt/mTOR kinase pathway in tumors. However, sustained mTOR inhibition paradoxically promotes Akt kinase hyperactivation due to loss of negative feedback regulation and tumors become drug resistant. Our data reveal that RABL6A, a novel oncoprotein amplified in PNETs, is a key regulator of this clinically relevant pathway.

Methods: RABL6A and Akt protein levels were manipulated using viral shRNAs in BON1 PNET cells. Transcript levels were assayed by microarray and qRT-PCR, proteins assessed by western blotting, and cell proliferation and survival measured by cell counts, trypan blue exclusion and EdU incorporation. Effect of RABL6A expression on sensitivity to clinically relevant drugs, MK2206 (Akt inhibitor) and everolimus, were tested.

Results: Silencing of RABL6A in PNET cells causes G1 and G2/M cell cycle arrest, and pathway analysis of microarray data suggested inactivation of Akt signaling in the arrested cells. Immunoblotting confirmed dramatic loss of Akt phosphorylation at Ser-473 along with impaired phosphorylation and activation of its targets, PRAS40 and FOXO-1/3. Phosphorylation of S6K, a downstream target of Akt-mTOR signaling, was also reduced by RABL6A deficiency. The mechanism by which RABL6A controls Akt-S473 phosphorylation is currently not known although we demonstrated that mTORC2 (the kinase that phosphorylates Akt at Ser473) remains active in RABL6A deficient cells since the phosphorylation of other mTORC2 substrates (SGK1 and PKCα) is unaffected. Given the central role of Akt1 in tumorigenesis, we hypothesized that reinstating its activity may rescue the arrest phenotype caused by RABL6A loss. Restoration of Akt1 in RABL6A-depleted cells partially rescued the G1 phase arrest and induced S phase entry but was insufficient to allow mitosis, suggesting RABL6A regulates other factors required for cell division. Finally, drug response assays showed that RABL6A loss desensitizes PNET cells to Akt and mTOR inhibitors.

Conclusion: Our previous work showed RABL6A promotes G1 progression in PNET cells by inactivating Rb1, an established suppressor of PNET pathogenesis. We now show that RABL6A also controls Akt phosphorylation and is essential for Akt-mTOR activation. Thus, RABL6A controls multiple cancer pathways necessary for PNET cell cycle progression and survival. We are testing if RABL6A status in PNETs predicts responsiveness to combination therapies targeting Akt and mTOR. Overall, this work identifies RABL6A as a new essential activator of Akt1-mTOR signaling, suggesting it is a new potential biomarker and target for anticancer therapy in PNET patients.

#1369

DNA, protein, and cell line reagents to facilitate RAS pathway drug discovery efforts.

Dominic Esposito. _Frederick National Laboratory for Cancer Research, Frederick, MD_.

The RAS Reagents Core within the NCI RAS Initiative produces and distributes qualified DNA vectors, cell lines, and protein production reagents to the external community to facilitate research into RAS biology and development of RAS cancer therapeutics. DNA vectors representing a variety of RAS mutations, as well as a complete collection of 180 genes in the RAS pathway are available as Gateway-compatible Entry clones. These clones represent the most commonly expressed transcript forms of the 180 genes, many of which were not previously available in full-length, wildtype forms. The vectors can be used with the FNLCR combinatorial cloning library to easily generate a large number of different types of expression clones with various promoters, fusion tags, and plasmid backbones. Cell line reagents include a variety of Ras-dependent mouse embryonic fibroblasts (MEFs) which have a defined mutant allele of KRAS and which serve as isogenic cell lines for investigating allele and mutant specific RAS biology, as well as for valuable screening tools. Protein reagents include E. coli and insect cell constructs and cell lines for production of a variety of Ras-related proteins. In particular, we have materials for the high yield production of properly processed, prenylated KRAS protein using a unique engineered insect cell expression system. This system can also be used to produce other prenylated small GTPases at levels not previously attainable. Many of the resources of the RAS Reference Core are readily available to the academic community through repositories or material transfer agreements, and can be licensed to industry scientists as well. The program is also interested in identifying the needs of the community for additional RAS-related resources, and is developing methods for vetting requests from the outside to provide additional reagents.

#1370

EGFR-mediated Spred1 phosphorylation inhibits NF1 to sustain constitutive Ras/MAPK signaling.

Evan Markegard, Ellen L. Mercado, Jillian M. Silva, Jacqueline Galeas, Marena I. Trinidad, Anatoly Urisman, Frank McCormick. _UCSF, San Francisco, CA_.

Spred proteins negatively regulate Ras/MAPK signaling following growth factor stimulation. Inhibition of Ras primary occurs through Spreds ability to bind and localize NF1, a RasGAP and major tumor suppressor, to the plasma membrane. Spred1 and NF1 loss-of-function mutations occur across multiple cancer types including non-small cell lung carcinoma, glioblastoma, melanoma, stomach carcinoma, and uterine carcinosarcoma. Here we demonstrate that EGFR signaling disrupts Spred1-NF1 binding. Mass spectrometry was performed on cells overexpressing EGFRL858R to identify potential phosphorylation sites on Spred1 and NF1 that could disrupt Spred1-NF1 binding by steric hindrance. A serine phosphorylation site on Spred1 was identified in which a phosphomimetic and phosphodeficient mutant decreased or increased Spred1-NF1 binding, respectively. Phosphomimetic Spred1 is unable to suppress Ras-GTP following EGF stimulation. Therefore, phosphorylation of Spred1 at this site by a serine kinase downstream of EGFR may disrupt Spred1-NF1 binding. To identify the Spred1 kinase we are performing an in vitro kinase assay and an unbiased CRISPRa screen. Our findings provide one potential mechanism by which EGFR signaling disrupts negative feedback to sustain constitutive Ras signaling. Furthermore, this work may elucidate a novel therapeutic target for restoring NF1-mediated inhibition of Ras.

#1371

Isoprenylcysteine carboxylmethyltransferase (ICMT) function is critical for epithelial malignant transformation by mutant-Ras.

Hiu Yeung Lau, Jingyi Tang, Patrick J. Casey, Mei Wang. _Duke-NUS Medical School, Singapore, Singapore_.

Purpose Limited progress has been made in the development of direct inhibitors of Ras. Therefore, targeting isoprenylcysteine carboxylmethyltransferase (ICMT), the final enzyme in the prenylation pathway for Ras and other proteins containing the so-called CaaX-motif, remains an important strategy to inhibit Ras function in cancers. In this study, we investigate the role ICMT plays in the initiation of human epithelial cancers involving constitutively-active mutant Ras using genetic manipulation strategies.

Methods Small T antigen and a shRNA against p53 (shp53) were sequentially expressed in human mammary epithelial cells immortalized by hTERT (HME1-hTERT) to create pre-malignant HME1-shp53 cells. All 4 isoforms of wild-type or mutant Ras, namely H-Ras, N-Ras, K-Ras-4A and K-Ras-4B, were independently introduced into HME1-shp53 cells to determine their transforming ability. Using CRISPR-Cas9 genome editing technology, ICMT-null HME1-shp53 cells were generated and mutant Ras isoforms were subsequently introduced; tumor formation abilities of thus generated cells were determined using soft-agar anchorage-independent colony formation assay and subcutaneous xenografts in mice. ICMT knock-out was also performed in MiaPaCa-2 and MDA-MB-231 cells, both expressing mutant K-Ras, and subcutaneous xenograft tumor formation in mice was measured.

Results Pre-malignant HME1-shp53 cells can be transformed by all mutant Ras isoforms but not by any wild-type Ras. ICMT loss-of-function dramatically reduced anchorage-independent colony formation caused by all mutant Ras isoforms in HME1-shp53 cells, and in vivo xenograft formation was completely abolished. ICMT loss-of-function also dramatically reduced in vivo xenograft formation for mutant K-Ras-expressing MiaPaCa-2 and MDA-MB-231 naturally-occurring cancer cells.

Conclusion ICMT function is essential for the malignant transformation of HME1 cells by all isoforms of Ras. Furthermore, ICMT function is also crucial for the maintenance of the malignant phenotype in mutant K-Ras-expresing cells such as MiaPaCa-2 and MDA-MB-231 cells.

### Epigenetics 1

#1372

Towards novel strategies of targeting specific vulnerabilities of T-PLL cells.

Sabine Puetzer,1 Emma Andersson,2 Alexandra Schrader,1 Lesley Varghese,1 M Hülsemann,1 Petra Mayer,1 Marc-Henri Stern,3 Sebastian Newrzela,4 L Frenzel,1 Satu Mustjoki,2 Marco Herling1. 1 _Center for Integrated Oncology (CIO) Köln-Bonn, University of Cologne, Cologne, Germany;_ 2 _University of Helsinki and Department of Hematology, Helsinki, Finland;_ 3 _Institute Curie, Paris, France;_ 4 _Institute of Pathology University of Frankfurt/M, Frankfurt/M, Germany_.

T-cell prolymphocytic leukemia (T-PLL) is a mature T-cell neoplasm usually presenting at an aggressive phase and a marked resistance to conventional cytostatics, i.e. alkylators. The monoclonal antibody alemtuzumab induces high response rates, but virtually all patients relapse and the mostly elderly individuals are often ineligible for allogeneic stem cell transplantation. Overall, the treatment options for T-PLL are scarce and its general prognosis with average survival times of <3 yrs. remains poor. In agreement with the pronounced amenability of T-cell tumors to intervention in the altered epigenetic code, recent reports also implicate a high potential of epigenetic targeting in T-PLL. In fact, profiling of anti-nucleoside sensitivities, including clinically used agents like fludarabine or nelarabine, revealed that the purine analog cladribine showed the highest T-PLL specific activity (LD50s: 0.16µM in T-PLL; 2.95µM in healthy-donor T-cells, 2.81µM in PBMC), potentially through its epigenetically active pyrimidine function. Fittingly, high-throughput genomic analysis of 95 T-PLL revealed alterations enriched in DNA repair genes and histone / DNA epigenetic modifiers. Extensive ex vivo drug screens (306 substances, 39 T-PLL) corroborated the genomic data and identified major clusters of T-PLL-active substances. The CDK inhibitor SNS-032 induced the highest T-PLL specific responses, followed by p53 activators, BCL2-family antagonists, and modulators of protein acetylation and chromatin accessibility, all surpassing the effects of JAK/STAT-inhibitors. The BCL2 inhibitor ABT199 (Venetoclax) profoundly affected T-PLL cell viability (LD50=6.9nM), although apoptosis was induced more efficiently in CLL cells (LD50=2.4nM). This slightly lower cytotoxicity in T-PLL might be explained by the more heterogeneous BCL2 expression in this disease than in CLL (flow-cytometry and gene expression arrays; 70 T-PLL). The best-performing substance of the set, SNS-032, selectively and profoundly induced apoptosis in T-PLL (LD50=0.19µM vs 0.47µM in normal T-cells vs 0.88µM in PBMC). Recognized as a CDK inhibitor, it antagonizes transcription via targeting CDK7 and -9. We identified associations of protein levels of the transcription factor MYC with the in vitro responses to SNS-032. Amplified cMYC is one of the genomic hallmarks of T-PLL and implicated in its aggressive phenotype. Hence it may be a target of SNS-032 or serve as a biomarker for SNS-032 sensitivity.In summary, we identified several substances with marked anti-leukemic activity in T-PLL. In vitro studies confirmed their potency and selectivity that could be aligned with identified protein and genomic-lesional target patterns. Follow-up studies of members of the major clusters of most T-PLL-active compounds including their best-informed combinations and pre-clinical in-vivo testing will guide in the design of promising clinical trials in T-PLL.

#1373

The role of the ASH1L SET domain in leukemogenesis.

David Rogawski,1 Hongzhi Miao,1 Katarzyna Kempinska,1 Lili Chen,1 Jen Chase,1 Ann Friedman,1 Xiaomei Yan,2 Kenichi Nishioka,3 Gang Huang,2 Patricia Ernst,4 Andy Muntean,1 Ivan Maillard,1 Tomasz Cierpicki,1 Jolanta Grembecka1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _Cincinnati Children's, Cincinnati, OH;_ 3 _Saga University, Saga, Japan;_ 4 _University of Colorado Denver, Aurora, CO_.

ASH1L (absent, small, or homeotic-like 1) is a histone lysine methyltransferase (KMTase) specific for histone 3, lysine 36 that has an emerging role in multiple types of cancer. During development, ASH1L activates HOX genes and regulates self-renewal potential of long-term hematopoietic stem cells. Interestingly, HOX genes are oncogenic drivers in a variety of cancer types. For example, overexpression of HOXA9 is highly associated with a poor prognosis in acute myeloid leukemia (AML). In addition, ASH1L is overexpressed in a variety of cancers, including breast and thyroid cancer, but its role in cancer remains poorly characterized. Recent data suggest that ASH1L promotes leukemogenesis in mixed-lineage leukemia by recruiting the MLL epigenetic regulator to target genes. However, ASH1L is a large protein with multiple chromatin-interacting domains, and which regions of the ASH1L protein are important in leukemia has not been reported. Specifically, the role of ASH1L's KMTase activity in leukemogenesis remains unknown.

To further investigate the function of ASH1L in leukemia, we knocked down expression of ASH1L in leukemia cell lines. We found that ASH1L knockdown slowed cell growth and caused a decrease in expression of HOXA genes and their collaborator MEIS1. To specifically investigate the role of ASH1L KMTase activity in leukemia, we used a mouse model with a targeted in-frame deletion of the ASH1L SET domain (ΔSET). ΔSET cells express a shorter version of ASH1L that does not contain the SET domain. We transduced bone marrow cells (BMCs) isolated from ΔSET and WT mice with several oncogenes relevant to AML to determine the role of the ASH1L SET domain in leukemic transformation. We found that ΔSET BMCs were significantly impaired in leukemic transformation compared to WT BMCs, as manifested by decreased colony-forming ability and reduced expression of HOXA genes. These findings suggest that the ASH1L SET domain plays an important role in leukemogenesis mediated by multiple different oncogenes. Future studies will further characterize ASH1L's role in leukemia by identifying the complete set of genetic loci that it methylates and regulates, and will determine whether the ASH1L SET domain represents a viable therapeutic target.

#1374

Frequent up-regulation of histone methyltransferase G9a contributed to liver carcinogenesis by epigenetically silencing of tumor suppressor RARRES3.

lai wei, Felice Ho-Ching Tsang, Dicky Cheuk-Ting Law, Sandy Leung-Kuen Au, Joyce Man-Fong Lee, Carmen Chak-Lui Wong, Irene Oi-Lin Ng, Jack Chun-Ming Wong. _The University of Hong Kong, Hong Kong, Hong Kong_.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide and is the most common type of cancer in sub-Saharan Africa and Southeast Asia area where hepatitis B infections are common. The initiation and progression of HCC was attributed to alternations in genetics and disruptions of epigenetic processes which lead to altered gene functions in both oncogenes and tumor suppressors. Recent advancements in the field of cancer epigenetics increasingly emphasize the important role of every component in the epigenetic machinery. However, the alternations of epigenetic mechanisms involved in HCC are still largely unknown. Using transcriptome sequencing, we examined the expression of 591 epigenetic regulators in hepatitis B-associated human HCC and found that most of the epigenetic regulators are deregulated especially the histone modification enzymes. Among all of them, we identified G9a (Euchromatic histone-lysine N-methyltransferase 2, EHMT2), a histone H3 lysine 9 (H3K9) specific histone methyltransferases, as one of the most significantly up-regulated epigenetic regulators in human HCCs. Previous studies about epigenetic alternations primarily focused on promoter DNA hypermethylation. However, little is known about the pathological implications of histone modifications. Herein, we hypothesis the frequent up-regulation of G9a causes epigenetic aberrations which contribute liver carcinogenesis, and targeting G9a could be a potential epigenetic therapeutic method for HCC treatment. In this study, we found that G9a was frequently up-regulated in different HCC sample cohorts. Up-regulation of G9a was significantly associated with HCC disease progression, cancer aggressiveness, and more malignant tumor phenotypes. Functionally, we demonstrated that shRNA knockdown and CRISPR/Cas9 knockout of G9a suppressed HCC cell proliferation in vitro and inhibited subcutaneously xenograft HCC tumorigenicity in vivo. Depletion of G9a significantly reduced HCC cell migration ability and induced cell senescence. Pharmacological inhibition of G9a by small molecule inhibitors, UNC0638 and BIX01294, also suppressed HCC cell growth and altered cell morphology. Mechanistically, we showed that the frequent up-regulation of G9a in human HCC was attributed to gene copy number gain at chromosome 6p21 and loss of miR-1. Furthermore, up-regulation of G9 also epigenetically repressed miR-1 expression and thus formed a feedforward regulation loop between them. By utilizing RNA-Seq and GSEA analysis, we identified a potential tumor suppressor RARRES that was epigenetically silenced by G9a and promoted tumor cells proliferation in human HCC. Taken together, we showed that G9a are novel oncogenes in human HCCs and G9a could be novel therapeutic targets for HCC treatment.

#1375

Loss of C-MYC and chromatin acetylation induce epigenetic reprogramming in acute lymphoblastic leukemia.

Elodie M. Da Costa, Gregory Armaos, Simon Jacques-Ricard, Annie Beaudry, Pascal St-Onge, Maxime Caron, Daniel Sinnett, Serge McGraw, Noël J. Raynal. _CHU Sainte-Justine research center, Montreal, Quebec, Canada_.

Epigenetic modifications play a key role in establishing and maintaining gene expression. In cancer, they are highly altered and responsable of gene expression deregulation. Epigenetic drugs have the ability to reset cancer cell epigenome producing cancer cell differentiation and apoptosis. In a drug screening initiative, we recently reported a series of FDA-approved drugs with unsuspected epigenetic and anticancer activities. Here, we tested in a secondary screen the activity of these drugs against acute lymphoblastic leukemia (ALL) cell lines (MOLT-4 and NALM-6). We found that Proscillaridin A, a cardiac glycoside used for heart failure treatment, was the most active with IC50 values in the low nanomolar range, suggesting drug repositioning potential. Proscillaridin treatments induced a significant decrease in RNA and protein levels of C-MYC, a master oncogenic driver in ALL. Shortly after proscillaridin A treatment, C-MYC exhibited a 75% reduction in lysine acetylation, a post-translational modification known to prevent its degradation. Loss of acetylation was associated with down-regulation of lysine acetyltransferases CBP, P300 and Tip60, which also correlated with a reduction in histone 3 and 4 acetylation levels (H3K14ac, H3K9ac, H3K27ac and H4K5ac). Preliminary analysis with si-RNA experiments reveal that independent HAT activities are not responsible of C-MYC downregulation. RNA sequencing and gene set enrichment analysis in proscillaridin-treated ALL cells (5 nM for 48h) showed that genes associated with cell differentiation and apoptosis pathways were up-regulated whereas down-regulated genes were associated with C-MYC target genes. Altogether, our findings show that acetylation through lysine acetyltransferase down-regulation simultaneously induces loss of C-MYC and H3 acetylation leading to epigenetic reprogramming in ALL cells. This drug repositioning strategy, using proscillaridin A, has the potential to reprogram cancer cells that are driven by MYC overexpression or hyperactivation.

#1376

Novel HDAC inhibitor SBAK-GHA: potential therapuetic molecule for lymophocytic leukaemia.

Javeed Ahmad Bhat,1 Mudassier Ahmad,1 Nawab John Dar,1 Aashiq Hussain,1 Rauf Ahmad Najar,1 Simmi Sharma,2 Mubashir Javeed Minto,1 Dilip Manikrao Mondhe,1 Vaqar M. Adhami,3 Bhahwal Ali Shah,2 Neena caplash,4 Abid Hamid*1. 1 _Cancer Pharmacology Division,Indian Institute of integrative Medicine,Canal Road, Jammu, India;_ 2 _Natural product Microbes, Indian Institute of integrative Medicine,Canal Road, Jammu, India;_ 3 _School of Medicine and Public Health University of Wisconsin-Madison 1300 University Ave MSC 4385 Madison, WI 53706., Wisconsin, WI;_ 4 _Department of Biotechnology,Panjab University, Chandigarh, U.T. – 160014 India, Chandigarh, U.T., India_.

In cancer cells, an imbalance exists between Histone acetylase(HAT) and Histone deacetylase(HDAC) activities and current research focuses actively on seeking competitive HDAC inhibitors (HDACi) for chemotherapeutic intervention.We have designed and synthesized a library of novel HDACi molecules from naturally occurring boswellic acids as an alternate CAP for already validated pharmacophore of suberanilohydroxamic acid (SAHA).Novel HDACi molecules were screened against a panel of cancer cell lines. A lead HDACi i.e.N1-hydroxy-N5-(3-α-hydroxy-11-oxo-24-norurs-12-en-4-yl) glutaramide (SBAK-GHA) having IC50 of 1.5µM against leukaemia HL-60 cell line and 6µM in enzyme based assay was obtained. Further HDACi, isoform evaluation was done for SBAK-GHA and it was found to be HDAC Class 1 Specific, especially against HDAC 2 (IC50 of 200 nM) and HDAC 8(IC50 of 250 nM). Acetylation level of different histone residues on H3 and H4 upon SBAK-GHA treatment in HL-60 cell line was studied by immunoblotting. HDACi, SAHA and Sodium Butyrate (NaB) were used as positive controls to compare acetylation levels of different H3 and H4 residues.SAHA and NaB showed significant increase in H3K9, H3K14, H3K27, H4K5, H4K12, and H4K16 acetylation levels while H3K18 showed mild change in acetylation levels. On the other hand SBAK-GHA showed significant increase in acetylation levels of all the lysine residues of histone H3 and H4 especially H4K16 in comparison to SAHA and NaB. In order to analyse varied expression of cyclins and their corresponding control of different phases of cell cycle, we performed chromatin immunoprecipitation assay (ChIP) and observed acetylation pattern of different histone (H3 and H4) residues regulates cyclins differently. In order to analyse the correlation of acetylation on different H3 and H4 residues and their varied pattern of regulation of cyclins, we next analyzed the effect of SBAK-GHA on different cell cycle events in HL-60 cells and found G1 phase cell cycle arrest at significantly low concentration (1µM).We further observed SBAK-GHA induced apoptosis at lower concentrations while as autophagy was found at higher concentrations. Furthermore,we conducted mitochondrial membrane potential studies of SBAK-GHA in HL-60 cells and observed significant loss in mitochondrial membrane potential. We took our findings from in vitro studies and analysed effect of SBAK-GHA on different in vivo murine models. SBAK-GHA showed significant activity especially against P388 lymphocytic leukemia compared to SAHA and 5-FU. Moreover, SBAK-GHA showed promising activity against Ehrlich Ascites Carcinoma (EAC). In conclusion our findings indicate that our lead molecule of interest have great therapeutic potential singly or in combination, however further mechanism of action needs to be elucidated before taking it from Bench to Bedside.

#1377

**Set7 is a novel histone methyltransferase in** Schizosaccharomyces Pombe **.**

Eric Di Luccio, Yunpeng Shen, Damiaan E.H.F Mevius, Yeon Jeong Noh, Jihyeon Kim, Masayo Morishita. _Kyungpook National University, Daegu, Republic of Korea_.

Dynamics and plasticity of chromatin regulation is mediated by a molecular ballet of writers, readers and erasers of epigenetic marks on both DNA and histones. The pattern of histone modifications may define a histone code, which is part of intricate networks that ultimately regulate transcriptional events. Dysfunction of histone methylation affects chromatin regulation and is involved in an increasing number of pathologies from cellular transformation to tumor progression and other diseases. However, histone methyltransferase (HMTase) pathways remain to be further explored and better understood.

Fission yeast (Schizosaccharomyces pombe) is an ideal model organism to investigate the fundamental mechanisms of chromatin dynamics. Fission yeast possesses 13 SET-containing proteins (Set1 to 13), several of which methylate histones or ribosomes. The catalytic SET domain is highly conserved across eukaryotes (e.g. Set2, a homologue of the oncoprotein NSD2/MMSET/WHSC1 in human). Structure-function studies of HMTases are essential from basic science to translational research. However, full-length structures of fission yeast methyltransferases are still scarce.

Here, in a bid to better understand histone methylation and to gain insight for drug-design, we report the identification and structural characterization of a novel histone methyltransferase Set7 (SPCC297.04c), in Schizosaccharomyces Pombe. In this study, we investigated Sp.Set7's cellular localization and the effect of Sp.Set7 knock-out on the cell cycle and sporulation. Next, we elucidated Sp.Set7's Lysine substrate specificity on histone H3 and H4 by biochemical assays and mass spectrometry. Finally, we solved the X-ray structure of the apo Sp.Set7 at 2.0 Å resolution.

In summary, we report the biochemical and structural characterization of a novel histone methyltransferase in fission yeast, which has implications for better understanding the fundamental mechanism of HMTases.

#1378

Identification of genomic regions associated with temozolomide resistance in glioblastoma through analysis of histone marks on chromatin.

Xiaoyue Chen,1 Haiyun Gan,2 Jeong Heong Lee,1 Dong Fang,2 Gaspar Kitange,1 Jann Sarkaria,1 Zhiguo Zhang2. 1 _Mayo Graduate School, Rochester, MN;_ 2 _Columbia University, New York, NY_.

Glioblastoma (GBM) is an aggressive and fatal primary brain tumor. Temozolomide (TMZ) is a critical component of the standard care of newly diagnosed GBM patients, but unfortunately preexisting primary resistance and rapid emergence of secondary resistance invariably limits the therapeutic benefit of TMZ in GBM. Prior studies have identified genetic and epigenetic alterations that can modulate TMZ sensitivity and treatment outcome. However, comprehensive analysis of histone marks and knowledge of epigenetic regulation of genes associated with TMZ sensitivity or resistance is lacking. To identify epigenetic states associated with TMZ resistance, we performed an epigenetic profiling of eight different histone marks in GBM xenografts. Using chromatin immunoprecipitation combined with high throughput sequencing (ChIP-seq), distribution of H3K4me1, H3K4me3, H3K9ac, H3K9me2, H3K9me3, H3K27ac, H3K27me3 and H3K36me3 histone marks was compared in a panel of GBM patient-derived xenograft sub-lines derived by treating TMZ sensitive GBM12 tumors with placebo (n=2 sublines) or temozolomide (n=6 sub-lines) and then propagating resulting recurrent tumors. Our analysis revealed that H3K4me1 and H3K27ac modification patterns varied globally across individual sub-lines, while distribution of H3K4me3, H3K36me3 histone marks was specifically altered in discrete genomic regions in resistant sub-lines depending on the mechanisms of resistance. To find how epigenetic modifications affect TMZ sensitivity, we analyzed ChIP-Seq data using Hidden Markov Model to test if one or a combination of histone marks relates to TMZ resistance. The effect of histone modifications on transcription was simultaneously determined by RNA sequencing. These analyses helped identify specific histone modifications which could be functionally related to TMZ resistance. Through these analyses we have identified 1142 genomic regions governed by a specific epigenetic pattern. We subsequently analyzed a candidate genomic region on top of the list. By using Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated nucleases 9 system (CRISPR/Cas9 system), we have generated clones from an intrinsically TMZ resistant SKMG3 cell line with deletion up to four-kilo base genomic region. Clonogenic growth assays showed that deletion of this genomic region enhanced TMZ sensitivity, reducing the IC50 from 186 μM to less than 60μM, p&lt;0.05. This finding indicates that this genomic region is functionally related with TMZ sensitivity. Taken together, our study reveals epigenetic modifications related to TMZ resistance in GBM cells and a specific genomic region involved in regulating TMZ sensitivity. The analysis of epigenetic state at this genomic region could potentially be useful in predicting treatment response and may help in designing TMZ sensitizing therapy in GBM.

#1379

Histone demethylase KDM6 as a target for glioblastoma radiosensitization.

Barbara Helen Rath, Marzia F. Shah, Kevin Camphausen, Philip J. Tofilon. _National Cancer Institute, Bethesda, MD_.

Radiotherapy is a primary treatment modality for glioblastomas (GBMs). Because post-translational histone modifications have been implicated in the regulation of radiosensitivity, as a potential strategy for enhancing the response of GBMs to radiotherapy we have evaluated the effect of radiation on histone methylation in glioblastoma stem-like cells (GSCs) and the established glioma cell line (U251). For these studies we focused on histone H3K27 tri-methylation (H3K27me3), whose opposing site H3K36me2 has already been linked to DNA repair. Exposure of GSCs and U251 to 10Gy decreased H3K27me3 by 30min, reaching a cell line depended maximum between 1h and 3h, followed by a return to control levels at 6h. According to immunoblot analysis, GSCs and U251 had significant higher levels of KDM6A compared to KDM6B, suggesting a more prominent role for KDM6A in the demethylation of H3K27me3 after irradiation. To determine whether KDM6 could serve as a target for GBM radiosensitization, we focused on GSKJ4, which is an inhibitor for KDM6A and KDM6B. GSKJ4 treatment (4uM) of GSCs and U251 increased the amount of H3K27me3 within 1h of exposure and was found to block the radiation-induced decrease of H3K27me3. Based on clonogenic survival analysis addition of GSKJ4 immediately prior to irradiation significantly enhanced the radiosensitivity of GSCs and U251. To begin to investigate the mechanism responsible for this radiosensitization, GSCs and U251 were irradiated (10Gy), treated with GSKJ4 and collected at 0.5-24h later for neutral comet assay, a measure of DNA double strand breaks. GSKJ4 had no effect on the initial comet-tail moment, yet significantly increased the comet tail-moment up to 24h after radiation, suggestive of an inhibition of DSB repair. To further investigate the potential role of histone demethylase as a potential target to radiosensitize GBM, siRNA was used to knock down KDM6A expression. Cells treated with siKDM6A showed a significant increase in H3K27me3 and the radiation-induced decrease of H3K27me3 was abolished. Analysis of γH2AX nuclear foci after irradiation (2Gy) of siKDM6A treated cells showed a significant delay in foci dispersal, consistent with an inhibition of DSB repair. To investigate whether similar changes in histone methylation occurred in an in vivo setting, H3K27me3 levels were determined in GSC orthotopic xenografts as a function of time after brain irradiation. Similar to in vitro conditions, radiation reduced H3K27me3 levels at 1h, with a decrease still present at 6h. Taken together, these results suggest that the histone demethylase KDM6A is a target for GBM radiosensitization.

#1380

Expression and functions of ASH1L in liver cancer.

Lai Wei, Felice H. Tsang, Daniel Ho, Cheuk-Ting Law, Mengnuo Chen, Long-Hin Tsang, Carmen Cl Wong, Irene Ol Ng, Chun-Ming Wong. _Univ.of Hong Kong, Hong Kong_.

Hepatocellular carcinoma (HCC) is a common cancer and ranks the third lethal cancer worldwide. However, the detail molecular mechanisms underlying the initiation and progression of HCC remain poorly understood. Liver carcinogenesis is a multistep process that is driven by the accumulation of genetic and epigenetic alterations. Mutational landscape and driver mutations have recently been delineated by high-throughput sequencing studies. On the other hand, our current knowledge about epigenetic deregulation in human HCC is limited. Histone modification is a major component of epigenetic regulation, and deregulation of histone medication could alter local chromatin structure and gene expression. We hypothesized that deregulation of histone methyltransferases might contribute to HCC development. In this study, we showed that trithorax-group protein ASH1L was frequently up-regulated in human HCC. Overexpression of ASH1L was detected in 40% of primary HCCs and significantly associated with larger tumor size, present of venous invasion and tumor microsatellite formation. Up-regulation of ASH1L also correlated with increased Ki67 expression. ASH1L is a histone methyltransferase specific for catalyzing transcriptional-active H3K4 and H3K36 methylation. We showed that stable knockdown of ASH1L significantly suppressed HCC cell proliferation and colony formation. We further demonstrated that knockdown of ASH1L inhibited HCC cell migration. In addition, we investigated the underlying mechanisms contribute to ASH1L up-regulation. We showed that up-regulation of ASH1L in human HCC was contributed by chromosome amplification at chromosome 1q22. Furthermore, we showed that ASH1L was negatively regulated by miR-142. Therefore, down-regulation of miR-142 contributed to ASH1L up-regulation in HCC. In summary, our findings suggested that ASH1L was frequently up-regulated in human HCC due to chromosome amplification and miRNA deregulation.

#1381

**Human CRL4** DDB2 **E3 ubiquitin ligase regulates post-repair chromatin restoration of epigenetic marker H3K56Ac through recruitment of histone chaperon CAF-1.**

Qianzheng Zhu, Nidhi Sharma, Jinshan He, Gulzar Wani, Altaf A. Wani. _Ohio State University, Columbus, OH_.

Epigenetic marker acetylated histone H3 lysine 56 (H3K56Ac) diminishes as an early DNA damage response but is restored in chromatin after DNA repair. Here, we report that human CRL4DDB2 ubiquitin ligase regulates the DNA repair-driven chromatin restoration of H3K56Ac by monitoring the recruitment of histone chaperon CAF-1. We show that the H3K56Ac accumulates at DNA damage sites. The post-repair H3K56Ac restoration depends on CAF-1 and CBP/p300 function. The CRL4DDB2 components DDB1, DDB2 and CUL4A are required not only for maintaining the H3K56Ac level in chromatin, but also for restoring H3K56Ac following the repair of DNA photolesions and strand breaks. Depletion of CUL4A decreases the recruitment of CAF-1 p60 and p150 to ultraviolet radiation (UVR)- and phleomycin-induced DNA damage. Inhibition of neddylation renders CRL4DDB2 inactive and concomitantly diminishes the recruitment of CAF-1 and prevents post-repair H3K56Ac restoration. Additionally, a PCNA-interacting protein (PIP) motif is shown to exist within DDB2. The mutation in the PIP box of DDB2 compromises its capability to maintain the H3K56Ac level but does not affect CRL4DDB2-mediated XPC ubiquitination. These results suggest a novel role of CRL4DDB2 in regulating post-repair chromatin restoration, which differs from its canonical role in the process of nucleotide excision repair.

#1382

A novel BET family bromodomain inhibitor NHWD-870 represents a promising therapeutic agent for a broad spectrum of cancers.

Nenghui Wang,1 Mingzhu Yin,2 Qin Yan2. 1 _Ningbo Wenda Pharma, Ninghai, China;_ 2 _Yale School of Medicine, New Haven, CT_.

Small molecule inhibitors of the bromodomain and extraterminal domain (BET) proteins have emerged as a promising option for cancer therapy. NHWD-870 is a potent and selective BET family bromodomain inhibitor and only binds bromodomains of BRD2/3/4/T. NHWD-870 exhibited robust single agent activity in cell viability assay across cancer cell lines derived from solid tumors, leukemia and lymphomas. Further characterization of cancer cell responses to NHWD-870 indicated that NHWD-870 manifested diverse mechanisms of action in different cancer settings. These include: 1) inhibition of tumor cell growth by downregulating the PDGFRβ, MEK1/2 and STAT1/MYC signaling in tumor cells; 2) inhibition of tumor angiogenesis by decreasing PDGF production in tumor cells and the PDGFRβ and MEK1/2 signaling in endothelial cells. Consistent with its broad spectrum of activities in vitro, NHWD-870 has potent tumor suppressive efficacies in xenograft mouse models of small cell lung cancer, triple negative breast cancer and ovarian cancer. These results support its further development for diverse solid tumor indications in the clinic.

#1383

Histone H3 lysine 9 methyltransferase SUV39H1 is associated with clinical outcome of renal clear cell carcinoma patients.

Wei Meng, Ri Cui, Saikh Jaharul Haque, Carlo M. Croce, Arnab Chakravarti. _The Ohio State University, Columbus, OH_.

Background: Epigenetic alternations have three important components including DNA methylation, histione modifications, and non-coding RNA. The epigenetic changes are highly dynamic and also reversible, which regulate a variety of biological processes. Euchromatin which is a loosely packed form of chromatin is highly associated with active gene expression. In contrast, heterochromatin is associated with high histone methylation and DNA methylation but low RNA transcription. SUV39H1 catalyzes the histone 3 lysine 9 trimethylation (H3K9me3) in heterochromatin regions, which also mediates DNA methylation at pericentric heterochromatin. Currently, the oncogenic role of SUV39H1 in renal cell carcinoma (RCC) pathogenesis is poorly understood. To this end, we assessed the role of SUV39H1 in renal RCC cells and association with clinical outcome of RCC patients.

Results: In SUV39H1 knocked down cells, we observed global H3K9me3 level reduction and cell growth inhibition. Proximal ligation assay was used to determine the H3K9me3 and 5-methylcytosine (5mC) interaction in RCC cells. Knocking down of SUV39H1 gene significantly reduced the co-localization of H3K9me3 and 5mC in pericentric repetitive sequences. Moreover, Southern blot demonstrated that expansion of a pentanucleotide (GGAAT) repeats was associated with upregulation of SUV39H1, which is linked to SUV39H1 function with microsatellite instability and rearrangement. In The Cancer Genome Atlas (TCGA) database, we found that SUV39H1 gene expression was upregulated in RCC tissues compared to normal tissues, and high SUV39H1 expression predicted a worse overall survival outcome (p = 0.002) in RCC patients.

Conclusion: SUV39H1 upregulation promotes cell proliferation and microsatellite instability. SUV39H1 expression could serve as a prognostic biomarker for RCC patients.

#1385

New insights into the roles of histone lysine-specific demethylase 2 (LSD2) in breast cancer.

Lin Chen, Shauna N. Vasilatos, Ye Qin, Steffi Oesterreich, Nancy E. Davidson, Yi Huang. _University of Pittsburgh, Pittsburgh, PA_.

Background: Epigenetic modifiers have been emerging as new players in breast cancer development. FAD-dependent histone demethylases have been proved to play important roles in regulating breast tumor initiation and development. While the role of histone lysine-specific demethylase 1 (LSD1) in breast cancer progression has been well characterized, the activity of its homolog, lysine-specific demethylase 2 (LSD2), in breast cancer is still a riddle that needs to be uncovered.

Methods: To explore the precise role of LSD2 in breast cancer, we overexpressed LSD2 protein in MDA-MB-231 cells (LSD2-OE). The phenotypic effect of LSD2-OE on MM231 cells was characterized by proliferation assay, soft agar assay (3D growth), trans-well cell migration and invasion assay and mammosphere formation assay. To extend stem cell-like features study of LSD2 overexpressed MM231 cells, we examined CD44+/CD24- subpopulation by flow cytometry and the expression of several stem cell markers by qPCR and western blot. In addition, we also investigated the potential altered phenotypes of LSD2 knockdown in MM231 cells via RNAi.

Results: Results from Oncomine database show that LSD2 expression level is greatly increased in invasive ductal carcinoma (IDC) vs. non-invasive breast cancers. The cellular proliferation assay revealed that LSD2-OE promoted MM231 cell growth. Similarly, LSD2 overexpressed MM231 cells were capable of forming large colonies in soft agar. Trans-well cell migration and invasion assay results indicated LSD2-OE significantly decreased both migration and invasion in MM231 cells. Mammosphere formation assay results showed LSD2 overexpression increased the number of primary and tertiary mammospheres in MM231 cells. Consistent with increase of mammosphere forming efficiency, NANOG and SOX2 were increased by LSD2 overexpression at the protein level. However, LSD2-OE significantly decreased CD44+/CD24- subpopulation. Moreover, siRNA mediated LSD2 depletion significantly decreased cell growth in multiple breast cancer cell lines without altering migration and invasion in MM231 cells. Finally, no obvious phenotypic changes were observed in MM231 cells expressing stable LSD2 shRNA.

Conclusion: The multifaceted effects of LSD2 on breast cancer phenotypes may reflect the profound influence of LSD2 activity on chromatin structure and gene expression in the breast cancer genome. Our studies suggest that overexpression of LSD2 facilitates growth and confers stem cell-like traits to breast cancer cells, and transient LSD2 inhibition by siRNA hinders breast cancer cell growth. These results suggest that LSD2 might be a valuable therapeutic target in breast cancer.

#1386

Effects of histone deacetylase inhibitors on differentiation markers and growth of colon and bladder cancer cells.

Michael A. Lea, Lauren Cué, Elizabeth Batista, Erik Lew, Charles desBordes. _Rutgers New Jersey Medical School, Newark, NJ_.

Alkaline phosphatase and dipeptidyl peptidase are markers of differentiation in colon cancer cells. In colon cancer cells differentiation can often be induced by treatment with inhibitors of histone deacetylase (HDAC) activity. In some colon cancer cells butyrate causes a several-fold induction of alkaline phosphatase but only modest effects on dipeptidyl peptidase. There is relatively little information on changes in activity of cell surface hydrolases in bladder cancer. However, there is some evidence for decreased alkaline phosphatase activity in bladder cancer. We tested the hypothesis that there may be retention of activity in more slowly growing cancer cells and the activity will be regulated by HDAC inhibitors in a manner similar to that in colon cancer. In our initial studies we screened seven human bladder cancer cell lines for alkaline phosphatase activity. We identified three cell lines (5637, HT1197 and HT1376) with activity comparable to that in more differentiated colon cancer cell lines. The response to incubation with the HDAC inhibitors butyrate and valproate was compared with effects in three colon cancer cell lines Caco-2, HT29 and SW1116. In all six cell lines there were growth inhibitory effects that tended to be a little greater with butyrate than with valproate. Induction of alkaline phosphatase activity was generally greater with butyrate than with valproate and was apparent even without normalizing the activity on a protein basis. After determining the activity per unit protein there were increases in dipeptidyl peptidase activity after treatment with the HDAC inhibitors that were smaller than for alkaline phosphatase. Growth inhibition was observed with all the HDAC inhibitors examined including LMK235, nicotinamide, RGFP966, romidepsin and tubacin but increased activity of alkaline phosphatase was not always seen. Activities of the enzymes were very low in CRL1790 human colonocytes of fetal origin and there was little or no induction with HDAC inhibitors. Although pyruvate and carnitine have been reported to be HDAC inhibitors, incubation with colon and bladder cancer cells did not cause induction of alkaline phosphatase activity. It was concluded that regulation of alkaline phosphatase and dipeptidyl peptidase by HDAC inhibitors can be similar in bladder and colon cancer cells.

#1387

Target RNA sequencing revealed the expression profile of cancer genes in response to deacetylase inhibitor in primary culture of prostate cancer cells.

Hong Yin, Adam H. Greer, Glenn Mills. _Louisiana State Univ. Health Sciences Ctr., Shreveport, LA_.

Histone acetylation is a dynamic event to optimize cell functions through the control of RNA transcription and gene expression. In cancer, the histone deacetylation is a therapeutic target. Suppression of histone deacetylase increased the growth arrest, apoptosis, and senescence in cancer cells. Several histone deacetylase inhibitors (HDACis) have been developed and applied as a target therapy of various types of cancer, including the prostate cancer. However, the altered gene expression in HDACis treated prostate cancer cells is less reported. Little is known about the gene expression profiles of primary cultures of prostate cancer. In this study, we examined the cancer gene expression profile in primary cultures of human prostate cancer. Cells were isolated from two patient's prostate cancer samples. Isolated cells were cultured in prostate epithelium culture medium CnT-52. After 24 hours of exposure to 400nM of Trichostatin A (TSA), an inhibitor of histone deacetylase I and II. RNA was extracted and quantified. The library was prepared with Illumina's TruSight RNA Pan-Cancer Panel. The sequencing was performed on NextSeq 500 platform with NextSeq 500/550 mid output v2 kit (150cycles). FastQ files were generated by BaseSpace Onsite and analyzed further by TopHat alignment (Illumina) and Partek Flow (Partek). The Trusight RNA Pan-Cancer panel targets 1385 cancer genes and covers 21043 exonic regions with 57010 probes. The results showed that 59 of the 1385 cancer genes were significantly changed (FDR p-value<0.05, fold change >4 fold) in TAS treated cells. In the treated cells 49 genes were unregulated and 10 genes were down-regulated. The altered expression of 59 genes is involved in multiple cell functions. Genes that were upregulated included apoptosis associated genes (MAP2K6 and BIRC3), tumor suppression genes (DIRAS3, PEG3, PTPRO, and DNM3), and P53-target gene (CYFIP2). The genes that were down-regulated included cell cycle regulation genes (CDK1 and CCNA2), DNA damage response genes (BRCA1 and BRIP1), as well as cell proliferation marker Ki67 gene (MKI67). However, the gene expression response to TSA is complicated. Some altered gene expression favors tumor growth and progression while other gene expression changes inhibit tumor growth and promote apoptosis. In conclusion, we identified the gene profile in primary culture of prostate tumor in response to a histone deacetylase inhibitor, TSA. The therapeutic effects of HDACis could be influenced by the summation of contradicting tumor promoting and suppression gene expression profile changes.

#1388

SphK2/S1P axis regulates hypoxia-mediated HIF-α synthesis in breast cancer cells.

Nitai C. Hait, Aparna Maiti. _Roswell Park Cancer Institute, Buffalo, NY_.

The hypoxia-inducible transcription factors (HIF)-1α and -2α play a critical role in cellular response to hypoxia in solid tumors. Elevated HIF-α expression correlates with poor patient survival in a large number of cancers including breast cancers. HIF-α activates expression of genes promoting angiogenesis, metastasis, increased tumor growth and resistance to treatments. Understanding transcriptional regulation HIF-α under physiologically relevant hypoxic conditions would be helpful to reduce breast cancer growth. Our recent studies identified that intracellular bioactive sphingosine-1-phosphate (S1P) generated from nuclear sphingosine kinase 2 (SphK2) acts as an endogenous modulator of histone deacetylases (HDACs) epigenetically regulates gene transcription in breast cancer cells. We wonder whether nuclear S1P epigenetically regulates acetylated histones, positive transcription marks in hypoxia and regulate transcription of HIF-α and target genes in breast cancer cells. We have used estrogen receptor positive human breast cancer MCF-7 cells, triple-negative human breast cancer MDA-231 cells, and lung metastatic LM-2-4 cells derived from MDA-231 cells cells for our studies. We found that hypoxic condition enhances nuclear bulk histone acetylation in breast cancer cells and enhances HIF-α synthesis. Down regulation of SphK2 with siRNA or using selective inhibitor of SphK2 (K145 compound) reduces nuclear S1P as well as bulk histone acetylation and consequently reduces nuclear HIF-α synthesis in breast cancer cells. Downregulation of cytosolic sphingosine kinase 1 (SphK1) with siRNA reduces cytosolic S1P does not alter nuclear bulk histone acetylation neither significantly alter nuclear HIF-α synthesis in breast cancer cells. We also found that targeting SphK2 with K145 compound dramatically reduces in vitro tumorosphere formation in breast cancer cells. Our data suggested that targeting the SphK2/S1P signaling could represent an attractive strategy for therapeutic intervention in hypoxic breast cancers. Supported by Health Research, Inc. (HRI) Grant 71-4084 (NCH), National Cancer Institute Grant R01CA160688 (KT).

#1389

Effects of geinstein on histone modifying enzyme expression in prostate cancer.

Lanni Aquila, Nick Smothermon, Lisa Morey. _Canisius College, Buffalo, NY_.

The present study sought to examine the expression of two histone modifying enzymes, SET8 and SIRT1, in human prostate cancer cell model when exposed to increasing concentrations of genistein. Genistein is a phytoestrogen found mainly in soy-based foods and is of importance due to the varying intake levels worldwide. This compound is thought to have chemopreventive properties and it is hypothesized to exert its affects by epigenetically modifying chromatin organization within cells. As an estrogen-like compound, genistein may exert effects similar to that of estrogen. For this reason, SET8, a histone methyltransferase, and, SIRT1, a histone deacetylase, were studied. Previously, these histone modifying enzymes were found to play a role in estrogen signaling. In the present study, cells were treated either for 24 hours (acute) or 5 days (chronic), total RNA was extracted, followed by an RT-PCR, and amplification of the genes of interest via PCR. Changes in gene expression were quantitated using ImageJ. Overall, it was concluded that the exposure to genistein impacts the gene expression of both SET8 and SIRT1 within human prostate cancer cells.

#1390

Deregulation of RNA N6-adenosine methylation contribute to liver carcinogenesis.

Mengnuo Chen, Larry L. Wei, Cheuk Ting Law, Felice HC Tsang, Iris MJ Xu, Joyce Lee MF Lee, Carmen Wong CL Wong, Irene OL Ng, Chun-Ming Wong. _The University of Hong Kong, Hong Kong, Hong Kong_.

Primary liver cancer, particularly the most common type hepatocellular carcinoma (HCC), is the third leading cause of cancer death worldwide. Case is even worse in Asia due to extremely high prevalence of HBV/HCV infection, which enhances the risk of HCC. However, the exact mechanism of how HCC develops is still not clearly defined. Previous studies have shown strong evidence for epigenetic alterations in human carcinogenesis, which mainly focus on abnormal DNA methylation, histone modifications, and chromatin remodeling. Recent studies suggest that diverse chemical modifications on RNAs, also known as "epi-transcriptome", constitute another layer of gene expression regulation. N-6-methyladenosine (m6A) is the most abundant modification on eukaryotic mRNA, which has been found to functionally influence mRNA stability, alternative splicing, and translation efficiency. However, the roles of m6A deregulation in human carcinogenesis remain unclear. Through whole-transcriptome sequencing, we identified that METTL3, the major component of m6A methyltransferase, was significantly up-regulated in human HCC. Up-regulation of METTL3 negatively associated with poor patient overall and disease free survival. Functionally knockdown/knockout of METTL3 dramatically inhibited HCC proliferation, migration and colony formation abilities in vitro and significantly suppressed in vivo tumorigenicity. In contrast, overexpression of METTL3 using CRISPR/dCas9 SAM system modestly but significantly enhanced HCC proliferation, migration, anchorage-independent growth in vitro and tumor formation in vivo. In order to find the downstream mechanisms which might attributed to METTL3 oncogenic function we performed RNA-Seq on two independent METTL3 knockdown cell lines to interrogated their transcriptome changes. Based on RNA-Seq data, we identified a prominent tumor suppressor SOCS2 as a potential down-stream target of METTL3. With m6A-RNA immunoprecipitation (RIP) assay, we confirmed that SOCS2 mRNA was subject to m6A modification in cancer cells. Inactivation of METTL3 by RNAi or treatment of methylation inhibitor (DAA) impaired m6A mediated mRNA degradation and thereby stabilized SOSC2 mRNA. We further confirm SOCS2 tumor suppressor function in HCC. Downregulation of SOCS2 was associated with poor patient overall survival and disease free survival of HCC patients. Knockdown of SOCS2 significantly promoted HCC proliferation, migration in HCC cells. In conclusion, our findings suggested that deregulation of METTL3 and its associated m6A modification could contribute to human carcinogenesis by imposing an epigenetic control on the stability and expressions of critical tumor suppressor genes.

#1391

The epigenetic regulator, G9a, is a KRAS-inducible protein and its inactivation inhibits PanIN formation by this oncogene.

Angela Mathison,1 Ann Salmonson,1 Brooke Paradise,1 Mckenna Missfeldt,1 Juan Iovanna,2 Daniel Billadeau,1 Raul Urrutia,1 Gwen Lomberk1. 1 _Mayo Clinic, Rochester, MN;_ 2 _Cancerology Research Center of Marseille (CRCM); INSERM U1068, Marseille, France_.

Pancreatic ductular adenocarcinoma (PDAC) ranks fourth as a cause of cancer death in the USA and is almost universally fatal, with the annual number of deaths equivalent to the number of newly diagnosed cases. Valuable research in the field has revealed genetic aberrations that contribute to PDAC development and progression, with KRAS being one of the most frequent mutations in more than 90% of patient samples. However, to date, any efforts to directly target KRAS have failed in the clinic. Thus, there is indisputably an urgent need to further improve our understanding of molecular mechanisms underlying PDAC development as to identify novel therapeutic targets, including druggable important downstream targets and nodes orchestrated by oncogenic KRAS. In particular, we are interested in epigenetic pathways involved in PDAC development and progression due to the potential reversibility of any alteration, unlike genetic mutation. In the current study, using a cell model that allows inducible expression of mutant KRASG12D, we find that protein levels of the dimethyl-K9H3 histone methyl transferase (HMT), G9a, and its complex partners are increased in response to activation of the oncogenic Kras pathway. Furthermore, the activation of this oncogenic pathway results in the formation of the G9a-GLP-Wiz trimer complex, as determined by affinity protein purification, combined with mass spectrometry. In vivo experiments involving the cross of the Pdx1-CRE/LSL-KRASG12D mice with G9afl/fl animals demonstrate that a loss of the H3K9Me2 mark in the nucleus of exocrine cells is accompanied by a significantly reduced number of PanIN lesions. RNA-Seq experiments from these animals reveal that these mice have reduced levels of typical molecular markers of PanINs. In addition, these experiments show changes in the levels in several genes, which have been previously been shown to synergize with Kras to mediate pancreatic cancer initiation. Congruently, pharmacological inhibition of G9a using BRD4770 displays an inhibitory effect on KRASG12D-induced cell proliferation. Combined, these data provide evidence for a key role of the meK9H3-G9a pathway as a mediator of the oncogenic Kras response and defines a novel point of potential therapeutic intervention for PDAC.

#1392

Development of a novel mouse model for intrahepatic cholangiocarcinoma.

Rebecca Marcus,1 Wai Chin Foo,2 Anirban Maitra,1 Sonal Gupta1. 1 _Sheikh Ahmed Bin Zayed Al Nahyan Center for Pancreatic Cancer Research, The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Cholangiocarcinoma (CCA) is a poorly understood cancer of the biliary epithelium and the second most common type of liver cancer. Surgery currently offers the only potential for cure; however, most patients present with advanced disease and are therefore unresectable. The Cancer Genome Atlas (TCGA) analysis and other recent genomic studies have revealed discrete epigenetic perturbations amongst CCAs originating from different anatomic sites. Intrahepatic cholangiocarcinoma (ICC) arises from the intrahepatic bile ducts, and a subset of ICCs is characterized by loss-of-function mutations in the gene encoding for the chromatin regulatory factor BRCA associated protein 1 (BAP1). Loss of this protein may be associated with global epigenomic and transcriptomic alterations that ultimately contribute to tumor progression and metastasis dissemination. While elucidating the molecular pathogenesis of ICC may identify potential targeted therapies and improve early detection, inquiry into this disease has been hampered by a lack of genetically faithful animal models. We developed a genetically engineered mouse model (GEMM) of ICC that incorporates an inactivating mutation in BAP1 combined with Kras activation. An Albumin-Cre promoter was used to induce hepatoblast-specific mutations. Mutant Kras cooperates with loss of BAP1 and results in lethal hepatic transformation and dose-dependent survival. Kras activation alone results in extended disease latency and survival > 50 weeks. Loss of BAP1 alone or heterozygous loss of BAP1 combined with mutant Kras shortens disease latency, with mice surviving 39 weeks on average. A significant reduction in survival is seen with homozygous loss of BAP1 and Kras activation (Kras BAP1L/L). These mice survive on average 23 weeks (p ≤ 0.0045). Histopathologic evaluation of Kras BAP1L/L mice demonstrates focal biliary precursor lesions, frank ICC, and hepatocellular carcinoma (HCC). Mice with heterozygous deletion of BAP1 and Kras activation, loss of BAP1 alone, or Kras activation alone develop HCC only. Given the bipotential nature of hepatoblasts, the ICC phenotype of our GEMM may be enhanced by inducing biliary tree-specific mutations. Adenoviral Cre enzyme (Ad-Cre) is used to achieve such combinatorial specificity, and a novel surgery was developed whereby retrograde biliary tree administration of this enzyme is performed. Surgery utilizing GFP-tagged adeno-associated virus confirms administration targeted to the biliary tree. Retrograde biliary tree injection of Ad-Cre into ROSAmT/mG mice demonstrates Cre recombinase expression within cholangiocytes, thereby establishing proof-of-principle. Ad-Cre injection in Kras BAP1L/L mice to induce cholangiocyte-specific BAP1 deletion and Kras activation is ongoing.

#1393

Chromatin remodelers as potential new targets for therapy of pediatric sarcoma.

Joana G. Marques,1 Berkley Gryder,2 Marco Wachtel,1 Javed Khan,2 Beat Schaefer1. 1 _University Children's Hospital, Zurich, Switzerland;_ 2 _National Institutes of Health, Bethesda, MD_.

Fusion-positive rhabdomyosarcoma (FP-RMS) is a pediatric malignancy driven by the fusion transcription factor PAX3-FOXO1, which generates an aberrant gene expression signature leading to cell transformation. Since FP-RMS cells are highly addicted to the fusion protein, it is in focus as target for alternative therapies. Nevertheless, PAX3-FOXO1, as a transcription factor, does not contain structural cavities and has a low druggability. We therefore hypothesize that we can affect this aggressive subtype of RMS by targeting the co-regulators that collaborate with the fusion protein in regulating transcription. Recently, we have identified the NuRD (Nucleosome Remodeling and Deacetylase) complex as a potential partner of PAX3-FOXO1 in gene expression modulation. The NuRD complex is unique among chromatin remodeling complexes due to its dual enzymatic activity (histone deacetylation through HDAC1/2 and nucleosome positioning by CHD4 - chromodomain-DNA-binding protein 4), offering new possible therapeutic targets. Silencing of two core members of NuRD, CHD4 and RBBP4, led to a drastic decrease in FP-RMS cell viability. Additionally, CHD4 depletion caused a complete regression of mouse tumor xenografts, but it did not affect proliferation of myoblasts, fibroblasts or fusion negative RMS cells, despite the fact that these cells also carry high CHD4 expression levels. We further investigated the nucleosome remodeler CHD4 and learnt that it affects the expression of approximately 50% of PAX3-FOXO1 target genes with most of these genes being upregulated, suggesting an activating role for CHD4 in these cases. Consistent with a positive effect of CHD4 on gene expression, ChIP-seq experiments with FP-RMS cell lines demonstrated that NuRD occupies promoter and enhancer regions of highly expressed genes and co-localizes with the fusion protein at regulatory regions of a subset of its target genes. Next, we studied the influence of this nucleosome remodeler on the chromatin status by DNase hypersensitivity assays and determined that the presence of a DNase signal at PAX3-FOXO1 binding sites is concordant with the presence of CHD4. Hence, we suggest a scenario where CHD4 plays an essential role on FP-RMS tumorigenesis by allowing chromatin to acquire an open architecture that enables PAX3-FOXO1 mediated gene expression. In summary, our data propose that CHD4 has a crucial role as a co-regulator of PAX3-FOXO1 driven gene expression. To our knowledge, CHD4 is the first identified chromatin remodeler associated with PAX3-FOXO1 transcriptional activity, thus highlighting the relevance of epigenetic regulation in FP-RMS tumor development and opening chromatin remodelling as a possible new field of action against this tumor, which is driving ongoing work aimed at finding first-in-class small molecules to inhibit CHD4 function.

#1394

Bioorthogonal Profiling of Protein Methylation (BPPM) identified MCM5 as a new substrate for SETD8 in DNA replication.

Fabio Pittella Silva, Gil Blum, Chamara Senevirathne, Luo Minkui. _Memorial Sloan Kettering Cancer Center, New York, NY_.

SETD8 is the only member of the SET domain containing methyltransferase family, which catalyzes mono-methylation of K20 on histone H4 (H4K20me1). Lysine residues of non-histone proteins such as p53 and proliferating cell nuclear antigen (PCNA) are also monomethylated, while lysine residues in Numb were found to be dimethylated. As a consequence, SETD8 methyltransferase activity is implicated in several fundamental cellular processes such as transcriptional regulation and heterochromatin formation as well as processes that ensures genomic stability including DNA replication and the DNA damage response. Although it has been suggested that SETD8 is involved in DNA replication as a positive regulator of origin licensing through H4K20 methyation and by supporting Okazaki fragment processing through PCNA methylation, to date, there is no evidence whether other key protein in the replication fork is directly modified by SETD8. To address this question, we used Bioorthogonal Profiling of Protein Methylation (BPPM) with engineered enzyme and synthetic SAM analogues to profile new substrates for SETD8. We genetically engineered SETD8 and identified mutants amenable to accommodate non-native SAM analogues containing a terminal alkyne moiety for click chemistry. The engineered SETD8 can transfer this distinct chemical moiety into target proteins for subsequent pulldown and identification of the modified substrates. Among the new substrates discovered, we identified MCM5, a subunit of the hexameric minichromosome maintenance (MCM) DNA helicase complex. MCM5 directly interacts with MCM2 and 3 to form the MCM2-7 hexamer which associates with the origins of DNA replication to form part of the pre-replicative complex (preRC), playing a key role during replication initiation and elongation. We found that SETD8 can mono-methylate MCM5 directly affecting its binding affinity to MCM2 and 3. In addition, MCM5 mutations at the methylated lysine further evidenced a stronger binding to its interacting partners contributing to the formation of the MCM hexamer. Taken together our results indicate that MCM5 methylation contributes to the assembly of the MCM complex suggesting an important role for SETD8 in replication initiation. Our findings may bring new perspectives on the biological importance of SETD8 during DNA replication.

#1395

Deciphering inosinome in glioblastoma versus normal cortex.

Angela Gallo,1 Alessandro Silvestris,1 Valeriana Cesarini,1 Valentina Tassinari,1 Nicolò Mangraviti,1 Ernesto Picardi,2 Graziano Pesole2. 1 _Ospedale Pediatrico Bambino Gesu, Roma, Italy;_ 2 _Università di Bari, Bari, Italy_.

Cancer is driven by alterations of the genomic information, which carries mutations in key genes providing selective advantage for clonal multiplication of cancer cells. However, mutations within DNA are not the only source for cell alteration. RNA molecules are targets of a series of post-transcriptional modifications, such as splicing and RNA editing, that can affect sequence, structure and stability. The most common type of RNA editing in humans converts Adenosine in RNA targets into Inosine (A-to-I) and is catalyzed by two adenosine deaminases that act on dsRNA (ADARs) family of enzymes (ADAR and ADARB1). Inosines are subsequently interpreted as guanosines by several cellular proteins and could ultimately lead to a genomic mutations (A-to-I/G).

At present, it has been estimated that over 4 millions editing sites exist in our transcriptome involving coding and non-coding RNAs. These huge amounts of Inosine at RNA level are necessary for our survival and their levels is highly regulated in different tissues and during development. Considering the importance of ADAR activity in our cells we believe that if ADAR are not well regulated they may contribute to cancer on set and/or progression.

The advent of high-throughput RNA sequencing has enabled identification of RNA editing sites and global analyses of cancer transcriptomes demonstrate that ADAR-mediated RNA editing dynamically contributes to genetic alterations in cancer, including high-grade gliomas.

Glioblastoma (GBM) is one of the most common and aggressive primary brain tumor in humans and despite advances in understanding the molecular mechanisms underlying these tumors, current treatments are ineffective.

In order to elucidate the glioma-specific RNA editing signature, we analyzed 146 RNA-Seq of primary glioblastomas from the TCGA dataset compared to 132 normal brain cortex RNA-Seq from the GTEx database and purified pools of normal cortex astrocytes. A-to-I editing events has been detected using a collection of more than 4 million annotated edited substrates and the REDItools suite of python scripts with stringent filters. By means of the Cuffquant/Cuffdiff tools, we have also compared global transcriptome profiles and ADAR genes expression patterns at gene level isoform level and using IHC at protein level.

We found that a general down regulation of editing events at both recoding and non -recoding (Alus) sites that correlate with a down expression of ADARB1 enzyme. No differences were observed with ADAR expression. Overall, we observed a strong editing landscape perturbation in glioblastoma that could be important for identifying the most effective target genes for possible therapeutic intervention.

#1396

Role of epigenetic reader and alternative mRNA splicing variant MBD2 variant 2 in triple-negative breast cancer.

Emily A. Girsch,1 Bin Bao,1 Cristina Mitrea,1 Gregory Dyson,1 Julie Boerner,1 Lisa Polin,1 Stan R. Terlecky,2 Aliccia Bollig-Fischer1. 1 _Wayne State University, Detroit, MI;_ 2 _Seton Hall University, South Orange, NJ_.

Background: Among all breast cancer patients, those diagnosed with the triple-negative breast cancer (TNBC) subtype have the worst prognosis, in part attributable to the fact that TNBC lacks targets for effective molecularly-targeted therapies. The concept that EGFR inhibitor drugs could be used as a targeted treatment against TNBC has been put forth because roughly 50% of TNBC express high levels of EGFR. However clinical trials targeting EGFR did not significantly improve patient outcomes. Our recent work offers a potential explanation as to why EGFR inhibitors failed and supports an innovative therapeutic approach combining an EGFR tyrosine kinase inhibitor with the novel antioxidant biologic, CAT-SKL. Our data indicate that inhibition of cancer stem cell-like cells depends on antioxidant-induced downregulation of an alternative mRNA splice variant of the methyl-CpG binding domain 2 gene, MBD2_v2.

Objective and Methods: The purpose of the present study was to evaluate the importance of MBD2_v2 in TNBC and better understand how CAT-SKL regulates its expression. Using TNBC cell lines we investigated the effects of MBD2_v2 expression on tumor initiation capacity. Furthermore we studied MBD2_v2 mRNA levels in an in vivo tumor xenograph mouse model of TNBC and in human TNBC samples using an existing database. Then by RNA-seq analysis we assessed how CAT-SKL regulates MBD2_v2 expression in TNBC cells through differential gene expression.

Results: The results support that MBD2_v2 expression is regulated by redox signaling in TNBC cells, which is tightly linked to TNBC cell tumor initiation capacity. Furthermore, MBD2_v2 was upregulated in tumors harvested from overweight mice, which also displayed increased tumor take rate, suggesting that a pro-inflammatory tumor microenvironment could play a role in promoting MBD2_v2 expression. In addition, RNA-seq analysis identified an overrepresentation of mRNA splicing factors with expression downregulated by CAT-SKL treatment of TNBC cells. From this gene set two splicing co-factors are highlighted: SRRM1 and CPSF3. These co-factors are critical for splicing factor function, 3' end formation and polyadenylation; and both have been linked via protein-protein interaction to a relevant exonic splicing enhancer site in exon 3 of the MBD2 _v2 transcript. Finally, according to the Kaplan Meier Plotter database high MBD2_v2 levels in BC were associated with shorter relapse free survival.

Conclusion: Our investigation sheds light on the relevance of MBD2_v2 expression in TNBC, and how a select antioxidant regulates its expression. The application of which holds promise of a novel, targeted therapeutic modality for TNBC, the subtype with the worst prognosis and highest need for treatment options.

#1397

Antisense oligo nucleotide of Annexin A4 improved platinum resistance in ovarian clear cell cancer.

Reisa Kakubari,1 Satoshi Nakagawa,2 Tadashi Iwamiya,1 Eiji Kobayashi,1 Shinnya Matsuzaki,1 Yutaka Ueda,1 Kiyoshi Yoshino,1 Yuya Kasahara,1 Satoshi Obika,1 Tadashi Kimura,1 Satoshi Serada,3 Tetsuji Naka,3 Minoru Fujimoto3. 1 _Osaka University, OSAKA, Japan;_ 2 _Sakai city medical center, OSAKA, Japan;_ 3 _National institute of Biomedical Innovation, OSAKA, Japan_.

Introduction: Ovarian cancer in Japan are classified as clear cell carcinoma (CCC) more than 20 %, this percentage is higher than in Europe and United States. Besides, it is well known that CCC of ovary is highly resistant to cancer chemotherapy including carboplatin and paclitaxel treatment. We reported that Annexin A4 protein was overexpressed in ovarian CCC tissues by immunohistochemical analysis. Elevated Annexin A4 level has been detected in various epithelial cancer cell lines and have reported associating with chemoresistance to platinum-based cancer drugs. To overcome the platinum chemoresistance, we thought antisense oligonucleotides (ASOs) to be a good therapeutic option in a way of highly specific therapy for improving chemoresistance by suppressing the expression of Annexin A4 in cancer cells.

Methods: We generated ASO targeting Annexin A4 with 2', 4'-bridged nucleic acid. And we analyzed suppression of Annexin A4 in ASO-transfected RMG-I cell line (CCC) in vitro using real time PCR and western blotting. In 16 types of ASOs targeting Annexin A4, 2 ASOs were eligible. Cells were seeded in 96-well plates (2,000 cells per well). Next day, cells were transfected with ASOs using lipofectamine 2000 and were exposed to various concentrations of cisplatin (0 - 100 μM) for 72 hr. Then, drug concentrations resulting in a 50% inhibition of cell growth (IC50 values) were calculated. Intracellular platinum accumulation in Annexin A4 overexpressing cells was analyzed. To assess the improvement of platinum resistance in vivo, we used ICR nu/nu mice xenografted subcutaneously with RMG-I cells. Intraperitoneal injection of cisplatin 3mg/kg after intratumoral administration of ASO 1mg/kg each twice a week were given to xenograft mice.

Results: By realtime PCR analysis, among strong 16 types of ASOs targeting Annexin A4, 2 ASOs showed strong knockdown efficiency (about 80% knockdown) compared to negative control ASOs. Western blotting analysis showing knockdown of Annexin A4 expression was observed in Annexin A4 ASO transfected cells compared to no treatment or control ASOs in vitro. ASO-transfected RMG-I cells was less resistant to cisplatin (IC50 = 3.3μM) compared with control cells (IC50 = 5.2μM) Same result were obtained with carboplatin. Platinum resistance was significantly improved in treated with Annexin A4 ASO and cisplatin compared to control ASO and cisplatin treated group in vivo.

Conclusion: By transfection of ASOs targeting Annexin A4, platinum resistance have improved in vivo and in vitro, Annexin A4 have associated with efflux of platinum anti-tumor drug. In conclusion, antisense oligonucleotides for Annexin A4 will be a therapeutic option for ovarian clear cell carcinoma with chemoresistance to platinum antitumor drug.

#1398

Loss of PIWIL4 and piRNAs attenuate somatic methylome and genome stability.

Syuan-Cih Lin,1 Chia-Chen Chiu,1 Shou-Tung Chen,2 Yao-Li Chen,3 Ping-Yi Lin,3 Shu-Huei Hsiao,1 Yu-Wei Leu1. 1 _National Chung Cheng Univ., Chia-Yi, Taiwan;_ 2 _Comprehensive Breast Cancer Center, Changhua, Taiwan;_ 3 _Transplant Medicine & Surgery Research Centre, Changhua, Taiwan_.

Piwi-like RNA-mediated gene silencing 4 (PIWIL4) is expressed in somatic tissues and is proposed to silence the transposition of repetitive elements like long interspersed nuclear elements (LINEs) through DNA methylation and maintain the genome stability. Through the knockout of PIWIL4 gene by CRISPR/Cas9 system in mesenchymal stem cells (MSCs), MDA-MB-231 (breast cancer cell line), HT29 (colon cancer cell line) and AGS (gastric cancer cell line), the piRNA biogenesis was decreased and so was the methylation within LINE1 target loci and global methylation. The recruitment of SUV39H1, a histone H3 methyl-transferase, into nucleus was blocked, leading to the reduction of silencing tri-methylation of histone H3 at lysine 9. Concurrently, the active H3K4me3 mark increased after knockout. Also, global CTCF bindings were distorted after the knockout of PIWIL4 especially at the bindings franking the piRNA clusters. This distortion implied the disorganization of genomic conformation which was validated by karyotyping. Through chromosomal G-banding, aneuploidy was observed which indicated the loss of genomic stability. The knockout of PIWIL4 also results in disorganized cytoskeleton, therefore, changed cell stiffness as measured by atomic force microscopy. Neural and hepatic induction of MSCs were also blocked by the knockout of PIWIL4. In correlation, tissue arrays detected abnormal PIWIL4 expression in gastric (n=45), colon (n=30) and breast cancers (n=30). In conclusion, we found that PIWIL4-piRNA interaction helps to maintain somatic DNA methylome, genome stability and stiffness in somatic stem cells. The loss of PIWIL4 expression might then a candidate for cancer biomarker. (Supported by: MOST-105-2314-B-371 -008, MOST-105-2320-B-194 -004, MOST Taiwan)

#1399

In vitro **characterization** **of several enzymes of the Fe(II)/2-oxoglutarate-dependent dioxygenase superfamily using a universal succinate detection assay.**

Juliano Alves, Said Goueli, Hicham Zegzouti. _Promega Corporation, Madison, WI_.

The covalent modification of histone proteins, DNA and RNA by Fe(II)/2-oxoglutarate-dependent dioxygenases are key to the modulation of biological processes such as epigenetics, hypoxic signaling and DNA/RNA repair. Of these, JumonjiC domain-containing histone lysine demethylases (JMJCs), the ten-eleven-translocation (TET) DNA dioxygenases, the ALKB DNA/RNA hydroxylases and the prolyl hydroxylases EGLN1-3 have generated increased interest as potential drug targets for the treatment of a number of pathological conditions, including cancer. Since succinate is a common product to all Fe(II)/2-oxoglutarate-dependent dioxygenases, we determined the substrate specificities, the apparent kinetic constants for several JMJCs and members of the dioxygenase superfamily (i.e.: TET1, EGLN1, ALKBH3 and FTO), as well as inhibition profiles of reported inhibitors using a novel bioluminescent and homogenous succinate detection assay. Our results demonstrate that succinate detection is a useful strategy for the characterization of multiple Fe(II)/2-oxoglutarate-dependent dioxygenases with distinct substrate requirements, enabling the investigation of a large number of enzymes that cannot be evaluated in a miniaturized or high-throughput manner with the methods currently available.

#1400

Using tumor spheroids to evaluate the efficacy of EZH2 inhibitors in clear cell renal cell carcinoma (ccRCC).

Darmood Wei, Youfeng Yang, Christopher J. Ricketts, Carole Sourbier, Laura S. Schmidt, William M. Linehan. _NCI, Bethesda, MD_.

Enhancer of zeste homolog 2 (EZH2) is a key component of the polycomb repressive complex 2 (PRC2). EZH2 is frequently overexpressed in a wide variety of human malignancies including non-Hodgkin lymphoma, gastric cancer, pancreatic cancer, and lung cancer. Thus it has potential to become a therapeutic target. Characterization of EZH2 as a therapeutic target in clear cell renal cell carcinoma (ccRCC) has not been fully explored. ccRCC have been defined by mutation of the von Hippel-Lindau (VHL) tumor suppressor gene in combination with chromosome 3p loss. Recent sequencing efforts have revealed that several chromatin remodeling genes encoded on chromosome 3p are often mutated, of which PBRM1 is the most frequent (41%). The PBRM1 gene codes for the BAF180 protein, a SWI/SNF chromatin remodeling complex subunit. Loss of BAF180 in ccRCC may disrupt the PBAF variant of the SWI/SNF complex. The SWI/SNF complex remodels the chromatin landscape by either sliding or evicting the nucleosomes from the chromatin. This chromatin remodeling modulates the accessibility to promoter regions by transcriptional machinery. It is through this mechanism that the SWI/SNF complex can regulate a range of cellular processes. It has been demonstrated that the SWI/SNF complex can act antagonistically to the PRC2 complex by evicting PRC2 complex from the promoters of tumor suppressors. Disruption of the SWI/SNF complex would impede the eviction of the PRC2 complex, similarly observed in SNF5-deficient malignant rhabdoid tumors. Therefore, we hypothesize that PBRM1 inactivation disrupts specific SWI/SNF complexes allowing EZH2 to bind and repress target tumor suppressor genes. Thus inhibition of EZH2 in ccRCC may present as a targeted therapeutic option in tumors with PBRM1 mutations. We have investigated EZH2 in ccRCC cell lines with PBRM1 mutations and observed that these cells lines have overexpression of EZH2 in comparison to RPTEC (renal cortex proximal tubule epithelium cell line). We examined the effects of two EZH2 inhibitors (GSK126 and EPZ6438) on ccRCC tumor spheroids. Our preliminary data suggests EZH2 inhibition results in reduced growth of PBRM1 mutant cell lines grown as tumor spheroids.

#1401

Deterring breast cancer progression. The critical role of a lipid-sphingolipid epigenetic mechanism of cell fate.

Stefano Rossetti, Vincenzo Gagliostro, Nicoletta Sacchi. _Roswell Park Cancer Inst., Buffalo, NY_.

Genetic and environmental factors cooperate to assure precise genome-wide epigenetic regulation of the mammary epithelia cell transcriptome. Our interest in regulatory epigenetic mechanisms that, by determining mammary epithelial cell decisions, are pivotal to deter the onset and progression of breast cancer, let us identify a regulatory epigenetic mechanism of mammary morphogenesis that links a physiological regulatory lipid of environmental origin (all-trans retinoic acid, RA) with sphingolipids capable of determining either cell death (ceramide, CER) or cell life (sphingosine 1 phosphate, S1P).

Whenever RA, for different reasons, fails to perform the epigenetic transcriptional control of neutral sphingomyelinase 2 (nSMase2/SMDP3), involved in the synthesis of pro-apoptotic CER, S1P fosters both pro-proliferative and pro-invasive activity. Apparently, in the absence of epigenetic control of CER production by RA, S1P contributes to determine RA tumorigenic action. This study provides the rationale for combination therapeutic approaches with epigenetic drugs and inhibitors of either sphingosine kinase, the enzyme involved in S1P synthesis, or S1P receptors.

This study was supported by the NCI R01 CA127614 grant (NS).

### Genomic Instability and Cancer Therapy

#1402

NME3 links mitochondrial fusion to DNA repair in nuclear genome.

Zee-Fen Chang. _National Taiwan University, Taipei, Taiwan_.

In mammals, there are 10 isoforms of NME (NDP Kinase), which catalyze the reversible reaction of NTPs formation from NDP and ATP. By RNA interference, NME3 was found critical for DNA repair. Data from analysis of the steady-state levels of rNTPs and dNTPs suggest that NME3 does not play a critical role in determining the total pools of these nucleotides. However, its mitochondrial localization and catalytic function are necessary for its function in DNA repair. Super-resolution microscopy analysis indicates the localization of NME3 at mitochondrial outer membrane. We further used APEX-mediated biotinylation to identify proteins interacting with NME3. Among them, Mfn1 is validated to associate with mitochondrial NME3 in vitro and in vivo. We provide evidence that NME3 is required for DNA damage-induced mitochondria fusion via GTP-mediated fueling of Mfn1. The lack of this process increased nucleotide oxidation and sustained DNA damage in nuclear genome. These data highlight the function of NME3 in mitochondrial fusion, which is required for the maintenance of genome integrity.

#1403

PCNA-dependent cleavage and degradation of SDE2 regulates response to replication stress.

Ukhyun Jo,1 Winson Cai,1 Jingming Wang,1 Yoojin Kwon,1 Alan D. D'Andrea,2 Hyungjin Kim1. 1 _Stony Brook University, Stony Brook, NY;_ 2 _Dana-Farber Cancer Institute, Boston, MA_.

Maintaining genomic integrity during DNA replication is essential for cellular survival and for preventing tumorigenesis. Proliferating cell nuclear antigen (PCNA) functions as a processivity factor for DNA replication, and posttranslational modification of PCNA plays a key role in coordinating DNA repair against replication-blocking lesions by providing a platform to recruit factors required for DNA repair and cell cycle control. Here, we identify human SDE2 as a new genome surveillance factor regulated by PCNA interaction. SDE2 contains an N-terminal ubiquitin-like (UBL) fold, which is cleaved at a diglycine motif via a PCNA-interacting peptide (PIP) box and deubiquitinating enzyme activity. The cleaved SDE2 is required for negatively regulating ultraviolet damage-inducible PCNA monoubiquitination and counteracting replication stress. The cleaved SDE2 products need to be degraded by the CRL4CDT2 ubiquitin E3 ligase in a cell cycle- and DNA damage-dependent manner, and failure to degrade SDE2 impairs S phase progression and cellular survival. Collectively, this study uncovers a new role for CRL4CDT2 in protecting genomic integrity against replication stress via regulated proteolysis of PCNA-associated SDE2 and provides insights into how an integrated UBL domain within linear polypeptide sequence controls protein stability and function. Knowledge on such mechanism will be useful to identify novel cancer therapeutic interventions exploiting deregulated ubiquitin signaling and SDE2 activities in cancer.

#1404

Regulation of 53BP1 by the structural nuclear protein NuMA.

Naike Salvador Moreno. _Wake Forest Baptist Medical Center, Winston Salem, NC_.

Cells repair DNA double-strand breaks (DSB) through two major pathways: homologous recombination (HR) and non-homologous end joining (NHEJ). The choice between these two pathways is critical to cell survival and is altered in cancers. 53BP1 is a protein with an important role in this choice: it promotes NHEJ by blocking CtIP-dependent DNA resection. Here, we present a new negative regulation mechanism of 53BP1 that relies on the structural protein NuMA. This protein participates in mitotic spindle assembly, influences chromatin organization during the interphase and modulates the chromatin response to DNA damage. We identified NuMA in a proteomics analysis of a 53BP1-interacting proteins. The interaction was confirmed by reciprocal immunoprecipitation and with a FRET assay. In response to DSB, we measured greater than 50% decreased 53BP1-NuMA binding, which suggests that NuMA may restrain 53BP1 diffusion in the absence of DNA damage. Fluorescence correlation spectroscopy (FCS) measurements in cells expressing GFP-tagged 53BP1 support this hypothesis. Moreover, NuMA overexpression inhibited 53BP1 recruitment at DNA damage sites in laser microirradiation assays, whereas NuMA silencing had the opposite effect. 53BP1 plays an essential role in B cell class switch recombination and mediates PARP inhibitor sensitivity in BRCA1-null cells. NuMA overexpression prevented immunoglobulin switching, and overwrote cancer cell sensitivity to PARP inhibitors to the same extent as 53BP1 loss of function. Our results help understand how 53BP1 is controlled, and shed light on the mechanisms regulating PARPi sensitivity.

#1405

A novel chemotherapeutic agent to treat tumors with DNA mismatch repair deficiencies.

Kyungjae Myung,1 Yongliang Zhang,2 Young-Un Park1. 1 _Institute for Basic Science, Ulsan, Republic of Korea;_ 2 _NIH, MD_.

Impairing the division of cancer cells with genotoxic small molecules has been a primary goal to develop chemotherapeutic agents. However, DNA mismatch repair (MMR)-deficient cancer cells, are resistant to most conventional chemotherapeutic agents. Here we have identified baicalein as a small molecule that selectively kills MutSα-deficient cancer cells. Baicalein binds preferentially to mismatched DNA and induces a DNA damage response in a mismatch repair-dependent manner. In MutSα-proficient cells, baicalein binds to MutSα to dissociate CHK2 from MutSα leading to S phase arrest and cell survival. In contrast, continued replication in the presence of baicalein in MutSα-deficient cells results in a high number of DNA double-strand breaks and ultimately leads to apoptosis. Consistently, baicalein specifically shrinks MutSα-deficient xenograft tumors and inhibits the growth of AOM-DSS-induced colon tumors in colon-specific MSH2 knockout mice. Collectively, baicalein offers the potential of an improved treatment option for patients with tumors with a DNA MMR deficiency.

#1406

Towards an RNA expression-based signature for oncogene-induced replication stress.

Sergi Guerrero, Rudolf Fehrmann, Marcel ATM van Vugt. _University Medical Center Groningen, Groningen, Netherlands_.

Introduction Certain tumor subgroups, including triple negative breast cancers (TNBCs), are characterized by high levels of genomic instability (GI). These tumors are typically very aggressive and difficult to threat. Increasingly, we realize that GI is fueled by replication stress (RS). RS can be induced by expression of oncogenes, including CCNE1, MYC and CDC25A, which perturb initiation, elongation and/or termination phases of DNA replication. RS poses a threat to cellular viability, and tumor cells with high levels of RS will therefore increasingly depend for their survival on DNA damage checkpoint and repair pathways. Possibly, this increased dependence creates therapeutic opportunities. Unfortunately, it is currently difficult to determine levels of RS in tumors, a requirement to efficiently select patients for novel treatments. The aim of this work is to develop an RNA expression-based signature for oncogene-induced replication-stress.

Material & Methods A panel of cell lines (RPE-1, RPE-1-P53-/-, MDA-MB-231, BT-549 and HCC-1806) was engineered to doxycycline-inducibly express a set of oncogenes (CDC25A, CCNE1 or MYC). Single DNA fiber replication analysis in combination with immunoblot analysis of RS markers was used to validate the cell line models. MTT assays were performed to assess sensitivity to ATR and Wee1 inhibitors. RNA-sequencing was used to uncover expression changes upon RS induction.

Results Treatment with doxycycline resulted in high expression levels of CDC25A, CYCLINE or MYC. Expression was heterogeneous but present in the far majority of RPE-1 cells (40% CDC25A, 88% CCNE1 and 93% MYC). DNA fiber analysis was subsequently used to study the alteration of DNA replication dynamics. Severe reduction on ongoing DNA synthesis velocity was shown in engineered cells (RPE-1 and MDA-MB231) under doxycycline treatment, confirming prominent RS. In line with elevated levels of RS, oncogene induction resulted in elevated levels of ATR activity, as judged by phospho-CHK1 and phospho-RPA levels. Importantly, oncogene-expression enhanced sensitivity to Wee1 and ATR checkpoint inhibitors. Subsequently, RNA samples were harvested under the presence and absence of doxycycline. In parallel to oncogene-expression, hydroxyurea, a chemical agent known to increase stalled replication forks was applied as a DNA damage control. RNA-sequencing analysis will be performed to identify differentially expressed genes upon oncogene expression. Commonly identified genes over multiple cell lines and shared between the different oncogenes will be used to build a 'RS' profile.

Conclusion Our results indicate that oncogene expression provokes RS in multiple cell line models and induces impaired replication fork stability and sensitivity to cell cycle checkpoint inhibitors. These models are therefore valid to develop an RNA expression-based qualifier in order to facilitate patient selection towards novel therapeutics.

#1407

Translational regulation of RPA2 via IRES by UNR and eIF3a.

Jia-Jia Cui, Lei-Yun Wang, Ji-Ye Yin. _Department of Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha, China_.

Purpose:

Translation is a critical step in the process of genetic information expression. Internal ribosome entry site (IRES) element is a RNA sequence with a complex structure, which plays an important role in cap independent translation regulation. The activation of RPA2 IRES element can cause its abnormal expression and finally effects DNA repair pathway. We conducted series of assays to explore the mechanism of translation initiation regulation of RPA2 via IRES by UNR and eIF3a

Methods:

Biotin pull down assay was taken to investigate the physical interaction between RPA2 IRES and UNR. UNR was knocked down and overexpressed in H1299, A549 and SK-MES cell lines. Western blot and real-time PCR were used to detect protein level and mRNA level respectively. For interaction of eIF3a with UNR, CO-IP assay and co-localization assay were conducted. And GST pull down assay was carried out to further identify their exact binding domains. RPA2 IRES-interacting domains of UNR and eIF3a were explored with EMSA assay.

Results:

UNR protein could directly bind to RPA2 IRES as well as to eIF3a, and UNR regulated the protein expression of RPA2 in H1299, A549 and SK-MES cells, while the mRNA level of RPA2 remained no change. UNR interacted with the first domain of eIF3a and with RPA2 IRES via its own first domain. However, we have not found a clue for the direct interaction of eIF3a with RPA2 IRES yet.

Conclusion:

UNR cooperated with eIF3a to regulate the RPA2 IRES activity and hence to regulate the RPA2 protein expression. This might be a regulation mechanism of cellular internal ribosomal entry site affecting translation initiation, the rate-limiting step of translation.

#1408

LIM protein Ajuba participates in the ATR response by direct interaction with RPA70.

Sandy Wan S. Fowler,1 Diego Loayza,1 Pascal Maguin2. 1 _Hunter College, Graduate Center City University of New York, New York, NY;_ 2 _Rockefeller University, New York, NY_.

DNA damage response (DDR) pathways are essential for genome stability and cell survival. Specifically, the ATR kinase is activated by DNA replication stress or UV irradiation. An early event in this activation is the phosphorylation and local recruitment of RPA, a single strand DNA binding protein. Our lab has shown that the LIM protein Ajuba associates with RPA, an OB fold-containing protein, and this interaction prevents an unscheduled ATR response. We hypothesize that Ajuba represses the ATR pathway through direct interaction with RPA, thereby preventing activation of ATR. To test this hypothesis, we employed E. coli and in vitro translation system for the production of recombinant proteins. Our data show that Ajuba directly associates with the large subunit of RPA, RPA70. The domains involved in the interaction were the C-terminal OB folds of RPA70, and the region between the PreLIM and LIM domains in Ajuba. We speculate that this interaction mediates the association of Ajuba with the RPA complex in cells. Understanding the implication of the Ajuba and RPA interaction will allow us to gain insights on the regulation of DDR, which is involved in early events of cellular transformation and tumorigenesis in human cells.

#1409

Analysis of 16,172 patient-derived tumor samples indicate TPX2 as being essential for survival of genomically instable cancer cells.

Stephanie E. van Gijn,1 Elles Wierenga,1 Anne Margriet Heijink,2 Rudolf S. Fehrmann,1 Marcel A. van Vugt1. 1 _University Medical Center Groningen, Groningen, Netherlands;_ 2 _European Research Institute for the Biology of Ageing, Groningen, Netherlands_.

Mutations in homologous recombination (HR) genes, including BRCA1 and BRCA2, compromise DNA repair and lead to genomic instability (GI). GI is lethal to normal cells but is a characteristic of many cancers. Apparently, these cancers are somehow re-wired to survive high levels of GI. Identification of the genetic alterations that allow viability of genomically instable tumor cells may uncover novel therapeutic targets.

To elucidate how these tumor cells are rewired, we analyzed publically available mRNA expression data of 16,172 human cancer samples. Functional genomic mRNA profiling (FGmRNA-profiling) was applied on these samples to infer levels of GI and to capture the downstream effects of somatic copy number alterations on gene expression. A genome-wide association analysis was subsequently performed to assess the correlation between FGmRNA signals of individual genes with the degree of GI. From the top 250 genes with strong positive correlation with GI, 11 genes were prioritized based on a co-functionality network in which genes are co-regulated and share similar predicted biological function. The 11 genes that were identified in this cluster were: BIRC5, UBE2C, CENPA, CDCA3, DEK, SKP2, TPX2, KIF2C, RAD21, MYBL2 and WDR67.

To validate these findings in genetically-defined models, we engineered a panel of 5 triple negative breast cancer (TNBC) cell lines with doxycycline-inducible shRNAs targeting BRCA2. BRCA2 depletion resulted in a failure of RAD51 foci to localize to DNA double strand breaks which generated isogenic cell line pairs proficient and deficient of HR repair. First, we depleted each of the identified 11 genes using RNA interference in BT-549 cells and observed that depletion of TPX2, a microtubule-associated protein, led to largest differential levels of cell death when comparing the BRCA2-deficient with the BRCA2-proficient context (86.6% vs 32.9% cell death in BRCA2-depleted vs controlled depleted cells respectively). Subsequently, we could replicate this decreased survival with TPX2 depletion in a BRCA2-deficient context in an additional 2 out of 4 other TNBC cell lines.

Furthermore, we investigated whether BRCA2-depleted cells were also more sensitive to depletion of Aurora kinase A, a substrate of TPX2. For this purpose, mouse mammary tumor cell lines, derived from Tp53-/- or Brca2-/-;Tp53-/- mice, or a Brca2-reconstituted version thereof were treated with an Aurora A inhibitor, Alisertib. Again, we found that the BRCA2-deficient cell line was more sensitive to Aurora A inhibition than the two BRCA2-proficient cell lines.

In conclusion, FGmRNA-profiling of mRNA expression data of human cancer samples identified TPX2 as an essential gene for survival of BRCA2-deficient breast cancer cells, when compared to BRCA2-proficient cells. Thus, targeting the TPX2/AURKA axis could potentially act as a novel therapeutic target in the treatment of genomically instable cancers.

#1410

Proliferation during epithelial-to-mesenchymal transition induces genomic instability.

Valentine Comaills, Lilian Kabeche, Robert Morris, Min Yu, Marissa Wells Madden, Joseph A. LiCausi, Nicola Aceto, Yu Zheng, David T. Miyamoto, Sridhar Ramaswamy, Lee Zou, Daniel A. Haber, Shyamala Maheswaran. _Massachusetts General Hosp. Cancer Ctr., Charlestown, MA_.

Epithelial to mesenchymal transition (EMT), a morphogenetic process required for proper embryonic development, is adopted by cancer cells during tumor progression. We show that induction of EMT by TGFß or other EMT-inducers such as Snail leads to genomic instability, associated with failed cytokinesis and chromosome missegregation resulting in aneuploidy and polyploidy. These defects are dependent on persistent proliferation of cells undergoing EMT and are absent in normal cells that growth arrest during EMT. While EMT and the associated mitotic abnormalities are reversible upon removal of the EMT-inducer, the resulting chromosomal abnormalities are inherited. TGFß-induced genomic instability is associated with the acquisition of tumorigenic phenotypes and the resulting tumors are enriched for a genomically-altered tumor cell population, which exhibits differential drug sensitivity. In breast and prostate cancer cells circulating in the blood, TGFß and EMT gene signatures are significantly correlated with aneuploid gene signatures. Analysis of single circulating tumor cells (CTC) from women with metastatic breast cancer reveals increased in genomic instability in mesenchymal CTCs. Together, these findings identify a novel mechanism whereby tumor microenvironment-derived signals impact heritable genetic changes within cancer cells.

#1411

Differential mismatch repair-dependent damage responses in subpopulations of cells from human intestinal organoids.

Bo Lin, Qingfen Yang, Abhijit Rath, Christopher D. Heinen. _University of Connecticut Health, Farmington, CT_.

Hereditary mutations in DNA mismatch repair (MMR) genes cause the cancer predisposition syndrome called Lynch syndrome. Patients predominantly develop colorectal cancer and some extracolonic cancers at a young age. The MMR pathway repairs mismatches created during DNA replication, but also can induce apoptosis and cell cycle arrest in response to certain DNA damaging agents as a protective mechanism to eliminate cells at risk of accumulating mutations. Our understanding of this MMR-directed DNA damage response has come primarily from studies in human cancer cell lines. We wished to examine the role of the MMR-dependent damage response in a model that may be more relevant to the human intestine. Therefore, we have created human intestinal organoids (HIOs) in vitro either by directing the differentiation of human embryonic stem cells (hESCs) or through culturing of adult intestinal tissue samples. We have used these HIOs to specifically study the MMR dependent damage response to DNA alkylation damage. We have found that whereas hESCs undergo rapid apoptosis in response to alkylation damage, the differentiated HIOs or those created from adult tissues undergo not only apoptosis, but also senescence and accelerated differentiation following damage. Using CRISPR-Cas9 gene editing in hESCs, we also created MMR-deficient HIOs that failed to respond to the drug indicating that these responses are MMR-dependent. We then determined that the cells undergoing apoptosis following DNA damage are most likely the intestinal stem cells suggesting that different cell types in the intestine have different MMR-dependent responses to damage. Together these results suggest that loss of MMR function in the intestinal crypts may provide a selective advantage particularly in an environment conducive with DNA damage that promotes tumorigenesis.

#1412

High EGFR somatic mutation frequency targeting the TK domain in colorectal cancer patients with microsatellite instability (MSI).

Safoora Deihimi,1 Michael Slifker,2 Eric A. Ross,2 Wafik S. El-Deiry1. 1 _Department of Hematology/Oncology and Molecular Therapeutics Program, Fox Chase Cancer Ctr., Philadelphia, PA;_ 2 _Department of Biostatistics, Fox Chase Cancer Ctr., Philadelphia, PA_.

CRCs arise through genetic changes that impact various driver genes and in some tumors increased mutation rate in microsatellite unstable tumors. The hypermutable phenotype associated with microsatellite instability (MSI) results from loss of the mismatch repair system (MMR) activity. MSI is detected in 15% of all CRCs, and such tumors have a better prognosis and different chemotherapeutic outcome patterns, including high clinical benefit from immune checkpoint therapy or relative resistance to 5-FU as compared to microsatellite stable (MSS) tumors. The epidermal growth factor receptor (EGFR) signaling pathway plays an essential role in carcinogenesis of CRCs and is known to be overexpressed in MSI CRCs while highly mutated in Lung cancer. We analyzed the mutation frequency of deregulated pathways including EGFR and its downstream genes KRAS and BRAF in CRCs. We found a significantly elevated EGFR mutation frequency in CRC MSI-H subtype (45.5% vs. 6.5% in MSS CRCs, p<0.0000001). Although KRAS and NRAS are mutated with high frequency in both MSI-H and MSS groups, BRAF corresponding to RTK-RAS pathways is more altered in MSI-H than MSS CRCs (32.67% vs 13.10%; p=0.001), consistent with the known association between MSI-H CRCs and BRAF mutations. We hypothesized that there might be a mutation pattern in EGFR in CRC subtypes that provide a rationale for EGFR-targeted therapy for a subtype of CRC. Of 1104 profiled CRCs in the COSMIC v73 database, somatic EGFR mutations were mapped for 101 MSI-High versus 916 MSS CRCs. EGFR mutations mapped on the protein structure revealed that mutations are mainly targeting tyrosine kinase (TK) domain while the extracellular domain remained mostly wild-type with potential for targeting by anti-EGFR monoclonal antibodies. It is known that downstream genes such as KRAS as well as expression levels of EGFR ligands can affect the sensitivity of CRCs to anti-EGFR therapy. We didn't detect any difference in mRNA expression level between MSI-H and MSS group but our analysis is indicative of the presence of G12D mutations in a high proportion of KRAS-mutated MSS CRCs, therefore resistance to anti-EGFR antibodies such as cetuximab would be expected. However, EGFR tyrosine kinase inhibitors such as erlotinib and gefitinib could be considered for further testing in patients with MSI-H tumors that have a lower frequency of G12D KRAS mutations and may have mutant EGFR.

#1413

Replication obstacles formed within common fragile sites under replication stress are targeted by the global genomic nucleotide excision repair pathway.

Martin Mistrik,1 Lucie Beresova,1 Eva Vesela,1 Ivo Chamrad,2 Jiri Voller,1 Masayuki Yamada,1 Tomas Furst,1 Rene Lenobel,2 Katarina Chroma,1 Jan Gursky,1 Jiri Bartek1. 1 _Palacky University in Olomouc, Faculty of Medicine and Dentistry, Olomouc, Czech Republic;_ 2 _Palacky University in Olomouc, Faculty of Science, Olomouc, Czech Republic_.

CFS's are genomic loci present in higher vertebrates that are particularly sensitive to various forms of replication stress and suffer from increased breakage and rearrangements in tumors. They have rather enigmatic evolutionary role and employ various DNA repair and checkpoint mechanisms promoting their stability. We used an original approach for identification of CFS's associated factors based on DNA probe designed to match the high flexibility island sequences typically present in some of the highly expressed CFS's. This probe was used as affinity bait for fishing specifically enriched proteins which were further identified using SILAC and quantitative mass spectrometry. Among already known CFS's stabilizers we identified also hits so far not implicated in CFS's maintenance. Interestingly, most of these novel hits are components of the global genomic nucleotide excision repair pathway (GG-NER). Knock down-based functional experiments revealed that GG-NER works most likely as an important trigger turning the CFS's-associated replication obstacles into DNA lesion further recognized by the ATR-promoted cellular checkpoint which function is to block an escape of DNA replication intermediates into mitosis and the next cell generation. Work was supported by the Grant Agency of the Czech Republic 13-17555S, Czech-BioImaging project (LM2015062 funded by MEYS, CR) and Ministry of Education of the Czech Republic (LO1304).

#1414

Chromatin remodeling histone chaperone FACT complex modulates AP site damage repair in chromatin and sensitizes cancer cells to chemotherapeutic drugs.

Heyu Song, Shrabasti Roychoudhury, Dan Feng, Kishor Bhakat. _University of Nebraska Medical Center, Omaha, NE_.

Recognition and repair of DNA lesions in the genome are critical for maintaining genomic stability and reducing the generation of mutations that lead to cancer development. The most frequently formed DNA lesion in the genome is the apurinic/apyrimidinic (AP) site which is mutagenic and blocks transcription and replication. The primary enzyme to repair AP sites in mammalian cells is the AP endonuclease (APE1), which often overexpresses in diverse cancer types and its overexpression is associated with patients' resistance to chemotherapeutic drugs. The repair of AP sites by APE1 through the Base Excision Repair (BER) pathway is extensively investigated in vitro. However, how AP site is recognized by APE1 in the context of highly complex nucleosome structure in chromatin is unknown. Because DNA is packaged tightly in nucleosome, the ability of repair proteins to access sites of DNA damage and facilitates repair of the damage requires chromatin remodeling activities. Here, we show that APE1 interacts with chromatin remodeling histone chaperone complex, FACT (facilitates of chromatin transcription) via its N-terminal domain in cells. By immunoprecipitation of endogenous APE1 from chromatin fraction and separation of protein bands in SDS-PAGE followed by MALDI-TOF-TOF analysis, we have identified both subunits (SPT16 & SSRP1) of FACT as the prominent APE1 interacting partners. Subsequently, we confirmed the interaction of APE1 with FACT complex by Co-IP and immunofluorescence analysis and found that both SPT16 and SSRP1 interact with APE1 in the nucleus and in chromatin. Interestingly, we found rapid eviction of histones with concomitant degradation of FACT complex upon induction of DNA damages. Downregulation of FACT complex abrogates the nucleosome eviction, the recruitment of repair protein APE1 in chromatin and the repair of AP sites, demonstrating the functional importance of nucleosomes disruption in BER pathway and identifying chromatin remodeling protein required for the process. Notably, knock-down of FACT showed increased sensitivity of cancer cells to many chemotherapeutic drugs. We also found that FACT down-regulated cells have much higher accumulation of AP site damages in the genome compared to control cells. Our study revealed a key role of nucleosome remodeling complex FACT in DNA damage repair in BER pathway. This study also suggests that histone chaperone complex could be a potential target for enhancing sensitivity of tumor cells to chemotherapy.

#1415

SAN1: a novel senataxin associated nuclease required for the repair of interstrand crosslinks.

Alex Andrews, Ian Macara. _Vanderbilt University, Nashville, TN_.

The DNA damage response (DDR) is a set of complex signaling pathways capable of sensing DNA damage, and activating a large number of enzymes involved in the remodeling and repair of the genome. Mutations in the genes involved in the DDR lead to DNA damage, genomic instability, and various cancers. One particularly dangerous type of DNA damage that can occur is an interstrand crosslink (ICL). ICLs can lead to the development of double strand breaks through the blockage of DNA replication and transcription. Although ICLs can arise endogenously from molecules such as aldehydes, most commonly they are induced from chemotherapeutic drugs such as Cisplatin and Mitomycin C (MMC). These drugs are commonly used in the treatment of breast and ovarian cancers. A better understanding of which proteins are involved in the repair of ICLs is critical for understanding resistance, toxicity, and response in patients treated with ICL inducing agents. The repair of ICLs requires the coordination of several DNA repair pathways including the Fanconi Anemia pathway, homologous recombination (HR), and nucleotide excision repair (NER). The Fanconi Anemia pathway is essential for the repair of these lesions as it is responsible for the recognition of the ICL lesion, as well as the recruitment of several nucleases responsible for unhooking and removal the cross-linked nucleotides. Recently, we identified an uncharacterized 5' nuclease that interacts with the RNA/DNA helicase Senataxin, which we have named senataxin-associated nuclease 1 (SAN1). Senataxin has been shown to act on R loops, RNA/DNA hybrids that are a source of endogenous DNA damage. Deletion of the SAN1 gene in HeLa cells or in mouse embryonic fibroblasts leads to the sensitization of cells to Cisplatin and Mitomycin C (MMC), but not to ionizing radiation that induces double strand breaks. Importantly, the defect in ICL repair can be restored using WT SAN1 but not with a mutant that is catalytically inactive. Treatment of SAN1 -/- HeLa cells with MMC also leads to radial chromosome formation, a characteristic of cells deficient in ICL repair. Additionally, SAN1 partially colocalizes with Fancd2 MMC induced foci, and treatment with MMC results in increased DNA damage and R loops in SAN1-/- cells. In conclusion, this study highlights the discovery of a novel nuclease involved in the repair ICLs, a process critical for understanding resistance and response to chemotherapies such as Cisplatin and MMC. Future work is aimed at determining the specific step of ICL repair that SAN1 participates in, how and when during repair SAN1 is recruited to ICL sites, and which other DNA repair proteins SAN1 functions with.

#1416

Development of small molecule inhibitors for cancer therapy by targeting RPA and XPA nucleotide excision repair proteins.

Navnath S. Gavande,1 Pamela S. VanderVere-Carozza,1 Tyler L. Vernon,1 Katherine Pawelczak,2 John J. Turchi1. 1 _Indiana University School of Medicine, Indianapolis, IN;_ 2 _NERx BioSciences Inc, Indianapolis, IN_.

Targeting DNA repair and the DNA damage response for cancer therapy has recently gained increasing attention with inhibitors of the PARP enzyme showing a therapeutic efficacy in various cancers. Solid tumors of the lung, pancreas, breast, and ovary represent a continuing clinical challenge in treatment and together account for over 250,000 deaths in the US alone, representing over 40% of all cancer deaths. There are limited therapeutic options for these patients, and targeted and combination therapies remain necessary for treating these aggressive cancers. The opportunity exists to exploit recent scientific advances in our knowledge of the underlying biology behind these cancers to create novel targeted therapeutics to dramatically enhance patient response to therapy and ultimately survival. To this end, we have developed a series of novel small chemical molecules that disrupt critical protein-DNA interactions in the nucleotide excision repair (NER) pathways. It is well understood that various cancer treatments like cisplatin, etoposide and ionizing radiation impart their chemotherapeutic effect by the formation of direct DNA damage which block DNA replication and transcription culminating in apoptosis. It is also well established that repair of this DNA damage by nucleotide excision repair (NER) or homologous recombination repair (HRR) reduces the effectiveness of chemo- or radio- therapy. Replication protein A (RPA) and Xeroderma Pigmentosum Group A (XPA) plays a crucial role in the NER pathway and makes them a novel drug target to develop novel cancer therapy. We anticipate both direct mechanisms of action on the repair pathways and synthetic lethal interactions can be exploited for therapeutic benefit. The series of novel small molecule inhibitors that we have developed targeting RPA and XPA proteins independently exhibit single-agent anti-cancer activity in cancer cell lines, and potentiate cellular sensitivity to chemotherapeutic agent. Data demonstrate that these novel inhibitors do not interact with DNA but directly bind the corresponding NER proteins. Our data demonstrate that this class of inhibitors can be further developed as an anti-cancer therapeutic with considerable potential to be used in conjunction with radiation therapy and other cancer therapies that induce DNA damage.

This works was supported by NIH grants R01-CA180710 and R41-CA162648 and the Tom and Julie Wood Family Foundation.

#1417

Expression of BARD1δ induces genetic instability and is associated with cervical carcinogenesis.

Maxim Pilyugin,1 Pierre-Alain André,1 Nicole Concin,2 Gerda Hofstetter,2 Irmgard Irminger-Finger1. 1 _University of Geneva, Geneva, Switzerland;_ 2 _University Hospital Innsbruck, Innsbruck, Austria_.

Cervical cancer is always linked to infection with the human papilloma virus (HPV), which causes hyper-proliferation of cervical epithelial cells and neoplasia and cervical cancer. The viral oncoprotein interaction with host tumor suppressor proteins partially explains the uncontrolled proliferation, which ultimately progresses to cervical cancer. However, little is known about the specific factors that promote or inhibit the malignant transformation. Since a portion of HPV infections never evolves into cancer, the identification of discriminatory factors would be an important step towards the development of prognostic and therapeutic tools.

Based on this notion, we investigated whether an isoform of BARD1, BARD1δ, which we found frequently expressed in cancer cells, plays a role in the malignant transformation of cervical epithelial cells. Using immunohystochemistry staining on tissue sections and RT-PCR we found that BARD1δ is expressed in proliferating cervical cancer cells, while normal BARD1 is repressed. BARD1 might be a driver of carcinogenesis and might be a critical event in the progression from hyper-proliferating cervical epithelium after HPV infection to cervical cancer.

Together, our results show that an isoform of BARD1, BARD1δ, is increasingly expressed in precancerous stages of cervical cancer and the specific inhibition of BARD1δ may represent a novel approach for therapeutic interventions to prevent cervical carcinogenesis after HPV infection.

#1418

G9a methyltransferase plays a role in ATM-dependent DNA damage response.

Lizahira Rodriguez-Colon,1 Vasudeva Ginjala,2 Atul Kulkarni,2 Safia Ansari,1 Shridar Ganesan2. 1 _Rutgers University, New Brunswick, NJ;_ 2 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ_.

Induction of DNA damage leads to a choreographed set of local chromatin changes that ensures an efficient recruitment of DNA repair factors. One principal regulator of the DNA Damage Response (DDR) signaling pathway is ATM kinase, which phosphorylates key factors at early stages of the response. G9a protein methyltransferase has been identified as a novel substrate for ATM. We have found that G9a localizes to sites of DNA damage in an ATM-dependent fashion and that inhibition of its activity affects recruitment of multiple DNA repair factors. Moreover G9a catalytic inhibition using UNC0638 leads to hyperactivation of ATM induced by DNA breaks. This was associated with an increased ATM-dependent "spreading" of pH2AX and MDC1 signals seen at regions of localized DNA breaks induced by UV-laser scissors. These data suggest that G9a activity is required for regulating the extent of ATM activation as well as for efficient recruitment of downstream DNA repair factors. Biochemical data will be presented that explore potential mechanisms for these findings. Overall our data suggests that G9a plays a critical role in the regulation of ATM-dependent signaling during the DNA damage response, and raises the possibility of using G9a inhibitors to target cancers with certain DNA repair defects.

#1419

Role of a novel Senataxin-associated nuclease in DNA repair.

Heather McCartney, Ian Macara. _Vanderbilt University, Nashville, TN_.

Yeast two-hybrid analysis has identified a novel, uncharacterized Senataxin-interacting protein with 5' exonuclease activity, and homology to the FEN1 nuclease domain. Senataxin is an RNA/DNA helicase that functions in resolving R loop structures which occur as a result of normal replication and transcription but can also persist resulting in genomic instability. We found that SAN1 (Senataxin-Associated Nuclease 1) is unable to cut dsDNA or bubbles, but is active against 5' overhangs and against ssDNAs of greater than 15 nucleotides. SAN1 cleaves 3 or 8 nt fragments from the 5' end of ssDNA and utilizes magnesium as a cofactor. As the definitive 5' nuclease responsible for unhooking ICLs has yet to be identified we speculate that SAN1 might function in excising crosslinked nucleotides that result from interstrand crosslink (ICL) damage. We generated a SAN1 -/- HeLa cell line through CRISPR/Cas gene editing and found that SAN1 -/- cells are sensitized to ICL agents mitomycin-c and cisplatin. Interestingly, colony survival assays have shown that SAN1 does not appear to be epistatic of FANCD2, a key component of the classical pathway for resolving ICLs, the Fanconi Anemia pathway. Additionally, although the nuclease domain of SAN1 is homologous to the FEN1 family of structure-specific nucleases, SAN1 also possesses a unique and conserved C terminus of unknown function. Our goal is to understand the relationship between the structure and nuclease activity of SAN1 in DNA repair and how SAN1 relates to other known ICL-repair pathways. Understanding the components that regulate genome stability is crucial in understanding how cancer occurs and what protein targets will be the most advantageous to pursue in developing potential therapeutics. This study may also provide a better understanding of a novel factor in resistance to ICL-inducing chemotherapeutics such as cisplatin and MMC which are still widely used in the treatment of breast and ovarian cancers.

#1420

The endonuclease Metnase promotes base excision repair of Clustered abasic DNA lesions.

Aruna S. Jaiswal, Elizabeth A. Williamson, Bhavita Patel, Gayathri Srinivasan, Satya Narayan, Robert A. Hromas. _University of Florida, Gainesville, FL_.

Metnase, a human SET-transposase fusion protein contains two functional domains: a SET domain and transposase domain. Transposase domain exhibit strand transfer and end joining activity while set domain is responsible for histone lysine methyltransferase activity (KMT). Metnase also increases the efficiency of double strand break repair by non-homologous end joining (NHEJ); however, its role in the excision of clustered DNA damage remains to be investigated. We hypothesize that 5'-3' endonuclease activity of metnase could possibly substitute for APE1 in BER and serve as an alternative initiator of BER in tumor cells lacking APE1. In the present study, we examined in vitro endonuclease activity of Metnase using abasic site containing artificial DNA, and describe its activity in BER. Its unique 5' endonuclease activity was selective only for abasic lesions, but not other base modifications such as 8-oxoguanine, uracil, hypoxanthine or xanthenes. Metnase specifically initiates removal of reduced abasic lesions from DNA, and allows completion of short-patch or long-patch BER. APE1 has difficulty cleaving 5' of clustered abasic lesions. This endonuclease that promotes BER in clustered oxidative DNA damage is not known. Metnase also cleaves multiple abasic lesions (clustered DNA damage) and facilitates the repair of DNA if the multiple DNA damages are 3 or more nucleotides apart on the opposing strands. However, repair of multiple DNA damages remains inefficient if these are in close proximity of each other (< 3nt apart on the opposing strands) and results in DNA double strand break (DSB). These results suggest that Metnase can promote BER of oxidative nucleotides, where APE1 is unable to function.

#1421

Evaluation of DDR-targeting strategy using ATR inhibitor in biliary tract cancer.

Ah Rong Nam,1 Ji Eun Park,1 Ju Hee Bang,1 Mei Hua Jin,1 Do Youn Oh,2 Yung Jue Bang2. 1 _Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea;_ 2 _Deparment of Internal Medicine, Seoul National University of Hospital, Seoul, Republic of Korea_.

Background: The DNA damage response (DDR) is a multicomplex network of signaling pathways involved in DNA damage repair, cell cycle checkpoints and apoptosis. The ataxia telangiectasia and Rad3-related (ATR) protein kinase is a key enzyme in the DDR that activates checkpoint kinase 1 (Chk1), resulting in cell cycle arrest. Tumor types with loss of ATM function and/or high replication stress are expected to be more susceptible to DDR targeting. In biliary tract cancer (BTC), DNA repair pathway, which includes BAP1, MSH6, BRCA1, ATM, MLH1, MSH2, is altered in about 20 % of cases. TP53 module is observed in 33.9% of BTC cases (Nat Genet 2015). The purpose of this study is to test DDR targeting strategy using ATR inhibitor in biliary tract cancer.

Methods: Using 9 kinds of BTC cells, MTT assay and colony formation assay were done for determining growth inhibitory effect of AZD6738, an ATR inhibitor. Cell cycle analysis was done by FACS Calibur flow cytometer and the methods described by Chou and Talalay were used to determine whether a synergistic effect existed between AZD6738 and cytotoxic chemotherapeutic agents (cisplatin, 5-FU, gemcitabine). The alkaline comet assay was done to measure of DNA damage in individual cells. Tumor xenografts model was used for in vivo test of AZD6738.

Results: Among 9 BTC cells, SNU478 and SNU869 were most sensitive to AZD6738, which showed low expression of both ATM and p53. AZD6738 blocked ATR-mediated Chk1 phosphorylation and increased rH2AX, a marker of DNA damage, in sensitive cells. AZD6738 significantly increased apoptosis (cleavage of PARP and caspase-7) and G2/M arrest, increased level of p21, and decreased cdc2. In addition, combination of AZD6738 and cytotoxic chemotherapeutic agents demonstrated synergistic effects in colony formation assay, cell cycle analysis and comet assay. In xenograft model of SNU478, AZD6738 monotherapy decreased tumor growth. The combination of AZD6738 and cisplatin showed more potent growth inhibitory effects, decreased Ki67, increased Tunel than monotherapy of each drug.

Conclusion: In BTC, DDR targeting strategy using ATR inhibitor demonstrated promising antitumor activity alone or in combination with cytotoxic chemotherapeutic agents. This supports further clinical development of DDR targeting strategy in BTC.

#1422

Enhancing the therapeutic effects of PARP inhibitors in combination DNA methyl transferase inhibitors, using low doses of ionizing radiation in non small cell lung cancers.

Christopher Biondi,1 Daniel Fontaine,1 Lora Stojanovic,1 Pratik Nagaria,1 Rena Lapidus,1 Eun Yong Choi,1 Javed Mahmood,1 Stephen Baylin,2 Feyruz V. Rassool1. 1 _Univ. of Maryland School of Medicine, Baltimore, MD;_ 2 _Johns Hopkins University, Baltimore, MD_.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths in the US. Treatment most commonly relies on ionizing radiation (IR) and platinum-based DNA damaging agents, but long-term survival is poor and patients tend to suffer chronic side-effects due to the high radiation dose to the surrounding normal tissues. Therefore, new treatments are needed that can be used in combination lower radiation doses.

We have recently reported that low, non-cytotoxic doses Poly ADP ribose polymerase inhibitors (PARPi) Talazoparib in combination with DNA methyltransferase inhibitors (DNMTi) Decitabine (DAC) or azacytidine (AZA) significantly increase cytotoxicity in acute myeloid leukemia and breast cancer models in vitro and anti-tumor effects in vivo. Simultaneous administration of both inhibitors result in increased PARP binding in DNA, leading to higher levels of DNA double strand breaks (DSBs), yielding increased cytotoxicity, compared with each agent treatment alone.

We first studied the efficacy of Talazoparib and AZA combination therapy in multiple NSCLC cell lines (A549, H358 and H838) in vitro through colony forming assays. Results showed, compared to single agent treatments, combination drug treatment significantly decreased colony formation. Cell viability was also significantly decreased with the drug combination in MTS assays (P<0.05). These results also showed synergistic activity between the two drugs, with a combination index of less than 1 for all tested NSCLC cell lines. Importantly, the drug combination increased PARP trapping in chromatin and DSB formation, as measured by immunofluorescent staining for γH2AX. We next determined whether AZA/Talazoparib in combination with a single dose of radiation (2Gy) had an increased anti-cancer effect compared to each modality alone in in vivo mouse xenografts of NSCLC A549. While the combination of PARPi+DNMTi with radiation treatment decreased tumor growth, compared to PARPi/DNMTi alone or RT alone, no significant enhancement with radiation was observed. We next determined whether low doses of fractionated IR (2Gy 3 fractions) would improve the efficacy of DNTMi and PARPi combination treatment. In vitro studies with colony forming assays of NSCLC cell line A549 show that IR doses in combination with PARPi and DNMTi decrease clonogenicity, compared with non-IR controls. Furthermore, A549 xenografts were treated with the drug combination and then irradiated 1 week later with 2Gy daily for 3 consecutive days. Mice treated with the drug combination followed by IR had significant decreases in tumor volume and survival (P<0.05). This suggests that low doses of PARPi and DNMTi therapy in combination with low dose IR can potentially target NSCLC tumors. This represents a novel treatment approach for NSCLC patients that may reduce chronic side-effects of high dose IR.

#1423

Nedd8-activating enzyme inhibitor pevonedistat synergizes with cisplatin and carboplatin through interference with nucleotide excision repair and interstrand cross-link repair mechanisms in non-small cell lung cancer.

Xiaozhen Liu,1 David Bouck,1 Khristofer Garcia,2 Jonathan Blank,1 Hugues Bernard,1 Allison Berger,1 Mike Kuranda,1 Erik Koenig,1 Eric Lightcap1. 1 _Takeda, Cambridge, MA;_ 2 _Genentec, San Francisco, CA_.

Platins are one of the most widely used classes of chemotherapeutic drugs. Although used as part of first-line standard of care regimens for advanced non-small cell lung cancer (NSCLC), their effectiveness remains limited, with a 5-year survival rate of 18%. Therefore, combination therapy to overcome resistance and improve survival is needed. Pevonedistat (TAK-924/MLN4924) is an investigational first-in-class Nedd8-activating enzyme (NAE) inhibitor that blocks activation of the cullin family of E3 ubiquitin ligases by preventing cullin conjugation with Nedd8, a ubiquitin-like protein. In vitro, pevonedistat is synergistic with carboplatin in a cell viability assay in 6 out of 20 NSCLC cell lines either with or without paclitaxel. In vivo studies using patient-derived xenograft (PDX) models showed a combination effect in a carboplatin-insensitive but not carboplatin-sensitive model. To evaluate the mechanism of the synergy between pevonedistat and platinum, RNAi of 320 DNA-damage response genes was performed in 4 cell lines in vitro. Depletion of genes involved in TC-NER (transcription-coupled nucleotide excision repair) and ICR (interstrand crosslink repair) reduced the synergy between pevonedistat and platinum, with the contribution of each pathway varying by cell line. Since TC-NER and ICR are also involved in the repair of DNA damage by platins, the results suggest that delaying completion of platin induced DNA repair by pevonedistat results in enhanced cell death. Pevonedistat may impede TC-NER by inhibition of neddylation of the E3 ubiquitin ligase CUL4-RBX1-DDB1-ERCC8, thereby possibly providing a direct reversal of resistance to platins.

#1424

DNA repair capacity in colon cancer patients - The effect on the response to treatment and long-term survival.

Sona Vodenkova,1 Michal Kroupa,2 Katerina Jiraskova,3 Alessio Naccarati,4 Alena Opattova,5 Pavel Vodicka5. 1 _Third Faculty of Medicine, Charles University, Prague, Czech Republic;_ 2 _Faculty of Medicine and Biomedical Center in Pilsen, Charles University, Pilsen, Czech Republic;_ 3 _First Faculty of Medicine, Charles University, Prague, Czech Republic;_ 4 _Human Genetics Foundation, Turin, Italy;_ 5 _Institute of Experimental Medicine, The Czech Academy of Sciences, Prague, Czech Republic_.

Colorectal carcinoma (CRC) is the third most common cancer worldwide with the highest incidence in Central Europe. It is the fourth leading cause of cancer related deaths mainly due to the late diagnosis and low efficacy of treatment. CRC diagnosed in early stage has a five-year survival rate about 90% which drops to near 12% once distant metastases occur. It is a heterogenous disease with different molecular and clinicopathological features depending on the tumor location. Therefore, different treatment strategies are required. A standard treatment of locally advanced rectal cancer includes neoadjuvant chemoradiotherapy followed by surgery, whereas colon cancer treatment consists of surgical resection of the tumor and/or subsequent adjuvant chemotherapy based on disease characteristics. 5-fluorouracil (5-FU) alone or in combination with other compounds is the most used treatment in CRC. The mechanism of 5-FU on molecular level is either its incorporation into DNA or it imbalances the synthesis of thymidine from uracil resulting in false uracil DNA incorporation. These DNA lesions are repaired by base excision (BER) and mismatch repair (MMR) pathways.

An effective DNA damage response (DDR) is essential for the maintenance of genome stability in healthy cells, whereas in malignant cells, the suppression of DNA repair capacity (DRC) would increase the effectiveness of chemotherapy through DNA damage accumulation and consequent apoptosis. In contrary the cells with high DRC may show better survival and therefore patients with these molecular characteristics may contend with poor response, resistance to treatment and decreased survival.

The aim of our present study was to investigate DRC of BER and MMR in target tissue as a predictive marker for a treatment strategy and long-term survival. In order to minimize a bias by heterogenous therapy we focused only on patients with newly diagnosed colon cancer. Our set of patients was selected based on the criteria of follow-up at minimum 30 months, subsequent treatment with 5-FU and microsatellite stable tumor tissue characteristics. Tumor and adjacent non-affected tissue samples were obtained from one hundred patients at surgical resection. Protein extracts from tissues were isolated both for protein expression analysis and for measurement of DRC. DNA repair and DDR protein expression levels were tested by Western Blot. Functional assessments of DRC were performed by comet assay-based in vitro DNA repair assay.

The analysis is running and the data will be statistically analyzed and compared with clinical data (TNM stage, type and course of treatment, presence of recurrence, patient´s performance, etc.). Understanding DRC effect on the treatment response might contribute to the concept assuming that targeted modulating of DNA repair processes can achieve clinical benefit in cancer treatment.

Acknowledgements: GA UK 800216, COST LD14050, GACR P303/15/14789S and AZV 15-27580A.

#1425

**Targeting cell cycle dependencies in** CCNE1 **amplified tumors.**

Kai Doberstein,1 Alison Karst,2 Paul Jones,2 Azra Ligon,2 Michelle Hirsch,2 Dariush Etemadmoghadam,3 William Hahn,2 David Bowtell,3 Ronny Drapkin1. 1 _Univ. of Pennsylvania School of Medicine, Philadelphia, PA;_ 2 _Harvard Medical School, Dana-Farber Cancer Institute, Boston, MA;_ 3 _University of Melbourne Peter MacCallum Cancer Center, Melbourne, Australia_.

Genomic instability is a hallmark of high grade serous ovarian carcinoma (HGSOC). Based on The Cancer Genome Atlas (TCGA), it is estimated that approximately 50% of HGSOCs harbor a defect in the homologous recombination (HR) pathway of DNA repair. In contrast, the 20% that harbor CCNE1 amplifications appear to have an intact HR pathway. These tumors are associated with shorter overall survival and resistance to chemotherapy. Cyclin E is the activating partner of cyclin-dependent kinase 2 (CDK2) which controls cell cycle progression from G1 to S phase. Our previous data showed that CCNE1 amplification and overexpression occurs early in serous tumorigenesis. Importantly, in immortalized human fallopian tube secretory epithelial cells (FTSEC), constitutive Cyclin E overexpression imparts malignant characteristics to these cells. This leads to an accumulation of DNA damage and altered gene expression of genes involved in DNA replication and fork protection. However, in the setting of hTERT expression and a p53 mutant, Cyclin E overexpression alone was not capable of fully transforming the FTSECs. Therefore, in order to identify cooperating genetic alterations, we performed an in vitro gain-of-function (GOF) screen. One of those identified hits was the RAD51 paralog XRCC2, which is known to be involved in the HR DNA repair pathway and in fork protection. We could show that XRCC2 expression is upregulated in response to Cyclin E overexpression in FTSECs and we found a strong correlation between RNAseq expression of XRCC2 and Cyclin E in the TCGA patient cohorts. We could further demonstrate that the knock down of XRCC2 is synthetic lethal in CCNE1 amplified ovarian cancer cell lines but not in cells that harbor no CCNE1 amplification, indicating that the upregulation of XRCC2 creates a dependency in CCNE1 amplified tumors. Since overexpression of Cyclin E leads to unscheduled S-phase entry and stress on the replication fork, we speculated that one of the roles of XRCC2 might be to stabilize the replication fork in Cyclin E overexpressing cells. We found that the knock down of XRCC2 in Cyclin E overexpressing cells leads to a strong reduction in fork speed and fork recovery. To further understand this mechanism we analyzed the binding partners of XRCC2 in CCNE1 amplified cells by mass spectrometry. Interestingly, we found that XRCC2 interacts with the minichromosome maintenance deficient 7 (MCM7) protein. MCM7 is part of the MCM complex that unwinds the DNA during replication. Surprisingly, the downregulation XRCC2 also led to a strong reduction in MCM7 protein expression indicating that XRCC2 may play an important role in stabilization the MCM complex. This is especially interesting since CCNE1 amplified cells are more dependent on active MCM complexes and are more sensitive to MCM complex reduction compared to normal cells. Further defining the factors that contribute to the XRCC2-MCM7 interaction at the replication fork may define novel vulnerabilities in CCNE1 amplified tumors.

#1426

Blinding the CYCLOPS - Cancer vulnerabilities unveiled by genomic loss.

Alica Torkov,1 Kai-Oliver Henrich,1 Chunxuan Shao,2 Moritz Gartlgruber,1 Frank Westermann1. 1 _DKFZ, Heidelberg, Germany;_ 2 _BioQuant, Heidelberg, Germany_.

Background:

Heterozygous deletions within distal 1p are observed in 30% of neuroblastomas. So far, several potential 1p tumor suppressor genes have been identified. However, in this study we are focussing on 1p genes whose inactivation is not necessarily linked to tumor development but which mediate cell-essential functions, rendering cells with copy number loss vulnerable to further impairment. These genes are candidate therapeutic targets according to the concept of CYCLOPS (copy number alterations yielding cancer liabilities owing to partial loss).

Methods:

To identify a subset of 1p genes for which heterozygous loss may be tolerated but further reduction leads to cell death, we performed siRNA screens mediating the systematic knock-down of distal 1p genes in five 1p-deleted versus five non-1p-deleted neuroblastoma cell lines. We used 3 different siRNAs per gene in a liquid forward approach. After 96h Hoechst stained nuclei were count. Among others, a neuron-related candidate gene has been identified as a potential CYCLOPS. The candidate gene was validated by viability assays, immunocytochemistry and cell cycle analysis via FACS.

Results:

We identified many potential CYCLOPS genes mapping on the distal end of chromosome arm 1p. One of these genes is involved in neuronal and embryonic development and has been further validated. Knock-down of the gene impaired cell viability in 1p-deleted cell lines but did not in 1p-non-deleted cells. G1/G0 phase arrest with corresponding S phase decrease was observed in both 1p-deleted and 1p-non-deleted cells. Additionally, neurite-like outgrowth could be observed in 1p-non-deleted cells indicating an induction of differentiation.

Conclusion:

This study identified a candidate CYCLOPS gene in neuroblastoma. Heterozygous deletions of chromosome arm 1p are also frequently observed in other cancers including melanoma, colorectal and breast cancer. We hypothesize that this proof-of-principle opens a new therapeutic window for tumors harbouring a heterozygous deletion of our candidate gene or other cell essential genes on chromosome arm 1p.

#1427

Investigating role of RECQ1 in response to gemcitabine treatment in triple negative breast cancer.

Swetha Parvathaneni, Xing Lu, Sudha Sharma. _Howard University, Washington DC, DC_.

The response of cancer cells to therapeutic drugs aimed at causing DNA damage is dependent on proteins that play roles in DNA repair. Single nucleotide polymorphism in RECQ1 (also known as RECQL or RECQL1), a DNA helicase involved in DNA damage and replication stress response, is reported to influence patient's response to gemcitabine in pancreatic cancer leading to poor survival. Gemcitabine is also used as a chemotherapeutic against triple negative breast cancer; however resistance to gemcitabine is reported. The mechanism by which gemcitabine chemoresistance is obtained is poorly understood. Given the importance of RECQ1 in replication fork restart and it being categorized as a breast cancer susceptibility gene, we wish to investigate the role of RECQ1 in modulating cellular response to gemcitabine in triple negative breast cancer cells. To study RECQ1 functions, we developed isogenic pairs of RECQ1-proficient and RECQ1-deficient cells by CRISPR-Cas9-mediated targeted inactivation of RECQ1 in breast cancer cell lines as in vitro model system. RECQ1-knockout MDA-MB-231 cells displayed reduced cell growth and proliferation, and increased DNA damage as compared to the MDA-MB-231 cells expressing wild type RECQ1. As compared to RECQ1-proficient MDA-MB-231 cells, RECQ1-deficient cells were more sensitive to gemcitabine treatment, incurred significantly greater load of double strand breaks upon gemcitabine treatment, and displayed aberrant cell cycle progression following recovery from gemcitabine treatment. Our ongoing experiments are investigating the detailed mechanism of RECQ1's role in response to gemcitabine and its implications in therapeutic response.

Funding: This work was funded by the NIGMS/NIH grant SC1GM093999 to Sudha Sharma. We also acknowledge support from the NIMHD/NIH award number G12MD007597 and NIA/NIH award number 1R25 AG047843-02. We thank Drs. Ritu Chaudhary, Xiaoling Li and Ashish Lal (NCI) for their help and support.

#1428

Single-cell transcriptomics reveals differential DNA repair signatures after chemotherapy in high-grade serous ovarian cancer.

Anniina Farkkila,1 Kaiyang Zhang,2 Katja Kaipio,3 Tarja Lamminen,3 Rainer Lehtonen,2 Johanna Hynninen,4 Seija Grénman,4 Olli Carpén,1 Sampsa Hautaniemi,2 Anna Vähärautio2. 1 _Univ. of Helsinki and Helsinki University Hospital, Helsinki, Finland;_ 2 _Univ. of Helsinki, Helsinki, Finland;_ 3 _University of Turku, Turku, Finland;_ 4 _University of Turku and Turku University Hospital, Turku, Finland_.

Resistance to chemotherapy poses a major clinical problem in the treatment of ovarian cancer. The efficacy of platinum- based chemotherapy relies on defective DNA repair mechanisms, and increased expression of homologous recombination (HR)/DNA repair genes in primary tumor samples is associated with improved chemoresponse. However, the effects of neo-adjuvant chemotherapy (NACT) on the expression DNA repair genes in cancer cells are poorly understood. In addition, high-grade serous ovarian cancers (HGSOCs) display significant inter- and intratumoral heterogeneity, and the identification of mechanisms of drug resistance has been hampered by the lack of data at the single cell resolution.

We collected 34 primary and 20 interval tumor samples from 15 patients treated for HGSOC at the Turku University Hospital, Finland. Tumor tissue total RNA was sequenced with Illumina HiSeq. For this study, we selected 363 genes selected due to their validated role in DNA repair pathways and compared their expression profiles between primary and interval samples. The treatment naïve primary tumor samples highly expressed genes related to HR, such as BRCA2, RAD54, PARPBP, FANCD2, and POLQ. Whereas after NACT the interval samples highly expressed genes regulating cell cycle, inflammatory response, and the drug efflux pump ABCB1. Moreover, higher expression of BRCA2 or POLQ in the interval samples correlated with poor primary therapy outcome (p<0.01).

To reveal the transcriptomics landscapes of treatment naïve and NACT treated HGSOCs at the single cell level, we performed single cell mRNA sequencing of primary and interval samples using the Fluidigm C1 Platform. Unsupervised clustering revealed eight distinct cell subpopulations, of which two clusters were annotated HGSOC cells based on enriched expression of cancer specific genes. In global gene expression analysis of 98 HGSOC cells, the most differentially expressed genes in primary HGSOC cells were heat shock proteins reflecting active protein synthesis. By contrast, the cancer cells from interval samples highly expressed genes regulating the cell cycle and immune response. Of the DNA repair pathway genes, the interval samples expressed lower levels of HES1, a transcription factor that is known to promote cancer progression and stemness.

The herein elucidated DNA repair pathway transcriptomics landscape at single-cell and whole tumor level provides novel targets for overcoming chemoresistance in HGSOC.

#1429

DNA damage response to dianhydrogalactitol (VAL-083) in p53-deficient non-small cell lung cancer cells.

Anne Steino,1 Guangan He,2 Jeffrey A. Bacha,1 Dennis M. Brown,1 Zahid Siddik2. 1 _DelMar pharmaceuticals, Inc., Vancouver, British Columbia, Canada;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Non-small cell lung cancer (NSCLC) treatment usually involves surgery and chemotherapy with tyrosine kinase inhibitors (TKI) in patients with EGFR mutations (10-15% in Western population, 40% in Asian populations) or with platinum-based regimens. Response to TKI treatment is short lived, and tumors recur with new mutations, primarily T790M. Recurrent NSCLC with T790M is sensitive to third generation TKIs, but resistance usually emerges through new mutations, including KRAS. Resistance to cisplatin and carboplatin, partly due to p53 mutation, is also a major clinical limitation and long-term prognosis in NSCLC is poor. Dianhydrogalactitol (VAL-083) is a bi-functional alkylating agent with demonstrated clinical activity against NSCLC in historical NCI-sponsored trials and VAL-083 is approved for lung cancer treatment in China. However, the mechanism-of-action of how DNA damage signals are propagated and their effects on NSCLC cells are not fully understood. Therefore, we examined VAL-083 in a panel of 11 human NSCLC cell lines harboring wild-type p53 (H460, A549, H226), mutant p53 (H1975, SkLU1, H2122, H157, H1792, H23) or null p53 (H838, H1299). Importantly, as determined by the 5-day MTT assay, VAL-083 was cytotoxic against all 11 cell lines at low μM concentrations and cytotoxicity was independent of p53 status. We chose 3 TKI-resistant cell lines with different mutation profiles for cell cycle kinetics studies: i) H1975 (p53 mut, EGFR-T790M mut, KRAS wt) with IC50 0.9μM, ii) A549 (p53 wt, EGFR wt, KRAS mut) with IC50 1.8μM, and iii) H157 (p53 mut, EGFR wt, KRAS mut) with IC50 4.5μM. In all 3 cell lines, early response at 18 hr showed dose-dependent increase in cells in S-phase, with continued slow cell cycle progression resulting in accumulation of cells in G2/M phase by 36 hr, suggesting persistent cell cycle arrest. DNA damage signaling was examined by immunoblot analysis in A549 and H1975 cells. In p53-wt A549, VAL-083 induced the phospho-Ser15 form of p53, total p53 and total p21, thus indicating that VAL-083 treatment activated p53 function. On the other hand, in p53-mutant H1975, VAL-083 treatment did not result in consistent p53 or p21 increases, but did readily induce phospho-Ser15 p53. This is consistent with a lack of p53 function, as anticipated for mutant p53 cells. Interestingly, H1975 was 2-fold more sensitive to VAL-083 than A549, suggesting, that in p53-mutant cells, VAL-083 acts through a p53-independent mechanism. Examination of ATR, ATM, Chk1 and Chk2 indicated that DNA damage by VAL-083 prompted phosphorylation of these kinases. Notably, the total anti-apoptotic Chk1 was more prominently reduced in H1975 than A549, which may partly explain the stronger cytotoxicity of VAL-083 in p53-mutant H1975. These preclinical data strongly support VAL-083 as a potential treatment of mutant p53 and TKI-resistant NSCLC, and indicate DNA damage signaling is mediated via ATM, ATR, Chk1 and Chk2.

#1430

The spliceosome U2 snRNP factors promote genome stability through distinct mechanisms; transcription of repair factors and R-loop processing.

Michihiro Tanikawa. _The University of Tokyo, Tokyo, Japan_.

(Objective)Recent whole-exome sequencing studies of malignancies have detected recurrent somatic mutations in U2 snRNP components of the spliceosome. These factors have also been listed as novel players of DNA damage response in several genome wide screens and proteome analysis for DDR genes. Although accumulating evidences have implied that the spliceosome plays an important role in genome stability and is an emerging hallmark of carcinogenic pathways, its precise role in genome stability still remains ambiguous. The aim of this study is to clarify the functions of U2 snRNP splicing factors, especially SNRPA1 (Small Nuclear Ribonucleoprotein Polypeptide A1) in DDR pathway.

(Methods)SNAPA1 and other U2 snRNP splicing factors were identified as HR repair genes by genome-wide screens based on homologous recombination and RAD51 immunofoci formation. Each splicing factor was depleted by siRNA knockdown in USOS cells. The functions of SNRPA1 in DNA repair were analyzed by HR assay (DR-GFP assay), immunofluorescence, real-time laser micro-irradiation and comet assay (single cell gel electrophoresis).

(Results)HR assay showed strong HR deficiencies in splicing factor's depleted cells. In these cells, accumulation of BRCA1 and Rad51, major HR factors, to DNA damage sites were severely impaired. Especially, live cell imaging showed recruitment of SNRPA1 to laser induced DNA damage sites and unveiled its direct involvement to DNA damage repair. Comet assay also showed that depletion of SNRPA1 markedly caused DNA damage with the tail of broken DNA fragments. This DNA damage was R-loop (DNA-RNA hybrid) mediated DNA damage and rescued by overexpression of RNAseH1.

(Conclusion) Here we unveiled two distinct pathways how spliceosome U2 snRNP factors contribute to genome stability. The main function is indirect, through transcription, to maintain the protein levels of essential repair factors and contribute to homologous recombination repair. Our data suggest that depletion of splicing factors can be one mechanism for HR deficiency (i.e. BRCAness). In addition real-time laser microirradiation analysis identified the rapid recruitment of SNRPA1 to DNA damage sites. Intensive functional analysis of SNRPA1 unveiled the other, more immediate and direct effect to process R-loop structure, deleterious transcriptional by-products for the genome, at sites of on-going transcription.

#1431

Therapeutic exploitation of mutant BRAF in childhood glioma.

Sudipa Saha Roy, Peter Houghton. _UTHSCSA, San Antonio, TX_.

Low grade gliomas (LGG) are the most common tumors of the central nervous system in children, accounting for about 50% of all brain tumors. They represent a heterogeneous group of grade I and II tumors according to the WHO classification. Pediatric LGG, is associated with activation of BRAF through a tandem duplication that results in the KIAA1549-BRAF fusion or through an activating point mutation of BRAF (predominantly V600E). More recent findings suggest that the KIAA1549-BRAF fusion is restricted to Grade 1 tumors (70-90%) whereas BRAF(V600E) occurs more frequently in Grade 2-4 tumors (~23%). Findings for BRAF mutation, similar to other tumors with activated BRAF (e.g. melanoma), and the phase I activity of MEK inhibitor in the PBTC-029 protocol, suggest that activated BRAF may provide a validated drug target. Previous studies from our lab indicate that, in the context of mutant BRAF, inhibition of MEK inhibits TORC1 signaling and may induce a 'BRCA-like' phenotype, through depletion of FANCD2. Potentially suppression of TORC1 could have effects on other DNA damage response pathways that could compensate for loss of FANCD2. To understand the consequences of MEK inhibition in the context of BRAF abreation in LGG we surveyed DNA repair genes that may be regulated via the MEK/TORC1 pathway in BRAF mutant cells. The objective of this particular study was to examine the mechanism and significance of MEK inhibition to the repair of DNA damage by the homologous recombination (HR) pathway. For this study, we have used glioma cell lines having BRAF(V600E) mutation; the BT40 cell line was developed from patient-derived astrocytoma xenograft (PDX) model in mice in our lab and AM38c1 and DBTRG-05MG cells were generously provided by T. Nicolades. Annexin binding assay demonstrated that MEKi treatment significantly increased the percentage of apoptosis and necrosis of glioma cells. Human DNA repair PCR Array analysis identified MEKi induced down regulation of genes involved in Base Excision Repair (BER), Nucleotide Excision Repair (NER), Mismatch Repair (MMR) and Double-Strand Break (DSB) repair pathways. DSB repair genes were further validated by real time qPCR analysis. Immunoblot analysis of glioma cell lines indicate that MEKi treatment enhanced the gamma-H2AX levels. To elucidate the mechanism of DSB repair pathway in presence of MEKi, clonogenic assay, nuclear foci formation assay, and GFP reporter assay for homologous recombination (HR) and non-homologous end-joining (NHEJ) are ongoing. Collectively, these findings demonstrate for the first time a previously unknown role for MEKi in treatment of glioma cells that involves inhibition of DNA double-strand break repair pathways. Further, we will examine the mechanism and significance of MEK/ERK and TORC1 signaling axis in regulation of DNA repair genes and its effect on LGG. Understanding how MEK plays a role in DNA repair pathways will be useful in maximizing treatment opportunities for childhood glioma. 

### Genomics in Inherited Susceptibility and Preneoplastic Conditions

#1432

Precision lung cancer medicine based on information on cancer and the host genomes.

Takashi Kohno,1 Kouya Shiraishi,1 Kazushi Yoshida,2 Motonobu Saito,1 Yukinori Okada3. 1 _National Cancer Ctr. Research Inst., Tokyo, Japan;_ 2 _National Cancer Ctr Hospital, Tokyo, Japan;_ 3 _Osaka University Graduate School of Medicine, Osaka, Japan_.

Aberrations of oncogene, such as EGFR mutation; and ALK, RET, and ROS1 fusions, function as a driver in the development of lung adenocarcinoma (LADC). They are a solid target of therapy using tyrosine kinase inhibitors, as represented by a high response rate of RET-fusion positive LADC to vandetanib (LURET study, Yoh et al., Lancet Resp Med, 2016). Our genome-wide sequencing study revealed that oncogene aberration positive LADCs carry less numbers of mutations in cancer-related genes than negative ones (Saito, Cancer Res, 2015), indicating a small mutation burden in the development of the formers. Consistently, a clinical trial of anti-PD-1 therapy indicated that EGFR-mutation positive cases do not benefit well from the therapy (Borghaei et al., N Engl J Med. 2015). LADC driven by somatic EGFR mutations is more prevalent in East Asians (30-50%) than in European/Americans (10-20%). We recently revealed that variations in HLA-class II loci underlie the risk of the disease, by conducting a genome-wide association study of 3,173 patients and 15,158 controls, combined with imputation of HLA alleles using a Japanese-specific panel (Shiraishi et al., Nature Comm, 2016). The result indicates that LADC develops in vivo through interaction between somatic mutations and germline variations that modulate immune reaction. Therefore, efficacy of immune checkpoint blockade targeting therapy might also be influenced by such germline variations. On the other hand, oncogene-negative LADCs show a high mutation burden associated with nonsynonymous mutations in chromatin remodeling and other cancer-related genes. Thus, these cases would respond well to immune checkpoint blockade targeting therapies. We would like to discuss here precision lung cancer medicine based on information on cancer and the host genomes.

#1433

Single cell RNA sequencing reveals smoking-associated alterations in bronchial airway epithelial subpopulations.

Grant E. Duclos, Joshua D. Campbell, Yaron Gesthalter, Patrick Autissier, Yves M. Dumas, Robert Terrano, Gang Liu, Marc E. Lenburg, Avrum Spira, Jennifer Beane. _Boston University School of Medicine, Boston, MA_.

Rationale: We have previously shown that bronchial airway epithelial gene expression reflects the physiologic response to cigarette smoke exposure. We have also shown that gene expression differences in cytologically normal airways cells can serve as a diagnostic biomarker for lung cancer. In this study, we use single cell RNA-seq to profile transcriptomes of individual bronchial epithelial cells from current and never smokers in order to detect smoking-associated alterations within specific epithelial cell types and to discover novel subpopulations that develop as a result of smoke exposure. This approach may be useful for identifying cell type-specific transcriptomic changes in the airways of cancer patients, which may lead to a better understanding of lung carcinogenesis and new approaches to early lung cancer detection.

Methods: We obtained bronchial brushings from current smokers (n=6) and never smokers (n=6) and isolated single cells by FACS. The CEL-Seq RNA library preparation protocol was used to sequence the transcriptomes of 1,140 cells (n=95/donor).

Results: Distinct populations of bronchial cells expressed known markers of basal (KRT5), ciliated (FOXJ1), secretory (SCGB1A1, MUC5AC) epithelial cells, as well as white blood cells (CD45). In the airways of smokers, we observed an increase in abundance of MUC5AC+ secretory cells as well as a decrease in abundance of KRT5+ basal cells and SCGB1A1+ secretory cells. A novel subset of KRT8+ cells that lacked expression of other known cell type markers was identified in the airways of smokers and may represent a population previously described as undifferentiated intermediate cells. Genes involved with metabolism of polycyclic aromatic hydrocarbons (CYP1B1) were detected in smoker secretory cells, whereas genes involved in the metabolic response to cigarette smoke toxins such as aldehydes (ALDH3A1) and quinones (NQO1), were most highly expressed by smoker ciliated cells. Interestingly, the novel KRT8+ cells identified in smokers expressed genes known to promote MUC5AC+ secretory cell differentiation (SPDEF), but did not express MUC5AC itself, suggesting that these may be pro-MUC5AC+ secretory intermediate cells. Furthermore, we found that genes previously associated with higher expression in the airways of lung cancer patients were enriched among genes most strongly associated with smoker ciliated and secretory cells, whereas genes with lower expression in lung cancer were enriched among genes most strongly associated with white blood cells.

Conclusion: We have identified cell type-specific transcriptomic alterations and shifts in epithelial cell population abundance in smoker airways. In future studies, profiling the transcriptomes of single cells from bronchial airways of smokers with and without lung cancer may lead to the identification of specific cellular subpopulations contributing to the airway field of lung-cancer associated injury.

#1434

Mutational landscape in the normal-appearing airway cancerization field of early-stage non-small cell lung cancer.

Smruthy Sivakumar,1 Yasminka Jakubek,2 Wenhua Lang,2 Tina McDowell,2 Melinda M. Garcia,2 Chi-Wan Chow,2 Zachary Weber,3 Carmen Behrens,2 Neda Kalhor,2 Cesar Moran,2 Randa El-Zein,4 Gareth Davies,3 Junya Fujimoto,2 Reza Mehran,2 Stephen G. Swisher,2 Jing Wang,2 Avrum E. Spira,5 Jerry Fowler,2 F Anthony San Lucas,2 Ignacio I. Wistuba,2 Erik Ehli,3 Paul Scheet,2 Humam Kadara6. 1 _The University of Texas Graduate School of Biomedical Sciences, Houston, TX;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _Avera Institute for Human Genetics, Sioux Falls, SD;_ 4 _Houston Methodist Research Institute, Houston, TX;_ 5 _School of Medicine, Boston University, Boston, MA;_ 6 _American University of Beirut, Beirut, Lebanon_.

Lung cancer, of which non-small cell lung cancer (NSCLC) is the most common form, is the second most prevalent cancer in the U.S. and the leading cause of cancer mortality. Field cancerization phenomenon establishes that normal appearing tissues surrounding a tumor will exhibit a field effect -- particularly in smokers -- with the tissues proximal to the tumor showing more, and even sharing, alterations with the tumor. Previous studies in NSCLC provide evidence for both complex progression trajectories, yet, precise mechanisms remain unknown. To investigate the field cancerization phenomenon, we conducted a genome-wide survey of acquired DNA alterations in normal-appearing tissues including small airways adjacent to NSCLC, mainstem bronchi (large airways), nasal epithelium, distant normal lung and blood, along with multiple samples from their surgically resected paired tumors, for a total of 500 samples from 48 patients with early-stage NSCLC (11 squamous cell carcinomas and 37 adenocarcinomas). Since we expect low mutation burdens and mutant cell fractions in pathologically normal tissues, we assessed somatic point mutations via deep targeted sequencing of 409 genes and paired these data with our recent study of acquired copy number alterations (CNAs) inferred from high-density whole-genome SNP microarrays, thus offering a deep and wide survey of somatic DNA alterations. After aggregating results from multiple mutation callers, we observed somatic mutations (exonic, splicing and UTRs) in 257 samples of which 65 (in 35 patients) were from normal-appearing field (non-tumor, non-blood) samples. Tumor and field samples in smokers showed a higher mutation burden and large proportion of C:G>A:T changes. We also observed concordance of mutations in the airway and corresponding tumor profiles. Further, a statistically significant field effect was established with mutational burden (measured by the variant allele frequency) increasing with proximity to the tumor. Among the 35 NSCLC cases with field mutations, the small adjacent (to tumor) airways in six patients exhibited mutations in lung cancer drivers such as KRAS, STK11, TP53 and KEAP1. We also identified mutations in the large airway (TP53, SETD2, CDKN2A), distant normal lung parenchyma (RB1, RET) and nasal epithelium (AKT1). We then correlated point mutation and CNA mutation profiles. Of eight cases showing a nonsynonymous or stopgain variants in established lung cancer drivers in the airways, four exhibited putative "two-hit" progression models, e.g. both KEAP1 and STK11 mutations with 19p loss, overlapping TP53 mutation/17p loss, or KRAS mutation/12p gain. Our findings in normal-appearing tissues of the respiratory epithelium offer insights into the earliest mutational events in their progression to NSCLC and, possibly, in tumor relapse and that may represent suitable targets for early detection and chemoprevention.

#1435

Immune/inflammatory polymorphisms predict lung cancer survival.

Donovan Watza,1 Chrissy Lusk,1 Angie Wenzlaff,1 Christine Neslund-Dudas,2 Gregory Dyson,1 Ayman Soubani,3 Shirish Gadgeel,1 Ann G. Schwartz1. 1 _Karmanos Cancer Institute, Detroit, MI;_ 2 _Henry Ford Hospital, Detroit, MI;_ 3 _Wayne State University School of Medicine, Detroit, MI_.

Lung cancer is the leading cause of cancer related mortality in the United States, with a median five year survival of ~16%. Recent advancements in the treatment of lung cancer focus on modulating the immune system to improve patient outcomes with markedly reduced toxicity profiles. This study aims to investigate single nucleotide polymorphisms (SNPs) within functional units of the immune system genome to identify genetic markers indicative of overall survival in a cohort of metropolitan Detroit lung cancer patients. This cohort presents a unique population of lung cancer patients for analysis, consisting of 40% African Americans, whom are known to have worse outcomes than their Caucasian counterparts. To assess SNPs within functional units of the immune genome, a hierarchical immune system gene and pathway list was constructed, incorporating genes and pathway identifiers from the Reactome database as well as previous genetic and inflammatory specific cancer studies to generate a well curated gene and pathway dataset. Genomic locations for the genes, together with proximal regulatory regions, were obtained from the UCSC genome browser for cross-referencing with the Illumina MEGA SNP array to generate SNPs for inclusion. Preliminary analysis of 848 NSCLC patients in association with 29,126 immune specific SNPs identified six SNPs as significantly associated with survival (p-value <10-4) in this lung cancer cohort, implicating the following six genes, PXN, GFRA3, HLA-DQA1, KSR2, BTBD1, and ATF2; although further gene/pathway analysis is necessary to elucidate how these associations interact in the context of genes and pathways of the immune system. PXN over expression has been previously implicated in both high risk lung epithelial dysplasia as well as in the development of lung adenocarcinoma in prior clinical studies. Additionally, GFRA3 has been identified as an activator of the RET kinase and increased GFRA3 activity in context of the Artemin pathway is a known promoter of NSCLC progression. As well, select polymorphisms in the HLA-DQA1 gene are known clinical modifiers of lung squamous cell carcinoma risk. Finally, ATF2 is a broadly active transcription factor and ATF2 upregulation has been associated with tumorigenesis and metastasis in both cell models and patient tumor samples in multiple cancer studies. The previous association of these genes in lung cancer is promising and further immune system gene/pathway analysis is warranted.

#1436

Identification of novel tumor suppressor candidates in familial cholangiocarcinoma using sequencing-based Megabase-scale haplotypes from germline and cancer genomes.

Stephanie Greer,1 Lincoln Nadauld,2 Billy Lau,1 Laura Miotke,1 Erik Hopmans,1 Christina M. Wood,1 John M. Bell,1 David A. Jones,3 Hanlee P. Ji1. 1 _Stanford Univ., Stanford, CA;_ 2 _Intermountain Healthcare, Salt Lake City, UT;_ 3 _Huntsman Cancer Institute, Salt Lake City, UT_.

Cholangiocarcinoma (bile duct cancer) is a rare epithelial malignancy with an extremely poor prognosis characterized by a 5-year survival of less than 10%. While this tumor type can be amenable to specific combination chemotherapy regimens, it is typically diagnosed at an advanced stage and inevitably progresses towards distant metastasis. We identified a family with a predisposition to cholangiocarcinoma, having multiple affected family members, and sought to identify the underlying germline mutation(s).

We employed linked-read sequencing, a microfluidic technology where high-molecular weight DNA molecules (>20 kb) are partitioned into droplets and labeled with unique barcodes, enabling computational reconstruction of the original large molecules following traditional short-read sequencing, to identify entire megabase-scale regions of the genome that segregate with the disease. We applied this technology to an entire generation of a family predisposed to cholangiocarcinoma, including a mix of 8 affected and unaffected siblings. We additionally utilized each affected individual's cancer genome to search for a common second hit that would serve as further confirmation. Our analysis yielded a small set of four candidate pathogenic variants. Functional validation of the candidate genes in a zebrafish model, using morpholino-based gene silencing, identified one candidate whose silencing led to a reproducible phenotype consistent with known aspects of the candidate gene's function. The candidate variant occurs in a TGF family-interacting domain, and has not been extensively characterized in other cancers. Cross-validation with TCGA datasets showed the gene was altered in 5% of cholangiocarcinomas, all of which displayed homozygous deletions. This identification of a germline allele associated with familial cholangiocarcinoma represents a unique discovery with clinical implications for the described family and warrants further investigation in other patients who display features of an inherited cholagniocarcinoma. This finding also raises the possibility of TGF pathway targeting as a therapeutic strategy in select cholangiocarcinomas, and highlights the power of whole-genome phasing to discover novel inherited tumor suppressor variants.

#1437

Differential role of mutations in clonal evolution in esophageal mucosa at risk for cancer.

Akira Yokoyama,1 Hiromichi Suzuki,1 Tetsuichi Yoshizato,1 Yuusuke Shiozawa,1 Yusuke Sato,1 Kosuke Aoki,1 Nobuyuki Kakiuchi,1 Yasuhide Takeuchi,1 Yoichi Fujii,1 Hideki Makishima,1 Kenichi Yoshida,1 Keisuke Kataoka,1 Yaichi Shiraishi,2 Masashi Sanada,3 Shigeru Tsunoda,1 Sachiko Minamiguchi,1 Satoru Miyano,2 Manabu Muto,1 Seishi Ogawa1. 1 _Kyoto university, kyoto, Japan;_ 2 _The University of Tokyo, tokyo, Japan;_ 3 _Nagoya Medical Center, nagoya, Japan_.

Background Esophageal squamous cell carcinoma (ESCC) develops in close association with a chronic exposure to heavy drinking and smoking, frequently in a metachronous manner, suggesting a strong field effect on ESCC carcinogenesis. However, it is poorly understood how the field effect promotes clonal evolution in esophageal mucosa to induce dysplasia, eventually leading to the development of ESCC.

Patients & Methods A total of 221 samples were obtained by multiple sampling from cancer tissues (n = 53) as well as dysplastic (n = 12) and non-dysplastic (n = 156) mucosae from 49 cancer-bearing patients and 2 high-risk and 10 healthy patients either by endoscopy or surgery with different sampling sizes (regular size: 4 - 8 mm2 and small size: 0.2 or 0.8 mm2). All the samples were subjected to whole exome (WES) (n = 99) and/or targeted-capture (TS) (n = 221) sequencing. Copy number alterations (CNAs) were also investigated using SNP array- or sequencing-based karyotyping.

Results WES and TS in regular size samples revealed clonal proliferation in most of the non-dysplastic samples (98%), although the number of accompanying mutations and their clone size were significantly lower compared to those in samples from cancer and dysplasia (respectively, P = 0.021 and P < 0.001). Non-dysplastic samples were most frequently accompanied by NOTCH1 mutations (75%), followed by TP53 mutations (50%). By contrast, cancer and dysplasia samples showed much lower frequencies of NOTCH1 mutations (19% and 66%), but instead characterized by higher frequency of TP53 mutations (100% and 92%), and CNAs (83% and 17%), especially those focally involving CCND1, CDKN2A, PIK3CA, and EGFR, respectively. Mutation signatures also differed significantly between samples from non-dysplastic mucosa, and cancer/dysplasia tissues; cancer/dysplasia samples exhibited an enhanced APOBEC-related and decreased transcription-coupled repair-related signatures, compared to non-dysplastic samples. In non-dysplasia samples, both NOTCH1 and TP53 mutations were significantly enriched in heavy drinkers compared to non- or occasional drinkers (p < 0.001 and p = 0.023, respectively). To decipher the clonal architecture in more detail, we also sequenced 58 samples collected from non-dysplastic mucosae with a smaller sampling size. Clonal proliferation was confirmed in all the samples analyzed; NOTCH1 and TP53 mutations were found in 79% and 55% of the samples, where 78% and 27% of the mutated area showed biallelic alteration, respectively.

Conclusions Clonal proliferation in non-cancer esophageal epithelia is quite common even in non- or occasional drinkers and could be extensive in heavy drinkers. NOTCH1 and TP53 mutations as well as CNAs play major roles in clonal evolution but may have differential impacts on esophageal carcinogenesis, where TP53 mutation and CNAs are likely to make major contributions possibly under the influence of a higher APOBEC activity.

#1438

The dynamics of genetic aberrations in Crohn's disease associated colorectal carcinogenesis.

Daniela D. Hirsch,1 Darawalee Wangsa,2 Yue Hu,2 Jack Zhu,2 David Petersen,2 Daniel C. Edelman,2 Paul S. Meltzer,2 Bao Tran,2 Kerstin Heselmeyer-Haddad,2 Thomas Ried,2 Timo Gaiser1. 1 _University Medical Center Mannheim, Mannheim, Germany;_ 2 _National Cancer Institute, Bethesda, MD_.

Crohn's disease, a condition of chronic inflammation of the intestine, significantly increases the risk for the development of colorectal cancer (CRC). Sporadic CRCs are characterized by a specific pattern of genomic imbalances and a landscape of acquired gene mutations. In this study we aimed to compare CRCs that arise as a consequence of chronic inflammation in Crohn's disease with sporadic CRCs.

We analyzed 26 Crohn's disease associated CRCs, four matched dysplastic lesions, six matched inflamed mucosa samples, and two matched lymph node metastases using array comparative genomic hybridization, targeted sequencing (564 cancer related genes) and gene expression profiling. As a control, we used normal intestinal mucosa from the resection margin of these CRCs and 24 sporadic CRCs.

In general, CRCs that developed in patients with Crohn's disease had a similar distribution of genomic imbalances compared to sporadic CRC. However, we identified distinct mutation signatures: in the Crohn's disease associated CRCs the most frequently mutated gene was TP53, which occurred in 65% of the samples. The second, third and fourth most frequently mutated genes were KRAS (27%), APC (23%) and PIK3CA (19%). In the control group of sporadically arising CRCs, the most commonly mutated gene was APC (75%), followed by KRAS (54%), TP53 (33%), SMAD3 (29%) and SMAD2 (25%). The genetic analyses of multiple lesions from individual patients revealed a high degree of intertumoral heterogeneity with diverse patterns of gene mutations and allowed the reconstruction of the sequence of genetic events during Crohn's disease associated tumorigenesis. In contrast to sporadic colorectal carcinogenesis, TP53 mutations were observed as early and common events while APC mutations occurred rather late and were infrequent.

Our comprehensive molecular profiling of Crohn's disease associated CRCs suggests that the genetic landscape of CRC is influenced by the activation of inflammation related pathways. Furthermore, our findings offer potential for establishing an early detection marker for dysplasia in patients with Crohn's disease.

#1439

**Genetic profiling of colitis-associated cancer indicates** de novo **carcinogenesis and the impact of chronic inflammation.**

Masashi Fujita,1 Nagahide Matsubara,2 Kazuhiro Maejima,1 Tomoki Yamano,2 Ikuo Matsuda,2 Seiichi Hirota,2 Hiroki Ikeuchi,2 Naohiro Tomita,2 Hidewaki Nakagawa1. 1 _RIKEN Center for Integrative Medical Sciences, Tokyo, Japan;_ 2 _Hyogo College of Medicine, Hyogo, Japan_.

Background: Chronic inflammation is one of the major causes of carcinogenesis. Ulcerative colitis (UC) and Crohn's disease (CD) increase the risk of colitis-associated cancer (CAC) development, and its biological and pathological characters are also distinct. However, the genetic mechanism of how chronic inflammation leads to CACs is still unclear.

Methods: We obtained frozen tumor tissues from 21 CAC patients and performed exome or whole-genome sequencing of the matched tumor-normal samples. We enumerated recurrently mutated genes and selected 43 genes among them for target capture. We then performed targeted resequencing of 84 FFPE specimens from CAC tissues. Totally, we made genomic profiles of 105 CACs.

Results: The most frequently mutated gene was TP53, whose mutation was found in 70 of 105 samples (67%), which is more prevalent in CACs than common colorectal cancers (CRCs). Somatic mutations of APC and KRAS were less prevalent (17 and 13 samples, respectively). Genes in the Wnt signaling pathway (APC, FBXW7, TCF7L2, and RNF43) were mutated in 31 samples, whereas those in the TGFβ pathway (TGFBR2, ACVR1B, SMAD2, SMAD4, LTBP4) were mutated in 25 samples. Mutational signature of CACs shows some differences from common CRCs.

Conclusion: Our results confirmed genetic differences for cancer development between CACs and common CRCs. Frequent TP53 mutations and less frequent APC mutations are consistent with the idea of "de novo" carcinogenesis than adenoma-carcinoma sequence. The association between the mutations and clinical information are also discussed.

#1440

Germline loss-of-function alleles in Finnish colorectal cancer patients.

Tomas Tanskanen, Ulrika Hänninen, Tatiana Cajuso, Johanna Kondelin, Riku Katainen, Jiri Hamberg, Niko Välimäki, Esa Pitkänen, Kimmo Palin, Lauri A. Aaltonen. _Genome-Scale Biology Research Program, Research Programs Unit, University of Helsinki and Department of Medical and Clinical Genetics, Medicum, University of Helsinki, Helsinki, Finland_.

Colorectal cancer, a major cause of cancer deaths worldwide, is often preventable by screening and surveillance, especially in patients at increased risk; however, its heritability is incompletely understood, and studies in isolated populations such as Finland may be valuable for gene discovery. We analyzed whole-genome or whole-exome sequencing data in 322 Finnish colorectal cancer cases of unknown etiology and 928 population-matched controls. To enrich for susceptibility alleles, cases were selected on the basis of early-onset colorectal cancer (age at diagnosis <50 years; 50/322, 16%), synchronous or metachronous colorectal cancer (30/322, 9.3%), familial colorectal cancer (134/322, 42%), personal history of noncolorectal cancer (93/322, 29%), or family history of at least two noncolorectal cancers (124/322, 39%). To increase the prior probability of pathogenicity of variants, the study was restricted to rare and low-frequency protein-coding loss-of-function variants (nonsense, frameshift, and essential splice site variants; allele frequency <3%). Single-variant and gene-level associations were tested, and genes with loss-of-function variants exclusively in cases were pinpointed. An independent set comprising 1,257 cases and 2,638 controls were genotyped using SNP arrays, and genotype imputation provided additional data on 225 (8.5%) of the 2,654 initially discovered loss-of-function variants. Principal component analysis was applied to correct for population structure. In association analyses, with a statistical power of at least 80% for a range of moderate-to-high effect sizes and low allele frequencies (e.g., odds ratio of 3 and allele frequency of 0.5%) at p <0.15, a total of 35 candidate colorectal cancer predisposition genes were identified, although none of them reached exome-wide significance. There were 21 genes with rare loss-of-function alleles in at least two cases but none of the sequenced controls. Whole-genome sequencing data from paired normal and tumor tissues were available in 243 cases, and potential second hits (wild-type allele losses or somatic point mutations) were found in 15 genes. These results may, through further analysis and replication, contribute to our knowledge of the genetic architecture and pathobiology of colorectal cancer susceptibility.

#1441

A comprehensive cis-eQTL analysis revealed target genes in breast cancer susceptibility loci identified in genome-wide association studies.

Xingyi Guo,1 Weiqiang Lin,2 Qiuyin Cai,1 Jiajun Shi,1 Yaqiong Sun,1 Xiao Pan,2 Mi-Ryung Han,1 Wanqing Wen,1 Bingshan Li,1 Jirong Long,1 Jianghua Chen,2 Wei Zheng1. 1 _Vanderbilt University School of Medicine, Nashville, TN;_ 2 _Zhejiang University School of Medicine, Hangzhou, China_.

Genome-wide association studies (GWAS) have identified approximately 107 common single nucleotide polymorphisms (index SNPs) for breast cancer risk. The target genes and underlying mechanisms for the large majority of the identified associations remain unknown. We conducted a cis-expression quantitative trait loci (cis-eQTL) analysis using transcriptome data from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) (N = 1,981), The Cancer Genome Atlas (TCGA) (N = 458), and the Genotype-Tissue Expression (GTEx) project (N = 183). We identified a total of 58 genes including SSBP4 at Benjamini-Hochberg adjusted P < 0.05 in at least one dataset and another gene, PAX9, at P < 0.05 with consistent association directions in all three datasets. Using data from the Encyclopedia of DNA Elements (ENCODE), we selected functional SNPs and showed that some alternative alleles could significantly change promoter activities of their target genes compared to reference alleles using luciferase reporter assays. The effects of target gene expression for those alleles were in line with eQTL observations. In addition, we validated the genes SETD9 and SSBP4 using Sequenom allelotype technique from 235 adjacent normal breast tissues and observed the allelic-specific expression associated with their index SNPs. This study revealed novel biological mechanisms for associations of genetic susceptibility risk loci for breast cancer.

#1442

**Analysis of** cis **-eQTLs in normal and tumor-derived pancreatic tissues reveals functional insights, including for the 9q34.1** ABO **pancreatic cancer risk locus.**

Laufey T. Amundadottir,1 Soren Lykke Andresen,2 Wenming Xiao,3 Jason Hoskins,4 Ashley Jermusyk,4 Lauren Rost,4 Irene Collins,4 Jinping Jia,4 Michael Mobaraki,4 Bin Zhu,4 Robert Kurtz,5 Hemang Parikh,4 Lei Song,4 Meredith Yeager,4 Torben Jensen,2 William Bamlet,6 Nilanjan Chatterjee,7 Brian Wolpin,8 Jill Smith,9 Sara Olson,10 Gloria Petersen,6 Jianxin Shi,4 Mingfeng Zhang4. 1 _National Cancer Inst., Bethesda, MD;_ 2 _Aarhus University, Denmark;_ 3 _FDA, AR;_ 4 _National Cancer Inst., MD;_ 5 _Memorial Sloan Kettering Cancer Center, NY;_ 6 _Mayo Clinic, MN;_ 7 _John's Hopkins Univeristy, MD;_ 8 _Dana Farber Cancer Institute, MA;_ 9 _Georgetown University, DC;_ 10 _MSK Cancer Center, NY_.

Objective: To elucidate the genetic architecture of gene expression in pancreatic tissues.

Design: We performed expression quantitative trait locus (eQTL) and allele specific expression (ASE) analyses using RNA-sequence data and 1000 Genomes (1000G) imputed GWAS genotypes from 95 fresh frozen histologically normal pancreatic tissue samples. Data from 115 pancreatic tumor-derived tissue samples from The Cancer Genome Atlas (TCGA) was included for comparison.

Results: We identified 38,615 cis-eQTLs (corresponding to 484 Genes) in histologically normal tissues and 39,713 cis-eQTL (corresponding to 237 Genes) in tumor tissues (FDR<0.1), with the strongest effects seen near transcriptional start sites (TSS). Approximately 23% and 42% of genes with significant cis-eQTLs (eGenes) appeared to be specific for tumor and normal derived tissues, respectively. Significant enrichment of cis-eQTL variants was noted in noncoding regulatory regions marked by modified histones, DNAse hypersensitivity and bound transcription factors, in particular for pancreatic tissues (1.53-3.12 fold, P≤0.0001), indicating tissue-specific functional relevance. A common pancreatic cancer risk locus on 9q34.2 in the ABO gene (rs687289) was associated with ABO expression in histologically normal (P=5.8x10-8) and tumor-derived (P=8.3x10-5) pancreatic tissues. The high linkage disequilibrium (LD) between this variant and the O blood group generating deletion variant in exon 6 of ABO suggested that nonsense-mediated decay (NMD) of the "O" mRNA could explain the eQTL. However, knock-down of crucial NMD regulators did not influence decay of the ABO "O" mRNA, indicating that a gene regulatory element influenced by pancreatic cancer risk alleles may underlie the eQTL.

Conclusions: We have identified cis-eQTLs representing potential functional regulatory variants in the pancreas and generated a rich dataset for further studies on gene expression and regulation in pancreatic tissues.

#1443

Identification of enhancer elements at kidney cancer susceptibility loci using MPRA.

Leandro Machado Colli,1 Lea Jessop,1 Mitchell Machiela,1 Jiyeon Choi,1 Timothy Myers,2 Stephen Chanock1. 1 _NCI, Rockville, MD;_ 2 _NCI, Bethesda, MD_.

GWAS is an important tool for discovering regions in the genome associated with cancer susceptibility. For renal cell carcinoma (RCC), only two loci have had their functional basis explained (11q13 and 12p12). Here, we used a massively parallel reporter assay (MPRA) to investigate enhancer activity in 20 GWAS regions that had an RCC association p-value of at least 10-7. We selected 784 SNPs with an r2>0.4 or D'>0.5 and MAF<0.05 that had evidence for enhancer activity based on available ENCODE transcription factor or histone ChIP-seq, FAIRE, and DNase I hypersensitive data. The MPRA library was composed of 47461 oligonucleotides 201 bp in length, compelled of 145 bps contained the SNP in the forward or reverse orientation followed by 10 bp of barcode sequence to allow for identification. Each of the 784 SNPs tested were tagged 10 separate times for both orientations. Controls were constructed by randomizing the 10 nucleotides centered on the SNP. The MPRA library was the cloned into a luciferase reporter vector that was transfected into HEK293T and ACHN cell lines. NGS was performed on 5 replicates using the the Illumina HiSeq 2500. After correcting for multiple comparisons, 50 SNPs from 16 regions showed significant p-values. From these 50 SNPs, 29 showed enhancer activity that was independent of the forward/reverse orientation and had the same effect in both cell lines. This analysis has identified possible functional variants at RCC risk loci and opens opportunities to discover new molecular mechanisms of genetic susceptibility of RCC.

#1444

Validation of prostate cancer risk variants by CRISPR/Cas9 mediated genome editing at the MSMB locus.

Xing Wang,1 James Hayes,1 Xing Xu,1 Dipti Mehtad,2 Xiaoni Gao,1 Hans Lilja,2 Robert Klein1. 1 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 2 _Memorial Sloan-Kettering Cancer Center, New York, NY_.

Genome wide association studies (GWAS) have identified nuermous single nucleotide polymorphisms (SNPs) associated with prostate cancer risk. While numerous in silico analyses suggest that the majority of these SNPs may function through regulation of gene expression, wet lab experiments to distinguish the causative variants from their linkage disequilibrium (LD) partners is still needed. Here, we demonstrate how genome editing can investigate the role of individual polymorphisms on gene expression.

Here, we focus on the prostate cancer-associated SNP rs10993994, which is in the promoter of the MSMB gene that encodes for the prostate-secreted protein beta-MSP. This SNP is notable because it associates with seminal and serum levels of beta-MSP, PSA, and hK2; mRNA levels of MSMB and the nearby androgen receptor co-activator NCOA4 in the prostate; and mRNA levels of MSMB in the stomach. Intriguingly, rs10993994 is not a gastric cancer risk SNP suggesting that there may be other variants with prostate-specific function responsible for the observed prostate cancer association.

Using data from the FANTOM5 project, we show that rs7098889, in LD with rs10993994, is located in one of the most prostate-specific enhancers. We had previously shown that this SNP is associated with beta-MSP independent of rs10993994.

To distinguish the effects of these two SNPs on gene expression, we generated a series of double gRNA mediated CRISPR/Cas9 deletions of the relevant regulatory elements. Removing the element containing rs7098889 flanking region results in ~10 to 20 fold increase of MSMB expression in the prostate cancer LNCaP cell line but not in the gastric cancer AGS cell line. There is no change in NCOA4 in either cell line. Removing rs10993994 flanking region results in over 10 fold increase of MSMB expression and ~50% decrease of NCOA4 in LNCaP.

As both LNCaP and AGS are heterozygous for both SNPs, we then generated single clones in which only one allele was removed. For rs7098889, an over 300 fold increase of MSMB expression was observed in two out of the three clones with the T but not the C allele. Transcripts from high expressers all came from one allele. Similarly, single clone analysis showed 40 to 60 fold up-regulation of MSMB expression in two out of three clones with the C but not the T allele of rs10993994. This is consistent with the observation for rs7098889 since rs7098889T and rs10993994C are on the same haplotype.

Our work demonstrates dramatic and locus specific effect of individual polymorphisms on gene expression, which strongly support the notion that an important function of disease risk variants is through regulation of gene expression.

#1445

Understanding the 8q24 colorectal cancer risk locus via CRISPR/Cas9 scarless genome editing.

Nicole Coggins, Jacob Stultz, David Segal, Luis Carvajal-Carmona. _Univ. of California, Davis, Davis, CA_.

Colorectal Cancer (CRC) is one of the leading causes of mortality in the US, being the third most common cancer among both men and women. While an exact cause of CRC has not yet been elucidated, the existence of a clear heritable genetic component has been established. With the recent surge of data from Genome-Wide Association Studies, a handful of CRC-associated loci have been identified, each containing numerous Single Nucleotide Polymorphisms (SNPs). We hypothesize that among the ~25 loci associated with increased risk for CRC, there are driver SNPs functioning to contribute to initiation or progression of CRC development. It is crucial, in order to enhance our understanding CRC tumorigenesis, to extract these functional variants from the pool of associated SNPs and validate their causative significance within the cell. To model these functional SNPs, we have developed a novel "scarless" isolation pipeline for selecting isogenic clones with the desired single-base edit without selection markers following CRISPR-Cas9 mediated HDR. With this pipeline, we have successfully modeled our first selected SNP, rs6983267, from the 8q24 risk locus in HCT-116 cells, producing the heterozygous genotype (G/T) from the parental homozygous risk (G/G) genotype. Rs6983267 is located within a known c-Myc enhancer and has been shown to exert risk-allele specific increase in enhancer activity. Oncogenic c-Myc is known to play a significant role in CRC pathogenesis. In line with previous studies, we observe a slight decrease in c-Myc expression in our heterozygous clones compared to wildtype homozygous risk. Moreover, we observe a preferential binding of the transcription factor in control of c-Myc transcriptional regulation, TCF7L2, to the risk allele over the reference allele while binding of proximal factors such as CTCF show no allelic preference. Interestingly, we also see significant changes in expression of target genes of the WNT signaling pathway. Dysregulation of WNT is a hallmark characteristic of CRC. These changes indicate there is indeed an effect of the risk allele of rs6983267 on overall WNT activity in CRC cells due to increased binding of transcription factor TCF7L2. This novel SNP editing pipeline provides a method to study precise effects of single base changes located anywhere in the genome. Utilizing it to model other risk SNPs will enable the enhancement of our current understanding of GWAS-identified risk loci in CRC pathogenesis at the molecular level.

#1446

Super-enhancer identification via Bru/BrUV-Seq and H3K27Ac ChIP-Seq.

Chunlao Tang, Vipin Yadav, Hui-Rong Qian, Jason B. Cunningham, Hong Gao, Yushi Liu, Jason C. Ting, Steven M. Bray, Philip J. Ebert, Yuhao Lin, Amit Aggarwal, John N. Calley, Bharvin K. Patel. _Eli Lilly and Company, Indianapolis, IN_.

Transcriptional super-enhancers (SEs) are large clusters of active enhancers that drive expression of genes critical to cell identity and function. In cancer and other human diseases, SEs can be acquired through somatic aberrations. Importantly, SEs and their target genes are particularly sensitive to perturbation, making this mechanism a promising target for both diagnosis and therapy. Histone modification H3K27Ac is a good single predicative marker of active enhancers, though additional marks would help. Accordingly, a common approach to SE identification is using H3K27Ac ChIP-Seq. The non-TSS (transcription start site) histone binding sites (e.g., principally excluding TSS±2.5kb regions) are mapped to genome and then subject to near-neighbor stitching (e.g., within 12.5kb). From the resulted relatively broad regions, SEs are identified by exceptional binding enrichment. Another potentially powerful approach is to use Bru/BrUV-Seq in combination. By directional sequencing of bromouridine(Bru)-labeled nascent RNAs, Bru-Seq has been developed to study nascent transcription. In the derivative BrUV-Seq, UV irradiation is additionally conducted to stabilize transcripts prior to labeling, thereby allowing more sensitive detection of TSSs and unstable transcripts, including enhancer RNAs (eRNAs). It has been shown that eRNAs indicate enhancer activity better than H3K27Ac binding. In this study, both approaches were used to identify SEs for three colon cancer cell lines, HCT-116, SW48, and SW620. We have demonstrated that Bru/BrUV-Seq has advantages in differentiating between mRNAs and eRNAs, and between overlapping transcripts; the reduced complexity helps prevent false identifications, which inflated our results obtained from ChIP-Seq.

#1447

**Identification of an oncogenic germline** KRAS **truncating mutation in hereditary cancers.**

Moloy T. Goswami,1 Daniel H. Hovelson,1 Anna Johnson,1 Scott A. Tomlins,1 Lucy Wang,1 Kimberly Zhulke,1 Bhavneet Singh,1 Sharath Kumar Anand,1 Andi Cani,1 Albert Liu,1 Steven Kamberov,1 Yi-Mi Wu,1 Dan Robinson,1 Arul Chinnaiyan,1 Kathleen A. Cooney2. 1 _University of Michigan, Ann Arbor, MI;_ 2 _University of Utah, Salt Lake City, UT_.

Somatic strongly activating KRAS mutations play an oncogenic role across numerous human cancers, while less activating germline KRAS mutations are associated with developmental disorders. KRAS encodes two splice variant products—KRAS-4A and KRAS-4B—differing in their C-terminus through alternative fourth coding exons. Though KRAS-4A is homologous to the original transforming transcript identified in Kirsten rat sarcoma virus, its role in human cancer is less characterized compared to KRAS-4B. Here, through genetic analyses of three cohorts of patients with hereditary and/or aggressive cancers, we identified a rare KRAS-4A specific C-terminal truncating germline mutation (KRAS-4A C180X; rs373169526) in affected men of three families with hereditary prostate cancer and a patient with hereditary melanoma (minor allele frequency [MAF] of 0.0014 in these combined cancer cohorts assessed vs. 0.000056 in the ExAC population database, odds ratio 24.6 [95% confidence interval 5.1-103.5], two sided Fisher's exact test p = 9.0E-5). The KRAS-4A C180X mutation truncates the C-terminus, removing the polybasic region and -CAAX motifs previously demonstrated to be necessary for Ras family member membrane association, MAP kinase signaling activation and transformation, suggesting a loss of function phenotype. However, in silico assessment of reported human variation demonstrates truncating germline variants only in KRAS-4A and not KRAS-4B, consistent with tolerance. Expression of KRAS-4A protein in NIH3T3 and MDCK leads to loss of exclusive membrane association and inhibits GTP loading, as expected, but paradoxically resulted in modest but significantly increased proliferation and soft agar colony growth compared to control or wildtype KRAS expressing cells. Pro-oncogenic phenotypes were not dependent on MAPK signaling, but showed sensitivity to AKT inhibition. In summary, we identified a germline truncating KRAS-4A mutation over-represented in hereditary cancers that defines a novel mechanism of KRAS activation not dependent on the C-terminal polybasic and -CAAX motifs.

#1448

Unraveling the oncogenic pathway of serrated polyposis syndrome driven by RNF43 germline mutation.

Helen Hoi Ning Yan, Jeffrey C W Lai, Siu Lun Ho, Anthony K W Chan, Wai Yin Tsui, Annie S Y Chan, Siu Tsan Yuen, Suet Yi Leung. _Dept. of Pathology, Univ. of Hong Kong, Hong Kong, Hong Kong_.

Serrated polyps can arise in a sporadic or familial polyposis setting and predispose to colorectal cancer (CRC). Recently, we have identified RNF43 germline mutation in a family with Serrated Polyposis Syndrome (Yan et al, GUT 2016). The presence of second somatic hit in all serrated polyps examined from members of this family further confirmed the pathogenicity of RNF43 germline mutation. In an attempt to further delineate the global genomic alterations in these serrated polyps, so as to understand the RNF43 driven serrated neoplasia pathway for the development of colorectal cancer, we performed whole exome sequencing (WES) on two sessile serrated adenomas (SSAs) resected from an RNF43 germline mutation carrier, together with the paired blood DNA. Consistent with the results from our previous Sanger sequencing study, WES detected complete inactivation of RNF43 through a 2nd hit somatic mutation, c.461 C>T P154L, or loss of heterozygosity (LOH) in the two SSAs, respectively. We also identified 51 and 58 somatic mutations in the two SSAs, respectively. BRAFV600E mutation was the only shared mutation between the two polyps. Interestingly, each of the SSAs carried a truncating mutation in a histone-methyltransferase gene, either PRDM9 or SETD1B, respectively. Other truncating or deleterious mutations included chromatin modifiers (CHD2, CHD4, TSPYL2) or other genes with methyltransferase activity (TRMT2B, METTL12, METTL14, ECE2). In addition, we found few instances of chromosomal aberration or LOH, apart from a region of LOH at chromosome 17 encompassing RNF43 in one of the SSAs.

Overall, we have revealed the genomic landscape of two sessile serrated adenomas resected from a germline RNF43 mutation carrier. The results confirmed somatic RNF43 2nd hit and BRAFV600E mutation as the key events, along with putative roles for histone methyltransferase, as well as other chromatin modifiers. These findings highlight the potentially important role of an altered chromatin in the oncogenic pathway of serrated neoplasia.

#1449

Germline mutation burden in an endometrial cancer cohort from an integrated health care system.

Raghu Metpally, Sarath Babu Krishnamurthy, Dokyoon Kim, Adam Cook, Ashlee Smith, John Nash, Marilyn Ritchie, David Carey, Radhika Gogoi. _Geisinger Medical Center, Danville, PA_.

Endometrial cancer (EMCA) is the most common gynecologic malignancy in the United States. We sought to explore germline mutations in 301 patients with a diagnosis of EMCA (EMCA cohort), who underwent whole exome sequencing as part of the Geisinger Health System's (GHS) MyCode project. We additionally compared these mutations to germline and somatic mutations in the TCGA array as well as a non-affected cohort at GHS. Whole exome sequencing was performed using 75 bp paired-end sequencing on an Illumina v4 HiSeq 2500 to a coverage depth sufficient to provide greater than 20x haploid read depth of over 85% of targeted bases in 96% of samples (approximately 80x mean haploid read depth of targeted bases). Control groups consisted of 1150 female patients over the age of 85 with no cancer diagnosis (Wellderly cohort) and the entire Geisinger's MyCode population of 50,000+ patients (GHS cohort). Somatic and germline mutations in EMCA were downloaded from the TCGA portal. We retrieved somatic mutations from 248 patients as a mutation annotation format (MAF) that consisted of 181,930 point mutations, while 2,931 indels were removed for the comparison. In addition to somatic mutations, germline mutations with the controlled access, obtained as a variant call format (VCF). Demographic characteristics, treatment and outcomes were identified through a manual chart review of the EHR. 2205 coding variants with 30 LOF and 802 missense mutations were noted in the germline. Approximately 24.67% of patients in the EMCA cohort had predictive pathogenic variants compared to control cohort. Genes with most predictive pathogenic variants among EMCA cohort are ATM, TSC2, ROS1, NOTCH2, NOTCH1. LOF mutations were found in 19 unique genes. When compared to pathogenic variants identified in other hormone related cancers or a Wellderly cohort; 26 unique variants were identified in the EMCA cohort. Overall 53.57% (2590/4836) of germline mutations were found in EMCA Cohort when compared to the overlapping mutations between DiscovEHR cohort and TCGA (5874 variants). In contrast, only (11/4836) 0.28% of germline mutations in EMCA Cohort overlap with TCGA somatic mutations (3164 variants). Based on the results, cancer predisposing germline mutations were found in a substantial number of MyCode participants with EMCA. Knowledge of these mutations and potential integration with somatic information will help to improve our understanding of the disease. This will lead to more specific communication of individual cancer risk and prognosis, cascade screening of their families and more personalized and disease-specific care, an important step towards precision medicine.

#1450

Alternative splicing analysis identifies mutation hotspots in hereditary breast and ovarian cancer genes.

Suzette Farber-Katz, Vickie Hsuan, Jayne Hoo, Sitao Wu, Huy Vuong, Dong Xu, Hsiao-Mei Lu, Phillip Gray, Aaron Elliott, Rachid Karam. _Ambry Genetics, Aliso Viejo, CA_.

Genetic testing for hereditary breast and ovarian cancer (HBOC) is becoming increasingly widespread in the era of precision medicine. The implementation of next-generation sequencing (NGS) has resulted in an explosion of genetic data. While the majority of patients receive definitive results, germline unclassified variants with unknown function are regularly detected in thousands of patients. In particular, variants of unknown significance (VUS) in the HBOC susceptibility genes BRCA1 and BRCA2 pose a quandary to medical providers and patients because these genes are clinically actionable. A large percentage of VUS in BRCA1/2 are predicted to affect splicing. Previous efforts have focused on interrogating splicing VUS using low-throughput and/or imprecise techniques. Therefore, we developed and validated a method for using RT-PCR NGS to accurately and efficiently characterize germline splicing defects. The ENIGMA (Evidence-based Network for the Interpretation of Germline Mutant Alleles) consortium recommends capillary electrophoresis and Sanger sequencing of subcloned transcripts as the gold standard to identify the BRCA1/2 transcripts that are present in patient samples. We followed the recommendations of the ENIGMA consortium and were able to largely replicate their results when we analyzed the cDNA of lymphoblastoid cell lines (LCLs) generated by the kConFab consortium from carriers of BRCA1 or BRCA2 variants known to be associated with splicing defects. In addition to LCLs, we analyzed RNA extracted from normal blood controls and normal breast tissue for the entire BRCA1 gene. We compared our capillary electrophoresis and Sanger sequencing results to our newly developed RT-PCR NGS assay. For this assay, we used RT-PCR NGS coupled to our custom in-house bioinformatics analysis to identify splicing events including exon skipping, novel intron insertion, and alternative 5' donor and 3' acceptor splicing sites. Capillary electrophoresis allowed us to roughly visualize the various transcripts present in our samples, though sequencing was needed to confidently identify the exact splicing event. Similar to Sanger sequencing, our RT-PCR NGS assay detected all major splicing events. Interestingly, we found that RT-PCR NGS allowed us to visualize more minor splicing events present in the samples due to our high coverage. We sequenced tens of thousands of reads with RT-PCR NGS, as opposed to Sanger sequencing, which was limited to low-throughput sequencing of single colonies containing subcloned RT-PCR products. In conclusion, RT-PCR NGS is a reliable high-throughput alternative to the gold-standard splicing assays. Our assay will greatly improve the interpretation of splicing VUS detected by clinical genetic tests for BRCA1/2.

#1451

Search for new hereditary breast cancer genes by whole exome sequencing.

Ekaterina Sh. Kuligina,1 Anna P. Sokolenko,1 Ilya V. Bizin,2 Aleksandr A. Romanko,3 Maria Anisimova,3 Evgeny N. Imyanitov1. 1 _N.N. Petrov Research Inst. of Oncology, St. Petersburg, Russian Federation;_ 2 _St. Petersburg State Polytechnical University, St. Petersburg, Russian Federation;_ 3 _St. Petersburg Pediatric Medical University, St. Petersburg, Russian Federation_.

Russian population is characterized by pronounced genetic homogeneity and the increased occurrence of recurrent germ-line mutations, therefore it is particularly suitable for the whole exome sequencing (WES) studies. Exome sequencing was performed for 43 breast cancer (BC) cases, which demonstrated evident clinical signs of the hereditary disease (family history and/or BC bilaterality and/or young onset) and lacked germ-line mutations in BRCA1, BRCA2, CHEK2, NBS1/NBN and BLM genes. Approximately 52070 nonsynonymous alternative variants were identified; 27503 (53%) of them were absent in 16 analyzed exomes from cancer-free controls. 2204 (4%) variants had sufficiently low allelic frequency (<1%; ExAC database) and appeared to be functional upon in silico assessment (CADD score >25); this list was composed of 186 frameshift insertions/deletions, 152 stop-gains, 74 essential splice-site disruptions and 1792 potentially pathogenic missense mutations. Presumably causative mutations in the BRCA1 network genes were revealed in 8 patients (truncating mutations: PALB2 (p.Tyr1055*, p.Arg170Ilefs), RAD51C (c.904+1G>A), FANCM (p.Ser497fs); overtly pathogenic missense mutations: FANCM (p.Arg100Trp, p.Gln891Pro), ERCC4 (p.Arg799Trp), RAD51L (p.Arg394Trp)). For the remaining 35 patients, 57 truncating variants and 109 presumably deleterious missense mutations were further analyzed in a case-control study involving 550 hereditary BC cases and 450 elderly tumor-free controls. The most promising results were obtained for several recurrent heterozygous mutations (PZP p.Arg680* [OR = 13.2; 95 % CI: 0.75 to 230.85]; LEPREL1 p.Pro636Ser [OR = 5.4; 95 % CI: 0.28 to 104.77]; ING1 p.Pro319Leu [OR = 3.8; 95 % CI: 0.18 to 80.20]) as well as for homozygous missense substitution in BRCA1 gene p.Gln356Arg [OR = 6.7; 95 % CI: 0.82 to 54.83]. Conclusion: Although being promising in the design, this study and several similar investigations failed to identify novel breast cancer gene, whose medical significance is comparable with that for BRCA1, BRCA2, etc. This failure can be attributed either to the lack of contribution of dominant highly-penetrant recurrent mutations in the missing BC heritability or yet non-identified deficiency in the experimental approach. This work has been supported by the Russian Scientific Fund (grant № 16-45-02011).

#1452

Interrogating the impact of pregnancy on breast cancer biology using DNA copy number profiling.

Bastien Nguyen,1 David Venet,1 Françoise Rothé,1 Christine Desmedt,1 Samira Majjaj,1 Giancarlo Pruneri,2 David Brown,1 Fedro Peccatori,2 Hatem A. Azim,1 Christos Sotiriou1. 1 _Institut Jules Bordet, Brussels, Belgium;_ 2 _European Institute of Oncology, Milan, Italy_.

Background

Epidemiological evidence indicates a clear relationship between pregnancy and breast cancer (BC) risk. However, little is known regarding the impact of pregnancy on BC biology. DNA copy number aberrations (CNAs) play an important role in breast carcinogenesis. BC during pregnancy is a rare disease, however it can be used as a model to study the impact of pregnancy on BC biology.

Methods

We retrospectively included 54 pregnant and 113 non-pregnant BC patients matched for age and stage with complete clinic-pathological, gene expression and 5-year follow-up data. CNAs were assessed using Affymetrix OncoScan FFPE arrays. First, we filtered genes whose expression was driven by CNAs. Then, we identified genes that were differentially affected by pregnancy both at the CNA and the gene expression level.

Results

After quality control, CNA profiles were obtained for 38 pregnant and 87 non-pregnant BC patients. There were no significant differences in classical clinic-pathological features (pT, pN, grade, PAM50). We selected 1981 genes whose expression was correlated with CNAs (ρ ≥ 0.4). Among these, 77 genes were altered at the expression level (p ≤ 0.01) between pregnant and control patients, whereas 171 genes were altered at the CNA level (p ≤ 0.05). Twenty-eight genes were concomitantly altered at the expression and CNA levels which was significantly higher than expected (p = 0.021, permutation test). These genes were mapped on three different chromosomes. Genes located on chr7q and chr11q were associated with copy number gains and were up-regulated in pregnant patients whereas genes located on chr22q were associated with copy number loss in control patients. Of interest we identified 6 putative oncogenes (CDK6, ESRRA, S6K2, LIMK2, MKL1) that were up-regulated in pregnant patients. A 'pregnancy-associated breast cancer signature' was computed from the expression of the 28 genes. There was a negative relationship between the signature and the expression of ESR1 and PGR genes. This signature was significantly more correlated with the proliferation-associated AURKA gene in pregnant patients as compared to control (ρ = 0.73 vs ρ = 0.24, p = 0.001).

Conclusions

In this study, we were able to identify several genomic alterations associated with pregnancy that could help elucidate the impact of pregnancy on BC risk. Moreover, by combining CNAs with gene expression, we were able to identify genes that could be responsible for the poorer prognosis seen in pregnancy-associated breast cancer and could be potential drivers of this rare disease.

#1453

Transcriptoma analyses in triple-negative breast cancer with BRCA1 germline mutation.

Kivvi Duarte Nakamura, Elisa Ferreira, Rodrigo Ramalho, Rafael Brianese, Dirce Carraro. _A.C. Camargo Cancer Center, São Paulo, Brazil_.

Triple-negative breast cancer (TNBC), characterized by lack of expression of the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), is associated with a higher prevalence of germline BRCA1 mutations, especially in younger women. TNBC comprises approximately 15% of breast cancer cases and is considered one of the most aggressive subtypes. BRCA1 protein plays a pivotal role in DNA damage repair, cell cycle control and transcriptional regulation. In this context, we aimed to investigate the transcriptional difference of paired tumor and adjacent normal tissue from young patients in two TNBC groups - with or without BRCA1 germline mutation. Fourteen early-onset patients (≤41 years), with TNBC previously evaluated for BRCA1 and 2 germline mutation, were included. From these, nine patients are BRCA1/2 wild-type and five patients were BRCA1 germline mutation carriers. RNASeq libraries were constructed from rRNA depleted total RNA extracted from paired tumor and normal adjacent samples and sequenced on Illumina NextSeq 500 platform. Approximately 20 million reads aligned to human genome were generated per sample, revealing about 14,500 expressed genes with at least 10 copies per sample. Expression values were normalized by library-size read counts followed by a gene-wise normalization. To explore similarity level between samples we used the full matrix of the log2-transformed gene expression values and calculated sample-to-sample distances for each tumor compared to each normal sample, based on the Euclidian distances between samples considering all available genes. We observed that 80% of the BRCA1-mutated and 67% of the BRCA1/2 wild-type samples were clustered together. Also, we detected that BRCA1-mutated TNBC showed, in general, less difference in transcriptional pattern related to normal breast tissue than the BRCA1/2-wild type TNBC. Moreover, we compared differential expression between normal and tumor samples of each group (BRCA1-mutated and BRCA1/2-wild-type samples) using a linear-based model in DESeq2 program. Based on the criteria of fold-change ≥ │4│and FDR ≤ 0.01 we identified 2,809 DEGs, in the group of BRCA1-mutated samples, being 722 up regulated and 2,087 down regulated in the tumor and 904 DEGs in the group of BRCA1/2- wild-type samples, 399 were upregulated and 505 downregulated in the tumor. Interestingly, both lists of upregulated genes in TNBC of BRCA1-mutated and BRCA1/2-wild type were significantly enriched in the same molecular pathway involved with the cell cycle and mitosis, according to Reactome Pathways software. Overall, our first observation based on this preliminary data was that, in terms of general gene expression analysis, BRCA1-mutated TNBC has less transcriptional modification in comparison to the normal tissue than the BRCA1/2-wild type and that the tumorigenic process of both groups are similar in terms of defective biological pathway.

#1454

Estrogen receptor signaling in FTE of BRCA mutation carriers.

Leah V. Dodds,1 Omar L. Nelson,1 Ramlogan Sowamber,2 Andres Rodrigues,1 Victoria de Castro,1 Wendell Henry,1 Guillermo Morales,1 Brian Slomovitz,1 Patricia Shaw,3 Sophia H. George1. 1 _LEONARD MILLER SCHOOL OF MEDICINE, UNIVERSITY of MIAMI, MIAMI, FL;_ 2 _Princess Margaret Cancer Center, Toronto, Ontario, Canada;_ 3 _University Health Network, Toronto, Ontario, Canada_.

Genetic and reproductive factors predicate epidemiological risk factors underlying epithelial ovarian cancer. The fallopian tube epithelia (FTE), the presumptive etiological site of high-grade serous ovarian cancer (HGSC), is a hormonal responsive tissue. Estrogen is known to promote cell proliferation and its metabolism produces reactive oxygen species that damage DNA and promote tumorigenesis. Estrogen receptor (ER) is rarely mutated, amplified or deleted in HGSC, yet only 10% of patients respond to anti-estrogen treatment, suggesting that intrinsic variables to the ER pathway contribute to this clinical outcome. TP53 mutations occur in almost 100% of HGSCs, indicating that mutated p53 supports a model as an early event in the pathogenesis of HGSC. We hypothesized that in the presence of dysfunctional p53, subsequent promiscuous binding of ER will yield aberrant signaling, ultimately significantly contributing to cellular transformation. Mutant p53 and ER co-localize in FTE cells, suggesting potential synergy. We established cell lines with p53 mutations and treated them with estradiol, an estrogen analog, to observe any changes in response. The genome binding sites of ER-regulated transcription factors were then identified and mapped by whole genome chromatin immunoprecipitation-deep sequencing (ChIP-Seq). The data generated will facilitate the development of gene signatures that will predict response to anti-estrogen therapy in serous ovarian cancer patients, and contribute to the discovery of biomarkers to more accurately identify patients who will benefit from hormonal therapies.

#1455

Vitamin D receptor (VDR) gene polymorphisms among Sudanese females with breast cancer attending Radio and Isotope Center of Khartoum.

Sara O. Yousif,1 Amar M. Ismail,2 Abdelrahim O. Mohamed,3 Abdelrahim O. Mohamed4. 1 _Faculty of Medical Laboratory Sciences, Sudan University of Science and Technology, Khartoum, Sudan;_ 2 _Faculty of Science and Technology, Al-Neelain University, Khartoum, Sudan;_ 3 _Faculty of Medicine, University of Khartoum, Khartoum, Sudan;_ 4 _Neelain Institute for Medical Research, Al-Neelain University, Khartoum, Sudan_.

Background: Vitamin D Receptor (VDR) gene polymorphisms has been reported to

be associated with different cancers, including breast cancer. geographical and

environmental factors have an impact on vitamin D (VD) metabolism. This study

aims to evaluate VDR-gene polymorphisms (Fok1, Bsm1, Taq1, Apa1) and its

association with breast cancer risk among Sudanese females.

Materials and Methods: this is a case control study done in the Radio and Isotope

Center in Khartoum, Sudan. Hundred thirty seven patients and 69 healthy controls

were enrolled after informed consent. Blood genomic DNA was extracted using

quinidine chloroform method, and single nucleotide polymorphisms (SNPs) Fok1

(T/C) [rs2228570], Bsm1 (G/A) [rs1544410], Taq1 (C/T) [rs731236] and Apa1 (C/A)

[(rs7975232] in VDR-gene were detected by PCR and restriction fragment length

polymorphism (PCR-RFLP) analysis.

Results: VDR-SNPs rs731236 correlated significantly with low risk of breast cancer

(OR=0.5, p= 0.002). There was no significant association observed with rs1544410

and rs7975232. Although our study did not show an overall statistically significant

association of VDR-SNPs rs2228570 with the risk of breast cancer, we found a 1.3

fold increased risk of breast cancer (OR= 1.3) among females with FF genotype when

compared with ff genotype, suggesting that females homozygous for F allele, might be

at more risk than females carrying homozygous f allele.

Conclusions: These results suggested that, VDR-SNPs rs2228570 may be related to

higher risk of breast cancer and VDR-SNPs rs731236 associated with lower risk of

breast cancer in Sudanese females.

Key words: Breast Cancer, VDR, Polymorphisms, SNPs

#1456

Identifying early genetic steps in malignant transformation of neurofibromatosis type 1- associated plexiform neurofibromas.

Alexander Pemov,1 Nancy F. Hansen,2 Rajesh Patidar,3 Christine Higham,4 Eva Dombi,4 Joseph F. Boland,5 Settara C. Chandrasekharappa,2 NIH Intramural Sequencing Center, James C. Mullikin,2 Margaret Wallace,6 Javed Khan,3 Eric Legius,7 Brigitte Widemann,4 Douglas R. Stewart1. 1 _Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD;_ 2 _Cancer Genetics and Comparative Genomics Branch, National Human Genome Research Institute, Rockville, MD;_ 3 _Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD;_ 4 _Pediatric Oncology Branch, Center for Cancer Research, NCI, Bethesda, MD;_ 5 _Cancer Genomics Research Laboratory, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Rockville, MD;_ 6 _Departament of Molecular Genetics and Microbiology, UF Genetics Institute, UF Health Cancer Center, University of Florida, Gainesville, FL;_ 7 _Department of Human Genetics, Catholic University Leuven, Leuven, Belgium_.

BACKGROUND: Neurofibromatosis type 1 (NF1) is a genetic tumor predisposition disorder caused by germline mutations in tumor suppressor NF1. Plexiform neurofibromas (PN) are benign tumors that arise prenatally or early in childhood and affect 30-50% of NF1 population. Somatic inactivation of second copy of NF1 is believed to be primary genetic event leading to PN initiation. NF1 patients have 8-12% lifetime risk of developing malignant peripheral nerve sheath tumor (MPNST), a highly aggressive soft tissue sarcoma, often arising from pre-existing PN and atypical NF (ANF). ANF are pre-malignant tumors that often arise within PN and can transform into MPNST. They are distinct from both PN and MPNST clinically and histologically, thus representing an intermediate step in malignant transformation. Several studies identified deletion of the CDKN2A/2B locus as the most frequent genetic event in ANF, however it is not clear whether other genes or pathways play role in PN transformation into ANF and further into MPNST. In this study, we performed genomic analysis of 16 ANF and 4 MPNST matched with normal DNA obtained from 14 and 4 patients, respectively.

METHODS: We performed whole exome sequencing and whole transcriptome RNASeq analyses on Illumina Hi-Seq 2500 platform and copy-number variant (CNV) analysis on Illumina HumanOmniExpressExome-8 SNP-arrays. In addition, we performed deep sequencing of NF1 and validation of select mutations on IonTorrent platform. For select tumors we estimated growth rate and metabolic activity by using volumetric MRI and FDG-PET.

RESULTS: We identified inactivation of NF1 in the majority of ANF and all MPNST. We also detected CDKN2A/B locus deletion in the majority of ANF and MPNST (heterozygous in ANF and mostly homozygous in MPNST). We determined that PRC2 genes (EED and SUZ12) were mutated in multiple MPNST but never in ANF. We identified a low number of point mutations and small indels in the genomes of ANF (median 1, range 0-4) and somewhat elevated mutation burden in MPNST (median 23, range 18-31), however none of these mutations were recurrent and none of the mutant genes (other than NF1 and CDKN2A) were present in multiple samples. We found 93 CNV per tumor (median) in ANF that constituted ~2% of their genomes. In comparison, we observed 2,249 CNV (median) in MPNST that comprised ~75% of their genomes. We didn't detect significant correlation between growth rate or metabolic activity and the degree of genomic instability or mutation burden in the tumors, however the size of the sample set was modest. RNAseq data analysis is pending.

CONCLUSIONS: It appears that PN-ANF transition is predominantly if not exclusively driven by heterozygous deletion of the CDKN2A/2B locus. Further progression to MPNST likely involves homozygous loss of CDKN2A/B and complete inactivation of the PRC2 complex. Widespread LOH in MPNST may accelerate inactivation of key gatekeepers.

#1457

**Somatic biallelic inactivation of** fumarate hydratase **(** FH **) in uterine leiomyomas.**

Jaana Tolvanen,1 Netta Mäkinen,1 Hanna-Riikka Heinonen,1 Simona Bramante,1 Miika Mehine,1 Nanna Sarvilinna,2 Jari Sjöberg,3 Oskari Heikinheimo,3 Annukka Pasanen,4 Ralf Bützow,4 Lauri A. Aaltonen1. 1 _Department of Medical and Clinical Genetics, University of Helsinki; Research Programs Unit, Genome-Scale Biology, University of Helsinki, Helsinki, Finland;_ 2 _Research Programs Unit, Genome-Scale Biology, University of Helsinki; Department of Obstetrics and Gynecology, Helsinki University Hospital, University of Helsinki, Helsinki, Finland;_ 3 _Department of Obstetrics and Gynecology, Helsinki University Hospital, University of Helsinki, Helsinki, Finland;_ 4 _Department of Pathology and HUSLAB, Helsinki University Hospital, University of Helsinki, Helsinki, Finland_.

Uterine leiomyomas (ULMs) are highly common benign tumors that affect the health of millions of women by causing a variety of symptoms. Furthermore, ULMs are the most common indication for hysterectomy, generating considerable economic costs for the health care system. Recurrent and mutually exclusive genetic aberrations leading to unique gene expression profiles have been identified in ULMs. Thus these tumors can be divided into distinct subclasses according to their molecular genetic background.

The majority of ULMs (80-90%) have specific mutations in MED12 or aberrations in HMGA2. Somatic biallelic inactivation of FH can also, albeit rarely, drive ULM tumorigenesis and has previously been detected in 1.3% of sporadic ULM samples (2/153; Lehtonen et al, 2004).

FH encodes the fumarase enzyme that catalyzes the hydration of fumarate to L-malate in the tricarboxylic acid cycle. Biallelic inactivation of FH leads to accumulation of fumarate and aberrant succination of proteins. Heterozygous germline FH mutations predispose to a rare autosomal dominant tumor predisposition syndrome, hereditary leiomyomatosis and renal cell cancer (HLRCC). This highly penetrant syndrome is characterized by uterine and cutaneous leiomyomas and in some cases renal cell cancer.

In this study, we re-evaluated the frequency of somatic biallelic inactivation of FH in ULMs in the largest sample set thus far. The study material consisted of 1167 fresh frozen ULM and their respective normal myometrium tissue samples from 375 hysterectomy patients. All samples were analyzed with high-throughput SNP array (Infinium Human Core-24+ Kit, Illumina Inc., San Diego, CA). All tumors detected with somatic deletions or loss of heterozygosity (LOH) in the FH locus were included in S-(2-succinyl) cysteine (2SC) immunohistochemistry (IHC) to indirectly detect biallelic FH inactivation. Also samples without FH double deletions were selected for Sanger sequencing of FH coding regions to detect inactivating mutations.

Somatic FH deletions or LOH were detected in 30 tumors, of which 10 were identified FH deficient by 2SC IHC. In these samples, another deletion (a double deletion) in three ULMs and a somatic nucleotide change in six ULMs was found to be the second hit causing biallelic inactivation of FH. In one sample no second hit was detected with these methods. From the six somatic nucleotide changes, four were different FH missense mutations predicted to be damaging (SIFT, PolyPhen-2, Provean) and two were synonymous substitutions in the last nucleotide of different exons. Studies to determine the impact of these synonymous changes on splicing are underway.

To conclude, in our sample set we detected 0.9% (10/1167) of the ULMs to harbour somatic biallelic inactivation of FH. MED12 exon 1 or 2 mutations and HMGA2 rearrangements were not detected in these tumors. Thus the previously published results were validated in this study with the largest sample set so far.

#1458

Genome-wide association study of unselected prostate cancer cases in Finnish population.

Csilla Sipeky,1 Teuvo L. Tammela,2 Anssi Auvinen,2 the PRACTICAL consortium, Johanna Schleutker3. 1 _University of Turku, Turku, Finland;_ 2 _University of Tampere, Tampere, Finland;_ 3 _University of Turku and Turku University Hospital, Turku, Finland_.

Prostate cancer (PrCa) is the second most prevalent cancer in men worldwide, representing the most common cancer in males in Finland with 5043 new cases and with over 850 annual deaths (2013). PrCa has a significant heritable component, with genetic factors accounting for 58% of the risk. Accordingly, genome-wide association studies (GWAS) identified 104 low penetrance PrCa susceptibility loci to date, predominantly in populations of mixed European ancestry. However, effect allele frequencies (EAF) and strength of association (Odds ratio, OR) are often variable across populations. Our aim is to identify common PrCa susceptibility alleles specific for Finnish population. Therefore, we conducted a meta-analyses of >200.000 single nucleotide polymorphisms (SNPs) in 2764 PrCa cases and 2401 controls of Finnish origin. Germline DNA samples were genotyped within the Collaborative Oncological Gene-Environment Study (COGS) using Illumina iSelect custom SNP genotyping platform. Statistical analyses of unconditional case-control logistic regression model was performed by using PLINK software. After FDR adjustment altogether 160 variants remain significant on GWAS level (p<5x10-8). The Finnish population specific PrCa associated signals are concentrated on chromosomes (chr) 7, 8, 11, 17, 19, and in close proximity to one another on the same chr. The strength of association varies between 0.59-1.86. In our study we identified 101 risk variants on chr 8, 11, 17 (OR 1.24-1.86) and 59 protective variants on chr 7, 8, 17, 19 (OR 0.59-0.80) with non-overlapping confidence intervals (CIs). The OR 1.86 is the highest by now found in GWAS studies examining common SNPs in non-familiar cases. Our data suggests that chromosome 11 seem to be risk conferring, whereas chromosomes 7 and 19 possess protective variants. The results facilitate population risk stratification for screening, clinical studies, treatment choices and functional research of prostate cancer in Finnish population

### MicroRNA Regulation of Cancer Biology 2

#1459

**Dual-strand tumor-suppressor** microRNA-145 **(** miR-145-5p **and** miR-145-3p **) are involved in castration-resistant prostate cancer pathogenesis.**

Mayuko Kato,1 Akira Kurozumi,1 Yusuke Goto,1 Nijiro Nohata,2 Takayuki Arai,1 Atsushi Okato,1 Keiichi Koshizuka,1 Satoko Kojima,3 Tomohiko Ichikawa,4 Naohiko Seki1. 1 _Department of Functional Genomics, Chiba University Graduate School of Medicine, Chiba, Japan;_ 2 _University of California, San Diego, CA;_ 3 _Department of Urology, Teikyo University Chiba Medical Center, Chiba, Japan;_ 4 _Department of Urology, Chiba University Graduate School of Medicine, Chiba, Japan_.

Prostate cancer (PCa) is the most frequently diagnosed cancer and the second leading cause of cancer-related death among men in developed countries. Androgen signaling through the androgen receptor (AR) is an important oncogenic pathway for PCa progression. Most patients initially respond to androgen-deprivation therapy (ADT), but eventually acquire resistance and progress to castration-resistant prostate cancer (CRPC). Although several clinical trials for CRPC have been carried out, resulting in the availability of novel chemotherapeutic agents, these treatments provide limited benefits and are not considered curative. Therefore, identification of effective biomarkers for detection of CRPC and understanding the molecular mechanisms of androgen-independent signaling and metastatic signaling pathways underlying PCa using current genomic approaches would help to improve therapies for and prevention of the disease. The discovery of microRNAs (miRNAs) has resulted in major advancements in cancer research. miRNAs are small noncoding RNAs that function to fine tune the expression of protein coding/noncoding RNAs by repressing translation or cleaving RNA transcripts in a sequence-depending manner. The unique characteristic function of miRNAs is to regulate RNA transcripts in human cells. Therefore, dysregulated expression of miRNAs can disrupt tightly regulated RNA networks in cancer cells. miRNAs play critical roles in various biological processes, and their dysregulation is shown in several human cancers. In this study, we constructed the miRNA expression signature of CRPC using clinical specimens because the development of therapeutic strategies is a central theme in the advancement of PCa treatments. Using CRPC expression signature data, we investigated the specific roles of miRNAs in PCa and CRPC oncogenesis by examining differentially expressed miRNAs. Based on the CRPC signature, we focused on the dual strand of pre-miR-145, miR-145-5p and miR-145-3p because these miRNAs were significantly reduced in cancer tissues, suggesting these miRNAs act as antitumor miRNAs in this disease. In miRNA biogenesis, it is the general consensus that processing of the pre-miRNA through Dicer1 generates a miRNA duplex (a passenger strand and a guide strand), and that the passenger strand has degradation and no regulatory activity and disintegrates in cells. Our present data showed that both miRNAs, miR-145-5p and miR-145-3p significantly suppressed cancer cell migration and invasion. Moreover, Kaplan-Meier survival curves showed that low expression of miR-145-3p predicted a short duration of progression to CRPC. Dual strand of pre-miR-145 functioned as tumor suppressors based on the miRNA expression signature of CRPC. Identification of miRNA-mediated cancer networks may provide novel molecular pathogenesis of the disease.

#1460

miRNome analyses reveal that activity of CAF expressed podoplanin in tumor microenvironment is modulated by exosome miRNAs involved in the PTEN-PI3K-AKT-mTOR signaling.

Anna Tejchman,1 Eric Mennesson,2 Isabelle Fixe,2 Alexandra Foucher,2 Simon Perera,3 Laura Artigas,3 Jose Manuel Mas,3 Catherine Grillon,1 Maciej Ugorski,4 Nadia Normand,2 Claudine Kieda1. 1 _Centre for Molecular Biophysics - CNRS, Orleans, France;_ 2 _tebu-bio, Le Perray en Yvelines, France;_ 3 _Anaxomics Biotech, Barcelona, Spain;_ 4 _Institute of Immunology and Experimental Therapy, Wroclaw, Poland_.

In breast cancer, cancer associated fibroblasts (CAF) express podoplanin but little is known about its mechanism of expression and modulation. This might be crucial for cancer cell movements and for the ability to escape from the tumor site. Podoplanin showed anti-adhesive activity towards tumor and immune cells. A key control of tumor microenvironment is exerted by microRNAs on gene expression. Therefore miRNAs profiling was performed with 3D-Gene microarray technology on a breast CAF model, on cell lysates and on secreted exosomes, in hypoxia versus normoxia culture conditions.

Systems biology enrichment analyses were carried out through bioinformatics using mathematical models that simulate in silico the behaviour of human physiology. Targets linked to the set of differential miRNAs, both up-regulated and down-regulated, have been analyzed with various methods including a hypergeometric distribution method. Processes linked to hypoxia, glycosylation, angiogenesis, extracellular matrix and pathways associated with PI3K-AKT, mTOR, P53 and chemokines signaling were studied.

We showed that podoplanin expression strongly affected miRNAs involved in these processes. Hypoxia increased expression of miR-210, miR-21 and miR-29b in cells over-expressing podoplanin. miR-21 is an oncogenic regulator in most cancer cells through its downstream target proteins among which stands the tumor suppressor PTEN, a candidate to alleviate hypoxia in tumors, regulating tumor angiogenesis and modulating tumor hypoxia. miR-29b is a tumor suppressor, it also correlates with PTEN repression.

We highlighted miRNA regulation of podoplanin expression through downregulation of upstream genes for tumor suppressor PTEN. The combination of miRNome analysis and systems biology bioinformatics uncovered potential miRNAs involved in the regulation of podoplanin activity in CAF. Such data analysis may be applied to protein and mRNA biomarkers and extended to other pathology pathways.

#1461

miR-424-cdc42, key signaling axis in hyperglycemic regulation of stemness in triple negative breast cancer.

Sushmita B. Nandy,1 Alexis Orozco,1 Gautham Prabhakar,1 Viktoria Stewart,1 Stephanie Jones,2 Paloma Munoz,1 Ramadevi Subramani,1 Diego Pedroza,1 Rajkumar Lakshmanaswamy1. 1 _Texas Tech Univ. Health Sciences Ctr. El Paso, El Paso, TX;_ 2 _University of Texas at El Paso, El Paso, TX_.

Introduction: Meta-analysis shows women with diabetes have a 20% increased risk of breast cancer. Diabetes increases the risk of breast cancer mortality. Additionally, patients with breast cancer and preexisting diabetes have an increased risk for distant metastasis compared with non-diabetic counterparts. The molecular mechanisms for distant metastasis in breast cancer patients with diabetes are not very well understood. Our preliminary data suggested that hyperglycemia enhances stem cell activity in triple negative breast cancer (TNBC) cells. Here, we report miR-424 to be a key regulator of breast cancer stem cell pool dynamics under hyperglycemic environment.

Materials and Methods: MDA-MB-231, TNBC cell line was maintained under euglycemic (5mM) and hyperglycemic (10mM) culture conditions. RT-PCR based microRNA array was performed followed by validation of significantly altered microRNA to confirm the effect of hyperglycemia on the global microRNA profile. Flow cytometric analysis for CD44+/CD24- was performed to assess the alterations in breast cancer stem cell population. In addition, sphere-forming assay were also performed. Mir-424 over-expressing or knocked down cell lines were established from parental MDA-MB-231 cells and maintained in hyperglycemic or euglycemic condition respectively. Promoter analysis for PR/SET domain 14 (prdm14) was done. Western blotting and immunofluorescence assays were performed for cdc42 (cell division control 42), pStat5 (phospho signal transducer and activator of transcription factor 5) and prdm14.

Results: Loss of miR-424 in TNBC cells under euglycemic conditions led to enhanced stem cell activity; whereas over expression of miR- 424 under hyperglycemic conditions resulted in suppressed stem cell activity. Introduction of cdc42, (a miR-424 target gene) in miR-424 over expressing TNBC cells under hyperglycemic conditions leads to increased stem cell activity demonstrating the significance of miR-424-cdc42 signaling in hyperglycemia. Over-expression of miR-424 in TNBC dramatically reduced its metastatic abilities in vivo. Mechanistically, we found that miR-424-cdc42 signaling in hyperglycemia promotes Prdm14 activation, a stem cell regulator through increased phosphorylation of Stat5.

Conclusions: Our findings establish a key molecular signaling cascade (miR-424→cdc42→prdm14) that promotes TNBC stem cell activity under hyperglycemic conditions.

#1462

Exosome-derived miR-429 contributes to prostate cancer chemoresistance.

Yang Liu,1 Liye Xie,1 Jing Li,1 Atsushi Mizokami,2 Evan T. Keller,3 Yi Lu,1 Jian Zhang1. 1 _Guangxi Medical Univ., Nanning, China;_ 2 _Kanazawa University, Kanazawa, Japan;_ 3 _University of Michigan, Ann Arbor, MI_.

Prostate cancer (PCa) is the most common cancer in men and the 2nd leading cause of cancer-related death in the United States. Certain miRNAs, act as tumor promoting or inhibiting factors in tumor development, function primarily by targeting specific mRNAs for degradation or inhibition of translation of their targeted proteins. Emerging evidence indicates that exosomes play a key role in tumor-host crosstalk, and exosome secretion, composition, and functional capacity are altered as tumors progress to aggressive phenotypes. From our prior report, we have constructed a potential network of 29 deregulated miRNAs, including 19 up and 10 down, in exosome samples derived from two kinds of paclitaxel resistant PCa cells (PC3-TXR and DU145-TXR) compared with their parental cells (PC3 and DU145). In this study, to further determine the roles of certain miRNAs derived from exosomes in chemoresistant cells, we firstly confirmed the lower expression level of miR-429 miRNA in the exosomes derived from the paclitaxel resistant PCa cells compared to their parental cells. In addition, when the miR-429 was over-expressed in PC3-TXR and DU145-TXR cells by stable transfection, epithelial-to-mesenchymal transition (EMT) markers significantly altered together with morphological changes. Overexpression of miR-429 decreased the tumor cell migration and invasion that associated with inactivation of PI-3K/AKT and Notch signaling pathways since the pathway specific inhibitors significantly diminished the transfected tumor cell migration and invasion. Finally, we found that overexpression of miRNA-429 significantly enhanced chemo-sensitivity of the chemoresistant PCa cells. These data suggested that exosome-derived miR-429 contributes to PCa chemoresistance and miR-429 may become a potential therapeutic target for PCa patients. This work was supported by Natural Science Foundation of China (NSFC) Key Project (81130046); NSFC (81171993; 81272415); Guangxi Projects of China (2013GXNSFEA053004; 2012GXNSFCB053004; 2013GXNSFBA019177; 1355004-5; 201201ZD004; GZPT13-35; 14122008-22; 11-031-05-K2; KY2015YB057; 14-045-12-K2).

#1463

Intravital discovery of miRNA drivers of human cancer cell directional invasion.

Konstantin V. Stoletov, Lian Willetts, Juan Jovel, Emma Woolner, John D. Lewis. _Univ. of Alberta, Edmonton, Alberta, Canada_.

Metastatic cancer cells often use directional ECM cues such as blood vessels or collagen fibers when invading through live tissue. Oncogenic miRNAs have been implicated as key regulators of cancer progression yet the systemic discovery of miRNAs that drive directional cancer cell invasion has not been achieved.

Here we describe the first in vivo quantitative whole human miRNAome screen for miRNA drivers of directional cancer cell invasion using an ex ovo avian embryo model of human cancer cell metastasis combined with high resolution intravital imaging. We identified more than twenty novel miRNAs that promote cancer cell invasion during the key rate-limiting step of cancer metastasis, the initiation and expansion of overt metastatic lesions. In silico miRNA target analysis (DIANA, Targetscan) showed significant enrichment in gene targets/pathways that control cancer cell-ECM adhesion and cancer cell contractility. These findings were further confirmed using Affymetrix gene expression analysis that showed significant changes in the expression of genes that regulate actin cytoskeleton rearrangement, cancer cell-ECM interaction and cell surface-receptor signaling. Public cancer gene expression database analysis (Oncoprint) showed that these miRNA-mRNA networks deregulated in several deadly cancers (pancreas, prostate, breast) and negatively correlate with the cancer patient survival.

To elucidate potential mechanisms of these pro-metastatic miRNAs, we utilized an intravital imaging approach. In vivo 4D cancer cell tracking revealed that these pro-metastatic miRNAs are required for successful invasion into collagen-rich tissue and for attachment to the outer surface of the vascular wall. Deregulation of these miRNAs led to formation of loose contacts with the vasculature and chaotic, non-directional cancer cell invasion patterns in living tissue. Intravital second-harmonic microscopy analysis showed that inhibition of the expression of these miRNAs blocked the ability of cancer cells to rearrange the disorganized fine collagen fiber network into thicker collagen bundles that guide directional tumor cell invasion. In contrast to parental tumor cells that persistently moved along the collagen bundles, miRNA-deficient tumor cells were observed to transiently associate with multiple collagen fibers without establishing persistent collagen fiber-tumor cell protrusion contacts. Moreover, miRNA-deficient tumor cells failed to attach and protrude along preexisting perivascular collagen bundles, resulting in the complete abrogation of cancer cell/blood vessel co-option.

In summary, we identified a novel panel of human miRNAs that are functionally involved in the regulation of directional invasion and metastasis. This work establishes these miRNAs as promising therapeutic targets to block the metastatic spread of lethal cancers.

#1464

miR-6734 induces cell cycle arrest and apoptosis via up-regulation of p21 expression in colon cancer cells.

Jong Soon Kang, Moo Rim Kang. _KRIBB, Chungbuk, Republic of Korea_.

Recently, microRNAs have been implicated in the regulation of gene expression in terms of both gene silencing and gene activation. Here, we investigated the effects of miR-6734, which has a sequence homology with a specific region of p21WAF1/CIP1 (p21) promoter, on cancer cell growth and the mechanisms involved in this effect. miR-6734 up-regulated p21 expression at both mRNA and protein levels and chromatin immunoprecipitation analysis using biotin-labeled miR-6734 confirmed the association of miR-6734 with p21 promoter. Moreover, miR-6734 inhibited cancer cell growth and induced cell cycle arrest and apoptosis in HCT-116 cells, which was abolished by knockdown of p21. The phosphorylation of Rb and the cleavage of caspase 3 and PARP were suppressed by miR-6734 transfection in HCT-116 cells and these effects were also reversed by p21 knockdown. In addition, miR-6734 transfection caused prolonged induction of p21 gene and modification of histones in p21 promoter, which are typical aspects of a phenomenon referred to as RNA activation (RNAa). Collectively, our results demonstrated that miR-6734 inhibits the growth of colon cancer cells by up-regulating p21 gene expression and subsequent induction of cell cycle arrest and apoptosis, suggesting its role as an important endogenous regulator of cancer cell proliferation and survival.

#1465

Regulatory role of miRNA-661 in triple-negative breast cancer of African American women.

Aline S. Fonseca,1 Selene Elifio-Esposito,2 Marilesia F. Souza,3 Akanksha Mahajan,1 Yara R. Zabala,1 Bruna M. Sugita,4 Luciane R. Cavalli1. 1 _Georgetown University, Washington, DC;_ 2 _Pontifícia Universidade Católica do Paraná (PUC-PR), Curitiba, Brazil;_ 3 _Universidade Estadual de Londrina (UEL), Londrina, Brazil;_ 4 _Universidade Federal do Paraná (UFPR), Curitiba, Brazil_.

Triple negative breast cancer (TNBC) disproportionately afflicts young African American (AA) women when compared to Non-Hispanic Whites (NHW), being usually diagnosed in advanced stage, likely to experience metastasis and often unresponsive to treatment. An increased number of studies have characterized the differences in the tumor biology between AA and NHW patients and have shown that breast tumors from AA patients present increased cell proliferation, elevated expression of angiogenesis markers and higher migration and invasive properties, the fundamental hallmarks of cancer. MiRNAs are short sequences of non-coding RNAs that act on gene expression regulation. In this study, our main objective was to determine the expression levels of miRNA-661 in the TNBC of AA when compared to the NHWs and to verify it is directly role in affecting the aggressive TNBC phenotype. MiRNA-661 expression analysis was conducted by RT-qPCR in the tumor tissue of 31 AA-TNBC patients and 17 NHW-TNBC patients. AA and NHW samples of 40 and 20 non-TNBC subtype, respectively, and 49 adjacent normal tissue (ANTs) were used as control for the subtype and tumor specificity, respectively. Our results showed a significant down-regulation of miR-661 expression in TNBC of AA in relation to the NWH (P<0.0001) and between the AA-TNBC and non-TNBC subtypes (P<0.001). MiR-661 was also significantly differentially expressed in the ANT when compared to the tumor tissue (P<0.001). In addition, ROC analysis showed a high power of this miRNA in discriminating the TNBC genome of AA and NHW women (AUC= 0.79 (0.65-0.92), 95%CI). Modulation of the expression of this miRNA in the HCC1806 and BT549 TNBC cell lines, derived from an AA and NHW patient respectively, using inducers/repressors systems, showed its direct effect in cell proliferation, migration and cytotoxicity. Interestingly these effects presented a higher impact in the HCC1806 cell line model. Altogether, our findings indicate that miRNA-661 dysregulation is associated with TNBC of AA women, and directly impacts their tumor phenotype. The uncovering of the molecular mechanisms that dictates these effects, specially the ones that modulate drug response, is critical for patient selection and stratification into novel and efficient target therapies. This much needed knowledge would lead to profound clinical benefits, particularly to young AA patients, that are commonly affected by the TNBC aggressive phenotype. Funding: This project was supported by the Georgetown University Center of Excellence in Regulatory Science and Innovation (CERSI U01FD004319), a collaborative effort between the university and the U.S. Food and Drug Administration to promote regulatory science through innovative research and education. This research does not necessarily reflect the views of the FDA. The authors thank the CNPq - Brazil for scholarship (ASF).

#1466

QKI, a miRNA-200 target gene, suppresses epithelial-to-mesenchymal transition in oral squamous cell carcinoma cells.

Yoon Ho Ko,1 Eun Ju Kim,2 Der Sheng Sun,1 Hye Sung Won,1 Young-Ho Ahn2. 1 _Catholic University, Uijeongbu-si, Republic of Korea;_ 2 _Ewha Womans University College of Medicine, Seoul-si, Republic of Korea_.

Objectives:

The microRNA (miR)-200 family plays a major role in specifying the epithelial phenotype by preventing expression of the transcription repressors, ZEB1 and ZEB2, which are well-known regulators of epithelial-to-mesenchymal transition (EMT) in epithelial tumors including oral squamous cell carcinoma (OSCC). Here, we elucidated whether the miR-200 family members control RNA-binding protein quaking (QKI) which is a newly identified tumor suppressor and is regulated during EMT.

Methods:

We predicted that miR-200a and miR-200b could recognize QKI 3'-UTR by analyzing TargetScan and TCGA head and neck SCC dataset. To further verify the role of miR-200 on QKI in HNSCC, we carried out the functional study in CAL27 and HSC3 cells.

Results:

Forced expression of miR-200b/a/429 inhibited the expression of ZEB1 and ZEB2, and decreased cell migration in CAL27 and HSC3 cells. QKI expression was also suppressed by miR-200 over-expression, and the 3'-UTR of QKI mRNA was directly targeted by miR-200 in luciferase reporter assays. Interestingly, shRNA-mediated knockdown of QKI led to pronounced EMT and pro-tumor effects in vitro and in vivo studies of OSCC.

Conclusion:

QKI increases during EMT and is targeted by miR-200; while, it suppresses EMT and tumorigenesis, contradictorily. We suggest that QKI and miR-200 could form a balancing feedback loop maintaining homeostatic responses to EMT-inducing signals.

#1467

A large set of miRNAs is deregulated since the earliest steps of human HCC.

Francesca Rizzo,1 Luca Di Tommaso,2 Pia Sulas,3 Elena Coviello,1 Chiara Novello,2 Alessandro Weisz,1 Massimo Roncalli,2 Amedeo Columbano3. 1 _University of Salerno, Salerno, Italy;_ 2 _Humanitas University, Rozzano, Italy;_ 3 _University of Cagliari, Cagliari, Italy_.

Introduction: Hepatocellular carcinoma (HCC) is the result of a stepwise process preceded by the development of premalignant lesions. To assess the involvement of microRNAs (miRNAs) in hepatocarcinogenesis it is critical to understand whether and which miRNAs are involved in the early stages of tumor development. To this end, changes in expression of miRNAs were investigated in low and high grade dysplastic nodules (LGDNs, HGDNs), early HCCs (eHCCs) and progressed HCCs (pHCCs).

Materials and Methods: Small RNA sequencing (smallRNA-Seq) was applied to search for liver miRNAs and to profile their expression patterns in 14 cirrhotic nodules (CNs), 9 LGDNs, 6 HGDNs, 6 eHCCs and 20 pHCCs) from 17 patients with liver lesions of different etiology (HCV, HBV and alcohol). To investigate miRNAs differentially expressed in pHCCs towards their matched cirrhotic tissue, we applied the Random-Variance Model (F-test) and Multivariate Permutation Test (Fold Change, FC ≥ 2.0). QRT-PCR analysis was also performed on 14 additional patients with the same etiology of the first cohort for validation of the smallRNA-Seq results.

Results: We identified a 62 miRNA expression signature that differentiates pHCCs from matched CNs. Notably, 52/62 miRNAs dysregulated in pHCCs were likewise altered in LGDN and HGDN, suggesting their possible role in HCC onset. Indeed, 28 miRNAs showed lower expression in CN that was significantly increased in LGDNs and remained high up to pHCCs. In contrast, 24 miRNAs displayed an opposite behavior, as their expression was very low or absent all throughout the tumorigenic process, compared to CNs. Interestingly, 2 miRNAs (let-7a-5p, miR-101-3p) showed a progressive decrease, suggesting that they are involved in later stages of the process. To further support the finding that dysregulation of miRNAs is a very early event in HCC development, we performed qRT-PCR in a series of LGDN, HGDN, eHCC and pHCCs from 14 additional patients. We selected for this analysis a handful of miRNAs found dysregulated by NGS analysis. The results demonstrate that all the 6 miRNAs examined (miR-429, miR-141-3p, miR-200a-3p and miR-200b-3p, miR-375 and miR-21-5p), were dysregulated at all stages of the tumorigenic process as compared to CNs.

Conclusion: The results demonstrate that miRNAs deregulation may be a critical player in human hepatocarcinogenesis, from early stages to pHCC. They also suggest that some miRNAs might represent biomarkers useful for differential diagnosis of dysplastic and neoplastic liver lesions.

#1468

MiR-200b targets ARHGAP18 and suppresses triple negative breast cancer metastasis.

Chengfeng Yang,1 Brock Humphries,2 Yunfei Li,1 Zhishan Wang1. 1 _University Of Kentucky, Lexington, KY;_ 2 _Michigan State University, East Lansing, MI_.

Triple negative breast cancer (TNBC) refers to a group of highly heterogeneous malignant tumors that lack estrogen receptor and progesterone receptor expression, and human epidermal growth factor receptor 2 amplification, accounting for 10-20% of newly diagnosed breast cancer cases. Compared to other subtypes of breast cancer, TNBCs usually show worse clinical features such as rapid tumor growth, earlier recurrence, and more aggressive metastasis. Unfortunately, the mechanism of TNBC metastatic behavior has not been well understood. Moreover, no efficient targeted therapies for TNBCs are currently available, representing a real unmet need for effective new therapies. MiR-200 family members are among the first miRNAs reported to function as potent inhibitors of cancer metastasis. However, the mechanism of miR-200 family on cancer metastasis has not been well understood. In this study, we investigated the effect of miR-200b, one member of the miR-200 family, on TNBC metastasis using cell culture and mouse orthotopic mammary xenograft tumor models. We found that the expression level of miR-200b is significantly lower in TNBC cells and tissues than that in other types of breast cancer. Stably expressing miR-200b significantly reduced TNBC cell migration and invasion and suppressed TNBC metastasis in a mouse orthotopic mammary xenograft tumor model. Mechanistic studies revealed that miR-200b overexpression in TNBC cells caused drastic changes in cellular actin cytoskeleton organization patterns as evidenced by reduced lamellipodia formation but increased stress fiber formation. In consistent with these findings, Rho GTPase pulldown assays demonstrated that stably expressing miR-200b significantly increased the Rho GTPase Rho A activation, but reduced the Rho GTPase Rac1 activation. Moreover, inhibition of Rho A signaling impaired the inhibitory effect of miR-200b on TNBC cell migration. Bioinformatics analysis indicated that ARHGAP18, a specific Rho A GTPase activating protein (GAP), is a predicate target of miR-200b. Further Q-PCR, Western blot and 3'UTR reporter analysis confirmed that ARHGAP18 is a target of miR-200b. Knocking down ARHGAP18 in TNBC cells using siRNAs significantly increased Rho A activation but reduced Rac1 activation. To further determine the role of ARHGAP18 in TNBC, ARHGAP knockout TNBC cells were generated using the CRISPR technology. It was found that knockout ARHGAP18 phenocopied the effect of miR-200b overexpression. Moreover, overexpressing ARHGAP18 in miR-200b stable expression cells overcome the inhibitory effect of miR-200b on TNBC metastasis. Together, these findings suggest that miR-200b suppresses triple negative breast cancer metastasis by targeting ARHGAP18 and enhancing Rho A activation.

#1469

**Integrated analysis of microRNA, mRNA, and protein expression utilizing MultiOmyx** TM **and NanoString** TM **from formalin-fixed paraffin-embedded, lung, head and neck, breast, and melanoma tumors.**

Qingyan Au, RaghavKrishna Padmanabhan, Nam Tran, Lakshmi Chandramohan, Nicholas Hoe. _NeoGenomics, Aliso Viejo, CA_.

Cancer is characterized as a loss of normal cellular regulation, due to accumulation of mutations and disruption of complex biological pathways. MicroRNAs (miRNAs) regulation of co-stimulatory and immune checkpoint pathways have been implicated as one of the potential mechanisms for cancer evasion in immuno-oncology. It is estimated that 30% of all mRNA expression may be regulated by miRNAs, and some are either oncogenic or tumor suppressive. Complexity of miRNA regulation highlights the need for integrated assays, providing direct correlation between miRNA and mRNA, and protein expression. From a single 4 µm FFPE section, MultiOmyxTM hyperplexed immunofluorescent assay (demonstrated to stain up to 60 protein biomarkers) is utilized to measure CD3, CD4, CD8, CD16, CD56, Granzyme B, FoxP3, ICOS, OX40, OX40L, PD1, PDL1, HLA-DR, and Ki67 protein expression. From an adjacent 10 µm section, NanoStringTM nCounter PanCancer Immune Profiling Panel and Human v3 miRNA expression panel were utilized to comprehensively profile the expression of 770 mRNA and 800 miRNA.

Integrating MultiOmyx and NanoString technologies, the current study measured miRNA, mRNA, and protein expression in lung, head and neck, breast, and melanoma samples. For each indication, three samples were selected from a larger sample set, based on high protein expression of lymphocytes and macrophage markers (CD3, CD4, CD8, CD56, CD16), co-stimulator markers (ICOS, OX40), and immune checkpoint markers (PD1, PD-L1). Protein expression results indicate positive correlation between expression of ICOS and OX40 with higher infiltration of Thelper (CD3+CD4+), Tcytotoxic (CD3+CD8+), and effector T cells (CD3+CD8+Granzyme B). NanoString normalized mRNA counts for the protein biomarkers profiled indicate that all markers except for HLA-DR belong to low expressers group with counts ranging from 20-700. Comparison of protein expression to mRNA counts revealed inconsistencies in modulated markers (PD1, PD-L1, ICOS, OX40) which are attributed to differences in population of cells between the two sections. Direct assessment of up regulation of miRNA and down regulation of target mRNA could not be made for miRNAs reported in literature as negative regulator of PD-L1 (miR-34a, 34b, 34c), PD1 (miR-28, miR-107), FoxP3 (miR-210, miR-24, miR-31), and ICOS (miR-101). Analysis of multiple miRNAs (combinatorial targeting) mapped in context to mRNA, and their respective protein expression will be presented from lung, head and neck, breast, and melanoma cancer samples.

#1470

Fine-tuning the expression of heterogeneous network of genes involved in androgen signaling, aerobic glycolysis, apoptosis and epithelial-mesenchymal transition by microRNA-644a in prostate cancer potentiation of AR signaling therapy by miR-644a: Selective manipulation of the prostate cancer transcriptome by miR-644a.

Jey Sabith Ebron,1 Jagjit Singh,1 Eswar Shankar,2 Crystal Weyman,1 Sanjay Gupta,2 Daniel J. Lindner,3 Girish C. Shukla1. 1 _Cleveland State Univ., Cleveland, OH;_ 2 _Case Western Reserve University, Cleveland, OH;_ 3 _Lerner Research Institute, Cleveland, OH_.

Intratumoral Prostate cancer (PCa) heterogeneity is characterized by multiple intrinsic mechanisms including Androgen signaling, aerobic glycolysis, aberrant transcriptional activation of c-Myc and dysregulation of miRNAs. The ability of prostate cancer cells to alter growth and metabolism in a manner distinct from benign cells is a major concern in the treatment of castration-resistant prostate cancer. Therefore, targeting the diverse oncogenesis promoting pathways involved in PCa progression and resistance at the posttranscriptional level using micro RNAs (miRNAs) offers a novel therapeutic option. Using a miRNA mimic screen, we identified a human-specific miRNA miR-644a, which modulates expression of a diverse set of oncogenic pathways in PCa including androgen signaling, AKT/IGF-1R signaling, c-myc mediated signaling and EMT pathways. We have also demonstrated a potent tumor suppressive function of miR-644a in mouse prostate cancer xenograft model. miR-644a also sensitizes the PCa tumors to current therapeutic options including AR antagonist Enzalutamide by regulating the key modulators in development of drug resistance. In conclusion, our study demonstrates that miR-644a is a novel tumor suppressor miRNA regulating key prostate carcinogenesis promoting pathways.

#1471

miR-148a-3p regulation of OTX2 and PRC2 in group 3 and 4 medulloblastoma.

Qingfu Xu,1 Kimberly Wang,1 Sama Ahsan,2 Fausto Rodriguez,2 Charles Eberhart,2 Yugang Jiang,3 John Laterra,1 Yunqing Li1. 1 _Hugo W. Moser Research Institute at Kennedy Krieger and Johns Hopkins School of Medicine, Baltimore, MD;_ 2 _The Johns Hopkins School of Medicine, Baltimore, MD, United States, Baltimore, MD;_ 3 _The Second Xiangya Hospital of Central South University, Changsha, China_.

Four different molecular subgroups are recognized in medulloblastoma (MB). Among these subgroups, Group 3 and 4 tumors are the most aggressive malignancies with few effective therapies. New therapies are likely to be most effective if they target molecular alterations that mediate the formation and growth of these malignancies.

We searched the public cancer microarray database Oncomine and found that the expression of OTX2, c-Myc, polycomb repressor complex 2 (PRC2) subunits EZH2 and SuZ12 are significantly higher in Groups 3 and 4 as compared to the SHH subgroups. Moreover, Immunohistochemical staining of a medulloblastoma tissue array showed that H3k27me3 expression is higher in Group 3 and 4 compared with the SHH groups. These findings suggest that these distinct molecular alterations could represent promising therapeutic targets. In this study, we identify miRNAs with the potential to serve as therapeutic agents for Groups 3 and 4 by targeting oncogene pathway OTX2 and c-Myc as well as targeting PRC2 and modulating H3k27me3-targeted tumor suppressors.

Using TargetScan, Pictar and miRanda prediction algorithms, miR-148a-3p is predicted to target OTX2. We show that enforced miR-148a-3p potently inhibits protein expression of OTX2 and c-Myc in medulloblastoma cells. Luciferase reporter analysis shows that miR-148a-3p expression downregulates OTX2 luciferase activities, and overexpressing OTX2 cDNA that lack the respective 3'-UTR region recognized by miR-148a-3p significantly rescues OTX2 downregulation by miR-148a-3p. Furthermore, we show for the first time that enforced miR-148a-3p expression can down-regulate the expression of the PRC2 methylase EZH2 and the PRC2 regulatory subunit SUZ12 in multiple MB cell lines. Conversely, silencing endogenous miR-148a-3p expression up-regulates the expression of EZH2 and SUZ12. Furthermore, enforced OTX2 expression attenuates the expression of miR-148a-3p-downregulated PRC2 subunit EZh2 and Suz12. Consistent with the effects of miR-148a-3p on EZH2 and SUZ12, miR-148a-3p was found to modulate H3k27me3 levels and restore the expression of H3k27me3-targeted tumor suppressors. We also show that enforced miR-148a-3p increases the sensitivity of Group 3 and 4 medulloblastoma cells to radiation and chemotherapy. Together, our findings show that miR-148a-3p has the potential to modulate Group 3 and 4 medulluloblastoma malignancy and chemo/radiation sensitivity by (1) reversing H3k27me3 modifications and thereby potentially restoring tumor suppressor expression; and (2) down-regulating OTX2 and leading to the inhibition of c-Myc-dependent oncogenes.

#1472

Alternate translation initiation regulation of PRDX5 mRNA by miR6855<->3p in basal<->like breast cancer cells.

Gautam Chaudhuri, Smita Misra. _Meharry Medical College, Nashville, TN_.

Human peroxiredoxins (PRDXs) are a superfamily of six thiol<->dependent peroxidases that are able to reduce hydrogen peroxide, alkyl hydroperoxides and peroxynitrite. Apart from the classical, apparently redundant function of mitochondrial and often peroxisomal peroxide neutralization shared by other peroxiredoxins, PRDX5 was initially discovered as a DNA binding transcriptional repressor regulating the biosynthesis of cytotoxic Alu RNAs through a mitochondrial<->localization signal<->truncated mature form (SPRDX5] of this protein that is accumulated in the nucleus and binds to a 60 bp nucleotide sequence at the Alu gene promoters. Our data suggest that SPRDX5 also binds to the Alu<->sequence<->containing BRCA2 gene silencer to prevent the binding of SLUG at the silencer thus enhancing the BRCA2 gene expression leading to the genotoxin and radiation<->resistance of aggressive SLUG<->high basal<->like breast cancer cells. PRDX5 mRNA contains two in<->frame start codons (AUGs) that are conditionally used as alternate translation initiation sites. Translation from the first AUG would result in the synthesis of a larger 214<->residue protein (LPRDX5) whereas the use of the second AUG would result in the production of a shorter 162<->residue polypeptide (SPRDX5). Since SPRDX5 lacks the mitochondrial localization signal, it is accumulated in the nucleus via its C<->terminal bipartite nuclear localization signal. We present evidence here that a miRNA, miR<->6855<->3p, binds the primary PRDX5 transcript between the two AUG codons and prevents the translation of the mRNA from the first AUG codon but not that from the second AUG codon thus favoring the initiation of translation from the second AUG codon. To characterize the effect of miR<->6855<->3p on Prdx5 mRNA translation, we developed three different PRDX5<->FLAG constructs in pCV3XFLAG14 (Sigma) that has Prdx5 ORF with or without mutation on the first or second AUG codon. Transfection of these constructs into BT549 cells followed by treatments with miR<->6855<->3p mimic or antagomiR and subcellular fractionation further supported our notion. We propose here that miR<->6855<->3p acts as a tsmiR by promoting the biosynthesis of the transcriptional regulator protein SPRDX5 which enhances the expression of the tumor suppressor protein BRCA2 leading to the genotoxin and radiation<->resistance of aggressive SLUG<->high basal<->like breast cancer cells. Supported in part by DOD-CDMRP IDEA Expansion Grant# BC103645 and NIH/NCI grant 1R21CA181920 01 to GC and 1U54RR026140 to SM.

#1473

miR-1207-3p regulates c-Myc in aggressive prostate cancer.

Dibash K. Das, Olorunseun O. Ogunwobi. _CUNY Graduate Center/CUNY Hunter College, New York, NY_.

Prostate cancer (PCa) is the most common non-skin cancer and the second leading cause of cancer-related death for men in the U.S. A major challenge is understanding the molecular mechanisms involved in the progression from the asymptomatic androgen-dependent PCa to the lethal castration resistant prostate cancer (CRPC). The chromosomal region 8q24 is associated with aggressive PCa and variants of this region have been identified to interact with the PVT1 non-coding gene in PCa. In previous work we demonstrated that microRNA-1207-3p (miR-1207-3p) has prognostic value in PCa, and directly binds to the 3' UTR of Fibronectin type III domain containing 1 (FNDC1) to regulate a novel FNDC1/fibronectin (FN1)/androgen receptor (AR) pathway upregulated in metastatic PCa. miR-1207-3p is encoded at the PVT1 gene locus, which is located downstream of c-Myc on the 8q24 human chromosomal region. Studies have suggested amplification at the 8q24 human chromosomal region, which includes C-Myc, as a possible prognostic factor for CRPC. c-Myc is a well-established proto-oncogene that is commonly found to be amplified in up to 72% of CRPCs significant amplication of c-myc has been a consequence of antiandrogen treatment, and c-Myc is downstream of AR in some PCa. miRNAs have also been implicated in the regulation of c-Myc. Nevertheless, the mechanisms regulating c-Myc remain unclear in PCa. Using a panel of five PCa cell lines we discovered that c-Myc expression is higher in aggressive CRPC cell lines compared to non-aggressive CRPC and androgen-dependent prostate cancer cell lines. Furthermore, our data reveals that c-Myc expression is higher in the aggressive E006AA-hT PCa cell line when compared to the indolent E006AA PCa cell line. This suggests that c-Myc is associated with aggressive PCa. Moreover, overexpression of miR-1207-3p suppresses the expression of c-Myc in E006AA-hT PCa cells but not E006AA PCa cell line in which c-Myc is underexpressed compared to the normal prostate epithelial cell line, RWPE-1. Next, using a synthetic biotinylated miR-1207-3p duplex, we discovered that overexpression of miR-1207-3p more effictively inhibited migration, inhibited proliferation and increased apoptosis in the aggressive E006AA-hT PCa cell line with increased c-Myc expression when compared to the indolent E006AA PCa cell line with decreased c-Myc expression. These data demonstrate that the miR-1207-3p/FNDC1/FN1/AR pathway may regulate c-Myc in aggressive PCa.

#1474

Epigenetic silencing of microRNA-137 enhances ASCT2 expression and tumor glutamine metabolism.

Xiao Daibiao,1 Junli Dong,1 Zihan Zhao,2 Ping Ren,3 Cong Li,4 Yufeng Hu,1 Jianguo Shi,5 Hexiu Su,1 Zhaojing Wang,1 Hudan Liu,1 Bo Li,4 Peng Gao,6 Guoliang Qing1. 1 _Medical Research Institute of Wuhan University, Wuhan, China;_ 2 _The Second Hospital of Dalian Medical University, Dalian, China;_ 3 _Hubei University of Science and Technology, Xianning, China;_ 4 _Zhongshan School of Medicine of Sun Yat-Sen University, Guangzhou, China;_ 5 _Affiliated Union Hospital of Tongji Medical College, Wuhan, China;_ 6 _Affiliated Dalian Sixth People's Hospital, Dalian, China_.

Cancer cells amplify the expression of ASC amino acid transporter 2 (ASCT2, also called SLC1A5), a high-affinity glutamine carrier protein, to coordinate metabolic reprogramming and malignant transformation. Yet genetic and/or epigenetic mechanisms underlying the control of ASCT2-mediated glutamine metabolism remain to be clarified. Combined in-silico algorithms with systemic experimental screening, we herein identify the tumor suppressor miR-137 as an essential regulator that targets ASCT2 mRNA and cancer cell glutamine metabolism. Metabolic analysis shows that miR-137 derepression, similar to ASCT2 inactivation, significantly inhibits glutamine consumption and TCA cycle anaplerosis. Mechanistically, methyl-CpG binding protein 2 (MeCP2) and DNA methyltransferases (DNMTs) cooperate to promote active methylation of the miR-137 promoter and its decreased transcription, which conversely enhances ASCT2 expression and glutamine metabolism. As such, expression between miR-137 and ASCT2 is inversely correlated in multiple human cancer types, including colorectal carcinomas, glioblastomas, prostate and pancreatic cancers. These findings thus elucidate a universal mechanism responsible for ASCT2 deregulation in human cancers, revealing a molecular link between miR-137, ASCT2 and tumor metabolism.

#1475

miRNA-450a suppresses adhesion but promotes invasion through targeting of TMEM182 in oral squamous cell carcinoma.

En-Wei Hsing,1 Shine-Gwo Shiah,1 Ching-Chuan Kuo,1 Jang-Yang Chang2. 1 _National Health Research Institutes, Miaoli, Taiwan;_ 2 _National Health Research Institutes, Tainan, Taiwan_.

Our microRNA (miRNA) expression signatures of oral squamous cell carcinoma (OSCC) revealed that miR-450a was significantly increased in cancer tissues compared with normal epithelium. In this study, we focused on the functional significance of miR-450a in cancer cells and identification of miR-450a-regulated novel targets in OSCC. Overexpression of miR-450a in DOK and SAS cells showed significant inhibition of cell adhesion and induction of cell invasiveness, suggesting that miR-450a functions as an onco-miRNA. We performed genome-wide gene expression analysis to search for miR-450a-regulated molecular targets. Gene expression data and luciferase reporter assays revealed that TMEM182 was directly targeted by miR-450a. The miR-450a-reduced cellular adhesion was blocked by TMEM182 restoration, suggesting that miR-450a exhibits its oncogenic activity through negatively regulating TMEM182 level. Furthermore, miR-450a expression could be induced by the cytokine TNF-α primarily through activating extracellular signal-regulated kinase (ERK) signaling pathway. ERK inhibitor prevented the TNF-α-induced miR-450a expression and enhanced adhesion ability. Taken together, these data indicate that TNF-α/ERK-dependent expression of miR-450a plays an important role in mediating cellular adhesion and invasiveness, and scavenging miR-450a function using antagomir may have therapeutic potential for the treatment of OSCC. (The study was supported by the following grants: MOST 103-2320-B-006-036-MY3 and MOST 105-2325-B-400-001 from the Ministry of Science and Technology of Taiwan, ROC)

#1476

Cell-free miR-141 as a molecular marker for prostate cancer metastasis.

Marilesia Ferreira de Souza,1 Ilce Mara de Syllos Cólus,1 Aline Simoneti Fonseca,2 Hellen Kuasne,3 Paulo Emilio Fuganti,4 Deepak Kumar,5 Silvia Regina Rogatto,3 Luciane Regina Cavalli2. 1 _State University of Londrina, Londrina, Brazil;_ 2 _Georgetown University, Washington, DC;_ 3 _AC Camargo cancer center, São Paulo, Brazil;_ 4 _Londrina cancer hospital, Londrina, Brazil;_ 5 _University of the District of Columbia, Washington, DC_.

Prostate cancer is the second most commonly diagnosed neoplasia in men. Currently, there is no available non-invasive tool to assist in the identification of the aggressive form of this disease. Cell-free nucleic acid has been tested as a potential new molecular tool that can be useful in the diagnosis and prognosis of prostate cancer. MicroRNAs are a class of non-coding RNAs, that are shown to impact the development and progression of prostate cancer and their detection in plasma have been indicated as a promising non-invasive tool for disease screening and prognosis assessment. Among the several deregulated miRNAs that are observed in prostate cancer, the miRNA-141-3p was observed with upregulated expression levels in prostate cancer, particularly in the metastatic lesions. Therefore, the main aim of this study was to verify the diagnostic potential of miR-141 as a circulating tumor marker for prostate cancer and determine its functional role in modulating tumorigenesis in prostate cancer metastatic cell lines. MiR-141 expression analysis was performed in plasma samples of 102 prostate cancer patients without treatment and of 50 health controls by RT-qPCR. The experimental assays were performed by transfecting the PC3 cell line using miRNA-141 mimic/ inhibitor systems and directly accessing their effect in classical tumor phenotypes. Expression of the miR-141 were found significantly upregulated in the metastatic patients' plasma specimens (FC=9.11, p=0.04; AUC=0.66) when compared to the patients without metastasis, showing its screening potential for prostate cancer metastasis. The in vitro ectopic expression of miR-141 in PC3 cell line showed a significant increased in cell proliferation, associated with changes in cell cycle (decreased number of cells in the G1 phase). Conversely, the inhibition of miR-141 showed an increase in the migratory ability, cell adhesion and in the Docetaxel cytotoxicity of the PC3 cells. Western-blot analysis showed that the inhibition of the miR-141 levels reduced the expression of the EMT inducers markers E-CADHERIN and CLAUDIN, and up-regulated the levels of ZEB-1, pAKT and VIMENTIN, consistent with their repression roles in the EMT process. These findings showed that miR-141 presents a direct function in controlling some of the in vitro metastatic phenotypes in prostate cancer, through the regulation of the epithelial-mesenchymal transition expression proteins. In conclusion, miR-141 presents a potential use as a minimally invasive molecular marker for prostate cancer diagnosis and can be used as a druggable target for therapy, particularly in the metastatic clinical setting.

#1477

Melatonin regulates the tumor suppressor miR-148a-3p involved in angiogenesis and metastasis of breast cancer.

Debora Zuccari,1 Jéssica Zani Lacerda,2 Lívia Carvalho Ferreira,2 Beatriz Camargo Lopes,3 Andrés Felipe Aristizábal-Pachón,4 Marcio Chaim Bajgelman5. 1 _Faculdade de Medicina de São José do Rio Preto - FAMERP, São José do Rio Preto, Brazil;_ 2 _Universidade Estadual Paulista - UNESP, São José do Rio Preto, Brazil;_ 3 _Laboratorio de Investigação Molecular do Cancer - LIMC/FAMERP, São José do Rio Preto, Brazil;_ 4 _Department of Genetics/Faculdade de Medicina - USP, Ribeirão Preto, Brazil;_ 5 _Laboratorio Nacional de Biociencias/Centro Nacional de Pesquisa em Energia e Materiais - CNPEM, Campinas, Brazil_.

Women with breast cancer has the tumor progression and angiogenesis-induced metastasis as the main cause of death. MicroRNAs (miRNAs) are small noncoding mRNA molecules that play an important role in gene regulation and once deregulated, these molecules may be involved with the progression of different human tumor types, including breast cancer. These miRNAs play an oncostatic role on tumor suppressor genes and regulate the process of angiogenesis, tumor growth and metastasis. So as new possible adjuvant treatment against breast cancer our group have shown melatonin, that is a hormone secreted by the pineal gland, by exhibiting several anti-tumor and antiangiogenic effects. Therefore, the aim of this study was to evaluate the potential therapeutic of melatonin on miRNAs regulation to verify breast cancer progression and potent tumor suppressor miR-148a-3p. In silico analysis was performed for selection of miRNAs involved in breast cancer. The MDA-MB-231 breast cancer cell line (metastatic negative estrogen receptor) was grown and separated in two different experimental conditions maintained for 24 hours: control group and melatonin-treated group (concentration of 1 mM). After this period the extraction of total RNA was performed (Qiagen®) and the total concentration of miRNAs of each sample was determined (NanoDrop Spectrophotometer 2000C - Thermo Scientific®). Differential expressions of these miRNAs was evaluate using miScript miRNA PCR-Array (Qiagen®) containing 84 miRNAs associated with breast cancer. The overexpression of miR-148a-3p in MDA-MB-231 cells was performed by bacterial cloning vector and the relative quantification of gene expression of their target IGF-1R and VEGF by real-time PCR. Moreover, the quantification of protein expression was performed by immunocytochemistry of IGF-1R and VEGF. Lastly, the migration and invasion cell assays were carried out to assess the potential capacity of these cells to undergo metastasis. The analysis of miRNAs MDA-MB-231 cell line by PCR-array showed 24 upregulated miRNAs and 8 miRNAs downregulated after the treatment with melatonin. Relative quantification shows that the melatonin treatment increases the gene expression of miR-148a-3p and decreases the gene and protein levels of IGF-1R and VEGF. The capability of migration and invasion of the breast cancer cells decreased after treatment with melatonin and overexpression of miR-148a-3p. Our results confirm the action of melatonin on the miR-148a-3p regulation known being involved in the progression of breast cancer. Thus the establishment of this therapeutic protocols can control these cellular events essential for the prognosis of patients with breast cancer.

#1478

Curcumin inhibits epithelial-mesenchymal transition and invasion in breast cancer cells by controlling miR-34a expression.

Marcela Gallardo, Richard Ponce-Cusi, Gloria M. Calaf. _Universidad De Tarapaca, Arica, Chile_.

Breast cancer in advanced stages tends to develop metastases and / or chemoresistance, in both cases therapeutic options are limited and have low probability of success, which represents the biggest obstacle in reducing mortality from this disease. There is a close connection between the Epithelial-Mesenchymal Transition (EMT) process of cancer cells and the acquisition of invasive and metastatic ability. Numerous EMT mediators have been described in cancer and among them miRNAs play a fundamental role in regulating such process, suggesting that it could be a therapeutic target to address this phenotype. Curcumin (diferuloylmethane) is a derivative compound of Curcuma longa that has therapeutic properties in various cancers as blocking initiation and tumor progression through its anti-inflammatory, antioxidant, proapoptotic, antiangiogenic and antimetastatic effects. The role of curcumin on EMT in non-cancerous breast cells MCF-10F and in breast cancer cell lines MCF-7 and MDA-MB-231 was evaluated. This work shows that in all these cell lines curcumin induced the expression of tumor suppressor microRNA miR-34a and repressed the expression of several genes involved in EMT and metastasis as Axl, Slug, Twist, N-cadherin, vimentin, fibronectin, among others. Consequently, curcumin inhibited the migration and invasiveness in these cells, irrespective of the expression of estrogen and progesterone receptors and p53 mutational status. Blockade of miR-34a by transfection with antagomiR-34a inhibited the effect of curcumin on EMT genes and on the migratory/invasive potential of cells indicating that miR-34a plays a central role in the Curcumin-mediated suppression of EMT and invasion. Therefore, results confirm the suppressive effect of curcumin on EMT and invasion in breast cancer cells, showing that such substance exerts this effect by inducing expression of miRNA miR-34a and consequently the repression of several of its target genes. Supported by Tarapacá University, Arica, Chile (GMC).

#1479

Differential expression of microRNAs in transformation of follicular lymphoma to diffuse large B cell lymphoma.

Katerina Musilova,1 Gabriela Pavlasova,1 Vaclav Seda,1 Eva Vojackova,1 Katerina Cerna,1 Veronika Svobodova,1 Robert Pytlik,2 Vit Prochazka,3 Zuzana Prouzova,3 Sarka Pospisilova,4 Lenka Zlamalikova,5 Heidi Mocikova,6 Lenka Kruzova,3 Marie Jarosova,3 Andrew Evans,7 Clive Zent,8 Leos Kren,5 Marek Trneny,2 Jiri Mayer,4 Andrea Janikova,5 Marek Mraz1. 1 _Ceitec MU and University Hospital Brno and Faculty of Medicine MU, Brno, Czech Republic;_ 2 _General University Hospital and Charles University in Prague, Prague, Czech Republic;_ 3 _Palacky University and University Hospital Olomouc, Olomouc, Czech Republic;_ 4 _University Hospital Brno and Faculty of Medicine MU, Brno, Czech Republic;_ 5 _University Hospital Brno, Brno, Czech Republic;_ 6 _University Hospital Kralovske Vinohrady and Charles University in Prague, Prague, Czech Republic;_ 7 _University of Rochester Medical Center, School of Medicine and Dentistry, Rochester, NY;_ 8 _Wilmot Cancer Institute, University of Rochester Medical Center, School of Medicine and Dentistry, Rochester, NY_.

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression, and are frequently aberrantly expressed in cancer. We aimed to understand their role in the transformation of indolent follicular lymphoma (FL) into an aggressive diffuse large B cell lymphoma. This happens in ~3% of cases per year during the course of the disease, and is associated with median survival of only 2 years. The NGS revealed number of aberrations associated with transformed FL (tFL), including frequent high-level activity of MYC (amplifications, translocations, and mutations) or loss of DNA damage regulators (p53, CDKN2A/B). Firstly, we performed a miRNA profiling (TaqMan miRNA Arrays) in paired FL and tFL samples (N=8 pairs). This revealed a relatively small group of 5 miRNAs that are consistently differentially expressed in tFL (P<0.05, fold-change >1.5). Since the most frequently acquired aberration in tFL is the high-level activity of MYC we performed a correlation analysis of MYC levels and expression of these miRNAs in additional samples of FL, tFL, and CLL samples with/without MYC duplication (N=40 FL/tFL, N=39 CLL). This revealed that at least one of these miRNAs is significantly down-modulated (P<0.05) in cases with high-levels of MYC. The MYC-mediated repression of miRNA levels was also observed (P<0.05) in B cells from transgenic MYC over-expressing mice (MYC controlled by an Ig-alpha enhancer) in comparison to wild-type animals (samples obtained from young animals before occurrence of any malignancy). We have further shown that the levels of this miRNA affect B cell proliferation in vitro, and its low-levels associate with percentage of Ki67 positive cells in FL samples (P<0.005). Moreover, low levels of tFL-associated miRNA were present in FL cases with a shorter overall survival (P<0.01), and its expression directly affected BCR signalling (calcium flux assay after anti-IgM). We have shown that the expression of this miRNA is not only down-modulated by high-level MYC expression, but also by B cell adhesion to stromal cells in co-culture in vitro (HS-5 stromal cells). This suggests that its normal physiological function might be related to regulation of B cell functions in the context of immune niches, and this might play a role in FL progression and transformation. It remains to be elucidated what other molecular mechanisms ensure low-level expression of the studied miRNA in cases that do not harbor MYC over-expression, and what pool of target mRNAs is regulated by this miRNA in FL cells.This work was supported by: the Ministry of Health of the Czech Republic, grant nr. 16-29622A. All rights reserved. contact: marek.mraz@email.cz

#1480

Mir-182 is involved in sulindac anticancer activity in colon cancer.

Hongyou Zhao, Bin Yi, Zhipin Liang, Ruixia Ma, Yaguang Xi. _Louisiana State University Health Sciences Center, New Orleans, LA_.

Nonsteroidal anti-inflammatory drugs (NSAIDs) display promising antineoplastic activity in many human solid tumors including colorectal cancer. Previous studies reported that sulindac sulfide (SS) can inhibit the growth of tumor cells through cyclooxygenase-2 (COX-2) dependent or independent pathways. Obviously, COX-2 independent pathway involves a low toxic property to support the clinical potential for using sulindac as a chemoprevention drug. However, the molecular mechanisms responsible for COX-2 independent pathway have not been completely elucidated. In this project, we employed two human colon cancer cell lines, referred to HCT116 and HT29. HCT116 cells are characterized for low COX-2 expression, while HT29 cells show relatively high COX-2 expression. We found that SS could unbiasedly inhibit the growth of HCT116 and HT29 cells by arresting cells in G1/G2 phases. CyclinG2 was found to be upregulated in response to SS treatment. FOXO3a has been reported to regulate CyclinG2 expression at the transcriptional level. Our results demonstrate that SS could also upregulate FOXO3a. By using the loss-of-function strategy, we found that SS could not efficiently upregulate CyclinG2 and lead to cell cycle arrest in cells with FOXO3a knockdown. In addition, we studied the mechanism that could be involved in SS regulation of FOXO3a. Our previous studies reported that a panel of miRNAs could be altered by SS treatment in both colon and breast cancer cells. In the downregulated miRNA list, we found that miR-182 could potentially target FOXO3a. By using luciferase assay, we validated the direct regulation of miR-182 on the expression of FOXO3a. When miR-182 was downregulated, SS could neither efficiently upregulate the expression of FOXO3a nor inhibit cell growth as it did in control cells. We gained highly consistent results in HT29 and HT29 cells with COX-2 knockdown. Therefore, our study demonstrates a pathway consisting of miR-182/ FOXO3a/ CyclinG2 as a novel mechanism responsible for SS anticancer activity in colon cancer, which may imply a COX-2 independent pathway.

#1481

Determination of microRNA profile in lung cancer cell line treated with chemotherapy cisplatin (C), pemetrexed (P) or PC with bevacizumab (B) and its implication in resistance.

Chao H. Huang,1 Hannah C. Motes,2 Siddarth Sharma,2 Jared Kevern,3 Emma Borrego-Diaz,4 Mukut Sharma2. 1 _University of Kansas Med Ctr, Lenexa, KS;_ 2 _Kansas City VA Medical Center, Kansas City, MO;_ 3 _University of Kansas Med Ctr, Kansas City, KS;_ 4 _Eurofins Lancaster Laboratoris, Lancaster, PA_.

Background: Cellular microRNAs (miRNAs) regulate gene expression through modulation of messenger RNA transcription and are involved in epigenetic regulation, metastasis and cancer immunity. They have prognostic and therapeutic significance. The miRNAs associated with resistance in lung cancer without EGFR mutation are not yet known. We sought to determine the profile of miRNA of lung cancer cell lines without EGFR mutation treated with chemotherapy.

Methods: lung cancer cell lines HTB177 were treated with PC or PCB (0.05mg/mL Cisplatin, 0.05 mg/mL pemetrexed, 6.25 mg/mL Bevacizumab) for 24, 48, 72 and 144 hours. We obtained baseline miRNA prior to therapy and after treatment to determine the changes in miRNA profile using RT PCR technique. After 144 hours, we obtained miRNA profile of the cells that survived treatment to assess change in the miRNA profile. We chose miRNA that has been reported to influence on the outcome of lung cancer: Let 7 modulates RAS, miRNA 27 is involved as tumor suppressor gene, mir212 negatively regulates antiapoptotic protein PED, miR 375 down regulates ASH1 a transcription factor. Mir21 targets anti-apoptotic genes including PDCD4 and PTEN.

Results: We observed down regulation of these miRNA after 24h of treatment with PC and these miRNA then became upregulated after 48 hours, especially let 7f. In contrast, the treatment of PC with B caused upregulation of these miRNA, especially miR 27b and let 7f at 24 h and then decreased levels of upregulation at 48hours. These miRNA were not upregulated at 72h for both groups. The expression of miRNA 21 is upregulated 50 times in cells that survived treatment with PC or PCB at 144h.

Conclusions: The treatment using PC and PC with B in lung cancer cell lines regulates change in the profile of miRNA at different time points. There is a difference of the miRNA profile in these 2 treatment groups. miRNA 21 is upregulated in cells resistant to therapy. miRNA could be involved in the activity of chemotherapy and development of resistance.

#1482

**Triptolide inhibits the growth of osteosarcoma by regulating microRNA-181a via targeting PTEN gene** in vivo **and** in vitro **.**

Chunming Jiang, Xiang Fang, Xuepeng Wang, Maoqiang Li, Wu Jiang, Liulong Zhu, Zhenyu Bian. _Hangzhou First People's Hospital, Nanjing Medical University, Hangzhou, China_.

Objective: We aimed to study the effect of triptolide on OS and the related molecular mechanism.

Methods: The cell viability, apoptosis portion, tumor size, tumor weight and invasion of OS cells were determined. The relative level of miR-181 in OS tissues and the adjacent tissues was determined by qRT-PCR. The target gene of miR-181a was determined and verified by luciferase report assay. At last, OS cells were treated with triptolide and triptolide + miR-181a mimics to verify the relationship between triptolide and miRN-181a.

Results: Triptolide inhibited the cell viability, promoted the apoptosis, decreased the tumor size and weight, and reduced the invasion of OS cells. The level of miR-181a in OS cells decreased significantly after treating with triptolide, and the relative level of miR-181a in OS tissues was markedly higher than that in the adjacent tissues. PTEN was reported and verified the direct target gene of miR-181a. The overexpression of miR-181a decreased the inhibition of triptolide on OS proliferation and promotion on OS apoptosis.

Conclusion: Triptolide inhibited the proliferation of OS by regulating miR-181a via targeting PTEN gene in vivo and vitro.

#1483

Alteration of miR-186 expression modifies inflammatory markers in normal epithelial and prostate cancer cell models.

Suman Suman,1 Dominique Z. Jones-Reed,1 M. L. Schmidt,1 Geoffrey J. Clark,1 Carolyn Klinge,1 Shirish Barve,1 Kevin S. Kimbro,2 La Creis R. Kidd1. 1 _University of Louisville, Louisville, KY;_ 2 _North Carolina Central University, Durham, NC_.

Dysregulation of miRNAs and chronic inflammation are strongly implicated in the development of various malignancies, including prostate cancer (PCa). Previously, our lab identified several inflammatory and immune response sequence variants in CCL5 and CCR5 significantly modified PCa risk. Recently, we propose an oncogenic role for miR-186-5p based on its up-regulation in serum from PCa patients and metastatic PCa cell lines. Moreover, miR-186-5p inhibition reduced proliferation, anchorage independent growth and invasion in metastatic PC cells (PC3, MDA-PCA-2b). We hypothesize dysregulation of inflammatory and immune response markers may enhance immune surveillance leading to a reduced aggressive tumor phenotype. Following modulations in miRNA-186 levels in normal prostate epithelial (RWPE1) and metastatic PCa (PC3) cell lines, we evaluated alterations in mRNA expression using micro-array analysis. Ectopic expression of miRNA-186 in the RWPE1 resulted in a 1.3-2.6-fold down-regulation in TLR2, IRAK2, CCL20, IL1RAP, IL1RAP, IL15, and IL17RD (FDR p-value ≥7.8x10-06). Whereas, inhibition of miR-186 in PC3 cells corresponded with a 1.4-fold up-regulation of IL18R1 (p=0.0095). Notably, these inflammatory markers are involved in cell invasion (TLR2), apoptosis (IL17RD), tumorigenesis (TLR2, IRAK2) and autoimmune diseases (CCL20). Validation of these targets is underway using qRT-PCR, western blots and luciferase reporter assays. We will assess the mechanism by which these miR-186 targets suppress aggressive cancer behavior using knock-out and knock-in cell and ultimately animal models. Future studies may identify inflammatory targets that may guide immune therapies for the effective treatment of aggressive prostate cancer.

#1484

Genome wide study reveal microrna based methylation phenotype in oral cancer.

Roshni Roy,1 Aniruddha Chatterjee,2 Navonil De Sarkar,3 Bidyut Roy1. 1 _Indian Statistical Institute, Kolkata, India;_ 2 _Dunedin School of Medicine, Dunedin, New Zealand;_ 3 _Fred Hutchinson Cancer Research Center, Seattle, WA_.

Introduction: Oral cancer is one of the leading causes of cancer burden in India. Studies over the years have enforced the role of deregulated microRNA expression in oral cancer development and progression. Incidentally not much is known about the cause of microRNA deregulation or to what extent can it be attributed to methylation. Established genome wide microarray platforms like 450K Illumina Bead Chip have probes for very limited number of microRNAs along with restriction on distance of the probes from microRNA transcription start site. We have used genome wide methylation sequencing approach RRBS to get deeper insight on microRNA methylation.

Aim: Genome wide methylation profile of 15 paired oral cancer and adjacent normal tissue was compared to generate a detailed map of microRNA methylation in oral cancer. The methylation results were further correlated to microRNA expression data generated on a subset of these samples.

Methodology: RRBS based global methylation sequencing of 15 pairs of samples was conducted on HiSeq 2500 with an average 10X depth. Analyses were done using DMAP software. Previously generated TLDA based microRNA expression data was used for correlation analysis.

Result: Similar to protein coding genes, microRNAs also showed methylation based clustering. Interestingly majority of methylation hits were more than 2kb upstream of miRNA transcription start site which is generally not considered in known microarrays. Distribution of hypo and hyper methylation sites is different across the various CpG features like cores and shelves as well as across chromosomes. MicroRNAs like mir-181c, miR-99b, miR-23a among others showed significant negative correlation between methylation frequency and expression level. IPA based pathway analysis emphasized repression of apoptosis and cell death pathways along with activation of cell proliferation and colony formation.

Conclusion: Understanding the role of methylation in microRNA deregulation could help us detect and possibly prevent a chain of events, which lead to disrupted pathways eventually leading to cancer, at the onset.

#1485

Measuring relative utilization of aerobic glycolysis in breast cancer cells by positional isotopic discrimination.

Da-Qing Yang, Margot Cleary. _University of Minnesota, Austin, MN_.

Cancer deaths, including breast cancer, are caused by metastasis of the malignant tumors to distant locations. However, current methods of detection cannot distinguish pre-invasive breast cancer from noninvasive breast tumor or benign breast disease. Population-wide mammographic screenings have led to increased detection of ductal carcinoma in situ or DCIS, noninvasive, proliferative cells contained by the basement membrane of the terminal ductal lobular unit. DCIS is usually not associated with metastasis and/or cancer death. Each year, in the US alone, about 1.5 million of women who have been diagnosed with DCIS or a suspicious lump/lesion by mammography will require resection or breast biopsy after diagnosis for further pathologic analysis. However, ~80-85% of biopsies result in noninvasive breast disease or benign findings. As a result, a considerable number of patients suffer from side effects caused by breast biopsy and/or overtreatment. Therefore, there is an urgent need to find a biomarker for pre-invasive breast cancer.

The ability of cancer cells to produce lactate through aerobic glycolysis (the Warburg effect) is a consistent hallmark of cancer, including breast cancer. Recent advancements in liquid chromatography-mass spectrometry (LC-MS) technology have significantly improved the sensitivity of this method compared to traditional NMR or GC-MS-based technologies, which make it feasible to detect very low concentrations of small molecules or metabolites. We have recently established a positional isotopic labeling and LC-MS-based targeted metabolomics method that can directly measure the conversion from [1-13C]glucose to [3-13C]lactate through glycolysis. Our results show that metastatic breast cancer cells exhibit a dramatically increased production of [3-13C]lactate from [1-13C]glucose even under aerobic conditions as compared to low- or noninvasive breast cancer cell lines. We found that the rate of aerobic glycolysis is closely correlated with glucose uptake and lactate production in breast cancer cells. We have also observed significantly elevated production of [3-13C]lactate in serum samples of early stage metastatic mammary tumors developed in mice. Since elevated levels of lactate are closely correlated to increased tumor aggressiveness, these results suggest that monitoring of lactate production from glycolysis by targeted metabolomics may provide a biomarker for pre-invasive breast cancer. These results will pave the way for further exploration of the elevated production of lactate as a promising biomarker for pre-invasive breast cancer and for assessment of therapeutic response in clinical trials. 

### Mitochondria, Autophagy, and Cancer Metabolism

#1487

ONC201 kills breast cancer cells by inhibiting mitochondrial respiration.

Yoshimi Greer,1 Samuel Gilbert,1 Celia Islam,1 Yun Ji,1 Luca Gattinoni,1 Christina Stuelten,1 Natalie Porat-Shliom,1 Roberto Weigert,1 Xiantao Wang,2 Markus Hafner,2 Kunio Nagashima,1 Donna Voeller,1 Stanley Lipkowitz1. 1 _NCI, Bethesda, MD;_ 2 _NIAMS, Bethesda, MD_.

Background: ONC201 is a small molecule originally identified as a TRAIL inducing compound currently being tested in phase1/2 clinical trials in multiple cancer types. Two recent studies reported that ONC201 also induces an atypical stress response mediated in part by ATF4 and CHOP.

Methods: ONC201 was obtained from Oncoceutics, Inc. Recombinant GST-TRAIL was prepared in the laboratory. Cell viability was tested with MTS assay and CellTiter-Glo luminescent cell viability assay. ATP level was measured with CellTiter-Glo 2.0 assay. RNAseq and western blotting were performed to investigate change of gene expression. Mitochondrial respiration was monitored by Seahorse XF analyzer. Live cell imaging was performed to examine the mode of cell death. Confocal microscopy and electron microscopy analysis were performed to study mitochondrial morphology.

Results: We tested the effects of ONC201 on 18 human breast cancer cell lines that represent ER+, HER2 amplified, TNBC basal A and TNBC basal B breast cancer. ONC201 reduced cell viability in breast cancer cell lines in all subtypes tested with IC50s ranging from 0.8-5 μM, similar to what has been reported for other cancer cell types. Unexpectedly, ONC201 toxicity was not dependent on TRAIL receptors or caspases and live cell imaging revealed ONC201 induces cell membrane ballooning followed by rupture. By contrast, GST-TRAIL induced TRAIL-receptor dependent caspase mediated death and classic apoptosis morphology. These results suggest that ONC201 kills breast cancer cells via a caspase-independent, TRAIL-receptor-independent mechanism distinct from TRAIL-induced apoptosis. Western blots revealed that ONC201 induces the stress pathway proteins ATF4 and CHOP, consistent with the recently published observations. ONC201 also induced phosphorylation of AMP-dependent kinase (AMPK) and depletion of cellular ATP in multiple breast cancer cell lines. Seahorse XF analysis found that ONC201 inhibited mitochondrial oxygen consumption rate but did not inhibit glycolysis as measured by the extracellular acidification rate. Both ONC201-induced toxicity and ATP depletion were enhanced when cells were cultured in non-glucose (galactose) medium. Supplementing glucose to cells grown in galactose medium partially prevented ONC201-dependent ATP depletion, induction of phospho-AMPK, ATF4 and CHOP, and cell death. These data are consistent with an inhibition of oxidative phosphorylation (OxPhos) by ONC201. RNAseq revealed ONC201 inhibits expression of multiple mitochondrial genes involved in OxPhos and other mitochondrial functions, and western blot confirmed those findings. Confocal and electron microscopic evaluation revealed abnormal mitochondrial morphology.

Conclusion: Our data demonstrate that ONC201 can kill breast cancer cells by a novel mechanism involving disruption of mitochondrial morphology and inhibition of mitochondrial respiration.

#1488

Altered skeletal muscle mitochondrial function and redox biology with chemotherapy and exercise in a colorectal cancer mouse model.

Malcolm Andrew West,1 Alexandrina Roman,1 Ronan Astin,2 Sian Pugh,1 Bernadette Fernandez,1 Annette Hayden,1 Judith Cave,3 Eleanor Jaynes,4 John N. Primrose,1 Sandy Jack,1 Michael P. Grocott,1 Andrew Murray,5 Martin Feelisch,1 Alexander Mirnezami,1 Timothy Underwood1. 1 _University of Southampton, Southampton, United Kingdom;_ 2 _University College London, London, United Kingdom;_ 3 _University Hospital Southampton, Southampton, United Kingdom;_ 4 _University Hospitals Southampton, Southampton, United Kingdom;_ 5 _University of Cambridge, Cambridge, United Kingdom_.

Background: Chemotherapy improves outcomes for patients with gastrointestinal cancer, but carries associated morbidity. Platinum-based chemotherapy adversely impacts fitness, invivo mitochondrial function, and cellular redox status in humans, however the mechanisms are poorly understood. Exercise interventions mitigate against this, but interactions between the effect of the cancer, chemotherapy, and exercise intervention are poorly understood. Using a colorectal cancer (CRC) mouse model we investigated changes in skeletal muscle oxidative phosphorylation (OxPhos) capacity and redox biology with chemotherapy and exercise.

Method: SCID mice (n=48) were randomized to laparotomy with (Ca) or without (NCa) orthotopic caecal injection of DLD-1 CRC cells. CRC established over 8 weeks then mice were randomized to 1) 3 cycles of intra-peritoneal (IP) chemotherapy (Chemo; 5FU 5mg/kg, Oxaliplatin 10mg/kg and Leucovorin 90mg/kg) followed by interval exercise-training 2) Chemo and no exercise 3) IP saline sham (Sal) with exercise or 4) Sal with no exercise. Blood was collected pre and post-Chemo and at death (post-exercise), and markers of oxidative stress measured. Soleus muscle mitochondrial function was analyzed using high-resolution respirometry. Measurements included maximum OxPhos capacity (PI+II+β), Complex I and II capacities and the ratio of octanoyl-carnitine to pyruvate-supported respiration (FAO). Histopathological caecal examination was undertaken to determine cancer response.

Results: Chemotherapy alone resulted in 37% lower OxPhos capacities in sham mice (p=0.011), whilst FAO was 40% lower (p=0.004). Cancer alone resulted in 38% lower OxPhos capacities (p=0.012). In combination with cancer, chemotherapy resulted in OxPhos capacities that were 42% lower than in mice with cancer but no chemotherapy (p=0.007), along with 51% lower Complex I (p<0.001) and 42% lower Complex II (p=0.007) capacities. In sham mice (NCaSal), exercise resulted in 33% greater Complex II activities (p=0.042), but exercise did not alter mitochondrial function in any other group. Total plasma protein content (TPPC) increased with time in the CaSal (+32%) and the NCaSal mice (+23%), whilst it decreased with chemotherapy (CaChemo -20%, NCaChemo -10%). Oxidative stress increased with chemotherapy and exercise, however exploratory interaction analyses are ongoing. Complete histopathological response was seen in 2 of 6 CaChemoEx animals. No other complete response was seen.

Conclusion: Chemotherapy and cancer alone were associated with a loss of oxidative capacity, with a greater reduction when combined. Chemotherapy alone and with cancer caused a shift away from FAO and reduction in TPPC. Though we demonstrated an exercise effect in sham mice, exercise did not increase OxPhos capacity, nor did it ameliorate the effects of cancer and chemotherapy. A more intense exercise intervention may be required.

#1489

Targeting the mitochondrial enzyme proline dehydrogenase with a mechanism-based irreversible inhibitor induces selective mitochondrial stress and enhances breast cancer cell death under hypoxia.

Gary K. Scott, Katya Frazier, Christina Yau, Beatrice Becker, Mauricio Ortega, Christopher C. Benz. _Buck Inst. for Research on Aging, Novato, CA_.

Proline dehydrogenase (PRODH) is a p53-inducible inner mitochondrial membrane flavoprotein functionally linked to electron transport for proline catabolism and intracellular ATP production, particularly under nutrient stress conditions. We have previously shown that in breast cancer cells, PRODH and glutaminase (GLS1) transcript levels are inversely correlated, supplying anaplerotic glutamate to triple-negative (GLS1) and luminal (PRODH) breast cancer subtypes by different means; and that siRNA knockdown or competitive inhibition of PRODH induces synthetic lethal interactions with both GLS1 inhibition and p53 upregulation in various malignant (ZR-75-1, DU4475, MCF7) but not normal (MCF10A) breast epithelial models. In the present study we have synthesized and structurally modeled a novel mechanism-based irreversible (suicide) inhibitor of PRODH, N-propargylglycine (PPG), that shows more than 2-fold greater capacity to inhibit PRODH activity in isolated mitochondrial assays when compared to competitive PRODH inhibitors (L-tetrahydrofuroic acid, THFA; or 5-oxo-2-tetrahydrofurancarboxylic acid, 5-oxo). Modeling human PRODH predicts a post-reactive PPG structure with PPG covalently linked to the enzyme pocket's FAD moiety, producing pocket distortion that does not occur with competitive PRODH inhibitors. Reflecting PPG's irreversible binding to PRODH, mitochondria isolated from PPG treated ZR-75-1 cells are unable to catabolize proline despite being able to efficiently catabolize malate; in contrast, isolated mitochondria from 5-oxo treated cells remain efficient at catabolizing both proline and malate. Unexpectedly, we observed that PPG but not the competitive inhibitors induces selective degradation of mitochondrial PRODH protein levels within 24 h of cell culture treatment followed by loss of other mitochondrial proteins like complex-I NDUFS1 but not by concomitant loss of cytosolic FAD-containing proteins like MTHRF. MitoTracker assays confirm the selective cellular loss of mitochondrial mass in ZR-75-1 cells within 24 h of PPG treatment. To confirm that suicide inhibition of PRODH can also induce synthetically lethal metabolic interactions, breast cancer cells (MCF7) were cultured under normoxic (20% O2) or hypoxic (1% O2) conditions and then treated with PPG and a p53 upregulator known to induce PRODH expression (MI-63). Cell viability demonstrated that during hypoxia PPG with p53 upregulation synergistically reduces cell survival at 48 h, confirming the synthetic lethality of this treatment combination. Altogether, these findings support the preclinical development of suicide PRODH inhibitors as potential cancer therapeutics capable of inducing mitochondrial stress, exploiting synthetically lethal metabolic conditions, and selectively enhancing cancer cell death.

#1490

Role of sub-cellular specific reactive oxygen species in heart regeneration after cancer therapy.

Salim Abdisalaam, Souparno Bhattacharya, Kalayarasan Srinivasan, Shibani Mukherjee, Hesham A. Sadek, Aroumougame Asaithamby. _UT Southwestern Medical Center, Dallas, TX_.

Introduction: Cardiovascular disease and cancer are the two leading causes of morbidity and mortality worldwide. As advancements in radiation therapy (RT) have significantly increased the number of cancer survivors, the risk of radiation-induced cardiovascular disease in this group is a growing concern. However, the molecular mechanism of radiation-induced heart failure is still elusive. Recently, it has been discovered that the reactive oxygen species (ROS)-mediated oxidative DNA damage is the primary upstream mechanism that prevents cardiomyocyte proliferation. Therefore, elucidating the spatial and temporal aspects of ROS production will lead to the development of countermeasures to prevent heart injury following chest radiotherapy.

Methods: We have generated GSH redox potential (Grx-roGFP) ROS probe targeted to cytoplasm, chromatin, nucleolus, telomere, nuclear inner membrane and heterochromatin. These probes have been inserted into cardiomyocytes specific AAV9 vectors which were used to infect cardiomyocytes both in vitro an in vivo. For in vivo study, we infected three months old mice with high-titer AAV particles via tail vein injection and then exposed to chest-only radiation (5-10 Gy). At different post-radiation times, fresh heart slices of 300-500 µM thickness were either mock- or treated with mitochondrial electron transport complex (ETC) inhibitors and then subjected to live tissue imaging using a confocal microscope.

Results: Interestingly, our results showed that the distribution of basal ROS levels is not uniform in different sub-nuclear compartments. Significantly, upon the induction of oxidative stress, the ROS levels was elevated in all the cellular compartments, but the extent of ROS level was significantly higher in the cytoplasm. Similarly, radiation altered ROS levels in all the cellular compartments; however the effect of radiation on the ROS levels was sub-nuclear compartment-specific. Intriguingly, we found that the complex IV of the ETC was critical for the maintenance of ROS levels in different cellular compartments as compared with complexes I and II.

Conclusion and Future Directions: Our data clearly indicate that the spatial and temporal levels of ROS are not uniform across the cell and the mitochondrial ETC plays a major role in regulation ROS levels in different sub-nuclear compartments. The results obtained from this study can be utilized to develop sub-cellular compartment specific targeted both genetic and pharmacological ROS scavengers that will help to regenerate adult heart by re-activating the proliferative capacity of cardiomyocytes following cancer therapy. Finally, our novel approach can be applied to assess alterations in ROS levels in different cancers.

Funding: This work was supported by the NASA (NNX13AD57G/NNX15AE06G) CPRIT (RP160520) and NIH R01AG053341 grants.

#1491

Mitochondrial dysfunction from defective Cytochrome oxidase complex induces tumorigenic phenotype.

Satish Srinivasan, Manti Guha, Gordon Ruthel, Hiroshi Nakagawa, Narayan Avadhani. _University of Pennsylvania, Philadelphia, PA_.

Mitochondrial defects that affect activity of the electron transport chain (ETC) complexes are associated with several diseases including cancer. Dysfunctional mitochondria resulting from mitochondrial DNA (mtDNA) alterations or exposure to xenobiotics have been shown to initiate retrograde signaling pathway characterized by disrupted membrane potential, elevated cytosolic calcium and activation of Calcineurin, a calcium dependent phosphatase. In some cell types these events form the basis for triggering transcriptional reprogramming that converts non tumorigenic cells to tumorigenic phenotype. Cytochrome oxidase (CcO) is the terminal enzyme of the ETC that catalyzes the transfer of electrons from reduced cytochrome C to oxygen. All the mutations of CcO subunits identified as prevalent in various cancers like prostrate, pancreatic, colon and ovarian cancers have been found in mtDNA encoded subunits. Effect of loss of nuclear subunits leading to reduced Cytochrome oxidase activity on tumor progression are not well studied. Nuclear subunits IVi1 and Vb of CcO are susceptible to various stress conditions like exposure to long term hypoxia, ischemia-reperfusion and treatment with ethanol resulting in selective degradation and loss of activity. Here we report the activation of retrograde signaling by loss of these subunits. Genetic silencing of the subunits IVi1 and Vb, and loss of activity resulted in metabolic shift to glycolysis and was accompanied by increased glucose utilization. Disruption of the CcO complex activated many of the hallmark factors of Ca2+/Calcineurin mediated retrograde signaling. Importantly, rescue of the CcO deficiency by overexpressing CcO subunits or by inhibitors of retrograde signaling pathway attenuated the phenotypic changes such as anchorage independent growth and increased invasive potential. Further, esophageal tumor sections from human patients revealed reduced CcO subunits IVi1 and Vb in the center of the tumor mass suggesting a pathological significance of these findings. Our results show that mitochondrial ETC defect initiates a retrograde signaling and induces genes with major roles in tumor development. (Supported by NIH grants CA-22762 and GM-34883)

#1492

Chemical approaches to development of mitochondrial-targeted Hsp90 inhibitor in anti-cancer therapeutics: Mechanism and structure change of mitochondrial Hsp90.

Sung Hu, Nam Gu Yoon, Byoung Heon Kang. _Ulsan National Institute of Science and Technology(UNIST), Ulsan, Republic of Korea_.

Tumor Necrosis Factor Receptor-Associated Protein 1 (TRAP1), a member of the heat shock protein 90 (Hsp90) which located in mitochondria, is thought to promote tumor cell survival and to reprogram energy metabolism in cancer. Previous studies have demonstrated that the inactivation of TRAP1 selectively induced mitochondrial dysfunction and cell death in cancer cells. Therefore, TRAP1 has been suggested as an emerging target protein for anti-cancer drug development. Isopropyl amine of the Hsp90 inhibitor, PU-H71, was replaced with the mitochondria-targeting moiety, triphenyphosphonium, to produce the mitochondria-accumulating TRAP1 inhibitor, SMTIN-P01. To further optimize SMTIN-P01, we have synthesized several SMTIN-P01 derivatives including TPP-PU-X03 with potent TRAP1 inhibitory activities. TPP-PU-X03 increased TRAP1 ATPase enzyme activity while suppressing chaperone activity, suggesting asymmetric effect on dimeric TRAP1 chaperone functions. Electron microscopic analyses showed TPP-PU-X03 shift TRAP1 conformation from open to close conformation, while other TRAP1 or Hsp90 inhibitors stabilizes open conformation. Importantly, TPP-PU-X03 showed improved mitochondria dysfunction and enhanced cytotoxicity to various cancer cells in vitro and in vivo. Collectively, the structure based optimization of current TRAP1 inhibitors may generate potent anti-cancer drugs with novel mechanisms of action.

#1493

LonP1 is required for tumor maintenance and growth.

Benjamin S. Amidon, Dongyun Wu, John Bradley, Jingya Ma, Christopher Tsu, Elizabeth Carideo-Cunniff, Vihren Kolev, James Garnsey, Pooja Shah, Erik Koenig, Hua Liao, Courtney Cullis. _Takeda Pharmaceuticals, Inc, Cambridge, MA_.

Proper mitochondrial function is critical for energy production and the cycling of metabolites required for tumor establishment and growth. LonP1 is an ATP-dependent serine protease that is critical for maintaining mitochondrial protein homeostasis by degrading misfolded and damaged proteins in the mitochondrial matrix. Knockdown of LonP1 has been shown to reduce tumorogenesis and is consistent with the model that requires properly functioning mitochondria for tumor establishment. It is unclear what the role of LonP1 is in tumor maintenance and growth from an established tumor, therefore the LonP1 gene was mutated and complemented by a doxycycline-inducible copy of LonP1. These complementation clones have the ability to establish tumors in vivo in the presence of doxycycline, however upon doxycycline withdrawal the tumors regress and show evidence of apoptosis. These xenograft lines permit unique insight into not only potential LonP1 substrates but also the direct and indirect effects from loss of mitochondrial matrix protein homeostasis. Comparison and contrast of these complemented cell lines in vitro and in vivo by expression and proteomic analysis will be discussed.

#1494

Interethnic germline variants in mitochondrial DNA polymerase (POLG1) induce mitochondrial dysfunction and confer tumorigenic properties.

Prachi Bajpai,1 Bhupendra Singh,1 Kjerstin M. Owens,2 Vinodh Srinivasasainagendra,3 Hemant K. Tiwari,3 Keshav K. Singh4. 1 _Department of Genetics, University of Alabama at Birmingham, Birmingham, AL;_ 2 _Roswell Park Cancer Institute, Buffalo, NY;_ 3 _Department of Biostatistics, University of Alabama at Birmingham, Birmingham, AL;_ 4 _Department of Genetics, Pathology, Environmental Health, Center for Free Radical Biology, Center for Aging, University of Alabama at Birmingham, UAB Comprehensive Cancer Center, Birmingham Veterans Affairs Medical Center, Birmingham, AL_.

Purpose: Germline variants contribute to large fraction of the diversity observed among different ethnic groups and could give better insights in genetic predisposition and susceptibility from one population to another for certain disease including cancer. Decreased mitochondrial OXPHOS is one of the most common phenotypes of cancer cells. Mitochondrial DNA (mtDNA) depletion impairs OXPHOS and adaptive metabolic responses. In this context, DNA polymerase γ (POLG1) is known to function in human mitochondria and performs critical function of mtDNA replication and repair. POLG1 is the most frequent target of gene mutation and is involved in variety of mitochondrial diseases. POLG1 protein consists of exonuclease, linker and polymerase domain, each with distinct functions. Mutations in exonuclease domain leads to erroneous proofreading and is responsible for increased mutations in mtDNA whereas mutations in polymerase domain leads to erroneous replication and hence depletion in mtDNA. Germline variants in these domains of POLG1 may serve as important signature markers in association studies of individuals in a population study. Through our present study, we identified germline variants, examined copy number variation, expression and regulation of POLG1 gene in human cancers. We provide evidence that altered POLG1 expression as well as germline variations in POLG1 gene contribute to tumorigenesis.

Methods: A comprehensive race based bioinformatics analysis of POLG1 gene in European-American and African-American was conducted and several unique germline mutations were discovered. To analyze functional contribution of these unique germline mutations in tumor progression, these unique prevalent mutations in the evolutionary conserved regions of exonuclease and polymerase domains we generated by site directed mutagenesis and functional and tumorigenic analysis was performed.

Results: We observed marked differences in copy number variation of POLG1 through cBioPortal and Cosmic databases and validated the analysis in primary tumors and cancer cell lines. Our results also indicate that mtDNA copy number in cancer cell is governed by regulation of POLG1 methylation and demethylation status, suggestive of epigenetic regulation of POLG1. We identified a mitochondrial disease causing missense variation in polymerase domain of POLG1 at amino acid 1143 (E1143G) to be 25 times more prevalent in European-Americans when compared to African-Americans population. Expression of this germline variant (E1143G) increased glucose consumption, decreased ATP production and increased matrigel invasion.

Discussion/ Conclusion: We demonstrate that POLG1 is epigenetically regulated and its unique germline variants contribute to disruption of mitochondrial function. Thus contributes to differences in increased predisposition to cancer in inter-ethnic population.

#1496

Connecting the metallome with metabolism in ovarian cancer.

Eric D. Shide, Lauren Amable. _NIMHD, Bethesda, MD_.

Ovarian cancer has historically been diagnosed late stage and lacks effective biomarkers for detection and prognosis. The deregulation of metabolism and signaling cascades inherent of cancer may contribute to aberrant levels of metal within cells. Biological processes essential for cell proliferation require metals for enzyme cofactors, signaling molecules, and structural components. Metallomics is the study of the comprehensive, dynamic metal profile within cells. The goal of our study was to characterize the ovarian cancer metallome at the level of whole cell and mitochondria in comparison to non-cancer. Additionally, cellular metabolism was analyzed to connect the mitochondrial (mito-)metallome to mitochondrial function. Ten human ovarian cancer cell lines were used for experiments: A2780, CAOV3, A2780/CIS, A2780/CP70, ES2, OVCAR3, OVCAR5, SKOV3, TOV112, and TOV21. Three non-cancer human ovarian cell lines of epithelial and fibroblast origin served as controls. Inductively coupled mass spectrometry (ICP-MS) was used to evaluate the metallome by measuring the following metals: calcium (Ca), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), molybdenum (Mo), phosphorous (P), sulfur (S), selenium (Se), and zinc (Zn). An XF24 Seahorse analyzer was used to measure cellular metabolism and mitochondrial function in the ovarian cancer and non-cancer cell lines. Our results demonstrated that there was a distinct metal profile at the level of the whole cell and mitochondria in ovarian cancer cells compared to non-cancer ovarian cells. The whole cell ovarian cancer metallome consisted of increased Mg, P, and Cu, and decreased Fe, Mn, and Se in comparison to non-cancer. The cancer mito-metallome consisted of increased Mg, P, Cu, Zn, Se, and Ca. Referencing established literature of the known metals that serve as cofactors for mitochondrial proteins, the relationship between the mito-metallome and functional metabolic pathways was established. Changes in cellular metabolism were reflected by changes in the mito-metallome. Our study was the first to evaluate an extensive panel of metals simultaneously in the mitochondria and bridge the unique metal signature of ovarian cancer to cell metabolism. Future proteomics studies will be used to further validate the connection of metals to cellular function. Metallomics and the ovarian cancer cell metallome provide further understanding of how cancer deregulates the metallome and how metallomics may provide an avenue for identifying new cancer biomarkers and allow for effective targeting of cancer with chemotherapy.

#1497

BPM31510 modulates mitochondrial complex activity to influence oxidative stress in effectuating cell death in multiple cancers.

Tulin Dadali, Katerina Krumova, Anne R. Diers, Pallavi Awate, Ryan Ng, Arleide Lee, Stephane Gesta, Vivek K. Vishnudas, Rangaprasad Sarangarajan, Niven R. Narain. _BERG, LLC, Framingham, MA_.

Dysregulated mitochondria play a multifaceted role in tumorigenesis through regulation of energy production, biomass, redox state, and engagement of cell death pathways. Perturbations in mitochondrial fluxes (i.e inhibition of electron chain complexes activity, impaired electron flow) have substantial effects on cell viability, suggesting that targeting mitochondrial function could be effective for therapeutic response in cancer. BPM31510, containing oxidized coenzyme Q10 elicits an anti-Warburg effect is currently in phase II clinical trials for solid tumors. Previously, we have demonstrated the anti-cancer properties of BPM31510 in breast and pancreatic xenograft models. Here, we examined the mechanism of action of BPM31510 in vitro. Using a multi-cancer cell panel, BPM31510 was shown to be consistently and selectively cytotoxic to cancer cells, compared to normal and non-tumorigenic controls, and sensitivity did not correlate to cell doubling time or mutational status. Treatment with BPM 31510 (EC50) in breast and pancreatic cancer cells resulted in a time- and dose-dependent decrease in mitochondrial membrane potential which preceded an increase in early and late apoptosis cells, suggesting BPM31510 initiates a mitochondrial mediated cell death pathway. Using a fluorescently labeled CoQ10 we were able to trace the subcellular location of the CoQ10, which predominantly accumulates in mitochondria and lipid droplets in a time dependent manner. Additionally, the mitochondrial enrichment of CoQ10 is accompanied by morphological changes that varied amongst the different cancer cell types. As CoQ10 is a redox molecule required for electron transfer activity between complexes, we hypothesized that disruption of Q-pool homeostasis would alter complex activity. To investigate this, Complex driven respiration was measured in cells treated with BPM31510 and compared to untreated. Alterations in mitochondrial respiration characterized by a dose-dependent decrease in succinate (Complex II) and glycerol-3-phosphate (Complex III)-fueled respiration were observed in cells treated with BPM31510, while no changes were seen in pyruvate driven respiration (Complex I), suggesting that BPM31510 specifically impairs respiration responses that are more dependent on Q-pool functionality. As impairment of the electron transport chain increases intracellular oxidative stress, we next investigated if BPM 31510 treatment increases ROS levels. After 24h treatment, BPM31510 significantly increased ROS levels in treated cancer cells compared to untreated. Furthermore, BPM31510 induced death could be in part prevented by co-treatment with antioxidants. Together, these data demonstrates BPM31510 has anti-cancer activity in multiple cancer cell types and define a unique and novel functional link between mitochondrial Q-pool disruption and the mechanism of action of BPM31510.

#1498

Melatonin inhibits mitochondrial transcription factor A expression in glioblastoma U87MG cell culture inducing an anti-tumorigenic effect.

Daiane G. Franco, Isabele F. Moretti, Suely K. Marie. _University of Sao Paulo, Sao Paulo, Brazil_.

Melatonin, the major hormone of pineal gland, has been described to induce intrinsic apoptosis in tumor cell, while it is inhibited in non-tumor cell. In tumor cell, melatonin stimulates intracellular production of reactive oxygen species (ROS) by a mechanism not completely understood. Here we propose that melatonin inhibits the expression of the mitochondrial transcription factor A (TFAM) in culture of glioblastoma (GBM) cell lineage, leading to an increase of the production of ROS. TFAM is an important mitochondrial protein that maintains the mitochondrial DNA (mtDNA) integrity and consequently the oxidative phosphorylation. Human U87MG GBM cell lineage was treated with melatonin (1 mM) for 72 h to evaluate the expression of TFAM and the mtDNA copy number through Real Time PCR. Proliferation, apoptosis, cell cycle and ROS production were also evaluated by cytometry (Muse - Millipore Corporation) and the results were normalized to control (vehicle: ethanol 0.3 %). Melatonin reduced the expression of TFAM in 17.5 % compared to the control group, but had no effect on the mtDNA copy number. Moreover, the incubation of U87MG with melatonin induced a reduction in cell proliferation in 14.24 % ± 4.3 compared to control, with an arrest of cell cycle in G0/G1 phase, which increased from 67.4 ± 3.71 % to 72.78 ± 3.18 % in the group treated with melatonin compared to the control. Production of ROS presented an increase of 7.1 %, with no significant effect on apoptosis. These results suggest that TFAM maybe a target for the melatonin function for an anti-tumorigenic effect through an increase of ROS production, inducing a delay in the cell cycle and a reduction of cell proliferation. The melatonin effect onTFAM maybe a novel signaling pathway that could be explored for the control of GBM progression.

#1499

Role of mutations and expression change of mitochondrial function related nuclear genes in oral gingivobuccal squamous cell carcinoma.

Esita Chattopadhyay,1 Richa Singh,1 Roshni Roy,2 Bidyut Roy1. 1 _Indian Statistical Institute, Kolkata, India;_ 2 _Baylor Research Institute, Dallas, TX_.

Background: Mitochondria have long been suspected to have contribution in progression and sustenance of cancer. But mitochondria related nuclear genes have not been well-studied in oral cancer. The aim of this study is deciphering the impact of somatic mutations and expression deregulation of mitochondria related nuclear genes in oral gingivobuccal squamous cell carcinoma (GBSCC).

Methods: Nuclear-encoded genes which are functionally involved with mitochondria were enlisted from Mitocarta 2.0 and IMPI databases. Whole exome sequencing was performed with 12 paired cancer-normal GBSCC samples and somatic mutations in mitochondria related nuclear genes were extracted from the data. Expression deregulation of mitochondria related nuclear genes were quantified from whole transcriptome data of 12 paired cancer-normal GBSCC samples. Expression was re-validated in another set of 12 cancer-normal paired samples. 5 cancer-normal paired samples with whole transcriptome data were also used for reduced representation bisulfite sequencing (RRBS). Probable impact of non-synonymous somatic mutations were predicted using SIFT and POLYPHEN2 tools. Genes with possible damaging mutations and expression deregulation were taken for literature search and KEGG pathway analysis to understand their contribution in the function of mitochondria in GBSCC.

Result: Total 1561 mitochondria related nuclear genes were identified from two databases and included in the study. A total of 977 somatic mutations were identified in 583 such genes in 12 oral GBSCC samples. These mutations were distributed in exonic (346 mutations), intronic (288 mutations), splicing (4 mutations), UTR3' (270 mutations) and UTR5' (69 mutations) regions. Ten recurrently mutated genes (AK3, AK4, ATP10D, CASP8, ERBB4, MRPL10, NT5DC3, RYR3, SMURF1and TP53) were identified in at least 25% (3/12) of samples. Significant expression deregulation in 147 mitochondrial related nuclear genes was found from whole transcriptome data. Differential methylation was found in some of these 147 genes in 5 oral GBSCC samples. Thirty-seven genes were selected from 147 genes for expression re-validation by qPCR and expressions of 32 genes were re-validated in another set of 12 GBSCC samples. Somatically mutated and expression deregulated genes were different except two genes, ACADM and NNT, which were mutated and deregulated. Literature survey and KEGG pathway analysis revealed that most of the mutated or deregulated genes were functionally involved with biological pathways like mitochondrial metabolism, oxidative phosphorylation, apoptosis and molecular transport. It indicates possible disruption of these pathways in oral GBSCC.

Conclusion: Mitochondrial function related nuclear genes may have significant impact on oral GBSCC in maintenance and progression, but needs to be validated in larger set of samples.

#1500

Evaluating the efficacy of limiting heme availability on growth and progression of lung tumor.

Poorva Ghosh, Sarada Preeta Kalainayakan, Sanchareeka Dey, Sagar Sohoni, Li Zhang. _UT Dallas, Richardson, TX_.

Numerous studies support the importance of mitochondrial respiration in tumorigenesis. Studies from our lab have demonstrated that mitochondrial respiration is intensified, and the levels of intracellular heme and hemoproteins are elevated in Non-small cell lung cancer (NSCLC) cells. We also demonstrated that lowering intracellular heme levels selectively decreases oxygen consumption in NSCLC cells and inhibits cell migration and colony formation. Heme is a central molecule for mitochondrial function and for all processes involved in oxygen utilization. It serves as a prosthetic group in several oxidative phosphorylation enzymes and other oxygen-utilizing hemoproteins. Most human cells can synthesize heme de novo and uptake heme from the circulation. Further, a number of epidemiological studies have shown that high heme intake is associated with increased risk of several cancers, including lung cancer. The purpose of the study is to determine the extent to which altering heme availability impacts NSCLC tumor growth and development.

To this end, we devised a two-pronged approach to target both heme intake and heme synthesis to limit the heme available to cells. Our lab synthesized a peptide to sequester heme from circulation and thereby limit heme available for uptake. A well-known inhibitor of heme synthesis is used to limit heme synthesis. NSCLC cells that express luciferase were implanted in NOD/SCID mice to generate subcutaneous and lung orthotopic xenografts. The mice were treated with the peptide alone and in combination with an inhibitor. The tumor growth was monitored by non-invasive bioluminescence imaging (BLI) using Perkin Elmer's IVIS Lumina III Imager.

In subcutaneously implanted xenografts, our BLI data show that there is a considerable reduction in radiance (total flux in photons per second) in the treatment groups that received the peptide and a combination of peptide and inhibitor. This trend was also observed in tumor volumes measured by calipers. This observation is further corroborated in lung orthotopic xenografts, where there is a significant reduction in radiance in the treatment groups that received the peptide and peptide-inhibitor combination. In all the cases the combination of the peptide and the inhibitor had a greater effect than the peptide alone.

Our results indicate that combination of heme lowering agents works effectively in limiting lung tumor growth and progression by limiting both heme uptake and synthesis. Further studies are underway to discern the role of heme in lung cancer growth and progression.

#1501

Sideroflexin4: A novel regulator of iron metabolism in ovarian cancer.

Bibbin Paul,1 Miranda Lynch,2 Frank Torti,1 Suzy Torti1. 1 _University of Connecticut Health, Farmington, CT;_ 2 _Los Alamos National Laboratory, Los Alamos, NM_.

The five year survival rate for women with ovarian cancer is 9-34%. This high mortality is due to late stage diagnosis, marginally effective treatment and frequent disease recurrence. Alterations in metabolism have been widely recognized as an important hallmark of cancer. Metabolism of the micronutrient iron is significantly altered in many cancers, including ovarian cancers. The expression of genes governing iron transport (TFR1 and FPN), iron storage (FTL and FTH) and iron regulation (IRP1 and IRP2) are significantly altered in ovarian cancer compared to normal ovarian epithelial cells, resulting in higher intratumoral iron retention. However, specific pathways of intracellular iron utilization that are altered in cancer cells, and the functional consequences of disrupting these pathways, remain poorly understood.

To identify pathways of iron utilization that are perturbed in ovarian cancer, we analyzed the expression of genes involved in iron metabolism using a publicly available microarray database of ovarian cancer and normal oviduct (GSE69428). We observed that sideroflexin4 (SFXN4), an inner mitochondrial membrane protein that is essential for mitochondrial respiratory homeostasis, was significantly upregulated (p= 0.002) in ovarian cancer patient samples. We screened several ovarian cancer cell lines and observed that SFXN4 was similarly up-regulated both at the mRNA and protein level when compared to normal human ovarian surface epithelial cells.

To test the role of SFXN4 in ovarian cancer cells we performed knockdown and overexpression studies. Knockdown of SFXN4 induced S and G2-phase cell cycle arrest and reduced the ability of MDAH2774 and SKOV3 ovarian cancer cells to form colonies. Further investigation revealed that modulation of SFXN4 alters mitochondrial respiration by affecting iron-sulfur cluster biogenesis. Thus, knock-out cells exhibited impaired respiratory activity and a phenotype similar to cells with defects in iron-sulfur cluster biogenesis, including increased IRP-IRE binding and a decrease in IRP1/ACO1 aconitase activity. Furthermore, SFXN4 knock-out decreased the activity of iron-sulfur cluster-containing enzymes such as mitochondrial aconitase and succinate dehydrogenase.

Based on these observations, we postulate that SFXN4 acts as a molecular mediator that channels the excess iron present in ovarian cancer cells to iron-sulfur cluster-dependent metabolic pathways that favor growth and metastasis. SFXN4 may be a potential druggable target in ovarian cancer.

#1502

The link between NRF2 and mitochondria through the regulation of miR-181c/mitochondria-encoded cytochrome c oxidase subunit-1.

Mi-Kyoung Kwak, Kyeong-Ah Jung, Donghyeok Kim, Sujin Lee. _The Catholic University of Korea, Gyeonggi-do, Republic of Korea_.

The nuclear factor erythroid 2-related factor 2 (NRF2) pathway is involved in the environmental resistance of cancers by enhancing the antioxidant capacity. In the current study, we investigated the potential link between NRF2 and mitochondrial function in cancer cells. Global miRNA expression analysis of HT29 and HCT116 identified miR-181c as an NRF2-silencing-inducible miRNA, and miR-181c elevation was associated with the decrease in mitochondria-encoded cytochrome c oxidase subunit-1 (MT-CO1), a mitochondrial genome-encoded complex IV subunit of the electron transport chain. As a result of decreased complex IV activity, NRF2-silenced cancer cells exhibited reduced levels of mitochondrial membrane potential (MMP), oxygen consumption rate, and ATP production. Notably, these changes induced adaptive activation of AMP-activated protein kinase-α (AMPKα) and thereby, NRF2-silenced cells were more vulnerable to AMPK inhibitor-induced growth suppression. Similarly, mouse tumor xenografts derived from NRF2-silenced HT29 exhibited MT-CO1 reduction and AMPKα activation, thereby increasing responsiveness to the AMPK inhibitor treatment. Collectively, we showed a novel link of NRF2 to cancer mitochondria and AMPK signaling by elucidating miR-181c/MT-CO1 signaling as an underlying molecular mechanism. These results also suggest that it may an effective strategy to inhibit both NRF2 and AMPK to control adaptive tumor responses.

#1503

A physiologically-based pharmacokinetic (PBPK) model of hydroxychloroquine.

Keagan P. Collins, Kristen M. Jackson, Daniel L. Gustafson. _Colorado State Univ., Fort Collins, CO_.

Hydroxychloroquine (HCQ) is a lysotropic autophagy inhibitor that is being used in over 45 clinical trials either alone or in combination with another chemotherapeutic. Pharmacokinetic (PK) and pharmacodynamic (PD) studies with HCQ have shown that drug exposure in the blood does not correlate with autophagy inhibition in either peripheral blood mononuclear cells (PBMCs) or tumor tissue (Autophagy 10:1415). HCQ exhibits primarily pH-driven PK and has been shown, by way of heightened levels of autophagy markers, to generate a therapeutic effect longer than PK data suggests. A physiologically-based pharmacokinetic model (PBPK) was developed for HCQ to describe the tissue-specific absorption, distribution, metabolism, and excretion as well as lysosome-specific sequestration. Physiologic parameters were adapted from literature, or obtained from experimental data when necessary, and used to simulate physiologically-based HCQ PK following designated dosing regimen in mice and rats. Experimentally derived PK data from these species were compared to simulation generated data to drive model development and subsequently determine model accuracy, achieving statistically-similar PK predictions of blood and tissues. Through allometric scaling and species-specific parameter modifications this model can be easily adapted for accurate prediction of HCQ PK in dogs and humans, as determined by comparison with respective blood levels. The value of this model lies in its ability to simulate HCQ PK in cancer patients with tumor types deemed autophagy-dependent. Model data simulating HCQ uptake in a neutral tumor compartment (pH = 7.2) shows that peak concentration in the lysosomes, the active site of the drug, is roughly four-fold higher than the peak concentration of lysosomes in an acidic tumor compartment (pH = 6.8), yet there is only a small change between whole-tumor concentrations. This suggests that the ability of HCQ to inhibit autophagy in acidic tumors would be significantly reduced, which is currently being investigated through in vitro and in vivo uptake of HCQ in MDA-MB-231 and MCF-7 tumor cell lines. Additionally, HCQ PK exhibits large interpatient variability, thus model utilization is beneficial in determining if therapeutic levels of HCQ are achieved at the tumor site and the impact of variability in the local tumor environment on HCQ disposition. The flexibility and simulation capabilities of the developed PBPK model also allows for investigation of how HCQ PK and subsequent autophagy inhibition may potentially be modified by other treatment modalities and suggest dosing schedules to optimize therapeutic response.

#1504

Tumor-treating fields (TTFields) interfere with biological key properties of glioma cells in vitro.

Manuela Silginer, Michael Weller, Roger Stupp, Patrick Roth. _University Hospital Zurich, Zurich, Switzerland_.

Tumor-treating fields (TTFields) are low amplitude alternating electric fields which are supposed to exert anti-tumor effects by targeting dividing tumor cells while sparing cells in the brain not undergoing cell division. Although this novel therapeutic approach has shown encouraging results in phase III trials in glioblastoma, its biological effects on tumor cells have only been poorly understood.

Here, we investigated the effects of TTFields on glioma cells in vitro using the inovitroTM system that allows the application of TTFields to cell cultures. Exposure to TTFields potently induce autophagy and necroptosis and interfere with the migration and invasion of long-term glioma cell lines, but also of glioma-initiating. The combination of TTFields with irradiation or temozolomide (TMZ) reduced viability and clonogenic survival in an additive or synergistic manner. Further studies suggest that the O6-methyl-guanine DNA methyltransferase (MGMT) status does not influence the efficacy of TTFields and TMZ-resistant glioma cells remain responsive to TTFields application, thus making TTFields particularly attractive for the majority of glioblastoma patients with tumors that are unlikely to benefit from TMZ treatment.

In summary, these findings demonstrate that the application of TTFields may interfere with various biological key properties of glioma cells and may allow for a more detailed clinical evaluation of TTFields beyond the clinical data available so far.

#1505

Breast cancer cells treated with mitochondria targeted redox active agents induce mitophagy.

Thomas Biel, Ashutosh Rao. _Food and Drug Administration, Silver Spring, MD_.

Triphenylphosphonium (TPP) conjugated agents induce mitochondrial dysfunction in cancer cells. However, the onset of mitophagy to remove the dysfunctional mitochondria is unknown. Here, a series of mitochondria targeted redox active agents (MTA) that contain TPP were used to investigate mitochondrial dysfunction and mitophagy in MDA-MB-231 cells as compared to MCF-12A cells. Three different MTAs were identified to induced mitochondrial depolarization and enhanced autophagic flux selectively in MDA-MB-231 cells. Mitochondrial reactive oxygen species generation and respiration indicated that MDA-MB-231 cells had a heighten sensitivity to MTA treatments. Using stably expressing mt-mKeima MDA-MB-231 and MCF-12A cell lines, a non-cell type selective decline in mitochondrial alkalinity and altered mitochondrial morphology was detected. Furthermore, FACS analysis of mt-mKeima revealed MTAs induced lysosomal dependent mitochondrial degradation in the presence of Bafilomycin, a lysosomal inhibitor. To confirm MTAs induced mitochondrial autophagy in MDA-MB-231 cells, MitoTracker Red preloaded GFP-LC3 expressing MDA-MB-231 cells were used to identify autophagosomes containing mitochondria using confocal microscopy, in addition to coimmunoprecipitation for the detection of an endogenous autophagy-related protein complex, and immunoblot to for mitochondrial PINK1 accumulation. To translate these in vitro studies to an in vivo rat SST-2 xenograph breast cancer model, tumor mitochondrial extracts from rats treated with MitoQ demonstrated an accumulation of mitochondrial PINK1. Collectively, these data suggest that mitochondrial agents selectively caused mitochondrial depolarization, PINK1 accumulation and mitophagy in MDA-MB-231 cancer cells as compared to MCF-12A healthy cells.

#1506

The Wnt signature of melanoma cells predicts their invasiveness, autophagy activity, and their response to pharmacologically mediated autophagy inhibition.

Abibatou M.N. Ndoye,1 Anna Budina,1 Amanpreet Kaur,1 Curtis H. Kugel,1 Marie Webster,1 Reeti Behera,1 Vito Rebecca,2 Ling Li,1 Meenhard Herlyn,1 Ravi Amaravadi,1 Maureen Murphy,1 Ashani Weeraratna1. 1 _The Wistar Institute, Philadelphia, PA;_ 2 _The University of Pennsylvania, Philadelphia, PA_.

Melanoma is the most aggressive type of skin cancer and the leading cause of death from skin cancer. As the incidence of melanoma continues to increase, it is crucial to investigate the cellular and molecular mechanisms that lead to invasion and metastasis. High autophagy has been shown to correlate with melanoma tumor aggressiveness and poor survival in patients, and is a common mechanism of resistance to therapy. Autophagy inhibition leads to reduced levels in Wnt5A in a breast cancer model, suggesting a cross-talk between Wnt5A and autophagy in cancer. Our laboratory showed that Wnt5A is a driver of invasion and metastasis in melanoma. β catenin, a canonical Wnt mediator, has been shown to negatively regulate autophagy in a colorectal cancer model. Given that Wnt5A downregulates β catenin, we hypothesized that autophagy promotes melanoma tumor aggressiveness through the regulation of Wnt signaling and that in turn the Wnt signaling status of melanoma cells affects their autophagy activity. Our results demonstrate that melanoma cells with high Wnt5A and low β catenin have higher autophagy levels compared to less aggressive melanoma cells that have low Wnt5A and high β catenin. To determine whether there is a feedback loop between Wnt signaling and autophagy, we inhibited autophagy in invasive melanoma cells using lentivirus-mediated shATG5 knockdown and evaluated the effects of autophagy inhibition on Wnt5A and β catenin expression. We found that the inhibition of autophagy results in a decrease in Wnt5A and an increase in β catenin. Our data also demonstrate that the Wnt signature of melanoma cells greatly affects their response to Lys05-mediated autophagy inhibition both in vitro and in vivo. Wnt5A decreased sensitivity to autophagy inhibition while β catenin increased sensitivity to Lys05 both in vitro and in vivo. Pre-clinical models and clinical trials demonstrate a potential of autophagy inhibition for melanoma therapy; however, the clinical effects are not consistent and do not yield significant anti-tumor effects. This study will provide a rationale for the development of therapeutic strategies that more efficiently target autophagy in aggressive melanoma by taking into account the Wnt signaling status of melanoma cells.

#1507

Fatty acid oxidation mediated autophagy regulation in triple negative breast cancer.

Kwang Hwa Jung, Jun Hyoung Park, Tirupataiah Sirupangi, Sajna Vithayathil, Lee-Jun Wong, Benny A. Kaipparettu. _Baylor College of Medicine, Houston, TX_.

Compared to hormone regulated/responsive (ER+) breast cancer (BC), triple negative BC (TNBC) patients have a worse overall survival, a significantly shorter disease-free survival, and a shorter post-recurrence survival. Using transmitochondrial cybrid (cybrid) (compares different mitochondria under a common defined nuclear background)-based discovery and validation in cell lines, patient derived xenografts (PDXs) and BC clinical tissues/data, we recently published that fatty acid oxidation (FAO) is a major energy pathway in TNBC. We also showed that FAO is a major regulator of Src activation by autophosphorylation at Src Y419 in metastatic TNBC. Our data suggest that inhibition of FAO induce cell growth inhibition. Short-term treatment of FAO inhibitors induces both cell cycle arrest and apoptosis. We observed cell cycle arrest in G1-S phase in FAO inhibitor treated TNBC cells. Apoptotic markers like cleaved Caspase-3 and cleaved PARP induced in FAO inhibited cells. Short-term treatment of FAO inhibitor also showed induction of AKT pathway. Further analysis on the role of FAO in TNBC suggests that FAO is a critical player in autophagy signaling in TNBC. Long-term treatment with FAO inhibitors or knockdown of FAO rate-limiting proteins carnitine palmitoyltransferase I (CPT1) induce autophagic cell death with beclin-1 and LC3-II induction in TNBC. However, no such phenomenon observed in estrogen receptor positive (ER+) breast cancer cell lines. Treatment with autophagy inhibitor 3-methyladenine (3-MA) abolished the FAO-induced cell growth inhibition and reduced beclin-1 and LC3-II induction. Ongoing studies using knock-down and over expression approaches focus on the role of specific pathways that are responsible for mitochondrial energy reprogramming regulated autophagy signaling in metastatic TNBC.

### Targeting Aberrant Transcription in Cancer

#1508

MTL-CEBPA activates the transcription factor CEBPalpha leading to inhibition of hepatocellular cancer growth.

Nagy Habib,1 Vikash Reebye,1 Xiaoyang Zhao,1 Jon Voutila,2 Robert Habib,2 Pål Sætrom,3 Hans Huber,4 Kai-Wen Huang,5 John J. Rossi,6 David C. Blakey2. 1 _Imperial College London, London, United Kingdom;_ 2 _MiNA Therapeutics Ltd, London, United Kingdom;_ 3 _Norwegian University of Science and Technology, Trondheim, Norway;_ 4 _BioTD Strategies, LLC, Lansdale, PA;_ 5 _National Taiwan University, Taipei, Taiwan;_ 6 _Beckman Research Institute of City of Hope, Duarte, CA_.

CCATT/enhancer binding protein alpha (CEBPA) is a transcription factor that can act as a tumor suppressor. Its expression is downregulated in a number of cancers including hepatocellular carcinoma (HCC). We have investigated the role of CEBPA in models of HCC using small activating RNAs (saRNA) to transcriptionally upregulate its expression.

Three liver tumor cell lines (HepG2, Hep3B, PLCPRF5) were transfected with 20nM CEBPA-saRNAs (CEBPA51). The transcriptional regulation of two key members of the CEBP family: CEBPA and CEBPB and their protein expression level were measured. The impact on cell growth was assessed by way of an SRB and WST-1 assay. To investigate the role of CEBPB in protecting cells from the activity of CEBPA, siRNAs were used to knock down CEBPB. Using i.v. delivery of CEBPA51 oligonucleotide, the impact on tumor growth was investigated in a DEN (N-nitrosodiethylamine) model of liver cancer. Rats were treated with DEN for 7 weeks, followed by a 2 week wash out and then treated with the CEBPA51 (3-4mg/kg) complexed with PAMAM-dendrimers or encapsulated in a nanoparticle formulation (SMARTCLES). The impact on CEBPA mRNA levels in the liver, tumor growth and liver functions (including, bilirubin, ALT and AST) were measured.

Transfection of CEBPA51 into HepG2, Hep3B or PLCPRF5 cells after 72hr led to a significant increase in both CEBPA mRNA (1.7-2.5 fold by qPCR) and protein expression measured by western blot in all 3 cell lines. A significant inhibition in cell growth compared to either PBS or control oligonucleotide was observed in HepG2 and Hep3B but not in PLCPRF5 cells measured by both SRB and WST-1 assays. The levels of CEBPB mRNA and protein, which may act as an antagonist of CEBPA, were found to be higher (1.4-2 fold) in PLCRF5 cells compared to HepG2 and Hep3B cells. Co-transfection of PLCPRF5 cells with siRNAs to CEBPB and CEBPA51 saRNA led to downregulation of CEBPB and senstized the PLCPRF5 cells to growth inhibition by CEBPA51. Administration of CEBPA51 complexed to either dendrimers or encapsulated in SMARTCLES nanoparticle at doses of 3-4mg/kg i.v. over 2 weeks led to a significant elevation of CEBPA mRNA in the liver. This was accompanied by a reduction (80-90%) in the size of DEN induced liver tumour nodules compared to a control oligonucleotide using both delivery vehicles. The antitumor effects following treatment with CEBPA51 using both delivery vehicles were also accompanied by reduction in markers of liver injury (bilirubin, ALT and AST).

These studies support an important role for CEBPA in suppressing progression of HCC. Activation of the CEBPα gene by saRNA leading to restoration of CEBPA levels in the liver represents a promising novel approach for inhibiting the growth of HCC whilst improving normal liver function. The SMARTCLES formulation, MTL-CEBPA, was chosen for clinical development and is currently in a Phase 1 trial in patients with liver cancer (NCT02716012).

#1509

**RUNX1 dosage dictates gene signature and** in vitro **response to glucocorticoids in acute myeloid leukemia.**

Laura Simon, Vincent-Philippe Lavallée, Marie-Eve Bordeleau, Jana Krosl, Irène Baccelli, Geneviève Boucher, Bernhard Lehnertz, Tara MacRae, Réjean Ruel, Sébastien Lemieux, Anne Marinier, Josée Hébert, Guy Sauvageau. _University of Montreal, Montreal, Quebec, Canada_.

Background: Poor prognosis subgroups of Acute Myeloid Leukemia (AML), such as RUNX1-mutated (RUNX1mut) AML, would greatly benefit from more efficacious therapies that target leukemic stem cells (LSC) and improve patient outcome. To understand factors that predict sensitivity to drugs, we used a chemogenomic approach to interrogate primary AML specimens.

Methods: We performed RNA sequencing of 415 primary AML specimens comprising various cytogenetic subgroups. Using culture conditions that support LSC activity ex vivo, we carried out a viability screen including 20 primary AML specimens and ~5,100 low molecular weight compounds. RUNX1mut specimens showed increased sensitivity to glucocorticoid compounds (GCs). Validation screens were done in 248 primary AML samples and 32 AML cell lines treated with selected GCs in a dose-response manner. The effect of RUNX1 dosage in the in vitro response to drugs was assessed by shRNA gene knockdown. GCs target, the glucocorticoid receptor (GR), was validated by chemical blockage and shRNA silencing.

Results: RUNX1mut specimens were associated with older age, French-American-British (FAB) M0 morphology, intermediate-risk cytogenetics with abnormal karyotype and poor patient survival. RUNX1mut gene expression signature showed the overexpression of previously described genes such as DNTT, BAALC and CD34, as well as novel genes such as PROM1 and EGFEM1P. Most interestingly, the expression levels of these genes was influenced by the nature of the RUNX1 mutations, with levels progressively increasing with mutations corresponding to decreased levels of functional RUNX1. Chemical screens comprising 33 RUNX1mut specimens confirmed that RUNX1mut are more sensitive to GCs than RUNX1 wild-type samples. In agreement with the RUNX1mut gene signature, the anti-proliferative effect of GCs anti-correlated with levels of functional RUNX1. Specimens harboring loss-of-function and dominant-negative RUNX1 mutations showed increased sensitivity to GCs when compared to samples carrying missense mutations expected to have little impact on RUNX1 function. Mutations in other genes, such as CEBPA and SRSF2, had an additive effect on GC sensitivity when combined with RUNX1 mutations. In accordance with our hypothesis, the downregulation of RUNX1 could reverse GC-resistance in AML cell lines, and the sensitivity to compounds was proportional to knockdown levels. Treatment of cell lines with the GR antagonist RU486 blocked the inhibitory response induced by GCs and GR silencing completely abrogated the anti-proliferative effects of GCs in GC-sensitive cells, confirming that GC operate through the GR in AML.

Conclusion: Altogether, these findings highlight the impact of RUNX1 dosage on gene expression and GCs sensitivity in AML cells in vitro. Further studies should investigate the benefits of repositioning GCs in the treatment of RUNX1mut AML patients.

#1510

Selection inhibition of acute myeloid leukemia by targeting Taf12.

Yali Xu, Joseph Milazzo, Yusuke Tarumoto, Chris Vakoc. _Cold Spring Harbor Laboratory, Cold Spring Harbor, NY_.

Recent studies have suggested that co-activators are good drug targets for cancer therapy, as exemplified by BRD4, the small molecule inhibitors of which have already entered clinical trials. The high potency of targeting BRD4 in leukemia cells mainly rises from suppressing the functions of multiple lineage specific transcription factors. However, such pleiotropic effects of Brd4 on different transcription factors might be problematic, considering many of the perturbed transcription factors are equally required by normal cells. This prompted us to explore more co-activators as potential drug targets, aiming to identify the candidates that discretely disturb the function of oncogenic transcription factors but not the general ones. In this study, we explored the roles of one of the largest co-activator complexes TFIID in the maintenance of acute myeloid cells.

To address this, we designed an shRNA library targeting each TAF subunit of TFIID and tested the growth effect of these shRNAs in both mouse leukemia cell line and primary bone marrow culture. We found that multiple shRNAs targeting Taf12 shows strong toxicity in leukemia cells but not normal cells in vitro. Knocking down Taf12 also inhibits leukemia progression in vivo. To gain insight into the toxicity of inhibiting Taf12 in different organs, we generated doxycycline inducible transgenic Taf12 shRNA mice, where Taf12 can be inhibited in different tissues specifically at adult stage. In consistent to our in vitro observation, normal myeloid cells are not affected by Taf12 knockdown. Instead, the lymphoid lineage cells are affected while removing doxycycline can reverse the phenotype. To our surprise, all the other tissues that have been examined and show significant Taf12 knockdown do not have any defects.

At molecular level, we found that inhibition of Taf12 suppresses the transcription of core targets of Myb, but not other transcription factors, like Pu.1, Erg, Fli-1, which is in contrast to the pleiotropic effects of Brd4. Interestingly, it has been shown before that leukemia cells are hypersensitive to Myb inhibition compared to normal cells. Furthermore, similar to Taf12 mice, mice with hypomorphic level of Myb show defects in lymphoid but not myeloid cells. Taf12 is a small protein with only one histone fold domain (HFD), which can dimerize with the HFD of Taf4a within TFIID complex. We found that the histone fold dimer of Taf12 and Taf4a can physically interact with the transactivation domain (TAD) of Myb. Finally, we showed that the HFD of Taf4a can be utilized to target Taf12 to inhibit growth of leukemia cells. Similar to Taf12 shRNA, function of Myb is disturbed by overexpressing Taf4a HFD. More strikingly, overexpressing Taf4a HFD is sufficient to cause regression of leukemia in vivo.

#1511

AML patient clustering by super-enhancers reveals an RARA associated transcription factor signaling partner.

Michael R. McKeown,1 Matthew L. Eaton,1 Chris Fiore,1 Emily Lee,1 Katie Austgen,1 Darren Smith,1 M. Ryan Corces,2 Ravindra Majeti,2 Christian C. Fritz1. 1 _Syros Pharmaceuticals, Cambridge, MA;_ 2 _Stanford University, Palo Alto, CA_.

Prior studies have shown that the RARA gene is associated with a super-enhancer (SE) and has upregulated mRNA expression in a subset of AML patients. Furthermore, this has been found to confer increased sensitivity to SY-1425, a potent and selective RARα agonist. We sought to better characterize the cell state and transcription factor circuitry in these RARA-high AML cells. Clustering of 62 primary AML patient samples based on their genome wide SE maps identified six discrete clusters. RARA-high patients partitioned principally into cluster 2, and to a lesser extent 1, suggesting that RARA upregulation is associated with a specific transcription factor (TF) network and cell state. To start unraveling the TF circuitry in the RARA-high cluster, we investigated which other TFs were SE associated with clusters 1 and 2. In particular, interferon regulatory factor 8 (IRF8) was found to be strongly associated with clusters 1 and 2 by SE and mRNA expression, similar to RARA. Moreover, the expression of both genes is correlated in primary patient samples. IRF8 is involved in interferon signaling and previous studies have shown crosstalk between interferon and retinoic acid signaling. Furthermore, aberrant IRF8 pathway signaling is implicated in AML and CML pathogenesis. The tight clustering of RARA and IRF8 in patient subgroups defined by genome wide enhancer maps suggests RARα and IRF8 may form an integrated transcriptional circuit. Indeed, treatment with SY-1425 was found to strongly induce interferon-like gene expression changes in AML cells with high RARA or IRF8 levels, including the tumor suppressive IFN responsive gene IRF1. While RARA-high AML cell line models have been previously shown to respond to SY-1425, we found that models with high IRF8 expression and low levels of RARA were also found to respond to SY-1425. Such IRF8-high, RARA-low AML cell lines showed activation of similar transcriptional pathways as RARA-high cell lines in response to SY-1425 based on GSEA. IRF8-high AML also had comparable low nM EC50 anti-proliferative effects following SY-1425 treatment. In addition, SY-1425 was found to elicit differentiation in both RARA-high and IRF8-high AML cell lines based on flow cytometry. While RARA and IRF8 expression appear correlated, this data suggests that IRF8 levels may predict for sensitivity to SY-1425 in addition to RARA levels, particularly in cases of AML with high IRF8 expression but low RARA levels. Insights derived from enhancer analysis, transcriptional profiling and differentiation response in preclinical models support the recently initiated Phase 2 trial of SY-1425 (NCT02807558) in which we are evaluating the SE based patient selection strategies and gene circuitry derived pharmacodynamics clinical measurements, including differentiation markers, in patients with AML and MDS.

#1512

Functional characterization of novel transcription-regulating cancer drug targets, CDK8 and CDK19, using CRISPR/Cas9 knockout and a highly selective CDK8/19 kinase inhibitor.

Mengqian Chen,1 Bing Hu,2 Hao Ji,1 Serena Altilia,1 Jiaxin Liang,1 Martina McDermott,1 Chang-uk Lim,1 Donald C. Porter,3 Eugenia Broude,1 Igor Roninson1. 1 _University of South Carolina, Columbia, SC;_ 2 _Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China;_ 3 _Senex Biotechnology, Inc., Columbia, SC_.

The Mediator complex-associated cyclin-dependent kinase CDK8 is an oncogenic transcription-regulating serine/threonine kinase that mediates multiple cancer-associated transcriptional pathways. Despite recent high-profile attention to CDK8 as a novel cancer drug target, very little is known about the function of CDK8's closely related paralog CDK19. Using CRISPR/Cas9n system we generated CDK8/CDK19 single-knockout (CDK8-KO and CDK19-KO) and double-knockout (CDK8/19-dKO) derivatives of HEK293 cells. RNA-Seq was used to characterize the effects of a highly selective small-molecule CDK8/19 kinase inhibitor Senexin B on gene expression in the parental, single-knockout and double-knockout cell lines. This analysis was conducted in the absence or in the presence of TNF-alpha, an inducer of transcription factor NF-kappa-B that we have previously shown to be potentiated by CDK8. The following results were obtained. (1) CDK8 and CDK19 have complementary functions in stabilization of their partner Cyclin C (independent of their kinase activity) and phosphorylation of transcription factor STAT1 at S727 (dependent on the kinase activity). (2) Senexin B treatment affected gene expression in wild-type 293 cells but had almost no effect in CDK8/19-dKO cells, indicating a very high degree of target selectivity. (3) In contrast to the results with CDK8/19-dKO, most of the genes affected by Senexin B in the wild-type cells were also affected in CDK8-KO and CDK19-KO cells, indicating complementary functions of CDK8 and CDK19. (4) Re-expression of either CDK8 or CDK19, but not of a CDK8 kinase-dead (D173A) mutant, in CDK8/19-dKO cells restored CDK8/19 kinase-dependent gene expression as well as the regulatory effects of Senexin B. (5) Many more genes were inhibited rather than induced by Senexin B, indicating that CDK8/19 act primarily as positive regulators of transcription. (6) The total number of genes affected by Senexin B was greatly increased in cells treated with TNF-alpha, suggesting that cooperation with other transcription factors (such as NF-kappa-B) is the primary role of CDK8/19. Our results indicate that complete suppression of cancer-relevant activities of CDK8 requires simultaneous inhibition of both CDK8 and CDK19.

#1514

A novel approach for targeting androgen receptor signaling axis in prostate cancer.

Salma Kaochar, Christopher Foley, Cristian Coarfa, Nicholas Mitsiades. _Baylor College of Medicine, Houston, TX_.

Background and Objectives: The androgen receptor (AR) is a ligand-activated transcription factor (TF) that plays critical oncogenic roles. Medical castration therapy is the mainstay treatment for advanced PrCa, but the AR signaling axis frequently remains active in castration-resistant PrCa (CRPC). Despite advances in AR targeting with abiraterone and enzalutamide, disease progression re-occurs in the form of CRPCs that still express AR-dependent genes. This highlights the need for novel approaches to block AR signaling and CRPC growth.

The GATA family of TFs contains six members in mammals, all of which bind a consensus DNA sequence (A/T)GATA(A/G) to regulate gene expression. GATA2 is the predominant family member in prostate luminal epithelial cells. In a search for TFs that control AR expression, we found that GATA2 induces AR gene expression as well as promotes the recruitment of coactivators to the AR transcriptional complex. We found that GATA2 protein expression was strongly correlated with AR protein expression (Spearman's correlation coefficient: 0.323, p-value <0.0001) in our PrCa specimens (n=383). The intensity of GATA2 immunostaining in our BCM patient cohort positively correlated with Gleason score (R=0.37, p-value <0.01) and was a significant predictor of biochemical recurrence (p-value <0.001). We also demonstrated that GATA2 directly promotes expression of both full-length and AR splice-variant, resulting in a strong positive correlation between GATA2 and AR expression in both PrCa cell lines and patient specimens. Conversely, GATA2 expression is repressed by androgen and AR, suggesting a negative feedback regulatory loop that, upon androgen deprivation, derepresses GATA2 to contribute to AR overexpression in CRPC. Simultaneously, GATA2 is necessary for optimal transcriptional activity of both full-length and AR splice-variants. Using ChIP-Seq we found that GATA2 colocalizes with AR and Forkhead box protein A1 (FOXA1) on chromatin to enhance recruitment of steroid receptor coactivators (SRC1, SRC2, and SRC3) and formation of the transcriptional holocomplex. Lastly, we identified a GATA2 small molecule inhibitor (SMI) that can suppress the expression and transcriptional function of both full-length and splice-variant AR and exerted potent anticancer activity against PrCa cell lines. We propose that the inhibition of GATA2 is a 'first-in-field' approach to target AR expression and function, including ligand-independent AR, for the treatment of CRPC.

#1515

Bromodomain and extra-terminal motif protein inhibitors (BETi) in metastatic castration resistant prostate cancer (mCRPC): A novel mechanism for regulating androgen receptor variant 7 (ARV7).

Jonathan Welti,1 Adam Sharp,1 Ines Figueiredo,1 Wei Yuan,1 Daniel Nava Rodrigues,1 Veronica S. Gil,1 Eleanor Knight,2 Jian Ning,2 Jeff Francis,2 Antje Neeb,1 Gunther Boysen,1 Amanda Swain,2 Johann S. de Bono1. 1 _Institute of Cancer Research and the Royal Marsden Hospital NHS Trust, Sutton, United Kingdom;_ 2 _Institute of Cancer Research, Sutton, United Kingdom_.

Persistent androgen receptor (AR) signaling is key to the development and progression of metastatic castration resistant prostate cancer (mCRPC). This is in part due to expression of constitutively active AR splice variants like AR variant 7 (ARV7), confering resistance to current anti-androgens including enzalutamide (E) and abiraterone (A). To improve the outcome for patients with mCRPC, new therapeutic strategies to overcome AR and ARV7 oncogenic signaling are urgently required. The inhibition of co-factors modulating AR signaling are currently being investigated as novel strategies to treat mCRPC. One promising candidate is BRD4, a member of the BET protein family, that binds the AR on androgen response elements and facilitates the recruitment of the transcriptional machinery. BET inhibitors (BETi) have been shown to regulate AR and ARV7 signalling, however, the exact mechanism of ARV7 regulation remains unclear. As BETi are currently being explored in clinical trials of unstratified patients with mCRPC, we investigated their potential mechanism of action in CRPC cell lines, patient derived organoids (PDOs) and a patient derived mouse xenograft (PDX). Here we demonstrate that nuclear expression of BRD4 and ARV7 increases as patients develop resistance to E and/or A and inhibition of BRD4 by BETi is sufficient to block AR and ARV7 signalling in mCRPC. Both inhibition of BRD4 by BETi and genetic knockdown of BRD4 reduced the growth of CRPC cell lines and led to down-regulation of AR and ARV7 at the mRNA and protein level. To further investigate whether BETi is sufficient to inhibit ARV7 activity in patients with mCRPC, we treated patient derived organoids (PDOs) and a mouse xenograft (PDX) grown from metastatic biopsies of patients resistant to E and/or A with BETi. In this study 5 out of 10 PDOs were sensitive to BETi. Consistent with the cell culture experiments, BETi treatment of the PDX led to down-regulation of both ARV7 mRNA and protein expression. Mechanistically, BETi might inhibit pre-mRNA splicing of AR resulting in the observed decrease of ARV7 expression. However, RNAseq analysis of the ARV7 expressing CRPC cell line LNCaP95 demonstrated an increase in total splicing events including skipped exons, retained introns, mutually exclusive exons, alternative 5' splice site and alternative 3' splice site after BETi treatment. Despite this, focused analysis of splicing factors and spliceosome components identified a subset of eight splicing factors being down-regulated by BET inhibition including one yet uncharacterized factor that is crucial for ARV7 expression in LNCaP95 cells. Based on our results we propose a model that BETi mediated inhibition of this novel ARV7-mRNA splicing factor may lead to decreased splicing and subsequent expression of ARV7 at both mRNA and protein level; providing a novel approach to target ARV7 in mCRPC.

#1516

Androgen-repressed and androgen-induced genes: challenging the traditional dogma of prostate cancer therapy.

Daniel P. Caley, Nasrin R. Mawji, Marianne Sadar. _BC Cancer Agency, Vancouver, British Columbia, Canada_.

Background: Benign and malignant prostate tissues are dependent upon the activity of androgen receptor (AR). The primary function of full-length AR is as a ligand activated transcription factor to increase or repress gene expression. Many androgen-repressed genes regulate the cell cycle and proliferation. With castration, the main therapeutic approach for advanced prostate cancer (PC), these genes are believed to play a role in the initial clinical response. Current approved therapies for advanced PC and castration-resistant PC (CRPC), target the AR C-terminal ligand-binding domain (LBD), such as antiandrogens. Recent antagonists of the AR N-terminal domain (NTD) have been described with EPI-506, the prodrug of EPI-002, now in Phase 1 clinical trials. EPI-002 binds tau-5 in activation function-1 (AF-1) of the NTD that is essential for AR transcriptional activity. As expected with an AF-1 antagonist, EPI-002 is an excellent inhibitor of androgen-induced gene expression and at blocking the transcriptional activities of truncated AR splice variants lacking LBD, such as AR-V7. EPI-002 blocks expression of genes regulated by truncated AR-V7 such as UBE2C while antiandrogens have no effect. Here we reveal that the major difference in gene expression regulated by full-length AR between EPI-002 and antiandrogens is their abilities to de-repress genes that are turned off by androgen.

Methods: The androgen sensitive human prostate cancer cell line LNCaP, which expresses full-length AR, was treated with antiandrogens (bicalutamide [BIC] and enzalutamide [ENZA]), EPI-002 and a control vehicle, with and without androgen. Gene expression was analysed using Affymetrix microarrays. Bioinformatical analysis was completed and a selection of androgen-repressed genes that were de-repressed with antiandrogens and/or EPI-002, were selected for validation using qRT-PCR.

Results: EPI-002 de-repressed known androgen-repressed genes including SPLTLC3, ST7, PSAT1, TMEM140 and TNFRSF21. EPI-002 was as effective or better than BIC or ENZA in de-repressing a subset of androgen-repressed genes. Importantly, EPI-002 failed to de-repress expression of many androgen-repressed genes that antiandrogens de-repressed, such as SLITRK3, GPR63 and DAB1.

Conclusions: EPI binds AR NTD which blocks the transcriptional activities of full-length AR and truncated AR splice variants. EPI-002 was excellent at inhibiting androgen-induced genes. However, EPI did not broadly de-repress expression of genes turned off by androgen when compared to antiandrogens. Such differences between EPI-002, a tau-5/NTD antagonist, and C-terminal LBD antiandrogens probably reflect the complexities of the mechanisms of repression of gene expression that may involve different domains of AR.

#1517

AKT inhibition induces neuroendocrine phenotype in prostate cancer cells.

RuiQi Chen, Yinan Li, Xuesen Dong. _University of British Columbia, Vancouver, British Columbia, Canada_.

Androgen receptor (AR) signaling pathway inhibition (ARPI) is the primary treatment for locally advanced, recurrent, or metastatic prostate cancer. While ARPI is effective in short term, a fatal relapse is inevitable. The AKT signaling pathway inhibition has been investigated under multiple ongoing clinical trials as a co-therapeutic target with ARPI. The AKT signaling pathway is targeted due to its role in tumorigenesis, prevalent over-activation, and reciprocal activation upon ARPI in prostate cancer. However, this novel combination therapy may in fact facilitate PCa to progress into one of the most lethal subtypes called neuroendocrine (NE) prostate cancer (NEPC). Specifically, evidence has shown that ARPI contributes to NEPC progression at least in part by down-regulating the expression of RE-1 Silencing Transcription Factor (REST). Loss of REST is a hallmark of NEPC progression because REST functions as a negative master regulator in neurogenesis by suppressing genes required for neural differentiation. Similar to ARPI, our studies found that AKT inhibition reduced REST protein expression and increased NEPC markers in multiple prostate cancer cell lines. We also showed that the loss of REST upon AKT inhibition was through protein degradation mediated by an E3-ubiquitin ligase TRCP, which recognizes a phosphorylated REST degron region to recruit ubiquitins. In vivo ubiquitination assays confirmed the elevated REST ubiquitination after the treatment of PI3K inhibitors. Furthermore, mutations within the REST degron region reversed the TRCP-mediated REST ubiquitination and degradation. Finally, co-treatment of AKT pathway inhibition and ARPI showed aggravated REST depletion and increased NE markers in the prostate cancer cell line LNCaP. Collectively, these findings indicate that AKT pathway inhibition can induce NE phenotype in prostate cancer cells via REST protein degradation. This study may provide a caution to the ARPI/AKT co-target strategy as this strategy can potentially facilitate NEPC development.

#1518

Mechanisms of bromodomain and extra-terminal motif inhibitor (BETi) sensitivity in triple-negative breast cancer (TNBC).

Johanna Schafer,1 Brian Lehmann,2 Phillip Liu,3 Matthew Stubbs,3 Peggy Scherle,3 Jennifer Pietenpol2. 1 _Vanderbilt University, Nashville, TN;_ 2 _Vanderbilt University Medical Center, Nashville, TN;_ 3 _Incyte Corporation, Wilmington, DE_.

Triple-negative breast cancers (TNBCs) lack expression of estrogen receptor (ER), progesterone receptor (PR), and amplifications in the human epidermal growth factor receptor 2 (HER2). With currently no FDA-approved targeted therapies for TNBC, patients with TNBC have a higher risk of local and distant recurrence, and an overall increased rate of mortality. Recent studies have demonstrated increased sensitivity of TNBC to the anti-proliferative effects of BETi compared to the other breast cancer subtypes. To determine mechanisms of sensitivity to BET inhibition, we analyzed the effect of two BET inhibitors, INCB054329 and OTX-015, across a panel of TNBC cell line models and identified cell lines that were both sensitive and insensitive to BETi. With the intent of identifying biomarkers of sensitivity, we performed RNA-seq and precision nuclear run-on and sequencing (PRO-seq) on both sensitive and resistant cell line models and data generated identified significant differences in key growth regulatory and apoptotic signaling pathways, including notable differences in Myc-dependent signaling. Our data suggest potential biomarkers of BETi sensitivity that may be of value in further pre-clinical studies. Further, our results provide mechanistic rationale for combinations of BETi with select, targeted therapies in a disease that is in need of new therapeutic intervention.

#1519

Estrogen receptor beta and p53 signaling crosstalk: implications for ER beta as a potential therapeutic target in triple negative breast cancer.

Christina E. Adams, Gokul M. Das. _Roswell Park Cancer Institute, Buffalo, NY_.

Despite over 20 years of research, there is no current consensus on whether estrogen receptor β (ERβ) is pro- or anti-tumorigenic. With its expression found among all breast cancer subtypes, ERβ is a potentially valuable therapeutic target for breast cancers that lack effective, targeted therapies, especially the triple negative breast cancer (TNBC) subtype. In the published literature, there has been no consensus as to whether ERβ functions as a pro- or anti-tumorigenic protein, although a bifaceted role for ERβ has been proposed. Previous studies from our lab have shown that ERβ can directly bind and interact with p53, resulting in inhibition of its functions. Knockdown of ERβ in a breast cancer cell line with wild-type (wt) p53 (MCF-7) elicits opposite functional effects compared to knockdown of ERβ in a TNBC breast cancer cell line with mutant (mut) p53 (MDA-MB-231). Therefore, our hypothesis is that ERβ functions are dependent on p53 context. Besides having different p53 statuses, these two cell systems also have many other genetic differences that may or may not contribute to the opposing functions of ERβ. Currently, we are using an isogenic cell system that allows for expression of different combinations of ERβ and p53 (wt or mut) proteins. Here, we used CRISPR/Cas9 technology to knockout (KO) the TP53 gene in MDA-MB-231 cells, resulting in a complete absence of the endogenous mut-p53 protein. By transiently transfecting back in wt-p53, we can induce a prototypical wt-p53 response, which causes an increase in p53 target genes accompanied by a decrease in cell growth. Meanwhile, the parental cell line with endogenous mut-53 protein expression shows downregulation of p53 target genes and increases cell growth. This demonstrates that wt-p53 is still functional in the context of a cell that originally expressed mut-p53, and that wt-p53 and mut-p53 have opposing functions. In this system, we demonstrate that ERβ decreases p53 target gene expression and apoptosis in the wt-p53 context. Conversely, in the mut-p53 context, ERβ increases p53 target gene expression, while decreasing cell cycle progression. Taken together, these data support a pro-tumorigenic role of ERβ in the wt-p53 context, and an anti-tumorigenic role of ERβ in the mut-p53 context in our isogenic system. Finally, we explore the ability of ERβ ligands to modulate the ERβ-p53 interaction and subsequently affect p53 functions. We have found that two ERβ antagonists, 4-hydroxytamoxifen and PHTPP, have opposite effects on p53 target genes, suggesting a difference in their ability to modulate the ERβ-p53 interaction. Our data show that p53 status of the tumor should be taken into consideration for therapeutic targeting of ERβ in TNBC.

#1520

Down-regulation of c-MYC and hTERT gene expression in triple negative breast cancer.

Elaine Stur, Shelia Thomas, Francine Rezzoug, Donald Miller. _University of Louisville, Louisville, KY_.

Breast cancer is the most common cancer affecting women, with about 1.4 million new cases diagnosed each year. Triple-negative breast cancer (TNBC) which is characterized by lack of expression of estrogen, progesterone and human epidermal growth factor receptors. Is the most aggressive group of tumors associated with a poor prognosis (15% of all mammary tumors). Previous studies have shown that specific G-rich genomic sequences in the promoters of multiple human genes can form G-quadruplex structures, resulting in decreased transcription and gene expression. These regions are generally found within gene promoters especially of oncogenes such as c-MYC, KRAS, VEGF, BCL2 or hTERT. Most of these genes are abnormally expressed in breast cancer. The c-MYC gene regulates a large array of genes essential for cell functions including proliferation, metabolism, differentiation, adhesion and apoptosis. We have recently shown that oligonucleotides encoding the G-quadruplex sequence of the c-MYC, VEGF or hTERT promoters down regulate expression of their respective genes and inhibit cell proliferation. We hypothesized that such oligonucleotides could be applied to breast cancer cell lines in order to inhibit cell proliferation and metastasis. In this study, we evaluated the effect of oligonucleotides targeted to c-MYC (Pu27 and Pu27 Palmi), KRAS (KRASq), VEGF (VEGFq), hTERT (Tert-FL) and BCL2 (BCL2q) in Breast cancer cell lines (MDA-MB-231, SKBR3, MCF7) and MCF10A (non-transformed mammary cells). The effect of each oligonucleotide on cell growth was evaluated using the MTT assay. Our results reveal that all oligos inhibit cell growth of MDA-MB-231. However, for SKBR3 and MCF7, only Tert-FL had growth inhibitory activity. The control cell line, MCF10A, did not respond suggesting that the oligonucleotides affect preferentially tumor cells. The effect of the oligonucleotides on gene expression (qRT-PCR) was performed on MDA-MB-231 exposed for 3 days. The analysis of gene expression shows the downregulation of c-MYC by Pu27 and hTERT by Tert-FL suggesting a direct effect on gene expression. In addition, Pu27 downregulated hTERT and VEGF which are both under c-MYC control. Since MDA-MB-231 is a TNBC cell line overexpressing c-MYC and is highly enriched in cancer stem cells, we evaluate the effect of Pu27 and Tert-FL on the tumorsphere formation. The results showed a very strong inhibition of tumorsphere formation in the cells treated with Pu27. Our findings suggest that oligonucleotides which target genes such as c-MYC, hTERT or KRAS very efficiently inhibit the growth of BC cell lines in particular MDA-MB-231 TN most likely by downregulating target gene expression. In addition, we demonstrate that downregulating c-MYC expression Pu27 dramatically reduces the cancer stem cell numbers. The use of G-quadruplex forming oligonucleotides targeted to c-MYC and /or hTERT may constitute a new therapeutic strategy especially for TNBC where targeted therapy is lacking.

#1521

hTERT **G-quadruplex-targeted oligonucleotides inhibit glioblastoma cell growth.**

Alex West. _University of Louisville, Louisville, KY_.

Glioblastoma is one of the most common and deadly forms of brain cancer, representing roughly 75% of all brain malignancies. These tumors generally have poor prognoses and are resistant to conventional therapy. It has recently been shown that as many as 80% of all glioblastomas contain mutations in a G-rich 68 base pair region of the hTERT promoter. hTERT is the catalytic subunit of telomerase, a holoenzyme responsible for lengthening the ends of chromosomes, thereby conferring immortality to cells. Normally, hTERT is not expressed in somatic cells and its expression is tightly controlled in stem cells. However, hTERT is upregulated in up-to 95% of human tumors and is considered a key activator of cancer progression and a sign of poor clinical outcome. Therefore, hTERT has been under investigation for the past decade as a potential therapeutic target. We have shown that the mutations in hTERT promoter occur in a G-rich region that is part of a silencer element which forms a secondary G-quadruplex structure required for function, and that these mutations destabilize the G-quadruplex structure, allowing hTERT expression. We have also demonstrated that oligonucleotides encoding the G-quadruplex forming sequence in the c-MYC promoter can stabilize the G-quadruplex structure and downregulate c-MYC expression. Therefore, we hypothesized that oligonucleotides targeted to the G-quadruplex of the hTERT promoter could downregulate this gene expression and inhibit glioblastoma cell proliferation in a similar manner. We designed several G-quadruplex-forming oligonucleotides covering the mutated sites in the hTERT promoter either in totality (68 nucleotides) or separately (25 nucleotides) to stabilize the G-quadruplex region. Two glioblastoma cell lines (A172 and U87) and one neuroblastoma cell line (CHP134) were exposed to these oligonucleotides and evaluated for growth inhibition using the MTT assay and for gene expression by QRT-PCR. All oligonucleotides tested were found to induce between 40 to 90% growth inhibition in the 3 cell lines. The cell growth inhibition was both time and dose dependent and showed effectiveness as early as 3 days suggesting that this effect is not solely due to telomere shortening. Four oligonucleotides with the most consistent efficacy in growth inhibition were evaluated for their effect on hTERT gene expression in the 3 cell lines at 4 day exposure and revealed that two of the G-quadruplex forming oligonucleotides significantly decreased hTERT expression compared to untreated cells. In conclusion, we have defined G-quadruplex oligonucleotides targeted to the hTERT promoter that downregulate hTERT gene expression and are effective growth inhibitors in glioblastoma cells. Our findings indicate that downregulation of hTERT with targeted oligonucleotides affects non-canonical functions of hTERT conferring an advantage to this therapeutic approach.

#1522

Targeted gene therapy · c-MYC and hTERT in the scope.

Francine Rezzoug, Shelia D. Thomas, Segen Tella, Donald M. Miller. _Univ. of Louisville, Louisville, KY_.

In the past decade immunotherapy and kinase inhibitors have revolutionized the approach for cancer therapy, particularly for melanoma which has benefit from this new therapy in the last 5 years. Although, new treatment increase patient survival, there is no cure and treatments are often limited to a subtype of patients. Therefore there is still an urgent need for efficient therapy for melanoma. Among potential targets for cancer therapy, c-MYC and hTERT which are overexpressed in up-to 85% of all cancers are the most promising. The overexpression of hTERT provides indefinite division to cancer cells. Deregulation of c-MYC affect a large array of genes involved in cell cycle, proliferation/differentiation and apoptosis. Notably, both c-MYC and hTERT genes have in their promoter area a G-rich sequence that form secondary structure called G-quadruplex. These G-quadruplex structures are part of the silencers and are required for the repression of c-MYC or hTERT transcription. Mutations in the G-rich sequences of hTERT promoter destabilize the G-quadruplex and are associated with incidence of melanoma. We have shown that oligonucleotides encoding for the G-quadruples sequences of these genes promoter downregulate their respective gene expression and inhibit cell growth in different cell lines. We investigated the effect of targeting c-MYC or hTERT gene expression in melanoma cell lines using oligonucleotides targeted to the silencer G-quadruplex sequence. Four cell lines where investigated (SK-Mel-2, SK-Mel-3, SK-Mel-28 and A375) for the effect of Pu27 (targeting c-MYC) and Tert-FL (targeting hTERT) on cell proliferation using MTT assay. The treatment for 6 days resulted in 60% growth inhibition in the presence of Pu27 and 50% in the presence of Tert-FL for SK-Mel-2, SK-Mel-3, SK-Mel-28 while A375 was less sensitive. Gene expression evaluated by QRT-PCR showed down-regulation of c-MYC and hTERT in cell exposed to Pu27 and hTERT in cells exposed to Tert-FL. hTERT expression is regulated by c-MYC and in our experiment the down-regulation of c-MYC correlate with down-regulation of hTERT suggesting a cascade effect. In addition, SOX2 gene expression was also down-regulated by Pu27 suggesting an effect on the cancer stem cells (CSC). Our data demonstrate that oligonucleotides targeted to c-MYC and hTERT down-regulate gene expression associated with melanoma cell growth inhibition and possibly on metastasis.

#1523

The mechanism of action of BCL6 in glioblastoma.

Nicole M. Jones,1 Marie-Sophie Fabre,1 Dinindu Sachindra Senanayake,1 Katerina Hatzi,2 Ari M. Melnick,2 Melanie J. McConnell1. 1 _Victoria University of Wellington, Wellington, New Zealand;_ 2 _Weill Cornell Medical College, Cornell University, New York, NY_.

Glioblastoma (GBM) is the most common and most deadly brain tumor to occur in adults. Initially patients respond to radiation and chemotherapy, which primarily work by causing large amounts of DNA damage, causing apoptosis of the cells. However, this process does not happen effectively in GBM and understanding how these cells resist cell death in response to therapy is key to improving the efficacy of treatment. BCL6 is a transcription factor that stops cell death in response to DNA damage. Recent work in our lab has shown BCL6 to be present in untreated GBM tumors and up-regulated in treated GBM cells. This evidence indicates that BCL6 may be used as a mechanism of therapy resistance by GBM cells. In this study, the objective was to determine the mechanism of action of BCL6 in GBM cells using luciferase reporter assays, quantitative chromatin immunoprecipitation (qChIP) and RNA sequencing. We observed that BCL6 was transcriptionally active in GBM as shown by a reduction in luciferase activity when BCL6 was present. qChIP experiments revealed that BCL6 binding changed over time and was different with different types of DNA-damaging treatment. Preliminary analysis of our RNA sequencing data has identified a unique subset of genes which are upregulated when BCL6 is inhibited and downregulated in response to chemotherapy. These changes indicate that these genes may be regulated by BCL6 in chemotherapy treated cells. All of these results illustrate that BCL6 appears to have an active and relevant function in GBM cells, which demonstrates that BCL6 is an attractive therapeutic target in GBM.

#1524

BCL6 modulates the TP53 and STAT pathways in glioma.

Ye Chen,1 Liang Xu,1 Marina Dutra-Clarke,2 Anand Mayakonda,1 De-Chen Lin,1 Lynnette Koh,3 Yuk Kien Chong,3 Edwin Sandanaraj,3 Vikas Madan,1 Henry Yang,1 Ngan Doan,4 Jonathan W. Said,4 William H. Yong,4 Markus Müschen,5 Beng Ti Ang,3 Carol Tang,3 Joshua J. Breunig,2 Phillip Koeffler1. 1 _National University of Singapore, Singapore, Singapore;_ 2 _Cedars-Sinai Medical Center, UCLA, Los Angeles, CA;_ 3 _National Neuroscience Institute, Singapore, Singapore;_ 4 _UCLA, Los Angeles, CA;_ 5 _UCSF, San Francisco, CA_.

Glioblastoma multiforme (GBM) remains the most aggressive brain malignancy with little improvement in prognosis or therapy for decades. Recently, we identified BCL6, also known as ZBTB27, to be a novel oncogene in GBM. In this study, we performed IHC analysis of 153 primary human glioma specimens and 8 normal brain samples. BCL6 expression is robustly elevated in tumor samples and positively correlated with glioma pathological grade. High BCL6 expression strongly predicts a worse prognosis of GBM patients. Depletion of BCL6 in human GBM cells reduced the incorporation of BrdU, promoted the cellular senescence and inhibited the growth of human GBM cells in vivo. Next, genome-wide occupancy of BCL6 in GBM cells was characterized by ChIP-seq assay. Genomic regions centered on BCL6 peaks are co-enriched with RNA-Pol II and flanked with strong H3K27ac and H3K4me3 modifications. MYC and two long non-coding RNAs MALAT1 and NEAT1 were identified as novel BCL6 targets in GBM. Moreover, pathway enrichment analysis of BCL6 peak-associated genes reveals a significant enrichment of JAK-STAT, TP53, ERBB and MAPK pathways. We demostrated further that BCL6 represses the TP53 pathway and promotes the JAK-STAT pathway activation in GBM cells. Together, our findings uncover potential downstream targets and provide a better understanding of BCL6 function in GBM.

#1525

Chemically induced degradation of the oncogenic transcription factor BCL6.

Nina Kerres,1 Steffen Steurer,1 Stefanie Schlager,1 Gerd Bader,1 Helmut Berger,1 Sophia Blake,1 Maureen Caligiuri,2 John Engen,3 Peter Ettmayer,1 Thomas Gerstberger,1 Daniel Gerlach,1 Roxana Jacob,3 Simon Lucas,1 Moriz Mayer,1 Mischerikow Nikolai,1 Rumpel Klaus,1 Scharn Dirk,1 Schnitzer Renate,1 Tilman Voss,1 Zoephel Andreas,1 Norbert Kraut,1 Darryl McConnell,1 Pearson Mark,1 Manfred Koegl1. 1 _Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria; _2 _FORMA Therapeutics, Watertown, MA;_ 3 _Northeastern University, Boston, MA_.

The transcription factor BCL6 is a known driver of oncogenesis in lymphoid malignancies, including diffuse large B-cell lymphoma (DLBCL). It is a DNA-binding protein that represses gene transcription through the recruitment of co-repressor proteins via its BTB domain. Disruption of the interaction of BCL6 with co-repressor proteins interferes with the physiological and oncogenic functions of BCL6. We have used structure-based drug desgin to develop potent compounds that block this interaction with half maximal inhibitory concentration values (IC50s) below 3 nM. In addition to inhibiting co-repressor binding, a subset of the identified inhibitors also caused rapid disappearance of BCL6 protein in cells. This effect was mediated by compound-induced multi-ubiquitylation of BCL6 and degradation by the proteasome and was dependent on the presence of a functional DNA binding domain on BCL6. Compounds that induced BCL6 degradation displayed significantly stronger induction of expression of BCL6-repressed genes than compounds that merely inhibited co-repressor interaction. BCL6-degrading inhibitors had anti-proliferative effects in several DLBCL cell lines, while non-degrading BCL6 inhibitors only had minor effects on proliferation. The fact that the magnitude of effects elicited by this novel class of BCL6 degrading compounds greatly exceeds that of our equipotent classical co-repressor interaction inhibitors offers exciting new opportunities for the development of BCL6-based lymphoma therapeutics.

#1526

Usp9x inhibition and depletion overcomes SOX2 dependent resistance to BRAF inhibition in melanoma.

Harish Potu. _Univ. of Michigan Comp. Cancer Ctr., Ann Arbor, MI_.

Melanoma tumors often show limited response to BRAF/MEK/ERK pathway inhibitors currently used in melanoma treatment. One mechanism of resistance to current melanoma treatments is upregulation of SOX2, a transcription factor that is essential for tumor growth and expansion, particularly in tumors with BRAF mutation in melanoma. Targeting drugs to transcription factors has been elusive and limits treatment options. Here we report that the deubiquitinating enzyme Usp9x controls SOX2. We show that ubiquitin-specific peptidase 9, X-linked (Usp9x), a deubiquitinases (DUB) enzyme, binds SOX2 in melanoma and deubiquitinates SOX2 in vitro. Usp9x knockdown results in increased levels of ubiquitinated SOX2 in melanoma, is coupled with depletion of SOX2 and promotes apoptosis synergistically with vemurafenib in melanoma cell lines. Protein levels in primary melanoma tumor samples demonstrated moderately elevated Usp9x/SOX2 protein expression in metastatic melanoma compared to primary melanoma patient tumors. Further, Usp9x knockdown or inhibition with G9, a DUB inhibitor, blocked BRAF/MEK kinase inhibitor-induced induction of SOX2 and fully suppressed in vitro colony growth of BRAF-mutant melanoma cells and promoted apoptosis. We discovered a novel mechanism of targeting SOX2, transcription factor by inhibition of Usp9x. Thus, development of DUB inhibitors will add to the limited repertoire of current melanoma treatments.

#1527

BRD3 as a specific vulnerable therapeutic target in neuroblastoma.

Kaat Durinck,1 Jolien Dewyn,1 Anneleen Beckers,1 Siebe Loontiens,1 Suzanne Vanhauwaert,1 Daniel Carter,2 Belamy Chueng,2 Glenn Marshall,2 Katleen Depreter,1 Frank Westermann,3 Frank Speleman1. 1 _Ghent University, Ghent, Belgium;_ 2 _Children's Cancer Institute Australia for Medical Research, Sydney, Australia;_ 3 _DKFZ, Heidelberg, Germany_.

Introduction: BET inhibitors have raised high expectations for cancer treatment given their anti-proliferative effect by inhibiting BRD4 controlled enhancer activity of highly transcribed genes such as MYC(N). However, current inhibitors also target BRD2 and BRD3 which are functionally nonredundant with BRD4. In neuroblastoma only MYCN amplified tumors respond well to these drugs.

Methods: We performed an integrated bioinformatics approach to scrutinize BET family genes as well as further candidate epigenetic regulators as targets for novel therapies in neuroblastoma.

Results: First we performed a time-resolved expression data analysis of week 1 and 2 hyperplastic lesions and tumors derived from the TH-MYCN transgenic mouse model and confirmed dynamic regulation during tumor development for established neuroblastoma oncogenes and tumor suppressor genes. Next, we filtered within the highest upregulated genes for Cancer Gene Census (CGC) genes and identified 21 upregulated CGC genes mainly involved in chromatin remodeling and DNA repair. Finally, after further selection based on expression in CCLE and survival in neuroblastoma patients, BRD3 was identified as the top-ranked candidate. BRD3 exhibits drastic upregulation during tumor formation. Elevated BRD3 expression is the highest expressed gene in neuroblastoma cell lines upon analysis of the CCLE panel and associated with very poor prognosis. To explore the nonredundant functions of BRD3 in relation to BRD4, we performed RNA-sequencing after stable knockdown of BRD3 in neuroblastoma cell lines and compared the downstream effects on the transcriptome as well as the impact on cell viability to knockdown of BRD4 and pharmacological treatment with BET-inhibitors (JQ1, OTX015). In addition, we dissected the BRD3 protein complex by means of label-free mass spectrometry analysis to gain further insights into the BRD3 specific functions in relation to control of gene transcription and putative interaction with transcription factors such as MYCN. Current efforts are ongoing to test cooperative interaction of BRD3 versus BRD4 in dbh-MYCN driven neuroblastoma formation in zebrafish as well as BRD3 and BRD4 ChIP-sequencing in neuroblastoma cells.

Conclusion: We identified BRD3 as a candidate novel driver gene in neuroblastoma and will present differential transcriptional control and protein interactions of BRD3 versus BRD4. This study can open the way towards developing BRD3 specific inhibitors for neuroblastoma and other BRD3 overexpressing cancers such as T-ALL and small cell lung carcinoma.

#1528

Forecasting novel therapies by understanding the role BRD4 in regulating the Notch3 signaling in ovarian cancer.

Alejandro Villar-Prados,1 Sherry Y. Wu,1 Jason Roszik,1 Anil K. Sood,1 Margaret I. Engelhardt2. 1 _UT MD Andersion Cancer Center, Houston, TX;_ 2 _McGovern Medial School, Houston, TX_.

Advances in cancer research have led to the rapid development of novel drug therapies designed for optimal personalized patient care. Despite this surge of treatments, there is still a challenge in accurately predicting the most effective targeted therapy for a specific cancer. Additionally, the potential of systematically identifying and re-purposing currently existing therapeutics designed for one specific cancer to treat other malignancies remains understudied. To address this problem, our group has developed an integrative computational therapy-forecasting algorithm. This algorithm incorporates data from The Cancer Genome Atlas (TCGA) as well as molecular and survival correlations to identify candidate anti-tumor drugs that can be re-purposed for other malignancies by targeting novel pathways. Our computational analysis predicted that bromodomain inhibitors, which inhibit bromodomain-containing proteins such as BRD4, would target the Notch3 pathway in high-grade serous ovarian carcinoma (HGSC). Upregulation of Notch3 plays a crucial role in HGSC tumorigenesis and is associated with worse patient survival. Current Notch3 targeted therapies are not effective, thus designing therapeutic strategies to target Notch3 are critical for HGSC. We hypothesize that inhibition of BRD4 is an effective therapeutic target in HGSC by downregulating the Notch3 pathway. Current in vitro results demonstrated that BRD4 inhibition either by using chemical inhibitors or siRNA results in a decrease in HGSC cell viability by both MTT and 2-D colony formation assays. Furthermore, inhibition of BRD4 resulted in a decrease in both Notch3 transcription and protein levels. Given that BRD4 regulates gene transcription, we performed chromatin immunoprecipitation (ChIP) and observed that BRD4 was present in the Notch3 gene promoter. These findings suggest that by inhibiting BRD4 in HGSC, we can target Notch3 and biologically validates our initial in sillico analysis. The successful completion of this project could have major implications for development of personalized therapies not only for HGSC but for other cancers as well.

#1529

Scutellaria baicalensis **targets the hypoxia-inducible factor-1α and attenuates ovarian cancer growth.**

Imran Hussain,1 Sana Waheed,1 Kashif A. Ahmad,2 John E. Pirog,3 Viqar Syed1. 1 _Uniformed Services Univ. of the Health Sci., Bethesda, MD;_ 2 _University of Illinois College of Medicine-Urbana Champaign, Urbana, IL;_ 3 _Northwestern Health Sciences University, Bloomington, MN_.

Hypoxia-inducible factor-1alpha (HIF-1α), a transcription factor important for maintaining cellular oxygen homeostasis, is aberrantly upregulated in many tumors and implicated in angiogenesis, metastasis and drug resistance. Therefore, developing treatments that target HIF-1α may be a viable therapeutic approach. For centuries, traditional Chinese medicine has used herbs, such as Scutellaria baicalensis (SB), as a natural remedy for tumors. However, the mechanisms by which SB inhibits tumor growth are not known. We have previously reported that SB inhibits cancer cell proliferation and targets NF-κB in endometrial and ovarian cancers and TGF-β in endometrial cancers. In this study, we examined the effects of SB on HIF-1α protein levels in three ovarian cancer cell lines, SKOV3, OVCA 429, and OVCA 420, grown under normoxic and hypoxic conditions. When treated daily with SB, cancer cells showed a decrease in HIF-1α expression after 48 hours. Investigation into the molecular mechanisms of SB-induced inhibition revealed a significant reduction of HIF-1α accumulation in cells treated with cycloheximide, a protein synthesis inhibitor. Further decrease of HIF-1α accumulation was seen when cycloheximide was combined with SB. Additionally, SB- induced HIF-1α proteasomal degradation was prevented by treatment of cells with proteasomal inhibitor MG132 and chloroquine, lysosome inhibitor, abrogated SB-induced inhibition of HIF-1α. Activation of PI3K/AKT and MAPK/ERK is shown in cancers. SB reduced levels of phospho-AKT and phospho-extracellular signal-related kinase (ERK) in ovarian cancer cells. Furthermore, pretreatment of cells with LY294002 (phosphoinositide 3-kinase inhibitor) and PD98059 (mitogen-activated protein kinase kinase inhibitor) reduced HIF-1α expression comparable to SB-treated cells. Hypoxia-induced cisplatin resistance is a major challenge in therapy of ovarian tumors. SB reduces the proliferation and reverses cisplatin resistance in ovarian cancer cells by attenuating the expression of HIF-1α. Taken together, the findings suggest that targeting HIF-1α with SB could be an effective treatment strategy for ovarian cancer and that SB can improve the sensitivity of cells to cisplatin.

#1530

RUNX1 positively regulates ErbB2/HER2 signaling pathway through modulating the expression of SOS1 in gastric cancer cells.

Yoshihide Mitsuda, Ken Morita, Shintaro Maeda, Kensho Suzuki, Gengo Kashiwazaki, Junichi Taniguchi, Toshikazu Bando, Hiroshi Sugiyama, Souichi Adachi, Yasuhiko Kamikubo. _Kyoto University, Kyoto, Japan_.

Although runt-related transcription factor 1 (RUNX1) is well known for its context-dependent oncogenic properties in various malignancies, its role in gastric cancers has been poorly defined. Up-regulation of receptor tyrosine kinase (RTK) ErbB2/HER2 signaling pathway is encountered in the vast majority of gastric cancer cases and contributes to the initiation and maintenance of these cancer cells. This signaling cascade is partly yet arbitrary mediated by son of sevenless gene 1 (SOS1), which functions as an adaptor protein in RTK cascades. Herein we report that RUNX1 regulates ErbB2/HER2 signaling pathway in gastric cancer cells through transcriptionally regulating SOS1 expressions, rendering itself an ideal target in anticancer strategies. Mechanistically, RUNX1 interacts with the consensus RUNX1 binding sequence (5'-TGTGGT-3') located in the proximal promoter region of SOS1 and positively regulates it. Short hairpin RNA (shRNA)-mediated knockdown of RUNX1 in the gastric cancer cell line MKN45 led to the decreased expression of SOS1 and of phosphorylated form of ErbB2/HER2 as well as the deactivation of its downstream targets such as AKT and ERK. RUNX1 knockdown subsequently induced cell cycle arrest at G0/G1 phase and successive apoptotic cell death in MKN45 cells. Silencing of HER2 or SOS1 in MKN45 cells unalterably suppressed the proliferation of these cancer cells, highlighting the importance of this ErbB2/HER2 signaling cascade in the maintenance of gastric cancer cells. We also found that SOS1 is one of the most consistently up-regulated genes in RUNX1-high expressing primary gastric cancer cells derived from previously reported human clinical samples. These data collectively indicates that inhibition of RUNX1 could be a legitimate therapeutic choice in the management of gastric cancers. Lastly, we examined the efficacy of a novel small molecule specifically binds and inhibits RUNX1 (we named it as CM). Intriguingly, CM was exceptionally effective against MKN45 cells (IC50 value at 403.5 nM). Besides, CM was well-tolerated in mice and fabulously suppressed the growth of xenotransplanted MKN45 cells in immunodeficient mice in vivo. Taken together, our work identified a novel interaction of RUNX1 and ErbB2/HER2 signaling pathway, paving a new way for the management of dismal-prognostic advanced stage HER2-positive gastric cancer patients.

#1531

Therapeutic strategies for osteosarcoma stem cells by regulating adipocyte differentiation based on actin dynamics.

Hiroyuki Nobusue,1 Nobuhiro Takahashi,1 Nobuyuki Onishi,1 Takatsune Shimizu,2 Eiji Sugihara,1 Yoshinao Oki,3 Koichi Akashi,4 Koichiro Kano,3 Hideyuki Saya1. 1 _Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan;_ 2 _School of Pharmacy and Pharmaceutical Sciences, Hoshi University, Tokyo, Japan;_ 3 _College of Bioresource Sciences, Nihon University, Tokyo, Japan;_ 4 _Kyushu University Graduate School of Medical Science, Fukuoka, Japan_.

We generated induced cancer stem cells that initiate osteosarcoma (OS) by overexpression of c-MYC and loss of Ink4a/Arf in bone marrow-derived stromal cells, and found that the loss of adipogenic potential is an essential event for OS tumorigenesis. Therefore, our understanding of regulatory mechanisms of adipocyte differentiateon would greatly contribute to control OS tumorigenesis. Here, we show that regulation of the transcriptional coactivator MKL1 (megakaryoblastic leukemia 1) by actin cytoskeleton dynamics drives adipocyte differentiation mediated by PPARγ. Exposure of preadipocytes to an adipogenic cocktail results in the rapid disruption of actin stress fibres as a consequence of downregulation of RhoA-ROCK signaling, which regulates the formation of actin stress fibers and focal adhesions, and the consequent increase in monomeric G-actin levels. The resulting increase in the amount of G-actin leads to the interaction of G-actin with MKL1, which prevents nuclear translocation of MKL1 and allows expression of PPARγ followed by adipogenic differentiation. We further found that depletion of MKL1 can drive adipocyte differentiation in preadipocytes and even in nonadipogenic fibroblasts in the absence of an adipogenic cocktail, and these data revealed that MKL1 functions as a gatekeeper that controls adipocyte differentiation. In addition, we found that treatment with ROCK inhibitors induced terminal adipocyte differentiateon even in OS stem cells through regulation of MKL1 by actin cytoskeleton dynamics, which resulted in a significant suppression of tumorigenesis. Our findings provide new insight into the regulatory mechanism of adipogenesis, and trans-differentiation approach by controlling actin cytoskeleton dynamics may provide a novel therapeutic strategy for targeting cancer stem cells.

#1532

Mithramycin induces the antiproliferative activity of chemotherapeutic agents in Ewing sarcoma cells.

Anish Ray,1 Bhavani Nagarajan,2 Umesh T. Sankpal,2 Sagar Shelake,2 W. Paul Bowman,2 Andras Lacko,2 Riyaz M. Basha2. 1 _Cook Children's Medical Center, Fort Worth, TX;_ 2 _University of North Texas Health Science Center, Fort Worth, TX_.

Ewing sarcoma (ES) is one of the most frequent primary bone and soft tissue tumors that occur in pediatric and adolescent population. ES is often diagnosed after the disease has already metastasized. With available treatment options, the prognosis of metastatic ES patients is poor and the 5-year survival rate is less than 20%. Therefore, identifying precise targeted therapies to induce therapeutic response in ES patients is urgently needed. An oncogenic fusion protein and transcription factor EWS-FLI1 is associated with more than 85% of ES tumors. Mithramycin has been identified as an effective agent to target EWS-FLI1 and is currently in clinical trials. In this study, we investigated the efficacy of Mithramycin to induce the anti-proliferative activity of chemotherapeutic agents commonly used for the treatment of this malignancy using human ES cell lines. TC71, TC32, CHLA32, CHLA10 and TC205 cells were treated with increasing concentrations of Mithramycin or Vincristine or Doxorubicin or Etoposide. Cell growth inhibition was evaluated at 24 and 48 h post-treatment using CellTiter Glo kit. Results showed a dose/time-dependent anti-proliferative effect for all agents. To confirm the effect of Mithramycin on selected cell lines, mRNA expression of downstream targets of EWS-FLI1 (ID2, LDB2, NROB1 and RCOR1 genes) was determined by quantitative PCR following treatment with Mithramycin. Mithramycin significantly down-regulated all tested genes and these results are in agreement with published work. In order to determine the combination response, cell growth inhibition of the chemotherapeutic drugs was assessed in the presence of added Mithramycin. The combination Index was evaluated by Chou-Talalay method. Further studies were performed for the combination of Mithramycin and Vincristine to investigate its effect on apoptosis and cell cycle arrest using CHLA10 and TC205 cells. Apoptotic markers such as the expression of cleaved Poly (ADP-ribose) polymerase (c-PARP) and survivin were determined by Western blot analysis. The apoptotic cell population was measured by Annexin-V staining using flow cytometry. When compared to individual agents, the combination of Mithramycin and Vincristine showed greater effect on apoptosis in both cell lines as evinced by an increase in Annexin-V positive cells, decrease in survivin expression and up-regulation of c-PARP. Overall, these results indicate the potential clinical advantage of combination treatment involving Mithramycin along with other chemotherapeutic agents for inhibiting the growth of ES cells. Molecular profiling analysis is underway to elucidate the candidate pathways associated with beneficial effect of this combination treatment.

#1533

Transcriptional inhibition of brachyury in chordoma is associated with adoption of quiescent phenotype.

Ashlee N. Seldomridge,1 Jinkyu Jung,1 Heather M. Sonnemann,1 Amber Giles,1 Kristan Meetze,2 Mark R. Gilbert,1 Claudia M. Palena,3 Deric M. Park1. 1 _Neuro-Oncology Branch, CCR, National Cancer Institute, NIH, Bethesda, MD;_ 2 _Tragara Pharmaceuticals, Carlsbad, CA;_ 3 _Laboratory of Tumor Immunology and Biology, CCR, National Cancer Institute, NIH, Bethesda, MD_.

BACKGROUND: Brachyury is a mesoderm specification transcription factor involved in notochord development and overexpressed in a variety of cancers, including chordoma. High levels of brachyury protein expression in cancers is associated with a poor prognosis in part due to its role in mediating epithelial-mesenchymal transition (EMT). TG02 is a multikinase inhibitor that targets transcriptional regulation of cyclin-dependent kinases (CDKs). Because chordoma is characterized by a relative paucity of genomic mutations and largely driven by the super-enhancer activity of brachyury expression, we investigated the downstream effect of a transcriptional inhibitor, TG02, in chordomas.

METHODS: Established chordoma cell lines, UCH-1 and UM-Chor1, were exposed to increasing concentrations of TG02 to determine effect on brachyury protein expression. qPCR was used to analyze brachyury mRNA expression as well as the mesenchymal proteins, vimentin and fibronectin, typically associated with brachyury expression in tumor cells. Cell migration was

evaluated using wound healing assay. CellTiterGlo Luminescence Assay was used to quantify cell viability.

RESULTS: TG02 down-regulated protein expression of brachyury in a dose-dependent manner. Attenuation of brachyury expression was seen within 4 hours and persisted for 5 days upon single exposure to a clinically relevant concentration of TG02. Brachyury downregulation did not affect cell count or viability, and was associated with a quiescent phenotype, including impaired migration. Expression of vimentin and fibronectin, both associated with EMT, was also suppressed within 4 hours of TG02 treatment and persisted for 24 hours.

CONCLUSIONS: Inhibition of brachyury expression and its downstream signaling is associated with quiescent behavior of chordoma cells. Pharmacologically induced transcriptional targeting of brachyury using TG02, a potent CDK9 inhibitor, represents a potential therapeutic strategy.

#1534

The significance of a p53 gain-of-function mutation in the acquisition of metastatic traits in the SW13 cell line.

McKale Montgomery, Elizabeth Hull. _Midwestern University, Glendale, AZ_.

The therapeutic potential of histone deacetylase inhibitors (HDACi), shown to epigenetically regulate a variety of genes, has yet to be fully realized due to their documented pleiotropic effects in vivo. Currently, four HDACi are FDA-approved for the treatment of hematological malignancies, but their effectiveness against other types of cancers have been shown to be limited at best and, as a result, most of these clinical trials have failed. A comprehensive review of the recent literature has identified that approximately 50% of all cell lines treated with an HDACi demonstrate an increase in metastatic potential as measured by increases in migration and invasion. Of these cell lines that respond with increased metastatic potential to HDACi treatment, 60% have a gain-of-function mutation within p53 (p=0.03). As HDACi have been shown to destabilize mutant p53, they have been used in studies to examine the effects of HDACi treatment on cancers with an identified p53 mutation. This is concerning as the specific p53 mutation in these cancers is not always known, and thus treatment could potentially result in an increase in cancer cell growth. Previous work in our lab demonstrates that the human SW13 adrenal adenocarcinoma cell line, which harbors a homozygous H193Y gain-of-function mutation, does have an HDACi-induced metastatic phenotype; treated cells demonstrate significant increases in MMP activity and ~50% reduction in p53 protein expression compared to untreated cells. To investigate the role that a decrease in mutant p53 expression plays in the acquisition of metastatic characteristics we utilized stable shRNA plasmids to knock-down p53 expression in the SW13 cell line. HeLa cells, which possess a wild-type p53 gene, were used as a positive control. We found that a decrease in p53 expression alone was not sufficient to induce changes in SW13 growth or invasiveness. In fact, even though p53 expression was knocked down by ~70% in the shRNA lines, HDAC inhibition still induced the acquisition of metastatic properties in these cells. Intriguingly, while the DNA binding activity of mutant p53 was correlated with protein expression and was significantly decreased following HDAC inhibition, the same response was not observed in HeLa cells. These findings suggest that p53 status may be an important determinant of therapeutic response to HDAC inhibition.

#1535

Angiogenesis-related gene expression profile of a novel antiangiogenic agent F16 in human vascular endothelial cells.

Khalid Alhazzani, Ali Alaseem, Thiagarajan Venkatesan, Appu Rathinavelu. _Nova Southeastern Univ., Davie, FL_.

Angiogenesis is a key process in tumor propagation, progression, and metastasis. This process is tightly modulated by a wide range of growth factors, receptor tyrosine kinases, cytokines, matrix metalloproteinases, and transcription factors. Distinctively, vascular endothelial growth factor (VEGF) and their receptors have been identified as prominent stimulators of tumor angiogenesis. Therefore, inhibiting VEGF-induced tumor angiogenesis has been explored extensively for cancer therapy. In this context, F16, a novel small molecule possessing a unique molecular configuration to block VEGFR2, has been subjected to intensive preclinical investigations. The F16 molecule exhibits in vitro antiangiogenic activity via inhibiting endothelial cell proliferation, migration, and tube formation. In addition, F16 was found to significantly inhibit the in vivo angiogenesis in chick chorioallantoic membranes as well as in athymic nude mice with xenograft tumors. As a consequence of the antiangiogenic effects, F16 was able to effectively control the tumor growth in mice with xenograft tumors. In the present study, we aimed to comprehensively interrogate the impact of F16 treatment on the expression of a panel of angiogenesis-related genes to gain insight into the underlying molecular mode of action. We analyzed the expression of 84 genes that are known to be associated with angiogenesis in human umbilical vein endothelial cells (HUVECs). Our pilot study identified upregulation of some key genes in response to F16 treatment which include tissue inhibitor of metalloproteinase 1 (TIMP1), angiopoietin-like 4 (ANGPTL 4), chondromodulin 1 (LECT 1), and placenta growth factor (PIGF). On the other hand, several genes involved in promoting angiogenesis were downregulated, which include matrix metallopeptidase 9 (MMP9), integrin subunit beta 3 (ITGβ 3), insulin growth factor 1 (IGF 1), transforming growth factor alpha (TGF α), interleukin 6 (IL 6), leptin (LEP), thrombospondin -1, -2 (THBS -1, -2), tyrosine kinase (TEK), VEGF B and C. The differential genes expression related to pro- and anti-angiogenic growth factors coincide very well with our previous observation of F16 inhibiting endothelial cell proliferation, migration and tube formation. Based on the Gene Expression Profile (GEP) observed in our experiments, we speculate that F16 can induce TIMP1 which in turn could suppress the MMP9 signaling leading to the inhibition of endothelial cell migration. This disruption of TIMP1/MMP9 pathway offers an interesting foundation for functional studies that can be performed to confirm the role of F16 as a potential MMP9 inhibitor, which may provide extended benefits during the use of this drug for treating highly metastatic cancers. (This project was supported by The Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida).

#1536

CRISPR/Cas9 biased mutagenesis in the mouse genome: Is it useful or harmful for cancer therapeutics.

Ha Youn Shin. _NIH, Bethesda, MD_.

Advances in CRISPR/Cas9 genome editing have provided unprecedented opportunities to interrogate the functional significance of any given genomic site. While this technology is rapidly spreading and extended to with almost 4,000 PubMed entries as of today, there is a paucity of data on the extent of unexpected and undesired molecular consequences established upon targeting the mouse genome. We have introduced deletions at more than 20 genomic sites including CTCF and STAT5 transcription factor binding sites, screened over 500 founder mice and established and analyzed more than 50 lines. These data sets revealed hitherto unknown deletion patterns, unexpected large deletions using single sgRNAs and the consistent deletion of sequences bridging distant loci that were targeted simultaneously. Our findings provide guidelines for the application of CRISPR/Cas9 in the mouse germline and provide the insight into designing CRISPR-based cancer therapeutics.

#1536A

Progesterone receptor regulation of interferon signaling in breast cancer.

Katherine Walter, Merit Goodman, Gloria Trinca, Jade Hall, Christy Hagan. _University of Kansas Medical Center, Kansas City, KS_.

Breast cancer is an extremely heterogeneous disease that affects close to two million women across the globe each year. Of these breast tumors, approximately 70% express the progesterone receptor (PR), a nuclear receptor that is activated in response to its ligand progesterone. Compelling clinical trial data has suggested that progestins have a role in breast cancer development, independent of the widely studied estrogen receptor. The mechanism by which this occurs, however, is vastly understudied.

Traditionally, PR exerts its effects following ligand activation by translocating to the nucleus and binding to DNA where it affects the transcription of a variety of genes involved in growth, survival and proliferation. Our lab, using microarray data combined with Gene Set Enrichment Analysis, has identified a novel subset of genes that have altered expression following PR activation. These genes are primarily involved in interferon signaling—a pathway normally utilized in response to viral infection. Our data show that many genes that are normally activated in response to interferon signaling (interferon stimulated genes, ISGs) are repressed when PR is activated by its ligand. Interestingly, this repression occurs even in the presence of interferon alpha—the activating ligand of the interferon signaling cascade. Our lab has also shown that when PR expression is transiently knocked down, ISG transcriptional repression is lost, indicating that the repression of these genes is PR dependent.

In attempting to elucidate a mechanism by which PR exerts this effect, we have performed chromatin immunoprecipitation experiments following PR activation and observed PR recruitment to ISG enhancer regions. This suggests PR is potentially interfering with normal transcription of these genes. We hypothesize that PR is functioning by either blocking normal transcriptional machinery and co-activators or through the recruitment of transcriptional co-repressors to these regions. Experiments to address these models are currently underway.

Evasion of the immune system has recently been added to the list of Hallmarks of Cancer and our preliminary data suggest a potential mechanism by which breast cancer is able to accomplish this. Activation of type I interferon signaling is an early step in marking tumors for immune clearance. By repressing ISG protein expression, it is possible that these tumors are able to avoid detection by host immune cells leading to tumor establishment and subsequent progression. Our preliminary data suggest that PR may be capable of aiding early breast malignancies in evading immune recognition. This novel role of PR and progestins offers insight that could aid in improving upon established estrogen only-based therapies for prevention and treatment of hormone dependent breast cancers. 

### Tumor Suppressors 1: Modeling, Metabolism, and Epigenetics

#1537

**3p13-14** FOXP1-SHQ1 **deletion spanning multiple potential tumor suppressor genes cooperates with** PTEN **loss in cancer.**

Haley Hieronymus, Philip J. Iaquinta, John Wongvipat, Anuradha Gopalan, Rajmohan Murali, Ninghui Mao, Brett S. Carver, Charles L. Sawyers. _Memorial Sloan Kettering Cancer Center, New York, NY_.

The recurrent 3p13-14 deletion spanning from FOXP1 to SHQ1 occurs frequently in prostate cancer and is broadly lost in a range of cancer types, but this deletion has unknown tumor suppressive potential. FOXP1-SHQ1 deletion significantly co-occurs with PTEN loss in prostate cancer and other cancers. We find that FOXP1-SHQ1 deletion cooperates with PTEN loss to accelerate prostate cancer development in mice, resulting in tumors with increased proliferation and highly anaplastic dedifferentiation. FOXP1-SHQ1 deletion in these PTEN null tumors results in selective mTORC1 pathway hyperactivation beyond that mediated by PTEN loss alone. FOXP1-SHQ1 deletion also partially rescues AR target gene inhibition caused by PTEN loss, circumventing the repression of the androgen axis seen upon PI3K pathway activation. Clinically, combined FOXP1 and PTEN loss is associated with increased prostate cancer recurrence, and this finding extends to other cancer types, most notably breast cancer.

#1538

A mouse model that mimics human MEN1 tumor is effective in preclinical neuroendocrine tumor therapeutics.

Chung Wong,1 Richard Clausen,1 Laura Tang,2 Christian Davidson,3 Evan Vosburgh,1 Arnold Levine,4 Eugenia Xu1. 1 _Raymond and Beverly Sackler Foundation, New Brunswick, NJ;_ 2 _Memorial Sloan-Kettering Cancer Center, New York, NY;_ 3 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ;_ 4 _Institute for Advanced Study, Princeton, NJ_.

Conditional knockout of Men1 in β cells leads to development of pituitary adenomas and pancreatic neuroendocrine tumors (PanNETs) by the age of eight to twelve months. This suggests that other events, like loss of additional mutations, may be required for development of tumors in the absence of MEN1. Since it was reported that mutations in genes of PI3K/mTOR pathway are found in human neuroendocrine tumors, we hypothesized that double deletion of PTEN and MEN1 may accelerate neuroendocrine tumorigenesis. Mice with double deletion of PTEN and MEN1 in β cells were produced by crosses among mice with Cre gene under the control of Rat Insulin promoter (Rip-Cre), floxed allele Men1 (Men1fl/fl), and Pten (Ptenfl/fl). Then mice with the genotype of Men1fl/fl Ptenfl/fl Rip-Cre were monitored for development of tumors. Consistent with our hypothesis, mice with conditional deletion of PTEN and MEN1 leads to fully developed PanNETs and pituitary adenomas by three to four months old, much earlier than that with Men1 deletion alone. These tumors resemble human MEN1 tumors at a histological and molecular level. Activation of phospho-AKT was observed in these tumors and Rapamycin treatments lead to delayed PanNETs and pituitary adenomas for at least two months. This mouse model (Men1fl/fl Ptenfl/fl Rip-Cre) directly supports that PI3K/mTOR pathway plays a critical role in neuroendocrine tumorigenesis, and also provides a platform to study new therapeutic opportunities for PanNETs and neuroendocrine tumors of the pituitary through the targeting of PI3K/mTOR pathway and MEN1 pathway. This model represents an important tool for studying the biology and preclinical therapeutics of human neuroendocrine tumors.

#1539

Investigating the mechanisms of tumor suppression mediated by PTEN PDZ-binding domain in a murine breast cancer model.

Mingfei Yan,1 Alexander Many,2 Hong Guan,3 Penelope M. Or,1 Andrew M. Chan1. 1 _School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong;_ 2 _Department of Oncological Sciences, Mount Sinai School of Medicine, New York, NY;_ 3 _Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI_.

PTEN (Phosphatase and tensin homolog) functions as a key negative regulator of PI3K-Akt pathway through its lipid phosphatase activity. Mutations or loss of PTEN is frequently observed in brain, breast, prostate and endometrial cancer. PTEN possesses a PDZ-binding domain (PDZ-BD) at the end of its carboxyl terminus. Functionally, PDZ-BD mediates PTEN's interactions with other PDZ domain-containing proteins, including ZO-1, PSD-95, DLG, etc, which are involved in the formation of cell junctions, post-synaptic densities, and serves as scaffolding proteins for multiple signaling complexes. However, whether the PTEN PDZ-binding domain is required to suppress tumorigenesis still remains obscure.

To study the role of PTEN PDZ-BD in breast epithelial tumorigenesis, a PTEN-ΔPDZ mice lacking this domain was generated. PTEN expression level and activity of PI3K-Akt pathway are not changed in the mammary epithelial cells, indicating that PDZ-BD is dispensable for PTEN stability and negative regulation of the PI3K-Akt pathway. Further, we crossed the PTEN-ΔPDZ mice with the MMTV-PyMT breast cancer model and observed increased total tumor burden as well as higher tendency of lung metastasis in PTEN-ΔPDZ mice. This data indicates that PTEN PDZ-BD is indeed important for tumor suppression during breast cancer progression.

Previously, PTEN has been reported to govern the integrity of tight junctions and apical-basal polarity of epithelial cells, which requires PTEN's interactions with other proteins through the PDZ-BD. However, through in vitro three-dimensional culture of primary mouse mammary epithelial cells, we failed to observe any disruption of either apical-basal polarity or tight junction. Meanwhile, we investigated the alteration of the transcriptome of breast cancer tissue in PTEN-ΔPDZ mice by performing RNA-sequencing. Several enriched signaling pathways were identified by Ingenuity Pathway Analysis (IPA), which seems to be mainly associated with cell skeleton signaling as well as signaling interaction between extracellular matrix and the cancer cell. A list of genes were validated by RT-PCR, and functional validation of these genes during the tumorigenesis in our animal model is to be carried out in the near future.

This work was supported by a General Research Fund grant (#460713) from the Hong Kong University Grants Committee to AC. YM was supported by a Hong Kong PhD Fellowship (PF12-13584), and a Lo Kwee-Seong Biomedical Research Seed Fund to AC.

#1540

Creating faithful genetic zebrafish models of pediatric high grade gliomas and MPNSTs.

Felix Oppel, Ting Tao, Shuning He, Mark W. Zimmerman, Dong H. Ki, Nina Weichert, A Thomas Look. _Dana-Farber Cancer Institute, Boston, MA_.

Pediatric high-grade gliomas (HGGs) are the leading cause of cancer-related death in children. Despite a slight improvement of patient prognosis over the past decades pediatric HGGs remain largely incurable. Thus, new experimental models are needed to understand the mechanisms of the disease and find more effective treatment options. We previously reported a model of HGGs and malignant peripheral nerve sheath tumors (MPNSTs) which is based on the combined deficiencies in the tumor suppressor genes tp53 and nf1. However, HGG penetrance is very low in this line and most fish develop MPNSTs starting at about 3 months of age. On top of the existing model we used CRISPR/Cas9 to incorporate knock-out mutations in the tumor suppressor genes atrx or suz12 which are described to be involved in pediatric HGG biology. Heterozygous atrx loss-of-function (lof) did not impact tumor onset or penetrance of neither HGGs nor MPNSTs. Since a total loss of atrx was lethal in development, we re-injected effective atrx targeting gRNAs and Cas9 mRNA into the atrx+/- line to create a mosaic atrx-/- genotype. Surprisingly, despite a high mutation efficiency of the remaining atrx allele the re-injection strategy still did not alter tumor onset and penetrance in that model. This suggests that loss of atrx is only effective in HGGs in cooperation with additional hits other than tp53 and nf1. In contrast, loss of suz12 cooperated well with the tp53/nf1-deficient background. As nf1, suz12 is duplicated in zebrafish (suz12a and suz12b) resulting in 4 alleles of each gene per cell. When at least 2 out of 4 alleles of either suz12a or suz12b were lost, MPNST onset was accelerated. This effect was much stronger in tp53-/-, nf1a+/-, nf1b-/- fish (5-7 weeks) compared to tp53-/-, nf1a+/+, nf1b-/- siblings (3-4 weeks). This indicates that the tumor supporting effect of suz12 lof increases the more nf1 levels decrease. However, HGG onset still remained unchanged. We hypothesize that efficient onset of HGGs in our model requires the presence of an activated oncogene. Specific missense mutations in H3F3A are reported to be implicated in HGG progression in children and young adults. Thus, we overexpressed zebrafish h3f3a-K27M or -G34R mutant sequences in the tp53/nf1/atrx/suz12-deficient line and are currently investigating possible changes in tumor biology. Our zebrafish models of pediatric HGGs and MPNSTs will be useful to dissect the mechanisms underlying the cooperation among driver mutations and for small molecule screens to identify specific inhibitors of cell growth and survival in these malignancies.

#1541

The tumor suppressor BAP1 promotes a developmental switch from pluripotency to differentiation.

Jeffim N. Kuznetsov, Tristan Aguero, Stefan Kurtenbach, Matthew G. Field, Mary Lou King, J William Harbour. _University of Miami Miller School of Medicine, Miami, FL_.

Introduction: Our lab discovered that mutations in the tumor suppressor BAP1 are strongly associated with metastasis and death in patients with uveal melanoma. Subsequently, other cancers have been found to harbor BAP1 mutations, including skin melanoma, kidney cancer, mesothelioma and others. Germline BAP1 mutations are responsible for a newly described genetic cancer syndrome. Therapeutic molecules that reverse the effects of BAP1 mutations could represent a potent new treatment strategy for BAP1-mutant cancers. Unfortunately, there are several obstacles to developing such therapies. First, BAP1 is a tumor suppressor that is inactivated by mutations, such that targeted therapy would need to be directed against downstream effectors that are deregulated by BAP1 loss. Second, the effectors of BAP1 that are relevant to cancer are not known. Interestingly, most known proteins that interact with Bap1 are developmental epigenetic regulators such as Asxl1/2, Cbx1/3 and Kdm1b. Third, BAP1 is difficult to study in cultured cells because BAP1 loss results in cell cycle exit and stem cell-like behavior. These obstacles led us to shift to Xenopus laevis as an in vivo developmental model to study the functions of BAP1. Results and conclusions: Loss of BAP1 during embryo development results in a failure to turn off pluripotency genes such as ventx 1, ventx2 (Xenopus orthologues of the mammal gene nanog), oct 25, oct 91 (Xenopus orthologues of the mammal gene oct3/4) and pax3, and a failure to induce lineage specification genes such as the prospective epidermis marker keratin1 and the melanocyte precursor marker sox10. This block in the shift from pluripotency to differentiation programs results in a delay in gastrulation, neural crest specification and migration, mesodermal differentiation and other phenotypes. The BAP1-deficient phenotype can be rescued by Xenopus or human wildtype BAP1 or by the histone deacetylase inhibitor SAHA (vorinostat). We conclude that BAP1 is a fundamental regulator of multiple developmental lineages, including the neural crest from which melanomas arises, and that this in vivo model can be used to screen for novel therapeutic compounds that reverse the phenotypic effects of BAP1 loss.

#1542

**Identifying genetic modifiers of** PTEN **using the Collaborative Cross mouse panel.**

Amanda Lanier, William Barrington, David Threadgill. _Texas A &M University, College Station, TX_.

Tumor suppressor genes code for proteins that limit cellular proliferation and cancer progression, and are often exploited in cancer therapies. One such tumor suppressor gene, Phosphatase and Tensin homolog deleted from Chromosome Ten (PTEN), mediates cell growth through negative regulation of phosphatidylinositol 3-kinase (PI3K) dependent pathways. PTEN is frequently deleted or mutated in a variety of human cancers. Mice carrying a PTEN transgene (Super-PTEN mice) were found to be resistant to tumor formation, and even small changes in PTEN expression and activity have been shown to influence cancer susceptibility and progression. We hypothesized that PTEN activity can be altered by modifier genes. To determine whether PTEN modifiers are influencing its activity, we are using the Collaborative Cross (CC), a panel of inbred mouse strains that has a similar amount of genetic variation as human populations. In CC-Super-PTEN crosses, we are utilizing an easily measured phenotype of Super-PTEN mice, reduced body weight, to identify CC strains with altered PTEN function. On average, weanlings carrying the PTEN transgene weigh 16% less than their wild type littermates, but this weight difference varies significantly across strains, from no weight reduction to as much as a 29% difference. This distribution of phenotypes has allowed us to use quantitative trait locus (QTL) mapping to identify potential modifiers of PTEN. Through association mapping, we have determined that the loci with the highest LOD scores reside on chromosomes 2, 12, 16, and X, and we are now reviewing genes within those loci to identify candidate modifiers of PTEN. Additionally, we are using several cancer models to determine the effects of said candidate genes and confirm that an altered PTEN weight phenotype correlates with changes in cancer susceptibility and progression. Ultimately, this research will demonstrate the use of the CC mouse panel to identify novel genetic modifiers and improve our understanding of PTEN regulation.

#1543

Caspase-10 suppresses tumorigenesis by targeting ATP-citrate lyase.

Rajni Kumari, Sanjeev Das. _National Institute of Immunology, New Delhi, India_.

p53 fosters metabolic reprogramming, which restricts metabolic adaptation of tumor cells under energy stress conditions. However, modulation and directionality of underlying cascades to induce or restrict metabolism are determined by the downstream target of p53. To explore the role of p53 in tumor metabolism upon metabolic stress, we performed a microarray screen in the presence or absence of p53 under glucose starvation. We observed caspase-10 to be upregulated by p53 upon metabolic stress. Caspase-10 belongs to the class of initiator caspases and is poorly understood in terms of substrate specificities. Emerging evidences also suggest intrinsic activation of caspase-10, distinct from its homologue caspase-8. Moreover, inactivating mutations as well as downregulation of caspase-10 in several carcinomas are suggestive of its role in tumorigenesis. Thus, we investigated the function of caspase-10 as a tumor suppressor. Our results indicated that caspase-10, upregulated in a p53-dependent manner in response to metabolic stress, was activated by homodimerization-induced intrinsic activation mechanism involving Adenylate Kinase 2 and FADD. We further performed a proteomic screen to identify novel caspase-10 substrates. We observed that several anabolic enzymes are caspase-10 interacting partners. However, in vitro and in vivo assays confirmed only ATP-Citrate Lyase (ACLY) as a novel substrate of caspase-10. Cleavage of ACLY by caspase-10 abrogates enzymatic conversion of cytosolic citrate to acetyl-CoA, leading to overall decrease in nucleocytosolic acetyl-CoA pool. Decrease in cytosolic acetyl-CoA levels suppress lipogenesis upon metabolic stress. Moreover, decreased levels of nuclear acetyl-CoA alter global histone acetylation. Further studies using RNAi established that alteration in histone acetylation is GCN5-dependent. Caspase-10-dependent abrogation of histone acetylation reprograms the epigenetic profile of metastatic and proliferative genes, leading to their diminished expression under metabolic stress. Concomitant suppression of both cytosolic lipogenesis and nuclear histone acetylation by caspase-10-mediated ACLY cleavage decreases proliferation capacity, migration potential and invasiveness of tumor cells. Further, studies in mouse tumor models confirmed that caspase-10 restricts tumorigenesis and metastases under metabolic stress via ACLY and GCN5. Taken together, our findings establish caspase-10 as a novel integrator of metabolism and epigenetics, by regulating acetyl-CoA levels. Hence, it functions as a tumor suppressor by disrupting lipogenesis-mediated metabolic reprogramming and GCN5-mediated epigenetic reprogramming.

#1544

MTAP insufficiency promotes metastasis in lung cancer via diminishing protein dimethylation.

Wen-Hsin Chang,1 Bing-Ching Ho,1 Kang-Yi Su,1 Jian-Wei Chen,2 Reen Wu,3 Pan-Chyr Yang,1 Sung-Liang Yu1. 1 _National Taiwan University, College of Medicine, Taipei, Taiwan;_ 2 _National Chung Hsing University, Taichung, Taiwan;_ 3 _University of California Davis, Davis, CA_.

Genome instability, metabolic switch, and activating metastasis are three of the cancer hallmarks, which always collectively contribute to cancer malignancy. We identified methylthioadenosine phosphorylase (MTAP) as a metastasis suppressor in a series isogenic lung cell lines with different invasion ability by array CGH. Using siRNA silencing and ectopic expression of MTAP, we found that MTAP not only inhibited invasion and colony forming in vitro, but also reduced metastasis and tumorigenesis in vivo. Clinically, patients with low MTAP expression associated with poor overall survival and progression-free survival. Furthermore, the metabolite analysis of clinical specimens showed that low MTAP expression leads to MTA substrate accumulation and product decrease. MTA accumulation in MTAP knockout cells reduced the level of symmetric arginine dimethylation of proteins. By using LC/MS, we identified a novel methyl-protein, whose level is elevated in MTAP knockout cells with the depletion of symmetric arginine dimethylation. Functionally, the unmethylated protein would be protected from proteolysis via caspase 3 cleavage. Moreover, the unmethylated mutant protein promoted migration and invasion in vitro strongly than the wild-type one did. In conclusion, we identify a novel marker in low MTAP expression or MTAP knockout cells, providing a potential therapeutic candidate in lung adenocarcinoma.

#1545

Loss of glycogen debranching enzyme (AGL) promotes rapid growth of non-small cell lung cancer cells.

David R. Meier, Benjamin Weinhaus, Darby Oldenburg, Sunny Guin. _Gundersen Medical Foundation, La Crosse, WI_.

Glycogen Debranching Enzyme (AGL) is involved in glycogen breakdown. Compromise in glycogen breakdown due to loss of function mutations in the AGL gene lead to metabolic disease - Glycogen Storage Disease Type III. We have previously shown that loss of AGL results in rapid growth of bladder cancer cells using extensive in vitro and in vivo experiments. Here we test whether AGL regulate growth of non-small cell lung cancer (NSCLC) cells. We show that loss of AGL promotes rapid growth of NSCLC cells using anchorage independent growth assay. Loss of AGL also resulted in rapid xenograft growth of NSCLC cells when injected subcutaneously in immunocompromised mice. Whereas overexpression of AGL reduce the growth of these NSCLC cells. Further loss of glycogen phosphorylase the other enzyme involved in glycogen breakdown does not promote aggressive growth of these cancer cells. This observation is similar to our findings in bladder cancer which confirms that AGL regulates tumor growth independent of its role in glycogen metabolism. Thus using in vitro and in vivo experiments we show that AGL can regulate the growth of multiple tumor types such as bladder cancer and NSCLC. The exact mechanism on how AGL regulates NSCLC growth needs further investigation.

#1546

Contribution of heterozygous loss of ribosomal protein L5 as general tumor suppressor in cancer.

Kim R. Kampen, Laura Fancello, Jelle Verbeeck, Kim De Keersmaecker. _KU Leuven, Leuven, Belgium_.

The association between ribosome defects and cancer became clear with the recent discovery of somatic mutations in ribosomal protein genes in several cancers, such as lymphoid leukemia's and glioblastoma (Nat Genet. 2013 Feb;45(2):186-90 & Blood. 2016 Feb 25;127(8):1007-16 & Nature. 2014 Jan 23;505(7484):495-501). To further delineate the role of ribosomal proteins in human cancer, we systematically screened the TCGA database for genetic lesions in ribosomal protein genes, confirmed the contributing capacity of the most common somatic ribosomal protein defect in cancer pathogenesis by molecular interference, and explored the mode of action of this ribosomal protein defect. Systematic analysis of TCGA mutation and copy number data of respectively 4926 and 7322 patients representing 16 cancer types for defects in all 81 ribosomal protein genes. After stringent filtering, six ribosomal protein encoding genes (RPL5, RPL11, RPL23A, RPS5, RPS20 and RPSA) were significantly altered and identified as candidate cancer driver genes. RPL5 was located at a significant peak of heterozygous deletion on chromosome 1p22 and showed significant mutations and deletions in 11% of glioblastoma (GBM), 28% of melanoma, 34% of breast cancer (BRCA), and in 20% of multiple myeloma cases, thereby supporting heterozygous RPL5 inactivation to be the most common somatic ribosomal protein defect in human cancer. Human specific RPL5 doxycycline inducible knockdown (sh-hRPL5) in TP53 WT and TP53 mutant human breast cancer cell lines (MCF7 and MDA-MB-231) and in a TP53 mutant human glioblastoma cell line (U-118 MG) proved that RPL5 knockdown accelerated in vivo tumor progression in NSG mice. This acceleration was associated with reduced phosphorylation of CDK1tyr15, which is required for cell cycle progression from G2 to mitosis. Whereas RPL5 has been implicated in TP53 and MYC regulation, no consistent effects of RPL5 downregulation on these proteins could be detected in TP53 WT and TP53 homozygous R280K mutant breast cancer models (sh-hRPL5). Interestingly, partial RPL5 inactivation was found to be associated with PTEN protein suppression in these tumors. This observation was confirmed in various cell models; in normal mouse neural stem/progenitor cells and mouse bone marrow cells (RPL5+/lox by INFβ and poly I:C), and also in GBM cancer pre-disposition mouse neural stem/progenitor cell model (sh-mRPL5 TP53 -/-). RPL5 downregulation also accelerated tumor formation in the PTEN deficient GBM cell line U-118 MG, underscoring undefined PTEN-independent mechanisms of RPL5. Overall, we identified RPL5 as a new tumor suppressor that shows heterozygous inactivation in 11-34% of multiple human cancer types. Partial molecular inactivation of RPL5 supported a tumor suppressor function for RPL5 in accelerating breast cancer and glioblastoma progression in vivo, which may be, in part, due to a new function of RPL5 in regulating PTEN.

#1547

BCCIPβ modulates the ribosomal and extraribosomal function of S7 through a direct interaction.

Qian Ba, Hui Wang. _Institute for Nutritional Sciences ,SIBS,CAS, Shanghai, China_.

Extraribosomal functions of ribosomal proteins (RPs) have gained much attention for their implications in tumorigenesis and progression. However, the regulations of transition between the ribosomal and extraribosomal functions of RPs are rarely reported. Herein, we identified a novel ribosomal protein S7-interacting partner, BCCIPβ, which could modulate the functional conversion of S7. Through the N-terminal acidic domain, BCCIPβ interacted with the central basic region in S7 and regulated the extraribosomal distribution of S7. BCCIPβ deficiency abrogated the ribosomal accumulation but enhanced the ribosomal-free location of S7. This translocation further impaired protein synthesis and triggered ribosomal stress. Consequently, BCCIPβ deficiency suppressed the ribosomal function and launched the extraribosomal function of S7, and eventually resulted in cell proliferation restriction. In conclusion, BCCIPβ, as a novel S7 modulator, contributes to the delicate regulation of ribosomal and extraribosomal functions of S7 and has implications in cell growth and tumor development.

#1548

Down-regulation of 4-hydroxyphenylpyruvate dioxygenate (HPD) contributes to the pathogenesis of hepatocellular carcinoma (HCC) through ERK / BCL-2 signalling activation.

Man Tong,1 Tin Lok Wong,1 Steve Tin-Chi Luk,1 Noélia Che,1 Kai Yau Wong,1 Tsun Ming Fung,1 Xin-Yuan Guan,1 Nikki P Lee,1 Yun-Fei Yuan,2 Terence K Lee,3 Stephanie Ma1. 1 _The University of Hong Kong, Hong Kong;_ 2 _State Key Laboratory of Oncology in Southern China, Sun Yat-Sen University Cancer Center, Guangzhou, China;_ 3 _The Hong Kong Polytechnic University, Hong Kong_.

Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies in the world. Metastasis, recurrence and therapy resistance remain major obstacles to the improvement of long-term survival and represent major causative factors contributing to the rising mortality rates and poor prognosis of HCC. Identification of key drivers important for clinical prognostic utility and elucidation of mechanisms involved in hepatocarcinogenesis is urgently needed to aid in the development of novel treatment modalities. To this end, we began our study with an analysis of a publicly gene expression dataset (GSE14520) comprising of transcriptomic profiles from a large cohort of human non-tumor liver and HCC clinical samples in hope to establish a prognostic gene signature associated with metastatic risk / recurrence status so to identify novel driver genes responsible for HCC development and progression. By this method, 4-hydroxyphenylpyruvate dioxygenase (HPD) was identified as a candidate tumor suppressor gene in HCC. HPD was found to be frequently down-regulated in primary HCC tumors as compared with peri-tumor liver tissues. Its expression negatively correlated with aggressive HCC pathological features, including tumor stage, metastasis, recurrence and survival. Notably, down-regulated HPD expression in HCC is in part a result of hypermethylation at the HPD promoter. The functional effect of HPD was then examined in HCC cells with or without HPD stably repressed or ectopically overexpressed. Upon knockdown of HPD, HCC cells displayed significantly enhanced abilities to form tumors, metastasize and confer sorafenib resistance in vitro and in vivo. Conversely, overexpression of HPD in HCC cells led to an opposing effect. Consistently, sorafenib-resistant HCC patient derived xenografts also displayed attenuated levels of HPD as compared with parental sorafenib-sensitive counterparts. Mechanistically, HPD down-regulation mediates aggressive cancer features in HCC through activation of an ERK and BCL-2 pro-survival signaling pathway, as evidenced by rescue experiments involving the ERK inhibitor U0126. Collectively, our findings suggest HPD down-regulation in HCC tumors to promote tumorigenicity, metastasis and sorafenib resistance through ERK / BCL-2 signaling activation and that HPD may represent a novel prognostic biomarker for HCC.

#1549

Prognostic significance and tumor suppressive functions of SDCT2 in renal cell carcinoma.

Andre R. Jordan,1 Martin Hennig,2 Daley S. Morera,3 Soum D. Lokeshwar,1 Asif Talukder,3 Lokeshwar Vinata3. 1 _University of Miami, Miami, FL;_ 2 _University of Lübeck, Lübeck, Germany;_ 3 _Augusta University, Augusta, GA_.

Introduction: Five-year survival of metastatic renal cell carcinoma (mRCC) patients is < 10% and African American (AA) males have the highest incidence. Identification of the molecular determinants of mRCC and racial disparity in RCC is critical for biomarker development and targeted therapy. SDCT2 is expressed in kidney epithelial cells and is a succinate and citrate transporter, but its role has not been examined in any benign diseases or cancer. We examined SDCT2 expression in normal and RCC tissues and correlated it with clinical outcome and racial disparity. We also evaluated the biological functions and molecular signaling regulated by SDCT2 in RCC cells.

Methods: Differential gene expression in the matched normal and RCC tissues (n=6/category) was evaluated by microarray analysis; results were validated by quantitative-PCR and immunoblotting in normal and RCC tissues from 53 patients (White=21; Hispanic=19; AA=13). VHL+ and VHL- RCC cells were stably transfected with a Flag-tagged SDCT2 construct. Transfectants were characterized for cell proliferation, cell cycle, motility, succinate/citrate transport and reactive oxygen species (ROS) measurement assays under normoxia and hypoxia (1% O2); cell death and senescence pathway markers were also evaluated. SDCT2 induction was evaluated following 5-azacytidine plus Trichostatin A treatment

Results: SDCT2 was 63- and 100-fold downregulated in low- and high-stage RCC tissues, respectively. Q-PCR validation showed that SDCT2 levels were 40-fold downregulated in tumor tissues when compared to normal kidney (P<0.0001 Mann-Whitney test). Downregulation was 40-fold in White and Hispanic patients, but 198-fold in AA patients (P=0.0049) and also correlated with tumor stage and metastasis (P=0.009). Under hypoxia, SDCT2 expression caused over 3-fold inhibition of proliferation, cell-cycle (G1-S block), and motility in both VHL+ and VHL- cells (P<0.01), only VHL+ cells were inhibited under normoxia. SDCT2 expression induced ROS levels and succinate transport by 3-fold in RCC cells (P<0.01). SDCT2 expression induced the p16INK4a-RB pathway and apoptosis (caspase-3 and PARP activation). 5-AZA+TSA treatment caused a 50-fold induction (P<0.0001) of SDCT2 expression.

Conclusion: This is the first study on a functional biomarker in RCC, SDCT2, that is a possible novel tumor suppressor gene. SDCT2 loss promotes RCC growth, survival and inhibits cellular senescence and its downregulation correlates with metastasis and racial disparity. Support: Grant NCI/NIH 5R01CA72821; 5R01CA176691

#1550

Epigenetic downregulation and growth inhibition of IGFBP7 in gastric cancer.

Jin Kim,1 Min A Kim,2 Sun-Ju Byeon,3 Woo Ho Kim4. 1 _Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea;_ 2 _Seoul National University Hospital, Seoul, Republic of Korea;_ 3 _Asan Medical Center, Seoul, Republic of Korea;_ 4 _Seoul National University College of Medicine, Seoul, Republic of Korea_.

Insulin-like growth factor-binding protein 7 (IGFBP7) has been found to be a tumor suppressor in several human cancers, but the role of IGFBP7 in gastric cancer has not yet been fully investigated. In this study, we examined the epigenetic downregulation of IGFBP7 expression in gastric cancer. Analysis by qRT-PCR, western blot, and methylation specific-PCR revealed that DNA methylation was inversely correlated with IGFBP7 expression, and the expression of IGFBP7 was restored after treatment with 5-aza-2'-deoxycytidine. Immunohistochemistry showed that IGFBP7 expression was downregulated in 47.5% of advanced gastric cancer patients. Univariate and multivariate analysis showed that IGFBP7 expression and tumor stage were independent prognostic factors. In cell culture, IGFBP7 knockdown increased gastric cancer cell growth, invasion, and migration, whereas IGFBP7 overexpression in gastric cancer cells showed cell growth inhibition and apoptosis. Our data suggest that IGFBP7 functions as a tumor suppressor in gastric cancer via an epigenetic pathway.

#1551

GULP1 **is an epigenetically altered and functional tumor suppressor in urothelial carcinoma through regulation of Nrf2-Keap1 signaling axis.**

Masamichi Hayashi, Elisa Guida, Rachel Goldberg, Yoshikuni Inokawa, Leonardo Reis, Akira Oki, Evgeny Izumchenko, Leonel Maldonado, Luigi Marchionni, Mariana Brait, Trinity Bivalacqua, Alexander Baras, George J. Netto, Wayne Koch, David Sidransky, Mohammad O. Hoque. _The Johns Hopkins University School of Medicine, Baltimore, MD_.

We identified GULP1 as a novel tumor suppressor gene (TSG) selectively silenced during urothelial cancer (UC) progression through promoter hypermethylation (PH) analyzed by quantitative methylation specific PCR (QMSP) and novel methylation specific digital droplet PCR (ddPCR) assay. Numerous cell based assays revealed that GULP1 silencing confers growth advantage to tumor cells. Correspondently, in vivo tumorigenicity after xenotransplantation of GULP1 knockdown T24 cells was significantly higher than control cells. Further mechanistic analysis revealed that GULP1 has a crucial role in the regulation of Nrf2-Keap1 axis, maintaining actin cytoskeleton architecture and helping Keap1 to scaffold Nrf2 in the cytoplasm. Moreover, GULP1 silencing induces constitutive activation of Nrf2 target signature, responsible for chemoresistance of UCs. Additionally, we analyzed GULP1 PH and expression in cisplatin-based therapy responsive and resistant primary UC samples, and in isogenic cisplatin sensitive and resistant T24 cell lines. Interestingly, GULP1 expression at transcription level was lower in both resistant primary UC samples and resistant T24 cell line. Cell lines with lower expressions of GULP1 (SW780 and UM-UC-3) also showed higher resistance to cisplatin than those with higher expression (T24 and BFTC905). Altogether, our findings determined that GULP1 is an epigenetically silenced potential TSG in UC and GULP1 expression and/or PH may guide in selecting candidate patients for cisplatin based neo-adjuvant therapy.

## BIOINFORMATICS AND SYSTEMS BIOLOGY:

### Functional and Genomic Analysis of Cancer

#1552

Integrative bioinformatics analyses of ChIP seq and RNA seq data reveals a complex regulatory landscape of NRF1 network involved in the pathogenesis of breast cancer.

Jairo Ramos, Deodutta Roy. _Florida International University, Miami, FL_.

The expression of redox sensitive transcription factor -nuclear respiratory factor 1 (NRF1) expression significantly correlates with histological grades and prognosis of breast cancer. However, the molecular mechanism by which NRF1 may contribute in the development of breast cancer is not clear. In this study we examined whether NRF1 is a molecular risk factor of breast cancer. We also identified regulatory landscape of NRF1 network involved in the pathogenesis of breast cancer. Steps followed for the investigation included the selection of mRNA microarray dataset from TCGA and Metabric, selection and analysis of ChIP-Seq datasets from different breast cancer cell lines to identify NRF1 target genes possibly involved in breast cancer through Molecular Pathway, Gene Ontology and statistical analysis. Analysis of mRNA expression in breast cancer patients showed that NRF1 was significantly overexpressed in breast cancer tissues compared to normal tissues. ChIP-Seq analysis of two different breast cancer cell line - MCF7 and T7D showed that there are more than 8,000 genes, which contain NRF1 binding DNA motif(s). The NRF1 binding to ChIP DNA sequences of target gene is cell context dependent. High percentage of known breast cancer (~50%) susceptibility genes are transcriptional targets of NRF1. Network analysis revealed that NRF1 regulates genes, which are key regulators of epithelial-mesenchymal transition (EMT), stemness, cell apoptosis, cell cycle regulation, chromosomal integrity, and DNA damage and repair. Furthermore, we observed that NRF1 regulates target genes of both breast cancer and cancer KEGG pathways. Many of the MicroRNAs involved in cancer and genes of signaling pathways involved in cancer initiation and progression such as MAPK, PI3K/AKT and Notch signaling are also transcriptional targets of NRF1. In summary, this study reveals a complex regulatory landscape of NRF1 network involved in the pathogenesis of breast cancer. Dysregulation of NRF1 signaling pathways may contribute in the development of breast cancer. Clinical confirmation of our study will have significant impact on our understanding of the role of NRF1 as a valuable biomarker for breast cancer diagnosis and prognosis and will provide strong rationale for the future studies to further develop NRF1 for breast cancer therapeutic target. This work was in part supported by a VA MERIT Review (VA BX001463) grant to DR.

#1553

Delineating the role of multiple copies of RNA binding domains in human nucleolin and its homologs using a computational approach.

Kamrun Begum, Ruchama Chaya Steinberg, Anjana Saxena, Shaneen Singh. _CUNY Brooklyn College, Brooklyn, NY_.

All eukaryotes have nucleoli, the sub-nuclear compartments where ribosomes synthesis occur. Nucleolin (NCL), an abundant RNA binding phosphoprotein,constitutes 5-10% of total nucleolar proteins. This multifunctional protein plays defined role/s in many critical cellular processes e.g. chromatin remodeling, ribosome biogenesis, transcriptional and translational regulation of various non-coding as well as coding RNAs. NCL binds to its target RNAs via two or multiple RNA binding domains (RBDs) to control gene expression during normal cell cycle as well as during cellular response to stress. A variety of tumors express elevated levels of NCL where NCL plays a direct role/s in increasing expressions of genes involved in cell survival, angiogenesis and metastasis. A comparison of NCL in various organisms shows that it is highly conserved, especially in its RNA binding domains that can vary in number (2 to 4 consecutive domains). Interestingly, the individual NCL-RBDs are more conserved across different species rather than within the same protein. RBDs are known to interact in pairs with RNA via RNA recognition motifs called RNP motifs, and complementary nucleic acid motifs or elements in RNA allow interaction with NCL. Vertebrate NCL proteins contains four RBDs, however most studies have focused only on the role/s of RDB 1,2; none probing the possible functional redundancy of the additional RBDs nor identifying any target-specificity for the RBD 3,4 in human NCL and other homologs. Earlier data from our lab suggested RBD 3,4 can bind RNA in a structurally analogous manner to RBD1,2 albeit with altered affinity. Here, we continue this research by investigating various homologs of human NCL to better understand the conserved mechanism of its interaction with RNA and whether the additional RBDs, are in fact, redundant in function. To test this hypothesis, RBDs from various homologs were modeled using template based methods and analyzed by a combination of sequence and structure analysis tools. We show that there are key residue differences in individual RBDs from various homologs that potentially manifest as differences in binding affinities. This study reveals new insights into NCL RBD function and describes the evolution of RBD function with respect to nucleic acid binding.

#1554

Development of a deconvolution algorithm for tissue-based gene expression data.

Sha Cao,1 Chi Zhang,2 Ying Xu1. 1 _University of Georgia, Athens, GA;_ 2 _Indiana University, Indianapolis, IN_.

Tissue data provide substantially more information than cell-line data, and offer new opportunities to study cancer biology and evolution in its actual microenvironment, when multiple tissue samples of the same cancer type are analyzed together. However, it is very challenging to do information discovery from tissue data because of their compositional complexity - each dataset represents a mixture of gene-expression data from multiple cell types. Hence, meaningful tissue-data analyses require to first sort out the detailed contributions to the observed tissue-level data by different cell types. However, the computational challenge in solving the tissue data deconvolution problem stems from the reality: each cell type has a very large number of complex relations among its expressed genes and pathways, which are preserved under different conditions. To make deconvolution results meaningful, the co-expressions among functionally closely related genes, must be captured and enforced in a deconvolution problem formulation.

We have a fundamentally novel formulation of the tissue-data deconvolution problem, which is pathway instead of gene based. It preserves co-expressions of genes of the same pathways through capturing and using an expression signature among such genes, and allows differential expressions of pathways by giving them varying weights in different tissues. In addition, this pathway-based model substantially reduces the number of free variables compared to gene-based models, making our problem formulation efficiently solvable. The unique ideas of our approach are: (i) estimate the proportion of each cell type within a mixture based on expression patterns of cell-type specific genes; (ii) identify co-expressed gene clusters among genes encoding each pathway (as defined by REACTOME) in each cell type based on cell line data; (iii) derive a condition-invariant expression signature for each of the ~2,000 REACTOME pathways in each cell type; (iv) demonstrate that each set of cell line gene-expression data can be uniquely represented as a weighted sum of such signatures; and (v) formulate the deconvolution problem as to estimate the weight of each pathway in each cell type that minimizes the Frobenius norm of deviance matrix of estimated and observed gene expression matrices. We demonstrated its effectiveness on simulated data, i.e., in silico mixtures of gene-expression data from different cell types with varying proportions of each cell type.

We anticipate that the successful development and deployment of the planned deconvolution method will enable and inspire a wide range of new ways to study tissue-based

expression data and uncover the very rich information hidden in cancer tissues about the fundamental biology of cancer.

#1555

Pan-cancer patterns of synthetic lethality: statistical modeling of gene dependency profiles.

Huwate Yeerna,1 Ramya Rangan,2 Andrew Aguirre,3 William Kim,3 Francisca Vazquez,3 Barbara Weir,3 Mahmoud Ghandi,3 Aviad Tsherniak,3 Jesse Boehm,3 William Hahn,3 Jill Mesirov,1 Pablo Tamayo1. 1 _University of California, San Diego, La Jolla, CA;_ 2 _Harvard University, Cambridge, MA;_ 3 _Broad Institute, Cambridge, MA_.

We present a methodology to fit statistical models of cell-viability profiles from RNAi-gene knockdowns. This method allows us to classify genes according to the degree of skewness in their viability distributions. The set of genes with the highest degree of skewness is highly enriched with many known oncogenes and tumor suppressors. We characterize many of these genes, compare them against the results of large sequencing efforts, and use them as inputs to a matrix-decomposition procedure that identifies the most salient cell viabilities shared by different cancer types. We catalog these pan-cancer patterns of synthetic lethality and characterize them by the genomic, transcriptional, and phenotypic features. This analysis provides a rich catalog of the most salient Achilles' Heels of Pan-Cancer that can be helpful to identify new therapeutic strategies across cancers.

#1556

Algorithms for discovery of somatic single nucleotide mutation display specific artifacts and different detection capabilities under the effect of read coverage and sample heterogeneity.

Wenming Xiao,1 Leihong Wu,1 Gokhan Yavas,1 Huixiao Hong,1 Baitang Ning,1 Weida Tong,1 Eric F. Donaldson,2 Zivana Tezak,3 Reena Philip,3 Howard Jacob,4 Louis M. Staudt5. 1 _FDA/NCTR, Jefferson, AR;_ 2 _FDA/CDER, Silver Spring, MD;_ 3 _FDA/CDRH, Silver Spring, MD;_ 4 _HudsonAlpha Institute, Huntsville, AL;_ 5 _NCI, Bethesda, MD_.

Understanding the performance and capability of different bioinformatics algorithms used for the discovery of somatic variants is very important for helping scientists choose an appropriate tool for cancer research. In this study, we developed a comparison approach and created a series of data sets that could be used to provide such guidance. We mixed reads from two well characterized individuals, NA12878 and NA24385, and generated a series of data sets with different coverages and sample heterogeneity. We then used these data sets to evaluate five commonly utilized somatic mutation detection tools. Our results indicate that read coverage has a significant impact on the accuracy and capability of mutation calling by individual bioinformatics algorithms. The mutation caller that performs well with high read coverage may perform poorly with low read coverage. On the other hand, the tool that performed well in calling variants in a relatively higher homogeneity sample may not have the same power to detect rare variants with low mutation allele frequency. In addition, we demonstrated that different mutation calling algorithms are associated with specific artifacts that were sensitive to read coverage. Furthermore, there were large numbers of false positives and false negatives shared by five callers, indicating that other factors, such as read alignment, library preparation, and even the properties of the sequencing platform could be the sources of false discovery for somatic variants. We observed similar behavior of the five variant calling algorithms using the sequencing data of a pair of matched tumor/normal cell lines, confirming the findings from the comparative analyses on the mixture of reads from the two normal individuals. Our findings are expected to facilitate selection of bioinformatics pipelines that fit for specific purposes in cancer research based on sequencing data.

#1557

**Longitudinal expression response of glycosylation related genes, regulators, and targets in cancer cell lines treated with** **eleven anti-tumor agents.**

Julia Krushkal,1 Yingdong Zhao,1 Curtis Hose,2 Anne Monks,2 James H. Doroshow,1 Richard Simon1. 1 _National Cancer Institute, Rockville, MD;_ 2 _Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD_.

Cancer cells have profound alterations in glycosylation processes, which affects the functioning of malignant cells and their interaction with surrounding environment. We examined the effects of treatment of cancer cell lines with eleven antitumor agents (bortezomib, cisplatin, dasatinib, doxorubicin, erlotinib, gemcitabine, paclitaxel, sirolimus, sorafenib, sunitinib, and topotecan) on transcriptional changes in 179 candidate genes involved in biochemical glycosylation pathways or encoding glycosylation targets, regulators of glycosylation processes, and components of cancer pathways affected by glycosylation. In order to achieve this goal, we analyzed time course gene expression information for the NCI-60 cancer cell line panel using the data from the NCI Transcriptional Pharmacodynamic Workbench (NCI TPW), a resource developed at the National Cancer Institute. Our analysis identified 145 glycosylation related genes that responded to drug treatment in a concerted manner. Many of these genes had a concerted response to multiple agents; however, the direction and the magnitude of concerted response were specific to individual drugs. Expression of multiple genes prior to treatment and/or transcriptional changes in response to treatment were significantly correlated with drug chemosensitivity, measured as log(GI50). Treatment with dasatinib resulted in the largest number of significant correlations of chemosensitivity with gene expression prior to treatment and with gene expression changes in response to treatment. Significant correlations of transcriptional changes after treatment and/or of pre-treatment gene expression levels of glycosylation related genes and glycosylation targets were also observed for other antitumor agents. Chemosensitivity to kinase inhibitors and DNA damaging agents was significantly correlated with expression of genes involved in N- and O-glycosylation, fucosylation, biosynthesis of poly-N-acetyllactosamine, removal of misfolded proteins, binding to hyaluronic acid and other glycans, and cell adhesion. In particular, tumor cell sensitivity or resistance to multiple agents were significantly correlated with changes in expression of C1GALT1C1 (COSMC), FUCA1, SDC1, MUC1, members of the MGAT, GALNT, B4GALT, B3GNT, MAN, and EDEM gene families, and other genes involved in glycosylation processes or encoding glycosylation targets and signaling regulators. We present a catalog of dynamic concerted changes in expression of glycosylation related genes, their ligands, regulators, and targets in response to chemotherapy, and provide a list of genes that influence the strength of response of cancer cells to treatment. These glycosylation related genes may be considered as potential candidates for drug targeting in combination therapy to enhance the effects of treatment. Funded by NCI Contract No. HHSN261200800001E.

#1558

Data-driven approach to detecting novel gene interactions in cancer with applications to drug response prediction and cancer stratification.

Assaf Magen, Avinash Das, Joo Lee, Sridhar Hannenhalli, Eytan Ruppin. _UMD, College Park, MD_.

The effect of a gene's activity on a phenotype depends on the context of a complex network of functionally interacting genes. Several genetic interactions (GIs), such as Synthetic lethality and Synthetic Rescues, have been reported to have a significant functional role in cancer progression and provide potential candidates for selective cancer treatments. However, numerous other types of GIs with potential clinical significance are yet to be explored. In this work we generalize the concept of GI and detect ~70,000 GIs of different types with both molecular and clinical signature. We demonstrate their clinical predictive value as well as their ability to stratify breast cancer patients into refined clinical subtypes that might allow for better diagnosis and modified course of treatment. These results compare favorably with previous sequence based approaches and provide evidence for the importance of context specific genomic events and their effect on tumor progression. Additionally, the GI network accurately predicts patients' drug response, where difference GI types are found to be predictive of distinct drugs in a complementary manner. This work provides the basis for future exploration of novel GI types as well as individual interactions with major impact on cancer progression.

#1559

Proteogenomic analysis of alternative splicing in colorectal adenoma-to-carcinoma progression.

Malgorzata A. Komor,1 Thang V. Pham,2 Sander R. Piersma,2 Anne S. Bolijn,1 Tim Schelfhorst,2 Pien M. Delis-van Diemen,1 Marianne Tijssen,1 Annemieke C. Hiemstra,1 Meike de Wit,1 Beatriz Carvalho,1 Gerrit A. Meijer,1 Connie R. Jimenez,2 Remond J. Fijneman1. 1 _Netherlands Cancer Insitute, Amsterdam, Netherlands;_ 2 _VU University Medical Center, Amsterdam, Netherlands_.

Background

Early diagnosis of colorectal cancer (CRC) and identification of its precursor lesions (adenomas) is crucial in reducing CRC mortality rates. The fecal immunochemical test (FIT) is a non-invasive CRC screening test that detects human protein hemoglobin. Although FIT is beneficial in its current form with a sensitivity of ~65% for detection of CRC and ~27% for adenomas, its performance is still suboptimal and needs to be further improved. Adenoma-to-carcinoma progression is accompanied by alternative splicing, which results in expression of tumor-specific protein variants. These may yield novel biomarkers suitable for improving detection of progressive adenomas and CRCs.

Aim

We aim to identify novel biomarkers to improve early detection of CRC.

Materials and methods

RNA was isolated from 3D organoid cultures derived from 5 adenomas and 4 CRC tissues. RNA and proteins were isolated from 18 healthy human colon tissues, 30 adenomas and 30 CRCs. Samples were analyzed by RNA sequencing (Illumina) and in-depth tandem mass spectrometry proteomics (QExactive). For both organoid- and tissue-datasets differential splicing analysis was performed on RNA level to enrich the sequence database, against which mass spectra were searched, with predicted protein isoforms.

Results

Comparative splicing analysis between CRC and adenoma organoids revealed ~90 differentially spliced genes, yielding candidate biomarkers from epithelial origin. In the tissues, differential splicing analysis between CRCs and controls and between CRCs and adenomas identified over 1000 of splice variants. These include known alternatively spliced genes involved in cancer such as CD44 and VEGFA and a number of candidates overlapping with the isoforms derived from the organoids. Proteomics analysis revealed that approximately 150 of the splice variants were expressed on protein level.

Conclusion and Discussion

We have confirmed that adenoma-to-carcinoma progression is accompanied by aberrant splicing. Analysis of the organoid cultures allowed us to identify gene isoforms from (neoplastic) epithelial origin. Tissue analysis yielded tumor-specific splice variants that represent novel protein candidate biomarkers for early detection of CRC. The diagnostic performance of these splice variant proteins will be validated in series of stool and FIT samples.

#1560

Differential sensitivity analysis for resistant malignancies (DISARM), a novel approach for drug screen analysis, identifies common candidate drugs across platinum-resistant cancer types.

Carl M. Gay,1 Pan Tong,1 Robert J. Cardnell,1 Xiao Su,2 Nene N. Kalu,1 Upasana Banerjee,1 Rasha O. Bara,1 Faye M. Johnson,1 John V. Heymach,1 Jing Wang,1 Lauren A. Byers1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _The University of Texas Health Science Center at Houston, Houston, TX_.

Resistance to therapy, including conventional chemotherapy, targeted therapy and immunotherapy, continues to plague cancer treatment. Moreover, mechanisms governing resistance are poorly characterized leading to a dearth of rational combinatorial and sequential treatment strategies. While drug response data is abundant across myriad tumor types and drug classes, there exists no high-throughput method to probe such data with a query as simple as "If tumors are resistant to drug X, to what drug(s) are they sensitive?"- a seemingly trivial problem beset by immense data sets and imprecise definitions of sensitivity and resistance. Here, we present DISARM, a novel approach designed specifically to screen for drugs that are active in spite of resistance to a reference drug. DISARM selects candidates based on the proportion of samples that are resistant to a reference drug but sensitive to a candidate drug with simultaneous consideration to relatively lower IC50 values for candidate drugs and higher IC50 values for reference drugs. As candidates may work in only a subset of resistant models and precise delineation between sensitivity and resistance may vary between experimental settings, DISARM permits flexibility in dichotomizing drug data and uses grid search to optimize specifications. To illustrate, we analyzed publically available cell line data (IC50 data) from several cancer types for which platinum-based therapy is a standard of care, identifying multiple drugs that demonstrate activity in cisplatin-resistant models across tumor types such as the BCL-2 inhibitor obatoclax in small cell lung cancer, lung adenocarcinoma, gastric adenocarcinoma and bladder cancer, and the farnesyltransferase inhibitor tipifarnib in small cell lung cancer, bladder cancer, esophageal cancer, colon adenocarcinoma and head and neck squamous cell carcinoma. Frequently, multiple drugs from the same class were selected by DISARM for a single tumor type and, in these cases, we found statistically significant similarity between sensitive cell lines suggesting a subset of cisplatin-resistant cell lines that are repeatedly sensitive to a drug class. While translating preclinical observations into approved clinical use is often thwarted by an inability to identify predictive biomarkers, DISARM also allows us to select cell lines that are especially sensitive to candidate drugs or drug classes on which to perform biomarker analysis. To demonstrate this approach, we chose drugs with activity in multiple cancer types and compared mRNA and protein expression data to highlight potentially novel common and tumor-specific biomarkers for concomitant candidate drug sensitivity and cisplatin resistance. Thus, DISARM offers a simple yet effective approach for both drug and biomarker discovery within a specified clinical niche.

#1561

**Gene expression analysis identifies heterogeneity in cutaneous melanoma subjects with disruptive** MC1R **alleles and** BRAF **hotspot mutations.**

Piyushkumar A. Mundra, Candelaria Bracalente, Lucas Trucco, Nathalie Dhomen, Richard Marais. _Cancer Research UK Manchester Institute, Manchester, United Kingdom_.

Background: Previous epidemiological and population sequencing studies have established that subjects with melanocortin 1 receptor (MC1R) germline mutations, associated with red hair and light skin phenotypes, have an increased risk of melanoma. However, several conflicting reports about the role of BRAF somatic mutations in MC1R subjects exist in the literature. We hypothesise that this conflict is due to biological process driven heterogeneity within MC1R subjects carrying disruptive alleles. To understand the heterogeneity, we analysed the TCGA cohort at the gene expression level using mRNA sequencing data.

Method: From the previously published studies, we identified 68 cutaneous melanoma subjects who were of white ethinicity, had at least one disruptive allele of MC1R, and had BRAF mutations. Associated clinical and mRNA sequencing data were downloaded from the Firebrowse website. An unbiased hierarchical clustering analysis was performed using the preprocessed mRNA data containing 14669 gene expressions. The resultant clusters were then characterised using available clinical variables. A differential expression analysis was performed to identify the genomic signatures of these clusters. The resultant p-values were corrected for false discovery rate (FDR) using the Benjamini-Hochberg approach. Genes with corrected p-values less than 0.01 were further analysed using gene set enrichment analysis (GSEA) preranked lists.

Results: An unbiased clustering identified two clear and separable clusters with 40 and 28 subjects respectively. The UV-signature (dipyrimidine C>T load) was correlated with these clusters (p-value = 0.047). However, no significant differences in age at the time of diagnosis, overall survival, and total mutation load between clusters were observed. At the genomic level, 2588 genes were significantly differentially expressed between two clusters (FDR corrected p- value less than 0.05). The top 10 differentially expressed genes included ATF2, MSH6, SP3, MAP3K2, and VEGFB. Further GSEA revealed gene sets playing roles in several pathways and biological processes, including oxidative phosphorylation, UV response in keratinocytes, DNA repair, cell cycle, and cellular response to stress.

Conclusion: Cutaneous melanoma subjects with disruptive MC1R alleles and BRAF hotspot mutations have heterogeneous gene expression profiles with several key oncogenes differentially expressed. This suggests potential roles of other pathways, either independently or in cooperation with BRAF mutations, in melanomagenesis in MC1R bearing patients.

#1562

Drug repurposing for hepatocellular carcinoma enabled via transcriptomics data from experimental models of sorafenib resistance.

Kelly Regan,1 Ryan Reyes,1 Samson Jacob,1 Philip Payne,2 Tasneem Motiwala1. 1 _The Ohio State University, Columbus, OH;_ 2 _Washington University in St. Louis, St. Louis, MO_.

Hepatocellular carcinoma (HCC) is often diagnosed in patients with advanced disease who are ineligible for curative surgical therapies. Sorafenib is the only approved drug for treating late stage HCC patients. However, patients rapidly become unresponsive due to inherent and acquired drug resistance. The promise of drug repurposing is that identifying new uses for existing drugs may reduce the high costs and time required for drug discovery. This is the first study employing connectivity mapping, a transcriptomics-based drug repurposing method, to identify drugs for use against sorafenib resistant (SR)-HCC via reversal of gene expression patterns. As a first step, we characterized gene expression signatures of different models of sorafenib resistance. We obtained gene expression signatures from an in vitro and an in vivo model of (SR)-HCC (publicly available microarray data) as well as from sorafenib-resistant (pool and clone) Huh7 cells generated in our lab. We determined the presence of the SR-HCC gene signatures across six patient-derived HCC gene expression datasets from the Gene Expression Omnibus (GEO) database using the nearest template method (FDR<0.05), and found that the gene signatures performed similarly in distinguishing tumor vs. normal liver tissue (FET p<0.05). We also analyzed RNAseq data from HCC patients (n=423) in The Cancer Genome Atlas (TCGA) for the presence of these SR gene signatures, and observed that patients harboring the SR-HCC gene signatures generated by our lab had significantly reduced survival (log-rank p=0.036 SR Huh7 pool; p=0.009 SR Huh7 clone). Utilizing drug-induced gene expression profiles (n= 3,740 drugs) in the HepG2 HCC cell line from the Library of Integrated Network-based Cellular Signatures (LINCS) database, we applied connectivity mapping analysis to the SR-HCC gene signatures. Dasatinib, a Src family kinase inhibitor, was prioritized as a top drug candidate from our LINCS analysis to reverse HCC sorafenib resistance. We confirmed up-regulated activity of Src family kinases in SR-Huh7 cells, as compared to sorafenib sensitive Huh7 cells (two-tailed t test, p<0.05). We validated the use of dasatinib against sorafenib-resistant HCC cells in vitro alone and in combination with sorafenib using cell viability and clonogenic survival assays. In summary, we provide physiological relevance of SR models and proof of concept evidence for the validity of this novel drug repurposing approach for SR-HCC with implications for personalized medicine.

#1563

A machine learning approach to predict platform specific gene essentiality in cancer.

Coryandar M. Gilvary, Neel S. Madhukar, Kaitlyn M. Gayvert, David S. Rickman, Olivier Elemento. _Weill Cornell Medical College, New York, NY_.

Loss-of-function (LOF) screenings across a set of diverse cancer cell lines has the potential to reveal novel synthetic lethal interactions, cancer-specific vulnerabilities, and guide treatment options. These were traditionally done using shRNAs, but with the recent emergence of CRISPR technology there has been a shift in methodology. The Achilles project is to date the largest cancer LOF screening effort undertaken, however we found a large amount of inconsistency between their shRNA and CRISPR-Cas9 essentiality results for the same set of cell lines. Here we characterize the differences between genes found to be essential in either CRISPR or shRNA screens. We found that certain features such as gene expression, network connectivity and conservation could accurately separate out essential genes that were found exclusively in either one of these screens. This information could be tremendously useful in understanding the differences in the CRISPR and shRNA screening results. Furthermore, one limitation with Project Achilles was that they conducted shRNA screens on 216 cell lines, but only 33 cell lines in CRISPR. Therefore we developed a model that integrates these genetic, network, and population features to predict CRISPR results from shRNA screenings, and found that our model can accurately identify CRISPR essential genes better than approaches just based on the shRNA results (p-value < 10-5, d-statistic =~0.5 ). This potentially eliminates the need for a costly CRISPR screen, predicts essential genes that would be missed in the shRNA screen, and provides new data on thousands of genes in almost 200 cell lines. Additionally we integrated prior screening results to build a second set of models to predict gene essentiality for untested genes with no LOF screening needed. We found this accurately predicted whether a gene would be marked as essential as well as what type of platform (CRISPR or shRNA) was more likely to accurately identify essentiality. When predicting genes which were exclusively essential in CRISPR we observed an area under the receiver operating characteristic curve (AUC) of 0.82. Overall, these methods allow for a more comprehensive essentiality analysis of genes; which is not possible by single screening platforms.

#1564

A computational framework for removing mouse contamination in tumors sequenced from patient-derived xenografts.

Ali Amin-Mansour,1 Judit Jané-Valbuena,2 Xinmeng Jasmine Mu,1 Levi Garraway2. 1 _The Broad Institute of MIT and Harvard, Cambridge, MA;_ 2 _Dana-Farber Cancer Institute, Boston, MA_.

Introduction: Using patient derived xenograft (PDX) models has become an effective way for investigating response to standard or new therapeutics in cancer. Human cancer cells injected in mice are allowed to establish tumors and subjected to desired treatments. The PDX tumors are later harvested and characterized, often by massive parallel sequencing. However, a major challenge with analyzing these data is the presence of stromal mouse genomic material, frequently resulting in artifacts in downstream variant detection. We present a computational method to eliminate mouse contamination in PDX.

Method: We used the Burrows-Wheeler Aligner to map reads obtained from sequencing the PDX samples to a combined human and mouse reference genome . We remove reads that are mapped to the mouse reference. The remaining reads are then processed through standard mutation calling pipelines for somatic mutation detection. To test the efficacy of our method, we created in silico mixtures of human and mouse whole-exome sequencing reads from a melanoma patient's tumor and an immortalized mouse cell line captured with human exome baits. We then carried out a sensitivity analysis to examine how changing the mean target coverage of sequencing, or mouse contamination levels affects our results. For each of the computational experiments, we evaluated somatic mutations detected from the synthetic samples in comparison to the original human sample.

Results: We calculated the sensitivity and specificity of detecting somatic mutations to determine our algorithm's performance. In all instances, we found greater than 99% for both sensitivity and specificity.

Conclusions: Our results demonstrate that our method works accurately towards removing mouse reads in PDX samples. This task could also be applied to separating sequence reads from other species.

#1565

OnPLS-based integrative proteogenomics analysis of lung squamous cell cancer.

Fredrik Pettersson, Paul A. Stewart, Robbert J. Slebos, Eric A. Welsh, Ling Cen, Yonghong Zhang, Zhihua Chen, Chia-Ho Cheng, Guolin Zhang, Bin Fang, Victoria Izumi, Sean Yoder, Katherine Fellows, Y Ann Chen, Jamie K. Teer, Steven Eschrich, John M. Koomen, Anders Berglund, Eric B. Haura. _Moffitt Cancer Center, Tampa, FL_.

Introduction: Multivariate projection methods such as PCA and PLS has been widely applied for analysis of biological and chemical data. OnPLS is a recent extension to these methods suitable for integrative analysis of omics data. With OnPLS it is possible to compare multiple omics datasets to identify joint variation and variation locally unique for each of the studied datasets. OnPLS is a new approach for truly integrative analysis of omics data to be contrasted to commonly applied approaches limiting analysis to 1) comparing findings from individually analyzed blocks of data 2) pairwise comparison of individual probes.

Experimental: A Java based implementation of OnPLS was used for the statistical modeling. 116 lung squamous cell cancer samples were characterized using gene expression profiling and global proteomics. The OnPLS model was applied to jointly model variation between mRNA and protein expression. Enrichment analysis of factor loadings was performed using the Enrichr tools to identify biological mechanisms explained by the different joint and unique components of the OnPLS model.

Results: Using a cross-validation procedure the model with the highest predictive ability was calculated having two joint components and one locally unique component for each of the proteomics and gene expression datasets. The model explained 21.9% of the variation in the expression data and 26.1% of the variation in the proteomics data. The first joint component captures the highest degree of common variation between mRNA and protein activity. From the mRNA data, this component is related to immune infiltrates, especially monocytes and B-cells, whereas this component is related to extracellular matrix activity from the protein data. This suggests covariance of mRNA immune-related gene expression and extracellular matrix-related protein expression. As expected, local variation specific to the protein measurements involved regulation of protein activation and processing. mRNA-specific variation is related to keratinization, a key process in squamous cell cancer.

Conclusion: OnPLS offers an interesting approach for integrative analysis of omics data. Applying this approach to proteo-genomics data of lung squamous cell cancers suggest similar patterns of activity is represented in protein and gene expression data, however the biological processes associated with this activity may be distinct.

#1566

Differential expression of Epstein Barr virus miRNAs in triple-negative breast cancer.

Alexander Blanchard,1 Tiffany Walls,1 Ramon Vidal,2 Stefan Bonn,2 Scott Harrison,1 Perpetua Muganda1. 1 _North Carolina A &T State University, Greensboro, NC; _2 _German Center for Neurodegenerative Diseases, Goettingen, Germany_.

Triple-negative breast cancers (TNBC) accounts for up to 20% of breast cancer cases. These tumors are negative for estrogen, progesterone, and HER2 receptors, which are markers used for diagnosis and treatment of other types of breast cancer. Clinically, TNBCs are notoriously aggressive, respond poorly to targeted therapies, have high rates of relapse, as well as poor prognosis. The molecular basis of TNBC oncogenesis is currently unknown, but it is possible that viral factors, such as Epstein-Barr Virus (EBV) miRNAs, play a role. EBV miRNAs have been implicated in the oncogenesis of several forms of cancer, including Burkitt's lymphoma and nasopharyngeal carcinoma. The objective of this study, therefore, was to determine the differential expression of EBV miRNAs in TNBC tumors as compared to normal breast tissue. We conducted a comprehensive profiling of viral miRNAs in 48 TNBC tumors as compared to 15 control normal breast tissues, utilizing deep sequencing analysis software and publically available deep sequencing data. Four EBV miRNAs (BART18-3p, BART 8-5p, BART15, BART22) were significantly expressed in 11-17% of the TNBC tumors, and not in control normal breast tissue. Four novel putative EBV miRNAs were found to be differentially expressed in TNBC tumors as compared to control normal cells. Two of the novel putative EBV miRNAs were differentially expressed above 125 reads per million (RPM) in 20% and 27% of the TNBC tumors as compared to 0% and 6.6% of normal breast tissues, respectively. One novel putative EBV miRNA was expressed above 125 RPM in 83% of TNBC tumors as compared to 13% of normal breast tissue. One novel putative EBV miRNA was expressed above 125 RPM in 66% of the normal breast tissue as compared to 2% of the TNBCs. These putative EBV miRNAs localize within genomic regions of EBV, including the LMP-2 region. Ongoing work has so far computationally validated one of these miRNAs as a novel EBV miRNA; in vitro validation studies are in progress. This is the first report on the differential expression of EBV miRNAs in TNBC tumors. Since TNBC tumors are extremely heterogeneous, it is intriguing that 20% of TNBC tumors specifically express the same EBV miRNA. Our findings suggest that these differentially expressed EBV miRNAs may potentially play a role in the pathogenesis of TNBC.

#1567

Oncogenesis may result from multiple different combinations of a small number of tumorigenic mutations.

Ramu Anandakrishnan, Robin Varghese, Nick Kinney, Harold R. Garner. _Edward Via College of Osteopathic Medicine, Blacksburg, VA_.

Cancer is known to result from genetic mutations, both inherited and somatic. Yet, decades of investigation and the availability of extensive genomic data, have failed to reveal the specific mutations that directly result in tumorigenesis for most cancers. One possible reason is that tumorigenesis may result from one or more of multiple different combinations of mutations, while investigators generally search for a single set of mutations. A mathematical model of the probability of "hitting" one of many possible combinations of a small number of mutations was compared to the actual distribution of accumulated mutations at diagnosis as reported on the cancer genome atlas (TCGA). This "multi-combination multi-hit" model reproduces the distribution of accumulated mutations from the TCGA database with surprising accuracy (RMSD = 1.5%) for a model where one or more of 100 different possible combinations of three mutations result in tumorigenesis. We speculate that each of the three mutations affects one of the several genes in three distinct cell proliferation control pathways, such as senescence, apoptosis, autophagy and necrosis, and different combinations of mutations affecting three of these pathways may result in tumorigenesis. We have identified several such combinations for further investigation.

#1568

Predicting stochastic proliferation and death in response to drugs with mechanistic models tailored to genomic, transcriptomic, and proteomic data.

Mehdi Bouhaddou, Anne Marie Barrette, Rick J. Koch, Marc R. Birtwistle. _Icahn School of Medicine at Mount Sinai, New York, NY_.

Over the past decade we have seen a shift in cancer therapy from broadly cytotoxic drugs to molecular therapies targeting "driver" mutations. Although targeted therapy has seen great success for some cancers (e.g. imatinib for leukemia), it has struggled with poor efficacy in treating other cancers that can sometimes possess multiple "driver" mutations. This highlights the complex, and at times non-intuitive, interplay between multiple players in a signaling cascade, which can be highly dependent on the biological context – that is, gene expression levels and mutational architecture – of a tumor or cell line. A quantitative, mechanistic, biologically-tailored understanding of how these signaling dynamics drive proliferation and death could improve precision pharmacology approaches to treat cancer.

Here, we constructed the first highly detailed, large-scale ordinary differential equation (ODE) mechanistic mathematical model depicting the most commonly mutated cancer signaling pathways across human cancers, as indicated by a pan-cancer analysis by The Cancer Genome Atlas (TCGA). The model includes the RTK/Ras/MAPK, PI3K/AKT/mTOR, CDK/RB cell cycle, p53/MDM2 DNA damage response, and BCL/Caspases apoptosis pathways. The adjustable parameters of the model can be informed by measurements from patients or cell lines, including copy number alterations, mutations, and gene expression levels. This single-cell model links stochastic gene expression processes to quantitative signaling dynamics, and once tailored to a biological context can be used to simulate the effect of various anti-cancer therapies on cell fate behavior such as proliferation and death for a population of cells.

The first instance of the model integrated genomic, transcriptomic, and proteomic data from the MCF10A cell line, a non-transformed cell line with predictable phenotypic behaviors. We trained the model using western blot and flow cytometry experiments to refine various biochemical parameters and phenotypic outcomes. Many fundamental questions in signal transduction arose during this process, such as how EGF and insulin synergize to drive S-phase entry or how a specific biological context confers sensitivity or resistance to inhibitors of the ERK and AKT pathways. Simultaneously, we are tailoring the model to patient-derived genetic information from primary glioblastoma tumors and screening brain-penetrable compounds in a patient-specific manner.

In conclusion, a quantitative, mechanistic, biologically-tailored mathematical model depicting the major cancer pathways allows us to probe the mechanisms that underlie how signaling dynamics drive proliferation and death in response to various perturbations, and gain insight into their dependence on the biological context of cell lines and patient tumors.

#1569

Optimizing transducer array configuration for treatment of pancreatic cancer using Tumor Treating Fields (TTFields).

Ariel Naveh,1 Ze'ev Bomzon,1 Noa Urman,1 Ofir Yesharim,1 Eilon D. Kirson,1 Uri Weinberg2. 1 _Novocure ltd., Haifa, Israel;_ 2 _Novocure GMBH, Lucerne, Switzerland_.

Introduction

TTFields is an antimitotic cancer treatment that utilizes low intensity (1-3 V/cm) alternating electric fields in the intermediate frequency (100-300 kHz) that are delivered in two orthogonal directions using 2 pairs of transducer arrays. TTFields are currently approved for Glioblastoma Multiforme (GBM). A phase II clinical trial (EF-20) showed that TTFields in combination with gemcitabine was safe in patients with locally advanced pancreatic cancer.

Preclinical studies show that the effect of TTFields is intensity-dependent with a therapeutic threshold of 1 V/cm. Simulation-based studies show that the field distribution changes with array placement. Treatment planning with arrays positioned on the scalp to maximize field intensity to the tumor is standard of practice when treating GBM. Studies to examine array layout on the abdomen to maximize the field distribution in this region have not been done. In the EF-20 study, a generic layout was used. We used computer simulations to test how altering the transducer array layout on the mid-body alters the field distribution within the abdomen and pancreas.

Methods

To simulate delivery of TTFields to the abdomen, we used a realistic computerized model of a human male (DUKE 3.0 from ZMT-Zurich). Eight different layouts utilizing combinations of arrays with either 13 or 20 disks placed at different locations on the abdomen were simulated. In order to generate TTFields, an alternating voltage difference with a peak to peak magnitude of 100V and a frequency of 150 KHz was imposed on the outer surfaces of the disks of each pair of arrays. The simulations were performed using ZMT's Sim4Life V3.0 electro-quasi-static solver. For each pair of transducer arrays, the mean field intensity in the abdomen and chest, and in the pancreas and liver, was calculated.

Results

All eight layouts delivered fields with mean intensities of about 2 V/cm to the abdomen. The average field intensity delivered to the pancreas and the liver exceeded the therapeutic threshold of 1 V/cm for all layouts. The highest intensities were delivered to the pancreas by layouts in which a 20-disk array was placed at the middle of the back, and either a 20 disk array placed at the middle of the abdomen or a 13 disk array placed on the front-left side of the abdomen over the pancreas. Generally, field intensities in the pancreas are lower than in the rest of the abdomen because of the high electric conductivity of this organ.

Conclusion

This work shows that TTFields can be delivered to the pancreas with intensities well above the therapeutic threshold of 1 V/cm, and that altering the location of the arrays on the abdomen can influence the field intensity and distribution within the body. This work forms the basis for developing optimal strategies for delivering TTFields to the pancreas.

#1571

Decrypting the transcriptome profile of Pak4 using next generation sequencing.

Chetan Rane, Misaal Patel, Li Cai, Audrey Minden. _Rutgers University, Piscataway, NJ_.

The PAK4 (p-21 Activated Kinase 4) protein kinase has long been associated with cancer because of its key roles in regulating cell proliferation, cell cycle progression and cell morphology. The PAK4 gene is amplified in different types of cancers, including breast cancer. PAK4 overexpression is associated with oncogenic transformation in several breast cancer cell lines, while PAK4 inhibition has been shown to significantly reduce their tumorigenic potential. However, there is limited information available on PAK4's mechanism of action in promoting tumorigenesis. To gain an insight into PAK4 downstream signaling pathways, we performed Next Generation Sequencing (NGS) on RNA samples collected from non-transformed immortalized mouse mammary epithelial cells (WT iMMECs) and iMMECs overexpressing PAK4. Research from our lab has shown that unlike WT iMMECs, iMMECs overexpressing PAK4 formed tumors when injected into mammary fat pads of mice, suggesting a central role for PAK4 in tumor formation. Previous studies to delineate PAK4 signaling pathways have mostly focused on substrates phosphorylated by PAK4. This study, however, takes into account genes which may act downstream of PAK4 and play an important role in mediating long term PAK4 function. RNA-seq analysis offers the ability to discover new genes and splice variants and measure transcript expression. mRNA was isolated in triplicates from WT iMMECs and iMMECs overexpressing PAK4; mRNA from each condition was then sequenced using Illumina NextSeq platform. The RNA-seq data generated was analyzed using Top Hat and Cufflinks software, which created a list of genes whose expression was significantly different in these two cell types, and previously unknown to be regulated by PAK4 (26 genes were up regulated and 51 genes were down regulated by more than a log2 fold change of 3). A qPCR analysis of 8 of these genes validated the sequencing data. Among the different genes we identified, we chose to focus on ParvB, which is consistently down regulated by almost seven fold in the iMMECs overexpressing PAK4. Previous studies have suggested a tumor suppressor role for ParvB in regulating breast tumorigenicity. We hypothesize that PAK4 mediates mammary tumor formation through ParvB downregulation. Additional studies investigating the role of ParvB in the PAK4 signaling pathway are under investigation. Future studies will also involve studying the other genes we found to be regulated by PAK4 overexpression and determining their role in tumorigenesis.

#1572

Transformation from melanocytes or nevi into melanoma: models based on a transcriptome meta-analysis.

Daniel Ortega-Bernal,1 Claudia Rangel-Escareño,2 Elena Arechaga-Ocampo,1 Claudia H. Gonzalez-De la Rosa1. 1 _Universidad Autonoma Metropolitana, Mexico, Mexico;_ 2 _Instituto Nacional de Medicina Genomica, Mexico, Mexico_.

Background

Melanoma is the most aggressive skin cancer due to its ability to rapidly metastasize and resist radio- and chemotherapy. The frequency of this disease has increased 400% worldwide in the last 30 years. There are 2 ways to develop melanoma: direct transformation of melanocytes into melanoma and the nevus as an intermediary. Here, we used a computational strategy based on transcriptome meta-analysis to identify the pathways involved in the 2 possible ways by which cells may transform to melanoma.

Methods

Affymetrix HU 133A Plus 2.0 microarray files (50 in total) were collected from the Gene Expression Omnibus database for the meta-analysis, representing melanocytes, nevi and stage I, II, and III melanoma. Differential expression was analyzed using linear models and the limma library. The analysis focused on 3 comparisons: nevi vs. melanocytes; melanoma vs. nevi; and melanoma vs. melanocytes. Transcripts with a log fold change of <-1/>1 (B-statistic >3) were included in the study. Functional enrichment analysis identified the most deregulated pathway. Genes resulting from the intersection of the melanoma vs. nevi and melanoma vs. melanocytes comparisons were used as possible biomarkers of transformation and analyzed by IPA.

Gene signature identification

In melanoma vs. nevi comparison, 1881 expressed genes were differentially expressed, of which only 888 were used to generate the transformation model and to perform functional enrichment. Consistent with the phenomenon of malignant transformation, we identified 3 pathways associated with cell cycle checkpoint control that exhibited decreased activity. Conversely, the pathways that exhibited increased activity, Estrogen-mediated S-phase Entry and Cyclins and Cell Cycle Regulation, promote cell cycle progression. In melanoma vs. Melanocytes comparison, 4112 expressed genes were differentially expressed (2310 downregulated and 1802 upregulated), 1099 of which intersected with the nevi vs. melanocytes comparison. The 682 genes intersecting the melanoma vs. nevi comparison were removed. Thus, 2331 genes were used to perform enrichment analysis. The main result was the decreased activity of the cAMP-mediated signaling pathway. The intersection of both comparisons (682 genes), was used to identify 22 genes upregulated whose product is a secreted protein. Two of these genes were previously reported as potential biomarkers in neoplasia, cytokines CXCL8 and CXCL16, supporting the quality of our analysis.

Conclusions

Our analysis, in agreement with previously reported results, identify CXCL8 as biomarker of melanoma, and also identified new biomarkers, including CXCL16, MMP19, BIVM, and FAM60A. A better understanding of the specific molecular alterations causally involved in this disease may lead to new biomarkers and pathways involved in the transformation for this currently incurable malignancy.

#1573

Detection of late-arising driver mutations using multi-sample tumor sequencing data.

Karen Gomez, Sayaka Miura, Sudhir Kumar. _Temple University, Philadelphia, PA_.

Whole-genome sequencing of tumors has identified genetic mutations that drive the origination, growth, and spread of cancer. Most driver mutations detected from sequencing tumors have been categorized as early-occurring and are believed to play a role in initial tumor formation. However, single-region primary tumor samples represent just one snapshot of the continuously changing mutational landscape of a tumor. Drivers of initial tumor formation, occurring early on in tumor development, are present in more tumor cells and are more likely to be identified from a single tumor sample than drivers of later events like metastasis, which are likely to be found in only one or a few tumor samples. We analyzed 101 multi-region and/or multi-tumor sequencing data sets using driver mutation annotation and phylogenetic inference to determine whether driver mutations occur preferentially early on. We found driver mutations frequently arise late in the course of tumor evolution, even when statistically accounting for small amounts of total drivers and many early total mutations. In addition, the timing of driver mutation occurrence varies widely depending on cancer type as well as among different individuals with a single cancer subtype. Finally, we identify known cancer genes that are enriched in either early or late driver mutations, reflecting different selective pressures at various stages of cancer development. These results illustrate how drivers of primary tumorigenesis must be distinguished from drivers of treatment resistance, metastasis, or recurrence in order to better understand the genetic changes behind cancer progression.

#1574

Computational approach for discovery of regulatory noncoding variants in cancer.

Yu Liu,1 Michael Edmonson,1 Xiaotu Ma,1 Michael Rusch,1 Yongjin Li,1 Benshang Li,2 Shuhong Shen,2 A. Thomas Look,3 Jinghui Zhang1. 1 _St. Jude Children's Research Hospital, Memphis, TN;_ 2 _Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, China;_ 3 _Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA_.

Noncoding mutations in the cancer genome have historically been poorly understood. Recent studies have shown however that mutations in intergenic regions may play an important role in cancer by introducing de novo regulatory elements and thus cis-regulating target gene expression. While mono-allelic expression of the dysregulated gene is an important signature of regulatory noncoding mutations, recognition of this feature has not been implemented by any methods evaluating noncoding mutations to date.

To perform genome-wide detection of noncoding mutations with a potential regulatory function, we developed a novel computational approach implementing this allelic specific expression signature (ASE) as a core component, using WGS and RNAseq data as input. We first searched for genes showing ASE across the tumor genome by comparing the minor allele frequency of heterozygous markers between WGS and RNAseq data. We excluded genes known to be in an imprinted locus, as well as sites matching a precomputed set of 5,576,857 problematic polymorphism markers (estimated by analyzing WGS data from 625 normal genomes). Next, the somatic mutations were filtered for regulatory candidates that could introduce a transcription factor (TF) binding motif absent from the reference sequence. We included 614 TF binding motifs from the HOCOMOCO database and performed motif analysis with the FIMO package. Finally, the somatic mutations were assigned to dysregulated genes in the same topologically associating domains in human defined by public Hi-C data and prioritized by open chromatin state defined by DNaseI hypersensitivity regions (ENCODE project).

We applied our approach to 22 T-ALL tumor samples collected at Shanghai Children's Medical Center in a pilot study consisting of 13 diagnosis and 9 relapse samples (9 of these were trios). WGS was performed on both tumor and normal samples and RNAseq was performed for the tumor sample only. We predicted 213 regulatory noncoding mutations (157 unique variants) in total from this cohort, potentially targeting 119 genes. Among the dysregulated targets we found TAL1 and LMO2, which are known TFs playing a key role in T-ALL. TAL1 mutations were identified in a total of 6 tumors (3 trios). Besides the known oncogenic insertion at ~7kb upstream of TAL1 (1 trio), we also discovered a novel mutation in the first intron of TAL1 (2 trios) that may potentially drive ectopic TAL1 expression. Novel LMO2 mutations were found in 6 tumors (3 trios) at 3 different sites. This approach also identified novel targets such as GPSM1 and PTPRN2, shedding light on heretofore unknown players in T-ALL targeted by noncoding mutations.

By integrating WGS and RNAseq data, our novel computational framework can identify somatic noncoding mutations in cancer that may introduce de novo regulatory elements, thus driving target gene expression. Wider application of this approach will expand our understanding of the role of noncoding mutations in the cancer genome. 

## ENDOCRINOLOGY:

### Prostate Cancer Biology and Therapy

#1575

Novel small molecule inhibitors of p300/CBP down-regulate AR and c-Myc for the treatment of castrate resistant prostate cancer.

Nigel Brooks,1 Neil Pegg,1 Jenny Worthington,2 Barbara Young,3 Amy Prosser,3 Jordan Lane,3 David Taddei,3 Matthew Schiewer,4 Renee deLeeuw,4 Jennifer McCann,4 Karen Knusden4. 1 _CellCentric Ltd, Cambridge, United Kingdom;_ 2 _Axis Bioservices, Coleraine, United Kingdom;_ 3 _Sygnature Discovery, Nottingham, United Kingdom;_ 4 _Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA_.

Background: E1A binding protein (p300) and CREB binding protein (CBP) are two closely related, paralogue histone acetyl transferase proteins that act as transcriptional co-activators of a variety of cancer related genes. We have developed potent, selective and orally active small molecule inhibitors of the bromodomain of p300/CBP and investigated their role in regulating androgen receptor expression and function. We have also examined their role in driving synthetic lethality in tumours. Loss of function mutations in either p300 or CBP (including in significant proportions of lung and bladder tumours), can lead to a dependency on the corresponding paralogue protein.

Methods: Binding affinity to p300, CBP and BRD4 was measured in a surface plasmon resonance (SPR) assay. Potency and functional activity was demonstrated in a panel of prostate cells lines representing hormone responsive (LNCaP), hormone independent (DU145, PC3) and castrate resistant disease (22Rv1, C4-2, VCaP, LNCaP-AR) as well as wildtype (A549) and CBP deficient (H520, H1703, LK2) lung cancer cells. Combination effects of p300/CBP inhibitors with a PARP or CDK4/6 inhibitor were determined in LNCaP and C4-2 cells. Effects of p300/CBP inhibitors (and by comparison, the BET inhibitor, JQ1), on AR, AR-V7 splice variant and c-Myc protein, as well as c-Myc, KLK3 and TMPRSS2 gene expression, were assessed in 22Rv1 cells in vitro. In vivo effects on biomarkers were measured in a 22Rv1 xenograft model.

Results: CCS1357, an in vitro probe compound, binds to p300 and CBP with high affinity (Kd=4nM) and selectivity (Kd=245nM; BRD4). It is a potent inhibitor of cell proliferation in castrate resistant cell lines (IC50=100nM in LnCaP-AR; 350nM in 22Rv1) with minimal effects in hormone independent lines. CCS1357 combined with palbociclib (CDK4/6) or olaparib (PARP) in LNCaP or C4-2 cells, showed reduced cell viability compared with any of these drugs given alone. In 22RV1 cells, CCS1357 significantly down-regulated AR-FL, AR-V7 and c-Myc protein by Western, an effect not seen with JQ1 at equivalent proliferation IC50s. CCS1357 effects were reversed by the proteasome inhibitor, MG132. CCS1357 also caused a profound inhibition of c-Myc, KLK3 and TMPRSS2 genes measured by qPCR in 22Rv1 cells in vitro. A preclinical candidate (CCS1477) given as a single oral dose (30mg/kg) inhibited plasma PSA and tumour AR, AR-V7 and c-Myc in a 22Rv1 xenograft model. In the lung cancer cell lines, we observed differential sensitivity to CCS1357; CBP deficient lines were more sensitive (cell proliferation) compared with normal.

Conclusions: Taken together these data support the clinical testing of p300/CBP inhibition in patients in two settings; firstly, castrate resistant prostate cancer by down-regulating of AR, AR-SV and c-MYC expression and function; and secondly in patients with loss of function mutations in p300 or CBP by driving synthetic lethality.

#1576

UGT2B17 promotes castration-resistant prostate cancer progression through enhancing ligand-independent AR signaling.

Haolong Li,1 Ning Xie,1 Ruiqi Chen,1 Mélanie Verreault,2 Ladan Fazli,1 Martin E. Gleave,1 Olivier Barbier,2 Xuesen Dong1. 1 _University of British Columbia, Vancouver, British Columbia, Canada;_ 2 _Laval University, Québec, Quebec, Canada_.

Background: Castration resistant prostate cancer (CRPC) is characterized by a shift of androgen receptor (AR) signaling from ligand-dependent to ligand-independent. Defining mechanisms that control AR signaling transformation is important to develop therapies for disease control. UDP-glucuronosyltransferase 2B17 (UGT2B17) is a key enzyme that maintains androgen homeostasis by catabolizing AR agonists into inactive forms. Although enhanced UGT2B17 expression by antiandrogens was reported in androgen-independent prostate cancers, its role in regulating AR signaling transformation and CRPC progression remain unknown.

Method and Results: We first evaluated the UGT2B17 protein expression levels by immunohistochemistry (IHC) on Vancouver Prostate Centre tissue microarrays. We show that higher UGT2B17 protein expression in prostate tumors is associated with higher Gleason score, metastasis and CRPC progression. The expression and activity of UGT2B17 are also higher in androgen-independent compared to androgen-dependent cell lines. Interestingly, enzalutamide-resistant MR49F cells expresses low UGT2B17 mRNA but strong protein expression, suggesting that posttranslational mechanisms may enhance UGT2B17 protein stability in these cells. We then constructed PCa cell lines with gain-of-function UGT2B17 by lentivirus. Functional analyses indicate that UGT2B17 stimulates cancer cell proliferation, invasion, and xenograft progression to CRPC after prolonged androgen deprivation. To further decipher the molecular mechanisms by which UGT2B17 enhance PCa cell growth independent of androgen, we performed gene microarray using LNCaP(mock) and LNCaP(UGT2B17) cells cultured under the regular serum condition or the prolonged androgen deprivation condition. Microarray analyses reveal that UGT2B17 suppresses androgen-dependent AR transcriptional activity, while enhancing androgen (ligand)-independent AR transcriptional activity. The latter targets genes associated with cell mitosis. These UGT2B17 actions in reprogramming androgen signaling are mainly mediated by activating the c-Src kinase. We confirmed that, in CRPC tumors, UGT2B17 expression is positively associated with c-Src activation.

Conclusion: These results indicate that UGT2B17 expedites CRPC progression by enhancing ligand-independent AR signaling that activates predominantly cell mitosis in cancer cells.

#1577

GRK3 is a direct target of ADT-induced CREB1 activation and it promotes neuroendocrine differentiation of prostate cancer cells.

Dayong Zheng,1 Mohit Hulsurkar,1 Meixiang Sang,1 Songlin Zhang,1 Jianming Xu,2 Martin Gleave,3 Michael Ittmann,2 Wenliang Li1. 1 _University of Texas Health Science Center at Houston, Houston, TX;_ 2 _Baylor College of Medicine, Houston, TX;_ 3 _University of British Columbia, Vancouver, British Columbia, Canada_.

Treatment-related neuroendocrine prostate cancer (t-NEPC) is an aggressive subtype of prostate cancer that is believed to arise through neuroendocrine differentiation (NED) from prostate adenocarcinoma (PAC) upon castration resistance to androgen deprivation treatment (ADT). t-NEPC is highly metastatic with poor prognosis. With the recent introduction of potent ADT drugs in clinic, the incidence of t-NEPC is expected to increase dramatically. No effective therapeutic is available for t-NEPC and its molecular mechanisms remain poorly understood. We reported that GRK3 (G protein-coupled receptor kinase 3) promotes prostate cancer progression. We further demonstrated that the ADT activates CREB1 (cAMP response element binding protein 1), which directly targets and induces GRK3. GRK3 expression is higher in t-NEPC than in PAC cells and mouse models, and it positively correlates with the expression and activity of CREB1 in human prostate cancers. Notably, overexpression of GRK3 in PAC cells increased the expression of NE markers. Conversely, silencing GRK3 blocked CREB1-induced NED in PAC cells, reversed NE phenotypes and inhibited proliferation of t-NEPC cells in culture and in xenograft. Currently, we are investigating the mechanisms underlying NEPC progression promoted by the CREB1/GRK3 axis. Taken together, these results indicate that GRK3 is a new critical activator of NE phenotypes and mediator of CREB1 activation in promoting NED of prostate cancer cells.

#1578

Distinct expression and biological functions of PMEPA1 isoforms in prostate cancer.

Shashwat Sharad, Hua Li, Allissa Dillman, Alagarsamy Srinivasan, Albert Dobi, Shiv Srivastava. _USU/Center for Prostate Disease Research (CPDR), Rockville, MD_.

Introduction and Objectives: The androgen receptor (AR) and TGF-β dysfunctions contribute to prostate cancer (CaP) development and progression. Our laboratory since the discovery of PMEPA1, has established PMEPA1/NEDD4 and AR negative feedback loop and its role in AR regulation in prostate cancer. PMEPA1 gene has also identified as a TGF-β responsive gene and inhibits TGF-β signaling via a negative feedback loop. Five isoforms are transcribed from distinct promoters within the PMEPA1 locus. PMEPA1 isoforms were shown to have variations at the N-terminus of the protein. Our previous study has shown the differential correlations of 2 PMEPA isoforms (PMEPA1 and STAG1) in context of AR expression in CaP. This study focuses on understanding of the expression and biological functions of PMEPA1 isoforms in CaP.

Methods: RNA-Seq data from LNCaP and VCaP cells, and from The Cancer Genome Atlas (TCGA) dataset, were used to evaluate the expression levels of PMEPA1 isoforms. LNCaP cells were treated with R1881 (0, 0.1, 1.0 nM) and DU-145 and PC-3 cells were treated with TGF-β (0, 5 and 25 ng/ml) for 24 hours. PMEPA1 isoform specific plasmids and siRNAs were transfected into LNCaP, DU-145 and PC-3 cells individually. Cell growth was analyzed by cell counting and BrdU incorporation assay. The protein levels of PMEPA1 isoforms and AR were detected by immunoblotting, and the transcript levels were evaluated by QRT-PCR.

Results: Since the annotation of PMEPA1/TMEPA1 isoforms has been empirical in the literature, here we propose a new structure/expression based nomenclature: PMEPA1 (reading frame of 252 amino acids (aa)); PMEPA2 (344aa); PMEPA3 (287aa; STAG1); PMEPA4 (259aa); and PMEPA5 (237aa). Expression of PMEPA1 and PMEPA2 was restricted to androgen responsive prostate cancer cells in comparison to broader expression pattern of other isoforms (PMEPA3-5). The expression of PMEPA1-2 was androgen regulated, whereas expression of PMEPA 3-5 was regulated by TGF-β. Only PMEPA1 inhibited cell growth of LNCaP, DU-145 and PC-3 cells. In contrast, PMEPA2-4 promoted cell growth of DU-145 and PC-3 cells. Of all the isoforms only PMEPA1 mediated AR protein degradation in CaP.

Conclusions: The PMEPA isforms appear to underscore distinct biological functions in the context of androgen and TGF-β signaling. Widely studied PMEPA1 was specific for AR degradation in prostate cancer cells and was consistent with previous observations of association of AR upregulation with loss of PMEPA1 in prostate cancer. The roles of PMEPA isoforms need to be better defined in prostate cancer and other cancers.

Funding: This study was supported by CPDR, USUHS, HU0001-10-2-0002 to DGM.

#1579

**Bicalutamide-induced expression of T-type Ca** 2+ **channels in prostate cancer cells undergoing neuroendocrine differentiation** in vitro **.**

Miguel Martin-Caraballo, Megan Hall, Bryan Todd, Yoon Jung Kwon, Vu Nguyen, Jennifer L. Hearne. _University of Maryland Eastern Shore, Princess Anne, MD_.

The growth of prostate cancer cells depends on the activation of androgen receptors. Anti-androgen therapies that inhibit androgen synthesis or receptor activation are effective in limiting tumor growth. However, prolonged treatment with anti-androgen therapies result in the transition of prostate cancers into an androgen refractory state. Neuroendocrine differentiation (NED) has been associated with the progression of prostate cancers to an androgen resistant phenotype. In this work we investigated the effect of the androgen receptor blocker bicalutamide in promoting NED of LNCaP cells and whether it is accompanied by increased T-type Ca2+ channel expression. The ability of bicalutamide to evoke morphological and biochemical changes associated with NED was assessed by PCR, western blot and immuno-histochemical analysis. Changes in the Cav3.2 T-type Ca2+ channel subunit expression was studied using PCR analysis, western blot and whole cell recordings. The role of T-type Ca2+ channels in promoting the differentiation of LNCaP cells was assessed by a cell viability assay and changes in cell morphology, specifically the development of neurite-like processes. PCR analysis of bicalutamide-stimulated cells indicates no significant changes in Cav3.2 mRNA, yet results in Cav3.2 protein expression and functional channels. Western blot and immuno-histochemical analysis of LNCaP cells simulated with bicalutamide for 4-10 day reveals biochemical changes consistent with NED including the expression of tubulin IIIbeta and neuron-specific enolase. Pharmacological inhibition of T-type Ca2+ channel function with nickel ions and NNC 33-0936 disrupts the morphological differentiation and cell viability of LNCaP cells treated with bicalutamide. These results suggest that bicalutamide treatment of LNCaP cells evokes significant morphological and biochemical changes associated with NED and results in increased expression of T-type Ca2+ channels, which can significantly alter Ca2+ homeostasis.

#1580

Combined neuroendocrine differentiation and androgen receptor hyperactivity in MAP37 and CHD1 null prostate cancer.

Leah C. Rider, Lindsey U. Rodrigues, Anis Karimpour-Fard, Lina Romero, Claire Gillette, James C. Costello, Scott D. Cramer. _Univ. of Colorado Anschutz Medical Campus, Aurora, CO_.

We recently functionally validated a clinically aggressive subtype of prostate cancer characterized by dual deletion of MAP3K7 and CHD1. This subtype accounts for up to 25% of prostate cancer deaths. Androgen signaling is a critical component of prostate tumorigenesis and disease progression, which is exploited in treatment of prostate cancer with androgen deprivation and anti-androgen therapies. Neuroendocrine differentiation is thought to provide a selective growth advantage of prostate cancer in androgen-depleted conditions such as surgical or chemical castration. REST is a transcriptional repressor of neuronal gene expression and has been implicated in neuroendocrine differentiation of prostate cancer. The impact of combined MAP3K7 and CHD1 loss on androgen signaling and neuroendocrine differentiation has not been determined. Here, we demonstrate that dual loss of MAP3K7 and CHD1 simultaneously stimulates enhanced androgen signaling and enhanced neuronal differentiation correlated with enhanced growth in both androgen-depleted and androgen-replete conditions. These changes occur via increased androgen receptor chromatin binding, loss of CHD1 chromatin binding, loss of REST expression, and reduced chromatin binding by REST. Suppression of CHD1 and MAP3K7 attenuates the antiproliferative activity of the anti-androgen Enzalutamide. Furthermore, we translate these findings to clinical samples and show that decreased expression of CHD1, MAP3K7, and REST is associated with previous therapeutic intervention, castrate-resistant disease, and progression to a neuroendocrine phenotype. Overall, patients with low expression of all three have poorer disease-free survival. Together these data support the lineage plasticity of prostate tumors with loss of CHD1 and MAP3K7 exhibiting both androgen-dependent and -independent phenotypes leading to poor clinical outcome, which poses challenges to conventional therapeutic approaches.

#1581

Androgen receptor action in prostate cancer partitions into distinct transcriptional codes that differ in clinical relevance.

Sangeeta Kumari,1 Varadha Balaji Venkadakrishnan,1 Dhirodatta Senapati,1 Qiang Hu,2 Song Liu,2 Hannelore V. Heemers1. 1 _Cleveland Clinic, Cleveland, OH;_ 2 _Roswell Park Cancer Institute, Buffalo, NY_.

With few exceptions the 30,000 prostate cancer (CaP) deaths annually in the US are due to failure of androgen deprivation therapy (ADT). ADT prevents ligand-activation of androgen receptor (AR). Despite remission, CaP progresses while continuing to rely on AR. AR's transcriptional output that controls CaP behavior is an alternative therapeutic target, but its molecular regulation is poorly understood. Here, we determined the androgen dependence of hundreds of direct AR target genes on 18 coregulators that are relevant to CaP progression, and uncovered that the AR-dependent transcriptome breaks down in coregulator-dependent gene sets. Pairwise comparison and unsupervised clustering showed limited overlap between the gene subsignatures. Neither ChIP nor qRT-PCR studies revealed differences in the kinetics of coregulator recruitment to Androgen Response Elements (AREs) or of androgen regulation of different gene sets. Rather, Cistrome analyses of AR binding sites demonstrated preferential enrichment in binding motifs for distinct transcription factors (TFs) between coregulator-dependent gene sets. Pathway and GSEA analyses of these signatures indicated associations with select biological processes and differential enrichments between normal prostate and CaP, CaPs of different stages, and CaP and bone marrow microenvironment. These results suggested that coregulators may unite the action of DNA-bound AR and TF(s) to control select aspects of androgen-dependent CaP cell biology. The presence and functionality of such novel predicted AR transcriptional codes, namely AR-WDR77-p53 and AR-STAT3-IRF1 with projected roles in CaP cell proliferation and stemness respectively, was confirmed in Co-IP and gene expression profiling. As AR and p53 are the major drivers of lethal CaP, the AR-WDR77-p53 code was explored further. Mass spectrometry after WDR77 and p53 IP independently identified PGAM5, a recently isolated serine/threonine protein phosphatase that regulates cell death and unknown to be relevant to AR signaling or CaP biology, as part of the AR-WDR77-p53 complex. Co-IP assays verified IP-mass spectrometry results and ChIP studies confirmed recruitment of PGAM5 to WDR77-dependent AREs. Expression profiling demonstrated significant overlap in genes for which androgen regulation was altered after silencing of WDR77, PGAM5 or p53. Co-IP and ChIP studies showed that androgen-dependent recruitment of p53 to ARE-bound AR requires WDR77 and PGAM5. Western blotting and flow cytometry analyses indicated that WDR77 and PGAM5 and their dependent AR target genes control G1/S cell cycle progression, which was maintained in presence of clinically relevant gain-of-function p53 mutants. These novel insights indicate that disrupting select protein-protein and protein-DNA interactions may be a viable strategy to inhibit AR action that drives CaP lethal progression.

#1582

Therapeutic potential of combination therapy using a next generation antisense oligonucleotide targeting the androgen receptor and AKT inhibition with AZD5363 in genetically engineered mouse models of prostate cancer.

Marco A. De Velasco,1 Yurie Kura,1 Naomi Ando,1 Koichi Sugimoto,1 Kazuko Sakai,1 Barry R. Davies,2 Youngsoo Kim,3 A. Robert MacLeod,3 Masahiro Nozawa,1 Kazuhiro Yoshimura,1 Kazuhiro Yoshikawa,4 Kazuto Nishio ,1 Hirotsugu Uemura 1. 1 _Kindai University Faculty of Medicine, Osaka-Sayama, Japan;_ 2 _AstraZeneca, Macclesfield, United Kingdom;_ 3 _Ionis Pharmaceuticals, Carlsbad, CA;_ 4 _Aichi Medical University, Nagakute, Japan_.

Prostate cancer is highly dependent on androgen receptor (AR) and PI3K/AKT signaling pathways for survival and disease progression. Preclinical evidence suggests that combinatorial approaches targeting both AR and PI3K/AKT activity improves treatment efficacy. However, sustained responses from traditional and next-generation anti-androgen therapies targeting AR remain elusive in clinical practice due to inherent/acquired resistance resulting in lethal castration-resistant prostate cancer (CRPC). Mechanisms for continued AR transcriptional activity may be ligand dependent or independent but still require AR gene expression. Persistent AR gene expression is a key feature of CRPC. Thus, blocking AR gene expression by antisense oligonucleotides (ASO) is a logical approach to CRPC. We previously showed that monotherapy with ISIS581088, a generation 2.5 ASO targeting mouse AR, demonstrated strong antitumor activity in a transgenic mouse model of PTEN-deficient prostate cancer. In this study we show the antitumor effects of combined therapy of ISIS581088 and AZD5363, a potent AKT inhibitor and demonstrate the therapeutic benefit of combination therapy in a clinically relevant mouse model of CRPC. Sixteen-week-old mice with PTEN-/- castration-naïve prostate tumors were treated with ISIS581088 and AZD5363 alone or in combination for four weeks. Tumor growth inhibition rates were 41.2%, 20.2% and 54.4% for ISIS581088, AZD5363 and ISIS581088/AZD5363 treatment groups, respectively, P<0.001. In a model of mouse CRPC, 16-week-old mice with PTEN-/- castration-resistant prostate tumors (eight weeks post castration) experienced reduced tumor burden with all treatments but no enhancement was observed when the compounds were administered in combination compared with monotherapy drug treatments. In a randomized trial of advanced CRPC in PTEN/P53 double knockout mice, combination therapy significantly increased overall survival. Median survival were 18, 17 21, 22, and 38 days for control vehicle, control ASO, ISIS581088, AZD5363 and ISIS581088/AZD5363 treatment groups, respectively, P=0.041. In conclusion, our data shows that combination therapy significantly reduced tumor burden in mice with castration-naïve tumors compared to those treated with monotherapy. Notably, combination therapy did not produce an additive effect in an early stage CRPC intervention model. Still, combination therapy demonstrated a clear advantage in prolonging overall survival in a long-term randomized mouse trial of PTEN/P53-deficient CRPC. Thus, our data provides preclinical evidence to support that next generation ASOs targeting AR in combination with AKT inhibition is a potentially effective treatment approach for CRPC.

#1583

Inhibition of proline isomerase Pin1 interrupts the function of the androgen receptor N-terminal domain and suppresses androgen-independent growth of prostate cancer cells.

Jacky K. Leung, Yusuke Imamura, Minoru Kato, Nasrin R. Mawji, Marianne D. Sadar. _British Columbia Cancer Agency, Vancouver, British Columbia, Canada_.

Patients with advanced prostate cancer continue to develop lethal castration-resistant prostate cancer (CRPC) despite hormone therapy and maintaining castrate levels of serum androgen. Most CRPC appears to be dependent on the androgen receptor (AR), but instead of the C-terminal ligand-binding domain, it is the N-terminal domain (NTD) harboring a powerful transactivation domain that drives AR transcriptional activity. This was supported by the discovery of AR splice variants (ARv567es and V7) which are constitutively active, do not encode a functional ligand-binding domain, and correlate with poor patient outcome. The AR NTD is intrinsically disordered, but it contains several putative binding sites for Pin1, a proline isomerase specific for phosphorylated-Ser/Thr-Pro motifs. Since the innate ability of the AR NTD to adopt multiple transient structures is important for transactivation of AR, we aimed to determine whether Pin1 regulates motifs within the AR NTD.

We tested several known inhibitors of Pin1 in cell-based assays that measure proliferation or transcription mediated by AR. Our results demonstrated that inhibition of Pin1 interrupted the function of the AR NTD. The Pin1 inhibitor juglone effectively and specifically blocked transcription mediated by AR induced by androgen, as well as transactivation of the AR NTD in the presence of IL-6. We found that Pin1 predominantly interacted with a specific region of the AR NTD containing two Pin1 binding sites, and by inhibiting Pin1 the interactions between endogenous AR and STAT3 became attenuated. Furthermore, Pin1 inhibitors were more effective than second-generation anti-androgens in blocking androgen-independent proliferation of LNCaP95 cells driven by AR variants.

Here we describe that Pin1 is a critical factor for transcription mediated by AR, regardless of ligand, by regulating the AR NTD. Understanding the molecular mechanisms that may promote AR signaling in the absence of androgen will aid the development of more effective therapies for CRPC.

#1584

Wnt/beta-catenin and Foxa2 axis activates AR signaling in castration resistant prostate cancer.

Zachary M. Connelly, Shu Yang, Jiahe Li, Robert Matusik, Xiuping Yu. _LSUHSC Shreveport, Shreveport, LA_.

Background: Prostate cancer (PCa) is the leading cancer among men in the world. Androgen deprivation therapy is a common treatment to cease prostate growth. However, androgen deprivation eventually fails and PCa relapses, evolving into castration resistant prostate cancer (CRPCa). It has been established that androgen receptor (AR) continues to be the central player in mediating CRPCa. However, the mechanism by which the AR signaling remains active is unclear. We have reported that activation of Wnt/beta-catenin signaling pathway promotes castrate-resistant progression. Our study has also shown that activation of the Wnt/beta-catenin pathway induces the expression of Foxa2, a fork-head transcription factor that is expressed in embryonic prostate & advanced stage PCa. Here, we study the Wnt pathway and the role of Foxa2 in retaining active AR signaling in advanced PCa in the absence of androgens. TRAMP mice, SV40 T-antigen transgenic mouse models for PCa, were used. It has been recognized that in this model, T-antigen, which is driven by androgen-responsive probasin promoter, remains expressed following androgen ablation. In this study, we identified a mechanism that sustains an active AR signaling in CRPCa.

Methods: To study whether the Wnt/beta-catenin pathway is active in advanced stage PCa, we crossed the TRAMP mice with GFP-Wnt reporter mice. Some of the TRAMP/Wnt-reporter mice were castrated to study how androgen deprivation affects Wnt/beta-Catenin signaling and promotes PCa progression. Prostates were analyzed using IHC and IF staining. We also established a prostate epithelial cell line that stably expresses Foxa2. Cell proliferation was examined with IncuCyte Zoom software. The expression of AR target genes was assessed by using quantitative RT-PCR and Western blotting. ChIP was conducted to assess the recruitment of transcriptional machinery to the promoters of AR-regulated genes.

Results: Utilizing the TRAMP/Wnt mouse model, we show that Wnt/beta-Catenin signaling is active in advanced stage PCa. Additionally, our data showed that Foxa2, the downstream target of Wnt/beta-Catenin signaling, drives the expression of androgen-responsive genes, regardless of androgen deprivation. Furthermore, cell proliferation assays revealed that the expression of Foxa2 enabled an androgen-independent growth of prostatic cells. Using this model system, we further explored the mechanism that activates the transcription of AR-responsive genes in the absence of androgens. Our data indicated that Foxa2 is capable of recruiting H3k27Ac to AR-responsive promoters.

Conclusion: Together, these data indicate that Wnt signaling is activated in advanced stage PCa, driving Foxa2 expression, and promoting AR signaling to persist. This represents a novel mechanism that enables advanced PCa to retain AR signaling pathway following androgen ablation. Targeting both Wnt signaling and Foxa2 may pose as novel therapeutics for CRPCa.

#1585

Endostatin inhibits androgen-independent prostate cancer growth by suppressing nuclear receptor-mediated oxidative stress.

Joo Hyoung Lee, Minsung Kang, Gurudatta Naik, James A. Mobley, Guru Sonpavde, W. Timothy Garvey, Victor M. Darley-Usmar, Selvarangan Ponnazhagan. _University of Alabama at Birmingham, Birmingham, AL_.

Androgen-deprivation therapy has been identified to induce oxidative stress in prostate cancer (PCa), leading to re-activation of androgen receptor (AR) signaling in a hormone-refractory manner. Thus, antioxidant therapies have gained attention as adjuvants for castration-resistant PCa (CRPC). Here, we report for the first time that human endostatin (ES) prevents androgen-independent growth phenotype in PCa cells through its molecular targeting of AR and glucocorticoid receptor (GR), and downstream pro-oxidant signaling. This reversal following ES treatment significantly decreased PCa cell proliferation through downregulation of GR, and upregulation of manganese superoxide dismutase and reduced glutathione levels. Proteome and biochemical analyses of ES-treated PCa cells further indicated a significant upregulation of enzymes in major ROS scavenging machinery, including catalase, glutathione synthetase, glutathione reductase, NADPH-cytochrome P450 reductase, biliverdin reductase and thioredoxin reductase, resulting in a concomitant reduction of intracellular ROS. ES further augmented antioxidant system through upregulation of glucose influx, pentose phosphate pathway (PPP) and NAD salvaging pathway. This shift in cancer cell redox homeostasis by ES significantly decreased the effect of protumorigenic oxidative machinery on androgen-independent PCa growth, suggesting that ES can suppress GR-induced CRPC phenotype upon AR antagonism, and dual targeting action of ES on AR and GR can be further translated to PCa therapy.

#1586

Developing novel inhibitors of S100A4 for neuroendocrine (NE) and metastatic prostate cancer: systematic testing using relevant models and drug development techniques.

Mohammad Saleem,1 Arsheed A. Ganaie,1 Reihana Maqbool,1 Firdous A. Beigh,1 Syed Umbreen,1 Natalya G. Dulyaninova,2 Badrinath R. Konety3. 1 _Univ. of Minnesota Hormel Inst., Austin, MN;_ 2 _Albert Einstein College of Medicine of Yeshiva University, Bronx, NY;_ 3 _Univ. of Minnesota, Minneapolis, MN_.

S100A4, a calcium binding protein has been well studied as a marker of fibrosis and metastasis. We recently showed that in addition of being a metastatic marker, S100A4 is in fact an oncogene that plays an important role in the development of prostate cancer (CaP) and is amenable of targeting for the treatment of this lethal disease particularly neuroendocrine CaP (NE-CaP). Using a genetically engineered transgenic mouse model of NE-CaP, we show that knocking down of S100A4 significantly inhibited growth of prostate tumorigenesis and metastasis. Our noticeable finding is that S100A4 is secreted by prostatic tumors, and extracellular/soluble S100A4 acts as a growth factor that has the ability to confer aggressive potential to less aggressive or indolent tumor cells. We show that serum-S100A4 level is highly elevated in human CaP patients with aggressive disease regardless of their serum-PSA levels. We next asked if small molecule inhibitors could be developed to inhibit the activity if intracellular as well as extracellular S100A4. Using a highly robust Blue/Gene supercomputer-based in silico method, we screened a library of 5000 molecules and based on their binding efficacy to S100A4, identified potential inhibitors (SMI1 and SMI2). We next tested if SMI1 and SMI2 bind to the S100A4 protein in biological solution. We generated recombinant S100A4 protein and standardized an isothermal titration Calorimetry (ITC) assay for S100A4 binding. The ITC analysis (Kcal vs time) shows that both SMI1 and SMI2 inhibitors significantly bind to the S100A4 protein nevertheless SMI2 exhibited higher binding affinity to S100A4 than SMI1. Next, we used Surface Plasmon Resonance (SPR) method (accurate and sensitive technique) for detecting binding of inhibitors to S100A4. The SPR data (sensogram) shows that SMI1 and SMI2 bind to the S100A4 protein. The binding of S100A4 to Myosin IIA (MIIA) is known to disrupt the latter's monomer-polymer equilibrium. This phenomenon is captured in solution (in terms of disassembly of MIIA filaments and change in turbidity). Using disassembly/ or turbidity assays as an index of S100A4 activity (where recombinant S100A4 and MIIA are incubated +/- inhibitors), we show that SMI1 and SMI2 inhibit the activity of S100A4 protein. Next, we tested efficacy of inhibitors in vitro and show that SMI1 and SMI2 therapies inhibit the growth, proliferation, migration and invasiveness of NE-CaP (TRAMPC2) and AI-CaP (DU145, PC3) cells. Notably, SMI1 and SMI2 therapies inhibited the growth-promoting effects of extracellular S100A4 and decreased activities of S100A4 downstream target proteins (MMP9 and NFκB). These data suggest that S100A4 inhibitors (SMI1 and SMI2) exhibit high anti-metastatic efficacy and are the potential candidates for treating NE-CaP and metastatic AI-CaP. The validation of SMI1 and SMI2 under in vivo models is underway in our laboratory.

#1587

Disruption of nuclear export with selinexor or KPT-8602 reduces androgen receptor expression and leads to potent anti-tumor activity in preclinical models of androgen-independent prostate cancer.

Christian Argueta, Boris Klebanov, Trinayan Kashyap, Hua Chang, Sharon Friedlander, Erkan Baloglu, Margaret Lee, Humphrey Gardner, Sharon Shacham, William Senapedis. _Karyopharm Therapeutics, Newton, MA_.

Metastatic castration resistant prostate cancer (mCRPC) is an advanced form of prostate cancer (PC) associated with poor prognosis. Approximately 20 - 40% of mCRPCs are androgen-independent and do not respond to treatment with abiraterone or enzalutamide, drugs that suppress androgen synthesis or directly target androgen ligand binding domains (LBD) respectively. Patients initially responding to these drugs eventually relapse, highlighting the need for alternative therapies. Resistance is attributed to the emergence of constitutively active AR splice variants lacking C-terminal LBDs such as AR variant 7. Although ARv7 activity cannot be mitigated by current therapeutic approaches, it is known that AR mRNA is exported from the nucleus by eIF4E and exportin-1 (XPO1). Selinexor and KPT-8602 are orally bioavailable SINE (Selective Inhibitor of Nuclear Export) compounds that specifically target XPO1. The purpose of this study is to provide mechanistic evidence for using SINE compounds as novel therapies for androgen-independent mCRPC.

Methods: In vitro, selinexor or KPT-8602 were tested on androgen-independent PC cell line 22Rv1. RNA and protein were analyzed by qPCR and immunoblot. Cell viability was examined using the Celltiter-Glo assay. In vivo, immune deficient SCID mice were injected subcutaneously with 22Rv1 cells. Tumors were grown to ~150 mm3 before treatment with selinexor (10 mg/kg qodx3) or KPT-8602 (15 mg/kg reduce to 10 mg/kg qdx5). Tumor growth and animal weights were monitored to determine tumor growth inhibition (TGI) and tolerability to treatment. Tumors were analyzed using immunohistochemistry (IHC).

Results: We found that expression of AR, ARv7 and prostate specific antigen (PSA; transcriptionally regulated by AR) proteins are reduced following 24-hour treatment with SINE compounds. Nuclear vs. cytoplasmic fractionation of RNA revealed that ARv7 and PSA mRNA localization was increased in the nucleus (4-fold and 3-fold, respectively) and reduced in the cytoplasm (5-fold and 3-fold, respectively). Moreover, KPT-8602 potently inhibited cell viability (IC50: 100 nM), while enzalutamide and abiraterone had no effect. Finally, mice bearing 22Rv1 xenografts, treated with selinexor or KPT-8602 exhibited a complete reduction in tumor volume (95% and 94% TGI, respectively), which coincided with prolonged overall survival. IHC analysis showed a reduction of proliferation markers and a concomitant increase in cell death makers in selinexor and KPT-8602 treated tumors.

Conclusion: SINE compounds show strong anti-tumor activity in androgen-independent prostate cancer models in vitro and in vivo by reducing AR, ARv7, and PSA expression. These findings highlight the promise of SINE compounds as treatment options for androgen-independent mCRPCs resistant to first line therapies.

#1588

Effects of the selective CYP17-lyase and androgen receptor (AR) inhibitor, seviteronel, and the cyclin-dependent kinase (CDK) 4/6 inhibitor, G1T38, on tumor growth in an AR-V7+ castration-resistant prostate cancer (CRPC) xenograft model.

Suzanne E. Wardell,1 Alexander P. Yllanes,1 John D. Norris,1 James P. Stice,1 Hannah White,2 Ronald A. Fleming,3 Jay C. Strum,2 William R. Moore,3 Donald P. McDonnell1. 1 _Duke University School of Medicine, Durham, NC;_ 2 _G1 Therapeutics, Research Triangle Park, NC;_ 3 _Innocrin Pharmaceuticals, Durham, NC_.

Background: Castration-resistant prostate cancers that express the constitutively active androgen receptor (AR) variant AR-V7 exhibit significantly reduced progression-free and overall survival in response to the AR targeting agents, enzalutamide (ENZ) and abiraterone. Inhibition of Phosphoinositide-3-Kinase (PIK3CA) or CDK4/6 has been proposed as alternative therapies that directly target the cell cycle pathway in these tumors. However, the combined role of AR plus cell cycle inhibition in this treatment setting has not been established. G1T38 is a potent, selective clinical stage CDK 4/6 inhibitor. Seviteronel (SEVI) is a selective CYP17-lyase and AR inhibitor that blocks the growth of CRPC tumors with clinically relevant AR mutations, including T878A and AR-F876L. The activity of SEVI, ENZ and G1T38, alone or in combination, was evaluated using the 22Rv1 (AR-V7+) CRPC model in vitro and in vivo.

Methods: Cellular proliferation (7 days) of 22Rv1 prostate cancer cells treated with SEVI, ENZ, and G1T38 alone or in combination with SEVI or ENZA was assessed by measuring DNA content. Orchiectomized male nu/nu mice bearing 22Rv1 were randomized to receive vehicle, SEVI (150mg/kg/day p.o.), ENZ (30 mg/kg/day p.o.), G1T38 (25-100 mg/kg/day p.o.), or SEVI or ENZ in combination with G1T38. A docetaxel (20 mg/kg weekly i.p.) group was included as a standard of care comparison. Time to progression (X-fold increase in tumor volume) and tumor volume changes were assessed over a 3-7-week dosing period. Blood and tissues were collected for analysis of exposure and pharmacodynamics markers of response.

Results: SEVI and G1T38 alone or in combination, significantly reduced the proliferation of 22Rv1 cells in vitro (p<0.05). Compared to vehicle, SEVI and G1T38 monotherapy significantly inhibited tumor growth and time to progression by 1.5 and 2.5 fold, respectively (p<0.05); the combination of SEVI and G1T38 further decreased tumor growth and resulted in a 4-fold delay in progression (p<0.001). In contrast, ENZ alone was without effect on tumor growth and did not increase response to G1T38 as compared to G1T38 alone.

Conclusions: SEVI and G1T38 mono- or combination therapy significantly decreased the growth of the AR-V7+ 22Rv1 CPRC model in vitro and in vivo, an activity that distinguishes SEVI from ENZ. Therefore, combining SEVI with a CDK4/6 inhibitor such as G1T38 may be an effective therapy for the treatment of CRPC that is resistant to current standards of care.

#1589

Zinc inhibits androgen receptor expression to inhibit prostate cancer cell growth.

Phuong Kim To, Young-Suk Cho, Se-Young Kwon, Taek Won Kang, Kyung Keun Kim, Chaeyong Jung. _Chonnam National Univ. Med. School, Hwasun-gun, Republic of Korea_.

Background: Prostate gland contains high level of intracellular zinc which is dramatically diminished during cancer development. Due to the obscure role of zinc in this process, therapeutic application using zinc and its supplement is very limited. This study aims to clarify the role(s) of zinc and its intervening mechanism.

Material and methods: Treated by zinc chloride (15-150 µM), several prostate cancer cell lines were applied to confocal microscopy for intracellular trafficking of exogenous zinc, in vitro proliferation assays for their growth, prostate specific antigen (PSA)-based reporter-mediated transactivation, and Western blot for detection of androgen receptor (AR), PSA and ubiquitination. Further in vivo studies were performed to demonstrate the effect of zinc (10-20 mg/kg) on xenograft cancer growth using syngeneic animals followed by tumor analyses.

Results: Zinc chloride suppressed androgen-dependent proliferation of human prostate cancer cells and accordingly zinc chloride dramatically inhibited androgen-mediated transactivation and several androgen target protein expressions, including PSA and p21. Further investigation showed that addition of zinc chloride strikingly downregulated AR protein levels after 4 hours up to 24 hours in both human LNCaP and murine TRAMP C2 prostate cancer cell lines. AR downregulation resulted from facilitated protein degradation instead of transcriptional control. Further in vivo study was carried out using syngeneic mice bearing C2 subcutaneous tumors. Peritoneal injection of zinc chloride significantly reduced tumor size. Analysis of these tumors revealed that there were reduced expression of AR and increased cell death.

Conclusions: Zinc has been shown to inhibit incumbent oncogenic NF-κB pathway. These results also suggest that intracellular zinc inhibits cell growth via downregulation of AR to inhibit growth of prostate cancer. Considering that AR functions as a major effector in prostate cancer development and progression into castration resistant prostate cancer, loss of zinc may be a critical step for this devastating disease and further studies can be performed to develop zinc-based cancer therapeutics.

#1590

High intranuclear mobility of AR-v7 reveals distinct mode of transcriptional activity in prostate cancer with important therapeutic implications.

Seaho Kim, Mohd Azrin Jamalruddin, Paraskevi Giannakakou. _Weill Cornell Medicine, New York, NY_.

It is well established that androgen receptor (AR) signaling, is a key driver of prostate cancer (PC) growth and metastatic progression. Therefore, androgen deprivation therapy (ADT) is the first line of treatment for PC. However, most patients develop castration resistant prostate cancer (CRPC). Interestingly, AR signaling remains active in CRPC, due to the expression of transcriptionally active AR splice variants (AR-Vs), which lack the ligand binding domain (LBD) and constitutively translocate to the nucleus even in castrate conditions. AR-v7 is the most prevalent AR-V expressed in about 60% of CRPC tumors. AR-v7 expression was clinically correlated with poor prognosis of CRPC patients and with resistance to next-generation AR signaling inhibitors, which are part of standard clinical care. Therefore, inhibition of AR-v7 function is urgently needed for the treatment of CRPC. Currently, there is no therapeutic modality that can inhibit AR-v7 expression or activity. Mechanistically, AR-v7 transcriptional targets largely overlap with those of AR-fl, with the exception of a few AR-v7 unique targets. However, the exact mechanism by which transcription is activated by AR-v7 is not known. In this study we sought to investigate the mechanisms underlying the transcriptional activity of the ligand-independent AR-v7 in comparison to liganded AR-fl. We used live cell imaging to monitor the dynamics and intranuclear mobility of fluorescently-tagged AR-v7 or AR-fl. Fluorescent recovery after photobleaching (FRAP) revealed that AR-v7 intranuclear mobility was significantly faster than that of liganded AR-fl, with t1/2 3s versus several minutes, respectively. To precisely map the spatial distribution and chromatin-binding kinetics of AR-fl and AR-v7, we generated expression plasmids with AR tagged with green-to-red mEos4 photo-convertible proteins. Photoconversion of a discrete, subnuclear pool of AR-fl in the presence of its ligand (R1881) followed by 1 hr of time-lapse imaging in 5 min intervals, revealed absence of mobility indicating tight chromatin binding and active transcription. In contrast, similar photoconversion experiment for AR-v7, revealed immediate redistribution throughout the nucleus in less than 9 s, suggesting a "hit-and-run" mode of interaction with DNA with uncertain transcriptional output. QRT-PCR of endogenous target genes and ARE-mcherry reporter assays showed similar transcriptional activity of the two proteins. We are currently investigating the relationship between rates of intranuclear mobility and transcriptional activity and the mechanisms underlying the distinct mobility patterns. These data suggest that AR-v7 has a distinct mode of interaction with DNA and gene promoters, which may identify novel targetable pathways for its inhibition in CRPC.

#1591

Inhibition of prostate tumor growth by an extract from the muscadine grape.

Patricia E. Gallagher, E. Ann Tallant. _Wake Forest School of Medicine, Winston-Salem, NC_.

Despite decades of advancements, cancer remains the second leading cause of mortality in the United States with an estimated 1.7 million new cases and over 600,000 deaths predicted for 2016, indicating a clear need for more effective chemotherapeutic and chemopreventive agents. While natural products derived from plants have served as an abundant source of anticancer agents, it is predicted that less than 2 percent of the plant species with medicinal value have been investigated. Muscadine grape (Vitis rotundifolia) skin and/or seed extracts are a popular nutraceutical supplement due to the high anti-oxidant, anti-inflammatory, and anti-proliferative effects of a distinct phytochemical composition of polyphenols and proanthocyanidins. The current study examines the effects of a proprietary muscadine grape extract formulation (MGE; Piedmont Research & Development Corp.) on the proliferation of prostate cancer cells and tumors. MGE inhibited the growth of human LnCaP prostate cancer cells, in both a time- and dose-dependent manner, as quantified using an IncuCyte Zoom Imaging system. Cell growth was reduced by 36.8% after a 5-day treatment with 20 µg/mL MGE compared to untreated cells (n = 6, p < 0.001). Athymic mice (male, 15-20 g, 5-6 weeks of age) were injected subcutaneously on the lower flank with 1.6 x 106 LNCaP cells, to assess the effect of the MGE in vivo. When the tumors reached 100 mm3, the mice were randomized and MGE was added to the drinking water at a concentration of 0.2 µg phenolics/mL (1.0 mg phenolics/mouse/day for a 25 g mouse); control mice drank untreated water. After 5 weeks, the mice were sacrificed and the tumors were weighed. The volume of prostate tumors growing in the flank of nude mice drinking water with MGE was markedly reduced compared to the size of the tumors from control mice [252.6 ± 61.73 mm3 (MGE) versus 765.2 ± 101.8 mm3 (control); n=6, p<0.001). Further, the weight of prostate tumors from mice drinking MGE was also significantly decreased [0.96 ± 0.14 g (control) as compared to 0.41 ± 0.11 g (MGE)], a reduction of 57% (p < 0.05). Five microns tumor sections were incubated with an antibody to CD34 and tumor vessels were identified by positive immunoreactivity and morphology. MGE reduced the number of blood vessels/field from 10.25 ± 0.9 to 7.10 ± 0.6 (p < 0.05), suggesting that the extract inhibits angiogenesis. Vascular endothelial growth factor (VEGF) and placental growth factor (PLGF) were measured by qPCR, to determine the effect of the extract on angiogenic factors. MGE reduced VEGF mRNA from 1.02 ± 0.06 to 0.72 ± 0.07 relative units (p < 0.05) and PLGF mRNA from 1.03 ± 0.11 to 0.63 ± 0.07 relative units (p < 0.05), providing additional evidence of an anti-angiogenic effect of the extract. Collectively, these results are the first to demonstrate that a novel MGE formulation reduces prostate tumor growth in a mouse model, by inhibiting angiogenesis, suggesting that MGE may be an effective treatment for prostate cancer.

#1592

Anti-cancer evaluation of various solvent extracts of blue honeysuckle berry (Lonicera caerulea L.) against prostate cancer cells.

Syed M. Ali,1 Alex Ourth,2 Chun-Tao Che,2 Mian-Ying Wang,1 Gnanasekar Munirathinam1. 1 _University of Illinois College of Medicine, Rockford, IL;_ 2 _University of Illinois at Chicago, Chicago, IL_.

Prostate cancer (PCa) is the second most common cancer among men in the United Sates. It is estimated 1 in 6 men will be diagnosed with PCa and approximately 180,890 new cases are predicted in 2016 alone. Standard treatments for PCa include surgery, radiation, chemotherapy and hormonal therapy or a combination of these treatments. However, factors like patient health, drug resistance, specificity, and toxicity can result in poor disease prognosis. In order to overcome these limitations and improve patient prognosis, there is an urgent need to identify new anti-cancer agents with minimal side effects. Current studies are being focused on natural products and their components for alternative therapeutics. Blue Honeysuckle (Lonicera caerule L.) a berry native to northeast Asia, is known to be rich in Vitamin C and polyphenols such as anthocyanins, flavonoids, and phenolic acids. Polyphenols are found to have several therapeutic effects such as anti-inflammatory, antioxidant and antimicrobial properties. Our present study used sequential solvent extracts of Blue Honeysuckle (BHS) berry using Hexane, Ethyl Acetate, Methanol, and Water respectively. These fractions were used to assess various therapeutic effects of BHS on DU 145, PC-3, C4-2 and LNCaP PCa cell lines. The goal was to identify the most potent BHS fraction that is effective against PCa using pre-clinical studies. MTT assays were used to identify the anti-proliferative effects of various BHS fractions. The above indicated PCa cells were treated with different doses (10-150 µg/mL) of BHS factions over various time periods (24, 48, 72 hr). Our data revealed that Hexane extract (HE) exhibited the highest inhibition of cell viability in a time and dose-dependent manner. HE fraction showed to have an IC50 of 89.6 μg/mL for DU 145, 117.4 μg/mL for PC-3, 163.3 μg/mL for C4-2 and 140.84 for LNCaP cells. Moreover, BHS HE fraction showed a decrease in migration capacity and colony formation ability of PCa cells in vitro. Cellular senescence assay was performed to assess β-Galactosidase activity in PCa cells treated with BHS HE extract. All the PCa cell lines treated with 100 µg/mL of BHS HE showed increased senescence. Western blotting was performed to identify the potential anti-cancer mechanism of BHS against PCa using DU 145 and PC-3 cell lines. Results from this study indicated that DU 145 and PC-3 cell lines after 24 hr treatment with 100 μg/mL of various BHS fractions especially BHS HE showed an increase in apoptosis (Caspase 3,8,9 and PARP-1) and autophagy (LC3 A/B) markers. Furthermore, cell cycle analysis of DU 145 and PC-3 cells treated with BHS HE extract showed increased apoptotic cells. Further analysis of apoptotic and autophagy pathways modulated by BHS treatments are required to illustrate its potential underlying anti-cancer mechanisms in PCa. In conclusion, results from our study warrant further evaluation of BHS berry for potential PCa management.

## IMMUNOLOGY:

### Cytokines: The First Modern Immunotherapies

#1593

Neutralization of IL-10 enhances antitumor efficacy of dendritic cell-targeting MIP-3α-gp100 vaccine by way of type-I interferons in B16F10 mouse melanoma model.

James Gordy, Kun Luo, Richard Markham. _Johns Hopkins School of Public Health, Baltimore, MD_.

The chemokine MIP-3α (CCL20) binds to CCR6 found on immature dendritic cells. Vaccines fusing MIP-3α to gp100 have been shown to be effective in therapeutically alleviating melanoma in mouse models. Other studies have provided evidence that IL-10 neutralizing antibodies enhance immunological melanoma therapies by modulating the tolerogenic tumor microenvironment. Here, we report that neutralizing IL-10 enhances the therapeutic anti-melanoma efficacy of a MIP-3α-gp100 DNA vaccine. The current studies utilize the B16F10 syngeneic mouse melanoma model system. The MIP-3α-gp100 DNA vaccine is administered intramuscularly (i.m.) into the tibialis muscle, followed immediately by i.m. electroporation. The standard therapeutic protocol was the following: challenge with lethal dose of B16F10 cells (5x104) on day 0; vaccinate with 50μg vaccine plasmid on days 3, 10, and 17; and administer 150μg of αIL10 antibody intra-tumorally beginning on day 5, once every three days for up to six doses. Tumor sizes, growth, and survival were all assessed. Treatment responses were characterized by flow cytometric analysis of tumor infiltrate. Vaccine-specific T-cells were delineated by gp10025-33 stimulation followed by intracellular cytokine staining for IFN-γ and assessment by flow cytometry. The mechanism of αIL-10 efficacy was explored by RT-PCR and confirmed with a knockout mouse model. With this therapeutic protocol, we demonstrate for the first time that a therapy neutralizing IL-10 additively enhances the anti-tumor efficacy of a MIP-3α-gp100 vaccine, leading to significantly smaller tumors, slower growing tumors, and overall increases in mouse survival. Surprisingly, the additive effects of αIL-10 were not shown to be directly mediated by any T-cell parameter tested, including vaccine-specific tumor infiltrating lymphocytes (TILs), total TILs of either CD4+ or CD8+ subset, regulatory T-cells, granzyme positive T-cells, and others. We discovered, however, that IFNα-4 transcripts in the tumor were significantly upregulated in mice given vaccine and αIL-10 compared to vaccine alone. Furthermore, infiltration into the tumor of plasmacytoid dendritic cells, known to be professional IFNα-producing cells, were enhanced with the combination therapy. A mouse model with IFNαR1 knocked out eliminated the protection provided by αIL-10, demonstrating that the additional therapeutic value of αIL-10 is primarily mediated by type-I interferons. In conclusion, efficient targeting of antigen to immature dendritic cells with a chemokine fusion vaccine offers a potential alternative approach to the ex vivo dendritic cell antigen loading protocols currently undergoing clinical investigation. Combining this approach with an IL-10 neutralizing antibody therapy that modulates the tolerogenic tumor microenvironment offers promise as a novel melanoma therapy.

#1594

Enhancement of the anti-tumor activity of CEA TCB via combination with checkpoint blockade by PD-L1 and interleukin-2 variant immunocytokine.

Marina Bacac, Sara Colombetti, Linda Fahrni, Tanja Fauti, Valeria Nicolini, Johannes Sam, Petros Papastogiannidis, Marine Le Clech, Xavier Miot, Inja Waldhauer, Karolin Rommel, Christian Gerdes, Christian Klein, Pablo Umaña. _Roche Innovation Center Zurich, Schlieren, Switzerland_.

Cancer immunotherapy represents a promising therapeutic approach to extend the overall survival of cancer patients. However, several mechanisms within the tumor microenvironment orchestrate the suppression of host immune response requiring combination strategies to prolong the durability of effect. Among suppressive pathways, up-regulation of PD-1 ligand (PD-L1) and its interaction with PD-1 receptor plays a key role in suppression of T-cell activity, a mechanism also called adaptive immune resistance. Therefore, approaches able to inhibit the tumor immune suppressive mechanisms along with those expanding the frequency of intra-tumor T-cells and enhancing/prolonging their functionality are required.

CEA TCB (RG7802, RO6958688) is a novel T cell bispecific antibody targeting carcinoembryonic antigen (CEA) on tumor cells and CD3 on T cells, currently being investigated as single agent and in combination with atezolizumab in Phase 1/1b studies in patients with advanced and/or metastatic CEA-expressing tumors (NCT02324257; NCT02650713). CEA TCB treatment leads to increased intra-tumoral T cell infiltration and T-cell activation along with up-regulation of PD-1/PD-L1 suppressive pathway. Here we show that combination of CEA TCB with PD-L1 blocking antibody in vitro enhances T cell activation as detected by increased CD3 signaling and secretion of pro-inflammatory cytokines. Combination in vivo performed in both stem cell humanized NOG mice (HSC mice engrafted with MKN45) and fully immunocompetent human CEA transgenic C56BL/6 mice (hCEA Tg mice engrafted with MC38-hCEA) demonstrated significantly improved anti-tumor activity of combination as compared to activity of single agents, yielding to increased number of tumor-free animals. Randomization of animals that progressed to CEA TCB monotherapy revealed that combination of CEA TCB with PD-L1 blocking antibody is required to control tumor outgrowth, as tumors treated with corresponding monotherapy arms progressed to treatment. Efficacy of CEA TCB was also potentiated when administered in combination with a half-life-extended IL-2 variant (untargeted (IgG-IL2v) or fibroblast-activating protein-targeted IL-2 variant (FAP-IL2v)), resulting in stronger tumor growth inhibition in MKN45-bearing HSC mice or prolonged survival in PanCO2-hCEA-bearing hCEA Tg C56BL/6 mice. Synergy likely reflects ability of IL2v to enhance anti-tumor efficacy by increasing number of effector T cells in tumors.

In conclusion, CEA TCB treatment leads to intra-tumoral T cell infiltration and T-cell activation. This is accompanied by up-regulation of PD-1/PD-L1 suppressive pathway, which can be overcome by combination therapy with a PD-L1 inhibitor. In vivo efficacy of CEA TCB is further potentiated when administered in combination with immunotherapies that increase the pool of available tumor-infiltrating effector cells.

#1595

IL15/IL15Rα heterodimeric Fc-fusions with extended half-lives.

Matthew J. Bernett, Christine Bonzon, Rumana Rashid, Rajat Varma, Kendra N. Avery, Irene W. Leung, Seung Y. Chu, Umesh S. Muchhal, Gregory L. Moore, John R. Desjarlais. _Xencor, Inc., Monrovia, CA_.

IL15 and IL2 are similar cytokines that stimulate the proliferation and differentiation of B cells, T cells, and NK cells. Both cytokines exert their cell signaling function through binding to a trimeric complex consisting of two shared receptors, the common gamma chain and IL2Rβ, as well as an alpha chain receptor unique to each cytokine: IL2Rα or IL15Rα. IL2Rα is highly expressed on Tregs, and the therapeutic benefit of IL2 for cancer treatment has been limited for this reason. IL15 is produced by monocytes and dendritic cells and functions as a stabilized heterodimeric complex with membrane-bound IL15Rα present on the same cells. This IL15/IL15Rα complex is presented in trans to NK cells and CD8+ T cells expressing IL2Rβ and the common gamma chain. It has been shown that recombinant IL15/IL15Rα heterodimer is highly active. Currently there are no approved versions of recombinant IL15 although several clinical trials are ongoing. As potential drugs, cytokines suffer from a very fast clearance that hinders favorable dosing. Consequently, a more druggable version of IL15 would consist of the IL15/IL15Rα complex coupled with slower clearance. We created various IL15/IL15Rα heterodimeric Fc-fusions in an effort to facilitate production, promote FcRn-mediated recycling of the complex, and thus prolong half-life.

We engineered IL15/IL15Rα heterodimeric Fc-fusions by either fusing IL15 to one side of a heterodimeric Fc-region, and the sushi domain of IL15Rα to the other side, or by creating a single-chain IL15/IL15Rα that was attached to one side of a heterodimeric Fc-region. These Fc-fusions were tuned for optimal activity by engineering the linker regions between IL15/IL15Rα and the Fc and/or by engineering substitutions on IL15 at the IL15/IL2Rβ or IL15/gamma chain interface. In vitro proliferation of T and NK cells in healthy PBMCs was monitored by counting Ki67+ cells after incubation with Fc-fusions for 4 days. In vivo activity was evaluated using a mouse model in which human PBMCs are engrafted into NSG mice (huPBMC-NSG) and measuring the extent of T cell engraftment by flow cytometry as well as IFNγ. PK was evaluated in C57BL/6J mice.

IL15/IL15Rα heterodimeric Fc-fusions were successfully produced with favorable yields. The Fc-fusions enhanced proliferation of T and NK cells in vitro. Treatment of huPBMC-NSG mice with weekly doses of IL15/IL15Rα heterodimeric Fc-fusions promoted enhanced T cell engraftment and elevated levels of IFNγ in a dose dependent manner. Severe graft versus host disease was observed in treated mice. Little activity was seen with a comparable weekly dose of recombinant IL15. PK in C57BL/6J mice indicated half-lives of several days for the IL15/IL15Rα heterodimeric Fc-fusions, which are significantly longer than the <1 hr half-life reported for IL15.

Together, these data indicate that IL15/IL15Rα heterodimeric Fc-fusions demonstrate the high activity of IL15 with a more favorable PK profile.

#1596

Phase I trial of recombinant human Interleukin 15 (rhIL-15) administered as continuous intravenous infusion (CIV) for 10 days (240 hours) in patients with refractory metastatic cancers.

Kevin C. Conlon, Sigrid Dubois, Chyi-chia Richard Lee, Thomas Fleisher, Jennifer I. Hsu, Thomas A. Waldmann. _National Cancer Inst., Bethesda, MD_.

Twenty-seven refractory metastatic cancer patients received continuous intravenous recombinant human Interleukin 15 (CIV rhIL-15) for 10 days (240 hours) in a phase 1 dose escalation trial. Six dose levels (0.125, 0.25, 0.5, 1, 2 and 4 micrograms/kilogram/day [mcg/kg/day]) were assessed and 2 mcg/kg was the maximum tolerated dose Common adverse events included fevers, myalgias, decreased albumin, anemia, decreased leukocytes, lymphocytes and platelets and elevations of ALT, AST and bilirubin most often grade 1 or 2 in severity. Serious adverse events were grade 5 (gr5) visceral ischemia, lymphocytic gastritis (gr2), hospitalization for abdominal pain (gr2), papilledema (gr3), duodenal hemorrhage (gr3), bronchial hemorrhage (gr3) and unexplained sudden death (gr5) at home post-treatment in a patient who stopped treatment early for toxicity and symptomatic disease progression. Patients treated at the higher doses developed a striking end of treatment peripheral blood lymphocytosis showing a 24X increase in absolute lymphocyte count, 10X increase in CD8 cells and 75X increase in NK cell numbers. No patients developed anti-IL-15 antibodies. Steady state serum IL-15 level was seen within the first 12 to 24 hours of therapy and was maintained for several days. Dramatically lower IL-15 levels (≈ 8% of early Cmax) were measured during the last 2 days of treatment suggesting a large reservoir of non-circulating IL-15 receptor positive cells. None of the patients achieved a partial response, but 2 patients met the criteria for treatment beyond cycle 2 (≥ 15% decrease in measurable disease). Evidence for tumor directed immune effects included several patients who developed unilateral pleural effusions in their disease involved hemithorax, complete regression of a cutaneous chondrosarcoma metastasis and decreased CA19-9 (125.7 → 47.3 U/mL) in a patient with pancreatic cancer. Histologic analysis of pleural fluid from these patients showed largely lymphocytes and other cells that were consistent with the patients known cancers. Lymphocytic infiltration of the regressing cutaneous sarcoma metastasis was predominantly CD4 cells, with some CD8 cells and a few NK cells. Immunohistochemical analyses of pre and on treatment tumor biopsies obtained from several other patients are currently being performed to better characterize the immunologic effects of this regimen. The results of this trial indicate CIVrhIL-15 treatment generates considerable in vivo immune activation and has important synergistic potential for combination therapy with other Immunotherapeutics.

#1597

Inhibition of the novel therapeutic target pregnancy associated plasma protein A (PAPP-A) in Ewing sarcoma enhances efficacy of IGF1R targeting in vivo.

Sabine Heitzeneder,1 John F. Shern,2 Lee J. Helman,2 Javed Khan,2 Crystal L. Mackall1. 1 _Stanford University, Palo Alto, CA;_ 2 _National Cancer Institute, Bethesda, MD_.

Background: Despite intensive treatment regimens, Ewing sarcoma (EWS) patients with metastatic or relapsed disease still face a 5-year overall survival of less than 20%, indicating a clear need for novel targeted therapies. Utilizing ribosomal depleted RNA sequencing of 122 EWS samples (49 cell lines and 73 tumor samples) and 96 normal samples of variable tissues, we previously reported PAPP-A as one of the top 5 genes overexpressed in EWS (> than 4-fold) compared to normal tissue. Notably, PAPP-A showed substantial expression in tumors (median log2FPKM = 3.933, n=122) and minimal expression in normal tissue (median log2FPKM = -1.564, n=96). Pregnancy Associated Plasma Protein A (PAPP-A) is a secreted zinc metalloproteinase, that anchors to cell surface of heparan sulfate proteoglycans and enhances local IGF signaling. Through the release of IGFs from IGFBP-4, IGFBP-2 and IGFBP-5, free, bioactive IGF is being increased in close proximity to its receptors. In pregnancy PAPP-A is highly expressed by placental trophoblasts and represents a key regulator of fetal growth. The impact of IGF signaling in EWS is illustrated by the modest activity of IGF-1R inhibition as a single agent treatment in clinical trials, an approach that is limited by the rapid development of resistance.

Methods: In an attempt to investigate the role of PAPP-A in Ewing sarcoma, we used CRISPR/Cas9 technology to target the PAPP-A locus in EWS cell lines and generated PAPP-A knockout clones (EW8, TC32) for further investigation in vitro and in vivo. Additionally, we tested inhibition of PAPP-A proteolytic function in a xenograft NSG mouse model, as a single agent treatment as well as in conjunction with IGF1R inhibition.

Results: Knockout of PAPP-A in EWS cell lines utilizing CRISPR/Cas9 technology completely abrogated PAPP-A secretion and metalloproteinase activity in single cell clones, resulting in significantly increased complexed IGFBP-4 and diminished bioactive IGF-1, together with decreased cell growth in vitro. This phenotype could be rescued by recombinant, soluble PAPP-A. In vivo experiments showed that treatment with a monoclonal PAPP-A neutralizing antibody that inhibits the IGFBP-4 proteolytic activity of PAPP-A delays tumor growth of xenografted EWS tumor cells (EW8) in NSG mice. Furthermore, in conjunction with IGF1R inhibition (mAB h7C10), PAPP-A neutralization significantly decreased tumor growth and resulted in prolonged survival of mice (p=0.021).Conclusions: PAPP-A is a novel biologically relevant and highly tumor specific cell surface target in Ewing sarcoma. Our data suggest that neutralization of PAPP-A is a new therapeutic option that merits further evaluation for the treatment of Ewing's Sarcoma.

#1598

Single agent NKTR-214, a biased IL2 pathway agonist, increases immune cell infiltrates in brain tumors and prolongs survival in rodent (rattus) glioblastoma (GBM).

Lawrence Recht,1 Seema Nagpal,1 Taichang Jang,1 Milton Merchant,1 Irene Choi,2 Ute Hoch,2 Deborah Charych2. 1 _Stanford U School of Medicine, Stanford, CA;_ 2 _Nektar Therapeutics, San Francisco, CA_.

Background: Immunotherapy is an attractive option for brain tumor therapy if a robust infiltrative T cell response can be elicited in the tumor. NKTR-214 is a CD122-biased cytokine agonist conjugated with multiple releasable chains of polyethylene glycol and designed to provide sustained signaling through the heterodimeric IL-2 receptor pathway (IL-2Rβγ) to preferentially activate and expand effector CD8+ T and NK cells over Tregs. To assess the potential activity of single agent NKTR-214 in GBM, we used an orthotopic rat glioblastoma survival model.

Methods:NKTR-214 was administered at 0.1 or 0.3 mg/kg q2w iv to Sprague-Dawley rats starting 2 or 7 days (D2, D7) post (p)-implantation of 106 C6 glioma cells into the right striatum. The model requires euthanasia by ~D14 due to tumor burden. Brain tumors were characterized by magnetic resonance (MR) and immunohistochemistry (IHC) for infiltration of CD4+ and CD8+ T cells and for retention of PEG polymer in brain tumor.

Results: Compared to rats receiving vehicle (n = 15), survival was significantly prolonged after NKTR-214 treatment (n = 43, mean 17.2 vs. 10.0 days (P < 0.001) with 15% of rats across all groups alive and tumor-free at Day 50 when the study was terminated. Both doses were equally effective and well tolerated. Surprisingly, treated rats bearing large MR-detectable D7 tumors survived significantly longer compared to rats bearing microscopic D2 tumors (6/21 or ~30% versus 0/21 or 0% respectively at D50, mean 23.1 vs. 12 days, P < 0.004). Concordantly, CD8+ T cells in D7 tumors were significantly increased after NKTR-214 therapy compared to vehicle and D2 tumors, while CD4+ remained low with no significant difference between groups. PEG polymer was detected in the tumor at least 72 hours p-injection.

Conclusions: NKTR-214 is well tolerated, prolongs survival and induces immunological activity in the brain when administered to rats harboring orthotopic GBM. While there was no significant dose dependence, a marked increase in survival was observed when larger D7 tumors were treated with NKTR-214 compared to microscopic D2 tumors, associated with increased intratumoral CD8+ T cells. Levels of CD4+ were unchanged, consistent with the mechanism of CD-122 biased activation of the IL2 pathway. While requiring further study, it is intriguing that the increased sensitivity of larger tumors also corresponds to onset of angiogenesis and rapid tumor growth in this model. NKTR-214 is currently being evaluated in an outpatient Phase 1 / 2 clinical trial for the treatment of solid tumors. The results presented suggest a potential role for NKTR-214 in the treatment of patients afflicted with GBM.

#1599

Antibody-based inhibition of CSF-1R as a component of combination immunotherapy in preclinical models.

David I. Bellovin, Nebiyu Wondyfraw, Barbara Sennino, Quinn Walker, Susan Johnson, Anita Levin, Emma Masteller, Susannah D. Barbee, Robert Sikorski, Tom Brennan. _Five Prime Therapeutics, Inc., South San Francisco, CA_.

The colony stimulating factor 1 receptor (CSF-1R) signaling pathway promotes tumor progression via the recruitment, differentiation, and survival of immuno-suppressive tumor-associated macrophages (TAMs). FivePrime has developed cabiralizumab (FPA008), an IgG4 antibody against CSF-1R that blocks the ability of both CSF-1 and IL-34 to bind and activate this receptor, thereby modulating the immune response to tumorigenesis. In order to investigate the impact of CSF-1R signaling inhibition in preclinical models, we generated a surrogate antibody, cmFPA008, which targets mouse CSF-1R and demonstrates equivalent affinity and ligand-blocking ability as FPA008. Utilizing a combination of flow cytometry and immunofluorescence analyses, we have identified alterations in the tumor microenvironment that occur upon CSF-1R inhibition, including significant reduction of immunosuppressive TAMs and an increase in tumor PD-L1 expression. Interestingly, we observe a transient increase in CD8+ T cell number and activation upon TAM depletion, followed by a subsequent increase in MDSC populations that corresponds with reduction of the CD8+ T cell numbers. Moreover, we have used murine syngeneic tumor models to examine the anti-tumor impact of CSF-1R inhibition in combination with other immuno-oncology agents. Our results show that, when added to PD-1/PD-L1 blockade, cmFPA008 can significantly enhance anti-tumor efficacy. We are currently exploring the effects of combining cmFPA008-induced TAM depletion with additional immuno-oncology agents, including T cell agonists. Our preclinical results demonstrate that inhibition of the CSF-1R pathway can combine with various immuno-oncology agents with distinct mechanisms of action. FivePrime has initiated a clinical trial in collaboration with Bristol-Myers Squibb (BMS) to investigate the use of cabiralizumab in combination with nivolumab (anti-PD-1, OPDIVO®) in six different tumor types.

#1600

Interleukin-8 (IL-8) in TGF-β immunotherapy and chemotherapies.

Larn Hwang,1 Kevin Ng,2 Osmond D'Cruz,2 Sanjive Qazi,2 Andrew Schmidt,2 Vuong Trieu1. 1 _Oncotelic Inc, Agoura Hills, CA;_ 2 _Autotelic Inc, Costa Mesa, CA_.

Background: Increased expression of IL-8 and/or its receptors has been characterized in cancer cells, endothelial cells, infiltrating neutrophils, and tumor-associated macrophages, suggesting that IL-8 may function as a significant regulatory factor within the tumor microenvironment. OT-101 is a phosphorothioate antisense oligodeoxynucleotide targeting transforming growth factor-beta 2 (TGF-β2). Herein, we examined the role of IL-8 in OT-101 treatment followed with chemotherapies during our Phase II trial in patients with advanced pancreatic cancer (PAC).

Methods: Time evolution of a panel of 31 cyto-/chemokine levels in plasma were tracked over 3 cycles of OT-101 administration (140 mg/m2/day) for 12 PAC patients. Samples were acquired before onset of OT-101 therapy and at 8 selected time points (Cycle 1 [Day 2 and 5], Cycle 2 [Days 1, 2, and 5], Final Visit, Cycle 3 [Day 5]) during the therapy. Samples were measured in duplicate and concentrations were expressed in pg/mL. Standardized log10 transformed values calculated from the mean and standard deviation of each cyto-/chemokine in each patient was utilized in an ANCOVA model to investigate the correlation with Overall Survival (OS).

Results: Clustering of correlation coefficients resulted in the identification of three highly correlated subsets of cyto-/chemokines (Cluster 1: EGF, MIP-1α, MIP-1β; Cluster 2: FGF-2, IL-1RA, MIG, IP-10, IL-15, IFN-α, IL-12A/IL-12; Cluster 3: HGF, IL-2R, IL-6, IL-8). Protein-Protein Interaction networks constructed using STRING10 algorithm identified a relationship between IL-8 and TGFβR2 inhibition. The ANCOVA model explained a significant proportion of the observed data for Cycle 1[Day 2] measurements of cyto-/chemokines (R2 = 0.3, F59,217 = 1.575, P = 0.0103). Other time points did not exhibit a significant model fit or significant relationships in the interaction term. IL-8 expression showed significant association with OS (positive association, N=12, T-value = 2.92, P = 0.0039) at Cycle 1[Day 2] measurements (7% False Discovery Rate). Linear regression of the increase in IL-8 levels during Cycle 1[Day 2] treatment with OT-101 was related to an increase in OS outcome (R2 = 0.54). To further define the appropriate chemoagent to combine with OT-101, we evaluated paclitaxel (PTX), gemcitabine (GEM), and dacarbazine (DTIC) in animal model. Synergy was observed with PTX and DTIC, but not with GEM. Both PTX and DTIC induced IL-8 expression whereas, surprisingly, GEM reduced IL-8 expression.

Conclusion: IL-8 expression during early phase of OT-101 treatment cycle was positively associated with OS across 12 patients. IL-8 is also induced by PTX and DTIC which synergized with OT-101. The data suggests that PAC and DTIC are acting on the same target as our TGF-β2 inhibitor; and potentially, the beneficial effect of these chemoagents is a result of chemoagent-induced immune/IL-8 response.

#1601

Anti-tumor effect of GAGomer-mediated intra-tumoral IL-2 expression following systemic administration.

Genia Alpert,1 David Altreuter,2 Sunil Anamandla,2 Arlyssa Birt,2 Guy Cinamon,1 Keren Cohen Merimi,1 Orli Even Or,1 Nir Gefen,1 Nadia Gurvich,2 Jeno Gyuris,2 Lorena Lerner,2 Adi Mondshine,1 Hong Wang2. 1 _Quiet Therapeutics, Ness Ziona, Israel;_ 2 _Quiet Therapeutics, Lexington, MA_.

Cancer immunotherapies are revolutionizing cancer treatment. Unfortunately, a large proportion of patients with solid tumors do not respond to currently available immune-therapeutics. The lack of response is due to a variety of mechanisms tumors adopt to avoid immune mediated clearance. The multiplicity of immunosuppressive mechanisms operational in the tumor microenvironment may not be overcome by single agents and require interventions at multiple control points. However, systemic exposure to combinations of immunoregulators may result in severe, dose limiting, acute and chronic toxicities that might be prevented if the effect of these agents is focused to the tumor microenvironment.

We are engaged in the discovery of a novel class of immuno-oncology drugs aimed at maximizing the effect of immunoregulatory molecules in the tumor microenvironment and minimizing systemic adverse effects. These drugs incorporate plasmids, engineered to program tumor cells to produce and secrete immune-regulatory proteins, within hyaluronic acid (HA) coated lipid nanoparticles, called GAGomers, which specifically target tumor cells that overexpress activated HA receptors (GAG-pDNA). GAG-pDNA based therapeutics promise highly potent but localized activation of the immune system exclusively in the tumor microenvironment following systemic administration, leading to the destruction of tumor cells by activated immune cells without debilitating toxic side effects.

To demonstrate the feasibility of the GAG-pDNA approach we have incorporated a plasmid directing the expression of murine IL-2 into GAGomers (GAG-pIL2) and assessed the anti-tumor activity of the construct after systemic delivery into tumor bearing mice. GAG-pIL2 administration resulted in statistically significant inhibition of tumor growth, which correlated with elevated IL-2 levels in the tumor and increased infiltration of T-cells into the tumor microenvironment.

These experiments demonstrate the feasibility of programming tumor cells using GAG-pDNA to produce and secrete immunoregulatory molecules into the tumor microenvironment and trigger robust anti-tumor immune responses.

#1602

Generation of anti-IL-17B antibodies neutralizing IL-17B-mediated alterations of the immune microenvironment, promotion of tumor cell initiating capacity and chemoresistance.

Emilie Laprevotte,1 Aurélie Docquier,1 Jeremy Bastid,1 Cécile Déjou,1 Marion Lapierre,1 Gilles Alberici,1 Armand Bensussan,2 Jean-François Eliaou,3 Nathalie Bonnefoy3. 1 _OREGA Biotech, Ecully, France;_ 2 _INSERM U976, PARIS, France;_ 3 _INSERM U1194, Montpellier, France_.

Interleukin 17B (IL-17B) is a pro-inflammatory cytokine that belongs to a family encompassing 6 interleukins (IL-17A to F) and binds to the IL-17 receptor B (IL-17RB). Recently, amplified IL-17B/IL-17RB signaling was found critical for breast and pancreatic tumorigenesis and elevated expression of IL-17RB has been associated with the shortest survival rates in patients with breast or pancreatic cancer. Using IL-17B knock-out (IL-17B KO) mice we demonstrate here that melanoma, fibrosarcoma and breast cancer cell tumorigenicity is strongly impaired in immunocompetent IL-17B KO mice compared to WT littermates, including a large number of tumor free mice. Reduced tumor incidence in IL-17B KO mice is associated with alterations of the immune tumor microenvironment especially within innate lymphocyte and myeloid sub-populations. We further demonstrate that IL-17B is a key cytokine shaping the tumor initiating cancer cell niche. Indeed, MDA-MB-468 human breast cancer cells overexpressing IL-17B exhibit 10 times higher frequency of tumor initiating cells when xenografted at a serial limiting dilution in nude mice. Tumor progression is, again, associated with alterations of NK cells within the tumor microenvironment and with increased percentages of CD44hi/CD24lo tumor cells, a phenotype associated with breast cancer stem cells (CSC). This is associated with resistance to conventional chemotherapeutic agents such as taxol, an effect that is totally abrogated by disrupting IL-17B-IL-17RB signaling with a neutralizing antibody. Altogether our results point out the key role of IL-17B in regulating the immune microenvironment as well as cardinal features of CSC, one of the alleged causes of resistance to therapy and tumor relapse. Thereby, IL-17B and its receptor appear as potential therapeutic targets for cancer immunotherapy. Collectively, these data support the ongoing development of IL-17B neutralizing antibodies.

#1603

NKTR-255 engages the IL-15 pathway driving CD8 T cell survival and CD8 memory T cell proliferation.

Peiwen Kuo, Mekhala Maiti, Phi Quach, Murali Addepalli, Arunasree Lanka, Poornachandra Mathamsetti, Christie Fanton, Ping Zhang, Peter Kirk, Takahiro Miyazaki, Jonathan Zalevsky. _Nektar Therapeutics, San Francisco, CA_.

Background: IL-15, an immunostimulatory cytokine, plays an important role in both the innate and adaptive immune system. Notably, this pleiotropic cytokine is a key regulator of homeostasis and survival of CD8 and CD8 memory T cells. Targeting the IL-15 pathway has therefore become a promising therapeutic approach in oncology through the induction of long-term T cell activation and durable memory responses. NKTR-255 is a polymer-engineered IL-15 that as a single-agent reduces tumor burden in various tumor models. Here, we show that the anti-tumor effects of NKTR-255 can be attributed to its modulation of survival and proliferation of CD8 and memory CD8 T cells.

Methods: Immunophenotyping of CD8 T cell subpopulations was performed in naïve and tumor-bearing Balb/c mice treated with NKTR-255. Cell surface staining of CD3, CD8, CD44, CD62L and Sca-1 was conducted to identify effector (Tem), central (Tcm) and stem (Tscm) memory T cells. Intracellular staining of Ki67 and Bcl-2 were also analyzed by flow cytometry. Human whole blood and PBMCs were stimulated with IL-15 (0.0001-1000ng/ml) or NKTR-255 (0.001-10,000ng/ml). At various time points pSTAT5 response in CD3, CD4, CD8 and NK (CD56 bright and dim) cells was monitored by flow cytometry.

Results: In naive mice, single dose NKTR-255 (dose range 0.06-1mg/kg) increased the proliferation of Tem, Tcm and Tscm CD8 T cells in a dose-dependent manner. Treatment at 0.3 and 1mg/kg dose levels increased proliferation at least 4 fold across the CD8 memory populations. The abundance of CD8 and CD8 memory T cells was still apparent 6 days post-treatment unlike single dose IL-15 which was ineffective. In a CT-26 lung metastasis model, NKTR-255 reduced the number of lung nodules in a dose-dependent manner. A 0.3mg/kg dose level increased Bcl-2 MFI 1.5 fold in CD8 T cells. Furthermore, administration of NKTR-255 at 0.3, 1 or 3mg/kg significantly increased CD8 proliferation in a dose-dependent manner in blood (1.7, 4.6 and 5.3 fold) and spleen (2.5, 5.7 and 6.9 fold) compared to vehicle. The enhanced Bcl-2 expression and CD8 proliferation were accompanied by elevated CD8 T cells in blood (1.5, 2.4, and 3.4 fold increase) and spleen (1.3, 1.7 and 2.3 fold increase). Consistent with increased proliferation and increased Bcl-2 levels observed in vivo, both IL-15 and NKTR-255 showed dose-dependent induction of pSTAT5, a modulator of Bcl-2 expression, in CD4 and CD8 T cells from human whole blood and PBMCs.

Conclusions: NKTR-255 effectively engages the IL-15 pathway as evidenced by its strong induction of CD8 and memory CD8 T cell proliferation and promotion of survival. Combined with sustained activity and potency in human blood and PBMCs, our results support NKTR-255 as a novel tumor immunotherapeutic with great potential.

#1604

NKTR-214 synergizes with radiotherapy to drive tumor regression.

Michael J. Mcnamara,1 Melissa Kasiewicz,1 Ian Hilgart-Martiszus,1 Ute Hoch,2 Deborah H. Charych,2 William L. Redmond1. 1 _Earle A. Chiles Research Institute, Providence Portland Medical Ctr., Portland, OR;_ 2 _Nektar Therapeutics, San Francisco, CA_.

The purpose of this study was to investigate therapeutic and mechanistic synergies between single-dose radiotherapy and systemic administration of NKTR-214. NKTR-214 is a CD122-biased cytokine agonist conjugated with multiple releasable chains of polyethylene glycol. NKTR-214 is designed to provide sustained signaling through the heterodimeric IL-2 receptor pathway (IL-2Rβγ) to preferentially activate and expand effector CD8+ T and NK cells over Regulatory CD4 T cells. Preclinical models demonstrated NKTR-214 preferentially expands effector CD8+ T cells and NK cells within the tumor resulting in marked tumor growth suppression as a single-agent and in combination with checkpoint inhibitors. A phase I/II trial is in progress to evaluate NKTR-214 safety and efficacy in an outpatient setting. Radiation therapy can induce antigen-release and epitope spreading, while NKTR-214 can active and expand antigen-specific effector populations. We hypothesized that the combination of systemic NKTR-214 and local radiotherapy would generate better therapeutic responses than either treatment alone. In this study, we evaluated the combination of systemic NKTR-214 treatment with single fraction high-dose radiotherapy (20 Gy) in multiple murine models. We used flow cytometry, multi-spectral histology, and whole tumor mRNA profiling to investigate local, systemic and potential abscopal immune responses. We used Nur77-GFP reporter mice to enable detection of T cell receptor ligation in vivo and to evaluate the effects of NKTR-214+RT on tumor-reactive T cells. The results from these studies indicate that the combination of NKTR-214 and radiotherapy is synergistic, providing significantly better anti-tumor responses than either monotherapy. Consistent with previous observations, NKTR-214 alone induces expression of a wide range of activation markers expressed by CD4 and CD8 T cells as well as NK cells in the blood, lymph nodes and tumor. The combination of radiotherapy and NKTR-214 was found to have several unique effects including a significant increase (>75%) in the absolute numbers of lymphocytes in the peripheral blood, increased expression of activation markers (CD25, PD-1) by CD8 T cells in the blood and tumor, and increased density of tumor-infiltrating NK cells. Evaluation of tumor infiltrating lymphocytes (TIL) in Nur77-GFP reporter mice revealed that the combination of NKTR-214+RT resulted in a higher frequency of recently activated (Nur77-GFP+) CD8 T cells in treated (irradiated) and abscopal (non-irradiated) tumors. Whole tumor mRNA profiling and multi-spectral histology of these tumors is being assessed to identify key differences in the tumor-microenvironment that may help to define the underlying mechanism of action. Taken together, these data provide evidence of synergy between localized radiotherapy and systemic NKTR-214 treatment via an expansion of activated, tumor-specific CD8 T cells.

#1605

Combined oral cytokine therapy effectively treats colon cancer in a murine model.

Neal Bhutiani, Qingsheng Li, Charles D. Anderson, Tao Gu, Nejat K. Egilmez. _University of Louisville, Louisville, KY_.

Background: Regardless of its etiology, colorectal cancer development is intimately associated with dysregulation of the intestinal immune system. Sustained release, orally delivered IL-10 not only inhibits colon tumor growth but restores T cell homeostasis both locoregionally and systemically. However, it does not induce tumor regression. We aimed to combine orally delivered IL-12, a potent immunostimulatory molecule with robust anti-tumor effect, with oral IL-10 therapy to induce tumor regression.

Methods: 6 week old APCmin mice were inoculated with enterotoxic Bacteroides fragilis (serotype 086) after five days of intestinal flora depletion with antibiotics dissolved in drinking water. 7-10 days after bacterial inoculation, mice were treated three times weekly with either 1) blank polylactic acid microspheres for 3 weeks, 2) IL-10 containing microspheres (0.5 microgram/dose) for 3 weeks, 3) IL-12 containing microspheres (0.25 micrograms/dose) for 3 weeks, or 4) IL-10 containing microspheres for 1 week followed by a combination of IL-10 and IL-12 containing microspheres for 2 weeks. After euthanasia, colons were assessed for polyp counts and select polyps were isolated for confocal microscopy analysis. Mesenteric lymph node lymphocytes were isolated and analyzed using flow cytometry.

Results: Mice treated with oral IL-10 and IL-12 demonstrated a reduction in polyp number compared to those mice treated with IL-10 alone, and both groups had fewer polyps than mice treated with either IL-12 or blank microspheres. Addition of IL-12 resulted in increased CD8+ T cell numbers and IFN-γ production compared to treatment with IL-10 alone. IL-10 retained its effect on CD4+ T cells when combining it with IL-12, resulting in a decrease in conventional regulatory T cells (FoxP3+ RORγT -) as well as pathogenic regulatory T cells (FoxP3+ RORγT+) and IL-17 producing CD4+ T cells.

Conclusions: The addition of IL-12 to IL-10 therapy augments the therapeutic effect of oral cytokine treatment on colon adenocarcinoma in the APCmin B. fragilis model. The effect appears additive and mediated by both IL-10's effect on regulatory T cell populations and IL-12's effect on CD8+ T cells. Further studies in the setting of more advanced disease, including liver metastases, will elucidate the therapeutic potential of combination oral cytokine therapy with IL-10 and IL-12 for treatment of colorectal cancer.

#1606

Targeting human T regulatory cells with novel Interleukin 2 alpha - IL2 complex-specific RNA aptamer.

Suresh Veeramani, Sue E. Blackwell, William H. Thiel, Paloma H. Giangrande, George J. Weiner. _University of Iowa, Iowa city, IA_.

Background: RNA aptamers are small RNA molecules that bind antigens like antibodies and are currently being explored as alternatives to antibodies for diagnosis and therapy. A potential merit of aptamers is that they can be generated against native cellular antigens, such as those with unique post-translational modifications or receptor-ligand complexes, for which antibody generation can be difficult. Here, we report the use of a cell-based systematic enrichment approach (SELEX) to develop a novel Treg-binding RNA aptamer specific to IL2Rα-IL2 receptor-ligand complex.

Methods and Results:

A. Generation of Treg-binding aptamers:

We designed a cell-based SELEX strategy to generate RNA aptamers specific to human T regulatory (Treg) cells. The starting library consisted of random RNA aptamers with a structural diversity of ~1012. Aptamers against common T cell antigens were pre-cleared using CD4+CD25\- Teff cells. Treg-binding aptamers were then positively selected using CD4+CD25+ Tregs from the same donor. After amplification of Treg binders by RT-PCR, the whole selection was repeated eight times, each time with T cells from a different donor. At the end, SELEX-enriched aptamer pools predominantly bound to Tregs, but not to Teff cells, which were then sequenced. The most prevalent Treg-binder, Tr-1, showed 63,875-fold enrichment by the end of SELEX (from 0.36 copies in the starting library to 22,995 copies in the eighth round per million total reads).

B. Tr-1 binds to IL2Rα- IL2 complex

We tested if Tr-1 recognized human IL2Rα by measuring Tr-1 binding to recombinant IL2Rα protein using RT-qPCR. Results indicated that Tr-1 bound to IL2Rα and showed ~3-fold higher binding to IL2Rα when IL2 was added indicating that Tr-1 recognizes either the receptor-ligand complex or a conformational change in IL2-bound IL2Rα. Binding of Tr-1 to IL2Rα did not significantly alter its affinity to IL2.

C. Tr-1 inhibits Treg induction

Transformed B cells can induce Treg development in the tumor microenvironment. Experimentally, this was evaluated by quantifying Tregs generated from autologous CD4+ T cells co-cultured with Epstein-Barr virus-transformed B (EBV-B) cells. Addition of Tr-1 resulted in ~30% reduction in EBV-B-induced Tregs (p=0.017).

Conclusion: We used a Treg cell-based SELEX strategy to derive a novel Treg-binding RNA aptamer (Tr-1) that preferentially binds to the IL2Rα-IL2 complex. Tr-1 reduced Treg induction by transformed B cells suggesting its potential as a therapeutic agent to reduce tumor-induced immunosuppression. Ongoing studies are further exploring its use in Treg inhibition and in targeting receptor-ligand complexes in cancer. While aptamers recognizing cellular receptors exist, to our knowledge, this is the first report of an aptamer recognizing a receptor-ligand complex. Our approach to generating aptamers against receptor-ligand complexes could have huge scientific impact.

#1607

Durable efficacy and anti-cancer immunity following intratumoral administration of messenger RNAs encoding IL-36γ, IL-23 and OX40L.

Josh P. Frederick,1 Ailin Bai,1 Susannah Hewitt,1 Russ Karp,1 John Zielinski,1 Kana Ichikawa,1 Ameya Apte,1 Dyane Bailey,1 Kristen Arnold,1 Sam Farlow,1 Darren Potz,1 Bo Ying,1 Graham MacLean,1 Kerry Benenato,2 Maja Sedic,2 Steve M. Kelsey1. 1 _Onkaido Therapeutics, Cambridge, MA;_ 2 _Moderna Therapeutics, Cambridge, MA_.

Immunologic responses to cancer involve complex interactions of cells within the tumor microenvironment (TME). These interactions are mediated in part through co-stimulatory and inhibitory transmembrane proteins, cytokines and chemokines. Direct intratumoral (ITu) administration of messenger RNA results in synthesis of proteins that can prime and enhance an anti-cancer immune response. Priming, expansion and migration of anti-cancer T-cell clones may also result in a systemic or 'abscopal' response in distal lesions. There is mounting preclinical evidence that OX40-OX40L signaling boosts anti-cancer immunity via co-stimulatory activity on T-cells. We generated mRNA constructs encoding OX40L with miR-122 binding sites that have been shown to suppress protein translation in hepatocytes yet not within tumor cells. A single ITu injection of OX40L mRNA, formulated in a lipid nanoparticle, resulted in transfection of up to 25% of live tumor cells, predominantly cancer and myeloid cells. OX40L protein persisted for 7 days after mRNA injection, and a resulting increase in tumor T-cells was observed. Repeat dosing of OX40L mRNA induced complete responses in a model that is sensitive to immune-meditated therapies, such as anti-PD1/PD-L1 blockade, yet was ineffective in a related model that might better represent an immunologically barren or immunosuppressive TME. The OX40L-resistant tumor model was also resistant to anti-PD1 treatment and was shown to contain fewer infiltrating immune cells. As this model may represent a subset of human cancers that do not respond well to checkpoint inhibitors, it was used to test various mRNA constructs designed to cooperatively harness components of both the innate and adaptive immune systems in igniting a productive anti-cancer response. Both IL-36γ and IL-23 have established roles in mediating immune responses in humans and have been implicated in driving various inflammatory diseases. IL-36γ activates innate immune cells and promotes Th1 responses, whereas IL-23 has been implicated in Th1/Th17 immunity as well as in the modulation of antigen presenting cells. Repeated weekly intratumoral co-administration of mRNAs encoding IL-23 and IL-36γ resulted in durable complete responses in both anti-PD1-sensitive and -resistant tumors in a synergistic fashion. A further modest improvement in outcomes was observed when the IL-23/36γ mRNA doublet was co-administered with OX40L mRNA. Further pharmacodynamic and mechanism of action studies with this combination are underway. ITu injection of mRNA encoding combinations of co-stimulatory proteins and cytokines represents a novel platform for cancer immunotherapy, and may be able to induce responses in patient tumors that are resistant to checkpoint inhibition. The co-formulated combination of mRNAs encoding human OX40L, IL-23 and IL-36γ is planned to enter clinical trials in 2017.

#1608

**CD122-selective IL-2/anti-IL-2 complexes reduce regulatory T cell function and promote CD8** + **T cell polyfunctionality for durable ovarian cancer immunotherapy.**

Justin M. Drerup, Sri Lakshmi Pandeswara, Aijie Liu, Curtis A. Clark, Alvaro S. Padron, Wanjiao Chen, Vincent Hurez, Tyler J. Curiel. _University of Texas Health Science Center at San Antonio, San Antonio, TX_.

The IL-2 receptor (IL-2R) is an attractive target for cancer immunotherapy as it controls both immune-suppressive regulatory T cells (Tregs) and anti-tumor T cells. We tested depleting Tregs as immunotherapy using anti-CD25 (high-affinity IL-2R subunit) antibodies (αCD25) in ID8agg mouse ovarian cancer (OC). αCD25 reduced ascites and Treg numbers but failed to reduce tumor burden, possibly because it depleted newly activated anti-tumor T cells in tumor-draining lymph nodes. Thus, αCD25 could be novel malignant ascites palliation, but has limited stand-alone efficacy. We then tested IL-2/anti-IL-2 complexes (IL-2c) that selectively stimulate medium-affinity (CD122/CD132) IL-2R thought to expand anti-tumor T cells preferentially, but with little Treg effects. In contrast to several single agents we tested that failed to treat ID8agg (e.g., αCD25, αPD-L1, IL-2 fusion toxin denileukin diftitox), IL-2c alone durably reduced ID8agg tumor burden despite lowering the tumor microenvironmental CD8+/Treg ratio. Thus, we hypothesized that IL-2c improved CD8+ function, reduced Treg function, or both. IL-2c increased polyfunctional IFN-γ+TNF-α+ anti-tumor T cells as expected, an effect that persists weeks after drug clearance. IL-2c also increased anti-tumor T cell CD25 expression that increased IL-2 sensitivity and STAT5 phosphorylation, a likely mechanism for increased polyfunctionality. Unexpectedly, IL-2c reduced the Treg functional mediators CD25, TIGIT and granzyme B, and reduced Treg suppressive function. Thus, favorable Treg modifications are a novel IL-2c mechanism of action. Adding αCD25 to IL-2c to deplete Tregs further unexpectedly worsened IL-2c efficacy in ID8agg and reduced effector memory T cells and polyfunctional T cells in the tumor microenvironment, suggesting a previously unappreciated role for CD25 in IL-2c therapy. Similar data were seen in B16 melanoma, suggesting αCD25 reduction of IL-2c efficacy is not tumor or compartment-specific (ID8agg is peritoneal and B16 is subcutaneous). αPD-L1, an ineffective monotherapy in ID8agg, combined with IL-2c to promote complete responses, suggesting potential for potent, novel combinatorial approaches. Our data suggest that antagonizing high affinity IL-2R (such as to deplete Tregs with αCD25) has limited cancer immunotherapy utility without more specific Treg targeting. In contrast, stimulating medium-affinity IL-2R with CD122-selective IL-2c has great translational promise by simultaneously improving beneficial anti-tumor T cells and reducing detrimental Treg function.

#1609

Oncolytic virotherapy for SCLC using immunocompetent mouse models.

Patrick Kellish, Daniil Shabashvili, Masmudur M. Rahman, Mary Reinhard, Grant McFadden, Frederic Kaye, Maria Zajac-Kaye. _University of Florida, Gainesville, FL_.

Background: Small cell lung cancer (SCLC) is an aggressive subtype of lung cancer with few treatment advances over the past 3 decades and poor survival. Locally targeted oncolytic virotherapy employs a viral vector that has selective cytotoxicity for tumor cells and non-toxic for normal cells and tissues. The therapeutic benefit is proposed to arise from an initial viral cytotoxic phase following by host immune responses. Myxoma Virus (MYXV) has been widely tested in Australia to control rabbit populations with no toxicity to humans. MYXV selectively infects mouse and human tumors cells with no cytotoxicity to normal tissues and is a promising oncolytic virotherapy agent that has not been tested in human lung cancer or in clinical trials.

Methods: To study MYXV infection and viral replication in vitro, we utilized human and mouse SCLC cell lines with MYXV engineered with fluorescent reporters. Using an optimized conditional genetically engineered mouse model (GEMM) (Ade-CRE mediated p53/Rb1/p130 null) we examined the effects of intrapulmonary MYXV treatment on SCLC tumors at 5 and 30 days post-MYXV treatment. Survival analysis was determined following intranasal MYXV 3 months post-Ade-CRE induction. We also tested intratumoral MYXV in patient derived xenografts (PDX) and subcutaneous syngeneic allografts in immunocompetent mice.

Results: We optimized a conditional SCLC GEMM using limiting dilutions of intratracheal Ade-CRE to reduce the SCLC foci to simulate human disease and generated mouse SCLC lines from individual clones. We observed efficient MYXV infection, late viral replication, and cytotoxicity in both human and mouse SCLC in vitro. In contrast, we did not detect productive infection nor cytotoxicity in non-tumor cells. Following intranasal MYXV instillation we observed MYXV localized exclusively within lungs at 3 days and no longer detected by 7 days' post treatment. TUNEL staining of SCLC lesions showed apoptosis and necrosis at 5 days within SCLC and the effect persisted with discrete foci of tumor necrosis 30 days' post-treatment. There was no toxicity to any mouse tissues. SCLC GEMM treated with intrapulmonary MYXV (n=30) showed a modest but statistically significant prolongation of survival compared to PBS control mice (n=30) (p < 0.05). Direct intratumoral MYXV injections performed on PDX tumors in immunodeficient mice showed efficient infection and late viral replication in all patient samples. We observed extensive tumor necrosis at 7 days, and persisting virus at 10 days' post treatment. Direct intratumoral MYXV injections performed on allograft tumors in syngeneic immunocompetent mice showed extensive necrosis accompanied by immune cell infiltration into the tumor, and the virus was undetectable 7 days' post treatment.

Conclusions: We show the potential for MYXV as an oncolytic virotherapy for SCLC with enhanced cytotoxicity in immunocompetent mice suggesting a role for concurrent immune checkpoint therapies.

#1610

**Targeted cancer immunotherapy with engineered** Salmonella typhimurium **secreting heterologous bacterial flagellin.**

Jung-Joon Min, Jin Hai Zheng, Yeongjin Hong, Hyon E. Choy, Joon Haeng Rhee. _Chonnam National University Medical School, Gwangju, Republic of Korea_.

Salmonella typhimurium is a facultative anaerobic bacteria and can selectively grow in tumors following systemic administration. Different strategies have been used to deliver payloads to tumor tissues using engineered Salmonellae. We report a novel method of cancer immunotherapy using an attenuated S. typhimurium strain engineered to secrete Vibrio vulnificus flagellin B (FlaB) in tumor tissues.Previously, we developed an attenuated strain of S. typhimurium, which is defective in ppGpp synthesis (ΔppGpp S. typhimurium), and manifested significantly increased LD50 by 100,000 to 1,000,000-fold. To generate an inducible vector system for bacterial expression of the therapeutic gene, we cloned the flaB gene into the pBAD plasmid vector in which the pelB leader sequence was cloned upstream of flaB to guide extracellular secretion; gene expression from the plasmid (pFlaB) was induced only in the presence of L-arabinose. To evaluate the antitumor activity of engineered S. typhimurium, C57BL/6 mice transplanted with MC38 tumors were injected intravenously (i.v.) with PBS, ΔppGpp S. typhimurium carrying an empty vector, or ΔppGpp S. typhimurium carrying pFlaB (+/˗ L-arabinose induction). To test whether the FlaB-secreting ΔppGpp S. typhimurium has inhibitory effects on human metastatic cancer, we next implanted HCT116-luc2 tumors into the intestinal wall of BALB/c athymic nu-/nu- mice using a surgical orthotopic implantation (SOI) procedure. Engineered FlaB-secreting Salmonellae significantly suppressed tumor growth and metastasis in mouse models and prolonged survival. By using TLR5-negative colon cancer cell lines (MC38 and HCT116), we have proved that the FlaB-mediated tumor suppression upon bacterial colonization should be associated with TLR5-mediated host reactions in the tumor microenvironment. These therapeutic effects were completely abrogated in TLR4 and MyD88 knockout mice, and partly in TLR5 knockout mice, strongly suggesting that TLR4 signaling is a requisite for the FlaB-secreting bacteria-mediated tumor suppression where TLR5 signaling augmented tumor suppressive host reactions. Tumor colonization by engineered Salmonellae appeared to induce the infiltration of abundant immune cells such as monocytes/macrophages and neutrophils via the TLR4 signaling. Subsequent secretion of FlaB from colonizing Salmonellae resulted in phenotypic and functional activation of intratumoral macrophages with M1 phenotypes and a reciprocal reduction in M2-like suppressive activities. Taken together, these findings provide evidence that non-virulent tumor targeting bacteria liberating multiple TLR ligands can be used as novel cancer immunotherapeutics.

#1611

Intravesical BCG induces CD4 T Cell expansion in an immune competent model of bladder cancer.

Max Kates, Thomas Nirschl, Nikolai Sopko, Noah Hahn, David McConkey, Alex Baras, Charles Drake, Trintiy Bivalacqua. _Johns Hopkins Medical Institutions, Baltimore, MD_.

Intravesical BCG Immunotherapy is the standard of care in treating non-muscle invasive bladder cancer (NMIBC), yet its mechanism of action remains elusive. Both innate and adaptive immune responses have been implicated in BCG activity. While prior research has indirectly demonstrated the importance of T cells and shown a rise in CD4+ T cells in bladder tissue after BCG, T cell subpopulations have not been fully characterized. We investigated the relationship between effector and regulatory T cells in an immune competent, clinically relevant rodent model of bladder cancer. Fischer 344 rats aged 7 weeks received 1.5mg/kg N-Nitroso-N-methylurea (MNU) every other week for 6 weeks (4 doses). Bladder dysplasia began by week 8 and by week 16 the majority of rats had a NMIBC phenotype. Beginning week 8 following the first MNU dose, rats were intravesically administered 0.3ml of BCG (Tice), cisplatin (1mg/ml), Mitomycin C (2mg/ml), MMC+ BCG, or saline (n=10 for all groups) weekly for 6 total doses. Animals were sacrificed at week 16, and bladders were processed for histopathology and digested into single cell suspensions for flow cytometry. Whole transcriptome expression profiling was then performed on sorted CD4 and CD8 cells of post-BCG tumors vs untreated tumors to assess T cell differentiation after BCG. Our data demonstrate that cancer progression in the MNU rat model of bladder cancer is characterized by a decline in the CD8/FoxP3 ratio, consistent with decreased adaptive immunity. By contrast, treatment with intravesical BCG leads to a large, transient rise in the CD4+ T cell population in the urothelium, and is both more effective and immunogenic compared to intravesical chemotherapy. Interestingly, whole transcriptome expression profiling of post-treatment intravesical CD4+ and CD8+ T cells revealed minimal differences in gene expression after BCG treatment. Together, our results suggest that while BCG induces T cell recruitment to the bladder, the T cell phenotype does not markedly change, implying that combining T cell activating agents with BCG might improve clinical activity.

#1612

Production of CD3+ cells using ferrofluids for cell isolation, activation, expansion and subsequent transfection for adoptive cell therapy.

Dustin W. Ritter, Todor R. Khristov, Paul A. Liberti. _BioMagnetic Solutions, State College, PA_.

The purpose of the study is to demonstrate whether magnetic nanoparticles functionalized with monoclonal antibodies in conjunction with solution-phase monoclonal antibodies can be used to activate T cells for expansion in vitro. The activation, expansion and transfection/transduction of immune cells—particularly T cells—has become an area of significant interest based on encouraging data generated from employing such genetically modified cells for cancer immunotherapies. The promise of soon being able to cure various types of cancers has resulted from years of methodical experimentation in the fields of oncology, immunology, and genetics, as well as the development of a vast array of enabling technological advances. BioMagnetic Solutions has developed colloidal magnetic nanoparticles—referred to as ferrofluids—which can be used to isolate a cell population through an immunomagnetic separation. These ferrofluids comprise a crystalline core of magnetite (ca. 100 nm in diameter) coated with multilayers of clinical-grade human serum albumin and further functionalized with a clinical-grade rat anti-mouse IgG1 monoclonal antibody. These "common-capture ferrofluids" (ca. 130 nm in diameter) are capable of binding cells which have been labeled with a monoclonal mouse IgG1 antibody; for example, CD3+ cells can be readily isolated from a complex cell mixture by labeling with a monoclonal mouse anti-human CD3 antibody, adding common-capture ferrofluid, and subjecting the sample to a magnetic field gradient. This technology can therefore be employed at the clinical scale to isolate T cells from an apheresis product. In this study, we investigated whether T cells isolated using common-capture ferrofluids could subsequently be activated and induced to expand in vitro. Based on preliminary experiments showing that T cell-bound common-capture ferrofluid has excess antibody-binding capacity, we designed experiments to test the hypothesis that solution-phase CD28 could be added to isolated T cells, which would bind to both CD28 determinants on the cell surface as well as to the cell-bound common-capture ferrofluid. Activation was quantified by measuring CD25 expression after four days in culture and expansion was monitored over 15 days. Following an immunomagnetic separation of CD3+ cells, magnetically isolated cells were re-suspended in expansion medium (STEMCELL) supplemented with 100 IU/mL IL-2 (Gibco) to a total concentration of 106 cells/mL and incubated at 37 °C. The following day, 0.5 µg/mL mouse anti-human CD28 monoclonal antibody (Mabtech) was added, and thereafter, cells were periodically agitated and diluted to 106 cells/mL with expansion media. After four days in culture, 88.4% of cells expressed CD25, while a 311-fold expansion was observed after 15 days. In summary, we have demonstrated that common-capture ferrofluids can be used to activate T cells for expansion in vitro.

#1613

Genetically modified neuronal stem cells expressing IL-12 and TNFSF14 attenuate highly metastatic mammary tumors in a mouse model.

Tej B. Shrestha, Dezhang Lu, Matthew T. Basel, Marla Pyle, Stefan H. Bossmann, Deryl L. Troyer. _Kansas State University, Manhattan, KS_.

Interleukin 12 (IL-12) is a heterodimeric cytokine encoded by two genes, p35 and p40. IL-12, also known as T cell-stimulating factor, induces proliferation of T-lymphocytes and natural killer (NK) cells, while also demonstrating an anti-angiogenic effect and inhibition of regulatory T cells. Similarly, LIGHT (TNFSF14) is a transmembrane protein which acts as a costimulatory factor for the activation lymphoid cells and stimulates the proliferation of T cells, leading to increased apoptosis of tumor cells. Expression of IL-12 and LIGHT specifically in the tumor region could activate T cells and stimulate the immune system to attenuate the tumor. Tumor homing neuronal stem cells C17.2 (NSC) were genetically engineered to express the mouse-LIGHT gene and IL-12 using a retrovirus. The expression of LIGHT and IL-12 genes in NSC was confirmed by PCR, ELISA and Western blot. To test the antitumor effects of the engineered cells, Balb/C mice bearing 4T1 mammary tumors were treated with NSC-LIGHT, NSC-IL-12 and NSC-LIGHT/IL-12 cells intratumorally (I.T.) at day nine. Repeated tumor measurement over time showed attenuation of mammary tumor growth in the both NSC/IL-12 and NSC/LIGHT groups in comparison with control groups PBS and NSC. Tumor growth was further reduced by treating the mice with NSC expressing both cytokines LIGHT and IL-12. Tumor metastasis was also evaluated by removing the primary tumor surgically and observing mice for any sign of metastatic burden. Treatment groups had less metastatic tumor burden and survived longer than the control group. Therefore, tumor homing NPC-LIGHT/IL-12 cells could be effective for the treatment of highly metastatic mammary tumors.

#1614

Intratumoral delivery of a P2A-linked bicistronic IL-12 construct leads to high intratumoral expression and systemic anti-tumor response.

David A. Canton, Christoph Burkart, Shawna A. Shirley, Anandaroop Mukhopadhyay, Richard J. Connolly, Arya Bahrami, Jean S. Campbell, Robert H. Pierce. _OncoSec Medical, San Diego, CA_.

The use of immunomodulatory cytokines has been shown effective in regressing a wide range of tumors. However, systemic delivery of recombinant cytokines can result in serious adverse effects, often life-threatening. DNA transfer via electroporation (EP) is a safe and effective method of delivering cytokines to target tissues.

Intratumoral (IT) electroporation of Interleukin 12 (IL-12), a potent immunomodulatory cytokine, is well tolerated with an acceptable safety profile and objective response rates of 34-40% and 33% in Phase II clinical trials in advanced melanoma and Merkel cell carcinoma, respectively. We sought to improve the systemic anti-tumor response of IT pIL-12-EP by improving IL-12p70 expression and electroporation conditions, which were evaluated in vitro and in vivo with a two-tumor syngeneic mouse model of melanoma. As functional IL-12 p70 is a heterodimer, we compared different expression constructs to achieve high levels of IL-12 protein expression. IL-12p70 protein expression from a plasmid that incorporated a picornavirus-derived co-translational cleavage site (P2A) was higher than constructs with an internal ribosomal entry sequence (IRES) or a fusion of the p35 and p40 subunits. In functional in vitro assays, pIL-12(P2A) was superior to pIL-12(IRES) and the fusion protein. Using the murine B16.F10 tumor model, we show that IT EP of pIL-12 (P2A) plasmid regresses the treated lesions in a dose-dependent manner compared to control treatments. Systemic effects of IT-expressed IL-12 was assessed by monitoring generation of an antigen-specific CD8 T cell response and regression of B16.F10 contralateral (untreated) tumors following primary tumor electroporation. IT-pIL12-EP treatment with the P2A-linked construct resulted in a significant increase in antigen-specific CD8 T cells, as well as enhanced contralateral tumor growth inhibition suggesting the induction of strong systemic anti-tumor immune response.

* Contributed equally to this work

#1615

C-Reactive protein and radiotherapy-induced skin toxicity in breast cancer.

Jennifer J. Hu,1 Doug Case,2 Mark O. Lively,2 Eunkyung Lee,1 Cristiane Takita,1 James J. Urbanic,3 Glenn J. Lesser,4 Edward G. Shaw4. 1 _Univ. of Miami Sylvester Comp. Cancer Ctr., Miami, FL;_ 2 _Wake Forest University School of Medicine, Winston-Salem, NC;_ 3 _Univ of California San Diego, San Diego, CA;_ 4 _Wake Forest Baptist Health, Winston-Salem, NC_.

Background: Post-surgery adjuvant radiotherapy (RT) for breast cancer significantly reduced the local recurrence rate. However, many patients develop early adverse skin reactions (EASRs) that impact quality of life.

Methods: In a large prospective study of 1,000 breast cancer patients undergoing RT, we evaluated an inflammatory biomarker, C-reactive protein (CRP) in predicting RT-induced EASRs. In each patient, we measured pre- and post-RT plasma CRP levels using a highly-sensitive ELISA CRP assay. RT-induced EASRs were assessed using the Oncology Nursing Society Skin Toxicity Criteria. Association between EASRs and CRP were assessed using logistic regression models after adjusting for potential confounders.

Results: The study population includes 405 non-Hispanic White, 280 African Americans, 218 Hispanic Whites, 52 Asians, and 45 others. RT-induced grade 3+ and 4+ skin toxicity at the end of RT were observed in 42% and 15% patients, respectively. CRP levels differ significantly by race/ethnicity at baseline and at the end of RT. RT-induced grade 4+ skin toxicity was significantly associated with: obesity and pre-RT CRP > 2mg/L (OR=3.27; 95%CI=1.88, 5.68), obesity and post-RT CRP > 2mg/L (OR=4.42; 95%CI=2.38, 8.23), or obesity and change of CRP > 1mg/L (OR=3.58; 95%CI=2.00, 6.39).

Conclusion: The current data validate our previous findings that the inflammatory biomarker CRP is associated with RT-induced EASRs, particularly combined with obesity.

Impact: Our current findings support the discovery and development of anti-inflammatory agents to protect normal tissue from RT-induced EASRs and improve quality of life in breast cancer patients undergoing RT.

#1616

S100A4 blockage alleviates agonistic anti-CD137 antibody induced liver pathology without disruption of anti-tumor immunity.

Jinhua Zhang, Kun Song, Zhihai Qin. _Institute of Biophysics, Chinese Academy of Science, China_.

Liver-related autoimmune toxicities triggered by anti-CD137 agonist antibodies have greatly limited their use in clinic applications. Here we found that anti-CD137 monoclonal antibody (mAb) treatment in mice induced the infiltration of a large number of S100A4+ macrophages into the liver. Depletion of these cells or deficiency of S100A4 decreased inflammatory cytokine profiles, and drastically reduced the number of liver pathogenic CD8+ T cells. Mechanistically, soluble S100A4 directly activated the Akt pathway and specifically prolonged CD8+ T cell survival. Interestingly, one S100A4 neutralizing monoclonal antibody selectively alleviated liver abnormalities but did not affect the anti-tumor immunity induced by anti-CD137 therapy. Thus, our study presents a novel molecular link to the liver pathology induced by an immune stimulatory antibody, and proposes that combinational immunotherapies targeting those pathways could potentially elicit optimal anti-tumor immunity with minimal side effects.

#1617

Mechanistic modeling of a new kinetically-controlled CD122 agonist for cancer immunotherapy: NKTR-214 pharmacokinetics, pharmacodynamics, and receptor pharmacology.

Samira Khalili, Aleksandrs Odinecs, Deborah H. Charych, Vidula Dixit, Peter Kirk, Thomas Chang, John Langowski, Werner Rubas, Steve Doberstein, Jonathan Zalevsky, Michael A. Eldon, Ute Hoch. _Nektar Therapeutics, San Francisco, CA_.

Introduction: NKTR-214 is a biologic prodrug currently in a Phase 1 / 2 clinical trial in patients with solid tumors, as a single agent and in combination with anti-PD1. It is a CD122-biased cytokine agonist conjugated with multiple releasable chains of polyethylene glycol and designed to provide sustained signaling through the heterodimeric IL-2 receptor pathway (IL-2Rβγ) to preferentially activate and expand effector CD8+ T and NK cells over Tregs. Here we describe a mechanistic mathematical model that quantifies conjugated aldesleukin (IL-2) species that are generated from NKTR-214, their sustained exposure, and biased receptor pharmacology in rodent models.

Methods: A mechanistic mathematical model, using ordinary differential equations (ODE), was developed using Matlab® programming language to describe the dynamics of NKTR-214 PEG release, clearance, distribution, and receptor occupancy in vivo. The model was conditioned using experimental data for in vitro binding of unconjugated IL-2 and active conjugated IL-2 derived from NKTR-214 to IL-2 receptors using surface plasmon resonance, in vitro PEG release data, and mouse pharmacokinetics data. Simulations were performed to 1) quantify the concentration-time profiles of the various conjugated IL-2 species derived from NKTR-214 and contribution of each of them to the NKTR-214 mechanism of action, and 2) compare occupancy at IL-2Rβγ and IL-2Rαβγ receptors after administration of therapeutic doses of NKTR-214 and aldesleukin.

Results: After NKTR-214 administration, PEG chains are released slowly from the prodrug and active conjugated IL-2 species gradually increase and reach maximum concentrations about one day post-dose, followed by sustained exposure for up to a week. Compared to an equivalent dose of aldesleukin, the active conjugated IL-2 species derived from NKTR-214 achieve a 26-fold higher area under the curve (AUC) of IL-2Rβγ occupancy, and a 0.34-fold lower AUC of IL-2Rαβγ occupancy. The significant difference is due to the combined effects of slow release of active conjugated IL-2 species from NKTR-214 and their favorable binding kinetics towards IL-2Rβγ. Aldesleukin, even when simulated at repeated daily doses or constant infusion, is incapable of increasing the receptor occupancy at IL-2Rβγ without simultaneously increasing the receptor occupancy at IL-2Rαβγ in this model.

Conclusions: The mechanistic model demonstrated how NKTR-214's intrinsic design enables both biased receptor pharmacology and sustained exposure in vivo. Such biased receptor binding could not be achieved by a sustained delivery of aldesleukin.

#1618

Immune-modulating effect of bevacizumab in EGFR mutated lung adenocarcinoma.

Po-Hao Feng, Kang-Yun Lee. _Shuang Ho Hospital, Taipei Medical University, New Taipei City, Taiwan_.

Background: Host immunity affect treatment effect of lung cancer. Peripheral blood S100A9+ monocytic myeloid derived suppressor cells (MDSCs) is a predictive factor for treatment response of chemotherapy and epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) of lung adenocarcinoma patients. Bevacizumab have immune-modulating effect, including decreasing MDSCs, and improve treatment effect in combination with immunotherapy. However, the effect of immune modulation in combination of bevacizumab and EGFR-TKI in EGFR mutated lung adenocarcinoma patients is not clear.

Methods: Stage IV lung adenocarcinoma harboring sensitive EGFR mutation patients receiving first line EGFR-TKI or combination EGFR-TKI and bevacizumab were enrolled. The peripheral blood mononuclear cell (PBMCs) were collected, and S100A9+ MDSCs percentage was calculated by flow cytometry from CD14+S100A9+ in PBMC. Clinical data was collected.

Results: Eight patients receiving EGFR-TKI and bevacizumab, as combination group, and twenty patients received EGFR-TKI alone, as control group, were enrolled. PBMC S100A9+ MDSC decreased in combination group (decrease 39±18% from baseline), but not in control group. Combination group had longer progress free survival (PFS) comparing with control group. (Median PFS combination vs control group: 15.2 vs 9.9 months, Log Rank test, p=0.05) Increased of peripheral blood cytotoxic T cells also had trend in combination group but not in control group.

Conclusion: Besides the anti-angiogenesis effect, bevacizumab had immune modulated effect, especially in decreasing circulating S100A9+ MDSC in EGFR mutated lung adenocarcinoma patients. This might partially explain the longer PFS in combination of EGFR-TKI of anti-angiogenesis agent treatment.

#1619

Gemcitabine-generated ROS promotes neutrophil transmigration and activation by synergistic CXCL8 production with PKC delta-activated macrophages.

Jinseon Jeong,1 Yong-Jae Kim,1 Kwang Hoon Yang,1 Sun Young Yoon,2 Ki-Young Sohn,2 Heung-Jae Kim,2 Jae-Uk Chung,2 Myung-Hwan Kim,3 Jae Wha Kim1. 1 _Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea;_ 2 _Enzychem Lifesciences, Daejeon, Republic of Korea;_ 3 _Asan Medical Center, Seoul, Republic of Korea_.

Chemotherapy-induced neutropenia (CIN) is a common side effect that necessitate dose reductions during treatment of cancer patients. Preventing CIN is critical in chemotherapy because a rapid decline of neutrophil counts increase susceptibility to infection of cancer patients. In spite of its importance, the mechanism responsible for CIN still remains unclear. The purpose of this study is to investigate the mechanism of CIN in an aspect of neutrophil activation by chemotaxis in in vivo and in vitro model systems. It is well known that protein kinase C δ (PKCδ) is endogenously activated in immune and cancer cells of tumor environments, and treatment of gemcitabine generates reactive oxygen species (ROS). Based on these phenomena, we hypothesized that gemcitabine-generated ROS synergistically amplifies PKCδ signaling and C-X-C Motif Chemokine Ligand 8 (CXCL8) production in macrophages and neutrophil recruitment to the chemokine gradient following cell death by activation. In breast tumor mouse model, we found that intracellular PKCδ is activated in both the peritoneal cavity and the tumor region. To investigate if neutrophils are transmigrated by gemcitabine treatment, blood and peritoneal leukocytes were analyzed at different times after intraperitoneal administration of gemcitabine (50mg/kg). The number of neutrophils significantly decreased in the blood but increased in the peritoneal cavity in the early phase (1-2h) after administration. However, both blood and peritoneal neutrophil counts fell by almost the normal range in the late-phase (15h). Similarly with the neutrophil migration, the level of macrophage inflammatory protein 2 (MIP-2) was highest at 1h in the peritoneal cavity. To check whether neutrophils elicit neutrophil extracellular trap (NET) formation in response to gemcitabine treatment, we employed peritoneal neutrophils and determined that neutrophils produce NET in response to gemcitabine as determined by immunofluorescence. To examine the synergistic effects in in vitro systems, we induced mild PKCδ activation using 0.1nM of phorbol 12-myristate 13-acetate (PMA) in human monocytic THP-1 cells, and co-treatment of gemcitabine significantly induced the increased activity of PKCδ and the level of CXCL8 production compared to their single treatments. The synergistic activation of PKCδ led to the increase transcriptional activities, including AP-1, NF-κB, and STAT3. In conclusion, regulation of PKCδ activity is critical for preventing cancer patients from CIN, and PKCδ regulators could be a potential therapeutic agent for CIN.

#1620

Oral IL-10 suppresses colon carcinogenesis via elimination of pathogenic CD4+ T-cells and induction of antitumor CD8+ T-cell activity.

TAO GU,1 Magdia DeJesus,2 Thomas P. Burris,3 Nejat K. Egilmez1. 1 _University of Louisville, Louisville, KY;_ 2 _University at Albany School of Public Health, Albany, NY;_ 3 _Saint Louis University, St. Louis, MO_.

An oral sustained-release formulation of Interleukin-10 suppressed tumor growth and enhanced survival in the APCmin/+ / Bacteroides fragilis spontaneous colon cancer model. Therapeutic benefit was associated with a 5-fold reduction in CD4+RORɣt+Foxp3-IL-17+ T-helper cell, CD4+RORɣt+Foxp3+IL-17+ pathogenic T-regulatory cell (pgTreg) and CD4+RORɣt-Foxp3+IL-17- conventional T-regulatory cell (cTreg) numbers; and a concurrent enhancement of CD8+ T-cell cytotoxicity in the colonic lamina propria. Selective subset depletion and functional blockade studies demonstrated that CD4+RORɣt+IL-17+ T-cell subsets and CD4+Foxp3+ cTreg supported tumorigenesis/tumor progression whereas CD8+ cytotoxic T-lymphocytes suppressed tumor growth. These findings establish the utility of oral particulate IL-10 as a potential new therapeutic in the management of colon cancer and shed light on the cellular mechanisms that underlie its antitumor activity.

#1621

Adoptively transferred B cells directly kill tumor cells via the CXCR4/CXCL12 & perforin pathways.

Leiming Xia,1 Yang Xia,1 Quanning Chen,2 Yi Wang,3 Yangyi Bao,3 Steven K. Lundy Lundy,1 Fu Dai,3 Alfred E Chang,1 Qiao Li1. 1 _Univ. of Michigan, Ann Arbor, MI;_ 2 _Tongji Hospital of Tongji University, Shanghai, China;_ 3 _The No.1 People's Hospital of Hefei, Hefei, China_.

Over the years, the role of B cells in the host immune response to malignancy has been overshadowed by our focus on T cells. The role played by B cells in cancer immunology is complex and controversial. The observation made by our lab that activated B cells alone can mediate tumor regression in the adoptive immunotherapy of solid tumors is innovative (JI 2009; CCR 2011; JSO 2012; EJI 2015). One novel mechanism by which activated B cells mediate tumor regression is via direct tumor cell cytotoxicity in the absence of antibodies. We previously reported that antitumor B cells directly kill tumor cells via the Fas/FasL pathway and are regulated by IL-10. In this study, we defined additional mechanisms involved in B cell antitumor immunity. Administration of IL-2 significantly augmented the therapeutic efficacy of adoptively transferred tumor-draining lymph node (TDLN) B cells which express IL-2R. Culture supernatant of purified B splenocytes harvested from the mice that received adoptive transfer of 4T1 TDLN B cells plus IL-2 administration produced larger amounts of IgG which bound to 4T1, resulting in 4T1 lysis. Furthermore, we detected CXCR4 expression on 4T1 TDLN B cells, and 4T1 tumor cells produced its ligand CXCL12. Transwell experiments demonstrated the chemotraction of CXCR4-expressing 4T1 TDLN B cells towards CXCL12-producing 4T1 cells. Blockade of CXCR4 using a CXCR4-specific inhibitor, AMD3100, significantly reduced the killing of 4T1 tumor cells by 4T1 TDLN B cells. Blockade of FasL and CXCR4 concurrently inhibited B cell-mediated direct killing of tumor cells in an additive manner, indicating that both Fas/FasL and CXCL12/CXCR4 pathways are involved in the direct killing of 4T1 cells by 4T1 TDLN B cells. TDLN B cells produced perforin. Additional transwell experiments showed that effector B cells could directly kill tumor cells in cell-cell contact via the Fas/FasL and CXCR4/CXCL12 pathways as well as perforin, while without cell contact, perforin secreted by B cells led to tumor cell cytotoxicity. These findings underscore the diversity of function by which B cells can play an important role in the host immune response to tumor, and clearly indicated that transferred effector B cells can act independently of T cells in causing tumor destruction in adoptive immunotherapy.

#1622

The role of the CCL-2 on lymphopenia-induced myeloid derived suppressor cells.

Pasquale P. Innamarato, Amy Weber, Shari Pilon-Thomas. _H. Lee Moffitt Cancer Center, Tampa, FL_.

The induction of lymphopenia prompts the expansion of CD11b+Ly6ChiLy6G- monocytic MDSCs (M-MDSCs) and CD11b+Ly6C+Ly6G+ polymorphonuclear MDSCs (PMN-MDSCs). Patients receiving adoptive cell therapy (ACT) require nonmyeloablative chemotherapy to induce lymphopenia and support anti-tumor immunity. However, the role of MDSCs in the setting of ACT is not fully understood. As high levels of CCL-2 can be measured in lymphopenic mice, in this study we investigate the role of CCL-2 in the expansion and function of lymphopenia-induced MDSCs using CCR2KO mouse models. Lymphopenia was induced in melanoma-bearing C57BL/6 (WT) and CCR2KO mice by 600rad of total body irradiation or combination therapy with cyclophosphamide and fludarabine. We first evaluated the percentages of splenic MDSCs in WT and CCR2KO mice after the induction of lymphopenia. At day 14, the expansion of total MDSCs were similar in WT (279% of normal) and CCR2KO (241% of normal). However, the mean percentages of M-MDSCs (3.5%) and PMN-MDSCs (30.3%) in CCR2KO mice were altered compared to M-MDSCs (22.2%) and PMN-MDSCs (21.7%) in WT mice. In addition, M-MDSCs in B16 tumors grown in CCR2KO mice were decreased compared to intratumoral M-MDSCs in WT mice (p<0.001). In contrast, the percentages of M-MDSCs and PMN-MDSCs were unchanged in the bone marrow (BM). To investigate the suppressive capacity of lymphopenia-induced MDSCs, OVA antigen-specific CD8+ T cells were co-cultured with OVA peptide in the presence of MDSCs purified from the spleens of CCR2KO and WT mice. Both CCR2KO and WT MDSCs potently suppressed T cell proliferation as measured by 3H thymidine incorporation. CCR2KO and WT MDSCs had significant arginase activity and production of nitrites. To investigate the efficacy of ACT, B16 tumor-bearing CCR2KO and WT mice were lymphodepleted and gp100 antigen-specific Thy1.2+CD8+ T cells were adoptively transferred followed by 3 days of IL-2 treatment. We found that donor T cell tumor infiltration was similar and no difference in tumor growth was observed between CCR2KO and WT mice. Collectively, these results suggest that the role of CCL-2 is important for the egress of lymphopenia-induced M-MDSCs from the BM, but has no effect on the expansion or function of PMN-MDSCs in the setting of lymphopenia. These data suggest that factors besides CCL-2 play an important role in the expansion and function of MDSCs after the induction of lymphopenia. 

### Tumor Microenvironment and Checkpoints

#1623

Minute variation in murine ovarian cancer preclinical models significantly impact immunotherapy results obtained for clinical trial.

AJ Robert Mcgray, Kristen Starbuck, Samar Masoumi-Moghaddam, Ariel Francois, Kunle Odunsi, Emese Zsiros. _Roswell Park Cancer Institute, Buffalo, NY_.

Cancer immunotherapies, particularly checkpoint inhibitors, have emerged as highly promising approaches for treatment of a variety of solid tumors, including recurrent ovarian cancer. While dramatic clinical benefit is observed in a subset of patients with ovarian cancer, the majority of ovarian cancer patients do not respond to checkpoint inhibitors, thus combinatorial treatments are sorely needed. Immune competent pre-clinical animal models are crucial to test emerging treatment strategies and for the identification of biomarkers to predict response to therapy.

As results from immune competent preclinical models are often translated into clinical trials, we sought to investigate whether slight variability related to animal housing and/or breeding practices in the same strain could impact experimental outcomes related to immunotherapy. We conducted animal studies using C57BL/6J mice purchased from Jackson Laboratories (Cat# 000664) 2 weeks prior to experimental use (Jax) and using the same mouse strain purchased from Jackson Laboratories but bred within our institution for a period of 6 months prior to use (Jax-R). Using the gold standard ID8 murine metastatic intraperitoneal ovarian cancer model we tested the therapeutic efficacy of anti-PD-1 monotherapy in both Jax and Jax-R animals. Aged-matched female mice were housed under the same conditions for 2 weeks prior to tumor inoculation and for the duration of study. Strikingly, we noted that while anti-PD-1 monotherapy had absolutely no effect on tumor growth or survival of Jax mice, the Jax-R animals showed dramatic tumor growth control and significantly improved long term survival. Tumor progression and survival was similar across sub-strains in untreated control animals, suggesting that the observed differences in treatment outcome following anti-PD-1 treatment were not simply due to intrinsic differences between the animals. Response to treatment was also associated with increased frequencies of circulating CD4+ and CD8+ T cells in the Jax-R mice compared with the Jax animals.

Although major variations in age, animal strain and gut microbiome can dramatically impact upon the efficacy of checkpoint inhibitors, our findings demonstrate that even minute changes within sub-strains recently derived from the same founder animals could result in significantly different survival and pre-clinical outcome. Thus, immunotherapy results obtained from immune competent animal studies need to be interpreted with caution when designing future immunotherapy trials.

#1624

NOTCH ligand-based therapeutics for immunomodulation in cancer and organ transplantation.

Elena I. Tchekneva,1 Anneliese E. Antonucci,1 Irina Chekneva,2 Nicholas Long,1 Jason V. Evans,3 Anwari Akhter,1 David P. Carbone,1 Thomas Magliery,1 Mikhail M. Dikov1. 1 _Ohio State University, Columbus, OH;_ 2 _1st Moscow State Medical University, Moscow, Russian Federation;_ 3 _West Virginia University, Morgantown, WV_.

We demonstrated in human and mouse studies that tumor-induced modulation of Notch ligand expression and Notch signaling in hematopoietic compartment contributes to tumor immune escape. Down-regulation of delta-like ligands (DLL) leads to defects in T cell development and T helper (Th1) cell differentiation with the prevalence of regulatory T cell (Treg) generation.

To determine the roles of Notch ligands in antigen-presenting dendritic cells in regulation of antitumor immune responses we generated a set of lineage-specific knock-out mice lacking one of the Notch ligands in CD11c+ dendritic cells (DC). We are developing and testing a set of reagents for clinical application for ligand-specific activation or inhibition of Notch signaling to stimulate or inhibit, respectively, various types of immune responses for applications in oncology and immune diseases.

Mice with DLL1 insufficiency in DC demonstrated remarkably accelerated growth of Lewis lung carcinoma (LLC) tumor, and reduced survival compared to wild type animals. This associated with impaired anti-tumor immune responses indicated by the decreased tumor infiltration by IFNγ-producing T cells. Jagged2 knockout did not cause any significant alterations. Notch ligand expression in antigen-presenting cells was identified as a "checkpoint" regulating the type of immune response. Data reveal that expression of Notch ligands by antigen-presenting cells is an important immune response specifying mechanism and that ligand-specific Notch signaling could be a valuable therapeutic target.

Reagents for the pharmacological modulation of immune responses based on Notch ligand constructs is proposed. Our cell-based study showed that pharmacological activation of Notch ligands required multivalent receptor-ligand interaction, whereas soluble ligands acted as competitive Notch inhibitors. We have generated reagents that comprise specific domains of the DLL1 in multivalent or monovalent form. Therapeutic inhibition of Notch by monovalent DLL1-based reagent accelerated LLC tumor growth and attenuated T cell-mediated anti-tumor immune response. In a heart transplantation mouse model, monovalent DLL1 reagent significantly prolonged allograft survival by inhibiting Th1 effector and memory T cell differentiation. Multivalent forms of DLL1 effectively stimulated Notch signaling in T cell cultures and enhanced IFNγ production, whereas monovalent reagent had opposite effects.

Pharmacological up-regulation of DLL1-mediated Notch signaling with multivalent forms of ligand represents an efficient strategy for the enhancement of anti-tumor immunity and targeting multiple mechanisms of tumor growth. Monovalent DLL1 forms could be utilized for therapeutic inhibition of Th1 responses in autoimmune diseases and organ transplantation. Reagents based on the mono- and multivalent forms of Notch ligands can be efficiently utilized for therapeutic modulation of Notch signaling.

#1625

NGS reveals specimen characteristics have minimal impact on immune gene expression signature.

Jeffrey Conroy,1 Sean Glenn,1 Blake Burgher,1 Sarabjot Pabla,1 Maochun Qin,1 Jon Andreas,1 Vincent Giamo,1 Marc Ernstoff,2 Mary Nesline,1 Ji He,1 Mark Gardner,1 Carl Morrison1. 1 _OmniSeq, LLC, NY;_ 2 _Roswell Park Cancer Institute, NY_.

Background: Therapeutic antibodies targeting immune checkpoint molecules have been approved by the FDA for the treatment of several types of cancer. Currently, evaluation of the tumor checkpoint blockade is limited to FDA-approved IHC assays measuring PD-L1 ligand status which is subjective and not analytically robust. As the number of antibodies targeting immune checkpoints expands, assays that can evaluate additional biomarkers in tumor specimens are needed to accurately predict patient response to these drugs. To address these issues, a custom immune response NGS assay was developed to measure the transcript level of 54 genes involving T-cell receptor signaling (TCRS) and tumor infiltrating lymphocytes (TILs) in solid tumors of various characteristics including heterogeneity, disease, biopsy type and age. As part of the study, we evaluated the impact these variable have on the immune gene expression signature and their role as possible assay interferents.

Methods: Studies were designed to evaluate the analytical performance of a targeted RNA-seq assay for FFPE samples from NSCLC, melanoma, RCC, HNSCC, kidney and bladder cancer. To assess degree of assay tolerance to the wide range of specimen characteristics that are inherent in tumors, samples and mixed samples of various histopathologic characteristics were included. PCA and unsupervised clustering was performed on samples with checkpoint inhibition, TCRS and TILs genes to reveal sample groups with three distinct immune signatures (low, indeterminate and high). Further correlation and over-representation analysis was performed to determine impact of specimen characteristics on these three immune signatures.

Results: Immune signatures were maintained for the majority of characteristics studied within a specified range. As expected, only TIL status was significantly associated with the high expression group. Other factors including architecture, neoplastic content, percent necrosis, stroma quality/quantity, T-Path, PMR, specimen type, tissue amount and specimen age were not over-represented in any immune signature.

Conclusion: Tumor samples harbor a mixture of potential assay interferents including variable benign, neoplastic and immune cells populations with both naïve and reactive stroma contributing to a complex tumor microenvironment that is difficult to catalogue prior to testing. Our study demonstrates that the immune signature present in the tumor microenvironment is sufficiently strong to withstand a wide range of tumor heterogeneity, thereby reducing the need of extensive tissue macrodissection and the exclusion of samples previously thought to be non-evaluable.

#1626

Technical variability in NGS immune gene expression and mutation profiling has a nominal effect on tumor classification.

Sean Glenn,1 Jeffrey Conroy,1 Blake Burgher,1 Sarabjot Pabla,1 Maochun Qin,1 Jon Andreas,1 Vincent Giamo,1 Marc Ernstoff,2 Mary Nesline,1 Ji He,1 Mark Gardner,1 Carl Morrison1. 1 _OmniSeq, LLC, Buffalo, NY;_ 2 _Roswell Park Cancer Institute, Buffalo, NY_.

Background: A custom NGS cancer immune gene expression assay was developed which measures the transcript level of >350 genes involved in T-cell receptor signaling (TCRS), tumor infiltrating lymphocyte (TILs) complement as well as other key targets expected to predict the likelihood of patient response to checkpoint inhibitors (CPI). In parallel to the gene expression assay, mutational profiling was carried out using the 409 gene Comprehensive Cancer Panel (ThermoFisher). As variability between runs is common when performing NGS assays a detailed comparison of specific technical variations were assessed for their ability to effect gene expression and mutation profiles of clinical FFPE samples.

Methods: Studies were designed to characterize the analytical performance of the immune response NGS assay using RNA and DNA from a subset of 300 FFPE tissues representing NSCLC, melanoma, renal cell carcinoma and bladder cancer. As part of the study, we tested the impact of variability in RNA and DNA input quantity at the library preparation step, sample batch size which affects mapped reads/sample and depth of coverage, and linearity of expression and sensitivity of mutation profiling through serial dilutions of pico-molar (pM) input of normalized library. PCA and unsupervised clustering was performed on samples with checkpoint inhibition, TCRS and TILs genes as well as mutational profiling to reveal sample groups with three distinct immune signatures (low, indeterminate and high). Further correlation and over-representation analysis was performed to determine impact of technical characteristics on these three immune signatures.

Results: Immune signatures including mutation profiles and gene expression levels were maintained throughout variable RNA/DNA input amounts at the library generation level as well as with diminution of pM levels of library pooled at the sequencing step. Increase in the number of mapped reads and sequencing depth through decreasing the number of batched samples per sequencing run also did not affect the gene expression and mutation profile signatures of the FFPE derived samples.

Conclusion: The gene expression and mutation profiles responsible for classifying FFPE samples using NGS are not affected by variation normally introduced in the technical workflow commonly associated with these platforms. The analytical assessment of input at the nucleic acid, library, and sample size level has shown the plasticity available when using amplicon based NGS technologies for classifying the immune gene expression signature as well as mutational profiles of FFPE derived clinical tumor samples. This flexibility increases the strength and utility of NGS-base gene expression profiling and mutational analysis of tumor samples for both basic research and clinical applications.

#1627

Enhancement of anti-cancer immunity by OMX, a novel oxygen carrier immunotherapeutic that ameliorates the hypoxic tumor microenvironment.

Kevin G. Leong, Yuqiong Pan, Jonathan A. Winger, Stephen P. Cary, Natacha Le Moan, Ana Krtolica. _Omniox, Inc., San Carlos, CA_.

Hypoxia is a hallmark of cancer and a driver of tumor progression and poor patient outcomes. By generating an immunosuppressive tumor microenvironment that limits cytotoxic T lymphocyte (CTL) infiltration and activation, hypoxia limits the effectiveness of cancer immunotherapy and thus promotes tumor cell evasion of the host immune response. Omniox has developed a first-in-class anti-cancer immunotherapeutic, OMX, specifically designed to reverse tumor hypoxia to enhance cancer immunotherapy efficacy. In preclinical models, we have demonstrated that OMX accumulates in rodent subcutaneous and orthotopic tumors, as well as spontaneous canine melanomas and brain tumors, resulting in significant tumor hypoxia reduction.Here, using multiple subcutaneous syngeneic mouse tumor models (MC38, CT26, 4T1), we assessed OMX effects on intratumoral CTLs and immunosuppressive regulatory T cells (Treg), as well as the anti-tumor potential of OMX as a single agent and in combination with established immunotherapies. Using quantitative immunohistochemistry, we confirmed reports that hypoxic tumor areas are devoid of CTLs. Accordingly, by flow cytometry we observed a negative correlation between tumor hypoxia and CTL infiltration. While OMX single agent treatment did not affect the overall CD45-positive leukocyte population, Treg cells were selectively depleted and the CTL:Treg ratio was substantially increased, suggesting that OMX induced a shift towards immunosensitization. Consistent with this finding, we observed OMX single agent anti-tumor efficacy in MC38 colon tumors. Impressively, anti-tumor effects of OMX single agent were equivalent to that of a single treatment of the checkpoint inhibitor anti-CTLA4. We next assessed whether OMX would enhance the efficacy of checkpoint inhibitors when used in combination. In CT26 colon tumors, OMX exhibited combination anti-tumor activity with anti-CTLA4, giving rise to faster cures and a greater number of complete and durable responders compared to anti-CTLA4 alone. Of note, this enhanced response was observed for both early-stage and late-stage CT26 tumors. In 4T1 breast tumors, known to be insensitive to checkpoint inhibitors, treatment of early-stage (~60mm3) tumors with combination OMX and anti-PD1 resulted in a 27% response rate, compared to a 0% response rate to anti-PD1 alone. Taken together, our data suggest that OMX, by delivering oxygen to hypoxic tumor areas, induces a microenvironmental change from an immunosuppressive to an immunopermissive state. Given that OMX is well-tolerated in both small and large animals, and that its mechanism of action is upstream of numerous major immunosuppressive pathways, OMX holds great clinical potential to synergize with multiple immunotherapeutic agents to enhance tumor control by restoring anti-cancer immune responses in cancer patients.

#1628

Early events in metastatic spread: new approaches using targeted therapeutics to disrupt formation of the pre-metastatic niche and development of lung metastases.

Khalid A. Mohamedali, Lawrence H. Cheung, Michael G. Rosenblum. _UT MD Anderson Cancer Ctr., Houston, TX_.

Solid tumors release soluble factors causing migration of a subset of normal bone marrow derived cells (BMDCs) - primarily VEGFR-1+ hematopoietic progenitor cells and VEGFR-2+ circulating endothelial progenitor cells - from the bone marrow to organ sites. These normal BMDCs self-assemble into a pre-metastatic niche- a micro-environment eventually hosting migratory tumor cells from the primary site. Disruption of this niche or selective targeting the migratory tumor cells may inhibit metastatic spread. The significance and specific characteristics of the BMDCs is still unclear. We have previously observed migration of VEGFR-1+ BMDCs to the lung and lymph nodes, and VEGFR-2+ BMDCs to liver, lung and lymph nodes. Using GFP+ BMDC's from genetically engineered mice, we characterized the role that recruitment of BMDCs may play in breast cancer metastasis. We assessed the BMDC recruitment profile in lethally irradiated female nu/nu mice transplanted with GFP+ BMDCs from donor mice, followed by orthotopic placement of MDA-MB-231/luc cells or injected with MDA-MB-231/luc conditioned media for 30 days. Flow cytometry results show a gradual increase in the recruitment of CD11b+VEGFR-1+ cells in all the tissues examined from tumor-bearing mice. Recruitment of these cells to the liver in mice treated only with MDA-MB-231/Luc conditioned media was also apparent, but the increase was not as high as in livers of mice with the orthotopically-placed tumors. Recruitment of CD11b+VEGFR-2+ BMDCs was also observed but only in liver and lung. Interestingly, conditioned media seemed to recruit this subset of cells more strongly to these two tissues than signaling from orthotopic tumors. Spleen and lymph node showed minimal recruitment of CD11b+VEGFR-2+ BMDCs. We have previously developed the GrB/VEGF fusion construct- a novel pro-apoptotic fusion protein which specifically targets cells harboring VEGFR2 and have utilized this agent to specifically target BMDCs which are VEGFR2 +. Our preliminary data indicate that treatment with GrB/VEGF does not significantly alter the recruitment of VEGFR-1+ or VEFGR-2+ cells to lungs, when assessed two weeks after the final treatment. We observed increased recruitment of F4/80+ macrophages to the lung. On the other hand, CD11b+Gr-1- BMDCs were significantly reduced following GrB/VEGF treatment, although the F4/80+ subpopulation carrying this signature increased. Studies are ongoing to determine whether systemic administration of this agent can disrupt the formation of the niche and the eventual establishment of metastatic tumors. Understanding the role of BMDCs in metastatic spread and the formation of the pre-metastatic niche and their role in the early development of metastasis will be critical in designing targeted therapeutic approaches to inhibit the metastatic process. Research sponsored, in part, by the Clayton Foundation for Research.

#1629

An integrated view of Notch signaling that regulates tissue renewal in response to enteric infection.

Badal C. Roy,1 Ishfaq Ahmed,1 Audrey Seamons,2 Shrikant Anant,1 Lillian Maggio-Price,2 Seth Septer,3 Shahid Umar1. 1 _Kansas University Medical Center, Kansas City, KS;_ 2 _University of Washington, Seattle, WA;_ 3 _Children's Mercy Hospital, Kansas City, MO_.

Background: Microbial dysbiosis and the associated gut inflammation unbalances epithelial renewal, potentially leading to cancer which is increasingly being recognized as a stem cell disease. Notch signaling is active in multipotent intestinal stem cells (ISCs); yet, how Notch signaling orchestrates communication between the gut microbes and ISC-dependent tissue renewal following a pathogenic insult, is poorly understood.

Aim: To investigate how Notch signaling contributes towards ISC regeneration and pathogenesis of infection.

Methodology: Rag-1-/- mice and wild type littermates were infected with Citrobacter rodentium (CR; 108CFUs) and treated with Notch blocker Dibenzazepine [(DBZ), ip at 10 µmol/kg body weight]. Whole distal colon or purified crypts were isolated for analyses. Fecal 16S rDNA analysis was performed. Transgenic mice expressing MHC-II either in IEC (EpithTg) or in dendritic cells (CD11cTg) were crossed to Rag2-/- mice and received Helicobacter bilis (Hb) to induce colitis. De-identified sections from control or Crohn's Disease (CD) and Ulcerative Colitis (UC) patients were stained for markers of ISCs and immune cells, respectively.

Results: Rag-1-/- mice but not WT littermates, exhibited dramatic increases in Dclk1 (Doublecortin-like kinase 1; an ISC marker) expression in the colonic crypts, measured via flow cytometry and IHC at 12-days post CR-infection that co-localized with Notch Intracellular Domain (NICD). CR-infected mice when treated with DBZ for 10 days exhibited: i) significant dysbiosis with Proteobacteria dominating (48% compared to 27% after CR infection), ii) increases in paracellular permeability concomitant with almost complete attenuation of Dclk1 expression and, iii) exacerbation of inflammation/colitis. Intriguingly, Dclk1 immunoreactivity shifted towards the stroma wherein, Dclk1 co-localized with NICD and with CD11c+ dendritic cells, CD11b+;F4/80+ macrophages and MHCII. Both EpithTg/Rag2-/- and CD11cTg/Rag2-/- mice when infected with Hb compared to uninfected mice, exhibited loss of crypt Dclk1 and its co-localization with NICD that coincided with severity of colitis. Sections prepared from the colons of Crohn's Disease (CD) or Ulcerative Colitis (UC) patients paralleled loss of crypt Dclk1 seen in mice while Dclk1 continued to co-localize with NICD and with markers of immune cells within the stroma. When CR infected and DBZ-treated Rag-1-/- mice were given a cocktail of antibiotics (500mg/l Vancomycin, 1g/l metronidazole and 0.2 g/l ciprofloxacin) for 7 days, we observed increased survival and decreases in colon myeloperoxidase activity that coincided with an elevated Dclk1 levels in the crypt.

Conclusions: 1. Bacterial dysbiosis following chronic Notch inhibition coupled with loss of crypt Dclk1 impairs crypt regeneration. 2. Co-localization of stromal Dclk1 with markers of immune cells and with MHCII, suggests a novel role for Dclk1 in antigen presentation.

#1630

Modulation of the neuroblastoma microenvironment via polyamine blockade.

Adriana D. Benavides, Annette Vu, Gabrielle M. Ferry, Michael D. Hogarty, Hamid Bassiri. _Children's Hospital of Philadelphia, Philadelphia, PA_.

Despite improvements in therapy over the last few decades, neuroblastoma (NB) still accounts for a considerable portion of childhood cancer-related mortalities and 5-year survival rates in patients with high-risk disease remains poor (40-50%). Amplification of MYCN and dysregulation in downstream Myc-related pathways result in NBs with high-risk features. Notably, MYCN-amplified NBs have elevated polyamine (PA) levels, and the gate-keeper enzyme in the PA synthesis pathway, ornithine decarboxylase (ODC1), is a direct target of Myc. Yet, ODC1 activity can be irreversibly inhibited by the FDA-approved drug difluoromethylornithine (DFMO), and exposure of NB cell lines in vitro to DFMO reduces cellular growth. In a mouse model of MYCN-driven NB (TH-MYCN+/+ transgenic mice), inhibition of PA synthesis with DFMO led to reductions in NB burden and extension of survival that appeared more profound than that predicted by the in vitro activity of the drug, suggesting that in addition to tumor-intrinsic effects, DFMO may have significant effects on the NB tumor microenvironment (TME). We therefore sought to characterize the tumor microenvironment of NB in TH-MYCN+/+ mice in the presence or absence of DFMO-mediated PA blockade. Tumors from DFMO treated and untreated mice were dissected, mechanically and enzymatically dissociated, and the number and frequencies of various TIL subsets were assessed using an optimized flow cytometry-based protocol. Our results indicate that DFMO reduces tumor growth and results in distinct and reproducible alterations in the cellular composition of the NB TME, the most profound of which was a significant increase in the frequency of NK cells. Moreover, we found an increase in the percentage of tumor cells expressing NK cell ligands. Concordantly, we also witnessed a shift in the NK cell expression of activating and inhibitory receptors. These findings are consistent with the hypothesis that PA blockade induces distinct TME changes that predispose to more efficient immune control of NB growth. To probe the contribution of NK cells to this process, we are now testing whether antibody-mediated depletion of NK cells in DFMO-treated TH-MYCN+/+ mice results in loss of the extension in survival afforded by PA blockade. In parallel, we are also performing transcriptomic analysis on tumor and NK cells sorted from DFMO-treated and untreated tumors, to ascertain whether specific signaling pathways may be being altered by exposure to DFMO. It is our hope that these studies will complement the data being accrued from phase I/II clinical studies using DFMO in various therapeutic strategies for NB, and will allow for an increased understanding of how to more effectively combine PA blockade with other immunotherapies for this disease.

#1631

**Sequencing the circulating and infiltrating T-cell repertoire on the Ion S5** TM **.**

Geoffrey Lowman,1 Elizabeth Linch,1 Lauren Miller,1 Denise Topacio-Hall,1 Timothy Looney,2 Alex Pankov,2 Yongming Sun,2 Xinzhan Peng,1 Mark Andersen,1 Fiona Hyland,2 Ann Mongan1. 1 _ThermoFisher Scientific, Carlsbad, CA;_ 2 _ThermoFisher Scientific, South San Francisco, CA_.

T-cell repertoire (TCR) sequencing by next-generation sequencing (NGS) is a valuable tool for building a deeper understanding of the adaptive immune system. As immunotherapies, particularly T-cell dependent therapies, show increasing potential in treating cancer, the ability to gain a detailed, unbiased view of the TCR becomes imperative for biomarker discovery, immune response to treatment, and study of tumor microenvironments. A key question the field seeks to understand is the relationship between circulating T-cells and infiltrating T-cells at the tumor site. Here, we present a novel approach for TCR sequencing using the Ion S5 ™ sequencer which leverages simplified library construction workflows and offers a more complete characterization of the entire V(D)J region of TCRΒ. This method can leverage mRNA as input, minimizing requirements in starting materials and focusing sequencing to productive TCRΒ arrangements. This approach targets the constant (C) and the FR1 regions, minimizing the potential for primer bias and greatly increasing the phylogenetic information content compared to techniques that exclusively characterize the CDR3 domain. Our results show that the observed circulating T-cell repertoire size is approximately 2 orders of magnitude higher than the infiltrating T-cell repertoire. Accordingly, while it is difficult to fully capture the complete repertoire of circulating T-cells due to its vast diversity, we show that it is possible to reliably capture the complete infiltrating T-cell repertoire with as high as 10 samples on the Ion 530 ™ chip. Replicate sequencing runs of infiltrating T-cells offers correlation of ~0.9, indicating that the results were reproducible, and the samples were sequenced to appropriate depth. In summary, we believe that this workflow will allow researchers to more routinely characterize the infiltrating T-cell repertoire and offers the field a better understanding of the impact of repertoire diversity on tumor elimination.

#1632

Identification and analysis of EGLN3 as tumor-associated peptide in ccRCC.

Anna Reustle,1 Moreno Di Marco,2 Florian Büttner,1 Stefan Winter,1 Daniel Kowalewski,2 Linus Backert,2 Steffen Rausch,3 Joerg Hennenlotter,3 Marcus Scharpf,4 Falko Fend,4 Arnulf Stenzl,3 Jens Bedke,3 Matthias Schwab,1 Elke Schaeffeler1. 1 _Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology and University of Tuebingen, Stuttgart/Tuebingen, Germany;_ 2 _Department of Immunology, University of Tuebingen, Tuebingen, Germany;_ 3 _Department of Urology, University Hospital Tuebingen, Tuebingen, Germany;_ 4 _Institute of Pathology and Neuropathology, University Hospital Tuebingen, Tuebingen, Germany_.

Introduction:

Kidney cancer is the 12th most common cancer world-wide with 338.000 newly diagnosed cases in 2012, according to the World Cancer Research Fund International. With around 70-80%, clear cell renal cell carcinomas (ccRCC) make up the vast majority of kidney cancers. Advanced and metastatic ccRCCs are marked by poor survival rates despite the availability of targeted therapies. Currently several immunotherapeutic approaches, including checkpoint-blocking antibodies and dendritic cell vaccines are undergoing clinical investigations. In this study we identified tumor-associated peptides which could be used in cancer vaccines to improve treatment of patients with advanced ccRCC.

Methods:

To identify suitable peptides for cancer vaccines we set up a tissue collection of paired tumors and normal kidney tissues from 60 ccRCC patients. From these tissues, HLA molecules were isolated by affinity chromatography and bound peptides analyzed by mass spectrometry. HLA ligand profiles of tissues excised from various organ sites of non-ccRCC patients were used to complement the data. Based on these analyses, Egl nine homolog 3 (EGLN3) was selected as a candidate for peptide vaccination and further investigated in our own data cohort comprising the 60 ccRCC patients. Therefore, mRNA and protein expression, as well as DNA methylation data were collected and correlated with HLA ligandomics and clinical data. In addition, we integrated RNA-Seq, DNA methylation, somatic mutation and clinical data from a ccRCC patient cohort from The Cancer Genome Atlas in our analyses. Finally, EGLN3 function was analyzed in 2D and 3D kidney carcinoma cell culture systems.

Results:

16 different peptides from EGLN3 were detected in 32 of the 60 analyzed tumor samples, with the most frequent peptide found in tumors of 10 different patients. Only two of the peptides were also detected in normal kidney or other non-ccRCC tissues, rendering EGLN3 peptides promising candidates to be used in ccRCC-directed vaccines. Most of our patients were HLA A*02:01 positive, however using NetMHCpan (http://www.cbs.dtu.dk/services/NetMHCpan/) only one of the peptides identified by HLA ligandomics was also predicted to be a strong binder for that particular HLA molecule. Expression of EGLN3 mRNA was not different in patients with and without HLA-presented peptides. Generally, EGLN3 mRNA expression was strongly induced in ccRCC tumors compared to normal kidney (p<10-6), whereas protein levels remained mainly unchanged in most patients. Functional investigations in different RCC cell culture models indicated involvement of EGLN3 in regulation of apoptosis and cell cycle progression.

Conclusion:

Advanced ccRCC is marked by poor survival rates due to ineffective therapeutic options. Peptides from EGLN3, as well as other ccRCC-exclusive peptides could represent activators of the immune system to specifically fight ccRCC tumors. The function and targetability of EGLN3 remains to be investigated.

#1633

Immunotherapy for malignant mesothelioma that combines a mesothelia-targeted immune-activating protein and CXCL12/CXCR4 blockade.

Huabiao Chen, Binghao Li, Yang Zeng, Patrick Reeves, Qiuyan Liu, Ann Sluder, Jeffrey Gelfand, Timothy Brauns, Mark Poznansky. _Massachusetts General Hospital, Charlestown, MA_.

Background and Purpose: There is a significant unmet need for new treatment strategies for malignant mesothelioma (MM). Despite relevant advances in many cancer treatment areas, including improvements in diagnosis, staging, and the clinical course of treated patients, MM remains a highly lethal disease. The purpose of this study is to develop a combination immunotherapy for MM, which involves a fusion protein to target and evoke a cellualr immune response to mesothelin (MSLN) and the blockade of CXCL12/CXCR4 pathway to mobilize cytotoxic effector cells into tumors.

Experimental Procedures: The efficacy of the MSLN targeted immune activating fusion protein (scFv-MtbHsp70), FDA-approved small molecule CXCR4 antagonist AMD3100 (plerixafor), and the combination were evaluated in two syngeneic and orthotopic murine models of MM in immune competent C57BL/6 mice. Mice received 4 intraperitoneal (i.p.) treatments from 7 days post i.p. injection of luciferase-expressing 40L and AE17 cells. Tumor growth was monitored by in vivo imaging of luciferase activity with an IVIS Spectrum. Survival time was calculated as life span from the day of tumor inoculation. In immunological studies, mice were sacrificed 4 weeks after tumor cell inoculation. Immune cells from spleens and tumors were labeled with antibodies against CD3, CD4, CD8, CD25 and Foxp3 antibodies, and examined by flow cytometry. Splenocytes were stimulated with MSLN and assessed for intracellular IFN-γ production by flow cytometry.

Results: In both murine mesothelioma models, the fusion protein scFv-MtbHsp70 alone delayed tumor growth and prolonged mouse survival, which was associated with increased tumor infiltration by CD3+CD8+ T cells. Treatment enhanced the cytotoxic function of tumor-specific CD3+CD8+ T cells by evoking dendritic cell activation as well as antigen presentation and cross presentation. AMD3100 alone reduced the proportion of CD4+CD25+Foxp3+ Treg cells in tumors and decreased PD-1 expression on CD3+CD8+ T cells. The combination of the fusion protein and AMD3100 further significantly slowed tumor growth and enhanced mouse survival while augmenting tumor-specific CD8+ T-cell immune responses and abrogating intratumoral immunosuppression.

Conclusion: Our findings demonstrated for the first time the synergistic effect of combination of MSLN-targeted immune-activating fusion protein scFv-MtbHsp70 and AMD3100 in treatment of MM in mice. This is a new therapeutic strategy which may significantly prolong survival of patients with this disease.

#1634

CDK4/6 inhibition directly enhances an anti-tumor immune response in breast cancer.

Molly J. DeCristo*,1 Shom Goel*,2 April C. Watt,2 Haley BrinJones,2 Jaclyn Sceneay,1 Ben Li,2 Jessalyn M. Ubellacker,1 Shaozhen Xie,2 Susanne Ramm,3 Hye-Jung Kim,2 Sandra S. McAllister,1 Jean J. Zhao2. 1 _Brigham and Women's Hospital, Boston, MA;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _Harvard Medical School, Boston, MA_.

The cyclin D:cyclin-dependent kinases 4 and 6 (CDK4/6) axis is one of the most frequently dysregulated pathways in human cancers, and CDK4/6 inhibitors have shown significant activity against a number of solid tumors, including breast cancer. Analogous to clinical experience, we found that the CDK4/6 inhibitor, abemaciclib, caused significant tumor regression in the MMTV-rtTA/tetO-HER2 mouse model of luminal breast cancer. However, as CDK4/6 inhibitors are known to block tumor cell proliferation, but not directly induce tumor cell apoptosis, as we confirmed in our study, the mechanisms by which CDK4/6 inhibition caused tumor regression were not clear. Notably, abemaciclib therapy increased total CD3+ T cells in these tumors, while decreasing the immunosuppressive CD4+ regulatory T cell population. Further investigation revealed that CDK4/6 inhibition directly suppresses regulatory T cells by inhibiting their proliferation without impacting natural regulatory T cell formation in the thymus, the differentiation of inducible regulatory T cells, or regulatory T cell apoptosis. We are currently exploring potential effects of CDK4/6 inhibition on regulatory T cell function and lineage stability. Analysis of CD8+ and CD4+ T cells in abemaciclib-treated tumors revealed a marked reduction in the expression of the inhibitory immune checkpoint receptors PD-1, Tim-3, CTLA-4, and LAG3. Specifically, the fraction of CD8+ T cells expressing known markers of T cell exhaustion (PD-1/Tim-3 double-positivity or high expression of PD-1) was decreased in abemaciclib-treated tumors. Importantly, antibody-mediated depletion of CD8+ T cells established that response of MMTV-rtTA/tetO-HER2 tumors to abemaciclib is dependent on CD8+ T cells. Finally, given the observed immunomodulatory effects of abemaciclib, we sought to determine if abemaciclib treatment would sensitize MMTV-rtTA/tetO-HER2 tumors to immune checkpoint blockade. Pretreatment with abemaciclib followed by combination abemaciclib and α-PDL1 significantly enhanced tumor regression compared to abemaciclib or α-PDL1 alone. Our studies reveal a novel mechanism by which CDK4/6 inhibitors directly elicit an anti-tumor immune response. These results provide strong rationale for further investigations into combining CDK4/6 inhibitors with immune checkpoint blockade in breast cancer.

#1635

Multiplexed analysis of fixed tumor tissues using imaging mass cytometry.

Franz Villarroel-Espindola,1 Daniel Carvajal-Hausdorf,1 Ila Datar,1 Amanda Esch,2 Narges Rashidi,2 Ala Nassar,1 Shelly Ren,1 Ruth R. Montgomery,1 Roy S. Herbst,1 David L. Rimm,1 Kurt A. Schalper1. 1 _Yale University, New Haven, CT;_ 2 _Fluidigm, San Francisco, CA_.

Introduction: Comprehensive evaluation of the tumor microenvironment with preservation of spatial context requires simultaneous in situ detection of multiple targets. Current fluorescence-based methods can accommodate up to 5-6 distinct markers and can be applied to formalin fixed, paraffin embedded tissue (FFPE). Imaging mass cytometry uses metal-conjugated antibodies and mass spectrometry to perform highly multiparametric, quantitative measurement of protein targets within tissue sections providing unprecedented detail of quantitative tissue labeling. To date, the IMC technology has not been widely available and its performance has not yet been compared with other validated methods.

Methods: Using multiplexed quantitative immunofluorescence (QIF, AQUA/Genoptix) and imaging mass cytometry (IMC, Fluidigm) we validated and optimized metal-conjugated assays to detect HER2 (29D8-CST, 176Yb), CD3 (DAKO, 170Er), Ki-67 (B56-BD biosciences, 168Er), Histone H3 (D1H2-CST, 176Yb), pancytokeratin (C11-Biorad, 162Dy), Vimentin (RV202-BD Pharmingen, 156Gd), LipoR (Fluidigm, 115In) and DNA/nuclei (DNA intercalator, Fluidigm, 191/193Ir). The markers were applied to FFPE samples from tumor cell lines, human tonsil, lymph node; and breast and lung carcinomas represented in tissue microarray format. Data was visualized using MCD viewer and ImageJ software and results from QIF and IMC were compared.

Results: Metal conjugation did not affect the performance of the primary antibodies studied here. The staining patterns of the epithelial/tumor and stromal compartments evaluated using the IMC platform and QIF were comparable. HER2 signal was higher in tumors and cell lines harboring HER2 amplification by FISH/IHC. The CD3 signal was higher in T-cell lymphoma cells and in the inter-follicular areas of human tonsil and lymph nodes. Ki-67 signal was nuclear, co-localized with Histone H3 and showed positivity in lymphoid germinal centers, cytokeratin-positive carcinoma cells and CD3+ tumor infiltrating lymphocytes. Results obtained using the IMC at different time points in serial tissue sections were comparable (P<0.05).

Conclusion: Quantitative and reproducible measurement of immune and non-immune targets with spatial resolution using the IMC platform is feasible in FFPE breast and lung carcinomas and the results are comparable to multiplexed QIF. Expansion of the current panel to include >30 markers for immune cell phenotypes/function and tumor markers is ongoing.

#1636

Increased expression of programmed death-ligand 1 (PD-L1) on infiltrating immune cells of hepatocellular carcinoma (HCC) tissues after sorafenib treatment.

Li-Chun Lu,1 Yi-Hsuan Lee,1 Chun-Jung Chang,2 Yu-Yun Shao,1 Tsung-hao Liu,3 Ann-Lii Cheng,1 Chih-Hung Hsu1. 1 _National Taiwan Univ. Hospital, Taipei, Taiwan;_ 2 _National Taiwan University, Taipei, Taiwan;_ 3 _National Taiwan Univ. Hospital Hsin-chu Branch, Hsin-chu, Taiwan_.

Background: PD-L1 expression in tumor microenvironment of HCC was reported to associate with tumor aggressiveness and recurrence. The impact of sorafenib treatment on PD-L1 expression on tumor cells (TC) or tumor-infiltrating immune cells (IC) of HCC has been unclear.

Patients and Methods: We reviewed patients with HCC who had received sorafenib for advanced diseases at National Taiwan University Hospital, Taipei, Taiwan. Patients with paired HCC tissues, obtained before and after sorafenib treatment, were included. Immunohistochemistry (IHC) assay with clone SP142 antibody (Spring Bioscience, Pleasanton, CA, USA) was performed to analyze PD-L1 expression on TC and IC in paired specimens obtained before and after sorafenib. PD-L1 expression was scored as IHC 0, 1, 2, or 3 if <1%, ≥1% but <5%, ≥5% but <10%, or ≥10% of cells were PD-L1 positive, respectively. Further IHC assay was employed to characterize the PD-L1-positive IC. Associations between the PD-L1 expression and overall survival (OS) or duration of sorafenib treatment of the patients were analyzed.

Results: Twenty-three advanced HCC patients (Male: Female= 20: 3, median age of 64 years) with paired HCC tissues were included. All of the post-sorafenib HCC tissues were obtained after disease progression. The median duration of sorafenib treatment was 4.3 months (range: 1.3 to 18.7). The PD-L1 expression on IC was significantly increased in post-sorafenib HCC tissues, compared with tissues obtained before sorafenib (pre-sorafenib vs post-sorafenib IHC 0/1/2/3: 10/5/5/3 vs 5/5/2/11, p=0.046). However, the PD-L1 expression on TC was not significantly different between pre- and post-sorafenib tissues (IHC 0/1/2/3: 19/2/0/2 vs 14/5/0/4, p=0.065). By using IHC staining of CD68, CD66b, CD11b, and CD3 on the consecutive slides, we found the PD-L1-expressing IC were mainly CD68-positive macrophages, but not neutrophils or T lymphocytes. Neither the PD-L1 expression levels on IC of pre-sorafenib or those of post-sorafenib tissues were associated with OS or duration of sorafenib treatment.

Conclusions: The PD-L1 expression on IC, especially on macrophages, in tumor microenvironment was significantly increased in post-sorafenib progression HCC tissues. Whether this increased PD-L1 expression contributes to treatment failure of sorafenib warrants further investigation (This work was supported by the grants of NTUH 105-M3232 and MOST 105-2314-B-002-180).

#1637

ICOS agonism induces potent immune activation and anti-tumor response in non-clinical models.

Sapna Yadavilli, Tianqian Zhang, Ashleigh Hahn, Laura M. Seestaller-Wehr, Hong Shi, Yao-Bin Liu, M.Phillip DeYoung, David J. Kilian, Meixia Bi, Michael P. Adam, Shu-Yun Zhang, Sabyasachi Bhattacharya, Yuliya Katlinskaya, Christina Blackwell, Christopher B. Hopson, Niranjan Yanamandra, Roopa Srinivasan, Patrick A. Mayes, Axel Hoos. _Glaxosmithkline, Collegeville, PA_.

Inducible T-cell costimulator (ICOS) is a costimulatory receptor that is upregulated on activated CD4 and CD8 T cells and plays an important role in T cell survival, differentiation, regulation of memory and regulatory T cell pools and humoral responses. Preclinically, augmenting signaling through the ICOS pathway has been reported to induce anti-tumor activity and enhance responses to CTLA4 blockade.

Here we present non-clinical data evaluating ICOS agonist antibody activity in human and mouse model systems using a different antibody for each species. GSK3359609 is a novel, selective anti-human ICOS agonist. GSK3359609 induces ICOS signaling through phosphorylation of intermediates in the Pi3K pathway leading to lymphocyte activation, proliferation and pro-inflammatory cytokine secretion in human PBMC in-vitro. A robust increase in CD4 effector T cell proliferation and Granzyme B secreting CD8 T cells was observed with GSK3359609 treatment in in-vitro assays utilizing PBMC from healthy donors, cancer patients or tumor infiltrating lymphocytes (TIL). Modest induction of regulatory T cell proliferation and IL-10 secretion were also observed. Significant increase in IFNγ (p<0.05) and TNFα secretion was observed in both primary PBMC and TIL based assays. Gene expression analysis of GSK3359609 treated human T cells confirmed changes in genes associated with T and B cell activation. In mice, an ICOS surrogate antibody was utilized in immune competent mouse tumor models. Tumor regressions were observed in 10-40% of mice and were associated with a robust increase in effector memory T cells in periphery as well as increases in T cell activation and proliferation in lymphoid tissues and tumor. Robust increases in PD1, PD-L1 and PD-L2 gene expression were observed in the tumors from ICOS antibody treated mice along with an increase in cytotoxic T cell signature and induction of an IFNγ gene signature. Changes in regulatory T cell proliferation were also observed in the blood and tumor of mice treated with the mouse ICOS agonist however changes were consistently less in magnitude than corresponding functional changes in cytotoxic CD8+ and effector CD4 cells.

We further explored treatment settings where a combination therapy may condition the tumor immune microenvironment to a more favorable context for ICOS agonist therapy. Treatment with an anti-PD1 antibody resulted in strong upregulation of ICOS expression on tumor infiltrating CD8, CD4 effector and regulatory T cells while decreasing ICOS+ Tregs relative to CD8 and CD4 effectors in the tumor microenvironment. Synergistic anti-tumor activity was observed for the combination of PD-1 with ICOS agonist antibodies in preclinical studies. These studies provide a strong rationale for the ongoing FTIH Phase I study of GSK3359609 administered alone and in combination with pembrolizumab to patients with selected advanced solid tumors.

#1638

Improved outcomes with drug-resistant immunotherapy in a human xenograft model of glioblastoma multiforme.

Samantha B. Langford,1 Harold T. Spencer,2 Anindya Dasgupta,2 George Y. Gillespie,1 Kathryn Sutton,2 Larisa Pereboeva,1 Lawrence S. Lamb1. 1 _Univ. of Alabama at Birmingham, Birmingham, AL;_ 2 _Emory University, Atlanta, GA_.

INTRODUCTION: Conventional treatment strategies for high-grade gliomas have been uniformly dismal. We have previously shown that both primary and Temozolomide (TMZ)-resistant glioblastoma (GBM) cell lines upregulate stress-associated NKG2D ligands (NKG2DL) during the first several hours following exposure to TMZ, thereby creating an opportunity for NKG2DL-directed cell therapy, particularly γδ T cells that directly recognize these stress-associated antigens. Using a human/mouse patient-derived tumor xenograft (PDTX) model, we combined TMZ chemotherapy and TMZ-resistant ex vivo expanded/activated γδ T cells as Drug Resistant Immunotherapy (DRI). Drug resistance in this example is conferred by O-6-methylguanine-DNA-methyltransferase (MGMT) gene transfer, thereby enabling cytotoxic lymphocyte function in a chemotherapy-rich environment when the tumor is maximally stressed.

METHODS: A total of five GBM PDTX were examined, three parent (X12P, X22P, X59P) and two TMZ-resistant (X12T, X22T). Tumor NKG2DL expression and cytotoxicity of DRI were assessed using flow cytometry with cultured human astrocytes as controls. Intracranial (IC) glioma xenografts were established using either an unmodified (P) or a TMZ resistant clone (T) of human GBM explants passaged exclusively in immunodeficient mice. Tumor-bearing mice received intraperitoneal 60mg/kg TMZ on days 6, 8,13, and 15 and received IC injection of 1.5 x 106 DRI 4 hours following TMZ injection. Control mice received DRI T cells alone, TMZ alone or no therapy. Survival was assessed using Kaplan-Meier analysis.

RESULTS: All xenografts were found to constitutionally express NKG2DL which were upregulated upon exposure to TMZ. DRI T cells were cytotoxic to all tumors in vitro, showing 65%-80% specific lysis at an E:T ratio of 20:1 with no evidence of toxicity against cultured human astrocytes. Median survival (MS) for all groups of untreated mice was approximately 25 days, and γδ T cell therapy alone did not improve survival in the absence of TMZ. For the parent tumors, TMZ therapy significantly improved MS over untreated controls for both X12P and X59P (29 vs. 59 days and 20 vs 51 days respectively, p =0.0001) and eliminated tumors in X22P. DRI γδ T cells + TMZ significantly increased median survival additionally over TMZ alone with 80% of animals in both X12P and X59P surviving long-term (p=0.0001 and 0.05 respectively). DRI had no clear effect over TMZ for X22T (p=0.46), however, for X12T, DRI + TMZ significantly increased median survival from 22 to 38 days (72.7%) over TMZ alone (p = 0.04).

CONCLUSIONS: Combined TMZ chemotherapy and drug-resistance modified γδ T cell therapy can produce a significant increase in time to progression and improvement in median and overall survival for both primary and TMZ-resistant GBM using a strategy is readily adaptable to the clinical setting.

#1639

Combination of PD1 blockade and T cell costimulation by bispecific antibodies promotes human T cell activation and proliferation.

Gregory L. Moore, Michael Hedvat, Matthew J. Bernett, Rajat Varma, Suzanne Schubbert, Christine Bonzon, Kendra N. Avery, Rumana Rashid, Alex Nisthal, Liz Bogaert, Irene W. Leung, Seung Y. Chu, Umesh S. Muchhal, John R. Desjarlais. _Xencor, Inc., Monrovia, CA_.

Tumor infiltrating lymphocytes (TILs) express multiple checkpoint receptors, in contrast to lymphocytes found in the periphery (Matsuzaki et al PNAS 2010, Fourcade et al Cancer Res 2012, Gros et al JCI 2014). Checkpoint blockade has demonstrated increased clinical response rates relative to other treatment options; however, many patients fail to achieve a response to checkpoint blockade. We sought to identify an additional therapeutic modality to stack with checkpoint blockade that could increase patient response rate. We hypothesized that engagement of T cell costimulatory receptors in combination with checkpoint blockade could further increase T cell activation and proliferation. The combination of checkpoint blockade with costimulation could be accomplished using a bispecific antibody format, with the potential benefits of reduced cost and more selective targeting of TILs to improve safety.

Antibodies binding to immune checkpoint PD1 and a T cell costimulatory receptor were assembled in a bispecific antibody platform with substitutions in the Fc domain to suppress effector function. PD1 x costimulation (PD1 x costim) bispecific antibodies were evaluated in vitro by measuring antibody binding and de-repression of Staphylococcal enterotoxin B (SEB) stimulated PBMCs. IL2 and IFNγ production was measured by immunoassay. In vivo activity was evaluated using a mouse model in which human PBMCs are engrafted into NSG mice (huPBMC-NSG) and the extent of T cell engraftment is monitored by flow cytometry.

We produced PD1 x costim bispecific antibodies that bound PD1 and a T cell costimulatory receptor monovalently. The bispecifics bound to SEB-stimulated T cells more tightly than monovalent controls, indicating that a single bispecific molecule was capable of avid simultaneous co-engagement of both PD1 and a costimulatory receptor. The bispecifics enhanced IL2 and IFNγ production in an in vitro SEB stimulation assay relative to control (p < 0.001, n = 19 donors). IL2 and IFNγ production was superior to anti-PD1 or anti-costim antibodies alone (p < 0.001), indicating productive and synergistic combination of checkpoint blockade plus costimulation. Treatment of huPBMC-NSG mice with checkpoint bispecifics promoted enhanced T cell engraftment relative to control. Engraftment levels promoted by bispecifics were superior to those found for anti-PD1 treatment alone. For example, in one run of the model, while anti-PD1 treatment alone promoted a 2-fold increase in human CD45+ cells, a PD1 x costim bispecific antibody induced a 16-fold increase.

Combination of checkpoint blockade and costimulation with bispecific antibodies is feasible and promotes strong T cell activation in vitro and in vivo. Compelling activity suggests clinical development is warranted for the treatment of human malignancies.

#1640

A novel peptide-suppressing M2 polarized tumor-associated macrophages enhances tumor response to chemotherapy.

Anghesom A. Ghebremedhin,1 Clayton Yates,1 Jesse Jaynes,1 George Martin,2 Henry Lopez,3 Charles Garvin2. 1 _Tuskegee Univ., Tuskegee, AL;_ 2 _Riptide Biosciences, Inc, Vallejo, CA;_ 3 _Murigenics Inc, Vallejo, CA_.

Introduction: Multiple reports have demonstrated that M2-polarized tumor associated macrophages (TAMs) play a significant role in promoting tumor metastasis, survival and resistance to chemotherapy. Thus, limiting the pro-tumorigenic activity of TAMs may hold great therapeutic promise. Objective: To determine whether targeting the Cluster of Differentiation 206 (CD206) receptor on TAMs with an engineered peptides having both CD206 specificity and apoptotic activity, will limit the protumorigenic effects of TAMs and enhance cancer cells response to chemotherapy. Method: A class of engineered peptides were designed to stimulate phagocytosis via the CD206 receptor, and to initiate apopotic cell death once endocytosed. Primary cells were isolated from C57BL/6J mice from Jackson Laboratory and were polarized to M1 and M2 phenotypes by treating with IL-4 (20ng/ml) and IFN-γ (10ng/ml) respectively from R&D Biosciences for 24 hrs. CD206 targeting peptide was biotin-labeled and utilized to treat M2-polarized cells. Precipitated proteins were analyzed by mass spectrometry analysis, and informatics software. In vitro proliferation and confirmation of apoptosis was assessed using Annexin/PI staining and Western blot analysis for Caspase 8 expression and activity. In xenograft models of triple negative breast cancer (MDA-MB-231), mice were treated with CD206 peptide and gemcitabine, singly or in combination Results: Mass spectrometry analysis of gel bands from the biotin / streptavidin pull down assay confirmed CD206 as a cell surface target. Bioinformatics analysis of the proteomics data from the pull down assay further confirmed that apoptotic proteins were highly expressed in the M2 populations than in the M1 populations. Treatment with CD206 peptide produced a significant decrease in cell proliferation only in M2 macrophages. FACS analysis of Annexin/PI staining and Caspase 8 activity, demonstrated by Western blot, showed that CD206 targeting peptide caused apoptosis specifically in M2 macrophages. In xenograft models of triple negative breast cancer (MDA-MB-231) mice treated with peptide alone (10 mg/kg qD subcu), there was a 39% reduction in tumor growth; gemcitabine @ 40 mg/kg q4D produced a 68% reduction; however, peptide in combination with gemcitabine completely blocked tumor growth. Moreover, immunohistochemistry staining of treated tumors showed a decrease in the expression of CD206 positive M2 macrophages and CD45/CD25 positive Treg cells, and an increase in CD86 positive M1 macrophages. These results were consistent with the in vitro results obtained. Discussion and Conclusion: These results suggest that a targeting CD206 peptide engineered to promote apoptosis, enhances the tumor response to chemotherapy by limiting the pro-tumorigenic activity of TAMs.

#1641

Dual antibody blockade of TIM3 and PD1 on NYESO1 redirected human T cells leads to augmented control of lung cancer tumors.

Edmund Moon,1 Soyeon Kim,1 Shaun O'Brien,1 Naomi Saint Jean,1 Raluca Verona,2 Linda Snyder,2 Yangbing Zhao,1 Steven Albelda1. 1 _Univ. of Pennsylvania, Philadelphia, PA;_ 2 _Janssen Research and Development, Springhouse, PA_.

Background: Immunotherapy using checkpoint blockade has demonstrated impressive, durable responses in select patients with solid malignancies. This is especially true for the blockade of programmed death 1 (PD1). Currently, efforts are focused on understanding non-response. One hypothesis is that multiple inhibitory receptors (IRs) are upregulated on hypofunctional tumor-infiltrating lymphocytes (TILs) and necessitates blocking multiple IRs to increase the response rate. We describe data from a unique xenograft model of human lung cancer where tumor-reactive human T cells become hypofunctional and upregulate multiple IRs including PD1 and TIM3 (T cell immunoglobulin and mucin-domain containing-3). Combining a single IV dose of tumor-reactive T cells with blockade of both PD1 and TIM3 led to greater control of tumor growth than combining it with blockade of either alone.

Materials/Methods: The A549 human lung cancer cell line was transduced to stably express the cancer-testis antigen, NYESO1, expressed in the contact of HLA-A2 (A549-A2-ESO). 5x106 A549-A2-ESO cells were injected subcutaneously into the flanks NSG mice. Activated T cells from healthy donors were lentivirally transduced with a TCR targeting NYESO1 (Ly95). After two weeks when tumors were established and measured ~150mm3, the mice were randomly assigned to one of the following treatments: 1) untreated, 2) non-transduced (NTD) T cells plus anti-PD1 and anti-TIM3 Ab, 3) Ly95 T cells, 4) Ly95 T cells + anti-PD1 Ab, 5) Ly95 T cells + anti-TIM3 Ab, 6) Ly95 T cells + anti-PD1 and anti-TIM3 Ab. T cells were injected IV at a single dose of 10x106/mouse. Abs were injected intraperitonealy (IP) at 10mg/kg every 5 days from the time of T cell injection. Tumor volumes were measured serially. At the end, the mice were sacrificed and the tumors were harvested, digested, and processed into single cell suspension. Flow cytometry was performed to look at degree of TIL infiltration and expression of surface markers. Ficoll gradient was used to isolate the T cells to conduct functional analyses.

Results/Conclusion: By twenty days post T cell injection, the tumors in the Ly95 T cell group (3) were ~30% smaller than that of the untreated (1) and the NTD T cell + Ab group (2). Anti-PD1 Ab augmented Ly95 T cell control of tumor (4) (further decrease in size by ~30%) (4). Anti-TIM3 Ab had no effect on Ly95 T cell tumor control (5). Anti-PD1 plus anti-TIM3 Abs had the greatest augmentation on Ly95 T cell control of tumor size (6) (greater than 50% reduction in tumor size compared Ly95 T cells alone) The greatest TIL infiltration and ex-vivo TIL function were seen in tumors that received Ly95 T cell + anti-PD1 and anti-TIM3 Abs. Flow cytometry analysis revealed dynamic, adaptive upregulation of one IR when another IR was blocked. This explained why augmentation of Ly95 T cell control of tumor was greatest with combinatorial Ab blockade.

#1642

Evaluation of ENMD-2076 in combination with anti-PD1 in syngeneic cancer models.

Graham C. Fletcher,1 Reza Kiarash,1 Mark R. Bray,1 Amanda S. Hu,2 Ken K. Ren2. 1 _Campbell Institute, Univ. Health Network, Toronto, Ontario, Canada;_ 2 _CASI Pharmaceuticals, Rockville, MD_.

ENMD-2076 is a clinical stage compound with potent activity towards Aurora A and angiogenic kinases. ENMD-2076 has shown promising activity in multiple Phase 1 clinical trials, as well as in a Phase 2 trial in advanced ovarian cancer. ENMD-2076 is currently the subject of several ongoing Phase 2 clinical trials including fibrolamellar carcinoma, triple-negative breast cancer (TNBC), advanced/metastatic soft tissue sarcoma (STS), and advanced ovarian clear cell carcinomas (OCCC). ENMD-2076 has been developed to date as a single agent, however ENMD-2076 inhibits a spectrum of targets including Aurora A, FAK, CSF1R, c-Kit, and KDR, that are potentially involved in immune evasion mechanisms. These kinases have been shown in published studies, when inhibited, to enhance or augment the activity of immune checkpoint inhibitors such as anti-PD1. A study was thereby conducted in syngeneic models to determine the utility of ENMD-2076 combined with immune checkpoint inhibition as a rational strategy for cancer therapy. The study evaluated the efficacy of ENMD-2076 administered daily by oral gavage in the MC38 and CT26 colon cancer models, alone and in combination with an anti-PD1 antibody. Xenografts were established in the appropriate mouse strain (C57BL/6 and BALBc, respectively) by the subcutaneous inoculation of MC38 or CT26 cells into the right flank of female mice. Treatment was initiated 7 days following inoculation when tumor volumes had reached a mean volume of approximately 85 mm3. All treatments were well tolerated, with no significant body weight loss seen during either study. While CT26 tumors were relatively refractory to single agent ENMD-2076, tumor regression was observed in several MC38-bearing animals suggesting an immune activating mechanism. In both models a trend was observed for an augmentation of anti-tumor response in combination relative to single agent ENMD-2076 and anti-PD1 alone. Further studies to evaluate mechanism and an assessment of re-challenge experiments in animals exhibiting complete regression of tumors will be discussed. These studies support the further evaluation of ENMD-2076 in combination with immune checkpoint inhibition as a strategy for cancer therapy.

#1643

Cytotoxic T lymphocyte CD3+, CD8+, and immunoscore as prognostic marker in patients after radical cystectomy.

Jose J. Mansure, Alice Yu, Shraddha Solanki, Ana B. Dias, Miguel M. Burnier, Fadi Brimo, Wassim Kassouf. _McGill University Health Center, Montréal, Quebec, Canada_.

Purpose: Prognosis after radical cystectomy for bladder cancer can be unpredictable. There is increasing evidence that the presence of cytotoxic T lymphocytes correlates with survival. The Immunoscore, a new approach to classification of cancer using the number, type and distribution of immune cells, is an independent predictor for survival in colon cancer. Our objective was to evaluate the prognostic impact of lymphocyte distribution and the Immnoscore in bladder cancer.

Methods: Cystectomy sections with core tumor (CT) involvement and identifiable invasive margin (IM) were selected and stained for CD3+ and CD8+ lymphocytes. Three non-contiguous areas of highest lymphocyte density were selected from both CT and IM. The number of lymphocytes in each area was used to calculate the Immunoscore based on previously defined criteria. Kaplan-Meier curves were used to determine differences between Disease-free Survival (DFS) and Overall Survival (OS). Multivariate Cox proportional hazards model was used to determine hazard ratios.

Results: Total of 67 patients who had cystectomy for invasive bladder cancer were included in the study. High concentration of CD8+ lymphocytes in the tumour margin was associated with better DFS (P=0.005) and OS (P=0.03). Similar results were found for CD3+ lymphocytes with regards to DFS (P=0.05). A higher Immunoscore was also associated with better DFS (P=0.04). On multivariate analysis, CD8+ in the invasive margin was independently associated with better prognosis.

Conclusions: The host's own immune system plays a valuable role in clinical outcome. Our data suggests that a strong immune response in the tumor margin is independently associated with better prognosis.

#1644

Combination of MT-3724 with sirolimus reduces anti-drug antibody response and prolongs drug exposure.

Jack P. Higgins, Garrett L. Robinson, Sangeetha Rajagopalan, Brigitte Brieschke, Jensing Liu, Erin K. Willert. _Molecular Templates Inc., Austin, TX_.

Molecular Templates is developing engineered toxin bodies (ETBs), potent recombinant immunotoxins that combine the specificity of an antibody fragment with the powerful direct cytotoxicity of the Shiga-like toxin A subunit. ETBs can induce their own internalization, route through the cell in a predictable manner, enzymatically and irreversibly destroy ribosomes to shutdown protein synthesis and induce apoptosis of tumor cells. This mechanism of action is distinct from that of other therapeutics, making ETBs an attractive treatment for patients who have become resistant to chemotherapy and other treatment modalities.

MT-3724 is Molecular Templates' first-generation ETB targeting CD20, a surface receptor that is highly expressed on malignant B cells in hematological malignancies. Pre-clinical and clinical studies have shown promising results for non-Hodgkin's lymphoma cell lines and patients; however anti-drug antibodies are formed in experimental animals and patients after repeat dosing. Sirolimus (rapamycin) is a macrolide compound approved to prevent organ rejection and in LAM, and has been used in tolerization protocols with replacement enzymes. In pre-clinical studies, we have co-administered sirolimus in combination with MT-3724 in rodents as well as in a non-human primate model in order to reduce the anti-drug antibody response to MT-3724. These studies demonstrated that the combination of sirolimus with MT-3724 decreased anti-drug antibody response, prolonged serum exposure of MT-3724, and improved B-cell depletion as compared to MT-3724 alone.

Molecular Template's ETB technology has resulted in potent and targeted therapeutic agents that have a unique mechanism of action in the field of oncology. Our first-generation, CD20-targeted therapeutic, MT-3724, has promising clinical results in the refractory setting for non-Hodgkin's lymphoma. The combination of sirolimus with MT-3724 is an attractive and feasible regimen which will be further explored in clinical studies.

#1645

CEACAM1-blockade for T-cell activation and antitumor T-cell response.

So-Young Eun. _MOGAM Institute for Biomedical Research, YongIn, Republic of Korea_.

Immune checkpoint blockade became the most powerful way to control cancer without causing tremendous side effects, since anti-PD-1 blocking antibodies proved their superiority in clinical outcomes to those of traditional chemo- or targeted therapeutics. CEACAM1 (CCM1) is one of the several immune checkpoint receptors expressed on T cells that mediate suppression of inflammatory T cell response. CCM1 caught our attention for a cancer immune-therapeutic target, because of its inhibitory role in TCR proximal signaling complex. The anti-cancer therapeutic potential of CCM1-blockade has already been studied in mouse models, and one of the anti-human CCM1 antibody clones recently emerged in clinical Phase I studies. CCM1-targeting antibody candidates were screened out of our in-house synthetic libraries, among which a few of the clones stood out showing their significant blocking activities against CCM1-CCM1 homophilic interaction, enhancement of T cell activation, cytokine release, NFAT signaling, and tumor lysis. One of the clones (C25) showed its strongest efficacy equivalent to that of a reference Ab in CCM1-dependent T cell activation, proliferation, and IL-2 release, in parental Jurkat cells as well as in CCM1-overexpressing stable Jurkat cells. Moreover, C25 also enhanced tumor lysis by CCM1+ NK cells or CCM1+ cytotoxic T cells in a CCM1-dependent manner. In addition, C25 shows distinct characteristics in binding to CCM family from those of a reference Ab. Thus, C25 is thought to have a strong therapeutic potential on CCM1+ cancer.

#1646

Highly cytotoxic, completely human constructs targeting HER2 and containing the immuno-oncology payload granzyme B.

Lawrence H. Cheung,1 Yunli Zhao,2 Khalid A. Mohamedali,3 Ana Alvarez-Cienfuegos,3 Walter N. Hittelman,3 Michael G. Rosenblum3. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Shenyang Pharmaceutical University, Shenyang, China;_ 3 _UT MD Anderson Cancer Ctr., Houston, TX_.

Recent immunotherapeutic approaches designed to augment T- and B-cell mediated killing of tumor cells has met with clinical success suggesting that immuno-oncology (IO) approaches have tremendous potential for treatment in a broad spectrum of tumor types. After complex recognition of target cells by T and B cells, delivery of the serine protease granzyme B (GrB) to tumor cells comprises the cytotoxic insult resulting in a well-characterized, multimodal apoptotic cascade. We designed a recombinant fusion construct composed of a human anti-Her2 scFv fused to active GrB for recognition and delivery of GrB to tumor cells simulating T and B-cell therapy. The GrB-Fc-4D5 dimeric construct (mw 160 kDa) was generated and expressed in stably-transfected CHO-S cells at ~60 mg/L and purified to homogeneity. The enzymatic activity of the fusion construct was similar to commercially-available GrB and the affinity of the construct for purified Her2 extracellular domain (ECD) was 0.328 nM, comparable to that of Herceptin (0.150 nM). The GrB-Fc-4D5 construct was highly cytotoxic to Her2-positive cells such as SKBR3, MCF7 and MDA-MB-231 with IC50 values of 56, 99 and 27 nM respectively. Using immunofluorescence, the fusion construct internalized rapidly into target (SKBR3 or SKOV3) cells within 1 h of exposure, rapidly delivering GrB to the cytoplasmic compartment in a similar manner to that of immune T and B-cell targeting but without the action of the transmembrane pore-forming agent perforin. Against a large panel of various tumor types, GrB-Fc-4D5 was highly cytotoxic to virtually all cells regardless of natural expression levels of the nominal endogenous GrB inhibitor PI-9. Contemporaneous studies with Kadcyla demonstrate similar levels of in vitro activity against virtually all HER2-positive cells tested. GrB-Fc-4D5 demonstrated activity against both log-phase and confluent tumor cells. In keeping with its relatively high molecular weight (~160 kDa), the construct demonstrated a terminal-phase half-life of 13.6 hrs. In vivo efficacy studies are currently ongoing in several orthotopic xenograft models. Also ongoing are pre-IND toxicology, histopathology and clinical chemistry studies. Research conducted, in part, by the Clayton Foundation for Research.

#1647

**Characterization of MiXeno** TM **humanized mouse model for immuno-oncological therapy evaluation.**

Juan Zhang, Qian Shi, Lan Zhang. _CrownBio, Taicang, Jiangsu Province, China_.

Backgrounds: Despite the unprecedented success of immune checkpoint antibody therapy in patients with various cancer types, lack of stable experimental immunotherapy models is still a big challenge in development of effective new immune-therapeutics. Different strategies of humanized mouse model system, including human hematopoietic stem cell (HSC) or human PBMC humanization in immune-deficient mice, or chimeric models harboring human immune checkpoint targets in immune-competent mice have been reported. CrownBio has successfully established a number of human PBMC-humanized xenograft models MiXenoTM for in vivo IO therapy evaluation, which has been shown to be a rapid and simple strategy of humanization. However, several outstanding questions, e.g. the optimal PBMC injection route and cell number, the donor dependence of the efficacy result and impact of donor HLA type on the efficacy, etc., need to be further addressed.

Results: PBMC number was titrated in one of our MiXenoTM model HCC827, and PBMC injection via i.p. vs. i.v. was compared. Tumor growth curve, graft versus host disease (GVHD), and blood immunophenotyping were followed under different settings. Furthermore, the antitumor activity of PD-1 antibody pembrolizumab in a panel of PBMC donor context that bears diversified HLA types was precisely analyzed. We found that the efficacy was highly donor dependent, and there was lack of correlation between efficacy and donor/tumor HLA match.

Conclusion: The MiXenoTM model provides an alternative to the full stem cell reconstitution approach. Better understanding on the characteristics of the model may allow a rational model selection and study design in the discovery IO therapeutics.

#1648

**ImmunoGraft** ® **platform for the evaluation of Immuno-Oncology agents in PDX tumors models.**

David Cerna,1 Daniel Ciznadija,1 Bhavna Verma,1 David Sidransky,2 Evgeny Izumchenko,2 Angela Davies,1 Maria Mancini1. 1 _Champions Oncology, Baltimore, MD;_ 2 _Johns Hopkins University School of Medicine, Baltimore, MD_.

Recent breakthroughs in Immunotherapy have given new hope to treating previously untreatable tumor types and provide a better tolerated alternative to standard agents. There is an unmet need for a pre-clinical platform to test potential immune-oncology therapeutics that would also provide a tool to examine the mechanisms of response to better predict clinical outcomes. We have previously presented the Champions ImmunoGraft®, an innovative pre-clinical model enabling immunotherapeutic agents to be evaluated for efficacy in solid tumors. This platform is more reflective of the human tumor microenvironment (both immune and non-immune cell-based) and may be one of the most translationally-relevant models to date for screening therapies targeting the immune system. However, optimization of humanization and PDX implantation protocols that allow a broader reconstitution of cell linage and higher engraftment rate are necessary to further improve the pre-clinical evaluation of immune-oncology therapeutics and enhance the value of this modality for patient's benefit.

To this end, immune-compromised NOG (PrkdcscidIl2rgtm1Sug) mice were reconstituted (humanized) with human CD34+ cells using optimized procedure and blood was collected at different time points post engraftment to check for the major leukocyte linages. At ten weeks after humanization, mature human CD45+ cells comprised close to 50% of the leukocytes detected in the circulation and secondary lymphoid tissues of the humanized animals. As a result of improved methodology of the reconstitution protocol we have achieved an 85% success rate of humanization with a shortened engraftment period utilizing fewer CD34+ cells that maintain humanization reconfirmed at 22 weeks post reconstitution.

Champions TumorGraft® database contains more than one-thousand clinically relevant well-annotated PDX models. We have complied data from extensive genetic and protein expression analyses to design applicable ImmunoGraft® studies for testing of Immuno-Oncology agents. We have identified tumors expressing key markers that may predict response to immunotherapy agents including PDL-1, CD40, IDO-1 and IDO-2 as well as tumors that harbor a high mutational load. Here we present the efficacy and clinically relevant endpoints of two well-studied checkpoint inhibitors Ipilimumab and Pembrolizumab in this highly translational PDX platform.

#1649

In vivo **CRISPR-Cas9 library screen for immune checkpoint regulators.**

Sascha Karassek, Min Long, Xi Li, Zhengang Peng, Qunsheng Ji, Yong Cang. _WuXi AppTec (Shanghai) Co., Ltd., Shanghai, China_.

Therapeutic antibodies blocking immune checkpoints such as programmed cell death protein 1 (PD1) have proven clinically beneficial in multiple cancer types. However, anti-PD1 drugs approved by FDA are neither effective in treating all cancer types nor in all patients in a responsive cancer type. Therefore, understanding the mechanism underlying the efficacy of immune checkpoint blockade is critical for our rational design of synergistic treatment combinations. Here we describe how we employ the latest CRISPR-Cas9 genome-wide knockout screening strategies to identify targets in cancer cells or in T lymphocytes that control the sensitivity or resistance to anti-PD1 treatment. We constructed a sgRNA library targeting the functional domains of 116 mouse methyltransferases and demethylases, which was infected into the mouse Cloudman S91 melanoma cell line. Syngeneic mouse xenograft models derived from these infected cells were treated with anti-mPD1 monoclonal antibody, and the xenograft tumors were harvest at different time points after antibody treatment. Deep sequencing of the integrated sgRNA sequences in the tumors has identified potential druggable targets to sensitize or antagonize the tumors resistance to anti-mPD1 immunotherapy. Alternatively, we introduced the same library to naïve T cells purified from the OT-1 transgenic mice. The T cell library was then injected to B16-OVA melanoma syngeneic mouse models, followed by anti-mPD1 treatment. Deep sequencing of the integrated sgRNA sequences in the tumor-infiltrating T cells would allow us to uncover novel signaling molecules in the immune system that can potentially be manipulated to enhance the anti-PD1 therapy. In summary, we provide in vivo knockout screen strategies that can be applied to identify and validate combination therapies with immune checkpoint antibodies.

#1650

Targeting CD47- SIRPα interaction by novel peptide-based antagonists.

Pottayil G. Sasikumar, Chennakrishnareddy Gundala, Nagaraj M. Gowda, Sudarshan S. Naremaddepalli, Archana Bhumireddy, Rashmi Nair, Wesley Roy Balasubramanian, Anirudha Lakshminarasimhan, Samiulla S. Dodheri, Kiran Aithal, Raghuveer K. Ramachandra, Girish Daginakatte, Murali Ramachandra. _Aurigene Discovery Technologies Ltd., Bangalore, India_.

Background: Cluster of differentiation (CD47) is a trans-membrane glycosylated protein which is upregulated in several cancers. Increased expression of CD47 on tumor cells is associated with immune evasion and cancer progression. CD47 through its interaction with signal regulatory protein alpha (SIRPα), a cell-surface molecule on macrophages inhibits phagocytosis of tumor cells. Disrupting CD47-SIRPα interactions by monoclonal antibodies targeting CD47 and recombinant SIRPα proteins have been used as therapeutic strategies for treating cancer. Our objective was to discover and develop peptide/peptidomimetic based CD47 antagonists for disrupting CD47-SIRPα interactions.

Methods: Through rational design based on crystal structure of CD47/SIRPα interacting interface, we designed peptides having potential to disrupt CD47-SIRPα interactions. FACS based cellular binding assay was developed to assess the binding affinity of CD47 antagonists. SIRPα protein labelled with fluorescent dye was incubated with Jurkat T cells expressing high levels of CD47 in the presence/absence of peptides. Binding affinity was measured by decrease in fluorescence. Functional activity of the peptides was evaluated in a FACS-based phagocytosis assays, in which tumor cells were incubated with human/mouse macrophages in the presence/absence of CD47 antagonists.

Results: We identified CD47 antagonists demonstrating disruption of CD47-SIRPα interaction in a cellular binding assay. These peptides significantly inhibited phagocytosis of different tumor cells by macrophages. The lead CD47 antagonist displaying good ADME properties including moderate oral bioavailability was evaluated in a B16F10 syngeneic mouse tumor model. The lead CD47 antagonist inhibited primary tumor growth as well tumor metastasis to lungs. Biomarker characterization and efficacy studies in additional tumor models are ongoing.

#1651

Targeting phosphatidylserine in combination with adoptive T cell transfer eliminates advanced tumors without off-target toxicities in a melanoma preclinical model.

Daniel Hirschhorn-Cymerman,1 Sara Sara Schad,1 Sadna Budhu,1 Zhong Hong,1 Xia Yang,1 Hutchins T. Jeff,2 Bruce D. Freimark,2 Michael J. Gray,2 Jedd Wolchok,1 Taha Merghoub1. 1 _Memorial Sloan-Kettering Cancer Center, New York, NY;_ 2 _Peregrine Pharmaceuticals, Tustin, CA_.

A viable strategy to treat advanced cancers includes transferring of tumor-specific T cells. T cells that recognize tumor antigens can be expanded and reinvigorated ex-vivo. Furthermore, autologous T cells can be genetically modified to express anti-tumor T cell receptors or chimeric antigen receptors. Although the potency and specificity of tumor-specific T cells can be manipulated ex-vivo, once re-infused into patients, the T cells are subjected to immunosuppressive mechanisms established by the tumor. An important immune checkpoint regulator within tumors is phosphatidylserine (PS). Innate immune cells exposed to PS secrete suppressive cytokines and chemokines that can significantly impair the function and activation of anti-tumor T cells. Therefore, monoclonal antibodies that block PS activity can increase the anti-tumor potency of transferred T cells to treat aggressive cancers. Here we show that a PS targeting monoclonal antibody in combination with CD4+ T cells that recognize the melanoma antigen Trp1 can regress very advanced melanomas in all treated mice. Combination of anti-Trp1 CD4+ T cells with other immunomodulatory modalities such as anti-OX40 antibodies, can achieve equivalent treatment rates but these are typically accompanied by severe immune related adverse events. In contrast, in this setting, PS blockade did not show any off-target toxicities. Flow cytometry analysis revealed lower levels of CD206 expression concomitant with higher activation markers in macrophages and neutrophils in tumors from anti-PS treated mice. These results suggest that diminishing suppressive mechanisms locally in adoptive transfer protocols is a highly desirable strategy that can eliminate tumors while minimizing related adverse events.

#1652

Optogenetic immunoengineering to develop improved anti-cancer immunotherapy.

Lian He,1 Nhung Nguyen,1 Gang Han,2 Yun Huang,1 Yubin Zhou1. 1 _Texas A &M University, Houston, TX; _2 _University of Massachusetts Medical School, Worcester, MA_.

The application of current channelrhodopsin-based optogenetic tools is limited by the lack of strict ion selectivity and the inability to extend the spectra sensitivity into the near-infrared (NIR) tissue transmissible range. Here we present an NIR-stimulable optogenetic platform (termed "Opto-CRAC") that selectively and remotely controls Ca2+ oscillations and Ca2+-responsive gene expression to regulate the function of model cellular systems, as well as cells of the immune system. When coupled to upconversion nanoparticles, the optogenetic operation window is shifted from the visible range to NIR wavelengths to enable wireless and remote photoactivation of Ca2+-dependent signaling and optogenetic modulation of immunoinflammatory responses. In a mouse model of melanoma by using ovalbumin as surrogate tumor antigen, Opto-CRAC has been shown to act as a genetically-encoded "photoactivatable adjuvant" to improve antigen-specific immune responses to specifically destruct tumor cells. In addition, we have extended similar engineering approaches to photomanipulate membrane contact sites that play critical roles in controlling lipid metabolism and calcium signaling in cancer cells. Our study represents a solid step forward towards the goal of improving spatial and temporal control of anti-cancer therapy through optogenetic immunoengineering.

### Tumor Models and Assays

#1653

Modeling tumor immuno-dynamics to predict patient survival and immunotherapy efficacy.

Nicholas K. Akers, Eric E. Schadt, Bojan Losic. _Icahn School of Medicine at Mount Sinai, New York, NY_.

State of the art models of cancer survival are increasingly utilizing molecular data that feature the number of non-synonymous mutations, or a network based stratification of mutations, as significant predictors. It is widely hypothesized that this association between mutations and survival is a result of neo-epitopes that induce a robust anti-tumor immune response. Naively this implies that patient neo-epitope burden should better predict survival than that of mutations alone, however this has generally been difficult to prove. Indeed, mutations and predicted neo-epitopes explain roughly the same survival variance, likely due to the intertwined difficulties of neo-epitope prioritization and the often immuno-suppressive tumor microenvironment.

Reasoning that integrating leading order immune response with novel neo-epitope prioritization will lead to superior performance, we built a statistical model to quantify the influence of tumor immune-dynamics on patient survival. Neoepitopes (MHC-I/II) were predicted from mutations and filtered on a self-ligandome. The clonal structure of these mutations and the distribution of resulting epitopes were assessed as predictive features. Additionally, the existing response to epitopes was assayed by incorporating TCR and BCR sequence counts and entropy as a proxy for clonal tumor infiltrating lymphocyte expansion.

Leveraging the cancer genome atlas (TCGA) for 9 major cancer types including bladder (BLCA), breast (BRCA), colorectal (COAD/READ), glioblastoma (GBM), liver (LIHC), lung (NSCLC), melanoma (SKCM), pancreatic (PAAD), and uterine (UCEC) cancer, we obtained omics data for a total of 2,866 patients. Random forest based recursive feature elimination was used to determine which features were most likely to be predictive while overfitting was controlled with k-fold cross validation. A cox proportional hazard model of survival as a function of mutation burden and functions of neoepitope and immune response predictors was constructed using the selected features. Our results confirm that neoepitope and immune response based predictors for survival often significantly outperform mutation burden alone and simultaneously suggest a quantitative classification of immunotherapy efficacy across cancer subtypes.

#1654

Development of subtype specific mouse models of bladder cancer.

Christof C. Smith,1 Ryoichi Saito,2 Lisa M. Bixby,2 Takanobu Utsumi,2 Jordan Kardos,1 Shengjie Chai,1 Sara E. Wobker,1 Bhavani Krishnan,2 Jeffrey S. Damrauer,3 Jonathan S. Serody,2 David Darr,1 Benjamin G. Vincent,2 William Y. Kim2. 1 _UNC School of Medicine, Chapel Hill, NC;_ 2 _Lineberger Comprehensive Cancer Center, Chapel Hill, NC;_ 3 _Duke University, Durham, NC_.

Introduction: High-grade, muscle-invasive bladder cancer has recently been shown to harbor intrinsic molecular subtypes with distinct biologic features. Current murine models of bladder cancer, including the prominent carcinogen induced model MB49, do not account for subtype specific characteristics, leaving a gap in available tools for understanding subtype specific differences in bladder cancer. We have developed and validated immunocompetent, subtype specific models of bladder cancer, and we have used these models to assess differential responses to immune checkpoint inhibition.

Methods: Two distinct models of murine bladder cancer were developed in a C57BL/6 background. The UPPL models were generated through Pten/Trp53 conditional knockout in Uroplakin3a expressing cells. BBN models were generated through exposure of wild-type C57BL/6 mice to the carcinogen N-Butyl-N-(4-hydmoxybutyl)nitrosamine and subsequent generation of cell lines from spontaneous tumors. RNAseq was performed on several BBN and UPPL tumors and cell lines, with findings validated with flow cytometry and T/B cell receptor (TCR/BCR) amplicon sequencing of tumor infiltrating lymphocytes (TILs).

Results: BBN and UPPL models reflected characteristics of human basal and luminal bladder cancers, respectively. BBN (basal) models demonstrated higher immune gene signature expression, with concordantly higher numbers of TILs compared to the UPPL (luminal) model (p < 0.0001). Two BBN and two UPPL models were assessed for response to anti-PD-1 therapy in vivo as syngeneic tumors grown in wild type C57BL/6 mice. One of the BBN lines (BBN963) demonstrated robust control of tumor growth in some animals, including multiple complete responses (p = 0.0003), but also tumors that progressed, leading us to characterize BBN963 as a mixed response model. The marked response to PD-1 blockade in BBN963 was associated with significantly higher sharing of TCR CDR3 sequences among TILs compared to sequences of the other tumors (p = 0.003). In addition, analysis of BBN963 tumors by flow cytometry demonstrated naïve and memory T cell phenotypes correlated with increased and decreased tumor sizes, respectively. Closer examination of individual BBN963 tumor responses to PD-1 blockade revealed distinct responder and non-responder infiltrating immune cell phenotypes. Responders demonstrated a less diverse B cell repertoire (p = 0.0043) with increased BCR CDR3 sequence sharing (p < 0.0001).

Discussion: We have developed two unique classes of murine bladder cancer lines, UPPL and BBN, with gene expression and TIL profiles that closely correlate with human luminal and basal bladder cancers, respectively. The BBN and UPPL subtype specific models can serve as a tool for elucidating bladder cancer responses to immunotherapy. The mixed response of BBN963 tumors to PD-1 blockade should be an asset for assessing pathways mediating response to checkpoint blockade as well as the value of combination therapy. [C.S., R.S, B.V, W.K contributed equally to this work]

#1655

Organotypic 3D models to characterize the molecular requirements for NK and T-cell infiltration.

Wolfgang Sommergruber,1 Annika Osswald,1 Viola Hedrich,1 Marta Majewska,1 Helmut Dolznig2. 1 _Boehringer Ingelheim RCV, Vienna, Austria;_ 2 _Medical University of Vienna, Vienna, Austria_.

Multiple studies have demonstrated that the presence of tumor infiltrating lymphocytes is strongly correlated with increased survival. However, the level of infiltration within a specific tumor entity and between various tumor types varies widely. Increasing the degree of lymphocyte infiltration in a tumor represents an innovative drug concept. Organotypic 3D models consisting of tumor spheroids, stromal fibroblasts and Natural Killer (NK) or T-cells could represent valuable models to appropriately model infiltration and establish novel therapeutic concepts in the area of immune-oncology. As a first step in the development of a model for lymphocyte infiltration, the cytotoxicity and infiltration of human NK cells in 3D tumor spheroids were analyzed. Different NK cell lines and primary NK cells had a toxic effect on 3D tumor spheroids upon co-culture. Cryosections of co-cultured tumor spheroids showed infiltrated NK cells inside tumor spheroids. Infiltrated NK cells recovered from tumor spheroids were characterized by flow cytometry. Results showed that a higher infiltration correlates with cytotoxicity on tumor spheroids and transcriptomes of infiltrated vs. non infiltrated NK cells differ substantially. IL-12 stimulation increased the killing effect of NK cells on tumor spheroids, inducing higher expression of Perforin, IFNγ and GranzymeB levels. 3D tumor spheroids were embedded in a Matrigel/Collagen mixture with fibroblasts and the effect of compounds on NK cell infiltration was monitored. It was demonstrated that not only IL-12 increased NK cell infiltration, but also several compounds. Moreover, encapsulated 3D models with different cell types (tumor cells, fibroblasts, NK or T-cells) were established at a bioreactor scale to mimic the in vivo situation of complex tumor tissues. We could demonstrate NK and T-cell infiltration into these encapsulated 3D tumor spheroids. These encapsulated model systems permit long-term cultures to study the effect of compounds and/or NK or T-cells on later stages of tumorigenesis. In conclusion, this study demonstrates that organotypic 3D models are a valuable tool for the analysis of the molecular mechanisms that regulate lymphocyte infiltration into tumors.

#1656

**Comprehensive evaluation of human immune system reconstitution in NSG** TM **and NSG** TM- **SGM3 toward the development of a novel Onco-Hu** TM **xenograft model.**

Aaron J. Middlebrook,1 Eileen Snowden,2 Warren Porter,2 Friedrich Hahn,2 Mitchell Ferguson,2 Brian Soper,3 James Keck,3 Joan Malcolm,3 Shannon Dillmore,2 Smita Ghanekar,1 Rainer Blaesius2. 1 _BD Biosciences, San Jose, CA;_ 2 _BD Technologies, Raleigh-Durham, NC;_ 3 _The Jackson Laboratory, Bar Harbor, ME_.

The recent successes of immunotherapeutic approaches to the treatment of melanoma and the promise of similar treatments in a variety of other cancers underscore the importance of the immune system in cancer. Indeed, effective therapeutic design and evaluation require a comprehensive understanding of the interplay between the immune compartment and the proliferating tumor cells that comprise the tumor microenvironment. A humanized mouse strain engrafted with cancerous tissue from a patient derived xenograft (PDX) tumor provides researchers with a highly sophisticated tool, ideally suited to facilitate the design of treatment strategies that prevent tumor evasion of immune cells and that improve cytotoxic responses. Severely combined immunodeficient mice such as NOD scid gamma (NSGTM) and triple transgenic NSG mice expressing human cytokines KITLG, CSF2, and IL-3 (NSGTM-SGM3) are proven hosts for the engraftment of human tumors and establishment of human immune system components following hematopoietic stem cell (CD34+) transplantation. The endogenous expression of cytokines that support the development of myeloid lineages and regulatory T cells potentially represents a substantial improvement over standard NSG mice. Here we employ four 14-color flow cytometry panels to perform a comprehensive and detailed analysis of the entire immune system. The four panels are designed to fully characterize specific branches of the immune system: 1) T cells 2) NK cells/dendritic cells/B cells 3) myeloid lineages, and 4) immune checkpoint markers. Blood, spleen, and bone marrow tissue from both NSG and NSG-SGM3 mice were evaluated at 9, 16, 21, and 31 weeks of age using each of the four phenotyping panels. Our results indicate that the triple transgenic NSG-SGM3 mice exhibit a more completely humanized immune system compared to NSG mice, with specific improvements in the distribution of T-cell subsets and overall representation of the myeloid lineage. NSG mice engrafted with allogeneic human tumors represent a valuable preclinical testing platform for immuno-oncology.

#1657

Evaluating anti-tumor activity of the human anti-PD-1 antibody pembrolizumab using humanized mouse models.

Douglas E. Linn,1 Razvan Cristescu,1 Kallol Ray,1 Shuli Zhang,2 Sripriya Dhandapani,2 Sarav Kaliyaperumal,2 Jennifer H. Yearley,2 Brian J. Long1. 1 _Merck & Co., Inc., Boston, MA; _2 _Merck & Co., Inc., Palo Alto, CA_.

Targeting immune checkpoints has proven to be an effective strategy for reactivating the immune system to elicit potent anti-tumor activity. For example, the anti-PD-1 antibody pembrolizumab has shown robust and durable clinical activity in numerous malignancies including melanoma and non-small cell lung cancer. Unfortunately, a subset of patients do not benefit from pembrolizumab treatment. Ongoing molecular analyses of patient samples are establishing gene signatures which aim to more accurately predict which patients will benefit from immunotherapy. However, there are currently no preclinical in vivo platforms that comprehensively translate these clinical findings. Therefore, we have explored the use of mice engineered with a human immune system to examine the anti-tumor activity of pembrolizumab against a panel of human tumor xenografts derived from both cultured cell lines as well as patient tissue. Tumor models were selected based on parameters including PD-L1 expression and mutational load in order to closely mimic responder patients in the ongoing pembrolizumab clinical trials. Our data confirm that pembrolizumab can induce significant anti-tumor efficacy in humanized mice inoculated with human tumor models derived from both cancer cell lines and patient tumors. In responsive models, pembrolizumab caused significant tumor growth inhibition and, in some instances, tumor regressions. FACS analyses confirmed effective targeting of PD-1+ T cell populations. Anti-tumor efficacy correlated with increased effector T cell to regulatory T cell ratios as well as an elevated T cell infiltrate presence. Nonresponsive models lacked robust immune infiltration detectable by FACS or IHC, consistent with clinical data. Furthermore, presence of cytolytic activity at baseline and T-cell inflamed environment were evaluated using transcriptome analysis and subsequently linked to pembrolizumab response and to somatic neoantigen burden of the tumors. Together these studies validate the utility of humanized mice in the preclinical development of novel cancer immunotherapies and may also be beneficial in better defining the patient responder hypothesis and its prognostic value. Additionally, humanized mice may help identify optimal combination treatment regimens to provide more meaningful clinical benefit to patients who do not respond to single-agent therapy with pembrolizumab.

#1658

Establishment of CD137 humanized mouse model for efficacy assessment of agonistic anti-CD137 therapeutic antibodies.

Davy Xuesong Ouyang,1 Gang Chen,2 Zhensheng Wang,1 Lei Zheng,1 Annie Xiaoyu An,1 Jean-Pierre Wery,1 Jay Liu,2 Xin Dong,2 Henry Q. X. Li1. 1 _Crown Bioscience, Inc., Taicang, Jiangsu, China;_ 2 _Nanjing Galaxy Biopharmaceutical Co. Ltd., Nanjing, Jiangsu, China_.

CD137 belongs to the TNF receptor super family, and its activation is essential to the function of T cells and NK cells. Multiple agonistic CD137 antibodies are currently being tested in humans with advanced cancer, which expected to become an important new immuno-oncology therapeutics in clinic. It is important to establish preclinical models for efficacy assessment of investigational CD137 antibodies before entering clinic. Currently available animal models cannot meet this need. Syngeneic mouse tumor models provide a useful platform for testing surrogate immuno-oncology therapies, but cannot be used for testing human therapeutic antibodies due to the species specificity; human immunity reconstituted models, by human PBMC or hematopoietic stem cell inoculation into immune deficient mice, suffering from highly variable responses, are not robust enough to provide a reliable system for efficacy studies. Establishing target humanized models by replacing mouse therapeutic target with human counterpart while maintaining normal mouse immunity is a practical approach to evaluate human therapeutic antibodies in vivo. Using CRISPR-Cas9 gene editing, we have engineered human CD137 knock-in model expressing chimeric human/mouse CD137 composed of human extracellular and transmembrane domains (Exon 4-7), with intact mouse signal peptide and intracellular domain. FACS analysis of splenocytes derived from homozygous knock-in mice showed that CD3/CD28 stimulated T cells only express chimeric CD137 reactive to human CD137 antibodies, but not mouse CD137; Yet the expression levels of the chimeric CD137 in the knock-in mice are generally lower than endogenous mouse protein in wild-type C57BL/6 mice. At present, we are testing the efficacy of reference human CD137 agonistic antibodies by treating these homozygous knock-in mice grafted with syngeneic MC38 tumor cells. Data will be presented at the conference.

#1659

Immunological characterization of the Oncopig model and detection of cell-mediated immune responses to cancer.

Nana H. Overgaard,1 Daniel R. Principe,2 Jeanne T. Jakobsen,1 Laurie A. Rund,3 Paul J. Grippo,2 Gregers Jungersen,1 Lawrence B. Schook3. 1 _Technical University of Denmark, Frederiksberg C, Denmark;_ 2 _University of Illinois, Chicago, IL;_ 3 _University of Illinois, Urbana-Champaign, Urbana-Champaign, IL_.

In recent years, immunotherapy has shown great breakthroughs; however, the majority of preclinical studies has been based solely on rodent models and tends to experience a troublesome translation to human patients often related to the differences between mice and humans. Here, we utilized the Oncopig model that, due to its similarity in the immunome, metabolism, and size between humans and pigs, can serve as a supplementary large animal model for translational cancer research. With the establishment of the immunoscore as a new approach for staging cancer patients, it has become evident that the immune status of the tumor microenvironment has a crucial impact on the patient's outcome and response to therapies. For this reason, we set out to perform an immunological characterization of Oncopig-derived tumors to elucidate the potential in using this model for testing cancer immune therapeutic approaches. Following injection of Cre recombinase, the transgenic Oncopigs express KRASG12D and TP53R167H, two mutations commonly found in human cancers, subsequently resulting in tumor formation at the site of injection. The expression of these two transgenes was confirmed using intracellular flow cytometry staining or immunohistochemistry (IHC). Subcutaneous, intramuscular, and testicular tumors all showed the presence of infiltrating CD3+ cells, which were found both in the periphery and in the core of the tumor as determined by IHC. With the establishment of a digestion protocol allowing isolation of the in vivo-generated tumor cells, we confirmed the CD3+ staining and found that the infiltrating T-cell pool was comprised of both cytotoxic and regulatory cells as indicated by positive flow cytometry staining for the CD8β chain and FoxP3, respectively. In order to investigate the immunogenicity of the tumor cells, we set up flow cytometry detection of porcine TNF-α, IFN-γ, and perforin in a PBMC-tumor cell co-culture. Despite the production of various cytokines, actual tumor cell killing is a desirable parameter to measure when evaluating both the immunogenicity and response to therapy. Therefore, we developed, to our knowledge, the first fluorescence-based in vitro porcine cytotoxicity assay and found a significant recognition and specific killing of autologous tumor cells in an effector-target titration dependent manner. Taken together, our results show that the established Oncopig tumors are infiltrated by T cells exhibiting an either cytotoxic or regulatory phenotype, thus indicative of a tumor microenvironment mimicking the complexity seen in human patients. Additionally, we were able to measure cell-mediated immune responses to cancer in this novel, large animal model, and both the cytokine production and tumor-specific killing hence underline the potential in using the Oncopig for future testing of immune therapies against human cancer.

#1660

Humanization of patient derived xenograft (PDX) cancer model mice with peripheral blood mononuclear cells (PBMCs).

Jens Henrik Norum, Dag Josefsen, Gunnar Kvalheim, Olav Engebråten, Therese Sørlie, Gunhild M. Mælandsmo. _Oslo University Hospital, Norwegian Radium Hospital, Oslo, Norway_.

Immunocompetent transgenic mouse models have for decades served as valuable tools to address the effect of oncogenes and tumor suppressors. Immunodeficient mice have been used to establish patient derived xenograft (PDX) models harboring human tumors. These types of models have been used to study cancer initiation and progression as well as preclinical evaluation of anticancer drugs. One major limiting factor for both these model systems is the immune system; the PDX models lack an immune system and the immune system in immunocompetent mouse models display differences compared to the human immune system. Thus, in cancer research there is a need for preclinical models to study the influence of human immune cells on tumor progression and response to cancer therapies. We have established and optimized protocols for intravenous (IV) or intraperitoneal (IP) injections of isolated human PBMCs, to generate humanized mice harboring human immune cells. Our protocols do not require irradiation or busulfan pretreatment of the animals. Flow cytometry analyses showed that mainly T helper cells, CD4+, and cytotoxic T cells, CD8+, were present in the humanized mice. Immunohistochemistry (IHC) analyses of humanized PDX cancer model mice showed that human lymphocytes were present in the tumor periphery and some very few cells within the tumor, in addition to the human leukocytes in the liver and spleen. The growth rates of the PDX tumors were not affected by the humanization. Our protocols require minimal preparations of the animals and generate humanized PDX mice harboring human lymphocytes locating in close proximity of, as well as inside the PDX tumors. The model systems are suitable for preclinical studies of human, adaptive immune responses in tumor progression and cancer therapies.

#1661

Humanized mouse models for immune-oncology biotherapeutics: Development, validation and applications.

Gang Chen, Bin Cai, Feng Zhou, Jerry Zhou, Jie Liu, Xin Dong. _Nanjing Galaxy Biopharma, Nanjing, China_.

With clinical success and FDA approval of Ipilimumab, Nivolumab, Pembrolizumab and Atezolizumab, developing biotherapeutics against immune checkpoint targets, like CTLA-4, PD-1/PDL-1 and others, becomes hot pursuits for big pharma and biotech. However, traditional syngeneic models with surrogate murine reagents or human immune cell re-constitution in immune deficient mice are useful but not reliable and robust due to the species specificity and highly variable responses. It is crucial to establish additional preclinical models for efficacy assessment of investigational immune-oncology antibodies before entering clinic. Establishing human therapeutic target engineered mouse models by replacing mouse gene with human counterpart while maintaining intact mouse immunity is a practical approach for in vivo studies.

Applying CRISPR-Cas9 and other gene editing techniques, we have engineered genetically a series of single and double human immune-checkpoint genes, like PD-1/PDL-1, CTLA-4, TIM3, CD137 and others, knock-in model expressing chimeric human/mouse targets composed of human extracellular domains, with intact mouse signal peptide and intracellular domain. FACS analysis of immune cells derived from homozygous knock-in mice showed that activated cells only express chimeric targets reactive to anti human target antibodies, but not mouse gene specific antibodies. We have been testing the efficacy of reference antibodies by treating these homozygous knock-in mice inoculated with syngeneic murine tumor cells. Other applications, like target validation, combination therapy and biomarker discovery, are also explored.

#1662

Development and validation of humanized mice models implanted with patient derived colorectal cancer xenografts.

Anna Capasso,1 Julie Lang,1 Todd M. Pitts,1 Sarah Lindsey Davis,1 Christopher Lieu,1 Scott Kopetz,2 Stacey Bagby,1 Paul Francoeur,1 John J. Tentler,1 Jill Slansky,1 Roberta Pelanda,1 Gail Eckhardt1. 1 _Univ. of Colorado Denver, Denver, CO;_ 2 _MD Anderson Cancer Center, Houston, TX_.

Introduction: Agents that reverse T cell inhibitory signals have reinvigorated the strategy of cancer immunotherapy and are leading to robust clinical responses. In colorectal cancer (CRC), objective responses to single-agent therapy with anti-PD-1/PD-L1 antibodies have largely been restricted to the small proportion of CRC patients with microsatellite unstable (MSI-high) disease. Although there are additional subsets of advanced CRC patients that are responsive to immune checkpoint inhibition in combination with tumor-targeted agents, preclinical models have been hampered by their immune-compromised status. In order to gain a better biological understanding of the context of immune responses and facilitate preclinical evaluation of combination strategies incorporating cancer immunotherapy, we developed a "hematopoietic" humanized mouse model utilizing patient-derived CRC xenografts with the intent of leveraging this model for the development of rational combinations.

Methods: BRG.NODSirpalfa newborn pups were humanized through transplantation of 1x105 CD34+ cells purified from umbilical cord blood. Mice were evaluated for chimerism at 8 and 12 weeks. At 16 weeks, tumor tissue from established PDX models was implanted on the right and left flanks of humanized mice. When the average tumor size reached a volume of ~150-300 mm3, the mice were randomized into vehicle or nivolumab (30 mg/kg twice weekly i.p.) treatment groups according to %chimerism. Mice were monitored for signs of toxicity and tumor size was evaluated twice weekly by caliper measurements (tumor volume= (length × width2) × 0.52). At the end of the treatment, mice were euthanized while sera, lymph nodes, spleen, bone marrow and tumors were collected for immunological assessment.

Results: As preliminary proof-of-concept, we successfully established 3 humanized CRC PDX models and one breast cancer cell line (MDA-MB-231). In one of the CRC (MSI-high) and in the breast model we observed tumor growth inhibition in the treated groups vs control. We detected differences in PD-1 expression among treated versus control mice, with lower expression in the nivolumab-treated groups. We also observed an increased number of TILs, CD8+ T cells and greater numbers of T cells in the lymph nodes of treated mice, suggesting T cell expansion. Mice were highly chimeric with high TILs whereas responder tumors exhibited an increase of CD44 high IFN+ T cells, high CD8% and a higher effector memory% (HLADR+, CD45RO+).

Conclusions: Humanized PDX models were successfully established and tumor engraftment occurred in all humanized mice with nivolumab-treated mice demonstrating the development of lymph nodes that were populated by activated T cells. These preliminary results demonstrate that human immunity and PD-1 expressing T cells exist in these models and provide the basis for planned immunotherapy combination studies.

#1663

Preclinical efficacy model to promote immunotherapy development for prostate cancer.

Mari I. Suominen, Tiina Kähkönen, Yvonne Konkol, Jenni Mäki-Jouppila, Jussi M. Halleen, Jenni Bernoulli. _Pharmatest Services Ltd, Turku, Finland_.

Immunotherapy for prostate cancer has recently emerged as an attractive treatment strategy. Yet, preclinical models where relationship between inflammation, stroma, tumor cells and prostate cancer progression can be studied are limited. GEMM models of prostate cancer are scarce and in xenograft models, even when in humanized mice, the role of immune system in the initiation and in progress of the malignancy cannot be studied. As the requirement to test novel immunotherapies and especially combination treatments is increasing, a preclinical model that takes into account tumor microenvironment and immune system would be highly useful to promote development of novel therapies to combat against prostate cancer. Aim of the present study was to reveal if there is role between the immune system and development of prostate cancer, and secondly, to validate a model to be utilized later in immunotherapy development.

Intact 10-12 weeks old male Noble rats were s.c. implanted with slow-releasing estradiol and testosterone pellets for 6, 13 and 18 weeks. Daily release for testosterone was 0.8 mg and for estradiol 0.08 mg. Control group animals received placebo hormone pellets without hormones. Serum samples were collected during the study to monitor hormone levels, and prostates were removed and processed for histopathological evaluation at the end of the study. Hormonal treatment caused an increase in estradiol to testosterone ratio, and the prostates were enlarged. Imbalance in hormone-milieu induced inflammation in the prostate, followed by formation of prostatic intraepithelial neoplasia (PIN)-like lesions and finally adenocarcinomas in the periurethral region. Inflammatory cells, mainly T-cells were noticed in the vicinity of PIN-like lesions. During the progression of prostate cancer, inflammatory cells disappeared from the adenocarcinoma sites. In the prostate, inflammation consisting of perivascular, stromal and periglandular T-lymphocytes and intraluminal neutrophils remained.

Results of this study indicate significance of hormonal milieu, especially estrogens and androgens, in the development of inflammation and progression of prostate cancer, with a key role for tumor microenvironment. Presence of lymphocytes in the proximity of PIN-like lesions during the early phases of prostate cancer, and their disappearance later in the adenocarcinomas, indicate interaction between innate and adaptive immune system and cancer. Therefore, this preclinical prostate cancer model that combines immune system and cancer can be utilized when new immunotherapies, combination treatments and prevention possibilities against prostate cancer progression are developed.

#1664

Inhibition of 4T1 mammary tumor growth in BALB/c mice by subcutaneous and intraperitoneal injection of a 4T1 whole cell vaccine containing IL-2 and GM-CSF as adjuvants.

Jonathan F. Head,1 Jeffrey T. Phillips,1 Xianpeng Jiang,1 Robert L. Elliott2. 1 _OncBioMune Pharmaceuticals, Baton Rouge, LA;_ 2 _SAB Breast Foundation, Baton Rouge, LA_.

Developing animal tumor models for human cancer vaccines creates a tool to investigate the mechanism of action, variations in formulation, dosing schedules, and combinations with other forms of cancer therapy. In this study we developed a mouse whole cell mammary cancer vaccine model with both subcutaneous and intraperitoneal injection of the vaccine. The vaccine, containing 4T1 mouse mammary cancer cells (1,000,000 cells), IL-2 (0.2 µg) and GM-CSF (0.1 µg) in a total volume of 150 µl, was injected either subcutaneously into the backs of BALB/c mice or intraperitoneally. There were six injections of the vaccine (weeks 1, 2, 3, 7, 11, 15) and one week after the last injection 100,000 4T1 cells from cell culture in a volume of 100 µl were injected into the scapular region of the mice. At 22 days post 4T1 tumor transplantation there was a 37% inhibition of the growth of the 4T1 tumor in the mice receiving the subcutaneous injection of the vaccine and a 42% inhibition of the growth of the 4T1 tumor in the mice receiving the intraperitoneal injection of the vaccine. The inhibition of 4T1 mammary tumor growth in this mouse model with subcutaneous injection of a whole cell vaccine gives further support for the previously reported efficacy in a Phase 1/2 clinical trial of our therapeutic breast cancer vaccine containing autologous and allogeneic breast cancer cells in the adjuvant setting. The inhibition of 4T1 tumor growth with intraperitoneal injection suggests a delivery method that may be applicable to ovarian cancer and cancer patients with malignant ascites.

#1665

**MuScreen** TM **: A well-characterized syngeneic model platform for rapid** in vivo **screening.**

Lan Zhang, Binchen Mao, Qian Shi. _CrownBio, Taicang, Jiangsu Province, China_.

Background: Syngeneic tumor models, many shelved for decades, have been revived as effective models for immunotherapy, accompanying the clinical success of the immuno-checkpoint inhibitors (e.g. anti-CTLA4, anti-PD1, anti-PD-L1 antibodies). In vitro cell-based screens that are frequently used in oncology to quickly identify responsive cells and assess PD effects, often fail in immuno-oncology, due to immunotherapeutics targeting the complex host immune system. Alternatively, an in vivo screen with a panel of models addresses many of these questions, e.g. PD and efficacy, but may be cost prohibitive.

Material and methods: Leveraging in-house detailed profiling data on our syngeneic models, including efficacy benchmarking with anti PD-1, PD-L1, CTLA-4, OX-40, GITR , LAG3 and TIM3 antibodies, RNAseq data on tumor samples, and FACS analysis on both baseline and post-treatment tumor samples, we created a new in vivo screening tool for immune-oncology: MuScreen. MuScreen includes up to 20 well-characterized syngeneic models in a 3 month screening run. Both PD and efficacy may be determined in the run, allowing researchers to make decisions based on results observed from a large dataset. To address the cost issue, test agents from multiple clients are pooled together for each run (sharing vehicle and other common groups) providing a significant reduction in the number of animals used and the associated costs.

Results: CrownBio has established the largest collection of syngeneic models with well-characterized immunotherapy data. With the three MuScreen runs, we have generated new data on common IO agents (e.g. aPD-1 antibody) and combinations treatments, with FACS analysis, IHC, and efficacy data. Based on these data, a bioinformatics

analyses were further explored to identify the makers/pathways that could be correlated with the efficacy or PD effect of the tested IO treatment.

Conclusions: MuScreen, the first in vivo screening tool for cancer immunotherapeutics, provides detailed response data on a panel of syngeneic models, and may help on biomarker discovery in a cost and time efficient manner.

#1666

Immune consequences of penfluridol treatment associated with inhibition of glioblastoma tumor growth.

Alok Ranjan, Nehal Gupta, Sharavan Ramachandran, Stephen Wright, Sanjay Srivastava. _Texas Tech Uni. Health Science Center, Amarillo, TX_.

Glioblastoma is the most common and lethal brain tumor associated with only 12% median survival rate of patients. Despite the development of advanced surgical, radiation or use of combinations of anti-cancer drugs, treatment for glioblastoma patients is still a challenge. The major contributing factor in glioblastoma progression and resistive nature is its ability to evade the immune surveillance. Hence, modulating the immune system in glioblastoma tumors could be an important strategy for anticancer therapeutics. Penfluridol, an antipsychotic drug has been shown to have anti-cancer properties in our recently published studies. The present study evaluates the immune response of penfluridol in glioblastoma tumors. Our results demonstrated that penfluridol treatment significantly suppressed glioblastoma tumor growth. Our current results demonstrated about 72% suppression of myeloid derived suppressor cells (MDSCs) with penfluridol treatment in mouse bearing U87MG glioblastoma tumors. MDSCs are known to increase regulatory T cells (Treg), which are immunosuppressive in nature and suppresses M1 macrophages that are tumor suppressive in nature. Our results also showed suppression of regulatory T cells as well as elevation of M1 macrophages with penfluridol treatment by 58% and 57% respectively. Decrease in CCL4 as well as IFNγ with penfluridol treatment was also observed indicating decrease in overall tumor inflammation. This is the first report demonstrating immune modulations by penfluridol treatment associated with glioblastoma tumor growth suppression prompting further investigation to establish penfluridol as a treatment option for glioblastoma patients. [Studies supported in part by R01 grant CA129038, awarded by National Cancer Institute, NIH]

#1667

Identifying intrinsic regulators of PD-L1 expression in cancer cells: A genome-scale CRISPR knock-out approach.

Jacqueline E. Mann, Megan L. Ludwig, Rebecca C. Hoesli, Aditi Kulkarni, Simmy Patel, Judy Kafelghazal, Alexey Nesvizhskii, J Chad Brenner. _University of Michigan, Ann Arbor, MI_.

Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common cancer in the United States and affects 600,000 people each year worldwide. With a five-year survival rate of only 50% and a recent rise in HPV-associated HNSCCs, improved treatment protocols are urgently needed. Evidence of immunosuppression is often reported in HNSCC, making immunotherapy an attractive strategy for the management of this disease. The immune checkpoint inhibitor Pembrolizumab was recently approved for the treatment of metastatic and recurrent HNSCC, but only 18% of initial participants in a trial of Pembrolizumab responded, and it remains difficult to predict patients likely to experience benefit. To increase the number of patients that respond to immune checkpoint inhibition, we sought to identify targetable genetic factors modulating PD-L1, a molecule that serves to dampen the anti-tumor immune response. We show that several HNSCC models exhibit mild (2-4 fold) induction of PD-L1 expression following treatment with interferon-γ (adaptive expression), and recent reports suggest that common oncogenic pathways, including the EGFR and PI3K pathways, may provide a novel strategy to regulate PD-L1 expression. To this end, we developed a high throughput screen to identify targetable pathways that may be used to regulate PD-L1 expression in patients with specific genetic lesions. We have employed a Genome-scale CRISPR Knock-Out (GeCKO) screening technique in HNSCC cell lines and selected for genetic knockouts exhibiting altered PD-L1 expression. Stable knockout pools with representation of approximately 300 gRNAs per target gene (>20,000 target genes in the library) were expanded and serially sorted to create stable sub-populations with enhanced PD-L1 expression. These sub-populations were sequenced to identify gRNAs whose knockout causes a change in PD-L1 expression (e.g. genes that repress or enhance PD-L1 expression). We expect that these large-scale screens, when performed over multiple HNSCC cell lines with diverse genetic lesions, will identify patterns of targetable regulators that may ultimately be manipulated in combination with PD-1/PD-L1 inhibitors.

#1668

Comprehensive 10 color flow cytometry analysis of the neuroblastoma intratumoral immune response using the murine syngeneic neuro-2a tumor model.

David D. Draper, Matt Thayer, Alden Wong, Dan Saims, Scott C. Wise. _MI Bioresearch, Ann Arbor, MI_.

Neuroblastoma is the most prevalent solid tumor in childhood and the most common tumor in infants less than 1 year of age. In spite of aggressive multi-modal therapeutic approaches, the 5-year survival rate in patients with high-risk neuroblastoma remains poor at <50%. Recent advances have focused on harnessing the immune system to combat neuroblastoma. Antibodies that abrogate the immune checkpoint signaling pathway such as anti-CTLA-4 and anti-PD-L1 suppress neuroblastoma growth in certain mouse models of disease. Furthermore, the dinutuximab antibody that targets ganglioside GD2 expressing cells has been demonstrated to trigger neuroblastoma regression using a mechanism that involves several immune subsets. Although evidence suggest the host immune response can be exploited to fight neuroblastoma, the key immune subsets, their relative contribution, and mechanism of action to suppress neuroblastoma growth have yet to be fully characterized. To this end, we used flow cytometry to immunophenotype the intratumoral immune response in the neuro-2a model of neuroblastoma. This was accomplished by developing two 10-color panels to quantify the proportions of various myeloid and T cell subsets respectively. Using the myeloid panel, our results showed that total neuro-2a tumor immune cells were predominantly myeloid lineage-derived (CD11b+). A combination of Ly-6G and Ly-6C antibodies demonstrated that myeloid-derived suppressor cells (MDSCs) comprised the bulk of the myeloid cells and were more heavily skewed towards the granulocytic phenotype versus monocytic. An MDSC exclusion gate and a combination of F4/80, MHC class II, and CD206 antibodies enabled the analysis of tumor-associated macrophage subsets. Our results revealed that neuro-2a tumors contained relatively few activated M1 macrophages, which are typically associated with anti-tumor responses, and instead were dominated with a pro-tumor M2 macrophage response. The T cell antibody panel revealed that CD4+ T cells outnumbered CD8+ T cells by 2-fold. Regulatory T cells (Tregs) in the CD4+ T cell fraction were identified by a combination of FoxP3 and CD25 antibodies. The frequency of Tregs varied but represented <50% of total CD4+ T cells. Finally, the activation state of CD8+ T cells was examined using Ki-67, CTLA-4, and PD-1 antibodies. Analysis demonstrated that the majority of CD8+ T cells in neuro-2a tumors were actively proliferating based on the expression of Ki-67. However, most of the proliferating CD8+ T cells also co-expressed PD-1 suggesting a loss of anti-tumor functionality and an exhausted phenotype. This study provides a comprehensive immune profile of neuro-2a tumors and supports a platform with which to test new single agent and combination therapies designed to treat neuroblastoma.

#1669

Multiplex immunohistochemical staining of PD-L1, PD-1, CD3, CD4, CD8, CD68, FoxP3, and Ki-67 and image analysis of tumor and invasive margin in human FFPE NSCLC tissue.

Lisa M. Dauffenbach, Gela C. Sia, Jianping Zheng, Natalia Jun, Eric P. Olsen, Christopher A. Kerfoot. _Mosaic Laboratories, LLC, Lake Forest, CA_.

Evaluating the density of immune cell subtypes and their relative spatial positioning in cancers has become an important tool in understanding response to immune checkpoint inhibitors. The purpose of this study was to investigate protein expression of PD-L1, PD-1, CD3, CD4, CD8, CD68, FoxP3, and Ki-67 with four multiplex immunohistochemical assays (Ki-67+CD8, CD3+CD4+Ki-67, FoxP3+PD-1+CD8, and CD68+PD-L1+CD3) using DAB, red, and green chromogens within formalin-fixed, paraffin embedded non-small cell lung (NSCLC) tissues. The Ki-67+CD8 multiplex was developed to evaluate the density of cytotoxic T cells and the percentage that are proliferating. The CD3+CD4+Ki-67 multiplex characterizes the frequency of CD4+ T cells and evaluate proliferation in this subset. The FoxP3+PD-1+CD8 multiplex evaluates the regulatory T cell subset and evaluates PD-1 expression. The CD68+PD-L1+CD3 multiplex characterizes PD-L1 expression on macrophages and T cells. Cell densities were evaluated in the center of tumor and invasive margin regions, and image analysis was performed to quantitate each stain as a single agent and co-expression within each multiplex assay.

#1670

Identification and characterization of novel mediators of tumor-induced T-cell dysfunction.

Geoffrey J. Markowitz,1 Mary Philip,2 Andrea Schietinger,2 Vivek Mittal1. 1 _Weill Cornell Medicine, New York, NY;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Immunotherapies targeting T-cell functionality have been shown to have efficacy in numerous cancers, including non-small cell lung cancer (NSCLC). However, treatment with currently available therapeutics, such as anti-PD-1 and anti-CTLA-4, induce responses only in a subset of patients. This raises the possibility that numerous other effectors of T-cell dysfunction remain to be elucidated. To evaluate possible novel mediators of T-cell dysfunction, we isolated CD8+ T-cells from orthotopic KrasG12D/p53-/- murine lung adenocarcinomas (HKP-1) that exhibited either stable or progressive disease as a function of tumor growth. RNAseq analysis revealed increased expression of several genes including Tim3, PD-1, Lag3, 2B4, and CISH, and decreased expression of GzmB and Eomes in CD8+ T-cells isolated from progressive tumors indicative of a dysfunctional state, as well as multiple novel genes not previously associated with T-cell dysfunction. To explore the functional role of the novel candidate genes, we have developed in vitro and in vivo assays leveraging Ova and GFP overexpression in our HKP-1 cell line (KP1-Ova-GFP). We have begun to target these unique set of candidate genes in tumor-specific T-cells associated with dysfunction through innovative RNAi/shRNA and adoptive transfer approaches to facilitate therapeutic reprogramming and enhanced T-cell-mediated immunity in lung cancer. We expect to provide novel strategies for designing rational immunotherapies targeting dysfunctional tumor-specific CD8+ T-cells to generate durable anti-tumor immune responses for the treatment of lung adenocarcinoma.

#1671

Characterization of the immune landscape in stage II-III melanoma using qIF.

Robyn Gartrell,1 Douglas Marks,1 Edward C. Stack,2 Yan Lu,1 Thomas D. Hart,1 Camille Gerard,3 Camden Esancy,4 Dan Tong Jia,1 Paul Armenta,1 Daisuke Izaki,4 Danielle Davari,4 Ashley White-Stern,1 Zoe Blake,1 Yichun Fu,1 Basil Horst,1 Yvonne Saenger1. 1 _Columbia University School of Medicine, New York, NY;_ 2 _PerkinElmer, Inc., Hopkinton, MA;_ 3 _Iuliu Hatieganu University, Romania;_ 4 _Columbia University, New York, NY_.

Background: The tumor microenvironment plays a crucial role in cancer progression, often supporting immune evasion. This is of particular importance in melanoma, where immune checkpoint therapies have resulted in significant clinical benefit, yet only in a subset of melanoma patients respond. Precise biomarkers are urgently needed to characterize the tumor immune micro-environment, both for prognostication and to predict the benefit of immuno-therapeutic intervention. HLA-DR on tumor cells and Ki67 on cytotoxic (CD8+) T cells have been proposed as biomarkers of anti-PD1 activity. Quantitative immunofluorescence (qIF) allows for automated quantitation of phenotypes and spatial distributions of immune cell populations within formalin fixed paraffin embedded (FFPE) tissues.

Methods: To characterize the tumor immune microenvironment, we screened databases at the Herbert Irving Cancer Center (HICC) at Columbia University for early stage melanoma patients with documented clinical follow up. We identified a preliminary population of 40 stage II-III melanoma patients diagnosed between 2000 and 2012. Clinical follow up was available on 21 patients, 12 of whom were alive with no evidence of recurrence, 1 who died of another malignancy, and 8 who died of melanoma. 19 patients had more than 24 months of survival information available but no detailed clinical information. 5µm slides from either primary biopsy or subsequent wide local excision procedure were stained using qIF for DAPI, CD3, CD8, CD68, SOX10, HLA-DR and Ki67. Cell phenotypes within representative fields selected by a trained dermatopathologist (BH), were visualized using multispectral imaging, and analysis of spatial distribution of cells were analyzed using inForm image analysis software (Perkin Elmer), and Spotfire software (TIBCO).

Results: We were able to quantify and identify coordinates for multiple immune cell subsets in melanoma tissues. In 21 patients with clinical follow up, we found that higher densities of CD3+CD8+ T cells in tumor and stroma trended towards correlation with non-recurrence. In addition, CD68+HLA-DR- predicts poor prognosis (p<0.05), whether in tumor or stroma. Assessment of spatial distribution across all 40 tumors demonstrated that CD3+CD8+ cells are closer to CD68+ cells and Sox10+ tumor cells when they express HLA-DR (p<0.001). Conversely, CD3+CD8+ cells are significantly farther from tumor cells when they express Ki-67, (p<0.001). Among patients with clinical follow up, CD3+CD8+ cells were closer to CD68+HLA-DR- cells in recurrent patients (p < 0.05).

Conclusion: Using qIF imaging and analysis we find that density and spatial relationships of immune cell subsets correlates with recurrence status. qIF may offer the potential for the development of prognostic biomarkers in stage II/III melanoma.

#1672

Workflow automation and parallelization improves the isolation and analysis of tumor-infiltrating immune subpopulations.

Janina Brauner, David Agorku, Anne Langhammer, Lukas Pohlig, Jan Drewes, Thomas Dino Rockel, Oliver Schilling, Stephan Mehlhose, Wa'el Al Rawashdeh, Andreas Bosio, Olaf Hardt. _Miltenyi Biotec GmbH, Bergisch Gladbach, Germany_.

Immunotherapy against cancer has proven clinical efficacy and tremendous potential in multiple tumor entities. Syngeneic mouse tumor models represent the gold standard to analyze effects of immunotherapy due to their fully competent immune-repertoire. However, the amount and composition of tumor infiltrating leukocytes (TIL) is highly variable, complicating the targeted analysis of subpopulations. In particular, small subpopulations cannot be analyzed properly but may be lost in the background noise. When working with large cohort sizes, even the immune-phenotyping of TIL by flow cytometry is time consuming and data processing highly work intensive making pre-enrichment methods for sample debulking attractive.

We have established an automated workflow combining tissue dissociation with TIL specific isolation to improve and accelerate downstream analysis. Tumor dissociation was automated using the gentleMACS™Octo Dissociator and optimized for epitope conservation to overcome bias in immune-phenotyping caused by dissociation with aggressive of impure enzymes. Next, isolation of TIL was improved by developing a new CD45-specific enrichment reagent for the magnetic cell sorting (MACS) based isolation directly from dissociated tumor tissue. The whole workflow takes only about 90 min. To validate this method on starting material showing variable frequencies of TIL infiltration, we used syngeneic mouse tumors derived by injection three independent tumor cell lines. Tumors derived from injection of B16.F10 melanoma showed TIL frequencies of 2-4%, CT26.WT colon carcinoma tumors 15-21%, and 4T1 breast carcinoma tumors 32-37% among total viable cells after dissociation. Using a manual separation system, TIL were enriched to purities above 90% at yields above 70% for CT26.WT and 4T1 tumors, and purities above 80% at high yields above 95% for B16.F10 tumors. To address the need of parallelization and automation for sample processing in large cohort sizes used in in vivo studies, a fully automated version of the MultiMACS™ Cell24 Separator was developed by integrating the instrument in a liquid handling robotic platform. This new system, the MultiMACS™ X, can process 24 cell separations in parallel with minimal hands-on time. When compared to the manual system, equal purities were achieved whereas the overall yield of target cells was increased from 70% to 90% as shown in the case of CT26.WT tumors. Importantly, while the TIL enrichment significantly reduced the time and reagent costs in immune subset analysis, the composition of infiltrating immune cells was not affected, excluding the risk of introducing a bias by this method.

Taken together, we have developed an automated workflow for the isolation of TIL from mouse tumors reducing time and costs of downstream analysis while standardizing and enhancing the detection and quantification of immune cell subpopulations.

#1673

Mass spectrometric characterization of peptides associated with molecules of the major histocompatibility complex.

Michael Ford,1 Richard Jones,1 David Allen,1 Ravi Amunugama,1 Paul Del Rizzo,2 Michael Pisano,2 James Mobley,2 Paul Domanski,2 Bill Ho,2 Daniel Bochar2. 1 _MS Bioworks, Ann Arbor, MI;_ 2 _Cayman Chemical, Ann Arbor, MI_.

The major histocompatibility complex (MHC) is a region of highly polymorphic genes encoding for glycoproteins (MHC molecules) that form part of the cell-mediated branch of the acquired immune system. In the cytosol, cellular self and foreign (non-self) proteins are constantly being degraded; it is the peptides generated that are presented, non-covalently bound to MHC molecules, on the surface of cells for inspection by cytotoxic T-lymphocytes (CTLs). Non recognition of the presented peptide ultimately leads to cell destruction.

Characterizing the factors associated with Non recognition is an attractive proposition for anyone interested in generating tools for targeted cell destruction. In the field of oncology the obvious application then is the targeted destruction of cancerous cells. To enable the molecular level characterization of peptides associated with molecules of the major histocompatibility complex requires a targeted protein complex enrichment, an unbiased peptide elution and finally a peptide analysis method. Most frequently an immunoprecipitation is used to isolate the target complex. The peptide elution is performed under conditions minimizing protein contamination and finally peptide analysis is accomplished by mass spectrometry.

Here we present a case study of our recent work optimizing and performing a workflow for the analysis of peptides associated with Class I MHC molecules. The goal of the assay optimization was to minimize the amount of antibody required for the assay, to minimize the amount of biological material needed from which the complex is isolated and to achieve the optimum sensitivity towards the hitherto unknown target peptides.

#1674

Quantifying tumor-infiltrating leukocytes in hematoxylin stained NSCLC tissue samples using morphometric features.

Elliott Ergon, Allison S. Harney, Nathan Martin, Will Paces, Famke Aeffner, Kristin Wilson, Janet Patterson-Kane, Karen Ryall, Daniel G. Rudmann, Brooke Hirsch, Joseph Krueger. _Flagship Biosciences, Westminster, CO_.

Quantification of tumor-infiltrating lymphocytes (TILs) in non-small cell lung cancers (NSCLC) is valuable for understanding patient prognosis and survival. TILs comprise a subset of tumor-infiltrating leukocytes that modulate immune evasion and response to therapy. Understanding the composition of TIL subsets, especially relative to the total tumor leukocyte population, may provide additional context for understanding NSCLC pathogenesis and patient response to treatment. However, availability of tissues and use of chromogenic assays can limit the number of TIL and leukocyte subset markers assayed in a tissue section. Therefore, this study evaluated the identification of total leukocyte component in NSCLC using morphometric parameters and routine TIL marker monoplex immunohistochemistry (IHC) assays to further identify the composition of TIL subsets. Computational Tissue Analysis (cTA™) tools were used to determine the morphometric parameters which could identify immune cells in the absence of biomarker stain. The morphometric features which characterized immune infiltrates were used to quantify the total immune cell population frequency in the tumor nests and surrounding stroma in hematoxylin-stained tissues. The leukocyte population identified with morphometric parameters was correlated with CD45+ cell frequencies identified by cTA based on biomarker staining in CD45-stained serial sections. This morphometric ruleset was then applied to CD3- and CD8-stained tissues to evaluate the frequency of CD3+ and CD8+ TILs in the context of total infiltrating leukocytes. The relative populations of CD3+ and CD8+ TILs were consistent with available literature demonstrating that the morphometric ruleset could be utilized to enable evaluation of TIL sub-types relative to total leukocyte population without the need for additional IHC stains. The approach could, therefore, provide an added dimension of analysis for tissues stained by IHC for identifying the total immune cell infiltrating component without requiring additional biomarker staining.

#1675

Parallel genome and transcriptome-wide profiling of PD-L1 expressing tumor and infiltrating immune single cells in NSCLC.

Stefano Casonato,1 Giulio Bassi,1 Chiara Bolognesi,1 Chiara Mangano,1 Valentina del Monaco,1 Genny Buson,1 Claudio Forcato,1 Alberto Ferrarini,1 Farideh Bischoff,2 Gianni Medoro,1 Nicolò Menaresi,1 Francesca Fontana1. 1 _Menarini Silicon Biosystems SpA, Castel Maggiore, Bologna, Italy;_ 2 _Menarini Silicon Biosystems Inc, San Diego, CA_.

Introduction Targeting immune checkpoint has demonstrated contrasting clinical response in non-small cell lung cancer (NSCLC) due to the lack of robust biomarkers for patients stratification. This is partially due to a poor understanding of the role of the different cellular players (tumor, stromal, infiltrating immune cells) within the tumor microenvironment. Here we present an innovative workflow using the DEPArray™ sorting system, to isolate different classes of epithelial and hematopoietic viable single cells from fresh tissue biopsies for integrated genome and transcriptome sequencing analysis.

Methods Freshly resected NSCLC tissue was dissociated mechanically and enzymatically down to single cell suspension, and labelled with immunofluorescent antibodies targeting PD-L1 (28-8), EpCAM, CD8, CD45 along with Hoechst 33342 for nuclei detection. Distinct pure single cells, along with pools of precise number of cells were digitally isolated using DEPArray™ platform based on marker expression. For each cell recovery, mRNA was physically separated from genomic DNA and then amplified using the Ampli1™ WTA Kit to generate a library of high quality, full-length double stranded cDNA. Amplified cDNA is used for NGS library preparation and sequencing on Illumina® MiSeq System. In parallel, genomic DNA is collected with magnetic beads and eluted directly into the reaction mixture for whole genome amplification by Ampli1™ WGA kit. The WGA products were used to profile genome-wide copy-number aberrations (CNA) using Ampli1™ LowPass kit.

Results As a first step, we identified two main cell populations, the CD45+ infiltrating hematopoietic cells and the EpCAM+ epithelial cells. The hematopoietic fraction was further investigated to evaluate the presence of different subpopulations. Interestingly the PD-L1+ population, corresponding to immune suppressive hematopoietic cells, represented 47% of the total CD45+ fraction while CD8+ cells corresponding to cytotoxic T cells represented 9% of the total CD45. In the EpCAM+ population the PD-L1+ fraction represented 29% of the cells. Single viable cells along with matching pools of precise number of cells from each population were digitally isolated with DEPArray™ system for downstream analysis. Results of RNA-Seq and genomic DNA copy-number aberrations profiling will be presented at the conference.

Conclusion We show here a new approach to isolate pure, single PD-L1+/- tumor and tumor infiltrating hematopoietic cells from NSCLC tissue for integrated genome and transcriptome sequencing. The direct comparison of gene expression data to its corresponding genomic data in the same tumor cell, along with the transcriptomic profiling of immune infiltrate and stroma, may add a powerful tool to disentangle the tumor microenvironment biology. This will potentially offer a novel tool to develop new biomarkers and identify therapeutic leads.

#1676

EZH2 inhibitors in immunotherapy of melanoma.

Peter Hersey,1 Stuart Gallagher,1 Jessamy Tiffen,1 Elena Shklovskaya,1 Stephen Wilson,2 Fabian Filipp3. 1 _Univ. of Sydney, Sydney, Australia;_ 2 _University of California Merced, Merced, CA;_ 3 _University of California Merced, Merced, CA_.

EZH2 contains the catalytic subunit of the PRC2 complex, which plays a role in maintenance of stem cell- like state of cancer cells and in the progression of melanoma. Our previous studies have shown that hyperactivation of EZH2 by somatic mutations or copy number gain occurs in 20% of melanoma patients and impacts overall survival. Elevated activity of EZH2 in cancer can be targeted by small molecule drugs (e.g. GSK126) and is associated with sensitivity to apoptotic cell death. Transcriptomic profiling showed that several well-known tumor suppressors such as BOK, NDRG1 and RASSF5 are down-regulated in cells with abnormalities in EZH2, which can be reversed by inhibitor treatment. By using our in vitro EZH2 drug targeting assay we validated genes associated with immune recognition and MHC class II antigen presentation such as HLA A and B,DR and CD74 (HLA DR invariant chain). In addition, ChIP-Seq studies identified adhesion molecules, chemokine ligands, and receptors involved in traffic of lymphocytes to tumors as well as the function of NK cells. Treatment of melanoma with EZH2 inhibitors showed increased killing by NK cells suggesting that an immunosuppressive effect of EZH2 involving both innate and adaptive immunity. In a mouse model of subcutaneous B16 melanoma, a course of daily treatment with an EZH2 inhibitor did not change tumour growth or immune responses in immunosufficient mice but caused a transient 9-fold increase in circulating Ly6G+ [granulocytic] myeloid-derived suppressor cells [MDSCs] suggesting the effect of EZH2 modifiers on the myeloid compartment. In summary, patients with somatic activation of EZH2 may benefit from treatment combinations that include inhibitors of EZH2. Supported by NHMRC program grant 633004, Sydney medical school foundation and Cancer Institute NSW. F.V.F. is grateful for the support of grants CA154887 from the National Institutes of Health, National Cancer Institute and CRCC CRN-17-427258.

#1677

Immuno-target selection of infiltrating immune cells and laser capture microdissection mediated transcriptional profiling.

Ross Haggart, Chaxiraxi Arzola-Donate, Elliott Harrison, Benjamin J. Reed, Saba Alzabin, Gino Miele. _Epistem Ltd., Manchester, United Kingdom_.

Immune checkpoints define an evolving class of inhibitory mediators that are expressed by tumour cells and infiltrating leucocytes to down-modulate the effector immune response against tumour cells. Therefore, the most recent efforts in cancer therapy centre on targeting immune-mediated mechanisms of tumour evasion. The ability to assess gene expression profiles from isolated populations of tumour-infiltrating lymphocytes (TILs) can assist in monitoring checkpoint inhibitor therapy efficacy and provide a better understanding of clinical outcomes. Ideally, transcriptional profiles should be obtained from isolated cell populations of interest, free from contaminating cell populations that may create adverse background noise. Micro sampling by laser capture microdissection is a powerful tool that allows for specific analysis from whole tissues, particularly in a heterogeneous microenvironment such as in a tumour, where less well represented target cells are dwarfed in abundance by tissue stroma and other cell types. However, whilst the application is potentially powerful for target discovery and mechanistic understanding, the process is notoriously difficult, particularly in clinical specimens. We developed a methodology to successfully overcome these challenges in analysing target cells, organelles or other tissue subsets by transcriptional profiling following immunostain-mediated laser capture microscopy (LCM). Thorough optimisation of traditional immunohistochemistry techniques enabled us to select target cell populations of interest from tissue sections whilst minimising degradation of mRNA and miRNA allowing us to perform genomic and pathway interrogation using microarray and/or RT-qPCR analysis. Our technique allows multiple capture types per slide, sample pooling when required, high capture throughput and capture image documentation. Multiple target selection, to differentiate lymphocytes, can be performed routinely using comparative serial sectioning. Here we exhibit simple staining and immunostaining histology images using methods sympathetic to RNA integrity which allow for target selection by specific staining or target morphology, and demonstrate comparative analysis of matched pairs of disease and healthy sections of colon tumours. We used a Palm MicroBeam 4 LCM platform to identify, cut and specifically capture CD8+ tumour-infiltrating lymphocytes from frozen embedded tissue. This approach enabled us to isolate discrete targeted cell populations free from contaminating cell populations. Epistem's Single Cell RNA-Amp™ was then used to provide robust and linear amplification of RNA and enable comparative analysis in applications such as target discovery and pharmacodynamics. The results indicated that transcriptional profiling was technically robust with a good replicate correlation despite the limited input obtained by micro-sampling.

#1678

A flow-based immunoprofiling strategy for interrogating system-wide leukocyte composition in response to glioblastoma.

Gregory J. Baker,1 P.S. Thiagarajan,1 Sucheendra K. Palaniappan,2 Stephanie H. Davis,1 Jodene K. Moore,1 Peter K. Sorger1. 1 _Harvard Medical School, Boston, MA;_ 2 _Systems Biology Institute, Tokyo, Japan_.

Glioblastoma (GBM) is a deadly brain cancer that suppresses cellular immunity through the production of anti-inflammatory cytokines/catabolites, expression of immune checkpoint molecules, and induction of tolerogenic immune cell subsets. While preclinical models have shown each of these mechanisms to negatively impact survival outcome, it remains unclear how these distinct processes manifest themselves within the context of a coordinated immune system and whether additional immunosuppressive mechanisms exist. To address these questions, we have developed an extensible in vivo screening platform to holistically determine how GBM affects cellular immune organization. The method allows for temporal assessment of leukocyte population dynamics in response to tumor progression. Leukocytes derived from lymphoid tissues of tumor-bearing mice are isolated and immunolabeled with a 12-color optimized immunofluorescence panel (OMIP) targeting several major leukocyte lineages. Single-cell data is acquired by flow cytometry and is clustered in an unsupervised manner based on cell-surface protein expression. A vector-based classification system is then used to map phenotypically distinct clusters onto known immune cell lineages. The platform allows for the extraction of immune signatures whose divergence from tumor-naïve standards may be quantified and statistically analyzed; such signatures may also be correlated with response/resistance to therapy. The approach has been validated on orthotopic GL261 glioma in syngeneic C57BL6/J mice, yet it is readily amenable to the study of diverse model systems, including de novo tumors driven by clinically-relevant oncogenes. Alternative flow cytometry antibody panels may easily be substituted to probe more specific immune cell subsets of interest. Systems biology approaches applied to the field of tumor-induced immunosuppression hold promise in identifying novel network-level immune signatures, facilitating a better understanding of immunotherapeutic drug action, and driving next generation high-dimensional biomarker discovery.

#1679

BET inhibitors suppress PD-L1 expression in pancreatic stellate cells.

Kazumi Ebine, Brian T. DeCant, Katharine A. Collier, Thao N. Pham, Krishan Kumar, Hidayatullah G. Munshi. _Northwestern University, Chicago, IL_.

Single agent treatment with T-cell checkpoint inhibitors has not been effective in pancreatic ductal adenocarcinoma (PDAC) patients. The PDAC stroma, which can account for as much as 80-90% of the tumor mass, can act as a physical and an immunologic barrier to T-cell-mediated therapies. Transgenic mouse models have shown that ablation of pancreatic stellate cells (PSCs), key regulators of fibrosis in vivo, can sensitize PDAC tumors to immune checkpoint therapies. Recently, inhibitors targeting bromodomains and extra-terminal (BET) proteins, a number of which are currently being evaluated in clinical trials for solid tumors, were shown to induce stellate cells to become quiescent and decrease collagen production. The BET family of proteins binds to acetylation motifs present in histones and enables recruitment of transcription factors and other chromatin regulators during RNA transcription. We have found that PSCs express significantly increased PD-L1 levels compared to pancreatic cancer cell lines. We show that BET inhibitors, and in particular specific knockdown of BRD4 protein, decrease basal and IFN-γ-mediated PD-L1 expression in primary stellate cells. We also show that, in contrast to a recent report using cancer cells, c-MYC does not mediate basal or IFN-γ-mediated PD-L1 expression in stellate cells. Instead, we show that the IFN-γ-mediated PD-L1 expression is regulated by IRF1, suggesting cross talk between BRD4 and IRF1 in the regulation of PD-L1 expression. Ongoing in vivo experiments will evaluate the role of BET inhibitors in the regulation of PD-L1 in mouse models of pancreatic cancer.

#1680

BPM31510, an anti-cancer agent selectively causes activation and proliferation of T cells, demonstrating potential utility in an immune-oncology setting.

Maria-D Nastke, Nidhi Gaur, Louisa Dowal, Samantha Fowler, Anne Diers, Stephane Gesta, Vivek K. Vishnudas, Niven R. Narain, Rangaprasad Sarangarajan. _BERG, LLC, Framingham, MA_.

BPM 31510, a clinical stage, nanodispersion of ubidecarenone has a unique mechanism of action that effectuates an anti-Warburg switch in cancer cell metabolism and activation of apoptosis. Given the observed central role of BPM 31510 in regulating mitochondrial function in cancer cells, it is of great interest to investigate the ability of BPM 31510 to modulate immune cells and their functionality. Therefore, the effects of BPM 31510 on peripheral blood mononuclear cells (PBMCs) were investigated to elucidate the immuno-metabolic mechanism of BPM 31510. Healthy donor PBMCs activated with PHA or PWM was used as model system, and the effect of BPM 31510 on immune cells viability was determined using flow cytometry. In addition, the effect of BPM31510 on immune cell function was evaluated by measuring a panel cytokines released in these cells, using a quantitative ELISA platform from Meso Scale Discovery.

Results: BPM 31510 has been shown in previous studies to effectively induce apoptosis on a variety of cancer cell lines while sparing normal cells. Interestingly, increasing concentrations of BPM31510 lead to an increased frequency of viable CD3+ cells. Further phenotypic analysis revealed that cytotoxic T cells (CD8+/CD3+) and T helper cells (CD4+/CD3+), as well as NKT cells (CD56+/CD3+) contributed to the observed increase in T cells frequency. On the other hand, B cells (CD19+), NK cells (CD56+/CD3-), and the monocytes (CD14+) showed a decrease in frequency, an effect reflected also by a reduction in viability with increasing BPM 31510 concentrations. Cytokine analysis indicated that effector cytokines IL-2, IFN-γ, and TNF- α were secreted at significantly higher levels with increasing concentration of BPM 31510. Interestingly, IL-10 level, an immuno-regulatory cytokine, was strongly decreased in the supernatant of PBMCs treated with BPM31510. Taken together, we demonstrated that BPM 31510 has a direct effect on immune cells and their functionality. Although BPM 31510 induced apoptosis of cancer cells, our data indicate that it supports cell proliferation of T cells, and effector function of adaptive immune cells, likely by providing a higher energy supply for effector T cells. Subsequently, a higher activation state of effector T cells may result in increased levels of cytotoxic effector molecules (perforin, granzymes, Fas ligand) and macrophage activating effector molecules (IFN-γ, IL-2, TNF-α) which supports and attracts other immune cells like NK cells to the tumor.

#1681

The antitumor effect of a soluble recombinant human thrombomodulin as growth suppression against gastrointestinal tumor in murine peritonitis model.

En Amada, Kazumasa Fukuda, Koshi Kumagai, Koichi Suda, Hiroya Takeuchi, Yuko Kitagawa. _Keio University School of Medicine, Tokyo, Japan_.

Introduction:

Severe postoperative inflammatory response (PIR) with CARS increases the risk of tumor recurrence after cancer surgery by Tregs suppressing antitumor immunity. Also, cancer recruits Tregs into its tissue by secreting CCL22 and TGF-beta that strengthen its survival.

We established a model to represent tumor dynamics undergo PIR by mice that are performed cecal ligation and puncture (CLP) followed by subcutaneous injection of cancer cells into the dorsum. In this model, we previously revealed significant tumor growth and increase number of Tregs both in tumor tissue and blood compared to the normal mice.

A soluble recombinant human thrombomodulin (rhTM) is developed as a treatment drug against DIC. It has anti-inflammatory effect and several reports showed that it prevents cancer metastasis.

We hypothesized that rhTM normalizes PIR and prevents cancer growth caused by inflammation.

Method:

Ten-week-old C57BL/6 mice were divided in CLP/rhTM, CLP/normal saline (NS), and control (simple laparotomy plus NS) groups. rhTM (3mg/kg/12hrs) was injected subcutaneously for 7 consecutive days from the day before CLP. CT26 cells (1×104) were implanted 4 hours after CLP. Mice were sacrificed 28 days after CLP. Then, the tumor tissue and blood were collected.

To analyze dynamics of Treg, peripheral blood leukocytes the tumor cells were isolated. CD4, CD25 and Foxp3 were stained by fluorescent antibody and stained cells underwent flow cytometric analysis. The quantity of Tregs is measured by the rate of CD25+/Foxp3+ population in CD4+ T cells.

The level of CCL22 and TGF-beta were measured both in serum and tumor tissue. The ELISA was used for analyzing serum samples. And for tumor tissue, immunostaing by DAB was performed and positively stained areas were calculated by image-editing software.

Results:

CLP/NS group mice exhibited significant enhanced tumor growth compared to controls (6.0±1.9 g vs. 3.5±1.6 g, p=0.03), while CLP/rhTM group produced significantly smaller tumors (2.2±1.1 g) than CLP/NS (p=0.01).

The Tregs in the blood stream was smaller in CLP/rhTM group compared to CLP/NS group. Intratumoral Tregs showed significant difference between CLP/rhTM group and CLP/NS group (8.89±3.5 % vs. 24.1±15.0 %, p=0.02).

We found significant elevation of serum TGF-beta level by CLP (4.7±1.2 pg/ml vs. 15.4±7.0 pg/ml, p=0.02) and relative reduction to 15.4±7.0 pg/ml by administration of rhTM (p=0.05). However, we did not recognize any change of serum CCL22 level in this study.

Intratumoral rate of CCL22 positive areas increased by CLP (41% vs. 50%), while rhTM made it decreased to 43%. We found little difference of TGF-beta positive areas among three groups in this study.

Conclusion:

Acute inflammation induced by CLP enhances the growth of implanted tumors, while administration of rhTM suppresses tumor growth by possibly affecting dynamics Tregs via TGF-beta and CCL22.

#1681A

Overcoming pd1 targeting antibody resistance using combination strategies.

Jean-François Mirjolet, Maxime Ramelet, Damien France. _Oncodesign S.A., Dijon Cedex, France_.

PD-1 targeting antibodies (nivolumab, pembrolizumab) are now approved in various tumor types either as second line treatment (locally advanced or metastatic urothelial carcinoma, advanced renal cell carcinoma, recurrent or metastatic head and neck squamous cell carcinoma, classical Hodgkin lymphoma) or even as first line therapy (metastatic melanoma, metastatic non-small cell lung cancer). Despite an increase in response rate as well as survival, there is still issue with resistance to PD-1 targeting therapies. Selection biomarkers as well as response biomarkers are still under intensive research. In addition, combination strategies are needed to increase response rate and overcome resistance.

Combination strategies were therefore tested in one well characterized syngeneic mouse model, i.e. EMT-6 breast carcinoma. The model is known to be highly sensitive to CTLA-4 targeting antibody (optimum tumor to control ratio (T/C) of 3%), a moderate responder to PD-1 targeting treatment (best T/C of 54%) and a well immune infiltrated tumor. Antibodies targeting co-stimulatory receptor such as OX40, GITR and 4-1BB as well as co-inhibitory receptor such as Tim3 were combined to anti-PD-1 antibody. In all cases, combination arm produces better therapeutic activity when compared to single treatment alone. Moreover, both strategies, meaning combination of two antibodies targeting co-inhibitory receptors (PD-1 + Tim3) and combination targeting one co-inhibitory (i.e. PD-1) and one co-stimulatory receptors (OX40, GITR or 4-1BB) worked. Detailed results will be presented including response rate, immune infiltrates and cytokines profiles in order to rationally choose the best therapeutic treatment.

## CLINICAL RESEARCH:

### Adaptive Immunity to Cancer

#1682

Correlation of immune co-stimulatory molecule OX40 and outcome in trastuzumab treated HER2-positive breast cancer patients in the NCCTG-N9831.

Saranya Chumsri,1 Daniel J. Serie,1 Afshin Mashadi-Hossein,2 Sarah Warren,2 Alvaro Moreno-Aspitia,1 Geraldo Colon-Otero,1 Keith L. Knutson,1 Edith A. Perez,1 E. Aubrey Thompson1. 1 _Mayo Clinic Cancer Center, Jacksonville, FL;_ 2 _NanoString Technologies, Inc., Seattle, WA_.

Background: Trastuzumab (H), a monoclonal antibody against HER2, has revolutionized the treatment for HER2-positive breast cancer. Besides inhibiting downstream signaling of HER2, several studies showed that H also exerts its anti-tumor activity via immune-related mechanisms. While H is quite effective in preventing recurrence, significant numbers of patients still develop recurrence despite adjuvant H-based chemotherapy. In this study, we analyzed immune-related genes that were associated with poor outcome in N9831.

Methods: NanoStringTM technology was used to quantify mRNA of immune-related genes in baseline samples from 1,280 patients in N9831. In N9831, patients in arm A were treated with chemotherapy alone (AC-T), arm B received chemotherapy followed by sequential H (AC-T-H), and arm C received H concurrently with chemotherapy (AC-TH). Cox proportional hazard ratio (HR) was used to determine the association of each immune-related gene with recurrence free survival (RFS). Different immune subset signatures, including CD45, CD8, cytotoxic-cells, and T-cells were analyzed using algorithms developed by NanoString.

Results: With the median follow up of 10.6 years, we identified a total 77 genes that were associated with improved outcome in arm C. Among these 77 genes, there were 20 tumor necrosis factor (TNF)-related genes. Of those, only OX40 (TNFRSF4) and its ligand TNFSF4 have interaction p < 0.10. Interestingly, we found uneven distribution of OX40 expression in the N9831 specimens. Approximately, 9.5% of HER2-positive breast cancer expressed OX40 at distinctly low level. Low expression of OX40 was significantly associated with HR positivity (OX40 low 61% vs. OX40 high 51%, p 0.003) and larger tumor size but not patients' age, tumor grade, and lymph node status. Low expression of OX40 was significantly associated with low mTIL-CD45, CD8, cytotoxic-cells, and T-cells immune signature scores. Tumors with low OX40 expression had significantly lower levels of CTLA4 (p 9.11e-71) and PD-L1 expressions (p 2.49e-92). Low expression of OX40 is associated with poorer outcome among patients treated with H in both sequential (HR 0.88, 95% CI 0.79-0.98, p 0.022) and concurrent arms (HR 0.86, 95%CI 0.76-0.98, p 0.027) but not in chemotherapy only arm (HR 0.9, 95%CI 0.90-1.09, p 0.8). Similar findings were observed with its ligand TNFSF4 in arm A (HR 1.02, 95%CI 0.92-1.15, p 0.68), arm B (HR 0.87, 95%CI 0.76-0.99, p 0.04), and arm C (HR 0.81, 95%CI 0.68-0.95, p 0.01).

Conclusion: Our study suggests that pre-existing expression of OX40 and its ligand TNFSF4 are prognostic and may also be predictive of adjuvant H benefit. Patients with distinctly low level of OX40 had poor outcome despite adjuvant H-based adjuvant chemotherapy. Our study provides a rationale to further evaluate the strategy to increase immune activation to improve outcome in this group of patients.

#1683

A novel adenosine A2A receptor antagonist optimized for high potency in adenosine-rich tumor microenvironment boosts antitumor immunity.

Erica Houthuys, Margreet Brouwer, Florence Nyawouame, Romain Pirson, Reece Marillier, Theo Deregnaucourt, Joao Marchante, Jakub Swiercz, Charlotte Moulin, Vanesa Bol, Gregory Driessens, Michel Detheux, Christophe Quéva, Stefano Crosignani, Bruno Gomes. _iTeos Therapeutics, Gosselies, Belgium_.

High levels of extracellular adenosine in the tumor microenvironment are known to play a significant role in tumor immune evasion and promote tumor growth and metastasis. We defined the receptor(s) required for mediating the effect of adenosine on immune cells within the tumor microenvionment and report the characterization of a novel Immuno-Oncology-dedicated adenosine receptor 2A antagonist that functions in the high adenosine concentration found in tumors.

We first explored the expression of the four adenosine receptors in primary human immune cells. A2A receptor was the main adenosine receptor expressed by CD4 and CD8 T lymphocytes and monocytes, and the only one in mature monocyte-derived dendritic cells and NK cells. A2B receptor was poorly detected in T cells and monocytes, while A1 and A3 receptors were never detected. Given these expression patterns, we further studied A2A functions in primary human T lymphocytes and monocytes. Selective A2A agonists such as CGS-21680 strongly suppressed cytokine production by activated primary human T lymphocytes, thus highlighting that A2A is the main effector receptor of the sensing of adenosine in tumors.

We further confirmed the elevated extracellular adenosine level in the tumor microenvironment in several mouse and human tumors. High adenosine levels correlated with strong tumoral expression of CD73, the enzyme that converts AMP to adenosine. Interestingly, we showed that A2A receptor antagonists designed for Parkinson's disease dramatically lost potency in a high adenosine environment ; our data indicated that a 30-fold dose increase may be required for full target inhibition within tumors. Therefore we developed a novel and potent A2A blocker with sub-nanomolar Ki and IC50 in a cAMP assay and a more than 100-fold selectivity over other adenosine receptors. Our lead compound kept a high potency in an adenosine-rich environment and restored cytokine production even in the presence of high concentrations of A2A agonists. iTeos inhibitor also efficiently reversed AMP-mediated T cell suppression. Furthermore, our compound rescued A2A receptor agonist-induced decrease of TNFα production by primary human monocytes, and was able to potently increase CD8 T cell cytotoxicity in a cytotoxicity assay with CD8 T cells as effectors and cancer cells as targets.

These results suggest that iTeos new generation of A2A receptor antagonist, designed to keep a high potency in the adenosine-rich tumor microenvironment, may offer a new therapeutic opportunity in Immuno-Oncology.

#1685

Effects of anti-ctla-4 and anti-pd-1 on memory T-cell differentiation and resistance to tumor relapse.

Stephen Mok, Colm R. Duffy, James P. Allison. _MD Anderson Cancer Center, Houston, TX_.

The FDA has begun to expand the approved uses of immune checkpoint blockade antibodies targeting CTLA-4 and PD-1. Blocking either checkpoint relieves the negative regulation of T-cells resulting in significant responses in patients with cancer. Data has now begun to emerge regarding differences between these two therapies. While αPD-1 therapy has a greater response rate (~30% vs. 11%) according to RECIST criteria, recent reports have suggested responses to αPD-1 may not be as durable as αCTLA-4. 25% of patients who initially responded to αPD-1 have tumor relapse within 24 months. In contrast, the 3-year survival rate of patients treated with αCTLA-4 is at least ~25% suggesting a durable response. Previous studies in bacterial or chronic LCMV infectious models have shown that αCTLA-4 can increase CD8+ memory T-cell formation, whereas genetic ablation of PD-1 on T-cells often promotes the terminally differentiated exhausted CD8+ T-cell phenotype, while attenuating memory T-cell formation. However, the mechanism which leads to relapse following αPD-1 treatment in tumor models is not clear. The goal of this project is to understand how immunotherapies shape memory T-cell formation and how that relates to the mechanism of tumor relapse.

To test whether αCTLA-4 or αPD-1 can induce a better memory T-cell response, mice were vaccinated with irradiated B16F10 murine melanoma cells and treated αCTLA-4 or αPD-1. Mice were re-challenged with B16F10 80 days after vaccination. Although both αCTLA-4 and αPD-1 improved tumor rejection compared with controls, αCTLA-4 treated mice exhibited superior tumor control compared to αPD-1 (p<0.0005) suggesting the memory T-cell response mediated by αCTLA-4 is more durable. In order to test whether this memory T-cell response is antigen-specific, mice were re-challenged with unrelated MC38 or 3LL cancer cells. The antibody treated groups did not show improved antitumor effect compared with vaccine control (p>0.5). To test whether the frequency of memory T-cells recruited to the re-challenged tumor could affect memory T-cell response, antigen-specific pmel-1 T-cells were infused to mice following vaccination with αCTLA-4 or αPD-1. Our result suggested that there were more tumor-infiltrating pmel-1 T-cells in the αCTLA-4 treated group compared to the αPD-1 treated group (p<0.05). In order to augment the durability of αPD-1 treatment, αPD-1 was combined with αCTLA-4 following vaccination. The combined treatment group has superior antitumor response compared to that with αPD-1 (p<0.000003) and overlapped with the αCTLA-4 treated group (P>0.05) during re-challenge, indicating that the effect of the combined treatment is dominated by αCTLA-4.

Collectively, our studies facilitate the design of combination immunotherapy treatments that enhance both response rates and generation of memory T-cells to prevent relapse.

#1686

Epigenetic modulation of the tumor microenvironment enhances vaccine induced T cell responses in a murine model of pancreatic cancer.

Brian Christmas,1 Blake Scott,1 Todd Armstrong,1 Nilofer Azad,2 Elizabeth Jaffee1. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _Johns Hopkins Sidney Kimmel Comp. Cancer Ctr., Baltimore, MD_.

This project aims to test the hypothesis that epigenetic modulatory drugs (EMD) and GVAX, a GM-CSF-secreting whole tumor cell vaccine, are capable of altering the inflammatory environment of pancreatic ductal adenocarcinoma (PDAC) and sensitizing it to checkpoint blocking agents.

Recently the use of antibody therapy targeting immune checkpoints, such as CTLA-4 and PD-1, has become a major focus of cancer immunotherapy. In responsive patients, these therapies have resulted in long-term control of chemotherapy-resistant disease. The most compelling activity has been seen in the minority of patients with immunogenic tumors where T cell infiltration naturally occurs. These benefits are not observed in non-immunogenic tumors, such as PDAC, with low expression of tumor-associated antigens (TAA) and a lack of intrinsic T cell infiltrates. Therapies that can alter the tumor microenvironment (TME) and allow infiltration of effector T cells, decrease immunosuppressive cells, and stimulate TAA expression may convert non-immunogenic tumors into cancers sensitive to checkpoint inhibitors. Recent work with EMDs has shown that they are capable of altering the immunogenicity of the TME by inducing the expression of cancer testis antigens as well as increasing tumor cell expression of MHC class II and decreasing Tregs in the TME. Additionally, GVAX has been shown to induce tertiary lymphoid aggregates within the TME of patients with PDAC.

We are testing the hypothesis that treatment with EMDs and GVAX can sensitize the inflammatory environment of PDAC to checkpoint blockade inhibition by evaluating changes in immune cell function within the TME via flow cytometry, immunohistochemistry, and gene expression array. We are using a murine model of hepatic metastases of pancreatic cancer which involves injecting syngeneic pancreatic tumor cells into the spleen followed by a hemisplenectomy, resulting in the consistent formation of hepatic metastases that can be monitored by ultrasound. We have evaluated entinostat, a histone deacetylase inhibitor, in combination with GVAX which induces T cell responses, and demonstrated a significant increase in survival when compared with either agent alone. Flow cytometric analysis of the cells infiltrating the TME shows that the combination of entinostat and GVAX causes a significant increase in CD4+ T cell infiltration as well as a shift from an M-MDSC dominant to a more G-MDSC dominant myeloid population. Current studies aim to elucidate the functionality of the MDSC population as well as identify potential changes in the T helper cell subsets via flow cytometry. Additionally, future studies will evaluate changes within checkpoint blockade pathways via flow cytometry and gene expression array to identify pathways that require further modulation to enhance antitumor responses.

#1687

Systemic immunological changes during first line chemotherapy in patients with high-grade serous ovarian cancer.

Fenne L. Komdeur, Florine A. Eggink, Ninke Leffers, Hagma H. Workel, Kim L. Brunekreeft, Annechien Plat, Marco de Bruyn, Hans W. Nijman. _University of Groningen, University Medical Center Groningen, Groningen, Netherlands_.

Ovarian cancer remains the most lethal gynecological malignancy and new therapeutic strategies are urgently needed. High-grade serous ovarian cancer (HGSC), in particular, is associated with a five-year survival of only 40%. A promising new approach for treating HGSC is immunotherapy, which has resulted in complete and durable responses, albeit in a minority of patients. One potential approach for improving these response rates is by combining chemo- and immunotherapy. Indeed, carboplatin/taxol chemotherapy was shown to augment immune responses in cervical cancer patients by depleting circulating myeloid suppressor cells. As patients with HGSC are treated in first line with similar carboplatin/taxol chemotherapy, we explored the effects of chemotherapy on systemic immunity in HGSC patients. Within this prospective observational study, 75 patients with suspected ovarian cancer were included. Blood was collected at three different time points during first line chemotherapy treatment, namely: prior to chemotherapy, between the third and fourth cycle of chemotherapy and 4-6 weeks after the sixth cycle of chemotherapy. All patients received 6 cycles of carboplatin/taxol chemotherapy and cytoreductive surgery (either as primary debulking, or after 3 cycles of neo-adjuvant chemotherapy). Peripheral blood mononuclear cells were isolated and analyzed for a total of 36 immune markers using 9 flow cytometry panels, in total analyzing 49 immune cell subsets. Results were compared to an age-matched cohort consisting of women surgically treated for a benign disease, and a cohort of healthy young volunteers. 75 patients have been included so far from which 22 were diagnosed with benign disease, 3 were diagnosed with another malignancy, and 50 were diagnosed with OC. Of the 50 OC patients, 18 were diagnosed with HGSC and from 9 HGSC patients, multiple time points were available for analysis of chemotherapy-dependent changes in the immune cell subsets. All patients developed leukopenia as a result of chemotherapeutic treatment. Age-related changes in lymphocyte and myeloid cell subsets were observed in all HGSC patients and patients with benign disease. Chemotherapy-dependent depletion of myeloid cells was observed in a subset of patients. Depletion of myeloid subsets was equally distributed among monocytes, macrophages and dendritic cells. No HGSC- or chemotherapy-dependent changes in T cell subsets or migration and activation markers were observed. Taken together, we observed no major systemic changes in immune cell subsets during carboplatin/taxol chemotherapy treatment, suggesting that combined chemo-immunotherapeutic strategies could be feasible during first line treatment of HGSC.

#1688

HERA-CD40L: A novel hexavalent CD40 agonist with superior biological activity.

Christian Merz, Jaromir Sykora, Meinolf Thiemann, Viola Marschall, Karl H. Heinonen, Harald Fricke, Christian Gieffers, Oliver Hill. _Apogenix AG, Heidelberg, Germany_.

Introduction: Targeted therapeutics for cancer treatment are mostly developed as antibodies, however, the natural signaling complexes of the members of the TNF superfamily and their receptors consist of clusters of trimers. Consequently, most of these bivalent agonistic antibodies depend on Fc receptor mediated crosslinking for biological activity. The HERA-Technology developed by Apogenix generates hexavalent fusion proteins targeting the TNF-receptor superfamily with high clustering capacity for the cognate receptor, which overcomes this disadvantage of antibody-based drugs. Here we compared the efficacy of different CD40 agonist formats, including the novel HERA-CD40L and the functional consequences of differential receptor clustering.

Materials & Methods: Biological activity of CD40 agonists was compared using an engineered reporter cell line and by flow cytometric analysis of CD40-induced IκBα degradation in Ramos B cells. T lymphocytes and monocytes were isolated from buffy coats and expression of CD markers upon CD40 ligation was analysed by multicolor flow cytometry (MC-FC). Secretion of cytokines in response to CD40 ligation was determined by ELISA. Monitoring of T cell-induced killing of tumor cells in direct co-cultures employed a real-time cell analysis system (xCELLigence).

Results: Direct comparison of bivalent CD40 antibodies with trivalent CD40L and the hexavalent HERA-CD40L in two independent bioactivity assay formats demonstrated that only the hexavalent agonist was fully active without additional crosslinking. In contrast to HERA-CD40L, neither the bivalent agonistic CD40 antibody nor the trivalent CD40L were able to upregulate expression of activation markers on B cells or to induce secretion of proinflammatory cytokines such as IL-12 and TNFα by PBMCs. In vitro generated M2-macrophages acquired an M1 phenotype and enhanced proliferation of naïve CD4+ T cells in direct co-culture. Similarly, direct co-culture of CD4+ T cells with Ramos B cells in the presence of HERA-CD40L induced cytotoxic activity of CD4+ cells against tumor cells. The activating effect was dependent on cell-cell contacts and was not observed in indirect co-cultures. Importantly, only the hexavalent HERA-CD40L showed full biological activity without additional crosslinking.

Conclusion: The hexavalent CD40 agonist HERA-CD40L produced by Apogenix HERA-Technology platform triggers CD40 signaling on B cells and cells of the monocytic lineage, leads to direct cytolytic activation and proliferation of CD4+ T cells and shifts the M1/M2 balance towards proinflammatory conditions. Unlike bivalent CD40 antibodies or trivalent CD40L_ based agonists, the hexavalent HERA-CD40L forms highly clustered signaling complexes and thus exhibits superior biological activity over other agonistic formats without the need for Fc receptor mediated crosslinking.

#1689

Dynamic monitoring of immune response and reagent efficacy through high throughput label-free impedance-based technology.

Fabio Cerignoli, Biao Xi, Garret Garret Guenther, Lincoln Muir, Leyna Zhao, Yama Abassi. _ACEA Biosciences Inc., San Diego, CA_.

In vitro characterization of reagents efficacy in the context of cancer immunotherapy is a necessary step before moving to more expensive animal models and clinical studies. However, current in use in vitro assays like Chromium-51, ATP-based luminescence or flow cytometry are either difficult to implement in high throughput environment or are mainly based on end point methodologies that are unable to capture the full dynamic of the immune response. Here we present the adaptation of an impedance-based platform to monitoring cytotoxic activity of immune cells activated trough different means. Impedance technology detects cell death and proliferation of adherent cells by measuring changes in conductance of microelectrodes embedded in 96 and 384-wells cell culture plates. We utilized adherent and B cell leukemia/lymphoma cell lines as well as primary tumor cells as in vitro models for immunotherapy reagents evaluation. We seeded the cells on electrodes coated 96-well plates and monitored cell adhesion and proliferation for 24 hours. The following day effector cells were added at multiple effector:target ratios in presence of BiTEs antibodies and/or anti PD-1/PD-L1 antibodies. Impedance signal was monitored for up to seven days. Control wells were set up with effector cells only or with target plus effector cells but without antibodies. We adapted such adhesion-based technology to monitor non-adherent B-leukemia/lymphoma cells, by developing a strategy where the wells are coated with an anti-CD40 antibody. The coating allows specific adhesion and retention of B cells and measurement of changes in impedance that are proportional to cell number. Using increasing concentrations of EpCAM/CD3 BiTE we demonstrated the suitability of such impedance-based approach to quantitatively monitor the efficacy of immune cells-mediated cancer cell killing both under different effector:target ratios and antibody concentrations. Combination treatments with checkpoint reduced timing and increased amount of killed cancer cells. Similar results were also obtained with engineered Car-T cells against CD19 or NK cell lines, we were able to demonstrate specific killing of tumor B cells at very low effector:target ratios. The results were also confirmed by flow cytometry. Overall, our results demonstrate the value of such approach in measuring the cytotoxic response across the temporal scale, an aspect that is otherwise very difficult to assess with more canonical end point assays. Furthermore, the availability of 384-wells format and minimal sample handling place the technology in an ideal spot for applications in large reagent validation screening or personalized medicine, like therapeutic protocol validation directly on patient samples.

#1690

Epitope-minigenes for optimal induction of the immune response against tumor associated antigens and neoantigens.

Silvio Bandini,1 Laura Luberto,1 Fabio Palombo,1 Giuseppe Roscilli,1 Emanuele Marra,1 Gennaro Ciliberto,2 Luigi Aurisicchio1. 1 _Takis, Rome, Italy;_ 2 _Istituto Tumori Regina Elena, Rome, Italy_.

We have recently established a workflow that allows the identification of T cell epitopes within Tumor Associated Antigens (TAAs) and the construction of genetic cancer vaccine based on the use of minigenes. The T-cell epitope in silico prediction approach is based on three criteria: 1) binding to MHC Class I alleles; 2) uniqueness to the antigen of interest; 3) increased likelihood of natural processing. The combination of in silico prediction and a biochemical binding/stability assay resulted in an accurate identification of novel TAA-derived epitopes. Predicted T cell epitopes were connected by furin sensitive linkers and linked to human tissue plasminogen activator (TPA) signal and E. Coli enterotoxin B subunit, to construct an optimal minigene scaffold used as vaccine candidate. The present study was aimed at evaluating HER2/neu and hTERT (telomerase) minigenes with the same technology platform. First of all, minigenes delivered via Electro Gene Transfer (DNA-EGT) were more immunogenic than genetic vectors encoding the full-length protein or peptides injected subcutaneously and they were able to break immune tolerance in wild type and HLA-A0201 transgenic mice. Moreover, this technology applied to epitopes selected within Mutated Tumor Antigens results in strong immunogenicity and significant antitumor effects in mouse models. In conclusion, we show that minigenes delivered via DNA-EGT and based on predicted and/or experimentally identified epitopes are powerful tools to induce immune responses and combat cancer. Combination studies of minigenes with peptide vaccination, chemotherapy and immune checkpoint blockade may define new therapeutic opportunities for cancer patients.

#1691

USP7 inhibitors impair Foxp3+ T regulatory cell function and promote antitumor immunity against solid tumors.

Suresh Kumar, Jian Wu, Liqing Wang, Feng Wang, Ivan Sokirniy, Hui Wang, David Sterner, Charles Grove, Joseph Weinstock, Michael Mattern, Wayne Hancock. _Progenra Inc, Malvern, PA_.

Tumor microenvironment (TME) plays critical role in suppressing the immune system's ability to recognize and destroy cancer cells. Although T cell checkpoint blockers such as anti-PD1 antibodies unleash anti-tumor immune response against a variety of tumors, the complex immunosuppressive tumor milieu necessitates development of additional therapeutic agents to potentiate active drugs and expand the realm of the revolutionary cancer immunotherapy. The presence of highly immunosuppressive Foxp3+ T-regulatory cells (Tregs) in the TME has been correlated with poor prognosis. Thus, depletion of Tregs or impairment of Treg function is considered an attractive therapeutic approach. USP7, a deubiquitylase (DUB) implicated as a critical node in several cancer signaling pathways has emerged as an essential factor in maintaining Treg functions. Treg specific deletion of USP7 impairs Treg function leading to lethal autoimmunity. Using the UbiProTM discovery platform, Progenra has identified selective covalent USP7 inhibitors that were subjected to lead optimization and preclinical evaluation. Potent and selective candidate USP7 inhibitors have been shown to impair Treg functions ex vivo and in vivo as well as exhibit powerful anti-tumor activity against syngeneic lung tumor models in immunocompetent mice. Most importantly, Progenra's USP7 inhibitors enhances the efficacy of anti-PD1 antibody and cancer vaccines. Along with the already established direct anti-tumor activities of USP7 inhibitors, these studies provide a strong rationale for combining USP7 inhibitors to suppress Treg functions and improve the efficacy of currently approved cancer immunotherapy agents.

#1692

KRAS- **mediated therapeutic resistance abrogates immunogenic cell death in colorectal cancer cells.**

Yi-Jun Wang,1 Dongshi Chen,1 Jingshan Tong,1 Jian Yu,1 Alberto Bardelli,2 Lin Zhang1. 1 _University of Pittsburgh Cancer Institute, Pittsburgh, PA;_ 2 _University of Torino, Torino, Italy_.

A hallmark of cancer is compromised immune surveillance, which is characterized by failure of the immune system to recognize tumor-associated antigens and specifically eliminate malignant cells. A number of studies showed the tumor microenvironment in colorectal cancer (CRC) patients is highly immunosuppressive. Conventional chemotherapy and recent targeted therapy can restore anti-tumor immune response through induction of immunogenic cell death (ICD) in tumor cells. However, the mechanism and functional role of ICD in CRC treatment remains vague. In this study, we found FOLFIRI (Leucovorin/5-Fluorouracil/Irinotecan) combined with the anti-EGFR antibody cetuximab, a commonly used regimen for CRC treatment, induces death of CRC cells with characteristics of necroptosis, including ATP depletion, release of HMGB1, and phosphorylation of RIP3 and MLKL. Cells subjected to the combination treatment exhibited features of ICD including plasma membrane exposure of calreticulin and phagocytosis by dendritic cells. We identified the BH3-only Bcl-2 family protein PUMA as a key mediator of necroptosis induced by the combination treatment in vitro and in vivo. Furthermore, we found that CRC cells with acquired cetuximab resistance due to KRAS mutation or amplification are deficient in ICD. Collectively, our results suggest that oncogenic mutations cause resistance of CRC cells to anticancer therapies in part by suppressing ICD.

#1693

T cell responses to peptide-epitopes can be boosted by immune complexes of circulating anti-tetanus antibodies.

Erika A. Fletcher,1 Wendy van Maren,2 Robert Cordfunke,2 Jasper Dinkelaar,3 Ricardo Castelli,3 Jeroen Codee,3 Gijs van de Marel,3 Cornelis J. Melief,2 Ferry Ossendorp,2 Jan Wouter Drijfhout,2 Sara Mangsbo1. 1 _Uppsala University, Uppsala, Sweden;_ 2 _Leiden University Medical Center, Netherlands;_ 3 _Leiden University, Netherlands_.

The ability of dendritic cells (DCs) to boost an antigen-specific immune response is utilized in several cancer immunotherapy strategies including therapeutic vaccination using long peptides. Naked peptides are rapidly degraded and oil-based delivery strategies trap immune cells to unwanted sites or have inappropriate adjuvant properties. To enhance the uptake of cancer or viral T cell epitopes and subsequent activation of DCs we make use of circulating antibodies to mount cellular responses against tumor antigens of interest by conjugating a B cell epitope to a T cell epitope. The conjugation of the two greatly improves antigen uptake and concomitant activation of the same DC in contrast to naked long peptides which are not conjugated. Our identified B cell epitope of choice is derived from tetanus toxin and can be targeted by tetanus-specific antibodies boosted by a standard tetanus toxoid vaccine. We have applied a modified chandler loop model preserving intact cascade systems to characterize how the tetanus-peptide conjugated vaccine is targeted to human immune cells. The B cell-T cell conjugate is taken up by human monocytes and blood DCs in an antibody-dependent manner. Rather than FcγRs, the internalization of the antigen appears to be partly mediated through the classical pathway of the complement system. Tetanus-CMV conjugates, containing a T cell epitope from the pp65 protein of cytomegalovirus (CMV), strongly reactivates memory T cells when analyzed in blood from donors with CMV-specific T cells. The CMV-specific T cells rapidly produce IFNγ and TNF in response to the conjugate illustrating that the uptake of the conjugate leads to activation of antigen-specific T cells. Uptake as well as T cell activation occurs at low concentrations of the SLP conjugate, superior to a conjugate lacking the tetanus-sequence as well as to SLPs with or without additional adjuvant (LPS). Of importance, when the B and T cell epitopes are separate entities but mixed, CMV-specific T cells are not activated, illustrating the requirement of conjugating the two. Our data show that we have a unique delivery system for peptide based vaccines that can aid induction of human T cell responses, and may potentiate immune responses in cancer patients.We are now actively working on a prostate cancer vaccine candidate using this novel loading/adjuvant technology.

#1694

Systemic granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment increases T cell receptor diversity in localized and metastatic prostate cancer patients.

David Y. Oh,1 Li Zhang,1 Jason Cham,1 Alan Paciorek,1 Mark Klinger,2 Malek Faham,2 Susan F. Slovin,3 Lawrence Fong1. 1 _University of Califonia, San Francisco, San Francisco, CA;_ 2 _Adaptive Biotechnologies, South San Francisco, CA;_ 3 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is frequently utilized as an adjuvant in cancer immunotherapies, and has known effects as a growth factor. However the extent to which GM-CSF modulates the adaptive immune response, including possible effects on the antigenic repertoire, remains unclear. We used next-generation sequencing of T cell receptor (TCR) beta chain sequences amplified from total RNA from peripheral blood mononuclear cells using consensus primers to assess changes in the circulating antigenic repertoire of prostate cancer patients treated with GM-CSF in multiple clinical trials. Administration of systemic GM-CSF monotherapy to patients with localized prostate cancer prior to planned radical prostatectomy (NCT00305669) results in a significant decline in clonality from the pre-treatment timepoint to the 2-week timepoint on treatment, indicative of increased early repertoire diversity (p=0.039 by Wilcoxon signed rank test). In a separate clinical trial (NCT00064129), the combination of systemic GM-CSF (250 μg/m2/day on days 1-14 of each cycle) with ipilimumab in metastatic castrate-resistant prostate cancer (mCRPC) patients also resulted in a significant decline in clonality from pre-treatment samples to the 2-week timepoint on treatment (p=0.002). In contrast, mCRPC patients who received ipilimumab alone in a separate study (NCT00323882) did not experience a similar decline in clonality after 3 weeks on treatment (p=0.625). In addition, comparison of the dynamics of specific clonotypes between the paired timepoints in the two mCRPC studies demonstrates that while there is no significant difference in the ratios of post-treatment to pre-treatment clonality between studies, patients treated with the combination of iplilimumab and GM-CSF show more repertoire change, with lower Morisita's distance for all clones found at either timepoint (p=0.023), smaller intraclass correlation coefficient (p=0.028), and a smaller proportion of clonotypes that remain unchanged (defined by +/- 2-fold change for clones found at both timepoints) (p=0.002). These results indicate increased repertoire turnover when GM-CSF is combined with checkpoint inhibition. Hence data from both localized and metastatic prostate cancer, and from monotherapy and combination therapy regimens, supports a role for GM-CSF in inducing early diversification of the TCR repertoire.

#1695

Exploiting the pancreatic cancer mutome for immune interception.

Heather Kinkead,1 Eric Lutz,2 Thomas W. Dubensky,3 Elizabeth Jaffee1. 1 _Johns Hopkins School of Medicine, Baltimore, MD;_ 2 _WindMIL Therapeutics, Baltimore, MD;_ 3 _Aduro Biotech, Berkeley, CA_.

This study aims to translate tumor-specific mutations identified by next-generation sequencing techniques into an effective anti-tumor vaccine for a classically non-immunogenic tumor, pancreatic ductal adenocarcinoma (PDA). Pancreatic ductal adenocarcinomas develop an average of 63 mutations during tumorigenesis, collectively referred to as the "mutome." Of these, approximately 45 are amino acid point mutations. These mutations are not present in normal cells and provide a source of tumor-specific neoepitopes for targeted immunotherapy. Next-generation sequencing technologies allow for rapid identification of mutations present in patient tumors; however, methods for rapid identification of immunogenic neoepitopes which can be effectively targeted for tumor clearance, and optimized vaccination strategies for targeting them, still need to be developed.

In this study, we are using a transplantable murine PDA model, Panc02, to develop vaccination strategies for inducing neoepitope-specific T cell antitumor responses, and to define parameters for selecting appropriate targets. Exome sequencing identified 878 nonsynonymous mutations in the Panc02 cell line. Three immunological epitope prediction servers identified 878 potentially immunogenic peptides in the Panc02 model, 29 of which were strong candidates (scores <50) and another 240 candidate epitopes within the limits of prediction (scores <1000). Panc02 neoepitope peptide vaccinations in mice confirmed 14% of the predicted epitopes to be immunogenic by IFNγ ELISPOT, which may include both CD8+ and CD4+ T cell responses. Tumor transplant experiments demonstrate that therapeutic vaccination against neoepitopes in combination with a human STING-activating cyclic dinucleotide adjuvant is capable of inducing a temporary regression of implanted tumors. After tumor escape and regrowth, flow cytometry reveals a large population of infiltrating lymphocytes, the majority of which are co-expressing the checkpoint molecules PD-1, Tim3, and Lag3, indicating that treatment of vaccinated animals with checkpoint blockade therapy may potentiate infiltrating lymphocytes and ultimately result in permanent tumor regression. Thus, a broad and unbiased approach for screening vaccine targets will allow for the evaluation of predictive algorithms and ultimately the development of more effective vaccination approaches for targeting the tumor mutome.

#1696

High-dose versus low-dose fractionated ionizing radiation to enhance antigen-specific antitumor immunity.

Megan V. Morisada, Ellen C. Moore, Jay A. Friedman, James W. Hodge, James B. Mitchell, Clint T. Allen. _National Institutes of Health, Bethesda, MD_.

Ionizing radiation (IR) has variable effects on the solid tumor immune microenvironment. While many report additive or synergistic effects between IR and immune activating therapies, most studies utilize single fractions or hypofractionated high-dose radiation of 8 Gy or greater. For head and neck squamous cell carcinoma (HNSCC), 30 or more low-dose fractions of 1.8-2Gy are utilized as standard-of-care for the up-front treatment of early stage or advanced disease. Preliminary experiments from our laboratory have demonstrated that 10 fractions of 2Gy IR decreased the rate of rejection of T-cell inflamed syngeneic carcinomas in wild-type B6 mice when combined with cyclic dinucleotide (CDN) compared to CDN treatment alone (10% rejection rate with low-dose fractionated IR plus CDN vs. 50% with CDN alone). Immune suppression following IR in mice is mediated by at least galectin-1. Further, HNSCC patients treated with standard fractionated low-dose IR develop durable neutropenia. Thus, we hypothesized that low-dose fractionated IR is immunosuppressive and does not result in additive or synergistic effects when combined with cyclic dinucleotide. To test this hypothesis, we performed a time-course analysis of tumor immune infiltration and tumor-draining lymph node antigen-specific T-lymphocyte activation in three syngeneic models of carcinoma with defined tumor rejection antigens (MOC1-p15E, B16-OVA and MC38-CEA) following exposure to two different IR regimens of 8Gy x 2 fractions or 2Gy x 10 fractions. Analysis of effector immune infiltration and antigen-specific activation pre-IR and at 5, 10, and 20 days post-IR revealed consistent trends of profound immunosuppression with the low-dose fractionated IR regimen (2Gy x 10) but preserved or enhanced immune activation with the high-dose hypofractionated regimen (8Gy x 2). Galectin-1 levels will be measured from whole tumor lysates. We plan to perform nano-string RNA array analysis on irradiated tissues to serve as correlative immune analysis and also to validate this technology against well-defined immune read-outs for potential use in the clinical trial setting. We also plan to combine both IR regimens with intra-lesional CDN (20 μg x 3 injections) to determine if either IR regimen results in additive or synergistic tumor control. This work has profound implications for future pre-clinical studies and clinical trials combining IR and immune activating therapies, such as CDN or checkpoint inhibitors.

#1697

Humanized immune-oncology mouse models.

Maria Stecklum, Annika Wulf-Goldenberg, Bernadette Brzezicha, Iduna Fichtner, Jens Hoffmann. _EPO GmbH, Berlin, Germany_.

The recent clinical success of immune checkpoint modulators has stimulated immune-oncology research leading to the identification of new tumor immunology targets. However both, target validation and drug development need better preclinical immune oncology models. Translational research further urgently needs such models for identification of clinically relevant biomarkers and defining rational combination strategies. Human patient-derived xenografts (PDX) from different tumor indications transplanted on immunodeficient mice have demonstrated strong predictive power for many drug development programs in cancer research. However, one caveat of PDX models is, that these mice lack a functional immune system, which allows tumor engraftment in the xenogenic host. To overcome these constraints our aim is the development of PDX models on mice with a functional human immune system. This strategy should allow implementing the highly predictable PDX in a functional human immune environment for better drug efficacy and safety studies. We reconstituted a human immune system in mice by engrafting human hematopoietic stem cells in immunodeficient mice. We demonstrate the engraftment of a full set of human immune cells, including T cells, B cells, NK cells, monocytes and dendritic cells in these mice. Tumor expression of PD-L1 is currently evaluated as first biomarker for immunotherapy with the PD1 checkpoint inhibitors. We screened our tumor cell lines and PDX models for PD-L1 expression to identify such a correlation. PD-L1 positive and negative models were transplanted on humanized mice. Most of them showed no difference in tumor growth compared to non-humanized mice (= fully immune resistant). However other PDX showed a delayed growth on the humanized mice (= partly immune resistant), whereas only one model did not grow at all (= immune sensitive), demonstrating a high sensitivity to the innate immune response of this PDX. To evaluate the function of the human immune cells and checkpoints, mice were treated with checkpoint inhibitors ipilimumab and nivolumab. Checkpoint inhibitors alone or in combination led to a minor tumor growth delay and an increased number of activated T-cells in the blood and in the tumor. Our results from experiments with n=19 models revealed a first correlation: stronger tumor growth inhibition on PD-L1 positive PDX and increased sensitivity of "partly immune resistant" PDX. Furthermore we were able to demonstrate, that treatment effects of checkpoint inhibitors could be increased by combination with radiation. Summary: Our humanized PDX models enable appropriate preclinical studies on tumor immune biology, evaluation of new immune therapies and combinations, as well as the identification and validation of biomarkers for tumor immune therapy.

#1698

Immunoprevention of KRAS-driven lung adenocarcinoma by a multi-peptide KRAS vaccine.

Jing Pan,1 Qi Zhang,1 Shizuko Sei,2 Robert H. Shoemaker,2 Ronald A. Lubet,2 Yian Wang,1 Ming You1. 1 _Medical College of Wisconsin, Milwaukee, WI;_ 2 _National Cancer Institute, Bethesda, MD_.

Lung cancer remains the leading cause of cancer death worldwide. Mutations in KRAS are detected in up to 30% of lung cancer cases, about 80% of which occur in codon 12, yet no effective therapies specifically targeting mutant KRAS have been developed. Boosting the host immune response to cancer cells by vaccinating against defined tumor-associated antigens (TAAs) as a means of treating established tumors or preventing the development of tumors in high risk individuals such as current or former smokers is an area of intense research. The present study evaluated both immunogenicity and anti-tumor efficacy of a newly formulated multipeptide (peptides 15-17 amino acids long) vaccine targeting multiple epitopes of the KRAS molecule in a mouse model of KRAS-driven lung tumor. Vaccination was performed in the prevention setting in a transgenic mouse model, where mutated mouse Kras (G12D) is conditionally over-expressed in the lungs of mice using a CCSP promoter. A multi-score prediction algorithm was used to identify likely immunogenic epitopes in the KRAS protein sequence. The identified peptides were synthesized and screened in naïve mice for immunogenicity. The formulated vaccine contained the top four peptides, which elicited the strongest immunologic response and showed 100% sequence homology between human and mouse. The multipeptide KRAS vaccine was then tested in an inducible CCSP-TetO-KRASG12D mouse model, where the vaccines mixed with the Freund's adjuvant or adjuvant alone were administered prior to activating the mutant KRAS protein. Our results show the KRAS peptide vaccine exhibited striking efficacy, reducing tumor number and tumor burden by >80% when compared with adjuvant alone. Splenocytes collected from vaccinated animals at the end of the study showed a robust immunologic response to the immunizing peptides. Furthermore, in vitro stimulation of these splenocytes by the vaccine peptides resulted in the secretion of cytokines indicative of Th1 (interferon gamma) and Th17 (IL17) responses but with minimal secretion of Th2 related cytokines e.g. IL3 and IL4. In the draining lymph nodes from vaccinated mice rejecting tumors, there was a substantial increase in CD4 cells but a limited increase in CD8 cells. In summary, the multipeptide KRAS vaccine was immunogenic and efficacious in the primary prevention of KRAS-induced lung cancer in mice, indicating that the approach can potentially be used for the prevention of KRAS-driven cancers either alone or in combination with other modalities.

#1699

Impact of whole blood interferon gamma production for patient selection in vaccination with autologous formalin-fixed tumor vaccine.

Naoyuki Sakamoto,1 Takeshi Ishikawa,1 Tetsuya Okayama,1 Tomoyo Yasuda,1 Toshifumi Doi,1 Mari Tanigawa,2 Yuji Naito,1 Yoshito Itoh,1 Toshikazu Yoshikawa1. 1 _Kyoto Prefectural University of Medicine, KYOTO, Japan;_ 2 _Iseikai Hyakumanben Clinic, KYOTO, Japan_.

Introduction:

Immune checkpoint blockade by anti-CTLA-4 antibody and anti-PD-1 antibody led to clinical breakthrough for the treatment of patients with solid tumors. Moreover, recent reports have shown that neoantigens are important functional targets mediating response to immune checkpoint inhibitors. Therefore, sequential combination of personalized cancer vaccines and immune checkpoint blockade has emerged as a potential therapeutic strategy. However, existing drawbacks is that it is difficult to identify robust biomarkers for patient selection in vaccination with cancer vaccine. In this study, we examined whether the development of delayed-type hypersensitivity (DTH) response, measured after vaccination with autologous formalin-fixed tumor vaccine (AFTV), correlates to parameters of immune function such as whole blood cytokine levels and peripheral regulatory T cells (Treg) in patients with solid tumors.

Methods:

Subjects comprised 52 patients with solid malignancy. AFTV treatment consisted of 3 courses of vaccination performed at intervals of 2 weeks. DTH test was performed before and after vaccination. A positive DTH response was defined as a ≥ 10mm diameter erythema/induration. We evaluated whole blood cytokine production after phytohemagglutinin (PHA) stimulation using the bioplex array system. We also assessed the number of peripheral Tregs as well as routine hematological and biochemical parameters.

Results:

DTH positive patients and DTH negative patients did not differ with respect to the number of peripheral Tregs, leukocytes, lymphocytes or granulocyte/lymphocyte ratio. Whole blood IFN-γ production levels after phytohemagglutinin (PHA) stimulation at baseline in DTH positive patients were significantly higher than those in DTH negative patients (p=0.0397).

Conclusion:

These data indicate that whole blood IFN-γ production at baseline is likely to be useful to assess immunologic competence in vaccinated patients with AFTV. Consequently, clinical utility of whole blood IFN-γ production, as immunomonitoring method for patient selection in vaccination with cancer vaccine, is required to investigate in larger clinical studies in the future.

#1700

Radiation-induced vegf-targeted 4-1bb costimulation enhances immune control of tumor growth.

Brett Schrand,1 Bhavna Verma,1 Agata Levay,1 Shradha Patel,1 Iris Castro,1 Ana Paula Benaduce,1 Randall Brenneman,1 Oliver Umland,1 Hideo Yagita,2 Eli Gilboa,1 Adrian Ishkanian1. 1 _University of Miami Miller School of Medicine, Miami, FL;_ 2 _Juntendo University School of Medicine, Tokyo, Japan_.

Radiotherapy (RT)-potentiated immunomodulation is an emerging field in solid tumor malignancies. RT induces peripheral antitumor immunity that can lead to the reduction of distant, non-irradiated tumor lesions (abscopal response). This has provided rationale for current early phase trials combining existing checkpoint blockade and co-stimulatory mAbs with stereotactic RT. Objective responses remain suboptimal with checkpoint blockade monotherapy, and dose limiting toxicity is widely observed with these modalities. We hypothesize that targeting immunotherapy to the tumor using oligonucleotide aptamers that bind to RT induced tumor stress products will significantly enhance the therapeutic index.

We used the aptamer platform to generate a bi-specific construct containing a costimulatory aptamer ligand specific to murine 4-1BB which was conjugated to an aptamer specific to products secreted into the tumor stroma (VEGF or osteopontin). Tumor targeting, local control, abscopal responses and toxicity were compared to unconjugated 4-1BB-VEGF constructs, or mAB based therapies, when used in combination with RT in murine subcutaneous and autochthonous MCA tumor models.

RT induced VEGF upregulation was demonstrated in null/low VEGF secreting tumors that led to preferential accumulation of VEGF-41BB aptamers in the irradiated tumor.12Gy x1 elicited optimal tumor targeting of bi-specific VEGF-4-1BB conjugate. Significant anti-tumor immune responses were observed in s.c. murine tumor models. This conjugate showed dramatic reduction in systemic toxicity compared to the equivalent gold standard 4-1BB mAb. RT-VEGF-4-1BB treatment significantly improved local control, overall survival and induced potent abscopal responses compared to unconjugated and non-irradiated controls in the Balb/c 4T1 and MCA murine tumor models. Furthermore, RT-VEGF-4-1BB treatment showed similar anti-tumor effects as CTLA-4 mAB with significantly less systemic toxicity. This approach increased intra-tumoral CD4+ and CD8+ T cells, the CD8+ to Treg ratio, and induced significantly more tumor cell death.

In conclusion, this strategy has been shown to induce potent anti-tumor immune responses in relevant murine tumor models and significantly improves the therapeutic index over non-targeted mABs. Furthermore, radiotherapy can expand the scope of tumor targeted immune modulation to virtually all solid malignancies and induce potent abscopal responses. These results provide the rationale for developing clinical grade bispecific aptamers for future testing.

#1701

The novel anti-CD205 antibody drug conjugate (ADC) MEN1309 shows strong antitumoral activity in diffuse large B cell lymphoma (DLBCL).

Eugenio Gaudio,1 Chiara Tarantelli,1 Francesca Guidetti,1 Maurilio Ponzoni,1 Roberta Pittau Bordone,1 Alessio Fiascarelli,2 Andrea Rinaldi,1 Ivo Kwee,1 Afua Adjeiwaa Mensah,1 Anastasios Stathis,3 Davide Rossi,3 Georg Stussi,3 Emanuele Zucca,3 Giuseppe Merlino,2 Mario Bigioni,2 Monica Binaschi,2 Francesco Bertoni1. 1 _Lymphoma and Genomics Research Program, IOR Institute of Oncology Research, Bellinzona, Switzerland, Bellinzona, Switzerland;_ 2 _Menarini Ricerche S.p.A., Pomezia, Italy, Pomezia, RM, Italy;_ 3 _Lymphoma and New Drugs Units, IOSI Oncology Institute of Southern Switzerland, Bellinzona, Switzerland, Bellinzona, Switzerland_.

Background. Up to one third of DLBCL patients still succumb to their disease: novel therapeutic approaches are needed. MEN1309 is a novel ADC consisting of an anti-CD205 monoclonal antibody conjugated to the DM4 maytansine derivative through a cleavable linker. Here, we assessed its anti-DLBCL antitumoral activity.

Methods 24 DLBCL cell lines were exposed to MEN1309 and, as control, to IgG-conjugated DM4 for 72h. Cell proliferation was measured with MTT. Apoptosis activation, defined by at least 1.5 fold increase in the caspase 3/7 signal respect to controls, was measured with the Promega ApoTox-Glo Triplex Assay. Xenografts (15 x 106 cells/mouse, 200 μL of PBS) were established s.c. into the left flanks of female NOD-SCID mice; i.v. treatments started with tumors of 250-350 mm3 volume.

Results. MEN1309 showed strong cytotoxic activity in DLBCL (median IC50 = 300 pM; 95%CI, 200-771), while the IgG-conjugated DM4 toxin was 100x less active (30 nM; 95%CI, 20-33). MEN1309 induced apoptosis in 17/24 (71%) DLBCL. No difference was seen based upon DLBCL cell of origin, MYC or BCL2 translocations or TP53 status. MEN1309 activity was highly correlated with its target expression with an inverse correlation between IC50 values and CD205 expression at flow cytometry or RT-PCR (R = - 0.79, P < 0.0001). No correlation was seen between CD205 expression and IgG-conjugated DM4 activity. MEN1309 anti-tumor activity was in vivo confirmed in a DLBCL model (OCI-Ly10) characterized by high CD205 expression. MEN1309 was administered at 3 different doses: 1.25, 2.5 and 5 mg/Kg once / 3 weeks. Groups of control treated with IgG-DM4 (5 mg/Kg, every 3 weeks) were also used. MEN1309 5mg/Kg eradicated tumors in all mice with a single dose, as also shown by highly significant differences versus control mice (D7, D21, D28; P < 0.01). While all other groups had to be stopped by D35, the MEN1309 5mg/Kg group skipped the second treatment at D21 because there was no palpable tumor, and mice resulted cured even 2 months later (Kaplan-Meier, survival analysis, P < 0.0001). MEN1309 2.5 mg/Kg delayed tumor growth versus control (D21, P 0.039). Much lower activity was observed with MEN1309 1.25 mg/Kg (D7, P 0.012) and with IgG-DM4 (D21, P 0.049; D28, P 0.046).

Conclusions. In DLBCL models, the novel ADC MEN1309 had strong in vitro and in vivo anti-tumor activity, which is highly correlated with the expression of its target.

#1702

Multimeric anti-DR5 IgM antibody displays potent cytotoxicity in vitro and promotes tumor regression in vivo.

Beatrice Wang, Tasnim Kothambalwala, Paul Hinton, Dean Ng, Avneesh Saini, Ramesh Baliga, Bruce Keyt. _IGM Biosciences, Inc., Mountain View, CA_.

Death receptor 5 (DR5) is a member of the TNF receptor superfamily that induces apoptosis upon receptor trimerization. It is expressed on many tumor types and has therefore been an important target for developing antibody based treatments of epithelial, solid tumors. However several agonistic Anti-DR5 IgG antibodies that have demonstrated efficacy in preclinical models have been unsuccessful in clinical trials, likely due to insufficient receptor crosslinking by bivalent IgGs. We have developed a multimeric anti-DR5 IgM antibody which has strong avidity for the receptor. The IgM binds Colo205 cells and triggers apoptosis, and is 1000-fold more potent than the respective IgG and 100-fold more potent than crosslinked IgG in vitro. Anti-DR5 IgM displays strong in vitro potency across a panel of tumor cell lines, including ones that are IgG-resistant. Anti-DR5 IgM causes tumor regression and delays tumor growth in various in vivo xenograft models including Colo205, HCT15 and MDA-MB231. These results support the development of an human anti-DR5 IgM therapeutic with the potential to treat solid tumors.

#1703

A novel tumor vaccine platform: direct conjugation of antigens to the β glucan PAMP Imprime PGG enhances antigen presentation and T cell priming.

Kyle S. Michel, Ross B. Fulton, Steven M. Leonardo, Keith B. Gorden, Jeremy R. Graff, Michael E. Danielson. _Biothera Pharmaceuticals, Inc., Eagan, MN_.

Significant clinical and pre-clinical research has shown that PAMPs- Pathogen Associated Molecular Patterns- can trigger an integrated anti-cancer immune response involving both innate and adaptive immunity. Imprime PGG is a soluble yeast β-1,3/1,6 glucan currently in multiple phase 2 clinical studies in combination with the immune checkpoint inhibitor (CPI), pembrolizumab. Preclinical mechanistic research has shown that Imprime PGG can re-polarize immunosuppressive myeloid cells in the tumor microenvironment and activate antigen presenting cells to prime antigen-specific CD8 T cells thereby boosting effector T cell function and expansion. Based on this ability to activate dendritic cells and induce type I interferon, we sought to explore the use of Imprime as an immune activating/antigen-directing scaffold onto which we could attach tumor antigen to drive an antigen-specific T cell based immune response. Imprime PGG has been safely administered intravenously to more than 400 human subjects and thus represents a safe starting point for the development of a cancer vaccine. Imprime's 1,3/1,6 β-glucan polymeric structure allows for the straightforward attachment of peptides and proteins via three different conjugation routes. To explore this function we covalently linked the chicken ovalbumin (OVA) protein to Imprime to generate a β-glucan-protein conjugate (Imprime-OVA). Using T cell receptor transgenic OT-I CD8 and OT-II CD4 T cells to track responses to OVA, we treated mice with Imprime-OVA intravenously and examined the expansion and functional quality of the T cell response 7 days later at the peak of expansion. Following Imprime-OVA treatment, both OVA-specific CD8 and CD4 T cells underwent vigorous expansion. OT-I CD8 T cells upregulated the transcription factor Tbet, which is central to developing effector functions, and were highly multifunctional in their ability to produce IFN-γ, TNF-α and IL-2. OT-I T cells responded similarly to vaccination with the minimal H-2Kb/OVA257-264 peptide covalently attached to Imprime. By comparison, vaccination with unconjugated OVA (protein or peptide) and Imprime was much less effective in driving T cell expansion and differentiation. The CD8 T cell response required Batf3-dependent cross-presenting DCs whereas the CD4 T cell response did not, and both CD4 and CD8 responses required the C-type lectin receptor Dectin-1. These data show that an Imprime PGG-protein conjugate can effectively elicit the expansion and functional activation of cytotoxic T cells and may have utility as a potential cancer vaccine platform. These data provide evidence that Imprime PGG not only serves as a combination therapy for CPIs, anti-angiogenics, and tumor targeting antibodies but may also be a robust platform for multiple tumor vaccine strategies.

#1704

UPR signaling promotes T-cell dysfunction to prevent immune-mediated cancer cell killing and immune checkpoint therapy resistance.

Yismeilin R. Feliz Mosquea, David R. Soto Pantoja, Adam Wilson, Pierre L. Triozzi, Katherine L. Cook. _Wake Forest University, Winston Salem, NC_.

A critical point in cancer progression is evading recognition by the immune system. Cancer cells accomplish this by stimulating immune checkpoint signals on effector T-cells. In patients with advanced melanoma treated with immune checkpoint inhibitors, 3-year survival increased by 20%. While immune checkpoint therapies are the new first treatment option for advanced melanoma in over a decade, their efficacy is limited because resistance often develops. Understanding the molecular mechanisms of immune checkpoint inhibitor resistance is critical to develop combinatorial drug therapy to potentiate therapeutic responsiveness. The unfolded protein response (UPR) is an endoplasmic reticulum (ER) stress pathway activated when unfolded/misfolded proteins accumulate within the ER. Highly secretory cell types, such as T-cells, have larger ER cell compartments and elevated UPR components to deal with the increased protein synthesis/folding required by these cell types. Therefore, these cell types may be highly sensitive to ER stress. Our data demonstrates elevated UPR signaling components as a driver of T-cell exhaustion/dysfunction. Using a previously established T-cell exhaustion protocol, we stimulated naïve T-cells with antibodies to CD3/CD28 for 5 days and co-cultured them with MDA-MB-231 breast cancer cells, Mun2b melanoma cell line, or CMI patient-derived melanoma cell line. Exhausted T-cells displayed an increased PD-1 and PERK expression, suggesting that UPR signaling is activated during T-cell exhaustion. Treatment of naïve T-cells with DTT, a chemical agent that stimulates ER stress, also induced PD-1 and PERK compared with vehicle-treated T-cells. Gene expression analysis of T-cells indicate that co-culture with cancer cells, not CD3/CD28 activation, elevates T-cell UPR gene expression. Furthermore, induction of ER stress through low-dose DTT treatment decreased cytotoxic T-cell mediated cancer cell death, further supporting our hypothesis of ER stress inducing T-cell exhaustion. Inhibition of PERK by RNAi in TALL-104, a human cytotoxic T-cell line, enhanced T-cell mediated cancer cell clearance when exposed to ER stress-inducing agents, suggesting that PERK may represent a novel target to prevent T-cell exhaustion or restore T-cell effector capabilities. PERK inhibition in the patient-derived melanoma cells did not negatively affect T-cell-mediated killing, suggesting that systemic PERK inhibition may be an effective therapeutic strategy to enhance anti-tumor immune responses. Matched PBMC from melanoma patients before treatment or after ipilimumab therapy resistance indicated increased UPR signaling components in PBMC samples from patients after ipilimumab resistance when compared with PBMC samples before therapy, supporting a novel role UPR signaling in anti-CTLA4 therapy resistance.

#1705

Slide-based multi-parametric immunophenotyping of human blood samples by cyclic immunofluorescence using the Accucyte-CyteFinder system.

Jia-Ren Lin,1 Lance U'Ren,2 Gregory J. Baker,1 Joshua J. Nordberg,2 Zoltan Maliga,1 Jackie L. Stilwell,2 Eric P. Kaldjian,2 Peter K. Sorger1. 1 _Harvard Medical School, Boston, MA;_ 2 _Rarecyte, Inc, Seattle, WA_.

Immunophenotyping is an approach to measuring the abundance and functional state of immune cells as a means to understand mechanisms of homeostasis, identify biomarkers of disease, and measure therapeutic and adverse responses to drugs such as immune checkpoint inhibitors. Such studies are conventionally performed using flow cytometry together with combinations of immunomarkers that allow for identification of cell type, maturation state, and activation status. Here we describe a method for profiling circulating human leukocytes with highly-multiplexed immunofluorescence microscopy. The method has distinct advantages over flow cytometry in that it has greater sensitivity for detecting rare cell populations, allows for repeat analysis and long-term storage of precious biological samples, and obviates the requirement for spectral deconvolution. Using an open-source multiplexed immunofluorescence protocol referred to as cyclic immunofluorescence (CycIF), and commercially available reagents and instruments (AccuCyte and CyteFinder; RareCyte Inc.), we show that imaging can provide 16-plex, single-cell intensity data in addition to information on cellular morphology. Analytic technologies borrowed from the field of mass cytometry, such as ViSNE and Phenograph, allow for downstream phenotypic analysis and high-dimensional biomarker discovery. Current efforts now aim to expand the number of validated immune targets and combine imaging with single-cell picking and sequencing technologies.

#1706

Neoantigen frequency as an independent prognostic factor in patients with clear cell ovarian carcinoma (CCOC).

Hirokazu Matsushita,1 Kosei Hasegawa,2 Katsutoshi Oda,1 Shogo Yamamoto,3 Akira Nishijima,1 Yuichi Imai,2 Kayo Asada,1 Yuji Ikeda,2 Takahiro Karasaki,1 Keiichi Fujiwara,2 Hiroyuki Aburatani,3 Kazuhiro Kakimi1. 1 _The University of Tokyo Hospital, Tokyo, Japan;_ 2 _Saitama Medical University International Medical Center, Saitama, Japan;_ 3 _The University of Tokyo, Tokyo, Japan_.

Aim: Neoantigens derived from tumor-specific somatic mutations are thought to be targets for antitumor immune responses. It has been reported that mutation/neoantigen burdens correlate with prognosis of certain cancers under checkpoint blockade immunotherapy. In clear cell ovarian carcinoma (CCOC), checkpoint inhibitors provided durable responses in a subset of patients. To test whether the same may apply in CCOC patients, who are not on immunomodulatory therapy, we investigated the number of mutations or neoantigens, and immune signature of CCOC by integrated molecular analysis.

Methods: We performed exome sequencing and expression array for 74 CCOC patients. Candidate neoantigens derived from mutations were identified by MHC class I binding prediction algorism (NetMHCpan v 2.8). Immune signature was assessed by gene ontology and gene set enrichment analyses for immune cell lineage or molecule genes. We also analyzed their relationships with clinical outcomes.

Results: Neither number of missense mutations nor neoantigens correlated with clinical outcomes in CCOC. However, we found neoantigen frequency defined here as the number of neoantigens per missense mutation correlated with clinical outcomes. A cox multivariate regression analysis demonstrated that high neoantigen frequency correlated with decreased progression free survival (PFS) and was an independent prognostic factor (p=0.032). Immune-related genes including those related to effector memory CD8 T cell were dominantly expressed in tumors with low neoantigen frequency. This may suggest that CD8 T cell-mediated immunity is constantly eliminating immunogenic subclones expressing neoantigens (immunoediting). In contrast, we observed increased ratio of CTLA-4 and PD-1 to CD8A expression in tumors with high neoantigen frequency. This may imply that restricted antitumor immunity by immunoinhibitory mechanism result in the limited immunoediting and poor prognosis

Conclusions: Neoantigen frequency underlying immunoediting in CCOC is an independent prognostic factor for clinical outcome and may become a potential candidate biomarker for an immunomodulatory-based treatment.

#1707

Longitudinal and quantitative imaging of the localization, expansion, and contraction of tumor targeted adoptively transferred T cells.

Yogindra Vedvyas, Enda Shevlin, Marjan Zaman, Irene M. Min, Moonsoo M. Jin. _Weill Cornell Medicine, New York, NY_.

Introduction:

Current methodologies for monitoring adoptive cell transfer (ACT) rely on soluble markers and T cell quantity in serum reflecting mixed responses of T cell efficacy or toxicity. Quantitative, longitudinal T cell visualization can directly probe T cell distribution, expansion, and off-tumor localization, and provide rational to predict therapy successes or failures. To enable real-time PET imaging of adoptively transferred T cells in a manner directly translatable to clinics, we utilized a genetic reporter, somatostatin receptor 2 (SSTR2) and a clinically approved radiotracer to quantitatively and longitudinally visualize whole body T cell distribution and anti-tumor dynamics. SSTR2 imaging was also applied to chimeric antigen receptor (CAR) T cells in a setting of solid cancer.

Methods:

We developed subcutaneous tumors with a mixture of SSTR2 positive and negative Jurkat T cells, ranging from 0 to 100%. From this model and PET/CT imaging of SSTR2 radiotracer uptake, diagnostic performance of our imaging technique was rigorously defined. To apply SSTR2 imaging to CAR T cells, a new lentivirus vector was designed to allow dual expression of SSTR2 and CAR. Longitudinal PET/CT and luminescence imaging was performed to concurrently measure the rate of

tumor growth/killing and T cell expansion and contraction. Blood was also collected to correlate serum cytokines with T cell distribution at on- and off-tumor sites. Histology was performed to confirm the validity of SSTR2 imaging of CAR T cells and relating it to T cell activity.

Results:

Our T cell imaging technique was found to provide 1% limit of detection (one T cell in one hundred neighboring cells or ~4x106 cell/cm3 in solid tissues) with 95% specificity and 87% sensitivity. When applied to CAR T cells in solid cancer, a biphasic T cell expansion and contraction was observed in survivors where a temporal change of T cell density closely followed the change in tumor burden with some time delay. In contrast, nonsurvivors displayed unrelenting increases in tumor and T cell burden, indicating that tumor growth was outpacing T cell killing. Cytokine release syndrome indicated by weight loss and elevated proinflammatory cytokines was also apparent in a cohort of nonsurvivors.

Conclusion:

Our study is the first high quality and quantitative, longitudinal imaging of T cells, correlating T cell dynamism with therapy and toxicity responses in subjects. Correlative changes in cytokines and T cell density can be further developed for early prediction of the onset of cytokine release syndrome as well as for screening adjuvant therapies to augment CAR T cell efficacy against solid cancer. Owing to the use of a human gene and FDA-approved radiotracer, our imaging technique can be directly applicable to CAR T cells and other T cell based therapy. We are currently preparing for a phase I clinical study for real-time imaging CD19 CAR T cells in patients.

#1708

**Effective and reversible control of anti-tumor activity** in vivo **with a drug-regulated CAR T cell platform (DARIC).**

Wai-Hang Leung,1 Michael Certo,2 Holly Horton,2 Joel Gay,1 Tracy VandenBerg,2 Jordan Jarjour,1 Alexander Astrakhan1. 1 _bluebird bio, Seattle, WA;_ 2 _bluebird bio, Cambridge, MA_.

Redirecting T cells against tumors by introducing antigen-specific chimeric antigen receptors (CAR) has shown promising clinical results as a potential treatment strategy for certain cancers. However, traditional CARs are constitutively active, resulting in the persistent loss of all target cells (including off -tumor, on-target activity against normal tissues that express the target antigen) and enhanced potential of excessive T cell activation to drive cytokine release syndrome. While "off switches" based on suicide cassettes or other depleting cell approaches are in development, such systems by definition result in the elimination of the therapeutic cells. Here we have developed a novel drug-regulated CAR-based antigen targeting approach termed Dimerizing Agent Regulated Immune-receptor Complex (DARIC) that aims to: i) minimize the long-term toxicity of CAR T treatment; ii) allow the targeting of previously inaccessible antigens; and iii) be amenable to multiplex antigen targeting. The DARIC platform separates the antigen recognition and signaling functions of a CAR into two distinct polypeptides that are further engineered to contain the FKPB12 and FRB small-molecule regulated dimerization domains. In the absence of the dimerizing drug (e.g. rapamycin or the non-immunosuppressive rapalog AP21967) the DARIC system lacks signaling activity. However, the addition of dimerizing agent drives the interaction of the two DARIC subunits, fully restoring CAR function. Using CD19 as a model system, we show that treatment of CD19-DARIC+ T cells with rapamycin or AP21967 results in equivalent cytotoxicity, cytokine production and proliferation compared to a standard CD19-targeting CAR. Importantly, CD19-DARIC T cells were activated by picomolar levels of rapamycin and exhibited a higher antigen sensitivity than standard CD19-CAR T cells in vitro. In an aggressive CD19+Nalm-6 xenograft tumor mouse model, CD19-DARIC T cells did not exhibit anti-tumor activity in the absence of dimerizing agent. However, CD19-DARIC treated mice that received either low-dose rapamycin or AP21967 showed an equivalent level of tumor control compared to standard CD19-CAR treated animals. This activity was dependent on the presence of the dimerizing drug, as cessation of drug treatment resulted in the loss of CD19-DARIC T cell activity and the expansion of Nalm-6 tumors cells in the DARIC T cell treated mice, consistent with the ability to switch off CD19-DARIC T cells in vivo by withdrawing drug. Taken together, these results highlight the potential of the DARIC platform to facilitate the regulation of CAR T cell function both in vitro and in vivo.

#1709

Targeting immune checkpoint therapy to the lung tumor microenvironment.

Allison S. Cohen,1 Michael L. Doligalski,1 Hong Zheng,1 Narges K. Tafreshi,1 Veronica Estrella,1 Nella Delva,1 Jonathan Nguyen,1 Amer Beg,1 Mark L. McLaughlin,2 David L. Morse1. 1 _H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL;_ 2 _West Virginia University, Morgantown, WV_.

Lung cancer is the leading cause of cancer deaths worldwide. Many lung cancer patients are diagnosed with advanced disease. These patients have a low 5-year survival rate and limited treatment options. Novel therapies, which target biomarkers that are overexpressed in lung cancer but have basal expression in benign tissue, are desperately needed. The goal of this work was to develop a targeted agent for immune checkpoint therapy of lung cancer. Recently, immune checkpoint inhibitors have been approved for use in lung cancer and many more are being tested in clinical trials. The current immune checkpoint inhibitor agents are not tumor-targeted. Targeting the immune checkpoint inhibitor to tumor cell-surface markers should concentrate the conjugate in the tumor microenvironment and enhance the immune response in the tumor while reducing the systemic dosages needed, resulting in lower systemic toxicity.

The delta opioid receptor (DOR) is expressed in some lung cancers, but is not expressed or is expressed only at basal levels in normal tissues outside the brain. We have previously synthesized fluorescently-labeled DOR-targeted imaging agents based on a synthetic peptide antagonist (DORL). These targeted fluorescent agents have high affinity and selectivity for DOR, and exhibit good pharmacokinetic (PK) and biodistribution (BD) profiles, i.e. specific tumor uptake with rapid systemic clearance and no uptake in tissues of concern, e.g. brain. We are now developing lung cancer-specific immunotherapy agents that target the DOR by conjugating DORL to immunomodulatory molecules. In the current work, we synthesized a fluorescently-labeled DOR targeting ligand and conjugated it to an anti-PD1 antibody (DORL-PD1). We synthesized immunoconjugates with several targeting ligand-to-antibody ratios (TARs). We engineered murine lung cancer cells to constitutively express the DOR. By lanthanide time-resolved fluorescence (LTRF) competitive binding assays, we have shown that the agents have high avidity for the DOR in vitro with higher TARs resulting in higher binding avidity. We characterized the uptake of DORL-PD1 in vitro using live-cell fluorescence microscopy. Using syngeneic engraftment tumor models in immunocompetent mice, we performed longitudinal fluorescence imaging studies to determine the agent circulation time (PK), tumor selectivity and tissue distribution (BD). Immune checkpoint efficacy studies were performed using the DOR negative mouse tumor models.

In conclusion, we have synthesized fluorescent DOR-targeted immune checkpoint therapy agents, DORL-PD1; demonstrated avidity and selectivity for the DOR in vitro and in vivo; and immune checkpoint therapy efficacy in vivo. Future studies will evaluate the efficacy of DORL-PD1 in immune competent mice bearing DOR positive tumors. These agents could be useful for increasing the efficacy and reducing systemic toxicity of immune-checkpoint therapy of lung cancer.

#1710

Providing confidence around computational tissue analysis using heterogeneity assessments.

Carsten Schnatwinkel,1 Daniel Rudmann,1 Famke Aeffner,1 Jasmeet Bajwa,1 Natalie Hutnick,2 Michael Sharp,2 Gerry Chu,2 JD Alvarez2. 1 _Flagship Biosciences Inc., Westminster, CO;_ 2 _Janssen R &D, Spring House, PA_.

Background: Though the techniques to interrogate the appearance of a biomarker in tissue sections have greatly advanced, there are limitations as to how representative an analysis of a tissue section is compared to the entire diseased tissue. Depending on the heterogeneous expression level of a biomarker, tissue sampling can result in different interpretations of the biomarker's appearance, and hence could potentially lead to a false therapeutic intervention.

Hypothesis: Digital image analysis has demonstrated tremendous value in quantifying many features related to biomarker distribution and expression in biological tissues. The information can be collected for various indications and biomarkers and a phenotypic signature can be established that describes a biomarker representation across indications. Moreover, the assessment of new samples can be compared to the established phenotypic signature and a confidence score applied in support to the determined endpoint.

Approach: For a proof of concept, 6 prostate cancer samples were processed and a single section was collected after every 100microns. A total of 7 sections per sample were stained for the lymphocyte marker CD3, and the number of positive target cells were determined in the tumor and tumor microenvironment using tissue Image Analysis (tIA™). To assess how indicative the evaluation of a single tissue section would be for the entire tumor, the heterogeneity level was determined on the section level as well as by random grid analysis on each individual section. Both criteria were utilized to define an indication and biomarker specific confidence interval and heterogeneity score.

Conclusion: The combination of IHC and tIA is a powerful tool to convert complex data into meaningful interpretations. tIA is also a capable tool to catalogue valuable information about the biomarker's expression pattern across different disease stages and hence could be used to evaluate how representative a single biomarker evaluation is in the grand scheme. Ultimately, we demonstrated a technique that can be applied to any biomarker and would assist in guiding therapeutic decisions.

### Liquid Biopsies 1: Circulating Tumor Cells

#1711

Detection of circulating tumor cells for use as prognostic, predictive and pharmacodynamic biomarkers in neuroblastoma.

Swathi Merugu, Deborah Tweddle. _Newcastle University, Newcastle, United Kingdom_.

Introduction: Neuroblastoma (NB) is the commonest extracranial childhood solid tumour. New treatments and a better understanding of drug resistance are needed to improve survival. Circulating tumour cells (CTCs) may provide a source of tumour cells for genetic biomarkers, give insights into tumour load and serve as pharmacodynamic (PD) biomarkers for new treatments. In clinical diagnostics, slide based FISH is used to detect MYCN amplification in neuroblastoma tumours. As it is not possible to conduct slide based FISH on an entire blood sample due to the infrequency of CTCs in peripheral blood (1 in 106 leucocytes), an alternative method FISH-IS (in suspension) was used in the present study to detect MYCN gene amplification using the Imagestream imaging flow cytometer (ISx).

Aims: 1) To detect CTCs from blood and disseminated tumour cells (DTC) from bone marrow (BM) from NB patients using the (ISx) 2) To use these cells for genetic and PD biomarker studies for novel targeted therapies.

Methods: A panel of NB cell lines (n=6) cloned into neuronal (N) and substrate-adherent (S) types were used to determine expression of NB cell surface markers (GD2 and NCAM) using the ISx. Western blotting and fluorescence activated cell sorting (FACS) were used to validate NCAM and GD2 expression respectively. 25 paired NB patient blood and BM samples were analysed for CTCs and DTCs, 4 unpaired blood samples were analysed for CTCs and one unpaired BM sample was for DTCs using the ISx to detect GD2 +ve and CD45-ve cells.

Results: Using the ISx 80-90% of N-type cells immunostained positive for GD2 compared to 0.8 -10% of S-type cells confirmed by FACS. CTCs were detected in 21/29 patient samples and DTCs in 19/26 cases. The numbers of CTCs ranged from 10-522/ml blood and DTCs from 57-35,515/ml of bone marrow. After validating FISH-IS protocol on ISx using NGP and SHSY5Y cell lines, 4 NB patient DTC samples were analysed for MYCN amplification. 2/4 cases showed MYCN gene amplification consistent with results obtained on the primary tumour.

Conclusion: This the first study to show that DTCs and CTCs are detectable in NB patient samples using the ISx. Future work will involve further genetic characterisation and development of PD biomarkers for Phase I clinical trials.

#1712

Detection of circulating tumor cells in stage IV non-small cell lung cancer.

Stephanie N. Shishido,1 Lyudmila Bazhenova,2 Anders Carlsson,1 James Hicks,1 Peter Kuhn1. 1 _University of Southern California, Los Angeles, CA;_ 2 _University of California, San Diego, La Jolla, CA_.

Non-Small Cell Lung Cancer (NSCLC) is the most common cause of cancer related deaths in both men and women. Some lung cancer patients have a unique challenge of a tissue access due to tumor location or baseline lung disorders such as emphysema and chronic obstructive pulmonary disease (COPD). Liquid biopsy technologies can overcome tissue assess difficulties. However, as of today there are no commercially approved assays for CTC enumeration in lung cancer. Multiple non-commercial assays exist and some show correlation of CTC enumeration and changes in CTC counts on therapy with prognosis and response to therapy. Outside of absolute number of CTC there is little published data regarding prognostic significance of CTC morphologic heterogeneity in late stage NSCLC and its ability to predict treatment outcomes. Here we investigate CTC counts in 81 patients with stage IV NSCLC using a fluid phase biopsy and high definition (HD) diagnostic pathology imaging of all nucleated cells. HD-CTCs were detected in 51 (63%) patients at initiation of therapy with a median of 2.20 (range 0-509.20) and a mean of 26.21 HD-CTCs/mL (±15.64). There was no correlation between the absolute number of HD-CTCs at the time of initiation of new therapy and patient outcomes. A subset of 25 patients was further analyzed to determine the significance of HD-CTC kinetics, which may follow three distinct patterns: an increase in HD-CTCs with therapy, unchanged HD-CTCs numbers (stable), and a decline in HD-CTCs numbers. Patients that experienced an increasing, stable, or decreasing HD-CTC profile had an overall change of 118.40, 0.54, and 81.40 HD-CTCs/mL respectively during the first 3 months. The overall survival (OS) for increasing profiles was 31.08 months (±13.08, median 32.46, range 15.11-43.50), for stable profiles OS was 8.82 months (±3.57, median 7.23, range 3.91-19.75), and for decreasing profiles it was 15.30 months (±6.39, median 14.06, range 3.81-28.06) months. CTCs are identifiable in patients with stage IV NSCLC, but correlation of absolute HD-CTC counts with disease progression was not statistically significant. However, change in CTC counts were predictive of OS in patients with metastatic lung cancer receiving chemotherapy.

#1713

Transferrin receptor 1 (TfR) as marker for circulating tumor cells (CTCs) identification in NSCLC.

Giuseppe Galletti,1 Galatea Kallergi,2 Ashish Saxena,1 Despoina Aggouraki,2 Christos Stournaras,2 Vassilis Georgoulias,2 Timothy E. McGraw,1 Nasser Altorki,1 Paraskevi Giannakakou1. 1 _Weill Cornell Medical College, New York, NY;_ 2 _University of Crete, Greece_.

CTCs are used as a surrogate source of tumor material in solid tumors. Clinical applications of CTCs as liquid biopsy comprise the monitoring of molecular alterations during tumor progression and dynamic evaluation of molecular markers of treatment response. The FDA-cleared method to isolate CTCs in cancer patients (Cell Search) is based on positive selection of EpCAM expressing cells. However, this approach performs poorly in non-small cell lung cancer (NSCLC) as it identifies CTCs in only 7% of the subjects, failing to show any prognostic relevance.

Down-regulation/loss of epithelial markers to isolate (EpCAM) and identify (cytokeratin, CK) CTCs could in part explain the low CTC yield obtained in NSCLC with approaches based on epithelial markers expression. To overcome this challenge we used size-based CTC enrichment (ISET filters) from NSCLC patients' peripheral blood. As a positive identifier of CTCs we used transferrin receptor 1 (TfR) which is a cell membrane-associated protein, that mediates intracellular iron uptake, and which is expressed at low levels in many normal tissues but over-expressed in cancer cells.

We first analyzed TfR protein expression by immunofluorescence in a panel of NSCLC cell lines and in healthy donor leukocytes. While all NSCLC cells lines analyzed were positive for TfR expression, none of the leukocyte expressed the receptor. Moreover, TfR expression was detected also in EpCAM negative NSCLC cell lines. To determine the clinical applicability of this novel CTC identifier, we determined TfR expression in CTCs isolated from peripheral blood of 35 metastatic NSCLC patients using the ISET filter technology. The isolated CTCs were stained for TfR, CK, CD45 and DAPI. For each patient, one additional ISET filter was stained with Giemsa for morphologic analysis by a pathologist. By using the classic panel of CTC identifiers markers (CK+/CD45-/DAPI+), CTCs were identified in 4/34 (11%) patients, while by using TfR as positive identifier (TfR+/CD45-/DAPI+) CTCs were identified in 31/35 (88%) subjects. The morphologic review of Giemsa stained filters confirmed the presence of tumor cells in 28/34 (82%) samples [0-217 CTCs/sample]. Interestingly, patients with > 6 TfR+ CTCs had a worse overall survival (OS) than patients with < 6 TfR+ CTCs [p=0.048 Log Rank (Mantel-Cox)]. OS did not significantly differ using the same cutoff with CTCs defined based on CK or Giemsa staining.

Overall, our data indicate that TfR is a promising biomarker for the detection of CTCs in NSCLC CTCs, superior to CK or EpCAM. Our data also suggest that TfR may potentially identify CTCs subpopulations with a significant prognostic role in NSCLC. We are currently isolating TfR+ CTCs from early stage and metastatic NSCLC patients for further molecular characterization and determination of clinical significance.

#1714

Translating CTCs for clinical use: melanoma patient-derived CTCs evolution in xenografts.

Monika Vishnoi,1 Haowen N. Liu,1 Debasish Boral,1 Wei Yin,1 Marc L. Sprouse,1 Jean P. Thiery,2 Isabella C. Glitza,3 Dario Marchetti1. 1 _Houston Methodist Research Institute, Houston, TX;_ 2 _Cancer Science Institute of Singapore, National University of Singapore, Singapore;_ 3 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Circulating tumor cells (CTCs) are known to disseminate from primary/metastatic tumors, survive in the circulation, and develop competency to colonize distant organs. Dissecting CTC heterogeneity is therefore crucial to provide improve prognostic and diagnostic tools (concept of "liquid biopsy"). However, the clinical significance of CTC heterogeneity is still unknown. We hypothesized that melanoma patient blood-derived, lineage-negative cells (CD45-/CD34-/CD73-/CD90-/CD105-) contain melanoma cell subpopulations (Melan A+, S100+, CD146+ and NG2+ CTCs) that evolve in vivo and achieve metastatic potency when reaching an organ-specific niche. We enriched Lin-negative cells from peripheral blood of melanoma patients clinically diagnosed with or without brain metastasis (MBM) by multi-parametric flow cytometry; and analyzed CTC heterogeneity in xenografts by longitudinal capturing and interrogation at the single-cell level (DEPArrayTM platform). First, we evaluated the presence of melanoma CTC subpopulations in Lin-negative cells and developed Lin-negative murine xenograft models. Human-cell derived melanoma metastasis in xenografts confirmed that CTCs require metastasis-potentiating factors present in the Lin-negative population. Second, we performed multi-parametric flow cytometry to isolate human-derived HLA+/Melan A+/- CTCs/DTCs population from murine blood and bone marrow at T0, Tmid and Tend phases of metastasis. We interrogated the differential levels of CTCs/DTCs in HLA+/Melan A+ cell population and established their neoplastic identity by genetic profiling and the expression of melanoma markers at single-cell level. Third, we dissected longitudinal transcriptional signatures of CTCs vs DTCs in association with MBM onset. We assessed the comprehensive EMT scores of CTCs and DTCs transcriptomics and identified the pathways associated with CTC/DTCs dynamic functional states depending upon melanoma progression. Collectively, this study provide important insights to identify novel EMT-related biomarkers of MBM progression. Further elucidation of these CTC biomarkers and properties may provide novel therapeutic strategies that can be clinically useful in melanoma patients which are yet to develop MBM.

#1715

EGFR mutational detection in ctDNA, Vortex-enriched CTCs and comparison to tumor tissue in non-small-cell-lung-cancer (NSCLC) patients.

Haiyan E. Liu,1 Meghah Vuppalapaty,1 Clementine A. Lemaire,1 Charles Wilkerson,1 Steve C. Crouse,1 Jonathan W. Goldman,2 Elodie Sollier-Christen1. 1 _Vortex Biosciences, Menlo Park, CA;_ 2 _David Geffen School of Medicine at UCLA, Los Angeles, CA_.

Background: Lung cancer is the leading cause of cancer-related mortality worldwide. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) therapies, based on the evaluation of EGFR mutation, have shown dramatic clinical benefits. EGFR mutation assays are mainly performed on tumor biopsies, which carry risks and expense and are not always successful. In order to identify the development of secondary EGFR mutations, which cause resistance to 1st and 2nd generation TKI's and an indication for therapy with a 3rd generation drug, effective and non-invasive monitoring is needed. Liquid biopsy biomarkers, such as circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), allow such monitoring over the course of the therapy. Interestingly, ctDNA or CTC analysis alone had less sensitivity vs. combining both, with a genotyping of 70% and 80% for CTCs and ctDNA respectively, but 100% when combined2. Vortex technology is a platform enabling label-free capture of CTCs from blood samples and genomic assays downstream3. The aim of this study is to demonstrate the sensitivity of a combined CTC and ctDNA assay through Vortex using blood samples spiked with molecularly-characterized lung cancer cell lines and then to apply this technique to matched blood and tumor samples from NSCLC patients.

Method: Lung cancer cell lines with different EGFR mutations (A549: wild type, H1975: L858R+ and T790M+, HCC827: 19del+) were used to validate our CTC workflow. Blood samples and matched tumor tissues were collected from NSCLC patients. Plasma was extracted first for ctDNA assay. CTCs were isolated from the plasma-depleted-blood using Vortex technology, immunostained (CK, Vimentin, CD45) and enumerated. DNA from CTCs, plasma and matched tumor tissue was analyzed for EGFR mutations 19del, L858R and T790M using the ctEGFR kit from EntroGen.

Results: Mutant DNA could be identified at a quantity as low as 0.5 ng (~83 cells), with a sensitivity ranging from 0.1% to 2% for a total DNA varying from 25ng (~4 CTCs among 4000 WBCs) to 1ng (~4 CTCs among 200 WBCs). We demonstrated the ability of Vortex technology to enrich CTCs from metastatic NSCLC patients. Processing of plasma-depleted-blood showed the same capture efficiency when compared to whole blood. This makes possible the detection of EGFR mutations on CTC samples collected by Vortex technology. > 10 NSCLC patients are being enrolled in this study and results will be presented at AACR.

Conclusion: The ctEGFR mutation assay performed well on both Vortex-enriched CTCs and ctDNA, enabling a low cost approach to analyze EGFR mutation from a single blood tube. This non-invasive EGFR mutation analysis will be potentially a useful tool for monitoring treatment and medication guidance of NSCLC patients. 1. Calabuig-Fariñas et al. Transl Lung Cancer Res. 2016. 2. Sundaresan TK et al. Clin Cancer Res. 2016. 3. Kidess-Sigal E et al. Oncotarget 2016.

#1716

**GSTP1 promoter methylation in in-** vivo **isolated CTCs from high-risk prostate cancer patients.**

Athina N. Markou,1 Panagiotis Paraskevopoulos,1 Marifili Lazaridou,1 Shukun Chen,2 Thomas Kroneis,2 Monika Świerczewska,3 Joanna Budna,3 Andra Kuske,4 Tobias M. Gorges,4 Maciej Zabel,5 Peter Sedlmayr,2 Catherine Alix-Panabieres,6 Klaus Pantel,4 Evi Lianidou1. 1 _Univ. of Athens, Athens, Greece;_ 2 _Institute for Cell Biology, Histology and Embryology, Graz, Austria;_ 3 _Poznan University of Medical Sciences, Poznań, Poland;_ 4 _University Medical Center Hamburg-Eppendorf, Hamburg, Germany;_ 5 _Poznan University of Medical Sciences, Poland;_ 6 _University Institute for Clinical Research (IURC), Laboratory of Rare Human Circulating Cells, University Medical Centre of Montpellier Saint-Eloi Hospital, EA2415, Montpellier, France_.

Introduction: Glutathione S-transferase 1 (GSTP1) has been reported to function as tumor suppressor gene in various types of human cancers. GSTP1 inactivation is associated with CpG island promoter hypermethylation in the majority of prostate cancers (PCs). The initiation of minimal residual disease (MRD) and especially the detection of circulating tumor cells (CTCs) in patients' peripheral blood represents a negative prognostic parameter for recurrence-free survival. The aim of the present study was to assess the methylation status of the GSTP1 gene in CTCs that were isolated, using the CellCollector® (GILUPI, GmbH), a novel clinical device designed for the in vivo isolation of EpCAM-positive CTCs. Patients and methods: In-vivo isolation of CTCs was performed by using CellCollector® from high-risk prostate cancer patients (n=97) and 20 healthy volunteers. For all these patients, the Ab coated region of the CellCollector® was washed in PBS , cut, and stored in Trizol reagent till analysis and DNA was further prior to the analysis isolated DNA was modified by sodium bisulfite (SB) and subjected to a real time MSP assay specific for GSTP1 methylation. In all cases, peripheral blood was also collected and used for CTC analysis by Immunostaining and the CellSearch® system. Results: All DNA samples were first checked for their quality. Based on the quality evaluation of all available DNA samples, only 63 DNAs were further qualified for analysis. GSTP1 promoter was found methylated in 12/63 (19%) the EpCAM positive fraction of in-vivo isolated CTCs. Moreover, in 5/12 (41.7%) patients for which GSTP1 promoter was found methylated, CTCs were also detected by the CellSearch® and 7 /12 (58.3%) for which GSTP1 promoter was also found positive, CTCs were also detected by the Immunostaining. Conclusion: GSTP1 promoter is methylated in in-vivo isolated CTCs from high-risk prostate cancer patients. GSTP1 promoter methylation in in-vivo isolated CTCs should be prospectively validated as a novel tumor biomarker for prostate cancer patients in a large cohort of patients. Acknowledgements: This research has been co-financed by the European Union (European Regional Development Fund - ERDF) and Greek national funds through the Operational Program ''Competitiveness and Entrepreneurship'' of the National Strategic Reference Framework (NSRF) - Research Funding Program: "ERA-NET on Translational Cancer Research (TRANSCAN) Joint Transnational Call for Proposals 2011 (JTC 2011) on: "Validation of biomarkers for personalised cancer medicine

#1717

Orthogonal identification of circulating tumor cells (CTCs) using single cell low pass whole-genome sequencing (WGS) and copy-number alteration (CNA) analysis.

Gareth Morrison,1 Valeria Sero,2 Yucheng Xu,1 Jacek Pinski,1 Sue Ingles,1 David Quinn,1 Claudio Forcato,3 Genny Buson,3 Chiu-Ho Webb,4 Kyle Horvath,4 Aditi Khurana,4 Gianni Medoro,3 Suman Verma,4 Matthew Moore,4 Philip Cotter,4 Nicolò Manaresi,3 Farideh Bischoff,2 Amir Goldkorn1. 1 _University of Southern California, Keck School of Medicine, Norris Comprehensive Cancer Center, Los Angeles, CA;_ 2 _Menarini Silicon Biosystems Inc, San Diego, CA;_ 3 _Menarini Silicon Biosystems Spa, Bologna, Italy;_ 4 _Research DX, Irvine, CA_.

Introduction: Presence of circulating tumor cells has prognostic value in multiple malignancies, and molecular analysis of CTCs is currently ongoing in numerous clinical trials. Most CTC enrichment methods rely on standard epithelial and leukocyte markers (CK+CD45-), so recovered cells are assumed to be of epithelial origin but never shown to be bona fide tumor cells. Conversely, atypical cells lacking the characteristic marker profile may not be analyzed, even though they may represent important tumor subpopulations. Here we evaluate a rapid, non-exhaustive, and cost-effective first-pass genomic analysis of individual candidate CTCs. This approach allows efficient upfront CNA-based confirmation that a given cell is of tumor origin, while leaving abundant DNA for deeper subsequent analysis in cells of interest.

Methods: Whole peripheral blood of metastatic prostate cancer patients was enriched for CTCs using the CellSearch® system (Janssen Diagnostics) under an IRB-approved protocol, and 5 samples with >5 CTCs were selected for further study. Next, the DEPArray™ v2 system (Menarini Silicon Biosystems) was used to identify and isolate single CTCs (CK+CD45-DAPI+) and paired white blood cells (WBCs; CK-CD45+DAPI+) from the enriched samples. In addition, cells negative for both cytokeratin and CD45 but with characteristic malignant morphology (large with high nuclear-cytoplasmic ratio) were isolated. Recovered single cells were whole-genome amplified with Ampli1™ WGA and quality controlled by Ampli1 QC. Ampli1 LowPass kit was then used to prepare NGS libraries for absolute CNA profiling by low-pass WGS.

Results: Thirty-three single CTCs (CK+CD45-DAPI+) and 30 WBCs (CK-CD45+DAPI+), as well as 47 putative CTCs with non-conventional phenotype (CK-CD45-DAPI+) were isolated. Single-cell WGA products with high Genome-Integrity Index (QC score ≥3) were prioritized for CNA analysis. Ampli1 LowPass data demonstrated copy number gains/losses confirming tumor origin of the CK+ cells, while WBCs showed a normal profile. In addition, a portion of the cells having non-conventional phenotype also demonstrated copy number alterations consistent with tumor origin.

Discussion: We demonstrate a WGA and low-pass WGS approach on single CTCs sorted from enriched peripheral blood, which offers a dual benefit: i) it allows rapid, non-exhaustive upfront identification of bona fide tumor cells for further study, and ii) it reveals genetic similarities and diversities (vis a vis copy number alteration) across CTCs of classical as well as non-conventional phenotypes, which may better represent clonal diversity. In a clinical setting, this molecular approach may be more effective for reliably identifying and characterizing heterogeneous CTCs, yielding profiles that more accurately reflect disease evolution and inform treatment strategies.

#1718

RT-qPCR gene expression analysis of CTCs isolated through an epitope-independent enrichment microfluidic device in patients with head and neck squamous cell carcinoma.

Martha Zavridou,1 Areti Strati,1 George Koutsodontis,2 Amanda Psyrri,2 Evi Lianidou1. 1 _Univ. of Athens, Athens, Greece;_ 2 _Attikon University Hospital, Athens, Greece_.

Background: Molecular characterization of circulating tumor cells (CTCs) is very challenging since these cells are rare, and the amount of available sample for their analysis is very limited. Moreover, CTC are highly heterogeneous and enrichment technologies based on EpCAM expression present the risk of missing EpCAM-negative CTCs. The Parsortix system (ANGLE plc, UK), is a novel microfluidic technology platform designed for marker-independent capture of CTCs. In this study we used for the first time the Parsortix system to isolate CTCs from patients with Head and Neck Squamous Cell carcinoma (HNSCC), and proceeded to downstream molecular characterization through RT-qPCR gene expression analysis.

Methods: Peripheral blood samples (10 mL) from head and neck squamous cell carcinoma (HNSCC) patients (n=19) and healthy donors used as a control group (n=10) were used for the isolation of CTCs using the Parsortix device. Enriched CTCs were harvested in Trizol reagent, followed by extraction of total RNA and cDNA synthesis. RT-qPCR was performed in the LightCycler (Roche) for the following gene targets: PD-L1, VIM, TWIST, EGFR, and B2M (used as a reference gene). The expression levels of PD-L1, VIM, TWIST and EGFR were normalized using the 2-ΔΔCt approach in respect to the expression of B2M.

Results: All samples analyzed were of excellent RNA quality as this was evaluated by B2M expression. According to our results, PD-L1 overexpression was detected in 5/19(26.3%) samples, VIM was overexpressed in 3/19 (15.7%) and TWIST-1 in 1/19 (5.3%) sample, while EGFR expression was not detected in any patient (0/19, 0%). These are preliminary results and these percentages may change, since the number of samples that we are analyzing is continuously increasing.

Conclusions: This preliminary study is showing for the first time that RT-qPCR can be successfully used for the molecular characterization of CTCs isolated by the label-free Parsortix microfluidic device in HNSCC. Overexpression of individualized immunotherapy important biomarkers such as PD-L1 in CTCs of HNSCC patients could be of significant clinical importance for the selection and follow up of these patients.

#1719

Dual-profiling of CTC and exosome from the cultured circulating tumor cells using stimuli-responsive degaradable hydrogels.

Yoon-Tae Kang,1 Young Jun Kim,1 Tae Hee Lee,2 Jae-Eul Shim,1 Young-Ho Cho1. 1 _KAIST, Daejeon, Republic of Korea;_ 2 _Cell Bench Research Center(SEMCELL), Daejeon, Republic of Korea_.

Introduction: Liquid biopsy based on sub-micron or nanosized particles in human body fluid have been received vast attention due to their non-invasive characteristics and enabling multiple check-up. Circulating tumor cells (CTCs), as well as exosome are the most promising markers in liquid biopsy, however, dual isolation and profiling have been hampered due to their size difference and limited quantity for analysis. We proposed the novel and simple methods for both isolation and study their similarity between them. Using the label-free CTC filtration device and anti-CD63 antibody-conjugated degradable hydrogel, the CTCs and the CTCs-derived exosome are specifically isolated, and each samples were followed by molecular study after recovery. This versatile platform facilitates the comprehensive study of two biomarkers with reflecting their inherent characteristics, thus paving the way for revealing their roles in cancer progression and metastasis.

Methods: In order to make stimuli-responsive degradable hydrogel, poly (vinyl alcohol) and alginate were mixed under constant stirring at 85 °C. The mixture was poured into the mold and dried for 24 hours. Then, the dried sheet was immersed into 100 mM calcium chloride solution to achieve gelation through ionic interaction. Subsequently, the anti-CD63 antibody was immobilized onto the prepared hydrogels via cross-linking. For the dual-profiling, the hydrogel and the filters containing the captured breast cancer cells by microfiltration were incubated with the exosome-depleted cell culture media for 6 hours. The captured cells were released from the device and the hydrogels were degraded by adding EDTA. The cell and exosome lysate were prepared using RIPA buffer at 4 °C. The supernatant was collected by centrifugation followed by western-blot assay. Four different markers, including exosome-specific marker (CD63), cancer-associated markers (EpCAM, vimentin), and a housekeeping marker (β-actin), were used.

Results: All exosome and cell samples highly expressed the housekeeping marker. Especially, two exosome samples dissociated from the hydrogel showed CD63 predominantly, which support the secretion of exosome from the cancer cells. The samples from the released cancer cells from the device did not express CD63 remarkably. To verify the phenotypical similarity between cell and exosome, expressions of the epithelial marker (EpCAM) and mesenchymal marker (vimentin) were examined. The exosome and cell from MCF-7, epithelial cancer cell, showed higher expression of EpCAM then vimentin. On the contrary to this, the samples from MDA-MB-231, mesenchymal cell, showed higher vimentin expression then EpCAM.

Discussion and conclusion: We showed that exosome follow the phenotypical characteristics of mother-cells. This dual profiling would be helpful for in-depth study of cancer with consideration of its heterogeneity and complexity.

#1720

Circulating tumor cell (CTC) enrichment and DNA mutation detection by sequencing.

Ruqin Kou,1 Eric Zhao,1 Conrad Leung,1 Priya Gogoi,2 Will Chow,2 Yixin Wang2. 1 _GENEWIZ, South Plainfield, NJ 08070, NJ;_ 2 _Celsee Diagnostics, Plymouth, MI_.

Molecular analysis of circulating tumor cells (CTCs) is hindered by low sensitivity and high level of background leukocytes of currently available CTC enrichment technologies. The Celsee PREP100 device captures CTCs with high sensitivity and allows the captured CTCs to be retrieved for molecular analysis. To evaluate the potential of utilizing PREP100 in CTC molecular analysis, we first prepared mock samples using cultured prostate cancer cells PC3 and LnCAP and analyzed captured cells by PCR amplicon sequencing. Using Celsee PREP100, we were able to recover the average of 79% (ranging from 40% to 100%) of 110 - 1,100 PC3 cells and 60 - 1,500 LnCAP cells and detect p.K139fs*3 deletion in PC3 cells and T877A mutation in LnCAP cells. In addition, we also spiked in the two cell lines into normal donor blood samples and analyzed the captured cells by PCR amplicon sequencing. Preliminary experiments showed that 40% of 250 spiked PC3 cells and 74% of 50 spiked LnCAP cells were captured. Further, we were able to detect p.K139fs*3 deletion in the retrieved 50 - 1,000 spiked PC3 cells. Our results illustrate that Celsee PREP100 system represents a promising technology for capturing and molecular characterization of CTCs.

#1721

Detection of CTCs in thoracic malignant tumors with "universal" CTC-chip.

Kazue Yoneda, Taiji Kuwata, Yasuhiro Chikaishi, Kenichi Kobayashi, Sakiko Yura, Hiroki Matsumiya, Masatoshi Kanayama, Akihiro Taira, Yusuke Nabe, Shinji Shinohara, Masaru Takenaka, Soichi Oka, Ayako Hirai, Yuko Tashima, Naoko Imanishi, Koji Kuroda, Fumihiro Tanaka. _University of Occupational and Environmental Health, Kitakyushu, Japan_.

Background: Circulating tumor cells (CTCs) are tumor cells shed from primary tumor and circulate in the peripheral blood. CTCs, as a surrogate of distant metastasis, can be potentially useful in diagnosis and monitoring therapeutic effects in malignant tumors. Among a variety of systems for detection of CTCs, the "Cellsearch" is the only approved system for clinical use. However, EpCAM-negative tumor cells, such as those originating from non-epithelial cells and those undergoing epithelial-mesenchymal transition (EMT) cannot be captured with the "CellSearch" that is an EpCAM-based isolation system. Therefore, we have developed a novel polymeric microfluidic device ("Universal" CTC-chip) that can capture CTCs with or without EpCAM expression (AACR 2015). In the present study, we examined CTCs-detection performance of the CTC-chip in patients with thoracic malignant tumors (lung cancer [LC] as an "EpCAM-positive" tumor and malignant pleural mesothelioma [MPM] as an "EpCAM-negative" tumor) in comparison with that of the CellSearch.

Methods: Peripheral blood sampled from each patient was divided and subjected to quantitative evaluation of CTCs with the CTC-chip as well as with the "CellSearch". The CTC-chip, coated with an anti-EpCAM antibody, was used to capture CTCs in the blood samples (n=19) from lung cancer patients. To capture CTCs in the samples (n=11) from MPM patients, the CTC-chip was coated with an antibody against podoplanin that is expressed on the mesothelioma. After immuno-staining for cytokeratin and CD45 on the chip, a captured cell containing Hoechst-positive nucleus and cytokeratin-positive/ CD45-negative cytoplasm was judged as a CTC. The CTC-count for each sample was represented as the number per 7.5mL of the blood.

Results: The median CTC-count detected with the CTC-chip in LC was 50 (range, 0-270), which was significantly higher than that (the median CTC-count, 0; range, 0-47) with the CellSearch (p<0.01). In the peripheral blood sampled from MPM patients, CTC was detected in only one patient using the CellSearch, but was detected in all 11 patients with the median CTC-count of 144 (range 0-470).

Conclusion: The "universal" CTC-chip achieved higher performance in detection of CTCs of thoracic malignant tumors as compared with the CellSearch. The updated data will be presented at the AACR annual meeting 2017.

#1722

**Novel multiplex liquid biopsy detection of ALK and ROS1 rearrangements and intratumoral heterogeneity in lung cancer using circulating tumor cells (CTCs) isolated from NSCLC patient bloods and TR-CytoTrapNano** TM **technology.**

sangjun lee. _Cytolumina, Los Angeles, CA_.

Introduction: ALK and ROS1 rearrangements are common gene alterations found in subsets of patients with NSCLC. Identification of ALK and ROS1 rearrangement in NSCLC is critical for highly active targeted therapies aiming for these two main tumorigenic drivers. Due to the similarity of tyrosine kinase domains between ALK and ROS1, many ALK inhibitors are also functional in treatment of ROS1 subtype NSCLC. For example, crizotinib, which is a standard therapy for advanced ALK-rearranged NSCLC is highly effective in advanced ROS1-rearranged genotype NSCLC patients. The mutational analysis of these genes requires serial tumor biopsying from primary lesions, which requires high risk clinical procedures. Further, the current tissue-based diagnostics, such as fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are known to be challenging technically with limited sensitivity, subjective interpretation and inaccuracy in monitoring the current tumor status. The TR-CytoTrapNanoTM CTC technology is a blood-based assay to utilize CTCs as a biosource for mRNA extraction to detect genetic alteration or gene expression profiles including ALK and ROS1 gene rearrangements. In this study, we determined the variant specific expression of ALK fusion transcripts (EML4-ALK, KIF-ALK, and TGF-ALK) and ROS1 fusion transcripts (EZR-ROS1, SDC4-ROS1, TPM3-ROS1, LRIG3-ROS1, GOPC-ROS1, CD74-ROS1, and SLC34A2-ROS1) using multiplex digital droplet polymerase chain reaction(ddPCR) technology.

Method: The Peripheral Blood Mononuclear Cells isolated from NSCLC patient bloods run through the TR-CytoTrapNanoTM microfluidic system. The CTCs are enriched and captured on the chip surface conjugated with Anti-EpCAM targeting antibody, which specifically binds to CTCs of epithelial origin. Following lysis step facilitate total RNA release from trapped CTCs and then pure mRNAs are isolated using oligo-dT conjugated magnetic beads. The mRNAs are used for following ALK and ROS1 specific reverse transcription reaction to make cDNAs for either ALK or ROS1 fusion transcripts. The specific subtyping of ALK and ROS1 rearrangement are then validated using ddPCR.

Results and Conclusion: We tested several liquid biopsies from ALK and ROS1 rearrangement positive patients and corresponding positive data to FISH assays using solid tumor biopsies. Interestingly, some of the CTCs harboring ALK and ROS1 rearrangement has shown high intratumor heterogeneity in the subtypes of fusion partners. we envision that our novel TR-CytoTrapNanoTM CTC ALK/ROS1 rearrangement Assay to monitor treatment response and recurrence in NSCLC patients will be used to monitor treatment response and disease recurrence following treatment with Tyrosine Kinase Inhibitors (TKIs).

#1723

Diagnostic leukapheresis results in a significant increase in CTC yield in metastatic breast and prostate cancer.

Kiki C. Andree,1 Anouk Mentink,1 Joost F. Swennenhuis,1 Leon W. Terstappen,1 Nikolas H. Stoecklein,2 Rui P. Neves,2 Rita Lampignano,2 Hans Neubauer,2 Tanja Fehm,2 Johannes C. Fischer,2 Elisabetta Rossi,3 Mariangela Manicone,4 Umberto Basso,4 Piero Marson,5 Rita Zamarchi,4 Yohann Loriot,6 Valérie Lapierre,6 Vincent Faugeroux,7 Marianne Oulhen,6 Francoise Farace,7 Gemma Fowler,8 Mariane Sousa Fontes,9 Berni Ebbs,8 Maryou Lambros,8 Mateus Crespo,8 Penelope Flohr,8 Johann S. de Bono9. 1 _University of Twente, Enschede, Netherlands;_ 2 _University Hospital of the Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany;_ 3 _IOV-IRCCS and University of Padova, Padova, Italy;_ 4 _IOV-IRCCS, Padova, Italy;_ 5 _University Hospital of Padova, Padova, Italy;_ 6 _Gustave Roussy, Université Paris-Saclay, Villejuif, France;_ 7 _Gustave Roussy, Villejuif, France;_ 8 _Institute of Cancer Research, Sutton, United Kingdom;_ 9 _The Institute of Cancer Research and The Royal Marsden NHS Foundation Trust, Sutton, United Kingdom_.

Introduction Frequently the number of CTC isolated in 7.5 mL of blood is too small to reliably determine tumor heterogeneity and to be representative as a 'liquid biopsy'. In the EU FP7 program CTCTrap we aimed to validate and optimize the recently introduced Diagnostic LeukApheresis (DLA; doi: 10.1073/pnas.1313594110) approach to screen liters of blood and thereby substantially increasing the number of CTC available for further characterization. Here we present the results obtained from 32 metastatic cancer patients subjected to DLA in the participating institutions.

Methods Before the DLA procedure, whole blood was drawn in a CellSave blood collection tube and a 7.5 ml aliquot was processed with the 'gold standard' reference CellSearch® (Janssen Diagnostics, USA). DLAs from metastatic cancer patients were performed for ≈90 minutes to obtain 40 mL of product containing ≈4x109 mononuclear cells (MNC) representing ≈1 liter of blood. The obtained DLA samples were then divided, fixed with CellSave preservative, prepared and processed with each of the analysis techniques as described in the Standard Operating Procedures developed for DLA in the CTCTrap consortium (https://www.utwente.nl/tnw/mcbp/protocolsandtools/).

Results DLAs were obtained from 20 metastatic prostate cancer patients and 12 metastatic breast cancer patients at four different European academic medical institutions. Using a SOP for the DLA procedure, similar DLA products (MNC concentration: 64x106/mL, SD = 38x106) could be generated without any noticeable side effects. CTC in 7.5 mL of blood ranged from 0 to 324 (mean = 61, median = 18). DLA processed with CellSearch represented 7 to 212 mL of blood (mean = 100, median = 97), CTC ranged from 0 to 2913 (mean = 330, median = 105). Resulting in a significant increase in CTC yield (p = 0.004) ranging from 0x to 40x (mean = 13, median = 9) when comparing 1mL of whole blood to 1mL of DLA. Filtration of 50x106 WBC of DLA, through 5um microsieves yielded only 0 to 12 CTC (mean = 2, median = 0, n = 16). Leukocyte depletion of 18 mL of DLA followed by filtration yielded 0 to 178 CTC (mean = 37, median = 4, n = 22) not yielding a relative increase versus CellSearch DLA. Leukocyte depletion followed by CellSearch yielded 271 to 1620 CTC (mean = 792, median = 484, n = 3) also not yielding a relative increase versus CellSearch DLA. In 7 patients 0 CTC were detected in 7.5mL of blood, in 4 out of these 7 patients CTC were detected in DLA.

Conclusion The yield of CTC can be significantly increased by the use of DLA in patients with CTC detected in 7.5 mL of blood. Technology to select CTC from DLAs will need to be further improved before one can make optimal use of the large processed blood volumes.

#1724

Genomic profiling of Vortex-enriched CTCs using whole genome amplification and multiplex PCR-based targeted next generation sequencing.

Haiyan E. Liu,1 Melanie Triboulet,2 Amin Zia,3 Meghah Vuppalapaty,1 Evelyn Kidess-Sigal,4 John Coller,3 Vanita S. Natu,3 Vida Shokoohi,3 James Che,1 Corinne Renier,1 Natalie Chan,2 Violet Hanft,2 Elodie Sollier-Christen,1 Stefanie S. Jeffrey2. 1 _Vortex Biosciences, Menlo Park, CA;_ 2 _Stanford University, School of Medicine, Stanford, CA;_ 3 _Stanford University, Stanford, CA;_ 4 _Charite University Hospital, Berlin, Germany_.

Background: Genomic characterization of circulating tumor cells (CTCs) provides insights into cancer genetic changes, and might be utilized for cancer prognosis, diagnosis, as well as monitoring of therapeutic efficacy. Targeted Panel Next Generation Sequencing (NGS) enables analyzing CTC genetic variants of a focused gene panel at a relatively lower cost1. However, CTCs are rare, often resulting in very limited DNA quantities available that require whole genome amplification (WGA). In previous studies, we introduced the Vortex technology, a platform enabling label-free enrichment of CTCs from blood samples of colorectal cancer (CRC) patients and their use for genomic assays downstream2. In this study, we developed a simple and efficient NGS workflow for CTC samples collected by this technology.

Method: An optimized workflow using the Qiagen GeneRead DNAseq targeted panel and Illumina MiSeq NGS was first verified on HCT116 CRC cell line before being applied on patient CTCs. For patient blood samples, CTCs were collected with the Vortex technology, immunostained (CK, Vimentin, CD45) and enumerated. Matched white blood cell (WBC) DNA was included to subtract germline background. Fresh frozen liver metastasis tissue was collected and analyzed using the same NGS workflow. DNA from CTCs was extracted and amplified using Qiagen REPLI-g single cell WGA kit. Mutation detection on the WGA amplified DNA was performed using the GeneRead DNAseq CRC targeted panel of 38 genes and MiSeq sequencing. The sequencing data were analyzed by QIAGEN NGS Data Analysis Web Portal and Ingenuity Variant Analysis software.

Results: The Vortex technology was validated for the capture of CTCs from CRC patients. REPLI-g performed a uniform, unbiased amplification on fresh rare cells with a coverage of 97.7%, which enabled further targeted panel NGS. Blood from 3 CRC patients (P1, P2, P3) and 2 healthy donors (HD1, HD2) was processed with Vortex platform. Less than 1 CTCs/mL blood were found in HD1 and HD2. P1 and P2 had 66 and 20 CTCs/ mL of blood respectively, with many vimentin positive CTC clusters. P3 had 2 CTCs/mL of blood. No somatic mutation was found in healthy donors. Somatic variants were only detected in the CTCs from patient samples that were not present in matched germline WBCs. For P1, more mutations were found in the CTCs than in the liver metastasis while it was the opposite for P2 and P3.

Conclusion: For each patient, variants in CTCs and germline WBCs were analyzed from one blood sample using an optimized targeted NGS workflow and compared to liver mets. Our optimized workflow, using the Qiagen REPLIg and GeneRead DNAseq Targeted Panel NGS enabled the detection of CTC mutations for 38 CRC-focused genes. The inclusion of a germline WBC control in the workflow allowed the detection of mutations from pooled CTC samples collected using the Vortex technology.

Altmüller J, et al. (2014). Biol Chem.

Kidess-Sigal E, et al. (2016). Oncotarget.

#1725

**Detection of** ESR1 **D538G mutation in circulating tumor cells (CTCs) and paired circulating tumor DNA (ctDNA) samples of breast cancer patients.**

Eleni Tzanikou,1 Athina Markou,1 Eleni Politaki,2 Giorgos Koytsodontis,3 Amanda Psyrri,3 Vassileios Georgoulias,2 Evi Lianidou1. 1 _University of Athens, Athens, Greece;_ 2 _University of Crete, Heraklion, Greece;_ 3 _Attikon University Hospital, Athens, Greece_.

AIMS: Molecular characterization of CTCs and ctDNA analysis holds promise as an extremely powerful tool for the molecular profiling of cancer patients in real time. Estrogen receptor alpha (ERα) is expressed in approximately 70% of all breast cancers and endocrine therapy represents a major treatment modality in ERα-positive disease. Recently, somatic mutations in the ERα gene (ESR1) were linked to acquired resistance to endocrine therapies in breast cancer. In this study, we analyzed the most frequent ERS1 mutation (D538G) in CTCs (DNA samples isolated from CellSearch cartridges), corresponding ctDNA from early and metastatic breast cancer patients and healthy donors.

METHODS: We first developed a highly sensitive and specific methodology for the detection of ESR1 D538G hotspot mutation, based on a combination of allele-specific PCR, asymmetric rapid PCR and high resolution melting analysis. We analyzed DNAs isolated from CTCs (CellSearch) and the corresponding ctDNA before and/or after therapy in: a) 25 patients with ER+ operable breast cancer, b) 11 patients with ER+ metastatic breast cancer, c) 13 patients with ER- early breast cancer, d) 5 patients with ER- metastatic breast cancer and e) 80 healthy female volunteers. In all cases ctDNA (extracted from 2 ml plasma) and DNA from CTCs were first examined for their DNA quality before analysis.

RESULTS: The assay is highly sensitive (analytical sensitivity: 0.05%) and specific (0/80 healthy donors). ERS1 D538G hotspot mutation was identified in ctDNA in 4/18 (22.2%) of ER+ metastasis-verified and in 5/33 (15.2%) of ER+ early breast cancer. In CTCs, ERS1 D538G mutation was identified in 6/18 (33.3%) of ER+ metastasis-verified and 5/33 (15.2%) of ER+ early breast cancer. In ER-pos metastasis-verified breast cancer, the concordance for D538G mutation between CTCs and ctDNA was 10/18 (55.6%), whereas the corresponding concordance for ER+ operable breast cancer was 25/33 (75.8%). Moreover, ERS1 D538G hotspot mutation was identified in ctDNA in 2/9 (22.2%) of ER- metastasis-verified breast cancer and 2/16 (12.5%) of ER- early breast cancer. ERS1 D538G hotspot mutation was identified in CTCs in 3/16 (18.8%) of ER- operable breast cancer, whereas none of ER- metastasis-verified breast cancer patients (0/9) were positive. In ER- metastasis-verified breast cancer patients, the concordance between CTCs and ctDNA for D538G mutation was 7/9(77.8%), whereas the corresponding concordance for ER- early breast cancer was 15/16 (93.8%).

CONCLUSIONS: We developed and validated an ultrasensitive and highly specific methodology for the detection of ERS1 D538G hotspot mutation. This mutation was detected not only in the ER+ group, but also in the ER- group of breast cancer patients. We will further evaluate our findings in a large cohort of patients before and after treatment, to evaluate response to endocrine therapies in breast cancer.

#1726

Evaluation of PD-L1/PD-1 on circulating tumor cells (CTCs) and on primary tumor in advanced non-small cell lung cancer (NSCLC).

Galaktea Kallergi, Eleni Kyriaki Vetsika, Despoina Aggouraki, Eleni Lagoudaki, Anastasios Koutsopoulos, Filippos Koinis, Panagiotis Katsarlinos, Maria Trypaki, Christos Stournaras, Vassilis Georgoulias, Athanasios Kotsakis. _University of Crete, School of Medicine, Heraklion, Greece_.

Introduction: Circulating tumour cells (CTCs) are responsible for the metastatic dissemination of the tumor. They have been shown to express Programmed Death-Ligand1 (PD-L1) to escape from the immune system surveillance through its ligation with the PD-1receptor on the surface of effector immune cells. We investigated the expression of PD-1/PD-L1 on CTCs isolated from NSCLC patients treated with chemotherapy.

Methods: CTCs were isolated based on their size using the ISET platform from 30 stage IV chemo-naïve NSCLC patients (before and after chemotherapy). CTCs were detected after staining with Giemsa and immunofluorescence (IF). Double and triple staining experiments with different combination of antibodies: [Cytokeratins(CK)/PD-1/CD45 and CK/PD-L1/CD45] were performed and the samples were analyzed with the ARIOL system.

Results Giemsa staining showed that twenty-three (77%) out of 30 and six (54.5%) out of 11 patients had detectable CTCs at baseline and after the 3rd cycle of front-line chemotherapy. IF staining revealed seventeen out of 30 (56.7%) patients positive for CTCs at baseline level and 8 out of 11 (72.7%) samples after the 3rd cycle of treatment. PD-1 and PD-L1 expression was observed in 53% (9/17) and in 47% of the CTC-positive patients at baseline; in addition, 13% (1/8) and 63% (5/8) patients had PD-1 and PD-L1, respectively after the 3rd cycle. Among the total number of detected CTCs, 67% were PD-1(+) at baseline and 25% after the 3rd cycle (p=0.069). In addition, 26% and 80% were PD-L1(+) at baseline and after the 3rd cycle, respectively. Patients with more than 3 PD-1 positive CTCs showed shorter PFS (p=0.022). Primary tissue from ten of the examined patients was also available. More than 5% of PD-L1 (+) tumor infiltrating lymphocytes (TILs) were observed in 20% (2/10) of the patients. More than 5% PD-L1 positive cells in the primary tumor were observed in 20%. However the two group of the patients were different. In addition both patients with PD-L1 (+) TILs harvested CTCs with PD-1 expression and one of them had also PD-L1 positive CTCs.

Conclusion: PD-1- and PD-L1-positive CTCs could be detected before and during 1st line treatment in metastatic NSCLC. This expression was related to patients' prognosis, implying that these molecules can be served as targets for metastasis restoration. Furthermore the expression of PD-1 on CTCs suggests a bilateral cross-talk between tumor and immune cells.

#1727

Circulating Tumor Cells (CTCs) in patients with extensive stage small cell lung cancer and their association with clinical outcome.

Chun Zhang,1 Ryon Graf,2 Adam Jendrisak,2 Amanda K. Anderson,2 Priscilla Ontiveros,2 Sarah Orr,2 Anne Chiang,3 David Spigel,4 Charles Rudin,5 Eric Holmgren,1 Jakob Dupont,1 Gretchen Argast,1 Leonardo Faoro,1 Lei Zhou,1 John Lewicki,1 Ann M. Kapoun1. 1 _OncoMed Inc., Redwood City, CA;_ 2 _Epic Sciences, San Diego, CA;_ 3 _Yale School of Medicine, New Haven, CT;_ 4 _Sarah Cannon Research Institute, Nashville, TN;_ 5 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Background: The NOTCH pathway has been identified as a key therapeutic pathway in SCLC. Tarextumab (TRXT, anti-Notch2/3, OMP-59R5) is a fully human monoclonal antibody that targets the Notch2 and Notch3 receptors. PINNACLE is a Phase 1b/2 trial of TRXT in combination with etoposide and platinum therapy (EP) in patients with untreated extensive stage small cell lung cancer (ES-SCLC). Baseline CTCs and post treatment changes in CTCs have previously been shown to predict the response to chemotherapy in SCLC. CTCs may also serve as pharmacodynamic biomarkers. Here we describe a study of baseline and longitudinal CTCs in ES-SCLC patients from the PINNACLE phase 1b trial (clinicaltrials.gov:NCT01859741).

Materials and methods: CTCs, CTC clusters, apoptotic CTCs and N-Cadherin+ CTCs were identified and enumerated from patient blood samples using Epic Sciences CTC technology. Baseline CTCs from 26 patients were correlated with clinical outcome: progression-free survival (PFS), overall survival (OS) and best overall response, as well as metastatic status. A mixed effects model was used to investigate the post treatment changes in CTCs among the dose groups. Association of CTCs with PFS/OS and best overall response, including CTCs at each time point, as well as temporal changes of CTC status, were studied. Multivariate analysis was performed to identify CTC numbers in a subset of time points to correlate with response to treatment.

Results: CTCs were present in 81% of the patients (21/26). CTC clusters and apoptotic CTCs were detected in 38% and 77% of the patients, respectively. At baseline, CTC counts ≥ 5/mL were significantly associated with poor OS (p=0.04). There was a trend that the presence of CTC clusters was associated with worse OS. With a cut-off of 3.4/mL, apoptotic CTCs showed a trend in association with overall survival. CTC numbers in patients with liver metastasis were significantly higher than in patients without liver metastasis. CTCs were also found to be correlated significantly with the number of metastatic sites. When measuring at Day 7 post dosing, CTC numbers were significantly decreased.

Conclusions: Our findings suggest that CTCs are frequently detectable in patients and are a prognostic factor in ES-SCLC. CTCs decrease with TRXT and platinum-based chemotherapy. Updated results will be presented. CTCs will be further evaluated in the phase 2 portion of the PINNACLE trial.

#1728

CTCs and CTM as prognostic factors in sarcoma patients.

Alexcia C. Braun, Celso A. Mello, Marcelo P. Corassa, Vanessa S. Alves, Emne A. Abdallah, Ulisses R. Nicolau, Mônica Taiane de Macedo Díaz, Bianca de Cássia Troncarelli de Campos Parra Flores, Marcelo F. Fanelli, Patrizia Paternili-Brechót, Ludmilla T. Chinen. _AC Camargo Cancer Center, São Paulo, Brazil_.

Background: Sarcomas represent 1% of all cancers in adults, 8% in adolescents and young people, and 10% in children. Even though it is a rare cancer, it contributes to a significant loss of years of life in comparison with other types of cancer (Amankwah, et al., 2013). The presence of isolated Circulating Tumor Cells (CTCs) or Circulating Tumor Microemboli (CTM) in the blood of patients with sarcoma may be early markers of tumor invasion, because it is known that these cells circulate in the blood for months or years before the development of metastases (Paterlini-Brechot and Benali, 2007; Klain, 2009; Rhim et al., 2012).

Objective: To isolate and quantify CTCs and CTM from sarcoma patients, evaluating their presence and correlation with progression free survival (PFS).

Methods: The blood (approximately 8mL) was prospectively collected from patients with different types of high degree sarcoma. Blood was collected before the beginning of chemotherapy. The samples were processed and filtered on ISET (Isolation by Size of Epithelial Tumor Cells, Rarecells, France) system for the isolation and quantification of CTCs and CTMs. Later, was performed immunocytochemistry (ICC) with anti-CD45 antibody and counterstained with hematoxylin-eosyn for leucocytes population exclusion. The cutoff was estimated using the maximum of the standardized log-rank statistic proposed by Lausen & Schumacher (1992).

Results: we analyzed 11 high degree sarcoma patients before the beginning of treatment (any treatment line). The median age was 53 years-old (18-77) and 54,5% were male. The median CTCs number was 2.0 CTCs/mL (1.0-11.0) at baseline. By Kaplan-Meier test, we observed that patients with CTC number below the established cutoff (4.5 CTCs/mL) had better PFS compared to those above the cutoff (7.4 vs. 1.0 months respectively; P= 0.006). Moreover, patients without CTM had also better PFS compared to those patients with the presence of CTM (7.6 vs. 2.7 months respectively; P= 0.13).

Conclusion: this is the first study to demonstrate, besides the CTC and CTM presence in sarcoma patients, its real prognostic value, even in a limited sample. Our next step is to characterize these cells to better understand its capability to promote tumor progression.

#1729

Detection of gene expression biomarkers from enriched CTC preparations.

Bruce Seligmann,1 Marilyn Marron,2 Milos Babic,1 Elliot Imler,1 Peter Shepard,1 Pavani Chalasani,2 Alison Stopeck,3 Joanne Yeakley1. 1 _BioSpyder Technologies, Inc., Tucson, AZ;_ 2 _University of Arizona, Tucson, AZ;_ 3 _Stony Brook Cancer Center, Stony Brook, NY_.

Circulating tumor cells (CTCs) offer an opportunity for non-invasive characterization of metastatic cancer, including the ability to follow subtype evolution, development of resistance, tissue homing biomarkers, and changes in cell signaling mechanisms required for metastasis and continued growth and survival of the tumor. Analysis of CTCs and translation of a CTC assay into a clinical test has been difficult due to issues of obtaining high purity, high yield preparations and/or isolating individual CTCs. This is further compounded by the heterogeneity of CTCs, hurdles in profiling their molecular signatures, and difficulty correlating CTC number/subtype to actionable therapy. We pursued a detailed analysis of CTC gene expression using whole transcriptome TempO-Seq targeted sequencing, a highly sensitive, direct lysis, addition only assay. As this assay does not require purified CTC isolation or RNA extraction, there is no risk of CTC nor RNA loss. We exploited the single cell sensitivity of TempO-Seq to profile enriched CTC preparations prepared by RosetteSep gradient centrifugation, allowing us to characterize the phenotypes of CTCs within a high background of normal blood cells. To handle this large and potentially overwhelming background we developed and exploited a proprietary method of sample normalization that increased the sensitivity and consistency of CTC gene signature measurements. We demonstrated that the TempO-Seq assay could detect a single MCF7 (EpCAM+) Luminal-like cell in a background of 1,000 MDA MB 231 (EpCAM-) Basal-like cells (positive for an Endothelial Mesenchymal Transition, EMT), and vice versa. Next, we prepared mixtures of different proportions of these cultured cells spiked into normal blood, enriched the model "CTCs" using RosetteSep, and profiled the bulk preparation. Proportional discrimination of cell-specific signatures were obtained with as few as 40 spike-in cells. Finally, we profiled enriched CTC preparations from the blood of patients with metastatic breast cancer. The results demonstrated that the expression of HER2, ER, and EpCAM could be measured as well as biomarkers of drug resistance, bone/brain homing, EMT, and all 50 Hallmark cell signaling pathways. TempO-Seq EpCAM gene level correlated with the percent EpCAM+ cells measured by flow analysis while EMT signature levels correlated inversely. Patients with HER2- primary tumor and HER2+ CTCs could be identified, as could patients with elevated chemotherapy resistance pathways. These data demonstrate that it is possible to profile CTC phenotypes that are important in metastases and useful for selecting therapy using a bulk CTC preparation combined with the sensitivity of the TempO-Seq whole transcriptome assay. This suggests the opportunity for the development of a predictive test using currently available purification platforms without need for expensive specialized equipment or separation into single cells.

#1730

ESR1 **methylation in circulating tumor cells, ctDNA and primary tumors of breast cancer patients.**

Sophia Mastoraki,1 Areti Strati,1 Eleni Tzanikou,1 Eleni Politaki,2 George Koutsodontis,3 Loukas Kaklamanis,4 Nikolaos Malamos,1 Amanda Psyrri,3 Vassilis Georgoulias,2 Evi Lianidou1. 1 _University of Athens, Athens, Greece;_ 2 _University of Crete, Herakleion, Greece;_ 3 _Attikon University Hospital, Athens, Greece;_ 4 _Onassis Cardiac Surgery Center, Athens, Greece_.

Background: Estrogen receptor (ER) is an important prognostic biomarker in breast cancer. Epigenetic silencing of ESR1 could be of important clinical significance especially for its potential impact on endocrine treatment efficacy. Liquid biopsy provides real-time monitoring of tumor evolution and response to therapy through analysis of CTCs and ctDNA. Our group has evaluated for the first time epigenetic silencing of tumor and metastasis suppressor genes in CTCs and corresponding ctDNA. In this study, we evaluated for the first time ESR1 methylation in CTCs, paired ctDNA and primary tumors of breast cancer patients.

Methods: We developed and validated a highly sensitive and specific real-time MSP assay for ESR1 methylation. We further applied the developed assay in sodium bisulfite (SB) treated DNA samples from: a) FFPEs from 40 patients with operable breast cancer, 25 patients with metastasis, 30 mammoplasties and 15 fibroadenomas, b) EpCAM+ immunomagnetically isolated CTCs fractions, from 74 early breast cancer patients, 48 patients with metastasis and 30 healthy donors, c) CellSearch® cartridges from 36 early breast cancer patients, 22 patients with metastasis, d) ctDNA isolated from plasma of matched samples and 54 healthy donors as a control group.

Results: By using this highly specific and sensitive assay (sensitivity 0.1%) we detected methylation of ESR1 in: a) FFPEs: 16/40(40%) early breast cancer patients, 9/25(36%) patients with verified metastasis, 7/30(23.3%) mammoplasties and 5/15(33.3%) fibroadenomas. A statistically significant negative correlation was observed between ESR1 methylation status and ER protein expression (56/65 samples, 86%, p<0.001). b) In EpCAM+ CTCs fraction samples: ESR1 was found methylated in 16/74(21.6%) operable breast cancer patients, 10/48(20.8%) patients with metastasis, but only in 1/30(3.3%) healthy donors. c) CTC+ CellSearch® cartridges: 3/13(23.1%) in early breast cancer and 2/7(28.6%) in patients with metastasis. d) In ctDNA: ESR1 methylation was observed in 3/36(8.3%) early breast cancer patients, 3/22(13.6%) patients with metastasis and 2/54(3.7%) samples in the control group. ESR1 methylation status was highly correlated when paired DNA from CellSearch® cartridges and corresponding ctDNA samples were compared; 36/36 (100%, p<0.001) in early breast cancer and 21/22 (95.5%, p<0.001) in metastasis.

Conclusions: ER expression and ESR1 methylation were found 100% inversely correlated in primary tissues. The EpCAM+ CTC fraction of patients with breast cancer was found methylated for ESR1. Interestingly, ESR1 methylation was detected exclusively in CTC+ samples as analyzed from CellSearch® cartridges but in none of CTC- samples. In paired plasma samples, ESR1 methylation showed a high concordance (p<0.001) with ESR1 methylation in CTCs. Additional studies are needed to further evaluate the clinical significance of our findings.

#1731

Analysis of prostate cancer circulating tumor cells at the mRNA and protein level using a microfluidic graphene oxide chip.

Molly Kozminsky, Kathleen C. Day, Guadalupe Lorenzatti Hiles, Shamileh Fouladdel, Ebrahim Azizi, Mark L. Day, Todd Morgan, Sunitha Nagrath. _Univ. of Michigan, Ann Arbor, MI_.

While the relative five-year survival rate for men with localized prostate cancer is nearly 100%, it plummets to 28% in the case of distant metastases. This underscores the importance of investigating those cells able to spread and form these metastases: circulating tumor cells (CTCs). Though rare and surrounded by other blood cells, CTCs can be isolated using the sensitive nanomaterial-based microfluidic technology the graphene oxide (GO) Chip. Whole blood samples from 54 prostate cancer patients were obtained with consent and processed on parallel chips for immunofluorescence and RNA analysis. All of the samples were analyzed for CTC enumeration, where captured cells were stained for cytokeratin 7/8 (CK), CD45, and DAPI. CTCs were identified as DAPI+/CK+/CD45- cells. CTCs were detected in all of the samples. Interestingly, 25 samples showed CTC clusters ranging from 2-10 CTCs per cluster. To explore the potential role of EGFR in metastatic progression and CTC survival, a subset of samples was also assayed for EGFR expression with 9/10 samples showing EGFR+ CTCs. RNA extracted from a parallel device was reverse transcribed to cDNA. The cDNA was analyzed via qPCR for expression levels of 96 genes of interest, including housekeeping genes; epithelial and mesenchymal genes; oncogenes and tumor suppressor genes; prostate specific genes; extracellular matrix and inflammatory genes; and others. The results show our ability to examine RNA from CTCs to examine the role of relevant pathways in the cells in transit. With analysis capabilities at both the RNA and protein levels, the GO Chip is an example of a clinically relevant microfluidic technology.

#1732

Accuracy of extrapolation of circulating tumor cell count from small blood volumes: statistical estimation using the AccuCyte - CyteFinder system.

Jeffrey L. Werbin,1 Paulina Varshavskaya,1 Arturo B. Ramirez,1 Jackie L. Stilwell,1 Daniel E. Sabath,2 Ping-Yu Liu,3 Eric Kaldjian1. 1 _RareCyte, Inc., Seattle, WA;_ 2 _University of Washington, Seattle, WA;_ 3 _Fred Hutch Cancer Research Center, Seattle, WA_.

Background: Baseline and post-treatment counts of circulating tumor cells (CTCs) are prognostic of patient outcome in cancer. The FDA-cleared CellSearch® system has defined CTC count as the number of cells per 7.5 mL of blood, with poor prognosis being 5 or more for breast and prostate cancers, and 3 or more for colon cancer. There are little reported data on whether it is reasonable to extrapolate CTC counts from smaller blood volumes, particularly when CTC count is low, given sampling variability. The AccuCyte - CyteFinder system (RareCyte) collects 7.5 mL of blood for analysis, isolates the buffy coat and smears it onto 8 slides, which are then stained with markers for CTCs that are identified by fluorescence microscopy. We investigated the statistical accuracy of extrapolation of total CTC count from fewer than 8 slides.

Methods: 172 blood samples from various cancers were processed to microscope slides, stained, and analyzed by CyteFinder. Counts were made on a per slide basis, with total "true" CTC count being the sum of all 8 slides. Samples with no CTCs were excluded. To extrapolate the total CTC count from k slides, all possible combination of k slides were used to estimate the total count of 8 slides. This was done for k = 1-7 for all 172 samples and extrapolated counts were rounded to the nearest integer. The proportion of times that extrapolated counts fell within a specified percentage of the true CTC count was calculated across the entire sample set, as well as for 6 sub-categories defined by true CTC count range.

Results: The 6 CTC count categories were: 1-4 (N=39), 5-10 (N=21), 11-25 (N=21), 26-50 (N=14), 51-100 (N=24), more than 100 (N=53). Cumulative proportions within 5, 10, 25 and 50% of the true total CTC count were determined for the entire sample set as well as by category. In the entire sample set, the proportion of extrapolated counts that fell within 25% of the true count ranged from 0.448 (1 slide) to 0.723 (4 slides) to 0.951 (7 slides). Generally, the lower the true total count, the lower the fraction of extrapolated counts that fell within the specified percentage of the true count. For example, for true count 5-10, the fraction that fell within 25% of the true count ranged from 0.208 (1 slide) to 0.513 (4 slides) to 0.951 (7 slides). In contrast, for true count more than 100, the fraction that fell within 25% of the true count ranged from 0.751 (1 slide) to 0.968 (4 slides) to 1.000 (7 slides).

Conclusions: In this sample set, nearly three quarters of cases could be estimated within 25% of the true CTC count by extrapolating from 4 AccuCyte slides. Estimating each slide to represent ~1 mL of processed whole blood, our analysis suggests that a ~4 mL sample will have this level of accuracy relative to a CTC count from 7.5 mL. At low true CTC counts the accuracy of extrapolation decreases, indicating that a larger volume of blood is required for this level of accuracy.

#1733

Automated identification of circulating tumor cells by image analysis.

Leonie L. Zeune,1 Guus van Dalum,2 François-Clément Bidard,3 Jean-Yves Pierga,3 Tanja Fehm,2 Hans Neubauer,2 Brigitte Rack,4 Marianna Alunni-Fabbroni,4 Mateus Crespo,5 Johann de Bono,5 Leon W.M.M. Terstappen,1 Christoph Brune1. 1 _University of Twente, Enschede, Netherlands;_ 2 _Heinrich-Heine-University of Duesseldorf, Duesseldorf, Germany;_ 3 _Institut Curie, Paris, France;_ 4 _Ludwig-Maximilians-University of Munich, Munich, Germany;_ 5 _The Royal Marsden NHS Foundation Trust and The Institute of Cancer Research, Sutton, United Kingdom_.

In the field of Circulating Tumor Cell (CTC) research many new technologies are emerging to isolate CTCs. Some of them provide accompanying automated image analysis tools that present possible CTCs to the user. Others need fully manual image analysis. For all CTC isolation technologies the definition of a CTC based on the immuno-morphologic criteria is either customized to the specific platform or subjective to the user causing high interreader differences – a problem which may condemn many CTC-based clinical studies to failure. Thus, an important issue that the field is confronted with is the lack of a unified and standardized definition to classify a cellular object as a CTC. This problem is addressed within the European FP7 consortium CTCTrap and the Innovative Medicines Initiative (IMI) consortium CANCER-ID by the development of an open-source image analysis toolbox for CTC identification and enumeration. This toolbox is baptized ACCEPT (Automated CTC Classification, Enumeration and Phenotyping) and can process images generated by various CTC isolation technologies. The main software components are the Marker Characterization, the Full Detection and the Automatic Classification. The Marker Characterization tool aims at quantifying the antigens expressed by previously selected CTCs. The Full Detection tool is based on advanced mathematical methods to reliably detect all objects in the images, visualize the objects in scatter plots and enable the user to classify the cell types by the use of gates or selection of specific objects in the scatter plots or on the actual images. The Automatic Classification tool first detects all objects in the images followed by an automated classification approach that – as a result – presents found CTCs to the user. We demonstrate the effectiveness of these tools on two different datasets.

The Marker Characterization tool was tested for Her2 expression on archived CTC images isolated and classified by the CellSearch system from patients with metastatic breast cancer. Investigators from three different institutes were asked to score these cells for Her2 positivity first on the images generated by the CellTracks Analyzer and afterwards using ACCEPT. We show that the improved CTC visualization provided in ACCEPT, combined with several measurements which we extract for each cell, can reduce the inter-user variability.

The Full Detection and Automatic Classification tools of ACCEPT were tested on archived samples of patients with castration resistant prostate cancer processed with the CellSearch system as well as on microsieves obtained after filtration of the blood discarded by the CellSearch system. Results were compared with manually scored CTCs and showed the improvement of CTC classification by the availability of quantitative image analysis tools.

The Open Source ACCEPT program will be available on the MCBP website (http://www.tnw.utwente.nl/mcbp).

#1734

Absolute quantification of circulating tumor cell RNA enables high specificity detection of hepatocellular carcinoma.

Mark Kalinich,1 Irun Bhan,1 Tanya T. Kwan,1 David T. Miyamoto,1 Sarah Javaid,2 Joseph A. LiCausi,1 John D. Milner,1 Xin Hong,1 Lipika Goyal,1 Srinjoy Sil,3 Melissa Choz,4 Ravi Kapur,5 Alona Muzikansky,1 Huidan Zhang,6 David A. Weitz,6 Lecia V. Sequist,1 David P. Ryan,1 Raymond Chung,7 Andrew X. Zhu,1 Kurt J. Isselbacher,1 David T. Ting,1 Mehmet Toner,5 Shyamala Maheswaran,1 Daniel A. Haber1. 1 _Massachusetts General Hospital Cancer Center, Charlestown, MA;_ 2 _Merck Research Laboratories, Boston, MA;_ 3 _New York University School of Medicine, New York City, NY;_ 4 _Boston College, Chestnut Hill, MA;_ 5 _Center for Bioengineering in Medicine, Charlestown, MA;_ 6 _Harvard University, Cambridge, MA;_ 7 _Massachusetts General Hospital, Boston, MA_.

Background: Although liver cancer has the second-highest mortality rate among cancers internationally, accurate and scalable assays for the early detection and longitudinal monitoring of hepatocellular carcinoma are lacking. Circulating tumor cells are released from invasive cancers into the blood stream, but the difficulty inherent in isolating, identifying, and characterizing these ultra-rare cells has precluded their widespread implementation as a biomarker. By combining a high-throughput microfluidic negative depletion CTC isolation strategy with the absolute quantification of lineage-specific RNAs, we report the highly specific detection of hepatocellular carcinoma CTCs from patient blood draws.

Methods: Blood draws from 48 hepatocellular carcinoma patients, 31 chronic liver disease patients, 25 healthy donors, and 44 patients with primary cancers other than hepatocellular carcinoma were processed through the microfluidic device (CTC-iChip). RNA was extracted, whole-transcriptome amplified, and quantified using droplet digital PCR. Transcript counts were used to fit a logistic regression model to integrate distinct transcript levels into a single CTC-score. The technical feasibility of utilizing RNA sequencing for identification of novel CTC transcripts of interest was also demonstrated with a liver cancer cell line spike-in study.

Results: 9 of the 16 untreated HCC patients were successfully detected, while only 1/31 chronic liver disease patients were incorrectly classified. HCC patients undergoing treatment showed a significant decrease in their CTC-score; only 9/32 patients actively receiving treatment were positive. The CTC-score was not correlated with the HCC serum biomarker alpha-fetoprotein, and combining these two orthogonal measures led to estimated positive and negative predictive values of 80% and 86%, respectively, in a high-risk cohort. RNAseq analysis of cell line spike-in data revealed the potential of RNA sequencing for uncovering novel transcripts of interest.

Conclusion: Coupling microfluidic depletion with droplet digital PCR allows for the highly specific detection of hepatocellular carcinoma. The CTC-score generated from these data tracks with clinical intervention and is orthogonal to the existing biomarker AFP; combining these two assays has the potential to provide superior detection compared to either individual approach.

#1735

Assessing circulating tumor cells with a nanotechnology-based capture system as a novel biomarker for treatment response and surveillance in patients with oligometastatic solid tumors.

Joseph M. Caster,1 Kyle Wang,2 Bhisham Chera,1 Sin-Jun Park,3 Seungpyo Hong,3 Andrew Z. Wang1. 1 _Univ.of North Carolina, Durham, NC;_ 2 _Univ.of North Carolina, Chapel Hill, NC;_ 3 _University of Illinois at Chicago, Chicago, IL_.

Purpose: To prospectively evaluate changes in circulating tumor cells (CTCs) following definitive treatment in patients with oligometastatic solid tumors using a novel nanotechnology-based biomimetric platform.

Experiment Procedures: Patients with biopsy proven oligometastatic disease with up to three lesions were eligible if all sites of disease were treated with definitive therapy. Definitive therapy could include fractionated 3D conformal radiation, SBRT, IORT, surgical metastatectomy, and ablative procedures including microwave ablation. Patients initially presenting with oligometastatic disease were eligible if they had less than 3 lesions including the primary tumor. At least one lesion had to be treated with radiation. CTCs were enumerated from whole blood using the Onco-Sense CTC capture system which utilizes E-selectin and dendrimers functionalized with three cancer-specific antibodies (aEpCAM, aHER-2, aEGFR). Whole blood was collected from patients prior to starting treatment (baseline), mid-treatment, end of treatment, within 12 weeks of finishing treatment, and then every 3 months in follow up. We then assessed changes in CTCs with treatment and looked for associations between baseline CTCs, changes during treatment, and post-treatment changes with clinical outcomes.

Results: We have currently enrolled 24 patients. The majority of patients (20) had a single site of disease treated with radiosurgery. Sites of metastasis include bone, lung, and lymph nodes. Primary histologies include lung, breast, melanoma, prostate, RCC, colorectal cancer, and SCC of the head and neck. CTCs were detected in 100% of patients at baseline and decreased with treatment from a mean of 45 CTCs/mL pretreatment (range 3-99) to 14 CTCs/mL post-treatment (range 3-59). Post-treatment CTCs are available for 14 patients with a median follow up of 10.3 months. Ten of 14 patients remain clinically NED and post-treatment CTCs are lower than baseline in 13/14 of these patients. There have been 4 clinical failures and CTCs increased with or before radiographic progression in all 4 patients. Three of the failures occurred at least 3 months after initial complete responses to treatment and CTCs increased by a median 3.4-fold over post-treatment levels in these patients. One patient progressed before the first post-treatment follow up and CTCs increased from 7 to 22 CTCs/mL from baseline to post-treatment.

Conclusions: We showed here that we could enumerate CTCs in 100% of patients with oligometastatic disease using a novel CTC capture system. CTCs decreased with treatment and all clinical failures were preceded by significant rises in CTCs. Our preliminary data suggest that enumeration of CTCs by Onco-Sense may provide a novel biomarker for assessing treatment response and/or post-treatment surveillance for patients with oligometastatic solid tumors.

#1736

A novel RNA-based assay for the detection and monitoring of circulating tumor cell signatures in breast cancer.

Tanya T. Kwan,1 Aditya Bardia,2 Tilak Sundaresan,1 Laura Spring,1 Mark Kalinich,1 David Miyamoto,1 Xin Hong,1 Joseph LiCausi,1 Uyen Ho,1 Sarah Javaid,1 Erin Silva,1 Lecia Sequist,2 Shyamala Maheswaran,1 Daniel Haber1. 1 _Massachusetts General Hospital, Charlestown, MA;_ 2 _Massachusetts General Hospital, Boston, MA_.

Examination of circulating tumor cells (CTCs) holds the potential of offering a real-time non-invasive window into tumor biology. Information gleaned from CTC detection and characterization can be used for early cancer detection, choice of therapy decisions and long-term monitoring for disease recurrence and the emergence of drug resistance mechanisms. However, technical difficulties with CTC isolation and the inherent limitations of imaging-based analysis have hindered the broad clinical use of CTCs as biomarkers. To overcome these concerns, we have combined our unbiased microfluidic CTC enrichment technology, the iChip, with a highly sensitive and specific multi-gene RNA-based biomarker panel to develop an assay that detects CTC signatures in patient blood samples in a high throughput and quantitative fashion.

This assay was applied to a cohort of breast cancer patients, including women with both localized and metastatic disease. It successfully identified CTC signal in 50-70% of metastatic and 20-40% of localized pretreatment patient samples. Patient CTC scores, assigned based on a multi-marker prediction algorithm, correlated with cancer stage and grade, but not with hormone receptor status, suggesting the applicability of the assay to a wide range of breast cancer subtypes. To determine if CTC scoring was useful for disease monitoring, we performed monthly blood collection from metastatic patients starting a new line of treatment. CTC score as early as one month after initiation of therapy, but not at pretreatment, was predictive of progression-free survival and treatment outcome, suggesting that real-time kinetic changes in CTC signatures are more clinically informative that one-time static evaluation of their presence in a given sample. In addition, a subset of high-risk genes showed expression patterns over the course of treatment that highly correlated with disease recurrence. This observation indicates that a multi-marker panel can parse out dynamic changes in cancer gene expression programs and provide a molecular insight into treatment responses that are not achievable by assays built around one or few markers.

In conclusion, our novel RNA-based method of identifying CTC signatures in liquid biopsies provides a sensitive platform for breast cancer detection and monitoring that goes beyond CTC enumeration. It offers a non-invasive quantitative molecular characterization of tumor gene expression that can be used to guide informed clinical decisions in both standard course of care and clinical trial settings.

#1737

ALK rearrangement analysis in circulating tumor cells of lung cancer patients.

Min Kyung Jeon,1 Young Hun Kim,1 Eunjoo Hwang,1 Hye Seon Lee,1 Ji-hyun Uh,1 Myoung Shin Kim,1 JooKyung Park,2 Byung Hee Jeon,1 Se-Hoon Lee2. 1 _Cytogen Inc., Seoul, Republic of Korea;_ 2 _Samsung Medical Center, Seoul, Republic of Korea_.

The evaluation of ALK rearrangement in non-small-cell lung cancer (NSCLC) is a significant tool when considering chemotherapy. It is not always possible to perform a tumor biopsy in patients. We suggest isolation and culturing of circulating tumor cells (CTCs) as an alternative tool to a tumor biopsy for the diagnosis of ALK rearrangement. From 22 patients with NSCLC harboring ALK rearrangement, blood samples were collected and divided into two parts: one for immunofluorescence staining of CTC marker and the other for culturing of CTCs. Both samples were processed by size-based filtration, and Cultured CTCs were analyzed for EML4-ALK translocation by fluorescence in situ hybridization (FISH) using Vysis ALK break apart FISH probe kit. CTC culturing was successful in 18 of 22 cases (81.8%). Among 18 cases of successful CTC cultures, 13 cases showed ALK rearrangement positivity (72.2%). Therefore, we suggest that the CTCs can be used as an alternative method to tissue biopsy for diagnosing ALK rearrangement. In addition, this method may have clinical applications including serial blood sampling for the development of personalized cancer therapy based on individual genomic information.

#1738

Platelet activation and heterotypic platelet leukocyte conjugate formation in the blood of glioblastoma patients.

Sascha Marx, Maximilian Splittstöhser, Heiko Paland, Carolin Seifert, Madlen Juettner, Andreas Boehm, Christoph A. Ritter, Sandra Bien-Moeller, Henry W. Schroeder, Bernhard Rauch. _University Medicine Greifswald, Greifswald, Germany_.

Objective

Glioblastoma patients (GBM) suffer from an increased incidence of cardiovascular events. Platelets are well known as main player of the primary hemostasis, but have a broad range of additional functions. The formation of heterotypic conjugates between platelets and leukocytes (PLC) represents a pro-inflammatory surrogate marker and is usually increased after platelet activation. The aim of the present study was to evaluate the platelet activation status and the rate of circulating PLC in GBM.

Methods

Blood samples were drawn of consecutive patients before surgery for a suspected glioblastoma. The formation of PLC and several parameters of platelet activation were determined by flow cytometry before and after stimulation with either ADP or the thrombin receptor-activating peptide (TRAP) in vitro: expression of P-Selectin, CD63, CD40L and fibrinogen-binding to the activated GPIIb/ IIIa. Blood samples from age and gender matched healthy volunteers were used as controls. Statistical analysis was done with the Mann-Whitney-Test.

Results

Final analysis included 22 patients with histopathological proven virgin glioblastoma (9f, 13m, mean age 67.5 years, range from 55 to 86 years) and their respective controls. Basal platelet activation and in vitro platelet reactivity was increased in GBM. The difference got significant in the basal expression of CD63 (2.8% versus 1.9%, p=0.008), the Fibrinogen-binding after ADP-stimulation (110.3 MFI versus 63.1 MFI, p=0.04) and the CD63 expression after TRAP-stimulation (38.4% versus 33.3%, p=0.04). Furthermore, a reduced number of circulating PLC and in vitro PLC formation was seen in GBM without getting statistically significant.

Conclusions

In this preliminary report, we show for the first time an increased level of platelet activation and agonist-induced platelet reactivity in GBM. Both could be a reflection of the pro-thrombotic status in these patients. Interestingly, the formation of PLC was not increased, but in tendency decreased. Whether this observation potentially mirrors the intratumoral, anti-inflammatory microenviroment in GBM remains unclear.

#1739

Analysis of EML4-ALK fusion transcripts in plasma and platelets to monitor response to crizotinib in EML4-ALK positive non-small cell lung cancer patients (NSCLC).

Cristina Aguado,1 Cristina Teixido,1 Ana Gimenez-Capitan,1 Maria de los Llanos Gil,2 Sonia Rodriguez,1 Santiago Viteri,2 Niki Karachaliou,3 Erika Aldeguer,1 Vicente Peg,1 Lidia Alonso,1 Miguel Angel Molina-Vila,1 Rafael Rosell2. 1 _Pangaea Oncology, Barcelona, Spain;_ 2 _Dr. Rosell Oncology Institute, Barcelona, Spain;_ 3 _Hospital Sagrat Cor, Barcelona, Spain_.

Background: Rearrangements in anaplastic lymphoma kinase (ALK) gene can be detected in 5-7% of EGFR and KRAS wild-type advanced NSCLC patients (p). Fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC) are currently used for screening but are unable to identify the specific fusion partner and are unpractical to monitor clinical responses due to difficulty of obtaining rebiopsies. The RT-PCR technique has the potential to overcome this pitfall and allow patient monitorization in blood.

Methods: A total of 405 formalin-fixed paraffin-embedded (FFPE) samples from advanced NSCLC were analyzed by ALK IHC (Ventana D5F3) and FISH (Vysis). Positive patients were confirmed by RT-PCR and submitted to Sanger in order to identify the variant. In a subset of 36 patients with EML4-ALK-rearranged tumors who were treated with crizotinib, fusion transcripts were analyzed by RT-PCR in mRNA purified from plasma and platelets and correlated with clinical response.

Results: ALK IHC was analyzed in 405 NSCLC patients and 37 tested positive (9.1%) whereas 25 (7.7%) were identify as translocated by FISH (n=323). ALK fusion transcripts were analyzed by RT-PCR and a new fusion variant of ALK was identified. A total of 36 p benefited from crizotinib treatment, including the p with the new variant. Monitoring of EML4-ALK fusion transcripts in the plasma ad platelets of 35 ALK positive patients revealed a good correlation with clinical outcome to crizotinib treatment, with the fusion transcripts becoming undetectable in p with good clinical responses.

Conclusions: Analysis of ALK fusion transcripts in mRNA purified from plasma and platelets can have a value in patients with no biopsy available and to monitor the course of the disease.

#1740

Phenotypic, genomic, and clinical associations of Circulating Tumor Cells (CTCs) lacking epithelial biomarkers in metastatic Castration Resistant Prostate Cancer (mCRPC).

Ryon P. Graf,1 Yipeng Wang,1 Nicole Schreiber,2 Brigit McLaughlin,2 Stephanie Greene,1 Angel Rodriguez,1 Adam Jendrisak,1 Jerry Lee,1 Mark Landers,1 Ryan Dittamore,1 Howard I. Scher2. 1 _Epic Sciences, Inc., San Diego, CA;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Background: Epithelial Plasticity (EP) is a proposed mechanism of immune escape, resistance to programmed cell death, resistance to specific drugs, and promotion of metastasis. EP has been extensively explored in mathematical, cell biological, and animal models, which predict the presence of these tumor cells in circulation. Most studies on CTCs in human subjects are based on enrichment of cells expressing EpCAM prior to enumeration or molecular interrogation, which precludes analysis of cells that might be EP. The non-enrichment Epic Sciences platform was utilized to identify CTCs in metastatic castration-resistant prostate cancer patient samples phenotypically consistent with EP: CTCs that do not express cytokeratins (CK), but do express malignant or EP biomarkers: Androgen Receptor (AR), AR splice variant 7 isoform (AR-V7) or N-cadherin. Phenotypic and genotypic analyses of individual CTCs were performed to assess the malignant origin of CK- CTCs and their prevalence associated with overall patient survival.

Methods: 221 pre-therapy mCRPC blood samples were collected at Memorial Sloan-Kettering Cancer Center and shipped to Epic Sciences. Patients represent a diverse number of previous therapies and tumor burdens. All nucleated cells were plated to glass slides and subjected to DAPI and immunofluorescent (IF) staining of cytokeratin (CK), CD45 and another tumor marker (AR, AR-V7, or N-cadherin), followed by CTC identification by fluorescent scanners and malignant morphology. All samples were stained for AR, with a subset of samples stained for AR-V7 or N-Cadherin as well. Additionally, 83 AR(+)/CK(-) and 9 AR-V7(+)/CK(-) CTCs were analyzed for copy number variation (CNV) by low pass whole genome single CTC sequencing. Abnormal genomes were identified by amplifications and deletions of known oncogenes and tumor suppressors.

Results: 3931 AR(+) CTCs were detected, of which 2996 (76%) expressed CK and 935 (24%) did not. The AR(+)/CK(-) CTCs had a lower median AR signal, less nucleoli, and more nuclear speckles by digital pathology vs. CK(+) CTCs. AR(+)/CK(-) and AR-V7(+)/CK(-) CTCs had gross genomic alterations consistent with malignant prostate origin (AR gain, PTEN loss, RB1 loss, 8q gain, 8p loss, etc), often sharing the same genomic alterations with CK(+) CTCs in the same patients, with some having unique CNV profiles Presence of AR(+)/CK(-) CTCs was associated with worse overall survival (median 13.7 mo vs. 33.9 mo, p < 0.0001) and remained additive and independent to treatment Line, pre-therapy PSA, and therapy type in multivariate models.

Conclusions: Similar to CTCs expressing epithelial markers, CK(-) CTCs have gross genomic aberrations consistent with metastatic prostate cancer and are a negative prognostic factor associated with worse overall survival in both iterative threshold and multivariable analyses. 

### Molecular Classification of Tumors

#1741

**Whole-transcriptome characterisation of** NRAS **and** BRAF **mutated primary melanomas associated with immune cell infiltration signatures and differential survival benefit.**

Joanna Pozniak, Jérémie Nsengimana, Sathya Muralidhar, Jonathan Laye, D Timothy Bishop, Julia Newton-Bishop. _University of Leeds, Leeds, United Kingdom_.

In 703 primary melanoma transcriptomes derived from formalin-fixed tumors removed from participants in the Leeds Melanoma Cohort, we used in silico methodology described by Bindea et al.1 to develop immune cell signatures. We showed these to be associated with survival2. There is some reported evidence that patients with NRAS mutated melanoma have a better response to checkpoint therapies3. Our aim was, therefore, to use the transcriptomic data to ask if immune cell infiltration (evidenced by the presence of the immune cell signature) differed between mutation subgroups. We saw that the survival benefit of the immune cell signature was strongest in wild-type (HR=0.31, P=3x10-5), intermediate in BRAF mutated tumors (HR=0.56, P=0.008) but absent in NRAS mutated tumors (HR=1.09, P=0.79). That is that even if the NRAS mutated tumors had a strong T cell signature that signature did not correlate with better survival. The results remained significant after adjusting for sex, tumor site, age at diagnosis, and AJCC stage. We observed no evidence of increased numbers of regulatory T cells (FOXP3), in NRAS mutated tumors, nor PDL1 or PD1 expression. The only difference in checkpoint molecule expression studied was a borderline lower expression of CTLA4 in the NRAS mutated tumors (P=0.09). Our data suggest that even when T cells invade NRAS mutated tumors some functional deficiency may be present in

those tumors such that a survival benefit is not seen. Using genes significantly differentially expressed between NRAS and BRAF mutated tumor groups, we tested biological pathway enrichment using Reactome FIViz plugin in Cytoscape. The most enriched pathways in NRAS mutated melanomas were the PI3K (AKT3, NRAS) and mitochondrial translation (CHCHD1, PTCD3). In the BRAF mutated the most enriched were: direct P53 effectors (TGFA, IGFBP3), β-catenin independent WNT signaling (WNT5A) and MHC class I antigen processing (CYBA). A number of pathways related to cell adhesion involving GRIN1 were also identified. Others have reported that β-catenin signaling inhibits transcription of the chemokine CCL4 which reduces the trafficking of CD141+DC to melanomas and therefore T cell infiltration4. We have evidence from the β-catenin candidate transcriptomic analysis that primary melanomas without immune infiltration signatures have upregulated β-catenin but the comparison between transcriptomic patterns in NRAS and BRAF tumors did not suggest that β-catenin signaling was more active in NRAS mutated tumors. It may be that a different immunosuppressive mechanism is more common in NRAS

mutated tumors than in others.

References

1. Bindea et al. Immunity 39, 782-795 (2013)

2. Newton-Bishop et al. PCMR in press

3. Johnson et al. Cancer Immunol Res. 3, 288-295 (2015)

4. Spranger et al. Nature 523, 231-235 (2015)

#1742

Cancer cell-selective transcriptome analysis reveals new colorectal cancer molecular subtypes with improved biological resolution and superior predictive and prognostic performance.

Claudio Isella,1 Sara E. Bellomo,1 Francesco Brundu,2 Francesco Galimi,3 Elisa Ficarra,2 Luigi Marchionni,4 Livio Trusolino,1 Andrea Bertotti,1 Enzo Medico1. 1 _University of Torino, Candiolo Cancer Institute, Candiolo, Italy;_ 2 _Politecnico di Torino, Torino, Italy;_ 3 _Candiolo Cancer Institute, Candiolo, Italy;_ 4 _Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD_.

Molecular classification of colorectal cancer (CRC) based on gene expression profiling of tumor samples is known to be heavily affected by transcripts of stromal origin. As a consequence, current CRC transcriptional subtypes reflect an admixture of cancer cell-intrinsic traits and tumor microenvironment features. Whether selective analysis of the cancer cell transcriptome could improve CRC subtyping, remains an open issue. In patient-derived xenografts (PDXs), human transcripts only originate from cancer cells, because stromal transcripts are of mouse origin. We therefore assessed cancer-cell intrinsic transcriptional features of CRC by generating human-specific expression profiles of 515 PDXs from 244 CRC patients, and performing unsupervised class discovery. We identified five "CRC intrinsic subtypes" (CRIS A-E) only partially overlapping with the current ones, and robustly enriched for distinct molecular, functional and phenotypic traits: (i) CRIS-A: mucinous, glycolytic, CIMP, and enriched for microsatellite instability or mutations in KRAS; (ii) CRIS-B: marked TGF-β pathway activity, epithelial-mesenchymal transition and poor prognosis; (iii) CRIS-C: MSS, elevated EGFR signaling and sensitivity to EGFR-targeted treatments; (iv) CRIS-D: MSS, WNT activation, and IGF2 overexpression and amplification; (v) CRIS-E: MSS, Paneth cell-like phenotype and higher frequency of TP53 mutation. CRIS subtypes successfully categorized independent sets of primary and metastatic CRCs and cell lines, for a total of over 3000 samples profiled by microarrays and RNAseq. The new subtypes displayed unprecedented predictive and prognostic performances, indipendent from known markers including stromal signatures, whose integration with CRIS further enhanced prognostic significance.

#1743

Comprehensive genomic characterization of a large cohort of platinum-sensitive, high-grade serous ovarian cancer (HGSOC) FFPE specimens.

Brian C. Haynes,1 Marie E. Fahey,1 Darcy Myers,1 Diane Ilsley,1 Gary J. Latham,1 Elizabeth B. Somers,2 Nicholas C. Nicolaides,2 Charles Schweizer,2 Daniel J. O'Shannessy2. 1 _Asuragen, Inc., Austin, TX;_ 2 _Morphotek, Inc., Exton, PA_.

Introduction: Ovarian cancer is a leading cause of cancer related death in women. A comprehensive genomic characterization of platinum-sensitive tumors is required to further refine the definition of molecular subtypes and identify targeted therapies for this patient population. To this end we have performed a large-scale genomic and transcriptomic characterization of 348 primary FFPE tissues from a cohort of platinum-sensitive HGSOC patients collected from multiple clinical sites.

Methods: Macrodissection of FFPE resected tumor slides or sectioned blocks was performed to enrich for tumor content. RNA and DNA were each isolated from 2x 5µm sections of FFPE material. RNA expression and gene fusions were profiled by whole transcriptome RNA-Seq. DNA variants were analyzed by the AmpliSeq™ Cancer Hotspot Panel (Thermo Fisher). A subset of tumor and matched germline (PBL) specimens (N=181) were assessed for TP53 mutations by the QuantideX® NGS TP53 Assay (Asuragen, Inc.). CNV analysis of FFPE tumor DNA was performed using the OncoScan® FFPE Assay Kit (Affymetrix) and microsatellite instability was characterized by comparing matched tumor and PBL specimens with capillary electrophoresis using the Bethesda panel. Germline BRCA1/2 mutation status was determined by profiling PBL specimens with a custom AmpliSeq™ NGS panel.

Results: The spectrum of DNA mutations and CNVs was consistent with other HGSOC cohorts with mutations in TP53 present in the majority of specimens (87% for specimens with full exon coverage of TP53). Germline mutations in BRCA1 and BRCA2 were identified at 10.3% and 6.8% respectively. Analysis of recurrent whole chromosomal arm gain and loss displayed a striking agreement with the TCGA HGSOC cohort. Unsupervised analysis of the RNA-Seq expression data through non-negative matrix factorization revealed 4 distinct transcriptional subtypes, corresponding to the 4 established CLOVAR subtypes: differentiated, immunoreactive, mesenchymal and proliferative. Patients classified as mesenchymal had the poorest prognosis. Further integrative analysis identified additional associations between the CLOVAR subtypes and other molecular indications.

Conclusions: Through integrative genomic analyses on this challenging set of clinical specimens we have reproduced and refined the molecular subtypes of platinum-sensitive HGSOC and highlighted alterations that may lead to improved diagnostic and precision medicine strategies. In contrast to previous large-scale molecular characterization efforts such as TCGA where fresh-frozen tissues were collected under highly controlled settings, this study is based on molecular profiling of FFPE tissues collected at dozens of clinical sites. Thus, our study serves as a model for future molecular characterization efforts of FFPE specimens collected in real-world clinical settings.

#1744

RNAseq identified immune signatures associated with adverse outcome in high-risk neuroblastoma.

Jun S. Wei,1 Shile Zhang,1 Igor Kuznetsov,2 Young K. Song,1 Shahab Asgharzadeh,3 Sivasish Sindiri,1 Xinyu Wen,1 Rajesh Patidar,1 Jaime M. Guidry Auvil,1 Daniela S. Gerhard,1 Robert Seeger,3 John M. Maris,4 Javed Khan1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _University at Albany, Bethesda, NY;_ 3 _The Children's Hospital Los Angeles, Los Angeles, CA;_ 4 _The Children's Hospital of Philadelphia, Philadelphia, PA_.

Despite the success of multimodal therapies, the mortality and morbidity remains substantial for patients with high-risk neuroblastoma (NBL). Sequencing of paired tumor/normal DNA of NBL has revealed a low somatic mutation burden and few recurrent somatically-mutated genes. Here we hypothesize that whole transcriptome sequencing (WTS) in patients with high-risk NBL tumor will yield valuable insights into the biology of this disease. We performed deep WTS of 150 NBLs (129 high-risk stage 3 and 4; and 21 stage 4S tumors) of which majority of them had whole genome sequencing or whole exome sequencing of case-matched tumor/normal pairs through the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative. We identified correlations between gene expression and clinical parameters of patients such as MYCN-amplification and survival to provide understandings of high-risk NBL biology.

Consensus clustering using gene expression profiles of tumors revealed 4 subgroups with distinct survival probability (p<0.01). Among them, the group with the best outcome consisted of all stage 4S and 4S-like young patients; whereas the worst survival group was made up mostly of high-risk patients with MYCN-amplification. In order to identify the gene expression patterns correlating with outcome in the high-risk patients without MYCN-amplification, we further examined if any other known gene expression signatures can further stratify these patients using Gene Set Enrichment Analysis (GSEA). Among several molecular signatures, a previously reported 157-gene MYCN activation signature was significantly correlated with the poor outcome in high-risk patients without MYCN-amplification. Of note, we identified CD8, cytolytic, and activated nature killer (NK) cell signatures that were significantly associated with outcome in these patients. These findings were validated in an independent dataset. Our data has an important clinical implication for a subset of high-risk neuroblastoma patients without MYCN-amplification that have an activated MYCN signature may benefit from immune-modulating agents such as immune check point inhibitors which should be tested in context of clinical trials.

#1745

Multifocal prostate cancer has high degree of genomic heterogeneity.

Marthe Løvf,1 Sen Zhao,1 Ulrika Axcrona,2 Bjarne Johannesen,1 Andreas M. Hoff,1 Anne Cathrine Bakken,1 Kristina Totland Carm,1 Ola Myklebost,3 Leonardo A. Meza-Zepeda,1 Agnes K. Lie,4 Karol Axcrona,5 Ragnhild A. Lothe,1 Rolf I. Skotheim1. 1 _Oslo University Hospital, Institute for Cancer Research, Oslo, Norway;_ 2 _Oslo University Hospital, Department of Pathology, Oslo, Norway;_ 3 _Norwegian Cancer Genomics Consortium, Oslo, Norway;_ 4 _Østfold Hospital Trust, Department of Pathology, Fredrikstad, Norway;_ 5 _Akershus University Hospital, Department of Urology, Lørenskog, Norway_.

Prostate cancer is the most common cancer type among men in the Western world. Most prostate cancers are multifocal with individual tumors harboring different aggressiveness. In recent genomics studies, the multifocal nature of prostate cancer has been investigated only in small sample cohorts. Here we have investigated the intra-organ mutational spectra of multiple tumors from a large cohort of prostate cancer patients. From radical prostatectomies performed between 2010 and 2012 at Oslo University Hospital, 43 prostatectomy specimens with multiple and clearly separated tumors were identified based on histology. From each of these prostatectomy specimens, DNA from frozen samples from 2-3 different tumor foci and corresponding normal tissue samples were analyzed by high-coverage whole-exome sequencing, adding up to a total of 159 samples. We identified 3093 somatic substitutions, insertions and deletions, with an average of 27 alterations per tumor sample. Both known and novel significantly mutated genes were identified and their distribution among different tumor foci from the same prostate was examined. Mutations in genes such as SPOP, MED12, and FOXA1 have previously been identified in prostate cancer and were also found to be frequently mutated in this cohort. However, the same mutations were rarely found in multiple tumor foci within the same prostate. In fact, for 13 out of the 43 examined patients there were no common mutations among tumors from the same prostate. For 12 patients, we found only one overlapping mutation among tumor foci. Whereas the overall list of mutated genes (n = 2101) overlap significantly with the 594 genes of the Cancer Gene Census (100/594; p=9.6e-06), this was not the case for 142 genes with overlapping mutations among different tumor foci (2/594; p=0.63). This indicates that the overlapping mutations are not typical cancer driving genes. To conclude, results from exome sequencing of multifocal prostate cancer show a large degree of heterogeneity in genomic alterations between different tumor foci within the same prostate. With very few common inter-foci mutations, an implementation of genome-based personalized prostate cancer medicine will require sampling of all tumor foci to tailor optimal treatment.

#1746

Rational molecular assessment and innovative drug selection (RAIDs): Paving the way to personalized medicine in cervical cancer.

Leanne de Koning,1 Bérengère Ouine,1 Aurélie Cartier,1 Els M. Berns,2 Kirsten Ruigrok-Ritstier,2 Corine Beaufort,2 Balazs Balint,3 Attila Kereszt,3 Gemma Kenter,4 Sanne Samuels,4 Ekaterina S. Jordanova,4 Emmanuelle Jeannot,1 Heiko von der Leyen,5 Marina Popovic,6 Windy Luscap-Rondof,7 Vonick Sibut,7 Choumouss Kamoun,7 Isabel Britto,7 Claudia Rincon,7 Philippe Hupé,8 Maud Kamal,1 Suzy M. Scholl1. 1 _Institut Curie, Paris, France;_ 2 _Erasmus MC Cancer Institute, Rotterdam, Netherlands;_ 3 _SeqOmics, Moraholem, Hungary;_ 4 _Center for Gynecological Oncology Amsterdam, NKI-AVL, Amsterdam, Netherlands;_ 5 _Hannover Clinical Trial Center GmbH, Hannover, Germany;_ 6 _Institute of oncology of Vojvodina, Vojvodina, Serbia;_ 7 _Institut Curie, INSERM U900, Paris, France;_ 8 _Institut Curie, INSERM U900, Mines ParisTech, CNRS UMR 144, Paris, France_.

Cervical cancer (CC) is the fourth most common cause of cancer deaths in women worldwide, for which prognostic and predictive biomarkers are largely lacking. RAIDs is a EU-funded project on cervical cancer that spans seven European countries. The main objective of the RAIDs project is to use this tumor type, which is easily accessible for repeated biopsies, to learn how to stratify patients into targeted therapies. The project includes: 1) a cognitive cohort study (BioRAIDs), one of the first prospective trials intended to define patient stratification for targeted therapies, 2) a targeted clinical trial using an HPV directed vaccine and 3) preclinical studies aiming at assessing new treatment strategies. Molecular analysis on quality controlled tumor and sera samples from 400 patients include Next Generation Sequencing at SeqOmics (Hungary), PIK3CA mutations detection in circulating tumor DNA at Erasmus MC (Netherlands), Reverse Phase Protein array and HPV insertion sites analyses at Institut Curie (France) and immune-microenvironment analyses at CGOA (Netherlands). In addition, 20 CC cell lines have been profiled pharmacologically using a panel of drugs which potentially synergize with "standard treatment".

The present poster will mainly focus on the Reverse Phase Protein Array (RPPA) results of BioRAIDs and how these relate to the genomic profiling and to patient outcome. More than 150 cryopreserved baseline (before treatment) samples and 23 CC cell lines have been screened by RPPA. From these, whole exome sequencing is available for 92 patient samples. Stratification of patients based on proteomics and genomics has evidenced different subgroups (clusters) of patients displaying specific molecular characteristics. Genomics data and proteomics data both demonstrate that these clusters differ notably in the pathways of oxidative phosphorylation, glycolysis and DNA repair. Patient response to treatment is assessed by the presence or absence of residual tumor at six months after treatment. The correlation of proteomics and genomics data with these clinical data is ongoing and will identify putative predictive biomarkers. Correlation of protein data with response to drugs treatment in 20 CC cell lines has identified several potential biomarkers, some of which again relate to (glucose) metabolism.

In conclusion, we here present new evidence for molecular subgroups of cervical cancer that could benefit from different treatment options. Notably, our data suggest that the metabolism/glycolysis pathways are a major effector in CC and constitute a potential therapeutic target in a subset of patients.

This project has received funding from the European Union's Seventh Program for research, technological development and demonstration under grant agreement No 304810.

#1747

A microenvironment gene signature predicts poor outcome in patients with luminal breast cancers subjected to local-regional treatment.

Maria Grazia Daidone, Giuseppe Merlino, Patrizia Miodini, Maurizio Callari, Vera Cappelletti. _Istituto Nazionale Tumori, Milan, Italy_.

Gene expression studies, mainly addressed at the tumor cell compartment of breast cancers (BC) contributed to unravel the molecular heterogeneity providing clinicians with new and reliable tools for therapeutic planning. Despite many investigations have focused on genetic abnormalities initiating and driving tumorigenesis, there is now evidence that the cancer cell behavior depends not only on intrinsic features, but also on the interactions with the microenvironment. However, notwithstanding the interest in investigating tumor-stroma cross-talks, the clinical relevance of subtype-specific molecular imprint of such interactions is largely unexplored. As such, we attempted to clinically and biologically validate the subtype-specific gene signatures derived from an in vitro model recapitulating the interaction between BCs and activated stromal cells. Gene expression signatures derived from luminal (T47D, MCF7, ZR75.1), HER2+ (SkBr3+, BT474, MDA-MB-361) and basal-like (MDA-MD-468, MDA-MB-231, BT20) cell lines treated by conditioned media from normal or cancer-associated fibroblasts (CAFs) showed common- and subtype-specific pathways according to GSEA. In fact, interferon, IL-6, IL-12 and IL-23 signaling, Toll Like Receptor pathway, pathways related to inflammation and metabolism of carbohydrates were positively enriched in HER2+ and luminal subtypes. Complement cascade and Tumor Necrosis Factor pathways were positively enriched in basal and luminal tumors; no common pathway was observed in HER2+ and basal subtypes. The association between microenvironment signatures specific for luminal, HER2+ and basal BC cell lines and disease-free survival was evaluated in silico on published gene expression profiles (GEPs) from 2048 patients homogeneously treated. BCs were classified as microenvironment-positive (µENV+ve, with GEP suggesting stroma activation), or negative (µENV-ve) by correlating tumor's GEP with the respective subtype-specific signature. Patients with luminal µENV+ve tumors had 2.5-fold higher risk of developing distant metastases (HR=2.546; 95% Confidence Interval, Cl: 1.751-3.701, P=9.84E-07), while µENV status did not affect or was only suggestive of metastases in women with HER2-enriched (HR=1.541; 95% Cl: 0.788-3.012, P=0.206) or basal tumors (HR=1.894; 95% Cl: 0.938-3.824; P=0.0747), respectively. In luminal tumors, the µENV status maintained its prognostic role (HR=2.098; CI: 1.214-3.624; P=0.00791) in multivariable analysis including size, age and genomic grade index. Validity of our in vitro model was also supported by biological endpoints such as proliferation (MTT-assay) and migration/invasion (Transwell-assay). In vitro-derived gene signatures trace a subtype-specific interaction with CAFs and add independent information to classical prognostic variables in women with luminal "microenvironment sensitive" tumors.

#1748

Organ-specific epigenetic reprogramming of leukemia cells: Clues to chemoresistance.

Isabel Cunningham,1 Diane Hamele-Bena,1 Audrey Papp,2 Yan Guo,3 Antony B. Holmes,1 Yu Shyr,3 Rory A. Fisher4. 1 _Columbia University College of Physicians and Surgeons, New York, NY;_ 2 _Ohio State University, Columbus, OH;_ 3 _Vanderbilt University, Nashville, TN;_ 4 _University of Iowa Carver College of Medicine, Iowa City, IA_.

We previously reported our observation that tumors composed of leukemic cells found in breast mimic solid breast cancers in gross and histologic appearance (single-filing of cells and distinctive keloid-like fibrosis), invasiveness, metastatic pathway, and high mortality. They are resistant to anti-leukemia drugs; only 4% treated without excision lived 4 years but survivals over 20 yrs resulted when tumor and its microenvironment were excised. The incidence of occult leukemic tumors, still unknown as scans are not yet routine, was estimated near 20% in 1970s autopsy studies. We hypothesized there may be similarly altered genes in leukemic and epithelial breast tumors directing clinical behavior and resistance that could provide targets for curative treatment. We performed RNA sequencing on 11 FFPE breast tumors from annotated AML cases, all IHC-negative for hormones and E-cadherin, compared to normal breast samples. Differential expression analysis identified 3647 genes that were significantly downregulated (adjusted p < 0.05 and log2 fold change < -2). Among the 100 genes downregulated 8-10 fold in leukemic breast, FOXA1, CA12, CXCL14, SFRP1, SFRP4, DCN, LUM, PHLDA1, NTN4, GABRP, TAGLN, AZGP1, and ARRDC3 have been associated with poor prognosis in breast cancer, mainly triple-negative (TNBC)/basal types. Compared to TNBC cases in the TCGA database, 851 genes were similarly deregulated; significantly enriched KEGG pathways include neuroactive ligand-receptor interaction, focal adhesion, ECM-receptor interaction, and regulation of lipolysis in adipocytes. A role for altered stroma is suggested as many of the similarly downregulated genes were reported in dissected breast cancer stroma and silenced SFRP1 and CXCL14 were found in keloid fibroblasts. Silencing of SFRP1 and E-cadherin in AML marrows was reported in 30-50% and each was correlated with poor survival. Reversal of SFRP1 silencing has been achieved by epigenetic-modifying drugs in cancers and keloid fibroblasts. We validated loss of SFRP1 and RGS6 in leukemic breasts by RT-PCR. Loss of RGS6 in breast and other cancers promotes doxorubicin resistance and DNMT1 upregulation leading to silencing of tumor suppressor genes. These data open a new view of resistance of AML cells to marrow-directed drugs through interaction with aberrant microenvironment. Solid tumors of epithelial and leukemic cells may share unrecognized treatable targets. Adding drugs directed at tumor, including available epigenetic-modifying agents, to marrow-effective protocols, may finally decrease the persistently high rate of AML treatment failure.

#1749

Stability of copy-number profiles defines two molecular subtypes during urothelial carcinoma's evolution.

Bishoy M. Faltas,1 Davide Prandi,2 Scott T. Tagawa,1 David Nanus,1 Ana M. Molina,1 Himisha Beltran,1 Francesca Demichelis,2 Mark A. Rubin1. 1 _Weill Cornell Medical College, New York, NY;_ 2 _University of Trento, Italy_.

Background: Somatic copy-number alterations (SCNAs) are distinct molecular events occurring in muscle-invasive urothelial carcinoma (UC). Defining the biologic role and the timing of SCNAs in UC's evolution is critical for understanding disease biology and for precision therapy.

Methods: To examine whether distinct SCNAs define specific subtypes of UC and characterize the evolutionary dynamics of SCNA acquisition, we conducted whole-exome sequencing (WES) of a discovery cohort of 44 advanced UC tumors and matching germline samples, including 13 pairs of matched primary and metastatic tumors prospectively collected before and after chemotherapy. We queried WES data from 285 UCs from The Cancer Genome Atlas (TCGA) as a validation cohort. We performed a novel in-depth allele-specific copy-number analysis in 2503 cancer genes using the CLONET computational framework, which accounts for tumor ploidy and cellularity to infer the clonal hierarchy of SCNAs.

Results: Analysis of SCNAs from tumor pairs within each patient revealed punctuated evolution of SCNAs occurring early in UC's development followed by minor changes (clonal stasis) throughout the tumor's lifetime. Two distinct clusters of UCs with strikingly different SCNA profiles in the discovery cohort emerged. Cluster A was defined by diploid copy number background and focal deletions in the 9p21 region (CDKN2A, CDKN2B and MTAP). Cluster B was defined by genome-wide duplications and several enriched amplifications including 1q21.1 (SETDB1 and MLLT11), (P = 0.0002) and 6p22.3 (E2F3), (P = 0.001). TP53 mutations (P = 0.0001) were enriched in the cluster B, consistent with the role of functional p53 as a "guardian of the genome" preventing chromosomal instability. SCNAs clusters A and B were confirmed in the TCGA validation cohort. Cluster A was enriched with tumors belonging to TCGA bladder cancer cluster III ("basal/squamous-like") (P = 0.02).

Conclusions: Using novel allele-specific SCNAs analysis, we define two distinct clusters of UC presenting a framework for understanding this aspect of UC's biology. In contrast to somatic mutations, SCNAs are clonally static during each tumor's evolution. This data has important implications for precision medicine for UC.

#1750

Analysis of non-metastatic HCC patient tumors revealed the significance of cell cycle regulation and tumor immunity in association with overall survival and identified clinically relevant druggable targets.

Pei-Zhen Miao,1 Jeff Cheng,2 Kwame Okrah,3 Bonnie Liu,3 Charlie Sun,2 Grace Cao,2 Tony Guo,2 Shijing Fu,2 Marie-Claire Wagle,3 Elizabeth Blackwood,3 David Shames,3 Garret Hampton,3 Lukas Amler,3 Astrid Kiermaier,4 Yevgeniya Fridlyand,3 Mark Lackner,3 Shih-Min Huang,3 Jian Zhou,1 Zhi Dai1. 1 _Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China;_ 2 _Genentech, Shanghai, China;_ 3 _Genentech, South San Francisco, CA;_ 4 _Genentech, Switzerland_.

The lack of efficacious therapies have hindered the progress in improving the survival of HCC patients worldwide. As a disease that associates with the underlying liver injuries caused by various etiologies and remains asymptomatic during most of its decades long progression, HCC presents challenges to accurately depict its biological landscape and determine suitable therapeutic intervention. An approach to tackle this issue is to define significant biological variants that impact patient survival in each disease stage, then attempt to chart paths of potential therapies.

To identify significant biological determinants associated with the overall survival (OS) of early stage, non-metastatic HCC patients, we constructed focused Nanostring panels that are represented by 1,164 genes of validated components and transcriptional outputs of multiple signaling pathways, cellular machineries, and tumor immunity. An unbiased analysis of patient survival and gene transcript levels revealed four distinct groups of genes that are significantly prognostic. The first two (Group 1 & 2) are associated with poorer prognosis when expression is high (hazard ratio > 2), whereas the remaining two (Group 3 & 4) correspond to significantly better OS with elevated transcript levels (hazard ratio<0.5). Of note, Gene Set Enrichment Analyses (GSEA) clearly indicated that while Group 2 was substantially enriched in genes positively regulating cell cycle and cell division machinery, Group 4 was overrepresented by genes participating in the positive modulation and feedback mechanisms of adaptive immune response, implicating the survival benefit of acquiring higher immunogenic tumor environment in early stage HCC.

Searching for druggable targets associated with poorer prognosis, we identified both EZH2 and CHEK1 as candidates from Group 2. In vitro treatment of three HCC cell lines with EPZ-6438 (EZH2 inhibitor) or GDC-0425 (CHEK1 inhibitor) resulted in significant modulation of H3K27me3 or pCHEK1 levels (s345) respectively. The growth of these cell lines was also dose-dependently suppressed by each inhibitor in colony formation assays . Furthermore, the combination of both inhibitors was able to achieve substantial cell growth inhibition at lower concentrations, suggesting a combinatorial effect of targeting both EZH2 and CHEK1 in HCC cell lines. Xenograft studies are ongoing to assess in vivo efficacy of this combination.

In conclusion, utilizing well constructed Nanostring gene panels, we identified significant association of cell cycle machinery and adaptive tumor immunity with HCC patient survival. Our findings not only provided potential rationale for expand testing of cancer immunotherapies to non-metastatic, early stage HCC, but also revealed the potential utility of combining EZH2 and CHEK1 inhibitors to treat HCC.

#1751

Systematic bias in genomic breast cancer classification due to selecting cases with high tumor percentage and good RNA quality.

Esther H. Lips, Antien L. Mooyaart, Maartje van Seijen, Lennart Mulder, Marlous Hoogstraat, Petra M. Nederlof, Lodewyk F. Wessels, Sjoerd Rodenhuis, Gabe S. Sonke, Jelle Wesseling. _Netherlands Cancer Institute, Amsterdam, Netherlands_.

Background

Cancer classification, prognostication, and prediction of treatment sensitivity increasingly rely on DNA and RNA-based tests. This approach requires sufficiently high tumor cell percentages to yield enough DNA or RNA for reliable test results. As a consequence, samples of insufficient quality may drop out, e.g. due to poor cellularity or high numbers of tumor infiltrating lymphocytes. We hypothesized that requiring a high tumor cell percentage and high quality RNA causes systemic bias when interpreting genomic test results in breast cancer, as specific breast cancer subgroups may be over- or underrepresented.

Patients and methods

For this analysis, we used pre-treatment frozen samples from patients included in neoadjuvant chemotherapy trials at the Netherlands Cancer Institute between 2004 and 2012. Histological features and tumor cell percentage were reviewed and assessed by a consultant breast pathologist. Gene expression profiling was done if the tumor cell percentage exceeded 50% and RNA quantity (>1 µg) and quality (RIN value≥ 6.5) were sufficient. We compared patient and tumor characteristics between patients in whom gene expression profiling could be performed and those in whom it could not be performed. In addition, we performed a systematic review on gene expression profiling for breast cancer, to assess the percentage of sample dropout in published studies.

Results

Frozen biopsies were available from 658 patients (79% of the total study population) and gene expression profiling could be performed in 60% of the cases, a percentage comparable to what is reported in the literature. Reasons for drop out were a low cellularity, poor quality and quantity of the RNA, or a too small biopsy for processing. These patients had more grade 3 tumors (43% versus 34%, p=0.04) and were more node positive (76% versus 69%, p=0.03). Analysis of neoadjuvant chemotherapy response and survival in these patients is ongoing and will be presented at the AACR Annual Meeting 2017.

Conclusion

Breast cancers for which gene expression data were successfully obtained were associated with a higher grade and with lymph node metastasis. Such tumors represent a more aggressive phenotype and have a relatively poor prognosis, compared to the patients for whom gene expression data could not be measured. As gene expression arrays are now broadly used in a clinical context, it is important to acknowledge this systematic bias, and to be cautious in applying gene expression based tests on different patient populations than a test was developed on.

#1752

Immune-related gene signatures in colorectal liver metastases: Exposing an opportunity for immune modulating therapy.

Vigdis Nygaard,1 Vegar J. Dagenborg,1 Olga Østrup,2 Einar A. Rødland,2 Veronica Skarpeteig,2 Laxmi Silwal-Pandit,2 Krzysztof Grzyb,3 Audun E. Berstad,4 Åsmund A. Fretland,5 Gunhild M. Mælandsmo,1 Anne-Lise Børresen-Dale,2 Anne H. Ree,6 Bjørn Edwin,5 Kjersti Flatmark1. 1 _Dept. of Tumor Biology, Institute for Cancer Research, Oslo University Hospital, The Norwegian Radium Hospital, Oslo, Norway;_ 2 _Dept. of Cancer Genetics, Institute for Cancer Research, Oslo University Hospital, The Norwegian Radium Hospital, Oslo, Norway;_ 3 _Dept. of Pathology, Oslo University Hospital, Rikshospitalet, Oslo, Norway;_ 4 _Dept. of Radiology, Oslo University Hospital, Norwegian Radium Hospital, Oslo, Norway;_ 5 _The Intervention Centre, Oslo University Hospital, Rikshospitalet, Oslo, Norway;_ 6 _Dept. of Oncology, Akershus University Hospital, Lørenskog, Oslo, Norway_.

Colorectal cancer (CRC) is one of the most common cancers and one of the leading causes of cancer death in the Western world. Up to 50% of patients with CRC develop metastatic disease and the liver is the most common site. The recently identified consensus molecular subtypes (CMS1-4) based on analyses of primary CRC have prognostic and therapeutic implications, but it is unclear whether these molecular subtypes are valid for metastatic disease. In this study, characterisation of CRC liver metastases (CLM) was performed at multiple molecular levels to identify characteristics relevant to metastatic disease. Molecular stratification of a defined metastatic CRC cohort may yield results of clinical relevance and novel treatment opportunities.

Surgically resected CLM and tumor-adjacent liver tissue from 46 patients were analysed for the presence of mutations (targeted deep sequencing), genome-wide copy number alterations (CNA), and transcription profiled. Molecular profiles of CLM and tumor-adjacent liver tissues were analyzed and associations with clinicopathological features and outcome were investigated. We found oncogenic mutations in all except one tumor. Both mutation and CNA profiles were similar to profiles reported for primary CRC. A CMS classifier tool applied to gene expression data, revealed the cohort to be highly enriched for CMS2. Unsupervised clustering based on genes with highly variable expression identified a 55-gene cluster that segregated the samples into two subgroups. The segregation pattern was replicated in relevant publicly available data sets, but the clinical significance of this observation is not evident. The 55 genes were associated with lipid metabolic and immune-related functions, revealing a role of the tumor-host microenvironment. The engagement of the immune system was further underlined by analyzing subgroups defined by neoadjuvant chemotherapy (NACT) administration, which revealed altered expression of inflammatory mediators and immune regulatory genes. The majority of treated patients had received Oxaliplatin based chemotherapy.

The uniform classification of CLM by CMS may reflect the patient composition in our cohort, but may also indicate that novel class discovery approaches need to be explored to uncover clinically useful molecular stratification of CLM. Identified immune-related gene expression signatures associated with molecular and clinical features underline the integration and importance of the immune interactome in resectable CLM. Specifically, the transcriptomic snapshot of NACT exposed CLM revealed altered genes associated with immunogenic cell death but also immune suppression. These results point to rational exploration of immune-modulating strategies in CLM in combination with NACT to increase efficacy and broaden treatment opportunities for this patient group.

#1753

**Refining breast cancer characterization through single-cell analysis of** ex vivo **reprogrammed tumor and adjacent normal cells.**

Harikrishna Nakshatri, Manjushree Anjanappa, Angelo Cardoso, Lijun Cheng, Safa Mohamad, Andrea Gunawan, Susan Rice, Yan Dong, Lang Li, Edward Srour. _Indiana Univ. School of Medicine, Indianapolis, IN_.

Discovery of inter-individual functional variations in gene regulatory elements and the observation that tumor and normal tissues of the same organ are in different differentiation states necessitate rethinking of gene expression based subclassification/characterization of tumors. To address this issue, we performed single cell genomics of breast tumors and adjacent-normal cells propagated using epithelial reprogramming growth conditions for a short duration. Epithelial cells analyzed were either unselected for specific subpopulation or phenotypically defined undifferentiated ALDH+/CD49f+/EpCAM+ luminal progenitors present in the normal breast, which express both basal and luminal-enriched genes. Expression of 93 genes that included PAM50 intrinsic subtype classifier and stemness-related genes was analyzed in 420 tumor and 284 adjacent-normal cells. ALDH+/CD49f+/EpCAM+ tumor and normal cells clustered differently compared to unselected tumor and normal cells. PAM50 genes-set analyses of ALDH+/CD49f+/EpCAM+ populations efficiently identified major and minor clones of tumor cells with the major clone resembling clinical parameters of the tumor. Similarly, stemness-associated gene set identified clones with divergent stemness pathway activation within the same patient sample. This refined technique distinguished genes truly deregulated in cancer from genes that identify cellular precursors of tumors. Collectively, assays presented here may enable precise identification of genes deregulated in cancer, early identification of therapeutically-targetable minor population of tumor cells, and eventually to refinement of precision therapeutics.

#1754

A new glycan biomarker for pancreatic cancer complements CA19-9 and identifies a distinct subpopulation of cancer cells.

Daniel Barnett,1 Ying Liu,1 Katie Partyka,1 Galen Hostetter,1 Herbert Zeh,2 Aatur D. Singhi,2 Ying Huang,3 Richard R. Drake,4 Randall E. Brand,2 Brian B. Haab1. 1 _Van Andel Institute, Grand Rapids, MI;_ 2 _University of Pittsburgh Medical Center, Pittsburgh, PA;_ 3 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 4 _Medical University of South Carolina, Charleston, SC_.

A major challenge in diagnosing and treating pancreatic cancer is the complex diversity between and within tumors in cellular morphologies and behaviors. Molecular markers to detect and classify specific variants of cancer cells could facilitate drug targeting of subtypes of cancer cells. We recently found that a glycan related to the CA19-9 antigen is a strong serological biomarker and is elevated in the plasma of about half of the patients with low CA19-9. We hypothesized that the cancer cells producing CA19-9 are a different subtype than those producing the new biomarker, referred to as sTRA. Using multimarker immunofluorescence on tissue microarrays, we found that sTRA was significantly elevated (p < 0.001) in tumor tissue relative to adjacent pancreatic tissue in 3 separate TMAs covering 38 patients. The sTRA and CA19-9 markers were present in 31/38 (82%) and 20/38 (53%) of the tumors, respectively, with 17/38 (45%) displaying both and 3/38 (8%) displaying neither. The morphologies of the cancer cells fell into distinct categories based on glycan expression. Cells with dual staining of both markers tended to be in well-to-moderately differentiated ducts with good nuclear polarization and foamy cytoplasm, or with flat and thin epithelium. Cells with predominant or exclusive CA19-9 staining tended to be part of well-differentiated ducts with mucinous cytoplasm or moderately-differentiated ducts with poor membrane integrity. In contrast, strong sTRA staining was present in small clusters of cells or single cells with poor differentiation and large vacuoles, or in small and invasive ducts. The phenotypes were consistent in xenograft models made with either cells lines or patient-derived material, as well as in 2D culture. Costaining with protein markers of differentiation showed that the CA19-9-positive/sTRA-negative regions colocalized more with MUC5AC, CK19, and E-cadherin relative to the sTRA-positive cells, indicating ductal differentiation, and the sTRA-positive/CA19-9-negative regions were more likely to colocalize with beta-catenin than any other marker, indicating potential for anchorage-independent growth or altered cellular cohesion. The time-to-progression after surgery was associated with the relative levels of the two markers, based on staining of tumors from 48 patients in an independent TMA. Patients with higher dual-staining of CA19-9 and sTRA had statistically longer (p = 0.01 by log-rank test) time-to-progression, and patients with higher exclusive staining of either marker relative to the dual staining had shorter time-to-progression. Thus the sTRA marker used in conjunction with CA19-9 could provide a basis for classifying and targeting subtypes of cancer cells.

#1755

The fate of germline BRCA related mutations in breast tumor tissues.

Vassiliki Kotoula,1 Florentia Fostira Fostira,2 Kyriaki Papadopoulou,3 Paraskevi Apostolou,2 Eleftheria Tsolaki,4 Georgios Lazaridis,5 Kyriaki Manoussou,5 Flora Zagouri Zagouri,5 Dimitrios Pectasides,5 Ioannis Vlachos,4 Ioannis Tikas,4 Sotiris Lakis,4 Irene Konstantopoulou,6 George Pentheroudakis,5 Helen Gogas,5 Pavlos Papakostas,5 Christos Christodoulou,5 Drakoulis Yannoukakos,6 George Fountzilas4. 1 _Aristotle Univ. School of Medicine, Thessaloniki, Greece;_ 2 _National Center for Scientific Research NCSR Demokritos, Athens, Greece;_ 3 _Hellenic Foundation for Cancer Research, Athens, Greece;_ 4 _Hellenic Foundation for Cancer Research, Greece;_ 5 _Hellenic Cooperative Oncology Group, Greece;_ 6 _National Center for Scientific Research NCSR Demokritos, Greece_.

Background - aim: Germline mutation carriers in BRCA1 and related genes frequently develop triple-negative breast carcinomas (TNBC) that presumably preserve the inherited mutation. Herein, we examined the clinicopathologic features and prognostic impact of paired germline and tumor genotypes in this disease.

Methods: We compared baseline germline and paraffin tumor genotype data (next generation [NGS] and Sanger sequencing) from 194 patients with operable TNBC who were treated with anthracyclines-taxanes based chemotherapy within adjuvant trials by our Group. Because tissue NGS did not target BRCA-related genes, we additionally interrogated the presence of germline mutations in tumor and non-cancerous breast tissues, in carriers with available tissue material (n=33). We validated all NGS germline mutations with Sanger sequencing and ensured matched germline/tumor identity with microsatellite testing.

Results: We identified 50 (26%) germline mutation carriers, 39 in BRCA1, 4 in BRCA2 and the rest in RAD51C, BARD1, RAD50, NBN, MRE11A and BRIP1. BRCA1 carriers were younger as compared to non-BRCA1 carriers (median 42y.o. vs. 56y.o, respectively; p=0.020), premenopausal (p=0.014), and had lower nodal status (p<0.001). Somatic mutations were found in 136 tumors, TP53 in 113 (81%). The germline mutant allele was lost in 14/33 (42%) tumor and matched normal tissues (mut-LOH). Loss affected 12 BRCA1 mutations, among them the common pathogenic p.Q1756fsX74 (c.5266dupC) and p.G1738R (c.5212G>A), and 2 BRCA2 mutations. In one such tumor, the germline mutation was replaced by a somatic BRCA1 (p.Q1811X [c.5431C>T]). Tumors with mut-LOH were more often TP53 mutant as compared to those with preserved germline mutations, which had mutations in other genes as well (p=0.034). Overall though, TP53 mutations were not associated with germline status. Germline mutations and tumor TP53 mutations were not individually associated with patient disease-free survival (DFS) but interacted with each other: in non-carriers, TP53 status did not affect outcome; in carriers, those with mutant TP53 had unfavorable DFS compared to those with wild-type TP53 tumors (interaction p, multivariate analysis: 0.053).

Conclusions: Germline BRCA-related TNBC may be distinguished into two groups with potentially different biological characteristics, those with preserved and lost inherited mutation. The interaction between germline status and tumor TP53 mutations may have treatment implications and seems worthy pursuing in larger studies. Combined germline/tumor genotype testing may be needed for TNBC patient classification, especially in the context of clinical trials.

#1756

Genomic reclassification of endometrial carcinoma predicts drug response and facilitates refinement of current management based on PDX-guided efficacy outcomes.

Chieh-Hsiang Yang,1 David K. Gaffney,2 Theresa L. Werner,3 Elke A. Jarboe,4 Jason Gertz,5 Katherine E. Varley,5 Matthew Peterson,1 Margit M. Janat-Amsbury1. 1 _Department of OB/GYN, University of Utah, Salt Lake City, UT;_ 2 _Department of Radiation Oncology, University of Utah, Salt Lake City, UT;_ 3 _Department of Medicine, Division of Oncology, University of Utah, Salt Lake City, UT;_ 4 _Department of Pathology, University of Utah, Salt Lake City, UT;_ 5 _Huntsman Cancer Institute, Salt Lake City, UT_.

Endometrial cancer (EC) is the most common gynecological malignancy among American women. Steadily increasing numbers of newly diagnosed cases and deaths emphasize the necessity of improving current management strategies. Rather than the commonly known two types of EC based on histological classification, the Cancer Genome Atlas (TCGA) Research Network recently reported a reclassification that categorizes EC into four subtypes based on genomic characterization. Molecular similarities between these subtypes and other cancers, including colorectal, ovarian, and breast cancers, were identified. But to date, no prospective validation has delivered an improved understanding on how to translate these findings to the clinic in the form of treatments adapting genomic guidance.

Objectives: To evaluate drug response according to TCGA classification using a panel of twenty-seven established endometrial cancer patient-derived xenografts (EC-PDXs).

Methods: Twenty-seven EC-PDXs representing various stages of disease and histological subtypes were orthotopically transplanted and propagated over multiple generations. Upon thorough characterization including histology, metastatic status, and molecular features, EC-PDXs were further categorized into the four TCGA molecular subtypes on the basis of somatic copy number variations (CNV), microsatellite instability (MSI), and POLE mutations. A PDX clinical trial (PCT) was performed to re-evaluate current standard treatments in comparison to therapeutic agents adopted from treatment regimens for other cancer types based on their molecular similarities.

Results: EC-PDXs established from patients with recurrent disease exhibited minimal activity to first-line chemotherapeutic drugs, as expected, but remained sensitive to other second-line drugs. None of the tested drugs exhibited sufficient activity in POLE EC-PDXs. MSI EC-PDXs were found to be less sensitive to platinum-based treatment and resistant to 5-FU as reported for MSI colorectal cancer. CNV-high EC-PDXs were more sensitive to drugs adopted from current ovarian and breast cancer treatment regimens.

Conclusions: This is the first study that demonstrates the utility of TCGA classification for treatment guidance based on a PCT. Patients with recurrent and persistent disease are in desperate need of new therapeutic options. Our findings open up the possibility to utilize drugs which are currently used in the treatment of other cancers for the treatment of EC based on molecular similarities. In addition, this is the first study that successfully demonstrated the use of molecular classification to enable the prediction of drug response, and thus may facilitate the refinement of current EC management in a more precise and personalized way.

#1757

Spectrum of BAP1 mutations identified in diverse cancer lineages.

Zoran Gatalica, Joanne Xiu, Jeffrey Swensen, Wangjuh Chen. _Caris Life Sciences, Phoenix, AZ_.

Background: Germline mutations in the tumor suppressor gene, BAP1, a deubiquitylase that regulates key cellular pathways, are associated with a recently-described tumor predisposition syndrome characterized by early onset benign melanocytic skin tumors, and a significant risk of cancers that include mesothelioma, cutaneous and uveal melanoma, renal cell carcinoma (RCC) and cholangiocarcinoma. Somatic or germline BAP1 mutations have been associated with an aggressive course and a poor prognosis in RCC and cholangiocarcinoma and may render the cancer cells more sensitive to HDAC inhibitors or Parp inhibitors. We investigated types and frequencies of BAP1 mutations in a large cohort of diverse malignancies and their associations with other molecular/genomic characteristics.

Methods: A total of 9782 tumor samples from over 40 cancer types were molecularly profiled at Caris Life Sciences by next generation sequencing (Illumina NextSeq platform and Agilent SureSelect XT panel, 592 genes). Microsatellite instability (MSI) was tested by PCR and fragment analysis (Promega MSI Analysis System).

Results: A pathogenic somatic or germline BAP1 mutation was identified in 20 cancer types, with a total of 129 tumors with mutations found (1.3% prevalence). As expected, BAP1 mutations were frequently seen in uveal melanoma (50% or 24 in 48), malignant pleural mesothelioma (29% or 6 in 21), RCC (8% or 12 in 150), cholangiocarcinoma (6.6% or 13 in 196), and cutaneous melanoma (2.2% or 7 in 319). In addition, pathogenic BAP1 mutations were detected in carcinomas arising in parotid gland (10.3% or 3 in 29), anus (8.3% or 3 in 36), cervix (3.4% or 4 in 117), stomach (3.3%, or 6 in 180), head and neck (1 in 97 or 1%), lung (14 in 1590 or 0.9%), and breast (0.8% or 7 in 887). A mutation was also noted in a meningioma (1 in 43) and a uterine sarcoma (1 in 96). Variants of BAP1 (pathogenic, presumed pathogenic, and variants of unknown significance) were more often seen in MSI-high tumors (compared to MSS) in both colorectal (12/61 vs. 18/1003) and endometrial carcinomas (5 of 69 vs. 5 of 321; both p<0.01). It was not determined whether the BAP1 mutations were somatic or germline in origin.

Conclusions: The study confirmed the presence of pathogenic BAP1 mutations in carcinomas commonly associated with BAP1 germline and somatic mutations. It also identified BAP1 mutations in additional cancer types (parotid gland, anal and cervical carcinomas), as well as its association with MSI-H cancers (colon and endometrium). Evaluation of mutations in non-cancerous tissues is underway to determine if these novel cancer associations are related to germline predisposition.

#1758

A more mature immunophenotypic makeup of multiple myeloma clone(s) at diagnosis correlates with a higher genomic instability.

Marina Martello,1 Rosalinda Termini,1 Barbara Santacroce,1 Enrica Borsi,2 Vincenza Solli,1 Lucia Pantani,1 Elena Zamagni,1 Paola Tacchetti,1 Francesca Ulbar,1 Gabriella Chirumbolo,1 Mario Arpinati,1 Giovanni Martinelli,1 Michele Cavo,1 Carolina Terragna1. 1 _University of Bologna, Bologna, Italy;_ 2 _Fondazione Umberto Veronesi, Milano, Italy_.

Intro The sequence of events underlying the process of Multiple Myeloma (MM) plasma cells (PC) differentiation have not yet fully elucidated, even if recent findings suggest that different cell subpopulations, with distinct phenotype, compose the MM clone(s), whose plasticity has emerged as a typical feature.

Aim To evaluate the phenotypic plasticity and genomic background of MM clone(s) at diagnosis in order to stratify patients (pts) according to the PC differentiation stages and possibly correlate with disease outcome.

P&M Phenotypic characterization of both CD138+/CD38+ and CD19+ populations was carried out on 44 newly diagnosed MM. Fresh BM samples was analysed by 6color multiparametric flow citometry analysis, combining CD138PE, CD38-PE-Cy7, CD20-APC, CD19-APC-Cy7, CD27-FITC, CD45-FITC, CD28-APC, CD44-FITC, CD54-APC, CD81-PerCP-Cy5.5, CD56-APC and SHH-PE, as a functional marker of Hedgehog pathway activation (Miltenyi Biotech). Cytoscan HD array were carried out in order to detect genomic copy number alterations (CNAs).

Results According to the CD19 and CD81 markers co-expression, pts were stratified in 3 subgroups, recapitulating a progressive PC maturation process: the most immature one, including pts with CD19+/CD81+ PC (11/44 = 25%); the intermediate CD19-/CD81+ phenotype one (19/44 = 43%), and the CD19-/CD81- PC one, whose clone was mainly composed by most mature PCs.

The two extreme subgroups were characterized by a differential expression of maturation markers: the more mature PCs displayed a higher expression of CD28 and CD44, which usually characterized advanced disease stages, as well as a reduced expression of CD20, CD27 and CD45, commonly associated to preceding PC differentiation stages (p<.05). Any differential expression of SHH pathway's ligand was observed. On the contrary, a higher Hedgehog pathway activation was detected in the immature CD19+ compartment of pts with immature plasma cells (median SHH expression CD19+/CD81+ vs. CD19-/CD81-: 98,1% vs. 11,3%; p<.05), which probably highlighted a less quiescent immature reservoir pool.

The CNAs analysis showed that mature PCs were characterized by a higher genomic instability, as compared to the more immature ones (total CNA: 306.9 vs. 171.5, p 0.08; genome changed: 36.96 vs. 13.55, p 0.01), including several alterations commonly associated to worse prognosis (e.g. del17p).

Finally, a higher frequency of baseline clinical characteristics associated to bad prognosis was observed in the more mature, as compared to the more immature subgroups of pts (e.g. n. PET lesions, k/l ratio; p<.05).

Conclusion MM clone(s) is a mixture of different cell populations endowed with an inner phenotypic plasticity. Various PC differentiation stages were appreciable already at diagnosis and genomic features associated to bad prognosis characterize pts carrying more mature clones.

Acknowledgements: AIRC, Berlucchi, Prog. Bolondi, FUV

#1759

Description of molecular marker disparities and KRAS mutational spectrum of colorectal tumors from Puerto Rican Hispanics.

Julyann Perez-Mayoral,1 Camila Rivera-Lynch,1 Marievelisse Soto-Salgado,2 Xavier Llor,3 Luis Tous-Lopez,4 Marla Torres-Torres,4 Jose S. Reyes,4 Carlos Torres,5 Ajay Goel,6 Segundo Rodriguez-Quillichini,2 Marcia Cruz-Correa1. 1 _University of Puerto Rico Comprehensive Cancer Center, San Juan, PR;_ 2 _University of Puerto Rico Medical Sciences Campus, San Juan, PR;_ 3 _Yale University, New Haven, CT;_ 4 _Ashford Presbyterian Hospital, San Juan, PR;_ 5 _Puerto Rico Cancer Registry, San Juan, PR;_ 6 _Baylor Research Institute, Dallas, TX_.

Colorectal cancer (CRC) causes 27% of all cancer deaths among Puerto Rican Hispanics (PRH). Most CRC cases are due to sporadic genetic or epigenetic events that lead to the development of the tumor. These events aggregate to cause CRC carcinogenesis and involve genes that regulate cell growth and differentiation. Improving understanding of the molecular events that lead to CRC carcinogenesis has the potential to reduce CRC mortality by risk stratifying individuals as well as developing prevention strategies. Currently, there are a set of molecular markers that are used to characterize CRC tumors and inform prognosis and treatment decisions. These markers include: microsatellite instability (MSI), CpG island methylation and mutational analysis of the KRAS and BRAF oncogenes. The objective of this study was to characterize the molecular markers present in the CRC tumors of PRH and understand the disparities present at the molecular level in these tumors. The frequency of the MSI and CIMP were evaluated in 488 CRC tumors from PRH. Mutations in the KRAS gene and the BRAF V600E mutation were evaluated using SuperArray technology. Additionally, information regarding sociodemographic and clinicopathologic characteristics of the study population was obtained. Of the 488 evaluated CRC tumors 1.6% (n=2) showed MSI and 90.2% of the studied tumors had CIMP low phenotype (n=102). MSI-High tumors were more likely to be distal tumors compared to MSS tumors (p=0.02). The V600E mutation in the BRAF gene were found in 9.17% (n=11) of the studied tumors. KRAS mutations were found in 31.25% (n=40) of tumors. The most common KRAS mutations found in the CRC tumors of PRH were: c.35G>A (KRAS COSMIC521) (n=12) and c.38G>A (n=8) (KRAS COSMIC532). Furthermore, the traditional CRC carcinogenesis pathway was the predominant pathway observed for the development of the CRC tumors of our study population. The following study highlights a distinct molecular signature for the CRC tumors from PRH descent. The disparities observed in the prevalence of the molecular markers (low MSI, CIMP Low and mainly wild-type for KRAS and BRAF) suggest that the drivers for CRC in this population might differ from other populations. Additional studies are required to fully elucidate the CRC carcinogenesis pathway in PRH.

#1760

High expression of CASZ1 is associated with poor prognosis in ovarian cancer.

Yuan-Jhe Chuang, Yu-Ling Liang, Yuh-Ling Chen, Tse-Ming Hong, Keng-Fu Hsu. _National Cheng Kung Univ. Hospital, Tainan, Taiwan_.

Epithelial ovarian cancer (EOC) carries the highest mortality rate of all gynecologic malignancies. This high mortality rate is attributed to the fact that most cases of ovarian cancer are detected at late stages when metastases are already present. We previously demonstrated that castor zinc finger 1 (CASZ1) is up-regulated in EOC cells and promotes EOC cells metastasis. However, the relationship between the ovarian cancer patient's prognosis and CASZ1 expression is not clear. In the present study, we examined CASZ1 and CA125 expressions in EOC using immunohistochemistry and correlated their expression levels with patient survival. From January 2008 to January 2012, 144 EOC patients who underwent staging or cytoreductive surgery at the National Cheng Kung University Hospital (NCKUH) were consecutively enrolled. Clinical and pathology information, including age, stage, cell type, chemo-response, and survival, was collected from medical charts. The staging met the criteria of the International Federation of Gynecology and Obstetrics Classification (FIGO). Histological classification was defined according to the classification standards of the World Health Organization. The overall survival (OS) was calculated from the date of diagnosis. Progression-free survival (PFS) was measured as the period from surgery to the date of confirmed recurrence or disease progression or to the date of the investigators' last note of a disease-free status. CASZ1, CA125 expression was evaluated by TissueFaxs microscopy system using an image analysis program. Five fields of the tumor in each slide (corresponding to a mean of 10000 tumors cells per case) were photographed. Images were acquired from using fluorescence-activated cell sorting (FACS)-like tissue cytometry. The percentage of positive cells in each sample was further quantified using TissueQuest software (TissueGnostics, Vienna, Austria). The CASZ1 was significantly upregulated in advanced EOC tissues, compared with early stage tumors. High CASZ1 expression levels were significantly associated with worse EOC clinical characteristics. By univariate survival analysis, high CASZ1 levels significantly correlated with decreased overall survival, progression-free survival. In addition, patients with both high expression with CASZ1 and CA125 carried the worst prognosis. In conclusion, we demonstrate that high expression of CASZ1 levels correlates with an aggressive EOC phenotype and may contribute for poor prognosis in EOC patient. Determination of CASZ1 could be clinically useful in identifying high-risk EOC patients for a more aggressive adjuvant therapy.

#1761

**A rapid and accurate nucleic acid amplification and detection method for** KRAS **mutation testing in colorectal cancer specimens.**

Yong Shin, Choong Eun Jin, Seung-Seop Yeom, Seok-Byung Lim. _University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea_.

Colorectal cancer (CRC) is one of the most common type of cancers both men and women in worldwide. Fortunately, overall death rates of CRC have been decreasing for the last two decades due to the improvement of screening test assays that detect early-stage cancer and pre-cancerous polyps. Nevertheless, the most common treatment for CRC is surgery, because it may completely eliminate the cancer region. In case of the cancer with systemic metastasis, chemotherapy is required before or after surgery for primary or metastatic lesions. Among the regimens for the chemotherapy, both monoclonal antibodies (Cetuximab and Pantitumumab) against the epidermal growth factor receptor have been shown to improve survival for only patients with lack of RAS mutations. Thus, the KRAS gene mutations (codons 12 and 13) in CRC patients have been extensively studied as a strong negative predictive biomarker to indicate whether a CRC patient responds to the treatment. Therefore, testing the KRAS mutational status of tumor samples is becoming an essential tool for managing patients with CRCs. Although a myriad of nucleic acid testing methods have been developed to analyze the mutation status in the key regions of the KRAS gene of CRC, several obstacles still remain related to low sensitivity, time consuming, and required large instruments including thermal cyclers. Here, we present a novel nucleic acid amplification and detection method for KRAS mutations (G12D and G13D) testing that enable rapid and accurate detection. This method is based on combination of isothermal DNA amplification method and bio-photonic silicon sensor that can be detected the mutations in a label-free and real-time manner. The proposed method can detect the mutant cell present at 1% in a mixture of wild type cells, while both PCR and sequencing can detect the mutations in a sample containing approximately 30% of mutant cells. We used 60 tissue samples from CRC patients (22 samples with G12D mutations, 23 samples with G13D mutation, and 15 samples with no mutation) to compare the clinical utility of three methods including PCR, Sanger sequencing and the proposed method. The proposed method with both G12D and G13D showed a value of 100% and 100% for sensitivity and specificity, respectively. One the other hand, the sensitivity and specificity of PCR (90.5% and 100%) and sequencing (95% and 100%) were lower than that of the proposed method. Therefore, the proposed method was found to be a rapid (< 30 min), highly sensitive and specific method for KRAS mutation testing. We believe that this rapid and accurate method will enable proper treatment for CRC patients.

#1762

Keratin 17 identifies prognostic subtypes of pancreatic ductal adenocarcinoma.

Luisa F. Escobar-Hoyos,1 Cindy Leiton,2 Elizabeth Vanner,2 Lucia Roa-Pena,2 Jinyu Li,2 Scott Powers,2 Ali Akalin,3 Jela Bandovic,2 Peter Bailey,4 David Chang,4 Richard Moffitt,5 Jen Jen Yeh,5 Andrew Biankin,4 Kenneth Shroyer2. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Stony Brook University, Stony Brook, NY;_ 3 _University of Massachusetts Memorial Medical Center, Worcester, MA;_ 4 _Wolfson Wohl Cancer Research Centre, Glasgow, United Kingdom;_ 5 _Lineberger Comprehensive Cancer Center, Chapel Hill, NC_.

Recent RNA sequencing (RNASeq) studies from Bailey et al., 2016, Moffitt et al., 2015 and Collisson et al., 2011 reported that mRNA expression from bulk tumor defines molecular subtypes of pancreatic ductal adenocarcinoma (PDAC) that are highly correlated with patient survival. These studies independently identified Keratin 17 (K17) mRNA overexpression as one of over 20 upregulated transcripts that define the RNA signature of the most lethal PDACs. Studies in our lab determined that neither K17 gene missense mutations nor copy-number alterations explain the upregulation of K17 mRNA expression by malignant cells. In addition, we found that K17 expression, measured at the protein level by immunohistochemistry or at the level of mRNA by RNASeq, is sufficient to stratify patients by short- vs long- term survival at baseline after resection. These retrospective survival analyses were performed in four independent patient cohorts using a uniform threshold to define low- vs high- K17 patients (total n= 558). High-K17 cases were twice as likely to die from this disease compared to stage-matched low-K17 cases (P values < 0.05). Furthermore, we determined that K17 expression is associated with outcome after Gemcitabine treatment using the Bailey et al., 2016 patient cohort (n= 94). This is the first study to show that the expression of a single gene, K17, can accurately subtype PDAC at initial diagnosis. In conclusion, K17 was identified as a robust and independent, clinically relevant, prognostic and predictive biomarker to stratify clinical outcome at the time of initial diagnosis and to potentially inform clinical decisions regarding chemotherapeutic intervention.

#1763

**A novel RNA** in situ **hybridization approach highly sensitive for detection of Merkel cell polyomavirus.**

Lisha Wang,1 Paul W. Harms,1 Nallasivam Palanisamy,2 Shannon Carskadon,2 Xuhong Cao,1 Javed Siddiqui,1 Rajiv M. Patel,1 Sylvia Zelenka-Wang,1 Timothy M. Johnson,1 Alison Durham,1 Fengyun Su,1 Sudhanshu Shukla,1 Rohit Mehra,1 Arul M. Chinnaiyan1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _Henry Ford Health System, Detroit, MI_.

Background: Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine tumor of the skin. Merkel cell polyomavirus (MCPyV) plays an oncogenic role in the majority of MCC tumors. Detection of MCPyV in MCC tumors has diagnostic utility, and potentially prognostic and therapeutic implications. We investigated whether RNAscope, a novel RNA in situ hybridization (ISH) method for detection of RNA transcripts in tissues at the single-cell/single-molecule level, is useful for MCPyV detection.

Methods: We applied an RNAscope probe targeting MCPyV T antigen transcripts on tissue microarrays (TMAs) and full tissue sections encompassing 91 MCC tumors from 75 patients, 14 carcinomas of other types, and various normal tissues. qPCR and immunohistochemistry (IHC) for MCPyV were performed on 58 and 88 cases, respectively. qPCR was also performed on 18 cases of normal skin to establish background levels of MCPyV in non-tumor tissues.

Results: RNA-ISH demonstrated the presence of MCPyV in 46.2% (42/91) of cases. A total of 58 samples had data from all the three detection modalities (qPCR, RNA-ISH and IHC). RNA-ISH demonstrated 100% concordance with qPCR results, whereas IHC had a slightly lower concordance (96.6%). RNA-ISH demonstrated higher agreement than IHC among TMA cores from the same case, and between TMA cores and matched full tissue sections. Of samples with background inflammatory cells, 5 of 37 (15.6%) showed moderate nonspecific staining of lymphocytes by IHC, whereas RNA-ISH lacked background.

Conclusions: RNA-ISH is comparably sensitive to qPCR for detection of MCPyV, and allows for correlation with tissue morphology. Our findings support RNA-ISH as a highly effective method for MCPyV detection in tumors.

#1764

Accurate and reproducible detection of fusions and exon skipping events in NSCLC-derived samples using a comprehensive, targeted RNA-Seq system across multiple laboratories.

Gary J. Latham,1 Richard Blidner,1 Brian C. Haynes,1 Shobha Gokul,1 Maria L. Aguirre,2 Stephen Hyter,3 Ziyan Y. Pessetto,3 Maria Curtis,4 Dan Su,4 Tom Halsey,4 Victor Weigman,4 Patrick Hurban,4 Andrew K. Godwin,5 Leon C. van Kempen2. 1 _Asuragen, Austin, TX;_ 2 _Jewish General Hospital, Montreal, Quebec, Canada;_ 3 _University of Kansas Medical Center, Kansas City, KS;_ 4 _Q2 Solutions, Morrisville, NC;_ 5 _University of Kansas Cancer Center and University of Kansas Medical Center, Kansas City, KS_.

Introduction: Reliable assessment of cancer-associated RNA markers in lung cancer produced by gene-fusions or exon skipping events by next-generation sequencing requires integrated reagents, protocols, and interpretive software that can harmonize procedures and ensure consistent results across laboratories. We evaluated a comprehensive system for targeted RNA-Seq that includes reagents for nucleic acid quantification, library prep, run controls, and companion bioinformatics software. The reproducibility of this system was evaluated in a multi-phase study design at 5 independent laboratories.

Methods: Total nucleic acid (TNA) was isolated from formalin-fixed, paraffin-embedded (FFPE) residual non-small cell lung cancer (NSCLC) tumor biopsies and cell-lines (RT-112, H596, HCC78). These TNA isolates were used to prepare a set of 30 test samples, including a dilution series to assess assay sensitivity. A non-template control and kit positive control were also included. The sample set was evaluated using the QuantideX® NGS RNA Lung Cancer Kit RUO (Asuragen) and sequenced on the MiSeq® system (Illumina) at Asuragen and 4 independent laboratories. Analyses were conducted using QuantideX® NGS Reporter RUO (Asuragen), a software suite that includes a FASTQ processing pipeline and incorporates pre-analytical QC information into the fusion-caller algorithm and reporting tool.

Results: Laboratories were trained on the assay workflow and companion bioinformatics software in less than two days followed by independent library preparation and sequencing workflows. A total of 266 sample libraries were evaluated from inputs down to <10 ng. A single library was excluded from sequencing due to a failed QC status. Specific targeted fusions (ALK, ROS1, FGFR3) and splice variants (MET exon 14 skipping) were detected in 132 libraries and were concordant at all sites. Designs to detect 3'/5' expression imbalances reported the presence of gene fusions for ALK and ROS1 in a total of 96 libraries with one aberrant call and one missed call, which occurred in libraries flagged as "at risk" by the interpretive software.

Conclusions: The accuracy of this novel targeted NGS assay for RNA fusions and splice variants in NSCLC was demonstrated in a multi-site laboratory evaluation using clinically-relevant specimens and low inputs of TNA. The ability of the panel to detect both common and rare gene fusion transcripts and exon skipping events within an integrated wet- and dry-bench workflow provides a foundation for the reliable detection of oncogenic RNA fusions and aberrant splicing events that can respond to current and emerging targeted therapies. This study highlighted the ease of implementation and consistent performance that can be achieved in different laboratories when the process from sample-to-report is highly integrated.

#1765

Molecular and immunological characterization of non-small cell lung cancer harboring cMET alterations.

Maria Saigi,1 Carolina Pereira,1 Eva Pros,1 Elisabeth Brambilla,2 Ernest Nadal,3 Montse Sánchez-Céspedes1. 1 _IDIBELL, Barcelona, Spain;_ 2 _Centre Hospitalier Universitaire de Grenoble, Grenoble, France;_ 3 _Catalan Institute of Oncology, Barcelona, Spain_.

Purpose: To explore the correlation between the expression of immunological biomarkers in a subset of NSCLC tumors presenting cMET activation.

Methods: We retrospectively reviewed 200 tumor samples from a cohort of patients diagnosed with NSCLC in 3 different hospitals, from Spain and France. cMET activation was defined by the presence of one or more of the following criteria: 1- cMET mutations inducing exon 14 skipping (METex14), 2- cMET amplification, assessed by FISH 3- cMET overexpression, assessed by immunohistochemistry (IHC). METex14 mutations were determined by genomic sequencing and RT-PCR. FISH was interpreted according to Capuzzo scoring system, considering 5 or more gene copies as the mean value for positive amplification. Concurrent genetic alterations in EGFR and ERBB2 tyrosine kinase receptors were also determined. PD-L1 expression and density of tumoral infiltrating CD8+ lymphocytes were assessed by IHC. Statistical correlation analysis was performed by Chi-square and Fisher's exact test.

Results: METex14, cMET amplification and cMET overexpression were detected in 2%, 7.8% and 31% of tumors, respectively. Histological subtypes included: 70% adenocarcinoma, 20% squamous cell carcinoma, 8% large cell, 2% pulmonary sarcomatoid. Curiously, one of the METex14 adenocarcinoma was concurrent with ERBB2 amplification. Those tumors harbouring METex14 or cMET amplification were correlated with positivity for PD-L1 expression (p = 0.02). However, this association was not observed in cMET overexpressed tumors.

Conclusions: We observed a direct correlation between PD-L1 positivity and METex14 or cMET amplification. Functional analysis and a large cohort of tumors are required to better characterize the potential role of cMET pathway in the regulation of PD-L1 expression.

#1766

Distinct pattern of alterations in tp53 mutated and wild type acute myeloid leukemia (AML) patients.

Anna Ferrari,1 Eugenio Fonzi,1 Maria Chiara Fontana,1 Andrea Ghelli Luserna Di Rorà,1 Marco Manfrini,1 Antonella Padella,1 Carmen Baldazzi,1 Cristina Papayannidis,1 Giovanni Marconi,1 Stefania Paolini,1 Viviana Guadagnuolo,2 Margherita Perricone,1 Valentina Robustelli,1 Enrica Imbrogno,1 Eugenia Franchini,1 Claudia Venturi,1 Elisa Zuffa,1 Maria Chiara Abbenante,1 Giorgia Simonetti,1 Nicoletta Testoni,1 Emanuela Ottaviani,1 Martinelli Giovanni1. 1 _Institute of Hematology, Bologna, Italy;_ 2 _CellPly srl, Bologna, Italy_.

Background: Mutations in TP53 gene predict a poor prognosis in patients with AML. The reported TP53 mutation rate in AML is low (2.1%) by contrast AML with a complex karyotype (CK) is higher (69-78%) and have a poor outcome. Quite common is to found paired TP53 mutation together and a segmental 17p deletion.

Aims: To investigate the frequency, the types of mutations, the associated cytogenetic, the correlation with known molecular alterations and the prognostic role TP53 mutations in adult AML pts. Moreover we would identify genes/pathways that are mainly affected in the mutated TP53 group compared to the wt one.

Patients and Methods: 258 adult AML pts with miscellaneous cytogenetic abnormalities and normal karyotype were examined in our Institution for TP53 mutations using several methods, including Sanger sequencing, NGS and HiSeq2000 platform and were correlated with cytogenetic analysis. 124/258 samples were genotyped with SNP arrays (Affymetrix, 3 250K, 43 SNP 6.0, 78 CytoScan HD). Copy Number Alterations (CNAs) analyses were performed using Chromosome Analysis Suite (Affymetrix) and Nexus Copy Number (BioDiscovery) software.

Results: Mutation analysis detected TP53 mutations on 39 patients with 48 different types of mutations (32 deleterious point mutations; 4 deletions); nine pts have 2 mutations. We found 34/48 (70%) missense mutations, 5 mutations in the splice sites, 4 deletions ,2 intronic and and 3 others mutations. The mutation rate is of 15.1%. Mostly (28/39) of the TP53 mutated pts (71.8%) had CK while only 11/39 (28.2%) mutated pts have "no CK" (P>0.0001). Alterations of TP53 were significantly associated with poor outcome (OS and EFS p<0.0001). To take advantage of different methodological characteristics, on TP53 locus, we matched two software and cytogenetic analysis results. We identify 16 mutated pts that were also deleted and one pt that presented only a deletion. Therefore 50% of mutated pts present a concomitant deletion. OS of TP53 mutated and deleted pts is statistically inferior respect to muted pts (p<0.0061). Comparing 32 TP53 mutated and 92 TP53 wt pts CNAs results that: a) chromosomes significantly alterated are 5q, 17p, 12p, 16q, 22q13.33 and 7q; b) over 900 genes are differentially involved (all in Loss); c) and that pathway categories mainly enriched are Signal Transduction, Metabolism, Immune System, Transmembrane transport of small molecules, Gene expression, Cell Cycle.

Conclusions: Our data demonstrated that TP53 mutations occur in 15.1% of AML with a higher frequency in the subgroup of CK-AML (p<0.0001). They predicted to be deleterious and significantly correlated with worse prognosis especially if TP53 is both mutated and also deleted. For these reasons, TP53 mutation/deletion screening should be recommended. Different pattern of alterations in mutated and wt groups have to be deeper investigated. ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project.

#1767

Analysis of biomarkers and anthracycline benefit for hormone receptor-negative breast cancer: results from a randomized phase 2 neoadjuvant study (KBOG 1101 Study).

Takashi Ishikawa,1 Kazutaka Narui,2 Mari S. Oba,2 Akimitsu Yamada,2 Kumiko Kida,2 Mikiko Tanabe,2 Yasushi Ichikawa3. 1 _Tokyo Medical University, Tokyo, Japan;_ 2 _Yokohama City University Medical Center, Yokohama, Japan;_ 3 _Yokohama City University, Yokohama, Japan_.

AIM: We compared 6 cycles of docetaxel and cyclophosphamide (TC6) with 3 cycles of 5-fluorouracil and epirubicin and cyclophosphamide followed by 3 cycles of docetaxel (FEC-D) as neoadjuvant chemotherapy for patients with hormone receptor (HR)-negative breast cancer (BC) to identify biomarkers requiring anthracycline treatment.

Methods: In total, 103 patients with operable HR-negative BC were administered TC6 or FEC-D. Triple-negative BC was subdivided by CK5/6 and EGFR into basal- and non-basal BCs. The primary endpoint was pathological complete response (pCR). Secondary endpoints were safety, breast-conserving surgery ratio, disease-free survival, overall survival, and predictive factors (Ki-67, P-53, ALDH1 and TOP2A by IHC and TOP2A by FISH) for each regimen.

Results: Of the 103 patients, 97 completed the study. Overall pCR was higher for patients treated with FEC-D (36%) than for those treated with TC6 (25.5%) (P=0.265). FEC-D was significantly superior to TC6 in basal BC (42.9% vs 13.6%; P=0.033), while no differences in HER2 and non-basal BCs. Aldehyde dehydrogenase 1 (ALDH1) positivity was inversely associated with pCR for both regimens, significantly for FEC-D (FEC-D: p=0.047, TC6: p=0.085). Patients who achieved pCR tended to have longer DFS (P = 0.287) and OS (P = 0.069). Patients with basal and non-basal BC treated with FEC-D had significantly better DFS (P = 0.016) and OS (P = 0.034) than those with TC6.

Conclusion: We found TC6 was less effective than FEC-D for HR-negative BC because it was not sufficient to treat basal-BC. This suggests that DNA damaging agents like anthracyclines are required for treating basal-BC. Additionally, ALDH1 could be a marker for resistance to conventional chemotherapy.

#1768

A practical tool for probing protein glycosylation in clinical samples: Application to characterizing subtypes of MUC5AC glycosylation in distinct subsets of pancreatic cancer patients.

Peter Y. Hsueh. _Van Andel Institute, Grand Rapids, MI_.

Glycans are critical to protein biology and are useful as disease biomarkers. Many studies of glycans rely on clinical specimens, but the low amount of sample available for some specimens limits the experimental options. We have a method to obtain information about protein glycosylation using a minimal amount of protein, and we applied it to the study of biomarkers in the blood plasma of pancreatic cancer patients. The method works by treating proteins that were captured by antibody microarrays (2.2 x 2.2 mm) with exoglycosidases to successively expose underlying features, and then probing the native or exposed features using a panel of lectins or glycan-binding antibodies. We use an algorithm to interpret the data and provide predictions about the glycan motifs that are present in the sample. We applied the method to the analysis of the glycosylation of a biomarker, MUC5AC, found in the plasma of patients with pancreatic cancer. An O-linked glycan on MUC5AC that is elevated in the plasma of about 75% of patient had mostly 2,3 linked sialic acid on type-1 N-acetyl-lactosamine (LacNAc), whereas other glycans on MUC5AC had a 2,6 linkage of sialic acid on type-2 LacNAc. Another glycan, fucosylated and sialylated/sulfated type-2 LacNAc, which is elevated in distinct subset of patients, was on N-linked glycans, representing an unusual finding of N-linked glycans on mucins associated with cancer. Further developments are enabling the testing for consistent structural features associating with subtypes of cancer, which could improve our ability to detect and target the subtypes. The on-chip glycan modification and probing (on-chip GMAP) method provides a platform for analyzing protein glycosylation in clinical specimens and could facilitate the characterization and discovery of glycan biomarkers.

#1769

The clinical outcome of stage I lung adenocarcinoma patients with or without activating EGFR mutation.

Yueh-Fu Fang. _Chang Gung Memorial Hospital, Taipei, Taiwan_.

Purpose: Advanced stage lung adenocarcinoma patients with activating EGFR mutation have better survival result after EGFR tyrosine kinase inhibitor developed. The prevalence of EGFR mutation is high in Asian lung adenocarcinoma patients. The difference of clinical outcome of stage I lung adenocarcinoma patients with or without activating EGFR mutation was unclear. Our aim of study is to evaluate the clinical outcome between wild and mutated stage I lung adenocarcinoma.

Patients and Methods: We had 343 Stage I lung adenocarcinoma patients who received surgical resection from January 2010 to April 2014. The preliminary data of EGFR mutation in 167 patients was done. Sanger method of direct PCR, scorpion & ARMS method or ABI allele specific TagMan PCR were used to check the EGFR mutation status of these patients' samples. The selection of these methods was depended on purity of tumor samples.

Results: There were 93 stage IA patients and 74 stage IB patients. Sixty-four (69%) patients had activating EGFR mutations in stage IA patients, and fifty (68%) patients had activating EGFR mutations in stage IB patients. The survival of stage IA and IB were similar in patients with or without EGFR mutations. The patients without mutated EGFR had better progression free survival in IA and IB patients, especially in the IB patients, although the significance did not be reached in small preliminary sample size.

Conclusion: The survival was similar in stage IA or IB lung adenocarcinoma with or without EGFR mutations. Patient without EGFR mutation had better progression free survival although there was no statistical significance between patients with or without EGFR mutations. We will collect more patients' data for analysis.

#1770

The association between sarcopenia and cellular senescence of cancer associated fibroblast in pancreatic cancer.

Kensuke Yamamura, Yo-ichi Yamashita, Yuki Kitano, Kota Arima, Takayoshi Kaida, Tatsunori Miyata, Shigeki Nakagawa, Kosuke Mima, Katsunori Imai, Daisuke Hashimoto, Akira Chikamoto, Hideo Baba. _Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan_.

Background: Sarcopenia is a syndrome that is characterized by progressive and generalized loss of skeletal muscle mass and strength. Several recent studies have shown that sarcopenia has prognostic significance for patients with malignant disease, including cancers of the esophagus, colon, pancreas and liver. On the other hand, cellular senescence is gaining increased attention from clinicians and researchers, yet incompletely understood role in the development of malignant disease. Previous studies reported that Caveolin-1 expression in cancer associated fibroblast is associated with cancer progression, and it is known in recent years that caveolin-1 plays a major role in controlling cellular senescence. Therefore, we hypothesized that sarcopenia causes cellular senescence of cancer associated fibroblast (CAF). The aim of this study is to clarify the between sarcopenia and cellular senescence of CAF through Caveolin-1 in pancreatic cancer.

Methods: All consecutive patients with pancreatic cancer underwent curative resection between January 2004 and December 2014 were enrolled in this retrospective study. Skeletal muscle and visceral fat amount at the third lumbar vertebra (L3) in the inferior direction were quantified using enhanced computed tomography. The patients were divided into two groups, with and without sarcopenia, based on Japan Society of Hepatology guidelines for sarcopenia. Next, Caveolin-1 expression was analyzed by immunohistochemistry (IHC). Moreover, we investigated the relationship between sarcopenia and Caveolin-1 expression.

Results: Forty-one (31.5%) of 130 patients were identified with sarcopenia. Kaplan-Meier analysis showed that patients with sarcopenia had a significantly worse cancer specific survival than patients without sarcopenia (3-year-survival: 37.2% vs 50.4%, respectively, p = 0.038). While, a high level of Caveolin-1 expression was detected in 29.2% (38/130) of our samples. High Caveolin-1 expression in CAF significantly correlated with poor prognoses with respect to overall survival (log-rank p = 0.014) and disease-free survival (log-rank p = 0.0015). However, there was no relationship between sarcopenia and IHC score of Caveolin-1 expression in CAF.

Conclusion: The present result suggested that sarcopenia and Caveolin-1 expression of CAF in patient with pancreatic cancer is respectively associated with poor prognosis, but not associated each other. Now, we focus on Caveolin-1 expression of CAF, and establish primary cultures of CAF from the pancreatic cancer tissues and examine its function.

### Predictive Biomarkers 1

#1771

Phase 3 trial of adjuvant sunitinib in patients with high-risk renal cell carcinoma: exploratory molecular analysis of tumor biomarkers.

Daniel J. George,1 Jean-Francois Martini,2 Yen-Hwa Chang,3 Michael Staehler,4 Jan Breza,5 Jean-Jacques Patard,6 Robert J. Motzer,7 Ahmed Magheli,8 Bernard Escudier,9 Giacomo Carteni,10 Paola Gerletti,11 Sherry Li,2 Michelle Casey,12 Brigitte Laguerre,13 Hardev S. Pandha,14 Allan J. Pantuck,15 Anup Patel,16 Maria Lechuga,11 Alain Ravaud17. 1 _Duke Cancer Center, NC;_ 2 _Pfizer Inc, CA;_ 3 _Taipei Veterans General Hospital, Taiwan;_ 4 _Hospital of Munich, Germany;_ 5 _Slovak Medical University, Slovakia;_ 6 _Bicêtre Hospital, France;_ 7 _Memorial Sloan Kettering Cancer Center, NY;_ 8 _Charité Universitaetsmedizin Berlin, Germany;_ 9 _Institut Gustave Roussy, France;_ 10 _Azienda Ospedaliera di Rilievo Nazionale A. Cardarelli, Italy;_ 11 _Pfizer, Italy;_ 12 _Pfizer Inc, PA;_ 13 _Centre Eugene Marquis, France;_ 14 _University of Surrey, United Kingdom;_ 15 _Ronald Reagan UCLA Medical Center, CA;_ 16 _Spire Roding Hospital, United Kingdom;_ 17 _Bordeaux University Hospital, France_.

Background: Adjuvant therapy with sunitinib (SU) compared with placebo (PBO) prolonged disease-free survival (DFS) in patients (pts) with loco-regional high-risk renal cell carcinoma (HR=0.76, 95% CI: 0.59-0.98; P=0.03; median[m] DFS, 6.8 vs 5.6 years). Here, we report the results of a retrospective exploratory molecular biomarker analysis using nephrectomy biospecimens from the S-TRAC trial.

Materials and Methods: Formalin-fixed paraffin-embedded tumor tissue blocks from patients who provided informed consent were used for immunohistochemistry (IHC) staining of PD-L1, CD4, CD8, and CD68. Biomarker quantification was done by automated image analysis of the regions of interest (ROI). The analysis algorithm utilized an immunoscore approach applied to ROI, reflecting assessment of both the center and invasive margin of tumors (for PD-L1 and CD8 staining). DFS was compared between biomarker stratum by < median vs ≥ median values of a particular IHC parameter using Kaplan-Meier (K-M) analysis. Receiver Operating Characteristics (ROC) curves were generated to further assess the potential clinical utility of biomarkers for which significant (P < 0.05) results were obtained in K-M analysis.

Results: In total, 191/615 (101, SU and 90, PBO) pts in the intent-to-treat population were included for IHC analysis. Baseline characteristics were similar in the subpopulations with and without IHC data. Shorter DFS was observed in the PBO group for pts with PD-L1+ vs PD-L1- tumors, although not statistically significant (HR=1.75; 95% CI: 0.89-3.46; P=0.103). In pts with PD-L1+ tumors, DFS was numerically longer for SU vs PBO (mDFS=6.17 vs 2.67 years) (HR=0.58; 95% CI: 0.26-1.29; P=0.175). In the SU group, pts with CD8\+ T-cell density ≥ median (cutoff=269.5 CD8+ cells/mm2) had longer DFS (mDFS=not reached [NR]; 95% CI: 6.83-NR) than pts with CD8+ T-cell density < median (mDFS=3.47 years; 95% CI: 1.73-NR), and the difference was statistically significant (HR=0.40, 95% CI: 0.20-0.81; P=0.009), while CD8\+ T-cell density showed no significant difference in DFS for PBO pts (HR=0.80, 95% CI: 0.42-1.50; P=0.484). The sensitivity and specificity for CD8+ T-cell density in predicting DFS were 0.604 and 0.658, respectively, and the optimal cutoff was 222.22 cells/mm2 with an area under ROC curve of 0.622.

Conclusions: Increased density of CD8\+ T-cells in tumor tissue was associated with longer DFS in SU-randomized pts but not PBO, suggesting this may be predictive of treatment effect. Further validation in an independent cohort is warranted. The prognostic value of PD-L1 expression in primary tumors from patients with high-risk non-metastatic RCC should be further explored.

#1772

PIK3CA **C2 domain deletions hyperactivate PI3K, generate oncogene dependence and are exquisitely sensitive to PI3Kα inhibitors.**

Sarah Croessmann,1 Jonathan Sheehan,1 Gregory Sliwoski,1 Nalin Leelatian,1 Jie He,2 Rebecca Nagy,3 Justin M. Balko,1 Ingrid A. Mayer,1 Richard B. Lanman,3 Vincent Miller,2 Lewis C. Cantley,4 Jonathan M. Irish,1 Jens Meiler,1 Carlos L. Arteaga1. 1 _Vanderbilt University, Nashville, TN;_ 2 _Foundation Medicine, Cambridge, MA;_ 3 _Guardant Health, Inc., Redwood, CA;_ 4 _Meyer Cancer Center of Weill Cornell Medical College, New York, NY_.

Background: A 63 year old postmenopausal woman with advanced ER+ breast cancer resistant to endocrine therapy exhibited an exceptional response to the PI3Kα inhibitor alpelisib (BYL719) and the aromatase inhibitor letrozole (Mayer et al. Clin Cancer Res 2016). Targeted capture next generation sequence (NGS) of DNA from a liver metastasis identified a P447_L455 deletion in the C2 domain of PIK3CA. About 80% of PIK3CA activating mutations are in 'hot spots' within the helical and kinase domains. C2 domain mutations make up ~10% of all PIK3CA mutations in breast cancer (TCGA, Foundation Medicine) and are frequently not reported by tumor and plasma cell-free DNA NGS panels. Deletions and mutations in this domain cluster in a region encompassing amino acids 446-460 of PIK3CA. We investigated herein the functional role of PIK3CA C2 domain mutations and their response to PI3K inhibitors.

Methods: V5-tagged lentiviral vectors encoding wild type, delP447-L455, and delH450-P458 were stably transduced into MCF10A non-tumorigenic human breast epithelial cells. Cell viability and acini formation in 3D Matrigel were examined in media ± EGF or insulin or alpelisib. Markers of PI3K activation were examined by immunoblot and phosphoflow analysis. The Rosetta software suite was used to construct a structural model that would predict the change in stability of the p85/p110α complex. Physical association of p85α and p110α was determined by precipitation with V5 antibodies and immunoblot analysis.

Results: MCF10A cells stably expressing V5-tagged PIK3CAdelP447-L455 and PIK3CAdelH450-P458 exhibited EGF- and insulin-independent growth and higher phosphorylation of AKT, ERK and S6 when compared to parental MCF10A cells. In 3D Matrigel, MCF10A cells with PIK3CA C2 domain deletions formed invasive acini with increased protrusions, spindling, and bridging between acini. All these changes were ablated upon the addition of 1 µM of alpelisib whereas parental MCF10A cells were unaffected by alpelisib. We hypothesized that delP447-L455 would reduce the binding affinity of p110α with the p85 regulatory subunit of PI3K. A structural model of PIK3CAdelP447-L455 in the context of the regulatory complex revealed specific favorable inter-residue contacts that would be lost as a result of the deletion, predicting a significant decrease in binding energy. Consistent with this structural analysis, coimmunoprecipitation of p85 with V5 antibodies showed reduced binding of the C2 domain deletion mutants with p85 compared to wild type p110α.

Conclusions: These data suggest that C2 domain deletions in PIK3CA are activating mutations and generate oncogene dependence. As a result, tumors expressing these mutations are exquisitely sensitive to PI3Kα inhibitors. Thus, in addition to PIK3CA 'hot spot' mutations, C2 domain mutations should also be considered biomarkers of sensitivity to PI3K inhibitors.

#1773

A baseline IFNG gene expression signature correlates with clinical outcomes in durvalumab-treated advanced NSCLC cancer patients.

Brandon W. Higgs, Chris A. Morehouse, Katie Streicher, Philp Z. Brohawn, Keith Steele, Marlon Rebelatto, Fernanda Pilataxi, Carlos Bais, Li Shi, Xiaoping Jin, Joyce Antal, Ashok Gupta, Koustubh Ranade. _MedImmune, Gaithersburg, MD_.

Durvalumab (D) is a human IgG1 monoclonal antibody which inhibits PDL1 binding to PD-1 and CD80, restoring antitumor immunity. In D-treated (tx) NSCLC patients (pts), we previously reported high baseline levels of tumoral PD-L1 protein and IFNγ mRNA expression associated with improved ORRs, PFS and OS. Here, a gene expression signature of baseline tumors associates with improved outcomes on D. CP1108/NCT01693562 was a nonrandomized phase 1/2 trial evaluating D in advanced previously tx NSCLC or other solid tumors. By 29APR2016, 304 NSCLC pts received 10 mg/kg Q2W of D ≤12 months with median 18.8 months follow up. RNA sequencing of frozen biopsies was conducted on 97 NSCLC tumors of sufficient quality with matched IHC for tumoral PD-L1 on 92 fresh or archival biopsies. Among 21 pre-identified immune-related genes, mRNAs for IFNG, LAG3, CXCL9, and PDL1 individually correlated best with outcomes in NSCLC after adjustment for sex, age, prior therapy, histology, ECOG and smoking. A signature was developed as mean mRNA levels of the four genes; signatures >upper tertile were IFNG signature positive (IFNGS+). Analysis was performed on NSCLC, then applied to 30 available urothelial bladder cancer (UBC) biopsies. NSCLC with ≥25% tumor cells stained for PD-L1 at any intensity were PD-L1+. 29 NSCLC had pre/post-treatment tumors for mRNA analysis. KM and Cox PH models were used. IFNGS+ D-tx NSCLC pts had higher ORR, median PFS and OS compared to PDL1+, PDL1-, and IFNGS- pts (Table 1); IFNGS+ UBC D-tx pts also correlated with these outcomes. Following D treatment, IFNGS was induced in NSCLC pts (FC=2; p=0.0046) regardless of clinical response. High levels of pre-treatment IFNGS in NSCLC pts associated with greater benefit from D. D induces IFNGS within the tumor microenvironment. Observations from other tumor types will be presented. Table 1. Clinical outcomes by IFNGS or PD-L1 status | # Pts (# events [OS;PFS]) | ORR % (95% CI) | Median OS months (95% CI) | OS adjusted HRa; p | Median PFS months (95% CI) | PFS adjusted HRa; p

---|---|---|---|---|---|---

NSCLC | |  | |  | |

IFNGS+ | 32 (16;21) | 37.5 (21.7,56.3) | 24.6 (10.3,NA) | 0.42; 0.0082 | 7.5 (3.6,NA) | 0.32; 0.00028

IFNGS- | 65 (40;51) | 6.2 (2.0,15.8) | 6.5 (4.8,15.7) | |

1.4 (1.4,2.6)

|

PDL1 TPS>=25% | 38 (18;25) | 28.9 (16.0,46.1) | 20.5 (6.6,NA) | 0.53; 0.0788 | 3.6 (1.7,14.6) | 0.60; 0.0823

PDL1 TPS<25% | 54 (35;43) | 7.4 (5.8,25.5) | 9.1 (5.4,21.1) | |

1.5 (1.4,3.9)

|

NR=Not Reached; NA=Not Applicable; aAdjusted for covariates. TPS= tumor proportion score Ventana SP263 assay

#1774

Progression-free survival curves suggest a dichotomous determinant of PD-L1 inhibitor efficacy.

David J. Stewart,1 Dominick Bosse,2 Stephanie Brule,1 Andrew G. Robinson,3 Michael Ong,1 John F. Hilton1. 1 _University of Ottawa, Ottawa, Ontario, Canada;_ 2 _Harvard University, Boston, MA;_ 3 _Queen's University, Kingston, Ontario, Canada_.

Background: PD-L1 expression varies across tumors but does not accurately predict PD-L1 inhibitor efficacy. Some negative tumors respond and some positive tumors fail. PD-L1 inhibitor progression-free survival (PFS) curve shape in non-small cell lung cancer (NSCLC) suggests that a dichotomous (present vs absent) factor might drive sensitivity rather than it being driven by a continuous variable like PD-L1 expression. PFS curves may follow first order kinetics, with a straight line if log % PFS is plotted vs time. If the population had 2 distinct subgroups with differing rates of progression then one would expect an inflection point on the log-linear curve, and the curve would fit a 2-phase decay model in nonlinear regression analysis (NLRA). A more homogeneous population would not fit a 2-phase model.

Methods: We used arohatgi.info/WebPlotDigitizer/app/ to digitize published PFS curves, then GraphPad Prism5 for 2-phase NLRA, with the constraints Y0=100, plateau=0. To generate standardized 2-phase curves, we utilized 1) unselected NSCLC patients treated with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) where we expected a high proportion of curves to fit a 2-phase model as only some patients would have a sensitizing EGFR mutation; 2) NSCLC EGFR mutant and wild type (WT) patients treated with EGFR TKIs, and patients treated with platinum-based chemotherapy, taxanes or placebo/best supportive care (BSC), where we expected a lower proportion of curves to fit 2-phase models; 3) PD-L1 PFS curves in NSCLC and other tumor types.

Results: With EGFR TKIs in unselected patients, 58 of 79 (73%) curves were fit by 2-phase models, vs 5 of 37 (14%) with EGFR TKIs in EGFR mutant patients (p<0.0001), 13 of 27 (48%) in EGFR WT patients (p<0.02), 4 of 37 (11%) with platinum-based therapy (p<0.0001), 15 of 47 (32%) with a taxane (p<0.0001), and 6 of 22 (27%) with placebo/BSC (p=0.0001). With PD-L1 inhibitors in NSCLC, 30 of 32 (94%) curves fit 2-phase models (p<0.0001 vs each of EGFR TKIs in EGFR mutants, EGFR WTs, platinum and taxane chemotherapy and placebo/BSC). In other tumor types, 27 of 32 (84%) PD-L1 curves fit 2-phase models.

Conclusions: Most PD-L1 inhibitor PFS curves fit a 2-phase model. This is similar to what we observed with EGFR TKIs in unselected patients and different from EGFR TKIs in EGFR mutant and WT patients, and from chemotherapy or placebo/BSC. This leads us to hypothesize the existence of a dichotomous (present vs absent) factor such as a gene mutation, deletion or silencing that sensitizes tumors to PD-1/PD-L1 inhibitors. If found, such a dichotomous factor could prove to be a highly useful biomarker that could permit accurate prediction of PD-1/PD-L1 inhibitor efficacy. Since PD-1/PD-L1 inhibitor efficacy is higher in tumors with high PD-L1 expression, any sensitizing dichotomous factor might also drive PD-L1 expression.

#1775

The interaction of PD-L1, TMB, and genomic alterations in NSCLC.

Michael E. Goldberg, Laurie M. Gay, David Fabrizio, Garrett M. Frampton, Julia A. Elvin, Jeffrey S. Ross, Vincent A. Miller, Philip J. Stephens, Lee A. Albacker. _Foundation Medicine, Inc., Cambridge, MA_.

Introduction: In a cohort of non-small cell lung cancers (NSCLC), we identify new potential biomarkers that may predict response to checkpoint inhibitors through an analysis of genomics, PD-L1 protein expression, and tumor mutational burden (TMB).

Motivation: In cancer therapy, anti-PD-1/PD-L1 checkpoint inhibitors provide long term responses in a subset of patients. The current biomarker, PD-L1 IHC, is only partially successful in identifying responders. TMB, a measure of the number of mutations in a tumor's genome, is an emerging biomarker that can predict response rates to checkpoint inhibition in addition to PD-L1 IHC. Higher TMB is thought to confer a higher probability of expressing a neoantigen protein recognizable by the immune system as non-self and therefore responding to checkpoint inhibition. While TMB represents the likelihood that tumor cells express immune-triggering neoantigens, PD-L1 expression represents one of many ways a tumor can evade immune response. We investigated the patterns of PD-L1 IHC staining and TMB in NSCLC.

Methods: We tabulated PD-L1 IHC stain statuses, TMB, and genomic alterations for 789 unique NSCLC tumor samples. Tumor cell PD-L1 expression was measured by IHC and scored for distribution (0, 1-24%, &gt; 24% of cells) and intensity (0, 1+, 2+) as Negative, Low Positive, or High Positive. Samples were tested with a comprehensive genomic profiling assay that identifies alterations in exons and select introns in 405 genes. TMB was calculated over 1.1 Mb by counting somatic, non-driver genomic alterations and scored as Low (&lt; 6 mut/Mb), Medium (&lt; 20, ≥ 6 mut/Mb), or High (≥ 20 mut/Mb).

Results: PD-L1 tumor staining was significantly correlated with TMB (P = 3.52e-4), although the correlation was not strong (Spearman's rho = 0.12). To better explain discordant TMB/PD-L1, we ran Fisher's exact tests to identify genes enriched for alterations in TMB-High/PDL1-Negative or TMB-Low/PD-L1-High Positive samples. The TMB-High/PD-L1-Negative subset preferentially harbored STK11 loss-of-function alterations with an odds ratio of 3.65 (P = 1.20e-3). The TMB-Low/PD-L1-High Positive subset exhibited enrichment for BRAF alterations with an odds ratio of 5.46 (P = 1.71e-3).

Conclusions: TMB-High tumors have high immunogenicity and often rely on PD-L1 expression as a means of immune evasion. Alterations in BRAF and other genes enriched in TMB-Low/PDL1-High Positive samples may trigger immune responses moderated by PD-L1 expression. Samples with these alterations, although often TMB-Low, may nevertheless respond to checkpoint inhibitors. Alterations in STK11 and other genes enriched in TMB-High/PD-L1-Negative samples may be implicated as non-PD-L1-based mechanisms for immune evasion. Although further ongoing analysis is required, our study suggests that the combination of TMB, PD-L1 staining, and genomics may provide the best prediction of response to checkpoint inhibitors.

#1776

Impact of homologous recombination deficiency (HRD) biomarkers on outcomes in triple-negative breast cancer (TNBC) patients treated with AC chemotherapy (SWOG S9313).

Priyanka Sharma,1 William Barlow,2 Andrew K. Godwin,1 Harsh Pathak,1 Kamilla Isakova,1 Anne R. Hartman,3 Kristen M. Timms,3 Hannah M. Linden,4 Debu Tripathy,5 Gabriel N. Hortobagyi,5 Daniel F. Hayes6. 1 _University of Kansas Medical Center, Westwood, KS;_ 2 _SWOG Statistical Center/Cancer Research and Biostatistics (CRAB), Seattle, WA;_ 3 _Myriad Genetics, Inc., Salt Lake City, UT;_ 4 _University of Washington/Seattle Cancer Care Alliance, Seattle, WA;_ 5 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 6 _University of Michigan, Ann Arbor, MI_.

Introduction: There is a critical need to develop biomarkers of response and resistance to adjuvant chemotherapy for TNBC. In preliminary studies, homologous recombination deficiency (HRD)-causing alterations have been reported in TNBC patients. HRD may impact response to standard chemotherapy as well as investigational therapies, such as PARP inhibitors and platinum agents. We report on the prognostic impact of two such markers in a large cohort of early stage TNBC patients who were uniformly treated with adjuvant doxorubicin (A) and cyclophosphamide (C).

Aims: To investigate BRCA1 promoter methylation (PM) and HRD score as prognostic markers in TNBC patients treated with adjuvant AC on S9313.

Methods: SWOG protocol S9313 accrued 3,125 women with early stage breast cancer to two alternative dose schedules of AC with no difference in outcomes between the two arms (J Clin Oncol 2007). We identified 425 (14%) patients with centrally determined TNBC status for whom tissue was available. BRCA1 PM (methylation specific PCR) and HRD score (composite of loss of heterozygosity, telomeric allelic imbalance and large-scale state transitions, Myriad Genetics Inc.) were performed on genomic DNA isolated from pre-treatment FFPE breast tumor tissue. HRD was classified as positive if there was either a deleterious tumor(t) BRCA1 or BRCA2 mutation and/or a pre-defined HRD score > 42. The markers were tested for prognostic effect on DFS and OS using a Cox regression model with adjustment for randomized treatment assignment.

Results: For 425 TNBC patients median age was 45 years (range 22-74) and at a median follow up of 10.2 years there are 166 DFS and 129 OS events (5 year DFS and OS = 74% and 82%, respectively; 10-year DFS and OS = 66% and 73%). BRCA1 PM was determined in 82% (348/425) and was detected in 32% of patients. Presence of BRCA1 PM was suggestive of better DFS, but not statistically significant (HR=0.74; 95% CI 0.50-1.08, p=0.12). HRD results were determined in 91% (379/425) and 67% were HRD positive (27% with tBRCA mutation and 40% tBRCA negative but HRD score >42). HRD positive status was associated with a better DFS (HR = 0.69; 95% CI 0.49-0.96 (p=0.027)) and OS (HR= 0.67; 95% CI 0.47-0.97 (p=0.032)). High HRD score (≥42) in tBRCA negative patients (n=274) was also associated with better DFS (HR = 0.62; 95% CI 0.42-0.92). tBRCA status (positive versus negative) did not impact DFS (p = 0.78).

Conclusions: Two thirds of TNBC patients receiving adjuvant AC chemotherapy had tumor HRD positivity. HRD was associated with better DFS and OS, perhaps due to high responses to AC. HRD status has the potential to be used as a selection criterion to identify TNBC patients who receive significant benefit from anthracycline chemotherapy, and may also be of value in selecting patients suitable for treatment with other DNA damaging agents like platinum salts/PARP-inhibitors.

#1777

Development of a pan-cancer 15 gene expression signature to detect a subgroup driven by MAPK signalling.

Laura A. Knight,1 Bethanie Price,1 Andrena McCavigan,1 Aya El-Helali,2 Charlie Gourley,3 Denis P. Harkin,4 Richard Kennedy,2 Nuala McCabe2. 1 _Almac Diagnostics Ltd, Craigavon, United Kingdom;_ 2 _Queens University Belfast, Belfast, United Kingdom;_ 3 _Edinburgh Cancer Research Centre, United Kingdom;_ 4 _Almac Diagnostics Ltd, United Kingdom_.

INTRODUCTION

Unsupervised hierarchical clustering of gene expression data from 265 High Grade Serous Ovarian Cancer (HGSOC) patients identified 3 major molecular subgroups. One subgroup is driven by activation of the MAPK pathway and is associated with poor prognosis and resistance to platinum chemotherapy. The MAPK pathway is currently being targeted by novel therapeutics and hence an assay to detect activation of the pathway across cancers would be highly valuable as a clinical trial enrichment tool. Using internal and publicly available gene expression datasets we have demonstrated that the MAPK subgroup also exists in other cancer types and is associated with poor prognosis. The aim of this study was to develop a gene expression signature to predict the MAPK subgroup across multiple cancer types.

METHODS

Evaluation of gene expression data in a range of tumours (ovarian, colon, lung, melanoma and prostate) identified a common gene list (CGL) of 7802 genes showing high variability and high expression across diseases. The CGL was input to different machine learning algorithms developing signatures under 10x5-fold cross-validation (CV), trained against the MAPK HGSOC subgroup. Filter-Feature-Selection removed 10% of genes under CV based upon ranked correlation adjusted t-scores and the final model selected to satisfy a number of key criteria: AUC for predicting the endpoint; association with survival (C-Index); and functional relevance of signature content.

RESULTS

A 15 gene signature was selected, yielding an AUC=0.87 [95% CI:0.84-0.89] with respect to the MAPK subgroup. This model has validated as a poor prognostic marker in several other cancer types

(Colorectal, Relapse free survival: HR=1.46 [95% CI:1.07-1.98]; Lung, Relapse free survival: HR=2.18 [95% CI:1.33-3.56]; Prostate cancer, Biochemical recurrence: HR 2.49 CI: 1.43-4.34), and is suppressed by MEK inhibition (p=0.0023) and elevated by KRAS, NRAS and MEK1 overexpression in cell line models (p=0.0443, <0.0001and <0.0001). Additionally we have demonstrated that the 15 gene signature strongly predicts response to the MEK inhibitors Trametinib and Selumetinib in established cell line models (p<0.001) and in primary cells isolated from breast and ovarian patients.

CONCLUSION

A 15 gene signature has been developed from formalin fixed paraffin embedded samples across multiple diseases to detect a molecular subgroup driven by MAPK signalling. This assay predicts sensitivity to MEK inhibitors in pre-clinical model systems and in primary cells derived from patients. Further work aims to validate the signature in clinical samples from patients treated with a MEK inhibitor. This assay may be helpful for clinical trial enrichment to select patients that are likely to benefit from MAPK targeted therapies.

#1778

A clinical genomic biomarker study of the CHK1 inhibitor prexasertib in advanced head and neck squamous cancer and squamous cell carcinoma of the anus.

Ricardo Martinez,1 Sameera R. Wijayawardana,1 David Hong,2 Johanna Bendell,3 Anna Maria Russell,4 Richard P. Beckmann,1 Aimee Bence Lin1. 1 _Eli Lilly and Company, Indianapolis, IN;_ 2 _MD Anderson Cancer Center, Dallas, TX;_ 3 _Tennessee Oncology, Nashville, TN;_ 4 _Eli Lilly and Company, San Diego, CA_.

CHK1 plays a critical role in DNA damage repair (DDR) pathways as well as in coordinating DNA replication. Selective CHK1/CHK2 compounds are being tested in clinical trials but predictive biomarkers of patient response are lacking. A phase 1b expansion cohort study (I4D-MC-JTJA, NCT01115790) with the CHK 1 inhibitor, prexasertib, included patients with advanced, metastatic head and neck squamous cell carcinoma (HNSCC) or squamous cell carcinoma of the anus (SCCA). To identify genomic biomarkers associated with single-agent drug response, pre-treatment tissues (archived or biopsy) from 71 consented patients (HNSCC=47, SCCA=24) were subjected to next-generation sequencing (NGS) using the FoundationOne gene panel. In this subset of patients, the disease control rate (DCR) (Complete Response (CR) + Partial Response (PR) + Stable Disease (SD) based on RECIST Criteria (v 1.1)) was 60% (28/47) and 75% (18/24), respectively. We present here the observed genetic alterations corresponding to three pathways, Cell Cycle, DNA Damage Repair (DDR) and PI3K. In addition, patients' human papillomavirus (HPV) carrier status was inferred from DNA sequencing using HPV-specific capture probes. HPV+ was 47% for HNSCC and 87% for SCCA. HPV+ and TP53 mutations were mutually exclusive across the two patient cohorts. In HNSCC patients with evaluable progression-free survival (PFS) data, greater clinical efficacy was observed in the HPV+ cohort (median PFS: 4.5 vs 1.4 months, log-rank p = 0.0008). Known or likely loss-of-function (LOF) mutations in FBXW7 and PARK2, two genes implicated in Cyclin E1 proteolysis, were noted in patients with favorable response in both tumor types. Across both HNSCC and SCCA cohorts, mutations and/or germline variants in the DDR genes BRCA1, BRCA2, MRE11A and ATR but not in Fanconi (FANC) pathway genes, were found in patients with treatment benefit. Whereas PIK3CA mutations were infrequent in the HNSCC cohort, in SCCA, mutations occurred in 5/8 (63%) patients with disease control vs 1/6 (17%) with PD. All 7 PI3KCA mutations observed in HPV+ HNSCC and SCCA patients mapped to the helical domain suggestive of Apobec-induced mutagenesis as their source of origin. The enhanced clinical benefit to prexasertib associated with HPV+ in HNSCC may reflect a prognostic effect. Alternatively, the clinical biomarker findings may support the hypothesis that oncogene-induced replication stress (RS) (i.e. arising from HPV E6/E7 and/or FBXW7 loss-dependent Cyclin E1 dysregulation) in the context of attenuated DDR (i.e. BRCA1/2, MRE11A mutations) may sensitize patients to prexasertib monotherapy.

#1779

**Chronic NSAID use increases survival in** PIK3CA **-altered head and neck squamous cell carcinoma.**

Matthew Louis Hedberg,1 Noah Peyser,2 William Gooding,1 Hua Li,2 Toni Brand,2 Victor Olivas,2 Trever Bivona,2 Simion Choisea,1 Lin Wang,1 Jonas Johnson,1 Uma Duvvuri,1 Robert Ferris,1 Daniel Johnson,2 Patrick Ha,2 Julie Bauman,3 Jennifer Grandis2. 1 _University of Pittsburgh School of Medicine, PA;_ 2 _University of California San Francisco, CA;_ 3 _University of Arizona Cancer Center, AZ_.

Background: PIK3CA is the most commonly altered oncogene in head and neck squamous cell carcinoma (HNSCC). Chronic non-steroidal anti-inflammatory (NSAID) use has been shown to reduce mortality in PIK3CA-mutated colorectal cancer through an unknown mechanism. In this retrospective cohort analysis, we evaluated the impact of NSAIDs on HNSCC survival, and sought a biologically plausible explanation for the observed effect.

Methods: The PIK3CA status of tumor tissue was determined by FISH and next generation sequencing for 266 HNSCC patients treated at the University of Pittsburgh Cancer Institute. Cox joint proportional hazards models were used to generate multivariate projections of disease-specific (DSS) and overall survival (OS). Prostaglandin E2 (PGE2) secretion and NSAID sensitivity were then assessed in HNSCC cell lines with or without PIK3CA mutation.

Results: Chronic NSAID use (≥2 days/week for ≥6 months) was associated with greater disease-specific (HR 0.24; 95% CI, 0.09 - 0.62; p = 0.0032) and overall survival (HR 0.31; 95% CI, 0.14 - 0.69; p = 0.0043) in patients whose tumors harbor PIK3CA mutation and/or amplification. Survival was unchanged by chronic NSAID use in patients with wild-type, unamplified PIK3CA. Chronic NSAID users with PIK3CA-altered tumors had an increase in estimated 5-year DSS from 25% to 72% and in OS from 45% to 78%. Benefit was independent of age, stage, and human papillomavirus (HPV) status; all of which were controlled for in the model. HNSCC cells with endogenous or engineered PIK3CA mutation secreted higher levels of PGE2 and were more sensitive to growth inhibition by NSAID treatment.

Conclusions: Chronic NSAID use was associated with markedly improved survival in patients with PIK3CA-altered HNSCC, but not in those with wild-type, unamplified PIK3CA. Preclinical studies implicate the PI3K-COX-PGE2 signaling axis as a potential mediator of NSAID response. A prospective, randomized controlled trial of NSAIDs in patients with PIK3CA-altered HNSCC is warranted.

#1780

Identifying preclinical predictive biomarkers to taselisib in breast cancer cell lines.

Heidi M. Savage, Heather M. Moore, Carol L. O'Brien, Wei Zhou, Ciara Metcalfe, Mark R. Lackner, Timothy R. Wilson. _Genentech, Inc., South San Francisco, CA_.

The phosphoinositol-3-kinase (PI3K) signaling pathway is one of the most frequently activated pathways in cancer, and controls critical cellular processes such as proliferation, transcription and survival. Taselisib (GDC0032) is an orally bioavailable, potent, and selective inhibitor of Class I PI3K alpha, delta, and gamma isoforms, with 30fold less inhibition of the PI3K beta isoform relative to the PI3K alpha isoform. Previously published data demonstrated that taselisib has increased activity against PIK3CA mutant cancer cell lines (Ndubaku CO et al, J Med Chem, 2013).

A panel of 50 breast cancer cell lines were profiled for sensitivity to taselisib using viability assays and subsequently correlated with PIK3CA mutations and expression of ER, PR, HER2 and PTEN. The PIK3CA mutant cell lines displayed an average of 30-fold greater sensitivity in IC50 compared to the PIK3CA wild-type cell lines. PTEN null cell lines were largely refractory to taselisib. Within the PIK3CA wild-type cell lines, the HER2 amplified cells showed an average of 18-fold greater sensitivity in IC50 over the HER2 wild-type cell lines. No differences in taselisib sensitivity were seen between the kinase, helical, or the C2 domain of the PIK3CA gene within the PIK3CA mutant cell lines. In HER2 amplified PIK3CA wild-type cell lines, taselisib did not fully suppress pAKT and pS6 signaling, however full suppression was observed in combination with the HER2 inhibitor, lapatinib. Increased apoptosis was observed with the combination of taselisib and lapatinib in the HER2 amplified, PIK3CA mutant cells, but not in HER2 amplified, PIK3CA wild-type cells. In PIK3CA mutant luminal cell lines, taselisib induced ESR1 transcriptional activity, but not in PIK3CA wild-type luminal lines, as assessed using PR, GREB1 and IGFBP4 gene expression. This induction was ER- and estradiol-dependent, and was suppressed using the estrogen degrader, fulvestrant. Lastly, as up to 40% of ER+, second line PIK3CA mutant metastatic tumors have been shown to harbor a mutation in the ESR1 gene (Spoerke JM et al., Nat Commun, 2016), we assessed the crosstalk between the PI3K and ER pathways in PIK3CA mutant MCF-7 cells that overexpress two ESR1 hotspot mutations. These ESR1 mutant cell lines displayed increased ER transcriptional activity, which was further activated by taselisib, an observation that was reversed with fulvestrant co-treatment. These data suggest that the combination of taselisib and an estrogen degrader can suppress crosstalk between PI3K and ER in both an ESR1 mutant and wild-type background, and supports the ongoing SANDPIPER study of taselisib in combination with fulvestrant in metastatic ER+, PIK3CA mutant cancers that have progressed following an aromatase inhibitor therapy (NCT02340221).

#1781

EGFR amplification-A candidate predictive biomarker of PARP inhibitor Talazoparib sensitivity in gliomas.

Shaofang Wu,1 Feng Gao,1 Xiaolong Li,1 Jie Ding,1 Siyuan Zheng,1 Emmanuel Martinez-Ledesma,1 Ningping Feng,1 Erik Sulman,1 Roel Verhaak,2 John de Groot,1 Dimpy Koul,1 W.K.Alfred Yung1. 1 _MD Anderson, Houston, TX;_ 2 _Jackson Laboratory, Farmington, CT_.

Poly (ADP-ribose) polymerases (PARP) are enzymes involved in DNA-damage repair. Inhibition of PARP is a promising strategy for targeting cancers with defective DNA-damage repair. Several PARP inhibitors are currently in trials in the adjuvant, neoadjuvant, and metastatic settings for the treatment of ovarian, BRCA-mutated breast, and other cancers. Glioma Stem Cells (GSCs) exhibit higher oxidative base damage, single-strand DNA breaks and genomic instability and is reliant on single-strand break repair/base excision repair to tolerate additional stress. PARP critical for maintaining genomic stability by regulating a variety of DNA damage repair mechanisms is constitutively activated in GSCs. In this study, we show that talazoparib -- a unique PARP inhibitor exhibits a strong single agent activity to inhibit proliferation of GSCs in vitro and suppress tumor progression in GSCs xenografts. Talazoparib cytotoxic activity was through inhibiting PARylation and by trapping PARP-DNA complexes resulting in accumulation of DNA damage during replication and ultimately leading to apoptosis. More importantly, we found EFGR amplification, which occurs in about 45% of GBM as a biomarker of response to PARPi. Gene expression and RPPA data showed that GSCs with EGFR amplification were associated with increased ROS, basal expression of DNA repair proteins and more DNA damage following talazoparib treatment, which may partially explain the sensitivity of this group of GSCs to talazoparib. EGFR enzyme activity was important for PARPi sensitivity since kinase dead mutant of EGFR or EGFR knock down cell lines were resistant to PARPi as was also shown by decreased PARP/DNA complex formation. Talazoparib showed strong inhibition of tumor growth in subcutaneous glioma model and 20% increase in median survival in an intracranial model with a 10% blood brain penetration of talazoparib. These data provide mechanistic insights into the anti-cancer activity of PARP inhibitors with mechanistic rationale of PARP inhibition and potential EGFR amplification as biomarker in the development for precision cancer.

#1782

Reactive oxygen species (ROS) and mitochondrial DNA (mtDNA) damage in tumor hypoxia, poor radiotherapy response, and metastatic progression of rectal cancer.

Paula A. Bousquet,1 Sebastian Meltzer,1 Linda Sønstevold,1 Ying Esbensen,1 Lars G. Lyckander,1 Svein Dueland,2 Kjersti Flatmark,2 Kathrine R. Redalen,1 Lars Eide,3 Anne H. Ree1. 1 _Akershus University Hospital, Lørenskog, Norway;_ 2 _Oslo University Hospital, Oslo, Norway;_ 3 _University of Oslo, Oslo, Norway_.

Background: Hypoxia is an important feature of the tumor microenvironment and correlates with radiotherapy resistance and metastatic progression. Additionally, as the malignant phenotype promotes aerobic glycolysis and contributes to the microenvironmental acidification, an underlying mechanism in radiation resistance and metastasis is likely linked to metabolic changes caused by mitochondrial reprogramming. We hypothesized that ROS and mtDNA damage might be indicators of poor therapy response and dissemination of rectal cancer.

Methods: Three colorectal cancer (CRC) cell lines (HCT116, HT29, LoVo) were cultured under hypoxia (0.2% O2) or normoxia (21% O2) for 24 hours. In a prospective rectal cancer trial, serum was sampled from patients at the time of diagnosis. Levels of ROS were determined by the ability to oxidize dihydrochlorofluoroscein into a fluorescent derivative. mtDNA damage was quantified by the ability of modification on template DNA to inhibit restriction enzyme cleavage. For the study patients, histologic tumor response to neoadjuvant radiotherapy was evaluated according to standard ypTN staging and tumor regression grade (TRG) scoring. Groups were compared using Student's t-test. Survival differences were assessed by the log-rank test.

Results: Compared to normoxic conditions, hypoxia significantly reduced ROS levels in all CRC cell lines. Moreover, in serum samples, ROS levels were lower for T4 cases than for patients with T2-3 disease (p = 0.037), and accordingly, low circulating ROS was associated with adverse metastasis-free survival (MFS; p = 0.004) and overall survival (p < 0.001). Hypoxic HCT116 and HT29 cell lines, but not the LoVo cells, showed significantly higher mtDNA damage than under normoxia. Given the indicated dependence of ROS and mtDNA damage to hypoxia, a low ratio of ROS to mtDNA damage turned out to be a promising indicator of hypoxic CRC cells. This ratio was lower in serum samples from patients who obtained poor histologic tumor response to radiotherapy (ypT3-4 versus ypT0-2 and poor versus good TRG; p = 0.044 for both). Accordingly, a high ratio of circulating ROS to mtDNA damage was associated with longer MFS (p = 0.021).

Conclusion: We showed that culturing of CRC cells under hypoxia caused decrease in ROS levels but increase in mtDNA damage. Interestingly, in serum samples from rectal cancer patients, low ROS levels were associated with T4 disease, representing a bulky and often hypoxic tumor, and thus adverse survival. Moreover, low ratio of ROS to mtDNA damage was correlated with poor histologic tumor response to neoadjuvant radiotherapy. These parameters may reflect an aggressive biological phenotype instigated by a hostile tumor microenvironment. The results are currently under validation in an independent patient cohort. Trial registration: NCT00278694

#1783

Early detection of thymidylate synthase resistance in non-small cell lung cancer with FLT-PET imaging.

Xiao Chen, Yizeng Yang, Sharyn Katz. _Hospital of the University of Pennsylvania, Philadelphia, PA_.

Background: Inhibition of thymidylate synthase (TS) results in a transient compensatory "flare" in thymidine salvage pathway activity which is measureable with 18F-thymidine (FLT) positron emission tomography (PET) at 2 hrs of exposure to therapy. Here we examine the predictive value of the FLT-PET measured "flare" for NSCLC sensitivity to pemetrexed, a commonly used TS inhibitor, in a preclinical model.

Experimental Design: Resistance to pemetrexed therapy was induced in two sensitive human NSCLC cell lines, H460 and H1299, by overexpressing TS. TS overexpression was confirmed with RT-PCR and Western blotting and pemetrexed-resistance confirmed with IC50 assays. The presence of a pemetrexed-induced DNA salvage pathway "flare" was then measured using 3H-thymidine in both pemetrexed-sensitive (H460 and H1299) and resistant (H460R and H1299R) lines in vitro, as well as inherently resistant NSCLC cell lines CALU6, H522, H650, H661, H820, H1838, and validated with FLT-PET in vivo using H460 and H460R xenografts.

Results: Overexpression of TS induced resistance to pemetrexed in the H460 and H1299 with IC50 for H460, H1299, H460R and H1299R measured as 0.141 μM, 0.656 μM, 22.842 μM, 213.120 μM, respectively. Significant increases in DNA salvage pathway activity ("flare") was observed at 2hrs of therapy in the pemetrexed-sensitive H460 and H1299 lines but not the resistant H460R and H1299R, CALU6, H522, H650, H661, H820, H1838 cell lines in vitro using 3H-thymidine assays. Similarly, FLT "flare" was observed in the pemetrexed-sensitive H460 xenograft tumors but not the pemetrexed-resistant H460R xenograft tumors with post-therapy increases in measured FLTmax of 58% (STD 12.1) and 10.8% (STD 7.3) respectively.

Conclusions: FLT-PET imaging of TS inhibition may provide an early indicator of NSCLC sensitivity to pemetrexed.

#1784

Early feasibility and development of multiplexed, single-reaction assays for ALK, ROS1 and RET novel ddPCR RNA fusions.

Hestia Mellert,1 Kristin Alexander,1 Leisa Jackson,1 Galen Roda,1 Samantha Cooper,2 Dianna Marr,2 Stephen J. Jones,2 Nia Charrington,2 Gary Pestano1. 1 _Biodesix, Inc., Boulder, CO;_ 2 _Bio-Rad, Inc., Pleasanton, CA_.

We have previously described a targeted genomic Laboratory Developed Test (LDT) that includes variant specific Droplet Digital™ PCR (ddPCR) testing for EGFR, KRAS and BRAF in plasma. This test supports the rapid delivery of molecular diagnostic test results, with >95% of tests results delivered in 72 hours of receipt in our Laboratory. This test then meets the key clinical need for the delivery of results that can result in faster treatment decisions. Additionally, the test may be of utility for those patients who need mutation results quickly or for whom tissue may be unavailable or insufficient for molecular testing. This is especially true for patients diagnosed with non-small cell lung cancer (NSCLC). In this report we will update on new test concepts created using the recently available design software engine for ddPCR assays. Specifically, we will describe studies on the development of single-reaction, multiplexed assays for the respective detection of ROS1 (up to 11 variants), RET (up to 8 variants) and EML4-ALK (v1 - v3). Design considerations, specificity and sensitivity, as well as reproducibility and robustness studies for these complex assays will be reviewed. Similar studies were conducted for the development of the commercially available test for the EML4-ALK fusion variants. EML4-ALK concordance studies compared the fusions found in blood with known positives and negatives found using FISH and PCR based methods (n=24 evaluable matched pair samples). Clinical sensitivity, specificity and concordance were 85%, 100% and 92% respectively. In this study we also report on test performance of the ALK RNA fusion test over 3 consecutive months of testing. Of note, we have delivered greater than 95% of tests (n = 272 samples) with an observed positive sample frequency of 2%. The robust detection of rare variant, RNA fusions from plasma within 72 hours represents a molecular testing option of value to patients with NSCLC and their physicians.

#1785

Technical validation of novel 325 RNA predictive biomarkers using gene expression data generated by Nanostring n-counter and Affymetrix microarray.

Marcia V. Fournier,1 Said Attiya,1 Joan Chen,2 John Obenauer,2 Anup Madan,3 Patricia Smith3. 1 _Bioarray Genetics, Inc., Farmington, CT;_ 2 _Rancho Biosciences, San Diego, CA;_ 3 _Covance Genomics Services, Redmond, WA_.

Background: With new developments of oncology drug combinations and targeted treatments, the ability to stratify categories of patient populations and to develop companion diagnostics has become increasingly important. A panel of 325 RNA-biomarkers was selected based on cancer-related biological processes of healthy cells and gene expression changes over time during non-malignant epithelial cell organization. This "cancer in reverse" approach in combination with empirically derived algorithms resulted in a panel of biomarkers having little overlap with 9 other widely-used commercial gene panels analyzed to date (e.g., overlap with FoundationOne was 2% (lowest) and with OncotypeDX was 14% (highest)), providing a more in-depth and comprehensive view of gene expression profiles and key cellular signaling pathways beyond mutations in "driver-genes", and drug associations including chemotherapies, immunotherapies, and targeted-therapies.

Objective: In order to technically validate an assay for the 325 RNA-biomarkers we compared gene expression profiles side-by-side using two technology platforms to address the reproducibility of the assay.

Methods and Results: We have mapped the 325 RNA transcripts and 7 housekeeping genes in a custom NanoString n-Counter expression panel to be compared to all potential probe sets in the Affymetrix Human Genome U133 Plus 2.0 microarray. The experiments were conducted with 10 unique biological formalin-fixed paraffin-embedded (FFPE) breast tumor samples. Each site extracted RNA from four sections of 10-microns thick FFPE tissue over three different days by three different operators using an optimized standard operating procedure and quality control criteria. Samples were analyzed using mas5 in BioConductor and NanoStringNorm in R. Pearson correlation showed reproducibility between sites for all 60 samples with r=0.995 for Affymetrix and r=0.999 for NanoString. Correlation in multiple days and multiple users were for Affymetrix r=(0.962-0.999) and for NanoString r=(0.982-0.991). The platforms were compared using relative expression fold changes using linear regression (lm). Concordant genes were defined to have gene expression levels within +/- 2 standard deviation (sd) of each other at 95% confidence interval, or to have greater than +/- 2 sd but have changes in the same direction in both platforms. The discordant genes were defined to have opposite direction of changes. By this definition, approximately 90% of the genes fell into the concordant category.

Conclusion: The 325 RNA-biomarkers showed reproducibility in two technology platforms with high concordance. With five predictive tests under development for breast (3), lung and pancreatic cancers future directions include performing clinical validation studies and generate rationale for patient selection in clinical trials using the technically validated assays.

#1786

Predictive value of LRP8, KPNA2 and GDF15 expression to anthracycline/taxane based chemotherapy response in patients with locally advanced breast cancer.

Hector Maldonado-Mtz,1 Alonso-Luna Oscar,2 Ali Flores-Perez,2 Meneses-Garcia Abelardo,1 Erika B. Ruiz-Garcia,1 Cesar Lopez-Camarillo,3 Horacio Astudillo-de la Vega4. 1 _INCan, Mexico, DF, Mexico;_ 2 _Nanopharmacia Diagnostica, Mexico, DF, Mexico;_ 3 _UACM, Mexico, DF, Mexico;_ 4 _IMSS, Mexico, DF, Mexico_.

Background: Chemotherapy plays a major role in its treatment, though overtreatment is frequent since its choice is based on clinicopathological prognostic data. The identification and validation of new predictive profiles or markers continues to be relevant. New findings may contribute to achieve better results in this area, as well as to provide new targets of the pathways involved in chemoresistance. Karyopherin α2 (KPNA2), low density lipoprotein receptor-related protein 8 (LRP8) and growth and differentiation factor 15 (GDF15), are three candidate predictive biomarkers in breast cancer, previously identified on DNA microarrays reports.

Methods: Using tissue microarrays technology and immunohistochemistry, we compared the expression of these three markers between cases of breast cancer with response to chemotherapy (absence of recurrence), and those with failure (with recurrence). All patients received chemotherapy based on anthracyclines and taxanes. Potential confounding factors, as known recurrence and progression risk factors, were controlled for during the comparison.

Results: 20 out of 63 patients had tumor recurrence. Potential confounding factors were similar among cases with response and failure to chemotherapy. For KPNA2 no differences were found between cases with response or failure. LRP8 showed higher expression in cases with failure to chemotherapy (mean 7.22 vs 14.01%, p =0.025). In Cox multivariable regression analysis, which included potential confounding factors as covariates, only LRP8 remained as a significant predictive factor (HR 1.28, p=0.016). GDF15 showed no expression in our cases, nor in an external set of cases.

Conclusions: We demonstrated increased expression of LRP8 in breast cancer cases recurring after chemotherapy. KPNA2 and GDF 15 showed no predictive value for chemotherapy response in our study. LRP8 is a potential predictive marker worth of further investigation for its role in chemoresistance as well as for its potential validation.

#1787

miR-150 as a predictor of hematopoietic reconstitution after myeloablation.

Marshleen Yadav, Arnab Chakravarti, Naduparambil K. Jacob. _The Ohio State University, Columbus, OH_.

The identification of biomarkers that allow evaluation of myeloablation and hematopoietic reconstitution time frame is important for tailoring the radiation or chemo therapy in cancer patients. The fate of hematopoietic cells is regulated at multiple stages by various genes and regulatory microRNAs. In this context, miR-150 has been demonstrated as a major regulator of lymphoid lineage differentiation and development. Our mouse model studies shows a novel role of miR-150 in imparting radiation resistance. We observed a prolonged survival benefit in mice where both the copies of miR-150 genes are deleted in comparison to that of wildtype mice in the same C57BL/6 background (p<0.05). The survival benefit after total body irradiation was found to be associated with lower apoptosis and early reconstitution in hematopoietic stem cells (lin-ckit+sca1+/-) in bone marrow. Furthermore, the recovery kinetics at sub lethal dose (6Gy) showed that HSCs in miR-150-/- mice recovers faster. Myeloid (Mac1+Gr1+) and lymphoid (CD45+CD19+) lineage cells reconstitutes within 14 days after radiation in KO mice compared to 21 days or more in WT mice. We observed a statistically significant upregulation in myeloid cells in KO mice which implicates an early recovery from radiation induced neutropenia and hence more survivability. In addition, the enhanced bone marrow reconstitution was reflected in peripheral blood counts where a noticeable difference (p<0.05) in blood RBCs lymphocytes, monocytes, neutrophils, and basophiles were noted when compared sub lethally irradiated KO with WT mice. Altogether we demonstrate that the level of miR-150 regulates the bone marrow cells distribution and hence could be targeted for achieving accelerated hematopoietic recovery after myeloablation. These findings may be translated to human cancer patients for therapy guiding based on miR-150 expression level. Besides its use in prediction of reconstitution, miR-150 in the circulating system could provide a direct readout of functional marrow and hence hematological acute radiation syndrome (H-ARS).

#1789

A gene expression-based nomogram predicts overall and progression free survival in PRRT-treated gastroenteropancreatic neuroendocrine tumors.

Mark Kidd,1 Lisa Bodei,2 Stefano Severi,3 Ignat Drozdov,1 Sylvia Nicolini,3 Giovanni Paganelli,3 Irvin M. Modlin4. 1 _Wren Laboratories, Branford, CT;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 3 _Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (I.R.S.T.), Meldola, Italy;_ 4 _Yale University, New Haven, CT_.

Background: Nomograms and grading/staging systems are fundamental in guiding oncology disease management. Histological grading has efficacy in the management of gastroenteropancreatic neuroendocrine tumors (GEP-NET) but tumor heterogeneity renders outcome unpredictable. Clinical parameters alone have limited prognostic ability. A previously described NET nomogram calculated risk for overall survival (OS). To augment accuracy, we developed a circulating NET multigene expression blood test. The NETest exhibits high level metrics as a molecular prognostic for progression free survival (PFS). We evaluated whether combining clinical information with the NETest would provide accurate prognostic information for PRRT treatment.

Aim: Create a combined clinical and gene expression nomogram to predict OS and PFS in PRRT-treated GEP-NETs.

Methods: 177Lu-based-PRRT-treated GEP-NETs (n=57; median age 65 yrs (31-83); 34M:23F; small bowel (47%), pancreas (35%), predominantly G2: 65%) followed for a median 15 months (range 1-33). The clinical nomogram comprised 10 variables (including: age, gender, grade, Ki67, stage symptoms, liver metastases, somatostatin analog use, surgery extent, Chromogranin A level) with a score range: 25-200. This data was interpolated with pre-therapy NET transcript analysis results (range 0-100) into a hybrid molecular nomogram (range: 25-300). Outcomes were OS and PFS (RECIST criteria). Receiver operating characteristics (ROC), Kaplan-Meier survival and non-parametric (Mann-Whitney) analyses were performed.

Results: Median OS was not reached (8 deaths); mPFS was 19 months (20/57 disease progression). Neither grade nor stage alone predicted a difference in OS or PFS. Clinical nomogram scores for non-survivors was significantly higher (139±20 vs. 87±6, p<0.001) than survivors. Pre-treatment NETest values were higher in non-survivors (56±10 vs. 36±3, p<0.04). Integration of clinical and molecular nomogram scores into a hybrid nomogram significantly amplified the prognostic efficacy. Non-survivors had higher scores than survivors (189±14 vs. 113±9, p<0.001). A cut-off score of >130 exhibited an AUC for OS prediction of 0.84±0.05, (p=0.002) with a Hazard ratio (HR) of 8.7 (2.1-35.4, p<0.002). A score of <130 predicted 100% survival. PFS prediction using the hybrid nomogram cut off of 130 had an AUC of 0.68±0.07, p=0.02. The Gehan-Breslow-Wilcoxon test value was 7.2 (p<0.008) demonstrating that high scores were prognostic of early progression (treatment failure). The HR was 3.1 (1.2-7.4, p<0.02) and the mPFS was 17 months vs. not reached.

Conclusions: Hybrid molecular nomogram data derived from a circulating gene expression signature combined with a clinical NET nomogram accurately predicts OS in PRRT-treated GEP-NETs. The nomogram also predicted PFS particularly individuals with elevated transcript values who will not respond to PRRT.

#1790

Evaluation of UCK2 protein expression as a potential predictive biomarker of RX-3117.

Young Bok B. Lee,1 Deog J. Kim,1 Christina George,1 Chang-ho Ahn,1 Julie Frank,1 Reza Mazhari,1 Lisa M. Dauffenbach,2 Eric P. Olsen2. 1 _Rexahn Pharmaceuticals, Inc., Rockville, MD;_ 2 _Mosaic Laboratories, Lake Forest, CA_.

Background: A novel, orally bioavailable nucleoside analogue, RX-3117, is a prodrug activated intracellularly by Uridine Cytidine Kinase 2 (UCK2) that is thought to be expressed predominantly in tumor tissue. RX-3117 is currently being evaluated in a Phase Ib/IIa multi-center, open-label clinical study in patients with advanced pancreatic and bladder cancer. In this study, we aimed to determine the relation between UCK2 tissue protein expression and the efficacy of RX-3117 in mice xenograft models and also UCK2 protein expression in a panel of human cancer tissues relative to normal tissue.

Methods: The UCK2 protein expression in tumor tissues was analyzed by immunoblotting using clone 22-1 rabbit monoclonal antibody. The validated procedure for the immunohistochemistry (IHC) of UCK2 with clone 22-1 was performed in a panel of human formalin-fixed paraffin-embedded (FFPE) cancer and normal tissues.

Results: The immunoblotting protein level of UCK2 normalized to β-actin and corresponding tumor growth inhibition (oral RX-3117 dose of 500mg/kg, TIWK) were 57 and 67% in MiaPaCa2, 30 and -5% in BxPC3, 199 and 92% in Colo-205, 21 and 90% in Caki-1, 2 and 39% in A549, and 146 and 79% in H460, respectively. This data indicates an anti-tumor efficacy trend in a UCK2-dependent manner. The IHC of UCK2 showed that positive staining of UCK2 in cancer tissues was observed in 20/20 bladder cancer tissues (100% frequency), 19/20 CRC tissues (95% frequency), 18/20 NSCLC tissues (90% frequency), and 19/20 pancreatic cancer tissues (95% frequency). Average H-Scores of UCK2 in cancer tissues vs. normal tissues were 104 vs. 9 in lung, 97 vs. 20 in bladder, 67 vs. 41 in pancreas and 39 vs. 21 in colon, respectively.

Conclusions: The current data showed a correlation trend between UCK2 protein expression level and degree of antitumor activity of RX-3117 in xenograft models. It also supports a higher UCK2 protein expression level in human cancer tissues compared to their normal tissues. This suggests that RX-3117 activity may be specific to tumor tissue, and quantification of UCK2 expression in human cancer tissues may be useful as a predictive biomarker to select patients for their sensitivity to RX-3117 in future clinical studies.

#1791

**Spatially positioned tumor infiltrating lymphocytes predict survival in metastatic** HER2 **positive breast cancer treated with trastuzumab.**

Jussi Pekka Koivunen, Tiia Honkanen, Tiina Moilanen, Peeter Karihtala, Juha Väyrynen, Markus Mäkinen. _Oulu Univ. Hospital, Oulu, Finland_.

Background: Disease outcomes of HER2+ breast cancers have dramatically changed after targeted therapies, such as trastuzumab, came to clinical practice but predictive factors for trastuzumab sensitivity and resistance are frequently unknown.

Methods: Metastatic breast cancer patients (n=48), who were treated with trastuzumab and had pre-treatment tumor samples available, were studied. The tumors were immunostained for T-cell (CD3, CD8) and NK-cell (CD56) markers and quantitative analysis of the immune cells was carried out using a computer-assisted image analysis in different tumor locations.

Results: High number of CD3 and CD8 positive T-cells was associated with significant survival benefit in the center of the tumor (CT) (p=0.007, p=0.001), but not in the invasive region. NK-cell tumor infiltration was infrequent and they could not be reliably analyzed. In a subgroup analyses, high density of CD8 CT cells was associated with significant survival benefit in non-bone only disease, in TX-3, and in ER+ tumors (p=0.006, p=0.003, p=0.001). Moreover, high CD8 CT cell density was associated with good trastuzumab responses (p=0.042).

Conclusion: High number of CD3 and CD8 positive tumor infiltrating lymphocytes in the CT area is associated with survival benefit in some patient groups with HER2+ breast cancer treated with trastuzumab. Furthermore, high number of CD8 CT cells predicts benefit from trastuzumab.

#1792

Immune system pathway activation for prediction of the response to neoadjuvant chemotherapy in triple-negative breast cancer.

Takeshi Sawada,1 Riet Hilhorst,2 Savithri Rangarajan,2 Masayuki Yoshida,3 Kenji Tamura,3 Rinie van Beuningen,2 Rob Ruijtenbeek,2 Hitoshi Tsuda,4 Fumiaki Koizumi1. 1 _Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Tokyo, Japan;_ 2 _PamGene International BV, 's-Hertogenbosch, Netherlands;_ 3 _National Cancer Center Hospital, Tokyo, Japan;_ 4 _National Defense Medical College, Saitama, Japan_.

Background: Triple-negative breast cancer patients (TNBCs) who receive neoadjuvant chemotherapy (NAC) generally show a response to NAC, but about 5% experience progressive disease (PD). Although TNBCs that respond to NAC have been well-studied, reports about TNBCs with PD during NAC are absent. We aimed to compare kinase activity profiles of TNBCs of both groups to identify a biological predictor of NAC non-responders.

Methods: Tyrosine kinase activity profiles of lysates of fresh frozen cancer tissues from NAC non-responders and responders, the latter represented by TNBCs who did not receive NAC, were determined with a peptide microarray system (Cancer Res. 2009; 69(14):5987-95). Tumor infiltrating lymphocytes (TILs) were counted in these tissues.

Results: Among 740 TNBCs, 20 NAC non-responders were identified. For the 7 samples from PD confirmed patients and 10 TNBCs not receiving NAC, no correlation was observed between NAC response and age, menopausal status, tumor size or axillary lymph node status. Kinase activity measurements showed large differences in overall tyrosine kinase activity between responders and non-responders. 35 peptides had significantly (p < 0.05) higher phosphorylation in the responder group. Kinases that phosphorylate these peptides (upstream kinases) were identified from literature. Among others, the immune related kinases Zap70, Syk and JAK2 were identified as upstream kinases. Pathway analysis yielded evidence for increased activity in the responder group in immune-related pathways and in pathways involved in development and signal transduction. Responders had a significantly higher (p = 0.0053) TILs count.

Conclusion: Kinases related to the immune system are less activated in NAC non-responders. The low TILs count in the non-responders confirmed that activation of the immune system in non-responders is lower than in NAC responders. These data merit a further investigation of the use of kinase activity profiling either alone or in combination with TIL scoring for the prediction of the response to NAC in TNBC with a larger patient cohort.

#1793

Telomere length and mitochondrial DNA copy number variations as predictors for survival in hepatocellular carcinoma patients treated with sorafenib.

Dong Yan. _Beijing Chaoyang Hospital & UT MD Anderson Cancer Ctr., China_.

Background: The study aims to evaluate the potential predictive value of relative telomere length (RTL) and mitochondrial DNA (mtDNA) copy number variations for survival in hepatocellular carcinoma (HCC) patients treated with sorafenib.

Methods: The study was conducted in 120 patients with pathologically or radiologically confirmed HCC who were treated with sorafenib as their first-line or second-line monotherapy. RTL and mtDNA copy number in peripheral lymphocytes were measured using quantitative real-time polymerase chain reaction (PCR)-based method. ANOVA and non-parametric tests were applied to compare the RTL and mtDNA copy number between groups. Progression-free survival (PFS) and overall survival (OS) time was analyzed using Kaplan Meier plot, log-rank test, and Cox proportional hazards regression model.

Results: A longer telomere was observed in patients with early stage disease or normal body weight and in Hispanics compared to their counterparts. Patients with longer RTL compared to those with shorter RTL had significantly better PFS (6.90 vs 4.40 months, P=0.048) and OS (16.53 vs 7.72 months, P=0.006). Cox regression analysis demonstrated that longer RTL was an independent predictor for OS (hazard ratio [HR]=0.47, 95% confidence interval [CI]=0.29-0.76, P<0.01) in all patients and for PFS in patients received sorafenib (HR=0.37, 95% CI=0.15-0.94, P=0.01) after adjusting for other clinical predictors. RTL was positively correlated to mtDNA copy numbers (r=0.312, P=0.001), but no significant association of mtDNA copy numbers and PFS or OS was observed.

Conclusion: RTL, but not mtDNA copy number, might be a potential predictor for OS and for response to sorafenib treatment in HCC.

#1794

Lymphocyte apoptosis as a predictive biomarker for radiotherapy de-intensification in EBV-associated nasopharynx cancer.

Kevin L.M. Chua,1 Shihabuddeen Waseem Ahamed,1 Ma Than Than Shwe,1 Li-lian Kwok,1 Joseph T.S. Wee,1 Terence W.K. Tan,1 Prakash M. Hande,2 Kai Rothkamm,3 Melvin L.K. Chua1. 1 _National Cancer Centre Singapore, Singapore, Singapore;_ 2 _National University Singapore, Singapore, Singapore;_ 3 _University Medical Center Hamburg-Eppendorf, Hamburg, Germany_.

Aims: Emerging evidence in viral-associated oro- and naso-pharynx cancers supports the concept of radiotherapy (RT) dose de-intensification in these radiosensitive tumours. This notion is particularly relevant in the background that a majority of head and neck cancer patients develop severe late toxicities despite modern precision RT. A reasonable strategy could thus entail stratifying patients at risk of late toxicities to dose de-intensification. Here, we investigated the utility of a lymphocyte apoptosis (RILA) assay to predict for late toxicities in EBV-positive nasopharynx cancer patients. We also test if a multi-modal approach incorporating DNA damage induction and repair improves the prediction of clinical radiosensitivity. Methods: Assays were assessed retrospectively among survivors of a randomised controlled trial (NCC-0902), where patients were assigned to weekly cisplatin-RT with or without neoadjuvant gemcitabine, carboplatin, and paclitaxel. Late toxicities were assessed by CTCAE v.2 at the following intervals - 2-mo year 1, 4-mo year 2, 6-mo years 3-5, and annually thereafter; and considered severe if CTCAE v.2 ≥Grade 2. RILA was based on a fluorogenic inhibitor of caspases assay (48 h post-8 Gy). DNA damage induction and repair were assessed by semi-automated scoring of ϒH2AX foci at 30 min post-1 Gy and 24 h post-4 Gy, respectively. Results: Median follow-up of patients was 5.7 (range 4.6-7.4) years. Clinical and treatment indices, including assigned study arm and RT dosimetry to normal tissue structures, were balanced between patients with and without late toxicities (p >0.05). We observed a trend between decreasing RILA scores and increased risks of late toxicities; odds ratio (OR) 1.27 (95% CI = 0.92-1.72) for lowest quartile cut-off, corresponding to an AUC of 0.608 for prediction accuracy (p = 0.049). A multi-modal approach incorporating DNA damage induction and repair did not improve upon the predictive accuracy of RILA for clinical radiosensitivity. Independently, DNA damage induction and repair were not associated with risks of late toxicities; OR 1.03 (95% CI = 0.69-1.53, p = 0.901) and 1.04 (95% CI = 0.73-1.49, p = 0.829), respectively. Interestingly, onset of late toxicities was correlated to an improved disease-free survival in our cohort (5-y DFS = 94.1% vs 73.3%, no late toxicity, p = 0.003), which suggests a common mechanism underlying tumour and normal tissue radiosensitivity in EBV-positive nasopharynx cancer. Conclusions: Herein, we report on the potential utility of a RILA assay in stratifying for patients at risk of late RT-induced toxicities. The clinical implication of this assay is further exemplified by the observation of a superior survival in these sensitive individuals, which supports the approach of selecting this subgroup for treatment de-intensification.

#1795

Hypermethylation status of PDX1 has potential of diagnostic and predictive biomarker for risk stratification in colorectal cancer patients.

Takashi Ichikawa, Yuji Toiyama, Yoshinaga Okugawa, Hiromi Yasuda, Hiroyuki Fujikawa, Yoshiki Okita, Shigeyuki Yoshiyama, Junichiro Hiro, Minako Kobayashi, Toshimitsu Araki, Masaki Ohi, Yasuhiro Inoue, Keiichi Uchida, Yasuhiko Mohri, Masato Kusunoki. _Mie University Graduate School of Medicine, tsu,mie, Japan_.

Background Abnormal DNA methylation is an important epigenetic mechanism that has been strongly implicated in the pathogenesis and progression of colorectal neoplasia. In contrast to other potential biomarker species such as mRNA and protein levels, measurement of DNA methylation has been shown to be stable, reproducible and consistent between individuals. Based on these standpoints, detecting aberrant methylation of cancer-related genes helps for the development of diagnostic and prognostic biomarkers in colorectal cancer (CRC). In this study, we comprehensively evaluated whole-genome methylation analysis using colorectal cancer tissues and normal colonic mucosa to discover candidate CRC-specific DNA methylation as potential diagnostic and predictive biomarkers for risk stratification in CRC patients.

Patients and Method The study design included an initial discovery phases, and followed by a subsequent clinical validation phase. In discovery phase, we performed whole-genome methylation analysis using CRC tissues and normal colonic mucosae from total fourteen patients to identify CRC-specific methylated CpG cites. Furthermore, we evaluated methylation levels of candidate methylated CpG cites by quantitative bisulfite pyrosequencing using one hundred six colorectal specimens from fifty-three CRC patients to validate the findings from discovery phase.

Result In discovery phase, we successfully identified several candidates of CRC-specific hypermethylated- and hypomethylated-CpG cites in CRC tissues compared with normal colonic mucosa. Promoter lesion of PDX1 is one of candidate methylated CpG cites in discovery phase, and we focus a specific CpG cite of PDX1 promoter lesion as a target cite to progress in validation phase. We quantified methylation status of PDX1 using 106 colonic tissues, and demonstrated that PDX1 were significantly hyper-methylated in CRC tissues compared with normal mucosa (p<0.0001, Wilcoxon rank correlation test). Receiver operating characteristic (ROC) curves analysis revealed that methylation levels of PDX1 remarkably discriminated CRC tissues from normal colonic mucosa (AUC:0.9, Sensitivity:85.4%, Specificity:91.5%). Interestingly, methylation levels of PDX1 tend to increase in stage-dependent manner, and hypermethylated status of PDX1 was significantly correlated with presence of distant metastasis in validation cohort.

Conclusion Assessment of the PDX1 methylation status could be used as potential of diagnostic and predictive biomarker for risk stratification in CRC patients.

#1796

A targeted RNA-seq assay to measure activating ER mutations and ER/PR-associated gene expression predicts sensitivity to endocrine therapy for metastatic breast cancer.

Rosanna Lau,1 Lily Fu,1 Michael Samuels,2 Rashmi K. Murthy,1 Bruno Sinn,3 Jane Yu,1 Rebekah Gould,1 Jennifer Litton,1 Alda Tam,1 Stacy Moulder,1 Daniel Booser,1 Debu Tripathy,1 Vicente Valero,1 Fraser Symmans1. 1 _M.D. Anderson Cancer Center, Houston, TX;_ 2 _RainDance Technologies, MA;_ 3 _Charité – Universitätsmedizin Berlin, Berlin, Germany_.

Background: We developed SET4 as a targeted, droplet-based, next-generation RNA sequencing assay to measure both the SETER/PR index of gene expression and the percent of estrogen receptor (ER) transcripts with point mutation in the ligand-binding domain (LBD) in metastatic biopsies of Stage IV breast cancer. The SETER/PR index (31 genes) is a cumulative measure of gene expression for transcripts associated with ER and progesterone receptor (PR), excluding those with a direct role in proliferation. High SETER/PR could indicate increased sensitivity to endocrine therapy, whereas LBD mutations indicate resistance but might also induce high SETER/PR.

Methods: Targeted needle biopsies from a metastatic site were prospectively obtained from 82 patients with HR+/HER2- breast cancer at time of any progression event, and preserved in RNAlater. Samples were prepared for targeted sequencing on a MiSeq by combining purified total RNA with SET4 primers and RT-PCR master mix into single molecule-formatted picodroplets using a RainDrop Source instrument, followed by thermal cycling and sample indexing. Calculated SETER/PR index and percent ER transcripts with LBD mutations were evaluated as continuous variables and compared to progression-free and overall survival using Cox regression analysis with log-rank test, if the next treatment after biopsy included endocrine therapy.

Results: The average read depth for the LBD of the ER transcript was 33,475X (range: 1230-180,889X), confidently detecting mutation at 1% frequency. LBD mutations were identified in 17% (14/82) of metastases (range of mutated transcripts 1%-98%). LBD mutations (>10% of transcripts) were only observed in metastases with higher SETER/PR (above the median). In patients who next received endocrine therapy (n=58), higher SETER/PR predicted longer progression-free (PFS) (HR=0.37, p=0.0004, Δ median PFS 9 months) and overall survival (OS) (HR=0.49, p=0.03). The predictions were more pronounced in patients without LBD mutation (PFS HR=0.32, p=0.0001, Δ median PFS 13 months; OS HR=0.42, p=0.01). Currently, there are insufficient cases with LBD mutation for reliable survival analysis.

Conclusion: The SET4 assay measured the percent of ER transcripts with activating LBD mutation (≥1% prevalence) and also downstream ER/PR-related transcription. High SETER/PR predicted longer PFS and OS with endocrine therapy. While activating LBD mutations may be associated with endocrine resistance of Stage IV cancer, they were associated with high SETER/PR index. Consequently, metastatic cancers with high SETER/PR index and no LBD mutation in ER transcripts were particularly sensitive to endocrine therapy. This single assay unraveled a possible interaction between genotype, phenotype, and treatment outcome; and is currently being evaluated in a larger cohort of patients.

#1797

EGFR as a marker for head and neck squamous cell carcinoma-derived small extracellular vesicles.

Jenna A. Dombroski,1 Damaris Kuhnell,2 Susan Kasper,2 Scott M. Langevin2. 1 _University of Buffalo, Buffalo, NY;_ 2 _Univ. of Cincinnati College of Medicine, Cincinnati, OH_.

Head and neck cancer will account for an estimated 61,760 new cancer cases and 13,190 cancer-related deaths in the United States in 2016, the majority of which (> 90%) will be squamous cell carcinoma (HNSCC). The prognosis for HNSCC is relatively poor, with an overall 5-year survival around 60%, and the outlook worsens with increasing stage at diagnosis, which is problematic since about two-thirds of patients present with advanced stage disease. This underscores the urgent need for discovery, development and translation of novel biomarkers to facilitate early detection of HNSCC and bolster the chance for positive patient outcomes. Exosomes are nano-sized (40-150 nm) membrane-encapsulated vesicles that may offer a new avenue for discovery and development of novel HNSCC biomarkers. However, they are released by essentially all cells, both normal and malignant, into the extracellular space, resulting in a noisy environment in biofluids from the perspective of biomarker measurement. As such, a membrane-bound surface marker that allows for enrichment for cancer-derived exosomes in patient samples would provide a clear benefit. Epidermal growth factor receptor (EGFR), which belongs to the ErbB/HER family of receptor tyrosine kinases, localizes to the plasma membrane and is over-expressed in as much as 90% of HNSCC tumors. Therefore, we hypothesized that EGFR will be over-represented on membranes of HNSCC-derived exosomes and thus may offer utility as a surface marker for detection and/or enrichment. To test this, we cultured an HNSCC cell line that over-expresses EGFR (FaDu), EGFR-deficient SW620 colorectal adenocarcinoma cells, and primary human oral epithelial cells pooled from 3 healthy donors. Cells were cultivated in supplier-recommended media with 10% fetal bovine serum (FBS) that was super-depleted of exosomes via 18 hour ultracentrifugation at 100,000g and 1% penicillin/streptomycin at 37°C with 5% CO2 in 150 cm2 flasks with 25mL media. After reaching 80-90% confluence, media was harvested, and exosomes were isolated and purified via differential ultracentrifugation. Exosome isolates were whole-mounted on carbon grids, Immuno-Gold labeled with anti-EGFR, and imaged on a JEOL JEM-1230 transmission electron microscope (Immuno-EM); anti-nucleoporin 153 (NUP-153), which localizes to the nucleus, was used as a negative control. Immuno-EM images indicate abundant EGFR expression on exosomes secreted by FaDu HNSCC cells, but EGFR is not detectable on the exosomes secreted by the oral epithelial cells from healthy donors. These results indicate that EGFR may offer utility for enrichment for HNSCC-derived exosomes in human biofluid isolates using magnetic beads or flow cytometry.

#1798

Mortalin precursor as potential marker for chemoprevention with SHetA2.

Elangovan Thavathiru, Vishal Chandra, Rajani Rai, Doris Benbrook. _University of Oklahoma HSC, Oklahoma City, OK_.

Introduction: The flexible heteroarotinoid (Flex-Het), SHetA2, is a novel anticancer drug that induces both intrinsic and extrinsic apoptosis and autophagy in cancer cells, but not in normal cells. Protein isolation / mass spectrometry analysis using SHetA2-coupled polystyrene magnetic beads yielded three SHetA2-binding proteins all belonging to HSPA family namely, HSPA9/mortalin, HSPA8/Hsc70 and HSPA5/BiP. Mortalin, in addition to its vital chaperoning roles in other organelles of the cell, is essential for import and folding of nuclear-encoded mitochondrial proteins. The precursor form of mortalin has a 46-amino acid N-terminal region that functions as a mitochondrial localization sequence (MLS) and is cleaved by proteases after import into the mitochondrial matrix.

Hypothesis: SHetA2 binding to mortalin causes alterations that can be measured to study mechanism and monitor drug effects in animal models and clinical trials.

Methods: Western blots of whole cell or subcellular protein extracts made from cultures of normal human fallopian tube secretory or mammary epithelial cells, rat mammary tumors or human ovarian cancer cell lines treated with SHetA2 or solvent were probed with an antibody that recognizes total mortalin, a custom-made rabbit polyclonal antibody specific for the mortalin MLS (PA8238) or antibodies to loading control proteins. Immunocytochemistry using these antibodies was performed with an automated (Leica Bond III) IHC work station on cells treated with SHetA2 or solvent.

Results: Using a commercial antibody against total mortalin, we observed a lower-mobility band closely moving above the specific (mature) mortalin band in Western blots. Our mortalin MLS-specific antibody recognized only the lower-mobility band confirming it as the precursor form of mortalin in the SHetA2-treated cell extracts. Subcellular fractionation of the drug-treated cells revealed that the precursor protein accumulated only in the cytoplasm and not in the mitochondria. Staining of whole cells with an antibody to total mortalin showed no effect of SHetA2 treatment on the punctate pattern of expression consistent with mitochondrial localization. In contrast, staining with the mortalin MLS-specific antibody demonstrated that SHetA2 increased the intensity of the diffuse cytoplasmic staining consistent with localization of mortalin throughout the cytoplasm. These results were consistently observed among the various cell types.

Conclusions: SHetA2 is the only known drug that blocks the translocation of the precursor mortalin to mitochondria and its processing to mature protein. Such cytoplasmic accumulation of the precursor form of mortalin can potentially serve as pharmacodynamic endpoint to study SHetA2's effect in laboratory experimental tumor models as well as in human clinical trials. Funding: NCI PREVENT Task Order HHSN26100002

#1799

CRM1 expression in pancreatic adenocarcinoma correlates with survivin expression and the proliferative activity.

David M. Saulino, Pamela S. Younes, Jennifer M. Bailey, Mamoun Younes. _University of Texas Health Science Center at Houston McGovern Medical School, Houston, TX_.

Introduction: CRM1 is a nuclear export chaperone that mediates the export of proteins essential to growth regulation and tumor suppression. Its overexpression in tumors was found to be associated with poor prognosis. Selective inhibitors of nuclear export are in phase I and II clinical trials for several tumor types. The expression of CRM1 in human pancreatic adenocarcinoma (PAC) and its relation to survivin expression and tumor proliferative activity is largely unknown.

Experimental procedures: Sections of tissue microarray containing 77 formalin fixed and paraffin embedded PAC were stained by immunohistochemistry (IHC) for CRM1, survivin, and Cyclin A. Expression levels of CRM1 and survivin in tumor cells was determined using a quantitative digital image analysis solution (OTMIAS). The tumor proliferative activity was determined by measuring the S-phase fraction (SPF) in sections stained for Cyclin A, also using OTMIAS.

Summary: Sixty-six of the 77 (86%) PAC showed at least some positive staining for CRM1, and 11 (14%) were completely negative. The mean CRM1 expression levels ranged from 0.3 to 53 units and the median from 0.3 to 45 units. There was significant positive correlation between the mean and median expression levels of CRM1 in tumor cells with the mean and median levels of survivin (p<0.001). Moreover, there was positive correlation between the mean and median CRM1 levels in tumor cells the SPF (p=0.005).

Conclusions: CRM1 is expressed in a significant proportion of PAC, and increased CRM1 expression correlates with increased survivin expression and increased proliferative activity, suggesting that selective inhibitors of nuclear export may be effective against PAC.

#1800

Noninvasive approach to assess metastatic lesion of advanced colorectal cancer by denoised deep target sequencing.

Jun-Kyu Kang,1 Hwang-Phill Kim,1 Seul-Ki Cheon,1 Ye-Lim Park,1 Yoojoo Lim,2 Sae-Won Han,2 Tae-You Kim2. 1 _Seoul National University, Seoul, Republic of Korea;_ 2 _Seoul National University Hospital, Seoul, Republic of Korea_.

The cell-free DNA (cfDNA) represents a minimally invasive and alternative source of tumor DNA for molecular profiling. Despite next-generation sequencing (NGS) technique is qualified for genotyping cancer using cfDNA as a noninvasive method, it has caused problems as sequencing error and reproducibility. cfDNA in plasma and gDNA of Peripheral Blood Mononuclear Cells (PBMC) were isolated from each 54 advanced colorectal cancer patients. 39 available tumor tissues were isolated from same patients. Deep target-sequencing was performed with paired-end library enriched exons of 10 genes which are recurrently mutated in colorectal cancer. To reduce sequencing error, we devised 'Denoising' and calculated concordance of somatic variants between cfDNA and tumor tissue sequencing data. In addition, correlation of concordance data was analyzed with the clinical information. As a result, we selectively could detect clinically important somatic alteration among low/high variant allele frequency (0.31%~79.42%). For somatic alteration of 10 genes, sensitivity, specificity and accuracy were increased from 84.5%, 74.6% and 76.9% to 87.6%, 92.0% and 91.1% respectively after 'Denoising'. On the other hand, patients with high cfDNA concentration(>50ng/ml) had higher somatic mutant fragments and larger metastatic lesion in liver than patients who have low cfDNA concentration. Our study showed that denoised deep target-sequencing is a suitable method for cfDNA genotyping and provides insights into strategies for monitoring metastatic lesion of advanced colorectal cancer. 

## TUMOR BIOLOGY:

### Angiogenesis and Vascular Biology 2

#1801

Antiangiogenic agents targeting different angiogenic pathways have opposite effects on tumor hypoxia in R-18 human melanoma xenografts.

Jon-Vidar Gaustad, Trude G. Simonsen, Lise Mari K. Andersen, Einar K. Rofstad. _Institute for Cancer Research, Oslo University Hospital, Oslo, Norway_.

Background: Studies comparing the effect of antiangiogenic agents targeting different angiogenic pathways are sparse. The purpose of this study was to compare the effect of properdistatin and sunitinib treatment in a preclinical model of malignant melanoma. Properdistatin is a small peptide derived from the thrombospondin-1 domain of the plasma protein properdin, and sunitinib is a tyrosine kinase inhibitor targeting several receptors including the vascular endothelial growth factor receptors.

Methods: R-18 human melanoma xenografts growing in dorsal window chambers were treated with properdistatin, sunitinib, or vehicle. Parameters describing the morphology of tumor vasculature were assessed from high-resolution transillumination images, and BST (blood supply time; the time needed for arterial blood to flow from the main supplying artery to downstream microvessels) was assessed from first-pass imaging movies recorded after a bolus of fluorescence-labeled dextran had been administered intravenously. Tumor hypoxia was assessed from immunohistochemical preparations of the imaged tissue by using pimonidazole as a hypoxia marker.

Results: Properdistatin treatment selectively removed small-diameter vessels and reduced BST, whereas sunitinib treatment reduced the density of small- and large-diameter vessel similarly and did not change BST. These observations imply that properdistatin treatment reduced geometric resistance to blood flow and improved vascular function, whereas sunitinib treatment did not affect vascular function. Accordingly, sunitinib-treated tumors showed higher hypoxic fractions than properdistatin-treated tumors.

Conclusions: Properdistatin and sunitinib both inhibited angiogenesis, but had distinctly different effects on vascular morphology, vascular function, and extent of hypoxia in R-18 human melanoma xenografts.

#1802

Apoptosis pathway-focused gene expression profiling of a novel VEGFR2 inhibitor.

Mohammad Algahtani,1 Khalid Alhazzani,1 Thiagarajan Venkatesan,2 Appu Rathinavelu1. 1 _Nova Southeastern University, College of Pharmacy, Davie, FL;_ 2 _Rumbaugh-Goodwin Institute for Cancer Research, Nova Southeastern University, Davie, FL_.

Sprouting of the new blood vessel (angiogenesis) is fundamental to tumor growth, invasion, and metastatic dissemination. Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen, promotes cell survival via opposing apoptosis. On the other hand, antagonizing VEGF pathway suppresses endothelial cell overgrowth and promotes apoptotic signals leading to the regression of the existing vasculature and prevents neovascularization of the tumors. In this respect, a new chemical entity, code named as F16, that was developed in our institute can effectively stop VEGF-driven angiogenesis by selectively blocking VEGFR2 and the downstream signals. Our previous studies have revealed that human umbilical vein endothelial cells (HUVECs) undergo apoptosis in response to F16 treatment. However, in the present study, our main focus was on investigating the status of the signal mediators of the apoptotic pathway after treating the HUVECs with F16. This was expected to outline the intracellular sequence of events involved in triggering apoptosis following F16 treatment. For this purpose, we analyzed apoptosis pathway-focused gene expression in HUVECs using the human apoptosis RT2 profiler which contains 84 key genes involved in regulation of programmed cell death. Our experimental results clearly identified up-regulated pro-apoptotic genes in response to F16 treatment which include tumor necrosis factor -8 and -10A (TNF-8 and TNF-10A), tumor necrosis factor superfamily member 9 (TNFRSF9), FAS cell surface receptor (FAS), FAS ligand (FASLG), lymphotoxin alpha (LTA), caspase 5 (CASP5), and cytochrome c (CYCS). Moreover, a few anti-apoptotic genes were also up-regulated which include B-cell lymphoma 2 related protein A1 (BCL2A1), caspase 14 (CASP14), Bcl2 associated athanogene 3 (BAG3), and interleukin 10 (IL-10). However, anti-apoptotic genes such as Bcl-2, Bcl-2 like 10 (BCL2L10), baculoviral IAP repeat containing -3 and -6 (BIRC-3, and BIRC-6), CASP8 and FADD like apoptosis regulator (CFLAR), and insulin like growth factor 1 receptor (IGF1R) were found to be significantly downregulated. Interestingly, no changes were found in the expression of genes related to DNA damage and repair pathways such as TP53, ABL1, CIDEA, and CIDEB. This indicates that F16 may not provoke DNA damage-induced apoptosis instead, it stimulates the intrinsic or the extrinsic pathway. In conclusion, the diverse gene expressions impacting apoptosis related factors shows that F16 can induce apoptosis via potentiation of TNF- and FAS- signaling. (This research was supported by the generous funds provided by the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida)

#1803

Combining anti-Ang-2/VEGF-A therapy with radio- and chemotherapy in glioma.

Gergely Solecki,1 Matthias Osswald,1 Weber Daniel,1 Malte Glock,1 Miriam Ratliff,1 Hans-Joachim Müller,2 Oliver Krieter,2 Yvonne Kienast,2 Wolfgang Wick,1 Frank Winkler1. 1 _German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 2 _Roche Innovation Center, Munich, Germany_.

Angiogenesis is a biological hallmark of malignant gliomas, but antiangiogenic strategies especially targeting the VEGF axis did not show striking antitumor activities in the majority of patients so far. Other pathways may be more relevant in selected tumor entities or patients. Further, it remains unresolved which antiangiogenic combination regimen with standard radio- and/or chemotherapy is most effective. Therefore, we compared the therapeutic effects of anti-VEGF-A, anti-Ang-2, and bispecific anti-Ang-2/VEGF-A antibodies, alone and in combination with radio- or temozolomide (TMZ) chemotherapy in a malignant glioma model using multi-parameter two-photon in vivo microscopy in mice. We demonstrate that anti-Ang-2/VEGF-A leads to strongest vascular changes including vascular normalization, both as monotherapy and when combined with chemotherapy. The latter combination regimen was accompanied by most effective chemotherapy-induced death of cancer cells independent of blood vessel proximity, indicative of a better distribution of TMZ throughout the tumor. Furthermore, the combination of anti-Ang-2/VEGF-A plus TMZ consistently resulted in decreased tumor cell motility, and decreased formation of resistance-associated tumor microtubes (TMs), which finally lead to best tumor growth inhibition. Remarkably, all these parameters were just reverted when radiotherapy was chosen as combination partner. In contrast, when anti-VEGF-A was combined with radiotherapy, vascular normalization was highest, and TM length, nuclear motility and tumor growth were concordantly reduced. In conclusion, while TMZ chemotherapy benefits most from combination with anti-Ang-2/VEGF-A, radiotherapy does from anti-VEGF-A. The findings imply that unexpected, even divergent effects can occur when a specific antiangiogenic therapy is added to chemo- or radiotherapy in glioma, and that uninformed combination regimens should be avoided.

#1804

Identification of novel NM23 inhibitors as potential anti-angiogenic agents.

Senny Nordmeier, Jon Evasovic, Suzann Duan, Ryan Wuebbles, Dean J. Burkin, Iain L. Buxton. _University of Nevada, Reno, NV_.

Exogenous nucleoside diphosphate kinase (eNDPK or NM23) has been shown to promote endothelial cell proliferation and migration and tumor-mediated angiogenesis. This is facilitated by its transphosphorylase activity, in which a gamma terminal phosphate group from a triphosphate nucleoside is transferred to a diphosphate nucleoside, resulting in elevated ATP levels. Increased levels of ATP in the tumor microenvironment can activate purinergic receptors (P2Y1) on adjacent endothelial cells to promote angiogenesis, independent of VEGF stimulation. Triple negative human breast cancer (MDA-MB-231) cells have been shown to elaborate exosomes that contain NM23. These exosomes have implications in targeting specific tissues/cells to promote angiogenesis and tumorigenesis. Our lab has shown that inhibition of eNDPK and the P2Y1 receptor reduces endothelial cell tubulogenesis (Rumjahn, et al., 2007) and breast cancer metastasis (Yokdang, et al., 2015). Therefore, targeted inhibition of eNDPK may have implications as a treatment for early tumor-mediated angiogenesis. For this study, we utilized a transphosphorylation activity assay to perform a drug screen on compounds from the Prestwick chemical library. Recombinant NDPK-B was added to FDA-approved drug compounds along with ADP and UTP substrates. Subsequent ATP generation was detected with a luciferase luciferin reaction. Decreased luminescence signal/inhibition of NDPK was compared to ellagic acid control, a known inhibitor of NDPK. From our initial screen, we identified eight potential compounds that inhibited NDPK at comparable levels to ellagic acid. These eight compounds were further examined to determine the dose response curve. We found that Chicago Sky Blue 6B inhibited NDPK at an IC50 value of 1.88µM. The results from this study demonstrate a drug screening method to discover alternative small molecule inhibitors of NDPK as a potential treatment of angiogenesis and breast cancer metastasis.

#1805

Antitumor activity of lenvatinib mesilate in human hepatocellular carcinoma preclinical models.

Masahiro Matsuki, Yuji Yamamoto, Taisuke Hoshi, Takayuki Kimura, Yasuhiro Funahashi, Junji Matsui. _Eisai Co., Ltd., Tsukuba-shi, Ibaraki, Japan_.

Hepatocellular carcinoma (HCC) is a one of the leading causes of cancer death worldwide; however systemic therapeutic option for unresectable HCC is limited. Liver carcinogenesis is a complex process and various pathways have been found to be deregulated. Of those pathways, aberrant activation of fibroblast growth factor 19 (FGF19)/FGF receptor 4 (FGFR4) axis has been hypothesized to participate in the development of HCC with poor prognosis. Lenvatinib mesilate (lenvatinib) is an orally administered multi-targeted tyrosine kinase inhibitor (TKI) that selectively inhibits vascular endothelial growth factor receptors (VEGFR)1-3, FGFR1-4, platelet-derived growth factor receptor (PDGFR)α, RET, and KIT. In this study, we investigated the activities of lenvatinib against human HCC cells with or without amplification/overexpression of FGF19 gene in vitro and in vivo. Lenvatinib inhibited in vitro proliferation of human HCC cell lines Hep 3B2.1-7 and HuH-7 with IC50 values of 0.23 and 0.42 μmol/L (0.086 and 0.16 μmol/L as unbound IC50), respectively, whereas it did not show potent antiproliferative activity against PLC/PRF/5 (IC50 >10 μmol/L). Both Hep 3B2.1-7 and HuH-7 were reported to have an amplification of FGF19 gene and their cell growth was dependent on FGF19/FGFR4 axis. Sorafenib inhibited in vitro proliferation of the 3 HCC cell lines with IC50 values of 2.2 - 5.3 μmol/L (0.59 - 1.4 μmol/L as estimated unbound IC50 based on previously reported protein binding). Next, antitumor activity was evaluated in 2 human HCC cell line (PLC/PRF/5 and Hep 3B2.1-7) xenograft models. Lenvatinib treatments at 1 - 100 mg/kg resulted in tumor growth inhibition with a maximal antitumor effect giving a minimum T/C value (minT/C) of 14% via its antiangiogenic activity in the PLC/PRF/5 model, and at 3 - 30 mg/kg with minT/C of 31% in the Hep 3B2.1-7 model. Sorafenib showed significant antitumor effect at doses ≥30 mg/kg with minT/C of 30% in the PLC/PRF/5 model, and at 10 and 30 mg/kg with minT/C of 47% in the Hep 3B2.1-7 model. Finally, we tested antitumor activity in HCC patient-derived xenograft (PDX) model (LI0050), and PDX-derived cell line (LIXC-012) xenograft model with FGF19 overexpression in mice. Lenvatinib significantly inhibited the tumor growth of LI0050 at 10 and 30 mg/kg with minT/C of 33%, and that of LIXC-012 at 3 - 30 mg/kg with minT/C of 25%. Treatment with sorafenib at 30 mg/kg was intolerable in LI0050 model, and sorafenib showed significant antitumor activity at 30 mg/kg with T/C of 55%, but not 10 mg/kg in the LIXC-012 model. In conclusion, these results indicate that lenvatinib has potent preclinical antitumor activity in human HCC xenograft models with or without amplification/overexpression of FGF19. Furthermore, the maximum antitumor effect of lenvatinib was greater than that of sorafenib in this study. Direct effect of lenvatinib on proliferation of HCC cells with an aberrant FGF signaling pathway in tumor warrants further investigation.

#1806

Endothelial cells activate the cancer stem cell-associated Nanogp8 pathway in colorectal cancer cells in a paracrine fashion.

Rui Wang, Rajat Bhattacharya, Xiangcang Ye, Delphine R. Boulbes, Fan Fan, Lee M. Ellis. _UT MD Anderson Cancer Ctr., Houston, TX_.

INTRODUCTION: Median survival of patients with metastatic colorectal cancer (mCRC) is ~2.5 years and patients often become resistance to systemic therapy within 1 year of the diagnosis of metastasis. There is accumulating evidence for the existence of cancer stem cells (CSCs) in CRC, which are now believed to mediate chemoresistance. The molecular mechanisms that regulate the CSC phenotype in CRC have not been fully elucidated. We have previously described that endothelial cells (ECs) from liver, the most common site of metastases of CRC, secreted soluble Jagged-1 peptides to activate Notch signaling and increase the CSC phenotype of CRC cells in a paracrine fashion. In the current project, we sought to further elucidate the paracrine roles of liver ECs and ECs from other organs in mediating specific CSC-associated pathways in CRC cells.

METHODS: Several EC lines from liver, lung, colon mucosa and kidney were established and used to determine if conditioned medium (CM) of ECs from distinct organs promotes the CSC phenotype and chemoresistance in CRC cells. CRC cells were incubated with either their own CM (control) or CM from ECs. The CSC phenotype was determined by sphere formation assays. Chemoresistance was determined by Western blotting for apoptotic markers and fluorescence-activated cell sorting (FACS)-based Annexin V-PI double staining assay for apoptosis after treating CRC cells with 5-fluorouracil (5-FU) either in control CM or CM of ECs. The effects of EC CM on CSC-associated genes were determined by unbiased qPCR array and validated by Western blotting.

RESULTS: CM from ECs from liver and other organs all significantly increased sphere formation in CRC cells. qPCR arrays and Western blotting showed that the CSC-associated Nanog and Oct4 genes were increased by EC CM. Moreover, CRC cells treated by liver EC CM became more resistant to 5-FU induced apoptosis. Luciferase reporter assays suggested that the Nanog proteins in CRC cells were encoded by the retrogene NANOGP8, and EC CM specifically induced the expression of NANOGP8 but not NANOG. The specific induction of NANOGP8 in CRC cells were confirmed by AlwNI digestion, an established technique to distinguish NANOG and NANOGP8 based on a single nucleotide polymorphism (SNP) between two genes. In addition, Akt was shown to mediate the induction of NANOGP8 in CRC cell, as the PI3K inhibitor wortmannin blocked liver EC CM-induced NanogP8 expression and blocked sphere formation in CRC cells.

CONCLUSION: Our studies demonstrated a paracrine role of ECs from several organs, including liver, in regulating the CSC phenotype in CRC cells, via increasing NanogP8 expression. This study elucidated the importance of ECs in the microenvironment in mediating the survival of metastatic CRC cells in the liver, and potentially other organs.

#1807

Exosomal NM23 facilitates pro-angiogenic communication between triple-negative breast cancer cells and their vascular targets.

Suzann Duan, Senny Nordmeier, Iain L. Buxton. _University of Nevada, Reno School of Medicine, Reno, NV_.

Functional interactions between primary tumor cells and their vascular targets orchestrate the formation of a metastatic milieu enriched with oxygen and metabolic substrates. These interactions have been largely attributed to the activation of VEGF receptor pathways through localized tumor-mediated release of VEGF, in addition to indirect receptor phosphorylation. However, pro-angiogenic events are known to occur in the absence of VEGF and alternative pathways resulting in dual activation of neovascularization and enhanced vascular permeability are of growing interest. Our lab has previously shown that triple-negative human breast cancer (MDA-MB-231) cells elaborate exosomes carrying NM23, a nucleoside diphosphate kinase (eNDPK) that is suggested to act as an upstream mediator of vasodilation and pro-angiogenic activity through its action of elevating extracellular ADP/ATP levels. To further elucidate the role of eNDPK in breast cancer signaling and metastasis, we show that 231-exosomes induce a pro-angiogenic phenotype in both human umbilical vein and murine pulmonary endothelial cells. Treatment with an NDPK-specific inhibitor and an antagonist to the ADP/ATP-activated P2Y1 receptor results in amelioration of pro-angiogenic characteristics. Further, we report the development and application of a novel murine implant model to demonstrate increased specificity of 231 exosome targeting to pulmonary endothelial cells over unrelated cell lines. These results suggest the involvement of exosomes and eNDPK in pro-angiogenic communication between metastatic breast cancer cells and their vascular targets. Exosomes elaborated by these cells specifically target pulmonary endothelium in vivo, mirroring the organotropic nature of breast cancer metastasis. Implication of eNDPK in angiogenesis and metastasis may challenge current and invasive treatment methods in favor of developing novel small molecule inhibitors of eNDPK signaling. Lastly, eNDPK can be explored as a potential biomarker for early detection of metastatic breast cancer as it uniquely appears in the serum of patients with breast cancer and not in the serum of individuals with unrelated pathologies.

#1808

**Vascular endothelial growth factor receptor-2 (VEGFR-2)** N **-glycosylation modulates angiogenic signaling.**

Kevin B. Chandler, Deborah R. Leon, Rosana D. Meyer, Nader Rahimi, Catherine E. Costello. _Boston Univ., Boston, MA_.

Angiogenesis, the formation of new blood vessels from pre-existing vessels, is required for tumor growth and metastasis. Vascular endothelial growth factor receptor-2 (VEGFR-2) is one of the most important receptor tyrosine kinases (RTKs) among the VEGF receptor subfamily, and activation of VEGFR-2 is essential for tumor angiogenesis. The extracellular domain of VEGFR-2 contains seven immunoglobulin-like (Ig) domains, each with multiple potential N-glycosylation sites. N-glycosylation is thought to play a central role in receptor stability, ligand binding and trafficking. However, to date the occupancy and glycoform distributions at each of the potential N-glycosylation sites and their putative role(s) in VEGFR-2 function remain largely unknown. The objective of this study is to investigate the functional importance of VEGFR-2 N-glycosylation in VEGFR-2 angiogenic signaling.

Porcine aortic endothelial (PAE) cells with ectopic expression of VEGFR-2 were treated with PNGase F to remove N-linked glycans or heat-denatured PNGase F as a control. Following PNGase F treatment for 4 hours, cells were treated with VEGF-A ligand for 0, 5, 10 or 30 minutes. VEGFR-2 phosphorylation (activation) was measured via Western blot with an anti-pTyr-1054-VEGFR-2 antibody. In addition, a polyclonal anti-VEGFR2 antibody was used to immunoprecipitate untreated and PNGase F-treated VEGFR-2 from PAE cell lysates. Evaluation of the N-glycosylation sites targeted by PNGase F was carried out by gel electrophoresis, followed by protease digestion and MS/MS analysis. MS/MS data were processed using Proteome Discoverer 1.4. To obtain site-specific glycosylation information we performed proteolysis of VEGFR-2, glycopeptide enrichment via hydrophilic interaction liquid chromatography (HILIC) and subsequent analysis of glycopeptides with an Agilent 6550 Quadrupole Time-of-Flight (Q-TOF) MS using collision-induced dissociation.

We detected a dramatic increase in ligand-mediated activation of VEGFR-2 after treatment with PNGase F, suggesting that certain N-linked glycans may hinder ligand access to the VEGF binding site, or that removal of N-linked glycans results in conformational changes that lead to increased activation of the receptor by VEGF-A. To explore this observation in greater detail, we have created a series of VEGFR-2 N-glycosylation site mutants, and we are now using the mutants to determine which glycosylation sites are involved in the observed modulation of VEGFR-2 signaling.

#1809

PKM2 promotes tumor angiogenesis by regulating HIF-1alpha through NF-kappaB activation.

Ninel Azoitei,1 Alexander Becher,1 Konrad Steinestel,2 Arefeh Rouhi,1 Kristina Diepold,1 Susanne Bobrovich,1 Thomas Seufferlein1. 1 _University of Ulm, Ulm, Germany;_ 2 _University of Muenster, Muenster, Germany_.

Initially identified as a molecule that catalyzes the phosphorylation from phosphoenolpyruvate to pyruvate in the final step of glycolysis, the M2 isoform of pyruvate kinase (PKM2) was recently reported to have a central role in the metabolic reprogramming of cancer cells as well as participating in the regulation of apoptosis and proliferation. In this study, we have identified PKM2 as a crucial molecule for progression of pancreatic cancer in which tumor microenvironment has been reported to be highly hypoxic. Our data show moderate to strong PKM2 expression in all examined human pancreatic adenocarcinoma samples. Depletion of PKM2 was associated with impaired proliferation and augmented tumor cell death in vitro, while the in vivo tumor xenograft experiments revealed a close association between impaired tumor growth and decreased blood vessel formation. Furthermore, abrogation of PKM2 prevented hypoxia-mediated HIF-1alpha accumulation and HIF-1alpha promoter activity, which negatively impacted VEGF secretion by pancreatic cancer cells deprived of oxygen. Interestingly, PKM2 expression arrest was also mirrored by impaired hypoxia-driven promoter activity of NF-kappaB which also drives the transcription of VEGF. Ectopic expression of p65 restored VEGF transcription after PKM2 ablation inferring that the kinase regulates VEGF via NF-kappaB/p65 subunit. Altogether, our study suggests that in hypoxic pancreatic tumors PKM2 interferes both with NF-kappaB/p65 and HIF-1alpha activation that ultimately triggers VEGF-A secretion and subsequent blood vessel formation.

#1810

Dysregulation of myristoylated alanine-rich C kinase substrate (MARCKS): A novel therapeutic target in renal cell carcinoma.

Ching-Hsien Chen,1 Wen-Hsin Chang,2 Eric Yu,1 Muhammad S. Arif,1 Reen Wu,3 Robert H. Weiss1. 1 _Division of Nephrology, Department of Internal Medicine, University of California Davis, Davis, CA;_ 2 _Institute of Molecular Medicine, National Taiwan University College of Medicine; Division of Nephrology, Department of Internal Medicine, University of California Davis, Taiwan;_ 3 _Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine and Center for Comparative Respiratory Biology and Medicine, University of California Davis, Davis, CA_.

Targeted therapeutics, such as those abrogating hypoxia inducible factor (HIF)/VEGF signaling, are initially effective against kidney cancer (or renal cell carcinoma, RCC); however, drug resistance frequently occurs via subsequent activation of alternative pathways. Through genome-scale integrated analysis of the HIF-α network, we identified the major protein kinase C substrate MARCKS (myristoylated alanine-rich C kinase substrate) as a potential target molecule for kidney cancer. In a screen of nephrectomy samples from 56 patients with RCC, we found that MARCKS expression and its phosphorylation are increased and positively correlate with tumor grade. Genetic and pharmacologic suppression of MARCKS in high grade RCC cell lines in vitro led to a decrease in cell proliferation and migration. We further demonstrated that higher MARCKS expression promotes growth and angiogenesis in vivo in an RCC xenograft tumor. MARCKS acted upstream of the AKT/mTOR pathway, activating HIF-target genes, notably VEGF-A. Following knockdown of MARCKS in RCC cells, the IC50 of the multi-kinase inhibitor regorafenib was reduced. Surprisingly, attenuation of MARCKS using the MPS peptide synergistically interacted with regorafenib treatment and decreased survival of kidney cancer cells through inactivation of AKT and mTOR. Our data suggest a major contribution of MARCKS to kidney cancer growth and provide an alternative therapeutic strategy of improving the efficacy of multi-kinase inhibitors.

#1811

A PEAK1/GATA2 signaling axis controls VEGFR2 expression to mediate angiogenesis.

Richard L. Klemke. _UCSD Moores Cancer Ctr., La Jolla, CA_.

Formation of new blood vessels (angiogenesis) is crucial for proper development, but can also contribute to many diseases including cancer. Vascular endothelial growth factor receptor 2 (VEGFR2) is a receptor tyrosine kinase that mediates angiogenesis in developing vertebrates and is frequently upregulated in many human cancers. However, the underlying mechanisms that regulate VEGFR2 expression in normal and diseased cells are poorly understood. Here we demonstrate that pseudopodium-enriched atypical kinase 1 (PEAK1) mediates developmental and tumor-induced angiogenesis by modulating VEGFR2 expression and downstream signaling. Knockout of peak1 in zebrafish using TALEN technology (Transcription Activator-Like Effector Nucleases) or knockdown by specific peak1 morpholinos (MO) induced severe pericardial edema, blood-pooling defects, and inhibited formation of intersegmental (ISV) and subintestinal vessels (SIV). In Peak1 knockout mice, neonatal retinal vessel development was severely delayed, and the ex vivo angiogenic ability of adult thoracic aorta was greatly reduced. Intravital time-lapse imaging of ISV formation in peak1 knockout zebrafish revealed major defects in endothelial cell migration and proliferation. Similarly, PEAK1 knockdown or overexpression in HUVECs modulated endothelial cell migration, proliferation, and vessel sprouting in vitro. Biochemical studies and quantitative proteomic profiling of PEAK1 knockdown HUVECs revealed a dramatic loss of VEGFR2 mRNA and protein expression, which attenuated downstream signaling to Akt and ERK, leading to loss of VEGF-mediated vessel formation. PEAK1 specifically regulated VEGFR2 levels and did not alter VEGFR1 expression, and reconstitution of PEAK1 knockdown cells with PEAK1 restored VEGFR2 mRNA and protein expression. Mechanistic studies revealed that PEAK1 specifically regulates VEGFR2 mRNA transcription by modulating expression of the transcription factor GATA2. Re-expression of GATA2 in PEAK1 depleted cells restored VEGFR2 mRNA and protein expression as well as downstream signaling, indicating that GATA2 is a necessary downstream component in this response. Also, introduction of gata2 mRNA into PEAK1 knockdown zebrafish restored normal ISV formation and vegfr2 mRNA expression in these animals. Finally, bioinformatics and interrogation of the TCGA RNAseq database revealed significant Pearson's correlations between PEAK1 and VEGFR2 expression in 17 of 32 different human cancers. Collectively, our findings indicate that PEAK1 regulates developmental and tumor-induced angiogenesis by regulating GATA2-dependent VEGFR2 expression and its downstream signals.

#1812

In vitro **and** in vivo **antiangiogenic activity of desacetylvinblastine monohydrazide through inhibition of VEGFR2 and Axl pathways.**

Minfeng Chen,1 Xueping Lei,1 Qiulin Nie,1 Jianyang Hu,1 Zhenjian Zhuo,1 Anita Yiu,2 Heru Chen,1 Nanhui Xu,1 Maohua Huang,1 Kaihe Ye,1 Liangliang Bai,1 Wencai Ye,1 Dong-Mei Zhang1. 1 _College of Pharmacy, Guanghzou, China;_ 2 _School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China_.

Background and Objective: Tumor angiogenesis process is regulated by multiple proangiogenic pathways, such as VEGFR2 and Axl. Inhibition of VEGF/VEGFR2 signaling alone fails to block tumor neovascularization, and anti-VEGF resistance is often associated with Axl. Hence, discovery of novel agents that target multiple angiogenesis pathways is in demand. Here, we describe desacetylvinblastine monohydrazide (DAVLBH), a derivative of vinblastine (VLB) that exerts a more potent antiangiogenic effect than VLB in vitro and in vivo by inhibiting VEGFR2 and Axl pathways.

Methods: The antiangiogenic effects of DAVLBH were studied in vitro (proliferation, migration, and tube formation assays) and ex vivo (aortic ring assay). In vitro pericyte migration to endothelial tubes was assessed using a three-dimensional co-culture assay. In vivo assay was performed in HeLa xenograft model. Western blotting, immunohistochemical and immunofluorescence assays were conducted to evaluate the key proteins of the VEGF/VEGFR2 and Gas6/Axl pathways.

Results: DAVLBH (1 nM) inhibited VEGF- and Gas6-induced angiogenesis in vitro. At 0.75 μmol/kg, DAVLBH significantly delayed tumor growth and reduced vascular density in vivo, which was associated with the inactivation of VEGF/VEGFR2 and Gas6/Axl signalling pathways. DAVLBH blocked the compensatory upregulation of Axl in response to bevacizumab treatment in HUVECs. DAVLBH also suppressed the recruitment of pericytes to well-established endothelial tubes and reduced pericyte coverage in vivo, which was accompanied by inhibition of Axl.

Conclusions: DAVLBH potently inhibited angiogenesis-mediated tumor growth by blocking the activation of VEGF/VEGFR2 and Gas6/Axl pathways. DAVLBH might serve as a promising antiangiogenic agent for cancer therapy.

#1813

Bevacizumab potentiates the proteomic response to neoadjuvant chemotherapy in breast cancer patients: Rppa exploration of consecutive tumor samples in the NeoAva randomized phase II trial.

Mads H. Haugen,1 Ole Christian Lingjaerde,2 Marit Krohn,1 Wei Zhao,3 Evita M. Lindholm,1 Laxmi Silwal-Pandit,1 Elin Borgen,4 Øystein Garred,4 Anne Fangberget,4 Marit M. Holmen,4 Ellen Schlichting,4 Helle K. Skjerven,5 Steinar Lundgren,6 Erik Wist,4 Bjørn Naume,4 Gunhild M. Maelandsmo,1 Yiling Lu,3 Anne-Lise Boerresen-Dale,1 Gordon B. Mills,3 Olav Engebraaten1. 1 _Oslo University Hospital - Institute for Cancer Research, Oslo, Norway;_ 2 _University of Oslo, Oslo, Norway;_ 3 _MD Anderson Cancer Center, Houston, TX;_ 4 _Oslo University Hospital, Oslo, Norway;_ 5 _Vestre Viken Hospital, Drammen, Norway;_ 6 _St. Olav's University Hospital, Trondheim, Norway_.

Antiangiogenic therapy using bevacizumab has proven effective for a number of cancers; however, in breast cancer there is an unmet need to identify patients that benefit from such treatment. Sampling of tumor biopsies before and during treatment, as well as at the time of surgery enables the assessment of response at multiple molecular levels. At the proteomic level reverse phase protein analysis (RPPA) support expression of numerous cancer associated proteins simultaneously, which can further be used to unravel molecular mechanisms associated with clinical response to bevacizumab treatment.

In this phase II clinical trial, patients with HER2 negative primary tumors of ≥25 mm were treated with neoadjuvant chemotherapy (4 x FEC100 + 12 weeks of taxane-based therapy) and randomized (1:1) to receive bevacizumab or not. Mammography, ultrasound and MR imaging were used for response evaluation, in addition to final pathology assessment. Tumor responses were evaluable in 132 patients; of which 66 received bevacizumab. Ratio of the tumor size at final pathology assessment, and at inclusion was calculated to obtain a continuous scale of response reflecting the percentage of tumor shrinkage in response to therapy. Tumor biopsies were removed before start of treatment, at week 12 at the start of taxane-based tharapy and at the time of surgery. Lysates from each sample was analyzed on reverse phase protein arrays (RPPA) for expression levels of 210 proteins of which 54 were phospho-specific.

The addition of bevacizumab to the chemotherapy do not alter proteomic response from week 0 to 25 to such extent that this patient group cluster naturally together. While the proteomic response from week 0 to 12 in both treatment arms had an overall similar profile regarding up- and down-regulated proteins, the combination treatment (FEC100 + bevacizumab) induced substantially more effect on the regulation of each protein. This suggests that bevacizumab treatment have the capability to potentiate the effects of the anthracyclin based chemotherapy from week 0 to 12. Conversely, from week 12-25 (taxane-based therapy + bevacizumab) this effect was lost or even reversed, possibly due to a de-vascularized and less accessible tumor. An exception to this observation was a few phospho-proteins that do seem to have sustained stronger regulation over the whole treatment period. We are in the process of analyzing in more detail the impact of phosphorylation and thus protein activation states on treatment response.

Deciphering molecular response and activity regulation at the proteomic level is a promising approach and may reveal novel knowledge with potential important clinical relevance.

#1814

IL11 mediates tumor resistance to anti-VEGF therapy in lung cancer.

Jie Li, Kun Xie, Shenglin Mei, Yuanjian Xu, Hua Gu, Dong Li, Kaiming Chen, Fei Tao, Jiya Eerdeng, Jianmin Fang. _Tongji University, Shanghai, China_.

Anti-angiogenic therapy has been used for the treatment of a number of solid tumors in clinic, however, survival benefit is often limited due to development of tumor resistance to the therapy. In previous studies, we developed a mouse Lewis lung carcinoma (LLC) tumor model that is resistant to anti-VEGF therapy. In this model, we identified that Interleukin 11 (IL11) was among the most significantly upregulated genes in the resistant tumors. In current study, we demonstrated that IL11 was a pro-angiogenic factor and played a key role in the development of resistance to anti-VEGF therapy in LLC tumor. IL11 could effectively induce HUVEC proliferation, migration and capillary tube formation in vitro. It enhanced expression of MMP9 in endothelial cells. The conditioned medium from the resistant LLC cell culture could induce HUVEC migration, even VEGF was blocked, and this effect was attenuated by recombinant IL11 decoy receptor, an IL-11 inhibitor. Furthermore, combination therapy of anti-VEGF agent and IL11 blockade exhibited enhanced anti-tumor activity in lung cancer model. In addition, based on bioinformatics analysis on over 500 RNA-seq data deposited to The Cancer Genome Atlas (TCGA), we found that IL11 was highly expressed in various tumor types in patients. Data showed that high IL11 can predict a poor outcome in the lung cancer. The patients with the lower IL11 expression have significantly longer median survival time than those with higher expression. These findings support a role of IL11 signal as a compensatory angiogenic pathway that might trigger tumor resistance to anti-VEGF treatment. Therapeutically targeting IL11 could have a value in overcoming tumor resistance to anti-VEGF therapy.

#1815

Extracellular vesicles derived from lung cancer cell may contribute to the formation of malignant pleural effusion via activating Stat3/Tissue factor/VEGF.

Chien-Chung Lin, Wei-Lun Huang, Wu-Chou Su. _National Cheng Kung Univ. Hospital, Tainan, Taiwan_.

Malignant pleural effusion (MPE) is a poor prognostic sign for patients with non-small-cell lung cancer (NSCLC) and is noted around 15% at the time of initial diagnosis and 50% in their later courses. Previously, we have shown that IL-6/Stat3/tissue factor (TF)/VEGF pathway enhances lung cancer angiogenesis, metastasis and the generation of MPE. In the current study, we demonstrated that EVs derived from lung cancer cells may also contribute MPE formation. We first used ultra-filtration (UF) method as well as the classical ultra-centrifugation (UC) method to isolate EV from lung cancer cell line and demonstrated that the EVs isolated by UF method were characteristic with exosome marker in Western Blotting (Alix, CD63, and Tsg101), morphology at Transmission electron microscopy (TEM) and size in Nanosight system (30 to 150 nm). After labeling these EVs with the green fluorescent dye Cellvue, EVs uptake in AS2 cell can be imaged by the fluorescent microscopy. After the EV uptake of recipient cell, the oncogenic signal such as Stat3 and AKT were activated with increasing phosphorylation at Western blotting comparing to those adding serum free medium. Using the ELISA method, we found the cargos in these EV were enriched in IL6. We also found these cargo derived from EVs, containing high VEGF and TF, induced more prominent extravasation than serum free medium did in Miles assays. In clinical sample, we also isolated cancer cell from MPE and the purity of the cells was confirmed by immunofluorescent staining with specific TTF-1expression of lung adenocarcinoma. After short-term culture, EV from MPE cancer cell have the characteristic of exosome marker and induced more prominent extravasation than serum free medium as EV from AS2 cell did. And we also isolated EV direct from MPE and pleural effusion from congestive heart failure (CHF) patients. And we found EV derived from MPE induced more prominent extravasation than those derived from CHF in Miles assays. In conclusion, we demonstrated the UF is clinical assessable method for isolating EV from body fluid and EV derived from lung cancer cell and MPE may contribute oncogenic and angiogenic signal transmission accelerating the formation of MPE.

#1816

Endogenous ADAMTS-13 regulates angiogenesis in cultured human endothelial cells.

Huiyuan Tang, Manfai Lee, Eun Ho Kim, Daniel Bishop, George M. Rodgers. _University of Utah, Salt Lake City, UT_.

ADAMTS-13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13, is a zinc-containing metalloprotease that cleaves von Willebrand factor (vWf). Previous publications by our laboratory have shown that ADAMTS-13 may also be involved in angiogenesis; for example; addition of exogenous ADAMTS-13 enhances endothelial cell (Lee et al. Microvascular Res 2012; 84:109) and glioblastoma cell (Lee et al. Blood 2013; 122:#2306) migration and proliferation. For this study, we report the successful transient knockdown of endogenous ADAMTS-13 in human umbilical vein endothelial cells (HUVEC) via siRNA. 30pmols of ADAMTS-13 siRNA in a six-well plate inhibited HUVEC ADAMTS-13 expression levels by 90% after 24hr incubation, whereas control siRNA did not affect endogenous ADAMTS-13 levels. The knockdown effect was decreased over time: 72hr after the initial knockdown, ADAMTS-13 expression was reduced by only 50%. The effects of reduced endogenous ADAMTS-13 on HUVEC angiogenesis functions were studied. Transfection of HUVEC with 10pmols of ADAMTS-13 siRNA in a 24-well plate resulted in a 21% and 22% decrease in proliferation after 24hr and 48hr incubation, respectively. The effects of ADAMTS-13 knockdown on migration of HUVEC across a scratch wound were also evaluated. 24hr after transfection with control siRNA, there was robust cell migration across the scratch wound. This dramatic migration did not occur with ADAMTS-13 knockdown cells. Decreased expression of endogenous ADAMTS-13 also affected angiogenesis as measured by endothelial cell tube formation using a Matrigel matrix method. The tube lengths, sizes and junction numbers of the ADAMTS-13 knockdown cells were all significantly lower compared to control cells by about 40%. Activity of the AKT pathway, one of the angiogenesis downstream signaling pathways was down-regulated by ADAMTS-13 siRNA while ERK, a component of the MAP kinase pathway was not affected upon knockdown of ADAMTS-13. These data indicate that in cultured endothelial cells, one role of endogenous ADAMTS-13 is regulation of angiogenesis, mediated through the AKT signaling pathway. Overall, our data suggest an additional model of endogenous ADAMTS-13 functionality, beyond that of cleaving von Willebrand factor.

#1817

Resistin induces angiogenesis and lymphangiogenesis in human chondrosarcoma.

Meng-Ju Chi, Chih-Yang Lin, Chih-Hsin Tang. _China Medical University, Taichung City, Taiwan_.

Chondrosarcoma is a common kind of bone cancers, and it may develop distant metastasis, followed by a significant decline in overall survival. However, there are still no specific therapeutic methods for it today. For tumors to metastasis, angiogenesis and lymphangiogenesis are both important in the early processes. Therefore, inhibiting the development of angiogenesis and lymphangiogenesis could be a method to decline tumor metastasis. Resistin was discovered as an adipocyte-secreting adipokine, which may play a critical role in modulating cancer pathogenesis. In our lab, we previously found that resistin appears to increase MMP-2 expression and then promotes metastasis in human chondrosarcoma cells. Nevertheless, the role of resistin in angiogenesis and lymphangiogenesis of human chondrosarcoma is still unknown. To examine angiogenetic and lymphangiogenetic effects of resistin, we used human endothelial progenitor cells (EPCs) and lymphatic endothelial cells (LECs) to mimic capillary and lymphatic vessels formation. The results indicated that resistin-treated chondrosarcoma cell lines promoted EPCs VEGF-A-dependent as well as LECs VEGF-C-dependent tube formation and cell migration. Then we confirmed that treating cells with resistin increased VEGF-A and VEGF-C expression in human chondrosarcoma cell lines. Moreover, we found resistin-induced VEGF-A and VEGF-C expressions are mediated by PI3K/AKT signaling and by the activation of c-Src separately. In addition, resistin decreased the expression of miR-16-5p via PI3K/AKT pathway, and so of miR-186 via c-Src. We also demonstrated that miR-186 directly targeted on VEGF-C 3' untranslated region, and regulated the VEGF-C production. Besides, we found the expressions of resistin, VEGF-A and VEGF-C was higher in human chondrosarcoma biopsy tissues than those in normal cartilage. Taken together, resistin not only promotes human chondrosarcoma angiogenesis through the activation of PI3K/AKT signaling pathway and down-regulating miR-16-5p expression, but also promotes human chondrosarcoma lymphangiogenesis through the activation of c-Src and down-regulating miR-186. Consequently, resistin may represent a potential novel molecular therapeutic target for human chondrosarcoma therapeutic treatment.

#1818

Enhanced antitumor and anti-angiogenic effects of Apatinib combined with chemotherapy in a zebrafish model of non-small cell lung cancer.

Yi Xiang,1 Wenchao Zhang,1 Zhuanbin Wu,2 Zijun Qian,1 Jianping Zhou,1 Xiaofei Wang,1 Weiqin Wang,3 Ling Zhou,1 Jing Liu,1 Yun Feng,1 Min Zhou,1 Guochao Shi,1 Beili Gao1. 1 _Rui Jin Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai, China;_ 2 _Shanghai Biomodel Organism Science & Technology Development Co., Ltd, Shanghai, China; _3 _Tong Ren Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai, China_.

Background: Apatinib is a highly selective VEGFR2 inhibitor, but its effects on non-small cell lung cancer (NSCLC) has not been widely reported and whether there is synergistic effect of Apatinib and conventional chemotherapeutic drugs is also unclear.

Methods: In vitro, MTT assay was used to evaluate the combination effects of Apatinib and chemotherapy agents (Gemcitabine, Paclitaxel and Pemetrexed) against A549 (EGFR negative mutation cell line) lung cancer cells. In vivo, we firstly assessed the safety of Apatinib and chemotherapy agent in zebrafish model by observing organ developmental malformation and zebrafish embryos mortality induced by drug toxicity. The inhibition effect on the development of blood vessel in zebrafish was used to assess antiangiogenic property of Apatinib. By means of microinjection, A549 cells stained with red fluorescent cell tracer CM-Dil fluorescence were grafted into the yolk sac of zebrafish embryo. Then, zebrafish were incubated with Apatinib, chemotherapy agents or Apatinib combined chemotherapy agents, respectively. The tumor xenografts volume was measured by estimating the relative fluorescence intensity. Q-RTPCR was used to detect the expression of some genes associated with angiogenesis in order to identify molecular mechanism of the combination effects of Apatinib and chemotherapy agent.

Results: Apatinib had a direct inhibitory effect on A549 cells with the IC50 values ranging from 2.693 to 5.384μmol. Apatinib combined with Pemetrexed gave the most optimal anti-tumor effect compared with Gemcitabine or Paclitaxel in vitro. In zebrafish model, the LC50 value of Apatinib to zebrafish embryos was only half it of Pemetrexed to zebrafish embryos, suggesting that Apatinib is a low toxicity agent. In addition, Apatinib strongly inhibits the process of angiogenesis but not the developed and mature vasculature, suggesting that Apatinib can effectively inhibit tumor angiogenesis in lung cancer patient, which is aided in suppressing tumor growth. As for antitumor effects, Apatinib or Pemetrexed, whatever alone or combination all significantly inhibited tumor growth and the co-treatment of Apatinib and Pemetrexed gave the most optimal anti-tumor effect, suggesting that the combination of apatinib and Pemetrexed may be a promising alternative therapy for lung cancer patients. Finally, the qRT-PCR showed that in addition to synergistically inhibiting VEGFR2 gene expression, co-treatment of Apatinib and Pemetrexed also synergistically inhibit Efnb2a, Robo4 and FGFR4 gene expression.

Conclusions: Apatinib combined with chemotherapies, especially Pemetrexed, synergistically enhanced anti-tumor and anti-angiogenic effects in zebrafish model of NSCLC mainly through inhibiting VEGFR2, Efnb2a, Robo4 and FGFR4 pathways.

#1819

Reprogramming the tumor microenvironment by targeting endothelial DNA repair.

Cristina Espinosa-Diez, RaeAnna Wilson, Rebecca Ruhl, Nathan Kanner, Namita Chatterjee, Clay Hudson, Sudarshan Anand. _Oregon Health & Science Univ., Portland, OR_.

The tumor microenvironment (TME) plays a critical role in orchestrating immune infiltration, tumor progression and response to therapeutics. Therefore, strategies to manipulate the TME including cytokine gene therapy, antibodies to reverse macrophage polarization etc are under active investigation. We have identified an alternative approach to target the TME by disrupting DNA repair in the tumor endothelial cells (ECs). We discovered a seven-microRNA (miR) signature induced in ECs in in vitro and in vivo by oxidative stress and DNA damage. The top miR candidate in this signature, miR-103 altered the TME by inducing DNA damage in ECs, eliciting type I interferons, upregulating immune costimulatory receptors and decreasing PD-L1 expressing tumor associated macrophages and granulocytes. Moreover, miR-103 treatment had a paracrine effect on the tumors and upregulated Fas and TRAIL receptors. Mechanistically, these functions of miR-103 were largely due to its downregulation of the three prime exonuclease TREX1. Local, systemic or vascular targeted delivery of miR-103 decreased both angiogenesis and tumor burden in multiple mouse tumor models.

Complementary to the role of miR-103, two additional miRs in the miR signature, miRs 494 and 99b each induced senescence in the vasculature by downregulating the Mre11a-Rad50-NBN (MRN) complex. Ectopic expression of miRs 494 or 99b decreased telomerase activity, increase p21 levels with a concomitant decrease in pRb levels. Vascular targeted delivery of miR-494 decreased angiogenesis in vivo whereas systemic delivery decreased tumor growth. Interestingly, both miR mimics and the MRN knockdowns induced the transcription of a number of senescence associated genes including CD44. Taken together these data suggest that miRs 494 and 99b targeting of the MRN complex induces senescence.

The MRN complex interacts with the ATM kinase, histone H2AX and TREX1 suggesting that the miRs we identified disrupt critical nodes of a DNA Damage Response (DDR) network. Our findings reveal a complex, miR mediated cross talk between EC DNA damage pathways, the TME and tumor cells. These interactions can be exploited for designing therapies that synergize with tumor cell killing to enhance host anti-tumor responses.

#1820

Synergistic lethality of mAbs with an EMT reversal agent, Nintedanib, in epithelial ovarian cancer.

Jocelyn Teo,1 Heng Liang Tan,1 Ruby Yun-Ju Huang,2 Andre Choo1. 1 _A*STAR, singapore, Singapore;_ 2 _Cancer Science Institute, singapore, Singapore_.

Epithelial-mesenchymal transition (EMT) has long been associated with cancer progression and metastasis. As a reversible process, it has the potential to be a target for cancer therapy. However, this concept of reversing EMT has not yet been widely explored in current treatment strategies. With the identification of Nintedanib as an EMT reversal agent, we aim to broaden the utility of this triple angiokinase inhibitor by identifying agents that show synergistic lethality with it, using monoclonal antibodies (mAbs) as a combinatory therapeutic. Nintedanib alone is not significantly cytotoxic to cells, but it causes cell cycle arrest and reverses EMT. By selecting for this sub-population of cells that are sensitive to Nintedanib, their subsequent eradication can then be more intricately directed. Since the anti-cancer application of EMT reversal agents are not fully developed, it is vital to investigate mechanisms that sensitize cancer cells to cytotoxic agents following EMT reversal.Our group has generated panels of mAbs that are able to bind differentially to various breast and ovarian cancer cell lines along the EMT spectrum. Some mAbs preferentially bind to Epithelial lines, while some have stronger affinity to Mesenchymal lines. This implies that mAbs have the ability to differentiate between epithelial and mesenchymal phenotypes, uncovering a new dimension to the capability of mAbs which have yet to be explored.Using a mesenchymal ovarian cancer cell line as a starting model, Nintedanib-treated cells were screened with the mAbs and those showing a >2-fold increase in binding were shortlisted for further validation. Based on this criteria, 26 mAbs were identified and further characterized in functional Antibody Drug Conjugate (ADC) assays with mAbs conjugated to a toxic drug, Saporin (or Zap). Mabs successfully internalizing and killing the cancer cells will then be further chosen for antigen characterization and in vivo functional studies. Ultimately, there is huge potential for mAbs to be discovered that shows synergy with Nintedanib to enhance lethality to various types of cancer cells. A novel clinical strategy, the application of EMT reversal in terms of utilising a synthetic lethality-like approach allows better design of combinatory therapeutics, increasing treatment efficacy that might revolutionalise cancer treatment.

#1821

Sympathetic nerves regulate a metabolic switch promoting angiogenesis through adrenergic signaling in prostate cancer.

Ali Zahalka,1 Anna Arnal-Estape,2 Maria Maryanovich,1 Fumio Nakahara,1 Cristian Cruz,1 Paul S. Frenette1. 1 _Albert Einstein College of Medicine, Bronx, NY;_ 2 _Yale School of Medicine, New Haven, CT_.

Nerves pattern the vasculature during development and regeneration. Recent studies have shown that the sympathetic nervous system (SNS) is coopted to promote carcinogenesis, and that depletion of SNS β-adrenergic receptors (βARs) in the prostate stromal compartment inhibits tumor growth. As the vasculature is in direct contact with the SNS, and regulates tumor glucose and oxygen delivery, we aimed to identify the stromal populations that mediate SNS signaling and the mechanisms by which loss of βAR signaling inhibits prostate cancer progression. In vivo xenograft tumor growth was measured in Adrb2-/-; Adrb3-/- mice using an orthotopic PC3-luciferase model. Prostate cancer progression in the transgenic autochthonous HI-myc model was assessed after conditional Adrb2 deletion in stromal populations by intercrossing Adrb2fl/fl line to stromal Cre lines. Metabolism was measured ex vivo by flow quantification of the lipophilic cationic dye TMRE whose accumulation reflects mitochondrial membrane potential, and by NADH autofluorescence under UV excitation. In vitro oxidative phosphorylation and glycolytic flux were measured in primary prostate endothelial cells (VeraVec) using the Seahorse bioanalyzer and targeted gas chromatography-mass spectrometry (GC/MS). Deletion of the SNS βAR neural receptors in the microenvironment synergized to arrest exponential tumor growth. Imaging the tumor vasculature revealed a defect in vessel branching, a phenotype also observed after chemical sympathectomy, suggesting altered angiogenesis in the absence of βAR stimulation. Histology revealed an increase in vascular innervation and density during the transition from low to high grade pre-neoplastic PIN. Conditional Adrb2 deletion (Adrb2cKO) in endothelial cells (ECs), but not myeloid cells or pericytes, not only inhibited angiogenesis, but also reduced disease progression throughout all histopathological cancer stages. Transcriptome analysis of flow isolated Adrb2cKO ECs suggested an increase in oxidative metabolism and upregulation of mitochondrial Coa6. Ex vivo analysis of ECs revealed a metabolic switch during the low to high grade PIN transition, and this switch was inhibited by Adrb2cKO. Further in vitro characterization by Adrb2 knockdown revealed a metabolic shift in ECs to oxidative phosphorylation, which was mediated by Coa6 upregulation. As Coa6 cooperates with Cox10 in respiratory complex IV assembly, conditional co-deletion of Adrb2 and Cox10 in the HI-myc mouse rescued aerobic glycolysis, angiogenesis, and cancer progression. Our data thus demonstrates that the SNS mediates a pro-angiogenic switch during the early stages of prostate carcinogenesis. Moreover, we identified ECs as SNS stromal targets, and that abrogation of βAR signaling inhibits the angio-metabolic switch and delays prostate cancer progression, suggesting a novel therapeutic target for prostate cancer treatment.

#1822

Antitumor activity of agents targeting the angiogenic factors angiopoietin-2 and thymidine phosphorylase in mouse models of lung and pancreatic neuroendocrine cancer.

Evelyn Aranda, Ziqiang Yuan, Steven K. Libutti, Edward L. Schwartz. _Albert Einstein College of Medicine, Bronx, NY_.

Angiogenesis is a critical component of the initiation, maintenance and progression of tumors, and has been the target of extensive drug development. In clinical practice there are several approved and investigational anti-angiogenesis drugs, the majority of which primarily target the VEGF pathway, however their long term effectiveness remains limited. In designing new drug regimens, it is important to consider other known angiogenic factors that likely contribute to the pro-angiogenic phenotype. We evaluated two such factors: thymidine phosphorylase (TP) (aka platelet-derived endothelial cell growth factor, PD-ECGF), an enzyme that can stimulate endothelial cell migration, and angiopoietin 2 (Ang2), a member of the Ang/Tie2 signaling pathway. To target TP, we used a novel, highly specific, orally available small molecule inhibitor, which we had previously designed, named AEAC. To target Ang2, we used an Ang2 neutralizing peptibody, L1-7[N] (provided by CTEP-NCI), which is a genetically engineered peptide-Fc fusion protein that specifically binds Ang2. In a mouse NCI-H460 NSCLC sc xenograft model, AEAC and L1-7[N] produced 33% and 67% reductions, respectively, in tumor growth, and the combination produced a small, but statistically significant further reduction, when compared to either agent used alone. Tumor growth inhibition was paralleled by a reduction in Ki-67 staining. L1-7[N] also produced a large infiltration of macrophages (F4/80+) into the tumors after treatment. We also tested the drug combination in a mouse genetic model of pancreatic neuroendocrine tumors (PNET). PNET are one the few human solid tumors that are clinically responsive to angiogenesis inhibitors used alone. These mice have a conditional knockout of the Men1 gene in the endocrine pancreatic cells (Pdx1-Cre;Men1 mice) mimicking a primary genetic alteration seen in human disease. Men1 KO PNET experiments were initiated when the mice were 10-11 months old, and drug treatment was daily (5 days per week) for 3 weeks. To date, we have not seen any antiangiogenic or antitumor effects of the drugs. Upon sacrifice, the mice were found to have extensive insulinomas, involving around 70% of the entire pancreas. CD31(+) and F4/80(+) cells in the pancreas showed that most of them concentrated inside the PNET, however there was no change after the treatments. Studies are currently underway to determine if higher doses and/or starting treatment at an earlier time would be more efficacious.

#1823

Characterization of the anti-angiogenic properties of merestinib (LY2801653), an oncokinase inhibitor.

Diane M. Bodenmiller, Julie A. Stewart, Glenn F. Evans, Victoria L. Peek, Jennifer R. Stephens, Xi Lin, Seema Iyer, Beverly L. Falcon, Sudhakar Chintharlapalli, Sau-Chi Betty Yan, Anthony S. Fischl. _Eli Lilly and Company, Indianapolis, IN_.

Merestinib (LY2801653) is an orally bioavailable small molecule inhibitor of several oncokinases, including MET, AXL, DDR1/2, MERTK, ROS1, Tie2 (aka TEK), and MKNK1/2. Merestinib has been extensively characterized in a wide range of preclinical tumor xenograft models and shown to potently inhibit MET driven and non-MET driven tumor growth. In addition to its direct antitumor activity, merestinib inhibits angiogenesis and induces a tumor vessel normalization phenotype in xenograft tumors1. While MET signaling is important for angiogenesis, the effect of merestinib on angiogenesis is likely not exclusively driven by MET inhibition. In co-culture angiogenesis assays, merestinib inhibited VEGF-dependent and VEGF-independent endothelial cell cord formation2,3 and sprouting4 with potencies in the low nM range (3-30 nM). In contrast, the MET-specific kinase inhibitor, PF04217903, only weakly inhibited cord formation and endothelial sprouting. In an established in vivo matrigel co-implant vasculogenesis model where VEGFR2 or MET selective inhibition had minimal effect, merestinib decreased vascular density by 69%. In addition, while MET antibody emibetuzumab (human anti-MET antibody) plus ramucirumab (human anti-VEGFR2 antibody) decreased vascular density by 64%, merestinib plus ramucirumab decreased it by 92%. In a mouse adenovirus-driven VEGF-A ear angiogenesis model5, treatment with DC101, a mouse anti-VEGFR2 antibody, or merestinib inhibited angiogenesis; however the combination of DC101 and merestinib appeared to inhibit it even more. Finally, in the MKN45 gastric tumor xenograft model, merestinib (T/C = 4.8%) and DC101 (T/C = 15.3%) each significantly inhibited tumor growth alone and the combination resulted in 27.6% tumor regression and was significantly better than either single agent alone. Together, these studies indicate that merestinib has greater effects on angiogenesis than selective MET inhibition and its actions are not dependent on VEGFR2. In addition, while in vitro studies show reductions in VEGFR2 phosphorylation with high concentration of merestinib, treatment with merestinib did not inhibit VEGF dependent phosphorylation of VEGFR2 in mouse lung tissue at clinically relevant exposures. These data suggest that the anti-angiogenic activity of merestinib includes activities of other kinases targeted by merestinib. These data provide rationale and support for the clinical evaluation of combination of merestinib with ramucirumab (NCT02745769). 1-Yan et. al. Invest New Drugs. 2013;31:833-844, 2\- Falcon et. al. J Hematol Oncol. 2013;6:31, 3\- Falcon et. al. PLoS ONE. 2014;9:e106901, 4\- Nakatsu et. al. Methods Enzymol. 2008;433:65-82, 5\- Nagy et. al. Methods Enzymol. 2008;444:43-64.

#1824

Endothelial mTORC2 deficiency inhibits tumor angiogenesis, tumor progression and metastasis in non-small cell lung cancer (NSCLC).

Shan Wang. _Vanderbilt University Medical Center, Nashville, TN_.

Vascular endothelial cells (ECs) are specialized components of the tumor microenvironment that orchestrates tumor growth and invasion. They form tumor-associated blood vessels (angiogenic) that supply nutrients and oxygen, remove waste products, and also provide an entry site for tumorigenic cells to spread to secondary sites in various organs. One of the major signaling events in tumor vessels is PI3K/PDK-AKT-mTOR activation. mTOR is a serine/threonine kinase that functions in two distinct complexes, mTORC1 and mTORC2,which regulate a diverse array of cellular processes including cell growth, survival and metabolism. Although mTORC1, and to lesser extent, mTORC2, has been broadly studied in cancers and diseases, little is known regarding the relative contributions of mTORC1 versus mTORC2 in tumor endothelium. Using mouse model of endothelium-specific Rictor (a mTORC2 specific cofactor) gene targeting, we discovered that endothelial Rictor deletion decreased tumor neovascularization, suppressed tumor growth, and prevented metastasis in vivo. Direct co-culture of endothelial cells and NSCLC tumor cells in vitro showed that knockdown of endothelial Rictor inhibited tumor cell proliferation while Raptor (a mTORC1 specific cofactor) depletion had a modest effect. However, loss of endothelial Rictor or Raptor both markedly decreased tumor cell extravasation in response to endothelial cells, and also inhibited tumor spheroid formation in a direct co-culture system. Furthermore, a screen of phospho-kinase arrays revealed that Raptor or Rictor depletion in endothelial cells decreased various phosphor-RTKs in tumor cells that were co-cultured with ECs, including members of the Eph receptor family, Insulin receptor family and FGF receptor family. Additionally, endothelial Rictor knockdown suppressed phosphorylation levels of nerve growth factor receptors TrkB and TrkC in tumor cells. Collectively, these data suggest that endothelial mTORC1 and mTORC2 play critical roles in regulating tumor progression through distinct signaling pathways.

#1825

Angiopoietin-Tie-2 functional axis in colorectal cancer liver metastasis (CRCLM) provides a new marker for stratification and evaluation of tumor progression.

Nisreen S. Ibrahim. _McGill University, Montreal, Quebec, Canada_.

Colorectal cancer (CRC) is the third leading cause of cancer in Canadians, with liver metastases being the major cause of death from this disease. Tumors induce angiogenesis, a phenomenon known as the 'angiogenic switch', which is an essential step in tumor progression whereby the balance of pro- and anti-angiogenic factors are important for active angiogenesis. Clinical efficacy of targeted VEGF (anti-angiogenic) treatment has been validated as a cancer therapy. Our group, together with others, has identified unique histological growth patterns HGPs (desmoplastic, replacement and pushing) within liver metastases that have different responses to anti-angiogenic therapy. The patients with Desmoplastic HGP (DHGP) that received anti-angiogenic plus chemotherapy prior to resection had a significantly better pathologic response and survival than patients with Replacement HGP (RHGP). The aim of this study was to explore the role of Ang-1, Ang-2, Tie-2 and VEGF in the development and progression of CRCLM tumors with distinct HGPs. Here, human CRCLM tumor samples were analyzed by quantitative real-time PCR (Q-PCR) and immunohistochemistry (IHC) staining. The Q-PCR results demonstrated that the expression of Ang-2 was lower in RHGP tumor samples compared with DHGP tumor samples. This data was validated by IHC, were IHC scoring results showed that the ratio of Ang-2: Ang-1 expression in DHGP tumors was higher compared to RHGP tumors. VEGF and Tie2 proteins were expressed in both tumor patterns. Thus vascular quiescence maintained by constitutive Ang-1/Tie-2 signaling, found in RHGP tumors, prevails over destabilization and pro-inflammatory Ang-2/Tie-2 signaling, which is higher in the DHGP tumor samples. Since vascular remodelling is driven by Ang-2/Tie-2 in DHGP, which is dependent on VEGF we would expect anti-angiogenic therapy to be effective on DHGP. Furthermore, the RHGP had low levels of Ang-2 and high levels of Ang-1, together with the presence of Tie-2, and then one would predict that VEGF is not required for the growth of these tumors and thus would not respond to anti-angiogenic therapy, as has been shown in our patients' cohort. Taken together, these data suggest that the Angiopoietin/Tie-2 functional axis is an important player in CRCLM tumor progression and can be a potential target for CRCLM cancer therapy with stratification of patients by HGPs.

#1826

Bioavailability, pharmacodynamics and safety profile of a novel anti-angiogenic compound JFD in pre-clinical models.

Sivanesan Dhandayuthapani, Thanigaivelan Kanagasabai, Khadija Cheema, Appu Rathinavelu. _Rumbaugh-Goodwin Institute for Cancer Research, Nova Southeastern University, Fort Lauderdale, FL_.

JFD, isoindole (1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl)-amide, is a small molecule that was developed using molecular modeling to specifically antagonize Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) and associated kinase activity, thereby producing anti-angiogenic and anti-cancer effects. Since the original form of JFD is hydrophobic, it was recently modified into a water soluble form (JFD-WS) to increase its water solubility and bioavailability. Initially, we investigated the in vitro pharmacodynamics effect of both JFD and JFD-WS by utilizing Matrigel® tube formation assays, scratch and cell migration assays followed by inhibition of VEGFR2 phosphorylation in human umbilical vein endothelial cells (HUVECs). In vivo tumor growth inhibition of JFD and JFD-WS was tested using GI-101A breast adenocarcinoma cells implanted xenograft animals. Subsequently, the blood samples and the tumor proteins were extracted from the control and experimental animals to measure the pro-apoptotic signals and the levels of plasma biomarker such as MUC1 (mucin 1). The safety profile was established using Balb-c mice that were injected intraperitoneally (i.p) with JFD (6 mg/Kg body weight) and JFD-WS (100 mg/Kg body weight) for 30 days. The pharmacokinetics (PK) of JFD including the plasma concentration, excretion, elimination and distribution in the experimental animals were analyzed using a simple HPLC method. As expected, the JFD effectively inhibited HUVECs migration, tube formation, and VEGFR2 phosphorylation. Moreover, both JFD and JFD-WS exhibited strong anti-tumorigenic effects in xenograft implanted experimental animals that were evidenced by the decreasing plasma levels of MUC1. The western blot analysis of apoptotic markers and DNA fragmentation analysis further confirmed the pro-apoptotic effects of JFD as well as JFD-WS in our experimental system. Following i.p. injection, there was a significant distribution of JFD original into the liver, kidney, and brain, which contained the highest concentration compared to other two organs. When hematological, hepatic, renal and cardiac function markers were analyzed in the experimental animals, that were treated with JFD-WS for four weeks, no significant alterations were found in the test group compared to the control animals. Thus, whether used as a single agent or in combination with the other anti-cancer drugs, JFD shows strong antitumor effects without producing severe toxicities. The efficient delivery of JFD original to the brain suggests that this drug would be useful in treating solid tumors of the brain with pro-angiogenic abilities. (This project was supported by the Community Foundation of Broward, Ft. Lauderdale, FL and The Royal Dames of Cancer Research Inc., Ft. Lauderdale, FL).

### Cell Culture and Animal Models of Cancer 2

#1827

**Using** Drosophila **to study the role of metabolic enzyme mutations in glioblastoma and leukemia.**

Julia Fabiano, Gabriela Chiaramida, Mira Magner, Meghan O'Connor, Joseph Stallone, Nicholas DiDuca, Kathryn Neville, Richard Tartarini, Marla Tipping. _Providence College, Providence, RI_.

Metabolic reprogramming is a common hallmark shared by nearly all proliferating cancer cells, and thus has emerged as an exciting new direction in cancer research. Many signaling pathways have been implicated in mechanisms leading to the shift of metabolic programs in tumors, but more recently a small number of metabolic enzymes have also been identified in this process. Genes encoding the metabolic enzymes Isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) were found to be mutated in up to 70% of low-grade and medium grade gliomas, and in 15-20% of adult acute leukemia samples. These findings were the first to link the IDH gene to tumorigenesis. IDH1 and IDH2 function to irreversibly catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG). We are studying how mutations in IDH affect the metabolism and cellular processes of the cell. To this end, we have expressed IDH-R132H in Drosophila glial cells and hemoctyes to learn more about the impact of neomorphic activity of IDH in gliobastoma and chronic myeloid leukemia. We have verified that these cells now produce 2-HG, as is known in human tumors harboring IDH mutations. We have observed different cellular responses to IDH mutations in glia versus hemoctyes and have begun to investigate why this may be. Ultimately, the goal of our research is to elucidate the mechanism(s) that cause IDH to contribute to oncogenic activity in specific tissues, and use this knowledge to design tailored therapeutics.

#1828

Are callipers obsolute? A novel 3D scanning technology to measure subcutaneous tumor volume.

Zena Wilson,1 Juan Delgado,2 Michael Davies,1 Rebecca Whiteley,1 Jennifer Hare,1 Amar Rahi,1 Stephen Marshall,1 Andrew Smith,3 Stephen Atkinson,3 Jarno Ralli,3 Adeala Zabair,3 Adeala Zabair,3 Jane Kendrew1. 1 _AstraZeneca Pharmaceuticals, Cheshire, United Kingdom;_ 2 _Glaxo Medicines Research Council, Stevenage, United Kingdom;_ 3 _Fuel 3D, Chinnor, United Kingdom_.

Most preclinical oncology studies (xenograft, PDX, GEMMS) involve monitoring tumour growth rates, measuring them with callipers, and calculating the volume. Volume is calculated from the width and the length to estimate a 3D volume and is directly used to assess treatment efficacy. Although this technique is useful, it is unable to accurately assess non-uniformly shaped or very small tumours and introduces a systematic bias by assuming that tumours present with spheroid shape. Furthermore callipers do not inform of the tumour condition, which is dependent upon a visual estimation.

Here we describe the development and validation of a 3D scanner as an alternative method to callipers to monitor tumour progression in rodents. The resulting 3D scanner solution made up of hardware and software, has the potential to impact on the 3Rs guiding principles underpinning the humane use of animals in oncology research. The 3Rs benefits identified are primarily through reduction of animals via improved data accuracy allowing reduction in group sizes or the ability to include irregularly shaped tumours to test. In addition the scanner system described will make it possible to record tumour measurements in a rapid, minimally invasive, morphology-independent, and human-bias-free way, removing interoperator variability. This photo-based technique captures external symptoms of redness, paleness, ulceration of tumours, etc., which could ultimately be used to detect early toxicities of compounds or determine scales of animal welfare.

We describe the development and early validation of the scanner system within our laboratories. Using the 3D scanner alongside tumour callipers to monitor tumour growth of Oncology tumour studies we demonstrated that we can accurately measure tumour size parameters (length, width and volume) in multiple mouse strains and across a range of tumour models. 3D scanning tumour data is comparable to tumour measures generated from tumour callipers If successful the introduction of this system to replace tumour callipers could have a large impact for groups running oncology in-vivo tumour studies.

#1829

Evaluation of checkpoint inhibitors on tumour infiltrating immune cells in the orthotopic and metastatic tumour microenvironment using bioluminescent syngeneic cell line models.

Andrew McKenzie,1 Nektaria Papadopoulou,1 Simon Jiang,1 Jane Wrigley,1 Sumanjeet Malhi,1 Jason King,1 Kelly Jones,1 Neil Williams,2 Rajendra Kumari1. 1 _Crown Bioscience UK, Loughborough, United Kingdom;_ 2 _KWS Biotest, Bristol, United Kingdom_.

Background: Subcutaneous syngeneic models are widely used to evaluate the impact of immunotherapy on tumor growth and tumor invading leucocytes (TILs). However, the tumor microenvironment of orthotopic models is more comparable to the patient as they form an organ-specific tumour, facilitate metastatic spread and, in the case of syngeneic models, support immune and stromal component interactions. Bioluminescent imaging (BLI) enables non-invasive longitudinal monitoring of orthotopic/metastatic tumor burden, allowing for optimal randomisation, reduction of false positives and continuous feedback on treatment mid-study. Here we describe the generation of several bioluminescent variants of syngeneic cell lines and assess the impact of orthotopic growth on response to immune checkpoint therapy.

Methods: Bioluminescent variants of syngeneic cell lines were established by lentiviral transduction for: 4T1/EMT6 (breast), B16-F10 (skin melanoma), H22 (liver) and Pan02 (Pancreas) murine cancer cell lines. Subcutaneous growth of wild-type and bioluminescent variants was compared to assess any impact of luciferase expression on tumor growth, inflammation and TILs. Orthotopic models were established for most cell lines, and a metastatic model for B16-F10 was developed. BLI was carried out to assess real-time tumor burden and at end stage (Spectrum CT; PerkinElmer). Response to immune checkpoint therapy was evaluated and TIL infiltration was assessed by FACs analysis and IHC.

Results: No significant differences in growth profiles were exhibited when the bioluminescent variants of B16-F10 and Pan02 (subcutaneous) or EMT6 (mammary fat pad) were compared with the wild-type equivalents. Bioluminescent 4T1 cells readily metastasised to the lungs from both the orthotopic and subcutaneous sites and B16-F10 cells formed metastases. Response to immunotherapy, such as anti-CTLA-4, in the subcutaneous setting between the bioluminescent and WT models was similar. Anti-CTLA-4 therapy resulted in a statistically significant impact on orthotopic Pan02 tumor growth (BLI) and final tumor weight; however, further characterization by IHC and refinement of implantation conditions is required as the tumors exhibited low basal TIL levels, poor stromal and blood capillary infiltration.

Conclusions: The growth and response to immunotherapy does not appear to be significantly impacted in the bioluminescent cell line models tested; as such they are a useful tool for further assessing the impact of complex orthotopic, spontaneous and experimental metastasis modelling in immunocompetent mice. However, the orthotopic growth and TIL infiltration does vary between subcutaneous and orthotopic/metastatic settings and requires characterisation and validation with immunotherapeutics in order to understand the relevance and utilisation of such models.

#1830

Targeted inhibition of EGFR and glutaminase induces metabolic crisis in EGFR mutant lung cancer.

Milica Momcilovic,1 Sean T. Bailey,1 Jason T. Lee,1 Daniel Braas,1 Thomas G. Graeber,1 Melissa Works,2 Francesco Parlati,2 Susan Demo,2 Tonya C. Walser,1 Steven M. Dubinett,1 Saman Sadeghi,1 Heather Christofk,1 David B. Shackelford1. 1 _UCLA, Los Angeles, CA;_ 2 _Calithera Biosciences, CA_.

Cancer cells exhibit increased use of nutrients including glucose and glutamine to support the bioenergetic and biosynthetic demands of proliferation. We tested CB-839, a small molecule inhibitor of glutaminase that impairs glutamine utilization, in combination with erlotinib on EGFR mutant non- small cell lung cancer (NSCLC) as a therapeutic strategy to simultaneously impair cancer glucose and glutamine utilization and thereby suppress tumor growth. Here we show that CB-839 synergizes with erlotinib to drive energetic stress and activate the AMPK pathway in EGFR (del19) lung tumors. Tumor cells undergo metabolic crisis and cell death resulting in rapid tumor regression in vivo in mouse NSCLC xenografts. Consistently, positron emission tomography (PET) imaging with 18F- fluoro-2-deoxyglucose (18F-FDG) and 11C-Glutamine (11C-Gln) of xenografts indicated reduced glucose and glutamine uptake in tumors following CB-839 + erlotinib treatment. Therefore, PET imaging with 18F-FDG and 11C-Gln can be used to non-invasively monitor tumor metabolic and therapeutic response to CB-839 and erlotinib combination therapy.

#1831

PET/CT imaging of interleukin-13 receptor alpha-2-targeted peptide to glioblastoma after locoregional delivery.

Anirudh Sattiraju,1 Ang Xuan,2 Frankis Almaguel,1 Denise Herpai,1 Waldemar Debinski,1 Akiva Mintz,1 Kiran Kumar Solingapuram Sai1. 1 _Wake Forest School of Medicine, Winston Salem, NC;_ 2 _The People's Hospital of Zhengzhou University, Zhengzhou, China_.

Glioblastoma (GBM) is the most aggressive and common primary malignant astrocytoma which is characterized by tumor heterogeneity, infiltrating margins. Radiotherapy, chemotherapy and experimental targeted therapies have been ineffective at meaningfully increasing patient survival. One significant shortcoming of systemically delivered therapies is their inability to cross the blood brain barrier (BBB) and access infiltrating tumor cells. Therefore, in this work, we tested the potential of locoregionally targeting GBM via IL13Rα2, which we discovered to be expressed on greater than 75% of GBMs. To accomplish this, we intracranially infused copper-64 (64Cu) radiolabeled IL13Rα2 specific peptide Pep-1L, previously developed by Pandya et al., into mice bearing IL13Rα2 expressing orthotopic GBMs. Small animal micro PET/CT imaging showed ~2-fold greater tumor specific localization and lower volume of distribution of 64Cu-Pep-1L within brains of mice. Post-PET biodistribution study showed greater retention of 64Cu-Pep-1L 4 hours (%ID/g = 20.85± 0.65) and 24 hours (%ID/g = 14.65± 0.30) post infusion when compared to similarly infused 64Cu radiolabeled scrambled control peptide 4 hours (%ID/g = 10.73± 2.02) and 24 hours (%ID/g = 5.97± 1.47) post infusion. These results demonstrate that Pep-1L efficiently targets IL13Rα2 expressing GBMs in vivo upon loco-regional delivery and can effectively deliver potential therapeutic agents to GBM tumors while sparing normal brain.

This work was supported by the American Cancer Society Mentored Research Scholar grant # 124443-MRSG-13-121-01-CDD (Mintz), 1R01CA179072-01A1 (Mintz), P30 CA012197 (Pasche), R01CA07414519 (Debinski) and the Translational Imaging Program (TIP) of the Wake Forest CTSA (UL1TR001420).

#1832

Bioluminescent pharmacokinetics of luciferin in preclinical brain metastases of breast cancer models.

Neal Shah, Chris E. Adkins, Afroz S. Mohammad, Paul R. Lockman. _West Virginia University, Morgantown, WV_.

Background: Approximately 20% of breast cancer patients with disseminated disease will develop brain metastases. Preclinical models of brain metastases of breast cancer rely on ex-vivo histology to evaluate drug efficacy. However, bioluminescence imaging allows more precise quantification of tumor burden and progression through counting photons per second without requiring animal sacrifice. This methodology has not readily been applied to hematogenously derived brain metastases models given the heterogeneity of tumor burden and growth. Herein we demonstrate repeatable methodology to quantify brain metastases of breast cancer progression which is verified with histology.

Methods: Approximately 1.75 x 105 JIMT-1 (n=3) and 231-BrLuc (n=4) brain seeking subclones were injected intracardially to produce metastatic brain lesions. Mice were given 150 mg/kg of luciferin IP and luminescence was captured every 2-minutes for 60 minutes starting 24 hours after cell implantation. Radiance (photons per second per square centimeter per steridian) was plotted to observe peak luminescence. Imaging was repeated twice weekly to evaluate progression until euthanasia.

Results: Following intracardiac injection, both brain seeking metastatic models produced a maximum luminescence signal between 14-20 minutes after luciferin injection. For all subsequent imaging, 5 minute imaging between 15-20 minutes after luciferin injection was used. Longitudinally, both tumor cell lines produce bioluminescence 24 hours after cell injection, which is used to ensure tumor implantation and randomization. The BLI signal decreases to undetectable limits in both models between 3-7 days after cell implantation. Re-emergence of signal occurs on day 14 for the JIMT-1 line and day 21 for 231-Br line. JIMT-1 bioluminescence on day 14 begins at ~105 photons/sec/cm2/sr and then over time signal increases with an ~slope of 5.6x105 until reaching a maximum of 107 units on day 28. 231-Br bioluminescence on day 21 begins at ~104 photons/sec/cm2/sr and then over time signal increases with an ~ slope of 1.1x106 until reaching a maximum of ~107 on day 53.

Conclusion: To accurately measure bioluminescence as a surrogate for tumor burden in hematogenously implanted metastases, the optimal circulation time is approximately 15 minutes with a 5 minute imaging period. Bioluminescence of the 231Br line initially starts at a lower magnitude and later in time than the JIMT-1 line, but slopes of both intracardiac models are similar and result in similar bioluminescent curves.

#1833

Imaging-guided brain tumor surgery strategy based on a triple-modality MRI-PET-fluorescence imaging probe and imaging analysis.

Hui Meng,1 Yushen Jin,1 Shaowei Zhang,2 Shuai Zhang,3 Xibo Ma,1 Jie Tian1. 1 _Institute of Automation, Chinese Academy of Sciences, Beijing, China;_ 2 _Chinese PLA General Hospital, Beijing, China;_ 3 _Shandong University, Jinan, China_.

Precise brain tumor resection is valuable for increasing the 5-year survival rate. However, the delineation of tumor margin is a major challenge. Transferrin modified nanoprobes had the property of passing the blood-brain barrier (BBB). Here, we endeavored to use transferrin to fabricate a triple-modality magnetic resonance imaging-PET-fluorescence imaging molecule agent (MPF) which could improve diagnostic sensitivity and specificity of the brain tumors in the imaging acquisition and analysis. The localization of the tumor were determined by the MRI before surgery. Glucose metabolism was derived from PET imaging which would be an important influence factor for surgery strategy. Tumor margin was delineated using the quick segmentation methods according to the fluorescence imaging after craniotomy which could reduce residual tumor after resection. Finally, tissues were sliced and stained with hematoxylin and eosin every 5 mm. The histology results were processed into a whole image which can be used to evaluate the accuracy of intraoperative tumor margin. This three modality imaging-guided surgery strategy is a promising method which will probably achieve its application in clinical trials in near future.

#1834

Constitutive Nedd9 null genotype promotes lung cancer aggressiveness.

Alexander Deneka, Meghan Kopp, Anna S. Nikonova, Anna Gaponova, Alexandra Nagele, Harvey Hensley, Erica Golemis. _Fox Chase Cancer Center, Philadelphia, PA_.

Non-small cell lung cancer (NSCLC) has a low survival rate, with metastasis contributing to the vast majority of deaths. The NEDD9 (HEF1/Cas-L) protein has been reported to be elevated in expression and to promote metastasis in a large subset of lung cancers and in other malignancies. NEDD9 functions as a scaffold for multiple critical effectors in integrin/FAK/SRC and receptor tyrosine kinase signaling cascades, as well as for the mitotic kinase Aurora-A, with overexpression thought to sustain signaling by these pro-oncogenic proteins. Previous studies demonstrated depletion of NEDD9 by RNAi reduced the growth and invasion of established lung cell lines and tumors, based on cell culture and xenograft assays. We have now investigated the consequences of a null genotype for Nedd9 from the earliest stages of tumor formation, crossing Nedd9 null mice to a 129S/Sv-Krastm3Tyj/Trp53tm1Brn (KP) model in which Kras mutation is induced specifically in lung tissue by inhalation of adenovirus bearing the Cre gene. Unexpectedly, based on in vivo imaging, the Nedd9 null genotype significantly accelerated tumor growth in KP mice. Pathological examination of tissues indicated Nedd9 null genotype also was associated with higher invasive capacity in vivo, including direct invasion to the heart, as well as elevated proliferation rate, decreased apoptotic activity, and changes in the expression of proteins such as vimentin, associated with mesenchymal status. Although Nedd9 function has been implicated in immune cell activity, we found minimal differences in tumor infiltration of myeloid cells or macrophages based on Nedd9 genotype. Rather, use of Reverse Phase Protein Array (RPPA) analysis to characterize signaling in isolated tumors suggested tumor intrinsic changes in glycolytic and proliferative signaling pathways, which were subsequently validated using direct functional assays. These results for the first time imply that NSCLC tumor progression beyond early stages in the absence of Nedd9 requires reprogramming of intrinsic tumor signaling to compensate for the absence of this protein, emphasizing activities distinct from those associated with elevated Nedd9 in late stage tumors.

#1835

Development of mammary hyperplasia, dysplasia, and invasive ductal carcinoma in transgenic mice expressing the 8p11 amplicon oncogene NSD3 (WHSC1L1).

Alex C. Rutkovsky, Brittany Turner-Ivey, Ericka L. Smith, Laura S. Spruill, Jamie N. Mills, Stephen P. Ethier. _The Medical University of South Carolina, Charleston, SC_.

Amplification within the 8p11-12 locus occurs in approximately 15% of breast cancer (BC) and is associated with poor survival and distant recurrence. Overexpression of many of the genes within the 8p11-12 region can confer a pathological gain of function to breast cells. We and others have previously demonstrated that NSD3 (WHSC1L1), a lysine methyltransferase, can act as a potent transforming oncogene. Predictions by the GISTIC algorithm of the Human Cancer Genome Atlas data suggest that NSD3 is a driving gene within 8p11-12 in multiple tumor types, including BC. We previously showed that NSD3 can regulate the expression of influential genes, such as estrogen receptor alpha (ESR1/ERα). To further explore the role of NSD3 in promoting BC, we generated FVB/N transgenic mice with targeted overexpression of NSD3 in the mammary gland and compared them to matched non-transgenic wild-type (WT) females. We observed that pups nursed by transgenic females were underdeveloped, regardless of genotype. Underdeveloped pups displayed delayed hair growth and eye opening, and were half the weight of pups nursed by WT females. To investigate this phenotype, we characterized thoracic and inguinal mammary glands from virgin, mid-pregnancy, lactating, and post-lactating mice by whole mount and histological analysis. Mammary glands from virgin transgenic females displayed increased branching and terminal bud formation. Alveolar buds from mid-pregnant transgenic glands were more numerous and densely packed. Glands from lactating transgenic females showed large areas in which the alveoli failed to undergo functional differentiation, resulting in a lactation defect. Mammary glands taken post-lactation exhibited areas of ductal and alveolar hyperplasia. At 40 weeks of age, multiple transgenic mice had palpable tumors which were excised at 1 cm3 size. Whole mount and histological analysis of glands from tumor bearing mice commonly demonstrated hyperplasia, dysplasia, and carcinoma in situ, contrary to age-matched WT glands. The areas of ductal dysplasia and carcinoma in situ closely resembled several patterns commonly observed in human breast cancer, including micropapillary, cribriform, and high grade areas of ductal carcinoma in situ. Mammary tumors were analogous to infiltrating ductal carcinomas, many of which were high grade tumors. In summary, overexpression of NSD3 in the mouse mammary gland elicited drastic deformation, inhibited functional differentiation, and caused tumor formation. Continued characterization of this mouse model, the oncogenic role of NSD3, and other mechanistic studies are essential to improve patient outcome in 8p11-12 altered cancers.

#1836

Patient-derived xenograft (PDX) models expressing HER2 reflect clinical responses to targeted HER2 inhibition.

Daniel Ciznadija,1 Amir Sonnenblick,2 Jennifer Jaskowiak,1 Angela Davies,1 David Sidransky3. 1 _Champions Oncology, Hackensack, NJ;_ 2 _Hadassah-Hebrew University Medical Center, Jersulaem, Israel;_ 3 _Johns Hopkins University School of Medicine, Baltimore, MD_.

Background While HER2-directed agents are most often used for treating breast cancer, there is increasing evidence that these therapies may be of value in other solid tumors. Sequencing efforts and immunohistochemistry (IHC) have identified mutations, amplifications, and overexpression of HER2 in ovarian, HNSCC, NSCLC, and GI cancers. PDX models could permit evaluation of HER2 response/resistance mechanisms to optimize therapeutic strategies. In this pilot study, we evaluated the response of PDX models to HER2-targeted therapies and correlated responses to clinical outcomes.

Materials and Methods PDX models were developed from a variety of patient solid tumors, evaluated by IHC for HER2 expression and next-generation sequencing for genomic alterations in HER2 (mutations, amplifications/deletions, and expression levels). Models were screened against single agent HER2-directed therapies including trastuzumab (n=15), trastuzumab emtansine (n=23), and lapatinib (n=10). Tumor regression (TR) values and RECIST criteria were determined and correlated with known literature-based response rates (RR) as well as individual patient outcomes.

Results 32 PDX models from 30 patients were interrogated (primarily breast and colorectal). Twenty (63%) models have been sequenced to date; 13 (65%) harbor amplification at ERBB2 gene locus. Further, 56% (18/32) have been evaluated by IHC for HER2 to date: 50% have 2+ HER2 staining, 17% 3+ staining, and 33% 1+/- staining. Based on PDX tumor growth, stable disease/regression was observed in 10% of models screened against lapatinib (CR/PR=0%), 50% screened against trastuzumab (CR/PR=8%), and 67% tested against trastuzumab emtansine (CR/PR=14%). Only models with +2/+3 HER2 staining showed regression with HER2-targeted treatment, with nearly 70% of +1/- HER2 models showing progressive disease. Finally, there were 4 correlations to patient clinical outcomes available, with 3/4 (75%) of the PDX model responses mimicking those of the patient to the same treatment.

Conclusion and Future Directions Extensive sequencing of human cancers has demonstrated HER2 amplification or mutation in numerous solid tumors, suggesting HER2-directed therapy could be applied more broadly in the clinic. Consistent with clinical findings, HER2 therapy responses depended upon the strength of HER2 expression (based on IHC). Nevertheless, response rates in PDX models varied depending on which HER2-targeted agent was deployed, highlighting the potential existence of differential mechanisms of de novo resistance/sensitivity. Comprehensive sequencing and drug testing of these PDX models is planned and could allow a deeper understanding of such mechanisms. In this context, application of PDX models for translational modeling of HER2 drug responses, particularly in the context of co-clinical trials, will continue to evolve.

#1837

High Ras activity promotes neoplasia in pancreatic ductal cells.

Kanchan Singh,1 Melissa A. Pruski,1 Wasim A. Dar,1 Kishore Polireddy,1 John S. Bynon,1 Mamoun Younes,1 Anirban Maitra,2 Craig D. Logsdon,2 Jennifer M. Bailey1. 1 _University of Texas Health Science Center, Houston, TX;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal malignancy and is the third leading cause of cancer-related deaths in the United States. Next generation sequencing efforts have revealed KRAS mutations occur in over 99% of pancreatic ductal adenocarcinomas and are thought to be the initiating mutation for pancreatic cancer. Despite the high prevalence of KRAS mutations in pancreatic cancer patients, murine models expressing endogenous levels of mutant Kras in pancreatic cells do not develop PDAC. A number of publications have revealed that acinar cells are more sensitive to early neoplastic lesion formation in the presence of Kras mutations than ductal cells. In these models, a second genetic hit (loss of tumor suppressor TP53, SMAD4 or CDKN) is required for the development of invasive PDAC, including transforming pancreatic ductal cells. Recent data has revealed a novel role for high levels of Ras activity in acinar cells in the tranformation of this cell type to PDAC (Ji, B., 2009). We hypothesized based on other preliminary data that high levels of Ras activity would transform ductal cells. To test this hypothesis, we studied the role of constitutively active high levels of Kras activity in acinar and ductal cells by expressing an inducible KrasG12V allele in Ptf1a:CreERT2 (acinar cell specific) and Hnf1b:CreERT2 (ductal cell specific) mice. Similar to previously published work, high levels of Ras activity in acinar cells resulted in PDAC and a remarkably low survival time of 10 days after mice were injected with tamoxifen. Mice with elevated Ras activity in ductal cells needed to be euthanized two weeks after tamoxifen injection and manifested a cancer cachexic phenotype. Histological analysis of these mice revealed high grade oncocytic intraductal neoplasia and the phenotype extended throughout the pancreatobiliary ducts. Western blot analysis of human PDAC and Cholangiocarcinoma tissue revealed similar levels of Ras activity to our murine models, confirming human equivalent high Ras expression to both animal models. Oncocytic tumors are characterized by an abundance of mitochondria. Thus, we are currently studying metabolic alterations and mitochondria accumulation in ductal and acinar cells expressing high Ras activity. Our goal is to determine how mitochondrial alterations play a role in progression or inhibition of tumors in ductal vs acinar cells.

#1838

**TGFalpha (an EGFR ligand) promotes growth of** EGFR **-mutant lung tumors in airway regions but not in alveolar regions in a transgenic mouse model.**

Koichi Tomoshige, Yutaka Maeda. _Cincinnati Children's Hospital Medical Center, Cincinnati, OH_.

Although an exogenous EGF is dispensable for EGFRL858R-mediated NIH3T3 cell transformation in vitro (Greulich et al., PLoS Med 2005), an exogenous EGF promotes growth of H1650 and H1975 lung adenocarcinoma cells that carry EGFR mutations in vitro (Sordella et al., Science 2004). However, it is unknown whether an exogenous EGFR ligand plays a role in growth of EGFR-mutant lung tumors in vivo. Here, using conditional transgenic mice expressing EGFRL858R or KRASG12D along with an exogenous TGFalpha (an EGFR ligand) in lung epithelium, we sought to determine the role of an EGFR ligand in EGFRL858R-lung tumors or wild type EGFR-lung tumors (KRASG12D-lung tumors) in vivo. As previously reported, conditional expression of TGFalpha (an EGFR ligand) in lung epithelium caused pulmonary fibrosis but not lung tumors (Korfhagen et al., J Clin Invest 1994; Hardie et al., AJP-LCMP 2004), indicating that the expression of an exogenous EGFR ligand is not sufficient to induce lung tumors. Conditional expression of EGFRL858R or KRASG12D in lung epithelium caused lung tumors as previously reported (Fisher et al., Genes Dev 2001; Politi et al., Gene Dev 2006). In our study using a new rtTA driver, Scgb1a1-rtTA line 2 (Perl et al., AJRCMB 2006), the majority of EGFRL858R-lung tumors occurred in alveolar regions while KRASG12D-lung tumors occurred in both alveolar and airway regions. Importantly, co-expression of EGFRL858R along with TGFalpha in lung epithelium induced the growth of lung tumors in airway regions but not in alveolar regions while the expression of EGFRL858R alone did not induce lung tumors in airway regions. Co-expression of EGFRL858R along with TGFalpha also shortened survival of the mice compared to the expression of either EGFRL858R or TGFalpha alone (median 32 days for EGFRL858R/TGFalpha vs 118 days for EGFRL858R alone or 248 days for TGFalpha alone). Co-expression of KRASG12D with TGFalpha did not influence tumor location or survival of the mice compared to the expression of either KRASG12D or TGFalpha alone. Consistent with the mouse study, TGFalpha expression in human EGFR-mutant lung cancer but not wild-type EGFR lung cancer was associated with worse overall survival (median 44 months for TGFalpha high vs 75 months for TGFalpha low; p<0.05) and recurrence free survival (median 21 months for TGFalpha high vs 61 months for TGFalpha low; p<0.05). Collectively, our results demonstrate that TGFalpha (an EGFR ligand) promotes not only the growth of EGFRL858R-lung tumors but also influences the regions where the EGFRL858R-lung tumors form.

#1839

TGFBR1*6A mouse model mimics human breast cancer development and progression.

Michael James Pennison, Minghui Wang, Hugo Jimenez, Boris Pasche. _Wake Forest Baptist Medical Center, Winston-Salem, NC_.

Transforming Growth Factor-ß (TGF-β) is one of the most commonly altered cellular signaling pathways in human cancer. Our group was the first to discover TGFBR1*6A (*6A), a common hypomorphic variant of the type I TGF-ß receptor (TGFBR1), which has been associated with increased risk for breast cancer in multiple epidemiological studies. Functional analysis also suggests that *6A is associated with decreased tumor suppressive TGF-β signaling in early tumor development and is involved with promoting tumorigenesis in advanced carcinomas. To better understand the functional impact of *6A and constitutively reduced TGF-ß signaling on breast cancer development and progression, we have recently developed a series of transgenic knock-in mouse model variants by replacing exon 1 of the mouse Tgfbr1 sequence with human exon 1 sequences harboring both *6A and wild-type (*9A) TGFBR1 variants and crossed them with MMTVneu mice.

We generated a total of 5 FVB double and/or triple transgenic mouse strains: MMTVneu;9A/9A, MMTVneu;6A/6A, MMTVneu;9A/6A, MMTVneu;*9A/-, and MMTVneu;6A/-. Following tumor development, measurements were performed three times per week to determine tumor volume. All mice were allowed to live up to 18 months (548 days) or until they died of natural causes. Specimens were collected for later analysis of blood, tumors, normal mammary tissue, and were evaluated for lung metastases. Statistical comparisons performed using Student's t-test.

Only 34.4% of MMTVneu;9A/9A mice (11/32) developed mammary tumors, while 80% of MMTVneu;6A/6A mice (24/30; p=0.0002) and 62.5% of MMTVneu;6A/- mice (19/32, p=0.046) developed tumors. The percentage of MMTVneu;9A/6A and MMTVneu;*9A/- mice that developed tumors was higher than MMTVneu;9A/9A mice (48.3% and 50%, respectively) but was not statistically significant. Additionally, 66.7% of MMTVneu;6A/6A mice (20/30) developed 2 or more tumors, while only 25% of MMTVneu;9A/9A mice (8/32) developed multiple tumors (p=0.0007).

Our data suggests that we have developed transgenic mouse strains that accurately mimic human breast cancer susceptibility associated with *6A and constitutively decreased TGFBR1 expression in humans, as evidenced by other studies. These mouse models may be a tool to provide invaluable insight to better understand the role of TGFBR1 variants in the development and progression of breast cancer.

#1840

Whole-exome somatic mutation analysis of mouse cancer models and implications for preclinical immunomodulatory drug development.

Bruno Zeitouni,1 Cordula Tschuch,1 Jason M. Davis,2 Anne-Lise Peille,1 Yana Raeva,1 Manuel Landesfeind,1 Sheri Barnes,2 Julia B. Schüler1. 1 _Oncotest GmbH, Freiburg, Germany;_ 2 _Charles River Discovery, Morrisville, NC_.

Experimental tumors raised in rodents represent an important preclinical tool to develop innovative anticancer compounds before clinical testing. Amongst others such models include solid tumors raised in syngeneic fully immunocompetent hosts and tumors spontaneously growing in genetically engineered mice (GEM) and derivate thereof. These model platforms have gained additional value since the manipulation of the immune system to fight cancer has led to tangible benefits for cancer patients. In the current study, we analyzed somatic mutation profiles from whole-exome sequencing (WES) data in a panel of 14 different mouse models covering 6 major cancer types. 4 models were GEM-derived, all other lines were developed by injection of established cell lines into the corresponding mouse strain. In parallel, these models were evaluated for their sensitivity towards checkpoint inhibitors (α-CTLA-4, α-PD-1 or α-PDL-1) in mono- or combined therapy with cytostatic and/or targeted agents.WES achieved an average-of-coverage of 165X in tumor models and normal DNA. A median mutation rate of 34 somatic mutations (m)/MB was detected, ranging from 7 m/MB (GEM derived NSCLC model KP) to 328 m/MB (syngeneic NSCLC line Lewis Lung) in exons. Mutation rates were markedly lower in GEM-derived models as in syngeneic lines (median of 9 vs 43 m/MB). This reflects very well the different underlying carcinogenic mechanism of these two types of models. The cross-comparison of tissue-transplants vs cell lines from GEM-derived model KP revealed that 75% of the mutations found in the primary KP could also be detected in the corresponding cell lines KP1 and KP4. Of note, the mutation count increased 1.3- (KP4) and 2.9-fold (KP1) during cell line establishment. Every model depicted a distinct profile against modulators of the immune system dividing the panel in responders and non-responders. In our hands no significant correlation could be determined between mutational load and sensitivity towards checkpoint inhibition in vivo. This might be related to the fact that the dataset was not broad enough and the number of models per entity was too small, rendering the subtype analysis within the panel not feasible. However, a strong tendency was observed when investigating the colon lines Colon26, CT26 and MC38 showing best response to the combination of PD-1+CTLA-4 inhibitors and in parallel the highest mutation rates (52, 64 and 59 m/MB, respectively) compared to non-responders B16-F10, CloudmanS91, 4T1 and KP1 (23 m/MB on average). Mouse models of cancer are a relevant tool for preclinical studies specifically for immuno-oncology. The molecular characterization of these models will help to optimize their use in drug discovery. They will support the development of innovative drugs and indentification of biomarkers to classify the patient cohort profiting the most from these new compounds.

#1841

Characterization of neuronal alterations in a new mouse model of low grade glioma.

Daniela Torres, Angeliki Mela, Sohani Das Sharma, Nicholas Hornstein, Peter Sims, Peter Canoll. _Columbia University, New York, NY_.

Diffusely infiltrating gliomas are the most common type of primary brain tumor seen in adults. Patients with low grade glioma (LGG) can survive for many years, but often suffer from seizures and cognitive impairment, and these symptoms are associated with an unfavorable prognosis. Glioma cells diffusely infiltrate cortical tissue, where tumor cells aggregate around neuronal cell bodies, a growth pattern referred to as perineuronal satellitosis, however the effects of tumor cells on neurons has not been well defined. To address this important issue we generated a mouse model that recapitulates the genetic and histological features of diffusely infiltrating LGG. In our model tumor growth is driven by PDGFA overexpression and p53 deletion in a RiboTag-Camk2a-Cre transgenic mouse. The RiboTag system allows for the isolation and sequencing of ribosome-bound transcripts from Camk2a neurons to obtain a measure of transcription and translation. Our analysis in non-tumor bearing brains identified neuron-specific genes that are highly regulated at the level of translation. mTOR signaling regulates cell growth, proliferation, and translation in response to diverse stimuli. To characterize mTOR signaling in our model we used immunohistochemical staining of pS6 as a marker of mTOR activity and found that neurons within the glioma infiltrated cortex have lower levels of pS6 staining compared to neurons in the surrounding cortex. To further assess these results we are using the RiboTag system to identify alterations in neuronal transcription and translation that occur in our model during glioma formation and in response to mTOR targeted treatments. These studies will provide new insights into the neuronal alterations contributing to seizures and cognitive impairment in glioma and enable the identification of potential molecular targets for novel therapies to treat the devastating consequences of cortical dysfunctions associated with glioma.

#1842

Breast cancer brain metastasis (BCBM) model for determination of therapeutic brain penetration.

Emily A. Wyatt, Mark E. Davis. _California Institute of Technology, Pasadena, CA_.

Purpose

Brain metastases are presenting an increasing problem in the clinic, and especially in treatment of patients with human epidermal growth factor receptor-2 (HER2)-amplified breast cancer. Although extracranial metastases respond well to HER2 inhibitors, human clinical data shows brain metastases hide behind an intact blood-brain barrier (BBB) and are largely untreatable. Many current preclinical models lack this barrier integrity, limiting their utility in understanding delivery of drugs to the brain. We present here the development of a new model suitable for evaluating therapeutic brain penetration in addition to efficacy.

Experimental procedures

Human HER2-amplified BT-474 breast cancer cells were inoculated intravenously (tail vein) in female Rag2-/-;Il2rg-/- mice (2 million cells/mouse) to induce multiorgan metastasis. Formation of metastatic brain lesions was monitored by magnetic resonance imaging (MRI). For comparison, BT-474 cells were inoculated intracranially for direct brain tumor implantation (50,000 cells/mouse). Response to a suite of HER2-targeted therapies known not to appreciably cross an intact BBB (trastuzumab, lapatinib, etc.) was monitored by MRI for both metastatic and implanted brain tumors. Mice were sacrificed following signs of prolonged distress or loss of >20% body weight. Organs were collected for standard histological and immunohistochemical analysis, as well as for CLARITY tissue clearing and large-scale 3D macromolecule mapping.

Results

Intravenous inoculation of BT-474 cells into Rag2-/-;Il2rg-/- mice consistently reproduced the full metastatic profile seen in humans, with metastases in the lung, bone, liver, ovary, lymph, and brain tissues. Brain metastases were detected in >90% of mice inoculated intravenously. Histological analysis of metastatic brain tumors showed different morphologies and invasive characteristics compared to those intracranially implanted. Additional differences in vasculature between metastatic and implanted brain tumors were identified by CLARITY. Importantly, HER2-targeted therapy markedly delayed progression of implanted brain tumors, but failed to control metastatic brain tumor growth, recapitulating the clinical situation.

Conclusions

These data, together with ongoing efforts to further characterize therapeutic transport to these brain tumors, suggest that intracranial inoculation disrupts the BBB and creates artificial routes for therapeutics to reach implanted brain lesions, resulting in anomalous tumor response. In contrast, this new metastatic model reproduces the discordant effects of HER2-targeted therapy in patients, and offers a platform for studying the efficiency of therapeutic delivery across an intact BBB as well as antitumor activity, both of which are critical to effective clinical translation.

#1843

Intratumoral heterogeneity of renal cancer is related to differences in drug response and development of therapy resistance.

Michael Becker,1 Burkhard Jandrig,2 Susanne Flechsig,1 Reiner Zeisig,1 Daniel Schindele,2 Martin Schostak,2 Christian Schmees,3 Annika Wulf-Goldenberg,1 Jörg Hennenlotter,4 Elke Schaeffeler,5 Matthias Schwab,5 Arnulf Stenzl,4 Jens Bedke,4 Jens Hoffmann1. 1 _EPO - Experimental Pharmacology and Oncology Berlin-Buch GmbH, Berlin, Germany;_ 2 _University Medical Center Magdeburg, Magdeburg, Germany;_ 3 _NMI University Tuebingen, Tuebingen, Germany;_ 4 _University Hospital Tuebingen, Tuebingen, Germany;_ 5 _Margarete Fischer-Bosch-Institut for Clinical Pharmacology, Stuttgart, Germany_.

Background:

Patients with advanced renal cell cancer (RCC) have a poor prognosis not least because of resistance towards standard drugs (SoC). Recently, pronounced intratumoral heterogeneity (ITH) in RCC was shown. We were interested whether this ITH is a potential cause for treatment failure. We developed a large panel of patient-derived xenograft (PDX) models from RCC, including subsets of models from different regions of one individual tumor. The PDX models were evaluated for response to SoC. To better understand correlations between inter- and intratumoral heterogeneity and treatment response, tumor models were panel sequenced and expression profiled.

Methods:

Specimens from primary and metastatic RCCs were collected from consenting patients and transplanted into mice. Tumor engraftment was monitored for up to 4 months. Tumor sections were examined histopathologically to assess concordance between patient tumor and model and were stained for RCC specific markers (Pax2, Pax8, CD31, and RCC). Stable growing PDX were treated with SoC (sunitinib, sorafenib, bevacizumab and everolimus). Global gene expression was analyzed in primary tumors and PDX models using microarrays (Affymetrix). In addition, sequence variations (Illumina NGS cancer panel) and MET and TERT gene copy numbers were analyzed in PDX models.

Results:

A panel of 34 RCC PDX models was established from more than 200 patient samples. Among these, 13 models were derived from different tumor regions of three advanced tumors.

Original patient tumor and PDX showed a very similar and characteristic RCC histopathology. Inter- and intratumoral heterogeneity was preserved for several passages.

We treated all PDX with standard targeted drugs and observed response rates comparable to results from clinical trials. One out of 8 regions obtained from one aggressive RCC clearly differentiated in regard to its response to bevacizumab and sunitinib. Genomic analysis revealed that this region has differences in global gene expression and sequence variation pattern. Besides a common MET mutation an additional variation in the HRAS gene was detected.

In the whole PDX set we found 34 sequence variations in 20 genes, e.g. ATM, MET, TP53 and VHL and copy number variations in the MET locus.

Conclusion:

We have shown that PDX derived from distinct regions within one individual tumor exhibit differences in targeted treatment response as well as in genetic profile. These differences and their correlation to their molecular heterogeneity is subject of ongoing investigations to explain failure in treatment response.

The available panel of renal cancer PDX provides an excellent source for translational research and for preclinical testing of new drug candidates.

#1844

Investigating the role of amphiregulin in breast cancer.

Serena P. Chiang, George S. Karagiannis, John S. Condeelis, Jeffrey E. Segall. _Albert Einstein College of Medicine, Bronx, NY_.

Breast cancer is the most prevalent cancer in women worldwide and afflicts more than 3.1 million women in the US alone. The epidermal growth factor receptor (EGFR) is frequently overexpressed in breast cancer and is associated with poor clinical outcome. Several therapies involving anti-EGFR agents have been developed to target breast cancer. However, clinical trials have shown mixed outcomes with these therapies. Amphiregulin (AREG) is a ligand for EGFR that mediates estrogen-induced mammary ductal morphogenesis during development. Expression of AREG is enriched in invasive breast carcinomas, particularly ERα-positive breast tumors. Studies have shown that AREG increases the in vitro invasiveness of breast cancer cells. Furthermore, suppression of AREG expression in transformed human breast epithelial cells reduces tumor formation when injected in nude mice. These observations suggest that AREG plays an important role in breast cancer development and progression. To investigate the role of AREG in breast cancer development and progression, we utilized a spontaneous mammary tumor mouse model where oncogenesis is driven by expression of the polyoma middle T oncoprotein (PyMT) through the mouse mammary tumor virus (MMTV) promoter. We have crossed AREG knockout mice with the MMTV-PyMT mice to generate AREG KO mice that spontaneously form mammary tumors. Tumors in AREG KO PyMT mice initially grow more quickly but eventually reach 2 cm in diameter at a relatively time as compared to tumors formed in AREG WT PyMT mice. Histologically, AREG KO tumors appear more differentiated and display a greater proportion of tumor stroma. This prompted us to further hypothesize that AREG may have diverse effects in the tumoral or stromal compartment of developing tumors. To individually assess the effects of AREG knockout in the tumor cells and the stroma, we utilized a transplantation model in which we prepared tumor chunks from AREG WT and KO PyMT tumors and transplanted them into AREG WT and KO mice. Through this model, we have found that AREG expression in the tumor has a greater impact on tumor growth and progression than the tumor stroma. Regardless of the AREG status of the recipient mice, AREG KO tumors display a significant growth impairment and reduced transplant efficiency. In summary, these results demonstrate that AREG is important for mammary tumor progression.

#1845

Mutant p53 promotes progression and metastasis of mouse oral tumors induced by 4NQO, associated with specific immune infiltrates.

Jin Wang, Yuanyuan Zhang, Bingbing Wang, Adel K. El-Naggar, Jeffrey N. Myers, Carlos Caulin. _UT MD Anderson Cancer Center, Houston, TX_.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide. The vast majority of the human HNSCCs contain p53 mutations, some of which acquire oncogenic gain-of-function (GOF) activities. Previous mouse models in which either the p53R172H GOF mutation or deletion of the p53 gene were co-activated with oncogenic K-ras showed that p53R172H can predispose to oral tumor initiation, accelerate tumor growth and promote progression to SCC. However, the impact of p53 gain- and loss-of-function (LOF) mutations in metastasis could not be assessed because of the low rates of progression to SCC in that model. To overcome this limitation, we used the tobacco-surrogate 4NQO to induce oral lesions that may progress to advance carcinomas following stepwise changes that resemble the gradual accumulation of histological and molecular abnormalities observed during human oral cancer progression. To examine the role of p53 mutations during SCC malignant progression we applied 4NQO to mice in which the p53R172H mutation or homozygous deletion of p53 were activated in oral epithelial cells. We observed that oral tumors appeared faster in the presence of p53 mutations than in mice with wtp53. No significant difference was observed between the p53R172H mutation and p53 deletion. However, survival was shorter in mice expressing p53R172H than in mice with loss of p53. Notably, regional and distant metastasis were only observed in mice with oral tumors expressing p53R172H. Therefore, these mice are excellent tools for pre-clinical studies designed to target early stages of tumor development or latest stages of progression and metastasis. As immunotherapy is becoming adopted as part of the standard treatment for head and neck cancer patients, we conducted a detailed characterization of the immune infiltrates of the lesions that developed in these mice in order to assess their immunogenicity. We observed a progressive increase in T-cell infiltration from normal oral mucosa to oral premalignant lesions (OPL) to SCC, in the presence or absence of p53 mutations. Similarly, the expression of immune checkpoint proteins, including PD1, PDL1 and CTLA4 increased during oral tumor progression, regardless of the p53 status. Interestingly, we observed a sharp increase in the presence of immune infiltrates expressing the co-stimulatory OX40 in SCCs relative to premalignant lesions, with different extent depending on the nature of the p53 mutation. The significance of these observations is currently being analyzed. In summary, p53R172H promotes metastasis in oral tumors induced by 4NQO. In addition, T-cell infiltration and activation of immune checkpoints during oral tumor progression suggest that this is an excellent model to test immunotherapy-based strategies. Furthermore, our findings suggest that OX40-based therapies may be tailored to patients according to their p53 status.

#1846

Immunocompetent mouse allograft models for development of therapies to target breast cancer metastasis therapies to target breast cancer metastasis.

Yu-an Yang,1 Howard Yang,1 Ying Hu,1 Peter Watson,2 Huaitian Liu,1 Thomas R. Geiger,1 Miriam R. Anver,3 Diana Haines,3 Philip Martin,3 Maxwell P. Lee,1 Kent W. Hunter,1 Lalage M. Wakefield1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _British Columbia Cancer Agency, Victoria, British Columbia, Canada;_ 3 _Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, Frederick, MD_.

Effective drug development to combat metastatic disease in breast cancer would be aided by the availability of well-characterized preclinical animal models that (a) metastasize with high efficiency, (b) metastasize in a reasonable time-frame, (c) have an intact immune system, and (d) capture some of the heterogeneity of the human disease. To address these issues, we have assembled a panel of twelve mouse mammary cancer cell lines that can metastasize efficiently on implantation into syngeneic immunocompetent hosts. Genomic characterization shows that more than half of the 30 most commonly mutated genes in human breast cancer are represented within the panel. Transcriptomically, most of the models fall into the luminal A or B intrinsic molecular subtypes, despite the predominance of an aggressive, poorly-differentiated or spindled histopathology in all models. Patterns of immune cell infiltration, proliferation rates, apoptosis and angiogenesis differed significantly among models. Inherent within-model variability of the metastatic phenotype mandates large cohort sizes for intervention studies but may also capture some relevant non-genetic sources of variability. The varied molecular and phenotypic characteristics of this expanded panel of models should aid in model selection for development of anti-metastatic therapies in vivo, and serve as a useful platform for predictive biomarker identification.

#1847

Evaluating the metastatic potential and the molecular heterogeneity of patient-derived orthotopic xenograft models of triple-negative breast cancer.

Vishnu C. Ramani,1 Rakhi Gupta,1 Gerald Quon,2 Melanie Triboulet,1 Corinne Renier,3 Cassandra Greene,4 Chad Sanada,4 Tracy Lu,4 Lukasz Szpankowski,4 Naveen Ramalingam,4 Ameen A. Salahudeen,1 Sean de la O,1 Ranjani Rajapaksa,1 Shoshana Levy,1 Anne A. Leyrat,4 Jay A. West,4 Elodie Sollier-Christen,3 Calvin J. Kuo,1 George W. Sledge,1 Stefanie S. Jeffrey1. 1 _Stanford University, Stanford, CA;_ 2 _University of California, Davis, CA;_ 3 _Vortex Biosciences, Inc., Menlo Park, CA;_ 4 _Fluidigm Corporation, South San Francisco, CA_.

We report an in-depth characterization of patient-derived orthotopic xenograft (PDOX) models of triple-negative breast cancer (TNBC) regarding their molecular profile at the single cell level, tumor heterogeneity, 3D organoid generation, and ability to generate circulating tumor cells (CTCs). A panel of seven TNBC PDOX tumors were grown orthotopically in Nod scid gamma mice and used in this study. Blood obtained via cardiac puncture from tumor bearing animals was processed on the Vortex microfluidic platform, for label-free, size-based enrichment of circulating tumor cells (CTCs). Enriched cell populations were stained for human-specific cytokeratin (CK) and Vimentin (Vim), mouse-specific CD45, and DAPI; CTCs were identified as cells that were CD45 negative and positive for CK or Vim. Bulk tumor growing in the mammary fat pads was dissociated to single cells and characterized using Fluidigm's® PolarisTM platform for single cell biological experimentation and cDNA generation within an integrated fluidic circuit (IFC). From the cell suspension, Polaris identified single cells that were then processed for mRNA-seq. The resulting cDNA libraries were then multiplexed using Nextera XT® (Illumina®) and sequenced on Illumina systems. Data generated from mRNA-seq was processed to correct for confounding factors such as cell size, cell cycle and read depth and then analyzed to screen for heterogeneity between different populations of cells. Tumors were analyzed by flow cytometry for both tumor and immune cells and additionally the single cell suspension was seeded into 3-D culture to generate organoids. Finally, organs from tumor bearing animals were analyzed for metastases. With the Vortex platform, we detected CTCs from a majority of our PDOX tumor-bearing mice. The total number of CTCs varied over a wide range between different PDOX tumors. There was a clear heterogeneity in CTCs in terms of CK and Vim expression. In CTCs from one of the PDOX tumors, we detected a small population of CTCs that were either CK+ or Vim+ but the major fraction that was double positive (Vim+ CK+). Probing the bulk tumor from different PDOX models revealed heterogeneity in the levels and number of cells positive for cell surface markers like EpCAM and a difference in the levels of infiltrating myeloid cells (CD11b+). mRNA-seq analyses of individual tumor cells from the bulk tumor belonging to different PDOX models will be described. Additionally, lung and brain metastases were identified. 3D organoid cultures from our PDOX models were successfully grown and their gene expression profiles will be analyzed. In summary, PDOX models of TNBC will help advance our understanding of the molecular basis of this deadly cancer.

#1848

Autocrine IGF1 signaling mediates the survival of pancreatic cancer cells following the ablation of oncogenes.

Nirakar Rajbhandari. _Univ. of Nebraska Medical Ctr., Omaha, NE_.

Pancreatic cancer is an extremely lethal disease, which is mostly associated with the gain-of-function mutation in KRAS gene. Due to a very high frequency of activating mutations within the KRAS gene, mutant KRAS is considered a rational therapeutic target in pancreatic cancer. In line with the existing idea that oncogenic KRAS is required for the maintenance of pancreatic cancer, we show a complete macroscopic regression of both primary and orthotopically transplanted tumors in our ligand-regulatable (Doxycycline) mouse model of KRASG12D-driven pancreatic cancer. This finding indicate that the expression, and provides a strong rationale for the development of KRAS targeted therapy for the treatment of pancreatic cancer.

However, in our current study, despite the complete macroscopic regression of PDACs upon downregulation of oncogenic KRAS, few cancer cells always survived and remained dormant for a protracted duration. These residual cancer cells were responsible for the rapid recurrence upon KRAS reactivation. Using a genome-wide gene expression (RNA-Seq) analysis of in vivo-derived bulk and residual cancer cells followed by biochemical analysis, we identified a significant increase in autocrine IGF-1/AKT signaling in residual cancer cells. We also identified IGF1 signaling as a common survival mechanism of residual cancer cells in c-MYC-driven pancreatic tumor model. Pharmacological inhibition of IGF-1R signaling significantly delayed the tumor recurrence in both the tumor models, suggesting the crucial role of IGF-1R signaling in the survival of cancer cells in the absence of oncogenic drivers. Our findings from animal models were also validated in human pancreatic cancer cell lines harboring KRAS mutations, by demonstrating a compensatory increase in IGF1 signaling in response to a conditional knockdown of KRAS in vitro and in vivo. Collectively, all our findings from mouse model and human cancer cell studies strongly indicate that residual cancer cells can survive in the absence of tumor driving oncogenes by upregulation of autocrine IGF1 signaling loop. Thus, targeting oncogenic drivers along with IGF-1R signaling might be an effective strategy for prevention of tumor recurrence in pancreatic cancer.

#1849

The role of leptin signaling in the promotion of obesity-associated tumorigenesis and cancer stem cell characteristics in a transgenic mouse model of basal-like breast cancer.

Subreen A. Khatib,1 Laura W. Bowers,1 Emily L. Rossi,1 Shannon B. McDonell,1 Claire G. Lineberger,1 David A. Cavazos,2 Linda A. deGraffenried,3 Stephen D. Hursting1. 1 _The University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 2 _The University of Texas Health Science Center at San Antonio, San Antonio, TX;_ 3 _The University of Texas at Austin, Austin, TX_.

Background: Epidemiological studies have linked obesity to a greater risk of breast cancer-specific mortality. This association may be mediated in part by enhanced cancer stem cell (CSC) enrichment, as CSCs are thought to be the primary drivers of tumor initiation, growth, and metastasis. We and others have have demonstrated that obesity promotes CSC enrichment in pre-clinical models of basal-like breast cancer, but only when leptin signaling is present. These findings, coupled with evidence that elevated serum leptin and breast tumor leptin receptor expression are linked to a worse breast cancer prognosis, suggest that leptin-induced CSC enrichment may be a key contributor to obesity-associated breast cancer progression.

Methods: MMTV-Wnt-1 transgenic mice, which develop spontaneous basal-like mammary tumors, were fed a control diet (10% kcal from fat) or a diet-induced obesity regimen (DIO, 60% kcal from fat). Mice were euthanized when tumors reached 1.5 cm in diameter. RNA sequencing was performed on 4 tumors/diet group. Serum hormones and cytokines were measured via luminex-based assay. ALDH activity, a putative CSC marker, was quantified by an ALDH Activity Assay Kit (abcam). Quantitative RT-PCR was used to determine expression of CSC-related genes, leptin, and the leptin receptor. Two mammary tumor cell lines isolated from the MMTV-Wnt-1 model (M-Wnt and E-Wnt cells) and human MDA-MB-231 were exposed in vitro to pooled serum from DIO or control mice (2% concentration), and cell viability, migration, invasion and expression of CSC related genes were measured.

Results: Tumor incidence and growth rate were greater in DIO versus control mice. RNA sequencing revealed an upregulation in a CSC gene signature in tumors from DIO versus control mice. Significant upregulation in several of these genes, including aldh1a, oct4, twist1, twist2, and akt3, was confirmed by qRT-PCR (P < 0.05). ALDH activity in the DIO (relative to control) tumors was also significantly increased (P < 0.05). Serum levels and mammary tissue expression of leptin and tumor expression of the leptin receptor were significantly elevated in DIO versus control mice (P < 0.05). Exposure to serum from DIO mice significantly increased (relative to control) M-Wnt, E-Wnt, and MDA-MB-231 cell viability, migration, invasion, and expression of CSC-related genes in vitro (P < 0.05). The role of leptin signaling in these effects and in vitro measures of CSC enrichment will be explored through additional in experiments.

Conclusions: Obesity promotes MMTV-Wnt-1 mammary tumor incidence and growth rate, possibly via CSC enrichment. Preliminary findings suggest increased leptin signaling may underlie obesity-associated CSC enrichment and may contribute to the breast cancer burden in obese women.

#1850

Development of a NOTCH1 loss of function and PIK3CA mutant transgenic model of HNSCC.

Samantha N. Devenport, Nicole L. Michmerhuizen, Elizabeth Leonard, Brittany Jewell, Jacob Swanson, Sunny Wong, Thomas E. Carey, J Chad Brenner. _University of Michigan, Ann Arbor, MI_.

Head and neck squamous cell carcinoma (HSNCC) is a debilitating disease with a poor 5-year survival rate. The use of in vivo models to study tumorigenesis and to identify potential targeted therapies, especially those that work in combination with immune checkpoint inhibitors, is essential in improving overall patient care. However, the overall number of HNSCC transgenic models with recurrent genetic lesions identified by the HNSCC TCGA project has been limited. For example, TCGA data has shown both Notch1 loss of function mutations and PIK3CA activating mutations as some of the most common in HNSCC, but bi-genic models of these lesions do not yet exist. Here, we have designed a model which utilizes K14-CreERT to drive a bi-genic model with Notch1flox and PIK3CAH1047R mutations in epithelial compartments. Characterization of this line both in the context of spontaneous and carcinogen induced tumor formation is a tool which can be utilized to better understand common mutations in HNSCC. Immunohistochemistry confirms the overexpression of PIK3CA in tamoxifen induced mice. Furthermore, early characterization of this model with chronic treatment of the smoking analogue 4-Nitroquinoline N-oxide (4NQO) in drinking water shows tumor formation on the tongue and surrounding head and neck regions in as little as 12-16 weeks. Hematoxylin and Eosin staining confirmed the squamous tumor formation, while Ki67 staining showed enhanced proliferation of the transformed cells. In the future, this model can be used to study tumor formation over time and in response to targeted inhibitors and common therapies that are being advanced as companion diagnostic strategies for either NOTCH1 deficient or PIK3CA aberrant HNSCC tumors. Further detailed characterization of this model will allow for a deeper understanding of the mechanisms which drive HNSCC and will translate to improved patient care.

#1851

A murine model of von Hippel-Lindau associated retinal hemangioblastoma.

Herui Wang, Qi Zhang, Lijin Dong, Liliana Guedez, Stanley Park, Yujuan Wang, Shida Chen, Chun Gao, Wai T. Wong, Henry Wiley, Emily Chew, Chi-Chao Chan, Zhengping Zhuang. _NIH, Bethesda, MD_.

Purpose: von Hippel-Lindau disease (VHL) is an autosomal dominant condition that features a constellation of cysts or highly vascularized tumors due to loss of heterozygosity (LOH) of the VHL gene, resulting in deregulated hypoxia-induced factors (HIFs). Clinical manifestations of VHL include hemangioblastomas in the central nervous system and retina. To date, no VHL animal model has reproduced retinal capillary hemangioblastomas (RCH), the hallmark lesion of ocular VHL. The RCHs may arise from arrested hemangioblast progenitor cells, with VHL LOH in the tumor stroma. The stem cell leukemia (SCL) gene encodes a basic helix-loop-helix (bHLH) transcription factor, a critical regulator of hematopoiesis and vasculogenesis. We aim to generate a murine model of VHL-associated RCH by conditionally inactivating Vhl in a hemangioblast progenitor population with the angioblast-specific Cre line, (SCL) Cre-ERt2.

Methods: We established a genetic mouse model in which the inducible Vhl gene is specifically "knocked-out" in angioblast cells using the stem cell leukemia (SCL) Cre-ERt2 transgenic mice. Two-week-old SCL-CreERt2;Vhl-F/F mice were administrated tamoxifen (2mg/40g body weight) for 5 consecutive days to activate the Cre recombinase that induces Vhl deletion in angioblast-derived cells. Fundoscopy was done monthly to detect retinal lesions. Fluorescein angiography (FA) was performed on affected mice. All retinas were analyzed by histology at 4 months of age. Genome typing of the Vhl conditional KO allele was conducted in retinal lesions using microdissection, nest-PCR and Sanger sequencing.

Results: About 64% (18/28) of the transgenic mice exhibited various retinal vascular defects following induction. Affected mice demonstrated retinal vascular lesions that were variably associated with prominent vessels, anomalous capillary networks, hemorrhage, exudates, and localized fibrosis. FA revealed vascular leakage from the lesions. Histological analyses showed RCH-like lesions of tortuous, dilated vessels surrounded by "tumorlet" cells, isolated foamy stromal cells, and glia, classically found in RCH. Vhl LOH was detected in the tumor-like area as verified by sequencing.

Conclusion: This is the first demonstration of VHL-associated RCH in a transgenic mouse model. This model may be useful for studying RCH pathogenesis, including HIF-dependent and HIF-independent pathways, and for testing potential therapies.

#1853

Inhibition of mTOR downregulates expression of DNA repair proteins and is highly efficient against BRCA2-mutated breast cancer when combined to PARP inhibition.

Florence Coussy,1 Rania El Botty,1 Rana Hatem,1 Franck Assayag,1 Ahmed Dahmani,1 Marine Huppé,1 Sophie Chateau Joubert,2 Jean -Luc Servely,2 Virginie Bernard,1 Sophie Vacher,1 Berangere Ouine,1 Aurelie Cartier,1 Leanne De Koning,1 Paul Cottu,1 Ivan Bieche,1 Elisabetta Marangoni1. 1 _institut Curie, Paris, France;_ 2 _Ecole nationale veterinaire d' Alfort, Maison Alfort, France_.

Purpose: DNA repair deficiencies and activation of PI3K/AKT/mTOR pathway are common events in breast cancer. BRCA1/2 mutations have been associated with sensitivity to PARP1 inhibitors (synthetic lethality), while alterations in component of the PI3K/AKT/mTOR pathway might confer sensitivity to PI3KCA and mTOR inhibitors. Here we explored the therapeutic benefit of combining a mTOR and a PARP inhibitor in BRCA2-mutated patient-derived xenografts (PDX) with alterations in the PI3K/AKT/mTOR pathway.

Experimental design: the combination of the mTOR inhibitor everolimus and the PARP inhibitor olaparib was tested in two BRCA2-mutated PDX established from a luminal B tamoxifen-resistant and a basal-like breast cancers. Both carried alterations in the PI3K/AKT/mTOR pathway: PIK3R1 mutation and PTEN loss for the luminal B PDX and PTEN loss for the basal-like PDX. To identify putative crosstalk events between mTOR and DNA repair, a Reverse Phase Protein Array (RPPA) analysis of multiple signaling pathways and DNA repair processes was performed on untreated and treated xenografts. Gene and protein expression changes were confirmed by RT-PCR and Western Blot analyses. The capacity to repair DNA damage was measured by P-H2AX immunostaining.

Results: in both PDX, everolimus and olaparib showed marked anti-tumor activity with a growth inhibition comprised between 78% and 86% in the monotherapy setting and 96% in the combination arm, where 100% of mice showed tumor regressions. In the luminal B tumor this combination was more efficient than the combination of everolimus and endocrine therapies (fulvestrant or tamoxifen). In both PDX, the fraction of P-H2AX (marker of unrepaired DNA double-strand breaks) positive cells was increased after everolimus treatment, suggesting a link between mTOR and DNA damage, and strongly increased in the combination setting. RPPA analysis of tumors treated by everolimus alone revealed a marked downregulation of different proteins involved in DNA repair, including FANCD2, RAD50 and SUV39H1, a chromatin compactor factor essential in homologous recombination. In the combination setting, expression of these proteins was almost completely abolished, suggesting convergence of PARP and mTOR in downregulation of DNA repair components.

Conclusions: our results suggest that combining mTOR and DNA repair inhibitors could be a successful strategy to treat a subset of breast cancer with BRCA2 mutation and alterations in the PI3K/AKT/mTOR pathway. Further experiments with mTOR and PARP inhibitors combinations are ongoing in sporadic breast cancer PDX showing a BRCAness phenotype.

#1854

Generation and characterization of mouse model of Pmepa1 conditional knockout in prostate epithelia.

Hua Li, Shashwat Sharad, Talai Barbiev, Yingjie Song, Denise Young, Taduru Sreenath, Albert Dobi, Shiv Srivastava. _USU/Center for Prostate Disease Research (CPDR), Rockville, MD_.

Introduction and Objectives: Prostate cancer (CaP) is the most common non-skin malignancy diagnosed in American men and desfunctions of androgen receptor (AR) plays an essential role in prostate tumorigenesis. PMEPA1 is an androgen and TGF-β -induced gene abundant in prostate, which was identified to degrade AR protein via NEDD4 E3 ligase mediated pathway. Reduced or loss of PMEPA1 expression, commonly detected in prostate tumors, led to increased AR protein and activated AR signaling. PMEPA1 inhibited TGF-β receptor 1 meditated signaling by a negative feedback loop. It was reported that loss of PMEPA1 facilitates bone metastasis of CaP through blocking TGF-β signaling. To further investigating the biological function of PMEP1 gene in CaP tumorigenesis, particularly via AR and TGF-β, we generated Pmepa1 prostate conditional knockout mouse model.

Methods: C57BL/6 mice were utilized for generation of Pmepa1 conditional knockout model. Pmepa1 gene was conditionally deleted in mouse prostate epithelium by ARR2PB-Cre-Lox system. Male mice of genotypes of wild-type, Pmepa1 flox/wild-type-ARR2PB-Cre, Pmepa1 flox/flox-ARR2PB-Cre were euthanized at the age of 3 months for analysis. The prostate tissue was collected for frozen and formalin fixation for histology analysis, and other major organs including heart, lung, liver, spleen, bladder and testis were also collected for control. The tissue were sectioned and stained with Hematoxylin & Erosin (H&E). Total RNA and protein were harvested by homogenizing tissue. The protein levels of Pmepa1, Ar and Nkx3.1 were analyzed by immunohistochemistry (IHC) and immunoblotting, and the transcript levels of these genes were evaluated by in situ hybridization (ISH) and quantitative-PCR (Q-PCR).

Results: The expression of Pmepa1 protein was found to focus on lateral lobe, consistent with Ar protein expression pattern. The data of Q-PCR and ISH showed that transcript level of Pmepa1 was dramatically suppressed in Pmepa1 flox/flox and flox/wild-type ARR2PB-Cre mice. Compared to wild-type ones, Pmepa1 protein was shown significantly decreased in heterozygous and homozygous knockout mice by immunoblotting and IHC staining. And the protein level of Ar was stronger in Pmepa1 conditional deleted mouse. There was no dramatic morphology change in mouse prostate gland by H&E staining.

Conclusions: Conditional deletion of Pmepa1 gene in mouse prostate epithelium led to enhanced Ar protein and activating Ar signaling. The effects of Pmepa1 gene loss in mouse prostate gland on other signaling such as TGF-β and prostate tumorigenesis are needed to be further evaluated.

Funding: This study was supported by CPDR, USUHS, HU0001-10-2-0002 to DGM.

#1855

Developed operation method for patient-drived orthotopic xenograft model of hepatocellular carcinoma.

Hye Rim Byeon, YunSung Seo, Sun-Young Kong, Seung Duk Lee. _National Cancer Center, Goyang-si, Gyeonggi-do, Republic of Korea_.

Patient-drived xenograft(PDX) model is a biological organized platform and a patient substitute. Recent studies use the ectopic model for hepatocellular carcinoma(HCC) but this model don't reflect likewise the tumor state of patient. For exact prognosis, it is need to establish patient-derived orthotopic xenograft(PDOX) mouse model of hepatocellular carcinoma using direct injection technique. Additionally, secretion of growth factor from bone marrow stem cells is promoted after partial hepatectomy. This mechanism is helpful to not only general liver regeneration but also tumorigenesis. Based on former PDX research, we develop the new protocol using direct injection technique. From January 2015 to June 2016, consent about getting tumor pieces to the patient of hepatocellular carcinoma. The pieces is stored in RPMI1640 and moved from operation room to research room immediately. For PDOX and PDX to subcutaneous model, 8~10weeks old NOD SCID and NSG male mouse is used. The tumor pieces cutted to the cube size 1mm. For stop the bleeding, inferior vena cava is blocked using Bulldog Clamp and the cube is directly injected median lobe to 3 places randomly. After clear the Bulldog Clamp, a left lobe is removed using tie and weighted. After 3 months, the PDOX mouse is checked for cancer growth using MRI or PET-CT. Successful PDOX or PDX to subcutaneous model is sacrificed and succeeded a generation to another Balb/C-nude male mouse 8~10weeks old. The rate of success of PDOX model and PDX to subcutaneous model is about 10% respectively. Formed tumor is verified by H&E staining. Based on protocol established, further study plan is required for improving on the rate of success to establish PDOX model and clinical application. 

### Imaging for Cancer Diagnosis and Image-Guided Therapy

#1856

**Dual-modality immunoPET/fluorescence imaging of prostate cancer utilizing** 89 **Zr- or** 124 **I-Cy5.5-anti-PSCA cys-minibody.**

Wen-Ting K. Tsai, Kirstin A. Zettlitz, Richard Tavaré, Felix B. Salazar, Robert E. Reiter, Anna M. Wu. _University of California Los Angeles, Los Angeles, CA_.

Prostate cancer patients can benefit from non-invasive and more accurate diagnosis, as well as improved visualization during surgery. ImmunoPET can provide information on location and extent of the disease, while fluorescent image-guided surgery can distinguish cancerous tissue during resection. Prostate Stem Cell Antigen (PSCA) is upregulated in the majority of prostate cancers and metastases and is therefore a promising target for imaging. Engineered antibody fragments, such as the minibody, exhibit ideal imaging characteristics due to fast blood clearance for high target-to-background images at short imaging times post-injection. A dual-labeled minibody can reveal the whole-body PSCA-expressing tumor burden by PET, followed by intraoperative visualization of margins by fluorescence.

The fully humanized anti-PSCA A11 minibody was engineered with a C-terminal cys-tag (A11 cMb) for site-specific labeling by thiol-directed chemistry. In order to radiolabel with 89Zr, a metal chelator desferrioxamine (DFO) was site-specifically conjugated to A11 cMb by maleimide chemistry, while direct iodination was used to label with 124I. Singly labeled 89Zr- and 124I-A11 cMb successfully imaged subcutaneous PSCA (+) and (-) 22Rv1 human prostate adenocarcinoma tumors in nude mice, resulting in positive-to-negative tumor ratios of 2.5:1 and 8:1, respectively. Both 89Zr-A11 cMb and 124I-A11 cMb cleared from blood by 22 hours. As expected for the residualizing radiometal, 89Zr-A11 cMb resulted in retention in the organs of clearance (liver and kidneys).

For dual-modality imaging, A11 cMb was site-specifically conjugated with Cy5.5-maleimide and radiolabeled with 124I. PET imaging with 124I-Cy5.5-A11 cMb in the subcutaneous 22Rv1 tumor model resulted in a positive-to-negative tumor ratio of 13:1. The PSCA (+) tumors were subsequently visualized by fluorescence in situ and ex vivo with high contrast in comparison to PSCA (-) tumors and tissues. In an orthotopic model, PSCA (+) 22Rv1 cells were implanted in the prostate, and therefore 89Zr was used in order reduce interfering signal from clearance to the bladder. A11 cMb was site-specifically conjugated with Cy5.5 and randomly labeled with SCN-DFO, then radiolabeled with 89Zr. 89Zr-Cy5.5-A11 cMb successfully imaged the prostate tumor, resulting in a 3:1 tumor-to-blood ratio. Fluorescence imaging clearly distinguished prostate tumor from adjacent tissues including seminal vesicles. In conclusion, a single injection of the dual-labeled A11 cMb can visualize tumor burden by immunoPET and fluorescence imaging. This humanized probe has the potential for clinical translation for primary and metastatic prostate cancer detection and surgical guidance that can ultimately enhance treatment success.

#1857

**Early detection of pancreatic intraepithelial neoplasms (PanINs) in transgenic mouse model by hyperpolarized** 13 **C metabolic magnetic resonance imaging.**

Prasanta Dutta, Yu Zhang, Michelle Zoltan, Marilina Mascaro, Erick Riquelme, Jaehyuk Lee, Anirban Maitra, Florencia McAllister, Pratip Bhattacharya. _UT MD Anderson Cancer Ctr., Houston, TX_.

Pancreatic cancer, one of the most lethal solid tumors, is an aggressive disease that develops relatively symptom-free. Pre-invasive pancreatic intraepithelial neoplasias (PanINs) have been identified as precursor lesions to pancreatic cancer. While there is growing evidence supporting PanIN's genetic links to pancreatic cancer, there is no non-invasive method to detect them. There is an unmet need of novel strategies for detection of pancreatic cancer at the earliest stages, preferably at the stage of PanINs. Hyperpolarized magnetic resonance imaging (HP-MRI) has shown potential for early detection of cancers and monitoring therapeutic efficacy. Here we have employed in-vivo 13C pyruvate metabolic imaging and ex-vivo nuclear magnetic resonance (1H-NMR) metabolomics to identify and understand metabolic changes, to enable detection of early PanINs and its progression to advanced PanINs and pancreatic cancer. Genetically engineered mouse (GEM) models with progression of PanIN lesions and control animals, with no pancreatic lesions, were employed in this study. Hyperpolarization of pyruvate and in-vivo 13C MRS were performed using Hypersense (Oxford Instruments) and 7T MRI scanner with a dual tuned 1H/13C volume coil respectively. Tissue alanine and lactate concentrations were determined using a Bruker 500 MHz NMR spectrometer coupled with cryo-probe. Histology and immunohistochemistry were performed on excised tissue samples. P48Cre mice were used as controls and P48Cre;LSLKrasG12D mice were used for detection of early and advanced PanINs, which usually develop in these mice between 12 and 20 months. The imaging experiments were performed at the different stages of the disease. Progression of disease from tissue containing predominantly low-grade PanINs to tissue with high-grade PanINs showed a decreasing alanine/lactate concentration ratio. Real time in-vivo 13C MRS was used to measure non-invasively changes of alanine and lactate metabolites with disease progression and in control mice, following injection of hyperpolarized pyruvate. The alanine-to-lactate (Ala/Lac) signal intensity ratio was found decreased as the disease progressed from low-grade PanINs to high-grade PanINs. These results demonstrate that there are significant alterations of alanine transaminase (ALT) and lactate dehydrogenase (LDH) activities which favor the transformation of aggressive pancreatic cancer from PanINs lesion. Our results suggest that real-time conversion kinetic rate constants kPA(pyruvate-to-alanine) and kPL(pyruvate-to-lactate) can be used as metabolic imaging biomarkers for assessing the early stage of pancreatic diseases. Findings from this highly promising HP-MRI technique could be rapidly translated to the clinic for early detection of pancreatic cancer in patients at high risk for developing the disease.

#1858

Theranostic nanoparticles for detection and treatment of pancreatic cancer.

Phillip Chuong, Neal Bhutiani, Lacey R. McNally. _Univ. of Louisville Brown Cancer Ctr., Louisville, KY_.

Background: Despite continued efforts to improve early detection and treatment of pancreatic adenocarcinoma, the disease still carries a poor prognosis. The combination of multispectral optoacoustic tomography (MSOT) and theranostic nanoparticles offers a possible solution by providing tumor targeting using fluorescent dyes and treatment through drug delivery. We describe a 40 nm colloidal mesoporous silica nanoparticle (CMS) with vermiform pores and a chitosan coating and tagged with the V7 peptide to provide pH-specific dual targeting to deliver a contrast agent in a tumor-specific fashion.

Methods: CMSs were synthesized using a modified Stober method, coated with chitosan, and conjugated with a V7 peptide, resulting in a dual acidic pH targeting system. Particle characterization was performed using transmission electron microscopy (TEM), UV-vis spectrophotometry, Zeta potential, and dynamic light scattering . To determine acidic pH specificity of V7-CMS, Panc1 and S2VP10 cell lines were incubated in cell culture medium at either pH 7.4 or 6.6 followed by treatment with V7-CMS. Particle uptake was determined using near infrared (NIR) fluorescence and tissue phantoms. Finally, for in vivo testing, the same CMSNs were injected into mice bearing S2VP10 pancreatic tumors. MSOT imaging was performed 8 hours after CMS injection.

Results: TEM images demonstrated successful synthesis of approximately 40 nm V7-CMSs with vermiform pores. On NIR imaging, V7-CMS demonstrated acidic pH specificity in both S2VP10 and Panc 1 cells at pH 6.6, with particle localization and signal intensity 8-fold and 5-fold higher, respectively, than that observed at pH 7.4. In tissue phantoms, increased pH specificity was also observed in both S2VP10 and Panc 1 cells at pH 6.6. Particle location and signal intensity on MSOT was 20-fold and 4-fold higher, respectively, than that observed at pH 7.4. In the murine orthotopic S2VP10 pancreatic tumor model, MSOT imaging 8 hours after IV CMS injection demonstrated V7-CMS accumulation specifically within pancreatic tumors.

Conclusion: The acidic pH specific dual targeting system using chitosan-coated and pHLIP V7-tagged CMSs results in tumor specificity. Preferential binding and dye release was increased as much as 20-fold at pH 6.6 as compared to pH 7.4 in tissue phantoms. MSOT was able to detect tumor specific accumulation of V7-CMS in vivo. These small, pH specific particles hold significant promise for effectively identifying pancreatic tumors with high sensitivity and specificity and for targeted drug delivery.

#1859

Hsp90 targeted near infrared molecular imaging to detect mammografically occult invasive lobular breast cancer.

Takuya Osada, Kensuke Kaneko, Zachary Hartman, Amy Hobeika, Philip Hughes, Timothy Haystead, Michael Morse, H. Kim Lyerly. _Duke University, Durham, NC_.

Background: Early diagnosis of Invasive lobular carcinomas (ILCs) is clinically challenging due to its histopathologic features making it difficult to detect using mammography. Because of the diffuse infiltration of ILC cells into the surrounding stroma, ILC is also associated with a higher incidence of positive resection margins after breast-conserving surgery. Therefore, there is a significant unmet need for improved imaging for early detection, clinical staging, and possibly intraoperative imaging to assess surgical margins. We propose to detect mammografically occult ILC by the in vivo detecting of malignant signaling pathways activated in ILC. Heat shock protein 90 (Hsp90) comprises 1-3% of the total cellular protein in most cells and acts as a molecular chaperone for more than 200 reported client proteins. Recently, we developed a series of tethered Hsp90 inhibitors that specifically target a tumor specific form of Hsp90 associated with poor outcomes. Using near infrared (nIR) probe-tethered Hsp90 inhibitors, we demonstrated that Hsp90 is actively re-internalized and can be used to image murine and human breast cancer in vitro and in vivo.

Methods and Results: We tested the imaging efficacy of the nIR-tethered Hsp90 inhibitor, HS196, in ILC models in vitro and in vivo. An inactive structural analog HS199 was used as a control to monitor for non-specific (non-Hsp90 dependent) uptake. Two ILC cell lines, MDA-MB-134 VI and SUM44-PE, and one patient-derived ILC xenograft, HCI-013 EI, were tested. nIR signals of HS196 in vitro and in vivo were detected by Odyssey (LI-COR) and Pearl Trilogy (LI-COR)/SPY ELITE imager (Novadaq), respectively. High uptake of HS196 was observed in vitro by both cell lines (> 1 µM), while the control HS199 resulted in weaker nIR signals in these cells. In vivo imaging efficacy of HS196 was tested using HCI-013 EI xenograft in SCID-beige mice. After injection of 10 nmol compounds via tail vein, quick and stronger accumulation and longer retention of the HS196 in the ILC xenograft was observed, while HS199 showed significantly weaker accumulation and faster clearance (by 24 h after injection). Significant uptake of HS196 by HCI-013 EI tumors was confirmed by ex vivo imaging of tumors 24 h after compound injection. Histological and flow cytometry analysis of HCI-013 EI tumors showed strong nIR signals in tumor cells.

Conclusions: The non-radioactive, nIR imaging strategy, using a novel nIR-Hsp90 inhibitor compound, HS196, was effective in the non-invasive imaging of ILC tumors. This finding suggests that new molecular imaging techniques, not dependent on micro calcifications or architectural distortion, may be a novel strategy to met the need of ILC patients to detect, clinically stage and assess margins during surgical resection. Ongoing pre-clinical models will be employed and first in human testing of this approach in planned.

#1860

**Ligand-conjugated superparamagnetic biosynthesized magnetite nanoparticles for specific targeting via** in vivo **imaging of triple negative breast cancer.**

John David Obayemi,1 Jingjie Hu,1 Vanessa Uzonwanne,1 Karen Malatesta,1 Winston Soboyejo2. 1 _Princeton University, Princeton, NJ;_ 2 _Worcester Polytechnic Institute, Worcester, NJ_.

This paper presents ligand-conjugated biosynthesized magnetite nanoparticles (BMNPs) that serve as molecular magnetic resonance imaging (MRI) contrast agents for specific targeting of cell-surface receptors on triple negative breast cancer cells. Superparamagnetic BMNPs were functionalized with Luteinizing Hormone Releasing Hormone (LHRH), a molecular recognition units (MRUs) that have been shown to have receptors that are overexpressed on MDA MB 231 cells. Early and metastatic tumors from triple negative breast cancer xenografts were targeted in vivo in a nude mouse model in the presence of both ligand-conjugated and unconjugated superparamagnetic BMNPs. A combination of transmission electron microscopy (TEM), Atomic Force Microscopy (AFM) and spectrophotometric analysis was used to investigate the specific accumulation of the functionalized superparamagnetic BMNPs tumor specific sites. The results obtained compliment the in vitro experiment that showed enhancement of MRI contrast by ligand-conjugated BMNPs. At a concentration of ~ 20mg/kg of both nanoparticles, the ligand-conjugated BMNPs reveal specific attachment to tumor tissue as well as metastases in the lungs. However, most of the unconjugated BMNPs were found to accumulate in the liver, spleen and kidney which showed their non-specificity. The implications of the results obtained is for the specific targeting and early detection of triple negative breast cancer.

#1861

LHRH-conjugated magnetite nanoparticles for breast cancer detection and contrast enhancement in MRI.

Jingjie Hu,1 John Obayemi,1 Karen Malatesta,1 Derek Adler,2 Sean Wang,2 Edward Yurkow,2 Winston Soboyejo3. 1 _Princeton University, Princeton, NJ;_ 2 _Rutgers University, Piscataway, NJ;_ 3 _Worcester Polytechnic Institute, Worcester, MA_.

Breast cancer is one of the most frequently occurring types of cancer in women. Furthermore, at the time of initial diagnosis, some tumors may have metastasized. There is, therefore, a critical need for novel procedures for the early detection of breast tumors and their metasteses. One of the specific targeting receptors for human breast cancer cells is Luteinizing Hormone Releasing Hormone (LHRH). Approximately 52% of human breast cancers express binding sties for LHRH. Within this context, LHRH-functionalized nanoparticles have been considered for potential applications in cancer detection and treatment. In this study, we present results of experimental and theoretical studies of the entry of Luteinizing Hormone Releasing Hormone (LHRH)-conjugated Magnetite Nanoparticles (MNPs) into MDA-MB-231 breast cancer cells. The internalization of LHRH-conjugated MNPs into breast cells is studied using confocal fluorescence microscopy. The receptor-mediated entry of nanoparticles into breast cancer cells is also elucidated using a combination of thermodynamics and kinetics models. The trends in predicted nanoparticle entry times and the size range of the engulfed nanoparticles are shown to be consistent with the experimental observations. The specific interaction between LHRH receptors and LHRH can be used to enhance the specific targeting of breast cancer cells. Furthermore, LHRH-MNPs demonstrated specific enhancement for in-vivo signal in magnetic resonance imaging (MRI) for breast tumor sites and their metastases in the body. Lastly, nanoparticle concentration in different organs is presented to show that the conjugated MNPs are accumulated mainly into the tumor sites and their metastasis. While unconjugated MNPs are shown to accumulate in the liver, kidney, spleen and heart. The current results show that LHRH-conjugated magnetite nanoparticles have the potential for the specific targeting and detection/treatment of breast cancer.

#1862

Direct observation of colorectal cancers using field-emission scanning electron microscopy with a thin polymer membrane, the NanoSuit.

Hirotoshi Kikuchi, Tomohiro Matsumoto, Takanori Hiraide, Yusuke Ozaki, Amane Hirotsu, Tomohiro Murakami, Toshiki Kawabata, Yoshihiro Hiramatsu, Kinji Kamiya, Takanori Sakaguchi, Yasuharu Takaku, Isao Ohta, Takahiro Hariyama, Hiroyuki Konno. _Hamamatsu Univ. School of Medicine, Hamamatsu, Japan_.

Background: Field-emission scanning electron microscopy (FE-SEM) enables us to observe nano-sized objects with great depth of focus and high resolution. However, the observation of biological samples including colorectal tissues using an FE-SEM has been difficult because it requires to evacuate its inside to prevent electron scattering, therefore all organisms containing ca. 70% water are rapidly evaporated and consequently caused structural disruption and collapse. To overcome the limitations of the conventional SEM, equipment such as low-vacuum scanning electron microscopy and environmental scanning electron microscope was developed. However, they are not reliable enough to investigate living organisms or wet tissues with high resolution level. We have recently reported that a simple surface modification consisting of a thin extra layer, coined the term 'NanoSuit®', can keep organisms alive in the high vacuum (10−3 to 10−7 Pa) of the SEM. We now modified the technique and developed a new solution, which enables FE-SEM observations of wet tissues. In this study, we utilized this technique to observe real images of colorectal cancers and their adjacent normal mucosae at high resolution.

Materials and methods: Colorectal cancer tissues and their adjacent normal mucosa were cut with a scalpel from specimens surgically resected. All patients enrolled in this study provided written informed consent. Observations were carried out with an FE-SEM (S-4800, Hitachi or JEM-7100F, JEOL, Japan) at acceleration voltage of 1.0 kV. The vacuum level of the observation chamber was 10-3 \- 10-7 Pa. The newly developed surface shield enhancer (SSE) solution was used to make NanoSuit® for wet tissue observation. To form the NanoSuit®, the specimens were dipped for 1 min into the SSE solution and blotted briefly thereafter put on dry filter paper to remove excess solution. Specimens were then introduced directly into an FE-SEM to form a NanoSuit® following irradiation of the electron beam.

Results: Fine structures of intestinal crypt and villi were observed in normal colon mucosa using FE-SEM with a NanoSuit®, whereas fixed specimens prepared with conventional method showed obvious structural damage. Comparing with the region of normal colon mucosa, colorectal cancer lesion had relatively amorphous surface, therefore the border between non-cancerous mucosa and cancer lesions in colon tissues are able to be distinguished under high magnification. In addition, fiber-like structure was observed at the border between noncancerous mucosa and cancer lesions, suggesting an invasive front of colorectal cancer.

Conclusions: We successfully observed the real mucosal surfaces and cancer lesions of colorectum with high resolution by an FE-SEM using a newly developed vacuum-proof suit, the "NanoSuit®". This novel technique will enable us to investigate further physiopathology of GI tract including cancers.

#1863

Non-invasive imaging of colitis using multispectral optoacoustic tomography.

Neal Bhutiani,1 William Grizzle,2 Susan Galandiuk,1 Dennis Otali,2 Gerald Dryden,1 Nejat Egilmez,1 Lacey R. Mcnally1. 1 _Univ. of Louisville Brown Cancer Ctr., Louisville, KY;_ 2 _UAB, Birmingham, AL_.

Background: Currently, several non-invasive modalities, including MRI and PET, are being investigated to identify early intestinal inflammation, longitudinally monitor disease status, or detect dysplastic changes in patients with inflammatory bowel disease (IBD). Currently, multispectral optoacoustic tomography (MSOT; often in combination with ultrasound) has been used to effectively image tumor xenografts as well as several orthotopic tumor models, including pancreatic adenocarcinoma, but not to specifically assess inflammatory or dysplastic changes in the bowel in murine models. Here, we assess the applicability and utility of (MSOT) in evaluating the presence and severity of colitis.

Methods: Nine 6-7 week old C57B/6 mice underwent antibiotic depletion of gastrointestinal flora before inoculation with enterotoxic Bacteroides fragilis to induce colitis. Mice were then anesthetized, depilated, imaged from their superior thorax to inferior pelvis using MSOT. Mice were evaluated prior to bacterial inoculation, 2 days after inoculation, and 7 days after inoculation. MSOT values for oxygenated and deoxygenated hemoglobin were determined using MSOT imaging software and compared using linear regression. At each timepoint, three mice underwent colonoscopy prior to euthanasia and colon processing for histology.

Results: Mice with bacterially-induced colitis demonstrated a temporally-associated increase in mesenteric and colonic vascularity, with an increase in mean signal intensity of oxygenated hemoglobin (1.150 vs. 2.716 MSOT a.u. compared to controls; p=0.004) by MSOT two days after inoculation. These findings were significantly more prominent 7 days after inoculation, with increased mean signal intensity of oxygenated hemoglobin (1.150 vs. 2.716 vs. 3.422 MSOT a.u. for controls vs. 2 days post-ETBF vs. 7 days post-ETBF, p=0.0002) and the development of punctate vascular lesions on the colonic surface. Compared to untreated controls, mice at 2 days and 7 days post ETBF inoculation demonstrated an increased colitis score on colonoscopy (1.5 vs. 2.5 vs. 5.5), which correlated well with MSOT findings of mean oxygenated hemoglobin signal intensity (r=0.82, p=0.013). These findings were also associated with inflammatory changes observed on histologic analysis.

Conclusions: MSOT represents a non-invasive diagnostic modality that effectively identifies colitis in a murine model. With improvements in depth of tissue penetration, MSOT may hold potential as a sensitive, accurate, non-invasive imaging tool in evaluation of both disease status and early detection of malignancy in patients with IBD.

#1864

Feasibility of magnetic relaxometry for early ovarian cancer detection: preliminary evaluation of sensitivity and specificity in cell culture and in mice.

Kelsey Mathieu, Zhen Lu, Hailing Yang, Lan Pang, Adam Kulp, John Hazle, Robert C. Bast. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Introduction: Most ovarian cancers are diagnosed in a late, incurable stage, which has prompted efforts towards earlier detection and more effective screening strategies. To be considered effective, screening must provide sufficient sensitivity and specificity to impact patient mortality while minimizing false positives. Magnetic relaxometry (MRX), which detects binding of targeted iron oxide nanoparticles (NPs) to cancer cells, offers the promise of improved sensitivity and specificity over conventional early detection modalities.

Methods: We investigated the sensitivity and specificity of MRX by scanning ovarian cancer cell samples containing 105, 106, and 107 cells incubated with a fixed amount (57 µg Fe3O4) of anti-HER2 antibody-conjugated, PEG-coated NPs or unconjugated, PEG-coated NPs (Senior Scientific LLC). To further evaluate specificity, we used cell lines with both high and low expression of HER2 (SKOV3 and HEY, respectively). To assess MRX under in vivo conditions, we subcutaneously injected 105, 106, and 107 anti-HER2 NP-labeled SKOV3 cells into nude mice (n = 9) and immediately performed MRX scanning. Prior to performing this study, we verified successful antibody-NP conjugation through an ELISA assay, which confirmed the presence of anti-HER2 antibody in NP pellets. Additionally, we performed flow cytometry to confirm a high level of specific binding between SKOV3 cells and anti-HER2-conjugated NPs.

Results: Our in vitro data revealed strong linearity between cell number and MRX signal for both SKOV3 and HEY cells incubated with anti-HER2 NPs (R2 = 0.99 and 1, respectively). Furthermore, there was little to no MRX signal for all cell samples incubated with unconjugated, PEG NPs regardless of cell number. The highest MRX signal was observed when 107 SKOV3 cells were incubated with anti-HER2 NPs, which was 2.1 ± 0.3 and 15.7 ± 1.4 times higher than when 107 Hey cells were incubated with anti-HER2 NPs or when 107 SKOV3 cells were incubated with unconjugated PEG NPs, respectively; significantly higher MRX signals (relative to the controls; p < .01) were also noted for samples containing 106 SKOV3 cells incubated with anti-HER2 NPs. When scanning live mice injected with 107 anti-HER2 NP-labeled SKOV3 cells, the MRX signal was significantly higher than the signal from the mice prior to the injection (p < .001). Additionally, MRX signal versus cell number in the injected mice was highly correlated (r = 0.99) with the MRX data from the corresponding cell sample scans.

Conclusion: MRX is sufficiently sensitive to detect 1 million cells in culture or 10 million cells in mice with a high level of specificity. Sensitivity may be improved by using nanoparticles coated with antibodies against antigens that are overexpressed by a larger fraction of ovarian cancers and will be the focus of future work.

#1865

Enhancing the in vivo detection of cancer by manipulating magnetic fields applied to tumor targeting superparamagnetic iron oxide nanoparticles.

Todor Karaulanov, Erika C. Vreeland, Andrew Gomez, Kayla E. Minser, Caroline L. Weldon, William H. Anderson, Christopher Nettles, Giulio Paciotti. _Imagion Biosystems, Albuquerque, NM_.

Superparamagnetic Relaxometry (SPMR) is combination technology for the early detection of cancer. Conceptually, PEGylated superparamagnetic iron oxide (Fe3O4) nanoparticles (NPs) coupled with a tumor targeting monoclonal antibody are systemically administered and target solid tumors by both passive (EPR) and active (receptor targeting) mechanisms. Once bound to the target cells, the NPs are magnetized by a brief, low field magnetic pulse to create macroscopic magnetization. Once the field is removed, the particle's magnetization decays and is measurable by superconducting quantum interference device (SQUID) detectors. The pattern of decay, known as Néel relaxation, exhibits a difference in latency specific to bound nanoparticles and differs from the Brownian decay exhibited by free/unbound particles. As a proof of concept, we developed PEGylated NPs that are covalently bound to an anti-Her-2 monoclonal antibody. In vitro, the nanoconstruct exhibits specific binding to the Her-2 overexpressing BT-474 breast cancer cells, with little to no binding to Her-2 negative MCF-7cells. Similar patterns of selective targeting were observed in vivo.

To improve the specific detection of SPMR signals in tumors we developed a novel system that selectively enhances the magnetic signal at tumor sites and reduces the contribution of similar signals at off target sites such as the liver and spleen. For these studies, we created phantoms of varying strength to mimic tumor and non-specific signals. We observed that by creating non-homogenous excitation magnetic field patterns, we reduced the contribution of the non-specific signals by an order of magnitude, effectively increasing the signal to noise ratio of the tumor signal. These results are clinically relevant and support the use of SPMR in the detection of small tumors in cancer patients.

#1866

**Ultrahigh resolution optical coherence microscopy for label-free imaging of human cervical lesions** ex vivo **.**

Xianxu Zeng,1 Canyu Li,2 Xiaofang Wang,1 Jason Jerwick,2 Tao Xu,2 Yuan Ning,2 Linlin Zhang,1 Yutao Ma,3 Xiaoan Zhang,1 Chao Zhou2. 1 _Zhengzhou University, Zhengzhou, China;_ 2 _Lehigh University, Bethlehem, PA;_ 3 _Wuhan University, Wuhan, China_.

Cervical cancer remains the fourth most common cause of cancer and the fourth leading cause of cancer death in women worldwide. In contrast to the decreasing prevalence in developed countries, incidence of cervical cancer remains high in developing countries, such as China, India and Chile. Effective screening of cervical cancer is crucial for early diagnosis of the disease. Ultrahigh resolution optical coherence microscopy (UHR OCM) is an emerging label free imaging technique that can be used to obtain three dimensional (3D) "optical biopsy" of biological samples with cellular resolution up to 2 mm beneath the tissue surface. The goal of this study is to assess the diagnostic value of UHR OCM for human cervical precancer and cancer screening and identify characteristic features in UHR OCM images that correlate with the gold standard histopathology. Six hundred and fifty eight (658) fresh cervical tissues from cervical biopsy, conization and panhysterectomy were obtained from 254 consenting volunteers. Multiple 3D UHR OCM images were obtained from each specimen before the sample was processed for standard histopathology evaluation. Of the 658 samples imaged, 474 had normal cervix or benign lesions, 110 had low grade squamous intraepithelial lesion (LSIL), 57 had high grade squamous intraepithelial lesions (HSIL) and 17 had invasive cancer. UHR OCM images were compared to histopathology slides obtained from the same specimens in order to identify diagnostic image features. Distinctive patterns for squamous epithelium, columnar epithelium, basement membrane, cervical inflammation and erosion, koilocytic cells, low grade and high grade squamous intraepithelial lesions and cervical cancer were clearly observed. Our results lay the foundation for future non-invasive optical evaluation of cervical tissue in vivo. This could lead to a less invasive and more effective screening strategy for cervical cancer based on OCM guided biopsies.

#1867

Near infrared fluorescent imaging of brain tumor with IR780 dye incorporated phospholipid nanoparticles.

Shihong Li,1 Jennifer Johnson,1 Anderson Peck,1 Qian Xie2. 1 _Van Andel Research Institute, Grand Rapids, MI;_ 2 _East Tennessee State University, Johnson City, TN_.

Background: Near-IR fluorescence (NIRF) imaging is becoming a promising approach in preclinical tumor detection and clinical image-guided oncological surgery. While heptamethine cyanine dye IR780 has excellent tumor targeting and imaging potential, its hydrophobic properties limit its clinical use. In this study, we developed nanoparticle formulations to facilitate the use of IR780 for fluorescent imaging of malignant brain tumor.

Methods: Self-assembled IR780-liposomes and IR780-phospholipid micelles were prepared and their NIRF properties were characterized. The intracellular accumulation of IR780-nanoparticles in glioma cells were determined using confocal microscopy. The in vivo brain tumor targeting and NIRF imaging capacity of IR780-nanoparticles were evaluated using U87MG glioma ectopic and orthotopic xenograft models and a spontaneous glioma mouse model driven by RAS/RTK activation.

Results: The loading of IR780 into liposomes or phospholipid micelles was efficient. The particle diameter of IR780-liposomes and IR780-phospholipid micelles were 95 nm and 26 nm, respectively. While stock solutions of each preparation were maintained at ready-to-use condition, the IR780-phospholipid micelles were more stable. In tissue culture cells, IR780-nanoparticles prepared by either method accumulated in mitochondria, however, in animals the IR780-phospholipid micelles showed enhanced intra-tumoral accumulation in U87MG ectopic tumors. Moreover, IR780-phospholipid micelles also showed preferred intracranial tumor accumulation and potent NIRF signal intensity in glioma orthotopic models at a real-time, non-invasive manner.

Conclusion: The IR780-phospholipid micelles demonstrated tumor-specific NIRF imaging capacity in glioma preclinical mouse models, providing great potential for clinical imaging and image-guided surgery of brain tumors.

#1868

Molecular imaging of human esophageal cancer for early detection.

Xibo Ma, Hui Meng, Jie Tian. _Institute of Automation, Chinese Academy of Sciences, Beijing, China_.

Esophageal cancer was the sixth common cancer for incidence and ranks fourth for death rate in China, and squamous cell carcinoma is the main histological type. It is usually in an advanced stage at the time of diagnosis, resulting in an overall 5-year survival rate of <15%. Early detection can lead to an improvement in patient outcomes but is limited by nonspecific contrast mechanisms. As all we know, antibody and peptides establish a biological basis for image contrast, which could achieve sufficient target-to-background ratio for in vivo detection. In this paper, an EGFR antibody (Cetuximab) conjugated with an NIR dye (NIRDye800) was used for early detection of esophageal cancer. We demonstrated our technology could identify esophageal cancer in small size (<1 mm) and high-grade dysplasia from low-grade dysplasia. And 4.2-fold greater fluorescence intensity for esophageal cancer compared with surrounding normal esophageal tissues. No toxicity was observed in our experiments. The result indicated that this targeted, antibody-based imaging agents have great potential in early detection of esophageal cancer and other epidermal cancer.

#1869

**Improved image-guided surgical resection of glioblastoma with [** 18 **F]-fluoroethyltyrosine Cerenkov luminescence imaging.**

David Y. Lewis,1 Richard Mair,1 Alan J. Wright,1 Kieren Allinson,2 Scott K. Lyons,1 Tom Booth,1 Robert Bielik,1 Dmitry Soloviev,1 Kevin M. Brindle1. 1 _University of Cambridge, Cambridge, United Kingdom;_ 2 _Addenbrooke's Hospital, Cambridge, United Kingdom_.

The ability of a surgeon to completely resect a tumor is directly related to outcome in high- and low-grade gliomas. However current intraoperative navigational tools are useful only in a subset of glioma patients and do not detect the full extent of disease.

We modelled orthotopic brain tumors by stereotactically implanting human U87, rat F98 and C6 glioblastoma cells into the forebrains of rats. We show here that a new hybrid PET-Cerenkov luminescence imaging approach using O-(2-[18F]fluoroethyl)-L-tyrosine (FET) can accurately delineate tumor margins pre- and intra-operatively. We demonstrate consistency in the successful localisation and quantification of tumor burden using PET and Cerenkov luminescence imaging. The Cerenkov signal in individual tumors was directly proportional to the signal detected in corresponding FET PET scans (y = 1.06x - 0.01; R2 = 0.98; p < 0.0001) and subsequent autoradiography indicated equivalence between modalities. Cerenkov luminescence was better able to discriminate tumor from healthy brain tissue than the current 'gold standard' for intraoperative mapping, 5-ALA (5-aminolevulinic acid), indicated by a greater area under the ROC curve in human xenograft (0.968 ± 0.003 vs. 0.893 ± 0.019; p = 0.003) and syngeneic rat glioma models (0.970 ± 0.010 vs. 0.774 ± 0.046; p = 0.006). The quantitative accuracy of Cerenkov luminescence enabled us to determine a threshold of 2.1 x 102 p-1 sec-1 cm-2 sr-1 MBq-1 that separated U87 human glioblastoma and normal brain tissue, more precisely guiding tumor excision than 5-ALA. At the optimal threshold the specificity for detecting syngeneic rat and human xenograft gliomas was better with FET Cerenkov than 5-ALA (91.1 ± 2.7 % vs. 67.2 ± 5.2 %; p = 0.007 and 91.2 ± 1.0 % vs. 82.9 ± 1.8 %; p = 0.005 respectively). We confirmed and extended these findings at higher spatial resolution on cryosections using FET autoradiography and 5-ALA confocal microscopy, with FET demonstrating higher specificity and sensitivity for tumor detection than 5-ALA.

FET PET-Cerenkov luminescence imaging has the potential for guiding resections in a much broader range of glioma patients than current approaches. Previous clinical experience with FET offers a facile route for clinical translation of this technology.

#1870

Fluorescence-guided soft tissue sarcoma surgery using a cetuximab-imaging probe conjugate.

Nicole K. Behnke, Jason M. Warram, Andrew C. Prince. _The University of Alabama at Birmingham, Birmingham, AL_.

BACKGROUND: Soft tissue sarcomas (STS) are a heterogeneous group of solid malignancies whose treatment includes margin-negative resection. Large tumor size and anatomic constraints make margin assessment challenging. Fluorescence-guided surgical resection can help delineate intraoperative margins; preclinical studies demonstrate improved oncologic outcomes in other malignancies using cancer-specific imaging probes. Recent literature describes cathepsin activated probes selective for STS, but no studies using disease-specific chemotherapeutic agents conjugated to imaging probes. This novel strategy has potential to decrease unnecessary healthy tissue resection and improve oncologic outcomes by reducing margin-positive resections.

Epidermal growth factor receptor (EGFR) is overexpressed in multiple subtypes of STS and is a potential target for fluorescence-guided surgery. Recent studies show fluorescently labeled cetuximab, an FDA-approved, anti-EGFR antibody, to be safe and useful for margin assessment in other malignancies. Our aim was to evaluate its potential for STS, examining tumor-targeting specificity of two drug-probe conjugates, and comparing them to the described cathepsin-activated probes. We hypothesize the drug-probe conjugates would successfully target STS, with superior tumor specificity. We also aimed to determine the smallest tumor detectable by our conjugate.

METHODS: Athymic nude mice with subcutaneous HT1080 fibrosarcoma tumors were injected with one of five probes: IRDye800CW fluorescent probe conjugated to either cetuximab (anti-EGFR) or DC101 (anti-VEGFR2), IRDye800CW conjugated to an isotype control (IgG), or a cathepsin-activated probe (IntegriSense 750 and Prosense 750). Fluorescence imaging was performed daily with open- and closed-field systems. Tumor-to-background ratios (TBR), signal washout times and normalized signal averages were evaluated. On day 9, smallest resectable game evaluation was performed to assess sensitivity for detecting residual post-resection tumor

RESULTS: At day 9 post-injection, the TBR of the cetuximab-IRDye800CW group (11.1) was significantly greater than Integrisense750 (6.88, p=0.005), the IgG-IRDye800CW control (4.44, p=0.00005), Prosense750 (2.35, p=0.00009), and DC101-IRDye800CW (1.87, p=0.00003). During in vivo imaging, cetuximab-IRDye800CW outperformed all other agents by several folds of contrast enhancement. The smallest resectable game evaluation demonstrated 1mm3 fragment detection using the cetuximab-IRDye800CW probe.

CONCLUSIONS: This study demonstrates superiority of cetuximab-IRDye800CW for disease-specific imaging in a subcutaneous animal model of STS. The novel strategy of coupling improved margin-negative surgical resection with established chemotherapy has considerable translational significance and is an avenue for exploration with other drugs used to treat STS.

#1872

Fluorescence-guided surgery in the tumor microenvironment in a syngeneic mouse model of EL-4 lymphoma.

Kosuke Hasegawa,1 Atsushi Suetsugu,1 Miki Nakamura,1 Takuro Matsumoto,1 Takahiro Kunisada,1 Masahito Shimizu,1 Shigetoyo Saji,1 Hisataka Moriwaki,1 Michael Bouvet,2 Robert M. Hoffman2. 1 _Gifu Univ. Graduate School of Medicine, Gifu, Japan;_ 2 _University of California, San Diego, CA_.

We report fluorescence-guided surgery (FGS) of the tumor microenvironment of the EL-4-RFP lymphoma cells growing subcutaneously in C57/BL6 GFP transgenic mice. Using a portable hand-held Dino-Lite digital imaging system, subcutaneous tumors were resected by FGS. The tumor surface contained both RFP cancer cells and GFP stromal cells. The tumor microenvironment was clearly visualized and resected with the Dino-Lite. Host stromal cells, including adipocyte-like cells and blood vessels with lymphocytes, were observed along the tumor by confocal microscopy in addition to cancer cells by color-coded confocal imaging. Stromal cells were within the tumor and that cancer cells also invaded the TME. Color-coded FGS, which distinguished cancer and stroma, significantly prevented local recurrence, which bright-light surgery or single-color FGS could not.

#1873

Intraoperative assessment of breast tumor margins using multimodal photoacoustic tomography (MarginPAT).

Kyle McElyea,1 George Sandusky,1 Rui Li,2 Lu Lan,2 Ji-Xin Cheng,2 Linda K. Han,3 Pu Wang4. 1 _Indiana University School of Medicine, Indianapolis, IN;_ 2 _Purdue University, West Lafayette, IN;_ 3 _Indiana University Health Simon Cancer Center, Indianapolis, IN;_ 4 _Vibronix Inc., West Lafayette, IN_.

Radical mastectomies are progressively becoming a surgery of the past. Women today are increasingly opting for lumpectomies, a less invasive treatment option. Clinical data has shown no difference in survival or clinical outcomes between the two surgical groups in early stages of breast cancer. There is, however, an undesired outcome yet to be adequately addressed. Lumpectomies have, in some cases, failed to remove marginal malignant tumor cells left as a product from surgery.

Following surgery, tumor biopsies are analyzed for marginal tumor cells. Biopsies are cut in, fixed, processed in a tissue processor, embedded into a paraffin block, and stained with H&E. After processing, presence of marginal cancerous tissue is determined by a pathologist. This process takes 3-5 days, ultimately requiring the patient to undergo reoperation if a positive margin is discovered. Presently, the reoperation rate is 20-30%.

A device capable of imaging removed tissue to determine remaining marginal tumor during surgery would greatly reduce the reoperation rate. Multiple intraoperative imaging tools existing or are emerging for breast tumor margin assessment. Current devices fail to meet acceptable clinical specifications due to either long procedure time (15 mins+), or low sensitivity (~70%), and low specificity (~68%). An unmet need exists in developing an intraoperative margin assessment device that is rapid, sensitive, capable of measuring the entire tissue surface, and images a depth of 2mm+.

The MarginPAT device presents a multi-modal photoacoustic/ultrasound imaging system for rapid and highly sensitive breast cancer margin assessment. After surgical removal of tumor, tissue is inserted, via cartridge, into MarginPAT for imaging. An automatic scan (<3 mins) provides 3D images of the excised tissue, providing margin status. Such immediate feedback allows the surgeon to re-operate immediately or allow the patient to return home based on the marginal results from MarginPAT.

Our preliminary study on 40 patient samples, showed 93% sensitivity and 90% specificity in margin assessment by precise localization of adipose tissue using PA imaging and further RF spectrum analysis of ultrasound signaling. Other than the superior sensitivity and specificity, the unique features of MarginPAT include deep tissue sensing (>3 mm) and high surface scanning speed (20 cm2/per min).

These achieved specifications meet the needs of intraoperative margin assessment, which are expected to surpass similar platforms. More importantly, MarginPAT supports conventional ultrasound imaging, allowing for use in ultrasound-guided wire localization. These multimodality functions allow MarginPAT to be utilized in multiple fields of breast cancer diseases, another advantage over other platforms. In conclusion, current clinical results show through both verification and validation that MarginPAT competently performs in the operating room.

#1874

First demonstration that hypoxia targeting with radiation improves tumor control in a mammal tumor model.

Howard J. Halpern, Boris Epe;, Matthew C. Maggio, Martyna Krzykawska-Serda, Gage H. Redler, Richard C. Miller, Eugene D. Barth, Ralph R. Weichselbaum. _University of Chicago, Chicago,, IL_.

For over a century, resistance of radiation to living tissues has been associated with hypoxia, a local lack of molecular oxygen, or low pO2. The focus of this research has been to validate the hypothesis that has been assumed, but not proven over the past century, that radiation treatment focused specifically on hypoxic regions of tumors would improve tumor curability. This research used Electron Paramagnetic Resonance (EPR) imaging to provide absolute pO2 images in volume elements or voxels of murine tumors with 1 torr pO2 resolution and 0.7 mm spatial resolution in FSa fibrosarcomas in the legs of C3H mice. We define hypoxic regions of tumors as those whose image subvolumes or voxels have low pO2, voxels less than 10 torr. We have established EPR pO2 imaging as a reliable locator of relevant radiobiologically relevant hypoxia. To determine if pO2 based dose painting improves tumor cure, we implemented the XRAD225Cx system to deliver gantry based x-ray treatments to mouse tumors accurately registered with EPR pO2 images. All tumors were pretreated with a dose of radiation that had been determined in separate experiments to cure 30% of tumors, a TCD30. Conclusive failure to demonstrate a difference between treating crude spherical volumes including ~ 85% of hypoxic voxels with an additional curative dose compared with treatment with shells of radiation avoiding hypoxia (anti-boosts) proved tenets of radiation biology: a few surviving hypoxic clonogens cause radiation treatment failure. In response, we implemented rapid 3D printing Tungsten loaded, highly conformal plastic blocks to compare treating ~100% of hypoxic tumor voxels with hypoxia avoidance, treating similar volumes of well oxygenated tumor. Only then did we observe significant (p<0.01) tumor control differences between hypoxic boosts and anti-boosts. This is the first validation of the curative effectiveness of focusing hypoxia based dose painting in mammalian tumors.

#1875

**[** 18 **F]HX4 shows potential as a predictive biomarker for the radiosensitizing capacities of metformin in a NSCLC xenograft model.**

Sven De Bruycker,1 Christel Vangestel,2 Tim Van den Wyngaert,1 Steven Deleye,1 Dominique Vanderghinste,2 Steven Staelens,1 Sigrid Stroobants1. 1 _University of Antwerp, Wilrijk, Belgium;_ 2 _Antwerp University Hospital, Edegem, Belgium_.

Introduction

Metformin (MET) may improve tumor oxygenation and thus radiotherapy (RT) response. However, appropriate imaging biomarkers for patient stratification, critically needed to be able to advance to clinical trials with MET as a radiosensitizer, are still lacking. In this proof-of-concept (POC) study, we first assessed the effect of acute MET administration on NSCLC xenograft tumor hypoxia with PET using the novel hypoxia PET tracer [18F]HX4. Second, we verified the influence of a single dose of MET prior to RT on treatment outcome.

Experimental procedures

A549 tumor-bearing mice (n=9; animals were inoculated in both hind legs) underwent a [18F]HX4 PET/CT scan to determine baseline tumor hypoxia. The next day, mice were administered 100 mg/kg MET IV. [18F]HX4 was administered IV 30 min. later, whereupon a second PET/CT scan was performed to assess changes in tumor hypoxia. Two days after this second scan, mice were randomized into three groups (n=3/group): a control group (1), a RT group (2), and a MET+RT group (3) with comparable tumor volumes (259±103; 218±81 and 247±121 mm3, resp.). Animals were administered 0.9% saline (groups 1-2) or 100 mg/kg MET (group 3) IV, followed by a single dose of 10 Gy 30 min. after administration (groups 2-3). Control animals of group 1 underwent sham RT. During the entire study, tumor growth was monitored triweekly by caliper measurements. Calculation of the relative tumor volumes (RTV) was initiated when tumors reached a mean baseline volume of 100 mm3 with the formula RTV=Vtime x/Vbaseline. Tumor growth inhibition (TGI) was calculated for each tumor in both treatment groups with the formula TGI=1-(RTVtreated/RTVcontrol).

Results

MET significantly altered A549 tumor hypoxia, as the mean [18F]HX4 tumor-to-blood ratio (TBR) was reduced from 3.03±0.27 to 2.82±0.25 (p=0.040) after MET administration. The tempering influence of MET on tumor hypoxia improved RT response, as fifteen days after irradiation TGI was 63±7% in the MET+RT group as compared to only 36±9% in the RT group, however significance was not reached. To date, tumors are still being measured triweekly and this will be continued until a volume of 1500 mm3 is reached. Then, animals will be sacrificed for histological validations on tumor tissue to assess differences in a.o. proliferation and hypoxia.

Conclusions

Using non-invasive imaging, we showed in A549 xenograft tumors that MET acts as a radiosensitizer possibly by decreasing tumor hypoxia. Our results imply that [18F]HX4 PET imaging holds potential as a predictive biomarker for the beneficial effect of MET in the treatment of NSCLC; however, the small group sizes in this POC study did not allow us to draw sound conclusions. These promising findings will be validated in a larger follow-up study in order to validate [18F]HX4 PET as a predictive biomarker for MET and RT response.

#1875A

Preclinical proof of concept for the first Nanocyclix TKI-PET radiotracer targeting activated EGFR positive lung tumors.

Philippe Genne,1 Cyril Berthet,1 Olivier Raguin,1 Sylvie Chalon,2 Xavier Tizon,1 Sophie Serriere,2 Peggy Provent,1 Marc Hillairet de Boisferon,1 Johnny Vercouillie,3 Céline Mothes,4 Fabienne Gourand,5 Louisa BARRE,5 Denis Guilloteau,2 Pétra Blom,1 Jan Hoflack1. 1 _Oncodesign S.A., Dijon Cedex, France;_ 2 _INSERM, Tours, France;_ 3 _INSERM / CERRP, Tours, France;_ 4 _Cyclopharma, Saint-Beauzire, France;_ 5 _CEA, LDM-TEP, Caen, France_.

Background: IMAkinib® program is an innovative approach, based on Nanocyclix® chemistry technology, which aims to develop new Tyrosine Kinase Inhibitors (TKIs) radiotracers used for Positron Emission Tomography (PET). The epidermal growth factor receptor (EGFR) is an established target for the treatment of advanced non-small cell lung cancer (NSCLC). Gefitinib (Iressa®), erlotinib (Tarceva®) and afatinib (Giotrif®), TKIs targeting EGFR have already been approved for treatment of advanced non-small cell lung cancer (NSCLC). Unfortunately, the majority of patients develop a resistance to the TKI, which is for most of them (> 50%) related to an acquired T790M mutation of EGFR. TKI PET-imaging can provide a tool to determine and predict the activity of EGFR and the responsiveness to EGFR TKI.

Methods: New compound ODS2004436 was characterized in vitro for its activity and metabolism. After radiolabeling with [18F], the compound was evaluated in vivo in three clinically relevant lung cancer cell lines (NCI-H441 – EGFR_wild-type (WT); NCI-H3255 - EGFR_L858R; NCI-H1975 – EGFR_L858R/T790M) xenografted in nude rats.

Results: The biochemical and cellular profiles were comparable to gefitinib on EGFR_WT and activated EGFR_L858R mutant. The cellular activity was in the nanomolar range (5 ± 2 nM) on NCI-H3255 and weak on NCI-H441 (42 ± 27 µM) suggesting that our compound might inhibit to a lesser extent non activated WT-EGFR. We observed a better biochemical inhibition (254 nM vs 1350 nM) and cellular activity on NCI-H1975, the EGFR double mutant (4.8 ± 0.7 µM vs 18 ± 8.7 µM) of ODS2004436 compared to gefitinib. These data suggest that our compound is suitable to bind in vivo activated EGFR. In vitro binding experiments showed that [18F]-ODS2004436 specifically bound to patient tumor samples expressing EGFR. The in vivo experiments in rats showed that [18F]-ODS2004436 was rapidly cleared from the blood. Nevertheless the tumor uptake is stable overtime (up to 180 min) with a mean tumor/muscle ratio > 4 in NCI-H3255 and > 2 in NCI-H1975 at 90 min instead no specific binding was observed in the WT tumor, NCI-H441. Moreover, we observed a good correlation between the radiotracer uptake in the tumors and pEGFR immunostaining, suggesting that [18F]-ODS2004436 is a good biomarker of activated EGFR, regardless of the mutation.

Conclusions: Based on this study, we proved in vivo that [18F]-ODS2004436 binds selectively to activated EGFR, and is a good candidate to evaluate the EGFR activity in NLCSC using PET imaging. Clinical evaluation of this novel radiotracer is ongoing (first in-man phase 0/I clinical trial NCT02847377).

### Migration and Invasion 2

#1876

Cell cycle-dependent front polarized cell migration requires Aurora kinase A.

Tony Lok Heng Chu, Lixin Zhou, Jennifer Won, Pooja Mohan, Oksana Nemirovsky, Abbas Fotovati, Torsten Nielsen, Nelly Pante, Christopher Maxwell. _University of British Columbia, Richmond, British Columbia, Canada_.

PROBLEM- Metastasis accounts for 90% of deaths from carcinomas.

BACKGROUND- To initiate migration, epithelial cells must front-rear polarize and reorganize the microtubule cytoskeleton directed toward the lamillipodia or leading edge. During mitosis, centrosome maturation, duplication, spindle pole assembly and chromosome segregation require Aurora kinase A (AURKA), which is optimally active when complexed with TPX2. Accumulating evidences identify non-mitotic functions for AURKA, such as cell migration and ciliary reorganization. Overexpression of AURKA leads to tumorigenesis in breast cancer. Mechanisms that activate non-mitotic AURKA and promote cell migration as well as metastasis are yet to be uncovered.

RESULTS- In a large breast cancer tissue microarray (n= 3,175), the abundance of phosphorylated RHAMM (a substrate of AURKA) predicted overall and relapse-free survival in ER-negative, basal-subtype, and triple negative breast tumors. We studied the AURKA-TPX2-RHAMM signaling axis in mammary (MCF10A and nMUMG-FUCCI) and HeLa cells. In scratch wound assays, migratory cells were found to have front-polarized centrosomes, which correlated with G2 cell cycle phase, phosphorylated AURKA and elevated microtubule nucleation at centrosomes. Small molecule inhibition of Aurora kinase activity impaired centrosome polarity and retarded the kinetics of cell migration. In parallel, silencing RHAMM altered TPX2 location as indicated by immunofluorescence and immuno-EM, which located TPX2 to the nuclear envelope and nuclear pore basket. Nuclear import of recombinant TPX2 was attenuated in RHAMM silenced cells and nuclear envelope accumulation was elevated.

CONCLUSION- The AURKA-TPX2-RHAMM axis regulates centrosome polarity, microtubule nucleation and directional cell migration while phosphorylated RHAMM predicts survival in aggressive forms of breast cancer.

#1877

Coordination of Rac1 and Cdc42 by centrosomal microtubules in focal adhesion dynamics and directed cell migration.

Jean-Cheng Kuo. _National Yang-Ming University, Taipei, Taiwan_.

Cancer metastasis causes the increase of mortality in most cancer patients, and abnormal cell migration contributes to this disease progression. To control cell migration, the dynamical control of a protein complex within focal adhesions has to be regulated appropriately. It is known that focal adhesions can be regulated dynamically by microtubules. To examine the effects of centrosomal microtubules and non-centrosomal microtubules in regulating focal adhesion dynamics and directed cell migration, we first generated centrosome-deficient cells. We find that centrosomal microtubules and non-centrosomal microtubules control focal adhesion dynamics through modulating focal adhesion composition, which dynamically control the activity of small GTPases Rac1 and Cdc42. Deficiency of centrosomal microtubules significantly disrupts the balance of Rac1 and Cdc42, thereby inhibiting directed cell migration. This study reveals the effects of centrosome in focal adhesions, and indicates an important role for Rac1 and Cdc42 activation that controls actin polymerization, cell polarization and directed cell migration.

#1878

PDZ-RhoGEF functions as a critical signaling effector for TROY.

Zonghui Ding,1 Alison Roos,1 Jean Kloss,1 Harshil Dhruv,2 Marc Symons,3 Nhan L. Tran,1 Joseph C. Loftus1. 1 _Mayo Clinic Arizona, Scottsdale, AZ;_ 2 _Translational Genomics Research Institute, Phoenix, AZ;_ 3 _The Feinstein Institute for Medical Research at Northwell Health, Manhasset, NY_.

Glioblastoma Multiforme (GBM) is the most common tumor of the CNS with a median survival of approximately 15 months. The highly aggressive invasion of malignant cells into the surrounding normal brain tissue renders complete surgical resection impossible, increases therapeutic resistance, and virtually assures tumor recurrence. We have previously demonstrated that TROY (TNFRSF19) has an important role in GBM invasion and resistance. TROY expression is correlated with glial tumor grade and inversely correlated with patient survival. Increased TROY expression stimulated cell migration/invasion in vitro and in vivo and confers resistance to both ionizing radiation- and temozolomide-induced apoptosis via activation of Akt and NF-κB. These data indicate that TROY stimulated migration/invasion is associated with signaling pathways that also increase survival and therapeutic resistance, however, the mechanistic basis of this signaling remains unclear. To investigate the mechanism by which TROY induces GBM cell invasion, we performed immunoprecipitation of the TROY receptor coupled with MALDI-TOF and MS/MS analysis and identified PDZ-RhoGEF as a binding partner for TROY. We validated the interaction of TROY with PDZ-RhoGEF by immunoprecipitation and western blotting, and demonstrated that PDZ-RhoGEF can exchange for both RhoA and RhoC in glioma cells and is implicated in GBM cell invasion, proliferation, and survival. In order to assess the specific role of PDZ-RhoGEF in TROY signaling, we examined the effects of PDZ-RhoGEF on TROY-mediated activation of NF-κB. While overexpression of TROY alone strongly stimulated NF-κB transcriptional activity in serum-free conditions, increased expression of PDZ-RhoGEF alone had no effect on NF-κB activity. Co-transfection of PDZ-RhoGEF with TROY synergistically promoted NF-κB activation. Mutation of the TRAF binding domain (TBD) in the cytoplasmic domain of TROY abolished its capacity to activate NF-κB, even in the presence of PDZ-RhoGEF, suggesting that the TBD is important for TROY induced NF-κB activation. Furthermore, knockdown of PDZ-RhoGEF by shRNA attenuated NF-κB activation in TROY expressing cells, blocked TROY mediated invasion of primary GBM xenograft cells and increased sensitivity to temozolomide. Collectively, these data indicate that PDZ-RhoGEF plays an important role in TROY signaling and provides insights into a potential node of vulnerability to limit GBM cell invasion and decrease therapeutic resistance.

#1879

Transcriptional regulation of KAI1 by p73 in colorectal cancer metastasis.

Woo Kyun Bae,1 Kyung Hyun Yoo,2 Keunsoo Kang,3 Lothar Hennighausen,4 Ik-Joo Chung1. 1 _CNUHH, Jeonnam, Republic of Korea;_ 2 _Sookmyung Women's University, Seoul, Republic of Korea;_ 3 _Dankook University, Choenan, Republic of Korea;_ 4 _NIH, Bethesda, MD_.

Background: p73 is a member of the p53 family transcription factors and known tumor suppressor. However, its transcriptional activity is complex owing to its multiple isoforms, and limited information is available regarding the transcriptional and functional regulation of the p73 gene in colorectal cancer. In this study, we examined the role of p73 in colorectal cancer.

Methods: We established a TAp73 overexpressing cell line using the Tet-on inducible expression system and analyzed global RNA expression by RNA-sequencing (RNA-seq). We studied the effects of TAp73 expression on the migration, invasiveness, and EMT of colon cancer cells. We conducted experiments in animals with liver metastasis of colorectal cancer. To investigate the roles of p73 in colon cancer, we examined the samples from patients, who had undergone surgical resection for colorectal cancer between 2009 and 2013. mRNA expression of TAp73 and KAI1 was analyzed in 108 colorectal cancer specimen and adjacent non-cancerous tissues by real-time RT-PCR.

Results: RNA-seq showed the association between TAp73 and KAI1 expression. Migration and invasiveness of colon cancer cells were reduced by TAp73 expression, but induced by KAI1 knockdown. TAp73 regulates KAI1 expression in colon cancer cell lines. TAp73-induced decrease in invasiveness and epithelial-mesenchymal transition (EMT) was abrogated by KAI1 knockdown in TAp73 overexpressing cells. Migration ability of TAp73 overexpressing cells was recovered by KAI1 knockdown. Hepatic metastases were significantly reduced in mice injected with TAp73 overexpressing cells and increased in those injected with TAp73 overexpressing cells with KAI1 knockdown. mRNA expression of TAp73 and KAI1 was higher in colorectal cancer tissues than in adjacent non-cancerous tissues. Significant correlation between TAp73 and KAI1 mRNA expression was detected in early stage primary colorectal cancer, but not in the advanced stage.

Conclusions: This study suggests that p73 (TAp73) acts as a tumor suppressor in the progression of colorectal cancer and regulates the expression of KAI1, which is a key target of p73 in colorectal cancer liver metastases.

#1880

Bulk lung cancer cell migration is more common than single cell migration and regulated by different genes.

Helmut H. Popper, Sylvia Eidenhammer. _Medical Univ. of Graz, Graz, Austria_.

Introduction: There are two different forms how tumor cells migrate: single cell or small cell cluster movement as it is seen in small cell carcinoma, and movement by large clusters of organized cells as seen most often in adenocarcinoma or squamous cell carcinoma. This latter migration form is not understood. Drosophila border cells forming wings migrate in large cell complexes similar to what is seen in the carcinomas and the genes identified in these cells might regulate the same process in carcinomas. Methods: 30 cases of pulmonary squamous cell and adenocarcinomas were selected based on the identification of large tumor cell clusters visible within the stroma as well as in blood vessels. Immunohistochemistry was done for receptor of activated C kinase (Rack1), brinker (brk), mother against dpp (mad), and saxophone (sax), proteins shown to be responsible for bulk cell movement in drosophila development. In addition immunohistochemistry was also done for Src-kinase, Twist, Snail, and TGFβ1 Tyrosine kinase substrate 5 (Tks5), E-cadherin, SARI (suppressor of AP-1), and vimentin, all known to be associated with epithelial-to-mesenchymal transition (EMT) and formation of invadopodia. Results and Conclusion: Most well differentiated pulmonary carcinomas migrate in large cell clusters, for example acinar adenocarcinomas form nicely structured acini deep within the stroma and even within blood vessels. These carcinomas do not undergo classical EMT. Proteins expressed by the four genes (Rack1, brk, mad, and sax) associated with border cell movement in drosophila could also be identified in pulmonary carcinomas and might coordinate bulk cell movement. Other members of TGF-beta signaling cascade were identified. Inhibition of Src by Rack1 may be important for border cell migration and cluster cohesion maintenance. Proteins usually seen in single tumor cell migration, such as vimentin (a sign of EMT) could not be proven in the tumor cell clusters. Findings of this study show that similar mechanisms are working in pulmonary carcinomas and that bulk cell migration is probably another way of metastasis. Further investigation using cell culture system and genetically designed adenocarcinomas expressing these genes should prove our findings and contribute in understanding migration of carcinoma bulk complexes.

#1881

STIM1 and Orai1 control non-Hodgkin lymphoma cells migration.

Simon Latour,1 Isabelle Mahouche,1 Floriane Cherrier,2 Jean-Philippe Merlio,2 Sandrine Poglio,2 Laurence Bresson Bepoldin1. 1 _INSERM U1218 ACTION, Bordeaux, France;_ 2 _INSERM UMR 1053 BaRITOn, Bordeaux, France_.

Non-Hodgkin lymphoma is one of the most common cancer in United States representing 4% of all cancer. Diffuse large B cell lymphoma (DLBCL) is the most common and aggressive non-Hodgkin B lymphoma. One of the characteristic features of this disease is the dissemination of the cancer cells, through the lymphatic system in the secondary lymphoid organs and extranodal sites, leading to the death of patients. Chemokines such as the Stromal Derived Factor 1 (SDF1) control the spread and the homing of the cancer cells. It is known that SDF1 activates various signalling pathways involved in cell proliferation, transcription or migration. Moreover, SDF1 induces an increase in intracellular calcium concentration whose role is still unknown in B cells. Store-operated Ca2+ entry (SOCE) is a major Ca2+ influx pathway in this type of cells. By definition, SOCE is activated by Ca2+ release from the endoplasmic reticulum. Two genes are responsible for SOCE activity: Stromal interaction molecule 1 (STIM1), an ER Ca2+ sensor that detects store depletion and ORAI1, the pore-forming subunit of Ca2+ release-activated Ca2+ (CRAC) channel. Several studies performed on adherent cells showed that Orai1/STIM1 proteins are involved in cancer cell migration. However, the molecular mechanisms involved in cell migration differ widely between adherent and non-adherent cells. We studied the role of both actors of calcium entry : Orai1 and STIM1 in DLBCL pathology and more precisely in basal and SDF1-induced of DLBCL cell migration. Using Tissue MicroArrays approach we revealed that both Orai1 and STIM1 are under-expressed in DLBCL tumoral tissue compared to normal lymphoid tissue. Next, using calcium imaging experiments we confirmed that SDF-1 triggered Ca2+ responses in two DLBCL cell lines (SUDHL4 and HLY1) involving intracellular Ca2+ store mobilization and extracellular Ca2+ influx. Based on these observations, we investigated the role of Orai1 and STIM1 on SDF1-induced Ca2+ influx using pharmacological and RNA interference approaches. The inhibition of Orai1 by BTP2 as well as the under-expression of Orai1 or STIM1 by shRNA, prevented Ca2+ influx induced by SDF1 suggesting the involvement of Orai1 and STIM1 in this process. Regarding this results , we studied the role of Orai1 and STIM1 on DLBCL cell migration in vitro and in vivo. Our results show that basal or SDF1-induced cell migration was significantly inhibited by underexpression of STIM1 or Orai1 in SUDHL4 and HLY1 cell lines. These results suggest that STIM1 and Orai1 play a key role in the DLBCL cell migration. The identification of STIM1 and Orai1 proteins as key players in the migration of DLBCL cells might provide new therapeutic targets for the treatment of this pathology.

#1882

Tankyrase-binding protein TNKS1BP1 regulates cancer cell invasion.

Tomokazu Ohishi,1 Haruka Yoshida,1 Masamichi Katori,2 Toshiro Migita,1 Yukiko Muramatsu,1 Mao Miyake,1 Yuichi Ishikawa,2 Akio Saiura,3 Shun-ichiro Iemura,4 Tohru Natsume,4 Hiroyuki Seimiya1. 1 _JFCR Cancer Chemotherapy Center, Tokyo, Japan;_ 2 _JFCR Cancer Institute, Tokyo, Japan;_ 3 _JFCR Cancer Institute Hospital, Tokyo, Japan;_ 4 _National Institutes of Advanced Industrial Science and Technology, Tokyo, Japan_.

Tankyrase, a member of poly(ADP-ribose) polymerase (PARP) family, catalyzes addition of long PAR chains onto the acceptor proteins, such as TRF1, a telomere-binding protein, and Axin, a repressor of Wnt/β-catenin signaling. Tankyrase-mediated PARylation of TRF1 and Axin represses the biological functions of these proteins, leading to enhanced telomere elongation by telomerase and upregulation of Wnt/β-catenin signaling, respectively. Thus, tankyrase is a multifunctional protein that supports oncogenic properties of cancer cells. Given that tankyrase has a large protein-protein interaction platform called ANK repeat clusters, and is broadly distributed to various intracellular loci, including telomeres, nucleoplasm, nuclear pore complexes, cytoplasm, Golgi, and spindle poles, this protein seems to have a yet unidentified function. In the present study, we explored the function of a novel tankyrase-binding protein called TNKS1BP1 and elucidated its contribution to regulating cancer cell invasion. Since TNKS1BP1 co-localizes with the actin filaments, we first examined the effect of genetic manipulation of TNKS1BP1 expression on cell invasion. We found that TNKS1BP1 depletion by small interfering RNAs increased the invasive capacity of human fibrosarcoma cells, whereas its overexpression decreased the cell invasion. In TNKS1BP1-depleted cells, actin filament dynamics, focal adhesion and lamellipodia ruffling were increased with activation of the ROCK-LIMK-cofilin pathway, which promotes cell motility. Importantly, tankyrase overexpression reproduced the phenotype of TNKS1BP1-depleted cells and enhanced cell invasion in a PARP activity-dependent manner. Under approval by Institutional Review Board of Japanese Foundation for Cancer Research, we performed immunohistochemical analysis of clinical pancreatic cancer. We found that TNKS1BP1 protein expression is decreased in invasive regions of the tumors. Taken together, these observations suggest that the tankyrase-TNKS1BP1 axis constitutes a post-translational modulator of cell invasion and its aberration promotes cancer malignancy.

#1883

Fibrillar 1D tumor microenvironment is the key driver associating high-speed tumor cell motility with nuclear shape in breast cancer.

Ved P. Sharma,1 James Williams,2 Edison Leung,1 Joseph Sanders,2 Robert Eddy,1 James Castracane,2 John Condeelis1. 1 _Albert Einstein College of Medicine, Bronx, NY;_ 2 _SUNY Polytechnic Institute, Albany, NY_.

Unlike normal breast stroma, which contains curly collagen fibrils, aligned collagen fibers oriented perpendicular to blood vessels are seen in both human breast tumors and mouse models of breast cancer. These linear collagen fibers provide "highways" for tumor cells to migrate toward blood vessels in a directional migration mode known as tumor cell streaming. Streaming is characterized by tumor cell migration at high speed and directional persistence on 1D collagen fibers. Previous studies utilizing linear ECM substrates have shown that tumor cells adopt elongated morphology and display increased speeds on linear 1D substrates compared to their 2D motility. However, the relationship between 1D geometry of ECM fibers in breast tumor microenvironment and the underlying mechanotransduction mechanism regulating high-speed migration of tumor cells is not well understood. Here, we analyzed in vivo ECM architecture by SHG intravital imaging and found a narrow peak of fiber diameters falling in the range 2-3 µm. These fibers were composed of collagen I and fibronectin. Based on these findings, we developed a high fidelity in vitro nanofiber system to study the molecular mechanisms underlying tumor cell streaming migration. Breast carcinoma cells plated on 2 µm ECM-coated fibers showed enhanced motility matching in vivo velocities averaging 1.2 µm/min. We varied 1D fiber diameter (0.7-20 µm) and found that tumor cells move the fastest with highest persistence on smaller fibers within a narrow range of diameters from 0.7-3 µm. High tumor cell speeds correlated with enhanced alignments of F-actin and focal adhesions along the fiber axial dimension. Unexpectedly, we also observed nuclear deformation during carcinoma cell migration on narrow fibers in vitro, similar to nuclear deformation observed in vivo. This was a surprising finding because nuclear deformation in vivo was assumed to be caused by squeezing through ECM pores. Thus, we hypothesized that actomyosin forces not only regulate cell motility parameters, but also nuclear deformation independently of ECM pore size. To test this hypothesis, we disrupted the transmission of cytoskeletal forces to the nucleus by knocking down LINC complex proteins - SUN1 and SUN2, and found increased nuclear elongation and cell motility parameters, through the upregulation of actin polymerization. These results indicate that in carcinoma cells, F-actin associated forces are shared between the leading edge (to maintain cell speed) and the nucleus (to dynamically regulate nuclear shape). LINC complex disruption releases F-actin forces acting on the nucleus to the cell front, leading to higher tumor cell motility speeds. In summary, our results provide new insights into the interplay between actomyosin contractility and the LINC complex in the regulation of nuclear shape and high-speed tumor cell motility during carcinoma cell metastasis.

#1884

The breast cancer gene CCDC170 regulates the Golgi-associated microtubule network and directional cell migration.

Pengtao Jiang, Yueran Li, Andrey Poleshko, Valentina Medvedeva, Natalia Baulina, Yongchao Zhang, Yan Zhou, Carolyn M. Slater, Trinity Pellegrin, Jason Wasserman, Michael Lindy, Mary Daly, Richard A. Katz, Xiaowei Chen. _Fox Chase Cancer Ctr., Philadelphia, PA_.

Background: Breast cancer (BCa) is a genetically heterogeneous disease and many genes contributing to BCa risk remain to be identified. Genome-Wide Association Studies (GWAS) and subsequent fine-mapping studies (>50) have strongly implicated genetic alterations at the CCDC170/C6ORF97-ESR1 locus (6q25.1) as being associated with the risk of BCa. ESR1, encoding the estrogen receptor, might be a more obvious candidate for influencing risk. Surprisingly, our analysis using genome-wide differential allele-specific expression (DASE), an indicator for BCa susceptibility, suggested that the genetic alterations of CCDC170, but not ESR1, accounts for GWAS-associated BCa risk at this locus. CCDC170 is a coiled-coiled domain-containing protein of poorly understood function. BCa-specific truncation and missense mutations in CCDC170 also have been detected, with the truncations being implicated in driving Luminal B subtype BCa. Together these findings demonstrate that the CCDC170 gene is involved in BCa, but the underlying molecular mechanisms for its role in tumorigenesis are unknown.

Experimental designs and results: By using the approaches of confocal microscopy and cell imaging analysis, here we report for the first time that CCDC170 is associated with the Golgi apparatus and perinuclear microtubules (MTs), and support a role for CCDC170 in the Golgi-associated microtubule network. We have shown that overexpression of CCDC170 triggers Golgi reorganization and stabilizes Golgi-associated MTs, accompanied by dramatically increased acetylation of α-tubulin that is driven by the acetyltransferase ATAT1. The Golgi-associated MT network has been proposed to regulate cell polarity and migration. In support of this concept, we have shown that CRISPR knockout increases, and overexpression of CCDC170 decreases, BCa directional cell migration in vitro. We also found that the BCa-specific truncations result in mislocalization of CCDC170 and/or diminished stability of Golgi-associated MTs. Lastly, we identified candidate CCDC170 functional binding partners (e.g. MAP4) that are consistent with its localization and proposed function. These partners may serve to mediate the acetylation and stabilization of MTs.

Conclusions: Taken together, our findings demonstrate that CCDC170 plays an essential role in Golgi-associated MT organization and stabilization, and provide a mechanism for how perturbations in CCDC170 could alter Golgi-mediated cell polarity, and thereby drive BCa and other abnormalities. This work was partially supported by the Susan G. Komen for the Cure (KG100274), NCI (CA186853), and Eileen Stein Jacoby Fund.

#1885

VEGFA/NRP1 signal contributes to filopodia formation in breast cancer cells.

Marina Kiso, Sunao Tanaka, Masakazu Toi, Fumiaki Sato. _Kyoto Univ. Graduate School of Medicine, Kyoto, Japan_.

Background and objective: Bevacizumab is an antibody of VEGF and suppresses tumor angiogenesis. Bevacizumab has exhibited some therapeutic efficacy for metastatic breast cancer in combination with chemotherapy, but its impact on overall survival has not been proved. VEGF is a multi-function molecule targeting cell members in tumor microenvironment. We aimed to reveal VEGF-related molecular mechanisms on breast cancer cells itself.

Methods and Results: VEGF-expressing breast cancer cell line, MDA-MB-231 cells (231 cells) were used. VEGFA of 231 cell was knocked out using Crisper-Cas9 system (VEGFA-KO). VEGFA-KO 231 cells showed small and rounded morphology. Migration assay demonstrated the impaired cell migration of VEGFA-KO 231 cells. Bevacizumab treatment did not induce this morphologic change and impaired migration. Exogenous VEGF addition to VEGF-KO 231 cells recovered WT 231 cell like morphology and induced cell migration. Next, we generated soluble neuropilin-1 (sNRP1) overexpressed 231 cells. sNRP1 traps VEGFA to function as an antagonist. sNRP1-231 cells exhibited small and rounded morphology and impaired cell migration similar to that of VEGFA-KO 231 cells. Knock down of NRP1 by using siRNA in 231 cells also caused impaired cell migration. We evaluated the cell morphology using scanning electron microscope. It revealed diminished filopodia formation in VEGF-KO 231 cells compared to WT-231 cells. Exogenous rhVEGF addition to VEGF-KO 231 cells recovered filopodia formation. Filopodia is a fingerlike protrusion that comes out from the cell periphery by an actin polymerization. Filopodia is required for cell development and functions such as cell migration. Our results indicate, because of the absence of VEGF/NRP1 signaling, filopodia formation is abrogated. As a result of diminished filopodia formation, VEGFA-KO 231 cells showed small and rounded shape and reduced migration. Bevacizumab and NRP1 bind VEGFA at amino acid motifs from exon 3-4 and exon 7 of VEGFA, respectively. Thus, our findings suggested that VEGFA could play a role in filopodia formation via NRP1 for 231 cells, and that bevacizumab could not block the VEGFA/NRP1 signal.

Discussion NRP1 is a receptor of VEGF, Semaphorine and other growth factors, and expressed in variety of cancer cells. Intracellular portion of NRP1 lacks kinase domain. Previous study showed that the VEGF stimulated to form a complex between NRP1 and GIPC1/Syx/RhoA, and promoted tumor cell proliferation independently from VEGFRs (Yoshida et al, 2015). However, the precise mechanisms regarding NRP1 signaling remain unknown. In conclusion, inhibition of VEGFA/NRP1 signaling caused rounded morphology with diminished filopodia formation, which impaired cell migration of 231 cells. These findings would provide profound insights regarding molecular mechanisms in resistance to bevacizumab treatment.

#1886

mGluR1 drives invasion of proneural subtype of glioblastoma cells.

Alena Gladwin, Sen Peng, Jeff Kiefer, Seugchan Kim, Michael Berens, Harshil D. Dhruv. _TGen (The Translational Genomics Research Institute), Phoenix, AZ_.

A major cause for the therapeutic failure and subsequent morbidity and mortality of glioblastoma (GBM) is the aggressive invasion of malignant cells into the surrounding normal brain that effectively renders complete surgical resection impossible and virtually assures recurrent tumor growth. Multi-omic profiling of GBM led to their molecular sub-classification into two distinct molecular subtypes: proneural (PN) and mesenchymal (MES). However, very little is known about shared or distinct invasion processes of cells in these genomically different subtypes. Using Microarray gene expression profiles of microdissected paired stationary core and invasive rim samples from 19 patients, we demonstrated that invasive gene signature of MES subtypes differs from PN. Specifically, using three orthogonal but intersecting bioinformatic approaches, i.e. gene set variation analysis, causal network analysis, and iRegulon analysis; we discovered that genes differentially expressed between PN core and rim could, to a meaningful degree, be accounted for based on their annotation as "regulated by the transcription factor REST". REST functions as a repressor of gene expression. Of the genes repressed by REST, mGluR1 (Metabotropic Glutamate Receptor 1) was significantly overexpressed at the rim of PN glioma cells as compared to the core. Finally, we also investigated the role of mGluR1 in glutamate induced glioma cell migration; our results show that glutamate stimulates migration of proneural-like glioma cells (A172) as compare to non-proneural-like glioma cell (T98G). mGluR1 activation by glutamate has shown to induce activation of Pyk2 and Src in astrocytes; knockout of mGluR1 is not embryonic lethal. In summary, our data demonstrate that glutamate-induced glioma cell migration of PN subtype of GBM is dependent on mGluR1 and thus raises the prospect that therapeutic targeting of mGluR1 may be a novel approach to controlling the invasion of this deadly disease.

#1887

Untethering heterochromatin: how loss of HP1a enhances cell invasion by altering nuclear envelope integrity.

Tracy K. Hale, Sarah Bond, David Wheeler. _Massey University, Palmerston North, New Zealand_.

Malignant cell invasion is accompanied by complex changes in nuclear shape and organisation. The onset of invasion correlates with the loss of HP1a in many solid tumours including those of the thyroid, kidney, colon and breast, while in cancer cell lines HP1a suppresses invasive potential. In normal cells HP1a maintains the condensation of transcriptionally silent heterochromatin and sequesters it at the inner periphery of the nuclear envelope; an event critical for the maintenance of nuclear stability and envelope rigidity. This role for HP1a leads us to hypothesise that the un-tethering of heterochromatin from the nuclear periphery that would occur after loss of HP1 causes a remodelling of nuclear envelope that enhances cellular invasion. If true this would explain how the nucleus, the largest and most rigid cellular organelles, becomes malleable enough to squeeze through interstitial spaces in the surrounding tissue during metastatic invasion. In support of this hypothesis we observed a 44% increase in the invasive potential of the poorly invasive MCF7 breast cells when HP1a is knocked down. Lamin A staining and electron microscopy also confirmed that these HP1a knockdown cells had the expected alterations in nuclear morphology and loss of heterochromatin at the nuclear periphery. Furthermore the loss of HP1 was correlated with a decrease in the mechanical stability of the nuclei. The significant enrichment (p-val 6.7x10-8) of nuclear envelope proteins shown by RNAseq analysis of transcripts that are differentially expressed in the MCF7 versus HP1a knock-down cells, suggests this loss of nuclear envelope integrity is associated with alterations to its composition. The most striking change identified in the nuclear envelope of cells lacking HP1a was the loss of two Nesprin1 isoforms. While reintroduction of HP1a in highly invasive MDA-MB-231 cells shows the return of these isoforms to the nuclear envelope. As the Nesprin1 isoforms act as signalling scaffolds connecting to the cytoskeleton or reaching into the nucleus, they represent a means of modulating nuclear malleability. Together these findings suggest that when loss of HP1a increases invasive potential there is a corresponding disruption to the nuclear periphery that is a weakness of these cells that could be exploited.

#1888

3D matrix confinement triggers vascular mimicry through a conserved migratory and transcriptional response.

Daniel Ortiz, Brian Tsui, Tyler Goshia, Colleen Ricker, Hannah Carter, Stephanie I. Fraley. _University of California, San Diego, La Jolla, CA_.

An initial step in solid tumor metastasis involves the migration of tumor cells through extracellular matrix. Several cancer cell migration strategies exist in vivo, and the local properties of collagen fibers are implicated in modulating migration behaviors. Yet, individual tumor cells also display heterogeneity in their intrinsic ability to migrate and metastasize. It remains unclear to what extent intrinsic and extrinsic heterogeneity contribute to the emergence of distinct migration phenotypes and whether certain migration phenotypes contribute more to metastasis than others. To study this, we generated 3D collagen matrices of varying densities and monitored single cancer cell migration in these matrices with time-lapse microscopy. We observed a collagen density threshold at 2.5mg/ml, above which 86% of MDA-MB-231 breast cancer cells transition from single mesenchymal migration to collective cell migration, with a 50% increase in persistence after cell division. After seven days, these collectively migrating cells created networks coated with basement membrane molecules resembling a clinical phenotype known as vascular mimicry (VM).The remaining 14% of cells migrated randomly and eventually formed spheroids. HT-1080 fibrosarcoma cells also responded similarly, migrating persistently and forming cellular networks. Next we sought to identify the physical feature of high-density collagen driving VM. Neither hypoxia or matrix stiffness was sufficient to induce VM. However, PEG-induced matrix confinement triggered VM network formation. RNA sequencing revealed collectively migrating cells up-regulated a conserved transcriptional program significantly enriched for annotations of vascular development and motility regulation processes. This gene module predicted survival in human tumor transcriptome datasets. Our results suggest that the VM phenotype arises in a subpopulation of cells from a conserved transcriptional and migratory response to confinement in 3D collagen.

#1889

**Formation and merger of H69 and H69AR small cell lung cancer (SCLC) tumor cell clusters (TCCs)** in vitro **.**

Nico O. Ruprecht,1 Martin Hungerbuehler,1 Philipp Kellmann,2 Nathalie Harder,3 Günter Schmidt,3 Carlos Wotzkow,2 Beat Haenni,2 Fabian Blank,2 Hendrik Von Tengg-Kobligk,1 Christof Granzow4. 1 _Inselspital, University Hospital Bern, Bern, Switzerland;_ 2 _University Bern, Bern, Switzerland;_ 3 _Definiens AG, Munich, Germany;_ 4 _FLACOD GmbH, Heidelberg, Germany_.

A novel manifestation of tumor cell invasiveness, termed mutual cellular pervasion (MCP), was observed in vitro. It involved TCCs formed by SCLC cells.

Suspension cultures of H69 and H69AR cells were grown in flavin-free RPMI 1640 medium with 10% fetal bovine serum as the only source of flavins. Experiments and subcultivations were performed under light with wavelengths above 520 nm. TCC morphology was studied by confocal microscopy and transmission and scanning electron microscopy. Interactions of TCCs were followed by video microscopy in phase contrast and fluorescence modes. Data were analyzed using the Definiens software suite. The interaction of H69AR TCCs with confluent monolayers of human umbilical vascular endothelial cells (HUVEC) was visualized.

Freshly subcultivated, singularized tumor cells rapidly formed aggregates. After two days, solid TCCs prevailed whose surface cells were connected by desmosome-like junctions. Apoptosis but not necrosis became obvious in central parts of clusters with diameters above 100 to 150 μm. TCCs enlarged through both cell proliferation and serial cluster mergers. In the course of TCC merger, four stages were distinguishable: (1) directed cluster movement leading to contact of clusters, (2) formation of a near circular, symmetric structure termed contact disc. It consists of planar surface areas to which both TCCs involved contribute equally. (3) Merger of TCCs took place in the contact disc, and was accompanied by active movement of adjacent cells. (4) Starting from the merger zone, major surface areas of newly formed clusters were mutually pervaded by cells from both participating cell clusters. After numerous merger events, only low numbers of TCCs reaching sizes of up to 2 to 3 mm were present at subcultivation.

No spontaneous bursting, shrinkage, degeneration, or decay of TCCs has been observed. However, when TCCs of H69AR cells were confronted with confluent layers of HUVEC, drastic changes occurred in both formations. First, intense oscillatory cellular movements were triggered on TCC surfaces, preceding the release of cell aggregates which moved away from TCCs to the HUVEC layer. At the same time, HUVEC were disconnected from each other and started moving either randomly or in an oscillatory fashion. Individual tumor cells attached to the much larger HUVEC and spread on their surface, resulting in full covering which secluded HUVEC from the medium.

To our knowledge, neither formation nor merger of malignant TCCs in vitro have been described so far. TCC merger involves mutual cellular penetration of the involved TCC surfaces, i. e. MCP. Contrary to the invasion of tumor cells into non-tumor tissue, pervasion does not lead to destruction but results in TCC growth. However, key features of tumor cell invasion in vivo, such as chemotaxis, spreading and TCC formation as well as disruption of endothelial layers, manifest under conditions in vitro, thus facilitating related experimental studies.

#1890

PLEK2 contributes to lung cancer progression through upregulation of NDRG1 expression.

Yi-Pin Hsieh, Hui-Chia Liu, Hao-Yu Huang, Wan-Jiun Tang, Tai-Lin Lee, Meng-Feng Tsai. _Da-Yeh University, Changhua, Taiwan_.

PLEK2 is one of the pleckstrin homology domain (PH domain) proteins. Recently studies suggested that PLEK2 may play a critical role in T lymphocyte migration through by PI3K/AKT dependent and independent pathways. However, the biological functions of PLEK2 in lung cancer is not clear. We detected the expression of PLEK2 in various lung adenocarcinoma cell lines using quantitative RT-PCR methods. The results indicated that PLEK2 expression significantly positive correction with cancer cell migration and invasion ability. Forced expression of PLEK2 in lung cancer cells exhibited markedly promoting in the proliferation rate, anchorage independent growth, cell motility and invasion, whereas knockdown of PLEK2 was found to suppress lung cancer cell progression. In addition, we also reported that PLEK2 expression induce lung cancer cell epithelial-mesenchymal transition (EMT) phenomenon, and involve in chemotherapeutic drug resistance through upregulation NDRG1 gene expression. Overexpression of NDRG1 in PLEK2-knockdown cell line restored cancer cell proliferation, migration, and invasion. These results will provide information and help us to understand the roles of NDRG1 in lung cancer cells.

#1891

Annexin A2-binding S100 proteins promote proliferation and cell cycle progression of EGFR positive cancer cells.

Naoko Ogama, Ryuuichi Nagashima, Nobuyuki Tanaka. _Miyagi Cancer Ctr. Research Inst., Natori, Japan_.

(a) Aim: The purpose of this study is to clarify an unknown function of Annexin A2-binding S100A proteins in tumor growth, cell cycle progression and invasion. Annexin A2 is a membrane bound protein originally identified as v-Src substrate, and often found to be overexpressed in many types of cancers. As a multicompartmental protein that orchestrates a spectrum of dynamic membrane-related events, it is associated with actin cytoskeleton control, vesicle formation and intercellular interaction.

(b) Methods and Results: During our proteomics analysis using solid tumor cell lines, we identified both Annexin A2 and S100A proteins in cancer stem cell-rich population. We next examined S100A proteins expression in several Head and Neck cancer PDX samples. Immunohistochemical data from in vivo specimens as well as western blots showed that S100A expression abundance was well associated with poor prognosis. Annexin A2 has been indicated to be involved in malignant phenotypes such as invasion and metastasis, and Annexin A2 and S100A form (S100-Annexin A2)x2 dimers, we next examined roles of S100A proteins in cancer, by using shRNA-mediated depletion experiments.

(c) New Findings: Depletion of S100A from PDX-derived cancer cells resulted in loss of both in vivo tumor formation and invasion. Furthermore, cell cycle progression was inhibited by the depletion, suggesting its potential role in proliferation controls. Similar to the mutant Ras-driven tumors, EGFR-overexpression increased S100A gene expresssion.

(d) Conclusion: These results strongly suggest that increases in S100A plays significant roles in cancer cell proliferation and invasion.

#1892

Small molecule NSC59984 prevents cancer cell migration and invasion.

Shengliang Zhang, Jessica Wagner, Lanlan Zhou, Wafik S. El-Deiry. _Fox Chase Cancer Center, Philadelphia, PA_.

Tumor metastasis is one of the main mechanisms leading to mortality of cancer patients. Searching for new therapeutic strategies to prevent tumor metastasis is one of the anti-tumor approaches in cancer therapy. Cell migration and invasion are crucial initial steps in the complex process and progression of tumor metastasis. Mutant p53 has been known as an important factor associated to cancer cell migration. Therefore, targeting mutant p53 is a potential strategy to avoid tumor metastatic spread. We previously reported an anti-tumor small molecule, NSC59984 which degrades mutant p53 and restores p53 pathway via activation of p73. Here we demonstrate that NSC59984 inhibits cancer cell migration and invasion. Treatment with NSC59984 partially attenuates cell migration in various cancer cells based on the scratch assay and Boyden chamber assay. The effect of NSC59984 on cell migration is partially blocked by knockdown of p73, a member of the p53 family, suggesting that restoration of p53 pathway signaling via p73 is required for NSC59984-inhibition of cell migration in cancer cells. We found that NSC59984 induces ERK2-dependent p53 restoration and mutant p53 degradation. To examine the role of ERK2 in the cell migration, ERK2 signaling is blocked by U0126 treatment in cancer cells. The effect of NSC59984 on cancer cell migration is partially inhibited by U0126 treatment, suggesting that NSC59984 inhibits cancer cell migration partially dependent on ERK1/2 pathway signaling in cancer cells. In addition, the gelatin degradation assay shows that NSC59984 inhibits cancer cell invasion. Consistent to the in vitro assays, NSC59984 decreases tumor invasion of xenograft tumors as compared to the xenograft tumors treated with DMSO as control. Our results, taken together, suggest that NSC59984 not only suppresses tumor growth, but also inhibits cancer cell migration and invasion. Our findings provide the potential administration of p53-restoring compound NSC59984 in cancer therapy by blocking tumor metastasis.

#1893

Alpha-tubulin acetyltransferase, MEC-17, regulates cancer cell morphology and migration through epithelial-mesenchymal transition suppression and cell polarity disruption.

Cheng-Che Lee,1 Chi-Yen Chang,2 Chi-Min Lin,2 Jang-Yang Chang1. 1 _National Cheng Kung University, Tainan, Taiwan;_ 2 _National Health Research Institutes, Tainan, Taiwan_.

Acetylation of α-tubulin has characterized as a key process for microtubule stabilization, and it can regulate cell spreading and modulate the dynamics of focal adhesion. MEC-17, a newly identified alpha-tubulin-N-acetyltransferase 1, serves as the major α-tubulin acetyltransferase to promote α-tubulin acetylation in vitro and in vivo. However, the physiological function of MEC-17 in cancer cells and its effect on MT acetylation during cell morphogenesis and migration has remained elusive. In the present study, our results showed that overexpression of MEC-17 induced increment of cell focal adhesion area, suppressed pseudopods formation in three-dimension collagen-embedded cultured environment and inhibited cancer cell migratory and invasive abilities. These changes were further proved to be caused by epithelial-mensenchymal transition (EMT) repression, cell polarity disruption caused by alteration of Golgi orientation and cdc42 activity and the decrease in the phosphorylation of extracellular signal regulatedkinase1/2 (Erk1/2). On the contrary, silencing of endogenous MEC-17 by specific shRNA accelerated the pseudopods formation and EMT process, which lead to facilitating the cell migration. Taken together, these results demonstrated that the important role of MEC-17 in the modulation of intrinsic cellular morphogenesis and migratory and invasive functions through regulation of EMT and polarization. (The study was supported by the following grants: MOST 103-2320-B-006-036-MY3 and MOST 105-2325-B-400-001 from the Ministry of Science and Technology of Taiwan, ROC)

#1894

Gremlin-1 is an important regulator of invasiveness in malignant mesothelioma.

Miao Yin,1 Mira Tissari,1 Emmi I. Joensuu,1 Jenni Tamminen,1 Irene Ylivinkka,1 Mikko Rönty,2 Kaisa Lehti,1 Marko Hyytiäinen,1 Marjukka Myllärniemi,3 Katri Koli1. 1 _University of Helsinki, Helsinki, Finland;_ 2 _HUSLAB, Helsinki, Finland;_ 3 _Helsinki University Hospital, Helsinki, Finland_.

The purpose of this study was to clarify the role of BMP inhibitor gremlin 1 in invasion and migration in mesothelioma.

Primary mesothelioma cells isolated from patient pleural fluid as well as mesothelioma cell lines were used for in vitro studies. Cells were transfected with siRNAs or transduced with lentiviral expression vectors. Invasive growth was analyzed in 3D Matrigel or collagen I matrices. mRNA expressions were analyzed using a commercial PCR array and quantitative RT-PCR. Migration assays were performed using scratch wound assay or transwell migration assay with fibronectin or collagen coating. TGF-β and BMP signaling activity was measured with luciferase reporter assays. For in vivo mouse xenograft experiment cells were additionally transduced to express a luciferase marker. Subcutaneous cell injection with Matrigel matrix was performed in the flank of nude mice.

Malignant mesothelioma is an aggressive cancer that develops from mesothelial cells, most often in the pleural lining of the lung. We have previously shown that the BMP inhibitor protein gremlin 1 is highly expressed in mesothelioma and induces a mesenchymal and chemoresistant phenotype in mesothelioma cells. Since mesothelioma tumors are locally very invasive and has poor prognosis, we analyzed the role of gremlin 1 in mesothelioma cell migration and invasive growth.

We found that mesothelioma cells expressing gremlin 1 showed invasive sprouting when tumor cell spheroids were imbedded into 3D collagen matrix. Silencing of gremlin 1 expression significantly reduced invasive growth. In addition, cells overexpressing gremlin 1 gained more invasive phenotype. This was associated with increased mRNA expression levels of Slug and matrix metalloproteinases (MMP) as well as reduced expression of E-cadherin. The cells were more migratory and exhibited increased expression of certain integrins, especially the αv subunit. Gremlin 1 induced invasive growth was dependent on MMP activity and associated with increased TGF-β activity. Intrapleural injection of mesothelioma cells overexpressing gremlin 1 isolated from a patient with epithelioid mesothelioma, produced tumors in 2/4 mice over 5 months after injection. Mice with cells transduced with vector only did not develop tumors (0/4). When cells were injected subcutaneously together with Matrigel gremlin 1 overexpressing tumors appeared more slowly, but exhibited comparable luciferase signal 2.5 months after injection. However, gremlin 1 tumors showed more vascularization and in contrast to control tumors some also developed metastases (2/6 mice).

As conclusion, we identified gremlin 1 as an important regulator of mesothelioma invasive growth and chemoresistance. Blocking gremlin 1 function may overcome drug resistance and reduce invasive behavior of mesothelioma.

#1895

A conserved E2F1-activated gene regulatory network encompassing monocarboxylic acid transporter-1, its co-operating antisense lncRNA SLC16A1-AS1 and their common downstream targets mediates bladder cancer invasiveness.

Berdien A. Jansen,1 Stella Logotheti,1 Dirk Heckl,2 Jan-Henning Klusmann,2 Dirk Kozcan,3 Laila Taher,4 Georg Fuellen,5 David Engelmann,1 Brigitte Pützer1. 1 _Institute of Experimental Gene Therapy and Cancer Research, Rostock, Germany;_ 2 _Hannover Medical School, Hannover, Germany;_ 3 _Institute of Immunology, Rostock, Germany;_ 4 _University of Erlangen-Nürnberg, Erlangen, Germany;_ 5 _Institute for Biostatistics and Informatics in Medicine and Ageing Research, Rostock, Germany_.

AIM: The therapeutic arsenal against muscle-invasive bladder cancer (BC) is poor, leading to fatal outcomes and necessitating next-generation targeted therapeutics. A causative event of BC progression is activation of the gene regulatory networks (GRN) of the transcription factor E2F1. LncRNAs are emerging integral components of the E2F1-regulated GRNs, but their implication in BC progression is unknown. Our study focuses on lncRNAs that participate in E2F1-activated GRNs underlying BC progression and the therapeutic exploitation thereof.

METHODS: We applied bioinformatics on BC patient cohorts to identify clinically relevant lncRNAs upregulated in invasive stages. Of those, the E2F1-responsive lncRNAs were defined by subjecting T24 clones inducibly overexpressing E2F1 to HTA2.0 RNA arrays. Candidates in close proximity to oncogenes/tumor-suppressors were further selected, based on an emerging "guilt-by-association" rule of lncRNA biology. LncRNAs were qPCR-validated and E2F1-transactivation was shown by ChIP and reporter assays. The SLC16A1-AS1 lncRNA was functionally characterized by CRISPR/Cas9 knockout in invasive BC cells, followed by migration/invasion/metabolic assays, transcriptomics and mice in vivo experimentation. SLC16A1-AS1 homologues across species were predicted by comparative genomics.

RESULTS: Our pipeline unveiled SLC16A1-AS1 lncRNA located nearby and antisense to SLC16A1 protein-coding gene. SLC16A1 synthesizes the membrane-bound Monocarboxylic Acid Transporter-1 (MCT1), a key metabolic regulator that mediates lactate uptake and glycolytic flux and favors BC invasiveness; while SLC16A1-AS1 function was unknown to date. SLC16A1 and SLC16A1-AS1 present a 'head-to-head' gene arrangement, with a bidirectional E2F1-responsive promoter. This 'operon-like' gene cluster is conserved across mammals, implying functional relevance of MCT1/E2F1/SLC16A1-AS1. In invasive BC cells, E2F1 transactivates SLC16A1-AS1, which remains in the nucleus exerting gene regulatory functions, as well as MCT1, which localizes in the cytoplasm to control lactate flux. SLC16A1-AS1 induces EMT-independent invasiveness by regulating expression of downstream cell migration-related targets which are co-modulated by MCT1.

CONCLUSIONS: MCT1 and SLC16A1-AS1 constitute a composite, E2F1-responsive functional unit, co-operatively regulating common cell-migration related downstream targets. Their conserved gene arrangement, which ensures simultaneous transcription upon E2F1 upregulation, could facilitate co-ordination between metabolism reprogramming and cell migration to establish aggressive phenotypes. Combining SLC16A1-AS1 suppression with formulated MCT1 inhibitors might provide a therapeutic basis against progressive BC.

#1896

Soluble lung-derived selectins promote breast cancer cell migration.

Sami U. Khan,1 Jenny E. Chu,1 Ying Xia,2 Alison L. Allan1. 1 _Western University, London, Ontario, Canada;_ 2 _London Health Sciences Centre, London, Ontario, Canada_.

Ninety percent of all breast cancer-related deaths are attributed to metastasis, as current therapies are largely non-curative in the metastatic setting. One of the most common sites of breast cancer metastasis is the lung. Previous work from our lab has demonstrated that there are over 70 soluble factors produced by murine lung tissue, many of which have been associated with cancer cell growth, migration, invasion and metastasis. However, the relative contribution of each individual factor in enhancing the metastatic behavior of breast cancer cells has not been examined in detail. Three of the identified soluble lung-derived proteins are E-, L- and P-selectin, and these were the focus of the current study. The selectins are a family of glycoproteins with known physiological roles in vascular/immune cell migration, and soluble selectins have been clinically observed to be elevated in the sera of cancer patients. We hypothesized that soluble lung-derived selectins enhance the metastatic behavior of breast cancer cells. We used a 2D ex vivo model of the soluble lung microenvironment, generated by isolating conditioned media from dissociated healthy murine lung. E-, L- and P-selectin were then individually immunodepleted from the lung-conditioned media (lung-CM) and the subsequent functional effect on breast cancer cell migration and proliferation was assessed using transwell migration and BrdU incorporation assays, respectively. MDA-MB-231 and SUM149PT human breast cancer cells demonstrated significantly enhanced cell migration toward lung-CM relative to basal media, and this migration was significantly reduced when E-, L- or P-selectin were depleted (p<0.05). In contrast, although MDA-MB-231 and SUM149PT human breast cancer cells demonstrated significantly enhanced cell proliferation in response to lung-CM relative to basal media (p<0.05), depletion of soluble E-, L- or P-selectin from lung-CM had no influence on reducing proliferation (p>0.05). Ongoing studies are aimed at elucidating the pathways through which soluble lung-derived selectins exert their pro-migratory function. If these results implicate a common receptor and downstream signalling pathways for all three selectins, these ligand-receptor interactions may have value as potential therapeutic targets for the prevention of pulmonary metastasis in breast cancer patients.

#1897

The role of nuclear deformation and rupture in breast cancer cell migration.

Alexandra L. McGregor, Joshua J. Elacqua, Emily S. Bell, Jan Lammerding. _Cornell University, Ithaca, NY_.

Invasion of tumor cells into the surrounding tissue is a critical step in the metastatic cascade, which is responsible for the vast majority of cancer-related deaths. Recent findings have begun to demonstrate that the biophysical properties of the cell nucleus play a critical role in this process. During invasion, cancer cells must often pass through interstitial spaces much smaller than the cell nucleus. While the cell cytoplasm easily maneuvers through confined spaces, the deformability of the nucleus, which is the largest and stiffest organelle, ultimately determines how efficiently a cell can pass through a tight space. Furthermore, as the nucleus deforms to move through a confined space, it can exhibit transient loss of nuclear envelope (NE) integrity, where the nuclear membranes rupture and nuclear and cytoplasmic components interchange. We previously showed that chromatin is exposed to the cell cytoplasm during NE rupture, and cells can incur DNA damage. While this work revealed that cancer cell migration requires nuclear deformation and can involve NE rupture, it is currently unknown whether such events correlate with metastatic potential. We used a panel of human breast cancer cells lines, including claudin-low, basal-like, and lumen-like cells. Cells were stably labeled with a green fluorescent protein fused to a nuclear localization sequence (NLS-copGFP) to investigate whether some cell lines were particularly well suited to migrate through tight spaces, and if any were particularly prone to NE rupture. The cells were loaded into microfluidic devices that were designed to mimic interstitial in vivo spacing and monitored by time-lapse microscopy. Devices contained constrictions 5 µm tall and 1, 2, or 15 µm wide. We found that claudin-low cell lines such as BT-549 migrated through confined spaces (1+2 µm wide constrictions) significantly faster and more efficiently than basal-like HCC70 cancer cells. In contrast, the various cell lines displayed similar migration rates in the 15 µm wide constrictions, which do not require nuclear deformation. Thus, the differences observed between the cell lines reflect specific defects in migration through confined spaces, and not general migration defects. We are currently expanding our studies to a larger panel of cancer cell lines and to mouse breast cancer models. If confirmed, these results could indicate that highly invasive cancer cells may benefit from more deformable nuclei that facilitate movement through tight spaces encountered in the tissue microenvironment.

#1898

MEG3 as a competing endogenous RNA binds with miR-27a to promote PHLPP2 protein translation, and consequently impairs bladder cancer invasion.

Chao Huang,1 Xin Liao,2 Jingxia Li,2 Chuanshu Huang2. 1 _New York University School of Medicine and Wuhan Union Hospital, Tuxedo Park, NY;_ 2 _New York University School of Medicine, Tuxedo Park, NY_.

Muscle invasion and metastasis of bladder cancer dramatically decrease survival rate, compared to 77% of 5 year survival rate of all bladder cancer, metastasis bladder cancer is only 5%. This truth implies that one of the therapeutic kernels of bladder cancer is to decrease, even block, its invasiveness. In the current study, we find that MEG3 is significantly downregulate in human muscle-invasive bladder cancer tissues compared with non-muscle invasive tissues. We also demonstrate the MEG3 strongly inhibits human bladder cancer cell invasiveness by acting as a ceRNA. We find that MEG3 competes with PHLPP2 mRNA for miR-27a binding, which leads to a decreased miR-27a activity, and subsequently reducing the translation inhibition of miR-27a to PHLPP2. The upregulated PHLPP2 decreases the c-Jun phosphorylation at Ser63/73, and in turn inhibits the c-Myc transcription and its mediated invasiveness of the bladder cancer cells. This is the first demonstration that MEG3 inhibits the invasiveness of human bladder cancer cells via negative regulation of c-Myc as a ceRNA. These findings not only provides a novel insight into understanding the mechanisms behind the MEG3 inhibiting the cancer cell invasion, but also reveals a potential targets for invasive bladder cancer prevention and therapy.

#1899

MKK4/JNK2 down-regulation in NSCLC suppresses tumor growth and metastasis.

Tamer Saad Kaoud,1 Nancy D. Ebelt,1 Lili Du,2 Sabrina Van Ravenstein,3 Kenneth Y. Tsai,2 Kevin N. Dalby1. 1 _UT Austin College of Pharmacy, Austin, TX;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _UT Austin, Austin, TX_.

Mitogen-activated protein kinase kinase-4 (MKK4) has been reported to either enhance or suppress oncogenesis. Evidences of its pro-oncogenic activities in breast, pancreatic, lung and skin cancer have been reported. Although the mechanism of its possible tumorigenic role is still unclear. Recent studies in glioblastoma and lung carcinoma have suggested important roles for the constitutive activation of its downstream substrate JNK2. JNKs require phosphorylation by both MKK4 and MKK7 to be fully activated. The JNK2 isoform shows a unique propensity among the JNKs to autophosphorylate in vitro. Here we show that JNK2 autophosphorylation contributes to the proliferation and migration of NSCLC cells under conditions of low serum through an MKK4-dependent mechanism. This suggests a pro-oncogenic role of MKK4 in NSCLC through a JNK2 autophosphorylation-dependent pathway. Evidence in several cell lines, including mouse embryonic stem cells lacking MKK4 or MKK7 suggests that autophosphorylation alone activates JNK2 weakly in cells. However, JNK2 autophosphorylates on Thr-183, to create a pool of JNK2 primed for activation by MKK4, which phosphorylates JNK2 on Tyr-185 to activate it. Under conditions of low serum the down-regulation/overexpression of MKK4 or JNK2 but not MKK7 or JNK1 suppresses/promotes proliferation of multiple NSCLC cell lines, through STAT3. Under the same conditions, A549 cell migration is inhibited upon down regulation of either MKK4 or JNK2. Further supporting the notion that active JNK2 results primarily from autophosphorylation of Thr-183, pan-JNK ATP competitive inhibitors (JNK-IN-8 and SP600125) showed a dose-dependent dephosphorylation of JNK2 in NSCLC cell lines and inhibited their proliferation under conditions of low serum. Moreover, the downregulation of MKK4 or inhibition of JNK2 autophosphorylation by SP600125 inhibited nuclear localization of JNK2 in A549 cells cultured in low serum. To further understand the pro-oncogenic role of MKK4 in the A549 cell line, we investigated tumor growth and lung metastasis in A549 Xenografts in which either JNK2 or MKK4 were stably knocked down. Either JNK2 or MKK4 down regulation showed a similar suppression of tumor growth and lung metastasis if compared to cells expressing an shRNA control. Sequencing of MKK4 in the A549 cells revealed no genomic deletion or somatic mutations. Taken together, the data suggest that MKK4 may exhibit pro-oncogenic properties in NSCLC by activating Thr-183-autophosphorylated JNK2. Targeting this pathway may reduce or block lung tumor progression and/or metastasis. 

### Normal and Neoplastic Stem Cells

#1900

Understanding the biological role of latexin in the normal and malignant prostate.

Robert I. Seed, Alberto J. Taurozzi, Giovanna Nappo, Holger H. Erb, Anne Collins, Norman J. Maitland, Fiona Frame. _University of York, York, United Kingdom_.

The retinoid-inducible protein Latexin (LXN) acts as a stem cell suppressor in normal murine hematopoiesis, and in human tumors where it can influence both tumor cell migration and invasion (Liang et al, Nature genetics 2007). We have shown that LXN is silenced in the stem cell fraction of primary prostate epithelial cells, and that expression increases as cells differentiate through the prostate basal epithelial hierarchy towards mature luminal cells. Furthermore, transient knockdown of LXN significantly increased clonogenic potential and promoted a more invasive phenotype in primary prostate epithelia (Oldridge et al, Oncogenesis 2013). LXN may therefore be involved in the differentiation of prostate epithelial cells, but could act as a tumor suppressor in Prostate Cancer (CaP), in addition to other tumours. In genome databases LXN is described as an inhibitor for Carboxypeptidase A4, but despite considerable molecular characterisation, the mechanism of action has remained an enigma since perhaps paradoxically LXN appears to be able to function independently of its classical CPA4 inhibitory role. To resolve this, we adopted an unbiased approach to determine if targeted re-expression of LXN or its binding partners could provide a viable novel drug target for the treatment of CaP. Our preliminary studies had revealed that LXN was an exclusively cytosolic protein in prostate epithelial cells. However, subsequent live cell imaging of primary prostate epithelial cells overexpressing YFP-LXN demonstrated for the first time that, whilst predominantly cytosolic, LXN was also present within the cell nucleus. To characterise the effects of LXN overexpression on gene transcription after LXN-expressing lentiviral transduction of primary prostate epithelial cells, we utilised Affymetrix HTA2 gene arrays. Perhaps surprisingly, short-term overexpression of LXN did not significantly effect global gene transcription compared to controls (n=4 P=NS). Additionally, we used IP/MS to investigate the potential interacting partners of LXN, using multiple approaches, including both overexpression of HA-tagged and WT LXN, and endogenous LXN as bait. These approaches failed to reveal any strong (and reproducible) intracellular protein interactors of LXN. From these data, we hypothesised that LXN may function as a secreted protein. We found that LXN was present in not only the conditioned media of LXN+ prostate epithelial cells, but also preliminary evidence that LXN is enriched in extracellular exosomes. Therefore, LXN may perform its biological roles extracellularly, or at the cell surface. Our current work is focusing on determining the functional consequences of secreted LXN on prostate epithelia and to establish potential protein: protein interactions of LXN in the extracellular space. Full characterisation of LXN function considering its potent biological effects on cancer cells will determine its suitability as a therapeutic in CaP and other cancers.

#1901

DNMT1 is involved in esophageal squamous cell carcinoma (ESCC) and self-renewal ability of ESCC-cancer stem cells.

Ying Teng, Xiying Yu, Hui Yuan, Liping Guo, Wei Jiang, Shih-Hsin Lu. _National Cancer Center/ Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China_.

DNA methylation mediated by DNA methyltransferase 1 (DNMT1) plays an important role in carcinogenesis and self-renewal ability of cancer stem cells (CSCs). However, the function of DNMT1 in esophageal squamous cell carcinoma (ESCC) carcinogenesis, especially self-renewal ability of ESCC-CSCs remains unclear. In this study, we found a high expression of DNMT1 in both side population (SP) cells and sphere formation cells that represented as substitutes for CSCs in KYSE150 and EC109 ESCC cell lines. In addition, the expression of DNMT1 was decreased during the differentiation from SP to None-SP (NSP) in these ESCC cells. These results suggested that DNMT1 might have a role in regulating self-renewal and/or differentiation of ESCC-CSCs. To further investigate DNMT1 in ESCC carcinogenesis and self-renewal ability of ESCC-CSCs, we silenced the expression of DNMT1 in KYSE150 and EC109 ESCC cells using lentivirus-mediated RNA interference (RNAi). Our results showed that ablation of DNMT1 expression in KYSE150 and EC109 ESCC cells resulted in decreased their CSCs by SP analysis and sphere formation assay. Meanwhile, ablation of DNMT1 expression inhibited malignant phenotypes in KYSE150 and EC109 cells, including cell proliferation, colony formation, migration and drug resistance abilities. Treatment of 5-aza-2'-deoxycytidine (5-aza-dC), a DNMT inhibitor that led to the degradation of DNMT1 protein by proteasome, revealed that numbers of CSCs and the malignant phenotypes of KYSE150 and EC109 ESCC cells were refrained significantly, including a dramatic inhibition of self-renewal ability of these ESCC-CSCs. Thus, our results indicated that DNMT1 was involved in ESCC carcinogenesis, especially in the maintenance of ESCC-CSCs, suggesting that DNMT1 could be a potential target for ESCC, especially ESCC-CSCs, therapy.

#1902

Enrichment of putative cancer stem cells during anti-angiogenic therapies promotes relapse formation in hepatocellular carcinoma.

Darko Castven,1 Carolin Czauderna,1 Diana Becker,1 Marcus A. Wörns,1 Snorri S. Thorgeirsson,2 Peter Grimminger,1 Hauke Lang,1 Peter R. Galle,1 Jens U. Marquardt1. 1 _University of Mainz, Mainz, Germany;_ 2 _CCR/NCI/NIH, Bethesda, MD_.

Background and Aims: Activation of neo-angiogenic processes in hepatocellular carcinoma (HCC) during disease progression is frequently associated with poor clinical outcome. Consequently, inhibition of neo-angiogenesis is an effective treatment strategy for advanced HCC. However, development of chemoresistance is observed in the majority of patients. Compelling evidence suggest that cancer stem cells (CSCs) may contribute to the acquisition of resistant properties in many solid tumors, but their exact role in this process for HCC remains to be defined. Here, we evaluate the importance of CSCs in the development of resistance and relapse formation after exposure to different anti-angiogenic therapies in HCC and define the concomitant adaptive molecular changes.

Methods: Five HCC cell lines and one primary HCC isolate were exposed to sorafenib and sunitinib for a total of 14 days. The treatment effects on CSCs were estimated by sphere forming capacity in vitro as well as the side-population (SP) approach. Expression levels of key oncogenic and CSC markers, such as EpCAM, CD133 and ABCG2 transporter, were assessed by qRT-PCR and flow cytometry. Furthermore, whole transcriptome analyses were performed at different time points.

Results: Both treatment regimens effectively reduced oncogenic properties in all investigated HCC cells. However, sustained anti-proliferative effect after treatments was observed in only one cell line. In three other hepatoma lines an initial treatment effect was subsequently followed by rapid re-growth thereby mimicking the responses observed in patients. Interestingly, two cell lines showed differential response to applied drugs, showing anti-proliferative effects to sorafenib, while relapse formation occurred after sunitinib treatments. While anti-oncogenic effects in sensitive cell lines were associated with significant reduction in sphere forming capacity, CSC marker EpCAM as well as SP cells, resistant cell line showed a transient increased in CSC properties. Importantly, acquired resistance to both drugs uniformly developed in the cell lines suggesting that common molecular mechanisms might be operative. These adaptive molecular changes involved signaling pathways known to be associated to cell survival and proliferation (RAS, AKT, MYC), as well as angiogenesis (VEGFR, PDGFR, HIF1a). Furthermore, the resistant cell lines showed compensatory upregulation of key oncogenic molecules such as EGFR as well as multidrug resistance ABC transporters.

Conclusions: Our in vitro model recapitulates features of drug resistance observed in human HCC patients. Resistance to anti-angiogenic therapies might be fueled by transient expansion of CSCs. Therefore, specific targeting of CSCs as well as pro-oncogenic compensatory signaling pathways might be an effective therapeutic strategy to overcome resistance in HCC.

#1903

Cancer stem cell vaccine significantly reduces local tumor relapse and prolongs survival in the adjuvant setting.

Fei Liao,1 Yang yang Hu,1 Xin Chen,1 Alfed E Chang,1 Robert E Hollingsworth,2 Elaine Hurt,2 John Owen,1 Jeffrey S Moyer,1 Mark E.P Prince,1 Joel Whitfield,1 Yuxin Chu,3 Qibin Song,3 Max S Wicha,1 Qiao Li1. 1 _Univ. of Michigan, Ann Arbor, MI;_ 2 _MedImmune INC, MD;_ 3 _Renmin Hospital of Wuhan University, Wuhan, China_.

Although surgical resection has been a standard treatment for solid malignancies, therapeutic efficacy is limited by both local and distant recurrence. Effectively preventing local tumor recurrence remains a significant challenge. The existence of micro metastasis at the time of tumor resection represents an even greater therapeutic challenge, since 90% of tumor deaths are due to tumor metastasis. There is increasing evidence that many cancers are driven and maintained by a subpopulation of cells that display stem cell properties. Cancer stem cells (CSCs) can self-renew, mediate tumor growth and contribute to tumor recurrence and metastasis. Targeting CSCs may thus increase the therapeutic efficacy of current cancer treatment. We previously described a strategy to target CSCs using CSC-dendritic cell (DC) vaccination. However, the efficacy of CSC targeted therapeutics may be greatest when they are deployed in the adjuvant setting. In this study, two mouse models were utilized: SCC7 subcutaneous (s.c.) tumors, and a D5 melanoma model. Established s.c. SCC7 tumors were surgically removed from mice followed by treatment using ALDHhigh SCC7 CSC-DC vaccine, which significantly reduced local tumor relapse and prolonged animal survival. This effect was significantly augmented by simultaneous administration of anti-PD-L1 mAb. In the minimal disease setting of D5, ALDHhigh CSC-DC vaccination significantly inhibited tumor growth, reduced spontaneous lung metastases resulting in increased survival. CCR10 and its ligands were down-regulated on ALDHhigh D5 CSCs and in lung tissues respectively in animals subjected to ALDHhigh D5 CSC-DC vaccination. Down-regulation of CCR10 by siRNA significantly blocked tumor cell migration in vitro and metastasis in vivo. T cells harvested from ALDHhigh D5 CSC-DC vaccinated animals selectively killed the ALDHhigh D5 CSCs. There was also evidence of humoral immunological targeting of CSCs. As a result, CSC-DC vaccination significantly decreased the percentage of ALDHhigh cells in residual tumors. These data indicate that, when used in an adjuvant setting, ALDHhigh CSC-DC vaccines effectively inhibit local tumor recurrence, reduce spontaneous lung metastasis, and prolong animal survival; compared with traditional DC vaccines and that simultaneous PD-L1 blockade can significantly enhance this effect.

#1904

Novel 5-fluorouracil-thymoquinone hybrid kills colon cancer stem cells.

Benardina Ndreshkjana,1 Aysun Çapcı Karagöz,2 Volker Klein,2 Pithi Chanvorachote,3 Julienne K. Muenzner,1 Kerstin Huebner,1 Chuanpit Ninsontia,3 Sara Steinmann,1 Abbas Agaimy,1 Maamoun Fatfat,4 Hala Gali-Muhtasib,4 Svetlana B. Tsogoeva,2 Regine Schneider-Stock1. 1 _University Hospital of Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany;_ 2 _Friedrich-Alexander University of Erlangen-Nürnberg, Erlangen, Germany;_ 3 _Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand;_ 4 _American University of Beirut, Beirut, Lebanon_.

Objectives: Recently, it has been shown that a subset population of cancer cells in a tumor, the so-called cancer stem cells (CSCs), majorly contributes to chemoresistance and the ability of a tumor to form recurrences and metastases. Since the response to the standard chemotherapeutic drug 5-fluorouracil (5-FU) in patients with colorectal cancer (CRC) leads only to an inadequate response, we aimed to synthesize new hybrid molecules by combining 5-FU with the plant-derived drug thymoquinone (TQ). Methods: The cytotoxic effect of hybrids, the single drugs and the combination was investigated by crystal violet assays. HCT116, HT29 and 5-FU-resistant colorectal tumor cells were used to assess the spheroid forming capability after treatment with different concentrations of the hybrid and the single drugs by staining with the fluorescent stem cell marker CDy1 and Hoechst 3324 after 21 days of incubation. Western Blot analysis for stem cell surface marker CD133 were performed to verify these results. We have analyzed the anticancer effects of the single drugs TQ and 5-FU and the most effective hybrid on deregulation of cancer signaling pathways utilizing the NANOSTRING cancer pathway panel. In addition, we performed a single cell cloning from spheroids to generate monoclonal cell lines with enriched CSC populations. In vivo effects were studied in the chick chorioallantoic membrane (CAM) assay and in mouse xenografts. Results: The 5FU-TQ hybrid has an increased cytotoxic effect towards CRC cells when compared to single drug treatments. The spheroid formation assay revealed the capability of the hybrid to reduce sphere number and size at fairly low concentrations (10, 20 µM), however at 50 µM there was a complete suppression of spheroid growth. NANOSTRING analysis showed that gene expression of main components of the PI3K/AKT signaling pathway was significantly down-regulated after treatment with all drugs but most prominently with the new hybrid. After data processing we found that AKT3, IGF1, COL11A1, FGF10, GNG7, EPO genes were downregulated in hybrid-treated cells, while CDKN1A, BAX, JUN, MDM2, FAS were upregulated. This was verified via Western Blotting for a panel of markers. Next, we investigated the effectiveness of the hybrid to kill clones having an enriched CSC population. Indeed, CD133 protein levels were significantly decreased in hybrid-treated clones. Histological analysis of in vivo CAM grafts showed that HCT116 control cells developed a well vascularized tumor mass whereas HCT116 cells that were treated with the hybrid showed no more vital tumor cells, but extended areas of necrosis, calcification, and desmoplasia. In vivo mouse xenografts verified the potential of this hybrid as an antitumor agent reducing tumor volume significantly. Conclusion: Our findings strongly suggest that the newly synthesized 5-FU-TQ hybrid might become a promising anticancer drug by directly targeting CSCs.

#1905

Possible cancer stem cells: Folate hydrolase-1 is expressed in a subset of Oct4-positive melanoma cells.

Marigdalia K. Ramirez-Fort,1 He Liu,2 Vicente Navarro,2 Barbara Meier,3 Mitch Levesque,4 Jonathan Moy,2 Jessica Vissicchio,5 Emmanuel Contassot,3 Sae Kim,2 Talal Syed,1 Michael Zhang,1 Vincent Tem,1 Paul J. Christos,2 Scott T. Tagawa,2 Neil H. Bander,2 Christopher S. Lange,1 Lars E. French3. 1 _State Univ. of New York, Brooklyn, NY;_ 2 _Weill Cornell Medical College, New York, NY;_ 3 _University Hospital Zurich, Zurich, Switzerland;_ 4 _University Hospital of Zurich, Zurich, Switzerland;_ 5 _Emory University, GA_.

Background: Folate hydrolase-1 (FOLH1; NAALADase; PSMA) is a type II transmembrane protein that binds substrates with terminal glutamates. The MXXXL motif on the cytoplasmic N-terminal domain of FOLH1 interacts with clathrin and caveolin-1 to facilitate constitutive internalization upon substrate binding. J591, an established monoclonal antibody (AB) to FOLH1, is highly specific to and is effectively endocytosed after binding to the extracellular domain of FOLH1; J591 is presently being developed in the clinical trial setting, as a vehicle for AB-based brachytherapy in cancers that express FOLH1.

Physiological FOLH1 is expressed in cellular regions that are protected by tight-junctions or the blood-brain-barrier, and are therefore not targeted by circulating J591. Functionally, FOLH1 is responsible for cerebral glutamate production. In the oncological setting, FOLH1 is upregulated throughout prostate cancer cellular membranes, and is luminally expressed by cancer neovessels.

We have characterized neovascular FOLH1 expression in malignant melanoma (MM), a solid tumor of neural crest (NC) origin. Comparative RTqPCR analysis of normal skin, primary (p), and metastatic (m) MM revealed respective 10.64 and 18.21 -fold increases in FOLH1 gene-expression in pMM (p=0.0041) and mMM (p=0.042) samples as compared to normal skin. Immunohistochemistry of paraffin-embedded MM tumors revealed FOLH1 protein expression in the neovasculature of 4/11 (36%) of pMM and 9/14 (64%) of mMM cases; we noted a subset of keratinocytic and melanocytic FOLH1 positive cells.

These results, and the known functional role of glutamate production in a subset of NC-derived tissue, suggest that MM cells may also express cellular FOLH1.

Methods: Six (3 BRAF+; 3 BRAF-) MM cell lines were evaluated for cellular FOLH1 and Oct4 expression with J591-based and anti-Oct4 AB immunofluorescence (IF). Oct4 is a homeodomain binding protein encoded by POU5f1, widely accepted as a stem cell marker. FOLH1\+ cell lines with permeablized and non-permeablized membranes were incubated with J591. The effect of γ-irradiation (0, 4.5, or 9 Gy) was also tested in FOLH1\+ cell lines with harvesting 6 or 24 hours post-irradiation.

Results: Ten to 40% of MM cells demonstrated intracellular FOLH1 expression in 2 BRAF-/6 MM cell lines, with a predominant perinuclear and terminal dendritic distribution (e.g., axonal morphology). IF co-labeling with J591 (cytoplasmic) and antibodies to Oct4 (nuclear) identified possible stemness of FOLH1+ / Oct4\+ MM cells. RT-PCR analysis of the irradiated cell lines demonstrated ~2-fold increased FOLH1 mRNA levels in 1/2 MM cell lines.

Conclusions: Herein is the first demonstration that MM cells express FOLH1. Given that FOLH1 glutamate production is closely linked to NC-originating glial cells, it can be reasonably postulated that this newly identified subset of FOLH1+/Oct4+ MM cells could be NC precursors, or cancer stem cells, for MM. Further, radiation-induced FOLH1 upregulation may increase the therapeutic ratio of AB-based brachytherapy.

#1906

The role of immunogenic SPANX antigens in distinct cancer stem cell subsets within triple negative breast cancers.

Lauren Shahin, Elena Cubedo, Ebony Coats, Jeff Boyd, Diana Azzam. _Florida International University, Miami, FL_.

Breast cancer is the leading cause of death in women, primarily due to metastatic disease rather than the primary tumor. Median survival with metastatic breast cancer is 3 years, with no statistically significant change in survival in over 20 years. Triple-negative breast cancer (TNBC) lacks estrogen and progesterone receptor expression and ERBB2 amplification and is the most lethal form of breast cancer. It is resistant to endocrine therapy and chemo-resistance and metastasis invariably emerge. Increasing evidence indicates that cancer stem cells (CSCs) are chemo-resistant and initiate metastasis. We have previously identified and characterized distinct subsets of CSCs within TNBC cell lines and patient-derived tumors. Within the surface CD44+ populations, CD24 expression defines two subsets of CSCs: mesenchymal-like CD24neg and epithelial-like CD24low+. Epithelial-like CD24low+ cells are more aggressive than CD24neg cells, with increased self-renewal, migration, invasion, tumor-initiation and metastatic potential. In addition, CD24low+ cells are enriched following chemo- and radiation therapies; greater than 80% of CD24neg cells die and the majority of cells that survive are CD24low+. Hence, therapies that selectively eliminate the CD24low+ population in TNBC have the potential to be of enormous benefit to cancer patients. Therapeutic targeting of surface CD24 using antibodies have not been successful due to the expression of CD24 on many different cell types including B cells. Thus, there is a need for more selective strategies to target CSC populations without affecting normal cells. Here, we show for the first time, increased expression of a family of cancer/testis antigens (CTAs) namely SPANX in the most aggressive CD24low+ CSC population. CTAs belong to a class of testis-derived proteins which are only expressed in germ cells in the male testis, and the expression of CTA genes is entirely silenced in the adult somatic tissues. Thus, SPANX may serve as a selective marker for targeting CSCs. In addition, we demonstrate a functional role for SPANX in mediating CSC phenotype. Knockdown of SPANX decreased the percent of CD24low+ CSCs in TNBCs. It also attenuated the proliferation, migration and invasion of CD24low+ cells. Radiation treatment increased SPANX expression levels and enriched for CD24low\+ cells. Loss of SPANX resulted in increased cell death of CD24low+ cells after radiation treatment. Hence, we show that SPANX may be promoters of the most aggressive CSC subset of TNBC. Since SPANX is highly immunogenic, our data provide rationale for further testing of combined radiation and immunotherapy approaches in the treatment of this deadly cancer. It also supports the use of protective and therapeutic SPANX vaccines against the most aggressive CSC subset in TNBC.

#1907

Identification of a cancer stem cell-specific function for the histone deacetylases HDAC1 and HDAC7 in breast and ovarian cancer.

Abigail E. Witt,1 Corrado Caslini,1 Chung-Wei Lee,2 Tong Ihn Lee,3 Diana J. Azzam,4 Bin Wang,1 Fabio Petrocca,5 James Grosso,1 Michelle Jones,1 Evan B. Cohick,2 Adrienne B. Gropper,3 Claes Wahlestedt,4 Andrea L. Richardson,2 Ramin Shiekhattar,6 Richard A. Young,3 Tan A. Ince1. 1 _Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami, FL;_ 2 _Brigham and Women's Hospital, Harvard Medical School, Boston, MA;_ 3 _The Whitehead Institute for Biomedical Research, Cambridge, MA;_ 4 _Center for Therapeutic Innovation, University of Miami Miller School of Medicine, Miami, FL;_ 5 _Boston Children's Hospital, Harvard Medical School, Boston, MA;_ 6 _University of Miami Miller School of Medicine, Sylvester Comprehensive Cancer Center, Biomedical Research Building, Miami, FL_.

Tumors are comprised of a highly heterogeneous population of cells, of which only a small subset of stem-like cells possess the ability to regenerate tumors in vivo. These cancer-stem-cells (CSCs) represent a significant clinical challenge as they are resistant to conventional cancer therapies and play essential roles in metastasis and tumor relapse. Multiple signaling pathways involved in the regulation of CSCs have been identified. However, the regulation of CSCs is unlike the reversible short-term changes in cellular phenotype induced by various extracellular factors, or the permanent changes induced by mutations. The hierarchical differentiation of CSCs to non-stem-tumor-cells (nsTCs) is long-lasting over many cell generations but it is also reversible, that is more akin to tissue differentiation, which suggests that epigenomic factors such as histone modifications may also be involved in the regulation of the CSC phenotype. Here, we present evidence that specific histone deacetylases (HDACs) play essential roles in the breast and ovarian CSC phenotype. HDACs are chromatin-modifying enzymes that are involved in regulation of differentiation, autophagy, apoptosis, migration, mitosis, and angiogenesis. Utilizing a novel breast CSC model composed of isogenic BPLER (predominantly CSC) and HMLER (predominantly nsTC) cell line pairs, along with conventional breast and ovarian cancer cell lines, we discovered that among the 11 family members, HDAC1 and HDAC7 are specifically over-expressed in FACS-sorted breast CSCs when compared to unsorted cells. Furthermore, we found that HDAC1 and HDAC7 are necessary to maintain breast CSCs and that overexpression of HDAC7 is sufficient to augment the CSC phenotype. We also demonstrate that members of the benzamide class of HDAC inhibitors (HDACi) targeting HDAC1/2/3 enzymatic activity, such as MS275 and MGCD0103, can be used to preferentially target breast and ovarian CSCs and we are now trying to elucidate the molecular mechanisms behind the CSC-restricted downregulation of HDAC7 and c-Myc transcript and protein levels found in response to MS275 treatment. So far, limited clinical studies have been completed with these HDACi, predominantly in Leukemia and Lymphomas. Our results indicate that these drugs should be tested more extensively in solid tumors. Furthermore, in standard trial design with cytostatic and cytotoxic drugs, the reduction in tumor size has been one of the main metrics for monitoring patient response. Since the CSC-specific actions of MS275 and MGCD0103 were previously unknown, CSC biomarkers were not used in patient selection or for monitoring therapy response. Our results suggest that biomarkers such as HDAC1 and HDAC7 are particularly compelling as a supplement to tumor size measurements. Therefore, it would be feasible to use HDAC7 immunohistochemistry to stratify patients in future clinical trials.

#1908

FGFR inhibitor, AZD4547, impedes the stemness of mammary epithelial cells in the premalignant tissues of MMTV-ErbB2 transgenic mice.

Qingxia Zhao,1 Amanda B. Parris,1 Erin W. Howard,1 Ming Zhao,1 Ying Xing,2 Zhikun Ma,1 Xiaohe Yang1. 1 _North Carolina Central University, Kannapolis, NC;_ 2 _Basic Medical College of Zhengzhou University, Henan, China_.

The fibroblast growth factor receptor (FGFR) family (FGFR1-4) of receptor tyrosine kinases (RTKs) regulates signaling pathways involved in cell proliferation and differentiation. In particular, FGFR1 and FGFR2, which are found in the terminal end buds of developing mammary ducts, play a role in mammary development and glandular morphogenesis involving the regulation of mammary stem cells (MaSCs) in mice. As such, a number of FGFR inhibitors are being tested in preclinical studies and clinical trials for anti-tumor properties. Nevertheless, reports on FGFR inhibitor-mediated breast cancer prevention are sparse. In this study, we aimed to investigate the anti-cancer benefits of AZD4547, a small molecule inhibitor of FGFR1-3, on ErbB2-overexpressing breast cancer models. We particularly focus on the effects of AZD4547 on MaSCs and tumor-initiating cells (TICs) in the premalignant tissues of MMTV-ErbB2 transgenic mice. We first demonstrated the anti-proliferative effects of AZD4547 (1-5 µM) on human ErbB2-overexpressing breast cancer cell lines. We further showed that AZD4547 confers potent inhibition of the stemness of these breast cancer cells, as indicated by significant depletion of ALDH+ cells and impaired tumorsphere formation. To study the in vivo effects of AZD4547 on the stemness of mammary epithelial cells (MECs), MMTV-ErbB2 transgenic mice were administered AZD4547 (2-6 mg/kg/day) for 10 weeks (weeks 8-18 of age) during the 'risk window' for mammary tumor development. Histopathological analysis indicated that AZD4547 significantly inhibited ductal branching and MEC proliferation. To examine the effect of AZD4547 on MEC subpopulations and tissue hierarchy dynamics in the premalignant mammary tissues of this model, we performed flow cytometry analyses on the primary MECs using CD24/CD49f and CD61/CD49f cell surface markers. The results showed that AZD4547 treatment substantially reduced MaSC-derived luminal and myoepithelial cell populations. AZD4547 also selectively suppressed the CD61highCD49fhigh cell population, which is enriched with luminal progenitor cells that give rise to TICs during MMTV-ErbB2 mammary tumor development. Mammosphere and colony-forming cell (CFC) assays on primary MECs demonstrated that the stemness of these cells was also blocked by AZD4547 prior to malignant transformation. Consistently, AZD4547 inhibited the anchorage-independent growth of cells from spontaneous tumors. Moreover, we demonstrated that AZD4547 downregulated multiple pathways, including the inactivation of FGFR, EGFR, and Wnt/β-catenin signaling. Collectively, the morphogenic, MaSC/TIC, and signaling regulation associated with AZD4547 treatment provides critical evidence for AZD4547 as a breast cancer preventative and therapeutic agent, which ultimately reveals clues for more effective eradication of refractory mammary tumors.

#1909

Non-alcoholic steatohepatitis promotes EpCAM positive cancer stem cells mediated tumorigenesis in immunocompetent mouse model of hepatocellular carcinoma.

Harshul Pandit, Yan Li, Guozhen Cui, Suping Li, Salina Li, Robert C. Martin. _University of Louisville School of Medicine, Louisville, KY_.

Introduction: Non-alcoholic Steatohepatitis (NASH) is one of the major risk factors for cirrhosis and its progression to Hepatocellular Carcinoma (HCC). Cancer stem cells (CSCs) are sub-population of cells that bear stem-like properties, and are believed to contribute in tumor initiation and recurrence in tumor microenvironment. Understanding role of CSCs in tumorigenesis was limited to immunocompromised nude/SCID mouse models, and should be investigated in immunocompetent mouse model(s) representing clinical conditions. In this study, (1) we have investigated and confirmed CSCs properties in HCC cells lines, and (2) investigated tumor initiation capability of EpCAM positive CSCs in novel immunocompetent mouse model of HCC. Methods: In vitro CSC enrichment was achieved by treating murine (Hepa1-6) and human (HepG2, Hep3B) HCC cells in serum-free condition. To confirm CSCs, we analyzed CSC biomarkers (EpCAM,CD90,CD44,CD133) using flow-cytometry and Immunocytochemistry (ICC), and functional markers using Aldeflour assay and Hoechst-33342 efflux. Drug resistance property of CSCs was studied using Doxorubicin (anthracycline antibiotic) and Sorafenib (multikinase inhibitor) by MTT assay. To study in vivo tumorigenesis, immunocompetent mouse model was established using C57L/J mouse and copGFP expressing Hepa1-6 cells. Capability of EpCAM CSCs for tumor initiation was tested in 3 diet induced groups, i.e. control (10% dietary fat), high-fat (60% dietary fat), and NASH (60% dietary fat MCD diet with 0.1% methionine). 2 million FACS sorted EpCAM+/+ or EpCAM-/- cells were orthotopically injected into left liver lob. Tumor growth was monitored using high-frequency ultrasound and animals were euthanized after 18 days. Histology and ICC analysis was performed to confirm the tumors and findings. Results: Spheroid forming HCC-CSCs showed significant higher EpCAM expression and significant higher chemoresistance compared with control HCC cells. Our in vivo findings confirmed that EpCAM CSCs required NASH microenvironment. In NASH group mice, EpCAM+/+ CSCs have shown significant tumorigenesis compared with no tumors in EpCAM-/- (n=9, p<0.005). Histology analysis of NASH liver tissue confirmed lobular and portal inflammation, hepatocellular ballooning, and fibrosis. Control and high-fat diet group mice failed to develop tumors. Conclusions: We have developed a novel mouse model to study CSCs to overcome confounding limitations of immunocompromised mouse models for lacking functional immune system components. Our findings using immunocompetent mouse model suggest that, in HCC, (1) EpCAM+ CSCs cannot initiate tumor by itself within normal liver microenvironment in the presence of functional immune components, (2) NASH microenvironment promotes EpCAM+ CSCs mediated tumorigenesis while EpCAM-/- non-CSCs subset failed to develop tumor.

#1910

NF-kappaB classical versus alternative pathways support distinct populations of ovarian cancer tumor-initiating cells.

Carrie D. House, Christina M. Annunziata. _National Cancer Institute, Bethesda, MD_.

Ovarian cancer is the most lethal gynecological malignancy in the United States with high morbidity and mortality due to recurrence and chemoresistance. Our data suggest tumor-initiating cells (TICs) play an important role in disease biology. We previously showed that a subset of ovarian cancer cells depends on NF-kappaB signaling, and that expression of NF-kappaB proteins is associated with poor survival. Given that NF-kappaB expression correlates with a poor outcome in ovarian cancer, and NF-kappaB activity supports drug resistance and tumorigenicity, we hypothesize that NF-kappaB supports a TIC program responsible for ovarian cancer relapse. To investigate this pathway in TICs, we designed a novel method to enrich for TICs from cell lines and patient samples by culturing non-adherent, floating cells in stem cell conditions defined by low attachment flasks and serum free media. Preliminary data confirm that these cells have higher stem cell marker expression, are chemoresistant, and are more tumorigenic in nude mice compared to their adherent counterparts. These TIC-enriched culture conditions enhance NF-kappaB expression and activity. More specifically, alternative NF-kappaB signaling through the RelB transcription factor supports TIC populations by regulating aldehyde dehydrogenase (ALDH), an enzyme with high activity in TICs. Using an inducible shRNA targeting RelB we show that spheroid formation, ALDH expression and activity, chemoresistance, and tumorigenesis in both subcutaneous and intrabursal xenograft models, significantly decreased in the absence of RelB. Interestingly, loss of classical NF-kappaB signaling, through an shRNA targeting the RelA transcription factor, was less effective in targeting TICs, but more effective at targeting a proliferative subpopulation with high Ki67 staining. We conclude that classical and alternative NF-kappaB signaling support ovarian tumor cells with distinct phenotypes and the collaboration of these pathways may be critical for supporting tumor repopulation following chemotherapy. Current studies will measure system level changes induced by these shRNAs, and identify a gene signature specific to each NF-kappaB pathway in ovarian TICs. Clarifying the nuances of NF-kappaB signaling in TICs will increase our understanding of ovarian cancer recurrence and further focus therapeutic strategies to prevent relapse.

#1911

R-SPONDIN3 antagonism sensitizes colorectal cancer to taxane treatment.

Marcus M. Fischer, V. Pete Yeung, Fiore Cattaruzza, Christopher Murriel, James W. Evans, Gilbert O'Young, Alayne Brunner, Min Wang, Belinda Cancilla, Ann Kapoun, Timothy Hoey. _OncoMed Pharmaceuticals, Inc., Santa Clara, CA_.

Introduction: Colorectal cancer (CRC) is the third most common cancer diagnosed in both men and women. Dysregulation of the WNT/β-catenin pathway is the most commonly mutated pathway in CRC. R-spondins (RSPO), a family of secreted proteins, are enhancers of WNT signaling. Previous preclinical studies have demonstrated that WNT inhibition results in superior antitumor efficacy in combination with taxanes. This superior combinatorial activity with taxanes has been identified in lung, ovarian, breast and pancreatic cancers. Taxanes have been found to be generally ineffective in the clinical treatment of colorectal cancer. Analysis of the local microenvironment, or stem cell niche, has identified stromal cells as an abundant source of RSPO. In patient-derived tumor xenografts (PDX), the murine stromal cells replace human stromal cells and provide the microenvironment that supports tumor cell growth. We have classified PDX tumors as human RSPO3 high or low/negative using RNA sequencing and by expression studies. Inhibition of RSPO3 with OMP-131R10, a clinical stage therapeutic antibody which binds to human and murine RSPO3, has demonstrated efficacy in human RSPO3 high, APCWT PDX.

Purpose: Common features in CRC are mutations in APC or β-catenin which are present in approximately 90% of cases. Tumors with RSPO3 genomic translocation and/or overexpression of RSPO3 are generally APC WT and β-cateninWT, and thus RSPO overexpression represents an alternative mechanism to upregulate the Wnt pathway. Alternatively, RSPO3 may be secreted by murine stromal cells, or by a rare population of tumor cells. In this study, we tested OMP-131R10 in combination with taxane treatment in human RSPO3 low CRC PDX models with APC or β-catenin mutations and in a PDX model with a RSPO3 translocation.

Results: Anti-RSPO3 was highly effective in combination with nab-paclitaxel in a CRC model with an RSPO3 translocation and RSPO3 overexpression. Furthermore, the combination of OMP-131R10 and taxane treatment resulted in synergistic inhibition of tumor growth in 8/10 PDX models with APC or β -catenin mutations and low human tumor cell RSPO3 expression. Tumor regression or durable stable disease was evident in 6/8 models. Responsive models contained either heterozygous inactivating mutations in APC (6/8) or homozygous activating mutations in β-catenin (2/8). Combination of OMP-131R10 with paclitaxel potentiated mitotic arrest, enhanced terminal differentiation and reduced the tumorigenic cell frequency by 40 fold based on serial transplantation studies.

Conclusion: Anti-RSPO3 (OMP-131R10) was active in the majority of CRC PDX models tested when combined with taxane treatment. These CRC models harbored mutations in APC and β-catenin. This data indicates that RSPO3 inhibition is an effective means to target the mutated Wnt pathway in CRC, and that antagonizing RSPO3 in combination with paclitaxel may improve survival for CRC patients.

#1912

**Actn4, a novel therapeutic target of dietary ellagic acid, promotes breast cancer metastasis** via **mediating cancer stem cells.**

Neng Wang,1 Zhiyu Wang,2 Xiaoming Xie3. 1 _Sun Yat-sen University Cancer Center; University of Guangzhou Traditional Chinese Medicine, Guangzhu, China;_ 2 _University of Guangzhou Traditional Chinese Medicine, Guangzhu, China;_ 3 _Sun Yat-sen University Cancer Center, Guangzhu, China_.

Purpose: Pharmacology-based target identification has become a novel stratey leading to the discovery of novel pathological biomarkers. Ellagic acid (EA), a dietary polyphenol compound, exhibits potent anticancer activities, whereas the underlying mechanisms remain unclear. This study sought to determine the role and regulation of ACTN4 expression in human breast cancer metastasis and EA-based therapy.

Experimental Design: The anti-metastasis ability of EA was validated by MMTV-PyMT mice and in vitro cell models. Drug affiity responsive target stability (DARTS) was utilized to identify ACTN4 as the direct target of EA in breast breast cancer stem cells (CSCs). The metastatic regulated mechanisms of ACTN4 were assessed by CSC-related assays including mammosphere formation, tumorigenic ability, reattachment differentiation and signaling pathway analysis. The clinical significance of ACTN4 was based on human tissue microarray analysis and TCGA database exploration.

Results: EA inhibited breast cancer growth and metastasis via directly targeting ACTN4 in vitro and in vivo, accompanied with limited CSCs population. ACTN4 knockdown resulted in blockage of malignant cell proliferation, colony formation and ameliorated metastasis potency. ACTN4 positive CSCs exhibited higher ESA+ proportion, increased mammosphere-formation ability, as well as enhanced in vivo tumorigenesis ability. Increased ACTN4 expression was directly associated with later cancer stage, increased incidence of metastasis, and poor overall survival period.

Conclusions: ACTN4 plays an important role in breast CSCs-related metastasis and is a novel therapeutic target of EA treatment.

#1913

SOX9 regulates cancer stem-like cells in non-small cell lung cancer.

Maria Voronkova,1 Sudjit Luanpitpong,2 Heimo Riedel,1 Yon Rojanasakul1. 1 _West Virginia University, Morgantown, WV;_ 2 _Mahidol University, Bangkok, Thailand_.

Lung cancer is the leading cause of cancer death worldwide in both men and women, every year killing more people than breast, prostate and colon cancers combined. Drug/radiation resistance and tumor relapse contribute to low patient survival, which has largely been attributed to the acquisition of stem-like cells (CSCs), or tumor initiating cells. Moreover, conventional therapies are not effective against CSCs. This signifies the need to identify mechanisms of CSC regulation which could lead to the discovery of CSC-specific drug targets and the development of more effective drug therapies. An embryonic transcription factor SOX9 has been implicated in CSC regulation in a number of cancer types, but little is known about its role in non-small cell lung cancer (NSCLC). We demonstrated that high SOX9 expression correlates with poor survival in lung cancer patients by analyzing The Cancer Genome Atlas (TCGA) data. We hypothesized that SOX9 plays a key role in tumor progression and chemoresistance by upregulating CSCs in NSCLC. To test this hypothesis, SOX9 expression was stably or transiently knocked down in NSCLC cells, and their effects on CSC formation and biomarkers, and on drug resistance were investigated. Our results showed that SOX9 knockdown decreased the number of tumor spheres in NSCLC cell lines. Furthermore, SOX9 knockdown downregulated the expression and activity of stem cell marker ALDH1A1 as determined by Western blot and flow cytometric assays. Consistent with this finding, overexpression of SOX9 in non-cancer lung epithelial Beas-2B cells elevated tumor sphere formation, supporting the role of SOX9 in CSC regulation. Analysis of the soft agar colony formation assay further showed that SOX9 knockdown inhibited anchorage-independent growth in NSCLC cells. More importantly, SOX9 knockdown resulted in an increased sensitivity of the cells to a chemotherapeutic drug cisplatin, whereas SOX9 overexpression decreased it. Together, our results indicate an important role of SOX9 in the regulation of CSCs in NSCLC and suggest its potential utility as a drug target for chemoresistant lung cancer.

#1914

GNS561 is a new quinoline derivative with high efficacy on cancer stem cells from colorectal liver metastasis and hepatocellular carcinoma.

Firas Bassissi,1 Elena Patricia Gifu,2 Sonia Brun,1 Jerome Courcambeck,1 Antoine Beret,1 Jean Marc Pascussi,3 Julie Pannequin,3 Eric Raymond,1 Philippe Halfon,1 Philippe Merle,4 Claude Caron de Fromentel2. 1 _Genoscience Pharma, Marseille, France;_ 2 _CRCL, INSERM U1052 - CNRS 5286, Université Lyon1 - Centre Léon Bérard, Lyon, France;_ 3 _Institut de Génomique Fonctionnelle (IGF), Montpellier, France;_ 4 _CRCL, INSERM U1052 - CNRS 5286, Université Lyon1 - Centre Léon Bérard, Hospices Civils de Lyon, Lyon, France_.

Background: In spite of wide application of sorafenib for advanced hepatocellular carcinoma (HCC) treatment, and systemic chemotherapy cocktails (5-fluorouracil, irinotecan, and oxaliplatin) for metastatic colorectal cancer, the prognostic for both cancers remains poor. In recent years, highly tumorigenic sub-populations of cancer cells named Cancer Stem Cells (CSCs) have been claimed as responsible of some tumor recurrences. Indeed, CSCs are resistant to chemotherapy, and they have the ability to regenerate all the tumor bulk with its heterogeneous cell type populations. For this reason, new drugs with original mechanism of action which target CSC properties would likely improve cancer treatment

Material and methods: Antitumor activity of GNS561 was tested on a panel of cancer cell lines and primary tumors. GNS561 impact on CSCs subpopulation in patient derived cells from colorectal hepatic metastatic tumors was assessed by flow cytometry (ALDH activity). In HCC, the effect of this drug was evaluated by sphere formation assay as readout to estimate CSC survival. Tolerance and plasma and liver pharmacokinetic were evaluated after single and repeated dosing in mice and rats. In vivo GNS561 activity was tested in orthotopic mouse model

Results: GNS561 demonstrated multiple cellular effects such as inhibition of autophagy, induction of apoptosis and cell cycle modulation. It showed antitumor activity against several human cancer cell lines. Furthermore, GNS561 was effective against a panel of HCC tumors even from patients harboring sorafenib resistance. GNS561 showed nonetheless an original dose-response cytotoxic activity against the whole tumor populations but also against a subpopulation displaying high ALDH activity in three CRC patient-derived cell lines established from fresh liver metastasis biopsies. Consequently, on the same model this compound induced a striking decrease of sphere formation. In HCC cell lines GNS561 was active on both whole populations (mean EC50 2µM) and subpopulations displaying CSC features (Epcam high). In addition, in the opposite of sorafenib, GNS561 decreases the HCC cell capacity to form spheroids. In mouse, GNS561 was found well tolerated and highly selectively trapped in the liver (exposure ratio liver/plasma about 170 animals). In HCC PDX mouse model, tumor growth was significantly reduced by GNS561 with a dose-response manner, this tumor regression was associated with AFP level decreases by 72% with GNS561 (p=0.002) and 54% with sorafenib (p=0.046) compared to control

Conclusions: GNS561 is a liver selective drug which offers great promise for HCC and liver metastatic tumors treatment. By simultaneously targeting the cancer stem cell subpopulation and tumor bulk, both cell heterogeneity, plasticity and recurrences could be overcome at least in colorectal cancer and HCC. GNS 561 is now aimed to further reach clinical development in patients in 2017

#1915

Deconstructing cancer stem cells: IL-6, G-CSF and Activin-A as mediators of stroma-orchestrated epithelial cells' dedifferentiation.

Carlos F. Rodrigues,1 Eurico Serrano,2 Marco Cunha,1 Maria I. Patrício,3 Mariana Val,4 João Fonseca,1 Célia Gomes,5 Antero Abrunhosa,6 Artur Paiva,7 Filomena Botelho,2 Lina Carvalho,2 Isabel M. Carreira,4 Alpoim Carmen1. 1 _Centre for Neursociences and Cell Biology, Coimbra, Portugal;_ 2 _Center for Research in Environment, Genetics and Oncobiology (CIMAGO), Coimbra, Portugal;_ 3 _Nuffield Laboratory of Ophthalmology, NDCN & NIHR Oxford Biomedical Research Centre, University of Oxford, Oxford, United Kingdom; _4 _Cytogenetics and Genomics Laboratory, Faculty of Medicine, University of Coimbra, Coimbra, Portugal;_ 5 _Institute for Biomedical Imaging and Life Sciences, University of Coimbra, Coimbra, Portugal;_ 6 _Institute of Nuclear Sciences Applied to Heath (ICNAS), Univers, Coimbra, Portugal;_ 7 _Centro Hospitalar Universitário de Coimbra (CHUC), Coimbra, Portugal_.

Intercellular communication in cancer is a deceiving and extremely efficient process through which the corporal machinery is hijacked by a panoply of cellular and molecular strategies turning the entire human body into an evolutionary arena. The extensive crosstalk mediated by cytokines and chemokines overcome the inefficiency of the invasion-metastasis cascade, thus allowing the development of the often-fatal metastatic disease. Cancer stem cells (CSCs) have recently been implicated in major steps of the tumorigenic process, namely in tumor initiation, metastasis formation and tumor relapse following therapy. Yet, despite their relevance, the exact mechanisms underlying their formation are still unclear. After observing that the malignant human bronchial epithelial RenG2 cells dedifferentiated following culture in the subcutaneous mouse lumbar region, co-cultures of surgically isolated mice lumbar stromal cells with RenG2 cells were established and the conditioned media studied by multiplex and ELISA. Consequently, Interleukin-6 (IL-6), Granulocyte colony-stimulating factor (G-CSF) and Activin-A were identified as the paracrine mediators of the intercellular communication process. Aiming to ascertain the individual role of each cytokine in the dedifferentiation process, as well as to access the involvement of exosomes as transport vehicle, the same co-cultures were reproduced in the presence of specific cytokine-communication blockers, either individually or in combinations of up to three blockers, and exosome-mediated communication inhibitors. Finally, exosomes were also collected from control co-cultures and their cargo screened for the target cytokines. ELISA showed that the three cytokines were present inside fibroblasts-secreted exosomes. Moreover, whenever exosomes' release was blocked, dedifferentiation was abrogated, further proving the role of the aforementioned cytokines and of exosomes in the dedifferentiation process. Additionally, the cytokine-blocking experiments revealed that only IL-6 and Activin-A were endowed with the potential to orchestrate dedifferentiation, as when at least one of these cytokines was present a stem cell population developed inside RenG2 cells. Finally, G-CSF appeared to be decisive in maintaining the undifferentiated phenotype, as a larger pool of CSCs was attained whenever this cytokine and either IL-6 or Activin-A were present. Altogether the attained results implicated IL-6 and Activin-A in the formation of CSCs by dedifferentiation, and G-CSF as a potent keeper of the dedifferentiation status. Subsequent studies are now required to access the use of these cytokines as therapeutic targets so the tumorigenic process may be abrogated in its initial steps, improving patients' prognosis and survival.

Work sponsored by FEDER, POFC-COMPETE and the FCT grants PTDC/BBB-BQB/2450/2012 and SFRH/BD/33884/2009.

#1916

Peptidylarginine deiminase IV (PADI4) is a novel tumor suppressor that may contribute to regulation of breast cancer stem cell dynamics.

Nellie Moshkovich. _NCI, Bethesda, MD_.

Breast cancer is a global problem that accounts for almost a fourth of all cancers in women. Despite the many therapeutic strategies currently in use, treated patients often suffer from cancer relapse and metastasis due to the presence of a subpopulation of tumorigenic cells capable of self-renewal and termed cancer stem cells (CSCs). To address the properties of this critical sub-population, we utilized our recently-developed lentiviral CSC reporter carrying an Oct4/Sox2 response element coupled to a green fluorescent protein (SORE6-GFP) to detect and isolate the CSC population in a low-grade ER+ human breast cancer model, MCF10Ca1h, by FACS. Whole transcriptome sequencing analysis (RNA-seq) on the sorted cells identified Peptidylarginine deiminase IV (PADI4) as differentially expressed between CSCs and non-CSCs. PADI4 was recently shown to regulate citrullination of histone H1, displacing it from chromatin and resulting in global chromatin decondensation and pluripotency induction in embryonic stem cells (Christophorou, et al., 2014, Nature 507:104). Thus, we hypothesized that PADI4 may also regulate the stemness phenotype in breast cancer CSCs, likely through effects on chromatin structure. We showed that PADI4 protein expression is higher in luminal breast cancer cell lines than other subtypes, and we did shRNA knockdown (KD) of PADI4 in the luminal MCF10Ca1h model. PADI4 KD increased tumorosphere formation and clonogenicity indicating increased CSC population/activity. Additionally, MCF10Ca1h cells treated with a PADI4 selective inhibitor showed an increase in CSCs as assessed using the SORE6-GFP stem cell reporter. In vivo limiting dilution studies demonstrate that PADI4 KD increased tumor incidence at low cell inoculum, reflecting a statistically significant 2-4 fold increase in CSCs upon PADI4 KD. Our findings suggest that PADI4 may function as a tumor suppressor in luminal breast cancer. In support of this conclusion, deep deletions of PADI4 locus are found in 1% of human breast cancers in the TCGA datasets. Analysis of the effect of PADI4 KD on chromatin organization in the CSC and non-CSC compartments is ongoing.

#1917

AXL inhibition in ovarian cancer stem-like cells increases chemo-sensitivity.

Jin-Young Kim, Hyewon Chung, So-Jin Shin, Eunyoung Ha, Chi-Heum Cho. _Keimyung University School of Medicine, Daegu, Republic of Korea_.

Cancer stem-like cells (CSCs), a small population of cancer cells with regenerative potential, may lead to the initiation and progression of secondary tumors. Long-term chemotherapeutic drug treatment can also result in the generation of a drug resistant population of cancer stem cells. In this study we have isolated CD44+ and CD117+ enriched ovarian cancer stem-like cells from primary cultured ascites cells removed from ascites fluid of naïve ovarian cancer patients. AXL, a type I receptor tyrosine kinase family member, is found to be over-expressed in ovarian cancer stem-like cells. Cell viability assays showed that ovarian CSCs are chemo-resistant to concentration dependent paclitaxel treatment. AXL was inhibited using a R428 and cell viability assays and apoptotic assays in the presence of paclitaxel were performed, which made the CSCs chemo sensitive. Si-RNA based inhibition of AXL also displayed a similar pattern of improving the sensitivity of the stem-like cells to the chemotherapeutic drug. PI3-kinase/Akt signaling pathway has also been shown to be involved in decreasing the cell viability during the inhibition of AXL. In conclusion, this study establishes the role of AXL, a receptor tyrosine kinase, as one of the key proteins influencing the chemo resistance in ovarian cancer stem-like cells.

#1918

**Gene characterization of lung-tumorspheres for their usage as an** in vitro **screening platform for testing new therapeutic strategies.**

Alejandro Herreros-Pomares,1 Ester Munera-Maravilla,1 Silvia Calabuig-Fariñas,2 Rut Lucas,3 Rosa Farràs,4 Ana Blasco,5 Susana Torres,1 Jose Ferri,5 Ricardo Guijarro,6 Miguel Martorell,7 Eloísa Jantus-Lewintre,8 Camps Carlos9. 1 _Molecular Oncology Laboratory, General University Hospital of Valencia Research Fundation, Valencia, Spain;_ 2 _Molecular Oncology Laboratory, General University Hospital of Valencia Research Fundation; Department of Pathology. Universidad de Valencia., Valencia, Spain;_ 3 _Department of Science History and Documentation. Universidad de Valencia., Valencia, Spain;_ 4 _Department of Oncogenic signalling Laboratory. Centro de Investigación Principe Felipe., Valencia, Spain;_ 5 _Department of Medical Oncology, General University Hospital of Valencia, Valencia, Spain;_ 6 _Department of Thoracic Surgery, General University Hospital of Valencia, Valencia, Spain;_ 7 _Department of Pathology. Universidad de Valencia., Valencia, Spain;_ 8 _Molecular Oncology Laboratory, General University Hospital of Valencia Research Fundation; Department of Biotechnology. Universidad Politécnica de Valencia., Valencia, Spain;_ 9 _Molecular Oncology Laboratory, General University Hospital of Valencia Research Fundation. Department of Medicine. Universidad de Valencia. Department of Medical Oncology, General University Hospital of Valencia, Valencia, Spain_.

Background: Lung cancer features like treatment resistance or tumor relapse have been linked to cancer stem cells (CSCs), a population of cells with self-renewal properties, and the ability to grow forming tumorspheres in non-adherent conditions. The aim of this study was to isolate and characterize tumorspheres from lung cancer cell lines and tumor tissue from resectable non-small cell lung cancer (NSCLC) patients and to use them as an in vitro platform for drug screening.

Methods: This study was performed on cells from seven NSCLC tumor samples and five cell lines (H1650, H1993, H1395, A549 and PC9) grown in monolayer and as spheroids. The expression of 60 genes, including CSC-markers, pluripotency inducers, cell cycle regulators, invasion promoters and components of Notch, Wnt and Hedgehog pathways was analyzed by RTqPCR. Drugs commonly used in clinical guidelines (Cisplatin, Paclitaxel, Gefitinib, Erlotinib, Afatinib, Pemetrexed and Vinorelbine) and specific inhibitors of Wnt and Hedgehog pathways (Iwp2, Xav939, LDE225 and Vismodegib and Salynomicin) were tested in triplicates at 4 different concentrations. Cell viability was measured after 48h and 72h using MTS Assay, normalized to the respective mock-treated control cells and presented as percentage of control. Statistical analyses were considered significant at p<0.05.

Results: Lung tumorspheres had significant increased expression of CSC-related genes EPCAM1, CD44, CCND1, KLF4 and CDKN1A, compared to their paired-adherent cells. Likewise, epithelial to mesenquimal transition (EMT) inducer SNAI1 and integrin ITGA6 were overexpressed in spheroids too. Regarding stemness pathway, Notch pathway ligands JAG1 and DLL4 and receptors NOTCH1, NOTCH2 and NOTCH3 showed higher expression in lungspheres. In Wnt pathway, higher expression levels of WNT3, CTNNB1 and GSK3B were found in tumorspheres. No significant differences were found for the rest of genes. Drug screening showed classical anticancer drugs, such as Cisplatin, Vinorelbine or Pemetrexed, had mild cytotoxic effects on lungspheres, obtaining significant differences with the adherent-cultured cells. In contrast, the stemness pathways inhibitors IWP2, XAV939, Salinomycin and Vismodegib showed higher cytotoxic effects on spheroids than in cells grown in monolayers.

Conclusions: Lung-tumorspheres derived from cancer cell lines and primary tumor tissues show increased levels of CSC markers and components of Notch and Wnt signaling pathways compared to the cells grown in adherence. Spheroids showed resistance to classical anticancer drugs, and a greater response to inhibitors from Notch, Wnt and Hedgehog pathways, strengthening its possible use as a short-term culture platform for a simple, and cost-effective screening to investigate novel therapeutic approaches. Supported by grants RD12/0036/0025 from RTICC-FEDER, PI12-02838/PI15-00753 from ISCIII and SEOM/2012.

#1919

Twist1 modulates paclitaxel-induced cancer stem cell enrichment in triple-negative breast cancer.

Aimin Yang, Xiaoyuan He, Bradley A. Schulte, Steven L. Carroll, Stephen P. Ethier, Gavin Y. Wang. _Medical University of South Carolina, Charleston, SC_.

Triple-negative breast cancer (TNBC), the most aggressive subtype of breast cancers, is highly refractory to chemotherapy and has a high incidence of metastasis and tumor relapse. Targeted therapies for TNBC do not exist because this subtype lacks druggable hormone receptors and HER2 amplification or overexpression. Although Taxane-based chemotherapy is a standard of care for TNBC treatment, paclitaxel (Taxol)-induced cancer stem cell (CSC) enrichment presents a significant challenge to the success of breast cancer treatment. Thus, there is a critical need to understand the mechanisms by which chemotherapy induces CSC enrichment in residual tumors following anticancer therapies. Here we report that Taxol-induced enrichment for CSCs correlates well with a marked upregulation of Twist1 expression in human TNBC cells. Knockdown of Twist1 inhibits the clonogenic capacity of TNBC cells in vitro and TNBC tumorigenesis in vivo. Mammosphere formation assays indicate that silencing of Twist1 greatly diminishes the tumor sphere-forming potential of breast CSCs. Furthermore, limited dilution assays and stem cell xenotransplantation reveal that knockdown of Twist1 expression decreases the frequency of CSCs by 72-fold in human TNBC cells. More importantly, we show that silencing of Twist1 blocks Taxol-induced CSC enrichment in residual tumor cells that have survived drug treatment. Together, these results demonstrate a previously unrecognized role for Twist1 in mediating Taxol-induced CSC enrichment in residual tumors of human TNBC and suggest that targeted inhibition of the Twist1 signaling pathway may represent a novel therapeutic strategy to tackle therapy-induced CSC enrichment in breast cancer treatment.

#1920

Prostaglandin E2 accumulation enhances the expansion of ALDH1-positive cells and Kras-driven tumorigenesis in the pancreas.

Kota Arima,1 Takatsugu Ishimoto,1 Masaki Ohmuraya,2 Keisuke Miyake,1 Tsugio Eto,1 Hirohisa Okabe,1 Yuki Kitano,1 Kensuke Yamamura,1 Takayoshi Kaida,1 Katsunori Imai,1 Daisuke Hashimoto,1 Akira Chikamoto,1 Hideo Baba1. 1 _Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan;_ 2 _Hyogo College of Medicine, Nishinomiya, Japan_.

Background: Chronic inflammation is known to be a risk factor of carcinogenesis and tumor development, and it was demonstrated that oral aspirin intake reduced cancer-related death including pancreatic ductal adenocarcinoma (PDAC) through inhibiting prostaglandin synthases. On the other hand, cancer stem cells (CSCs) refer to a subset of tumor cells that have self-renewal ability and generate plenty of non-CSC cells that comprise a tumor. Prostaglandin E2 (PGE2) and inhibition of 15-PGDH, which is an enzyme degrading PGE2, promoted hematopoietic and tissue stem cell fraction, however, little is known about the role of PGE2 accumulation for CSC fraction. A number of CSC marker candidates have been explored to date, and Aldehyde dehydrogenase 1 (ALDH1) was identified as one of CSC markers in PDAC. The aim of this study is to elucidate the functional role of PGE2 in ALDH1 positive CSC fraction during tumor development in pancreas.

Methods: The clonogenic growth potential of ALDH1-positive PDAC cells was assessed in vitro by growth assays and sphere formation assays. We next investigated the expression of ALDH1 and self-renewal related genes in PDAC cell lines with PGE2 or 15-PGDH inhibitor treatment. We further conducted functional experiments using siALDH1 in PDAC progression. Furthermore, we examined the effect of PGE2 for pancreatic tumorigenesis using Kras-driven genetic mouse model treated with 15-PGDH inhibitor. Finally, ALDH1 and Ki67 expression was examined by immunohistochemistry in 121 primary surgical specimens of PDAC and analyzed a relationship with clinicopathological factors and clinical outcomes.

Results: The number of ALDH1-positive cells was significantly increased by PGE2 treatment, and PGE2 promoted growth and sphere formation potential in PDAC cells. In addition, 15-PGDH inhibitor induced PGE2 accumulation and gave rise to ALDH1-positive cells harboring high proliferating potential in PDAC cells. The growth and sphere formation potential were inhibited by silencing ALDH1 expression in PDAC cells. We next found that the expression of Nanog and Oct-4 genes was regulated by PGE2-ALDH1 signaling. Furthermore, 15-PGDH inhibitor induced ALDH1 expression and promoted PanIN formation in Kras-driven genetic mouse model. Finally, a high level of ALDH1 expression was significantly associated with large tumor size and high Ki67expression, and poor prognosis in PDAC patients.

Conclusion: Current findings suggested that PGE2 positively regulated ALDH1 expression, and the growth and sphere formation potential were promoted by regulating self-renewal related genes expression, resulting in poor prognosis of PDAC patients. Inhibiting PGE2-ALDH1 signaling could lead to the suppression of tumor growth in PDAC patients.

#1921

**Stem-like clusters of CD44** \+ **circulating tumor cells seed metastases in breast cancer.**

Xia Liu, Wenjing Chen, Rokana Taftaf, Huiping Liu. _Case Western Reserve Univ., Cleveland, OH_.

Background: Circulating tumor cells (CTCs) are cells shed from primary tumor and circulate in the peripheral blood. They are considered the seeds of metastasis. Compared to single CTCs, clusters of multiple CTCs possess 50 times higher metastatic capacity in mouse breast cancer models. However, the mechanisms underlying the metastatic promotion effect of CTC clusters are unclear. In addition, whether clustered CTCs have cancer stem cell (CSC) properties and what stem cell markers they express have not been determined.

Methods: Immunohistochemistry (IHC) was used to detect CTCs in vascular structures, and the stem cell markers in single and clustered CTCs. Lung metastases were either monitored by bioluminescence imaging (BLI) or visualized by fluorescence microscopy. Stem cell properties were examined by mammosphere assays in vitro and tumorigenic assays in vivo. Clustering assay was performed by culturing cells in Poly-HEMA coated plates (for cell lines) or collagen-coated plates (for primary cells derived from patient-derived breast tumor xenografts, PDXs), and then monitored by IncuCyte live cell dynamic imaging.

Results: CD44+ enriched circulating tumor cell (CTC) clusters were found in the lung/liver vascular structures in vivo in both metastatic breast cancer patients and PDXs that develop spontaneous lung metastases. Comparing to single CD44+ cells, clustered CD44+ cells formed more mammospheres, increased tumorigenic potential, and promoted lung colonization. Combining siRNA-mediated knockdown and CRISPR/Cas-based knockdout, we found that CD44 is required for breast tumor cell cluster formation and lung colonization upon tail vein seeding. During cluster formation, EGFR was activated, which improved both stemness and survival of clustered CTCs. Anti-EGFR antibody mimicked CD44 knockdown to inhibit cluster formation of PDX-derived tumor cells. Administration of EGFR inhibitor Erlotinib efficiently inhibited CD44+ cells-mediated spontaneous metastases to the lungs without affecting primary tumor growth.

Conclusions: Our data provide new insights into the cellular and molecular mechanisms of stem-like clustered CD44+ cells-seeded metastasis, and implicate that targeting CD44\+ CTC clusters by inhibition of EGFR activity could be a new therapeutic strategy to treat metastatic breast cancer.

#1922

S-SHIP promoter expression identifies a putative cancer stem cell population in C3(1)/Tag murine mammary tumors.

Lu Tian,1 Chann Lagadec,2 Eric Adriaenssens,2 Emmanuel Bouchaert,3 Hélène Bauderlique-Le Roy,4 Xuefen LeBourhis,2 Roland P. Bourette1. 1 _CNRS UMR 8161 - Institut de Biologie de Lille - SPES team, Lille, France;_ 2 _Cell Plasticity and Cancer - Inserm U908 - Université Lille 1, Lille, France;_ 3 _Plateforme des modèles animaux du SIRIC ONCOLille, Oncovet Clinical Research, Lille, France;_ 4 _BioImaging Center Lille Nord de France - Institut Pasteur de Lille, Lille, France_.

Breast cancer is the most common cancer in women worldwide. The isolation and characterization of breast cancer stem cells (CSC) are crucial for understanding cancer biology and revealing potential therapeutic targets. One of the major issues in the study of CSC is the lack of reliable markers. A transgenic mouse model (Tg 11.5kb–GFP) was generated using the 11.5kb s-SHIP (stem-SH2-containing 5'-Inositol Phosphatase) promoter that specifically expressed enhanced green fluorescent protein (GFP) in embryonic and various tissue stem cells. In the mammary gland, previous experiments showed that GFP labels puberty cap cells and pregnancy basal alveolar bud cells, and it has been demonstrated that these mammary GFP+ cells are activated tissue stem cells. In order to determine if s-SHIP promoter expression could also mark mammary cancer stem cells, we generated a bi-transgenic mouse model by crossing Tg 11.5kb-GFP mice with Tg C3(1)/Tag mice. Tg C3(1)/Tag mice express SV40 T antigen under the regulatory control of the rat prostatic steroid binding protein C3(1) gene. In female mice, the transgene is expressed primarily in the mammary gland. Mice develop mammary hyperplasia by 3 months of age with subsequent development of mammary adenocarcinoma by 6 months of age.

Here we show the presence of a rare population of GFP+ cells in mammary tumors of female bi-transgenic mice by using immunohistochemical and flow cytometry analysis. The GFP+ mammary cancer cells are also CD24+/CD49f+/CD29+. As compared to GFP- cells, GFP+ cells exhibit both a higher tumor sphere-forming potential, and a higher tumorigenicity when transplanted into SCID and FVB recipient mice. Moreover, upon subsequent transplantation, the GFP+ cells generated heterogeneous tumors that displayed properties similar to the primary tumor. Currently, we are conducting transcriptomic analysis of these GFP+ and GFP- cells in order to reveal differentially expressed genes. Altogether, these results suggest that s-SHIP promoter expression may be a new marker of mammary CSC that enables their identification and isolation via a single consistent parameter. Furthermore, our bi-transgenic C3(1)/Tag x 11.5kb-GFP mice provide a novel model for study of mammary cancer stem cells and investigation of potential therapeutic targets for breast cancer.

#1923

Paired isolation and expansion of CSC and CTC from primary small cell lung cancer patient tissue and blood using the 3DKUBE bioreactor platform.

Melissa Millard,1 Alina Lotstein,1 Lillia Holmes,1 David Schammel,2 Ki Chung,3 Jeff Edenfield,3 Hal E. Crosswell,1 Tessa DesRochers1. 1 _KIYATEC, Inc., Greenville, SC;_ 2 _Pathology Consultants, Greenville, SC;_ 3 _GHS, Greenville, SC_.

Surgical resection is rarely an option for small cell lung cancer (SCLC) patients as the majority present with extensive disease at diagnosis. This scarcity of patient samples suitable for research presents a significant road block for the development of SCLC targeted-therapeutics. To address the problem of tissue scarcity, we have developed a method for the isolation and expansion of cancer stem cells (CSC) and circulating tumor cells (CTC) from primary tissues and blood of SCLC patients using the 3DKUBE™ perfusion microbioreactor. We have established a label-free, combined chemical and functional selection method for the isolation of CSCs from SCLC samples, solid tumor as well as blood, that does not rely upon the bias imposed by marker-based selection. Cells enriched in this manner were further purified and expanded under optimized conditions (growth factors, ECM, scaffolding and oxygen tension) within the 3DKUBE™ perfusion microbioreactor. These isolated and expanded CSCs have maintained resistance to cisplatin and etoposide, stabilized the expression of traditional CSC markers, and been validated in vitro through serial spheroid formation assays. These CSCs are currently being characterized and compared to parental tissue through correlative genomic and phenomic analysis and validated through in vivo tumorigenesis models. These cells will be utilized to generate 3D microtumors to accurately predict SCLC drug response in vitro, a determination that is not accurately performed in conventional 2D cell culture and is inhibited by both cost and time in patient-derived xenografts (PDX)

#1924

**Caveolin-1 inhibits mammary carcinogenesis** via **suppressing c-myc-induced metabolism reprogramming in breast cancer stem cells.**

Zhiyu Wang,1 Neng Wang,2 Shengqi Wang,1 Yifeng Zheng1. 1 _Guangzhou Univ. Of Chinese Medicine, Guangzhou, China;_ 2 _Sun Yat Sen University, Guangzhou, China_.

Purpose: Caveolin-1(CAV1), a membrane constituent protein, exhibits tumor suppressor activities in multiple malignancies, whereas the underlying mechanisms remain unclear. The current study aimed to determine the significance of CAV-1 in regulating cancer metabolism and its relation to breast cancer stem cells (CSCs).

Experimental Design: The anti-carcinogenic function of CAV1 was evaluated by in vitro cell model, CAV1 knockout mice and MMTV-PyMT spontaneous breast cancer xenografts. Glycolysis activity and mitochondrial metabolism were assessed by immunoblotting, oxygen consumption and mitotacker staining analysis. Mammosphere formation, tumorigenic ability, reattachment differentiation and signaling pathway analysis were applied to study the regulation effects of CAV1 on breast CSCs. The clinical significance of CAV1 was also analyzed by human tissue microarray.

Results: In both mammary transformed cells and spontaneous breast cancer xenografts, CAV1 is found significantly downregulated and positively correlated to increased glycolysis activity and impaired mitochondrial metabolism. Cav1 knockout results in increased ductal hyperplasia, associating with increased stem cell population and glycolysis metabolism. Breast CSCs exhibits glycolytic phenotype and decreased CAV1 expression compared to normal mammary stem cells, and CAV1 overexpression significantly limits CSCs' self-renewal via inhibiting c-myc induced glycolysis. Clinical investigation suggests that high CAV1 expression is revealed with better overall survival and decreased CSCs population.

Conclusions: CAV1 loss facilitates mammary carcinogenesis via enhancing glycolytic activity in breast CSCs, and CAV1 based therapy might become a novel strategy for breast cancer prevention.

#1925

Modeling physiologic microenvironments in three-dimensional microtumors facilitates brain tumor initiating cell maintenance.

Ashley Gilbert,1 Kiera Walker,2 Anh Tran,2 Yancey Gillespie,2 Raj Singh,1 Anita B. Hjelmeland2. 1 _Vivo Biosciences, Birmingham, AL;_ 2 _Univ. of Alabama at Birmingham, Birmingham, AL_.

Development of effective novel anti-tumor treatments will require improved in vitro models that incorporate physiologic microenvironments and maintain intratumoral heterogeneity including tumor initiating cells. Brain tumor initiating cells (BTIC) are a target for cancer therapy because they are highly tumorigenic and contribute to tumor angiogenesis, invasion, and therapeutic resistance. Current leading studies rely on BTIC isolation from patient-derived xenografts followed by propagation as neurospheres. As this process is expensive and time-consuming, we determined whether three-dimensional microtumors were an alternative in vitro method for modeling tumor growth via BITC maintenance and/or enrichment. Brain tumor cells were grown as neurospheres or as microtumors produced using a human-derived biomatrix HuBiogelTM and maintained with physiologically relevant microenvironments. Percentages of BITCs were determined based on cell surface marker expression (CD133), label retention (carboxyfluorescein succinimidyl ester; CFSE), and tumorsphere formation capacity. Our data demonstrate that expansion of brain tumor cells as hypoxic and nutrient restricted microtumors significantly increased the percentage of both CD133+ and CFSE+ cells. We further demonstrate that BTIC-marker positive cells isolated from microtumors maintain neurosphere formation capacity in the in vitro limiting dilution assay and tumorigenic potential in vivo. These data demonstrate that microtumors can be a useful three-dimensional biological model for the study of BTIC maintenance and targeting.

#1926

The effect of Beta-Catenin upon the tumor molecular biology and growth in HNSCC: role of cancer stem cells.

Austin Maas, Anirban Chatterjee, Lisa Sudmeier, Kwang Nickel, Randall J. Kimple. _Univ. of Wisconsin-Madison, Madison, WI_.

Overview: Head and neck squamous cell carcinomas (HNSCC) are the sixth most prevalent type of malignancy worldwide. Recent reports have demonstrated that nearly every tumor contain a small subpopulation of cells called cancer stem-like cells (CSCs), which are responsible for tumor maintenance, metastasis relapse and therapeutic resistance. Recent studies demonstrate that even established HNSCC cell lines contain a definite sub-population of CSC providing us with the opportunity to discerning their roles in progression, treatment, and relapse of the disease.

Background: We have previously demonstrated that a definite subpopulation of CSCs exists in most HNSCC cell lines. Significant differences were seen between the HPV+ and HPV- subtypes, confirmed by analyses of stem-ness genes and EMT markers. In vivo tumor xenograft formation has established the heightened tumorigenicity of CSCs. We investigated the molecular and functional roles of Beta-catenin in HNSCC CSCs by shRNA knock down and then subjecting the knocked down (KD) cells to various molecular analysis and in-vivo tumor formations in nude mice. We have also identified SOX2 as a down-stream effector gene that could exert the tumor promoting effects of Beta-catenin. The promoter region of the Sox2 gene was found to contain several putative Beta-catenin binding sites.

Methods & Results: After identifying CSCs by in-vitro Sphere Forming (SFA) capabilities and molecular gene signatures, we have knocked down both Beta-catenin and Sox2 in both HPV+ and HPV- HNSCC cell lines (UMSCC47 and UMSCC6). SFA with the Beta-catenin KD-cells depicts a 50% decrease in sphere formation ability. Molecular analyses show lower expression of stemness genes: Sox2 (~ 3 fold) and Oct4 (~ 2 fold); metastatic genes: N-cadherin (~ 2.5 fold), Vimentin (~ 3 fold), Twist (~ 4 fold), Snail (~ 2.5 fold), Slug (~ 2.5 fold) and Hif-1alpha (~ 1.5 fold). Two tissue microarrays (TMA) were stained for Oct4, Sox2, and CD44 gene expression by multiplex RNA in situ hybridization. Nearly all tumor cells expressed high levels of CD44. Cells high in both Oct4 and Sox2 were quantified within duplicate cores and correlated with local control (TMA-1) and radiation response (TMA-2).

Conclusions: Initial molecular analyses depicted that upon loss of Beta-catenin, the crucial stem-ness genes are negatively affected and the loss of stem-ness is also reinforced by decreased sphere formations. Both the metastatic and hypoxic gene signatures are markedly altered. We expect similar outcomes with the in-vivo tumor formations in nude mice.

#1927

The significance of calreticulin in pancreatic cancer: a molecule highly expressed in pancreatic cancer stem-like cells.

Satoshi Matsukuma,1 Kiyoshi Yoshimura,2 Atsunori Oga,1 Moeko Inoue,2 Takuya Fujimtoto,1 Atsuo Kuramasu,1 Masanori Fuse,2 Ryouichi Tsunedomi,1 Hidetoshi Eguchi,3 Hiroto Matsui,1 Shinsuke Kanekiyo,1 Yukio Tokumitsu,1 Shinobu Tomochika,1 Michihisa Iida,1 Yoshihiro Tokuhisa,1 Kazuhiko Sakamoto,1 Nobuaki Suzuki,1 Tomoko Furuya-Kondo,1 Hiroshi Itoh,1 Shigeru Takeda,1 Shigeru Yamamoto,1 Shigefumi Yoshino,4 Shoichi Hazama,5 Tomio Ueno,1 Hiroaki Nagano1. 1 _Yamaguchi University Graduate School of Medicine, Ube, Japan;_ 2 _National Cancer Center of Japan, Tokyo, Japan;_ 3 _Osaka University Graduate School of Medicine, Suita, Japan;_ 4 _Yamaguchi University Hospital, Ube, Japan;_ 5 _Yamaguchi University School of Medicine, Ube, Japan_.

Cancer stem-like cells (CSLCs) in solid tumors are thought to be resistant to conventional chemotherapy or molecular targeting therapy and to contribute to cancer recurrence and metastasis. In this study, we aimed to identify a biomarker of pancreatic CSLCs (P-CSLCs). P-CSLC-enriched population was generated from pancreatic cancer cell lines using our previously reported method and its protein expression profile was compared with that of parental cells by two-dimensional electrophoresis and tandem mass spectrometry. The results indicated that a chaperone protein calreticulin (CRT) was significantly upregulated in P-CSLCs compared to parental cells. Flow cytometry analysis demonstrated that CRT was mostly localized to the surface of P-CSLCs and did not correlate with the levels of CD44v9, another P-CSLC biomarker. Furthermore, the side population in CRThigh/CD44v9low population is much higher than that in CRTlow/CD44v9high population. CRT expression was also assessed by immunohistochemistry in pancreatic cancer tissues (n = 80) obtained after radical resection and was found to be associated with patients' clinicopathological features and disease outcomes in the Cox's proportional hazard regression model. Multivariate analysis identified CRT as an independent prognostic factor for pancreatic cancer patients, along with age and post-operative therapy. Our results suggest that CRT can serve as a biomarker of P-CSLCs and a prognostic factor associated with poorer survival of pancreatic cancer patients. This novel biomarker can be useful for detecting P-CSLCs independently, which had been detectable by multiple surface markers like CD24, CD44 and ESA. We will present CSLCs properties of CRThigh population in P-CSLCs.

#1928

The lung microenvironment promotes stem-like phenotype and behavior of breast cancer cells.

Ashkan Sadri, Alison L. Allan. _Western University, London, Ontario, Canada_.

Ninety percent of breast cancer-related mortalities result from metastasis, a process whereby the primary tumour disseminates and targets distant secondary organs. We have previously demonstrated that stem-like breast cancer cells expressing high levels of aldehyde dehydrogenase (ALDH) and CD44 are critical for metastasis. These ALDHhiCD44+ cells preferentially migrate and/or metastasize to the lung and bone microenvironments, where secondary tumours can severely impede organ function. However, the specific role of the lung and bone marrow (BM) microenvironments in supporting and/or promoting metastasis of ALDHhiCD44+ cells remains poorly understood, and this is the purpose of the current study. Using a 2D ex vivo model, lung and bone marrow (BM) conditioned media (CM) enriched in tissue-specific soluble proteins were generated from murine tissues and utilized in cell culture to assess their ability to influence the stem-like phenotype and behavior of MDA-MB-468 human breast cancer cells. Exposure to lung-CM promoted the generation of a viable, non-adherent ("floating") breast cancer cell subpopulation that was not observed in response to BM-CM or basal media (p<0.05). Phenotypic analysis by flow cytometry and gene expression analysis by RT-qPCR revealed notable differences in ALDH/CD44 phenotype in whole cell populations, adherent subpopulations, and/or non-adherent subpopulations in response to 72 hour exposure to lung-CM relative to basal media. In particular, the non-adherent breast cancer cell subpopulation generated in response to lung-CM demonstrated increased CD44 and ALDH1A3 gene expression and decreased ALDH1A1 gene expression relative to the adherent subpopulation (p<0.05). No significant phenotypic or gene expression differences were observed in MDA-MB-468 breast cancer cells in response to BM-CM. Taken together with our previous studies, these findings suggest that while both the lung and bone microenvironments support breast cancer cells with a pre-existing ALDHhiCD44+ phenotype, the lung microenvironment may additionally help to even further promote stem-like phenotype and behavior in the secondary metastatic site. Ongoing studies are aimed at elucidating the mechanisms by which aggressive ALDHhiCD44+ breast cancer cells interact with and/or are influenced by the lung microenvironment during metastatic progression.

### Pediatric Cancer 1: Biomarkers, Preclinical Models, and New Targets

#1929

A novel orthotopic mouse model in sarcomas.

Silvia Garcia Monclús, Juan Huertas Martínez, Laura Lagares Tena, Santiago Rello Varona, Olga Almacellas Rabaiget, David Herrero Martin, Roser López Alemany, Oscar Martinez Tirado. _IDIBELL, Hospitalet de Llobregat, Spain_.

Pediatric sarcomas are a heterogeneous group of bone and soft tissue malignancies affecting children and young adults. One of the most important prognostic factors of those diseases is the presence of metastasis at diagnosis. In that context, we have developed a novel orthotopic model which consists in injecting Ewing Sarcoma (ES) or Rhabdomyosarcoma (RMS) tumor cells at the gastrocnemius muscle of mice and extracting the tumor at a of 10 mm x 13 mm volume. After surgery, animals maintain a complete functional extremity and can live until lung metastasis detection (about 60 days post-injection).

Moreover, we have validated the suitability of the model with an Ewing Sarcoma EphA2-low expression cell line. Epha2 is a tyrosine kinase receptor that has been found overexpressed in a wide variety of tumors and correlated with malignant phenotype. In this study we report that EphA2 receptor is phosphorylated at S897 in a panel of ES cell lines, which is related to the oncogenic properties of the receptor. Stable silencing of EphA2 in two different ES cell lines resulted in a decrease in the clonogenic, proliferation, migration and invasion capacity in vitro. Moreover, we performed an experimental metastasis assay, injecting tumor cells through the tail vain of mice and observed a reduction in the lung metastasis incidence in EphA2 silenced cells. We then used this new orthotopic metastasis model to validate the impairment in the invasion capacity of EphA2 silenced cells and confirm the decrease in lung metastasis incidence, indicating an oncogenic role for EphA2 in ES. This novel orthotopic metastasis model is a valuable tool both for the study of spontaneous metastasis and also for evaluating therapeutic index in the onset of metastasis, which can also be applied to the study of other pedriatric sarcomas.

#1930

Evaluating the effect of PARP inhibitors in combination with nicotinamide phosphoribosyltransferase inhibitors in Ewing sarcoma.

Joshua T. Baumgart,1 Christine Heske,1 Mindy I. Davis,2 Kelli Wilson,2 Xiaoha Zhang,2 Rajarshi Guha,2 Marc Ferrer,2 Arnulfo Mendoza,1 Craig J. Thomas,2 Lee J. Helman1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _National Center for Advancing Translational Sciences, Rockville, MD_.

Previous research has shown that Ewing sarcoma cells are sensitive to PARP inhibition, but in vivo studies and early phase clinical trials have failed to demonstrate meaningful responses to PARP inhibitors when used as single agents. For this reason, studies combining PARP inhibitors with other agents to enhance their effects in Ewing sarcoma are of particular interest. PARP, an enzyme involved in DNA damage repair, relies on NAD to function. In tumor cells, NAD production occurs primarily through the salvage pathway, in which nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme. Hence, the combination of PARP and NAMPT inhibition would be expected to enhance the activity of PARP inhibition through depletion of cellular NAD. The aim of this study was to assess the synergistic potential of PARP inhibition in combination with NAMPT inhibition in Ewing sarcoma cell lines and xenograft models. Synergistic drug combinations in Ewing sarcoma cells were initially identified using a high-throughput matrix drug screen. In vitro activity was further assessed using proliferation assays of multiple Ewing sarcoma cell lines treated with the PARP inhibitor niraparib and the NAMPT inhibitor GNE-618. In vitro measurement of PAR activity was performed to further understand the mechanism of action of the combination. For in vivo studies, female SCID-biege mice were orthotopically injected with Ewing sarcoma cells and randomized into treatment groups of vehicle, niraparib, GNE-618, or the combination. The initial screen revealed synergy between NAMPT inhibitors and PARP inhibitors using multiple combinations of different agents in each drug class. In vitro assays of the combination of niraparib with GNE-618 confirmed the results of the screen. Assays measuring PAR activity yielded results supporting the hypothesis that dual inhibition of NAMPT and PARP depletes PARP more than PARP inhibitors alone. In vivo studies with short term (5 day) dosing showed no activity of single agent nirapirib and only temporary disease stabilization with single agent GNE-618. However, mice treated with the combination underwent tumor regressions resulting in prolonged survival. When treatments were extended to 2 cycles (5 days each), the effect persisted for longer. Mice tolerated the agents well, with no signs of toxicity. Preclinical data suggest that PARP inhibition in combination with NAMPT inhibition may be a promising therapeutic strategy for Ewing sarcoma patients.

#1931

The first circulating tumor cell detection technique from frozen PBMCs.

Heming Li,1 Izhar Singh Batth,1 Xueqing Xia,1 Frank J. Hsu,2 Neeta Somaiah,1 Keila Enitt Torres,1 Ruoyu Wang,3 Shulin Li1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Immune Design, South San Francisco, CA;_ 3 _Zhongshan Affiliated Hospital of Dalian University, Dalian, China_.

Circulating tumor cells (CTCs) enter the vasculature or lymphatic system after shedding from the primary tumor. CTCs may serve as "seed" cells for tumor metastasis. The utility of CTCs in clinical application is not fully investigated, partly due to the necessity for fresh blood samples and the lack of CTC specific antibody. Of note, there is no CTC detection tool for pediatric tumors. To overcome these drawbacks, we developed a protocol for CTC capture from frozen peripheral blood mononuclear cells (PBMCs) based on the cell-surface vimentin (CSV) antibody 84-1. CSV is a unique marker compared to others such as EpCAM because it is only specific to tumor cells and has been demonstrated to be a viable target for isolation and enumeration of CTCs across tumor types. In this study, we report the first CTC isolation technology from frozen PBMCs of osteosarcoma patients using the CSV antibody 84-1. The CTCs captured using this new protocol were validated by single cell gene sequencing analysis and mesenchymal marker α-SMA staining. Moreover, spiking analysis was also performed to ensure the specificity and sensitivity of this unique CTC capture analysis. Linear regression analysis yielded a positive correlation between the number of detected sarcoma cells and the number of sarcoma cells spiked in both fresh and frozen PBMCs. In summary, our results demonstrate for the first time, a technology to specifically detect and isolate mesenchymal origin CTCs from frozen PBMCs. This technology can be easily expanded to other types of cancers including pediatric tumors because the antibody used is universal and CTC specific. Such a technology will boost the feasibility and utility of CTC-based diagnosis and therapeutic treatment monitoring in a centralized laboratory.

#1932

Mutations in ATRX increase genetic instability and sensitivity to PARP inhibitors in paediatric glioblastoma cells.

Janat Fazal-Salom, Mara Vinci, Diana Carvalho, Helen Pemberton, Stephen J. Pettitt, Christopher J. Lord, Alan Mackay, Lynn Bjerke, Chris Jones. _Institute of Cancer Research, London, United Kingdom_.

Paediatric glioblastomas (pGBM) are amongst the most common causes of cancer-related deaths in children, and are defined by highly recurrent mutations in H3 histones. Mutations affecting the chromatin remodeling protein ATRX have been reported in 30% of pGBM cases, and are strongly associated with the alternative lengthening of telomeres (ALT) phenotype, but their precise interaction with histone mutations and their role in tumorigenesis remain unclear.

We collected sequence data from 262 published and 64 unpublished cases of pGBM and identified somatic ATRX mutations in 54/326 (17%) of cases. ATRX mutations are mainly loss of function mutations, with the majority of frameshift mutations (37/54, 68,5%) found upstream of the helicase domain resulting in truncation of the main functional domain of ATRX. Missense mutations (16/54, 29,6%) reside almost exclusively in the helicase domain (11/54, 20,4%), whereas nonsense mutations are a less common event (7/54, 13%) but present in both the helicase (4/7, 57,1%) and ADD domains (3/7, 42,9%). ATRX mutations commonly co-segregate with H3.3 G34 (16/54) and TP53 (42/54) mutations, and define a subgroup of patients with a longer overall survival (16 months median overall survival in mutant ATRX cases versus 11 months in wild-type ATRX cases, COXPH p = 0.079), though with a greater number of somatic mutations (MWU p = 0.023) and copy number alterations (MWU p = 0.0011) than wild-type cases.

We screened a series of 21 primary patient-derived pGBM cell cultures for histone and ATRX mutation status in addition to ATRX protein expression and ALT, and subjected the panel to a high-throughput in vitro cell viability screen of >400 chemotherapeutics and small molecules. We identified a specific genetic dependency for ATRX mutation and sensitivity to distinct PARP inhibitor chemotypes, including olaparib and rucaparib (PARP catalytic inhibitors), and talazoparib (PARP trapper inhibitor). These data were validated using CRISPR-Cas9-engineered ATRX knockout, targeting either the ADD or helicase domain, in SF188 pGBM cells. Gene editing was confirmed by IonTorrent sequencing and Western blot. ATRX mutant clones were also more sensitive to drugs targeting DNA damage response pathways such as bleomycin and sapacitabine.

Gene expression analysis of ATRX mutant pGBM samples confirmed an intact homologous recombination pathway and overexpression of PARP1, suggesting an underlying mechanism distinct from that observed in BRCA-mutant breast and ovarian cancers.

Ongoing work is aimed at unravelling the specific pathways involved, and evaluating the utility of PARP inhibition in orthotopic pGBM xenografts in vivo. These data suggest a synthetic lethality for PARP inhibitors in ATRX-deficient pGBM cells, and may represent a novel therapeutic strategy for these highly aggressive tumours in children.

#1933

Discovery of first-in-class small molecule CD99 inhibitors for targeted therapy of Ewing sarcoma.

Haydar Celik,1 Marika Sciandra,2 Bess Flashner,1 Elif Gelmez,1 Neslihan Kayraklıoğlu,1 David V. Allegakoen,1 Jeff R. Petro,1 Erin J. Conn,1 Sarah Hour,1 Jenny Han,1 Lalehan Oktay,1 Purushottam B. Tiwari,1 Mutlu Hayran,3 Maria Cristina Manara,2 Jeffrey A. Toretsky,1 Katia Scotlandi,2 Aykut Uren1. 1 _Georgetown University Medical Center, Washington, DC;_ 2 _Rizzoli Orthopaedic Institute, Bologna, Italy;_ 3 _Hacettepe University, Ankara, Turkey_.

Ewing sarcoma (ES) is an aggressive bone and soft tissue malignancy that affects predominantly children and adolescents with a high propensity to metastasize and poor prognosis. CD99 is a transmembrane cell surface protein that is highly expressed on ES cells, and routinely used as a marker for histological diagnosis of ES. We screened small molecule libraries for their binding to recombinant CD99 protein and subsequent selective inhibition of ES cell growth. We identified two structurally similar FDA-approved nucleoside analogues, clofarabine and cladribine that selectively inhibited the growth of ES cells in a panel of 14 ES vs. 28 non-ES cell lines. A significant negative correlation was found in human cell lines between CD99 expression and IC50 values for clofarabine and cladribine. Both drugs inhibited CD99 dimerization and its interaction with downstream signaling components cyclophilin A and PKA-RIIα as well as led to reduced ROCK2 protein expression and migration in ES cells. A membrane-impermeable analog of clofarabine showed similar cytotoxicity in ES cells, suggesting that it can function through inhibiting CD99 alone without any effect on DNA metabolism. Clofarabine and cladribine led to a significant increase in hypodiploid DNA content of ES cells, which was diminished by suppression of CD99 expression. Both drugs drastically inhibited anchorage-independent growth of ES cells, but clofarabine was more effective in inhibiting ES xenografts. Finally, the screening of a set of chemotherapy drugs revealed a synergy for the combination of anti-CD99 drugs and dasatinib in ES cells, which may translate into increased survival and reduced toxicity. Overall, our findings suggest that clofarabine is a good candidate for early phase clinical trials in children with ES.

#1934

Retinoic acid and DFMO induce differentiation and inhibit tumor formation in neuroblastoma.

Austin Voydanoff,1 Ping Zhao,2 Abhinav Nagulapally,2 Jeff Bond,2 Giselle L. Sholler2. 1 _Michigan State University, Grand Rapids, MI;_ 2 _Helen DeVos Children's Hospital, Grand Rapids, MI_.

Background: A key component of high risk neuroblastoma (NB) therapy involves cis-retinoic acid (RA) for differentiation of minimal residual disease. DFMO induces differentiation and inhibition of tumor formation through the targeting of cancer stem cell (CSC) pathways via reversal of the Lin28/Let7 axis. Preventatitve DFMO therapy is currently in a phase II clinical trial at the end of therapy and in a Pilot study in combination with RA and ch14:18 antibody. We hypothesize that the combination of cis-RA and DFMO will induce greater differentiation, inhibition of tumor formation, and reduction of cell proliferation of NB.

Methods: NB cell lines SMSKCNR, BE2C and CHLA90 were incubated in 96 well plates for 24 and 48 hours with low doses of DFMO (2.5 and 5 mM), RA (5 and 10 μM), and the four combinations of dosages. A Calcein AM Cell Viability Assay and BrdU Cell Proliferation Assay Kit were used to determine cell viability and cell proliferation, respectively. Western blot analysis was used to measure protein levels of CSC and differentiation markers. A neurosphere assay was used to assess inhibition of CSCs and tumor formation within wells. Cells were plated 2 cells/well in 96 well plates, drugged with single agents, cominbation, or DMSO and the percentage of wells per plate that formed neurospheres was determined after 1 and 2 weeks. IncuCyte ZOOM Live-Cell Imaging system was used for kinetic monitoring of neurite length to assess differentiation of NB cells.

Results: Low dose RA and DFMO combination treatment (2.5-5mM DFMO and 5-10 μM RA) resulted in decreased cell viability as demonstrated through calcein AM. DFMO and RA combination treatments reduced cell viability by 60-71%, 75-78%, and 83-91%, in Be2C, CHLA90, and SMSR cells, respectively. BrdU incorporation demonstrated a reduction in cell proliferation at 48 hours of was 69-70.%, 60.2-64.5%, 62.7-71.1% in Be2c, CHLA90, and SMSR, respectively . Western blot analysis showed that DFMO, RA, and their combination reduced the CSC and increased the differentiation markers at 48 hours compared to control. The combination treatment also decreased tumor formation; the relative reduction in neurosphere formation at 2 weeks was 34.5% with 2.5 mM DFMO, 48% with 5 µM RA, and 73.2% with combination treatment. Lastly, differentiation was shown by neurite length increased by a factor of 1.4-1.6 and 5, in SMSR and BE2C cells, respectively with combination treatment.

Conclusion: This study indicates that the combination retinoic acid and DFMO effectively cause a decrease in cell viability with a reduction in cell proliferation. Further, the combination results in differentiation of NB cells as well as targeting of CSC pathways and inhibition of tumor formation. Preventative DFMO therapy has been initiatied with RA in a pilot study for the treatment of high-risk neuroblastoma patients.

#1935

Molecular characterization of orthotopic patient-derived xenograft models of pediatric brain tumors.

Sebastian Brabetz,1 Susanne N. Gröbner,1 Huriye Seker-Cin,1 Florian Selt,2 Till Milde,2 David T. Jones,1 Madison T. Wise,3 Jessica M. Rusert,4 Kyle Pedro,3 Andy Strand,3 Olaf Witt,2 Sarah E. Leary,3 Xiao-Nan Li,5 Robert J. Wechsler-Reya,4 James M. Olson,3 Stefan M. Pfister,2 Marcel Kool1. 1 _German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Heidelberg, Germany;_ 2 _German Cancer Research Center (DKFZ), German Cancer Consortium (DKTK), Center for Individualized Pediatric Oncology (ZIPO) and Pediatric Brain Tumors, Department of Pediatric Oncology, University Hospital and National Center for Tumor Diseases (NCT), Heidelberg, Germany;_ 3 _Fred Hutchinson Cancer Research Center and Seattle Children's Hospital, Seattle, WA;_ 4 _Sanford Burnham Prebys Medical Discovery Institute, Heidelberg, CA;_ 5 _Baylor College of Medicine, Houston, TX_.

[Introduction: Solid tumors of the nervous system are the most common childhood cancers after leukemias. Even though we might be able to cure more and more patients, survivors still severely suffer long-term from the intensive treatments. Therefore, new treatment strategies are urgently needed. Orthotopic patient-derived xenograft (PDX) models are an excellent platform for biomarker and preclinical drug development. However, the rarity of pediatric brain tumors and the multitude of different sub entities hinder the generation of large collection of PDX models of specific entities within single institutions. In order to generate an overview about existing PDX models in the community, we started collecting established PDX models from various centers all over the world and performed extensive molecular characterization to precisely determine the distinct molecular subgroup and constellation of genetic alterations for each PDX model, and thus identify its targetable oncogenic drivers.

Material and Methods: PDX models were established and maintained by dissociating tumor material into a single cell suspension and then orthotopically injecting it into the brain of immunodeficient animals. All PDX models and matching primary tumors (if available) have been analyzed by whole-exome and low-coverage whole-genome sequencing, as well as DNA methylation and gene expression profiling at the German Cancer Research Center (DKFZ).

Results and Discussion: Thus far, we have collected and characterized 70 established PDX models from 6 ATRTs, 8 ependymomas, 16 high-grade gliomas, 38 medulloblastomas, and 2 CNS-PNETs. PDX models always retain their molecular subtype and in the vast majority of cases also the mutations and copy number alterations when compared to their primary tumors. Only in rare cases do we observe additional aberrations, which most likely represent outgrowths of subclones from the primary tumor. Analysis of our entire cohort identified an overrepresentation of the most aggressive tumor subtypes, but also subtypes which have not been available for preclinical testing before due to lack of genetically engineered mouse models or suitable cell lines, such as Group 4 medulloblastoma. Based on our current analysis, the PDX models within the community are not yet covering the entire heterogeneity within the patient population. As a follow up, we aim to make these models and data accessible in a user-friendly manner so that the community can use them for preclinical research.

Conclusion: PDX models of pediatric brain tumors are very rare. Our molecular characterization allows researchers all over the world to find the right models for their specific scientific question. Therefore, this work will provide an unprecedented resource to study tumor biology and pave the way for improving treatment strategies for children with malignant brain tumors.

#1936

Epigenetic challenges in derivation of the first cell-based model of Beckwith-Wiedemann Syndrome.

Stella K. Hur,1 Joanne L. Thorvaldsen,1 Suhee Chang,1 Carolyn Lye,1 Alice Yu,2 Monserrat C. Anguera,3 Marisa S. Bartolomei,1 Jennifer M. Kalish2. 1 _Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA;_ 2 _Children's Hospital of Philadelphia, Philadelphia, PA;_ 3 _University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA_.

Beckwith-Wiedemann Syndrome (BWS) is a cancer predisposition syndrome that affects at least 1 in 10,500 children. Up to 25% of children with BWS develop tumors, primarily Wilms tumor and hepatoblastoma. BWS is due to genetic or epigenetic changes that affect imprinted loci on chromosome 11 and these same changes are also found in other types of cancer. There are no cell-based models of BWS and most mouse models do not recapitulate the tumor phenotype. To understand more about the mechanisms leading to tumor formation in BWS, we developed the first human cell-based model of BWS. Human induced pluripotent stem cell (iPSC) models are commonly used to study disease mechanisms in tissue types that are not normally accessible for study. In the case of BWS, we plan to use such models to study how the genetic and epigenetic changes in BWS lead to tumor formation in hepatic and renal cells. Using skin fibroblasts from four BWS patients, we derived the first iPSC models of BWS. Prior to iPSC derivation, we characterized different tissues available from these four patients, and demonstrated genetic mosaicism in different tissue types, including blood, skin, and pancreas. During the derivation process, we demonstrated that both normal and BWS iPSC lines could be derived from the same patient fibroblast sample and the number of clones of each type from each sample approximated the initial level of mosaicism in the original sample. For each patient, these lines are isogenic except for the BWS critical region. The BWS and isogenic normal iPSCs were characterized for pluripotency markers and demonstrated to have normal karyotypes. Following this analysis, BWS and isogenic normal lines were characterized extensively for DNA methylation at specific imprinted loci in both early passage and extended culture. Methylation analysis was performed by both pyrosequencing and COBRA assays. Methylation was maintained at some imprinted loci but not at others in extended culture. Importantly, relatively stable methylation levels were observed at the BWS critical imprinted regions (H19/IGF2 and KvDMR), regardless of methods of reprogramming, indicating a relatively stable state of DNA methylation in this region. Additionally, normal methylation was seen at the SNRPN locus. In contrast, another imprinted locus, IG/MEG3, displayed abnormal hypermethylation in iPSCs. These data indicate that reprogramming and extended culture of iPSCs can affect stability of DNA methylation at certain imprinted loci. Therefore caution should be used in interpreting studies using iPSCs as these aberrant methylation states at imprinted loci can affect the downstream functionality of iPSC models. BWS iPSCs and isogenic controls with normal methylation will be used for further study of the mechanism of tumor formation in BWS.

#1937

Evolution of neuroblastoma patient-derived orthotopic xenografts through space and time.

Noémie Braekeveldt,1 Susanne Fransson,2 Kristoffer von Stedingk,3 Ingrid Öra,3 Rosa Noguera,4 Tommy Martinsson,2 David Gisselsson Nord,5 Sven Påhlman,1 Daniel Bexell1. 1 _Translational Cancer Research, Lund University, Lund, Sweden;_ 2 _Department of Clinical Genetics, Sahlgrenska University Hospital, Gothenburg, Sweden;_ 3 _Department of Paediatrics, Lund University, Lund, Sweden;_ 4 _Department of Pathology, Medical School, University of Valencia, Valenica, Spain;_ 5 _Department of Clinical Genetics, Lund University, Lund, Sweden_.

Background Recently, we established orthotopic neuroblastoma patient-derived xenografts (PDXs) which maintain the phenotypic, genomic, and stromal hallmarks of patient tumors. Here we examined how PDXs evolve following years of in vivo growth.

Materials and Methods We established up to eight in vivo generations of neuroblastoma orthotopic PDXs through serial passaging in NSG mice. RNA sequencing, exome sequencing and SNP array analysis were used to analyze patient tumors and PDXs from different in vivo generations.

Results Using SNP analysis, we found mostly a remarkable genomic stability at chromosomal level between patient tumors, early and late PDX generations. RNA-seq revealed that patient tumors expressed higher levels of genes involved in immune responses and ECM metabolism compared to PDXs. Different PDX samples clustered correctly into their respective tumor type. PDXs from all early generations did not separate from PDXs from late generations. Thus, gene expression levels are surprisingly often quite stable despite years of in vivo growth. To shed light on neuroblastoma intratumor heterogeneity, we implanted 10 different tumor fragments from a single patient tumor into mice. We classified the 10 mice into three groups based on the time periods required for tumor growth. RNA-seq showed that each of these groups had a distinct gene expression profile and pathways involved in neuroblastoma progression have been identified.

Conclusions Neuroblastoma orthotopic PDXs are often very stable at chromosomal and gene expression levels despite years of in vivo growth. We utilized multiple PDXs to show functional intratumor heterogeneity coupled to distinct gene expression profile.

#1938

Exportin-1 (XPO1) is a novel therapeutic biomarker for patients with neuroblastoma.

Basia Galinski,1 Marcus Luxemburg,1 Michelle Ewart,2 Yosef Landesman,3 Daniel Weiser1. 1 _Albert Einstein College of Medicine, Bronx, NY;_ 2 _Montefiore Medical Center, Bronx, NY;_ 3 _Karyopharm Therapeutics, Newton, MA_.

Background: Half of patients with high-risk neuroblastoma succumb to disease, yet these patients with inferior outcome cannot be identified at diagnosis despite contemporary risk stratification that integrates MYCN copy number status, tumor histology, and patient age. We have shown that overexpression of Exportin-1 (XPO1) is associated with poor survival in neuroblastoma, affirming what has been identified across a range of malignancies. Selinexor (KPT-330, Karyopharm Therapeutics), an XPO1 inhibitor in early phase clinical trials, inhibits the nuclear to cytoplasmic translocation of tumor suppressor and growth regulatory proteins implicated in oncogenesis. We hypothesized that protein expression level of XPO1 in neuroblastoma cell lines predict the effective response of treatment with selinexor.

Methods: We used a panel of early passage patient derived neuroblastoma cell lines including IMR5, NLF, KELLY, NB-EBC1, and SKNSH that are representative of the spectrum and genetic diversity of human disease. Cell lines were treated with varying concentrations of selinexor. RNA and whole cell protein extract as well as nuclear and cytoplasmic fractionated protein was obtained from untreated and treated conditions. Treated and untreated cells were also pelleted, and, along with patient tumor samples, formalin-fixed and paraffin-embedded for microscopic and immunohistochemical (IHC) evaluation.

Results: Compared to noncancerous cell lines, neuroblastoma cell lines have high protein and mRNA expression of XPO1; higher expression correlates with greater sensitivity to selinexor. Selinexor treatment reduced XPO1 protein and increased XPO1 mRNA expression, and it lead to nuclear retention of XPO1 cargo such as p53 and survivin. Comparison of microscopic features and protein staining intensity of patient samples with known XPO1 expression is ongoing.

Conclusions: This study provides rationale for XPO1 protein abundance as a potential biomarker of therapeutic response to the drug selinexor. By using patient derived neuroblastoma cell lines, we are able to evaluate the response of selinexor and XPO1 expression in the context of this enigmatic disease. These data suggest that initial drug response to selinexor can be predicted early in the course of disease and more investigation of the high expression of XPO1 seen in patients with poor outcome will provide strategies for combinatorial treatment approaches.

#1939

Loss of MST/Hippo signaling promotes tumorigenesis in a genetically engineered mouse model of fusion-positive alveolar rhabdomyosarcoma.

Kristianne Oristian, Lisa E. Crose, Rex Bentley, Nina Kuprasertkul, David G. Kirsch, Corinne Linardic. _Duke University, Durham, NC_.

While improvement in survival for pediatric cancer patients over the last 40 years has been encouraging, certain cancer types evade cure. One such example is fusion-positive alveolar rhabdomyosarcoma (aRMS), a pediatric soft tissue sarcoma of mesenchymal origin with skeletal muscle features and a 5-year survival rate of <50%. A hallmark of this aggressive malignancy is the t{2;13} translocation fusion gene PAX3-FOXO1 (PF). Primary cell-based modeling experiments in our laboratory and others have shown that PF is necessary but not sufficient for aRMS tumorigenesis, indicating additional molecular alterations are required to initiate and sustain tumor growth. Previously we showed that PF-positive aRMS tumorigenesis relies on alterations in Hippo pathway signaling, including upregulation of the YAP transcriptional co-activator, and downregulation of the MST/Hippo kinase, which occurs when the scaffold protein RASSF4 (itself a PF transcriptional target) binds and inhibits MST1/Hippo. We hypothesized that ablating MST/Hippo signaling in an existing genetically engineered mouse model (GEMM) of aRMS would accelerate tumorigenesis and provide insight into the role of this pathway in aRMS.

To assess the role of MST/Hippo signaling in aRMS, MST1/2-floxed (Stk3F/F;Stk4F/F) mice were crossed with an established MSTWT aRMS GEMM driven by conditional expression of Pax3-Foxo1 from the endogenous Pax3 locus and conditional loss of Cdkn2a in Myf6-expressing cells.

Statistical analysis revealed that compared to MSTWT aRMS control animals, MSTNull have significantly accelerated tumorigenesis (median survival 112 vs. 224 days, p< 0.0001) and increased tumor penetrance (76% vs. 27%). MSTNull animals developed tumors disproportionately in the head and neck as compared to control, and incurred multiple tumors per animal. Tumors were analyzed via immunohistochemistry for MyoD and myogenin, markers of RMS, as well as Myf5, an early marker of myogenesis. Tumors in both cohorts were for positive for aRMS markers. Tumor-derived cell lines were used for in vitro cell-based assays and molecular interrogation.

We have identified the MST/Hippo signaling axis as an important tumor suppressor mechanism in aRMS. The rapid onset and increased penetrance of tumorigenesis in this GEMM provides a powerful tool for interrogating aRMS biology and screening novel therapeutics.

#1940

Prenatal stress increases malignancy of neuroblastoma tumors in TH-MYCN animal model.

Sung Hyeok Hong, Larissa Wietlisbach, Susana Galli, Akanksha Mahajan, Shiya Zhu, Jason Tilan, Yichien Lee, Olga Rodriguez, Chris Albanese, Joanna Kitlinska. _Georgetown University Medical Center, Washington, DC_.

Neuroblastoma (NB) is a pediatric malignancy arising due to defects in sympathetic neuron differentiation. NB is a heterogeneous disease, with phenotypes ranging from spontaneously regressing to highly aggressive, incurable tumors. This clinical variability cannot be explained solely by genetic aberrations. Even in families with hereditary NB the penetrance of the disease is incomplete and the same genetic mutation often results in tumors with phenotypes varying from differentiating ganglioneuromas to undifferentiated, highly aggressive NBs. Thus, other, perhaps non-genetic factors can contribute to the disease development and modify its phenotype. Strikingly, the two factors promoting de-differentiation of NB cells and their malignant phenotype, hypoxia and glucocorticoids, are elevated in the fetus during maternal stress, suggesting a role for prenatal stress in NB tumorigenesis. Previously, using TH-MYCN mice as a model of aggressive NB, we have shown that an increase in maternal corticosterone levels during pregnancy attained by inserting slow release pellets resulted in increased tumor frequency in TH-MYCN offspring. The goal of the current study was to determine the effect of prenatal stress on NB metastasis. To this end, pregnant mice carrying TH-MYCN hemizygous offspring were subjected to chronic stress at embryonic days 10-17, the time of sympathetic neuroblast proliferation and differentiation. Two established stress paradigms were used - chronic unpredictable stress, in which mice were subjected daily to various stressors, and chronic cold stress comprising of daily 30 min exposure to cold. The phenotypes of the disease and its dissemination were compared between offspring of control and stressed mothers. The offspring from both prenatally stressed groups presented with more malignant disease, as manifested by the presence of advanced lung metastases disseminating from small primary tumors (<200 mm3). This phenotype was associated with increased mortality in prenatally-stressed TH-MYCN offspring (p<0.01). In contrast, no advanced lung metastases and no disease-related deaths were observed in TH-MYCN offspring of control mothers despite the presence of large primary tumors (>1,000 mm3). Although not common, lung metastases occur preferentially in NB patients with MYCN amplification and are associated with significantly worse prognosis, as compared to patients with metastatic disease, but no pulmonary involvement (14 vs 43% 3-year event-free survival, respectively). Thus, the profound pulmonary dissemination observed in prenatally-stressed TH-MYCN mice mimics one of the most malignant NB phenotypes observed in human disease. Altogether, our data implicate maternal stress during pregnancy as a potential environmental factor modifying the effects of genetic aberrations and promoting malignant phenotype of NB.

#1941

Cell surface vimentin is a novel marker for CTC detection in neuroblastoma.

Izhar S. Batth,1 Heming Li,1 Giselle Saulnier Sholler,2 Shulin Li1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Helen DeVos Children's Hospital, Grand Rapids, MI_.

Among children in the United States, brain cancers now account for most number of cancer-related deaths. Neuroblastomas (NB) and sarcomas account for 13% of all childhood cancers in the United States. Despite significant progress yielding increased 5-year survival, 1250 deaths are expected this year from childhood cancers and cancer incidence has been steadily increasing. One of the key contributors to cancer mortality is tumor metastasis. It is generally believed that CTCs are shed into the circulation from primary tumors and contain unique driver mutations enabling their aggressive phenotype. However, detection of CTCs from NB is difficult as there is no direct method. Our lab has developed a monoclonal antibody targeting vimentin on the cells' surface. Cell surface vimentin (CSV) is only observed in tumor cells; it remains intracellular in normal cells. We have published data demonstrating the superior specificity and sensitivity of our approach in breast, colon, and prostate cancer. Here, we report direct CTC detection in NB using our CSV mAb. As part of our collaborative effort analyzing NB blood samples from a multicenter Phase II trial, we present novel discoveries regarding treatment of NB with difluoromethylornithine (DFMO) and its effects on CTC release into the circulation in patients under remission. Among our observations, approximately ~1/3 patients have low or no CTCs while ~1/4 with high numbers of CTC are responding to DFMO treatment. Remaining patients show oscillating CTC numbers. Further, treatment with DFMO showed a statistically significant decline in CTCs after the beginning of therapy. Ongoing work including whole genome analysis can possibly reveal new insight into CTCs' entering the circulation. These data demonstrate that our CTC capture using CSV is a novel and unique approach for detection in NB.

#1942

**The nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, OT-82, exhibits** in vitro **and** in vivo **efficacy against patient-derived xenograft models of high-risk acute lymphoblastic leukemia.**

Kathryn Evans,1 Tara Pritchard,1 Michelle J. Henderson,1 Klaartje Somers,1 Mawar Karsa,1 Leanna Cheung,1 Raymond Yung,1 Stephen W. Erickson,2 Lioubov Korotchkina,3 Olga Chernova,3 Andrei Gudkov,4 Malcolm A. Smith,2 Richard B. Lock1. 1 _Children's Cancer Institute, Sydney, Australia;_ 2 _National Cancer Institute, MD;_ 3 _Oncotartis, Inc., Buffalo, NY;_ 4 _Roswell Park Cancer Institute, Buffalo, NY_.

Cancer cells are highly dependent on nicotinamide phosphoribosyltransferase (NAMPT) for the biosynthesis of nicotinamide adenine dinucleotide (NAD). Besides its role in energy metabolism, NAMPT influences the activity of NAD-dependent enzymes, including poly (ADP-ribose) polymerase-1 (PARP-1) and sirtuins, and thereby regulates cellular survival and stress response. Disruption of NAD synthesis through NAMPT inhibition represents a potential therapeutic strategy for treating cancer. The aim of this study was to evaluate the efficacy of the novel NAMPT inhibitor OT-82, initially isolated for its selective toxicity against a panel of adult leukemia cell lines, in a diverse panel of leukemia cell lines in vitro and pediatric acute lymphoblastic leukemia (ALL) patient-derived xenografts (PDXs) in vivo, and to identify potential biomarkers predictive of OT-82 response. OT-82 demonstrated low nanomolar IC50 values (0.9 – 3.4 nM) in 3 ALL and 3 acute myeloid leukemia cell lines as determined by resazurin reduction assays. In vivo efficacy of OT-82 (40 mg/kg x 3 days x 3 weeks, p.o.) was evaluated as a single agent against pediatric ALL PDXs, including B-cell precursor ALL (n = 3), Philadelphia chromosome (Ph) positive ALL (n = 2), Ph-like ALL (n = 2), T-cell ALL (n = 3), and early T-cell precursor ALL (n = 3) in immune deficient (NSG) mice. Response to treatment was assessed by time to event or stringent objective response criteria modeled after the clinical setting. OT-82 was well tolerated, significantly increased event-free survival (EFS) relative to control mice in 11/13 ALL PDXs, and elicited objective responses in 11/13 (85%) PDXs [3 Partial Responses, 4 Complete Responses (CRs) and 4 Maintained CRs (MCRs)]. Analysis of basal protein expression revealed elevated levels of poly (ADP-ribosyl)ated (PARylated) PARP-1 in 4/5 responders versus 0/2 non-responders. In vitro studies examining various chemotherapeutic agents used for childhood leukemia showed synergy between cytarabine (AraC) and OT-82 in an ALL cell line. In an OT-82 sensitive Mixed Lineage Leukemia (MLL) PDX, treatment with AraC (25 mg/kg x 5 days x 2 weeks, i.p.) and OT-82 (40 mg/kg x 3 days x 2 weeks, p.o.) significantly increased EFS compared to OT-82 (P<0.0001) or AraC (P<0.0001) alone. Moreover, the OT-82/AraC combination elicited an MCR compared to OT-82 (CR) and AraC (Progressive Disease) alone. The results herein demonstrate significant activity of OT-82 against leukemia cell lines and a range of pediatric ALL subtypes in vivo, and the potential for PARylated PARP-1 expression as a biomarker for predicting OT-82 response. Moreover, the enhanced activity of OT-82 in combination with AraC over single agent therapy further suggests NAMPT inhibition as an attractive strategy for treating high-risk pediatric ALL. Supported by U01CA199000 from the NCI.

#1943

High-throughput chemical screening identifies Focal Adhesion Kinase and Aurora Kinase B inhibition as a synergistic treatment combination in Ewing sarcoma.

Brian Crompton,1 Sarah Wang,1 Elizabeth Hwang,1 Rajarshi Guha,2 Matthew Boxer,2 Crystal McKnight,2 Min Shen,2 Nicole Melong,3 Chansey Veinotte,3 Amy Conway,1 Jason Berman,3 Matthew Hall,2 Mindy Davis,2 Kimberly Stegmaier1. 1 _Dana-Farber/Boston Children's Cancer and Blood Disorder's Center, Boston, MA;_ 2 _National Center for Advancing Translational Sciences, Bathesda, MD;_ 3 _Dalhousie University, Halifax, Nova Scotia, Canada_.

Ewing sarcoma is the second most common bone malignancy of childhood. Current treatment employs chemotherapy, surgery, and radiation. Although this approach cures approximately 70% of patients with localized disease, treatments are largely ineffective for patients with metastases or relapse. Furthermore, these treatments are associated with an alarming rate of long-term toxicities. New treatment combinations are necessary to improve cures and lower toxicities for these patients.

We recently found that Ewing sarcoma is dependent on focal adhesion kinase (FAK) for cell viability and tumor proliferation. In order to identify candidate treatment combinations for Ewing sarcoma, we performed a screen of 1912 compounds to identify those with synergistic anti-Ewing activity when combined with FAK inhibition. The A673 Ewing cell line was treated with PF-562271, a FAK-specific inhibitor, in combination with compounds from the Mechanism Interrogation PlatE (MIPE) 4.0 library. Cell viability was measured after 48 hours of treatment. Multiple computational metrics were utilized to identify and rank all compound combinations for synergistic impairment of cell viability. Multiple Aurora kinase inhibitors scored as synergistic with FAK inhibition in this screen. Aurora kinases are important in the regulation of mitosis and are highly expressed in Ewing sarcoma tumors and cell lines. We found that Aurora kinase B inhibitors were synergistic across a larger range of concentrations than Aurora kinase A inhibitors when combined with FAK inhibition in multiple Ewing cell lines. We found that AZD-1152, an Aurora kinase B-selective inhibitor, and PF-562271 when used in combination induced apoptosis in Ewing cells at concentrations that had minimal effect on cell survival when either drug was used alone. We also found that the combination significantly impaired tumor proliferation in zebrafish xenograft models of Ewing sarcoma and prolonged survival in murine xenografts compared to either single-agent treatment alone. Interestingly, treatment with AZD-1152 alone also significantly impaired tumor proliferation and prolonged survival compared to vehicle treatment in a mouse xenograft model of Ewing sarcoma.

Our data demonstrate that FAK and Aurora kinase B inhibition synergistically impair Ewing sarcoma cell viability in vitro and significantly inhibit tumor proliferation in vivo. With multiple FAK and Aurora kinase inhibitors in early phase trials for adult malignancies, these results have the potential to be translated into clinical trials for patients with Ewing sarcoma. Previous studies have also suggested a dependency of Ewing sarcoma on Aurora kinase activity; our data further supports a role for Aurora kinase B inhibitors as therapeutic candidates in this disease.

#1944

Tazemetostat displays synergistic antiproliferative activity with backbone therapies in preclinical models of AT/RT and MRT.

Christine R. Klaus, Jeffrey A. Keats, Jesse J. Smith, Richard Chesworth, Robert A. Copeland, Scott Ribich, Alejandra Raimondi. _Epizyme, Inc., Cambridge, MA_.

Malignant Rhabdoid Tumors (MRT) and Atypical Teratoid Rhabdoid Tumors (AT/RT) are typically pediatric cancers which are rare and aggressive with extremely high unmet medical need. At a molecular level MRT and AT/RT tumors are almost universally characterized by loss of the INI1 protein (also known as SNF5 or SMARCB1), a component of the Switch Sucrose Non-fermentable (SWI/SNF) multimeric chromatin modifying complex. In many cell types, the SWI/SNF complex and the PRC2 complex have an antagonistic relationship in the regulation of tumor suppressor genes, cell cycle checkpoints, hedgehog and myc pathway genes, among others [Wilson et al, Cancer Cell. 2010 Oct 19;18(4):316-28.]. This explicated the possibility of a novel treatment modality for these tumors, based on inhibition of EZH2 - the catalytic subunit of PRC2. Tazemetostat (EPZ-6438) is a potent and orally bioavailable small molecule inhibitor of EZH2 currently in phase 2 clinical trials in adult patients with non-Hodgkin lymphoma, INI1-negative tumors and mesothelioma. Dependency of INI1-negative tumors on EZH2 catalytic activity was demonstrated in preclinical models of MRT, where robust tumor regressions were induced with tazemetostat treatment [Knutson et al, Proc Natl Acad Sci USA. 2013 May 7;110(19):7922-7]. Consistent with this, tazemetostat demonstrated activity in relapsed or refractory patients with INI1-negative tumors enrolled in the adult phase 1 clinical trial. In addition, a phase 1 dose escalation study in pediatric patients with INI1-negative solid tumors is currently ongoing. Backbone therapies for MRT and AT/RT are comprised of cytotoxic chemotherapy which may or may not be administered in the context of stem cell transplant, in addition to surgical resection and radiation therapy. In support of combination clinical scenarios including EZH2 inhibition in this setting, we sought to explore the antiproliferative effects of combining tazemetostat with current small molecule treatment therapies in cell line models of AT/RT and MRT. Synergistic activity was observed when tazometostat was combined with individual components of chemotherapeutic regimens and targeted therapies such as vincristine, doxorubicin, alisertib and HDAC inhibitors.Further, we investigated the effects of ionizing radiation together with tazemetostat treatment. Application of X-ray irradiation concomitantly or after tazemetostat treatment, induced robust antiproliferative activity and reduction in clonogenic potential of both AT/RT cell lines tested.Taken together these results suggest that pharmacological inhibition of EZH2 enhances the activity of backbone therapy and may have an advantage over monotherapy in INI1-negative cancers supporting the therapeutic potential of combination regimens that include tazemetostat in these tumors.

#1945

PPM1D/Wip1, promising new target in childhood cancers neuroblastoma and medulloblastoma.

Jelena Milosevic,1 Nina Eissler,1 Diana Treis,1 Malin Wicktröm,1 Susanne Fransson,2 Baldur Sveinbjornsson,3 Ninib Baryawno,1 Subazini Kosalai,2 Chandrasekhar Kanduri,2 Kazuyasu Sakaguchi,4 Tommy Martinsson,2 John Inge Johnsen,1 Per Kogner1. 1 _Karolinska Institutet, Stockholm, Sweden;_ 2 _University of Gothenburg, Gothenburg, Sweden;_ 3 _University of Tromsö, Tromsö, Norway;_ 4 _Hokkaido University, Sapporo, Japan_.

Background:

The most common cytogenetic lesions in the embryonal neural tumors medulloblastoma (MB) and neuroblastoma (NB) affect chromosome 17, with 17q+ or isochromosome 17q, in approximately one-third of MB with these aberrations being a significant indicator of poor clinical outcome. Similarly, in NB gain of 17q is the most powerful genetic predictor of adverse clinical outcome. 17q+ correlates with poor survival in our population-based material where we found aberrations of chromosome 17 in 85% of primary neuroblastomas, specifically, gain of PPM1D/Wip1 at 17q23. Wip1 is a serine/threonine phosphatase encoded by the gene PPM1D, described as a gatekeeper in the Mdm2-p53 regulatory loop involved in genetic stability, inflammation and a potential oncogene contributing to carcinogenesis.

Methods:

Comparative genomic hybridization (CGH), immunostaining, mRNA arrays, qPCR, exome- and RNA-sequencing was used to examine PPM1D/Wip1 in neuroblastoma and medulloblastoma. Genetic and pharmacological inhibition was used to analyze the function of Wip1 in preclinical neuroblastoma and medulloblastoma models.

Results:

CGH-array analysis detected PPM1D/Wip1 extra copies in all tumors and cell lines containing 17q-gain. Expression arrays and immunostaining showed high expression of Wip1 in neuroblastoma corresponding to poor survival. RNA sequencing confirmed PPM1D-gain and revealed truncated isoforms with oncogenic potential. Exome-sequencing detected a mutation leading to constitutive PPM1D/Wip1 activation in an aggressive metastatic infant neuroblastoma. Wip1 knockdown experiments showed significant decrease of cell viability, proliferation and colony formation as well as substantial increase of DNA-damage response in neuroblastoma and medulloblastoma cells. Tumor neuroblastoma xenograft development was significantly delayed showing median tumor development (0.10 mL) to be more than doubled (median 15 days, vs. 33 days, p<0.001) after Wip1 downregulation compared to scrambled controls. A novel Wip1 inhibitor was highly potent in cytotoxic/cytostatic effect in neuroblastoma and medulloblastoma cell lines. Furthermore, this Wip1 inhibitor significantly inhibited growth of established human neuroblastomas and medulloblastomas in nude mice after treatment (P<0.01).

Conclusions:

Our results show that PPM1D/Wip1 is oncogenic in neuroblastoma and medulloblastoma development. We propose three different ways on how PPM1D/Wip1 is activated: due to chromosomal gain, alternative RNA-isoforms and/or DNA-mutation. PPM1D/Wip1 provides a novel therapeutic target in neuroblastoma and medulloblastoma.

#1946

Synergistic effects of the XPO1 inhibitor selinexor with proteasome inhibitors in pediatric high-grade glioma and diffuse intrinsic pontine glioma.

John DeSisto,1 Patrick Flannery,1 Trinayan Kashyap,2 Rakeb Lemma,1 Shelby Mestnik,3 Andrew Kung,4 Rajeev Vibhakar,1 Yosef Landesman,2 Adam Green1. 1 _University of Colorado Anschutz Medical Campus, Aurora, CO;_ 2 _Karyopharm Therapeutics Inc., Newton, MA;_ 3 _Rocky Vista University College of Osteopathic Medicine, Parker, CO;_ 4 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Background: Pediatric high-grade gliomas (HGG) and diffuse intrinsic pontine gliomas (DIPG) account for the majority of pediatric brain tumor deaths and respond poorly to chemotherapy. Selinexor, a nuclear export inhibitor, is effective against HGG and DIPG in in vitro and in vivo models, but resistance to treatment develops. We previously identified the NF-κB pathway as a likely mediator of selinexor's activity in these tumors. NF-κB transcriptional activity is regulated by an inhibitor, IKB-α, whose levels are in turn regulated by ubiquitination and proteasomal degradation. IKB-α is a client of exportin-1 (XPO1); its nuclear levels are increased by selinexor treatment, leading to inhibition of NF-κB. We subsequently identified proteasome inhibitors as potentially synergistic with selinexor in HGG and DIPG through a screen of all FDA-approved chemotherapy agents. Proteasome inhibition has also been shown to synergize with selinexor treatment in multiple myeloma and osteosarcoma.

Methods: We treated HGG cell lines (BT245 and GBM1) and DIPG cell lines (DIPG4, DIPG7 and SF7761) for five days with selinexor in combination with each of three proteasome inhibitors, bortezomib, carfilzomib and marizomib, and assayed cell viability at the conclusion of treatment. In each experiment, cells were treated with selinexor, a proteasome inhibitor, and a combination of the two drugs at several constant ratios. IC50 values were computed for each drug acting alone, and the combination index (CI) of the two drugs acting together was computed using the Chou-Talalay method. We also treated SF7761 cells with a combination of radiation (8Gy), selinexor, and a proteasome inhibitor.

Results: The proteasome inhibitors had widely varying IC50 values in the cell lines treated, ranging from 1nM to 5µM. The CI for the combination of selinexor and each proteasome inhibitor was consistently less than 1 (indicating a synergistic relationship) in the cell lines tested. We found that radiation and proteasome inhibition had an antagonistic relationship (CI>1), radiation and selinexor a synergistic relationship (CI<1), and the combination of all three was mixed, with some combinations being synergistic and others antagonistic.

Conclusions: Selinexor and proteasome inhibitors show promise as a combination therapy for HGG and DIPG. We are conducting in vivo experiments to further explore this combination for subsequent clinical trial use.

#1947

Combination of Vincristine and Clotam induces antiproliferative response in medulloblastoma cells.

Shruti V. Patil,1 Don Eslin,2 Robert Sutphin,2 Umesh T. Sankpal,1 Yazmin Hernandez,1 Areeba Hafeez,1 W. Paul Bowman,1 Riyaz Basha1. 1 _University of North Texas Health Science Center, Fort Worth, TX;_ 2 _Arnold Palmer Hospital for Children, Orlando, FL_.

Medulloblastoma (MB) is the most common pediatric malignant brain tumor and usually originates in the cerebellum. These tumors have the propensity to disseminate throughout the central nervous system and are often difficult to treat. Chemotherapy is widely accepted as part of the multimodality treatment approach for MB. However, it is associated with debilitating toxicity and potential long term disabilities. Vincristine is a commonly used chemotherapeutic agent for MB treatment. This drug is known to induce some toxic effects including peripheral neuropathy. The aim of this study was to test a combination treatment involving Vincristine and an anti-cancer non-steroidal anti-inflammatory drug, Clotam (Tolfenamic acid) against MB cell lines. Previously, we showed that Clotam inhibited MB cell proliferation and tumor growth in mice by targeting the transcription factor, Specificity protein1 (Sp1) and an inhibitor of apoptosis protein, BIRC5 (baculoviral inhibitor of apoptosis repeat-containing 5). The overexpression of BIRC5 is associated with aggressiveness and poor prognosis in several cancers. MB cells, DAOY and D283 cells were treated with vehicle (dimethyl sulfoxide) or low dose of Vincristine (DAOY: 2ng/ml; D283: 1ng/ml) or Clotam (DAOY & D283: 10 µg/ml) or combination of Vincristine + Clotam and the cell viability was measured at 1 and 2 days post-treatment using Cell-TiterGlo kit. Flow cytometry was employed to analyze apoptotic cells using Annexin-V staining and cell cycle phase distribution using propidium iodide staining. The activation of apoptotic pathways was further investigated by assessing the levels of effector caspases with CaspaseGlo kit and the expression of apoptotic markers [cleaved Poly (ADP-ribose) polymerase (c-PARP), B-cell lymphoma 2, and BIRC5] by Western blot analysis. The expression of key proteins associated with cell cycle (Cyclin A, B, D, CDK4/6, and p21) was also determined by Western blot analysis. When compared to individual agents, the combination of Clotam and Vincristine increased MB cell growth inhibition which is accompanied by an induction of apoptotic markers and the modulation of (Cyclin A, B, D and CDK4/6: down-regulation; p21: up-regulation) proteins associated with cell cycle phase distribution. These results suggest that Vincristine and Clotam combination treatment is effective for inducing anti-proliferative effect in MB cells. The experiments to evaluate the effect of this combination in animal model for MB are currently under study.

#1948

Tumor-specific copy number alterations uncover therapeutic opportunities in osteosarcoma.

Leanne C. Sayles,1 Marcus Breese,2 Amanda L. Koehne,2 Stanley Leung,1 Aviv Spillinger,1 Alex Lee,1 Avanthi Shah,1 Krystal Straessler,2 Sheri Spunt,2 Neyssa Marina,2 Damon Jacobson,3 Raffi S. Avedian,2 David G. Mohler,2 Steven DuBois,4 Douglas S. Hawkins,3 E. Alejandro Sweet-Cordero1. 1 _UCSF, San Francisco, CA;_ 2 _Stanford, Stanford, CA;_ 3 _University of Washington, Seattle, WA;_ 4 _Dana-Farber Cancer Institute, Boston, MA_.

Osteosarcoma (OS) is a highly malignant cancer for which no targeted therapies are currently available. Current treatment modalities are limited to intensive, highly toxic chemotherapy and surgical resection. OS is characterized by wide spread copy number alterations and structural rearrangements. In contrast, no recurrent point mutations in protein-coding genes have been identified, suggesting that copy number alterations (CNAs) are key oncogenic drivers in this disease. However, as copy number alterations are highly heterogeneous, it is likely that each tumor has a distinct set of oncogenic drivers, making a unified treatment approach difficult to define. To identify candidate patient-specific drivers, we used a simple heuristic based on degree of amplification (as assessed by Whole Genome Sequencing) and changes in gene expression (as assessed by RNA sequencing). Using patient-derived tumor xenografts (PDTXs), we assessed if individual OS tumors respond to targeted therapy selected based on this approach. We rank-ordered CNAs in 9 PDTXs by the amplitude of the copy number gain and identified 5 pathways for targeted therapy including CCNE1, MYC, CDK4, PTEN/AKT and AURKB. Next, we prioritized drug choices to those that are in clinical trials and that are readily available and identified 5 drug matches for 9 PDTXs. We used the CDK2 inhibitor Dinaciclib for CCNE1 amplification and observed TGI of 85.5% and 67.8% for 2 different CCNE1 amplified PDTXs tested. CDK9 inhibitor AT7519 was used to treat 2 different MYC amplified PDTXs resulting in TGI of 104% and 83.9%. CDK4 amplified PDTXs were treated with CDK4/6 inhibitor, Palbociclib, resulting in TGI of 82.7%. AKT1 inhibitor MK-2206 was used to treat either AKT1 gains or PTEN loss resulting in TGI of 65.6% and 60.8% respectively. AURKB inhibitor AZD-1152 for AURKB amplified PDTXs in combination with cisplatin resulting in TGI 85.8%. These results support the hypothesis that specific genes within CNA regions serve as oncogenic drivers and that these represent therapeutic opportunities in OS. Our studies provide a roadmap for personalized genome-informed therapy of osteosarcoma, a cancer in which no new therapies have been identified in over 30 years.

#1949

Salvage pathway enzyme HPRT as a molecular marker for Burkitt's Lymphoma.

Michelle H. Townsend, John Ellis Lattin, Michael D. Anderson, Abigail Felsted, Edwin Velazquez, Evita Weagel, Richard Robison, Kim L. O'Neill. _Brigham Young University, Provo, UT_.

The aim of this study is to investigate the potential of Hypoxanthine phosphoribosyltransferase (HPRT) as a surface biomarker and target for future immunotherapies in Burkitt's B-cell Lymphoma. B-cell malignancies are the most common type of childhood cancer. Development of immunotherapies could improve current treatment. HPRT is a cytosolic transferase involved in nucleotide production via the purine salvage pathway. Altered expression of TK1, a salvage pathway enzyme, is an indicator of prognosis and diagnosis in multiple cancer types due to active proliferation of cells and the resulting elevated nucleotide demand. It has previously been reported that some salvage pathway enzymes are found on the surface of Burkitt's Lymphoma cells. HPRT presence on the surface of this B-cell lymphoma subtype could provide a target for adoptive cell transfer and other immunotherapies. The potential surface presentation of HPRT was assessed using flow cytometry, scanning electron microscopy (SEM), and cytoplasmic staining on both healthy lymphocytes and Burkitt's Lymphoma cells (Raji). Flow cytometry experiments with HPRT antibodies and fluorescent-labeled secondary antibodies show that Raji cells exhibit an 81.4% (p-value .0001) positive fluorescence shift when compared to IgG controls (1.5%). Healthy lymphocytes had a fluorescence shift of 2.38% (p-value .9787). The presence of HPRT on the surface of both Raji cells and healthy lymphocytes was further confirmed using gold-labeled antibodies. Utilizing a scanning electron microscope, the presence of the protein on the surface was evaluated and quantified via increases in gold weight percentage of the sample. When treated with antibodies against HPRT, there was a significant increase in gold binding along with an increase in gold weight percentage. These results suggest a direct relationship between HPRT and the surface of Burkitt's lymphoma cells, indicating HPRT as a potential target for future immunotherapeutic treatment in Burkitt's B-cell lymphoma pediatric patients.

#1950

Introducing c-Myc into transformed human mesenchymal stem cells and osteoblasts to recapitulate the osteosarcoma phenotype.

Sajida Piperdi (Thein),1 Wendong Zhang,1 Daria Ivenitsky,1 Yidan Zhang,1 Yunjia Zhang,1 David S. Geller,2 Bang Hoang,2 Rui Yang,2 Jonathan B. Gill,1 Michael Roth,1 Richard Gorlick1. 1 _Albert Einstein College of Medicine and The Children's Hospital at Montefiore, Bronx, NY;_ 2 _Montefiore Medical Center, Bronx, NY_.

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and young adults. It remains unclear at what point in the pathway of differentiation between human mesenchymal stem cells (hMSCs) and osteoblasts (OBs), OS originates. Since high grade OS frequently demonstrates histologic variability, as well as the potential for multi-lineage differentiation, some consider the hMSC as the cell of origin in OS, whereas others believe the osteoblast to be the most likely cell of origin. Identifying the primary cell of origin is crucial in understanding the molecular pathogenesis of OS. To address the potential for hMSCs or OBs to transform into OS, we have performed insertional mutagenesis in hMSCs and OBs with defined genetic elements which have the potential to lead to tumorigenesis and whose pathways have been altered in OS patient specimens. Serial introduction of the viral constructs hTERT (T), SV40Tag (S), and H-RAS (R), led to spindle cell tumor formation in mice. However, MSC-TSR tumors did not form osteoid, whereas OB-TSR tumors showed only scant production of osteoid. Also the addition of β-catenin to MSC-TS cells failed to lead to tumor formation in mice. Based on these findings, MSC-TS and OB-TS cell lines may be ideal platforms for further analysis of the impact of the genetic transformation of hMSCs and OBs into OS. c-Myc has been shown to be overexpressed in OS cells and hence has been suggested as an oncogene. It has been also suggested as a novel target of RUNX2 through rescue from the apoptosis.

MSC-TS and OB-TS cells were transformed with a retrovirus containing human c-Myc. Drug resistant colonies were picked up 21 days after selection to obtain stably transformed cell lines. Quantitative PCR and western blots were carried out to detect both gene and protein expression patterns in transformed cell lines, respectively. Furthermore, in order to determine the expression pattern of c-Myc in OS, quantitative PCR and western blots were also performed on human osteosarcoma primary samples, xenografts and cell lines. All functional assays outlined below will be performed to determine the tumorigenic properties of transformed cell lines.

Six out of seven (86%) osteosarcoma primary samples showed a significantly greater c-Myc gene expression level compared to the positive control cell lines. Selection of stably transformed clones and comparison between cell lines and characterizations are underway. Further ongoing characterization includes: soft agar assays, in-vivo tumorigenic assays, histological examination for osteoid production and other OS specific immunohistochemical markers, proliferation, invasion and migration, and differentiation assays. It is our hope that this study will highlight the model that closely recapitulates the human osteosarcoma phenotype by forming a malignant spindle cell tumor that produces aberrant osteoid, and has the potential for multi-lineage differentiation.

#1951

RORα activation opposes MYCN signaling and restores the circadian clock in neuroblastoma.

Myrthala Moreno-Smith,1 Ling Tao,1 Ronald Bernardi,1 Kathleen De Preter,2 Mario Capasso,3 Sanjeev Vasudevan,1 Jason M. Shohet,1 Eveline Barbieri1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Ghent University, Houston, Belgium;_ 3 _University of Napoli, Italy_.

Background. MYCN activation is a hallmark of advanced tumor stage in neuroblastoma (NB), characterizing high-risk patients prone to resistant disease. MYCN is also a potent regulator of metabolic reprogramming that favors NB adaptation to its microenvironment. The retinoic acid receptor-related orphan receptor α (RORα) is a key regulator of cell metabolism, immunity, as well as the circadian rhythm. Importantly, RORα activates the transcriptio of BMAL1, a master circadian transcription factor frequently deregulated in human cancers. Moreover, synthetic ligands RORα agonists (i.e. SR1078) have been recently optimized with great therapeutic potential.

Methods. Multivariate logistic regression analysis identified low levels of RORα as independent predictors of EFS and OS survival in large NB patients' cohorts (n=890 patients). MYCN inducible over-expression and knock-down NB lines were generated and Q-PCR assays used to assess MYCN-mediated disruption of central components of the clock machinery. Growth-suppressive and pro-apoptotic effects of genetic RORα over-expression and RORα activation (via SR1078) were tested in a panel of MYCN-amplified and non-amplified lines, as well as in MYCN-inducible MYCN-3 cells (Tet-ON). MYCN-amplified xenografts were used to test in vivo therapeutic response to SR1078.

Results. Ectopic MYCN expression upregulates the circadian repressor REV-ERBα and suppresses the circadian activator RORα and the central clock BMAL1. In contrast, MYCN silencing effectively restores their expression levels. Interestingly, the expression of the main regulators of the clock machinery (RORα, REV-ERBα, and BMAL1) are also profoundly altered in MYCN-amplified NB patient samples. Specifically, RORα and BMAL1 are uniformly repressed in MYCN-amplified NB and their reduced levels independently correlate with poor survival in large patients' cohorts (n=890 patients total, p<0.0001). Importantly, re-activation of RORα (via SR1078) strongly suppresses MYCN transcription and restores BMAL1 levels, inhibiting cell survival. Moreover, SR1078 reduces cell viability and induces apoptotic cell death to a higher extent in MYCN-amplified compared to MYCN-non amplified lines. Lastly, both SR1078 treatment and inducible RORα over-expression significantly (p=0.020) inhibit tumor growth in MYCN NB xenografts. Together, our data suggest that RORα loss promotes MYCN-induced disruption of molecular clock, contributing to NB tumorigenesis. Restoration of RORα opposes these MYCN functions, representing an effective strategy for MYCN-amplified NB.

#1952

Epigenetic modifiers outperform chemotherapy in prolonging survival in patient-derived xenograft models of Down syndrome AML.

Sonali P. Barwe, E A. Kolb, Anilkumar Gopalakrishnapillai. _Nemours/A. I. duPont Hospital for Children, Wilmington, DE_.

Children with Down syndrome (DS) are at a 500-fold increased risk for developing acute myeloid leukemia (AML) before they reach five years of age. DS-AML blasts have somatic mutations in the gene encoding the essential hematopoietic transcription factor GATA-1 resulting in hypersensitivity to chemotherapeutic drugs such as cytarabine and daunorubicin. However, therapy-induced toxicity results in greater morbidity and remains a major barrier in attaining higher survival rate. Thus, alternate therapy approaches to minimize toxicity and increase efficacy are needed. Trisomy 21 and GATA-1 mutations in DS-AML are known to alter the epigenetic landscape in multiple ways. Therefore, we evaluated the efficacy of epigenetic drugs in comparison to chemotherapy in two patient-derived xenograft (PDX) models of DS-AML.

We developed two distinct PDX models by successfully engraftment and serial passage of primary DS-AML cells in NSG-B2m mice. Both PDX lines possessed GATA-1 mutation resulting in the expression of a truncated form of GATA-1. NTPL-60 had a nonsense mutation generating a premature stop codon after the initiation codon, while NTPL-386 had a 136 bp deletion in exon 2 resulting in the loss of the initiation codon. The mouse passaged cells were intravenously injected into 6-8 week old NSG-B2m mice. Once disease establishment was confirmed based on the presence of human cells in mouse peripheral blood, five mice per group were treated with vehicle or DNA methylation inhibitor azacitidine and histone deacetylase inhibitor panobinostat either singularly or in combination at a previously determined maximally tolerated dose of 2.5 mg/Kg each. NTPL-386 xenografted mice treated with azacitidine or panobinostat survived 29 and 21 days longer than the vehicle-treated mice respectively, while the mice treated with the combination survived the longest (35 days). Similarly, azacitidine and panobinostat extended the survival of mice transplanted with NTPL-60 by 48 and 31 days respectively compared to the vehicle-treated mice. NTPL-60 mice treated with the epigenetic drug combination are alive at 57 days. Thus, we observed that the azacitidine-panobinostat combination showed statistically significant (p < 0.0001) differences in leukemic burden and mouse survival compared to treatments with either drug alone in both NTPL-386 and NTPL-60 PDX models.

We also tested the efficacy of epigenetic therapy followed by chemotherapy. The inclusion of epigenetic therapy before chemotherapy prolonged survival by 39 days compared to vehicle-treated mice. Taken together, our data indicate that epigenetic therapy may be of benefit for the treatment of children with DS-AML.

#1953

Accelerating prediction of pediatric and rare cancer vulnerabilities using next-generation cancer models.

Yuen-Yi Tseng,1 Andrew Hong,1 Paula Keskula,1 Shubhroz Gill,1 Jaime Cheah,2 Grigoriy Kryukov,1 Aviad Tsherniak,1 Francisca Vazquez,1 Glenn Cowley,1 Sahar Alkhairy,1 Coyin Oh,1 Anson Peng,1 Rebecca Deasy,1 Abeer Sayeed,1 Peter Ronning,1 Samuel Ng,3 Steven Corsello,1 Corrie Painter,1 David Sandak,4 Levi Garraway,3 Mark Rubin,5 Calvin Kuo,6 Sidharth Puram,7 David Weinstock,3 Adam Bass,3 Nikhil Wagle,3 Keith Ligon,3 Katherine Janeway,3 David Root,1 Stuart Schreiber,1 Paul Clemons,1 Aly Shamji,1 Aly Shamji,1 William Hahn,3 Todd Golub,1 Jesse Boehm1. 1 _The Broad Institute of MIT and Harvard, Cambridge, MA;_ 2 _Massachusetts Institute of Technology, Cambridge, MA;_ 3 _Dana-Farber Cancer Institute, Boston, MA;_ 4 _Rare Cancer Research Fundation, Durham, NC;_ 5 _Weill Cornell Medical College, New York, NY;_ 6 _Stanford University, Palo Alto, CA;_ 7 _Massachusetts General Hospital, Boston, MA_.

Ongoing pre-clinical efforts aim to deploy genome-scale CRISPR/Cas9 technology and large collections of small molecules to catalog maps of cancer vulnerabilities at scale. However, such efforts in pediatric and rare cancers have lagged behind comparable efforts in more common cancer types due to the dearth of cell models. Here, we present an update from our "Cancer Cell Line Factory" project on efforts to overcome key laboratory and biologistics challenges precluding progress in pediatric and rare cancers. This effort, now in it's 3rd year, represents an industry scale pipeline aiming to generate, characterize and share novel cancer models of many tumor types with the scientific community. Overall, we have processed 1153 samples from 818 patients across over 16 cancer types through this pipeline with a 28% success rate overall, including over 350 patient samples from rare and pediatric cancers. To optimize conditions for each tumor type, we have systematically compared published methods including (1) next-generation 2-dimension, (2) organoid and (3) standard approaches and have captured all information with a data management system that should enhance the ability to predict optimal ex vivo propagation conditions for future samples. Among the successful cell models verified already as part of this effort, we have generated a series of over 30 unique pediatric and rare cancer models, many of which represent the first of their kind. We screened these and other models against a library of highly annotated 440 small molecules that were previously tested against 860 existing cancer cell lines. Our results suggest that dependency data generated with novel next-generation cell cultures is potentially backwards-compatible with existing small molecule dependency datasets. Furthermore, we tested the novel Broad Institute Drug Repurposing library consisting of 4100 approved therapeutics, or those under investigation for any disease, against the first cell line models of several of these rare next generation models including angioimmunoblastic T-cell lymphoma and renal medullary carcinoma, leading to several novel drug repurposing hypotheses for rare cancers. Given these proof-of-concept studies, in partnership with the Rare Cancer Research Foundation, we launched an online matchmaking platform to connect patients with rare cancers to available research studies, facilitate online consent and provide biologistics support to enable fresh tissue donation to support cancer model generation from any clinical site in the United States. We will present results from this novel direct-to-patient approach to facilitate the generation of even larger numbers of next generation models from rare and pediatric cancers, propelling the generation of pre-clinical dependency maps of these tumors for the scientific community.

#1954

Metabolic serum signatures as potential prognostic biomarkers for neuroblastoma patients.

Richa Jain, Kirandeep Gill, Leon Qingliang Li, Amrita K. Cheema, Joanna B. Kitlinska. _Georgetown Univ., Washington, DC_.

Neuroblastoma (NB) is a pediatric malignancy with phenotypes varying from spontaneously regressing to metastatic tumors. Thus, disease stratification and the subsequent treatment decision is of utmost importance for NB patients. The methods, which are currently available for prognosis, require complex genetic analyses and access to the tumor tissue. Thus, there is a need for new prognostic and predictive markers that reflect the NB biology and rely on simple tests and easily accessible material. Serum metabolite profiles reflect a combination of factors released by the tumors and the general metabolic state of the patients. Thus, identifying the differences in metabolites between patients with various tumor phenotypes and responses to treatment may lead to discovery of biologically relevant prognostic biomarkers. The goal of our study was to determine blood-based metabolic signatures characteristic for patients with low and high-risk NB and identify potential biomarkers of high-risk disease. Plasma samples from NB patients at different stages of the disease were obtained from the Children's Oncology Group. Initially, 50 samples were analyzed by Ultra Performance Liquid Chromatography (UPLC) in conjunction with electrospray-quadrupole-time of flight mass spectrometry (ESI-Q-TOF). A set of 26 independent plasma samples was then used for validation study. Statistically significant differences in metabolic profiles were observed between serum from patients with low and high risk disease, various stages of the disease (stage 1-2 vs stage 3 and/or stage 4), as well as undifferentiated vs. differentiating tumors. For all comparisons, the metabolic signatures consisting of metabolites significantly different between the groups exhibited high test sensitivity and specificity (the area under the ROC curves 0.92-1). Metabolites that were dysregulated in high risk NBs included those involved in energy and choline metabolism, as well as amino acid and lipid biosynthesis. Of particular interest was a significant increase in serum levels of oxoglutaric acid observed in patients with high-risk disease (p<0.01) and metastatic tumors (p<0.001). As oxoglutaric acid is a key metabolite of the tricarboxylic acid (TCA) cycle that is involved in regulation of the balance between glutamine and glucose metabolism, as well as lipid synthesis and hypoxia response, these data warrant further investigations into the biological role of these pathways in biology of high risk NB. In summary, using a metabolomics approach we were able to delineate blood based biomarkers that could potentially be used for patient stratification following large scale validation studies.

#1955

Targeting ribonucleotide reductase (RNR) in Ewing sarcoma.

Kelli Goss, Stacia Koppenhafer, Kathryn Harmoney, David Gordon. _University of Iowa, Iowa City, IA_.

Ewing sarcoma is a highly aggressive bone and soft tissue cancer that is caused by the EWS-FLI1 fusion protein. The EWS-FLI1 oncoprotein functions, in part, as an aberrant transcription factor and is required for tumor growth and survival. In order to identify downstream targets of EWS-FLI1, we used human embryonic stem cells that express inducible EWS-FLI1 to model the initiation and development of Ewing sarcoma in a genetically defined system. We then used this model system and a gene expression based approach to identify that Ewing sarcoma cells are uniquely vulnerable to inhibitors of ribonucleotide reductase (RNR), which impair DNA replication by blocking the synthesis of deoxyribonucleotides. Here we report that the treatment of Ewing sarcoma cells with gemcitabine, an irreversible inhibitor of the RRM1 subunit of RNR, results in impaired DNA replication, cell cycle arrest, and apoptosis in Ewing sarcoma cells. Additionally, we have found that the effect of gemcitabine on the viability of Ewing sarcoma cells is sustained even after removal of the drug from the cell culture medium. Moreover, ataxia telangiectasia and rad3-related protein (ATR) and checkpoint kinase 1 (CHK1) inhibitors increase the toxicity of gemcitabine in Ewing sarcoma cells by blocking the adaptive response to impaired DNA replication. Currently, ongoing work is focused on the in vivo testing of gemcitabine, alone and in combination with CHK1 and ATR inhibitors, as a novel therapeutic approach for the treatment of Ewing sarcoma.

#1956

ABCE1 regulates the translational profile of neuroblastoma to drive tumor progression.

Jixuan Gao,1 Klaartje Somers,1 Katherine M. Hannan,2 Jamie I. Fletcher,1 Bing Liu,1 Ross D. Hannan,2 Richard B. Pearson,3 Michelle Haber,1 Murray D. Norris,1 Michelle J. Henderson1. 1 _Children's Cancer Institute, Randwick, Australia;_ 2 _The Australian National University, Canberra City, Australia;_ 3 _Peter MacCallum Cancer Centre, East Melbourne, Australia_.

Neuroblastoma is the most common extracranial solid tumor in children. A key driver of high-risk neuroblastoma is the MYCN transcription factor which fuels neuroblastoma progression by enhancing metabolic processes including protein translation (1). ABCE1, a member of the ATP-binding cassette (ABC) superfamily of transporters, is a translation factor directly up-regulated by MYCN (2). It is thought to dissociate the 80S ribosome into free 40S and 60S subunits so they can re-initiate translation and provide the protein building blocks required for rapid cell growth and migration (3). High ABCE1 expression is associated with poor clinical outcome in neuroblastoma patients (2). Therefore, we hypothesized that ABCE1 suppression may inhibit the aggressiveness of neuroblastoma by disabling protein synthesis. To test this, we suppressed ABCE1 using siRNAs in MYCN-amplified neuroblastoma cell lines, SK-N-BE(2) and CHP-134, which resulted in severely impaired cell migration (P<0.001) and proliferation (P<0.005). In mice xenografted with SK-N-BE(2) cells, ABCE1 suppression delayed tumor growth (P=0.001) and metastasis (P<0.001). Mechanistically, when polyribosome analysis was used to monitor the impact of ABCE1 expression on protein translation in SK-N-BE(2) cells, loss of ABCE1 reduced the proportion of actively translating ribosomes (P<0.001), leading to lower global protein synthesis (P=0.018). This is the first report of ABCE1 acting as pro-tumorigenic factor in neuroblastoma. Our data imply that targeting the translational machinery through ABCE1 may be an effective therapeutic approach for the treatment of MYC-driven cancers.

1.

Boon, K., Caron, H. N., van Asperen, R., Valentijn, L., Hermus, M. C., van Sluis, P., Roobeek, I., Weis, I., Voûte, P.A., Schwab, M. & Versteeg, R. (2001) EMBO J. 20, 1383-1393.

2.

Porro, A., Haber, M., Diolaiti, D., Iraci, N., Henderson, M., Gherardi, S., Valli, E., Munoz, M.A., Xue, C., Flemming, C., Schwab, M., Wong, J.H., Marshall, G.M., Della Valle, G., Norris, M.D. & Perini, G. (2010) J. Biol. Chem. 285, 19532-19543.

3.

Pisarev, A.V., Skabkin, M.A., Pisareva, V.P., Skabkina, O.V., Rakotondrafara, A.M., Hentze, M.W., Hellen C.U. & Pestova, T.V. (2010). Mol. Cell, 37, 196-210.

#1957

Molecular pathogenesis and drug synergism in a zebrafish model of high risk neuroblastoma.

Shuning He,1 Marc R. Mansour,2 Mark W. Zimmerman,1 Hillary M. Layden,1 A. Thomas Look1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _University College London, London, United Kingdom_.

We have developed a transgenic zebrafish model that overexpresses MYCN and harbors loss-of-function mutations of the nf1 tumor suppressor. In this model, loss of nf1 leads to aberrant activation of RAS-MAPK signaling, promoting both increased tumor cell survival and rapid tumor cell proliferation. These neuroblastomas are very aggressive in that almost all of the fish develop neuroblastoma by 3 weeks of age. Three-week old juvenile fish are very small, making it feasible to test the effectiveness of many drugs and drug combinations in vivo for activity against the primary tumors. We demonstrate these advantages of the model by showing marked synergistic anti-tumor effects of a MEK inhibitor (trametinib) and a retinoid (isotretinoin) in vivo at several different dosage combinations by in vivo isobologram analysis. Thus, inhibition of RAS-MAPK signaling can significantly improve the treatment of this very aggressive form of neuroblastoma when it is combined with the inhibition of other key pathways. Because of the very high penetrance and rapid onset of neuroblastoma in our nf1-deficient, MYCN-transgenic zebrafish model, it is one of the only model systems in which extensive analysis of the synergistic activity of two or more drugs can be evaluated in primary tumors in vivo. This capability is especially valuable given that mutations causing RAS-MAPK pathway hyperactivation have been shown to arise frequently at the time of relapse of childhood neuroblastomas, indicating the need to eliminate these mutated tumor cells as a component of the primary treatment. 

### Regulation and Imaging of Tumor Metastasis

#1958

Opposing roles of Smad2 and Smad3 in the regulation of growth and invasion of TNBC through TMEPAI.

Prajjal K. Singha, Srilakshmi Pandeswara, Manjeri A. Venkatachalam, Pothana Saikumar. _UT Health Science Ctr. at San Antonio, San Antonio, TX_.

The dependency of triple negative breast cancers (TNBC) on TGF-β signaling activity for their growth and metastasis is well documented. Breast tumors develop resistance to TGF-β-induced growth inhibition, which helps in tumor progression. Additionally, TGF-β is enriched in the tumor microenvironment and is a key inducer of epithelial to mesenchymal transition (EMT) in breast cancer. In an effort to understand the mechanisms by which TGF-β induces breast tumorigenesis, we sought to identify the relative individual contributions of Smad2 and Smad3 in mediating cancer cell growth and metastasis by TGF-β. Screening of several TNBC cell lines showed increased Smad3/Smad2 expression ratio compared to normal mammary epithelial cells, which suggested breast cancer cells may escape TGF-β mediated cell cycle arrest by altering their relative expressions of Smad3 and Smad2. Since overexpression of R-Smads may not always accurately reflect the functional status of the endogenous molecules, we individually knocked down each R-Smad and tested its effect on pro-oncogenic behavior of TGF-β. Although Smad2 deficiency has no effect on breast cancer cell behavior, Smad3 deficiency reduced growth and invasion capacity of breast cancer cells. Interestingly, while Smad3 deficiency was associated with reduced TMEPAI/PMEPA1 and EMT gene expressions and increased expression of cell cycle inhibitors, Smad2 deficiency had opposite effect on these regulators. Moreover, the decreased growth, invasion and associated gene expressions were largely reversed by overexpressing TMEPAI in Smad3 knockdown cells, suggesting that Smad3-TMEPAI axis may be involved in subverting growth suppressive effects of TGF-β into growth promotion. Compared to control and Smad2 deficient cells, Smad3 deficient cells had markedly elevated PTEN protein levels and suppressed Akt phosphorylation both under basal conditions and TGF-β stimulation. We identified that Smad3 but not Smad2 plays an important role in stimulating TMEPAI expression, growth and EMT in triple negative breast cancer cells by promoting TGF-β dependent non-canonical signaling by decreasing the cellular content of PTEN and p27 in a TMEPAI dependent manner. Taken together, the results demonstrate a novel role for Smad3 in cancer transformation and cancer progression through TMEPAI. Current therapeutic strategies are aiming at antagonizing the whole TGF-β signaling thereby completely blocking both Smad2 and Smad3 signaling pathways. Selective targeting of TGF-β-Smad3-TMEPAI axis may be more beneficial in triple negative breast cancer therapy and prevention.

#1959

MTA1 promotes tumor progression and bone metastasis in prostate cancer via positive regulation of MUC1 and CTSB.

Avinash Kumar,1 Swati Dhar,2 Gisella Campanelli,1 Nasir A. Butt,2 Christian R. Gomez,2 Jason M. Schallheim,2 Anait S. Levenson1. 1 _Long Island University, Brooklyn, NY;_ 2 _University of Mississippi Medical Center, Jackson, MS_.

About 80% of the time that prostate cancer cells metastasize, they spread to the bones. Metastasis involves activation of invasive programs through genetic and epigenetic alterations, including changes in expression of chromatin remodeler proteins. We have previously identified metastasis-associated protein 1 (MTA1), a chromatin remodeler, as a component of vicious cycle of bone metastasis and confirmed significantly higher expression of MTA1 in bone metastatic samples from patients. In an attempt to dissect molecular mechanisms of MTA1 action, we performed integrative analysis of our bone metastasis signature data along with MTA1 ChIP-Seq data, which revealed mucin 1 (MUC1) and cathepsin B (CTSB) as strong potential candidates responsible for MTA1-driven invasiveness.Here, we show that loss of function studies with MTA1 in PC3M cells exhibit reduced expression for MUC1 and CTSB. We also demonstrate that depletion of MTA1 in PC3M aggressive prostate cancer cellsdoes not affect cell proliferation but leads to decrease in colony forming ability, invasive and migratory property of these cells. When PC3M cells silenced for MTA1 and tagged with luciferase are used to generate subcutaneous or intracardiac xenografts, it leads to reduced tumor progression or decreased metastasis to bone, respectively as evident by bioluminescent measurements and histology of tumors. Ongoing experiments aim to validate the role of MUC1 and CTSB in MTA1-driven invasiveness and metastasis in prostate cancer.

#1960

Upregulation of MCP-1 regulates invasiveness in triple negative breast cancer.

Pranabananda Dutta, Kimberly Paico, Inez Yuwanita, Yanyuan Wu, Marianna Sarkissyan, Jaydutt Vadgama. _Charles R. Drew University, Los Angeles, CA_.

Background: Triple negative breast cancer (TNBC) poses a critical problem for targeted therapy due to lack of significant expression of estrogen, progesterone receptor or Her2/neu oncogene. Hence, it is imperative to identify novel therapeutic strategies to target TNBC. Our study is aimed to examine whether Monocyte Chemoattractant Protein -1 (MCP-1) is a specific marker for TNBC metastasis. Experimental

Design: We employed ELISA to determine secreted MCP-1 in cell conditioned media, as well as Real-time PCR to determine the status of MCP-1 in TNBC cell lines. Boyden chamber assay was used to determine the effect of recombinant MCP-1 on cellular metastasis. Cellular proliferation was measured with MTT assay. Immunofluorescence staining was utilized for protein of interest in breast cancer cells. MCP-1 knockdown was performed using lentiviral vector with shRNA targeting MCP-1 coding regions.

Results: Our data show that the key inflammatory chemokine MCP-1 is upregulated in TNBC cell lines both transcriptionally as well as in terms of secretion compared to ER-positive cell line, MCF-7. MCP-1 stimulation in MDA-MB231 and MCF-7cells does not affect cellular proliferation. However, MCP-1 enhances metastatic properties of MDA-MB-231 cells along with BT-549 cells. Inhibiting Chemokine receptor 2/4 (CCR2/4), cognate receptor for MCP-1, with small molecule antagonists negatively affects invasiveness in MDA-MB-231 cells as evidenced by Boyden chamber assay. Knocking down MCP-1 by shRNA decreases cell invasion in TNBC cell line, BT-549 along with downregulation of key epithelial to mesenchymal transition markers, N-cadherin and Vimentin. MCP-1 induced cell invasion in TNBC may involve activation of p44/p42 MAPK Thr202/Tyr204.

Conclusion: Our study suggests that high MCP-1 levels in TNBC is driving up metastasis potential in cells. Thus MCP-1 and its mediated pathways could be potential therapeutic targets for the treatment of TNBC.

#1961

Downregulation of proteasome activity promotes epithelial-mesenchymal transition.

Daniel A. Garcia, Asoka Banno, Eric D. Van Baarsel, Patrick J. Metz, Christella E. Widjaja, Stephanie H. Kim, Jack D. Bui, Jing Yang, John T. Chang. _UCSD, La Jolla, CA_.

The epithelial-mesenchymal transition (EMT) is a developmental program that is aberrantly activated in cancer cells, producing an invasive phenotype that can lead to metastases. Inducers of EMT are largely known and have been widely studied. However, the mechanisms that regulate the link between extracellular stimuli and EMT phenotypes remain poorly understood. Preliminary evidence from our laboratory suggests that downregulation of proteasome activity may be responsible, in part, for driving EMT. Using immortalized human mammary epithelial (HMLE) cells as a model, we show that (1) EMT is associated with decreased proteasome activity and increased polyubiquitinated substrates, (2) pharmacologic inhibition of proteasome activity leads to increased EMT phenotypes and functionality, and (3) pharmacologic inhibition of proteasome activity leads to increased EMT via stabilization of the TGF-β signaling pathway. Together, these data suggest that proteasome activity may be an unappreciated regulator of EMT.

#1962

Regulation of invasion by lysine demethylase 5B in non-small cell lung cancer cells.

Elizabeth L. Zoeller, Jessica Konen, Joshua Bell, Emily Summerbell, Jeanne Kowalski, Adam Marcus, Paula Vertino. _Emory Univ., Decatur, GA_.

Histone modifying enzymes are often dysregulated during carcinogenesis and are major contributors to the development of oncogenic features, including proliferation, drug resistance, and metastasis. Given these roles, histone modifiers are promising new targets for oncological therapeutics. One of the enzyme families at the center of this area of research is the lysine demethylase family KDM5, for which several inhibitors are in development. KDM5A and KDM5B are frequently overexpressed or mutated in human non-small cell lung cancer (NSCLC). In overexpression studies, KDM5B promotes invasion and migration of NSCLC cells, whereas invasion and migration of NSCLC cells were decreased following knockdown of KDM5B. Furthermore, in patients with NSCLC, KDM5B is expressed at higher levels in brain metastasis sites when compared to both normal tissues and primary tumors. These findings suggest a role for KDM5B in lung cancer and metastatic spread. However, the precise role of KDM5 family members in lung cancer invasion and metastasis is not known. Recently, we discovered that KDM5B is differentially expressed in cell subtypes within a 3D model of NSCLC collective cell invasion. In this model isolated single cells at the forefront of invasive branches (leader cells) express more 2-4 fold more KDM5B protein than the cells following (follower cells), while KDM5A and KDM5C are evenly expressed across both cell types. KDM5B mRNA expression is similar across cell types suggesting that the differential protein expression is mediated at the posttranscriptional level. Interestingly, global H3K4me3 levels are decreased in leader cells as compared to follower cells, supporting the idea that the lysine demethylases targeting this residue may be expressed at higher levels in leader cells. Collectively, these data suggest that KDM5B could be contributing to initiation of invasion at the primary site and thus, promoting metastasis. Given the rising prominence of therapeutic inhibitors of KDM5 family members, a better understanding how KDM5B contributes to cell invasion may lead to a new approach to the prevention of metastasis.

#1964

Identification of a novel molecule target for the detection, prediction, and treatment of peritoneal metastasis of gastric cancer.

Mitsuro Kanda, Haruyoshi Tanaka, Dai Shimizu, Daisuke Kobayashi, Chie Tanaka, Hideki Takami, Masamichi Hayashi, Naoki Iwata, Yukiko Niwa, Suguru Yamada, Tsutomu Fujii, Goro Nakayama, Hiroyuki Sugimoto, Masahiko Koike, Michitaka Fujiwara, Yasuhiro Kodera. _Nagoya Univ. Graduate School of Medicine, Nagoya, Japan_.

Background: Advanced gastric cancer (GC) frequently recurs because of undetected micrometastases even when disease is localized and patients undergo curative resection. Moreover, peritoneal metastasis is fatal. We aimed to develop novel diagnostic and therapeutic targets specific for peritoneal metastasis of GC to improve management.

Methods: We conducted a metastatic pathway-specific transcriptome analysis to identify candidate biomarkers comprising 340 patients allocated to discovery and validation sets (1:2) to evaluate the diagnostic and predictive value. The mRNA and protein levels in primary GC tissues were compared with patients' clinical characteristics and survival. The effects of siRNA-mediated knockdown on phenotype and fluorouracil sensitivity of GC cells were evaluated in vitro, and the therapeutic effects of siRNAs were evaluated using a mouse xenograft model.

Results: Synaptotagmin VIII (SYT8) was identified as a candidate biomarker specific to peritoneal metastasis. SYT8 levels were elevated in the validation set comprising patients with peritoneal recurrence or metastasis. High SYT8 levels were significantly and specifically associated with peritoneal metastasis, and served as an independent prognostic marker for peritoneal recurrence-free survival of patients with stage II/III GC. The survival difference between high and low SYT8 levels was associated with patients who received adjuvant chemotherapy. Inhibition of SYT8 expression by GC cells correlated with decreased invasion, migration, and fluorouracil resistance. Intraperitoneal administration of SYT8-siRNA inhibited the growth of peritoneal nodules and prolonged survival of mice engrafted with GC cells.

Conclusions: SYT8 expression represents a promising diagnostic and predictive biomarker for peritoneal metastasis of GC.

#1965

A novel biocompatible fluorescent nanoparticle enables enhanced live cell tagging and tracking of cancer cells.

Nick Asbrock,1 Vi Chu,1 Kevin Su,1 Ben Zhong Tang,2 Bin Liu3. 1 _MilliporeSigma, Temecula, CA;_ 2 _The Hong Kong University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong, China; _3 _National University of Singapore, Temecula, CA_.

Metastasis is the leading cause of cancer mortality. Metastasis is a multi-step process which includes local tumor cell invasion, cell migration into the vasculature, exit of cells from the circulation and colonization at the distal tissue sites. Long-term noninvasive cell tracking by fluorescent probes is of great importance to life science and biomedical engineering. Current methods used to fluorescently tag cancer cells have been limited by short signal duration, high background auto-fluorescence or lengthy cell line generation using GFP. We have developed a biocompatible fluorescent nanoparticle which relies on Aggregation Induced Emission (AIE) technology that are highly resistant to fluorescent signal quenching. These particles enable highly efficient live cell fluorescent tagging while retaining fluorescent signal for up to 10 days in vitro and 21 days in vivo. These nanoparticles will open new avenues in the development of fluorescent probes for following biological processes such as carcinogenesis.

#1966

Cell-surface major vault protein is a novel marker for circulating tumor cells with nonepithelial phenotypes in hepatocellular carcinoma.

Hyun Min Lee,1 Jae Won Joh,2 Won Tae Kim,1 Min Kyu Kim,1 Se Ri Seo,1 Hong Seo Choi,1 Hee Jin Chang,3 Young Joo Jang,4 Dae Shick Kim,2 Chun Jeih Ryu1. 1 _Sejong University, Seoul, Republic of Korea;_ 2 _Sungkyunkwan University, Seoul, Republic of Korea;_ 3 _National Cancer Center, Goyang, Republic of Korea;_ 4 _Dankook University, Cheonan, Republic of Korea_.

Hepatocellular carcinoma (HCC) is currently is the fifth most common malignancy worldwide and the poor because of frequent metastasis and resistance to chemotherapy. Circulating tumor cells (CTCs) in blood have attracted attention as potential seeds for metastasis and an important indicator of treatment outcome. However, the biological properties of CTCs are largely unknown due to rarity and lack of CTC-specific surface markers. Major vault protein (MVP) is upregulated during malignant progression and drug resistance development in various cancer cells, although it is ubiquitously expressed in many normal tissues. Here, we found for the first time that MVP was expressed on the surface of various cancer cells including HCC cell lines, although MVP has been known as a cytoplasmic and nuclear protein. To investigate the role of cell-surface MVP (csMVP) on HCC cell lines, MVP expression was knocked down in Huh7 cells by small interfering RNA. MVP knockdown decreased cell growth and increased apoptotic cell death. When Huh7 cells were treated with a polyclonal anti-MVP antibody (α-MVP) recognizing csMVP, Huh7 cell proliferation was decreased without apoptotic cell death. Cell sorting revealed that csMVP-positive Huh7 cells showed a higher proliferation and survival rate than csMVP-negative Huh7 cells under the stress of low density seeding. Huh7 cells treated with α-MVP also inhibited cell invasion and migration in vitro. Thus, csMVP is positively associated with HCC cell proliferation, survival, invasion and migration. Analysis of signaling molecules showed that MVP knockdown caused a significant decrease in the levels of phosphorylation of FAK, ERK, AKT and S6K. MVP knockdown also increased E-cadherin but decreased vimentin, indicating that MVP promotes the process of epithelial-to-mesenchymal transition. To further analyze the role of csMVP during HCC metastasis, blood samples from 62 HCC patients and 10 healthy volunteers were stained with csMVP, EpCAM, HSA, EGFR, CK, and/or vimentin antibodies after the depletion of red blood cells and CD45-positive cells. csMVP-positive CTCs were detected in approximately 85.5% (> 0.46CTCs/ml) of patients, and the cell count measured in ml of blood ranged between 0.48 and 37.8. Double staining showed that almost all csMVP-positive cells were panCK-negative while approximately one fifth of csMVP-positive CTCs was HSA-positive. Triple staining further showed that among csMVP-positive cells, EpCAM+Vimentin-, EpCAM+Vimentin+, EpCAM-Vimentin+ and EpCAM-Vimentin- cells were approximately 0, 0, 49 and 51%, respectively, suggesting that a half of csMVP-positive cells are EMT-phenotypic cells and the rest of them are both EpCAM- and vimentin-negative cells. The results suggest that csMVP is a novel marker on CTCs in patients with HCCs, where it is expressed predominantly on EMT phenotypic and EpCAM-vimentin- intermediate CTCs.

#1967

A novel 40kDa CPE-ΔN isoform promotes proliferation and invasion in pancreatic cancer cells.

XuYu Yang, Cong Ling, Hong Lou, Loh Yoke Peng. _National Institutes of Health, Bethesda, MD_.

Carboxypeptidase E (CPE) is a prohormone processing exopeptidase that cleaves C-terminal basic residues from peptide hormones liberated endoproteolytically from prohormones. It is a 476- amino acid protein with a signal peptide in its N-terminus and is mainly expressed in neuropeptide-rich areas of brain and endocrine tissues. As a multifunctional protein, CPE plays many non-enzymatic roles such as prohormone sorting, vesicle transport, and secretion in addition to its carboxypeptidase activity. Moreover, high expression of CPE is found in metastatic tumor tissues, including liver cancer, renal clear cell carcinoma, colorectal cancer, cervical cancer and melanoma, suggesting CPE may be involved in tumor metastasis and progression. In this study, a novel CPE splice isoform mRNA was identified by Northern blotting, which is ~1.83kb in size. RACE assay and sequence analysis confirmed existence of this CPE isoform mRNA, which partially lacks a N-terminus region, and has a shorter 3'-UTR compared to WT-CPE mRNA, by removal of 188 nucleotides within the first exon and 589 nucleotides out of the 3'-UTR, respectively. Bioinformatics analysis revealed that this CPE isoform mRNA has a shortened ORF, which starts coding from the 3rd ATG relative to wild-type (WT) CPE mRNA and encodes a 40kDa CPE-αN protein with the N-terminus truncated. Western blot analysis showed that 40kD CPE-αN is expressed in multiple cancer cell lines and tumor tissues. In Panc-1pancreatic cancer cells, knockdown of endogenous CPE(s) inhibited cell growth and invasion; in contrast, ectopic expression of 40kD CPE isoform significantly promoted proliferation, colony formation, invasion and migration, whereas overexpression of WT-CPE had only a small effect. These results suggest that the 40kD CPE-αN isoform plays a role in cell growth and metastasis.

#1968

A novel mechanism of metastasis: Extracellular ATP promotes invasion and metastasis independent of purinergic receptor signaling.

Yanyang Cao, Xuan Wang, Xiaozhuo Chen. _Ohio Univ., Athens, OH_.

Cancer is one of the top deadliest diseases in the US and worldwide. Metastasis, the dissemination of cancer cells from the primary tumors to distant organs, is responsible for 90% of solid tumor-related deaths. Tumor invasion and metastasis is a multistep process in which loss of cell-cell adhesion, increased proteolysis, and cell motility has been shown to be critical steps. Extracellular ATP is potentially a very important factor involved in tumor invasion and metastasis because both cell detachment and migration require energy, which is known to be mainly provided by ATP. Recent studies have indicated that the extracellular microenvironment of tumors contains much higher concentrations of ATP than normal tissues of the same cell origins. Our recent studies demonstrated that extracellular ATP is taken up by cancer cells through macropinocytosis and other endocytosis and promotes cancer cell growth, survival, and drug resistance (1-3). Based on all these, we hypothesized that extracellular ATP (eATP) plays critical roles in the regulation of tumor cell detachment, motility, invasion and tumor metastasis initiation. It was previously shown that ATP-induced purinergic receptor (PR) signaling is involved in metastasis. Our hypothesis is different in that eATP also mediates invasion and metastasis independent of PR signaling. Various bioassays were used in human non-small cell lung cancer (NSCLC) A549 cells to test our hypothesis. Our results show that eATP treatment led to a substantially increased number of floating cancer cells, and these cells were viable and formed clones in a clonogenic assay. This indicates that eATP induces cancer cell detachment. Moreover, treatment of eATP also induces cell migration in cell wounding and Transwell migration assays. In vitro invasion assay showed that eATP induced a dose-and time-dependent increase in the invasive capacities of the A549 cells. Western blot analysis indicates that eATP treatment reduced the expression of cell-cell adhesion molecule E-cadherin. PR inhibitors only slightly attenuated these effects. All these suggest novel ATP mechanisms independent of the PR signaling that are unreported before and imply novel targets for inhibiting/preventing metastasis.

References

1. Qian et al. Cancer Letters 351; 242-251 (2014).

2. Chen, Qian, and Wu. Free Radical Biol Med 79; 253-263 (2015)

3. Qian et al. Mol Cancer Res 14(11); 1087-1096 (2016)

#1969

PKCα-induced Twist1 phosphorylation is a novel regulator of Twist1 stabilization.

Roslyn Tedja,1 Ayesha B. Alvero,1 Carlos Cardenas,1 Mary Pitruzzello,1 Gang Yin,2 Yang Yang-Hartwich,1 Cai Roberts,1 Carlotta Glackin,3 Gil G. Mor1. 1 _Yale University School of Medicine, New Haven, CT;_ 2 _Central South University, Hunan, China;_ 3 _City of Hope Medical Center, Duarte, CA_.

Background:

Epithelial - mesenchymal transition (EMT) is a pre-requisite for cancer metastasis. Twist-1 is a transcription factor with a central role in the process of EMT and therefore metastasis formation. Using ovarian cancer models, our group has previously shown that although the stationary epithelial phenotype express Twist1 mRNA, this phenotype is enforced by mechanisms that constitutively ubiquitinates and degrades the Twist1 protein. The objective of this study is to identify molecular mechanisms that promote Twist1 protein stabilization and therefore confer a mesenchymal and mobile phenotype.

Materials and methods:

Novel phosphorylation sites on Twist1 protein, which are adjacent to ubiquitination sites, were identified using NetPhosK and Ubpred. Further analysis demonstrates that most of these sites contain the consensus sequence for PKCα. Constitutively active PKCα (PKCαcat) was ectopically expressed in HEK293T cells and patient-derived epithelial ovarian cancer cell lines. Protein expression and phosphorylation status were determined using western blot analysis using either whole cell lysates or cellular fractions. Protein-protein interaction was determined by immunoprecipiation. The specific Twist1 domain required for PKCα interaction was determined by co-transfection with Twist1 deletion mutants.

Results:

Ectopic expression of PKC⍺cat in HEK293T cells and ovarian cancer cells resulted in increased levels of Twist1 protein compared to empty vector control. In ovarian cancer cells, PKCcat-induced upregulation of Twist1 protein is not associated with an increase in Twist1 mRNA but instead is associated with Twist1 phosphorylation, decreased ubiquitination, and enhanced stabilization. We identify the twist box (WR) domain of Twist1 as a pre-requisite for PKCα binding and PKCαcat-induced Twist1 stabilization. Moreover, we identify TGFβ1 as a potent activator of endogenous PKCα in epithelial ovarian cancer cells. TGFβ1 (1 ng/ml) is able to: promote the activation and membranal translocation of PKCα; increase Twist1 protein levels; and induce spheroid formation and EMT in epithelial ovarian cancer cells. Interestingly, TGFβ1 had no effect on Twist1 mRNA suggesting that this mechanism is independent of the classical TGFβ-Smad pathway.

Conclusions:

We demonstrate for the first time a novel TGFβ-PKCα signaling pathway that specifically targets Twist1 protein for phosphorylation and stabilization resulting in EMT. This mechanism is independent of the classical TGFβ-Smad pathway that controls EMT via transcriptional regulation. Since TGFβ is a pleiotropic cytokine that can affect multiple cell types, the identification of PKCα as a novel target in ovarian cancer cells may aid in the development of better therapeutic modalities that can prevent EMT and curtail metastasis formation.

#1970

Glucose-regulated protein 94 is the novel progression biomarker in esophageal squamous cell carcinoma.

Yu-Jia Chang,1 Chien-Yu Huang2. 1 _Taipei Medical Univ., Taipei, Taiwan;_ 2 _Department of Surgery, Shuang Ho Hospital, Taipei Medical University, Taipei, Taiwan_.

Background

Esophageal cancer is a worldwide health problem with a very poor prognosis as most patients present very late when the disease is advanced and difficult to cure. It is urgent to figure out a diagnostic and therapeutic strategy. Glucose-regulated protein 94 (GRP94) has been found highly correlation with cancer progression. However, the role of GRP94 in the progression and metastasis of esophageal squamous cell carcinoma (ESCC) is still unclear.

Methods: We investigated the GRP94 expression level of ESCC cells and generated GRP94-KD cells by shRNA technique. The proliferation assay, migration, invasion assay were performed. The tissue array was performed to see the correlation of GRP94 and clinical outcome on ESCC. Mitochondrial bioenergetics were assessed using a Seahorse XF24 flux analyzer. The Transmission Electron microscopy was applied to see the structure of mitochondria.

Results: We found that ESCC specimen that expressed higher GRP94 showed the lower overall survival in tissue array results. The ESCC cells expressed high level of GRP94. Silencing GRP94 reduced the proliferation, migratory and invasion activity. Transmission Electron microscopy revealed the impaired mitochondrial in GRP94-KD cells. GRP94-KD cells also displayed reduced basal respiration, spare of respiratory capacity and ATP production. GRP94-KD caused the accumulation of oxidative damage. In molecular mechanism, we found that silencing GRP94 may cause a decrease COX2 expression and alter EMT expression pattern.

Conclusion

We found that silencing GRP94 in ESCC cells suppressed cancer growth and metastasis ability through COX-2 and EMT molecules expression. In addition, GRP94 may mediate the functional mitochondria in ESCC. GRP94 may be a new target to develop the therapeutic strategy for ESCC.

#1971

ITGBL1 is a novel epithelial mesenchymal transition-associated prognostic biomarker in colorectal cancer.

Takatoshi Matsuyama,1 Toshiaki Ishikawa,2 Naoki Takahashi,3 Yasuhide Yamada,3 Masamichi Yasuno,2 Tatsuyuki Kawano,2 Hiroyuki Uetake,2 Ajay Goel1. 1 _Baylor Scott & White Research Institute and Charles A Sammons Cancer Center, Dallas, TX; _2 _Tokyo Medical and Dental University, Tokyo, Japan;_ 3 _National Cancer Center Hospital, Tokyo, Japan_.

Purpose: Colorectal cancer (CRC) ranks as the third leading cancer worldwide, and its incidence continues to rise gradually, highlighting the need to stratify the risk of recurrence after curative surgery. Recently, several genes have been identified which appear to associate with metastasis, as they mediate epithelial-to-mesenchymal transition (EMT) in cancer. This study aimed to identify novel EMT and cancer recurrence-associated biomarkers through systematic and comprehensive discovery and validated strategy in multiple, independent CRC cohorts.

Experimental Design: Two independent gene expression microarray datasets (n =173 and n =307 respectively) were used to identify novel metastasis-recurrence biomarkers for CRC. Following carefully selection and prioritization of biomarkers, we selected a candidate gene and validated its performance as a recurrence marker in a large testing cohort (n=566), and two independent clinical validation cohorts (n=201, n=475, respectively). To confirm the protein expression of ITGBL1 in cancer, immunohistochemistry (IHC) was performed in paired 33 primary CRCs and adjacent normal mucosa, as well as a subset of liver and lung metastases tissues. In addition, we used Gene Set Enrichment Analysis (GSEA) to determine the functional role of ITGBL1 in CRC.

Results: During the discovery step, gene expression profiles from differentially expressed genes between recurrence positive and negative primary CRCs, as well as evaluation of the metastatic sites compared with primary CRC, identified ITGBL1 as a most promising candidate biomarker. High expression of ITGBL1 associated with poor overall survival (OS) in stage I-IV patients and worse disease-free survival (DFS) in stage I-III patients. Subgroup validation of these results in two large and independent patient cohorts confirmed these findings and demonstrated that high ITGBL1 expression correlated with shorter DFS in stage II and III CRC patients. In addition, high ITGBL1 expression emerged as an independent prognostic factor for DFS in stage II and III patients. IHC analysis revealed that both early stage CRCs and adjacent normal colonic mucosa displayed low ITGBL1 expression, while ITGBL1 expression gradually increased from tumor surface to the invasive front in late stage cancer, indicating that ITGBL1 may facilitate EMT process and promote a more aggressive phenotype in CRC.

Conclusions: High expression of ITGBL1 in primary tumors was associated with tumor recurrence in CRC patients after curative surgery. Collectively, we have identified ITGBL1 as a novel EMT-associated biomarker which could be used for risk stratification for metastatic potential in CRC.

#1972

Identification of novel candidate driver genes of colorectal cancer on chromosome 7p.

Yuta Kouyama,1 Yushi Ogawa,2 Takaaki Masuda,1 Yukihiro Yoshikawa,1 Miwa Noda,1 Hiroaki Wakiyama,1 Kuniaki Sato,1 Sho Nambara,1 Qingjiang Hu,1 Shinya Kidogami,1 Tomoko Saito,1 Shotaro Sakimura,1 Naoki Hayashi,1 Yohsuke Kuroda,1 Shuhei Ito,1 Hidetoshi Eguchi,1 Koshi Mimori1. 1 _Kyushu University Beppu Hospital, Japan, Japan;_ 2 _Showa University Northern Yokohama Hospital, Japan, Japan_.

Background

Colorectal cancer (CRC) is one of the most prevalent types of cancer. The high mortality rate of CRC is a serious problem. Hence it is urgently necessary to identify novel molecular target to improve the mortality rate. Amplification of chromosome 7p is frequent in CRC, and it has been considered to harbor driver genes that promote tumorigenesis or tumor progression by the gain of function. The aim of this study is to identify novel candidate driver genes on chromosome 7p and to clarify the clinical significance of their expression in CRC.

Material and Methods

1. We selected the candidate genes that satisfied the following criteria using CRC data from The Cancer Genome Atlas (TCGA). 1) The DNA copy number and mRNA expression is positively correlated with each other, 2) overexpressed in the tumor tissues compared to the normal tissues. 2. The mRNA expression of the candidate genes was measured in 108 surgically-resected CRC tissues and the paired normal tissues in our hospital by quantitative RT-PCR. The differences of mRNA expression between CRC tissues and normal tissues were analyzed by Mann Whitney U-test. 3. Survival analysis between high and low expression group of the candidate genes was performed by Kaplan-Meier method. Correlation between the mRNA expression of the candidate genes and the clinicopathological factors were analyzed by Fisher's exact test. 4. We performed Gene Set Enrichment Analysis (GSEA) in CRC data from TCGA to clarify the correlation between the candidate genes and gene sets that are associated with tumorigenesis or tumor progression.

Results

DEAD Box Helicase56 (DDX56), ATP-dependent RNA helicases involved in several aspects of RNA metabolism including mRNA splicing and transport, transcription, translation and remodeling of ribonucleoprotein complexes, was satisfied with the criteria. The expression of DDX56 was significantly higher in CRC tissues than in normal colon tissues (p<0.005), and it correlated with lymphatic invasion (p=0.02), and distant metastasis (p=0.03). The high DDX56 expression group had a significantly poorer prognosis than the low expression group (p=0.03). On multivariate analysis, high DDX56 expression was an independent prognostic factor affecting OS (p=0.013) with hazard ratios (95% CI) of 2.32 (1.20-4.48) among clinicopathological factors. GSEA showed that DDX56 expression was positively correlated with mitotic cell cycle progression and spliceosome.

Conclusions

We identified DDX56 as a promising driver gene of CRC on chromosome 7p. DDX56 expression was positively associated with lymphatic invasion and distant metastasis, and was an independent poor prognostic factor. Furthermore, DDX56 may be involved in tumor progression through stimulating cell-cycle. DDX56 could be a therapeutic target as well as a poor prognostic biomarker in CRC.

#1973

Novel relationships of expression of methylxanthine alkaloid receptor genes and risk of breast carcinoma recurrence.

Seth B. Sereff, Michael W. Daniels, James L. Wittliff. _University of Louisville, Louisville, KY_.

Consumption of methylxanthine alkaloids such as caffeine, theophylline and theobromine may induce breast pain. Caffeine appears to induce its biological activities by antagonizing adenosine receptors, which have been implicated in breast cancer cell behavior in vitro. Our goal is to evaluate expression of genes for methylxanthine receptors and metabolizing enzymes for assessing risk of breast cancer recurrence.

Procedures: De-identified primary cancers previously collected, stored and analyzed under stringent conditions were employed to amass an IRB-approved, de-identified comprehensive Database. Patient-related properties (e.g., nodal status, clinical outcome) and results from estrogen (ER) and progestin receptor (PR) analyses and gene expression assays guided selection. To decode clinical utility of gene expression profiles, Laser Capture Microdissection (PixCell IIe™ Arcturus/Thermo Fisher) was used previously to non-destructively collect carcinoma cells. RNA was extracted, amplified and analyzed by microarray (~ 22,000 genes).

Results: Gene expression levels of 8 methylxanthine receptors, 8 metabolizing enzymes and various phosphodiesterases were retrieved from microarray results of 247 breast biopsies. Univariable Cox regressions and Kaplan Meier plots were determined for each candidate gene with R software. Kaplan-Meier plots of PDE4A, CYP2A6 or CYP2E individually indicated lower expression was related to decreased progression free (PFS) and overall survival (OS) while PDE1A over-expression predicted decreased PFS and OS. ADORA2B and RYR1 over-expression was associated with lower OS. Gene expression was examined with ER or PR status without regard to clinical outcome. Breast cancers that were ER+ exhibited lower ADORA1, ADORA2B and RYR1 expression and elevated expression of PDE4A, CYP2A6 and CYP2E. PR+ carcinomas also exhibited decreased expression of ADORA2B and RYR1 expression. However, over-expression of ADORA3, PDE4A, CYP2C8 and CYP2E was observed in PR+ cancers. Analysis of variance (ANOVA) of carcinomas according to ER/PR status indicated ADORA2B expression was elevated in ER- cancers regardless of PR status. ANOVA also showed PDE4A was over expressed in ER+ carcinomas regardless of PR status. LASSO analysis revealed CYP2E, PDE1A and PDE4A expression taken jointly predicted PFS which was validated externally (Breast Cancer Meta-base) with SurvExpress.

Conclusions: Collectively, results suggest expression of genes involved in methylxanthine action and metabolism may be used to predict breast cancer behavior. Significantly, we identified gene expression signatures in single cell types that were highly associated with clinical outcome suggesting promise for development of novel prognostic tests for breast carcinoma management and design of new therapeutics.

#1974

Novel function of PCNA in mammary tumor development and distant metastasis through tyrosine phosphorylation.

Yuan-Liang Wang,1 Lanie Chang,2 Susan E. Waltz,3 Shao-Chun Wang1. 1 _China Medical University & Hospital, Taichung City, Taiwan; _2 _China Medical University Hospital, Taichung City, Taiwan;_ 3 _University of Cincinnati, Cincinnati, OH_.

Breast cancer is the most common cancer besides skin cancer and is the second leading cause of cancer-related death in women in the US. Despite intensive study, metastasis and tumor dormancy continue to be major hurdles to eradicate this disease. PCNA forms a homotrimer ring encircling the DNA double helix and acts as a sliding platform indispensable for DNA replication, damage repair, and chromatin remodeling. While a positive role of PCNA in is conceivable, how PCNA functions in tumor progression remains elusive due to the embryonic lethal phenotype in PCNA-deficient mice. We have shown that PCNA is regulated by phosphorylation at tyrosine 211 (Y211) meditated through multiple growth factor-stimulated signaling pathways. Y211 phosphorylation enhances chromatin-bound PCNA to promote cell proliferation and is frequently detected in multiple human cancers including breast cancer. To further dissect its biological functions, we have generated the PCNA211F/211F "knock-in" mice in which Y211 is replaced with a structurally similar but non-phosphorylatable amino acid phenylalanine (F). Comparing to wild-type mice, PCNA211F/211F mice have significantly reduced mammary tumor formation upon induction by the carcinogen DMBA. To understand its biological role in oncogene-induced mammary tumorigenesis, the PCNA211F/211F mice were crossed with the MMTV-PyMT mice in which mammary gland expression of the oncogene polyoma virus middle T antigen (PyMT) is driven by the mouse mammary tumor virus (MMTV) enhancer/promoter. PCNA211F/211F;MMTV-PyMT mice have statistically significant but only moderate decrease in mammary tumor development. However, their lung metastasis is dramatically ablated compared to PCNAWT/WT;MMTV-PyMT mice harboring primary tumors of similar size. These results suggest that pY211-PCNA has an essential function not only in tumorigenesis but more importantly in progression to distant metastasis. Consistently, mammary tumors derived from PCNAWT/WT;MMTV-PyMT mice exhibits more pronounced stromal activity than tumors of PCNA211F/211F;MMTV-PyMT mice. The suppression of metastasis and stromal activity in PCNA211F/211F;MMTV-PyMT mice is associated with reduced number of tumor-initiating cells (TICs) as determined by in vitro and in vivo analyses. Further study using syngeneic orthotopic model of transplanting murine cancer cells into the mammary glands of PCNAWT/WT and PCNA211F/211F mice unveils an unexpected function of pY211-PCNA in the stromal compartment on tumor development. Thus, our study reveals a novel tumor-promoting function of PCNA in both parenchymal and stromal compartments besides its traditional role in cell proliferation. Given our prior findings that the signaling pathways leading to Y211 phosphorylation can also be inhibited by FDA-approved drugs, our results suggest that pY211-PCNA may be a new druggable mechanism to target tumor metastasis.

#1975

FAM83B: A novel regulator of cell plasticity in breast cancer.

Courtney A. Bartel, Damian J. Junk, Mark W. Jackson. _Case Western Reserve University, Cleveland, OH_.

Epithelial to mesenchymal transition (EMT) is an important process in both metastasis and cancer stem cell (CSC) enrichment. Recent studies suggest that instead of simply transitioning from one cell state to another, cancer cells exist along a continuum of epithelial and mesenchymal cell states. Identifying novel regulators of tumor cell plasticity will be critical to fully understand this continuum and to target metastasis and CSC phenotypes in the future. FAM83B is a novel oncogene identified by the Jackson Laboratory in a forward genetic screen for drivers of Human Mammary Epithelial Cell (HMEC) transformation. Previous work showed that FAM83B expression is elevated in triple negative breast cancers and that FAM83B also activates key cell signaling pathways (EGFR, MAPK, and PI3K) in breast cancers. Here, we implicate FAM83B as a novel regulator of epithelial-mesenchymal plasticity using a HMEC transformation model. Transformation of primary HMEC by expressing shp16, shp53, c-Myc, and RAS results in the generation of two distinct transformed cell populations. One population retains epithelial characteristics, while an emergent population spontaneously acquires a mesenchymal morphology and a CD24LOCD44HI (CSC) cell surface marker profile. Moreover, isolation of epithelial, CD24HICD44LO cells (non-CSC) and exposure to Oncostatin M (OSM), an EMT-inducing cytokine, can also generate cells with a mesenchymal/CSC phenotype. Interestingly, FAM83B expression was significantly elevated in the epithelial/non-CSC population compared to the mesenchymal/CSC population. Moreover, treatment of purified epithelial/non-CSC cells with OSM resulted in decreased FAM83B expression levels, indicating FAM83B expression is suppressed as the cells move through EMT. FAM83B expression also correlated with an epithelial phenotype in a panel of breast cancer cell lines. shRNA-mediated knock-down of FAM83B from epithelial/non-CSC resulted in increased spontaneous EMT, concomitant with elevated expression of the mesenchymal marker Vimentin and master EMT transcription factor Zeb1. Following treatment with OSM, sh83B cells maintain prolonged, elevated p-Stat3 and p-ERK signaling, known contributors to the mesenchymal/CSC phenotype. Conversely, OSM-induced EMT was blunted in epithelial/non-CSC expressing exogenous FAM83B. Thus, FAM83B expression is a determinant in maintaining an epithelial phenotype, while suppression of FAM83B is important during EMT. Future work will seek to define novel FAM83B-mediated signaling pathways important for regulating cellular plasticity.

#1976

Dried blood imaging and immunoassay: Spot formation, particle migration, and tumor cell detection.

Quanxu Shen,1 Bin Hong2. 1 _The Fifth Central Hospital of Tianjin, Tianjin, China;_ 2 _TeloVISION LLC, West Lafayette, IN_.

Blood droplet, when dried, forms a structured sedimentation pattern with central flat, corona ring and peripheral rim. This pattern can be fine-tuned with the blood formula. Surfactant is critical to promote the gelation process, eliminate the film cracking and delamination, and hence, suppress the coffee-ring effect. Additionally, surfactant restrains the shrinkage of the dried film. Without surfactant, the contraction was 100~150µm for 20µL of blood; decreased with surfactant added; fully stopped at 10% of concentration. Drying at room temperature with surfactant formed a flat disc-shaped deposit, while at 37°C, volcanic protuberance was developed. The size of the protuberance depended on the concentration of the surfactant primarily, and the anticoagulant type, evaporation velocity, and blood volume. To monitor the dynamic desiccation process, video recording of the evaporating drop was employed. It revealed a quick dislocation of the leukocyte-resembling beads to the outer region of the blood spot, reportedly at velocity of 10µm/sec. The use of 1 and 10µm sized beads further demonstrated layered convective flow below the surface. Beads migrated outward primarily before gelation, inward slightly during solidification, and ultimately settled down across the spot. The movement of beads was found in the range of a few hundred microns to millimeters before and after desiccation for a 50µL drop. Notably, plasma proteins and saline have influenced the bead distribution more than the surfactant. After plasma removal and mixing with surfactant, the majority of beads were dragged to the periphery, compared with many beads for no plasma and surfactant, some for whole blood with surfactant, and none for whole blood without surfactant. For a thick blood film, detection of embedded cells and beads can be difficult. Experiments showed that beam of longer wavelength, 532nm, was capable of visualizing all fluorescent cells and beads. Dried blood spot (DBS) assay has been widely used for newborn metabolic testing and DNA analysis. To detect rare tumor cells in DBS, the dried pattern was optimized to eliminate the artifacts, stabilize the cells, and enrich the targeted cell. DBS assay holds greater potential for cancer detection when the medical resource is limited.

#1977

Tracking cancer colonization in xenografts using ultrasensitive accelerator mass spectrometry methods.

Nicholas R. Hum, Kelly A. Martin, Michael Malfatti, Kurt Haack, Bruce A. Buchholz, Gabriela G. Loots. _Lawrence Livermore National Laboratory, Livermore, CA_.

Background: The inability to effectively treat metastases is the main reason for the limited progress in reducing the rates of cancer morbidity and mortality. One major drawback is the lack of quantitative assays for assessing the size and tissue prevalence of tumors in newly diagnosed individuals. Current methods for quantifying tumor burden are mainly qualitative and include measuring the gross weight of the affected organ, counting tumors on the surface of the organ, or evaluating a small sample of the organ using histologic sections. These methods are crude measures of tumor burden and size distribution, and in the case of histology, they are time consuming, difficult to process an adequate sample size and non-quantitative.

Methods: Animal models of metastasis have been useful in identifying genes that regulate susceptibility to the development and progression of metastasis and have helped to highlight potential novel targets for drug development. In particular several small animal imaging technologies including magnetic resonance imaging, high frequency ultrasound, and optical imaging have been recently applied to this task. Each of these methods may be useful for specific research projects, based on their unique combination of resolution, image acquisition time, animal throughput, and cost-effectiveness, yet none of these modalities adequately address the need for rapid quantification of tumors across the entire organism, nor do they assess therapeutic effectiveness in eradicating cancer in xenograft models. We have developed an Accelerator Mass Spectrometry (AMS)-based high precision quantitative method for assessing the metastatic potential of primary tumors isolated from newly diagnosed patients.

Results: Our AMS-based methodology to study metastasis uses xenograft cancer cells labeled with 14C-labeled thymidine that are delivered intravenously into NSG mice and allowed to develop metastatic cancer over the course of up to 10 weeks. At the end of the experiment, all vital organs are collected; the DNA is isolated and is examined by AMS for the presence of 14C-signal. The labeling was optimized to achieve sufficient signal such that a tumor derived from a single cell could be detected by AMS, in secondary tumors, in vivo, independent of histological data.

Conclusions: Using this approach we have determined that tissue colonization by tumor cells is a very rare event, where most metastatic tumors are initiated by less than 10 cells delivered into NSG mice. Further optimization of these techniques will allow us to explore the metastatic potential of primary tumors, isolated from biopsies and expanded in Avatar mice.

This study was supported in part by NIH P41MI03483 and was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). IM number: LLNL-678306

#1978

Identification of the metastatic cell populations in a spontaneous mouse model of melanoma.

Xiaoshuang Li, Raul Torres, Lidia Kos. _Florida International University, Miami, FL_.

Melanoma is the deadliest form of skin cancer due to its high propensity to metastasize and resistance to current therapies. We have created a spontaneous mouse model of metastatic melanoma (Dct-Grm1/K5-Edn3) where metastasis to the lungs is 80% penetrant. The primary tumors of these mice present cellular heterogeneity with cells at varying levels of differentiation. The main goal of this study is to determine the metastatic potential of the primary tumor resident Tyrosinase positive cells and evaluate the dynamic pattern of gene expression as those cells move from the primary tumors to the sites of metastasis. To accomplish this aim we crossed the Dct-Grm1/K5-Edn3 mice to CreERT2/ ROSAmT/mG mice to indelibly label Tyrosinase cell populations within the primary tumor by topical application of 4-hydroxytamoxifen at the tumor site. In vivo lineage tracing and characterization of those labeled cells was performed in the metastatic lesions. We found that Tyrosinase positive cells enter the circulation before the appearance of any noticeable tumor or nevus and establish close interactions with the vasculature as single cells. Metastatic cells in close association with the inner wall of the vasculature lose pigmentation and do not express melanocytic markers. Interestingly, those intravascular cells mimic endothelial cell properties with the expression of platelet endothelial cell adhesion molecule (PECAM-1, also known as CD31). In the lung tissue, the primary tumor derived Tyrosinase positive cells or their progeny can survive and establish successful metastases with pigmentation. This in vivo lineage tracing system in mouse will be a powerful model to evaluate and help us understand the etiology and pathogenesis of melanoma metastasis. Further characterization of those more aggressive cells in melanoma will allow for the development of new prognostic tests and novel therapeutic strategies to eliminate metastasis.

#1979

Cell lines that genomically resemble high-grade serous ovarian carcinomas display differences in growth and invasive capacities in an orthometastatic nude mouse model.

Sarah S. Alghamdi, Alicia A. Goyeneche, Zu-hua Gao, Carlos M. Telleria. _McGill University, Montreal, Quebec, Canada_.

Ovarian cancer (OC) is a complex disease that can be divided into various histopathological subtypes. High-grade serous OC (HGSOC) is the most abundant histotype, accounting for up to 70% of all OC cases. Whereas the majority of patients are diagnosed when the disease is already disseminated in the peritoneal cavity, orthometastatic mouse models of peritoneal OC—needed to perform preclinical studies—are not well developed. The human OC cell lines mostly studied for in vivo tumor development in immunosuppressed mice have been implanted subcutaneously, not intra-abdominally, thus not reflecting the tumor niche this disease actually encounters when advanced. Furthermore, most studies utilize cells which do not have the genetic fidelity of HGSOC. Thus, we set up a study to track the development of intra-abdominal disease induced by 3 human OC cells lines with the genetic fidelity of HGSOC: OVCAR-4, OVSAHO, and PEO14. The 3 cell lines showed abundant expression of ARID1A, PAX8 and mutant p53, with varied levels of expression of CA125 and WT1. We contrasted among them, the time needed for the disease to develop, the tissues being targeted, and the histopathological abnormalities detected. We found that the elapsed time for the disease to develop for the 3 lines ranged between 4 to 11 months. This result stands in stark contrast with the less than 3 months it takes for the disease to evolve in the peritoneal cavity when injecting cells of unlikely HGSOC genomic fidelity, such as SKOV-3, IGROV-1, or A2780. Within the cell lines with HGSOC genotype we found commonalities and differences in terms of their in vivo behavior. Among the commonalities, all cells homed to the omental-spleen-pancreatic area, the liver base, the mesentery, and the diaphragm. Among the differences, PEO14 cells developed into sizable masses not invading the parenchyma of the abdominal organs. OVSAHO cells developed discrete masses closely surrounding the organs, while OVCAR-4 cells displayed full invasiveness into the parenchyma of several abdominal organs in addition to developing micro-metastases within the lung stroma. Whereas OVCAR-4 cells caused accumulation of large volume bloody ascites carrying abundant multicellular structures (MCS), OVSAHO and PEO14 cells led to the formation of low volume bloody ascites carrying scarce MCS. These phenotypic heterogeneities were further reflected in the different end-of-wellness endpoints criteria reached, which were: the increase in the abdominal circumference caused by the accumulation of ascites in animals carrying OVCAR-4 cells, the size of the solid abdominal masses in animals carrying PEO14 cells, and the dramatic weight loss in animals carrying OVSAHO cells. In conclusion, we show that, despite having similar genetic fidelities, HGSOC cell lines are heterogeneous in terms of their phenotypic behavior when ortometastatically implanted in nude mice.

#1980

Fusion of cancer and stromal cells imaged by color -coded imaging of metasasis.

Miki Nakamura,1 Atsushi Suetsugu,1 Kousuke Hasegawa,1 Takuro Matsumoto,1 Hitomi Aoki,1 Takahiro Kunisada,1 Masahito Shimizu,1 Shigetoyo Saji,1 Hisataka Moriwaki,1 Robert M. Hoffman2. 1 _Gifu Univ. Graduate School of Medicine, Gifu, Japan;_ 2 _AntiCancer,Inc., San Diego, CA_.

We report here imaging of stroma during the metastatic process. RFP-EL4 lymphoma cells were injected subcutaneously in C57/BL6 GFP transgenic mice. Subcutaneous tumors were resected and immediately transplanted to the abdominal cavity of BALB/c nude mice. Metastases to the liver, perigastric lymph node, ascites, bone marrow and as well as the primary tumor were imaged. Metastases expressed GFP at low magnification. At higher magnification, yellow-remaining cancer cells resulting from fusion of GFP stromal cells and RFP cancer cells were observed. Metastasis contained fusion of caner and stromal cells which may be a feature of metastasis.

#1981

Tissue-engineered bone for longitudinal intravital microscopy of solid tumor growth and therapy response.

Eleonora Dondossola,1 Stephanie Alexander,1 Boris Holzapfel,2 Christopher J. Logothetis,1 Dietmar W. Hutmacher,2 Peter Friedl1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Australia_.

Bone metastases are the initial site of progression and account for many of the complications experienced by men with metastatic prostate cancer (PCa), including therapy resistance. Besides cell intrinsic mechanisms, the interaction between PCa cells and the bone microenvironment critically contributes to lesion expansion and drug sensitivity. We here developed a mouse model amenable to intravital multiphoton microscopy (iMPM) to longitudinally study PCa-stromal cell interactions and therapy response in a partially humanized neobone, established in the dermis of the mouse. Tissue engineered bone constructs, TEBCs, were generated by functionalizing polymeric polycaprolactone scaffolds with human mesenchymal stem cells (hMSCs) differentiated to osteoblasts followed by cell-derived calcification of the inter-scaffold space. 30 days after implantation of functionalized scaffolds under the skin of the mouse, bone and bone marrow maturation resulted in a 30mm3 cavity surrounded by optically transparent cortical bone of 50-60 μm thickness, as monitored by microCT and iMPM. Intra-bone tumor growth and osteolysis dynamics caused by human fluorescent PCa cells (PC3) were three-dimensionally reconstructed by multi-parameter detection through a body window, including collagen and bone matrix (SHG), calcified bone (fluorescent bisphosphonates), osteoclasts (cathepsin K), bone surface (THG), blood vessels and stromal phagocytes (fluorescent dextran), and PC3 cells (nuclear H2B/eGFP, cytoplasmatic DsRed2). As a proof of concept, the efficacy to halt bone remodeling during bisphosphonate therapy was detected, revealing the composition and kinetics of the bone-tumor interface. Combining engineered neobone with an optical window is suited to detail the 3D organization and dynamics of cancer lesions in bone and provides mechanistic insight and efficacy predictions for therapy response. Thus, this strategy will be implied to dissect the therapeutic effect of radium 223, a radioisotope with bone seeking properties, and the first agent that improves the duration and quality of life of advanced metastatic PCa patients. By using this model we will test the hypothesis that 223Ra alters stromal niches, structurally and molecularly, in a manner that accounts for the clinical observations in established macroscopic lesions.

#1982

β-arrestin 2 mediates tumor growth and metastasis in renal cell carcinoma cells.

Jude Masannat, Yushan Zhang, Hamsa Purayil, Yehia Daaka. _University of Florida, Gainesville, FL_.

Renal Cell Carcinoma (RCC) is one of the most lethal urological cancers worldwide, with incidence and mortality rates increasing in the past two decades. The disease does not present early clinical symptoms and is commonly diagnosed at the metastatic stage, leaving the 5-year survival rate at ≈ 10-20%. For a wide array of neoplasms, many key molecular determinants involved in mediating the process of tumor cell growth, invasion, and colonization at a secondary site (i.e. metastasis) have been reported. However, few molecular predictors have been identified for RCC, rendering the mechanism(s) underlying RCC metastases poorly understood. This results in the lack of effective treatment for patients with advanced RCC.

G protein-coupled receptors (GPCRs) and their effectors, such as the arrestin proteins, have been implicated in tumor growth, metastasis, and angiogenesis. Arrestin proteins are well known for their function in the desensitization and trafficking of GPCRs, but have also been implicated in unique signaling pathways to regulate fundamental cellular functions, including cell cycle progression, cell migration, and survival. Furthermore, arrestin involvement has been identified in a number of breast, colorectal, lung, and hematological malignancies. However, the role of arrestin proteins in RCC is yet to be determined. Our preliminary data show that β-arrestin 2 (βarr2) protein levels correlate with growth and metastatic potential in several RCC cell lines, including ACHN and SN12C. We hypothesize that βarr2 regulates RCC tumor progression, specifically through involvement in proliferation, invasion, and metastatic processes. To test our hypothesis, we used genetic-based loss of function approaches such as interfering RNA and CRISPR/Cas9. βarr2 knockdown results indicated a role in RCC malignancy as it significantly reduces the migration and invasion of RCC cell lines in vitro. βarr2 knockout (KO) impaired 3D spheroid formation of these cells and induced morphological changes compared to control cells as observed under confocal microscopy imaging. Moreover, epithelial marker E-cadherin expression levels were elevated while mesenchymal markers twist1, twist2 and vimentin levels were decreased in the βarr2 KO cells compared to control, indicating a mesenchymal to epithelial transition. In vivo data support our hypothesis that βarr2 plays a critical role in tumor growth and metastasis. Our data suggests a role for βarr2 in RCC malignancy and present a possible target in development of therapies for patients with advanced RCC.

#1983

CD117 expression and activation induce prostate cancer metastasis.

Koran Harris,1 Lihong Shi,1 Taylor Peak,1 Stephanie Sanders,1 Aleksander Skardal,2 Bethany Kerr1. 1 _Wake Forest University School of Medicine, Winston Salem, NC;_ 2 _Wake Forest Institute for Regenerative Medicine, Winston Salem, NC_.

Tyrosine kinase receptors have been shown to drive prostate cancer progression and metastasis. In particular CD117/c-kit expression is upregulated during tumor progression with the highest levels being expressed in bone metastases. In addition, the numbers of circulating CD117+ cells was higher in advanced prostate cancer patients. The presence of these cells in patients' circulation after radical prostatectomy was also associated with biochemical recurrence. Further, the expression of CD117 can be upregulated on prostate cancer cells after repeated culturing in the bone microenvironment. These data indicated that CD117 expression and activation may be associated with bone metastasis. To test this, LNCaP-C4-2 cells were sorted into CD117+ and negative cell populations. Proliferation, matrigel invasion, and gene expression were compared in the two cell populations in the presence and absence of the ligand stem cell factor (SCF) or CD117 and Akt inhibitors. To track cells during coculture and competition experiments, sorted cells were infected to express mCherry (CD117+) or ZsGreen (negative). Using these cells, competition was tracked using an IncuCyte ZOOM live cell imager for in vitro studies and an IVIS imager for in vivo xenograft studies. To better visualize transendothelial migration, a 3D microfluidic metastasis-on-a-chip was developed to track tumor cell migration through the ECM and across an endothelial cell layer. Here, we examine the activation of CD117 in prostate cancer cell progression and migration. CD117 expression was associated with increased proliferation, beta1 integrin expression, and EMT marker expression. Interestingly, treatment with the CD117 ligand stem cell factor (SCF) reduced proliferation but further enhanced invasion indicating that CD117 activation may drive metastasis. CD117 activation stimulated Akt expression and drove the cancer stem cell phenotype as demonstrated by increased Oct4 and Sox2 expression. CD117 expression was associated with increased expression of cancer progression and EMT signaling pathways. Using live cell imaging, competition between CD117+ and negative cells was visualized in proliferation, scratch healing, invasion, trans-endothelial migration, and sphere formation. Xenograft models also demonstrated competition in vivo. Sectioning of tumors demonstrated the localization and composition of co-injected cells. In vivo fluorescent imaging was used to examine tumor initiation capabilities of the two cell populations. Using 3D microfluidics, we have modeled the competition between CD117+ and negative cell populations during transendothelial migration. CD117 expression on prostate cancer cells drives a more aggressive cell phenotype and may be involved in metastasis. Further, CD117+ cells represent a possible cancer stem cell population.

#1984

CtBP1 and metabolic syndrome induce breast cancer progression and lung metastasis.

Paula Lucía Farré, Georgina Scalise, Nicolás Guillermo Dalton, Rocío Belén Duca, Karen Daniela Graña, Cintia Massillo, Juliana Porretti, Adriana De Siervi, Paola De Luca. _Instituto de Biología y Medicina Experimental - CONICET, Buenos Aires, Argentina_.

Breast cancer (BrCa) is one of the most important public health problems in the entire world. Metabolic syndrome (MeS) increases the incidence and aggressiveness of BrCa. C-terminal binding protein 1 (CtBP1) is a co-repressor of tumor suppressor genes that is activated by low NAD+/NADH ratio. Recently, we generated a MeS-like experimental mice model by chronically feeding animals with high fat diet (HFD) and we found that CtBP1 and MeS modulated breast carcinogenesis and tumor growth. We also showed that CtBP1 and MeS decreased BrCa cell adhesion, a crucial event in the beginning of metastasis. Considering metastasis is still the main cause of death, and around 30 % of women with BrCa diagnosed at early stages will progress to metastatic stage, it is crucial to understand the impact of non-inherited factors and the mechanism underlying this process. The aim of this work was to explore CtBP1 and MeS role in BrCa cell migration and metastasis. By wound healing assay, we found that CtBP1 increased cell migration of MDA-MB-231 and 4T1 BrCa cells. To study CtBP1 and MeS effect in tumor progression, MeS nude mice induced by chronically feeding animals with HFD; and control diet fed animals, were injected with CtBP1-depleted expression or -control MDA-MB-231 cells. Six weeks post-injection primary tumors were surgically removed. After two weeks, mice were sacrificed and the presence of metastasis in lung, liver and ascites was analyzed by histology and/or quantified by RT-qPCR using specific primers for human GAPDH. Consistently with the onset of metastasis, MeS increased the number of mice that developed neoplastic ascites (20% in MeS vs. 50% in control) with presence of tumor cells (TC) detected by RT-qPCR. In addition, histological analysis and RT-qPCR quantitation revealed that CtBP1 hyperactivation by MeS significantly increased BrCa lung metastasis. Interestingly, human Vimentin mRNA was induced in TC from ascites compared to primary TC; while it was diminished in lung, suggesting the crucial role of EMT/MET processes in metastasis. Finally, we analyzed expression of cell adhesion and EMT-related genes in primary tumor tissue by RT-qPCR. We found that CtBP1 and MeS modulated cell adhesion and EMT expression genes: Vimentin, Slug, ITGB4, ITGB6, Col17A, FABP4 and PRSS2. Altogether, these results suggest a key role for MeS and CtBP1 inducing BrCa EMT and metastasis.

#1985

Tbk1 **loss in pancreatic cancer leads to changes in epithelial plasticity.**

Victoria Haley Burton, Rolf A. Brekken, Melissa Gross, Alberto Bremauntz. _UT Southwestern Medical Ctr., Dallas, TX_.

Effective therapies are needed to enhance the long-term survival of patients with pancreatic ductal adenocarcinoma (PDA), which is the fourth leading cause of cancer-related deaths in the United States and eighth worldwide. Initial stages of PDA are commonly characterized by an activating mutation in K-RAS, yet direct inhibition of K-RAS through pharmacological means remains a challenge. Higher levels of TANK Binding Kinase 1 (TBK1) mRNA, a critical downstream effector of mutant active K-RAS in lung cancer, are associated with poorer overall survival in a cohort of human PDA patients. We hypothesize that TBK1 is also an effective mediator of K-RAS driven pancreatic cancer.

Here we report that TBK1 is expressed and more active in human PDA cell lines relative to immortalized pancreatic epithelial lines (HPNE) and fibroblasts. We found that human PDA cell lines are sensitive to a small molecule inhibitor of TBK1 in the low micromolar range. Further mice engineered to express a mutant kinase dead form of TBK1 (Tbk1Δ/Δ) are viable and fertile yet display smaller tumors at early time points in a genetically engineered mouse model of PDA. Interestingly, tumors from Tbk1Δ/Δ: PDA mice are more epithelial in gene expression and show significantly less collagen deposition compared to Tbk1+/+: PDA tumors. Additionally, cell lines isolated from Tbk1Δ/Δ: PDA tumors are more epithelial in morphology and less migratory and invasive relative to Tbk1+/+: PDA cell lines. Current work is focused on delineating the functional contribution of TBK1 to tumor cell motility in vivo as well as mechanistic understanding of the epithelial phenotype in Tbk1Δ/Δ: PDA tumors. These results will further our understanding of Ras signaling in pancreatic cancer and are critical for exploring a new avenue of targeted therapy.

### Tumor Microenvironment 2

#1986

Determining the signaling pathway of epithelial-IKKα deletion-mediated symbiotic bacterial and fungal infection in carcinogenesis.

Na-Young Song, Jami Willette-Brown, Feng Zhu, Yinling Hu. _NCI-Frederick, Frederick, MD_.

Bacteria and fungi, two major components of the microbiota, generally share niches and develop both antagonistic and symbiotic relationships, regulating the pathological impacts on the host. The epithelium is where the bacterial-fungal interaction occurs most abundantly, but the relationship between the epithelium, bacteria, and fungi on the pathogenesis, particularly tumorigenesis, is poorly understood. IKKα is one of the crucial factors regulating the homeostasis of squamous epithelial tissues. Recently, our lab has established a mouse model that develops esophageal squamous cell carcinomas associated with IKKα reduction, inflammation and chronic fungal infection. Cladosporium cladosporioides was a major type of fungi identified in this mouse model. Because IKKα deletion in the keratinocytes causes impaired skin barrier, we hypothesized that loss of epithelial IKKα may control fungal colonization through regulating the barrier integrity and inflammation. We generated IKKαf/f mice with inducible K15.Cre (IKKαf/f/K15.Cre) specifically expressed in keratinocytes in hair follicles which is considered as skin stem cells. After deleting IKKα in K15 cells in oral mucosa and skin, IKKαf/f/K15.Cre mice were orally inoculated with Cladosporium cladosporioides. We found that epithelial IKKα deletion increased bacterial colonization in oral mucosa and skin. Moreover, Cladosporium infection further promoted bacterial and fungal colonization in oral cavity and development of skin tumors. Taken together, our data suggest that loss of epithelial IKKα induces the bacterial-fungal symbiosis in oral mucosa, promoting skin tumors. This study will shed light on the importance of the epithelial-bacterial-fungal interaction in the pathogenesis, proposing epithelial IKKα as a novel regulator of the bacterial-fungal interaction.

#1987

Inhibition of collagen receptor discoidin domain receptor-1 (DDR1) reduces gastric cancer cell motility and metastasis.

Ryo Yuge,1 Yasuhiko Kitadai,2 Hidehiko Takigawa,1 Shinji Tanaka,1 Kazuaki Chayama,1 Wataru Yasui1. 1 _Hiroshima Univ., Hiroshima, Japan;_ 2 _Hiroshima Prefectual Univ., Hiroshima, Japan_.

Accumulating evidences suggest that a unique set of receptor tyrosine kinases, known as discoidin domain receptors (DDRs), play a role in cancer progression by interaction with their surrounding collagen matrix. Although abnormal expression of DDRs is reported in some human cancers, little is known about the expression and function of DDRs in gastric cancer progression. In this study, we investigated expression and the role of DDR1 in the tumor progression of human gastric cancer. The expression and distribution of DDR1 was analyzed by immunohistochemistry in 127 human gastric cancer cases, and the expression level of DDR1 in 7 human gastric cancer cell lines was analyzed by RT-PCR and western blotting. Proliferation, migration, invasion, and tube formation assays were conducted in DDR1 silenced gastric cancer cells. The effect of DDR1 on the tumor growth and metastasis were examined in orthotopically implanted nude mice model and a model of liver metastasis. DDR1 was expressed in human gastric cancer cell lines, and expression of DDR1 in human gastric tumor was associated with poor prognosis. Among 7 gastric cancer cell lines, MKN74 expressed DDR1 at highest level. DDR1-silenced MKN74 cells showed unaltered proliferation activity. In contrast, migration, invasion, and tube formation were significantly reduced. When examined in an orthotopic nude mouse model, DDR1-silenced implanted tumor significantly decreased angiogenesis and lymphangiogensesis, thereby leading to reduction of lymph node metastasis and liver metastasis. We showed that treatment of DDR1 inhibitor also inhibited lymph node metastasis in an orthotopic nude mouse model These results suggest that DDR1 is involved in GC tumor progression and metastasis. DDR1 could be not only an independent prognostic classifier, but also a promising therapeutic target in patients with GC.

#1988

Exosome-mediated ovarian cancer tumorigenesis mediated by miR1246/Rb/Cav1 axis.

Pinar Kanlikilicer, Recep Bayraktar, Mohammed Rashed, Burcu Aslan, George A. Calin, Anil K. Sood, Gabriel Lopez-Berestein. _MD Anderson Cancer Center, Houston, TX_.

Exosomes are secreted from many cell types and play an important role in the tumor microenvironment. The most impressive breakthrough in exosomes research was that they contain the genetic material of the host cell. However, whether cancer cells use their exosomes to transfer their oncogenic material to recipient cells, or to get rid of their tumor suppressor material is not well understood. We previously identified that miR-1246 was hundreds of folds higher expressed in six different ovarian cancer exosomes compared to their originating cells. Here, we showed that miR-1246 co-localized in the exosomes in ovarian cancer cells. miR-1246 act as an oncogenic miRNA and the levels were elevated in ovarian cancer patients compared to health donors. We also demonstrated that miR-1246 inhibitor treatment in combination with paclitaxel was significantly inhibited tumor burden in SKOV3-ip1 orthotopic ovarian cancer model. Our results suggest that miR-1246 inhibited RB tumor suppressor protein and regulate Cav-1 and platelet-derived growth factor receptor beta precursor signaling in ovarian cancer. In addition Inhibiting miR-1246 led a significant decrease in exosome release. Together, our findings provide strong evidence that oncogenic miR-1246 can be targeted as a potential novel therapeutic approach in the treatment of ovarian cancer.

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#1989

Characterization and integration of mantle cell lymphoma microenvironments are determinant for the development of rational targeted therapies.

David Chiron,1 Celine Bellanger,1 Antonin Papin,1 Benoit Tessoulin,1 Christelle Dousset,1 Sophie Maiga,1 Philippe Moreau,2 Cyrille Touzeau,2 Steven Le Gouill,2 Martine Amiot,1 Catherine Pellat-deceunynck1. 1 _CRCNA, INSERM, CNRS, Université de Nantes, Nantes, France;_ 2 _Service d'Hématologie Clinique, Unité d'Investigation Clinique, CHU,, Nantes, France_.

Mantle cell lymphoma (MCL) accumulates in lymph nodes (LN) and disseminates early on in extranodal tissues. Recent effort has been invested on the identification of intrinsic MCL abnormalities but little attention has been paid to the importance of the surrounding cells and soluble factors in this pathology. Further investigations that integrate the key role of the microenvironments are now needed to overcome drug resistance in this incurable malignancy. Despite a significant level of the proliferation index Ki67 in LN, we did not detect any proliferating peripheral blood (PB) MCL cells, suggesting a major role of the tumor ecosystem. To determine whether stromal (hMSC) or lymphoid-like (CD40L) interactions could support survival and proliferation, primary circulating MCL cells were cocultured ex vivo. In all the 21 samples tested, CD40L, but not hMSC, induced cell-cycle progression that was amplified by a MCL-specific cytokine cocktail (Ck). To determine the microenvironment dependent molecular modulations, RNA-seq in MCL cells from PB or cocultured was performed (n=8) and compared with genes expressed in MCL cells from LN and PB (Geo, PB n=77, LN n=107). More than 65 % of genes induced by "CD40L+Ck" were also upregulated in the LN. Our model recapitulated molecular signatures that are characteristic of MCL i.e., cell cycle, BCR, NFkB, NIK and survival, confirming the relevance of the coculture. We further studied the coculture-induced regulation of the genes belonging to the survival signature. The major regulation was an increase in expression of Bcl-xL protein associated with a striking downregulation of Bcl2 and Bim. Using the functional BH3-profiling assay, we demonstrated that, whereas PB MCL cells were dependent on Bcl2 for survival, Bcl-xL upregulation was responsible for loss of mitochondrial priming and drug resistance. Consequently, whereas clinically available Bcl2 BH3-mimetic efficiently triggered apoptosis in PB MCL, cells protected by the microenvironment were resistant. We then hypothesized that targeting BclxL could increase treatment efficacy. By integration of microenvironment modulations using our coculture model, we developed efficient targeted strategies (i.e.: BTK inhibitor, Type II anti-CD20), which counteract BclxL overexpression and overcome drug resistance in primary cells ex vivo. This strategy should target cells protected into their niches and our ongoing OAsIs Trial (NTC#02558816) will rapidly determine in vivo efficacy in MCL. In summary, we reported here the development of a relevant model that provides new insights into the microenvironment-dependent regulation of proliferation and Bcl-2 family expression. Our increased understanding of intrinsic abnormalities and the integration of extrinsic signaling offer new opportunities to design mechanism-based strategies to overcome drug resistance in MCL and other B cell malignancies.

#1990

Single cell co-expression profiles of immunotherapy targets in the tumor microenvironment of NSCLC samples.

Jeffrey Kim, Nan Su, Xiao-Jun Ma, Emily Park. _Advanced Cell Diagnostics, Newark, CA_.

Cancer immunotherapies have made great strides in recent years, yet identifying key molecules in the tumor microenvironment is necessary to develop improved approaches to achieve more durable responses in a greater number of patients. Obstacles such as adaptive resistance to individual checkpoint marker blockade, such as PD1 and CTLA4, have led researchers down the path of pursuing multiple immune targets that may be upregulated on the same lymphocyte as a consequence of mono-antibody therapies. While several clinical trials taking a combinatorial approach are currently in progress, including targeting multiple checkpoint markers, it is clear that these new strategies may rely on the pre-existing immune system, regardless of being treatment naïve or previously treated. The challenge now is to identify the relevant biomarkers and to stratify patients to provide the most appropriate treatment. As a proof of concept, here we evaluate single cell co-expression profiles of key therapeutic targets in archived tissues of non-small cell lung cancer (NSCLC) samples using the RNAscope® Duplex Assay, an advanced in situ hybridization platform that allows for the visualization of single cell gene expression of mRNA targets directly in tissue.

In our investigation of 60 archived formalin-fixed paraffin embedded NSCLC tissues, co-expression profiles of PD1 or PD-L1 coupled with another therapeutic target (IDO1, LAG3, 4-1BB, GITR, and TIM3) were assessed, bringing to light substantial variations in the tumor microenvironment between tissue samples. These marked differences of co-expressed targets on tumor infiltrating lymphocytes were scrutinized through quantitative image analysis to further reveal both the diversity and degree of individuality between tissue samples. Quantitatively measuring the expression of multiple targets in single cells within intact tissues exposed the distinct milieu of targetable therapeutic molecules, leading to the potential identification of responsive tumors which could help guide in stratifying patients for different combinatorial approaches.

#1991

Enhanced role of the extracellular matrix in ovarian cancer recurrence.

Zhiqing Huang, Brad Foster, Carole Grenier, Gregory Sfakianos, Regina Whitaker, Andrew Berchuck, Susan K. Murphy. _Duke Univ. Medical Ctr., Durham, NC_.

Purpose: Despite improvements in care and advancements in the understanding of ovarian cancer (OC) pathobiology and genetic alterations, the survival rate is disappointingly low when compared to that of other types of cancer. One factor contributing to the poor survival rate is the development of chemotherapeutic resistance and a high rate of recurrent tumor growth in the months and years following primary OC treatment. Our objective was to identify potential new therapeutic targets for prevention of recurrence through examination of changes in gene regulation that occur in recurrent versus matched primary tumors.

Procedures: We used 16 primary-recurrent tumor pairs from patients with stage III/IV serous epithelial OC from the Duke Gynecologic Oncology Tissue Bank. Illumina Infinium HumanMethylation450 BeadChips and Affymetrix Human Genome U133A arrays were used to quantify methylation and gene expression, respectively. Expression and methylation of POSTN and COL11A1 were analyzed using an independent OC dataset comprising 38 women with <3 years survival and 26 women with >7 years survival, 3 normal ovarian surface epithelium (OSE) and 2 fallopian tube fimbriae epithelium (FTFE) samples. siRNA knockdown of POSTN and COL11A1 in OC cells was followed by expression and proliferation assays.

Results: Paired student t tests showed a >2-fold difference for 13 of the 1,569 significantly differentially expressed genes, seven with increased expression in recurrent OC (POSTN, COL11A1, MMP1, MMP13, TNC, ASPN, and EPYC), and six with decreased expression (GATA6, PEG3, SST, MAOB, TSPAN8, and C7). Interestingly, all seven up-regulated genes are involved in extracellular matrix (ECM) functions, an important component of the tumor microenvironment. With an independent OC microarray dataset, we found elevated COL11A1 mRNA in OCs as compared to OSE and FTFE tissues (p<0.05). There are inverse correlations between COL11A1 expression and methylation at five promoter CG sites (R=-0.58 to -0.65, p<0.05). Immunohistochemistry showed higher expression of POSTN and COL11A1 in recurrent OC. Knockdown of POSTN and COL11A1 in OC cell lines led to significantly reduced proliferation (p<0.05).

Conclusion: Our findings indicate that the genes involved in extracellular matrix function that contribute to the composition and regulation of the tumor microenvironment play critical roles in OC recurrence, and may offer new therapeutic opportunities.

#1992

Changes in gene expression of stromal and epithelial cells during prostate cancer progression.

Milos Babic,1 Elliot Imler,1 Peter Shepard,1 Joanne Yeakley,1 Raymond Nagle,2 Bruce Seligmann1. 1 _BioSpyder Technologies, Inc., Carlsbad, CA;_ 2 _University of Arizona, Tucson, AZ_.

Prostatic intraepithelial neoplasia (PIN) is a histologic abnormality which arises within the secretory epithelium of prostate glands, without disrupting the layer of basal cells which separates the epithelium from the surrounding stroma. PINs are generally considered to be a step towards development of prostatic adenocarcinoma (PCA). Molecular steps in the transition from normal glands to PIN to PCA are poorly understood, in part due to the difficulty of accessing and characterizing individual PINs without contamination from surrounding tissue. Furthermore, stromal cells surrounding pre-cancerous glands may play an active role in cancer development and progression, but are difficult to profile.

We utilized the ultra-sensitive and quantitative TempO-Seq gene expression assay of the whole transcriptome to profile microdisections of stromal vs. epithelial cells in archival prostatectomy FFPE tissue. TempO-Seq is highly insensitive to RNA degradation (measurements of intact RNA RIN=9.1 to degraded RNA RIN=3.0 correlate with R2=0.97) and does not require RNA extraction, allowing measurement of both soluble and insoluble cross-linked RNA. This allowed deparaffinization and H&E staining of prostate sections prior to the assay, so that morphology was clearly visible and subsections could be readily extracted and profiled. Microdissections were performed on individual 5 µm FFPE sections of prostatectomies from patients with Gleason sum scores of 6, containing PIN alongside PCA. We removed an ~300 µm diameter region of stroma surrounding individual normal, PIN, and PCA glands, followed by targeted dissection of the gland epithelium itself which was lysed and assayed separately. Stroma and epithelial samples from 3 adjacent sections of the same glands were pooled together (adding up to ~0.005 mm3 tissue/replicate), and then profiled.

We compared the whole transcriptome expression profile of stroma vs. epithelium at every stage of progression. Our results indicate that stroma and epithelium undergo separate and distinct changes in gene expression patterns throughout the development of PCA, most notably in genes involved in cell-cell signaling. Specifically, we found that changes in cAMP signaling in stromal cells accompanied the shift from normal glands to PIN. Furthermore, changes in androgen signaling pathways were present in stromal cells as well as in epithelium during the transition from PIN to invasive carcinoma.

These results shed light on the molecular progression to PCA and demonstrate the feasibility of profiling gene expression at the level of tissue microstructure to elucidate changes within the tissue microenvironment. Since archival FFPE can be used, studies of progression can be carried out and the findings translated to diagnosis and prognosis as well as to the investigation of new therapeutic approaches.

#1993

Acidic tumor microenvironment stimulation of GPR4 alters cytoskeletal dynamics and migration of vascular endothelial cells.

Elizabeth A. Krewson, Li V. Yang, Lixue Dong. _East Carolina University, Greenville, NC_.

The acidic tumor microenvironment results from aberrant vasculature, insufficient oxygen delivery and aerobic glycolysis of cancer cells. As cancer progresses, the acidic microenvironment can lead to structural changes of endothelial cells (ECs) resulting in increased vascular permeability which may increase metastasis. Extracellular acidification can activate cellular signaling molecules, including G-protein coupled receptor 4 (GPR4), which is prominently expressed in ECs. We have previously reported that acidic activation of GPR4 induces the transcription of several pro-inflammatory and ER-stress related genes in ECs. The purpose of the study is to identify the functional response of acidosis-induced GPR4 activity using Human Umbilical Vein Endothelial Cells (HUVECs) as a model system. The expression of GPR4 was overexpressed (HUVEC/GPR4) or knocked down (GPR4 shRNA) using several genetic constructs. HUVECs were treated with media buffered to pH 6.4 or to physiological pH 7.4. Permeability of a HUVEC cell monolayer was assessed by quantifying gap formation as one indicator. Cell migration was assessed through wound-healing assays. Cytoskeletal dynamics were also investigated using Rhodamine Phalloidin to investigate actin stress fiber formation and immunocytochemistry to investigate focal adhesion dynamics. Our results showed acidic pH induced GPR4 activation changed the cytoskeletal phenotype, decreased cellular migration, and proliferation in HUVECs compared to the physiological pH treatment. There was also a further increase in gap area of a HUVEC/GPR4 monolayer and formation of actin stress fibers in response to acidic conditions. Additionally, altering expression of phosphorylated paxillin and focal adhesion kinase were observed under acidosis/GPR4 mediated stress. In conclusion, the proton sensor GPR4 induces cytoskeletal variations, reduces cellular migration, and proliferation in response to an acidic tumor microenvironment.

#1994

Defining peritumoral and tumoral stromal gene signatures associated with response to neoadjuvant chemotherapy in breast cancer.

Celine Pourreyron,1 Violet Warwick,1 Patsy Whelehan,1 Philip Coates,2 Lee Jordan,1 Colin Purdie,1 Sarah Vinnicombe,1 Alastair Thompson,3 Andrew Evans,1 Frances Fuller-Pace1. 1 _University of Dundee, Dundee, United Kingdom;_ 2 _Regional Centre for Molecular Oncology, Brno, Czech Republic;_ 3 _MD Anderson Cancer Center, Houston, TX_.

Neoadjuvant chemotherapy (NACT) is used in breast cancer to decrease tumor and lymph node burden and the extent of surgery. However, response is variable and prediction poor. Stromal cells are involved in breast cancer progression and drug resistance although very little is known about the mechanisms. Our research aims to characterize breast cancer-associated fibroblasts (CAFs) and peritumoral fibroblasts (PTFs) that predict response to NACT and could be used to influence treatment choices.

Pre-NACT core biopsies from cancer-associated and peritumoral tissues were collected under ultrasound guidance. To date, mRNAs from early passage (passage ≤ 5) fibroblasts isolated from cores of 23 matched pairs (CAFs and PTFs) (8 triple-negative (TNBC), 7 ER+Her2+ and 8 ER+Her2- tumors) and an additional 8 PT samples (4 TNBC, 1 ER+Her2+ and 3 ER+Her2-) have been analysed by whole-genome expression array (Illumina beadchip HT12v4). Data were analysed with Partek® Genome SuiteTM 6.6 and Pathway softwares (Partek Inc., St. Louis, MO, USA). Differentially expressed genes (DEGs) were identified (t-test, p-value ≤ 0.05, fold-change (FC) ≥ 1.3) between CAFs and PTFs and between NACT responders (Residual Cancer Burden (RCB) 0/1) and non-responders (RCB 2/3).

No significant difference between CAFs and PTFs was seen by principal component analysis and t-test. PTFs showed similar mRNA levels of major myofibroblast markers (e.g. FAP, ASMA) with only FSP1 being lower in PTFs compared to CAFs (FC = -1.4, p-value = 0.023), suggesting that PTFs, like CAFs, are activated myofibroblasts. We then tried to identify a gene signature in CAFs or PTFs that could predict response to NACT. Comparing RCB 0/1 vs RCB 2/3 resulted in very few DEGs due to cancer subtype variation in both CAFs and PTFs. Similar to other studies, only 2/19 patients in our cohort with ER+ tumours achieved RCB 0/1, preventing analysis of these samples. However, 45% (5/11) TNBC responded to NACT and we identified 566 DEGs between RCB 0/1 and RCB 2/3 CAFs, with changes in metabolism, Pi3K/AKT pathway, proteoglycan synthesis or ECM-receptor interaction. For PTFs, 186 DEGs were identified, with changes in Wnt signalling and ECM-receptor pathways.

Microarray data from our uniquely large sample set of 23 pairs of CAFs and PTFs from breast cancer patients suggest that PTFs have a similar gene expression profile to CAFs. Although others have demonstrated that CAF phenotype is breast cancer subtype

dependent, we are the first to show similar observations in PTFs. Finally, we identified gene signatures from both CAFs and PTFs that relate to NACT response in TNBC. We are currently in the process of confirming the most highly dysregulated genes by qPCR and/or immunohistochemistry analysis.

#1995

The omentum promotes ovarian cancer cell survival by increasing cell cycle duration and chemoresistance.

Carlos Cardenas, Ayesha B. Alvero, Mary Pitruzzello, Roslyn Tedja, Gil G. Mor. _Yale University School of Medicine, New Haven, CT_.

Background:

Ovarian cancer is the most lethal of all gynecologic cancers. It is characterized by the presence of carcinomatosis at time of presentation and chemoresistant micrometastatic seedings at the time of recurrence. The omentum, an adipocyte-rich organ, is a frequent site of ovarian cancer metastasis in patients and the most common site of post-chemotherapy residual disease in animal models. Whereas previous studies have shown how the adipocyte microenvironment can induce metabolic re-programming in cancer cells, it is not clear if it can regulate pathways that affect chemoresponse. The objective of this study is to determine the effect of the adipocyte microenvironment on pathways that control cell cycle and apoptosis.

Methods:

In vitro: Patient-derived epithelial ovarian cancer cell lines were cultured in the presence of absence of omentum conditioned-media. Cell growth was determined by measuring culture confluence using IncucyteTM. Protein expression was determined by western blot analysis, and flow cytometry. In vivo: Human ovarian cancer xenografts were established intra-peritoneally in nude mice. Tumor implants from adipocyte-rich organs such as the omentum, pelvic fat, and mesentery and tumor implants from non-adipocyte-rich organs such as liver, ovaries, and GI tract were collected and analyzed independently.

Results:

Compared to cells cultured in growth media, epithelial ovarian cancer cells cultured in omentum CM demonstrated a more fibroblastic morphology characterized by elongated shape and bipolarity, significantly slower growth kinetics (p < 0.0001) accompanied by upregulation of the cell cycle inhibitor, p27, and enhanced secretion of the pro-angiogenic factor, VEGF. More importantly, these cells express higher levels of the anti-apoptotic proteins Bclxl and Mcl-1. Consequently, ovarian cancer cells in growth media treated with Carboplatin had a IC50 of 14.5 μg/ml. In contrast, cells cultured in omentum CM demonstrated enhanced resistance to carboplatin with IC50 of > 50 μg/ml.

Analysis of tumor implants in vivo showed similar results as observed in vitro. Thus, tumor implants isolated from adipocyte-rich organs express higher levels of p27, Bclxl, and Mcl-1.

Conclusion:

We demonstrate in this study that the adipocyte microenvironment induces major changes in the phenotype of ovarian cancer cells characterized by morphological changes chemoresistance and growth rate. These findings highlight the importance of the adipocyte microenvironment in the progression of ovarian cancer. Further studies that can identify specific therapeutic targets in the adipocyte-educated chemoresistant ovarian cancer cells may aid in the development of novel therapies and improve patient survival.

#1996

CXCL12 drives selective expansion of AKT-induced quiescent, pre-neoplastic breast cells.

Zahra S. Masoud,1 Lisa Kim,1 Kelsey J. Weigel,2 Matthew M. Champion,2 Zachary T. Schafer,2 Cheuk Leung1. 1 _University of Minnesota, Minneapolis, MN;_ 2 _University of Notre Dame, Notre Dame, IN_.

A critical stage of premalignant development in breast tumorigenesis is the emergence of preneoplastic cells that carry key oncogenic drivers yet remain quiescent. Preneoplastic cells maintain a state of quiescence, but may be triggered to proliferate through largely unknown factors. Expansion of these cells is key to driving clonal evolution, which is evident in early lesions and tissue fields surrounding tumors. Although preneoplastic development is determinant to successful evolution and formation of tumor growth, the cellular mechanisms controlling preneoplastic development is unclear. Our limited mechanistic understanding of quiescent, preneoplastic cell outgrowth highlights a major knowledge gap in the early control of tumor development. Advancements in detecting molecular changes in precancerous lesions have provided opportunities for devising early intervention strategies. Aberrant AKT activation has emerged as a founding oncogenic alteration in preneoplastic cells. While molecular changes predispose these cells to develop tumors and presumably favors their selection during early tumor development, how these alterations contribute to the cellular control of quiescent preneoplastic cells remains unknown. A major barrier to studying quiescent preneoplastic cells is the lack of physiologically relevant experimental models. Here, we utilized a novel quiescent, preneoplastic cell model that reconstructs the molecular and cellular contexts of preneoplastic breast tissue. We combined a three-dimensional (3D) organotypic cell culture on reconstituted basement membrane with cells that can be induced to activate defined oncogenic pathways. Using this model, we investigated the contribution of aberrant AKT activation in preneoplastic breast tumor development. We found that aberrant AKT signaling in quiescent, preneoplastic mammary cells does not stimulate cells to proliferate under normal conditions, but confers cell sensitivity to stroma-derived CXCL12 through upregulating the receptor CXCR4. Using heterotypic co-culture to recapitulate a tumor-promoting environment and quantitative proteomic analysis, we found that stromal-derived chemokine CXCL12 selectively promotes expansion of preneoplastic cells with hyperactivated AKT signaling. Mechanistically, we showed that AKT signaling induces the expression of the chemokine's receptor, CXCR4, which confers the selective sensitivity to CXCL12. This CXCL12-CXCR4 axis serves as an alternate mitogenic signal through PKA and ERK to drive the downregulation of CDKN1C and promote cell proliferation in AKT-induced preneoplastic cells. Our studies uncovered a distinct role of AKT signaling in promoting preneoplastic development. Furthermore, our studies suggest a model of preneoplastic clonal expansion where oncogene signaling confers quiescent preneoplastic cell sensitivity to alternate mitogenic stimuli in a permissive tissue microenvironment.

#1997

Charting the DNA methylation landscape of prostate cancer associated fibroblasts.

Mitchell G. Lawrence,1 Ruth Pidsley,2 Elena Zotenko,2 Birunthi Niranjan,1 Aaron Statham,2 Jenny Song,2 Nicola Armstrong,2 John Pedersen,3 Mark Frydenberg,1 Renea Taylor,1 Clare Stirzaker,2 Gail Risbridger,1 Susan Clark2. 1 _Monash University, Melbourne, Australia;_ 2 _Garvan Institute of Medical Research, Sydney, Australia;_ 3 _TissuPath Pathology, Melbourne, Australia_.

Most studies of the genome and epigenome landscape of tumors focus on the aberrations in epithelial cancer cells. However, the growth and progression of solid tumors involves dynamic cross-talk between cancer epithelium and the surrounding microenvironment, including cancer-associated fibroblasts (CAFs). The molecular landscape of these cells is still poorly understood, so it is important to define the epigenetic features that underpin the functional differences between CAFs and non-malignant prostate fibroblasts (NPFs) and influence cancer progression. Therefore, we used whole-genome bisulphite sequencing to chart the epigenome landscape of CAFs at single base resolution. Our data revealed that NPFs and CAFs from localized prostate cancer have remarkably different epigenome profiles characterized by locus-specific rather than global changes in DNA methylation. CAFs exhibited more than 7500 differentially methylated regions compared to NPFs. In contrast, no recurrent genomic aberrations were detected in CAFs, emphasising the importance of epigenome changes in reinforcing their pro-tumorigenic phenotype. Many differentially methylated regions occurred at known regulatory loci and were associated with differentially expressed genes measured using RNAseq. The methylation and gene expression changes were highly consistent across patients when validated using an independent cohort of patient-matched NPFs and CAFs. Remarkably, a subset of differentially methylated regions in CAFs is shared with prostate cancer epithelial cells, revealing the new concept of tumour-specific epigenome modifications in the tumour and its microenvironment. Collectively, these data demonstrate that CAFs are defined by an enduring epigenetic signature that comprises a distinct set of locus-specific DNA methylation alterations in cancer-related regulatory loci. The distinct methylome of CAFs provides a novel epigenetic hallmark of the cancer microenvironment and promises new biomarkers to improve interpretation of diagnostic samples.

#1998

Transcriptome analysis of hypoxic head and neck cancer cells uncovers intron retention in EIF2B5 as a mechanism to reduce protein synthesis.

Lauren K. Brady,1 Hejia Wang,1 Caleb Radens,1 Milan Radovich,2 Amit Maity,1 Cristina Ivan,3 Mircea Ivan,2 Yoseph Barash,1 Constantinos Koumenis1. 1 _University of Pennsylvania, Philadelphia, PA;_ 2 _Indiana University, Indianapolis, IN;_ 3 _University of Texas, MD Anderson Cancer Center, Houston, TX_.

Hypoxia is a key feature of solid tumors that contributes to resistance to therapy and reduced overall survival. Cellular adaptation to the hypoxic tumor microenvironment involves attenuation of energy-consuming processes such as macromolecular synthesis. We have identified RNA processing as another major process which is globally downregulated under hypoxic stress. By sequencing RNA of normoxic and hypoxic head and neck cancer cells, we observed widespread repression of genes that regulate RNA splicing and processing. As a result, we observed over 1,000 changes in relative mRNA isoform expression, including a significant increase in hypoxia-induced intron retention in nearly 100 genes. Genes observed to undergo intron retention in hypoxia included major regulators of RNA processing and protein synthesis, such as the translation initiation factor EIF2B5.

Surprisingly, hypoxia-induced intron retention in EIF2B5 creates a premature termination codon, which results in a truncated protein isoform of eIF2Bε. We provide evidence that this isoform acts in opposition to the full-length protein to inhibit translation under stringent hypoxic conditions. Moreover, this intron is expressed in solid cancers known to contain hypoxic fractions and is overexpressed in head and neck cancer in a stage-dependent manner compared to normal tissue.

To investigate how this intron is retained, we are examining the role of hypoxia-mediated changes in phosphorylation of the C-terminal domain (CTD) of RNA polymerase II. Our data suggest that increased phosphorylation of the CTD may lead to transcriptional pausing and subsequent retention of intron 12 in EIF2B5. Furthermore, distinct sequence features at this locus, such as a weak splice site at the intron12:exon13 junction, may explain how this intron is selectively retained in hypoxic cells. These results implicate mRNA splicing as a key mechanism to induce alternate transcripts and subsequent protein isoforms under hypoxia that influence cellular adaptation to stress.

#1999

Cofactor Paragon: a novel tool to analyze the tumor microenvironment using RNAseq.

Ryan Bloom, Raman Talwar, Jeff Hiken, Jon Armstrong. _Cofactor Genomics, St. Louis, MO_.

The impact of cancer immunotherapy on clinical cancer care is growing rapidly. However, there are several mechanisms by which a tumor can avoid recognition and/or elimination by the immune system. Immune recognition, activation, and infiltration are each required for effective clearance of a tumor by the immune system. Impairment of any one of these mechanisms requires a different therapeutic strategy or combination of strategies to significantly address. The development of new multi-modal biomarker technologies that have the ability to provide insight into each of these factors in a cost-effective manner is essential to effectively guide treatment choice for patients and to discover new therapeutic targets.

We have developed Paragon, which is an RNA sequencing based panel that utilizes novel informatics technologies to provide a comprehensive picture of a tumor's transcriptomic and immune micro-environment. There are three distinct outputs of Paragon: measurement of the expression levels of ten immune checkpoint genes, quantification of the total mutational burden of the tumor, and the levels of infiltration of 24 different immune cell subtypes within the tumor microenvironment. These outputs collectively inform the various mechanisms that tumors use to evade recognition and clearance by the immune system.

We show that Cofactor Paragon accurately reports on gene expression, mutational burden and infiltrating leukocytes compared to gold standard methods from just 20 ng of FFPE tumor RNA. We further analyze the data to show the correlation between mutational burden and infiltrating CD4+ and CD8+ T-cell levels. Finally, we show how Paragon can identify expression of genes involved in inhibitory tumor metabolism that correlate with resistance to PD-1 blockade therapy. These results position Cofactor Paragon as a novel tool that can help identify ideal therapeutic strategies in immuno-oncology.

#2000

Targeted characterization of tumor heterogeneity through RNA-Seq analysis of phenotypically defined subpopulations.

Warren Porter, Eileen Snowden, Friedrich Hahn, Mitchell Ferguson, Frances Tong, Shannon Dillmore, Rainer Blaesius. _BD Technologies, RTP, NC_.

Cancer progression and metastases have been linked to the complex heterogeneity found within many solid tumors. In order to understand the complexities within solid tumors there is a need for in-depth characterization of the tumor and the surrounding microenvironment, including the identification and measurement of the subpopulations found within the diseased tissue.

Here we describe a method to interrogate the heterogeneity within patient derived xenograft (PDX) derived solid tumors through tissue dissociation, single cell flow sorting, and expression analysis of the resulting subpopulations. Cell sorting enables the identification and capture of phenotypically defined subpopulations within the tumor microenvironment for additional downstream analysis. Whole transcriptome RNA-Seq was performed on samples collected throughout the workflow to characterize the expression changes in cells during the different steps of the process. Expression differences were measured before and after tissue dissociation, before and after incubation, and before and after cell sorting. Expression differences were also measured between phenotypically defined subpopulations sorted by the CD133 and CD49f surface markers.

Several significant differentially expressed gene combinations between samples were observed. These differences involved unique pathway characteristics between the different subpopulations sorted based on expression of CD133 and CD49f markers. In contrast, changes induced by the workflow process alone were restricted to a small and well-defined gene subset of immediate early genes (IEGs). Within the workflow, these changes were restricted to the tumor dissociation step and no changes were detected after incubating and/or cell sorting the dissociated cells.

Our results indicate that solid tumor dissociation followed by flow cytometric analysis and sorting enables the interrogation of phenotypically defined subpopulations found within solid tumors. Since workflow induced changes are minimal and defined, these methods reveal biological differences between subpopulations within and between cancers. Differential expression of numerous surface marker genes were identified by our method allowing discovery of other candidate surface markers that may extend the subpopulation structure. Once verified these additional surface markers could serve to elucidate the dynamic changes cancer tissue undergoes during progression as well as in response to selective pressures such as drug therapy.

#2001

Preclinical investigation of a 15 gene hypoxia profile in different prostate cancer cell lines.

Thomas R. Wittenborn,1 Brita S. Sorensen,1 Morten Busk,1 Mathilde B. Thomsen,2 Steffen Nielsen,1 Michael R. Horsman1. 1 _Aarhus University Hospital, Aarhus C, Denmark;_ 2 _Aarhus University Hospital, Aarhus N, Denmark_.

Purpose: Hypoxia is known to reduce the response to radiotherapy and has been found to be a common feature in prostate cancer. Identifying patients with hypoxic tumors will allow us to select those patients that require specific hypoxic modifiers. Previously our department has developed a gene profile consisting of 15 genes, which demonstrated prognostic and predictive impact for hypoxic modification in head and neck squamous cell carcinomas (HNSCC). In the current study we investigated the 15 gene profile in vitro in different prostate cancer cell lines, and subsequently in vivo in the PC3 xenograft model.

Methods: For the in vitro experiments the prostate cancer cell lines investigated were PC-3, LNCaP and DU-145. Cell lines were cultured under normoxic (21% O2) or varying hypoxic conditions (0%, 0.5%, 1.0%, 2.0%, and 5.0% O2) for 24 hours, totRNA was extracted and gene expression levels measured by qPCR. Individual reference genes were selected (PSMC4, TBP, NDFIP1) and applied in the normalization of the relative expression levels, together with the reference genes previously used in the HNSCC study. For in vivo experiments, the PC3 cell line was inoculated on the flank of female NMRI nu/nu mice. Two hypoxia-sensitive tracers (18F-FAZA and Pimonidazole) were administered in order to determine hypoxic and non-hypoxic regions on excised tumor sections. These regions were isolated by laser-assisted microdissection, after which totRNA was extracted and gene expression levels measured by qPCR.

Results: In the in vitro experiments, all three prostate cancer cell lines had 14 of the 15 genes induced by hypoxia, when comparing the anoxic cells (0% O2) to the normoxic.cells (21% O2). The only discrepancy was ALDOA, which was not upregulated in the hypoxic cells. In the PC3 and DU-145 cell lines genes were investigated at additional hypoxic levels (0.5%, 1.0%, 2.0%, and 5.0% O2), giving a more intricate expression of the 15 genes. In vivo experiments are still ongoing but preliminary results show a hypoxia induced upregulation in 10 out of the 15 genes, of which 4 were significantly upregulated (ADM, ANKRD37, FAM162A, and LOX).

Conclusion: We investigated the 15 gene hypoxic profile in different prostate cancer cell lines, both in vitro and in vivo. A hypoxia dependent induction of genes was observed in both types of experiments, however with a differential gene expression with regard to hypoxia level. From the performed experiments, and looking only at oxygen dependency, it appears that the gene profile could be suitable for prostate cancers as well as HNSCC.

Funding sources: Danish Cancer Society ; The Danish Council for Independent Research: Medical Sciences ; EC FP7 project METOXIA (project no. 222741) ; CIRRO: The Lundbeck Foundation Center for Interventional Research in Radiation Oncology and The Danish Council for Strategic Research.

#2002

Disrupting immunosuppressive tumor microenvironment via stealthy nanobombs to enhance T-cell anticancer efficacy.

Hongwei Chen, Hayley J. Paholak, Xin Luan, Joseph P. Burnett, Nicholas O. Stevers, Kanokwan Sansanaphongpricha, Duxin Sun. _University of Michigan, Ann Arbor, MI_.

Despite the fact that the immunosuppressive tumor microenvironment (ISTME) has long been recognized as a major hurdle that significantly limits the anticancer efficacy of various therapeutic strategies, including T-cell-based immunotherapy, there is currently no effective way to address this challenge. In this work, we prove that sequential photothermal therapy (PTT), mediated with stealthy laser-triggerable nanobombs composed of iron oxide nanoparticles (IONPs) following systemic delivery, can remove both pre-resident and also newly triggered immunosuppressor cells in established tumors to enhance the efficacy of T-cell anticancer therapy. Using flow cytometry to study various T-cell populations in tumor tissues 48 h post PTT in a 4T1 mouse model, our data suggest that IONP-mediated PTT could significantly reduce the CD4+FoxP3+ regulatory T-cell population. Our data also suggest that CD8+ cytotoxic T-cell populations in PTT-treated tumor tissues return to those of non-treated tumors, and are believed to be newly activated at the tumor site or recruited from periphery blood following PTT. Our data further suggest that PTT twice spaced by a 24 h interval has the best anticancer efficacy, compared to PTT once or three times at 24 h increments. This enhanced anticancer effect from the sequential IONP-mediated PTT (twice) is believed to be due to an enhanced ability to eliminate triggered immunosuppressor cells that respond faster than cytotoxic CD8+ T-cells. Furthermore, our Luminex analysis of chemokine serum concentrations indicates that IONP-mediated PTT can decrease suppressor-cell-attractive protein secretion, such as G-CSF. In addition, our immunohistochemistry data from tumor tissue staining indicate that IONP-mediated PTT down-regulates tumor cell PD-L1 expression to disrupt tumor-cell-mediated immunosuppression. The capability of IONP-mediated PTT to disrupt ISTME can significantly enhance T-cell anticancer efficacy toward both treated established tumors and distal tumor cells. When combining IONP-mediated PTT with anti-CTLA-4 therapy to block CD8+ T-cell inhibition, our data suggest that combination treatment significantly inhibits tumor growth (p < 0.01), while control treatments, including the antibody alone, IONP-mediated PTT alone, and mock treatment, fail to do so. Our data further show that in a metastasis-mimic model, mice (6/6) that had primary tumors treated with PTT/anti-CTLA-4 reject pre-inoculated cancer cells at a distal site. Our data also show that the majority of the PTT/anti-CTLA-4 treatment-cured mice can reject subcutaneously (8/12) or tail-vein injected 4T1 cancer cells (9/9), indicating memory T-cell immune surveillance. In conclusion, this study provides a nanotechnology-based novel strategy to effectively disrupt not only pre-existing but also treatment-induced ISTME to boost T-cell-based cancer immunotherapy.

#2003

Antibody against TIGIT (T cell immunoreceptor with Ig and ITIM domains) induces anti-tumor immune response and generates long-term immune memory.

Angie Inkyung Park, Minu Srivastava, Erin Mayes, Hyun-Bae Jie, Rui Yun, Christopher Murriel, Ming-hong Xie, Andrew Lam, May Ji, Fumiko Axelrod, Jorge Monteon, John Lewicki, Tim Hoey, Austin Gurney. _OncoMed Pharmaceuticals, Inc., Redwood City, CA_.

T cell immunoreceptor with Ig and ITIM domains (TIGIT) is a co-inhibitory molecule containing an immunoreceptor tyrosine-based inhibition motif (ITIM) within its cytoplasmic tail, and is highly expressed on regulatory T cells and activated CD4+ T, CD8+ T, and NK cells. TIGIT competes with CD226, which contains an immunoreceptor tyrosine-based activation motif (ITAM) within its cytoplasmic tail for ligands poliovirus receptor (PVR) and poliovirus receptor-related 2 (PVRL2), with higher affinity to PVR. The ligands are expressed on the surface of antigen presenting cells and at high levels on most tumors. Therefore, when TIGIT is present, the ligands preferentially engage TIGIT rather than CD226, leading to cell suppression. We have generated antibodies against TIGIT that blocks ligand binding and inhibits TIGIT signaling. The clinical candidate, OMP-313M32 binds human TIGIT but not rodent and non-human primate TIGIT. Therefore, a surrogate antibody was generated for pre-clinical assessments in mice. Antibody 313R12 is an anti-mouse TIGIT antibody that can block mouse PVR ligand binding and inhibit TIGIT signaling in a manner similar to the clinical candidate OMP-313M32. 313R12 inhibited the growth of syngeneic colon and kidney tumors in immune competent mice. In some cases, anti-TIGIT antibody 313R12 caused complete tumor regression and a potent anti-tumor immune memory response as demonstrated by the lack of tumor growth upon re-challenge of mice that remained tumor-free after prior anti-TIGIT treatment. Mechanistically, anti-TIGIT antibody 313R12 was shown to induce a Th1 response and increase cytotoxic T lymphocyte (CTL) activity. By in vivo depletion of T cell populations, we have shown that CD8 T cell depletion completely abrogated the anti-TIGIT therapeutic effect, whereas CD4 T cell depletion led to partial reversal of efficacy of anti-TIGIT. Therefore, both CD4+ and CD8+ T cells are critical for anti-TIGIT-mediated immune responses. Using mice reconstituted with human hematopoietic stem cells, we also demonstrated that the clinical candidate OMP-313M32 inhibits patient-derived melanoma tumor growth. Taken together, these data demonstrate that anti-TIGIT therapy suppresses tumor growth and generates long-term immunological memory against multiple tumors.

#2004

Epigenetic targeting of CD276 expression in medullolbastoma.

Maheedhara R. Guda,1 Jose R. Castellanos,1 Collin M. Labak,1 Ramadevi Sanam,2 David J. Daniels,3 Sarah E. Martin,1 Julian J. Lin,1 Andrew J. Tsung,1 Kiran K. Velpula,1 Swapna Asuthkar1. 1 _University of Illinois College of Medicine at Peoria, Peoria, IL;_ 2 _GVK Biosciences, Hyderabad, India;_ 3 _Mayo Clinic, Rochester, MN_.

Medulloblastoma (MB) is the most common malignant brain tumor in children and accounts for 20% of pediatric CNS tumors. Following surgery and treatment with chemotherapy and radiation, MBs recur in a significant proportion of patients. Moreover, survivors experience comorbidities related to the tumor and/or its treatment. New insights into MB tumor pathogenesis are needed for improving the survivorship and outcome of MB. Epigenetic silencing, including histone modification and DNA methylation, is an important tumorigenic mechanism in MB. However, its role in cancer immunopathology is poorly understood. Using datamining and immunohistochemistry analysis, we show that B7-H3 (CD276), an important immune checkpoint member of the B7 and CD28 families, is highly upregulated in all four molecular subtypes of MB compared to normal tissue. Based on the clinical success of inhibitory immune checkpoint blockades (CTLA-4, PD-1, and PD-L1), CD276 appears to be a promising therapeutic target for cancer immunotherapy. Moreover, we observed that increased expression of CD276 correlates significantly with the expression of Enhancer of Zeste Homologue 2 (EZH2), a histone H3-methylating (H3K27me3) catalytic subunit in the human MB specimens. We hypothesize that combined targeting of CD276 and EZH2 may have greater impact on tumor progression and immune cell response in MB. Simultaneously, we performed LC/MS analysis to identify novel interaction partners of CD276 in MB tumor. These findings taken together suggest that CD276 is strongly associated with several epigenetic markers, indicating a strong relationship between cancer immune evasion and epigenetic mechanisms.

#2005

Synergistic effect of phenyl alpha-galactosylceramide C34 and chemotherapy on 4T1-tumor bearing mouse model.

Jung-Tung Hung, Jen-Chien Wu, Tsai-Hsien Hung, Jing-Rong Huang, John Yu, Alice L. Yu. _Institute of Stem Cell & Translational Cancer Research, Taoyuan City, Taiwan_.

Invariant natural killer T (iNKT) cells represent a unique subpopulation of innate immune cells which have immune modulatory effects on many immune effector cells such as CD4 T cells and CD8 T cells. Alpha-galactosylceramide (a-GalCer) is a well-known iNKT-stimulating glycolipid. Using a-GalCer as a template, we have generated a more potent a-GalCer analog C34, which contains two aromatic rings at the acyl chain and demonstrated its anti-cancer effect on tumor bearing mice model. It has been reported that NKT cells killed cancer cell lines treated with chemotherapy agents through TRAIL- and FasL-mediated pathways. In this study, we evaluated the anti-cancer effects of chemotherapy and C34 on tumor-bearing mouse model. First, BALB/c mice bearing 4T1 breast cancer were injected with various dose of chemotherapy agents weekly x 5 and the sub-optimal dose of docetaxel and doxorubicin was determined to be 0.5 mg/Kg. Similarly, sub-optimal dose of cisplatin and docetaxel in TC1 lung cancer-bearing C57BL/6 mice was determined to be 1 mg/Kg and 2 mg/Kg, respectively. In both tumor models, the suboptimal dose of C34 was found to be 0.1 ug per mouse. The treatment schedules of combination of chemotherapy and C34 were examined by administration of C34 at the same day (0D), three days (-3D) and one day (-1D) before chemotherapy and one day (+1D) after chemotherapy. We observed a survival benefit in mice injected with C34 one day after docetaxel treatment in 4T1-bearing mice as compared to those treated with the other schedules. Moreover, the survival of mice was significantly prolonged when C34 was injected three days after cisplatin treatment in TC1-bearing mice (median survival: 46 days for cisplatin and 55 days for cisplatin+C34, log-rank test p = 0.041). In the 4T1 tumor microenvironment, we found the number of CD3+CD8+ T cells was significantly greater in mice treated with docetaxel+C34 (23,536 +/- 901.4) than those treated with docetaxel only (2,270 +/- 1,536, t-test P < 0.0001). In addition, the number of PD1+ CD8 T cells was slightly higher, but did not reach statistical significance (t-test p = 0.3), in mice treated with docetaxel+C34 (23.3 +/- 8.5) than in mice treated with docetaxel only (14.6 +/- 10.1). In short, we have established an optimal treatment schedule for the combination of C34 and chemotherapy that showed synergistic anti-tumor efficacy. We also demonstrated that C34 could increase the number of CD8+ T cells in the tumor microenvironment in mice receiving chemotherapy. These results indicate that combination of C34 and chemotherapy administered with an optimal treatment schedule might have synergistic therapeutic efficacy for patients with lung cancer and breast cancer.

#2006

AMX-513 polyamine depletion therapy inhibits tumor growth and reverses immunosuppression in cancers including MYC-driven neuroblastoma and pancreatic cancer.

Mark R. Burns,1 Kathy Fosnaugh,2 Michael G. Palfreyman,1 Laura Gamble,3 Jayne Murray,3 Sophie Allan,3 Georgina Eden,3 Sara Sarraf,3 Murray Norris,3 David Ziegler,3 Michelle Haber3. 1 _Aminex Therpeutics, Inc., Kirkland, WA;_ 2 _Aminex Therpeutics, Inc., Kenmore, WA;_ 3 _Children's Cancer Institute Australia, Sydney, Australia_.

Tumorigenesis is associated with increased polyamine levels and involves the induction of ornithine decarboxylase (ODC), the initial rate-limiting enzyme in polyamine biosynthesis, and increased uptake of polyamines from the blood and diet. As well as contributing to proliferation, polyamines are reported to exert an immunosuppressive effect. Amplification of the MYC/MYCN oncogenes has been shown to directly induce ODC activity and inhibition of this enzyme by α-difluoromethylornithine (DFMO) markedly delays tumor development. Aminex Therapeutics is developing a polyamine depletion approach targeting both biosynthesis and transport of polyamines with AMX-513, a combination of the approved ODC inhibitor, DFMO, together with AMXT 1501, an alkylated polyamine mimetic which blocks polyamine uptake.

In the syngeneic CT26.CL25 mouse model of colorectal cancer, AMX-513, dosed daily for four weeks, reduced tumor growth > 75% compared to vehicle-treated control in immunocompetent Balb/C mice. There was no effect in athymic nude mice indicating that tumor growth inhibition by AMX-513 is T-cell-dependent. In the induced transgenic K6/ODC squamous tumor mouse model, stable regression was sustained 10 weeks after treatment ended and was accompanied by tumor infiltrate increases in IFNɣ and in CD3+ and CD8+ T-cells. Tumor infiltrates from AMX-513-treated KPC pancreatic cancer transgenic mice with tumor regressions showed >90% reductions in myeloid-derived suppressor cells (MDSCs; CD11b+ Gr-1+) but no changes in mature myeloid cells (CD11+Gr-1neg) by FACS analysis. AMX-513 treatment did not impact the percentage or number of CD4+CD25+FoxP3+ Tregs, but did significantly increase the percentage of activated CD8+ T cells in tumors.

Neuroblastoma is an aggressive childhood cancer frequently associated with MYCN and ODC deregulation. In neuroblastoma cell lines, the AMX-513 combination was highly synergistic (CI<0.5). Prophylactic treatment of neuroblastoma-prone TH-MYCN transgenic mice with AMX-513 significantly extended survival compared to either agent alone (median survival time = 81.0±11.8 days versus DFMO alone = 57.1±7.1 days; P<0.0001). Treatment of mice with small palpable tumors with AMX-513 in combination with cyclophosphamide/topotecan significantly improved survival compared with either AMX-513 or cyclo/topo alone (5/9 long term survivors compared to 0/10 and 0/9 for cyclo/topo and AMT-513, respectively; P<0.001 in each case). Polyamine levels were significantly decreased in mice undergoing AMX-513 treatment compared to DFMO or AMXT 1501 alone.

In conclusion, AMX-513 treatment alone or in combination with other cancer therapies results in significant tumor growth reduction in multiple cancer models and demonstrates novel immunotherapeutic potential. Clinical evaluation of AMX-513 is planned in 2017.

#2007

MHC class I polypeptide related sequence A (MICA) in context of aggressive prostate cancer.

Marcelo J. Sakiyama, Ingrid Espinoza, Amit Reddy, Jack R. Lewin, Xinchun Zhou, Xu Zhang, Jesus Monico, Charles R. Pound, Christian R. Gomez. _Univ. of Mississippi Medical Center, Jackson, MS_.

Prostate cancer (PCa) is the second most common cancer in American men, with higher incidence and death rates in African American men (AAM) relative to Caucasian American men (CAM). MHC class I polypeptide-related sequence A (MICA) is a protein expressed in the membrane of tumor cells that binds to the NKG2D receptors in NK cells and subtypes of CD8+T cells, activating its cytotoxic effects. Aggressive tumors cleave MICA from the membrane, release the soluble form (sMICA) into the plasma and downregulate the NKG2D receptor from the immune cells. Reports in PCa show loss of MICA expression from the cell surface is associated to a more aggressive phenotype. Likewise, increased levels of sMICA were linked to patients with Gleason score (GS) greater than 7. We hypothesized that conditions related to tumor aggressiveness in the microenvironment, would modify the levels of MICA in PCa. We also speculated that, when compared to CAM, MICA would be differentially expressed in tumors from AAM. To access the different expression of MICA in AAM and CAM PCa patients we stained a TMA containing 41 CAM and 44 AAM tumor cores for MICA expression. Our results showed that CAM PCa tumor cores had higher proportion of high expression of MICA than the AAM (38% vs 7% respectively, p=0.008). Moreover, the association between race and MICA expression was more profound with GS greater than 7 (p=0.001). Next, we explored the effect of hypoxia, a condition known to be associated with tumor aggressiveness, on MICA surface expression. We stained our TMA for hypoxia-inducible factor 1-alpha (HIF1-α), the master regulator of cellular and developmental response to hypoxia. Similar to MICA expression, HIF1- α expression was observed in tumor cores compared to normal tissue cores (43% vs 17% respectively, p=0.021). In CAM there was a positive correlation between MICA and HIF1-α immunostaining (rs= 0.50, p= 0.004), while in AAM correlation was weak (rs= 0.32, p= 0.081). FACS analysis on C4-2B cells revealed that 1% O2 (hypoxia) reduced MICA expression by 24.1%, relative to 20% O2 (normoxia). Likewise, incubation of PCa cells in the presence of 100µM cobalt chloride, a compound that mimics hypoxia by inducing HIF-1/3 α, reduced MICA by 16.7%, relative to control conditions. Altogether, our results indicate that expression of MICA is modified by disease aggressiveness and hypoxic conditions. Experiments to validate our results in minorities are currently in progress. Of equal interest is the progress of experiments focused on elucidation of the mechanistic involvement of tumor microenvironment on MICA-mediated tumorigenesis and immunoevasion. Funding acknowledgements: PCRP W81XWH-14-1-0151 (CRG), UMMC Department of Pathology (CRG), CAPES (MJS).

#2008

Intestinal microbiota may dynamically facilitate the anti-PD-L1 immunotherapy.

Jia Xue,1 Zhun Wang,1 Sheng Guo,1 Jie Cai,1 Davy Ouyang,1 Bin Cai,2 Gang Chen,2 Jie Liu,2 Xin Dong,2 Henry Li1. 1 _Crown Bioscience, Inc., Beijing, China;_ 2 _Nanjing Galaxy Biopharmaceutical Co. Ltd., Nanjing, China_.

Cancer immunotherapies, e.g. the antibody against programmed death ligand 1 (PD-L1), a checkpoint inhibitor, have witnessed great successes in treating certain cancers in recent years. Recent data have also demonstrated that gut microbiota are important modulators on anticancer immunotherapy1,2. Heterogeneity in patient outcome seems to suggest complex communications between microbiota and host antitumor immunity. To this end, we tested an engineered chimeric MC38 mouse cell line, hPDL1-MC38-HuCELL™ via human PD-L1 knock-in procedure, where MC38 CRC syngeneic cell is derived from C57BL/6. After treatment of PD-L1 antibodies (cD7A8 and BMS PD-L1 of different dose regimens), we observed significantly different drug responses among the mice from three different Chinese rodent suppliers: the mice from Vendor 1 showed no tumor progression after treatment with a variety of doses while no favorable response was observed in mice from Vendor 2 and Vendor 3. To deeply study the gut microbiota of these mice, we performed 16S ribosomal RNA sequencing on untreated mice from three groups (5 replicates for each). Global diversity analysis by Quantitative Insights Into Microbial Ecology (QIIME) tool revealed a clear separation in the three sources of mice: the microbiota composition of Vendor 2 and Vendor 1 are relatively closer to each other whereas the Vendor 3 mice are different, suggesting that the main difference seen between Vendor 1/2 and 3 in the original composition of gut microbiota is not the key impact for the observed anti-PD-L1 efficacy, and there should be other complex dynamics impacted anti-PD-L1 treatment, which remains unknown. Moreover, a group of 27 taxa were identified with significant difference in abundance (Kruskal-Wallis test, p-value < 0.05) across the groups, such as Bacteroidaceae, Lachnospiraceae, and Ruminococcaceae, which confirmed the previous data1. In conclusion, intestinal microbiota dynamically facilitate anti-PD-L1 efficacy and reversely anti-PD-L1 treatment could influence reconstruction of gut microbiota.

References 1. Sivan A, et al. Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy. Science 2015;350(6264):1084-9. 2. Vétizou M, et al., Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota. Science 2015;350(6264):1079-84. 3. Caporaso JG, et al., QIIME allows analysis of high-throughput community sequencing data. Nat Methods. 2010;7(5):335-6.

#2009

CC-90002 (anti-CD47 antibody) in vivo anti-tumor activity is associated with an increase in M1-polarized macrophages.

Bing Zheng,1 Piu Wong,1 WenQing Yang,2 Rama Narla,2 Michael Burgess,1 Laure Escoubet,2 Heather Raymon,2 Kandasamy Hariharan,2 John Boylan,2 Kristen Hege,1 Victoria Sung1. 1 _Celgene Corporation, San Francisco, CA;_ 2 _Celgene Corporation, San Diego, CA_.

CD47, also known as integrin-associated protein, is over-expressed in several tumor types, including AML, NHL, breast cancer and multiple myeloma. Elevated expression of CD47 on the cell surface protects tumors from phagocytosis by macrophages through binding to signal regulatory protein alpha (SIRPα) on the surface of macrophages. The CD47-SIRPα interaction triggers events that culminate in the inhibition of the phagocytic process. Macrophages are one of the immune cell types frequently found in the tumor microenvironment and exist as a heterogeneous population that includes both M1 and M2 macrophages. While the spectrum of macrophage subpopulations are likely quite diverse, traditionally, it is thought that M1 macrophages are pro-inflammatory, enhancing immune responses against tumor cells, while M2 macrophages are pro-tumor, since they express a wide array of anti-inflammatory molecules, cytokines and growth factors that promote tumor growth, angiogenesis and an immunosuppressive microenvironment. We evaluated CC-90002 efficacy in the RPMI-8226 multiple myeloma and MDA-MB-231 breast cancer xenograft models and enumerated mouse M1/M2-like macrophage populations within in the tumor before and after C-90002 treatment. In both RPMI-8226 and MDA-MB-231 models, M2 macrophages were the primary resident macrophage. Our studies show that when mice bearing RPMI-8226 multiple myeloma xenografts were treated with CC-90002 (a humanized anti-CD47 antibody), tumor regression was preceded by infiltration of macrophages into the xenograft. In the MDA-MB-231 breast cancer model, resident macrophages appeared to mediate the CC-90002 anti-tumor efficacy in vivo without additional macrophage trafficking. Interestingly, in both models, M1 macrophages appear to mediate CC-90002 efficacy regardless of whether macrophages infiltrate the tumor or are tumor-resident. In the MDA-MB-231 breast cancer model where CC-90002 did not induce infiltration, it is possible that resident tumor macrophages were re-educated to an M1 phenotype. In in vitro experiments using human monocyte-derived macrophages, both M1 and M2 macrophages are able to promote phagocytosis and additionally, we observed that CC-90002 selectively inhibited migration of M2 macrophages toward tumor cell conditioned media. This would presumably shift the overall balance of tumor-associated macrophages toward the M1 phenotype and suggests that inhibiting CD47 can both promote tumor phagocytosis and skew tumor macrophage subpopulations toward an anti-tumor phenotype. CC-90002 is currently being tested in two ongoing Phase I clinical studies in subjects with advanced solid and hematologic cancers (NCT02367196, NCT02641002).

#2010

BTK inhibitor BGB-3111 demonstrates anti-tumor activity in solid tumor models.

Nan Hu, Jiye Zhang, Min He, Shuo Zhang, Yong Liu, Lai Wang. _BeiGene Bio, Ltd., Beijing, China_.

Background BGB-3111 is a novel, irreversible, second generation BTK inhibitor with better selectivity profile and DMPK property compared to ibrutinib. It has demonstrated promising anti-tumor activities in patients with advanced B cell malignancies. Given BTK is expressed in all hematopoietic lineages except for T lymphocytes, it is reasonable to explore the immune modulatory effect of BGB-3111. In this study, we sought to investigate the anti-tumor activity of BGB-3111 in solid tumor models.

Methods Cell viability was assessed with Cell Titer Glo® assay and half maximal inhibitory concentrations (IC50) were estimated. In vivo activity was assessed in subcutaneous mouse xenograft models. Treatments were administered by oral gavage and individual body weight and tumor volume was recorded twice weekly.

Results In vitro studies showed that MDA-MB-436 cells are insensitive to BGB-3111, which do not expresse BTK. In both human breast cancer MDA-MB-436 and human A431 epidermoid carcinoma subcutaneous xenograft models, the tumor cells were co-injected with peripheral blood mononuclear cells (PBMCs) of healthy donors. In MDA-MB-436 model, BGB-3111 (15 mg/kg, BID) single agent significantly induced tumor growth inhibition (TGI) of 55%, whereas showed no anti-tumor effect if no PBMCs co-injected. Furthermore, co-treatment of BGB-3111 (15 mg/kg, BID) and anti-PD-1 antibody BGB-A317 (10 mg/kg, QW) demonstrated enhanced therapeutic effect in A431 allogeneic model. Interestingly, in BCLU200 and BCLU054 NSCLC primary tumor xenograft models (without PBMC co-injection), BGB-3111 (15 mg/kg, BID) also demonstrated significant anti-tumor activity.

Conclusions In summary, BGB-3111 showed interesting activity in solid tumor models, suggesting BTK inhibitor might have potential to be used beyond hematology malignancies. .

#2012

UVB-induced ULBP1 upregulation in melanocytes depends on HMGB1-mediated activation of NF-κB and IRF3.

Zhaohui Wu, Wei Wang. _Univ. of Tennessee Health Science Ctr., Memphis, TN_.

Anti-tumor immune surveillance mediated by innate immunity cells such as NK cells plays a critical role in eliminating "damaged" cells at precancerous stage and preventing cancer progression. As a key epidemiological factor associated with melanoma, solar UV radiation also elicit immunosurveillance by upregulating ligands for NK cell activating receptor NKG2D. Nevertheless, how UVR upregulates NKG2DL in melanocytes is not completely understood. We found UVB exposure induced secretion of damage-associated molecular patterns molecule HMGB1 from skin cells. HMGB1 activates transcription factors NF-κB and IRF3 in melanocytes which synergistically enhances NKG2D ligand ULBP1 transcription. Moreover, RAGE is essential for HMGB1-induced activation of NF-κB and IRF3, which serves as the HMGB1 receptor and mediates activation of TBK1. Consistently, HMGB1 deficient melanoma cells are less susceptible to NK cells mediated cytotoxicity. Our study provides a mechanistic link between environmental UV radiation and antitumor immunosurveillance induced in melanocytes, which may play a critical role in the early stages of melanomagenesis.

#2013

Increased MMPs activity in MDM2 overexpressing cancer cell lines.

Ali Alaseem, Thiagarajan Venkatesan, Thanigaivelan Kanagasabai, Khalid Alhazzani, Saad Alobid, Priya Dondapati, Appu Rathinavelu. _Rumbaugh-Goodwin Institute for Cancer Research-Nova Southeastern Univ., Davie, FL_.

The matrix metalloprotenases (MMPs) are a family of zinc-dependent endopeptidase and have proteolysis activity which found to impact several physiological and pathophysiological conditions including cancer. In cancer, over 20 types of MMPs promote invasion, angiogenesis, metastasis, and proliferation. For instance, MMP-9 is involved in many processes such as wound healing, tissue remodeling and regulation of inflammation. So far, several studies have also shown that MMP-9 is associated with poor clinical outcome. MMP-9 can facilitate angiogenesis and metastasis by releasing cytokines, pro-angiogenic and pro-metastatic factors. Conversely, the tissue inhibitor of metalloproteinases (TIMP-1) is a well-known negative regulator of MMPs which can also modulate other biological functions independent of MMPs regulation. TIMP-1 dysregulation has been shown to impact ECM integrity and potentiate metastatic ability also. Similarly, MDM2 (Murine Double Minute) has been recognized as an important multi-domain protooncogene which is overexpressed in many types of cancer and is associated with poor prognosis in different tumor types including sarcomas and carcinomas. Previously, our group reported that MDM2 overexpression is regulating MMP-9/TIMP-1 axis in LNCaP and LNCaP-MST (MDM2 transfected) prostate cancer cells. In order to further analyze the status of MMP in MDM2 overexpressing cancers the fluorescence activity assay and the zymography assay were used for measuring the activity of MMPs including MMP-9. Immunoblotting analysis were also used to correlate the expression levels of MDM2, MMP-9, and TIMP-1 in MDM2 overexpressing cell lines with and without Nutlin-3 (20 uM) treatment. Our results indicated that MMP activity is elevated by 97.3 % in LNCaP-MST cells compared to LNCaP. This may be possibly due to the near knockdown levels of TIMP-1 that was observed in LNCaP-MST cells. However, the Nutlin 3 treatment was able to decrease the MMP-9 levels and activity only marginally, without elevating TIMP-1 levels. This suggests that, in addition to MDM2, the PTEN loss in LNCaP-MST cells may also play a role in regulating the levels of TIMP-1 and consequently the activity of MMP-9. Furthermore, in order to verify our original speculation, we analyzed the levels of TIMP-1 in SJSA1 and GI-101A cell lines, which are known to overexpress MDM2 and impart aggressive metastatic abilities. Interestingly, the levels of TIMP-1 were found to be elevated in both SJSA1 and GI-101A cell lines compared to LNCaP-MST cells. This finding indicates that TIMP-1 may paly dual role depending on cancer types and the gene expression status in the tumor microenvironment. Further studies are required to fully delineate the interplay between MDM2 and pro-metastatic mechanisms, including the expression of TIMP-1 and MMP-9. (The financial support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale is gratefully acknowledged).

#2014

Genitourinary cancer-derived cells produce microenvironment proteinases that regulate proteinase activated receptors (PARs) to drive oncogenic signaling.

Stacy G. Gibson,1 Koichiro Mihara,1 Andries Zijlstra,2 Matthew E. Hyndman,1 Morley D. Hollenberg1. 1 _University of Calgary, Calgary, Alberta, Canada;_ 2 _Vanderbilt University, Nashville, TN_.

Background:

Thrombin-triggered activation of proteinase-activated receptor-1 (PAR1) is recognized as a key stimulus for driving epithelial malignancies (IUBMB life.63:397, 2011; PMID:21557443). However, the enzymes for regulating PAR1 in the tumour microenvironment are not known.

Hypothesis:

We hypothesize that prostate (PC) and urinary bladder cancer (UBC) cells can regulate microenvironment signaling to drive cancer progression via the secretion of proteinases that cleave/regulate proteinase-activated receptors (PARs).

Aims:

1.) Visualize the cleavage status of N- and C-terminally-dual-labelled PARs in receptor-transfected UBC- and PC-derived cells.

2.) Measure PAR cleavage by UBC and PC-secreted proteinases.

3.) Identify the UBC & PC-derived proteinases that cleave/regulate PAR function.

Methods:

The cleavage status of dual-labeled PARs (N-terminus-mCherry/C-terminus-YFP) expressed in PC & UBC cells was determined by confocal image analysis as already described (JBC 288:32979, 2013; PMID: 26957205). Intact receptors look 'yellow' and cleaved receptors look 'green'. The cleavage of PARs in UBC and PC cells was studied for dual-tagged-PAR-expressing UBC and PC cells exposed or not to cell-derived culture supernatants, enzyme agonists for PAR1 and 2 (thrombin and trypsin, respectively), and proteinase inhibitors (e.g. for MMPs and other enzymes). The impact of CRISPR-mediated elimination of MMPs from PC3 cells on PAR1 cleavage status was also monitored.

Results:

UBC- and PC-produced enzymes can cleave PAR1 as efficiently as thrombin, an established PAR1 agonist. None of the UBC cell lines produce proteinases that cleave PAR2. Inhibition of MMP activity partially prevents PAR1 cleavage by UBC cells and completely prevents PAR1 cleavage in PC3-PC cells.

Conclusions:

We conclude that UBC and PC cells secrete PAR-regulating proteinases that can directly regulate PAR1 in the tumour microenvironment so as to drive cancer progression.

Funding:

Alberta Innovates CRIO Grant, Prostate Cancer Canada Discovery Grant, Motorcycle Ride for Dad, Johnson & Johnson Alberta Health Partnership and CIHR

#2015

Mesenchymal stem cell correlates oral leukoplakia malignant transformation through regulate T cell response.

Yichen Chen, Bailin He, Da Ma, Jingjing Song, Xi Wang, Bin Cheng, Zhi Wang. _Sun Yat-sen University, Guangzhou, China_.

Introduction

Leukoplakia is one of the most common clinically presented oral potential malignant disorders (OPMD). Detailed understanding of leukoplakia-associated molecular or cellular changes would help us better undertand about the progress of leukoplakia malignant transformation. In this study, we examined the potential involvement of MSC in this very progress, and demonstrated lesion resident MSCs may favour leukoplakia malignant transformation through its strong immunoregulation on T cell.

Methods

Eighteen six-week-old female Sprague-Dawley rats (180-220g) were given 0.05g/L 4NQO in drinking water for 22 weeks for development of lesions. Lesions classfied by histological HE stainnig. MSCs were obtained from single cell suspesion of lesions, and cultured with splenocyte to study its immunoregulate ability.

Results

1. MSC-like cells are enriched in carcinogen induced leukoplakia and cancers. Representative results showing the lesions on tongue of rats. And relative proportion of MSC phenotype marker (CD29+, CD31-, CD45-, CD90+) in oral lesions (OMSC) were counted by flow cytometry.

2. Leukoplakia-derived MSC (L-OMSC), cancer-derived MSC (C-OMSC), and the normal mucosa-derived MSC (N-OMSC) share the similar stemness properties.

3. Cancer lesion have less infiltrating T cells (quantified by IHC and FCM of CD3 ). Cancer-derived MSCs suppress T cell proliferation, but not promote T cell apoptosis and inhibit T cell migration to the lesions.

4. Higher number of lesion derived MSCs associates with higher celluar proliferation in the lesions.

Summary

Our study demonstrated MSCs could migrate to pre-malignant leukoplakia lesions prior to tumor establishment. Further, MSCs play an important role in leukoplakia malignant transformation induced by chemicals, which via its strong immunomodulatory activities.

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

### Cell Death Targets

#2016

Novel dual cIAP1/XIAP antagonist ASTX660 activity in preclinical models of human papillomavirus(+) and (-) head and neck squamous cell carcinoma.

Roy Xiao, Yi An, Adeeb Derakhshan, Zhong Chen, Nicole C. Schmitt, Carter Van Waes. _National Institutes of Health, Bethesda, MD_.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer with a five-year survival of ~50%. HNSCC are linked to human papillomavirus (HPV+) or tobacco carcinogenesis (HPV-). The Cancer Genome Atlas recently analyzed 279 HNSCC, revealing that 30% overexpress FADD (Fas-Associated Death Domain), with or without BIRC2/3 genes encoding cellular Inhibitor of Apoptosis Proteins 1/2 (cIAP1/2), which are critical components of the Tumor Necrosis Factor (TNF) Receptor signaling pathways that determine cell death or survival. The frequency of such mutations provides a window for potential therapeutics, as IAP antagonists have been shown to switch cancer cell TNFα signaling from being pro-survival to pro-apoptotic. We recently showed that birinapant, a second mitochondrial activator of caspases (SMAC) mimetic that promotes IAP degradation, sensitizes HNSCC to cell death by TNFα and eradicates tumors in combination with radiation in HPV- HNSCC models overexpressing FADD+/-BIRC2. ASTX660 (developed by Astex Pharmaceuticals) is a novel dual cIAP1/XIAP antagonist currently in clinical trials for treating advanced solid tumors and lymphomas. The objective of the present study is to determine the therapeutic effects of ASTX660 in HPV+/- HNSCC preclinical models. ASTX660 at nanomolar concentrations was found to potently inhibit cell proliferation (measured using XTT assays) and induce apoptosis (Annexin/7-AAD flow cytometry), and displayed combinatorial activity with TNFα, TRAIL, or cisplatin in multiple human HPV+/- HNSCC cell lines. Western blotting showed near complete degradation of cIAP1 expression at nanomolar concentrations in human HNSCC cell lines. Flow cytometry revealed that ASTX660 in combination with either TNFα or cisplatin reduced MHC-I expression and increased PD-L1 expression, suggesting that potential synergistic efficacy could be observed in combination with immune checkpoint inhibitors. Our results demonstrate that ASTX660 is a potential therapeutic agent for both HPV+/- HNSCC. Experiments evaluating the anti-tumor effects of ASTX660 as a monotherapy and in combination with radiation, cisplatin, and anti-PD-1 therapies are ongoing, using both human HNSCC xenograft and syngeneic mouse oral cancer (MOC) models. Supported by NIDCD intramural projects (ZIA-DC-000016, 73 and 74).

#2017

The role of ceramide in the dual cytotoxic and metabolic stimulative effects of sigma-2 receptors.

Cheri Z. Liu, Ellen Sukharevsky, Wayne D. Bowen. _Brown University, Providence, RI_.

Sigma-2 receptors are highly overexpressed in cancer cells compared to normal cells, as well as in proliferative versus quiescent cells. Sigma-2 receptors have been of interest as an antineoplastic target, as sigma-2 agonists cause cell death via a variety of pathways. Recently, a new class of sigma-2 agonists has been discovered, with ligands such as CM764, that do not induce cell death but that affect cellular metabolism, as shown by increased MTT reduction, increased ATP level, and HIF-1α stabilization. Previously, our lab has shown that treatment of breast tumor cells with sigma-2 receptor agonists CB-184 and BD737 caused dose dependent increases in ceramide and simultaneous decreases in sphingomyelin, suggesting that sigma-2 receptors modulate the sphingolipid pathway. The ceramide pathway has been shown to play both prosurvival and pro-apoptotic roles in cancer cells. Here, we examine the role of ceramide in sigma-2 receptor mediated pathways in human SK-N-SH neuroblastoma cells. A 24 h treatment of SK-N-SH neuroblastoma cells with 20 μM exogenous C6-ceramide caused less than 20% cell death. Interestingly, this compared to over 50% cell death in MCF-7 breast adenocarcinoma cells and PANC-1 pancreatic cancer cells, indicating a differential role of ceramide in cell death pathways across cell lines. SK-N-SH cells were treated with 10 μM of either CM572, a classic sigma-2 agonist that causes cell death, or CM764, a sigma-2 agonist that stimulates metabolism. Fumonisin B1 is a potent inhibitor of ceramide synthase. Treatment of cells with fumonisin B1 (10 μM) did not significantly alter CM572-induced cytotoxicity. Conversely, treatment with fumonisin B1 did significantly inhibit the MTT reduction caused by CM764. Taken together, these results suggest that ceramide formation may play more of a role in the metabolic stimulative effect of sigma-2 receptors, as opposed to the cytotoxic effect. Decreased levels of ceramide have been shown to increase cellular resistance to apoptosis. Therefore, it has been of interest to increase intracellular ceramide levels in drug resistant cancer cells in order to sensitize the resistant cells to chemotherapeutics. Multidrug resistance (MDR) in cancer cells is often attributed to the overexpression of particular ABC transporters that effectively pump drugs out of the cells. One such protein is the multidrug resistance-associated protein 1 (MRP1). It has been shown that increased expression of drug efflux pumps also decreases cell sensitivity to ceramide. Treatment with CM572 caused a dose-dependent decrease in MRP1 protein levels, while treatment with CM764 had little effect on MRP1. We will examine how this modulation of MRP1 affects sensitivity to C6-ceramide. We will also examine whether combination of C6-ceramide with sigma-2 receptor ligands augments the cytotoxic and/or metabolic stimulative effects of sigma-2 receptors.

#2018

**Synergistic interactions between PLK1 and HDAC inhibitors in non-Hodgkin's lymphoma cells occur** in vitro **and** in vivo **and proceed through multiple mechanisms.**

Tri Nguyen,1 Rebecca Parker,1 Elisa Hawkins,1 Victor Yazbeck,1 Akhil Kolluri,1 Maciej Kmieciak,2 Mohamed Rahmani,1 Steven Grant3. 1 _Department of Internal Medicine, Virginia Commonwealth University and Massey Cancer Center, Richmond, VA;_ 2 _Massey Cancer Center, Virginia Commonwealth University, Richmond, VA;_ 3 _Department of Internal Medicine, Biochemistry, Pharmacology, Virginia Commonwealth University and Massey Cancer Center, Richmond, VA_.

The mitotic kinase polo-like kinase 1 (PLK1) is over-expressed in neoplastic cells, including non-Hodgkin's lymphoma (NHL) cells, but not in normal cells, prompting development of PLK1 inhibitors e.g., volasertib (Boehringer-Ingelheim). PLK1 plays key roles in cell cycle progression, mitosis, cytokinesis, centriole duplication, and the DNA damage response (DDR). Notably, volasertib demonstrated preliminary signs of activity in NHL patients in a phase I trial. HDAC inhibitors (HDACIs) modify chromatin structure and gene expression, but also induce multiple mitotic abnormalities including mitotic slippage and DNA damage checkpoint disruption. Given these complementary mechanisms, we sought to examine volasertib interactions with the pan-HDACI belinostat (Spectrum Pharmaceuticals) in diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) cells in vitro and in vivo. Exposure of DLBCL cells to extremely low (e.g., 5-30 nM), minimally toxic concentrations of volasertib and low (e.g., 100-400 nM), marginally toxic belinostat concentrations for 48 hr synergistically increased apoptosis in SU-DHL16, SU-DHL4, SU-DHL8 (GC subtype), HBL-1, U2932 (ABC-subtype), and OCI-LY18, Carnaval (double hit) DLBCL cells. Similar interactions occurred in primary lymphoma cells, Granta-519 and Rec-1 (MCL), SU-DHL4/BR and SU-DHL16/BR (bortezomib-resistant cells exhibiting up-regulation of the proteasome sub-unit PSMB5 and bortezomib binding site mutation (Ala49Val). In contrast, identical regimens were minimally toxic to normal hematopoietic progenitors. Volasertib/belinostat induced massive M-phase arrest, p-histone H3 up-regulation, a marked increase in mitotic errors, and pronounced M-phase cell death associated with striking DNA damage (γH2A.X formation). Belinostat diminished c-Myc mRNA and protein in DLBCL cells, an effect significantly enhanced by volasertib. Notably, PLK1 knock-down in DLBCL cells significantly increased belinostat-induced M-phase accumulation, phospho-histone H3 accumulation, γH2AX generation, and apoptosis. Analogously, c-Myc knock-down increased DNA damage and apoptosis in response to volasertib, arguing that c-Myc down-regulation plays a functional role in lethality. Volasertib/belinostat co-administration dramatically reduced tumor growth in an ABC-DLBCL flank model (U2932) and significantly increased survival (56.2 vs 39.7 days; p < 0.001) without weight loss or other toxicities. The regimen also enhanced survival and reduced tumor cell bone marrow engraftment in a systemic double-hit lymphoma model (OCI-Ly18). Together, these findings indicate that PLK1/HDAC inhibition warrants attention as a therapeutic strategy in NHL. Accordingly, based on these findings, a phase I volasertib/belinostat trial in patients with aggressive NHL is planned (NCT02875002).

#2019

Synergistic anticancer activity of the RAD51 inhibitor IBR2 with inhibitors of receptor tyrosine kinases and microtubule protein.

Peter J. Ferguson,1 Mark D. Vincent,2 James Koropatnick1. 1 _Lawson Health Research Institute, London, Ontario, Canada;_ 2 _London Regional Cancer Program, London, Ontario, Canada_.

Although cancer cell genetic instability contributes to characteristics that mediate tumorigenicity, it also contributes to the selective toxicity that some chemotherapy drugs have for cancer cells. This "synthetic lethality" (Nature 434:913, '05) can be enhanced by agents that inhibit DNA repair (Mol Onc 8:1429, '14; Proc AACR 57:3718, '16). To exploit this potential "Achilles heel", we tested the ability of a small molecule inhibitor of RAD51 to potentiate the cytotoxicity of established chemotherapy drugs. 2-(benzylsulfonyl)-1-(1H-indol-3-yl)-1,2-dihydroisoquinoline (IBR2) was obtained from Drs. J-W Zhu and W-H Lee (Univ. California - Irvine). IBR2 inhibits RAD51-mediated double-strand DNA break repair, but also enhances induction of apoptosis by the ABL inhibitor imatinib against K562 cells (EMBO Mol Med 5:353, '13). There is potential value of such synergistic interaction among other tumor types and with other drugs. IBR2-drug combinations were therefore examined across a spectrum of cancer cell lines from various tissues (AML, CML, carcinoma of breast, colon, stomach, lung, and head) representing a range of oncogenic drivers (ABL, c-kit, Raf, Ras, ER, mutant p53). Cells were exposed to IBR2 simultaneously with inhibitors of various tyrosine kinase receptors, DNA-damaging agents, or inhibitors of microtubule function. Cells were cultured in 96-well plates, exposed to drugs alone and in combination, and cell density determined by viability staining (alamarBlue or neutral red) 4 days later. Inhibition of proliferation by drug combinations was normalized to that of IBR2 alone. Depending on the drug sensitivity of the cell line, IBR2, at concentrations that inhibited proliferation between 0% and 75% as a single agent, enhanced toxicity of imatinib by up to 80%. IBR2 also greatly enhanced antiproliferative activity of regorafenib (targets RAF, kit, others), EGFR inhibitors erlotinib, gefitinib, afatinib and osimertinib, and microtubule inhibitor vincristine (VCR). However, IBR2 was antagonistic with VP-16, cisplatin, irinotecan, melphalan, and olaparib. To determine a possible mechanism of the observed synergy, the interaction between IBR2 and imatinib or VCR was compared with that between verapamil, a P-glycoprotein inhibitor, and the latter 2 drugs. The VCR-resistant head and neck cell line HN-5a/V15e was not cross-resistant to imatinib, but IBR2 enhanced imatinib toxicity in this cell line, its HN-5a parent, and HT-29 by up to 60%, much better than verapamil (up to 40% at similar concentrations, P<0.05). IBR2 enhanced VCR toxicity in these 3 lines to degree similar to verapamil, decreasing the IC50 by up to 90%. IBR2 appears to enhance drug toxicities via mechanisms other than just inhibition of RAD51 and may potentially interfere with microtubule function. The results indicate that this agent may be useful as a clinical adjuvant to numerous cytotoxic drugs.

#2020

Synergistic induction of apoptosis of combined mesupron and auranofin treatment in human breast cancer cells.

Joo-Eun Lee, Yeo-Jung Kwon, Kyung-Soo Oh, Young-Jin Chun. _Chung-Ang Univ. College of Pharmacy, Seoul, Republic of Korea_.

Urokinase-type plasminogen activator (uPA) system may play a crucial role in cancer cell invasion and metastasis. uPA has been validated as a predictive or prognostic biomarker protein and considered as a therapeutic target in human breast cancer. Mesupron is a uPA inhibitor blocking uPA enzymatic activity to reduce tumor cell invasion, migration and cell growth. Auranofin has been known as an antirheumatic drug and a thioredoxin reductase inhibitor and recently its anticancer activity in ovarian and breast cancers has also been identified. To study whether cotreatment with mesupron and auranofin shows a significant anticancer activity, the synergistic induction of apoptosis of mesupron with auranofin was determined. Auranofin or mesupron alone inhibited cancer cell growth in MCF-7 cells with IC50 of 0.25 μM or 25 μM, respectively. Flow cytometric analysis also showed an increased apoptosis. When cells were treated with mesupron (0.125 μM) in combination with auranofin (10 μM), we found a significant induction of apoptosis although the cytotoxic effects of mesupron or auranofin alone at those concentrations were not quite strong. Interestingly, combined mesupron and auranofin treatment significantly suppressed mitochondrial antiapoptotic proteins including Bcl-2 and Bcl-xL. We also found the increase of caspase-3 and PARP cleavages. The combination index (CI) also indicated the synergistic induction of apoptosis by auranofin and mesupron. Taken together, these data suggest that the use of mesupron and auranofin in combination can be valuable to achieve higher anticancer activity.

#2021

Ablation of caspase-7 promotes solar-simulated light-induced mouse skin carcinogenesis through upregulation of keratin-17.

Mee-Hyun Lee,1 Do Young Lim,2 Sung Young Lee,2 Jung Hyun Shim,3 Xuejiao Liu,1 Ran Zhao,1 Hai Huang,1 G.Timothy Bowden,4 Young-Joon Surh,5 Yong-Yeon Cho,6 Ann M. Bode,2 Zigang Dong2. 1 _China-US (Henan) Hormel Cancer Institute, Zhengzhou, China;_ 2 _The Hormel Institute, Austin, MN;_ 3 _Department of Pharmacy, College of Pharmacy and Natural Medicine Research Institute, Mokpo National University, Mokpo, Republic of Korea;_ 4 _University of Arizona Cancer Center, Tucson, AZ;_ 5 _Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, College of Pharmacy, Seoul National University, Seoul, Republic of Korea;_ 6 _College of Pharmacy, The Catholic University of Korea, Bucheon, Republic of Korea_.

Solar UV irradiation is an environmental carcinogen that causes skin cancer. Caspase-7 is expressed at reduced levels in many cancers. The present study was designed to examine the role of caspase-7 in solar-simulated light (SSL)-induced skin cancerr and to elucidate its underlying molecular mechanisms. Our study revealed that mice with genetic ablation of caspase-7 are significantly susceptible to SSL-induced skin carcinogenesis. Epidermal hyperplasia, tumor volume and tumor numbers were increased in caspase-7 knockout (KO) mice compared with SKH1 wild-type mice irradiated with SSL. The expression of cell proliferation biomarkers, such as survivin and Ki-67, was elevated in SSL-irradiated skin of caspase-7 KO mice compared with those observed in SSL-exposed wild-type SKH1 mouse skin. Moreover, SSL-induced apoptosis was abolished in skin from caspase-7 KO mice. 2-DE gel electrophoresis, followed by matrix-assisted laser desorption/ionization-time-of-flight analysis of skin tissue lysates from SSL-irradiated SKH1 wild-type and caspase-7 KO mice revealed an aberrant induction of keratin-17 in caspase-7 KO mice. Immunohistochemical analysis of skin tumors also showed an increase of keratin-17 expression in caspase-7 KO mice compared with SKH1 wild-type mice. Furthermore the expression of keratin-17 was also elevated in SSL-irradiated caspase-7 KO keratinocytes as well as in human basal cell carcinomas. The in vitro caspase activity assay showed keratin-17 as a substrate of caspase-7, but not caspase-3. Overall, our study suggests that genetic loss of caspase-7 promotes SSL-induced skin carcinogenesis by blocking caspase-7-mediated cleavage of keratin-17.

#2022

Hsp90 inhibitor (NVP-AUY922) enhances anti-cancer effect of Bcl-2 inhibitor (ABT-737) in small cell lung cancer.

Hannah Yang, Kang-Seo Park, Junyoung Choi, Sang-We Kim Kim, Dae Ho Lee. _Asan Medical Center, Seoul, Republic of Korea_.

Background: Bcl-2 family is a group of apoptosis regulators that plays an anti-apoptotic role for cell survival and is known to contribute to induce chemotherapy resistance in small cell lung cancer (SCLC). ABT-737 is an anti-cancer drug that induces apoptosis by selectively blocking the activities of Bcl-2 and Bcl-xL, but not Mcl-1. Consequently, the efficacy of ABT-737 is largely restricted in the presence of Mcl-1. Heat-shock-protein 90 (Hsp90) is highly expressed in most tumors and Hsp90 inhibitors induce the proteasomal degradation of Hsp90 client proteins. In addition, Hsp90 inhibitor can reduce Mcl-1 expression, a down-stream of Akt and Erk pathway. Furthermore, Hsp90 inhibitor inhibits activities of NF-κB by degradation of IKK. Thus, we show that downregulation of Mcl-1 and NF-κB by Hsp90 inhibitor can lead to synergistic pro-apoptotic effects with ABT-737.

Materials and methods: The proliferative activity, apoptotic activities, and expression of apoptotic proteins were assessed in SCLC cell lines after treatment with ABT-737, NVP-AUY922, or both drugs. The synergy effects of ABT-737 and NVP-AUY922 were analyzed by cell viabilities with different concentrations in SCLC cell lines, and the combination index values were < 1. In addition, the synergy effects of the drugs were showed with xenograft model with human SLCL cell line in vivo.

Results: Here, we show that NVP-AUY922, an Hsp90 inhibitor, can potentiate the pro-apoptotic effects of ABT-737 not only by reducing the levels of Akt but also by inhibiting pNF-κB, both of which are proteins regulating apoptosis signaling. Western blot analysis revealed that proteins associated with apoptosis such as PARP, Caspase 3 and 7 were more upregulated in SCLC cells exposed to the drug combination than in cells exposed to NVP-AUY922 or ABT-737 alone. In addition, Annexin V & dead cell assay showed dual inhibition of the Hsp90 and Bcl-2 signaling pathways more increased apoptotic cells and dead cells in SCLC. In our result, ABT-737 induced apoptosis by blocking Bcl-2 activation, and NVP-AUY922 blocked the levels of Hsp90 client proteins, Akt and pErk, ultimately leading to decreased level of Mcl-1. In addition, NVP-AUY922 induced degradation of IKK, and increased IκB-α inhibited activation of NF-κB. And this combination treatment showed higher BIM and BID expression, pro-apoptotic proteins, than single treatment. Furthermore, synergy effects of combination were verified in xenograft model with human SCLC cell line.

Conclusions: Consequently, NVP-AUY922 synergizes with ABT-737 to induce apoptosis by reducing activities of Mcl-1 and NF-κB in SCLC. This study suggests that adopting an appropriate combination of drugs can lead to better outcomes compared with monotherapy in SCLC.

#2023

Artemisinins enhanced the antileukemic efficacy of BCL2 inhibitors.

Blake S. Moses,1 Jennifer M. Fox,1 Xiaochun Chen,1 Jeffrey W. Tyner,2 Gary H. Posner,3 Patrick Bailey,1 Curt I. Civin1. 1 _University of Maryland Baltimore, Baltimore, MD;_ 2 _Oregon Health and Science University, Portland, OR;_ 3 _Johns Hopkins Univesity, Baltimore, MD_.

More effective, less toxic treatments are needed to improve longevity and quality of life for patients with acute leukemias. Artesunate (AS), the current WHO-recommended drug for severe malaria, is a semi-synthetic derivative of the natural compound Artemisinin. AS has broad antineoplastic activity in vitro, and is well tolerated, inexpensive, and can be parenterally or orally administered in humans. Both AS and a more potent synthetic derivative, artemisinin-derived trioxane diphenylphosphate dimer 838 (ART-838), substantially inhibited acute leukemia growth in vivo and in vitro, at doses where normal CD34+ hematopoietic stem-progenitor cell clonogenicity was essentially unaffected. Moreover, AS and ART-838 synergized with several current antileukemic drugs (Fox et al, Oncotarget, 2015). To identify additional potential antileukemic drug combinations, we screened for the growth inhibitory effects of AS and ART-838 against 3 acute leukemia cell lines co-treated with a panel of 122 antileukemic compounds (mainly emerging antineoplastic drugs). Both AS and ART-838 showed synergistic or additive growth inhibition with several compounds. Two BCL2 protein family inhibitors demonstrated among the highest levels of synergy. One of these, ABT-199 (venetoclax), was recently approved by the FDA for the treatment of chronic lymphocytic leukemia (CLL) and received breakthrough designation for acute myeloid leukemia (AML). Therefore, we decided to further investigate the combination of ABT-199 with AS or ART-838. Synergy between AS (or ART-838) and ABT-199 was confirmed across several acute leukemia cell lines with varying sensitivities to each drug. Both leukemia cell proliferation and survival were affected synergistically by exposure to the combination of AS (or ART-838) plus ABT-199. Utilizing in vivo imaging, we assessed changes in growth kinetics of tail vein-injected acute leukemia cell lines and primary patient-derived samples constitutively expressing luciferase in mice treated with AS, ABT-199, or the combination. The combination of AS plus ABT-199 had the greatest antileukemic effect. Based on these data, we propose that Artemisinins plus BCL2 inhibitors comprise an antileukemic drug pair with high potential for incorporation into acute leukemia therapeutic regimens.

#2024

Reactivating apoptosis in BFL-1/A1 driven cancer with cysteine-reactive stapled peptide inhibitors.

Rachel M. Guerra, Annissa J. Huhn, Edward P. Harvey, Gregory H. Bird, Loren D. Walensky. _Dana Farber Cancer Institute, Boston, MA_.

BFL-1/A1 is a BCL-2 family protein implicated in the progression and chemoresistance of melanoma, lymphoma, and other cancers, yet it remains undrugged. Anti-apoptotic BCL-2 family proteins block cell death by trapping the critical α-helical BH3 domains of pro-apoptotic members in a surface groove. Cancer cells hijack this survival mechanism by overexpressing a spectrum of anti-apoptotic members, mounting formidable apoptotic blockades that resist chemotherapeutic treatment. Drugging the BH3-binding pockets of anti-apoptotic proteins has become a highest-priority goal, fueled by the clinical success of venetoclax, a selective BCL-2 inhibitor, in reactivating apoptosis in BCL-2-dependent cancers. A natural juxtaposition of two unique cysteines at the binding interface of the NOXA BH3 helix and BFL-1/A1 pocket informed the development of stapled BH3 peptides bearing acrylamide warheads to irreversibly inhibit BFL-1/A1 by covalent targeting. Here, we describe the development and application of cysteine-reactive stapled peptides that, compared to their non-covalent analogs, trigger rapid mitochondrial cytochrome c release, caspase 3/7 activation, and apoptosis induction of BFL-1/A1 driven cancers such as melanoma. Mechanism of action studies demonstrated the exquisite BFL-1/A1 selectivity and mitochondrial localization of the in situ covalent reaction. Given the frequent proximity of native cysteines to regulatory binding surfaces, covalent stapled peptide inhibitors provide a new therapeutic strategy for targeting oncogenic protein interactions.

#2025

A distinct role of RhoB in simvastatin-induced cytotoxicity in breast cancer cells.

Serena Seah, Tingting Wang, Soo Chin Lee. _National University of Singapore, Singapore, Singapore_.

Purpose Statins, a class of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, are initially developed as cholesterol-lowering drugs by inhibiting the mevalonate pathway. They have recently gained attention for their potential anticancer properties, but the mechanisms of their anticancer effects remain elusive. This study aims to investigate the antiproliferative effects of statins in breast cancer cell lines.

Methods We screened a panel of breast cancer cell lines (Estrogen receptor positive (ER+): CAMA1, MCF7, T47D, ZR-75-1 and Hcc1428; triple negative (TNBC): MDA MB231, MDA MB468, BT549, Hs578T and Hcc1806) and assessed the sensitivity of these cells to simvastatin in relation to their hormone receptor status using cell proliferation assay. Next, to elucidate the mechanisms underlying simvastatin sensitivity, we evaluated the (i) expression of key enzymes in the mevalonate pathway using qPCR and western blot analysis and (ii) the role of sterol biosynthesis metabolites by exogenously adding these compounds and testing their effects on simvastatin-induced cytotoxicity. Finally, we performed functional assays following siRNA knockout to identify the key small GTPase involved.

Results We found TNBC cell lines to be more susceptible to simvastatin compared to ER+ cell lines (mean IC50 of 7.98μM [95%CI 2.75-13.22] versus 41.74μM [95%CI 6.71-76.78]). Simvastatin treatment induced robust apoptosis in TNBC but not the ER+ cells. There was no significant difference in mRNA expression of HMGCR, HMGCS, MVK, MVD, IDI1 enzymes involved in the mevalonate pathway between TNBC and ER+ breast cancer cell lines. Supplementation with geranylgeranyl pyrophosphate but not farnesyl pyrophosphate, inhibited simvastatin-mediated cell death, suggesting the involvement of geranylgeranylated proteins such as Rho family of GTPases. Here, we identified RhoB to be a key player in simvastatin-mediated cytotoxicity. RhoB showed a significant increase in transcript and protein levels in response to simvastatin treatment in the TNBC cells. Interestingly, the increase in protein expression was not associated with an increase in its prenylation as the accumulation of RhoB was observed only in the cytosolic fraction. Finally, silencing RhoB was able to abrogate the cytotoxic effects mediated by simvastatin in TNBC cells.

Conclusions Taken together, our data reveals a critical role of RhoB in the anticancer activity of simvastatin, which appears to be exclusive to TNBC cells suggesting that HMG-CoA reductase inhibitors may provide new therapeutic strategies for TNBC treatment.

#2026

CHK1 inhibition synergizes with cisplatin to promote mitotic cell death via activation of caspase 2 and downregulation of E2F1 in small cell lung cancer.

Wei-Hsun Hsu,1 Xiaoliang Zhao,1 In-Gyu Kim,1 Guanhua Rao,1 Justine McCutcheon,1 Shuo-Tse Hsu,1 Afshin Dowlati,2 Yu-Wen Zhang,1 Giuseppe Giaccone1. 1 _Georgetown Univ. Medical Ctr., Washington, DC;_ 2 _Case Western Reserve University, Cleveland, OH_.

Platinum-based chemotherapy remains the standard treatment for patients with small cell lung cancer (SCLC), one of the deadliest cancers, but the benefit of such therapy is often hampered by rapid emergence of drug resistance. Thus far, there is no targeted therapy available for SCLC, mainly due to the lack of targetable oncogenic driver alterations; nevertheless, over 90% of SCLC tumors harbor inactivating mutations in tumor suppressor p53, an important checkpoint regulator for the control of cell cycle progression in the event of DNA damage. SCLC cells with p53-deficiency heavily rely on the checkpoint kinase 1 (CHK1), a key checkpoint regulator, to prevent the progression of cell cycle through G2/M in the presence of damaged DNA. Using CHK1 inhibitors and siRNA knockdown approaches, we explored the potential therapeutic benefit of CHK1 inhibition in SCLC models in combination with cisplatin, a DNA damaging agent. We demonstrated that CHK1 inhibition was able to release G2/M cell cycle arrest induced by cisplatin, and synergized with cisplatin to induce mitotic cell death especially in the p53-deficient SCLC cell lines. CHK1 inhibition activated caspase 2 and downregulated the expression of E2F1 transcription factor in these cells. We showed that activation of caspase 2 was required for the mitotic cell death induced by the combination of cisplatin and CHK1 inhibition. Interestingly, the upregulation of E2F1 induced by CHK1 inhibition was mediated in part by caspase 2, and E2F1 played a role in the DNA damage checkpoint control as knockdown of E2F1 enhanced cytotoxicity induced by cisplatin or CHK1 inhibitor in GLC4 cells. However, ectopic overexpression of E2F1 also led to increase of phospho-histone H3, γH2AX and cleaved PARP, and significantly enhanced cisplatin cytotoxicity, indicating that proper dosage of E2F1 is critical for DNA damage checkpoint response. These data provide a mechanistic insight of CHK1-mediated DNA damage checkpoint control and the anticancer effect of CHK1 inhibition against SCLC. In tumor samples derived from SCLC patients treated with platinum based chemotherapy, we found that high expression of CHK1 and E2F1 significantly correlated with poorer overall survival, further suggesting a link between CHK1 and E2F1 and potentially collaborative roles of these two factors in DNA damage checkpoint response. Moreover, we showed that CHK1 inhibition resensitized cisplatin-resistant cells to cisplatin treatment and induced mitotic cell death via caspase activation in these cells. These data indicate that CHK1 inhibition may enhance the benefit of cisplatin treatment for SCLC and overcome cisplatin resistance. We are presently confirming the in vitro results of CHK1 inhibition in combination with cisplatin treatment in xenograft tumor models in athymic nude mice.

#2027

Preclinical evaluation of AMG 176, a novel, potent and selective Mcl-1 inhibitor with robust anti-tumor activity in Mcl-1 dependent cancer models.

Sean R. Caenepeel, Brian Belmontes, Jan Sun, Angela Coxon, Gordon Moody, Paul E. Hughes. _Amgen Inc., Thousand Oaks, CA_.

Evasion of apoptosis represents an essential hallmark in the progression of many cancers. The Bcl-2 family of proteins plays a central role in regulating the apoptotic process. Targeting pro-survival Bcl-2 family members like Mcl-1 with small molecule inhibitors represents a viable therapeutic approach for the treatment of cancer. This study evaluated the in vitro and in vivo activity of AMG 176, a potent and selective Mcl-1 inhibitor currently in Phase I clinical development. AMG 176 binds with high affinity and selectivity to the BH3-binding groove of Mcl-1. In a cell based split-luciferase complementation assay, AMG 176 disrupted the interaction between Mcl-1 and Bak, leading to downstream activation of the intrinsic apoptosis pathway as measured by increased caspase activity and subsequent effects on viability. Oral administration of AMG 176 to mice bearing OPM-2 multiple myeloma xenografts resulted in a dose-dependent increase in activated Bak with a clear PK/PD relationship. Dosing regimens (20-60 mg/kg PO, QD) evaluating discontinuous and continuous administration of AMG 176 in established OPM2 xenografts demonstrated robust tumor growth inhibition with complete tumor regression at an elevated dose. Efficacy in this model was achieved at doses in agreement with those eliciting induction of apoptotic markers. Treatment of tumor cell lines with Compound A, a close structural analog of AMG 176, revealed a dose- and time-dependent increase in Mcl-1 protein levels that was reversible upon compound washout. Subsequent experiments performed with cycloheximide suggested that elevations in Mcl-1 protein levels were due to an increase in Mcl-1 protein half-life, likely driven by the compounds ability to disrupt proteasome-mediated degradation. Compound A was also used to characterize the kinetics of activating apoptosis. These studies revealed a rapid induction of apoptosis and loss of viability in Mcl-1 dependent multiple myeloma and AML cell lines. In cell lines highly dependent on Mcl-1, treatment with Compound A for as little as two hours was sufficient to achieve complete cell killing. Cell line profiling studies (>200 lines) revealed robust effects on cell viability in a subset of solid tumor cell lines and cell lines of hematological origin, including multiple myeloma, acute myeloid leukemia and non-Hodgkin lymphoma. Subsequent association analysis with molecular profiling endpoints identified an inverse correlation between BCLxL expression and sensitivity to Mcl-1 inhibition. Combination screens with Compound A revealed multiple highly synergistic combinations including compounds targeting the MAPK pathway, standard of care chemotherapeutics and agents targeting additional pro-survival members of the BCL-2 family. In conclusion, AMG 176 is a potent and selective Mcl-1 inhibitor, with significant in vitro and in vivo activity in Mcl-1 dependent cancer models.

#2028

PDE3A modulation for cancer therapy.

Xiaoyun Wu,1 Timothy Lewis,1 Luc de Waal,2 Galen Gao,1 Jian Zhang,1 Monica Schenone,1 Colin Garvie,1 Brett Diamond,3 Selena Lorrey,1 Andrew Cherniack,1 Steven Corsello,4 Alex Burgin,1 Todd Golub,1 Stuart Schreiber,1 Matthew Meyerson,4 Heidi Greulich1. 1 _Broad Institute, Cambridge, MA;_ 2 _C4 Therapeutics, Cambridge, MA;_ 3 _University of Connecticut, Cambridge, MA;_ 4 _Dana-Farber Cancer Institute, Boston, MA_.

In a differential cytotoxicity screen, we identified a novel small molecule modulator of phosphodiesterase 3A (PDE3A) that kills cancer cells expressing elevated levels of PDE3A and SLFN12 (de Waal, Nat Chem Biol, 2016). Treatment with this cell-selective cytotoxic small molecule, DNMDP, induces complex formation between PDE3A and SLFN12, resulting in apoptosis. Inhibition of PDE3A enzymatic activity is not sufficient for cancer cell killing, and expression of both PDE3A and SLFN12 are required. Although the mechanism of signaling to the apoptosis machinery remains unclear, we examined more closely the role of the PDE3A-SLFN12 complex in cancer cell killing mediated by DNMDP. We found that cancer cell lines made resistant to DNMDP by persistent exposure downregulated SLFN12 expression and that re-expression of SLFN12 was sufficient to restore sensitivity. Furthermore, ectopic expression of PDE3A and SLFN12 are sufficient to sensitize cancer cells to DNMDP. These data underscore the tight correlation of PDE3A-SLFN12 complex formation and cancer cell killing mediated by DNMDP.

#2028A

Glutamine deprivation-elicited sensitization of multiple myeloma to venetoclax is associated with electron transport chain inhibition.

Richa Bajpai, Changyong Wei, Mala Shanmugam. _Winship Cancer Institute, Emory University, Atlanta, GA_.

Cancer cells exhibit altered glucose and glutamine metabolism to sustain survival and proliferation. Altered nutrient utilization also contributes to the development of resistance to apoptosis via regulation of pro and anti-apoptotic BCL-2 family members. We previously reported that glutamine deprivation enhanced binding of BIM to BCL-2 thereby sensitizing myeloma cell lines and patient samples to Venetoclax (ABT-199). In this study we investigated the metabolites regulated by glutamine withdrawal that contribute to enhanced BIM-BCL-2 association and ABT-199 sensitivity. Metabolite profiling and isotope tracing flux analyses of glutamine deprived myeloma cell lines revealed specific reduction of TCA cycle metabolites including succinate, that were not maintained by glucose present in the medium. Cellular bioenergetics end points i.e. OCR and ATP were also suppressed upon glutamine withdrawal correlating with a suppression of the TCA cycle and oxidative phosphorylation. Supplementation of cell permeant dimethyl-α-ketoglutarate (DMK), a TCA cycle metabolite derived from glutaminolysis, to glutamine deprived cells reversed sensitivity to ABT-199. This ability of DMK to reverse ABT-199 sensitization in glutamine deprived cells was associated with a replenishment of TCA cycle intermediates without restoration of ATP levels. Therefore, we sought to further interrogate the TCA cycle and electron transport chain (ETC) in facilitating glutamine withdrawal-associated ABT-199 sensitization. TCA cycle metabolites are linked to mitochondrial respiration through ETC complexes. Inhibition of ETC complexes I, II, III or V in myeloma cell lines enforced similar effects as glutamine deprivation and sensitized cells to ABT-199. Among all five complexes, complex II is more explicitly linked with the TCA cycle through succinate dehydrogenase (SDH). SDH facilitates the oxidation of succinate to fumarate in the TCA cycle through its subunit SDHA, and transfers the released electrons to ubiquinone via its SDHB, C and D subunits supporting the ETC activity of complex II. Competitive inhibition of succinate dehydrogenase by 3-nitro propionic acid (3NPA) induced BIM expression and sensitized myeloma cells to ABT-199, similar to that detected upon glutamine deprivation. Our observations thus narrow down a role for ETC inhibition in facilitating ABT-199 sensitivity and underscore the importance of further interrogation of ways to metabolically perturb and sensitize cancer cells to the highly potent BH3 mimetic ABT-199.

### Combination Therapy 2

#2029

Precision therapeutic combinations are synergistic against triple negative breast cancer using compensatory pathways.

Jeffrey P. Solzak,1 Brad Hancock,1 Robin Paul,1 Patrick Kiel,2 Todd Skaar,1 Bryan Schneider,1 Milan Radovich1. 1 _Indiana Univ. School of Medicine, Indianapolis, IN;_ 2 _IU Health, Indianapolis, IN_.

Introduction: Triple negative breast cancer (TNBC) accounts for 15% of all breast cancer cases in the United States, and despite its lower incidence, contributes to a disproportionately higher rate of morbidity and mortality compared to other breast cancer subtypes. No FDA-approved targeted therapies are currently available. It has been hypothesized that drug combinations could potentially be chosen based on a tumor harboring two or more druggable genomic mutations. However, previously published data has noted that single agent treatment can change the genomic landscape and may be responsible for the lack of synergy of dual genomic drug targeting. Herein we show that targeting a compensatory pathway after treatment with a genomically-directed agent, results in synergistic combinations and can outperform choosing two drugs a priori based on genomic mutations.

Methods: Nine TNBC cell lines were chosen based on the presence of two or more clinically actionable genomics targets. Drug combinations based on targeting two genomic mutations were chosen using DNA-seq data from CCLE and a board consisting of oncologists and researchers. Compensatory therapies were found using RNA-seq data from untreated versus single-agent treated TNBC cell lines. The merged transcript RPKMs were transformed and analyzed for differential expression. Statistically significant genes were imported into Ingenuity Pathway Analysis (IPA) to identify therapeutics based on compensatory targets using the Causal Network Analysis and Upstream Regulator functions. Cell viability was assessed via Celltiter-Fluor. Synergy of the combinations was calculated using the Chou-Talalay method.

Results: Using two-drug combinations based on genomic mutations alone in all nine cell lines resulted only in additive or antagonistic responses. However, using RNA-seq data of each cell line treated with one genomically-directed agent, IPA analysis identified several compensatory targets that were upregulated or activated. Treating each cell line in combination with one genomically-directed agent plus one compensatory agent resulted in a striking increase in synergy. To understand why targeting compensatory pathways was superior to targeting two genomic mutations, we re-evaluated the RNA-seq data. This analysis revealed that treatment with the first genomically-directed agent, in many cases, resulted in down-regulation or inactivation of the second genomic-target. This may explain the lack of synergy when targeting cells with two drugs based only on genomic mutations.

Conclusion: Targeting TNBC cell lines based on drug combinations informed by compensatory pathways results in significant synergy and is superior to choosing drug combinations based on genomic alterations alone.

#2030

**Fisetin, a dietary flavonoid for the prevention and treatment of** PIK3CA **-mutant colorectal cancer.**

Naghma Khan, Farah Jajeh, Devon Miller, Rachel Van Doorn, Richard B. Halberg, Hasan Mukhtar. _Univ. of Wisconsin-Madison, Madison, WI_.

Colorectal cancer (CRC) is one of the most frequent and deadliest cancers worldwide with patients often diagnosed in advanced stages of the disease. Approximately 15-20% of advanced colorectal cancers harbor activating mutations in PIK3CA, which has been identified as an important oncogene in multiple cancers. Therefore, modeling the effects of this type of mutation in the mammalian colon is significant. Fisetin is a naturally occurring flavonoid in strawberry, apple, persimmon, grape, onion and cucumber. 5-Fluorouracil (5-FU) is the most used chemotherapeutic agent in CRC; however, it has serious side-effects. Therefore, augmentation of the 5-FU therapeutic effect could lead to lower effective doses and subsequently fewer side effects. We conducted in-vitro and in-vivo studies to determine the effect of fisetin, 5-FU and their combination on PI3K/AKT/mTOR signaling in PIK3CA-mutant colon cancer cells (HCT116 and HT-29), PIK3CA wild-type colon cancer cells (SW480), and newly developed mouse models. We found that there was more pronounced decrease in cell-viability and number of colonies in PIK3CA-mutant colon cancer cells than PIK3CA wild-type colon cancer cells. Apoptotic genes and proteins are promising targets for cancer treatment as they provide several theoretical basis to influence pathways causing greater tumor cell death. We observed an increase in the protein expression of Bax and decrease in Bcl2 on treatment with combination of fisetin and 5-FU than either agent alone. The full size PARP (116 KD) protein was also cleaved to yield an 85 KD fragment after treatment of cells with fisetin, 5-FU and their combination. Apoptotic effects of fisetin and 5-FU combination were also confirmed by flow cytometry in PIK3CA-mutant colon cancer cells. The PI3K/AKT pathway is frequently activated in CRC leading to tumorigenesis and the resistance to chemotherapy. Treatment of PIK3CA-mutant colon cancer cells with fisetin and 5-FU caused decrease in the expression of (i) PI3K (p85 and p110), (ii) phosphorylation of Akt (Ser473 and Thr308), (iii), phosphorylation of mTOR, its target proteins, and constituents of mTOR signaling complex. Treatment with fisetin and 5-FU also led to an increase in the phosphorylation of AMPKα. Next, we performed studies to investigate the effect of the treatment of combination of fisetin and 5-FU on colorectal tumorigenesis in FC13K1ApcMin/+ mice. These animals form tumors in the distal small intestine and colon that have lost APC activity and express constitutively active PI3K as often occurs in humans. Interestingly, the effect of fisetin was much stronger than that of 5-FU and comparable to the fisetin and 5-FU combination. Both fisetin only and combination treatment groups had significantly lower incidence relative to the control group. We suggest that fisetin could be used as a preventive agent as well as an adjuvant with 5-FU for the treatment of PIK3CA-mutant CRC.

#2031

Antitumoral synergy of iron chelators and chemotherapies in triple-negative breast cancer cell lines and patient-derived xenograft.

Sandrine Tury,1 Sophie Vacher,1 Véronique Becette,2 Franck Assayag,1 Sophie Chateau-Joubert,3 Jean-Luc Servely,3 Elisabetta Marangoni,1 Ivan Bièche,1 Céline Callens1. 1 _Institut Curie, Paris, France;_ 2 _Institut Curie, Hôpital René Huguenin, Saint Cloud, France;_ 3 _National Veterinary School of Alfort, Maison-Alfort, France_.

Introduction: Tumor cells present an iron metabolic disorder with high proliferation rate, increased iron storage (ferritin and Labile Iron Pool - LIP) and high sensibility to iron deprivation, which could be a therapeutic target. Anticancer effect of iron chelators deferoxamine (DFO) and deferasirox (DFX) has been revealed in several types of cancers. In breast cancer (BC), the development of new therapeutic approaches is urgently needed for triple negative (TN) subtype which presents poor prognosis and lacks targeting therapy. We investigated the therapeutic potential of iron chelators combined with chemotherapeutic agents in TNBC cell lines and patient-derived xenografts (PDX).

Methods: Anticancer effects of iron chelators combined with chemotherapeutic agents (doxorubicin, cisplatin or carboplatin) were evaluated in vitro in 4 TNBC cell lines by MTT assay, annexin V/PI staining and assessment of caspase 3/7 activity. Assessment of LIP, transferrin receptor 1 (TfR1) expression level, Reactive Oxygen Species (ROS) production and mitochondrial membrane potential variations were performed by flow cytometry, and ELISA assay for ferritin level. The activity of DFX alone or combined with doxorubicin/cyclophosphamide (AC) was tested in the HBCx-10 TNBC PDX selected because of its relapse to AC. Iron homeostasis, hypoxia and PI3K pathway were analyzed by immunohistochemistry (IHC) and Western-blot in both cell lines and PDX tumors. In DFX+/-AC treated and untreated tumors, induction of apoptosis was performed by TUNEL assay and a transcriptome analysis is ongoing.

Results: Iron chelators acted in synergy effect with three chemotherapies in all cell lines which were tested to inhibit cell proliferation and induce apoptosis. Chelators increased cytotoxicity until 60% compared to chemotherapies alone. In all cell lines, chelators treatment increased TfR1 expression level and decreased LIP and ferritin. Furthermore, down-regulation of PI3K pathway, hypoxia, mitochondrial membrane potential, and the production increasing of ROS were observed. In HBCx-10 PDX, a trend for antitumoral activity of DFX alone was observed (p=0.09) at the end of the experiment (day 81). A significant difference of Relative Tumor Volume (RTV) was observed since day 18 between the AC group and the DFX+AC group (tumor growth inhibition: 37 to 61%, tumor growth delay: 10 to 14 days, 0.005<p<0.04). Same as the observing in vitro, modulations of PI3K pathway and hypoxia are involved in this antitumoral synergy. Except neutropenia (due to chemotherapy), no other hematologic toxicity was observed in both AC and DFX+AC groups.

Conclusions: Iron chelators may increase the effectiveness of conventional chemotherapies for TNBC treatments. This antitumoral synergy involves PI3K pathway downregulation, ROS production and decrease mitochondrial membrane potential.

#2032

Combination of epigenetic regulation via LSD1 inhibition with signal transduction inhibitors significantly enhances anti-tumor activity in models of hematologic malignancies.

Sang Hyun Lee, Matthew Stubbs, Ashish Juvekar, Melody Diamond, Antony Chadderton, Robert Collins, Xiaoming Wen, Holly Koblish, Chunhong He, Liangxing Wu, Richard Wynn, Andrew Combs, Chu-Biao Xue, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Bruce Ruggeri. _Incyte Corporation, Wilmington, DE_.

Combinatorial therapeutic strategies have achieved improved response rates and durability of responses in several malignancies either by selectively targeting distinct and non-overlapping oncogenic signaling pathways (e.g. PARP and phosphoinositide 3-kinase (PI3K) inhibition in subsets of breast and ovarian cancers), or alternatively, inhibiting distinct nodal points of regulation in common oncogenic signaling pathways (e.g BRaf and MEK inhibition in subsets of melanoma). Recent data suggest that deregulated epigenetic modifications may be just as significant as genetic mutations in driving cancer development and growth by inhibition of tumor suppressor activity and activation of oncogenic pathways. We therefore hypothesized that an epigenetic regulator could potentiate the efficacy of a protein kinase inhibitor to result in robust tumor growth inhibition. We previously reported that the potent and selective LSD1 inhibitor INCB059872 potently inhibited tumor growth in multiple tumor xenograft models of AML and SCLC as a single agent and in a combination with standard of care of agents. In this study, we explored the anti-tumor effect of combining INCB059872 and various signal transduction pathway inhibitors, including the PIM kinase inhibitor INCB053914, the JAK1/2 inhibitor ruxolitinib, or the PI3K delta-selective inhibitor INCB050465 in models of human hematologic malignancies. Each of these therapeutic combinations significantly inhibited tumor growth in the Molm-16 human AML xenograft model. Mechanistic studies suggested that MYC expression levels were downregulated by these combinations both in vitro and in vivo. Treatment with INCB059872 alone or in combination with signal transduction kinase inhibitors significantly downregulated cytokines levels, particularly IL-10, sCD40L, and MCP-1 in Molm-16 tumors. These data suggest that the combination of an LSD1 inhibitor and signal transduction inhibitor can co-regulate key tumor intrinsic and extrinsic pathways involved in paracrine or autocrine signaling in AML. In addition to the improved efficacy observed in AML models, the combination of INCB059872 with the PI3Kdelta inhibitor INCB050465 enhanced tumor growth inhibition in the Will-2 xenograft model (GCB subtype, double hit lymphoma), whereas the activity of these single agents were modest in this particular subtype of lymphoma. Additional mechanistic studies are ongoing to further understand the molecular bases of these observations. Taken together, these data suggest that targeting distinct epigenetic and oncogenic signaling pathways may potentiate anti-tumor efficacy and overcome intrinsic resistance mechanisms in specific hematologic malignancies.

#2033

Testing the combined effects of low dose olaparib plus ibrutinib on MCL cytotoxicity.

Adam D. Curtis, Todd A. Hoffert, Sheila S. Rajan, Jens Rueter, Lindsay S. Shopland. _Eastern Maine Medical Center, Brewer, ME_.

Mantle cell lymphoma (MCL) presents a therapeutic challenge, with frequent relapses after therapy due to high levels of DNA damage and disease evolution. One currently approved MCL therapeutic, ibrutinib, is a tissue-specific agent with relatively few adverse effects. However, ibrutinib is administered at high dose for MCL and provides only a short progression-free survival time. To potentially improve ibrutinib therapy for MCL, we tested the effects of combining ibrutinib with the DNA damage response inhibitor, olaparib, on MCL cells in vitro. We confirmed that ibrutinib monotherapy is cytotoxic to MCL cell lines only at high doses (> 25 μM). In contrast, single-agent olaparib treatment is cytotoxic at lower concentrations (10 μM). Importantly, the addition of low dose olaparib to ibrutinib has an additive cytotoxic effect. Notably, the effects of ibrutinib alone and in combination are dependent on cell medium composition. Our findings support clinical testing of ibrutinib plus olaparib therapy to reduce MCL cell burden initially and to potentially slow MCL cell evolution in the longer term. Our data also suggest the possibility for more effective ibrutinib-based therapy via manipulation of the MCL cell niche.

#2034

LSD1 inhibition alone and in combination with chemotherapy in Ewing sarcoma cell lines.

Darcy Welch, Elliot Kahen, Christopher L. Cubitt, Damon R. Reed. _Moffitt Cancer Center, Tampa, FL_.

Background: Ewing Sarcoma (ES) is the second most common primary bone cancer affecting children and young adults. Despite advances in treatment that have led to survival rates of approximately 73% for localized disease, outcomes for patients with metastatic or recurrent ES remain poor. A distinguishing feature of ES is the presence of the EWS/FLI1 fusion in 85% of cases. The fusion has been shown to alter expression of a number of oncogenic genes. Mechanistic studies have demonstrated that the NuRD co-repressor complex interacts with EWS/FLI1. The associated protein LSD-1 contributes to the repressive function by histone modifications. While reversible LSD1 inhibitors demonstrate single agent activity, in preclinical models, a system to evaluate combinations may be needed for optimizing effect in clinical trials.

Methods: Here, we seek to confirm promising single drug activity and evaluate combination therapies using active chemotherapies currently utilized in ES care (4-HC, etoposide, SN-38, vincristine and doxorubicin) along with the LSD1 Inhibitors SP2509 and SP2577 and romidepsin, an HDAC inhibitor. We evaluated these combinations in high-throughput screening platforms and well-established cell line models for ES (A-673, TC-32, RD-ES, TC-71). Taking into consideration past lessons learned from in vitro experiments, we designed stringent screening conditions that assess the candidate compounds and combinations at clinically-relevant concentrations and exposure times that mimic the in vivo pharmacokinetics in an effort to maximize the translational potential of these results to the clinical setting. All combinations of agents were studied in two-drug combinations to evaluate for synergy in addition to efficacy.

Results: IC50 for SP2509 was found to be in the submicromolar range across cell lines with SP2577 being more potent. A-673 and TC-71 were 5-10 fold less sensitive than RD-ES and TC-32. Agents currently utilized in clinic were universally active at clinically achievable concentrations and exposure times. Combinations showed additivity frequently and demonstrated promising activity that can be used to inform further decision making once LSD1 inhibition toxicities are better known. These findings suggest potentially promising opportunities for developing combination clinical trials to maximize development of LSD1 inhibitors.

#2035

MGMT inhibition leads to CDK4/6 inhibition and enhances palbociclib and abemaciclib activity in breast cancer.

George C. Bobustuc, Amin B. Kassam, Richard A. Rovin, Deborah L. Donohoe, Maxwell Albiero, Tarun Jella, Olivia Fukui, Cameron Piron, Santhi D. Konduri. _Aurora Health Care, Milwaukee, WI_.

Background: MGMT (O6 methylguanine DNA methyltransferase), a DNA repair protein leading to chemotherapy resistance, increasingly studied for its cell cycle regulatory functions, also known to control ER expression and function, is overexpressed in a majority of cancers, including breast cancer. MGMT inhibition has been reported to restore ER function and sensitivity to hormonal therapy in tamoxifen resistant breast cancer. CDK4/6 is a cell cycle regulator targeted by a new class of drugs in the treatment of breast cancer in patients who had progressed during prior endocrine therapy. We investigated a potential correlative role between MGMT and CDK4/6 expression/activity. In this therapeutic context MGMT inhibition would have the dual role of increasing/restoring effect of endocrine therapy and facilitate activity of CDK4/6 inhibitors (Palbociclib and Abemaciclib).

Methods: We have tested the effect of Antabuse (disulfiram, DSF), as an MGMT inhibitor, at nontoxic doses, on the expression of CDK4/6, or in combination with Palbociclib (PB) or Abemaciclib (LY2835219 - LY) on ER+ breast cancer cells.

Results: DSF at very low doses (achievable in human serum with standard DSF clinical dosing) decreases ER+ breast cancer cell growth (MCF7, T47D and ZR75) in a dose-dependent manner. DSF further sensitizes breast cancer cells to PB or/and LY and significantly inhibits breast cancer growth without causing unwanted side effects on the normal breast epithelial cells. Dose effect and isobologram studies confirm synergistic activity of DSF + LY and moderate synergism for DSF + PB. DSF, alone or in combination with PB (DSF ± PB) and/or LY (DSF ± LY), significantly inhibits expression of MGMT, CDK4/6, ERα and aldehyde dehydrogenase activity - all involved in breast cancer cell cycle proliferation and tumorigenesis. Furthermore, PB and LY dose dependently decreased MGMT and CDK4 expression in breast cancer cells and significantly accumulated breast cancer cells in G1 phase of the cell cycle. DSF, alone or in combination with PB (DSF ± PB) and/or LY (DSF ± LY) caused significant apoptosis in breast cancer cells. DSF inhibited colony formation which was further enhanced by addition of PB/LY (DSF ± PB/LY). Similarly, DSF alone or in combination with PB (DSF ± PB) and/or LY (DSF ± LY) decreased the metastatic potential of breast cancer cells.

Conclusions: Our findings suggest that DSF as an MGMT inhibitor significantly enhances the antitumor effect of CDK4/6 inhibitors (PB or LY) in ER+ breast cancer.

#2036

In vitro **and** in vivo **effect of carmustine and selenite combination on EGFR signaling in androgen-independent prostate cancer.**

Vijayalakshmi Thamilselvan, Mani Menon, Sivagnanam Thamilselvan. _Vattikuti Urology Institute, Henry Ford Health System, Detroit, MI_.

Introduction: Despite the use of androgen deprivation therapy, the majority of prostate cancer patients will progress to castration resistant disease within 2-3 years, driven by aberrant androgen receptor activation. We have previously shown that combination of carmustine and selenite effectively induces apoptosis by completely reducing AR and AR-variants in AR-dependent castration resistant prostate cancer cells both in vitro and in vivo. Although studies have demonstrated that AR signaling is a central mechanism of castration resistant prostate cancer progression, the cell growth mediated by AR-independent signaling is also operative. EGFR signaling has been implicated in the survival, invasion, and metastasis of prostate cancer cells in an AR-independent manner. Therefore, in this study, we tested whether the combination of carmustine and selenite could inhibit EGFR signaling, induce apoptosis, and inhibit growth of AR-independent prostate cancer cells in an in vitro cell culture and in vivo xenograft models.

Methods: AR negative PC-3 prostate cancer cell line was used to study the effect of carmustine and selenite alone or in combination on EGFR signaling. Cell viability, proliferation, and apoptosis in the presence or absence of EGF (50ng/ml) were determined. Dose and time dependent effect of EGF on the activation of EGFR was studied. The carmustine and/or selenite effect on EGF stimulated phospho-EGFR, and its downstream signaling pathways, such as AKT and MAPK, were studied in comparison with inhibitors of EGFR (AG1478) or PI3 kinase (LY294002) or MAPK (PD98059). The in vivo effect of carmustine and selenite was examined on PC-3 tumor growth in athymic nude mice.

Results: Combination of carmustine and selenite treatment in the presence of EGF markedly suppressed cell viability and proliferation of PC-3 cells. EGF exposure increased phospho-EGFR (Tyr845, Tyr992, Tyr1068, and Tyr1045), pAkt (Ser473), and pERK1/2 (Thr204/Tyr202) protein expression levels in PC-3 cells in a dose and time dependent manner. The combination treatment exhibited a strong inhibitory effect on EGF stimulated EGFR, Akt, and ERK1/2 phosphorylation. Combination treatment was able to induce apoptosis even in the presence of EGF. Consistent with in vitro results, combination treatment for 3 weeks reduced PC-3 xenograft tumors in nude mice by 88% without any toxicity. Whereas individual agent treatment showed only partial effect both in vitro and in vivo.

Conclusions: Our pre-clinical data demonstrate that the combination treatment of carmustine and selenite substantially inhibits EGFR signaling, induces apoptosis, and reduces tumor growth of AR-independent prostate cancer cells in vitro and in vivo. Our novel findings suggest that the combination of carmustine and selenite is an effective therapeutic agent for successful treatment, survival, and improved quality of life for patients with prostate cancer.

#2037

Oral nitroxoline in combination with intravesical bacille calmette-guerin (BCG) shows synergistic antitumor efficacy in mouse bladder cancer orthotopic xenograft.

Qiang Li,1 Kevin Pan,1 Xiezhao Li,2 Naijin Xu,3 Peng Huang3. 1 _Asieris Pharmaceuticals Co., Ltd, Shanghai, China;_ 2 _Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong Province, China;_ 3 _Okayama University, Okayama, Japan_.

Nitroxoline, an antibiotic used to treat urinary tract infection, had previously been identified as a MetAP2 inhibitor and found to show antitumor efficacy in mouse models of human breast cancer xenograft and bladder cancer orthotopic xenograft [1]. Our purpose in this study was to evaluate the feasibility of combining oral nitroxoline with intravesical BCG, the first-line therapy for intermediate- and high-risk non-muscle invasive bladder cancer (NMIBC), in mouse bladder cancer orthotopic xenograft. The luciferase-expressing murine bladder cancer cell line MBT-2-Luc cells was transurethrally implanted in the bladder of syngeneic female C3H/He mice. The animals were randomly divided into groups and treated with phosphate-buffered saline (PBS), oral nitroxoline, intravesical BCG alone, and a combination as described in methods. The IVIS spectrum images analyses of luciferin-stained tumor at 21 days after inoculation revealed that combining oral nitroxoline with intravesical BCG showed synergistically enhanced antitumor efficacy and prolonged long-term survival, implicating the potential of combining bladder cancer treatment using oral nitroxoline with intravesical BCG in the clinical setting. The mechanism behind this observation is currently under investigated.

#2038

Reactive oxygen species-mediated synergism of fenretinide and romidepsin in preclinical models of T-cell lymphoid malignancies.

Monish Ram Makena, Balakrishna Koneru, Min H. Kang, C. Patrick Reynolds. _Texas Tech Univ. Health Science Ctr., Lubbock, TX_.

Introduction: T-cell lymphoid malignancies (TCLMs) are in need of novel and more effective therapies. Romidepsin is a histone deacetylase (HDAC) inhibitor that achieved FDA registration as 2nd line therapy for peripheral and cutaneous T-cell lymphomas. The cytotoxic retinoid fenretinide achieved durable complete responses against T-cell lymphomas in early-phase clinical trials and T-cell lymphoma patients who failed prior romidepsin treatment responded to fenretinide. Fenretinide is currently being evaluated in a Phase IIa clinical trial for relapsed/refractory PTCL patients (NCT02495415). We investigated the potential for using these two agents in combination in TCLMs. Methods and Results: Using the DIMSCAN assay, we demonstrated cytotoxic synergy between romidepsin and fenretinide in fifteen TCLM cell lines at clinically-achievable concentrations that lacked cytotoxicity for non-malignant cells (fibroblasts and blood mononuclear cells). In vivo, romidepsin + fenretinide + ketoconazole (enhances fenretinide exposures by inhibiting fenretinide metabolism) showed greater activity in subcutaneous (COG-LL-317m and TX-LY-183x PDX) and disseminated (COG-LL-317m Luc expressing the luciferase gene) TCLM xenograft models than single agent romidepsin or fenretinide + ketoconazole. Fenretinide + romidepsin caused a reactive oxygen species (ROS, measured by DCFDA dye)-dependent increase in pro-apoptotic proteins (Bim, tBid, Bax and Bad), apoptosis (via TUNEL assay), and inhibition of HDAC enzymatic activity, which achieved a synergistic increase in histone acetylation. The synergistic cytotoxicity, apoptosis, and histone acetylation of fenretinide + romidepsin was abrogated by antioxidants (vitamins C or E). Romidepsin + fenretinide activated p38 and JNK via ROS, and shRNA knockdown of p38 and JNK1 significantly decreased the synergistic cytotoxicity and apoptosis. Romidepsin + fenretinide also showed synergistic cytotoxicity for B-lymphoid malignancy cell lines, but did not increase ROS, acetylation of histones, activation of p38 + JNK, or cytotoxicity in fibroblasts and blood mononuclear cells. Conclusion: Romidepsin + fenretinide achieved synergistic activity in preclinical models of TCLMs, but not in non-malignant cells, via a novel molecular mechanism. These data support conducting clinical trials of romidepsin + fenretinide in relapsed and refractory TCLMs.

#2039

The use of self nano-emulsifying drug delivery systems to improve bioavailability of combination therapy and overcome erlotinib resistance in non-small cell lung cancer.

Ebony Nottingham, Vasanth Sekar, Arindam Mondal, Mandip Sachdeva. _Florida Agricultural and Mechanical University, Tallahassee, FL_.

Background:Erlotinib, an EGFR selective reversible inhibitor, improved response of patients diagnosed with non-small cell lung cancer (NSCLC) with 70% of patients showing significant tumor regression upon treatment. Unfortunately,most patients eventually relapse due to development of resistance. Our research goal is to quantify the effects of combination treatment of erlotinib with a glycyrrhetinic acid analogs and to improve the oral bioavailability of this treatment using self nano-emulsifying drug delivery systems.

Methods: NSCLC cell lines HCC827, HCC827 (4µM erlotinib resistant), HCC827 Cl4 (second site EGFR mutated), HCC827BEAS2B, and H1975 (4µMerlotinib resistant) were treated in combination with CDODA-Me, CF3CDODA-Me and Erlotinib. The cell viability assays were performed and combination index (CI) values were calculated by isobolographic analysis. Self nano-emulsifying drug delivery systems (SNEDDs) were formulated and characterized through in vitro and in vivo studies.

Results: CF3CDODA and improved efficacy for erlotinib in all cell lines with IC50 values of 6.0µM, 7.8µM, 4.6µM and 4.2±1.56µM for HCC827(RESISTANT), H1975(RESISTANT), HCC827CL4 and HCC827 respectively compared to IC50values of erlotinib alone (25µM, 21µM, 23µM, and 8µM). This was superior when compared to IC50 values of CDODA-Me combination treatment (6.66µM, 7.32µM, 12.06µM, and 5.45µM respectively). Combination treatment showed strong synergism with a constant concentration of 0.5µM CF3CDODA and CI values of 0.461, 0.548, 0.389, 0.550 for HCC827 4µM, H1975µM, HCC827CL4, and HCC827 respectively and antagonism in HCC827BEAS2B (1.508) cells. This is comparable to the synergism observed at a minimum concentration of 2µM CDODA-Me with CI values of 0.337, 0.67, 0.64, and 0.657 respectively and antagonism CI of 1.175 for HCC827BEAS2B. CF3CDODA Combination therapy showed a 2.5-fold decrease in colony formation in resistant cells making them comparable to wild type. This was comparable to CDODA-Me combination treatment with a 1.46-fold decrease in colony formation. Both combination treatments showed little effect on HCC827BEAS2B. Western Blot analysis showed decrease MET expression and decreased apoptosis inhibiting markers BCL2 and Survivin. SNEDD formulations increased maximum drug plasma concentrations for Erlotinib (281ng/ml to 442ng/ml) and circulating time as well (12hrs - 21hrs).

Conclusions: Based on the results of these studies, CDODA-Me and CF3CDODA have shown to overcome erlotinib resistance in non-small cell lung cancer and when combining these treatments with the use of SNEDDS, they have shown to be a promising treatment regimen.

#2040

Superior therapeutic efficacy of nanoparticle albumin-bound paclitaxel over cremophor-bound paclitaxel in experimental esophageal adenocarcinoma.

Md Sazzad Hassan, Niranjan Awasthi, Roderich E. Schwarz, Margaret A. Schwarz, Urs von Holzen. _Indiana University School of Medicine-South Bend, South Bend, IN_.

Introduction: Esophageal adenocarcinoma (EAC) has become the dominant type of esophageal cancer in United States. EAC is the fastest growing cancer in the western world and the overall 5 year survival rate of EAC is below 20 percent. Most patients with EAC present with locally advanced or widespread metastatic disease, where current treatment is largely ineffective. Prognosis for EAC patients remains poor even with combination therapies due to high resistance to chemotherapy. Therefore, new therapeutic approaches are urgently needed. Paclitaxel (PTX) has been used in combination with carboplatin (CP) as a standard combination therapy for advanced EAC. PTX required emulsification with solvents to allow intravenous administration which has resulted in hypersensitivity reactions and potentially dramatic side effects in patients. Nanoparticle albumin-bound (nab) PTX is an albumin-stabilized, cremophor-free and water soluble nanoparticle formulation of PTX. Nab-PTX is a novel microtubule-inhibitory cytotoxic agent and the potential role of nab-PTX has not been tested yet in experimental EAC.

Methods: We explored the antiproliferative and antitumor efficacy with survival advantage following CP, PTX and nab-PTX as monotherapy and in combinations in in-vitro, and in murine subcutaneous xenograft and peritoneal metastatic survival models of human EAC.

Results: Nab-PTX inhibited in-vitro cell proliferation with significantly lower IC50 (0.25 µM in OE19 and 49 nM in OE33) than that of PTX (0.74 µM in OE19 and 98 nM in OE33) and CP (5.21 µM in OE19 and 1.05 µM in OE33) in OE19 and OE33 EAC cell lines. Nab-PTX treatment resulted in significantly higher antitumour efficacy and survival benefit compared with PTX or CP treatment. After two-week nab-PTX, PTX, CP, nab-PTX+CP or PTX+CP treatments, the average in-vivo local tumor growth inhibition rate was 73, 60, 35, 81 and 68 percent respectively (p=0.025). Nab-PTX treatment increased expression of the mitotic-spindle associated phospho-stathmin, decreased expression of proliferative marker Ki-67 and enhanced apoptosis as confirmed by increased expression of cleaved-PARP and cleaved caspase-3. There was an increase in median animal survival after nab-PTX treatment (65 days) compared to controls (46 days, p=0.0023), PTX (57 days, p=0.0034) or to CP therapy (53 days, p=0.0034).

Conclusion: In conclusion, the present study demonstrates that nab-PTX had stronger antiproliferative and antitumor activity in experimental EAC than the current standard chemotherapeutic agents. This strong antitumor activity supports the rationale for clinical evaluation of nab-PTX as promising microtubule-inhibitory agent in EAC.

#2041

High-throughput chemical screening for sensitization of bladder cancer to gemcitabine and cisplatin chemotherapy.

Yuki Kita, Takashi Kobayashi, Ryouichi Saito, Toshinari Yamasaki, Takahiro Inoue, Osamu Ogawa. _Kyoto University, Kyoto, Japan_.

Introduction and Objectives Gemcitabine and cisplatin chemotherapy (GC) is the current standard regimen for locally advanced and metastatic bladder cancer (BC). Despite a relatively high initial response rate, some cases do not regress (intrinsic resistance) and the remaining cases often show regrowth after initial shrinkage (acquired resistance). To identify novel therapeutic agents for overcoming these resistances, we applied a high-throughput screening of chemicals administered in combination with GC.

Methods As a high-throughput screening, 2100 compounds were administered alone or in combination with GC to human BC cell lines (J82, UMUC-3). Cell viability was determined after 3-day incubation and chemicals that enhanced inhibitory effect of GC were screened. The in vivo effect of disulfiram (DSF) was studied in UMUC-3 cell xenografts, and western blot, immunofluorescence, induced coupled plasma spectrometry and measurement of reactive oxygen species (ROS) were done in vitro for mechanistic exploration.

Results The initial screening identified 26 compounds and further validation narrowed them into the most synergistic agent DSF, an FDA-approved drug for alcoholism. Combination index assay showed synergistic effects of DSF with cisplatin but not with gemcitabine in J82, UMUC-3, T24, HT1197 and HT1376 cells. Co-administration of DSF significantly increased DNA-platinum adducts by regulating cisplatin efflux transporter ATP7A and enhanced apoptosis by GC treatment in UMUC-3 cells, with significant increase of ROS production. Use of DSF in combination with GC (GCD) significantly inhibited tumor growth of UMUC-3 subcutaneous xenograft on athymic mice (by 39% compared with GC alone, p = 0.02). GCD regimen was as tolerable as GC and no significant differences were observed in body weight of treated mice between the two regimens.

Conclusions Repositioning of DSF to a chemotherapy sensitizer is a promising treatment strategy, which can be translated rapidly in the future.

#2042

Nano-tetraiodothyroacetic acid (NDAT) potentiates gefitinib-induced antiproliferation in colorectal cancer cells by inhibiting EGFR sialylation and PI3K activation.

Yu-Tang Chin,1 Chi-Yu Lin,1 Chun A. Changou,1 Jacqueline Whang-Peng,1 Paul J. Davis,2 Hung-Yun Lin1. 1 _Taipei Medical Univ., Taipei, Taiwan;_ 2 _Albany Medical College, Albany, NY_.

The resistance of gefitinib has been revealed to complicate cancer therapy. Tetraiodothyroacetic acid (tetrac) and its nanoparticulate derivative (NDAT, nano-tetrac) have been proved in vitro and in vivo xenograft demonstrating anti-proliferative and anti-angiogenetic activities. It also indicates that they potentiate anti-cancer agent-induced anti-proliferation in cancer cells. In this study, we investigated the effects of NDAT on gefitinib-induced anti-cancer activities in human colorectal cancer cells. Gefitinib inhibited cell proliferation at concentration 1 μM in K-ras wild type HT29 cell and NDAT enhanced the anti-proliferation-induced gefitinib significantly. Meanwhile, both inhibited proliferative and metastatic genes expression in HT29 cells. On the other hand, 10 μM gefitinib inhibited cell proliferation in K-ras mutant HCT116 cell which was further enhanced by NDAT. Different from results in HT-29 cells, only NDAT inhibited proliferative and metastatic genes expression significantly and enhanced the effect of gefitinib in HCT116 cells. ST6Gal-1 catalyzes sialylation of EGFR and induces gefitinib-resistant in colorectal cancers. In addition, NDAT did reduced not only ST6Gal-1 gene expression, but also its protein production. However, the inhibition of ST6Gal-1 expression may not be sufficient to induced anti-proliferation in colorectal cancer cells. PI3K inhibitor, LY294002, was able to potentiate the gefitinib-induced anti-proliferation in

HCT116 cells suggesting that constitutive activation of PI3K may play a key role on gefitinib-resistance in HCT116 cells. In summary, NDAT potentiated gefitinib-induced anti-proliferation via inhibiting the activity of ST6Gal-1 and PI3K activation in gefitinib-resistant colorectal cancer cells.

#2043

Entinostat in combination with Cladribine synergistically induces apoptosis in multiple myeloma cells.

Bolun Wang, Hui Lyu, Bolin Liu. _University of Colorado Anschutz Medical Campus, Aurora, CO_.

Cladribine (2-CDA) is a well-known purine nucleoside analog against lymphoproliferative disorders. Entinostat, a selective class I histone deacetylase (HDAC) inhibitor, exerts anti-tumor activity in various cancers, including multiple myeloma (MM). We sought to determine whether cladribine and entinostat may exhibit synergistic anti-proliferative/anti-survival effects on MM cells. Here we showed that, in cell growth MTS assays, either cladribine or entinostat alone inhibited cell proliferation in a dose-dependent manner. The combinations of cladribine and entinostat significantly induced growth inhibition in MM cells tested. The combination index (CI) curves showed a synergistic effect between cladribine and entinostat. An apoptotic-ELISA and western blot analyses of caspase-3 and PARP revealed that entinostat in combination with cladribine exhibited a much more potent activity than either agent alone to promote the MM cells undergoing apoptosis in a dose and time-dependent manner. Collectively, our data suggest that combinations of entinostat and cladribine possess synergistic anti-proliferative/anti-survival activity in MM cells. Regimens consisting of entinostat and cladribine may represent novel therapeutic strategies against MM. 

### Drug Resistance: Other Topics

#2044

Development of stable and brain-penetrating disulfiram nanoparticles, characterization and efficacy in glioma cell culture and xenograft models.

Hanumantha Rao Madala,1 Surendra R. Punganuru,1 Francis Ali-Osman,2 Kalkunte S. Srivenugopal1. 1 _Texas Tech Univ. Health Sciences Ctr., Amarillo, TX;_ 2 _Duke University, Durham, NC_.

Blood brain-barrier entry, tumor heterogeneity, need to eliminate the tumorigenic stem cells and inhibit the DNA repair protein MGMT are all essential considerations in glioma treatment. Previously, we showed that alcohol aversion drug disulfiram (DSF) shares all these properties [Carcinogenesis 35, 692, 2014; Oncotarget 4,502, 2013]. DSF was shown to inhibit the MGMT activity in the same way as ALDH by conjugating with the active-site cysteine 145 and sensitize brain tumors to alkylating agents. Although DSF exerts significant anticancer effects in vitro, the drug undergoes rapid decomposition and metabolism. To overcome this problem, we engineered DSF nanoparticles by encapsulating the drug in PEG: PLGA polymer by solvent evaporation method. The formulation was optimized for various process formulation variables. The optimized nano-formulation (NP) ranged from 70-80 nm in particle size as confirmed using dynamic light scattering and transition electron microscopy. Drug loading, encapsulation efficiency, in vitro release were also optimal and the particles showed excellent BBB permeability in vitro. The time course of internalization of particles using a cyanine dye (HITC-1) showed a lysosomal accumulation followed by mitochondria. Live animal imaging after IV injections of HITC-labeled DSF-NPs revealed a selective accumulation in the brain and subcutaneous tumors. Further, the cytotoxicity elicited by DSFNPs against SF188, DAOY, UW18, UW28 and T98G brain tumor cells was extended and greater than obtained with the free drug. When combined with DSF-NPs, temozolomide showed an 8-10-fold increased cytotoxicity against glioma cell lines. The DSFNPs also induced significant degradation of MGMT and other redox-sensitive proteins such as the NF-κB, MDR1 and GSTP1 in a concentration-dependent manner in glioma cells. ROS induction by the encapsulated drug was confirmed through DCF-DA and DHE staining of live cells; co-staining these cells with the mitochondrial membrane potential markers DiOC6 and DHE confirmed the altered integrity. GSTP1 inhibition and ROS induction by DSFNPs led to an activation of JNK/MAPK pathway, culminating in autophagy and apoptosis as verified by western blotting, FACS and immunofluorescence of appropriate markers. Finally, the antitumor efficacy of DSFNPs was tested in intracranial glioblastoma developed in nude mice by injecting luciferase-expressing SF-188 GBM cells. A superior tumor regression was evident in DSFNP-administered animals compared with the free drug after IVIS bioluminescence and histochemical staining of brain tissue. Collectively, our efforts provide strong evidence that DSF nanoparticles can cross the BBB and offer a sustained drug supply to eliminate CNS cancers. Further refinement of DSFNPs by incorporating brain targeting strategies is underway (supported by CPRIT grants RP130266 & RP170207 to KSS).

#2045

Exploring the potential of cellular thermal shift assay (CETSA) to study drug resistance during cancer therapy.

Lekshmy Kunjamma Usha Sreekumar, Yan Ting Lim, Saranya Veerappan, Par Nordlund. _Nanyang Technological University, Singapore, Singapore_.

The aim of this project is to understand the various mechanisms contributing to drug resistance development in cancer therapy.

The efficacy of therapeutics is dependent on a drug binding to its target. We have developed a method that allows for the first time to directly evaluate drug binding to target proteins in cells and tissue samples the cellular thermal shift assay (CETSA) (Martinez Molina et al. Science, 341:84). CETSA is based on the biophysical principle of ligand-induced thermal stabilization of target proteins. By monitoring the drug occupancy in the target protein, CETSA can be used to study processes of drug transport and metabolism in cancer cells. We have used CETSA to study the acquired drug resistance of, antifolate and fluropyrimidine drugs in pairs of parental and resistant cell lines. CETSA shifts and isothermal dose response fingerprint (ITDRF) were used to study the relative drug target engagement in these cells. Quantitative mass spectrometry was used to monitor differences in protein expression levels across the cell lines.

Based on the CETSA measurements, resistant cells clearly showed a higher drug dose threshold as compared to the parent cell lines, typically requiring 8-50 times higher dose to establish similar target engagement. Several potential mechanism for drug resistant emerged - we, for example, observed up-regulation of thymidylate synthase and down regulation of reduced folate carrier (RFC) protein associated with antifolate transport, in some resistant cell lines.

The data supports that CETSA is a potential valuable tool to dissect various mechanisms those contribute to resistant development in cancer cells.

#2046

Investigation of MDR1 in ovarian cancer using biodynamic imaging.

Gayatri Narayanan,1 David D. Nolte,1 Daniela Matei,2 Ran An,3 John J. Turek1. 1 _Purdue University, West Lafayette, IN;_ 2 _Northwestern University, Chicago, IL;_ 3 _Animated Dynamics, Inc, Indianapolis, IN_.

Multidrug resistance is one of the main causes of ineffective treatment of ovarian cancer. Almost 80% of advanced cases have recurring malignancies after treatment, even after initial responsiveness to the usual platinum-based chemotherapeutics. One of the many mechanisms for resistance is the transmembrane drug efflux pump p-glycoprotein, encoded by the gene MDR1. It has been shown that overexpression of this protein in ovarian cancer is associated with poor chemotherapeutic response and multidrug resistance due to low intracellular accumulation of the drug. Its wide substrate specificity allows it to pump out several common drugs such as vincristine, paclitaxel, doxorubicin, and etoposide. Biodynamic imaging (BDI) is a low-coherence form of digital holography that provides deep 3D functional information on intratumor subcellular motility and provides spectral signatures related to drug mode of action and for mitosis, apoptosis, and necrosis. BDI is currently being used in clinical trials to identify drug resistance in patient tumor biopsies. It was hypothesized that ovarian cancer spheroids with varying degrees of platinum resistance and p-glycoprotein would have identifiable p-glycoprotein signatures that could serve as a spectral biomarker for drug resistance.

Ovarian cancer tumor spheroids from the A2780 and SKOV3 cell lines were cultured with varying degrees of cisplatin to select for increasing platinum resistance and p-glycoprotein as detected by immunofluorescence and western blot analysis. Cells were grown as 3D spheroids and treated with anticancer drugs that are known p-glycoprotein substrates such as doxorubicin and paclitaxel. Treatments were used in combination with and without p-glycoprotein inhibitors such as verapamil and zosuquidar. For BDI, background spectral data was collected for 3 hrs and then for 9 hrs after drug treatment. Parallel experiments were done in 2D cultured cells in 96-well plates and cell viability was assessed with the XTT assay.

A suppression of average intracellular motion in the low-frequency range (0.01 Hz) was observed when 20 µM verapamil was used in combination with 10 µM paclitaxel in A2780 cells. This result may be due to the microtubule stabilizing effect of paclitaxel. SKOV3 spheroids were treated with verapamil plus 10 µM doxorubicin. Doxorubicin treated spheroids have increased high (10 Hz) and low (0.01 Hz) spectral signatures indicative of apoptosis. Addition of MDR1 modulators resulted in frequency changes associated with a greater apoptotic response in SKOV3 spheroids.

Modulation of p-glycoprotein alters the spectral response to drugs in a manner associated with the drug mode of action. A specific MDR1 spectral signature was not observed in these experiments but experiments with MDR1 silenced spheroids are underway to identify a possible signature.

#2047

The muscarinic receptor antagonist, benztropine, blocks the expansion of a temozolomide resistant subpopulation of glioma cells.

Damian A. Almiron Bonnin, Joseph M. Howard, Alison L. Young, Mark A. Israel, Matthew C. Havrda. _Geisel School of Medicine at Dartmouth, Lebanon, NH_.

Virtually all malignant gliomas become drug-resistant and recur after initial treatment, recurrent tumors are fatal within two-years, and no therapies exist that cure gliomas. The recognition that subpopulations of primitive cells with an oligodendrocyte precursor-like (OPC-like) phenotype capable of resisting standard therapy and contributing to recurrence, frames the question of whether adjuvant therapies designed to modify or eliminate OPC-like cells could slow the progression of glioma. Agents that force the differentiation of treatment-resistant OPC-like glioma cells, a phenomenon that requires cessation of proliferation, are compelling candidates for targeting OPC-like GSCs. Recently, it was observed that OPC proliferation was driven by activation of the M1, M3 and M4 muscarinic receptors. In another report, a large-scale drug screen designed to seek drugs to induce OPC differentiation in the context of multiple sclerosis identified a muscarinic receptor antagonist, benztropine (a.k.a. Cogentin), as a promoter of OPC differentiation and cell cycle exit. We have conducted studies in glioma cell lines supporting a role for benztropine in suppressing the development of drug-resistance to the standard glioma chemo-therapeutic temozolomide (TMZ). Upon treatment with TMZ, we identified the emergence of an OPC-like subpopulation of glioma cells identifiable by the co-expression of CD44, CD15 and PDGFRa. Treatment with pre-inhibitory doses of benztropine significantly reduced the TMZ IC50 in all glioma lines examined in association with blocking emergence of the CD44/CD15/PDGFR-positive subpopulation. Ongoing studies are designed to determine the tumor initiating potential of the CD44/CD15/PDGFR-positive subpopulation and whether benztropine will suppress the development of TMZ resistance in animal models of glioma.

#2048

Saturated fatty acids in cell membrane phospholipids play an important role in chemoresistance in colorectal cancer.

Takanori Hiraide, Takanori Sakaguchi, Satoru Furuhashi, Tomohiro Matsumoto, Yusuke Ozaki, Ryota Kiuchi, Makoto Takeda, Yasushi Shibasaki, Yoshifumi Morita, Hirotoshi Kikuchi, Megumi Baba, Mitsutoshi Setou, Hiroyuki Konno. _Hamamatsu University School of Medicine, Shizuoka, Japan_.

[Background and Aim] Resistance to chemotherapy represents the major obstacle to survival in patients with chemoresistant colorectal cancer. Various factors, such as drug efflux, autocrine survival signaling, and alterations in DNA damage repair mechanisms, can contribute to chemoresistance; however, the actual underlying mechanism is yet to be elucidated. The aim of this study was to elucidate the mechanism behind anticancer drug resistance based on lipidomics.

[Materials and Methods] 1) Spheroids were derived from three colorectal cancer patient-derived xenograft models (A, B, and C) that were incubated with chemotherapeutic agents, such as 5-fluorouracil (5-FU). We analyzed the response rate of spheroids under 5-FU treatment. Global lipid analysis was performed on 5-FU resistance spheroids (A) and sensitive spheroids (C) using a Q Exactive LC-MS/MS mass spectrometer, and lipid identification was performed using the Lipid Search software. 2) We tried to identify genes that were responsible for changes in phospholipid profiles in the cell membrane. 3) To determine phospholipid profile changes in the cell membrane that occur in colon cancer cell lines (HCT116, SW480) during anticancer drug resistance, the genes responsible for such resistance were knocked down using siRNA.

[Results and discussion] 1) Response to 5-FU was different in individual spheroids. Global lipid analysis and chemosensitivity assay revealed that phospholipids of 5-FU resistant spheroids were rich in saturated fatty acids (SFAs), while those of the 5-FU sensitive spheroids were rich in monounsaturated fatty acids (MUFAs). 2) The enzyme stearoyl-CoA desaturase (SCD) converts SFAs to MUFAs in the phospholipids. Based on these findings we hypothesized that decreased SCD expression is possibly associated with chemoresistance. 3) Cell lines with SCD knock down demonstrated increased SFA containing phospholipids and chemoresistance towards 5-FU. Viable cells after the administration of the anti-cancer agent confirmed that the expression of CD44 was upregulated. Therefore, changes in the phospholipid composition of cell membranes regulate 5-FU resistance in colon cancer.

[Conclusions] Identification and analysis of the role of membrane lipids has become possible using high-sensitivity and high-resolution mass spectrometers and the relevant software, such as Lipid Search. Changes of phospholipid composition regulates chemoresistance. In future, investigation of the pathophysiology of membrane lipids in cancer may help to reveal novel strategies for cancer therapy.

#2049

The role of epigenetic mark profile, cell cycle alteration and DNA repair in resistance of glioblastoma cells to photodynamic therapy.

Somayeh Shahmoradi Ghahe,1 Karolina Kopania,1 Agata Ciuba,1 Marek Foksinski,2 Ryszard Oliński,2 Barbara Tudek3. 1 _Institute of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland;_ 2 _Department of Clinical Biochemistry, Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, Poland;_ 3 _Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland & Institute of Genetics and Biotechnology, Faculty of Biology University of Warsaw, Warsaw, Poland_.

Glioblastoma is the most aggressive primary brain tumor. Treatment regimens for glioblastoma tumors, such as surgery, radiotherapy, and chemotherapy, are very invasive and can only prolong the median patient life to several months. It was shown that 5-aminolevulinic acid (5-ALA)-based photodynamic therapy (PDT) is a promising and less aggressive adjuvant modality for diagnosis and treatment of glioblastoma.

Limiting point of PDT outcome is appearing of cells with intrinsic or acquired PDT resistance that finally results in repopulating the tumor and short-term survival of glioma patients. Therefore, our study was designed to determine therapy resistance markers in PDT resistant glioblastoma cell line. Potential markers subsequently can be used for sensitization of glioblastoma to PDT.

Glioblastoma (U-87) cell line resistant to PDT (U-87R) was isolated from parental, sensitive line (U-87P) by applying several cycles of ALA-PDT, and further growing of surviving cells. U-87R cells subsequently were characterized from different aspects such as epigenetic markers, cell cycle events, oxidative stress and DNA repair capacity.

Assessment of nucleotide modifications and epigenetic marks in PDT resistant glioblastoma and its parental cell line showed significant higher level of two epigenetic marks including 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in U-87R compare to U-87P, before and after PDT.

Doubling time of U-87R cells was considerably longer than that of U-87P. Cell cycle analysis showed longer G1 phase in U-87R cells compare to U-87P. Moreover, accumulation in G2 phase following PDT was observed earlier in resistant cells than in parental line. That suggests the role of early activation of DNA damage response in resistant cells. Resistant glioblastoma cells were then sensitized to PDT by applying inhibitor of one of the main kinases of DNA damage response, ATM kinase. In comparison with parental cells, U-87R cells also showed higher activity of some DNA base excision repair (BER) enzymes including glycosylases and AP-endonuclease1 (APE1).

Studies of the whole protein profile of PDT resistant and parental cells demonstrated that the level of superoxide dismutase (SOD) was considerably higher in U-87R than in U-87P. That data was then confirmed by detecting lower oxidative stress in U-87R following PDT.

PDT resistant glioblastoma cells as a model of resistant cells in glioblastoma tumor population demonstrated the significant role of epigenetic mark profile, cell cycle alteration, higher DNA repair capacity, and antioxidant defense in conferring resistance to photodynamic therapy.

This work was supported by the Polish National Science Centre (NCN) grants: DEC-2012/07/B/NZ1/00008, UMO-2014/15/B/NZ5/01444, UMO-2014/13/N/NZ3/00863

#2050

Polyploid tumor cells: a chemoresistant cell type in triple-negative breast cancer.

Rekha Gyanchandani, Adrian Lee. _University of Pittsburgh, Pittsburgh, PA_.

Background: Triple-negative breast cancers (TNBC) are associated with an extremely poor prognosis due to their aggressive behavior and rapid resistance to chemotherapy. Chemotherapy may select for resistant subclones and lead to tumor recurrence. Although, DNA aneuploidy has been a long known prognostic biomarker in breast cancer, the biological role of aneuploid or polyploid cancer cells in tumorigenesis and chemoresistance is largely undefined. Polyploid cells arise due to repeated rounds of DNA duplication in the absence of mitosis. Chemotherapeutic DNA-damaging agents or mitotic inhibitors can also induce formation of polyploid cells and subsequent senescence. However, recent reports indicate that polyploid cells represent a viable and proliferating subpopulation within a tumor that may escape chemotherapy and contribute to tumor recurrence.

Methods: In this study, we examined the prevalence and functional significance of polyploidy occurring de novo or induced by chemotherapy in TNBC cell lines. DNA ploidy was analyzed in Hoechst 33342 stained cells using FACS. Cells with greater than 4N DNA content were defined as polyploid. Live-cell imaging was done to observe cell division patters in polyploid cells using IncuCyte Zoom. Cell proliferation was assessed in the absence and presence of chemotherapy drugs with distinct modes of action (docetaxel and paclitaxel: mitotic inhibitors, cisplatin: DNA cross-linking agent, and etoposide or VP16: topoisomerase inhibitor). RNAseq was performed to compare gene expression profiles between treatment-naive diploid and polyploid cells.

Results: Hoechst 33342 staining determined the prevalence of polyploid cells in HCC1395(6.7%) and HCC1937 (5.8%) cells lines. Live-cell imaging in polyploid cells showed formation of mitotic structures suggesting multi-polar cell division. Cell proliferation assay revealed that polyploid cells grow slower than diploid cells and show reduced sensitivity to all four chemotherapy drugs. Docetaxel treatment resulted in induction of polyploidy and drug-induced senescence in parental TNBC cells. Drug-induced polyploid cells were resistant to subsequent docetaxel treatment. Time-lapse imaging showed budding of small daughter cells from polyploid senescent cells. Majority of senescent cells died, some cells survived and regenerated the diploid and polyploid subpopulations similar to those present in parental cells. RNAseq identified differentially expressed genes involved in G1-S and G2-M checkpoint pathways (BRCA1/2, RAD51), cell proliferation (Aurora kinase A/B) and apoptosis (BIM, BIRC3).

Conclusions: Above findings indicate that there are molecular and functional differences between diploid and polyploid cells that are both naturally occurring or induced by chemotherapy. In our future studies, we will examine combinatorial approaches to overcome polyploidy-associated chemoresistance.

#2051

LSTRA cell line as a model for large granular lymphocytic leukemia in drug screening.

Fu-Shin Chueh,1 Fu-Yu Chueh,2 Chao-Lan Yu2. 1 _Asia University, Taichung City, Taiwan;_ 2 _Chang Gung University, Taoyuan City, Taiwan_.

Large granular lymphocytic (LGL) leukemia is a rare form of leukemia that is caused by the increase in cytotoxic T-lymphocytes or natural killer (NK) cells. A subcategory of T-LGL leukemia that co-expresses T and NK cell surface markers is very aggressive and has a very poor prognosis. Current treatment of aggressive T-LGL is based on non-specific immunosuppressive therapy. Lack of model cell lines contributes to slow progress in designing targeted therapy and clinical trials. Our recent studies demonstrate that the mouse T-leukemic cell line LSTRA reproduces some important characteristics of aggressive T-LGL leukemia. This is the first report of a cell line model mimicking the deadly human leukemia and has great potentials for drug screening in treating T-LGL leukemia.

Bortezomib is one of the drugs that show promising results in treating LGL leukemia patients in clinical trials. Bortezomib is the first FDA-approved reversible proteasome inhibitor in treating multiple myeloma and mantle cell lymphoma patients. One of the major concerns in using single-agent bortezomib is that patients develop resistance during the course of treatment and cancers relapse. Therefore, it is important to develop novel strategies in overcoming LGL leukemic cells' resistance to bortezomib and its derivatives.

In our current study, we showed that bortezomib inhibited cell proliferation and induced apoptosis in LSTRA leukemic cells. Partial reversion after 48-72 hr of bortezomib treatment suggested development of resistance to bortezomib. We further established bortezomib-resistant LSTRA cells that tolerated significantly higher levels of bortezomib. Both carfilzomib (FDA-approved second generation irreversible proteasome inhibitor) and MLN 4924 (proteasome pathway upstream inhibitor) failed to overcome bortezomib resistance. Only luteolin, a common flavonoid found in plants, can effectively overcome bortezomib resistance.

Metabolic reprogramming has been linked to drug resistance in cancer cells. Indeed, We found that bortezomib-resistant LSTRA cells had smaller cell size as well as reduced rate of glucose uptake, lactate secretion, and mitochondrial respiration. These results suggest that reduced mitochondrial activity may be an important molecular marker for LSTRA cell's resistance to bortezomib. All together, we identified luteolin as a potential small molecule in overcoming LGL leukemia's resistance to proteasome inhibitor therapy. Altered mitochondrial activity and cell metabolism in bortezomib-resistant cells provide additional targets in developing novel combined chemotherapy for cancer patients.

#2052

Chemotherapy sensitivity, cytokine IL-8 level, and genetic expression mRNA IL-8 in gastric cancer treatment response prediction.

Sirikan Limpakan (Yamada). _Chiang Mai Univ. Faculty of Medicine, Chiang Mai, Thailand_.

Aims: To determine chemotherapy sensitivity in primary gastric cancer cell isolated from Thai gastric cancer patients against anticancer drugs. To search for the correlation of interleukin-6 and interleukin-8 levels in primary gastric cancer cell culture in comparison with tissue mRNA IL-8 gene expression and drug response or resistance.

Method: The control background of corpus normal epithelial and gastric adenocarcinoma biopsy tissues were treated for primary cell culture with single cell isolation technique. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed to ex vivo examine the chemotherapy sensitivity to three anticancer drug, cisplatin, oxaliplatin, 5-Fluorouracil , and Irinotecan. Biopsy tissues from tumor site were collected before chemotherapy treatment in 63 patient who received neo-adjuvant chemotherapy by FOLFOX IV regimen due to advance stage III and IV in both resectable and un-resectable cases. The IL-8 mRNA gene expression was reported by relative quantitation real time RT-PCR. ELISA technique was performed to investigate the cytokine level from primary cultures taken from 41 gastric cancer patients who undergone biopsy from Maharaj Nakorn Chiang Mai Hospital during year 2014 to 2016. The clinical data base was parallel collected and analysed in correlation with clinical response and ex-vivo response.

Results: Each patient has different sensitivity pattern to the standard anticancer drugs. There are no significantly direct correlations or difference between cytokine IL-8 levels or mRNA IL-8 expression, and the chemotherapy sensitivity to the actual drugs that are being used for treatment of gastric adenocarcinoma in individual patients. However, the chemotherapy resistance to all drugs in primary gastric cancer cell culture has trend to increase after the third cycle of chemotherapy treatment when compared to prior cell treatment with fluctuation of cytokine level. Different cytotoxic drug gives the same response result for chemrsensitivity test in this population. The author observed the down regulation of tissue mRNA IL-8 expression and cytokine level in the good responders.

Conclusion: The results provide an additional basis of information for primary chemotherapy selection in the individualised treatment for advance gastric adenocarcinoma patients. Therefore, the author suggest to select the less toxicity cytotoxic drug for being primary chemotherapy of choice. Cytokine gene level and mRNA IL-8 gene expression show individually significantly change after chemotherapy treatment in good responder. This result may lead us to find some new targeted therapy or additional treatment for its suppression.

#2053

Mechanisms of resistance to palbociclib and aromatase inhibitors in hormone receptor positive breast cancer.

Armina A. Kazi,1 Antony Sare,1 Saranya Chumsri,2 Angela Brodie3. 1 _Loyola University MD, Baltimore, MD;_ 2 _Mayo Clinic, Jacksonville, FL;_ 3 _University of Maryland, Baltmore, Baltimore, MD_.

Estrogen receptor-positive (HR+) breast cancer is the most common form of breast cancer that is accountable for the majority of breast cancer mortality. Currently, the cyclin dependent kinase inhibitor palbociclib in combination with endocrine therapy represents the new standard first and second line therapy for patients with metastatic HR+ breast cancer. While palbociclib has been shown to significantly improved progression free survival in combination with aromatase inhibitor (AI) and fulvestrant, resistance will inevitably occur in patients with metastatic breast cancer. However, the mechanisms of resistance to the combination of palbociclib and AI remain largely unknown. It is also unclear whether resistance mechanisms would be the same if palbociclib was given as first line treatment in combination with letrozole vs. if palbociclib was given as second line treatment after letrozole resistance had already occurred. To address this, previously well established and well characterizied letrozole-sensitive MCF7Ca cells and letrozole-resistant LTLTCa cells were subjected to continuous, long term treatment with increasing doses of letrozole and palbociclib until resistance to both drugs was achieved (MCF7Calet+palb and LTLTCapalb, respectively). Preliminary MTT cell viability assays indicate palbociclib IC50s of 750 nM in MCF7Ca and LTLTCa cells vs. 21 uM in palbociclib and letrozole-resistant cells. First line and second line palbociclib resistance correlated with changes in morphology, protein expression, and cancer stem cell characteristics. Under phase contrast microscopy, first line and second line palbociclib-resistant MCF7Ca and LTLTCa cells were larger in size, more irregular in shape, and tended not to grow in epithelial cell-like groups compared to palbociclib-sensitive MCF7Ca and letrozole cells, with second-line palbociclib resistant cells exhibiting these characteristics the most. Western blot showed that ER protein expression in ER+/HER2- MCF7Ca cells decreased with first line palbociclib and letrozole resistance, and that both ER and HER2 protein expression were decreased with second line palbociclib resistance in ERlow/HER2+ LTLTCa cells. Lastly, mammosphere assays demonstrated increasing percentage of cancer stem cells with letrozole resistance alone (4 per 1000 cells plated MCF7Ca vs. 58 per 1000 planted LTLTCa) and with palbociclib and palbociclib resistance (317 per 1000 cells plated MCF7Ca and 202 per 1000 cells plated LTLTCa cells). Overall, these results indicate that 1) resistance to palbociclib, whether as first line or second line treatment has significant effects on breast cancer cells that may be relevant to patient diagnosis and treatment.

#2054

Cerivastatin overcomes the acquired resistance to crizotinib in EML4-ALK positive non-small cell lung cancer by controlling YAP activation.

Miran Yun,1 Hun Mi Choi,2 Kyoung Ho Pyo,2 Byoung Chul Cho2. 1 _Jeuk, Seoul, Republic of Korea;_ 2 _Yonsei University College of Medicine, Seoul, Republic of Korea_.

Crizotinib is highly effective in patients with non-small cell lung cancer (NSCLC) harboring the echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion. However, its efficacy has been limited by the development of acquired resistance, and the mechanisms of such resistance remain largely unknown. Herein, we investigated a possible candidate for circumventing the acquired resistance to crizotinib.

We established a model of acquired resistance to crizotinib (H3122-CR) by exposing EML4-ALK-positive H3122 lung cancer cells to increasing doses of crizotinib, and performed MTT screening using a library of FDA (Food and Drug Administration)-approved drugs composed of a collection of 640 clinically used compounds.

Our MTT screening identified that cerivastatin, a drug targeting 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, significantly inhibited the cell growth in H3122-CR cells, which was completely restored by addition of geranylgeranyl pyrophosphate (GGPP), a key metabolite of mevalonate pathway. We also found that yes-associated protein (YAP), a major effector of the Hippo tumor suppressor pathway, was robustly accumulated in nucleus with a concomitant decrement of YAP phosphorylation in H3122-CR cells compared to parental cells. Importantly, inhibition of geranylgeranylation (GGylation) by GGTI-298 or cerivastatin markedly increased YAP phosphorylation, led to cytoplasmic translocation of YAP, and subsequently induced YAP inactivation. Moreover, the enrichment of EGFR signaling pathway and cell cycle signature including transcriptional targets of YAP was enhanced in H3122-CR cells, whose induction contributed YAP-mediated resistance to crizotinib. Inhibition of YAP function with siRNA or verteporfin as a YAP inhibitor greatly abrogated cell growth of H3122-CR cells by modulating cell cycle regulators and EGFR activation. Finally, we confirmed up-regulation of nuclear YAP expression in crizotinib-acquired resistant patient derived tumor xenograft (PDTX) models established from EML4-ALK positive NSCLC patients. Collectively, our findings define the GGylation-mediated YAP transcriptional activation as a new mechanism of resistance to crizotinib, providing a rationale for further exploring statins as a possible anticancer agent to overcome the acquired resistance to crizotinib in EML4-ALK positive NSCLC cells.

#2055

Therapeutic resistance to BET bromodomain inhibition in prostate cancer.

Carl G. Engelke, Rohit Malik, Steven Kregel, Irfan A. Asangani, Kari Wilder-Romans, Xia Jiang, Xuhong Cao, Corey Speers, Arul M. Chinnaiyan. _University of Michigan, Ann Arbor, MI_.

Prostate cancer is the most common cancer among men and a leading cause of cancer-related death in North America. Current therapies include radical prostatectomy and radiotherapy for localized disease, and androgen deprivation therapy (i.e. surgical or medical castration) for metastatic disease. Recently, we described the use of inhibitors of bromodomain and extraterminal (BET) proteins for the treatment of castration-resistant prostate cancer (CRPC), which have since undergone investigation in clinical trials. In anticipation of patients refractory to treatment with BET inhibitors, we explored the development of resistance to a clinical-grade BET inhibitor in four cell line models—VCaP, LNCaP, LNCaP-AR, and 22Rv1—by serially passaging cells in increasing concentrations of drug with matched controls in DMSO. All resistance models displayed several-fold increased IC50 values relative to control in in vitro cell viability assays. Additionally, we have modeled resistance to BET inhibition in vivo by serially passaging cell line- and patient-derived xenografts in animals treated with BET inhibition. Interrogating these models through traditional molecular techniques and next-generation sequencing, we discovered several putative mechanisms of resistance to BET inhibition that we are currently validating in our laboratory.

#2056

MYC and HIF-2alpha mediates resistance to Epidermal Growth Factor Receptor (EGFR) antagonism in oral squamous carcinoma cells.

Sukanya Gayan,1 Reza Bayat-Mokhtari,1 Bidisha Pal,1 Anupam Sarma,2 Joyeeta Talkudar,3 Sorra Sandhya,3 Rashmi Bhuyan,1 Seema Bhuyan,3 Jaishree Garhyan,1 Debabrat Baishya,4 Amal Kataki,2 Rakesh Bhatnagar,5 Herman Yeger,6 Bikul Das1. 1 _Forsyth Institute, Cambridge, MA;_ 2 _B. Borooah Cancer Institue, Guwahati, India;_ 3 _KaviKrishna Laboratory, Guwahati Biotech Park, IIT, Guwahati, India;_ 4 _Gauhati University, Guwahati, India;_ 5 _Jawaharlal Nehru University, New Delhi, India;_ 6 _Hospital for Sick Children, Toronto, Ontario, Canada_.

Background: Oral squamous cell carcinoma (OSCC) is a devastating disease. Recent findings suggest that the EGFR antagonist including erlotinib may induce tumor regression alone or in combination with chemo/radiation. Our preliminary clinical study conducted at KaviKrishna laboratory indicates that many poor patients are taking the highly expensive drug Erlotinib, and or equivalent EGFR antagonists. However, oral cancer lesions are often hypoxic, and the hypoxia-induced cellular mechanisms might contribute to drug resistance. Here, we investigated, whether resistance to EGFR antagonists may involve MYC and HIF-2alpha, two transcription factors upregulated during hypoxia by using established oral cancer cell lines. We have also obtained primary oral cancer cells from patients living in the Kamrup district of Assam, where KaviKrishna laboratory is located. We intend to evaluate the drug sensitivity of these oral cancer cells towards EGFR antagonists.

Method: We used SSC-25 and SCC-9 cell lines, as well patient derived primary oral cancer cells (n=5) for the study. Immunomagnetic sorting was performed to obtain ABCG2+ population. The self-renewal was studied using in vitro clonogenic and in vivo serial transplantation assay in NOD/SCID mice.

Results: First, we identified a rare ABCG2+ expressing, highly tumorigenic cell population in SSC-25, and SCC-9 having cancer stem cell (CSC) like characteristics. Second, we found that the ABCG2+ cells exhibited sensitivity to PD158780 (10 uM; 62% inhibition within 48 hours), and AG1478 (10 uM; 56% inhibition within 48 hours), two small molecular inhibitors of EGFR tyrosine kinase. These small molecular inhibitors significantly inhibited the clonogenic capacity of the ABCG2+ cells. Next, we found that ABCG2+ cells also showed sensitivity to Erlotinib in the in vitro clonogenic assay. Third, the ABCG2+ cells, when exposed to hypoxia (<0.1% O2, 24 hours), exhibited enhanced expression and transcriptional activity of MYC, and HIF-2alpha. The post-hypoxia ABCG2+ cells exhibited complete resistance to PD158780, AG1478 and Erlotinib treatment, which could be reversed by siRNA silencing of MYC and or HIF-2alpha. ChIP assay revealed that HIF-2alpha directly binds to MYC in ABCG2+ cells. We found similar results in ABCG2+ cells obtained from primary oral cancer samples (n=4). Importantly, ABCG2+ cells directly isolated from patients exhibited hypoxic phenotype, including the high expression of HIF-1alpha, and HIF-2alpha, as well as resistance to erlotinib. Furthermore, erlotinib enhanced the stemness of ABCG2+ cells by activation of the MYC/HIF-2alpha self-renewal pathway (1).

Conclusion: These data indicate that MYC and HIF-2alpha co-operate to mediate intrinsic resistance of oral squamous cancer cells to EGFR antagonist in the hypoxia microenvironment.

(1). Bhuyan et al. Cancer Research, volume 76 (14), abstract 935; 2016

#2057

Evaluation of anti-tumor efficacy of EC1456 in low-passage and pre-treated patient-derived xenograft models of triple-negative breast cancer.

Yingjuan Lu,1 Nikki L. Parker,1 Haiyan Chu,1 Michael R. Pugh,1 Satish I. Rao,1 Patrick J. Klein,1 Michael F. Ritchie,2 Lonnie D. Myer,2 Jennifer Jaskowiak,2 Christopher P. Leamon1. 1 _Endocyte, West Lafayette, IN;_ 2 _Champions Oncology, Baltimore, MD_.

Triple negative breast cancer (TNBC) patients are insensitive to hormonal or anti-HER2 therapy and have a higher recurrence rate among all breast cancer subtypes. There is a lack of common therapeutic targets in TNBC due to its six distinct molecular characteristics. Recently, ~50% of TNBC cases were found to express the folate receptor alpha (FRα) on tumor cells. FRα is a GPI-anchored membrane glycoprotein capable of bringing folate-targeted small-molecule drug conjugates (SMDCs) inside the cell. EC1456 is a folic acid-tubulysin B hydrazide (TubBH) SMDC that specifically binds to the membrane FRα and is internalized by endocytosis. While encapsulated within the early endosome, EC1456 releases TubBH into the cytosol where it inhibits the polymerization of tubulin into microtubules, thus blocking spindle formation to arrest cells in metaphase which ultimately induces apoptosis. EC1456 is currently under Phase 1 clinical investigation in patients with common solid tumors [IND# 118,859]. The purpose of this study is to evaluate EC1456 activity in Champions TumorGraftTM TNBC patient-derived xenograft (PDX) models to help guide our drug development strategies. These PDX models were derived from patients who were treated with multiple lines of standard-of-care agents. A total of six low-passage, FR-positive TNBC models were tested against two different treatment regimens of EC1456 (once or twice a week for 2 weeks only). Plasma and tumor drug concentrations were quantified by LC-MS/MS using satellite study animals. The tumor-bearing animals were monitored for up to 60 days to assess both short-term (i.e. % TGI) and long-term (%PR, CR, TFS) anti-tumor responses. Using a stringent efficacy criteria (≥60% CR/TFS), 3 of the 6 TNBC models were found highly sensitive to EC1456 and 3 were found resistant. To identify potential gene signatures of EC1456 response, bioinformatics analysis was performed using existing RNA-seq data and compared across a broad panel of TumorGraftTM TNBC models, regardless of FR expression status. Specific biomarkers of interest were further analyzed by qRT-PCR using control tumors from the current study. Together, our analysis revealed potential resistance mechanisms associated with microtubule dynamics as well as a cancer cell's ability to undergo apoptosis.

#2058

Co-treatment of NVP-BSK805 highly sensitizes drug-resistant cancer cells by increasing P-gp inhibition.

Yu Jin Park, Ji Hyun Cheon, Ji Yeon Son, Sung Pil Yoon, Hyung Sik Kim. _School of Pharmacy, Sungkyunkwan University, Suwon, Republic of Korea_.

The high expression of P-glycoprotein (P-gp) is consistently observed in multidrug resistance (MDR) cancer cells. The purpose of our study is to identify conditions that could increase the sensitivity of P-gp-overexpressing the drug-resistant KBV20C cancer cell line. We have used the commercially-available epigenetic library, which includes 128 compounds. Using the cellular viability test, we also identified that NVP-BSK805 is highly sensitized to KBV20C-resistant cells and vincristine treatment. However, NVP-BSK805-induced sensitization was not observed in vincristine-treated sensitive KB parent cells, suggesting that the effects are specific to resistant cancer cells. Using FACS analysis, western blot, and Annexin V staining, we identified that NVP-BSK805 sensitized vincristine-treated KBV20C cells arrest via apoptosis and G2. Furthermore, sensitization potency of NVP-BSK805, in combination with vincristine, was measured in KBV20C cells when comparing them with P-gp inhibitor verapamil or another Jak2 inhibitor CEP-33779. We further investigated the mechanisms of NVP-BSK805 against sensitization of vincristine-treated KBV20C cells. The sensitization mechanism of NVP-BSK805 was mainly dependent on the inhibition of P-gp. In addition, ATPase activity was conducted to identify its potential role in NVP-BSK805 for P-gp inhibition. Both NVP-BSK805 and CEP-33779 showed high binding affinity docking scores -9.1 and -7.4, respectively, against the ABCB1 receptor protein. Collectively, inhibition of P-gp by NVP-BSK805 can increase vincristine-sensitivity in MDR cancer cells through increased apoptosis and G2 arrest. Our findings indicate that the Jak2 inhibitor may be a promising target in the treatment of patients resistant to antimitotic drugs.

#2059

Co-treatment of XL019, a selective Jak2 inhibitor, highly sensitizes drug-resistant cancer cells by increasing P-gp inhibition.

Ji Hyun Cheon, Yu Jin Park, Ji Yeon Son, Sung Pil Yoon, Hyung Sik Kim. _School of Pharmacy, Sungkyunkwan University, Suwon, Republic of Korea_.

1

Co-treatment of XL019, a selective Jak2 inhibitor, highly sensitizes drug-resistant cancer cells by increasing P-gp inhibition

Ji Hyun Cheon1, Yu Jin Park, Ji Yeon Son, Sung Pil Yoon, and Hyung Sik Kim

Lab of molecular of toxicology, School of Pharmacy, Sungkyunkwan University, Suwon, South Korea

A high expression of P-glycoprotein (P-gp) is consistently observed in multidrug resistance (MDR) cancer cells. The purpose of our study was to identify conditions that would increase the sensitivity of P-gp-overexpressing drug-resistant KBV20C cancer cell line. We used commercially available epigenetic library, which includes 128 compounds. Using cellular viability test, we identified that XL019 highly sensitized KBV20C-resistant cells to vincristine treatment. However, XL019-induced sensitization was not observed in vincristine-treated sensitive KB parent cells, suggesting that these effects are specific to resistant cancer cells. Using FACS analysis, western-blot, and annexin V staining, we identified that XL109 sensitized vincristine-treated KBV20C cells via apoptosis and G2 arrest. Furthermore, sensitization potency of XL109 in combination with vincristine was measured in KBV20C cells when comparing with P-gp inhibitor verapamil or another Jak2 inhibitor CEP-33779. We further investigated the mechanisms of XL109 against sensitization of vincristine-treated KBV20C cells. The sensitization mechanism of XL109 was mainly depends on the inhibition of P-gp. In addition, ATPase activity was also conducted to identify as potential role of XL019 in P-gp inhibition. In the docking modeling, XL019 and CEP-33779 showed high binding affinity docking score against ABCB1 receptor protein by -10.590 and -9.104, respectively. Collectively, inhibition of P-gp by XL109 can increase the vincristine-sensitivity in MDR cancer cells through increased apoptosis and G2 arrest. Our findings indicate that Jak2 inhibitor may be a promising target in the treatment of patients resistant to anti-mitotic drug.

#2060

Characterizing acquired resistance to palbociclib in breast cancer.

Smruthi Vijayaraghavan, Iman Doostan, Jason P.W. Carey, Khandan Keyomarsi. _UT MD Anderson Cancer Center, Houston, TX_.

The CDK4/6 inhibitor palbociclib is currently being used in combination with endocrine therapy to treat advanced ER positive breast cancer patients. While this treatment has shown great promise in the clinic, about 25% of the patients do not respond, and almost all patients eventually acquire resistance to palbociclib treatment. Hence, understanding the mechanism(s) of acquired resistance to CDK4/6 inhibition is crucial to devise alternate treatment strategies. To interrogate this, we developed MCF7 and T47D resistant cells by treating them with increasing doses of palbociclib over a 6-month period. After confirming that these cells were resistant to palbociclib, we performed genome-wide expression analysis via RNA-seq, in comparison with the parental (sensitive) cells. RNA- seq analysis revealed 2888 differentially expressed genes (p<0.05) in the resistant cells when compared to parental. Further, gene set enrichment analysis (GSEA) revealed enrichment of immune pathways (interferon alpha and gamma response, immune response) and pathways known to regulate EMT and cancer stem cells (IL-6/Stat3, IL-2/STAT-5, Notch, Wnt) in the resistant cells. Additionally, GSEA analysis revealed downregulation of G2/M checkpoint, estrogen response and DNA repair pathways (double strand break repair). Thus, this data suggests that combined targeting of two or more pathways that are altered in the resistant cells can provide a novel therapeutic strategy to combat CDK4/6 inhibitor resistance.

#2061

Non-covalent EGFR T790M targeting TKIs inhibit AZD9291 resistant EGFR C797S mutants.

Jacqulyne P. Robichaux, Monique Nilsson, John V. Heymach. _UT MD Anderson Cancer Center, Houston, TX_.

Approximately 10-15% of non-small-cell lung cancers (NSCLC) have epidermal growth factor receptor (EGFR) mutations resulting in increased sensitivity to 1st generation tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib. For common mutations in EGFR, treatment with 1st generation TKIs results in approximately 70% overall response rate, increased progression free survival, and increased quality of life compared to chemotherapy alone. However, resistance to 1st generation TKIs typically develops within ~12 months, and approximately 55% of patients acquire a secondary mutation in EGFR Exon 20, T790M. To overcome resistance, 2nd and 3rd generation covalently binding TKIs targeting T790M mutations have been developed. Recent studies show that approximately 40% of acquired resistance to 2nd and 3rd generation TKIs can also occur via a third acquired EGFR mutation at the site of covalent binding, C797S. To date, there are no standard approved targeted therapies for treating EGFR C797S mutant NSCLC. Moreover, with proposed increased use of covalent inhibitors in the first line setting, C797S mutations are expected to become more prevalent and new strategies to overcome therapeutic resistance will be required. To this end, we have generated stable Ba/F3 and HCC827 NSCLC cell lines expressing C797S mutant EGF receptors with common mutations in EGFR including, L858R/T790M/C797S, and Ex19del/T790M/C797S. EGFR mutant cell lines expressing C797S were screened against 1st, 2nd, and 3rd generation EGFR TKIs and cell viability was determined using Cell Titer Glo. Triple mutant cell lines containing T790M and C797S mutations were not sensitive to any 1st, 2nd, or 3rd generation inhibitors with IC50 values of >10µM, 7.0µM, and 7.6µM, respectively. However Ba/F3 and HCC827 cell lines transfected with EGFR triple mutants were inhibited by non-covalent inhibitors: CUDC-101, an EGFR, HER2 and HDAC inhibitor, and PKC412, a FLT3 inhibitor. In triple mutant Ba/F3 cells, CUDC-101 and PKC412 had IC50 values of 470nM and 250nM in Ex19del/T790M/C797S EGFR mutants, respectively, and IC50 values of 690nM and 270nM in L858R/T790M/C797S EGFR mutants. In HCC827 cells, PKC412 had IC50 values of 610nM and 520nM in L858R/T790M/C797S and Ex19del/T790M/C797S triple mutants, respectively. In addition, western blot analysis of triple mutant Ba/F3 cells showed decreased phosphorylation of EGFR in presence of 50nM PKC412 and 500nM of CUDC-101. Moreover, PKC412 partially decreased p-EGFR expression at 50nM in HCC827 triple mutant cell lines. In conclusion, unlike other EGFR TKIs, the non-covalent EGFR inhibitors CUDC-101 and PKC412 inhibit both T790M and C797S EGFR mutants at low concentrations. Currently, we are establishing additional osimertinib resistant HCC827, H1975, PC9 and H4006 cell lines for further in vitro and in vivo studies.

#2062

Inhibition of STAT3 overcomes gemcitabine resistance in pancreatic cancer cells.

Chi-Wei Chen, Ruiqin Wu, Yunxiao Meng, Wenge Zhu. _The George Washington University, Washington DC, DC_.

Gemcitabine is used as the first line treatment of pancreatic ductal adenocarcinoma (PDAC), however, either innate or acquired resistance to gemcitabine limit its therapeutic efficacy. In this study, several DNA damage agents including DNA repair inhibitors, and kinase inhibitors were examined for suppression of cell growth of gemcitabine resistant cells. The results show that most of these compounds are cross-resistant to gemcitabine-resistant pancreatic cells. By using STRING protein network database, we found that STAT3 signaling pathway plays a connectional role to link targeted proteins we tested. Interestingly, gemcitabine-resistant pancreatic cells have higher level of phosphor-STAT3 (pSTAT3) and are more sensitive to STAT3 inhibitor, S3I-201. Furthermore, combined S3I-201 with gemcitabine synergically suppresses the growth of both cell lines in vitro. We also confirmed the therapeutic efficacy of combined treatments using xenografted model. Taken together, inhibition of STAT3 overcomes gemcitabine resistance in pancreatic cancer cells. This study may provide us with a new approach to treat pancreatic cancer as well as gemcitabine resistance of pancreatic cancer.

#2063

**The farnesyltransferase inhibitor tipifarnib causes dramatic tumor regression and increases survival in murine Hras** G12V **driven aggressive thyroid cancers: Consequent adaptive and acquired resistance mechanisms inform combination treatments with improved responses.**

Brian R. Untch, Vanessa Dos Anjos, Maria ER Garcia-Rendueles, Jeff A. Knauf, Alan L. Ho, James A. Fagin. _MSKCC, New York, NY_.

Of the three RAS oncoproteins, only HRAS is delocalized and functionally inactivated by farnesyltransferase inhibition (FTI), an approach that has yet to be exploited clinically. We treated a murine model of poorly differentiated and anaplastic thyroid cancer (Tpo-cre/HrasG12V/p53flox/flox; Hras;p53) with the FTI tipifarnib, and observed sustained tumor regression and increased survival; however, tumors eventually recurred. Following HRAS delocalization by tipifarnib in vitro, ERK phosphorylation was only inhibited transiently in HRAS-mutant cell lines, which was associated with increased GTP loading of wild-type RAS proteins in the setting of RTK ligand stimulation. This adaptive reactivation of RAS-MAPK signaling was abrogated by selective RTK (i.e. EGFR, FGFR) inhibitors, or by MEK inhibitors. Importantly, tipifarnib combined with the MEK inhibitor AZD6244 led to improved responses in Hras;p53 mouse tumors, whereas combination with the EGFR/FGFR inhibitors erlotinib and ponatinib did not, suggesting heterogeneity of upstream inputs. In order to identify acquired resistance mechanisms, tumor-bearing Hras;p53 mice were treated with tipifarnib until resistance developed (6 months). Whole exome sequencing of resistant tumors identified a truncating NF1 mutation and an activating mutation in GNAS at high allelic frequency. Upon NF1 knockdown in the human HRAS- mutant cell line C643, tipifarnib failed to inhibit pERK in vitro and caused resistance to tipifarnib in xenografts. By contrast, activating GNAS mutations transduced into Hras;p53 mouse tumor cell lines produced tipifarnib-resistant xenografts that activated the GNAS-cAMP-CREB pathway and demonstrated signs of redifferentiation. These data show that pharmacological targeting of RAS in a genetically accurate, mouse model of a RAS-driven virulent cancer leads to objective major responses and improved survival. We identified adaptive and acquired resistance mechanisms, and show that combined treatment with selective MEK inhibitors are beneficial. These data should also inform a currently enrolling clinical trial of tipifarnib for HRAS-mutant malignancies.

#2064

Tumor cells treated with a PBD-based antibody-drug conjugate targeting 5T4 develop acquired resistance due to 5T4 down-regulation and response to alternate therapeutics.

Shenlan Mao,1 Andrew Garcia,1 Tracy Chen,1 Raghothama Chaerkady,1 Allison Marreno,1 Helen Zhong,1 Dorin Toader,1 Nazzareno Dimasi,1 Maureen Kennedy,1 Philip Howard,2 Changshou Gao,1 Herren Wu,1 Ronald Herbst,1 David Tice,1 Jay Harper1. 1 _MedImmune, Gaithersburg, MD;_ 2 _MedImmune, London, United Kingdom_.

Resistance to antibody-drug conjugates (ADC) has been observed in both preclinical models and in the clinic. It has been previously reported that an ADC targeting CD33 with a DNA cross-linking pyrrolobenzodiazepine (PBD) dimer was active in models of multidrug-resistant (MDR) AML, suggesting that PBDs, delivered as part of a molecular-targeted cancer therapy may be effective in MDR settings. However it could still be possible to develop resistance to PBD-conjugated ADCs though other mechanisms that remain unknown. Studies were conducted to determine if tumor cells have innate resistance to PBDs or an anti-5T4 ADC delivering PBD warhead. It was found that both the ADC and its PBD warhead, SG3199, completely eradicated MDA-MB-361 breast cancer cells and NCI-N87 gastric cancer cells in culture, suggesting a lack of intrinsic resistance in these cell lines. The potential emergence of acquired resistance was investigated by culturing MDA-MB-361 cells in gradually-increasing concentrations of either the ADC or SG3199 for over a year and the variants resistant to either the ADC (361-ADCr) or the PBD warhead (361-PBDr) were established as determined by decreased sensitivity in cytotoxicity assays. Transcriptional profiling characterization of these two cell models demonstrated that neither of them had significant upregulation of P-glycoprotein (P-gp) or other drug efflux transporter genes. Significant 5T4 antigen down-regulation was observed in the 361-ADCr cells but not the 361-PBDr cells and these cells also remained sensitive to alternate treatments including ADCs targeting a different surface antigen with the same PBD warhead, suggesting that down-regulation of target was likely the main driver of resistance in 361-ADCr cells. Further transcriptional profiling analyses conducted on 361-PBDr cells suggest a multi-factorial process may be needed to develop resistance to PBD warheads as differential expression of genes associated with DNA damage and repair, drug metabolism and cell cycle was observed. Similar gene expression profiles were observed in both cultured cells and in tumors derived from these cells. Studies to validate these differentially expressed genes, including proteomic and phosphoproteomic profiling are currently being conducted. Collectively these data could identify how resistance to ADCs conjugated with PBD payloads may develop in the clinic and more importantly, provide insight into developing strategies to overcome such resistance.

#2065

Targeting the crosstalk between MET and Notch signaling in Rhabdomyosarcoma.

Rossella Rota,1 Cristina Cossetti,1 Silvia Pomella,1 Alberto Gualtieri,1 Elena Carcarino,1 Carola Ponzetto,2 Riccardo Taulli,3 Lucio Miele,4 Franco Locatelli1. 1 _Ospedale Pediatrico Bambino Gesù, Roma, Italy;_ 2 _Univerisity of Turin, Turin, Italy;_ 3 _University of Turin, Turin, Italy;_ 4 _Louisiana Research Cancer Center, New Orleans, LA_.

Rhabdomyosarcoma (RMS) in a pediatric soft tissue sarcoma expressing myogenic markers. RMS includes two histological subtypes, embryonal and alveolar, which differ clinically and molecularly. The majority of alveolar RMS expresses fusion oncoproteins such as PAX3/7-FOXO1. Fusion positive (FP) RMS are at high risk showing a dismal prognosis while fusion negative (FN) ones have a better outcome. The MET protooncogene is a target gene of PAX3-FOXO1 and is overexpressed in FP RMS. However, it is upregulated/overactivated in FN RMS as well. It concurs to disease pathogenesis and is a potential target of therapy. We have shown that MET is down-regulated by the myomiR cluster miR-1/206 that promotes myogenic-like differentiation and inhibits proliferation of RMS cells. Notch signaling is also deregulated in RMS and inhibition of either Notch1 in FN or Notch3 in both subtypes leads to tumor growth blockade. Some preliminary data from our lab indicate that the expression of miR-1/206 is induced in Notch3-silenced RMS cells. Therefore, we sought to investigate the crosstalk between MET and Notch signaling in RMS.

We detected a down-regulation of MET transcript and protein in Notch3 knocked down RMS cells. In turn, MET silencing reduced Notch1 and Notch3 activation together with the levels of Notch ligands, JAG1 and DLL1. In line with this, forced expression of an activated form of MET (Tpr) resulted in Notch1 over-activation and up-regulation in FN RMS cells. Notably, a FN cell line treated for long time (more than 6 months) with growing doses of EMD, a clinically relevant MET inhibitor, showed resistance to treatment, over-activation of MET, i.e., hyperphosphorylation, and Notch1 signaling hyperactivation. These preliminary results suggest that MET and Notch signaling could crosstalk in RMS and that their co-targeting could be used to overcome drug resistance to single agent therapy. To this end, combination treatments of MET inhibitors with clinically relevant gamma-secretase inhibitors are in course.

The work is supported by Associazione Italiana Ricerca sul Cancro project IG15312 to RR.

#2066

Delivery of 2`F-PS2 PTGER3 siRNA-DOPC enhances anti-tumoral activity in cisplatin resistant ovarian cancer model.

Emine Bayraktar, Cristian Rodriguez-Aguayo, Zahid Siddik, Gabriel Lopez-Berestein. _MD Anderson Cancer Center, Houston, TX_.

RNAi-based therapeutics approaches holds a big promise for clinical translation. However, siRNAs are very weak and highly susceptible to degradation by nucleases. Nanoparticle delivery systems and chemical modifications of small interfering RNA (siRNA) can enhance efficient intracellular delivery and resistance to degradation for various siRNAs resulting in an increase in their clinical therapeutic potential. Here, we hypothesized that DOPC nanoparticles incorporated with a chemically fluoro phosphorodithioate (2`F-PS2) modified-PTGER3 siRNA will enhance the stability, prolong the silencing effect in vitro and increase the anti-tumoral effect when is combined with cisplatin in vivo. We screened 51 different chemically modified PTGER3-siRNAs which have 2'-F-PS2 or 2'-O-methyl modification on selectively protected nuclease-sensitive sites. We determined PTGER3 gene expression after siRNA-2'-F-PS2 or 2'-O-methyl modified mediated silencing, stability, time-response silencing effect and the therapeutic effect in xenograft OC in vivo model. We demonstrated that siRNA-2'-F-PS2 prolong the stability for more than 24 hours and enhance the silencing for more than 96 hours. Moreover, we found that PTGER3-silencing effect can control the expression of CFTR multidrug resistant pump by regulating ERK-ELK1-ETS1-CFTR axis, resulting in an increase of the cisplatin uptake, decreased the tumor-associated angiogenesis and tumor growth in xenograft OC in vivo model. SiRNA-PTGER3-2'-F-PS2 modification increase the stability and time-response silencing effect, leading to a synergistic anti-tumor in combination with cisplatin in ovarian cancer models.

#2067

Tumor-infiltrating PMN-MDSCs mediate sorafenib resistance in hepatocellular carcinoma through immune suppression.

Chun-Jung Chang,1 Li-Chun Lu,1 Cher-wei Liang,2 Chih-Hung Hsu,1 Ann-Lii Cheng1. 1 _Graduate Institutes of Oncology, College of Medicine, National Taiwan University, Taipei, Taiwan;_ 2 _Department and Graduate Institute of Pathology, National Taiwan University Hospital, Taipei, Taiwan_.

Background]: The mechanisms underlying the inherent or acquired resistance to sorafenib in advanced hepatocellular carcinoma (HCC) are not fully understood. We studied the role of myeloid-derived suppressor cells (MDSCs), a subset of bone marrow cells with immune-suppressive and proangiogenic activities, in sorafenib-treated mice and patients.

Materials and Methods: An orthotopic mouse liver cancer model was established by implanting BNL cells to BALB/cByJNarl mice. Liver tumor-bearing mice were treated with sorafenib (5 mg/kg/d) for 1 week or 3 weeks from the day 8 after tumor cells implantation. For combination therapy, mice were treated sorafenib and anti-IL-6 Ab or its isotype control Ab (500 μg/every 3 days starting from day 4 for 4 doses). Tumor-infiltrating leukocytes (TILs) and leukocytes of other tissues including liver, spleen, peripheral blood, and bone marrow were isolated, and characterized by flow cytometry analysis. Morphological characteristics of MDSCs were determined by cytospin and Liu staining. Pro-inflammatory cytokines were detected by ELISA or mouse inflammatory cytokines beads array (BD). Paired HCC tissues from advanced HCC patients treated with sorafenib (before and post-sorafenib progression tissues) were evaluated for the expression of CD11b, CD15, and CD66b in TILs by immunohistochemistry.

Results: In mice treated with sorafenib, the CD11b+Gr1+Ly6G+ MDSCs were significantly increased in liver tumors, but not in others tissues. Morphologically, these MDSCs were segmented nuclear PMN type cells. The analysis of TILs revealed that the percentage of total immune effectors (CD4+ or CD8+ IFN-γ-expressing T cells) versus total immune suppressors (IL-10 or TGF β-expressing CD4+ T cell, FOXP3-expressing CD4+ T cells) was lower in the tumors of sorafenib-treated mice than those of the vehicle-treated mice. The T cell proliferation capability was also significantly decreased in TILs of sorafenib-treated mice versus TILs of vehicle-treated mice. Multiple inflammatory cytokines including VEGF, G-CSF, MCP-1, and IL-6 were significantly increased in mouse liver tumors treated with sorafenib. When combined with sorafenib, anti-IL-6 antibody alleviated the early tumor-infiltration of Ly6G+ MDSCs, restored the proliferative activity of TILs, decreased the levels of pro-inflammatory factors in tumors, and suppressed

the liver tumor growth synergistically. In 6 paired human HCC tissues, we found that 3 exhibited higher proportion of CD11b, CD15, or CD66b+ cells in post-sorafenib progression tissues than their pre-sorafenib treatment tissues.

Conclusions: Our data indicate that tumor-infiltrating PMN type MDSCs and associated immunosuppression may be an important mechanism impeding the therapeutic efficacy of sorafenib in HCC. (This works was supported by MOST 105-2314-B-002-180).

#2068

Development of a rational triple combination therapy for castration-resistant prostate cancer.

Yuanyuan Qiao, Todd M. Morgan, Arul M. Chinnaiyan. _University of Michigan, Ann Arbor, MI_.

Background The androgen receptor (AR) signaling pathway drives prostate cancer development and progression, making it a major target for drug development. However, resistance to AR targeted therapies invariably develops and eventually leads to an aggressive, castrate-resistant prostate cancer (CRPC). Although CRPC often remains dependent on AR signaling, anti-androgen therapies can lead to the development of AR independent disease and metastatic CRPC. Understanding the molecular basis of this transition and resistance to current anti-androgen therapy will provide important insight and reveal novel therapeutic strategies for both AR-positive and -negative disease pathways.

Methods and Results We analyzed in silico data of AR (signaling)-positive or -negative human CRPC tissue samples and discovered that MET expression is specifically increased only in AR-negative CRPC samples. When AR-positive CRPC models are subjected to AR signaling inhibition (by the AR antagonist enzalutamide or androgen deprivation), MET is increased and susceptible to activation by its ligand HGF. Therefore, we postulate that dual targeting of AR and MET signaling pathways may be a better approach to prevent and overcome resistance-related disease progression. Our preliminary AR ChIP-seq data suggested that MET transcription is not affected by AR directly rather, MET protein is modulated by AR signaling at the post-translational level. Therefore, we hypothesize that a combination therapy of a proteasome inhibitor and MET inhibitor may have potential therapeutic benefit for some CRPC patients. By using in vitro and in vivo models of AR-dependent CRPC, we have showed that combination of the dual MET/VEGFR2 inhibitor cabozantinib and enzalutamide treatment is more efficacious than either inhibitor alone. In addition, Bortezomib, an FDA-approved proteasome inhibitor, showed significant synergistic effect when paired with cabozantinib in our preliminary data using in vitro and in vivo CRPC models. In vitro results suggest that triple combination of anti-androgen, MET inhibitor and proteasome inhibitor therapies will maximize inhibition in CRPC.

Conclusion MET is a compensatory survival pathway in AR+ CRPC upon anti-androgen therapy. The rational for triple combination of anti-androgen, MET inhibitor and proteasome inhibitor therapies is a feasible approach to maximize inhibition in CRPC while minimizing development of drug resistance to any single agent.

#2069

Overcoming drug resistance by targeting melanoma dedifferentiation through information-theoretic analysis and single cell proteomics.

Yapeng SU,1 Wei Wei,2 Lidia Robert,2 Jennifer Tsoi,2 Min Xue,1 Jungwoo Kim,1 Thomas Graeber,2 Raphael Levine,2 Antoni Ribas,2 James Heath1. 1 _Caltech, Pasadena, CA;_ 2 _UCLA, Los Angeles, CA_.

Despite impressive initial response of BRAF targeted therapy in mestastic melanoma patients, acquired drug resistance, often times derived from tumor heterogeneity, always limit its clinical outcome. A dedifferentiation process named melanocyte to neural crest transition (MNT) has shown to be critical in the early stage resistance development. The dedifferentiation could induce a whole cell state change resulting a drug tolerant state of melanoma cells, which will lead to initial drug resistance. Mathematic modeling indicates the nature of the transition as results of both stochastic phenotypic transition and non-genetic drug selection, which indicates and further proved that the whole cell state transition should be reversible during drug removal. Information theoretic analysis reduced the dimensional of thousands of gene expression level changes as results of only two unbalanced processes onto a cellular steady state. Reversible transition trajectory defined through two processes indicates it to resemble a critical transition in physical systems. Refined single cell proteomic analysis of those proteins that associate with major unbalanced processes identify the critical points of the transition as early as day6, which cannot be resolved from bulk proteomic measurement. By co-targeting the drivers at the critical points in combination with original brafi targeted drug successfully arrested the transition and induced a sustained tumor growth inhibition in multiple patient-derived melanoma cell lines. This study provides us a noval methodology of mining whole transcriptomic transition with information theoretic analysis to reduce the dimensionality and then using single cell proteomic analysis for refining critical points and driver proteins which eventually rationalized a effective combination therapy to stop the drug resistance development early on.

#2070

HMGB1 governs p53 to autophagic degradation via its nucleus-to-cytoplasm transport.

Qiaojun He, Peihua Luo, Xiaochun Yang, Qinjie Weng, Ling Ding, Bo Yang. _Zhejiang Univ. College of Pharmaceutical Sci., Hangzhou, China_.

P53 is under exquisitely fine regulation and acts as a transcription factor that regulates the expression of thousands of genes that control apoptosis, necroptosis, ferrotosis, cell cycle arrest, senescence, metabolism and fertility. Regulation of p53 turnover is essential not only for the response to chemotherapeutic drugs or radiation in cancer cell but also to keep p53 activity under control in a normal cell. Moreover, .p53 is found to be overactivated in various diseases including idiopathic pulmonary fibrosis (IPF), alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD). Promoting hyperactivated-p53 degradation can combat these diseases. However, the strategies that based on known proteasome-dependent degradation only can stabilize the expression level of p53 such as nutlins (inhibit the interaction between mdm2 and tumor suppressor p53). Autophagy-lysosomal degradation is the other pathway for controlling cellular protein stability. More and more researches have established that numerous proteins can be degraded through both proteasome and autophagy-lysosome pathways. However, very little is known to date about p53 turnover through autophagy-lysosome pathway. Here we discover that the activation of p53 autophagic degradation during an oral small molecule receptor tyrosine kinase inhibitor sunitinib in p53 wt cancer cells and normal cells. Mechanistically, the nucleus-to-cytoplasm shift is essential for the autophagic degradation of p53 induced by sunitinib, however, does not require p53 nuclear export signals. Indeed, p53 degradation is achieved by the nucleus-to-cytoplasm transport of its nuclear binding target HMGB1, shifting the distribution of p53 from the nucleus to the cytoplasm. Then cytoplasmic p53 directly interacts with the autophagy cargo receptor p62 to promote degradation. Importantly, inhibition of HMGB1 sensitizes cancer cell to sunitinib. Taken together, our study identified an alternative p53 protein turnover mechanism induced by sunitinib, thus not only elucidating the underlying mechanisms that limit sunitinib efficacy in cancer therapy but also opening an avenue for expanding the clinical indications of sunitinib.

#2071

Sub-clonal heterogeneity and risk of treatment failure in Philadelphia-positive leukemias.

Michael W. Schmitt,1 Justin R. Pritchard,2 Lan Beppu,3 J. Graeme Hodgson,2 Victor M. Rivera,2 Lawrence A. Loeb,1 Jerald P. Radich3. 1 _University of Washington, Seattle, WA;_ 2 _ARIAD Pharmaceuticals, Inc., Cambridge, MA;_ 3 _Fred Hutchinson Cancer Research Center, Seattle, WA_.

Molecularly targeted therapies have been exceptionally successful in the treatment of some malignancies, most notably chronic myeloid leukemia (CML). However in other cancers, e.g. Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL), drug resistance is a frequent occurrence. Resistance mutations may pre-exist as a consequence of intra-tumor heterogeneity; thus the extent of heterogeneity may determine the likelihood of treatment failure. Intra-tumor heterogeneity is challenging to quantitate, as heterogeneous mutations are likely to occur below the background error rate of conventional approaches for mutation detection. To overcome this limitation and explore the relationship between intra-tumor heterogeneity and drug resistance in Ph+ leukemias, we utilized single-molecule Duplex Sequencing (DS) of the ABL1 gene. DS eliminates sequencing errors by independently tagging the two strands of individual DNA molecules; true mutations are present at the same position in both strands, while PCR and sequencing errors are only present in one strand. DS has an error rate below 0.00001%, and thus enables quantitation of heterogeneous mutations with unprecedented resolution. We find that CP-CML, a disease that is often well controlled with targeted therapy, has an extremely low burden of intra-tumor heterogeneity at the time of diagnosis, with a sub-clonal mutation burden similar to that of normal individuals. Simulations which take into account the low cancer stem cell fraction of CP-CML and the sub-clonal mutation burden we measured reveal that only a minority of CP-CML patients will harbor pre-existing drug resistance, which may explain the unusual success of targeted therapy in this setting. Next, we studied patients with advanced Ph+ leukemias (refractory BP-CML and Ph+ ALL), which have a high rate of treatment failure and poor outcomes. We find that patients with BP-CML and Ph+ALL have a significant elevation in sub-clonal heterogeneity relative to those with CP-CML. We demonstrate that the higher extent of intra-tumor heterogeneity correlates with a significantly higher rate of drug resistance mutations and compound resistance (i.e. two concurrent resistance mutations) in patients. Unlike CP-CML, pre-existing mutations conferring resistance to targeted therapy are nearly certain in refractory BP-CML and Ph+ ALL as a consequence of the load of intra-tumor heterogeneity and high cancer stem cell fraction. We support this concept by three distinct approaches: (i) detection of pre-existing sub-clones that drive treatment failure; (ii) modeling the growth kinetics of resistant clones; and (iii) extrapolation of the mutation burden measured by DS. Our results suggest that intra-tumor heterogeneity influences the likelihood of resistance to molecularly targeted therapy. Quantitation of sub-clonal mutation load may be broadly applicable in other malignancies for predicting the likelihood of response to targeted therapy and monitoring disease progression.

#2072

Combination of CDODA-Me, a glycyrrhetinic acid derivative, and Erlotinib overcomes chemo-resistance in NSCLC PDX spheroids and 3D bio-printed cells.

Arindam Mondal,1 Aragaw Gebeyehu,1 Ebony Nottingham,1 Arvind Bagde,1 Subramanian Ramakrishnan,2 Arun K. Rishi,3 Mandip Singh1. 1 _Florida A &M University, Tallahassee, FL; _2 _Florida State University, Tallahassee, FL;_ 3 _Wayne State University, Detroit, MI_.

Patient-derived Xenografts (PDXs) are considered as relevant preclinical model for anticancer drug development due to original recapitulation of patient genetic profile, gene expression patterns and tissue histology. In this study, we investigated combination efficacy of CDODA-Me (Methyl 2-cyano-3,11-dioxo-18-olean-1,12-dien-30-oate) and TKI inhibitor Erlotinib (ERL) against Lung NSCLC PDX spheroids and 3D bio-printed PDX cells. NSCLC PDX cells (EGFR T790M mutants) were obtained from Dr. Rishi's Laboratory. PDX spheroids were grown in DMEM/ F12 media supplemented with L-glutamine, B27 supplement, recombinant human epidermal growth factor (EGF) and recombinant human basic fibroblast growth factor (bFGF). Spheroids were treated with CDODA-Me, ERL alone and in combination. Cell viability was measured by MTT assay. Western blot analysis was used to study the modulation of Bcl-xL, MDR1 and ABCG2 in treated PDX spheroids. For 3D bio-printing of PDX cells, hydrogels were prepared by partial cross-linking of sodium alginate (4.5% w/v) and gelatin (1% w/v) mixture with 40mM CaCl2 solution. PDX Cells were mixed with partially cross-linked hydrogel and printed with Inkredible 3D bio-printer (CELLINK, Sweden). Bio-printed scaffolds were fully cross-linked by 160 mM CaCl2 solution and then incubated overnight with cell culture media. The scaffolds were treated with CDODA-Me and ERL alone and in combination. After 48 h cell viabilities were determined by live/dead assay using fluorescence microscopy.

MTT assay showed that approximately 65% and 74% viability was observed at 10 µM ERL and 2.5 µM CDODA-Me respectively. Decreased spheroid cell viability was observed in ERL and CDODA-Me combination treatment. Our western blot studies showed down-regulation of Bcl-xL, MDR1 and ABCG2 in combination group. Further, 81.04 ± 5.65, 78.65 ± 3.98 and 74.35 ± 4.24 percent viable PDX cells were observed in the bio-printed scaffolds after 48, 72 and 96 h respectively. Higher percentage of dead cells (52.62 ± 1.66) were found in the combination group than CDODA-Me (28.39 ± 1.60) and ERL (29.62 ± 4.91) alone.

In conclusion, CDODA-Me in combination with ERL was found to be effective against human lung PDX spheroids and bio-printed PDX cells by decreasing the cell viability and overcoming drug resistance. Partially cross-linked sodium alginate-gelatin hydrogel enhances the possibility of PDX cell bio-printing with high cell survival rate. CDODA-Me can be considered as an effective neo-adjuvant to improve ERL efficacy in human NSCLC.

#2073

A systematic investigation of the effect of scheduling of targeted combination therapies on response and dynamics of relapse in triple negative breast cancer cells.

Gauri A. Patwardhan, Vikram B. Wali, Lajos Pusztai, Christos Hatzis. _Yale University, New Haven, CT_.

Cancer treatment typically involves administration of combination of targeted therapies, but initial response is often followed by disease relapse. The efficacy of a treatment regimen depends on the complex interplay between cancer growth dynamics, drug specificity and kinetics, treatment dose and its scheduling. In standard high-throughput drug screening assays, cells are treated with a single drug cocktail bolus and cell viability is assessed after 2-3 days, thus not considering treatment interactions and long-term effects. Recently we have reported a promising synergistic combination of crizotinib (ALK/MET inhibitor) and ABT-263 (BCL2/BCL-XL inhibitor) against triple negative breast cancer cells. To understand the effect of the sequence and combination doses of crizotnib and ABT-263, we designed a comprehensive experimental plan that involved a total of 567 treatment regimens by varying treatment duration with the first drug (1, 2, or 3 days), followed by drug withdrawal and recovery period (2, 5 or 10 days) and then by a second cycle of treatment and recovery periods over a 26-day period. Cell viability was assessed by the CellTiter-Glo luminescence assay. Interestingly, ABT-263 alone induced higher cytotoxicity than an equivalent dose of crizotinib, but the remaining viable cells recovered much faster after ABT-263 withdrawal than cells after crizotinib withdrawal. Furthermore, cells exposed to higher doses of ABT-263 eventually become less sensitive to crizotinib. Among sequential regimens, crizotinib followed by ABT-263 was significantly more effective than ABT-263 followed by crizotinib, and combinations that included lower doses of ABT-263 were most effective. Taken together, our results show a significant interaction between the two targeted therapies, and suggest that it may be possible to select treatment scheduling that can delay drug resistance and tumor relapse in vivo.

### Growth Factor and Hormone Receptors as Therapeutic Targets

#2074

Targeting SHP-1 mediated VEGF signaling to block the migration of human triple negative breast cancer.

Jung-Chen Su,1 Ai-Chung Mar,2 Chun-Yu Liu,3 Kuen-Feng Chen,4 Chung-Wai Shiau5. 1 _National Yang-Ming University Faculty of pharmacy, Taipei, Taiwan;_ 2 _Institute of Biomedical Sciences, Academia Sinica, Taipei 11521, Taiwan, Taipei, Taiwan;_ 3 _Division of Hematology and Oncology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan, Taipei, Taiwan;_ 4 _Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan, Taipei, Taiwan;_ 5 _National Yang-Ming University, Taipei, Taiwan_.

Patients with triple-negative breast cancer (TNBC) had an increased likelihood of distant recurrence and death, as compared with those with non-TNBC subtype. Regorafenib is a multi-receptor tyrosine kinase (RTK) inhibitor targeting oncogenesis and has been approved for metastatic colorectal cancer and advanced gastrointestinal stromal tumor. Recently, scientists have suggested that regorafenib also acts as a SHP-1 phosphatase agonist. In this study, we aimed to investigate the role of regorafenib in suppressing metastasis of TNBC cells through targeting the SHP-1/p-STAT3/VEGF-A axis. We first observed a significant correlation between migration ability and SHP-1/p-STAT3/VEGF-A expression in several human TNBC cell lines. Importantly, we also demonstrated that VEGF-A expression is correlated with worse disease-free and distant metastasis-free survival in clinical TNBC patients. Furthermore, we showed that regorafenib could inhibit the migration of human TNBC cells, which was related to downregulation of p-STAT3 and VEGF-A. To exclude the role of RTK inhibition in regorafenib-induced anti-metastasis, we synthesized a regorafenib derivative, SC-78, that had minimal effect on VEGFR2 and PDGFR kinase inhibition, while having more potent effects on SHP-1 activation. SC-78 demonstrated superior in vitro and in vivo anti-migration to regorafenib. This study indicated that the SHP-1/p-STAT3/VEGF-A axis is a potential therapeutic target for metastatic TNBC, and SC-78 may be a promising lead compound for suppressing metastasis of TNBC. (Grant supported by MOST 104-2628-B-075-001-MY3, MOST 105-2325-B-010-007, 105F026 )

#2076

Targeting the FGFR pathway in androgen receptor negative castration resistant prostate cancer.

Colm Morrissey,1 Eva Corey,1 Lisha Brown,1 Ilsa Coleman,2 Holly Nguyen,1 Michael Schweizer,1 Peter Nelson2. 1 _Univ. of Washington, Seattle, WA;_ 2 _Fred Hutchinson Cancer Research Center, Seattle, WA_.

Metastatic castration-resistant prostate cancer (CRPC) has a poor prognosis and remains a significant therapeutic challenge. The continued importance of androgen receptor (AR) signaling as a growth and survival pathway in most advanced prostate cancer (PC) has prompted the development of therapeutics directed toward further suppressing AR ligands or the AR itself. However, total androgen blockade can give rise to AR-negative PC and currently there are no effective therapies for AR-negative PC. We have developed two novel patient-derived xenograft (PDX) models from a liver metastasis and a rib metastasis of a patient with CRPC: LuCaP 173.1 (Neuroendocrine; NE) and LuCaP 173.2 (Double negative: DN (AR-negative and NE-negative)). Using RNA-Seq we observed that both of these lines express high levels of fibroblast growth factors (FGF's) and downstream effectors. We hypothesized that FGF signaling promotes survival and proliferation of NEPC and DNPC. To determine if blocking the FGFR survival pathway can attenuate the growth of NEPC/DNPC tumors, to characterize the molecular response and resistance to FGF signaling inhibition, and to support further evaluation in the clinic, we treated both PDX models with the FGFR inhibitor CH5183284. CH5183284 significantly reduced LuCaP 173.2 tumor volume (TV) (p<0.0001). The reduction in TV in CH5183284-treated animals bearing LuCaP 173.1 tumors was not as significant. To address this difference, we analyzed FGFR1 expression at the transcript and protein levels. Analysis of the FGFRs in the LuCaP 173.1 PDX model revealed a loss of FGFR1 expression. FGFR2 and FGFR3 transcripts were expressed at similar levels in both PDXs. The loss of FGFR1 expression may explain the low efficacy of CH5183284 in the LuCaP 173.1 PDX model. We are currently assessing the molecular pathways impacted by FGFR inhibition and mechanisms of resistance to treatment in these studies. We have demonstrated that FGFR inhibition attenuated tumor growth in FGFhigh AR-negative PC. We intend to target the FGFR survival pathway in additional NE and DNPC PDX models to investigate tumor heterogeneity in response to therapy and identify mechanisms of resistance. These studies will provide translational evidence that FGFR inhibitors could represent a new therapy for CRPC patients with AR-negative PC.

#2077

Selective MET kinase inhibition in MET-dependent glioma models.

Corina van den Heuvel,1 Anna Navis,1 Houshang Amiri,1 Kiek Verrijp,1 Arend Heerschap,1 Karen Rex,2 Isabelle Dussault,2 Sean Caenepeel,2 Angela Coxon,2 Pieter Wesseling,1 William Leenders1. 1 _Radboudumc, Nijmegen, Netherlands;_ 2 _Amgen Inc., CA_.

Background - Grade IV diffuse gliomas (glioblastomas) are notoriously difficult to treat. Many studies aim at targeting tumor-specific aberrations, such as mutations in genes encoding oncogenic receptor tyrosine kinases (RTKs). Of high interest as a tumor target in diffuse glioma is the RTK MET, which is amplified in a significant proportion of glioblastomas, and a number of MET inhibitors have been developed. However, most RTK inhibitors available in the clinic today, including those inhibiting MET, are not entirely selective and inhibit additional kinases at the doses used. They therefore may induce potentially undesired off-target effects, such as blood-brain barrier normalization in the case of concomitant VEGFR2 inhibition.

Methods – We studied the effects of the novel, selective MET-kinase inhibitor (Compound A) and the combined VEGFR2/RET/MET inhibitor cabozantinib on MET activation and proliferation in the MET-amplified E98 astrocytoma cell line in vitro, using western blot analysis and MTT proliferation assays. Effects of compound A were also studied in mice carrying orthotopic xenografts of the same E98 cell line. Survival was monitored and effects of MET inhibition were investigated by immunohistochemistry using phospho-specific antibodies.

Results – E98 cells were highly sensitive to treatment with Compound A in vitro (IC50~9.4 nM). Furthermore, Compound A effectively inhibited phosphorylation of MET in orthotopic E98 xenografts. In contrast, we have previously shown that after treatment with cabozantinib in the same model the high levels of phosphorylated MET were maintained. While treatment with Compound A significantly prolonged the survival of E98-xenograft bearing mice, tumors still developed and showed extensive AKT phosphorylation in the absence of MET phopshorylation, suggesting in vivo therapy resistance mechanisms. Interestingly, resistance to Compound A was not seen in vitro in an E98 cell line derived from Compound A-resistant tumor xenografts.

Conclusions – Compound A is a promising, highly selective MET kinase inhibitor with activity against gliomas with constitutive MET signaling. Selective MET kinase inhibitors may be more suited for treatment of glioma than combined VEGFR2/MET inhibitors, as the latter may induce vessel

normalization resulting in poor tumor penetration. Similar to clinical experience, E98 tumor-bearing mice ultimately develop resistance to TKIs. Combination therapies targeting both MET and potential resistance pathways may be required to allow long-term tumor treatment.

#2078

CK-101 (RX518), a mutant-selective inhibitor of EGFR that overcomes T790M-mediated resistance in NSCLC.

Xiangping Qian,1 Yong-Liang Zhu,1 Edward Cullen,2 Robert M. Niecestro,2 James F. Oliviero2. 1 _NeuPharma, Inc., Foster City, CA;_ 2 _Checkpoint Therapeutics, Inc., New York, NY_.

Patients with non-small cell lung cancer (NSCLC) with activating epidermal growth factor receptor (EGFR) mutations initially respond to small molecule inhibitors of the EGFR tyrosine kinase such as gefitinib and erlotinib. However, secondary mutations in the kinase domain lead to acquired resistance to these inhibitors and limited clinical efficacy, with the EGFR T790M mutation being the most common mechanism of acquired resistance in more than half the patients that experience disease progression. CK-101 (also known as RX518) is a novel, irreversible, orally administered EGFR kinase inhibitor that specifically targets the mutant forms of EGFR, including T790M, while exhibiting minimal activity toward the wild-type (WT) EGFR. WT EGFR inhibition is believed to drive the commonly observed side effects of skin rash and diarrhea. The 50% inhibitory concentrations (IC50s) of CK-101 and 2 reference compounds, afatinib and AZD-9291, were determined in cell proliferation assays in human cancer cell lines in vitro after incubation for 72 hours. Xenograft studies were conducted in BALB/c nude mice or SCID/Beige mice using once daily oral administration of CK-101 or afatinib for 14 or 21 days. CK-101 selectively inhibited cell proliferation of cell lines expressing both the activating (e.g. HCC827, IC50 <0.015 µM) and resistance mutations (NCI-H1975, IC50 <0.005 µM), but was much less potent at inhibiting proliferation of cell lines expressing the WT form of the receptor (e.g. A431, IC50 >0.5 µM; i.e., CK-101 was over 100 fold less potent against A431 than against NCI-H1975). Single agent CK-101 significantly inhibited tumor growth in EGFR-mutated NSCLC tumor xenograft models, with no activity in a WT EGFR tumor xenograft model. In a xenograft study of PC-9 cells (exon 19 deletion) in SCID/Beige mice, CK-101 inhibited tumor growth by up to 90% (p <0.001). In a xenograft study of NCI-H1975 cells (L858R/T790M double mutant) in BALB/c nude mice, CK-101 inhibited tumor growth by up to 95% (p <0.001). The pre-clinical findings from this work strongly supported the clinical development of CK-101, and a first-in-human study of CK-101 was initiated in September 2016.

#2079

Osimertinib, an irreversible mutant selective EGFR tyrosine kinase inhibitor, exerts anti tumor activity in NSCLC harbouring exon 20 insertion mutant-EGFR.

Nicolas Floc'h, Susan Ashton, Ambra Bianco, Nicola Colclough, Darren AE Cross, Maria Emanuela Cuomo, M. Raymond V. Finlay, Matthew J Martin, Ludovic Menard, Darren McKerrecher, Daniel J O'Neill, Jonathan P Orme, Anna D Staniszewska, Richard A Ward, James W T Yates. _AstraZeneca, Cambridge, United Kingdom_.

EGFR exon 20 insertion (Ex20Ins) mutations represent a combination of in-frame insertions and/or duplications that account for 4-10% of all EGFR mutants in non-small cell lung cancer (NSCLC). To date, more than one hundred different Ex20Ins mutations have been identified. With the notable exception of the rare A763_Y764insFQEA insertion (<1%), EGFR Ex20Ins mutations are clinically unresponsive to early generation EGFR inhibitors, the standard of care for NSCLC patients with EGFR Ex20Ins mutations is chemotherapy. Therefore, a significant unmet need remains requiring the development of an EGFR TKI agent that can more effectively target NSCLC with EGFR Ex20Ins mutations. Osimertinib is a newly developed EGFR TKI approved for the treatment of advanced NSCLC with EGFR T790M tumors. This work describes the potential of osimertinib and AZ5104, a circulating metabolite of osimertinib, in Ex20Ins tumours.

Using CRISPR-Cas 9 genome editing technology, we engineered cellular disease relevant models carrying the most prevalent Ex20Ins mutations, namely Ex20Ins D770_N771InsSVD (22%) or Ex20Ins V769_D770InsASV (17%).

Using these models, we show that osimertinib and AZ5104 inhibit signalling pathways and cellular growth of Ex20Ins CRISPR-Cas9 engineered cell lines in vitro. This translates into sustained tumor growth inhibition in vivo in both the Ex20Ins SVD (65%, p<0.001 & 95%, p<0.001 respectively at day 14) and Ex20Ins ASV (82%, p<0.001 & 95%, p<0.001 respectively at day 14) xenograft models when compared to the control group. Importantly in vivo osimertinib was dosed at exposures consistent with the 80 mg osimertinib clinical dose. Moreover, a dual EGFR blockade strategy combining osimertinib or AZ5104 with cetuximab (an EGFR antibody) improved the anti tumor effect further. We also describe the anti-tumor activity of osimertinib and AZ5104 using a series of patient derived xenograft models harbouring the rarer Ex20Ins H773-V774insNPH and M766-A767insASV mutations. In addition, we performed pharmacodynamic studies to explore the relationship between efficacy and target/pathway modulation. These studies establish a clear relationship between depth and duration of phospho-EGFR inhibition and anti tumor efficacy. Interestingly, we observed that downstream signalling molecules displayed a more transient inhibition than the phospho

EGFR signal. Two patients from the AURA Phase 2 osimertinib trials with plasma positive Ex20Ins (concurrent with Ex19del and T790M) had durable PFS responses of 6.4 and 13.9 months, supporting the premise that osimertinib has the potential to be clinically beneficial in tumors harboring Ex20Ins.

The work presented herein demonstrates that osimertinib has the potential to improve upon the current treatment options for NSCLC patients whose tumors harbour an Ex20Ins mutation, and warrants its further clinical investigation.

#2080

Polycomb-mediated disruption of an AR-feedback loop drives castration-resistant prostate cancer.

Ka-Wing Fong,1 Jonathan Zhao,1 Jung Kim,1 Shangze Li,1 Angela Yang,1 Bing Song,1 Laure Rittié,2 Ming Hu,3 Ximing Yang,1 Bernard Perbal,4 Jindan Yu1. 1 _Northwestern Univ., Chicago, IL;_ 2 _University of Michigan Medical School, Ann Arbor, MI;_ 3 _New York University School of Medicine, New York, NY;_ 4 _Université Côte d'Azur, CNRS, GREDEG., GREDEG, France_.

Androgen receptor (AR) is an androgen-stimulated transcription factor that critically promotes prostate cancer development and progression. In addition to its conventional role in transcriptional activation, AR also acts as a transcriptional repressor to directly inhibit target gene expression. Through meta-analysis of androgen-regulated expression microarray data, we nominated CCN3/NOV, a CCN family protein, as one of the top AR-repressed genes in prostate cancer, implicating that CCN3 may play a tumor suppressive role in prostate cancer development. Therefore, we sought to investigate the molecular functions of CCN3 and its roles in prostate cancer progression especially in castration-resistant prostate cancer (CRPC). We first showed that CCN3 protein physically interacts with AR. The CCN3 interaction domain was mapped in the N-terminal domain of AR which is also present in constitutively active AR variants. Using immunofluorescence microscopy as well as subcellular fractionation technique, we demonstrated that CCN3 sequesters AR or AR variants into the cytoplasm to inhibit AR nuclear translocation, which in turn suppresses AR chromatin targeting and transcriptional activation. However, this negative feedback loop between AR and AR-repressed gene CCN3is disrupted in CRPC through CCN3 persistent epigenetic silencing by the Polycomb group protein EZH2, rendering AR transcriptional activation and CRPC progression. Taking advantage of this repressive mechanism, we restored CCN3 in CRPC cell model, either through ectopic overexpression or treatment with EZH2 inhibitor, which results in strong suppression of CRPC cell proliferation and anchorage-independent cell growth. Consistently, application of EZH2 inhibitor remarkably reduced tumor size in a castrated mice model. Taken together, our data highlight CCN3 as a novel regulator of AR signaling and support the promise of EZH2-targeting agents in treating CRPC.

#2081

Robust activity of BLU-285, a potent and highly selective inhibitor of mutant KIT and PDGFRα, in patient-derived xenograft (PDX) models of gastrointestinal stromal tumor (GIST).

Yemarshet K. Gebreyohannes,1 Agnieszka Wozniak,1 Madelina-Elena Zhai,1 Jasmien Wellens,1 Jasmien Cornillie,1 Erica Evans,2 Alexandra K Gardino,2 Christoph Lengauer,2 Maria Debiec-Rychter,3 Raf Sciot,4 Patrick Schöffski1. 1 _Laboratory of Experimental Oncology, Department of Oncology, KU Leuven, and Department of General Medical Oncology, University Hospitals Leuven, Leuven Cancer Institute, Leuven, Belgium;_ 2 _Blueprint Medicines Corporation, Cambridge, MA;_ 3 _Department of Human Genetics, KU Leuven and University Hospitals Leuven, Leuven, Belgium;_ 4 _Department of Pathology, KU Leuven and University Hospitals Leuven, Leuven, Belgium_.

Objective: Patients with advanced and treatment-resistant GIST are treated with tyrosine kinase inhibitors (TKI) such as imatinib (IMA), sunitinib (SUN) or regorafenib (REG). Resistance to these TKI is mainly caused by the emergence of on-target secondary KIT mutations in exons 13 and 17. We tested the activity of BLU-285 (Blueprint Medicines), a potent and highly selective inhibitor of mutant KIT and PDGFRα, in three PDX models of primary and refractory GIST that respond differently to the approved TKI used in the treatment of GIST. Methods: NMRI nu/nu mice (n=93) were transplanted bilaterally with human xenografts UZLX-GIST3 (KIT: exon 11 p.W557_V559delinsF; IMA-sensitive), GIST2B (KIT: exon 9 p.A502_Y503dup, IMA dose-dependent sensitive) and GIST9 (KIT: exon 11 p.P577del; W557LfsX5; exon 17 D820G; IMA and SUN-resistant). We performed the following experimental comparisons: BLU-285 (10, 30 and 100 mg/kg/qd) vs. control and IMA (50 mg/kg/bid) [GIST3]; BLU-285 (10, 30 and 60 mg/kg/qd) vs. control and IMA (50 and 100 mg/kg/bid or SUN 40 mg/kg/qd) [GIST2B]; and BLU-285 (10 and 30 mg/kg/qd) vs. control, IMA (50 mg/kg/bid) or REG (30 mg/kg/qd) [GIST9]. Animals were dosed orally for 16 days. Activity was assessed by tumor volume measurement, histopathology and Western blotting of the KIT signaling pathway (WB). Mann Whitney U test was used for statistical analysis. A p value <0.05 was considered significant. Results: In all models, BLU-285 resulted in a dose dependent reduction of tumor volume, significant inhibition of proliferation (p < 0.005 compared to the control in all models, at all doses tested), and inhibition of KIT signaling as assessed by WB. In two models, BLU-285 led to a higher histologic response (graded as described previously by Antonescu et al. 2005), a significant increase of apoptosis (p < 0.005 compared to the control in both models, at all doses tested), and a pronounced decrease in MAPK phosphorylation as compared to the control. The activity of BLU-285 was similar (GIST3) or better (GIST2B and GIST9) than a standard dose of IMA. In the IMA-resistant model (GIST9), the anti-tumor effects of BLU-285 were significantly better those seen with either IMA or REG. BLU-285 was well tolerated at all administered doses. Conclusions: BLU-285, an investigational agent, has significant anti-tumor effects in GIST PDX models characterized by different KIT mutations and variable sensitivity to established TKI therapies. These data support the therapeutic rationale for the ongoing Phase I clinical trial being conducted by Blueprint Medicines (NCT02508532) and provide further evidence that BLU-285 has the potential to be an important treatment option for patients with PDGFRα or KIT-driven GIST.

#2082

NMS-E668, a potent and selective RET kinase inhibitor characterized by specificity towards VEGFR2 and high antitumor efficacy against RET-driven models.

Elena Ardini, Patrizia Banfi, Nilla Avanzi, Marina Ciomei, Paolo Polucci, Alessandra Cirla, Antonella Ermoli, Ilaria Motto, Elena Casale, Giulia Canevari, Cinzia Cristiani, Sonia Troiani, Federico Riccardi Sirtori, Nadia Amboldi, Dario Ballinari, Francesco Caprera, Eduard Felder, Arturo Galvani, Daniele Donati, Antonella Isacchi, Maria Menichincheri. _Nerviano Medical Sciences, Nerviano, Italy_.

RET, a receptor tyrosine kinase (RTK) expressed mainly in neural crest-derived tissues, plays a role in cell growth and differentiation and its physiological activation depends upon binding to the GDNF family. Genetic aberrations leading to constitutive RET activation are well-established as oncogenic events. Activating point mutations of RET, for example, are present in ca. 70% of medullary thyroid carcinoma patients including all hereditary cases, while RET gene rearrangements resulting in production of activated RET fusion proteins occur in approximately 10% of sporadic papillary thyroid carcinomas. More recently, recurring RET gene rearrangements have also been found in 1-2 % of lung adenocarcinomas and subsets of other solid tumors including colorectal and salivary gland carcinomas. Thus RET kinase represents an actionable therapeutic target in multiple clinical settings with high medical need. Consequently several small-molecule inhibitors targeting this kinase have been explored in clinical settings. A common feature of most advanced agents is their lack of selectivity and in particular their potent cross-reactivity against VEGFR2, an RTK whose inhibition is associated with serious, dose-limiting cardiovascular toxicity. Indeed, the high homology between the two kinases renders identification of ATP competitive compounds that selectively inhibit RET over VEGFR2 a highly challenging task. Here we describe the preclinical activity of NMS-E668, a potent and selective ATP-competitive RET inhibitor characterized by favorable activity, selectivity and ADME profiles. Biochemically, NMS-E668 has an excellent selectivity profile against a panel of >50 kinases, notably including >10-fold selectivity over VEGFR2. Selectivity of NMS-E668 for RET vs. VEGFR2 was confirmed in NIH-3T3 cells engineered to express activated forms of these kinases. NMS-E668 potently (IC50 circa 50 nM) and selectively inhibited proliferation of RET-dependent tumor cells, including TT medullary carcinoma cells harboring a RET C634W activating point mutation and LC2/ad lung carcinoma cells bearing the oncogenic fusion protein CCDC6-RET. NMS-E668 also potently inhibited IL3-independent growth of Ba/F3 cells expressing KIF5B-RET, the RET rearrangement that is most commonly found in lung adenocarcinomas. Cellular mechanism studies confirmed that NMS-E668 inhibits RET autophosphorylation and downstream signaling at doses consistent with antiproliferative activity. Tested in vivo against KIF5B-RET-driven Ba/F3 tumors, NMS-E668 displayed >90% tumor growth inhibition accompanied by target modulation following oral administration at 10 and 20 mg/kg, with prolonged tumor regressions observed at the higher dose. Thus NMS-E668, a potent and VEGFR2-sparing RET inhibitor is an innovative and highly promising candidate for further development.

#2083

Development of novel targeted adjuvant therapy for triple negative breast cancer.

Nidhi Bansal,1 Eduardo Farias,1 Veronica Gonzalez,2 Garry Nolan,2 Samuel Waxman1. 1 _Icahn School of Medicine at Mount Sinai, Manhattan, NY;_ 2 _Stanford University School of Medicine, Stanford, CA_.

There is an unmet clinical need for targeted adjuvant therapy in Triple Negative Breast Cancer (TNBC) to overcome its poor prognosis, short disease-free interval and metastatic dissemination. We previously reported that blocking interactions between the PAH2 domain of chromatin regulator Sin3 and Sin3 interaction domain (SID) containing proteins like PF1 and TGIF1 by SID decoys (peptides and small molecule, C16) decreased the cancer stem cell population, invasion, EMT, and metastases. This, programmed upregulation of retinoid signaling that sensitized TNBC cells to AM80, a novel clinically available RARα-specific agonist. Here we report preclinical investigations on effects of SID decoys and AM80 treatments on cellular heterogeneity, primary tumors, metastatic dissemination, minimum residual disease (MRD) and host microenvironment. Using CyTOF2, we made single cell measurements of markers of differentiation, proliferation and stemness in CSC-enriched 4T1 tumorspheres. Treatment with SID peptide decreased cell populations expressing nanog, sox2, vimentin and β-catenin with increase in γH2AX. Addition of AM80 in combination with C16, resulted in populations with increased expression of differentiation marker CD24 with decrease in vimentin, β-catenin and Ki-67. To interrogate the neo-adjuvant effects of C16-AM80 treatments, primary 4T1 tumors in Balb/c mice were treated with C16 and AM80 alone or in combination. Compared to DMSO, ~40 % decrease in tumor weight, 60% decrease in ALDH activity and 60% decrease in lung metastasis was seen in mice treated with C16-AM80 combination. In post-surgical adjuvant settings, in both 4T1 and MMTV-myc xenografts, we observed 100% disease-free survival and absence of macrometastasis in mice receiving minimally toxic adjuvant therapy with the C16-AM80 combination for 90 days. However, MRD was found consisting of a small number of single CK8+ cells which failed to form colonies when recovered from the bone marrow and selectively cultured in vitro. Upon stopping the treatments, 20-40% animals developed macrometastases within the first three months. To test the influence of C16-AM80 to condition the host microenvironment to prevent macrometastases mice received only pretreatment with C16 and AM80 followed by 4T1 cells injected in the tail vein. The percentage of parenchyma occupied by metastatic nodules were: DMSO = 50%; AM80 = 25%; C16 >10% and C16-AM = <<10%; the mitotic features were greatly reduce from 0-5 in DMSO to 0-1 per 400x/field in the C16-AM80 combination. The predominant effects were observed with C16 treatment, which was enhanced to a small degree in combination with AM80. These results suggest that C16 can potentially induce changes in the host microenvironment to prevent the colonization and metastatic growth of cancer cells in TNBC. Altogether, our preclinical studies merit expansion of a pre-clinical program for development of C16-AM80 combination as a targeted adjuvant therapy to treat TNBC.

#2084

Conformational activation and allosteric inhibition of SHP2 in RTK-driven cancers.

Michael G. Acker,1 Ying-Nan P. Chen,1 Matthew J. LaMarche,1 Ho Man Chan,1 Peter Fekkes,1 Jorge Garcia-Fortanet,1 Jonathan R. LaRochelle,2 Brandon Antonakos,1 Christine Hiu-Tung Chen,1 Zhuoliang Chen,1 Vesselina G. Cooke,1 Jason R. Dobson,1 Zhan Deng,1 Fei Feng,1 Brant Firestone,1 Michelle Fodor,1 Cary Fridrich,1 Hui Gao,1 Huai-Xiang Hao,1 Jaison Jacob,1 Samuel Ho,1 Kathy Hsiao,1 Zhao B. Kang,1 Rajesh Karki,1 Mitsunori Kato,1 Jay Larrow,1 Laura R. La Bonte,1 Gang Liu,1 Shumei Liu,1 Dyuti Majumdar,1 Matthew J. Meyer,1 Mark Palermo,1 Minying Pu,1 Edmund Price,1 Subarna Shakya,1 Michael D. Shultz,1 Kavitha Venkatesan,1 Ping Wang,1 Markus Warmuth,1 Sarah Williams,1 Guizhi Yang,1 Jing Yuan,1 Ji-Hu Zhang,1 Ping Zhu,1 Stephen C. Blacklow,2 Timothy Ramsey,1 Nicholas J. Keen,1 William R. Sellers,1 Travis Stams,1 Pascal D. Fortin1. 1 _Novartis Institutes for BioMedical Research, Cambridge, MA;_ 2 _Dana-Farber Cancer Institute, Boston, MA_.

The non-receptor protein tyrosine phosphatase (PTP) SHP2 is an important component of RTK signaling in response to growth factor stimulus and sits just upstream of the RAS-MAPK signaling cascade. The first oncogenic phosphatase to be identified, SHP2 is dysregulated in multiple human diseases including the developmental disorders Noonan and Leopard syndromes, as well as leukemia, lung cancer and neuroblastoma where aberrant activity of SHP2 leads to uncontrolled MAPK signaling. Cancer-associated activating mutations in SHP2 impart an "auto-on" state of the enzyme, boosting basal activity by shifting the equilibrium away from the auto-inhibited state. Reduction of SHP2 activity through genetic knockdown suppresses tumor growth, validating SHP2 as a target for cancer therapy. SHP099, a recently reported potent and selective allosteric inhibitor of SHP2, stabilizes the auto-inhibited form of SHP2 through interactions with the N-terminal SH2 and C-terminal PTP domains of the protein. SHP099 suppresses MAPK signaling in RTK amplified cancers resulting in suppressed proliferation in vitro and inhibition of tumor growth in mouse tumor xenograft models. Together, these data demonstrate the therapeutic potential of SHP2 inhibition in the treatment of cancer and other RAS/MAPK-linked diseases.

#2085

Development of a novel targeted therapy for malignant mesothelioma carcinoma by a midkine inhibitor.

Takuya Fukazawa,1 Yuitaka Maeda,2 Tomoki Yamatsuji,1 Munenori Takaoka,1 Masakazu Yoshida,1 Naomasa Ishida,1 Miki Iwai,1 Etsuko Yokota,1 Takuro Yukawa,1 Minoru Haisa,1 Noriko Miyake,1 Tomoko Ikeda,1 Nagio Takigawa,1 Jeffery Whitsett,2 Yoshio Naomoto1. 1 _Kawasaki Medical School, Okayama, Japan;_ 2 _Cincinnati Children's Hospital Medical Center, Cincinnati, OH_.

Malignant pleural mesothelioma is an aggressive tumor of mesenchymal origin and is increasing worldwide as a result of widespread exposure to asbestos. The median survival of patients with mesothelioma from time of diagnosis ranges between 1 and 2 years. The mortality is expected to increase, at least until 2020, which is mainly due to the long latency (30-50 years) of the disease. Despite considerable advances in the understanding of its pathogenesis and etiology, malignant mesothelioma remains largely unresponsive to standard modalities of cancer therapy. Thus, there is an urgent need for new therapeutic options for mesothelioma.Midkine (MDK) is a heparin-binding growth factor that is highly expressed in many malignant tumors, including lung cancers. We have previously reported that a MDK inhibitor, iMDK, suppresses non-small cell lung cancer expressing MDK without harming normal cells. Importantly, iMDK inhibits the PI3 kinase / Akt pathway and induces apoptosis in MDK expressing non-small cell lung cancer cells. In the present study, we have investigated the antitumor effect of iMDK against malignant mesothelioma both in vitro and in vivo. 48 hours after treatment, iMDK dose-dependently inhibited cell growth of MDK expressing malignant mesothelioma cells. iMDK also suppressed colony formation of MSTO-211H mesothelioma cells. TUNEL positive cells were significantly increased in MSTO-211H cells 48 hours after iMDK treatment in a dose-dependent manner, confirming the induction of apoptosis in mesothelioma cells by iMDK. Combination treatment of iMDK and Bcl-2 inhibitor ABT-263 is more effective than each drug alone in MSTO-211H mesothelioma cells. Moreover, systemic administration of iMDK significantly inhibited tumor growth in a mesothelioma xenograft tumor in vivo. Inhibition of MDK with iMDK provides a potential therapeutic approach for the treatment of malignant mesothelioma that is driven by MDK.

#2086

Dominant role of receptor tyrosine kinase in the activation of MAPK signaling in BRAF non-V600 mutant cancers.

Hiromichi Ebi, Hiroshi Kotani, Hidenori Kitai, Yuta Adachi, Seiji Yano. _Kanazawa Univ. Cancer Research Inst., Kanazawa, Japan_.

BRAF mutations are found in ~8% of all cancers. In addition to the commonest mutation in kinase domain at the V600, a wide range of other missense mutations (non-V600) have been reported. Non-V600 mutant BRAF protein was also shown to enhance MAPK signaling, suggesting the effectiveness of MEK inhibitors. However, the existence of feedback mechanism in MAPK singling and the roles of receptor tyrosine kinases (RTKs) in these cells are not fully understood. To interrogate this, BRAF non-V600 cell lines with intermediate/impaired kinase activity were treated with MEK inhibitor. RTKs involved in the upregulation of P-MEK following MEK inhibition in BRAF non-V600E mutant cell lines were determined by phosphor-RTK arrays. The effect of combinatorial inhibition of RTKs and MEK in BRAF non-V600 cancer was assessed in vitro and in vivo. By analyzing drug screen data, we first found cells with BRAF non-V600E mutation were less sensitive to MEK inhibition compared with BRAF V600E mutant cell lines and almost comparable to RAS/RAF wild-type cell lines. MEK inhibition did not induce P-MEK suppression and it resulted in significant upregulation of p-MEK compared to BRAF V600E mutant cell lines. Phospho-RTK arrays in the presence or absence of trametinib displayed basal phosphorylation of RTKs including EGFR, MET, and IGF1R, although the phosphorylation level was not consistently changed following MEK inhibition. Among these RTKs, treatment with the EGFR inhibitor erlotinib led to more complete suppression of P-ERK upon trametinib treatment. Furthermore, we found that erlotinib monotherapy downregulated P-ERK in BRAF non-V600 mutant cell lines in contrast that inhibition of RTKs had no effect on MAPK signaling in BRAF V600 mutant cells. While erlotinib treatment led to MAPK inhibition both in intermediate active and impaired kinase active BRAF mutant cells, p-MEK and p-ERK were more strongly suppressed in impaired type BRAF non-V600 mutant cells. The combination of EGFR inhibitor and MEK inhibitor suppressed cell growth and achieved tumor shrinkage in mice xenograft. These results suggest that RTKs, especially EGFR, are dominantly involved in the regulation of MAPK signaling in BRAF non-V600 cancer cells.

#2087

Pan-HER inhibitor, varlitinib, disrupts HER/ERK signaling and causes apoptosis in triple-negative breast cancer cells.

Chun-Yu Liu,1 Tzu-Ting Huang,1 Chun-Teng Huang,2 Hsiu-Ping Yang,1 Ling-Ming Tseng,1 Chung-Wai Shiau,3 Kuen-Feng Chen4. 1 _Taipei Veterans General Hospital, Taipei, Taiwan;_ 2 _Yang-Ming Branch of Taipei City Hospital, Taipei, Taiwan;_ 3 _National Yang-Ming University, Taipei, Taiwan;_ 4 _National Taiwan University Hospital, Taipei, Taiwan_.

Background:

Triple-negative breast cancer (TNBC), characterized by aggressive behavior and poor prognosis, represents an important clinical challenge because there is no well-established target therapy. Therefore, the identification and validation of a targeted therapy for TNBC is an urgent need. Molecular profiling studies have shown some TNBC tumors harboring aberrant epidermal growth factor receptor (EGFR) or human epidermal growth factor receptor (HER) signaling, suggesting therapeutic potential with EGFR inhibitors. Varlitinib (ASLAN001) is a small molecule reversible pan-HER inhibitor of EGFR (HER1), HER2 and HER4. To-date, varlitinib has been extensively investigated in several tumor types, including HER2 positive metastatic breast cancer. TNBC is known to demonstrate expression of EGFR. Since varlitinib also targets EGFR signaling, we hypothesized that it may also have antitumor efficacy in TNBC.

Methods:

MDA-MB-231 and MDA-MB-468 TNBC cell lines were used for in vitro studies. Cell viability was examined by MTT assay. Apoptotic effects were examined by flow cytometry and Western blot. Signal transduction pathways in cells were assessed by Western blot.

Results:

We first examined the protein expression of EGFR in a panel of TNBC cell lines. We identified MDA-MB-468 and MDA-MB-231 as EGFR-expressing cell lines. We found that varlitinib significantly inhibited cell viability and induced cell apoptosis in MDA-MB-468 cells but not in MDA-MB-231 cells. MDA-MB-231 cells carry the KRAS G13D mutation that drives downstream ERK signaling. We therefore examined the downstream signaling proteins of EGFR, including PI3K/Akt and MAPK/ERK signaling. Results showed that the protein levels of p-MEK and p-ERK were decreased in varlitinib-sensitive MDA-MB-468 cell lines, but there was no significant change in these phospho-proteins in varlitinib-resistant MDA-MB-231 cells. Furthermore, we found that ERK inhibition resensitized varlitinib-resistant cells to varlitinib-induced cell death. In addition, ectopic expression of ERK reduced the varlitinib-induced apoptosis on varlitinib-sensitive MDA-MB-468 cells. In addition, MDA-MB-468 cells are known to harbor p53-R273H gain-of-function mutation that may activate EGFR-signaling, whether the mutation is associated with varlitinib sensitivity needs further investigation.

Conclusions:

In this study, we identified TNBC as another tumor type that may be sensitive to varlitinib's antitumor activity through the inhibition of HER/MAPK signaling and subsequent increase in apoptotic activity.

#2088

The activity of the FGFR selective inhibitor Debio 1347 is correlated with high mRNA expression.

Franck Brichory,1 Anna Pokorska-Bocci,1 Paolo Nuciforo,2 Stefania Rigotti,1 Nathalie Lembrez,1 Grégoire Vuagniaux,1 Corinne Moulon,1 Anne Vaslin1. 1 _Debiopharm International SA, Lausanne, Switzerland;_ 2 _Vall d'Hebron Institute of Oncology, Barcelona, Spain_.

Dysregulation of the fibroblast growth factor receptor (FGFR) signaling pathway due to receptor overexpression, gene amplification, point mutations or fusions/chromosomal translocations is associated with cancer development and progression.

Debio 1347 (CH5183284) is an oral selective FGFR inhibitor (FGFR1, 2 and 3) currently in clinical development. The aim of this study was to investigate Debio 1347 activity in patient derived xenograft (PDX) mouse models harboring diverse FGFR alterations in multiple indications.

The trial was conducted in 66 PDX models of diverse histotypes selected according to their FGFR1, 2 and 3 alteration status. Debio 1347 was administered orally once daily at 40 up to 80 mg/kg for 10 to 22 consecutive days (N=3/group). Tumor volume was compared to the vehicle control group and measured by caliper twice weekly. Treatment response was determined by relative treatment-to-control ratios (ΔT/ΔC) of which a responding model was defined as ΔΤ/ΔC <0. PDX tumors were collected at the end of the treatment period and extensively characterized using FISH, nCounter Gene Expression and immunohistochemistry (IHC). Effect on downstream Dual Specificity Phosphatase 6 (DUSP6) signaling was also investigated by qPCR and RNA in situ hybridization (ISH).

Debio 1347 induced tumor regression in 33% of PDX models that exhibited a gene copy number gain and/or the presence of a FGFR fusion gene and/or an FGFR mutation. In addition, Debio 1347 treatment led to tumor regression in 29% of models that did not harbor any FGFR genetic alteration. In contrast, all models that responded to Debio 1347 were shown to display a high expression level (mRNA) of at least one FGFR gene. These findings suggest that high expression of at least one FGFR might be a better predictor of sensitivity to Debio 1347 than genetic alteration. Furthermore, response to Debio 1347 was associated with a decrease in DUSP6 mRNA levels, suggesting that it could be a reliable pharmacodynamic biomarker in clinical trials.

These results provide new mechanistic insights into the predictive sensitivity to Debio 1347 and will help refine patient selection for FGFR-targeted therapy.

#2089

Evaluation of fruquintinib, a potent and selective oral VEGFR inhibitor, in combination with targeted therapies or immune checkpoint inhibitors in preclinical tumor models.

Yongxin Ren, Qiaoling Sun, Jingwen Long, Shiming Fan, Renxiang Tang, Wei Zhang, Xuelei Ge, Jianxing Tang, Linfang Wang, Dongxia Shi, Hongbo Chen, Min Cheng, Weiguo Qing, Weiguo Su. _Hutchison MediPharma Ltd., Shanghai, China_.

The development of therapies targeting tumor angiogenesis, tumor driver gene alterations and tumor immune evasion has made tremendous advancement in improving overall survival. However, efficacy may be limited and resistance often develops rapidly when targeting a single axis of tumorigenesis. Therefore, it is worthwhile to explore rational combination of therapies based on tumor-specific features. Fruquintinib is a potent and selective oral VEGFR inhibitor currently in Phase III clinical trials for non-small-cell lung cancer (NSCLC) and colorectal cancer (CRC). We report here the evaluation of anti-tumor effect of fruquintinib in preclinical animal tumor models in combination with therapies targeting tumor driver gene alterations such as EGFR and c-MET or with immune checkpoints. In NSCLC xenograft models with EGFR activation such as activating mutations, gene amplification or protein overexpression, fruquintinib plus an EGFR tyrosine kinase inhibitor such as gefitinib or theliatinib (HMPL-309) was found to be more efficacious than either monotherapy. For instance, in PC-9 subcutaneous tumor model carrying EGFR exon 19 deletion, single agent treatment with fruquintinib at 2 mg/kg and gefitinib at 5 mg/kg produced the tumor growth inhibition (TGI) of 58% and 63%, respectively, while the combination treatment resulted in a TGI of 100% and tumor regression was observed in 11 of 16 mice treated with combinational therapy. In multiple xenograft models derived from lung cancer or renal cell cancer with c-MET activation (amplification or over-expression), addition of fruquintinib to a c-MET inhibitor savolitinib (AZD6094, HMPL-504) also improved the tumor growth inhibition substantially. At the end of the efficacy studies, CD31 and phosphorylation of EGFR, c-MET, AKT and ERK were analyzed with immunohistochemistry and western blotting method in tumor tissues. The results suggested that the enhanced anti-tumor effect in combination therapy could be attributed to the simultaneous blockade of cell signaling in tumor cells (EGFR or c-MET) and VEGFR suppression in the tumor microenvironment. Up-regulation of the immune inhibitory checkpoints induced by VEGF is one of the important mechanisms for tumor cells to escape immune surveillance. In a syngeneic murine tumor model, co-administration of fruquintinib and anti-PD-L1 antibody was found to provide improved anti-tumor effect compared to fruquintinib or anti-PD-L1 single agent alone. Studies to understand the mechanism responsible for the combination effect are under way. All combinations with fruquintinib described above were well tolerated. The efficacy observed in these models suggested that simultaneous blockade of tumor angiogenesis and tumor cell signaling or immune evasion may be a promising approach in improving treatment outcomes.

#2090

Targeting estrogen receptor negative breast cancer cells using diarylthiourea analogs of SHetA2.

Hongye Zou,1 Emily Ginn,1 Sabah M. Francis,2 Shengquan Liu,2 Maggie Louie1. 1 _Dominican University of California, San Rafael, CA;_ 2 _Touro University of California, Vallejo, CA_.

Breast cancer is one of the most common cancers that occur in women in the United States. Depending on the expression of estrogen receptor α (ERα), breast cancer can be classified as ER-positive or negative. ER-negative (ER-) breast cancers are often more aggressive and have poorer prognosis compared with the ER-positive (ER+) subtype. Consequently, there is a great need to develop more targeted therapeutic options for ER- breast cancer. One such compound is SHetA2, a flexible heteroarotinoid (flex-hets) that has been shown to inhibit growth of multiple cancer types including breast cancer. However, the clinical utility of SHetA2 is limited by its high lipophilicity (LogP). The goal of this study is to screen eleven 2nd generation analogs of SHetA2 with lower LogP values— SL1-22, 24, 27, 29, 30, 32, 36, 37, 38, 39, and 40— on different breast cancer cell lines to identify potential lead compounds that show the highest anti-growth activities against ER- breast cancer cells and further delineate the mechanism by which these compounds affect breast cancer cell growth. Results from this study demonstrate that SL1-38 [1-(3-chloro-4-methylphenyl)-3-(4-nitrophenyl)thiourea] and SL1-39 [1-(4-chloro-3-methylphenyl)-3-(4-nitrophenyl)thiourea], inhibit ER-negative breast cancer cells (MDA-MB-231, MDA-MB-453 and MDA-MB-468) effectively at micromolar concentrations. In order to understand the mechanisms of action, we evaluated the effects of SL1-38 and 39 on the expression of key cell cycle regulators. Our results show that these two analogs down-regulate the expression of cyclin A, cyclin B, cyclin D1, cyclin E and cdk2 and block S-phase progression. Taken together, these preliminary results suggest that SL1-38 and 39 may be further developed as anti-cancer agents for treating ER-negative breast cancer.

#2091

PRN1371, an irreversible, covalent inhibitor of FGFR1-4 exhibits sustained pathway inhibition in cancer cell lines.

Eleni Venetsanakos, Yan Xing, Natalie Loewenstein, J. Michael Bradshaw, Dane Karr, Jacob LaStant, Philip Nunn, Jin Shu, Abha Bommireddi, Jens Oliver Funk, David M. Goldstein, Stefani Wolff, Ken A. Brameld, Steven G. Gourlay. _Principia Biopharma, South San Francisco, CA_.

Introduction: Multiple human cancers harbor alterations in FGFRs that drive tumor growth, including mutations, translocations and amplifications. PRN1371 is an irreversible, covalent FGFR1-4 inhibitor that exhibits highly selective and sustained inhibition of FGFR which extends well beyond circulating drug concentrations in preclinical models. The duration of inhibition of the FGFR signaling pathway is dependent on protein turnover of FGFR, which may vary depending on the type of FGFR alteration. Thus, we set out to investigate whether the duration of target inhibition differs across cancer cell lines of various lineages harboring different FGFR alterations, including fusions and mutations. Furthermore, as FGFR inhibitors exhibit hyperphosphatemia via on-target pharmacology, we also investigated the duration of target inhibition in primary renal epithelial cells which are wild-type for FGFR.

Materials and Methods: Cancer cell lines from several lineages harboring different FGFR alterations were treated with increasing concentrations of PRN1371 in vitro for 1 hour, before washing out the compound. Cells were harvested at various time-points post-washout, protein lysates were generated and assessed for modulation of the downstream signaling pathway by western blot analysis.

Results: Dose-dependent inhibition of phospho-ERK was observed in the cancer cell lines tested in response to compound treatment for 1 hour in vitro. Dose dependent partial or full rebound of phospho-ERK back to baseline levels were detected in cancer cell lines after a prolonged period post-washout. In contrast, full rebound of phospho-ERK was observed at 1 hour post washout in response to a non-covalent inhibitor. Studies are on-going to assess duration of pathway inhibition in the primary renal proximal epithelial cells.

Conclusions: PRN1371 is a potent, highly selective irreversible inhibitor exhibiting sustained inhibition of FGFR signaling across cancer cell lines harboring different FGFR alterations. The duration of inhibition of the downstream signaling was comparable across cancer cell lines harboring different FGFR alterations, including mutations, fusions and amplification of FGFR and was prolonged when compared to a non-covalent inhibitor. A Phase 1 clinical trial of PRN1371 for the treatment of solid tumors harboring FGFR alterations is ongoing (NCT02608125).

#2092

A potent and selective RET inhibitor with efficacy in RET-driven mouse models of medullary thyroid carcinoma and lung adenocarcinoma.

Mandy Watson,1 Helen Small,1 Ben Acton,1 Habiba Begum,1 Samantha Hitchin,1 Allan Jordan,1 Paul Kelly,1 Rebecca Newton,1 Ian Waddell,1 Gina Paris,2 Donald Ogilvie1. 1 _Cancer Research UK Manchester Institute, Manchester, United Kingdom;_ 2 _Champions Oncology Inc., Baltimore, MD_.

Background: The aim of this CRUK-MI Drug Discovery project is to deliver a RET-selective inhibitor for the treatment of cancers with RET activating mutations, which include 1-2% of lung adenocarcinomas and medullary thyroid cancers (MTC). Recent data supports the hypothesis that the efficacy of vandetanib and cabozantinib, clinically approved multi-kinase inhibitors, is limited by toxicities associated with potent activity against KDR. Therefore, a RET-selective inhibitor would represent a best-in-class agent for the treatment of these cancers.

Methods: We have established a robust screening cascade to develop a potent, selective RET inhibitor and developed several in vivo models to evaluate compound PKPD and antitumor efficacy. Tumor growth inhibition and PKPD studies were carried out in BaF3 mouse allograft models overexpressing KIF5B-RET or RETV804M and other disease relevant models, including an MTC xenograft (MZ-CRC-1), a KIF5B-RET lung cancer patient derived xenograft (PDX) model (CTG-0838, Champions Oncology) and a lung cancer control xenograft (Calu-6).

Results: Two orally bioavailable compounds displaying nanomolar RET potency and >10 fold selectivity over KDR in cellular assays were selected from the lead series and further evaluated in our in vivo PD and efficacy models. Both compounds demonstrated efficacy in the BaF3 KIF5B-RET driven model (71% and 103% tumor growth inhibition (TGI), respectively), accompanied by reduced levels of pRET in the tumor tissue. Following further lead optimisation; a compound displaying an improved DMPK profile and additional nanomolar potency versus the gatekeeper mutation (RETV804M) was identified and accelerated through our DMPK/in vivo cascade. We consider this additional activity versus RETV804M beneficial since mutations at the gatekeeper residue in other tyrosine kinases (e.g. EGFR) have been shown to mediate acquired drug resistance in the clinic. This compound demonstrated significant TGI of 58% and 82% respectively in the BaF3 KIF5B-RET and BaF3 RETV804M allograft models. Moreover, tumor growth in the lung cancer PDX model was strongly inhibited (95% TGI) and tumor regression induced in the MTC xenograft model (109% TGI). As expected, this potent and selective RET inhibitor was not active in the Calu-6 model, which is sensitive to KDR inhibition, whereas vandetanib, a potent KDR inhibitor, significantly inhibited tumor growth (84% TGI). Additional in vitro and in vivo DMPK analyses further support the nomination of this compound as a preclinical candidate.

Conclusions: The identification of selective RET inhibitors with significant in vivo activity and minimal toxicity may overcome the limitations of the currently available clinical compounds. We have made considerable progress towards this goal and show here the compelling data supporting our nomination of a preclinical development compound.

#2093

HEC73543 is a novel potent, selective Flt3 receptor tyrosine kinase (RTK) Inhibitor for the treatment of refractory acute myeloid leukemia (AML).

Cliff Cheng. _HEC Pharm Group, Dongguan, China_.

Background: Adults acute myeloid leukemia (AML) account for ~30% of leukemia and ~40% of leukemia-related deaths. FLT3 is a receptor tyrosine kinase that is normally expressed on immature hematopoietic cells and functions in the development of both stem cells and the immune system. FLT3 is the most frequently mutated gene in AML, with an estimated 30 % of AML patients harboring FLT3 mutations. FLT3-ITD is a common mutation in AML and correlates with a poor prognosis and higher risk of relapse of AML patients.

Methods and Results: HEC73543 is a potent, orally bioavailable small molecule tyrosine kinase inhibitor (TKI) against FLT3 which have been implicated in acute myeloid leukemia (AML) pathogenesis. In cell-based assays, HEC73543 showed IC50s = 1.5 and 1.4 nM against MV-4-11 and MOLM-13 cells in proliferation (both cell lines are FLT3-ITD+). Induction of apoptosis following treatment of FLT3-ITD leukemic MV-4-11 cells for 48 h was assayed by flow cytometry. In MV-4-11 cells, a concentration of 10 nM HEC73543 induced 64.8% of the cells to undergo apoptosis and had a dose dependent increase. When single dosed orally (4.5mg/kg) to MV-4-11 tumor-bearing nude mice, HEC73543 potently inhibited the phosphorylation of FLT3 and its downstream signaling kinases STAT5, AKT and ERK1/2 for up to 8 hours in tumor tissues. The antitumor activity of HEC73543 was evaluated using an FLT3-ITD subcutaneous tumor xenograft model (MV-4-11 and MOLM-13) in athymic mice. Groups of 8 tumor-bearing mice were treated with vehicle alone or with HEC73543 administered orally at 0.5, 1.5, or 4.5mg/kg/day. The compound was well tolerated and no significant body weight loss or lethality was observed. The antitumor activity of HEC73543 was dose dependent. At the 1.5 mg/kg/day dose level all animals had a complete regression (CR) in MV-4-11 model, and the CR induced by 4.5mg/kg/day in MOLM-13 model. HEC73543 can significant prolong mice survival time at 1 mg/kg/day dose level in MOLM-13 systemic xenograft model. We have studied cytotoxic interactions of HEC73543 with conventional antileukemic agents cytarabine using two leukemia cell lines carrying FLT3-ITD (MOLM-13, MV-4-11) The combination of HEC73543 with cytarabine produced additive effects in MV-4-11 and MOLM-13 cell lines. HEC73543 displayed excellent oral bioavailability and desirable drug exposures in mice, rats, dogs and monkeys.

Conclusions: Together, HEC73543 exhibits potent target inhibition and efficacy in FLT3-ITD models suggests that this compound may have a therapeutic benefit for patients with FLT3-ITD leukemia.

#2094

Dual inhibition of FLT3 and Src pathways by ON150030, a type 1 inhibitor, as a novel strategy for relapsed and refractory AML therapy.

Helya Ghaffari, M.V. Ramana Reddy, Stephen C. Cosenza, Rodrigo Vasques del Carpio, E. Premkumar Reddy. _Icahn School of Medicine at Mount Sinai, New York City, NY_.

Approximately one third of patients suffering from Acute Myeloid Leukemia harbor a FLT3 Internal tandem duplication mutation (FLT3-ITD). When mutated, this receptor tyrosine kinase increases the activity of pathways for proliferation and blocks apoptosis. Quizartinib is a 2nd generation FLT3 inhibitor that inhibits FLT3-ITD in AML, but has a median duration response of 12.1 weeks. Studies revealed a secondary mutation in FLT3 at the aspartate of codon 835 (D835X) is responsible for relapse. The D835 substitution renders FLT3 constitutively active. Type 2 inhibitors like Quizartinib bind to FLT3 in its inactive state and fail to inhibit FLT3-ITD harboring a D835 mutation. Here, we tested the utility of ON150030, developed by our group, as a novel therapeutic agent to treat AML. Structural studies suggest ON150030 binds to the active form of FLT3 (Type 1 inhibitor) so mutations such as D835X do not affect the inhibitory activity of the compound. In vitro kinase assays demonstrate that ON150030 potently inhibits Wildtype and FLT3-D835Y forms, while Quizartinib fails to inhibit FLT3-D835Y. Additionally, ON150030 demonstrated time and temperature dependent inhibition of FLT3, suggesting that the compound is an irreversible inhibitor of FLT3. Biological studies reveal that ON150030 specifically inhibits the growth of MV4-11 cells harboring the FLT3-ITD mutation (GI50: 10nM). Western blot analysis demonstrates that MAPK and PI3K/AKT pathways in these cells are inhibited with increasing dose of ON150030. The JAK independent phosphorylation of STAT5 seen in the context of FLT3-ITD is also reduced in response to ON150030. Future goals are to introduce FLT3 and its various mutant isoforms into the mouse myeloid cells (32Dcl3) and examine how it affects proliferation and differentiation, and then compare the effects of ON150030 and Quizartinib. In addition to strongly inhibiting FLT3, ON150030 inhibits SRC, which was shown to induce resistance to targeted therapies in several leukemias including AML. We will introduce SRC into the 32D-FLT3-ITD cell lines sensitive to ON150030 and test whether these cells retain their sensitivity to the drug. Next, we will perform cytotoxicity and biochemical assays on patient-derived primary AML cells using ON150030. Mouse xenograft models will be used to determine if ON150030 synergizes with standard chemotherapy agents and inhibits cancer progression in vivo. At the conclusion of this project, we hope to demonstrate that ON150030 can be used in combination therapies in all AML patients harboring a FLT3 mutation, and result in sustained remission of disease.

#2095

E7090, a novel and selective FGFR inhibitor, for the treatment of cholangiocarcinoma cells harboring FGFR2-fusion genes.

Saori W. Miyano,1 Yasuhito Arai,2 Junji Matsui,1 Tatsuhiro Shibata3. 1 _Eisai Co., Ltd, Tsukuba-shi, Ibaraki, Japan;_ 2 _Division of Cancer Genomics, National Cancer Center Research Institute, Tokyo, Japan;_ 3 _Laboratory of Molecular Medicine, Human Genome Center, The Institute of Medical Science, The University of Tokyo, Division of Cancer Genomics, National Cancer Center Research Institute, Tokyo, Japan_.

Genetic abnormalities (gene fusion, mutation and amplification) of fibroblast growth factor receptor (FGFR) family members are known to cause constitutive activation of their signaling pathways, which play an important role in proliferation, survival and migration of cancer cells, as well as tumor angiogenesis. Several recent studies have identified that FGFR2 gene fusions were found in about 10% of intrahepatic cholangiocarcinoma and acted as oncogenes. E7090, an orally available FGFR1, 2 and 3 inhibitor, whose chemical-structure and basic kinase inhibitory profile have been disclosed at AACR2015, is currently under a first-in-human study (NCT02275910). The antitumor activity of E7090 against NIH3T3 and human cholangiocarcinoma cells harboring FGFR2-fusion genes was investigated. NIH3T3 cells stably infected of five FGFR2-fusion genes (FGFR2-AHCYL1, FGFR2-BICC1 type1, FGFR2-BICC1 type2, FGFR2-TXLNA, or FGFR2-KCTD1), all of which were discovered in human intrahepatic cholangiocarcinoma patients, were successfully established and exhibited anchorage-independent cell growth in soft agar assay. E7090 revealed an inhibitory activity on soft agar colony formation of these cells with IC50 values of 0.9 to 17.3 nmol/L in a dose dependent manner. E7090 also reduced activation of downstream pathways of FGFR signaling (MAPK and STAT3) in these cells. Subcutaneous transplantation of FGFR2-BICC1 type2, FGFR2-TXLNA, or FGFR2-KCTD1 expressing cells into immune-deficient mice produced tumor growth, and then oral administration of E7090 at 12.5, 25 and 50 mg/kg showed significant tumor growth inhibition. Finally, antitumor activity of E7090 was examined in a patient derived xenograft (PDX) model of human cholangiocarcinoma harboring FGFR2-BICC1 fusion gene. E7090 also showed dose-dependent antitumor activity and more than 50% of tumor regression was achieved at a dose of 50 mg/kg. In conclusion, E7090 showed potent antitumor activity against FGFR2-fusion genes found in intrahepatic cholangiocarcinoma both in vitro and in vivo models, suggesting that E7090 may provide therapeutic opportunities for cholangiocarcinoma harboring FGFR2-fusion genes.

#2096

Bozitinib, a highly selective inhibitor of cMet, demonstrates robust activity in gastric, lung, hepatic and pancreatic in vivo models.

Joe Shih,1 Boyu Zhong,1 Hepeng Shi,2 David Xue,2 Gavin S. Choy,3 Sanjeev Redkar3. 1 _Crown Bioscience, Taicang City, Jiangsu Province, China;_ 2 _Beijing Pearl Biotechnology Limited Liability Co., Chaoyang District, Beijing, China;_ 3 _CBT Pharmaceuticals, Inc, Santa Clara, CA_.

Background: cMET is a receptor tyrosine kinase that is located on the cell surface and is activated by the binding of its ligand, hepatocyte growth factor (HGF). In cancer cells, MET can be aberrantly active and cause abnormal signaling, which leads to tumor growth, angiogenesis, and metastasis. In vitro studies have demonstrated that bozitinib (CBT-101, PLB-1001) is a highly selective and specific inhibitor (8 nM) of tumor cell proliferation.

Methods: In-vivo PD studies of gastric (MKN45), lung (LUM858, LU1901, LU2503), hepatic (LIM0612, LIM0801), and pancreatic (KP4) were evaluated. These models covered both the HGF-dependent and HGF-independent mechanisms. Among these models, LUM858, LU1901, LU2503, LIM0612 and LIM0801 are PDX models. In particular, in the LU1901 model, bozitinib (BT) was compared to capmatinib (INC280). Groups included: BT at 1, 3 and 10 mg/kg QD×21 and INC280 at 1, 3, and 10 mg/kg QD×21 and 10 mg/kg BID×21 via IG, CDDP 5 mg/kg, Q7D×3 as a positive control via IP and the vehicle control (QD×21 via IG). Each group (n=8 mice) and the tumor volume was evaluated on D21.

Results: In MKN45, LU2503, LIM0612 and LIM0801, the effect of BT seemed superior than that of crizotinib; in LUM858, its effect was higher than that of erlotinib; in LU1901, its effect was higher than that of crizotinib and INC280. In the LU1901 model, the strongest activity was observed at BT 10 mg/kg with a T/C ratio of 2%, compared to an equi-dose of INC280 (T/C of 22%). All doses of BT and INC280 were well tolerated; no mouse experienced weight loss. In MKN45 model, BT showed a PK/PD correlation and dose-dependence. BT inhibited the phosphorylation of c-Met protein; the rate of target inhibition exceeded 90% at >7 mg/kg. The plasma concentration for BT decreased over time with a significant decrease 16h after its administration, conferring at least 16h of phosphorylation inhibition of the c-Met protein.

Conclusions: In conclusion, BT was well-tolerated, with no animal death nor major weight loss. The in vivo experiments demonstrated that BT is a viable candidate with effective anti-tumor activities. BT is currently under evaluation in cMet dysregulated NSCLC (NCT02896231) with additional trials planned.

#2097

A novel J-series prostamide induces ER stress-mediated apoptosis and upregulates ER oxidoreductase 1 alpha (ERO1α) in human colon cancer cells.

Hussam M. Albassam, Daniel A. Ladin, Rukiyah Van Dross. _East Carolina University, Greenville, NC_.

Colon cancer is the third most common cancer and the third leading cause of cancer-related death in the United States. The endoplasmic reticulum (ER) is a cellular organelle responsible for protein synthesis and oxidative folding. ER stress occurs when the protein folding load exceeds the protein folding capacity. Low levels of ER stress promote survival while excessive ER stress causes cell death. An important regulator of the cytotoxic ER stress pathway is the transcription factor, C/EBP homologous protein 10 (CHOP10). It has been shown that CHOP10 regulates the transcription of ER oxidoreductase 1α (ERO1α). ERO1α promotes ER luminal oxidation and under conditions of excessive ER stress releases H2O2 into the cytoplasm, resulting in oxidative stress-mediated apoptosis. Previous studies from our laboratory showed that 15deoxy, Δ12,14 prostamide J2 (15d PMJ2) induced ER stress-mediated apoptosis, leading to a reduction in cell viability in tumorigenic keratinocytes and melanocytes. In addition, 15d PMJ2-induced ER stress-apoptosis was decreased in the presence of ER stress inhibitors, 4-phenylbuterate (PBA) and salubrinal. However, the specific pathway involved in 15d PMJ2-induced ER stress-apoptosis have not been identified. In this study, we hypothesize that 15d PMJ2 causes ER stress-mediated apoptosis in colon tumor cells by activating CHOP10 and its downstream transcriptional target, ERO1α. To examine the anti-proliferative effect of 15d PMJ2, tumorigenic colon cells (HCT116) and non-tumorigenic colon cells (FHC) were treated with different concentrations of 15d PMJ2 or 10µM thapsigargin (TG) for 24 hours and cytotoxicity was measured by lactate dehydrogenase (LDH) assay. A significant increase in cell death was observed in HCT116 cells treated with 5µM 15d PMJ2 and this cytotoxic effect was 3-fold greater in HCT116 compared to FHC cells. Apoptotic measurements showed a significant increase in caspase 3/7 activity and annexin-V positivity following 15d PMJ2 treatment. The expression of CHOP10 was significantly enhanced in the 15d PMJ2-treated group. In addition, 15d PMJ2-induced apoptosis was decreased in the presence of ER stress inhibitors, PBA and salubrinal, suggesting that ER stress is essential for 15d PMJ2-induced apoptosis. Western blot analysis revealed an increase in ERO1α protein expression following 15d PMJ2 treatment. Apoptosis measurements showed a significant inhibition in 15d PMJ2-mediated apoptosis in the presence of ERO1α inhibitor, EN460, suggesting that ERO1α is important for 15d PMJ2-induced apoptosis. These findings suggest that 15d PMJ2-induced apoptosis is mediated via the ER stress pathway. CHOP10 and its transcriptional target, ERO1α, play a potential role in 15d PMJ2-induced ER stress-apoptosis, suggesting that 15d PMJ2 could be a potential anti-neoplastic agent with a unique mechanism for colon cancer.

#2098

NVP-FGF401, a first-in-class highly selective and potent FGFR4 inhibitor for the treatment of HCC.

Diana Graus Porta,1 Andreas Weiss,1 Robin A. Fairhurst,1 Markus Wartmann,1 Christelle Stamm,1 Flavia Reimann,1 Alexandra Buhles,1 Jaqueline Kinyamu-Akunda,2 Dario Sterker,1 Masato Murakami,1 Youzhen Wang,3 Jeffrey Engelman,3 Francesco Hofmann,1 William R. Sellers3. 1 _Novartis, Basel, Switzerland;_ 2 _Novartis, East Hanover, NJ;_ 3 _Novartis, Cambridge, MA_.

Hepatocellular carcinoma (HCC) is the seventh most common cancer worldwide and the third leading cause of cancer-related death. Sorafenib is the only targeted agent to show a marginal improvement in overall survival (OS) for patients with advanced HCC. Recent data have implicated aberrant activation of the FGF19-FGFR4/KLB axis as the driver of certain forms of HCC, making this pathway a novel therapeutic target in this disease. The first evidence for this is the finding that aberrant expression of FGF19, as a consequence of gene amplification and other not yet known mechanisms, occurs in subsets of HCC's and HCC cell lines leading to constitutive FGFR4 activation. In this setting, conditional knock down of FGF19, as well as its receptors FGFR4 and KLB, suppresses proliferation of HCC cell lines, supporting the notion that FGF19 activates FGFR4 in an autocrine fashion. Secondly, in transgenic mouse models, FGF19 produced by non-tumor cells at an ectopic site (skeletal muscle) acts in a paracrine fashion on the liver hepatocytes leading to liver dysplasia and HCC. In these mice, tumorigenesis is abolished in an FGFR4 null background, as well as upon treatment with anti-FGF19 and anti-FGFR4 blocking antibodies. Thus, we anticipate that targeted therapies aimed at blocking the FGFR4 pathway might be efficacious in subsets of HCC's. We have identified and developed NVP-FGF401, a first in class, highly selective and potent FGFR4 inhibitor that is currently in PhI/II clinical testing. NVP-FGF401 binds in a reversible covalent manner to the FGFR4 kinase domain and it inhibits FGFR4 with an IC50 of 1.1 nM. In biochemical assays, it shows at least 1,000 fold selectivity against of panel of 65 kinases and in a kinome wide scan, consisting of 456 kinases, FGFR4 was the only target of NVP-FGF401. In xenograft animal models in vivo, NVP-FGF401 showed a consistent pharmacokinetic / pharmacodynamic (PK/PD) relationship with phospho-FGFR4 over total FGFR4 (p/tFGFR4) levels in tumor robustly inhibited in a dose dependent manner. The data support a lowest observed trough concentration (Ctrough) driven PD/efficacy relationship. The anti-tumor activity was confirmed across several xenograft animal models, as well as in patient-derived tumor xenografts (PDX) established in mice. The excellent drug-like properties of NVP-FGF401 drove us to test its efficacy in HCC patients in a PhI/II study, being the first selective FGFR4 inhibitor to ever enter into clinical trials (NCT02325739).

#2099

In vitro characterization of the effect of nazartinib against non-small cell lung cancer activating clinically relevant EGFR mutants.

Keita Masuzawa, Hiroyuki Yasuda, Toshiyuki Hirano, Shigenari Nukaga, Hanako Hasegawa, Junko Hamamoto, Katsuhiko Naoki, Kenzo Soejima, Tomoko Betsuyaku. _Keio University, School of Medicine, Shinjuku-ku, Tokyo, Japan_.

Purpose: Multiple EGFR-TKIs are available and under development to treat patients with lung cancer harboring EGFR mutations. Nazartinib is one of the 3rd generation EGFR-TKIs targeting EGFR T790M as well as common mutations such as L858R and exon 19 deletions. Today, there is no clear guideline regarding which EGFR-TKIs should be used for which mutations. The purpose of this study is to clarify which EGFR-TKIs including nazartinib are best for each EGFR mutation, especially exon 20 insertion mutations using in vitro modeling.

Experimental design: We evaluated drug sensitivity and downstream signaling of human lung cancer cell lines (PC9, H3255, H1975, PC9ER, BID007) and Ba/F3 cells harboring multiple types of EGFR mutations for 1st- (erlotinib), 2nd- (afatinib) and 3rd- (osimertinib and nazartinib) generation EGFR-TKIs by MTS assay and western blotting. An in vitro model of mutation specificity was created by calculating the ratio of IC50 values between mutated and wild type EGFRs.

Results: The model of mutation specificity identified a wide therapeutic window of each EGFR-TKIs for exon 19 deletions and L858R. On the other hand, osimertinib and nazartinib have wide therapeutic window for T790M positive mutations in human cell lines and Ba/F3 cells. In human cell lines and Ba/F3 cells harboring exon 19 deletions or L858R, afatinib dramatically inhibited the phosphorylation of EGFR, AKT, and ERK. Afatinib and 3rd generation EGFR-TKIs, osimertinib and nazartinib, effectively inhibited the phosphorylation of EGFR, AKT, and ERK in T790M positive cells. For EGFR exon 20 insertion mutations, althogh afatinib, osimertinib and nazartinib effectively inhibited the phosphorylation of EGFR, AKT, and ERK in several exon 20 insertion mutations, osimertinib and nazartinib were potent and presented a wide therapeutic window.

Conclusion: Nazartinib as well as osimertinib has wide therapeutic windows for exon 19 deletions, L858R, T790M and some exon 20 insertions .

#2100

Selective inhibition of FGFR4 by INCB062079 is efficacious in models of FGF19- and FGFR4-dependent cancers.

Phillip C.C. Liu, Liang Lu, Kevin Bowman, Matthew C. Stubbs, Liangxing Wu, Darlise DiMatteo, Sindy Condon, Ronald Klabe, Ding-Quan Qian, Xiaoming Wen, Paul Collier, Karen Gallagher, Michael Hansbury, Xin He, Bruce Ruggeri, Yan-ou Yang, Maryanne Covington, Timothy C. Burn, Sharon Diamond-Fosbenner, Richard Wynn, Reid Huber, Wenqing Yao, Swamy Yeleswaram, Peggy Scherle, Gregory Hollis. _Incyte, Wilmington, DE_.

Aberrant signaling through Fibroblast Growth Factor Receptors (FGFR) has been reported in multiple types of human cancers. FGFR4 signaling contributes to the development and progression of subsets of cancer: in approximately 10 percent of hepatocellular carcinoma (HCC), genetic amplification of FGF19, encoding an endocrine FGF ligand that activates FGFR4-KLB receptors, has been reported. In models with this alteration, FGF19-FGFR4 signaling is oncogenic and antagonism of the FGF19-FGFR4 axis has been shown to be efficacious suggesting that selective targeting of FGFR4 may be an effective strategy for malignancies with FGFR4 activation.

We describe the preclinical characterization of INCB062079 a potent and selective inhibitor of the FGFR4 kinase. In biochemical assays INCB062079 inhibited FGFR4 with low nM potency and exhibited at least 250-fold selectivity against other FGFR kinases and greater than 800-fold selectivity against a large kinase panel. This selectivity derives from the ability of INCB062079 to bind irreversibly to Cys552, a residue within the active site of FGFR4 that is non-conserved among other FGFR receptors. Covalent binding of INCB062079 to Cys552 was demonstrated using a LC/MS/MS-based proteomic analysis that confirmed specificity for the target Cys. In assays using HCC cells with autocrine production of FGF19, INCB062079 inhibited the autophosphorylation of FGFR4 and blocked signal transduction by FGFR4 to downstream markers of pathway activation. Cancer cell lines that have amplification and expression of FGF19 are uniquely sensitive to growth inhibition by INCB062079 (EC50 less than 200 nM) compared with HCC cell lines or normal cells without FGF19-FGFR4 dependence (EC50 > 5000 nM) confirming selectivity for FGFR4. In vivo, oral administration of INCB062079 inhibited the growth and induced significant regressions of subcutaneous xenograft tumors dependent upon FGFR4 activity at doses that were well-tolerated (10-30 mg/kg BID) and did not result in a significant increase in serum phosphate levels which is observed with FGFR1/2/3 inhibition. Suppression of tumor growth correlated with pharmacodynamic inhibition of FGFR4 signaling. Collectively, these preclinical studies demonstrate that INCB062079 potently and selectively inhibits models of FGF19-FGFR4-dependent cancers in vitro and in vivo, supporting clinical evaluation in patients harboring oncogenic FGFR4 activation.

#2101

**Ormeloxifene suppresses the growth of prostate tumor** via **inhibition of β-catenin induced AR signaling.**

Aditya Ganju, Bilal Bin Hafeez, Mohammed Sikander, Vivek Kumar Kashyap, Murali Mohan Yallapu, Subhash C. Chauhan, Meena Jaggi. _Univ. of Tennessee Health Science Ctr., Memphis, TN_.

Background: Prostate cancer (PrCa) first manifests as an androgen-dependent disease and can be treated with androgen-deprivation therapy. Despite the initial success of androgen ablation therapy, resistance to anti-androgen therapy displays by progression to hormone refractory (androgen-independent) advanced stage PrCa which is primary cause of patient's death. Main underlying cause for the onset of hormone refractory cancer is ligand independent activation of AR signaling in PrCa cells. It has been shown that β-catenin acts as a non-androgen activator of AR which enhances AR transactivation in PrCa cells. Thus, identification of agents with excellent pharmacokinetics and pharmacodynamics parameters that can inhibit ligand independent activation of AR signaling might be highly useful for the treatment of advanced stage PrCa. Herein, we identified a synthetic molecule, ormeloxifene (ORM), which efficiently represses β-catenin mediated ligand independent activation of AR signaling, thus, inhibits growth and metastatic features of PrCa cells.

Methods: Androgen-refractory but AR positive PrCa cell (C4-2) was used as an in vitro and in vivo model systems. Effect of ORM on AR and PSA protein levels was determined by Western blot analysis. Effect of ORM treatment was analyzed on AR and PSA luciferase activities by transiently transfecting the C4-2 cells by AR and PSA luciferase plasmids. Renilla construct was used as an internal control. C4-2 cells nuclear and cytoplasmic lysates were prepared using Active Motif kit. Immunoprecipitation analysis was performed to determine if ORM inhibits physical interaction of β-catenin with AR. Therapeutic efficacy of ORM was evaluated in cell lines and PrCa xenograft mouse models.

Results: ORM dose-dependently (10, 15 and 20 µM) inhibited the protein levels of AR and its downstream target protein PSA. ORM (10 and 20μM) treatment also inhibited AR transactivation as determined by decreased promoter activities of AR and its target gene PSA. ORM (10 and 20μM) treatment inhibited protein levels of nuclear β-catenin and physical interaction of β-catenin with AR in PrCa cells. ORM administration dose- dependently (intra-peritoneal; 100 and/or 500µg/mouse; thrice/week) significantly (P<0.01) inhibited growth of C4-2 cells derived xenograft tumors in athymic nude mice. ORM treatment significantly (P<0.01) inhibited the expressions of nuclear AR and β-catenin expressions in xenograft tumor tissues. These ORM treated mice did not show any apparent toxicity in our study.

Conclusion: Our study demonstrates that ORM is a potent inhibitor of β-catenin-mediated activation of AR signaling. Based on its safety profile, ORM might be an ideal candidate for repurposing to treat advanced stage PrCa alone or in combination with other therapies.

#2102

ONC201 targets AR and AR-V7 signaling pathways, reduces PSA and synergizes with everolimus in castration resistant prostate cancer.

Avital Lev, Amriti R. Lulla, David T. Dicker, Wafik S. El-Deiry. _Fox Chase Cancer Ctr., Philadelphia, PA_.

Androgen receptor (AR) signaling plays a key role in prostate cancer progression; thus Androgen Deprivation Therapy (ADT) is a mainstay therapy for patients with advanced prostate cancer. However, in most cases the tumor becomes androgen-independent and resistant to ADT with patients ultimately progressing to metastatic castration resistant prostate cancer (mCRPC). Constitutively activated AR splice variants (AR-Vs) have emerged as major mediators of resistance to AR-targeted therapy and progression of mCRPC, representing an important therapeutic target for mCRPC. Out of at least 15 AR-Vs described thus far, AR-V7 is the most abundant in prostate cancer and its expression correlates with ADT resistance. ONC201 is the founding member of the imipridone class of small molecules that induces apoptosis in a variety of tumor types tested. It is currently in Phase I/II clinical trials for advanced solid tumors and hematological malignancies, including mCRPC with encouraging activity observed in patients in early clinical testing (Stein et al., ASCO, 2016). We demonstrate here that ONC201 treatment induces apoptosis in both castration-resistant and –sensitive human prostate cancer cell lines. Furthermore, an in-vitro synergistic effect is observed with ONC201 and FDA approved drugs for prostate cancer such as enzalutamide, docetaxel and everolimus. Mechanistically, we found that ONC201 inhibits both AR and AR-V7 signaling pathways. Analysis of AR+ cell lines stimulated with DHT showed a significant decrease in both RNA and protein levels of AR, AR-V7, PSA and other AR-target genes following ONC201 treatment. Ongoing experiments are aimed at better understanding the molecular and cellular mechanisms that enable ONC201 to inhibit the AR signaling pathway. In-vivo studies with AR-negative hormone refractory prostate cancer xenografts demonstrate single agent ONC201 anti-tumor efficacy. Ongoing studies are further evaluating the in-vivo efficacy of ONC201 as a single agent or in combination with enzalutamide and everolimus using mouse models of both castration-resistant and -sensitive prostate cancer. These preclinical results indicate that ONC201 is well suited to address mCRPC, including tumors that harbor AR-V7, as a single agent that may be combined synergistically with enzalutamide or everolimus . With these observations we envision further development a combination of ONC201 plus everolimus in CRPC with or without enzalutamide resistance.

#2103

NVP-FGF401: Cellular and in vivo profile of a novel highly potent and selective FGFR4 inhibitor for the treatment of FGF19/FGFR4/KLB+ tumors.

Andreas Weiss,1 Diana Graus Porta,1 Flavia Reimann,1 Alexandra Buhles,1 Christelle Stamm,1 Robin A. Fairhurst,1 Jacqueline Kinyamu-Akunda,2 Dario Sterker,1 Masato Murakami,1 Markus Wartmann,1 Youzhen Wang,3 Jeffrey A. Engelman,3 Francesco Hofmann,1 Wiliam R. Sellers3. 1 _Novartis Institutes for BioMedical Research, Basel, Switzerland;_ 2 _Novartis Institutes for BioMedical Research, East Hanover, NJ;_ 3 _Novartis Institutes for BioMedical Research, Cambridge, MA_.

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and a deadly disease. Treatment options are limited and prognosis generally is poor. Aberrant signaling through the fibroblast growth factor 19 (FGF19) - fibroblast growth factor receptor 4 (FGFR4) axis has been implicated in the development of HCC, and recently FGF19 has been determined as a specific driver gene amplification in a subset of liver tumors and cancer cell lines. Here, we describe the cellular and in vivo profile of NVP-FGF401, a highly potent and selective, first in class, reversible-covalent small-molecule inhibitor of the kinase activity of FGFR4. NVP-FGF401 is exquisitely selective for FGFR4 versus other FGFR family members and all other kinases. We show that among the FGF19-amplified liver cancer cells in the cancer cell line encyclopedia (CCLE), only those with concomitant expression of β-klotho (KLB), a co-receptor for FGF19 that facilitates its binding to FGFR4, are sensitive to NVP-FGF401. NVP-FGF401 has good oral PK properties and shows an excellent in vivo PK/PD relationship. NVP-FGF401 has remarkable anti-tumor activity in mice bearing HCC tumor xenografts and PDX models that are positive for FGF19, FGFR4 and KLB. NVP-FGF401 is the first FGFR4 inhibitor to enter clinical trials, and a PhI/II study is currently ongoing in HCC and other types of solid tumors. 

### Mechanism of Drug Action

#2104

In vitro **drug effects on cancer cell morphology and functional state revealed by multiparameter imaging mass cytometry.**

Olga Ornatsky, Alexandre Bouzekri, Bedilu Allo, Jessica Watson. _Fluidigm Canada, Markham, Ontario, Canada_.

Results of in vitro drug testing are correlated with clinical response to chemotherapy: Accuracy to predict clinical drug resistance was found to be as high as 90%. The benefit of using in vitro models lies in the ability to probe cellular response in a controlled closed system, where effects of drug concentrations, treatment duration, drug efflux kinetics and multidrug combinations can be assessed by a variety of cell biology techniques.

Cisplatin is a widely used chemotherapy drug that targets genomic DNA of the cells, forming both interstrand and intrastrand cross-links that lead to cell death. One limitation of its clinical use is in predicting the development of resistance and severe side effects in patients. Mechanisms of resistance such as reduced drug accumulation, increased detoxification through cisplatin binding to cellular thiols, reduced DNA platination, and increased DNA repair have been reported, however (tamoxifen enhancement of cisplatin).

In vitro models of different cancer types (SKOV3, HeLa, A431, MCF-7) were used to study the effects of cisplatin on cell morphology and phenotypic and functional characteristics with a large panel of metal-tagged antibodies and Imaging Mass Cytometry (IMC) at the single-cell level (1). Proteins involved in DNA damage repair (γH2AX, PP2A, pHistone H3), apoptosis (CD98, caspase-3, cleaved PARP), cell proliferation (cyclin B1, Ki-67), metastasis (vimentin, β-catenin, VEGF, CD63, CD9), substrate adhesion (CD29, CD49e, CD49b, CD51, CD54, CD47, CD61), organelle morphology (CD107a, Mito, histone H3), and signaling pathways (STAT3, pERK1/2, pS6), as well as surface receptors (EGFR, HER2, BRCA, MUC1, CD44, EpCAM, CD142, CD59, beta-catenin) and structural markers (CK5, CK8/18, β-actin, β-tubulin), were identified simultaneously in each individual cell with specific metal-conjugated antibodies. S-phase cells were visualized by detection of 127I in 5-iodo-2′-deoxyuridine (IdU) added to culture media. Presence of cisplatin in cell nuclei and cytoplasm was registered by IMC of platinum stable isotopes.

Combination therapy of cisplatin and paclitaxel is a standard chemotherapeutic regimen to treat recurrent or metastatic cervical cancer. Cell lines from various tumors may develop resistance to cisplatin. Reduced cisplatin uptake has been observed in cervical cancer cells with cisplatin resistance. The cisplatin-resistant HeLa cells and A431 (A431/Pt) cells show 50% and 77% reduction in cisplatin uptake, respectively, compared with the parental cell lines. Human ovarian cancers (in vitro model SKOV3), for which cisplatin is a mainstay of treatment, develop drug resistance and pose an important clinical challenge.

(1)

Chang, Q et al. Nature Scientific Reports 6 (2016): 36641

#2105

Evaluation of antitumor properties of metformin over human multidrug-resistant chronic myeloid leukemia cells.

Ligia P. Oliveira, Débora L. Renó, Rodrigo S. Curvello, Luana P. Lima, Ana Carolina S. Galvão. _UFABC, Santo André, Brazil_.

Besides the progress in the knowledge of cancer biology, the rates of mortality due to this disease are still considerably elevated. Therefore, application of new drugs in antitumor therapies has been constantly studied. However, any of them have achieved a considerable success so far. In this work we evaluated the ability of metformin, a traditional hypoglycemic prescribed to treatment of type II diabetes along decades, over programmed cell death and modulation of metastatic potential in multidrug resistant (MDR) human myeloid chronic leukemia cells (Lucena), through MTT reduction test, flow cytometry, western blotting and gelatin zymography. Metformin was able to reduce the cell viability in a concentration and time-dependent manner, but was not able to induce Lucena cells to apoptosis or necrosis, though activation of the apoptotic machinery. Indeed, it was not observed activation of caspase 3, in contrast to an expressive activation of PARP. Interestingly, proteins associated to autophagy were overexpressed by Lucena cells, and an arrest of cell cycle was observed after exposition to the drug. Finally, it was verified that metformin was able to modulate activity of MMP2 and MMP9, enzymes associated to the metastatic process. Taken together these results suggest that the hypoglycemic is able to acts over the first steps of cancers in more aggressive stages. Finally, inhibition of MMPs indicates a promissory action of metformin for the treatment of metastatic disease.

#2106

In-depth profiling of the energy depletion-mediated cancer cytotoxicity by passively delivered zinc complexes via PARP1 activation.

Jinhyuk F. Chung,1 Tae Jung Park,2 Yoko Norihisa3. 1 _Xylonix Pte Ltd, Singapore, Singapore;_ 2 _Chung-Ang University, Seoul, Republic of Korea;_ 3 _Cheongshim International Medical Center, Gyeonggi-do, Republic of Korea_.

Dysfunctional apoptotic function from impaired p53 tumor suppressor activity leads to poorer prognosis in cancer patients by compromising drug sensitivity and promoting drug resistance development. Programmed necrosis following energy crisis is a theoretically proposed cytotoxic mechanism for circumventing this problem. Herein we demonstrate that a biodegradable zinc complex is capable of triggering the programmed necrosis via activation of PARP1/PARG/MPTP, which consistently resulted in p53-independent broad spectrum cytotoxicity in vitro across all 20 cancer cell lines tested including many with reported drug resistance phenotypes. Additionally, we investigated its possible interaction with agents that influence p53 for possible antagonism/synergism. Supporting in vivo evidence in both animal models and clinical case studies involving terminal chemo-resistant gastric cancer patients upon oral administration (30 mg Zn/day) are presented. These results implicate macromolecular biodegradable zinc complexes as promising antitumor agents of simple compositions for overcoming the current hurdles in clinics, and warrant further clinical investigations. We plan to advance the studies through non-invasive smart devices reporting real-time blood glucose, blood pressure, microperfusion, and other hemodynamic parameters.

#2107

Tetra-arsenic hexoxide induces G2/M cell cycle arrest, apoptosis, and autophagy via p38 MAPK- and AKT-mediated pathways in SW620 human colon cancer cells.

Won Sup Lee,1 Jeong Won Yun,1 Min Jeong Kim,1 Arulkumar Nagappan,1 Jing Nan Lu,1 Seong-Hwan Chang,2 Jae-Hoon Jeong,3 GonSup Kim,1 Jin-Myung Jung,1 Soon Chan Hong1. 1 _Gyeongsang National Univ. Hospital, Jinju, Republic of Korea;_ 2 _Konkuk University School of Medicine, Seoul, Republic of Korea;_ 3 _Korea Institute of Radiological and Medical Sciences, Seoul, Republic of Korea_.

Tetraarsenic hexoxide (As4O6) has been used in Korean folk remedy for the treatment of cancer since the late 1980's. Evidence suggests that As4O6 show anti-cancer effects, whose mechanisms are different from those for As2O3. However, the detailed anticancer mechanism is still unclear. Here, we investigated the anticancer effects of As4O6 on SW620 human colon cancer cells. As4O6 induced cell death in a dose-dependent manner. Flow cytometry analysis revealed that As4O6 increased the sub-G1 (apoptotic cell population) and G2/M phase population in a dose-dependent manner. Further, nuclear condensation and cleaved nuclei were also observed upon staining with Hoechst 33342 in As4O6-treated SW620 cells. Western blot revealed that As4O6 significantly down-regulated the cyclin B1, cdc 2, pro-caspases -3, -8 and -9, and up-regulated p21 and cleavage of PARP in SW620 cells. In addition, As4O6 increased the expression of death receptor 5 (DR5), suppressed Bcl-2 and XIAP family proteins, and promoted the conversion of LC3-I to LC3-II in a Beclin-1 independent manner. Interestingly, As4O6 dephosphorylated Akt and JNK and phosphorylated p38 MAPK, and the cell death was inhibited by p38 MAPK inhibitor; whereas Akt inhibitor augmented the As4O6 induced cell death, suggesting p38 MAPK and AKT were associated with As4O6-induced cell death. Taken together, these findings suggest that As4O6 induced G2/M arrest, apoptosis and autophagic cell death at least in part through p38 MAPK and AKT pathways in SW620 human colon cancer cells. This study may explain the anecdotal anticancer effects showing central necrosis with dormant status of cancers when treated by As4O6 as folk remedy.

#2108

Imatinib-induced downregulation of Skp2 inhibits cell growth in human squamous cell carcinoma.

Hyo Jin Jeong. _Institute of Life science and Biotechnology, Daegu, Republic of Korea_.

It is apparent that Skp2(S-phase kinase associated protein) plays a crucial role in tumorigenesis of various human cancers. Imatinib, inhibitor of BCR-ABL tyrosine kinase, KIT and PDGF receptor, has been approved for the treatment and investigation of chronic myeloid leukemia, gastrointestinal stromal tumors and additional various solid tumors. But An area of skin cancer has not been yet investigated. The aim of this study was to explore the role of the Skp2 in Imatinib antitumor effect on human skin cancer. To explore Imatinib effect on growth of A431 cells (squamous cell carcinoma / skin cancer) we conducted CCK8 assay and Flow cytometry. Western blot assay was performed for determining the functions and molecular mechanism of Imatinib in A431 cells. we found that Imatinib significantly inhibited cell growth and induced cell cycle arrest at G0/G1 phase. Furthermore, we observed that depletion of Skp2 triggered cell cycle arrest and colony formation inhibition by Imatinib. Mechanistically we identified that Imatinib markedly downregulated Skp2 protein expression and subsequently upregulated p21 expression via increased protein stability. In conclusion, Imatinib exerts its antitumor activity via inhibition of Skp2 - p21 axis and these findings suggest that targeting Skp2 by Imatinib could be promising therapeutic approach for Squamous cell carcinoma therapy.

#2109

Transcriptional analysis of human leukemia cells treated with the experimental anticancer drug laromustine.

Amanda J. Loya,1 Dylan J. Cincotta,1 Abdel Elkahloun,2 David Bodine,2 Kevin P. Rice1. 1 _Colby College, Waterville, ME;_ 2 _National Human Genome Research Institute, Bethesda, MD_.

Laromustine is an experimental anticancer prodrug that demonstrated significant activity against acute myeloid leukemia in human clinical trials. Upon base-catalyzed activation, laromustine yields two reactive, electrophilic species in situ: 90CE, which can chloroethylate the O6 position of guanine in DNA causing lethal interstrand crosslinks, and methyl isocyanate, which can carbamoylate biochemically relevant nucleophiles, such as cysteine sulfhydryl groups. Previous in vitro and ex vivo investigations into laromustine's molecular mechanism of action reveal several biochemical consequences of methyl isocyanate, many of which may explain either the acute cytotoxicity of the drug's carbamoylating activity or the observed synergism with the cytotoxicity of guanine O6 chloroethylation. The combined activities of the electrophilic subspecies of laromustine are believed to induce apoptosis in leukemia cells. Presented herein are extensive flow cytometry experiments using FITC-AnnexinV and propidium iodide suggesting a dose-dependent cytotoxicity that includes apoptotosis. So as to gain a more complete understanding of the drug's toxicity, an investigation into how laromustine modulates gene transcription in HL60 cells is also carried out and reported here. Total mRNA was harvested from cultured HL60 cells treated with sub-lethal doses of laromustine or analogs lacking either carbamoylating or chloroethylating activity. These samples were subjected to GeneChip analyses against control mRNA. More than 4,000 genes were significantly dysregulated upon the cells' exposure to laromustine. The analog of laromustine possessing only carbamoylating activity caused the dysregulation of nearly 3,000 genes, the vast majority of which were also dysregulated in response to laromustine. 90CE, which lacks carbamoylating activity but retains chloroethylating activity, understood to be principally responsible for therapeutic cytotoxicity, demonstrated very little effect on gene transcription in HL60 cells. Preliminary analysis of gene pathways affected by laromustine suggests that among the altered pathways, those associated with cancer progression, the G1/S checkpoint, and hematological disorders are particularly stressed. The carbamoylating activity of laromustine is responsible for most of these effects. Identifying genetic pathways disrupted by this potentially useful drug may inform clinical strategies or identify potential targets for co-therapeutic agents.

#2110

Anti-invasive and antimetastatic actions of omega-3 polyunsaturated fatty acids in glioblastoma cells.

Soyeon Kim,1 Soyeon Shin,2 Soyeon Jeong,1 Seung-Hyeon Han,3 Yoon-Seon Yoo,3 Prashanta Silwal,3 Young-Joo Jeon,3 Jun-Young Heo,4 Gi-Ryang Kweon,4 Seung-Kiel Park,5 Jong-Il Park,5 Kyu Lim6. 1 _Dept. of Biochemistry, Infection Signaling Network Research Center, Chungnam National University, Daejeon, Republic of Korea;_ 2 _Dept. of Biochemistry, Cancer Research Institute, Chungnam National University, Daejeon, Republic of Korea;_ 3 _Dept. of Biochemistry, Dept. of Medical Science, School of Medicine, Chungnam National University, Daejeon, Republic of Korea;_ 4 _Dept. of Biochemistry, Dept. of Medical Science, School of Medicine, Infection Signaling Network Research Center, Chungnam National University, Daejeon, Republic of Korea;_ 5 _Dept. of Biochemistry, Chungnam National University, Daejeon, Republic of Korea;_ 6 _Dept. of Biochemistry, Dept. of Medical Science, School of Medicine, Cancer Research Institute, Infection Signaling Network Research Center, Chungnam National University, Daejeon, Republic of Korea_.

Glioblastoma (GBM) is the most aggressive and deadliest brain malignancy in adults. Despite of surgical techniques, radiotherapy and chemotherapy, GBM has remained an invariably lethal tumor with a median survival less than 15 months. Although the anticancer mechanisms of omega-3 polyunsaturated fatty acids (ω3-PUFAs) have been reported in several cancers, it is still unclear in brain cancer. Here, our data show the anti-invasive and anti-metastatic effects of ω3-PUFAs in GBM. Invasiveness using transwell chamber was inhibited by docosahexaenoic acid (DHA) treatment in D54MG and GL261 cells. In zymography, MMP-2 activity was suppressed by DHA in a dose dependent manner. MMP-2 promoter activity was also decreased. DHA inhibited both p-STAT3 and β-catenin levels that contribute to invasion by stimulating pro-invasion factors such as MMP-2. Additionally, fat-1 (ω3-desaturease) stable D54MG cell was established and the effect of the high level of ω3-PUFAs was investigated endogenously. The invasiveness were significantly inhibited in the fat-1 stable cells compared to control cell. Moreover, the metastasis in vivo was significantly reduced when GL261 mouse glioma cells were injected through tail vein into the fat-1 transgenic mice. In immunohistochemistry, intensity of p-STAT-3 and β-catenin were significantly weak in metastatic lung tumor from Fat-1 mice compared to wild type mice. These results indicate that anti-invasive and anti-metastatic actions of ω3-PUFA may be correlated with inhibition of MMP-2 through decrease of STAT3 and β-catenin in GBM cells. Thus, these findings provide important preclinical evidence and molecular insight for utilization of ω-3 PUFAs for the chemoprevention and treatment of human GBM. [This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (2007-0054932, NRF-2015R1D1A1A01056887) and by the framework of international cooperation program managed by National Research Foundation of Korea (2015K2A2A6002008)].

#2111

Ciclopirox inhibits tumor cell motility by suppressing protein expression of small GTPases and phosphorylation of paxillin.

Shile Huang, Tao Shen. _Louisiana State Univ. Health Sciences Ctr., Shreveport, LA_.

Ciclopirox olamine (CPX), an off-patent antifungal drug, is used for the treatment of superficial mycoses. Recent studies have demonstrated that CPX also possesses potent anticancer activity by inhibiting cell proliferation and inducing cell death in tumor cells. However, it is unknown whether CPX inhibits cell motility. In the present study, we found that CPX potently inhibited cell motility in a concentration-dependent manner in rhabdomyosarcoma (Rh30 and RD) cells, which was independent of the reduction of cell viability. As small GTPases (RhoA, Cdc42, and Rac1) and focal adhesion proteins (paxillin) play critical roles in the regulation of cell motility, we further studied whether CPX affects these proteins. Our Western blot analysis revealed that CPX did inhibit cellular protein expression of RhoA, Cdc42 and Rac1. However, by RT-PCR analysis, CPX did not alter the mRNA levels of the small GTPases. As microRNAs (miRNAs) can regulate protein expression at transcriptional and post-transcriptional levels, we investigated the effect of CPX on the expression of miRNAs. Our miRNA PCR array revealed that treatment with 10 μM of CPX for 24 hours upregulated the expression of 27 miRNAs by 2.04-6.10 fold, and downregulated the expression of 7 miRNAs by 2.00-7.89 fold in Rh30 cells. Further research is ongoing to identify whether these up/downregulated miRNAs are linked to the reduced expression of small GTPases and potentially more proteins. In addition, we found that CPX inhibited the phosphorylation of paxillin, despite no effect on the total cellular protein expression. The results suggest that CPX inhibits tumor cell motility at least by suppressing the protein expression of small GTPases and the phosphorylation of paxillin. Supported by the Feist-Weiller Cancer Center, LSU Health Sciences Center, Shreveport, LA.

#2112

FoxO3a activation by HDAC class IIa inhibition induces cell cycle arrest in pancreatic cancer cells.

Makoto Usami, Shohei Kikuchi, Kohichi Takada, Yusuke Sugama, Yohei Arihara, Naotaka Hayasaka, Hajime Nakamura, Yuki Ikeda, Yusuke Kamihara, Masahiro Hirakawa, Makoto Yoshida, Masayoshi Kobune, Koji Miyanishi, Junji Kato. _Sapporo Medical University, School of Medicine, Sapporo City, Japan_.

Pancreatic cancer is highly chemo-resistant associated with oncogenic mutations such as KRAS and/or p53. The effect of conventional chemotherapy is not sufficient and new target and strategy is urgently needed.

The forkhead box (Fox) proteins are multidirectional transcriptional factors strongly implicated in malignancies. Although Fox O (FoxO) protein, and particularly FoxO3a, works as negative regulator of cell proliferation by repressing cyclin proteins and inducing cell cycle inhibitors such as p21Waf1/Cip1, its expression is consistently suppressed by several oncogenic pathways including phosphatidylinositol-3 kinase (PI3K) / AKT pathway, constitutively activated in pancreatic cancer. Thus, upregulating FoxO3a activity could be a promising target of pancreatic cancer treatment without impact of underlying oncogenic mutations.

Class IIa Histone deacetylase (HDAC) is a subgroup of HDAC. Though HDAC inhibitors (HDACi) have been extensively investigated as a cancer target, its action mechanism is considered due to histone modification by class I. Biological significance of class IIa HDACs which have minimal histone deacetylation activity have not been elucidated yet. Recent studies show class IIa HDACs act as a transcriptional regulator including FoxO3a. In this study, we investigate the biologic impact of HDAC class IIa inhibition on FoxO3a and anti-tumor effect against pancreatic cancer cell line using selective class IIa HDACi TMP269.

TMP269 treatment showed increased FoxO3a expression in a dose dependent manner with immunoblotting and modest cell growth inhibition effect at 57.5 μM of IC50 dose for 48-hour treatment against AsPC-1 in MTT. G1/S arrest was observed with cell cycle assay. Upregulated p21Waf1/Cip1 and downregulated CDK2 and 4/6 and cyclin D1 and D2 expressions were further observed, consistent with inducing G1/S arrest and transcriptionally activated FoxO3a. Importantly, upregulated p21Waf1/Cip1 was observed in AsPC-1 p53 null cell line, suggesting independent with p53 pathway. These findings suggest upregulated FoxO3a induced by HDAC class IIa inhibition activated its transcription and resulted in cell growth inhibition.

Because PI3K/AKT leads FoxO3a to the ubiquitylation-mediated proteasome degradation, we examined irreversible proteasome inhibitor carfilzomib (CFZ) combined with TMP269, aiming synergistic FoxO3a upregulating. As expected, FoxO3a expression was further increased in TMP269 combined with CFZ compared with TMP269 or CFZ alone. Following the activated FoxO3a, p21Waf1/Cip1 expression was upregulated and cell growth inhibition was dramatically enhanced.

In conclusion, HDAC class IIa inhibition modified FoxO3a transcriptional activation and upregulating FoxO3 by dual inhibition of HDAC class IIa and proteasome is promising target against pancreas cancer.

#2113

Mechanism of action of pixantrone in non-Hodgkin's lymphoma cells.

Serina Ng, Ruben Muñoz, Daniel Von Hoff, Haiyong Han. _Translational Genomics Research Institute, Phoenix, AZ_.

Background: Pixantrone (PIX), an anthracenedione analog, has shown promising results with reduced cardiotoxicity as compared to structurally similar mitoxantrone and doxorubicin (DOX) in early phase clinical trials in patients with non-Hodgkin's lymphoma (NHL). The exact mechanisms of action on how PIX induce cell death by targeting DNA replication through intercalating DNA and/or inhibiting Topoisomerase II has not been clearly illustrated.

Methods: A short-term cell proliferation assay (3 days treatment) and a long-term clonogenic assay (cells were treated with the drugs for 24 hours and then allowed to grow into colonies for 10-14 days) were used to assess and compare the effects of PIX and DOX on cell growth in three NHL cell lines, OCI-Ly8, Z138, and Raji. Immunofluorescent assays using γH2AX antibodies, and DAPI staining were used to examine the effect of PIX and DOX on the formation of double-strand breaks and micronuclei.

Results: In the short-term cell proliferation assay, PIX was not as potent as DOX (IC50 values ranging from 22 nM to 2737 nM for PIX and 2.8 nM to 67 nM for DOX). The two drugs were significantly more potent in the long-term clonogenic assay with IC50 values in low nanomolar range (5.1 nM to 82.8 nM) and the difference between PIX and DOX was much smaller. Cells treated with PIX displayed less increases in DNA double-strand breaks compared to those of DOX as quantified by the number of γH2AX positive foci formed in the cells. Furthermore, PIX treatment significantly increased the formation of micronuclei in a drug concentration and time dependent manner.

Conclusions: The findings from the current study indicate that PIX induces DNA damages that impair the chromosomal segregation but do not significantly trigger double-strand breakage checkpoint response. Overall, our results suggest that PIX may exert its antitumor activity via mechanisms that are different from those of DOX.

#2114

Targeting invasive and drug-resistant PDAC with photodynamic therapy.

Gwendolyn Cramer, Seyedehrojin Jafari, Hamid El-Hamidi, Dustin Jones, Jonathan Celli. _University of Massachusetts Boston, Boston, MA_.

Pancreatic ductal adenocarcinoma (PDAC) is associated with an extensive desmoplastic reaction and a characteristic extracellular matrix (ECM)-rich stromal microenvironment which is a major determinant of disease progression and therapeutic response. Using 3D cell culture models of PDAC, we recently showed that an invasion-promoting ECM composition leads to differential response to chemotherapy agents and photodynamic therapy (PDT), in which cytotoxic response is triggered by light activation of a photosensitizing agent. In particular, ECM-infiltrating populations with characteristic mesenchymal phenotype display a marked increase in sensitivity to PDT (using the photosensitizer verteporfin), while the same cells exhibit increased chemoresistance. Here, motivated by these findings we systematically disentangle influences associated with the physical and biochemical properties of ECM as regulators of PDAC growth behavior and treatment response. In 3D tumor spheroids transplanted into invasion-promoting type 1 collagen gels, we use riboflavin-mediated photocrosslinking as a tool to regulate ECM stiffness (independent of its concentration), combined with high-content imaging to co-register invasive progression and differential response to chemotherapy and PDT. Increased extent of photocrosslinking correlates with increased collagen stiffness as confirmed by bulk oscillatory rheology, and is associated with a decrease in the invasive velocity (migration speed directed away from the primary transplanted spheroid) of ECM-infiltrating PDAC cells. Preliminary comparisons of PDT and oxaliplatin chemotherapy in low and high collagen crosslinking conditions show that PDT is significantly more effective than oxaliplatin in ECM-invading populations regardless of collagen stiffness. We also compare oxaliplatin chemotherapy and PDT before and after PDAC invasion into Matrigel ECM, and find that PDT efficacy is only enhanced after extensive invasion into the surrounding matrix. These results support the efficacy of PDT in targeting locally invasive PDAC while stressing the importance of elucidating how both biochemical and biophysical ECM interactions regulate therapeutic susceptibilities.

#2115

Therapeutic potential of fingolimod against prostate cancer cells is partly attributed to interrupting the cross talk between estrogen and sphingolipid metabolisms.

Rasha M. Allam,1 Ahmed M. Al-Abd,1 Alaa Khedr,2 Ola A. Sharaf,1 Salwa M. Nofal,1 Amani E. Khalifa,3 Hisham A. Mosli,4 Ashraf B. Abdel-Naim3. 1 _National Research Centre of Egypt, Giza, Egypt;_ 2 _King Abdulaziz University, Jeddah, Saudi Arabia;_ 3 _Faculty of Pharmacy, Ain Shams University, Cairo, Egypt;_ 4 _Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia_.

Sphingolipids influence tumor microenvironment in different malignancies. Estrogen and estrogen metabolites are involved in prostate cancer. Fingolimod is Sphingokinase-1 (SphK1) inhibitor which possesses anticancer properties in various tumor types. The aim of the current study is to investigate the interference of Fingolimod with the cross talk between sphingolipid and estrogen metabolisms within prostate cancer cells as a novel therapeutic strategy. The design is based on determining the mutual influence between estrogen metabolism/signaling and sphingolipid metabolism within prostate cancer cells. Cytotoxicity using SRB-U assay; cell cycle analysis using DNA cytometry and intracellular caspase-3 concentration were assessed. Estrogen metabolites and estrogen metabolizing enzymes were determined using LC/MS and RT-PCR, respectively. Estrogen receptor (ERα and ERβ) expression and their downstream signaling (CXCR4 and Cyclin-D1, respectively) were measured using immunocytochemical staining and RT-PCR, respectively. Fingolimod showed antiproliferative/cytotoxic effects against different prostate cancer cells (LNCaP, DU145 and PC3) with IC50 ranging from 3.0±0.3 to 6.8±1.7 µM. Fingolimod induced significant decrease in estradiol, estrone, 4-hydroxyestradiol and 16α-hydroxyestrone concentrations with reciprocal increase in 2-methoxyestrone and 2-methoxyestradiol concentrations. This was mirrored by significant down expression for the enzymes, aromatese, CYP1A1 and CYP1B1 and over expression for the enzyme COMT. Additionally, Fingolimod suppressed the expression of ERα and ERβ within prostate cancer cells as well as their downstream signaling (CXCR4 and Cyclin-D1, respectively). Reciprocally, it was found that estradiol and catechol estrogens induce the expression of SphK1 while methoxylated catechol estrogen suppresses its expression. Our results give compelling evidence that Fingolimod has therapeutic potential by modulating the estrogenic micromilieu and interrupting its cross talk with sphingolipid metabolism in prostate cancer cells.

#2116

Combining the power of different profiling approaches to better understand the activity of kinase inhibitor drugs.

Alastair J. King,1 Jesse J. Parry,1 Samantha C. Burkart,1 Lee R. Cavedine,1 Alyssa M. Cracchiolo,1 Brogan A. Epkins,1 Steven M. Garner,1 Timothy J. Sindelar,1 Charles R. Wageman,1 Stephen P. Davies,2 Andrew Plater,2 Anna M. Woodward,2 Usha Warrior1. 1 _Eurofins Panlabs Inc., St. Charles, MO;_ 2 _Eurofins Pharma Discovery Services UK, Ltd., Dundee, United Kingdom_.

Profiling of inhibitors for eventual therapeutic use is an essential part of drug discovery and development. Evaluating the in vitro potency of kinase inhibitors in a wide panel of enzyme assays is critical, not only to guiding SAR knowledge for improving drug design, but also for assessing selectivity, which has long-reaching consequences for both biological activity and safety. Evaluation of the same inhibitors in cellular models yields information on the activity of these inhibitors in a more physiologically relevant environment. However, while some selective kinase inhibitors show predictably selective profiles in cell proliferation assays, others do not. Thus, the biological context in which a kinase inhibitor functions, and the signaling networks influenced by inhibition of both the desired target and off-target activities, help to define how that inhibitor will work in a cellular setting and, ultimately, in a therapeutic application. We demonstrate the power of profiling several well-known kinase inhibitors in complementary panel assays, to predict behavior that has implications for clinical activity. The in vitro profiles of selective (trametinib, vemurafenib) and less selective (dasatinib, sorafenib) kinase inhibitors are shown and compared. With careful evaluation of their in vitro enzyme activity profiles in Eurofins' KinaseProfiler™ panel, cellular response profiles in OncoPanel™, and predictive biomarker identification by univariate genomic analysis, a fuller picture of these inhibitors' biological activity can be generated, and may be applied to predicting the therapeutic potential of such inhibitors. While, in some cases, clinical response is seen as expected, this kind of comprehensive analysis opens up the possibility for expanding clinical utility of an asset by potential repurposing for other indications. Collectively, this demonstrates not only the need to understand an inhibitor's fundamental activity(ies) at its designated target(s), but also the importance of considering cellular context, when assessing its maximum potential clinical utility.

#2117

TRAIL-induced apoptosis in TRAIL-resistant breast carcinoma.

Jasmine M. Manouchehri, Michael Kalafatis. _Cleveland State University, Cleveland, OH_.

Recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL), the optimized form of the endogenous death ligand TRAIL, shows therapeutic potential for cancer due to its ability to induce apoptosis in cancer cells independent of p53, while exhibiting minimal toxicity to normal cells. Despite this, a majority of breast cancers display resistance to rhTRAIL treatment due to up-regulation of pro-apoptotic proteins, down-regulation of anti-apoptotic proteins, and/or up-regulation of death receptors (DR) 4 and 5. To overcome rhTRAIL resistance, natural compounds have been investigated as sensitizing agents. This study considered the application of the naturally occurring flavonol Quercetin (Q). Q has been shown to have the ability to up-regulate DR5 and down-regulate anti-apoptotic proteins in cancer cells, and thereby, making Q a favorable choice to be employed as a sensitizing agent. The intention of this study was to ascertain the capacity of Q to sensitize rhTRAIL-resistant triple negative breast cancer BT-20 cells and hormone-dependent breast cancer MCF-7 cells to rhTRAIL-induced apoptosis and elucidate the underlying mechanism for Q's sensitization. Q demonstrated the ability to intensify rhTRAIL's pro-apoptotic effects in the breast cancer BT-20 and MCF-7 cell lines as detected through Annexin V/PI assays followed by FACS analysis. In comparison to single agent treatments, the cotreatment of Q and rhTRAIL enhanced the induction of the extrinsic pathway of apoptosis as marked by PARP cleavage (a hallmark of apoptosis), activation of caspase 8, and activation of the executioner caspases 3 and 7. The mechanism for Q's augmentation in breast cancer was determined to be through the down-regulation of c-FLIPL (caspase 8 inhibitor) in a dose-dependent manner. Furthermore, Q promoted the ubiquitination of c-FLIPL facilitating the proteasome-mediated degradation of c-FLIPL in breast cancer. An additional mechanism for Q's sensitization was displayed in breast cancer BT-20 cells. Q up-regulated DR5 membrane and protein expression in breast cancer BT-20 cells in a dose-dependent manner. RT-PCR analysis revealed that Q's influence on DR5 expression occurred at the transcriptional level in those breast cancer cells. Thus, these data suggest that the cotreatment of Q and rhTRAIL possesses the therapeutic potential to be an effective anti-breast carcinoma regimen.

#2118

The food additive agent potassium bromate prevents growth and aggressive phenotypes by targeting multiple molecular signatures in breast cancer cells.

Priyanka Ghosh, Gargi Maity, Snigdha Banerjee, Sushanta Banerjee. _VA Medical Center, Kansas City, MO_.

Potassium bromate (KBrO3) is by-product of ozone that has found multifunctional purpose in modern society. It is used as disinfectant in drinking water, a bleaching agent to improve flour, a component of cold-wave hair lotion and an ingredient in the production of fish paste and fermented beverages. Potassium bromide has also been used in America for bread-baking as a safe food additive since 1914. Despite the commercial value of this chemical, some studies suggest that KBrO3 could be a carcinogen. During the bread-baking process, Potassium bromate is normally converted into a stable and inert compound, potassium bromide (KBr). However, due to incomplete reduction, the residual KBrO3 remains in the bread, which eventually acts as a potential carcinogen to humans. Interestingly, our studies, in breast cancer cells, found contrasting results. We found that KBrO3 delays growth of ER-α positive luminal type breast cancer cells and triple-negative breast cancer cells (TNBC) via inducing apoptosis in a dose-dependent manner. KBrO3-induced apoptosis is mediated via targeting BCL-2/Bax and Caspase-3 signaling pathway. Moreover, aggressive phenotypes such as migration, invasion and sphere-forming ability of breast cancer cells are significantly impaired by KBrO3-treatment via targeting multiple molecular signatures in breast cancer cells. The growth inhibition effect of KBrO3 is also documented in a tumor xenograft model. Collectively, our findings provide a rationale for the basic and pre-clinical evaluation of the role of KBrO3 in breast cancer progression and therapy.

#2119

Acetylated secretory APE1/Ref-1 induces apoptotic cell death in orthotopic xenografts of triple-negative breast cancer.

Yu Ran Lee,1 Hee Kyoung Joo,1 Eun Ok Lee,1 Myoung Soo Park,2 Byeong Hwa Jeon,1 Sunga Choi1. 1 _Chungnam National Univ. College of Med., Daejeon, Republic of Korea;_ 2 _Chungnam National University Hospital, Daejeon, Republic of Korea_.

The anticancer properties of acetylated secretory apurinic/apyrimidinic endonuclease-1 (Ape1/Ref-1) was suggested in triple-negative breast cancer (TNBC) cells; Posttranslational modification, hyperacetylation in MDA-MB-231 cells caused extracellular secretion of acetylated-APE1/Ref-1 (Ac-APE1/Ref-1) and initiated apoptotic cell death by auto-, paracrine binding to the receptor for advanced glycation end products (RAGE). In the present study, we observed potential therapeutic efficacy of Ac-APE1/Ref-1 in preclinical orthotopic models of TNBC in response to hyperacetylation. The extracellular Ac-APE1/Ref-1 was confirmed by proximity ligation assay in hyperacetylated tumor tissue, showing direct binding of Ac-APE1/Ref-1 and RAGE. Treatment of orthotopic TNBC xenografts with acetylating agents induced a strong growth inhibition in the tumor development as observed in computed tomography: it caused an increase of RAGE expression and activation of caspase-3 and PARP. The tumors also exhibited markedly higher count of apoptotic bodies and reduced proliferation index and neovascularization compared with control tumors. However, the Ac-APE1/Ref-1-stimulated apoptotic cell death was remarkably retarded in RAGE-knockout tumor even in hyperacetylation compared with RAGE-overexpressed one. The functional role of secreted Ac-APE1/Ref-1 in hyperacetylated TNBC was confirmed in vivo, demonstrating its relevance to the anticancer agent. Our findings suggest that Ac-APE1/Ref-1 protein possesses potent chemotherapeutic efficacy against TNBC, resistant to standard chemotherapeutic agents, warranting further evaluation as an anticancer agent

#2120

Distinct molecular effects of chemotherapeutic agents on choline phospholipid metabolism of triple-negative breast cancer cells.

Menglin Cheng, Zaver M. Bhujwalla, Kristine Glunde. _Johns Hopkins University School of Medicine, Baltimore, MD_.

The magnetic resonance spectroscopy (MRS)-detected total choline (tCho) signal is a promising non-invasive surrogate marker able to predict chemotherapy response in breast cancer patients early on. However, the molecular mechanisms by which common chemotherapeutic drugs affect the tCho signal, which consists of glycerophosphocholine (GPC), phosphocholine (PC), and free choline (Cho), are mostly unknown. Here we have employed some widely used cancer chemotherapeutic drugs such as doxorubicin, paclitaxel, and vinorelbine to treat triple-negative human MDA-MB-231 breast cancer cells to elucidate their molecular effects on choline phospholipid metabolism. High-resolution (HR) 1H MRS of water-soluble cell extracts was employed to detect changes in cellular choline metabolite profiles, and quantitative RT-PCR (qRT-PCR) to assess the corresponding changes in the expression levels of choline-metabolizing enzymes. After 48 hours of doxorubicin treatment, the GPC levels in MDA-MB-231 cells significantly increased, while PC level decreased, and the tCho concentration remained unchanged. Vinorelbine treatment displayed a comparable effect to doxorubicin, but the GPC increase and PC decrease occurred to a lesser extent. Paclitaxel treatment caused an increased GPC level along with an unaltered PC level, leading to a slightly increased tCho level. In all these cases, the PC/GPC ratio decreased from 3.5 in the control to about 2.0 in paclitaxel- or vinorelbine-treated samples, to about 1.0 in doxorubicin-treated samples. qRT-PCR-detected mRNA expression levels showed that GDPD6 and Chkα were downregulated by doxorubicin. GDPD5, GDPD6, and Chkα genes displayed a reduced mRNA expression level following vinorelbine treatment, while no significant change in these three genes was detected after paclitaxel treatment. For comparison, the known GDPD6 inhibitor dipyridamole significantly increased cellular GPC levels, but, as expected, did not affect GDPD6 mRNA expression levels as it acts as an enzyme inhibitor. Our study demonstrates that choline containing metabolites change differently depending on the type of drug used for breast cancer treatment. However, all tested chemotherapeutic treatments resulted in metabolic alteration away from the 'cholinic phenotype', which was caused in some cases by decreases in expression of Chkα, GDPD6, and GDPD5 following drug treatment. The choline metabolite concentrations of GPC and/or PC and/or the PC/GPC ratio may serve as non-invasive surrogate makers of therapeutic response in triple-negative breast cancer patients undergoing chemotherapy.

#2121

Deguelin-derivative, L80 suppresses tumor growth and metastasis via inhibition of STAT3 activation in triple-negative breast cancer.

Tae-Min Cho, Eunhye Oh, Daeil Sung, Yoon-Jae Kim, Ji Young Kim, Jeewoo Lee, Jae Hong Seo. _Korea University, Seoul, Republic of Korea_.

Triple-negative breast cancer TNBC is associated with aggressive metastasis and poor clinical outcomes due to a lack of effective treatment options. Therefore, new drugs that effectively target both cancer cell proliferation and metastasis are needed to improve clinical outcomes. The 90-kDa molecular chaperone heat-shock protein, HSP90 has been implicated in cancer progression and metastasis by modulating the stabilization and maturation of many oncogenic proteins. Our objective was to investigate the mechanism of action of C-terminal HSP90 inhibitor, deguelin-derivative L80 on TNBC proliferation and metastasis in vitro and in vivo. L80 induced apoptosis and suppression of cell viability in TNBC cell lines via inhibition of Akt activation with concomitant nuclear accumulation of p27. L80 treatment also caused a marked suppression of cell migration and invasion. These responses were associated with inhibition of STAT3 phosphorylation (Tyr705) as evidenced by downregulation of STAT3 downstream target genes including cyclin D1 and survivin. The syngeneic orthotopic mouse model with 4T1 cells, L80 administration resulted in significant reduction in tumor growth together with decreased number of Ki-67-positive cells and downregulation of phospho-STAT3. Double-label immunofluorescence analysis showed that individuals receiving L80 exhibited a marked reduction in co-localization and expression of HIF-1α and HSP90 compared to their control groups in vivo. Finally, we observed that L80 administration significantly suppressed lung metastasis, as determined by in vivo bioluminescent imaging system. Our findings suggest that L80 may be potentially effective for the treatment for metastatic TNBC patients.

#2122

Pharmacologic interconversion of EMT to MET for prostate cancer.

Zheng Cao,1 Shahriar Koochekpour,2 Stephen E. Strup,1 Natasha Kyprianou1. 1 _University of Kentucky Medical Center, Lexington, KY;_ 2 _Roswell Park Cancer Institute, Buffalo, NY_.

Introduction and objection: Dysregulation of transforming growth factor-β (TGF-β) and insulin-like growth factor (IGF) axis has been linked to reactive stroma dynamics in the tumor microenvironment during prostate cancer progression. IGFBP3 induction is initiated by stroma remodeling and represents a potential therapeutic target for advanced prostate cancer. A lead quinazoline-based Doxazosin® derivative, DZ-50, generated in our laboratory (US Patent # 8377948), inhibits prostate tumor growth via inducing anoikis and disrupting focal adhesions. Molecular profiling revealed that the process of epithelial-mesenchymal-transition (EMT) is targeted by DZ-50. In this study, we investigated the effect of DZ-50 on EMT landscape, EMT to mesenchymal-epithelial-transition (MET) conversion, and invasive properties of prostate cancer cells.

Methods: Human prostate cancer cells LNCaP, LNCaP overexpressing TGF-β type II receptor (TβRII), and cancer associated fibroblasts (CAFs) derived from human prostate cancer specimens, were used. The antitumor effect of DZ-50 against prostate cancer epithelial cells and CAFs was evaluated using cell viability assays. Effect of the drug on EMT key regulators (including IGFBP3) was determined using RT-PCR and Western blot analysis. Drug-induced phenotypic conversions of EMT were evaluated by confocal microscopy. Impact of TGF-β from the stroma microenvironment or exogenous cytokine, on prostate tumor cell migration and invasion, was assessed in co-cultures with CAFs. The functional contribution of IGFBP3 to EMT-MET interconversion in response to DZ-50 was assessed using siRNA approaches.

Results: DZ-50 induced cell death in prostate cancer epithelial cells and CAFs, in a concentration-dependent manner. DZ-50 downregulated IGFBP3 mRNA and protein expression and promoted EMT-MET conversion in both LNCaP and LNCaPTβRII cells. IGFBP3 knockdown in LNCaPTβRII cells led to E-cadherin upregulation and MET induction, implicating IGFBP3 as a potential target of DZ-50 to reverse EMT to MET. Moreover exposure to TGF-β reversed DZ-50-induced MET by upregulating IGFBP3 in LNCaPTβRII cells. Co-cultures of LNCaPTβRII with CAFs promoted prostate cancer cell invasion via TGF-β and IGFBP3, an effect that was inhibited by the drug.

Conclusions: Treatment of prostate cancer cells with the novel agent DZ-50 inhibits cell migration and invasion and causes reversal of EMT to MET by regulating IGFBP3. This study integrates IGFBP3 as new signaling effector driving the antitumor action of DZ-50 via targeting the EMT-MET phenotypic landscape in the prostate tumor microenvironment. Ongoing work in pre-clinical models will establish the therapeutic value of this novel compound in advanced metastatic prostate cancer.

#2123

Inhibition of AURKA induces Raf1-independent activation of MAPK pathway in breast cancer cells.

Malgorzata Gil,1 Archana Chidambaram,1 Thaer Khoury,1 Kazuaki Takabe,1 Igor Puzanov,1 Irwin Gelman,1 Antonio D'Assoro,2 Mateusz Opyrchal1. 1 _Roswell Park Cancer Institute, Buffalo, NY;_ 2 _Mayo Clinic College of Medicine, Rochester, MN_.

Background: Aurora A (AURKA) is a mitotic kinase responsible for centrosome segregation and mitotic spindle formation. In normal cells, expression of AURKA is highly regulated and is predominantly restricted to G2/M phases of the cell cycle. Unlike healthy cells, cancer cells overexpress AURKA through all phases of the cell cycle resulting in the acquisition of alternate non-mitotic functions. Little is known about cellular functions regulated by AURKA and its interaction with other signaling molecules. Here, we report a novel interaction between AURKA and the mitogen-activated protein kinase (MAPK) pathway in wild type BRAF breast cancer cells as well as demonstrate an additive cytotoxic effect of AURKA- and MEK1/2-specific inhibitors against estrogen positive (ER+) and triple negative (ER-, PR-, HER2-) breast cancer cells.

Results: We show that treatment of ER+ HER2- MCF-7, ER- HER2+ SKBR3 and ER- HER2- BT549 cells with AURKA specific inhibitors alisertib, MK8745 and Aurora A Inhibitor I resulted in over 2-fold increase in the levels of both pMEK1/2 and pERK1/2 compared to the untreated controls. The activation of the MAPK pathway was rapid with changes seen within 5 min after treatment with AURKA inhibitors and was sustained for at least 48 hours. No differences in phosphorylation of MEK1/2 or ERK1/2 were observed in BRAFG464V triple negative MDA-MB-231 cells. Treatment with AURKA inhibitors resulted in downregulation of pAURKA and a significant increase in levels of total AURKA protein. The pull-down assay with Ras-binding domain coated agarose beads followed by western blot analysis with anti-pan-RAS Ab revealed no changes in active GTP-bound RAS in alisertib-treated MCF-7 cells compared to the untreated control. Consistently, no significant changes were observed in RAS-inducible phosphorylation of RAF1 activation site at Ser338 as demonstrated by western blot. Treatment with the pan RAF inhibitor TAK-632 did not diminish alisertib-induced pERK and pMEK1/2. Alternatively, treatment with the MEK1/2 specific inhibitor PD0325901 completely abrogated alisertib-induced phosphorylation of MEK1/2 and ERK1/2. Furthermore, combined treatment of alisertib and PD0325901 in vitro revealed significant additive cytotoxic effect in MCF-7 and BT549 cells when compared to either agent used alone (p< 0.008 and p<0.011; p <0.04 and p<0.028).

Conclusions: Our data suggests that AURKA is a RAF1-independent negative regulator of MAPK activity in breast cancer cells. The in-depth analysis of the AURKA-MEK1/2 interaction is currently under investigation. The results reveal a promising new strategy for the treatment of wild type BRAF, TNBC patients using a combination of AURKA and MEK1/2 inhibitors.

#2124

Differentiation of myeloid and multiple myeloma cell lines by inecalcitol involves a marked decrease in CD44 and CD49d cell surface labeling.

Susan Benjamin, Cecile Planquette, Remi Delansorne. _Hybrigenics, Paris, France_.

Inecalcitol is a vitamin D receptor (VDR) agonist characterized by a high antiproliferative activity on various cancer cell lines (Okamoto et al., 2012; Ma et al., 2013) and a low calcemic potential as demonstrated by the high maximal tolerated oral daily dose of 4 mg in human patients (Medioni et al., 2014). On myeloid cells, inhibition of proliferation by vitamin D derivatives is paralleled by induction of differentiation into a monocyte/macrophage phenotype, mostly described as gain of functions (increase in lysozyme activity, phagocytosis and cell motility; Abe et al., 1981) and of the cell surface CD11b and CD14 biomarkers. The stimulation of the secretion of cathelicidin, a natural human antimicrobial peptide, is another specific activity of vitamin D and its derivatives (Wang et al., 2004).

During a screening of 48 CD biomarkers on 4 human myeloid cell lines (HL-60, U-937, MOLM-13 and THP1) and 4 human multiple myeloma cell lines (MM.1R, MM.1S, L-363 and RPMI-8226) treated for 72 hours at 10 nM, we recently discovered that inecalcitol reproducibly induces CD38 expression (Benjamin et al., 2016), another gain of function. We now report the decrease in CD44 and CD49d cell surface labeling in all 8 cell lines (except CD44 in L-363 cells). This loss of functions reached -60% to -70% in MOLM-13 and HL-60 cells. We have then determined the EC50 (mean ± SEM; n = 3 or 4) of inecalcitol for several differentiation parameters on HL-60 cells. The most sensitive gains or loss of functions were the secretion of cathelicidin (0.2 ± 0.1 nM), the induction of CD38 (0.3 ± 0.1 nM) and the decrease in CD44 (0.8 ± 0.4 nM). Inhibition of proliferation (1.9 ± 0.8 nM) was exactly parallel to induction of CD11b (1.8 ± 0.8 nM). Decrease in CD49d (6.6 ± 1.1 nM) and increase in CD14 (8.0 ± 0.5 nM) occurred as final markers of differentiation by inecalcitol.

CD44 is the receptor for hyaluronic acid; it is found on the surface of acute myeloid leukemia stem cells and suspected to favor the spread and relapse of the disease. CD49d is the integrin α4-subunit involved in the interactions of tumor cells with their microenvironment resulting in their "hiding" and survival. Therefore, decreasing both CD44 and CD49d represents newly identified potential beneficial pro-differentiation effects of inecalcitol.

#2125

Flavokawain A, a kava chalcone, inhibits growth and invasion of human osteosarcoma cells by targeting Skp2.

Yidan Zhang,1 Wendong Zhang,1 Nikolas Zaphiros,1 Xiuquan Du,1 Pratistha Koirala,1 Michael Roth,1 Jonathan Gill,1 Sajida Piperdi,1 David Geller,1 Rui Yang,1 Jinghang Zhang,2 Richard Gorlick,1 Xiaolin Zi,3 Tao Ji,4 Bang H. Hoang1. 1 _Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, NY;_ 2 _Albert Einstein College of Medicine, Bronx, NY;_ 3 _University of California, Irvine Medical Center, Orange, CA;_ 4 _Peking University People's Hospital, Beijing, China_.

Purpose: Osteosarcoma (OS) is the most common primary bone malignancy with a high propensity for local invasion and distant metastasis. Flavokawain A (FKA), a major chalcone from kava extract, has been reported to have antitumor effects on multiple cancer cell lines. The consumption of kava-containing beverage has been associated with a low cancer incidence. In a previous report, mice treated with high-dose FKA did not demonstrate any significant major organ toxicity. However, the efficacy and anticancer mechanisms of FKA in OS is still to be elucidated.

Experimental Design: OS cell lines were treated with increasing dosage of FKA and tested for cell motility, proliferation, and invasion by MTT assay and Matrigel invasion assay. Cell cycle analysis was performed using flow cytometry. We examined Skp2 expression in several OS cell lines using western blot and in patient tissue array by immunostaining. Kaplan-meier analysis and log rank test were used to compare overall survival. Both Skp2-dependent cell cycle progression and Skp2-related RhoA expression were also examined after FKA treatment. The effects of FKA on lung metastasis were evaluated after orthotopic injection of OS cells into the tibia.

Results: We show that FKA inhibits the growth and motility of multiple OS cell lines in vitro. Flow cytometry analysis confirms cellular apoptosis and arrest in G2/M phase after FKA treatment, whereas cellular invasion is also inhibited in a dose-dependent manner. Skp2 is expressed in several OS cell lines and is associated with a poor prognosis in OS patients. Skp2 levels in OS cell lines decreased after FKA treatment. The expression of cell cycle regulators including p21 and p27, which are downstream of Skp2, was upregulated. Moreover, Skp2-related RhoA expression is inhibited by FKA and confirmed at protein level.

Conclusions: Taken together, the evidence suggests FKA exerts anti-invasive effects in association with Skp2-dependent cell cycle progression and Skp2-related RhoA expression. Since Skp2 is a negative prognostic factor for OS patients, FKA should be investigated further as an anti-Skp2 therapeutic strategy for OS.

#2126

**CA102N, a conjugate of hyaluronic acid (ha) and Nimuselide derivative (H-Nim) interferes with PI3K/Akt/mTOR signaling pathway in colorectal cancer (CRC) cells and inhibits tumor growth** in vivo **.**

Eskouhie H. Tchaparian, Louis Lin. _Holy Stone Healthcare Co., Ltd., Taipei, Taiwan_.

Aberrant PI3K/Akt/mTOR signaling pathway is common in several human cancers, including CRC. The pathway regulates many major cellular processes and promotes tumorigenesis and inhibition of apoptosis. Targeting the PI3K/Akt/mTOR network could be important in resistance mechanisms in cancer. CA102N, is a conjugate of Hyaluronic Acid (HA) and H-Nim, a nimuselide (COX-2 inhibitor) derivative, currently under investigation for preclinical development in CRC. Studies have suggested that the anti-proliferative effects of COX-2 inhibitors could be attributed to modulation of the PI3K/AKT signaling pathway; therefore, in this study we aimed to examine the overall molecular mechanism of the antitumor activity of CA102N and evaluate its impact on the PI3K pathway proteins. Cell based studies indicated that in addition to cell cycle arrest; the antitumor activity of CA102N is also related to apoptosis. Administration of CA102N to nude mice bearing established HT29 subcutaneous tumor xenografts caused significant tumor growth inhibition with no signs of systemic toxicity. Immunohistochemical analyses of xenograft tumors demonstrated inhibition of VEGF and CD31, suggesting a role for CA102N in angiogenesis. Disruption of angiogenesis and apoptosis was correlated with a decrease in VEGF and the antiapoptotic protein BCL-2. Suppression of PI3K downstream signaling was a key observation in HT29 cells in vitro and in xenograft tumors treated with CA102N. As compared to untreated vehicle control, a significant decrease in phosphorylation of AKT, mTOR, p70S6K, PRAS40, 4EBP1 and FOXO1 was detected by Western blot analyses. The reduction observed may be caused by both specific protein dephosphorylation/deactivation and by ubiquitin-mediated proteasomal degradation of pathway proteins. These results suggest that the antitumor activity of CA102N may be at least partly related to the modulation of the PI3K/Akt pathway proteins. CA102N represents a promising anticancer agent with minimal toxicity, the insights provided into the mechanisms of its anticancer activity may be the basis for advancing this candidate into clinic.

#2127

p53-independent Noxa induction by cisplatin is regulated by ATF3/ATF4 in HNSCC cells.

Kanika Sharma,1 Thien-Trang Vu,1 Mitra Naseri,1 Wataru Nakajima,2 Kevin Zhan,1 Hisashi Harada1. 1 _VCU Massey Cancer Ctr., Richmond, VA;_ 2 _Nippon Medical School, Tokyo, Japan_.

Head and neck cancer is the sixth leading cancer worldwide and head and neck squamous cell carcinoma (HNSCC) accounts for more than 90% of incident cases. The long-term prognosis of patients with advanced HNSCC has shown little improvement over the last three decades. Induction chemotherapy with platinum-based compounds, taxanes, and 5-fluorouracil is beneficial for head and neck cancer patients, but the prolonged use of chemotherapeutic drugs is limited by their toxicity and by the development of resistance. Tumor cell death induced by both conventional and targeted chemotherapy is often mediated by the BCL-2 family-dependent mitochondrial apoptotic pathway. However, initiators of this apoptotic pathway, such as p53, are frequently mutated or deleted in HNSCC rendering it refractory to treatment. To counter such resistance, direct therapeutic targeting of the BCL-2 family is conceptually appealing. We have investigated the cytotoxic effects of a DNA damaging agent, cisplatin that is used for a standard therapy for locally advanced HNSCC. Cisplatin produces anticancer effects mainly by generating DNA lesions and activating DNA damage response followed by inducing BCL-2 family-dependent mitochondrial apoptosis. Cisplatin induces the expression of a pro-apoptotic BH3-only protein, Noxa that binds to the pro-survival BCL-2 family protein, MCL-1 to inactivate its function and induces cell death. To examine the contribution of Noxa in cisplatin-induced apoptosis, shRNA for Noxa was introduced in p53-null HNSCC cells, since p53 is mutated or deleted in more than 50% of HNSCC. We found that the up-regulation of Noxa is critical for cisplatin-induced apoptosis in p53-independent HNSCC cells. This Noxa induction is regulated at the transcriptional level. Thus, we examined the regulation of the Noxa promoter with a series of luciferase reporter assays. We found that the CRE (cAMP responsive element located at -66~-59 from the transcription start site) on the promoter plays a critical role in Noxa induction. The CRE is known to be regulated by ATF/CREB transcription factors. Downregulation of ATF3 or ATF4 by shRNAs clearly reduced Noxa induction. Furthermore, the binding of these transcription factors to the CRE on the Noxa promoter was increased by cisplatin treatment, determined by chromatin-immunoprecipitation assays. In conclusion, ATF3 and ATF4 are important regulators for cisplatin-induced Noxa in a p53-independent pathway. The results suggest that modulation of the signaling pathways that activate ATF3/ATF4 could be an alternative approach to induce Noxa followed by apoptosis in HNSCC.

#2128

PDGFR beta signaling is required for hypoxia induced mitophagy in glioblastoma survival.

Jinkyu Jung, Mark R. Gilbert, Deric M. Park. _NCI, NIH, Bethesda, MD_.

BACKGROUND: Platelet-derived growth factor (PDGF) signaling plays a key role in gliomagenesis. Glioblastomas express all PDGF ligands and both cell membrane receptors, PDGFRα and PDGFRβ, that are involved in pro-survival autocrine and paracrine loops. Our previous work demonstrated that expression of PDGFRβ is enriched in hypoxic glioblastoma tumor tissues. Also, induction of mitophagy is a well-known critical coping response to tissue hypoxia. We therefore investigated the role of PDGFRβ signaling cascade in regulating hypoxia-induced mitophagy of glioblastoma.

METHODS: Protein detection was performed by immunoblotting and immunofluorescence to determine changes in levels of target proteins upon RNA interference, treatment with PDGFRβ inhibitor, and ectopic gene transfection. MitoSox assay was used to measure mitochondrial reactive oxygen species (ROS). Tumor growth assays were performed in vitro and in vivo using subcutaneous tumor implantation model.

RESULTS: PDGFRβ signaling was essential for induction of hypoxia-mediated mitophagy in glioblastoma cells. Nix, a hypoxic responsive protein and regulator of mitophagy, was necessary for PDGFRβ-mediated glioblastoma survival. Inhibition of PDGFRβ signaling pathway killed glioblastoma cells by suppressing mitophagy and leading to subsequent elevation of mitochondrial ROS. Furthermore, interference of mitophagic response by blocking PDGFRβ signaling enhanced cell death even in chemotherapy-resistant tumor cells.

CONCLUSION: This work suggests a potential mechanism by which PDGFRβ inhibition could kill glioblastoma cells through suppression of NIX-mediated mitophagy. Manipulation of NIX and PDGFRβ pathway may provide novel therapeutic opportunities in chemoresistant tumor models.

#2129

Assessing the efficacy of targeting mitochondrial respiration in delaying lung tumor growth by using subcutaneous xenografts in mouse models.

Sarada Preeta Kalainayakan, Poorva Ghosh, Sanchareeka Dey, Li Zhang. _UT Dallas, Richardson, TX_.

Previously, studies have focused mainly on the precept that tumors depend on glycolysis for energy and growth (the Warburg effect) and that the mitochondria are dysfunctional in cancer cells. However, there is mounting evidence that some cancer cells exhibit elevated mitochondrial respiration. Recent studies in our lab have demonstrated that Non-Small Cell Lung Cancer (NSCLC) cells exhibit intensified mitochondrial respiration and oxygen consumption. We further demonstrated that targeting increased mitochondrial respiration by a therapeutic agent effectively hampers proliferation of Non-Small cell lung cancer (NSCLC) cells in vitro. The purpose of the study is to determine to what extent mitochondrial respiration is affected by the therapeutic agent in vivo.

In order to achieve this goal, NSCLC cells were transduced with lentiviral particles carrying luciferase sequence. NSCLC cells expressing luciferase cells were implanted on the right flank of 4-6 weeks old female NOD/SCID mice to generate subcutaneous xenografts. The therapeutic agent was administered intravenously. Tumor implantation and growth were monitored by bioluminescence imaging (BLI) using Perkin Elmer's IVIS Lumina III Imager. On sacrificing the mice, the tumors were harvested, formalin fixed and paraffin embedded for Immunohistochemistry (IHC).

Our BLI data suggest that there is a considerable reduction in radiance (total flux in photons per second) in the group of mice that were treated with therapeutic agent in comparison to control. Further, there is a significant reduction in tumor volume in mice that received treatment. Our IHC data suggest that there is a reduction in expression of a vital enzyme involved in heme synthesis and hemoproteins involved in mitochondrial respiration.

Our BLI and IHC data suggest that the therapeutic agent affects mitochondrial respiration by affecting mitochondrial complexes in vivo. Additional experiments to corroborate the results are underway.

#2130

Antitumor activity of marin natural product derived Psammaplin A analogs in human lung cancer cells.

Woong sub Byun, Yoonho Shin, Sang Kook Lee, Hyeung-geun Park, Suckchang Hong. _College of pharmacy, seoul national university, Seoul, Republic of Korea_.

Natural products are important sources for bioactive molecules, which have been developed to treat various human health-related symptoms. In our continuos efforts to search for anticancer agents from natural sources, Psammaplin A (PsA), isolated from marine natural products, was found to be a potential candidate in the growth inhibition of cancer cells. PsA, a unique symmetrical bromotyrosine contained in a number of marine sponges of the order Verongida, has exhibited a variety of bioactivities including antimicrobial activity, cytotoxicity against the leukemia cell line, and inhibitory activity of DNA gyrase and DNA topoisomerase. PsA is also known to be a potent inhibitor of DNA methyltransferase and histone deacetylase with growth-inhibitory activity of cancer cells. However, its underlying mechanism of action and the structure-activity relationship (SAR) with PsA analogs have not been elucidated yet. In the present study, twenty-eight synthetic analogs of PsA were newly synthesized and examined the potential of cytotoxicities against cancer cells. A SAR study revealed that the presence of free oxime and disulfide functional groups was responsible for high cytotoxicity. Furthermore, the bromotyrosine component in PsA was relatively tolerable and hydrophobic aromatic groups preserved the cytotoxicity. The aromatic groups were dependent on their size and spatial geometry. Among them, a β-naphthyl derivative of PsA showed a potential cytotoxicity and was comparable to that of PsA. The compound also exhibited a potential antitumor activity in a nude mouse xenograft model. These findings indicated that free oxime and disulfide linker in the chemical structure play an important role for cytotoxicity and PsA analogs might be provided as potential antitumor agents. This work was supported by the National Reseach Foundation of Korea (NRF) Grant funded by the Korean Government (MEST) (NRF-2016M3A9B6903499) and also supported by a National Research Foundation of Korea (NRF) grant funded by the Korean Government (MEST) (No.2009-0083533).

#2131

Tanshinone IIA can decrease growth factor receptors expression and dural-block both Ras/Raf/MEK/ERK and Ras/PI3K/Akt/mTOR pathways to inhibit human breast cancer BT-20 cells.

Chin Cheng Su. _Changhua Christian Hospital, Changhua City, Taiwan_.

Background : Tanshinone IIA (Tan-IIA, C19H18O3) with anti-inflammatory activities and antioxidant properties, is one of the diterpine quinones extracted from Salviae miltiorrhizae radix (Danshen). Tan-IIA can inhibit many human cancer cell lines through different molecular mechanisms. The phosphoinositide-3-kinase (PI3K)/AKT/ mammalian target of rapamycin (mTOR) and RAS/RAF/MEK/ERK pathways are two of the most frequently dysregulated kinase cascades in human cancer. Transmembrane tyrosine kinase has been strongly implicated in the proliferation, survival, and metastasis of human tumors. Both pathways represent important signal transduction mechanisms that facilitate the proliferation and survival of cancers driven by growth factor receptors, such as vascular endothelial growth factor receptor (VEGFR), insulin-like growth factor-I receptor (IGF-IR), or epidermal growth factor receptor (EGFR). Targeting both the Ras/Raf/MEK/ERK and Ras/PI3K/Akt/mTOR Pathways for suppressing inhibitor resistant cells is necessary because the individual downstream components of these signaling cascades either through epigenetic modification or somatic mutation are also known to be frequently altered in cancer, thus contributing to resistance to anticancer therapies and tumorigenesis.

Material and methods: In the present study, the human breast cancer BT-20 cells were treated with Tan-IIA in vitro. The cytotoxicity of Tan-IIA was evaluated by MTT assay. The effects of Tan-IIA on the protein expressions of EGFR, IGF-IR, VEGFR, PI3K, AKT, mTOR, Ras, Raf, MEK and ERK and β-actin in the BT-20 cells were examined by western blot analysis.

Results: The results showed that Tan-IIA can induce the proliferation inhibition with time and dose dependent and inhibit the activity of the Ras/Raf/MEK/ERK and Ras/PI3K/Akt/mTOR pathways. In addition, it was showed that Tan-IIA treatment inhibited the protein expression levels of EGFR, VEGFR and IGF-IR significantly.

Conclusions: These findings indicated that one of the molecular mechanisms for Tan-IIA to inhibit BT-20 cells maybe through inhibiting the protein expression levels of EGFR, VEGFR, IGF-IR and both Ras/Raf/MEK/ERK and Ras/PI3K/Akt/mTOR pathways. The use of Tan-IIA for breast cancer may become a feasible novel therapy option. Further studies are warranted to elucidate its mechanisms fully.

### New Targets 2

#2132

Novel therapeutic approach through systemic depletion of L-cyst(e)ine with engineered cyst(e)inase enzyme for suppression of prostate tumor growth.

Achinto Saha, Shira L. Cramer, Sabin Kshattry, Stefano Tiziani, Everett Stone, George Georgiou, John DiGiovanni. _University of Texas at Austin, Austin, TX_.

Prostate cancer (PCa) is the most common non-skin neoplasm and second leading cause of cancer death in men in the USA. The major drawbacks of PCa treatment is the development of resistance to androgen ablation therapy. Due to abnormal growth and genetic alterations, cancer cells experience higher oxidative stress from reactive oxygen species (ROS) than the normal cells. The L-cysteine (Cys) containing tripeptide, glutathione (GSH) is the major intracellular antioxidant and is essential for the survival and proliferation of cancer cells. Under conditions of elevated ROS, endogenous Cys production is insufficient for GSH synthesis. This necessitates uptake of Cys that is predominantly in its disulfide form, L-cystine (CSSC), via the xCT(-) transporter. Cys is a non-essential amino acid in animals; therefore, eliminating Cys and CSSC uptake should selectively impact tumors that display increased ROS production, without causing an adverse effect on normal physiology. However, inhibition of xCT(-) alone is insufficient because free Cys is still imported via other transporters. A superior approach is the elimination of Cys and CSSC through the action of an enzyme that converts these amino acids into non-toxic products.

Based on the idea that enzyme mediated systemic depletion of the serum Cys/CSSC pool would constitute a powerful and completely novel therapeutic approach, we developed a genetically engineered and pharmacologically optimized human enzyme called cyst(e)inase. We show that administration of cyst(e)inase mediates sustained depletion of the extracellular Cys and CSSC pool in mice. Treatment with this enzyme selectively causes cell cycle arrest and death in cancer cells due to depletion of intracellular GSH and ensuing elevated ROS; yet no apparent toxicities in mice even after months of continuous treatment. Cyst(e)inase suppressed the growth of prostate carcinoma allografts and reduced tumor growth in PCa xenografts. Mechanistically, cyst(e)inase treatment increased AMPK phosphorylation, reduced mTORC1 activity, formation of LC3 II as well as modulation of several cell cycle proteins including p27, c-Myc, CDK2, CDK4, pRB, E2F4 and cyclins A, D1 and E1. Further studies showed cyst(e)inase produced synergistic effects with a GSH synthesis inhibitor, buthionine sulfoximine and the natural compound, curcumin for cell growth inhibition and ROS production in vitro. Cyst(e)inase also showed synergistic tumor growth inhibition with curcumin in a xenograft model of human castrate resistant PCa cells. Collectively, enzyme-mediated depletion of serum Cys and CSSC pool suppresses the growth of prostate tumors, and is very well tolerated. These results suggest that cyst(e)inase represents a potentially safe and effective therapeutic modality as a single agent or in combination for the treatment of prostate and possibly other cancers.

#2133

Pre-clinical studies of EC2629, a highly potent FR targeted DNA crosslinking agent.

Joseph A. Reddy, Melissa Nelson, Christina Dircksen, Theresa Johnson, Marilynn Vetzel, Spencer Hahn, Longwu Qi, Iontcho Vlahov, Christopher Leamon. _Endocyte, Inc., West Lafayette, IN_.

Folate receptor (FR) targeted small molecule drug conjugates (SMDCs) have shown promising results in early stage clinical trials with vintafolide and EC1456. In our effort to develop FR targeted SMDCs with varying mechanisms of action, we have now built EC2629, a folate conjugate of a DNA crosslinking agent based on a novel DNA-alkylating moiety . This agent was found to be extremely potent with an in vitro IC50 ~ 100 x lower than any other folate SMDC we have created to date. Treatment of nude mice bearing FR positive human xenografts led to cures in 100% of the mice at very low doses (300 nmol/kg) using a convenient once a week schedule. The observed activity was not accompanied by any noticeable weight loss (up to 20 weeks post end of dosing) or major organ tissue degeneration. Complete responses were also observed in other FR-positive drug resistant (paclitaxel and cisplatin) models. When evaluated against FR-positive PDX models of ovarian, endometrial and triple negative breast (TNBC) cancers, EC2629 showed significantly greater anti-tumor activity than EC1456 or standard of care (SOC) treatments. Taken together, these studies demonstrated that EC2629 with a distinct DNA reacting mechanism has significant anti-tumor growth activity in numerous models, including those which were drug resistant, thus lending support to our planned clinical development of this novel FR-targeted agent.

#2134

Use of methionine gamma-lyase-loaded erythrocytes to induce effective methionine depletion in cancer therapy.

Fabien Gay,1 Karine Aguera,1 Karine Senechal,1 Philip Lorenzi,2 Alexander Scheer,1 Françoise Horand,1 Vanessa Bourgeaux1. 1 _ERYTECH Pharma, Lyon, France;_ 2 _MD Anderson Cancer Center, Houston, TX_.

Methionine (Met) dependence is a cancer-specific metabolic defect that has emerged as a target during the last two decades. The use of methionine gamma-lyase (MGL; EC number 4.4.1.11), a bacterial Met-catabolizing enzyme, is a promising strategy for treatment of Met-dependent cancers. However, one challenge is that MGL has a very short half-life (~2 hours), resulting in a short-term Met depletion in vivo. Additionally, its cofactor, pyridoxal 5'-phosphate (PLP) is rapidly eliminated from plasma (Yang et al., 2004). PEGylation extends the MGL half-life in mice to up to 38 hours. Nevertheless, frequent injections are still necessary for maintaining an effective Met depletion over time (Sun et al., 2003). In addition, the low bioavailability of PLP remains a major hurdle due to both scavenging by plasma proteins and very short half-life (< 15 minutes) in the blood stream (Zempleni, 1995). Studies showed that pyridoxine (PN), a vitamin B6 vitamer, can easily cross red blood cells (RBCs) membrane to be rapidly converted to PLP via an enzymatic reactions cascade (Anderson et al., 1971). Considering those particular characteristics of RBCs, we propose that RBC-encapsulated MGL (ERY-MET) will protect the enzyme from degradation/immune reactions and therefore overcome the pharmacodynamic limitations for its use in the treatment of Met-dependent cancers. Pharmacokinetics, pharmacodynamics, and safety parameters of MGL-loaded RBCs were evaluated in healthy mice. In parallel, the role of RBC in PLP biosynthesis from exogenous uptake of PN was investigated in vivo. Finally, we investigated the antitumoral effect of repeated injections of MGL-loaded RBCs combined with daily intragastric administration of PN in two subcutaneous xenografted mouse models for human gastric and glioblastoma tumors. The MGL half-life increased from < 24 hours to ~ 10 days when encapsulated in RBC, with no toxicity reported after one injection of ERY-MET. Following intragastric administration (3.2 mg/kg), PN was rapidly (15 minutes) converted into PLP within the RBC, resulting in an increase of MGL activity (holoenzyme). Combining a weekly single intravenous injection of ERY-MET for 5 days, in association with daily PN supplementation by gavage led to a sustained Met depletion in plasma and induced a 85% inhibition of tumor growth 45 days following implantation of glioblastoma cells. In the gastric mouse model, tumor growth was inhibited by 72% at the same time point (45 days).This study clearly demonstrated that encapsulation of MGL in erythrocytes both strongly improved the half-life and contributed to provide active cofactor. In parallel, repeated injections of ERY-MET were effective against tumor growth in mouse models. Thus, due to the RBC intrinsic characteristics, ERY-MET represents a new promising treatment against a broad scope of cancers that rely on Met metabolism.

#2135

Selectivity and specificity of engineered T cells expressing KITE-585, a chimeric antigen receptor targeting B-cell maturation antigen (BCMA).

Gregor B. Adams, Jun Feng, Atefeh Ghogha, Armen Mardiros, Ruben Rodriguez, Tassja J. Spindler, Jed Wiltzius, Tony Polverino. _Kite Pharma, Santa Monica, CA_.

Background: Immunotherapy has provided treatment options for cancers that are otherwise refractory to standard approaches. One such technique is to use adoptive transfer of engineered autologous T cells expressing a chimeric antigen receptor (CAR) directed against a tumor antigen. The efficacy of CAR T cells directed against hematological malignancies, particularly CD19-expressing B cell leukemia and lymphomas, has been demonstrated in multiple clinical studies. The success of this approach has prompted development of CAR T cells directed to different tumor antigens for other tumor types. To ensure the selectivity and specificity of the CAR T cells against their intended target, screening methods need to be employed. Multiple myeloma is an incurable malignancy of plasma cells. B-cell maturation antigen (BCMA), also known as tumor necrosis factor superfamily member 17 (TNFRSF17) is nearly ubiquitously expressed on multiple myeloma cells, plasma cells and subsets of mature B cells.

Methods: In order to screen for the specificity of novel CAR T cells directed against BCMA, we utilized a cell microarray platform developed by Retrogenix. In this screen, approximately 4500 human plasma membrane proteins (representing up to 75% of the human plasma membrane proteome) are individually expressed in human HEK293 cells. Fluorescently labeled CAR T cells, which showed cytolytic activity against MM cell lines expressing BCMA, were applied to the cell microarray and specific binding of the CAR T cells to target cells was determined.

Results: Primary hits were sequenced to confirm identity and secondary specificity screens were performed on the identified hits. Specific binding of both mock transduced and BCMA CAR transduced T cells were confirmed for different plasma membrane proteins expressed from the HEK293 cells. These included known T cell interactors, such as ICOSLG, CD244 and CD86, where binding is proposed to be independent of CAR expression. Subtracting the hits of the mock transduced T cells from the BCMA CAR T cells demonstrated specific binding of the CAR T cells to BCMA. Utilizing the fully human IgGs directed against BCMA from which the single-chain variable fragments (scFvs) of the CARs were derived, we further confirmed specific binding to BCMA in additional secondary screens. Additionally, a lack of off-target binding of the fully human IgGs to normal tissue was demonstrated in a tissue cross reactivity screen.

Conclusions: These studies highlight the tractability of this cell microarray approach for determining the specificity of novel CAR constructs expressed in T cell. Demonstrating the selectivity and specificity of anti-BMCA CAR T cells further supports the progression of KITE-585 towards Phase 1 clinical studies in MM patients.

#2136

Inhibiting cancer growth by targeting the TNFR2 oncogene with TNFR2 antagonistic antibodies.

Heather Torrey, John Butterworth, Toshiyuki Mera, Yoshiaki Okubo, Limei Wang, Danielle Baum, Audrey Defusco, Sara Plager, Sarah Warden, Daniel Huang, Eva Vanamee, Rosemary Foster, Denise L. Faustman. _Massachusetts General Hospital & Harvard Medical, Charlestown, MA_.

Background: Although antibody targeting of the HER2 oncogene represents an outstanding clinical treatment and a success story showing direct cancer killing with antibodies, many oncogenes are intracellular and are not expressed broadly, whether on a single or multiple tumor types. The recently discovered TNFR2 oncogene is broadly expressed on many human tumors. Colon cancer cells, multiple myeloma cells, renal cell carcinoma cells, Hodgkin's lymphoma cells, ovarian cancer epithelial cells and cutaneous non-Hodgkin's lymphoma cells can aberrantly express the TNFR2 receptor as an oncogene for growth. For non-cutaneous T cell lymphomas, the genetic basis of the TNFR2 deregulation has recently been tied to constitutive overexpression of TNFR2 from frequent gene duplications or cytoplasmic TNFR2 mutations that confer constitutive agonism, i.e. tumor expansion. These features make TNFR2 an advantageous molecular target for direct tumor targeting.

Methods: We designed monoclonal antibodies to target the TNFR2 oncogene and directly kill human tumor cells. TNFR2-directed antibodies were screened for their ability to induce the death of rapidly growing tumor cells, such as ovarian cancer cells (i.e., OVCAR3).

Results: Novel dominant anti-TNFR2 antibody candidates (TNFR2 antagonistic antibodies) did not require Fc binding for activity, expressed dominance over TNF-mediated agonism, and hampered intracellular NF-ĸB activation and phosphorylation obligatory for TNFR2 signaling and cell growth of tumor cells. Even low doses of TNFR2 antagonists rapidly and directly killed TNFR2 oncogene-expressing ovarian cancer cells. Examination of the structural biology of these dominant TNFR2 antagonist antibodies uncovered a unique and stabilizing TNFR2 receptor formation, anti-parallel dimeric TNFR2, which inhibits intracellular signaling, cannot bind TNF, cannot be cleaved to create soluble TNFR2 and is exponentially more active on the dividing cells of cancer.

Conclusions: TNFR2 is a unique and broadly expressed human oncogene that can potentially be targeted to directly stop the growth of cancer cells (including ovarian cancer cells) by antibody-induced cell death. The creation of dominant TNFR2 antagonism provides a unique, non-signaling complex that has implications for the therapeutic targeting of TNF superfamily receptors, especially TNFR2.

#2137

JAK2 as a novel therapeutic target in anaplastic thyroid cancer.

Nicole C. Pinto,1 Kara Ruicci,1 Stephenie Prokopec,2 Karlee Searle,1 Matthew Lowerison,1 John Yoo,1 Kevin Fung,1 Danielle MacNeil,1 Hon S. Leong,1 Alessandro Datti,3 Paul C. Boutros,2 John W. Barrett,1 Anthony C. Nichols1. 1 _Western University, London, Ontario, Canada;_ 2 _Ontario Institute for Cancer Research, Toronto, Ontario, Canada;_ 3 _Lunenfeld-Tanenbaum Research Institute, Toronto, Ontario, Canada_.

Introduction: Thyroid carcinoma is the most common endocrine malignancy. Anaplastic thyroid cancer (ATC) is rare (1.3%) and represents arguably the most lethal human malignancy with 1-year survival rates of only 10%. There are currently no effective treatments for the majority of patients, highlighting an urgent need for novel therapeutics to manage this disease.

Objective: To validate the functional importance of JAK2 as a therapeutic target in ATC.

Methods: In this study, we have used siRNA knockdown to interrogate the JAK2 signaling pathway as a therapeutic target in ATC. We investigated the mechanism of action and cell death, and assayed for migration and invasion, in vitro. The chick chorioallantoic membrane (CAM) model was also utilized for drug testing, whereby 1x106 Cal62 ATC cells were on-planted to the chick embryo membrane. Two days post on-plant, CAM models were treated with the vehicle (DMSO) or lestaurtinib to measure outcomes including tumor volume and vascularity.

Results: We identified the JAK2 inhibitor lestaurtinib as an inhibitory agent controlling cell line proliferation at submicromolar mean inhibitory concentrations. Immunoblotting revealed the inhibition of phosphorylation of the downstream signaling molecule STAT5 in a dose-dependent manner. Treatment of Cal62 cells resulted in a decrease in cell migration using the scratch-wound assay. The anti-proliferative effective of lestaurtinib did not cause apoptosis, autophagy or cell senescence. CAM models treated with a 4 uM dose of lestaurtinib showed a significant decrease in both tumor volume and vascularity.

Conclusions: Lestaurtinib was found to provide potent control of ATC cell proliferation and migration, and was also found to decrease tumor growth and vascularity in a CAM model. Knockout studies are underway to confirm that the anticancer effect of this drug is indeed mediated through JAK2 signaling. If validated, JAK2 represents a novel therapeutic target for the treatment of aggressive thyroid cancers.

#2138

Tumor treatment fields downregulate the BRCA1/FA pathway genes leading to reduced DNA repair capacity, the inhibition of mitophagy and enhanced cell death.

Narasimha Kumar Karanam, Lianghao Ding, Brock Sishc, Debabrata Saha, Michael D. Story. _UT Southwestern Medical Center, Dallas, TX_.

The application of new physical cancer treatment modality utilizing alternating electric fields termed tumor treatment fields (TTFields) has revolutionized the treatment of recurrent and newly diagnosed glioblastoma. This non-invasive exposure to low-intensity, intermediate frequency, alternating electric fields to the region of the tumor has resulted in a significant increase in overall survival when compared to standard therapy with very minimal side effects. Clinical trials are recruiting or ongoing at additional tumor sites including lung, pancreatic, and ovarian cancer. The primary mechanism of TTField cell killing is thought to be the disruption of mitosis; however, other potential mechanisms are under investigation. Using a panel of five NSCLC cell lines we found that TTFields treatment alone inhibits cell proliferation, and decreases survival, though the degree of inhibition varies between cell lines. To understand the molecular mechanisms underlying the biological effects of TTField exposure we studied temporal gene expression changes in the NSCLC cell lines after TTField treatment. We observed that most differentially expressed genes are part of cell cycle and proliferation pathways which is in agreement with earlier findings. Interestingly we found that the expression of BRCA1 DNA damage repair pathway genes were significantly downregulated (P < 0.05) upon TTField treatment. We confirmed the downregulation of BRCA1/FA pathway proteins by western blot. When examining the nuclear role of the BRCA1/FA pathway genes we found that TTField treatment slowed the repair of ionizing radiation-induced DNA damage compared to radiation alone which is evident by an increased number of DNA double strand break repair foci at any given time. Moreover, we found that TTField treatment increased the incidence of chromatid aberrations. We also examined the newly identified BRCA1/FA pathway genes cytosolic role in mitophagy where we observed alterations in mitophagy related gene (PINK1, OSCP1, ATP5 and DAPIT) expression and confirmed the same at the protein level by western blot. We hypothesized that TTFields disrupt the clearance of damaged mitochondria due to the downregulation of BRCA1/FA pathway players, causing an imbalance in oxygen metabolism leading to the production of high levels of radical oxygen species (ROS) and as a result, cell death. Using CellROX dye we found that TTField treatment did result in increased ROS production suggesting a new mechanism of action for TTField exposure. Novel chemotherapy agents, particularly PARP inhibitors, in combination with DNA damaging agents like radiation and TTFields may be advantageous through the conditional vulnerability of down-regulated BRCA1.

#2139

The effects of cyclodipeptide on the transport of doxorubicin and its cytotoxicity on tumor cell.

Ikumi Sugiyama, Yasuyuki Sadzuka. _Iwate Medical Univ. School of Pharmacy, Shiwa-Gun, Japan_.

Purpose: Chemotherapy using antitumor agents plays an important role in clinical cancer therapy. Among the treatments involving antitumor agents, the enhancement of antitumor activity was observed by combined chemotherapy. We have shown previously that some amino acids included theanine, taurine and anserine et al. as food components, increased doxorubicin (DOX) induced antitumor effect in vitro and in vivo. These effects by combined amino acids have depended on the increased DOX concentration in the tumor cells. Furthermore, these suppressions of DOX efflux by theanine and taurine were caused by the inhibitions of glutamate transporter and taurine transporter, respectively. The effect of anserine on DOX influx might connect with dipeptide transporter. Thus, other amino acid and unique peptides may act to DOX transport. In this study, the effects of cyclodipeptide on the transport of DOX and its cytotoxicity on tumor cell were clarified.

Methods: Cyclo-leucine-proline (cyclo-Leu-Pro), cyclo-phenylalanine-proline (cyclo-Phe-Pro) and cyclo-glycine-proline (cyclo-Gly-Pro) as cyclodipeptide was used in this study. In transport of DOX, DOX influx or efflux was determined with combined cyclodipeptide in M5076 ovarian sarcoma cells or P388 leukemia cells. DOX concentration in tumor cell was measured using spectrofluorometer (Ex. 470 nm, Em. 580 nm). The cytotoxic study of DOX with combined cyclodipeptide was performed using WST-8.

Results and Discussion: Cyclo-Phe-Pro and cyclo-Gly-Pro did not affect DOX influx and efflux in M5076 ovarian sarcoma cells and P388 leukemia cells. By combined cyclo-Leu-Pro, DOX influx into both M5076 ovarian sarcoma cells and P388 leukemia cells changed, compared to that of DOX only group. Into M5076 ovarian sarcoma, cyclo-Leu-Pro had strong effect in increased DOX influx in particular. And cyclo-Leu-Pro showed a tendency to suppress DOX efflux from M5076 ovarian sarcoma. In this studies of influx and efflux, concentration of cyclo-Leu-Pro existed optimum amount for increased DOX concentration in tumor cells. It is expected that cyclo-Leu-Pro induces the increment of DOX concentration in the tumor in vivo. DOX had the cytotoxic effects on M5076 ovarian sarcoma cells or P388 leukemia cells in culture. The combined cyclo-Leu-Pro with DOX increased cytotoxic effect, compared to that in the DOX alone group. In conclusion, cyclo-Leu-Pro has increased effect on DOX influx and inhibited effect on DOX efflux, and DOX induced cytotoxicity. It is expected that the combined cyclo-Leu-Pro will improve cancer chemotherapy by DOX. Moreover, cyclo-Leu-Pro may be help to prevent adverse effect since it will be able to decrease DOX dose.

#2140

Understanding molecular mechanisms underlying heme function in non-small cell lung cancer cells.

Sagar Sohoni, Chantal Vidal, Li Zhang. _University of Texas at Dallas, Richardson, TX_.

Heme is a central molecule for mitochondrial respiration and for all processes involved in oxygen utilization. Heme serves as a prosthetic group or as a cofactor for a number of oxidative phosphorylation enzymes and other oxygen-utilizing hemoproteins. Heme directly regulates the synthesis, translocation and assembly of these enzyme complexes. Most, if not all, human cells can synthesize and uptake heme from the circulation. A number of epidemiological studies have shown that high heme intake is associated with increased risk of cancer, including lung cancer. To assess the status of heme metabolism in lung cancer cells, we performed a series of experiments in non-small-cell lung cancer (NSCLC) cell lines such as HCC4017, H1299, A549, H2009, H1395, H460 and Calu-3. We then compared results with an immortalized normal lung cell line, HBEC30KT and observed significant increase in rates of heme synthesis and heme uptake in NSCLC cells compared to HBEC30KT. Previous studies carried out in our lab showed intensified mitochondrial respiration and increased levels of heme and hemoproteins in NSCLCs. We examined the effect of heme depletion on mitochondrial biogenesis and function. We observed that mitochondrial functions such as ATP production, NAD/NADH ratios and ROS production are also affected by heme depletion. Proteins involved in mitochondrial biogenesis are significantly affected when cells are grown in heme depleted medium. Studies are currently underway to check how heme depletion affects the interactions of various transcription factors involved in mitochondrial biogenesis and function.

#2141

Development of a novel miR-129 mimic with enhanced therapeutic potential for treatment of resistant colorectal cancer.

Andrew T. Fesler,1 Ning Wu,1 Hua Liu,2 Jingfang Ju1. 1 _Stony Brook University, Sound Beach, NY;_ 2 _Stony Brook University, Stony Brook, NY_.

Treatment of advanced stage colorectal cancer remains a clinical challenge associated with resistance to fluoropyrimidine based chemotherapy. There is an urgent need to discover and develop new strategies to enhance treatment efficacy in order to improve outcomes for these patients. Non-coding microRNAs (miRNAs) have important functions as oncogenes or tumor suppressor genes in the regulation of cancer development and progression. Recently, miRNAs have emerged as potential therapeutic options. We have previously identified miR-129 as a tumor suppressor miRNA and potential therapeutic candidate in colorectal cancer. The expression of miR-129 is progressively lost in colorectal cancer patients and is an important regulator of apoptosis through the targeting of genes such as BCL-2. miR-129 was also found to enhance 5-flurouracil (5-FU) cytotoxicity in vitro and in vivo. To further developing miR-129 based novel therapeutics in colorectal cancer, we have designed a modified version of miR-129 to enhance stability and efficacy. The miR-129 mimic is significantly more potent in inhibiting proliferation of a panel of colon cancer cell lines than the native miR-129 precursor, with 79% reduction by miR-129 mimic compared to 38% for native precursor. The miR-129 mimic induces profound cell cycle arrest at the G1/S checkpoint. The G1/S ratio increased 3.8 fold compared to control when cells were transfected with miR-129 mimic. We also demonstrated that the miR-129 mimic retains its target specificity to BCL-2, TS and E2F3. The therapeutic potential of miR-129 mimic was demonstrated in vivo mouse colon tumor models as a potent inhibitor of tumor growth and metastasis. As a result, miR-129 mimic has a great potential to be further developed as a novel therapeutic drug for treatment of advanced colorectal cancer.

#2142

Role of TRAIL signaling through the development of carcinogen-induced colorectal cancer.

Yumin Oh,1 Seulki Lee,1 Kang Choon Lee2. 1 _Johns Hopkins School of Medicine, Baltimore, MD;_ 2 _Sungkyunkwan University, Suwon, Republic of Korea_.

TNF-related apoptosis-inducing ligand (TRAIL/APO-2L) is a death ligand that can induce apoptosis by its cognate death receptors (DRs). Due to its unique ability to selectively induce DR-mediated apoptosis in cancer cells while showing no apparent toxicity to normal cells, the rhTRAIL has been actively studied for cancer therapy. Clinical studies of TRAIL revealed a broad tolerability in humans but failed to demonstrate a robust therapeutic benefit in oncology. The main factors responsible for the disappointing results of TRAIL used in cancer patients are 1) its short half-life (less than 30 min in humans) and 2) heterogeneous primary cancers are generally TRAIL-resistant. To overcome the short half and low potency of TRAIL in vivo, we have developed an engineered PEGylated TRAIL (TRAILPEG). PEGylation is a gold standard to extend the half-life of protein drugs and a highly efficient commercial strategy. While investigating the improved therapeutic potential of TRAILPEG in colitis-associated cancer (CAC), we validated that TRAILPEG can ameliorate an inflammatory bowel disease (IBD), the high risk for CRC. Therapeutic efficacy of systemically administered long-acting TRAIL was validated in vivo in IBD and CAC animal models showing an anti-inflammatory and anti-cancer effects. Dimethylhydrazine metabolite azoxymethane (AOM) used to induce CAC in A/J mice, while a chronic inflammation model of colitis/IBD was induced with DSS without the AOM. Most of the mice administered with AOM/DSS developed adenocarcinoma on the distal part of the colon, but the number of adenocarcinomas per colon was significantly lower in the TRAILPEG treated mice than in the vehicle-treated groups. The shortening of the colon length, a measurable characteristic of colitis severity was more recovered in mice treated with TRAILPEG compared to disease control. The TRAILPEG treatment significantly decreased the number of large neoplasms in total tumor and led to an over 30~40% reduction in the total tumors. In addition, the symptoms of rectal bleeding in the AOM/DSS mice was rare to detect. The elevated MPO activity was significantly suppressed by TRAILPEG treatment. In parallel, we found that TRAILPEG treatment resulted in the activation of Caspase-8 and decrease of phosph-STAT3 suggesting the dual roles of TRAIL signaling for apoptosis and anti-inflammation by western blotting. The mRNA levels of inflammatory cytokines including of IL-6, IL-17, MCP-1, ICAM-1, and TNF-β were recovered by TRAILPEG in the AOM/DSS model. Our studies demonstrated that TRAILPEG could effectively treat IBD and CAC in AOM/DSS mice model. The mechanisms involved in this effect of TRAILPEG on IBD and CAC were associated with an increase of cell death, as well as, intervention inflammatory responses.

#2143

**Occupational exposure to automobile exhaust fumes: Genotoxicity, clastogenicity and cell growth assessment in** E. coli **PQ37 and** Allium cepa **models.**

Solomon Eduviere Owumi, Michael A. Gbadegesin, Nkemdilim Anyaoku, Oyeronke A. Odunola. _University of Ibadan, Ibadan, Nigeria_.

Epidemiological studies have established links between occupational exposures to automobile exhaust fumes (AEF) and lung cancer, the propensity for systemic toxic responses. Street hawking and begging between automobiles, on various highways in Nigeria is on the increase. This occupational hazard is further worsen by dire economic recession in Nigeria, and growing population with limited prospect of improving their economic situation. Ages of vulnerable population ranges: 4 to 60yrs, exposure to AEF and susceptibility to health hazard maybe age related.

We examined the effect of AEF exposure on E. coli PQ37 deoxyribonucleic acid (DNA) damage, cell growth and chromosomal aberration in Allium cepa. The effective concentration of AEF was determined as EC50: 0.43mg/ml from A. cepa root length growth against AEF concentrations. Furthermore, A. cepa exposed to AEF at EC50 (0.43mg/ml), EC25 (0.108mg/ml) and EC10 (0.043mg/ml) for 12, 24 and 48hrs mitotic index and chromosomal aberration were evaluated at various endpoints. Chromosomal abnormalities were observed in dividing cells and decreases in A. cepa cell growth occurred dose and time dependently.

Exposure of E. coli to AEF (EC100, EC50, EC25, EC10) resulted in DNA damage (SOS inducing potency (SOSIP) with increasing concentration of AEF and compared to the positive control 4- nitroquinolone oxide (4-NQO). Decreases in the SOSIP (at EC200) indicate a threshold beyond which E. coli SOS response gene may have been extensively damaged to produce a response.

Taken together we conclude that constituents of AEF are genotoxic in E. coli, induce chromosomal aberration and increases cell growth in A.cepa. We speculate on the possibilities of toxic health outcomes including carcinogenesis in humans persistently exposed to AEF fumes either occupationally or by any other means as observed in both street hawkers and beggars in Nigeria

#2144

A novel method for high-throughput discovery of neo-antigens and corresponding T-cell receptors.

Songming Peng,1 Jesse Zaretsky,2 Michael T. Bethune,1 Alice Hsu,1 David Baltimore,1 Antoni Ribas,2 James Heath1. 1 _California Institute of Technology, Pasadena, CA;_ 2 _UCLA, Los Angeles, CA_.

Immunotherapies that boost the ability of endogenous T cells to destroy cancer cells have demonstrated therapeutic efficacy in several human malignancies. Cytotoxic T cells actively recognize peptide antigens, which are displayed on major histocompatibility complexes (MHCs) on the surface of the malignant cells, to play a central role in cancer immunotherapy. However, efficient methods for identifying the tumor antigens and the corresponding T cell receptors are still limited. We report here a DNA-barcode nanoparticle and microfluidics-based method to sort and enumerate neoantigen-specific T cells, at an unprecedented level of sensitivity. The method also permits the matching of a single neoantigen to a specific CD8+ T cell with the TCR a/b gene sequence. The resultant information informs an immunotherapy regimen that is specific for that patient, which can be used to custom-design effective ACT therapies.

#2145

Regulation of YAP1 during hypoxia and its novel role in vascular mimicry and angiogenesis.

Namrata Bora Singhal, Srikumar Chellappan. _H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL_.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality. As in other solid tumors, angiogenesis is necessary for the growth and progression of NSCLC. Vascular mimicry is a phenomenon in which non-endothelial tumor cells form angiogenic vascular structures. We have shown in the past that NSCLC cancer stem-like cells (CSCs) have unique ability to undergo vascular mimicry. The present study shows that transcriptional co-activator YAP1, the oncogenic component of the Hippo pathway, enables the NSCLC CSCs to form angiogenic tubule-like structures in matrigel. Inhibition of YAP1 or depletion of YAP1 suppressed vascular mimicry of CSCs as well as angiogenic tubule formation by HUVECs in matrigel as well as in a fibrin gel bead assay (FIBA) assay. The NSCLC CSCs were found to have higher mRNA expression of VEGF receptor II (KDR) and Angiopoietin-2 (AngPT-2) and depletion of YAP1 reduced the expression of VEGF, KDR and AngPT-2 mRNA; these promoters were induced by YAP1 in transient transfection assays, suggesting a possible molecular mechanism by which YAP1 regulates angiogenesis. Hypoxia is a strong inducer of tumor angiogenesis, cancer progression and metastasis. Our experiments showed that NSCLC cells grown in hypoxic conditions or treated with hypoxia mimetic DMOG have higher YAP1 mRNA and protein expression. However, such a change was not observed in the expression of YAP1 orthologue, TAZ, nor in the canonical Hippo pathway proteins like LATS1&2, Mst1&2, Sav or Mob. We identified a novel regulation of YAP1 by prolyl hydroxylase PHD2, which is mainly known to regulate HIF1α. PHD2 was found to hydroxylate proline residue(s) in YAP1 in a region between aa 284 to aa 289 as seen by mutational analysis. YAP1 was found to directly bind to PHD2 and depletion of PHD2 or treatment with DMOG which is an inhibitor of prolyl hydroxylases, elevated YAP1 protein levels in the nucleus. Further, YAP1 was found to associate with HIF1α as detected by co-immunoprecipitation experiments and could enhance HIF1α-mediated induction of the VEGF promoter. Proximity ligation assays performed on TMA showed enhanced YAP1 and HIF1α interaction in lung tumor tissues compared to normal cells. Our data suggest a novel regulation of YAP1 in hypoxic environment that supports angiogenesis and tumor growth.

#2146

Expression of SLC22A23 gene in laryngeal carcinoma.

Nur Buyru, Seda Ekizoglu, Emin Karaman, Turgut Ulutin. _Istanbul University, Istanbul, Turkey_.

Laryngeal squamous cell carcinoma (LSCC) accounts for approximately 30-40% of head and neck cancers. Although tobacco and alcohol consumption are considered the major risk factors for LSCC, various genetic and epigenetic factors have also been associated with LSCC development and progression. Since early detection and diagnosis of LSCC can greatly increase the chance of a successful treatment and survival, several studies have addressed the identification of target genes involved in LSCC pathogenesis. In this study we aimed to identify differentially expressed genes involved in the pathogenesis of LSCC using the Genefishing technique. As a result of this investigation we identified the SLC22A23 gene as one of the differentially expressed gene.

Solute carriers (SLC) comprise the largest family of membrane transport proteins with more than 300 members which have been divided into 52 families. The main functions of these proteins is to transfer a wide range of substrates such as amino acids, lipids, inorganic ions, peptides, saccharides, metal ions, proteins, xenobiotics and drugs. It is well known that cancer cells need extra metabolic requirements during rapid cell cycles. One of the known functions of the SLC proteins is to facilitate the uptake of nutrients and removal of metabolites. SLC22A23 (solute carrier family 22, member 23) belongs to the SLC family of organic ion transporters that are responsible for the uptake or excretion of many compounds including drugs, toxins and endogenous metabolites in a variety of tissues. SLC22A23 is expressed in various tissues but no substrates or functions have yet been identified for SLC22A23. Altough the exact function is unknown, single-nucleotide polymorphisms which are located in the SLC22A23 gene have been associated with inflammatory bowel disease, endometriosis-related infertility and the clearance of antipsychotic drugs. On the other hand, SLC22A23 was identified as a prognostic gene to predict the recurrence of triple-negative breast cancer.

In this study, to confirm the GeneFishing assay data, we investigated expression of the SLC22A23 gene in laryngeal tissue samples obtained from 83 patients by quantitative RT-PCR. We detected statistically significant upregulation of the SLC22A23 mRNA in laryngeal tumor tissues (55.4%) (p<0.001). However, no significant correlation was found between SLC22A23 expression and clinicopathological parameters including age, sex, stage, histological grade and smoking (p>0.05).

To understand the association between the SNPs in SLC22A23 and LSCC we also investigated the frequency of different genetic variants (rs4959235, rs6923667, rs9503518). 77.2% of patients were homozygote for the rs9503518-AA genotype and %52.2 of these patients had increased SLC22A23 expression (p=0.046).

Our results indicate that there is a clear need for further studies investigating the SLC22A23 gene to identify its exact function and way of action in LSCC.

#2147

IDH1 mutation-inspired α-ketoglutaric acid mimics for epigenetic therapy of higher grade gliomas.

Hanumantha Rao Madala, Surendra Reddy Punganuru, Kalkunte S. Srivenugopal. _Texas Tech Univ. Health Sciences Ctr., Amarillo, TX_.

Mutations at the active site of isocitrate dehydrogenase 1 gene (IDH1; R132H) occur at a high frequency (>70%) early in the oncogenesis of lower grade malignant gliomas, and result in a dramatic accumulation of the oncometabolite D-2 hydroxyglutarate ( D-2HG), that effectively replaces the normal metabolite α-ketoglutarate (α-KG) in cell physiology. Surprisingly, IDH1 mutations bestow superior therapeutic responses to alkylating agents and better patient survival. D-2HG, effectively competes with α-KG and potently inhibits various dioxygenase reactions, including the TET1, TET2 DNA -demethylases and histone demethylases (5m-cytosine to 5-OH cytosine, H3-K-meX to H3-K-meX-1) thereby, re-shaping and reprogramming the epigenetic landscape, and consequent transcriptional silencing; the DNA repair protein O6-methlguanine DNA methyltransferase (MGMT), which confers drug resistance is one major target for such repression. As a novel and innovative strategy of turning the tide against GBMs by exploiting the mechanistic aspects of the oncometabolite, we hypothesized that D-2HG and α-KG derivatives that can replace the natural metabolite in epigenomic dioxygenase reactions will serve as potent anti-glioma drugs either by themselves or in combination with the alkylating agents. To this end, we first synthesized a D-2HG diethyl ester to enhance its cellular uptake and tested its effects on four human brain tumor cell lines (DAOY, T98G, SF188 and UW18). This compound, at 5-10 mM and 24 h treatments, moderately inhibited the DNA repair activity of MGMT, increased temozolomide cytotoxicity by 1.5 to 3-fold, and induced histone H3-methylations as determined by western blot analyses. To increase the potency and design better compounds mimicking the oncometabolite and validate its impact on epigenesis, we synthesized a 2,4-dimethyleneglutaric acid (DMG), a α-KG mimic with methylene groups inserted at the C2 and C4 positions. The hydrophobic DMG ester, by itself, was cytotoxic with IC50 values up to 500 µM against brain tumor cell lines, however, when combined at 100 µM with TMZ resulted in a great synergistic cell killing (generally-9 fold, but 28-fold with UW-18 GBM cells). 0.25 mM DMG inhibited the cellular MGMT activity by >80%, induced degradation of TET1 protein and highly increased the methylation levels of histones (H1K25me1, me2 and H2BK25me2). Currently, experiments to determine the BBB-penetrance of DMG, its ability to induce genomic and MGMT-specific methylations in glioma cells and GBM regression in intracranial xenograft models developed in nude mice are underway. Collectively, these data reveal that acute treatments of α-KG analogs can alter the cellular epigenetic makeup in a manner ascribed to D-2HG, and open up the much-needed novel and exciting avenues of oncometabolite therapy for brain tumors (supported by CPRIT grants RP130266 & RP170207 to KSS).

#2148

Phenyl butyrate inhibits pyruvate dehydrogenase kinase 1 and contributes to its anti-cancer effects.

Wen Zhang, Kin Yip Tam. _University of Macau, Macau, China_.

In cancer cells, the metabolic features are significantly different from those of normal ones, which are hallmarks of several malignancies. Recent studies brought atypical cellular metabolism, such as aerobic glycolysis or the Warburg effect, into the scientific limelight. Pyruvate dehydrogenase kinase 1 (PDK1), a key enzyme in the pathway of glucose metabolism, could inactivate the pyruvate dehydrogenase (PDH) by phosphorylating it and preserving the substrates pyruvate, lactate and alanine for gluconeogenesis. Overexpression of PDK1 could block the oxidative decarboxylation of pyruvate to satisfy high oxygen demand in cancer cells, while inhibition of PDK1 could upregulate the activity of PDH and rectify the balance between the demand and supply of oxygen, which could lead to cancer cell death. Thus, inhibitors targeting PDK1 might be a promising strategy for cancer treatment by acting on glycolytic tumors while showing minimal side effects on the oxidative healthy organs.

Phenyl butyrate (PB) has been proved to decrease PDH phosphorylation level and increase PDH activity by inhibiting PDK1 in fibroblast cells, PDH deficiency zebrafish and wild type mice. PB has also shown efficacy in many cancers and so far, all of its anti-tumor activity has been attributed to the histone deacetylase (HDAC) inhibitor mode of action. As PDK1/PDH controls the critical switch between oxidative phosphorylation and glycolysis in cancer cells, PDK1 is a key target in tumor metabolism for anti-cancer treatment. We hypothesize that the therapeutic effects of PB in cancers might depend on suppressing PDKs and promoting PDH activity, in addition to its proposed role as HDAC inhibitor. We showed that PB directly inhibited the kinase activity of PDK1 in a purified system. In several different cancer cell lines, PB reduced the phosphorylation level of PDH, increased the mitochondrial respiration, decreased glycolysis in cytoplasm, reversed mitochondrial hyperpolarization, activated some proteins in apoptotic signalling pathway and then induced the apoptosis of cells. In summary, this is the first study indicated that PB could exert its anti-cancer effects through inhibiting PDK1, altering the mitochondrial bioenergetics and inducing apoptosis.

#2149

Unusual expression of HPRT on the surface of the colorectal cancer cell lines HT29 and SW620.

Evita G. Weagel, Michelle H. Townsend, Michael D. Anderson, Edwin J. Velazquez, K Scott Weber, Richard A. Robison, Kim L. O'Neill. _Brigham Young Univ., Provo, UT_.

Colorectal cancer is one of the most common cancers in the world, affecting nearly 1.2 million people in the United States alone. The aim of this study is to investigate the salvage pathway enzyme HPRT as a possible biomarker in two colorectal cancer cell lines: HT29 and SW620. HPRT is a transferase in the purine salvage pathway that functions primarily by catalyzing the conversion of hypoxanthine to inosine monophosphate and guanine to guanosine monophosphate. Because of its role in proliferation and cell cycle regulation, we hypothesized an increase in HPRT expression within cancer cells, which could potentially lead to presentation on the surface of the cell. HPRT surface localization was assessed utilizing confocal microscopy, flow cytometry, and scanning electron microscopy. These techniques allowed us to visualize HPRT on the plasma membrane and quantify expression. There was statistically significant expression of HPRT on the surface of both HT29 and SW620 cells with a 28% and 58% fluorescent shift in the population, respectively. Confocal microscopy images revealed overlap between cells stained with a membrane dye and anti-HPRT FITC antibody, which indicates a direct relationship between HPRT and the plasma membrane of SW620 cells. To visualize the location of HPRT on the plasma membrane, anti-HPRT antibodies were labelled with gold and protein presence was quantified using an electron microscope, which measured the gold elemental weight percentage of each sample. When exposed to anti-HPRT antibody, the gold weight percentage of the samples significantly increased with 12.4% gold in SW620 cells and 11.2% gold in HT29 cells, indicating significant HPRT surface presence. The observed localization of HPRT to the plasma membrane was variable between the two different cancer cell lines. SW620 cells, which are faster growing and more aggressive, had significantly higher HPRT presentation that HT29 cells. These data collectively suggest that HPRT may be a possible biomarker target for the identification and possible treatment of colorectal cancer cells.

#2149A

CRISPR/Cas9 mutagenesis invalidates a genetic target of clinical trials in cancer.

Ann Lin, Christopher Giuliano, Nicole Sayles, Joan Smith, Jason Sheltzer. _Cold Spring Harbor Laboratory, Cold Spring Harbor, NY_.

The Maternal Embryonic Leucine Zipper Kinase MELK has been described as a genetic dependency in several cancer types, most notably in the highly-aggressive basal subtype of breast cancer; MELK inhibition through the use of both RNAi and small-molecule approaches appears to block the growth of cancer types with such dependency. Based on these results, the MELK inhibitor OTS167 is currently being tested as a novel chemotherapy agent in multiple clinical trials. Here, however, we report that mutagenizing MELK with CRISPR/Cas9 has no effect on the fitness of basal breast cancer cell lines or cell lines from other cancer types. Through seven guide RNAs targeting the kinase and kinase-associated domains of MELK, we demonstrate that mutagenesis of MELK causes no defect in proliferative ability or anchorage independent growth in these cancer types. Additionally, cells with mutagenized MELK remain sensitive to OTS167, suggesting that this drug blocks proliferation through an off-target mechanism. Finally, the patient tumor gene expression data that initially identified MELK as being significantly upregulated in patients with poor survival was reexamined. As MELK is thought to play a role in mitosis, we compared MELK expression to a set of well-known cell proliferation markers and show significant correlations of MELK with the proliferation genes; this suggests a role of MELK in representing the mitotic activity of a tumor, rather than possessing a transformative role in itself. In total, our results undermine the rationale for a series of current clinical trials based on MELK inhibition and provide an experimental approach for the use of CRISPR/Cas9 in preclinical target validation that can be broadly applied.

### Targeting p53, Apoptosis, and the Cell Cycle

#2150

Oncogenic role of GAEC1 and its potential modulation with p53 in pathogenesis of colon cancer.

Riajul Wahab, Farhadul Islam, Vinod Gopalan, Alfred King-Yin Lam. _Griffith University, Southport, Gold Coast, Australia_.

Introduction: GAEC1 (Gene amplified in esophageal cancer 1), is frequently amplified and overexpressed in colon cancer tissues. In the present study, we aimed to unveil the oncogenic potential of GAEC1 in carcinogenesis of colon cancer by studying the underlying cellular functions and molecular interactions by in vitro and in vivo experiments.

Method: Transient overexpression of GAEC1 with pcDNA3.1-GAEC1 and silencing with GAEC1-siRNA was performed and several downstream assays were done such as migration, clonogenic and apoptotic assay. Analysis of cell kinetics was done using flow cytometry and cell counting kit-8 was used for cell proliferation assay. Immunofluorescence and Western blot assay were used to determine the expression of different target proteins. Co-immunoprecipitation was used to confirm the protein-protein interaction. For xenotransplantation the severely combined immunodeficient (SCID) mice (4 groups, 6 in each group) were injected subcutaneously with GAEC1 shRNA and control shRNA transfected (stable) SW480 and SW48 colon cancer cells.

Result: The overexpression of GAEC1 increased cell proliferation, migration, reduced apoptosis in colon cancer cells. Also, these cells showed cell cycle arrest at the synthetic phase, activation of Bcl-2, K-ras, pAKT proteins as well as inhibition of p53, PUMA, p21 and BAX proteins. Conversely, knockdown of GAEC1 reduced cell proliferation, migration, decreased the phosphorylation of AKT, and induced apoptosis, G2/M phase arrest and cleavage of poly (ADP-ribose) polymerase (PARP). Co-immunoprecipitation revealed GAEC1's interaction with p53. In addition ectopic over expression and silencing of GAEC1 lead to reciprocal effects of p53 protein expression. Moreover, knockdown of GAEC1 reduced the nuclear translocation of murine double minute 2 protein (mdm2) indicating that GAEC1 is responsible for the degradation of p53 through the direct interaction between mdm2-p53 in nucleus. Furthermore, our in vivo data demonstrate that the loss of GAEC1 inhibits the tumor formation in xenograft model.

Conclusion: Collectively our study demonstrates that GAEC1 exhibits the oncogenic role in colon cancer by reducing expression of p53 through protein-protein interaction which leads to the inhibition of PUMA, p21, BAX, and activation of Bcl-2, K-ras and pAKT protein expression.

#2151

Inhibition of WIP1/PPM1D phosphatase by GSK2830371 potentiates the growth inhibitory and cytotoxic activity of MDM2 antagonists (nutlin-3, RG7388 and HDM201) in cutaneous melanoma cells.

Chiao-En Wu,1 Arman Esfandiari,1 Yi-Hsuan Ho,1 Colin Shepherd,1 Ahmed Khairallah Mahdi,1 Erhan Aptullahoglu,1 John Wen-Cheng Chang,2 Penny Lovat,3 John Lunec1. 1 _Northern Institute for Cancer Research, School of Medicine, Newcastle University, United Kingdom;_ 2 _Division of Hematology-Oncology, Department of Internal Medicine, Chang Gung Memorial Hospital at Linkou, Chang Gung University College of Medicine, Taoyuan, Taoyuan, Taiwan;_ 3 _Dermatological Sciences, Institute of Cellular Medicine, Newcastle University, United Kingdom_.

Cutaneous melanoma is the most serious skin malignancy. The current study aimed to investigate the WIP1 inhibitor GSK2830371 and MDM2-p53 antagonists (nutlin-3, RG7388 and HDM201) alone and in combination treatment in cutaneous melanoma cell lines and explored the mechanistic basis of these responses in relation to the genotype and induced gene expression profile of the cells. A panel of three p53WT (A375, WM35, C8161) and three p53MUT (WM164, WM35-R5R1, CHL-1) melanoma cell lines were used. GSK2830371 (≤10 μM) alone had no growth-inhibitory or cytotoxic effects on the cells, measured by sulforhodamine B (SRB) and clonogenic assays. In combination treatment GSK2830371 significantly potentiated the growth-inhibitory and clonogenic cell killing effects of MDM2 inhibitors in p53WT but not p53MUT melanoma cells, indicating the potentiation worked in a p53-dependent manner (Table). Western blotting demonstrated GSK2830371 increased p53 stabilization through Ser15 phosphorylation and consequent Lys382 acetylation when it was combined with MDM2 inhibitors. These changes were ATM-mediated, shown by reversal with the ATM inhibitor (KU55933). Furthermore, GSK2830371 was demonstrated to slow down p53 degradation when de-novo protein synthesis was inhibited by cycloheximide. In qRT-PCR, nutlin-3 or RG7388 induced p53 transcriptional target genes (CDKN1A, MDM2, BAX, FAS, PUMA, TNFBSF10B, TP53INP1) and GSK2830371 enhanced the induction in p53WT but not p53MUT cells. In conclusion, GSK2830371, a WIP1 inhibitor, at doses with no growth-inhibitory activity alone, potentiated the growth-inhibitory and cytotoxic activity of MDM2 inhibitors, by increasing phosphorylation, acetylation, and stabilization of p53 in cutaneous melanoma cells in a functional p53-dependent manner. Further studies in vivo are warranted to investigate the efficacy of this combination treatment.

GI50 by sulforhodamine B (SRB) and LC50 by clonogenic assays in p53WT melanoma cells

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|

Cell lines

GI50 (Mean + SEM) | A375 | WM35 | C8161

GSK2830371 | - | + | - | + | - | +

Nutlin-3 (µM) | 5.1 + 0.6 | 1.5 + 0.4 | 8.3 + 1.5 | 5.3 + 1.4 | 1.7 + 0.3 | 1.0 + 0.5

p value | 0.012 | 0.010 | 0.049

RG7388 (nM) | 228 + 39 | 62 + 2 | 377 + 58 | 169 + 57 | 46 + 0 | 15 + 5

p value | 0.025 | 0.028 | 0.009

HDM201 (nM) | 167 + 25 | 41 + 9 | 165 + 98 | 77 + 59 | 81 + 11 | 49 + 10

p value | 0.0046 | 0.1048 | < 0.001

LC50 (Mean + SEM) | A375 | WM35 | C8161

GSK2830371 | - | + | - | + | - | +

Nutlin-3 (µM) | 2.0 + 0.9 | 0.8 + 0.5 | 0.5 + 0.1 | 0.4 + 0.1 | 0.9 + 0.2 | 0.4 + 0.1

p value | 0.040 | 0.0714 | 0.003

RG7388 (nM) | 166 + 95 | 54 + 37 | 186 + 78 | 68 + 23 | 50 + 22 | 21 + 10

p value | 0.006 | 0.070 | 0.026

HDM201 (nM) | 198 + 70 | 15 + 4 | 11 + 3 | 8 + 3 | 139 + 85 | 19 + 5

p value | <0.001 | 0.1062 | 0.010

#2152

**Protein phosphatase Mg** 2+ **and Mn** 2+ **dependent 1F promotes smoking-induced breast cancer by inactivating phosphorylated p53-induced signals.**

Li-Ching Chen. _Taipei Medical University, Taipei City, Taiwan_.

Previously we demonstrated that the activation of α9-nicotinic acetylcholine receptor (α9-nAchR) signaling by smoking promotes breast cancer formation. To investigate the downstream signaling molecules involved in α9-nAChR-induced breast tumorigenesis, we used real-time polymerase chain reactions and Western blotting to assess expression of protein phosphatase Mg2+/Mn2+ dependent 1F (PPM1F), a Ser/Thr protein phosphatase, in human breast cancer samples (n=167). Additionally, stable PPM1F-knockdown and overexpressing cell lines were established to evaluate the function of PPM1F. The phosphatase activity of PPM1F in nicotine-treated cells was assessed through Western blotting, confocal microscopy, and fluorescence resonance energy transfer. Higher levels of PPM1F were detected in the breast cancer tissues of heavy smokers (n=7, 12.8-fold) greater than of non-smokers (n= 28, 6.3-fold) (**p=0.01). In vitro, nicotine induced PPM1F expression, whereas α9-nAChR knockdown reduced the protein expression of PPM1F. A series of biochemical experiments using nicotine-treated cells suggested that the dephosphorylation of p53 (Ser-20) and BAX (Ser-184) by PPM1F is a critical posttranslational modification, as observed in breast cancer patients who were heavy smokers. These observations indicate that PPM1F may be a mediator downstream of α9-nAChR that activates smoking-induced carcinogenic signals. Thus, PPM1F expression could be used for prognostic diagnosis or inhibited

for cancer prevention and therapy.

#2153

Elephant p53 (EP53) expression induces apoptosis of human cancer cells.

Lisa M. Abegglen,1 Lauren N. Donovan,1 Genevieve Couldwell,1 Rosann Robinson,1 Cristhian Toruno,1 Mor Goldeder,2 Aleah F. Caulin,3 Katrin P. Guillen,1 Bryan E. Welm,1 Carlo C. Maley,4 Avi Schroeder,2 Joshua D. Schiffman1. 1 _Huntsman Cancer Institute, University of Utah, Salt Lake City, UT;_ 2 _Technion - Israel Institute of Technology, Haifa, Israel;_ 3 _Driver, San Franscisco, CA;_ 4 _Biodesign Institute and School of Life Sciences, Arizona State University, Tempe, AZ_.

The goal of our study was to determine if elephant TP53 (EP53) proteins contributing to increased apoptosis and possible cancer resistance in elephants could translate into human cancer cells as a future effective cancer treatment. We previously reported that elephants have a lower than expected rate of cancer, 20 copies of TP53 (1 ancestral gene with introns [EP53-anc] and 19 retrogenes [EP53-retro1-19]), and increased p53-mediated apoptosis induced by DNA damage in elephant cells compared to human cells (Abegglen JAMA 2015). For the current study, we expressed various EP53 proteins in human cancer cells with different p53 status, including osteosarcoma (U2-OS, Saos-2), glioblastoma (T98G), and breast cancer (MCF7). Western blot analysis confirmed EP53 expression. We compared apoptosis in the human cancer cells transfected/transduced with negative control vectors vs. epitope or protein-tagged EP53 exposed to doxorubicin (to induce DNA damage). Apoptosis was measured by cell viability, caspase activity, Propidium Iodide/Annexin V staining, and fluorescence microscopy. We observed a significant increase in caspase activity (normalized to cell viability) of U2-OS and T98G cells expressing EP53 compared to negative control treated cells as shown in Table 1, and apoptosis with p21 restoration in Saos-2. In U2-OS, which overexpress MDM2, EP53 was more effective at inducing apoptosis compared to human TP53. Taken together, we found that EP53-anc restored p53-mediated apoptosis and EP53-anc / EP53-retro9 enhanced p53-mediated apoptosis. These data suggest for the first time that EP53 functions in human cancer cells to promote cell death. Ongoing efforts are exploring the EP53 mechanism of action that leads to increased apoptosis, including expression of EP53 in additional cancer types (lung, melanoma, colon, prostate, and others) with a variety of genetic backgrounds to characterize its functional context. These results support the further exploration of EP53-based cancer therapeutics.

Table 1: Increase in apoptosis with EP53 expression relative to EP53 empty vector control cells

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Cell Type | TP53 Status | EP53-anc | EP53-retro9 | Construct | Assay Results | No Treatment (fold difference) | P-value | 1uM doxorubicin (fold difference) | P-value

U-2 OS (osteosarcoma) | WT | Apoptosis | Apoptosis | myc-EP53-retro9 | Increase in caspase relative to control | 1.73 | 0.0091 | 1.79 | 1.7x10^-5

eGFP-EP53-retro9 | Increase in caspase relative to control | 1.68 | 6.7x10^-5 | 2.59 | 0.0041

eGFP-EP53-retro9 | Percent decrease in GFP positive cells | 23% percent decrease | 4.8x10^-5 | - | -

dyk-EP53-anc | Increase in caspase relative to control | 14.22 | 5.4x10^-7 | 3.31 | 1.0x10^-5

dyk-huTP53 | Increase in caspase relative to control | 10.9 | 1.2x10^-7 | 2.47 | 5.8x10^-5

MCF7 (breast cancer) | WT | - | Apoptosis | eGFP-EP53-retro9 | Cell death by Propidium Iodide staining | 0.15% GFP/surface area | 0.011 | - | -

T98G (glioblastoma) | Null | Apoptosis | No Apoptosis | dyk-EP53-anc | Increase in caspase relative to control | 3.07 | 7.9x10^-9 | 1.55 | 2.6x10^-5

myc-EP53-anc | Increase in caspase relative to control | 3.46 | 0.00015 | 1.52 | 6.8x10^-5

dyk-huTP53 | Increase in caspase relative to control | 2.82 | 1.2x10^-5 | 1.8 | 8.6x10^-5

#2154

P53 pathway restoring compound P306 inhibits colorectal cancer growth.

Xiaobing Tian, Shengliang Zhang, Amriti Lulla, wafik S. El-Deiry. _Fox Chase Cancer Ctr., Philadelphia, PA_.

We recently reported that P01 is a potent p53 pathway-restoring small molecule that acts through increasing levels of TAp73. Further, it also interferes with the p73-mutant p53 protein-protein interaction and by downregulation of ∆Np73 (Tian, Zhang and El-Deiry, abstract# 3830, AACR 2016). P01 is a member of the natural products that have been shown to have potent anti-cancer activity against tumors with mutated p53. Based on the structure of the pharmacophore of compound P01, we designed and synthesized new analogs based on published structure-activity relationship and organic synthesis papers. The newly synthesized analogs were potent in reducing both short-term and long-term proliferation in a broad panel of mutant p53 cell lines such as HT29, SW480, DLD-1, MDA-MB-231and H1975 with EC50s in the range of 0.16 μM to 0.26 μM. We are currently evaluating the anti-cancer effects of three analogs P301, P304 and P306, of which only two of these analogs induce TAp73 as assayed by western blot. We are currently characterizing the analog P306, which is the most potent compound among them. P306 engages the apoptosis pathway by upregulation of pro-apoptotic proteins like PUMA, DR5, and BIM and downregulation of anti-apoptotic markers such as Mcl-1 in a time-and dose-dependent manner in colorectal cancer cell lines. P306 treatment also downregulates both mRNA and protein level of MET and EGFR in HT29 cells that overexpress MET and EGFR receptors. We are currently investigating the mechanisms of induction of both PUMA and DR5 in mutant p53 cells and determining whether either gene is necessary for the apoptotic effects of P306 in colorectal cancer cells. Our preliminary data indicates that upregulation of DR5 is through the ATF4/CHOP pathway post-P306 treatment. It has been reported that Puma is a target gene of ATF4, CHOP and p73, however, we found that none of them is responsible for P306-induced upregulation of PUMA in p53 mutated DLD1, SW480 or HCT116 p53-null cell lines. Thus, our ongoing in vitro studies are focused on further understanding the mechanism of action of P306 in mutant p53 cells and potentially exploring combinations with FDA-approved therapies. We are also in the process of conducting, first-in animal studies of P306 as single agent or in combination with chemotherapy or targeted therapy.

#2155

Small molecule NSC59984 suppresses cancer cell growth under hypoxia.

Shengliang Zhang, Lanlan Zhou, Wafik S. El-Deiry. _Fox Chase Cancer Center, Philadelphia, PA_.

Hypoxia is one of the main features of solid tumors, contributes to drug resistance and is associated with poor prognosis. We have reported that a small-molecule NSC59984 restores p53 signaling and degrades mutant p53 protein in cancer cells. We find that NSC59984-mediated p53 signaling restoration and mutant p53 degradation is significantly blocked by NAC, an inhibitor of reactive oxygen species (ROS), while, further enhanced by BSO, a chemical compound increasing ROS in cells. These results suggest that ROS is a factor required for the effect of NSC59984 on cancer cells. Cellular ROS is produced by hypoxia in solid tumors. We investigated the effect of NSC59984 on p53 pathway and cell death in p53 mutant colorectal cancer (CRC) cells cultured under hypoxia. NSC59984 increases p53 signaling in p53 mutant cancer cells under hypoxia, as it does under normoxia. The effect of NSC59984 under hypoxia is blocked by NAC. Moreover, NSC59984 decreases cancer cell viability in a dose-dependent manner under hypoxia similar to normoxia. Our results suggest that NSC59984 inhibits cancer cell growth under hypoxia. Hypoxia has been known to increase cancer stem cells in tumors, which is one of the reasons causing drug resistance. We then evaluate the effect of NSC59984 on the stem cell growth. NSC59984 significantly reduces colonosphere formation of SW480, DLD-1 and HT29 cells. CRC stem cell markers such as CD44 and ALDH are decreased in colonospheres upon NSC59984 treatment, suggesting that NSC59984 inhibits stem cell self-renewal or/and growth. Our results provide the rationale for administration of NSC59984 in solid tumors with or without hypoxia and in combination treatment with ROS activators.

#2156

NSC59984 induces mutant p53 degradation via activating ERK2 pathway-MDM2 axis.

Shengliang Zhang, Lanlan Zhou, David Dicker, Wafik S. El-Deiry. _Fox Chase Cancer Center, Philadelphia, PA_.

Mutant p53 protein is highly expressed in most cancer cells due to its protein stabilization. Mutant p53 loses wild-type p53 function but gains new oncogenic functions in driving tumor growth and drug resistance. Depletion of mutant p53 is an attractive strategy for cancer therapy. We reported a small-molecule NSC59984 destabilizes mutant p53 protein via protein degradation (Zhang et al., Cancer Research, 2015). In this study, we demonstrate that the ERK2 pathway plays a specific role in NSC59984-induced mutant p53 degradation via MDM2-mediated ubiquitination. We observe a sustained-phosphorylation of ERK1/2 in cancer cells treated with NSC59984. ERK1/2 pathway is negatively regulated by MKP-1. We find that MKP-1 activity is partially inhibited by NSC59984 in a dose dependent manner in vitro. These results suggest that NSC5998 sustains ERK1/2 phosphorylation partially via disruption of the feedback-loop between MKP-1 and ERK2 pathway. Knockdown of ERK2 rescues mutant p53 from NSC59984-mediated degradation, and inhibits NSC59984-restoration of p53 signaling in mutant p53-expressing cells. On the contrary, the effect of NSC59984 on the mutant p53 is not inhibited by the blockage of JNK and P38, two pathways negatively regulated by MKP-1. These results suggest that the ERK2 phosphorylation is specifically required for NSC59984-mediated mutant p53 degradation. We further find that NSC59984 induces ERK2-dependent MDM2 phosphorylation. The effect of NSC59984 on MDM2 phosphoryation is partially inhibited by the blockage of ERK2 signaling (via U0126 treatment or Knockdown of ERK2), not by the attenuation of the JNK pathway. Furthermore, NSC59984-mediated mutant p53 protein degradation is inhibited by MDM2 knockdown, and enhanced by MDM2 overexpression in cancer cells. NSC59984-increased ubiquitination of mutant p53 is attenuated by U0126. Our results suggest that the ERK2 signaling pathway-MDM2 axis is a major determinant of NSC59984-mediated mutant p53 degradation. We further demonstrate that NSC59984 induces cell death via apoptosis, and NSC59984-induced cell death is mainly rescued by inhibition of the ERK2 pathway in p53 mutant cancer cells. Taken together, our results suggest that phosphorylated-ERK2 is an important factor required for NSC59984-mediated mutant p53 degradation which may further contribute to NSC59984-induced cell death in cancer cells.

#2157

P73 isoforms regulate cellular survival and response to treatment in diffuse large B-cell lymphoma.

Hesham M. Hassan,1 Michelle L. Varney,2 Pamela A. Althof,2 Gabriel C. Caponetti,3 Kai Fu,2 Dennis D. Weisenburger,4 Rakesh K. Singh,2 Bhavana J. Dave2. 1 _Assiut University, Egypt;_ 2 _Univ. of Nebraska Medical Ctr., Omaha, NE;_ 3 _Univ. of Pennsylvania, Philadelphia, PA;_ 4 _City of Hope, Duarte, CA_.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin's lymphoma (NHL) and is curable in more than 60% of the cases. Despite long-term remission achieved with current therapies, relapse occurs in almost one-third of the cases. Rearrangements of chromosome locus 1p36 with ensuing deletion or disruption of TP73, one of the most distally located putative tumor suppressor genes, is frequent in many NHL subtypes, including DLBCL, and confers inferior prognosis. Recent reports in normal B-cells and lymphoma animal models suggest an important role of p73 in B-cell lymphomas; however, the biological significance of p73 isoforms in DLBCL is not clear. TP73 is a member of the TP53 family that shares structural homology and hence is capable of transactivating p53 target genes. We investigated the relationship between rearrangements of chromosome locus 1p36 and p73 expression and examined whether differential expression of p73 isoforms (TAp73 and ∆Np73) correlate with proliferation and apoptosis in DLBCL. We utilized molecular techniques in conjunction with fluorescence in situ hybridization (FISH) in untreated histologically confirmed DLBCL patient specimens. In our data set, the 1p36 chromosomal locus was disrupted (mainly heterozygous deletion) in 35% of the cases and was significantly associated with ∆Np73 expression. Subsequent immunohistochemical analysis showed a significant positive correlation between TAp73 and cleaved caspase-3 expression and between ∆Np73 and Ki-67 expression. Additionally, we observed a negative correlation between TAp73 and the anti-apoptotic molecules Bcl-2 and Bcl-6. We then performed in vitro studies in DLBCL cells and modulated the expression of TAp73 and ∆Np73 using expression vectors and siRNA. TAp73-transfected cells were more susceptible to serum deprivation and doxorubicin treatment, conversely the ∆Np73-transfected cells were more resistant to serum deprivation and doxorubicin treatment compared to control cells. Thus, rearrangements of 1p36 consequently deregulates p73 isoform expression in DLBCL. Our data emphasizes the importance of delineating the functional aspect of genetic alterations and underscores the significant role of p73 isoforms in DLBCL pathogenesis and treatment.

#2158

Targeted intracellular disruption of oncogenic p53/HDM2 and p53/HDMX complexes underlies the therapeutic activity of a stapled peptide drug.

Loren D. Walensky, Franziska Wachter, Ann Morgan, Marina Godes, Gregory Bird. _Dana-Farber Cancer Inst., Boston, MA_.

The first anti-cancer stapled peptide drug has now been advanced to clinical testing in relapsed human cancers that retain the expression of wild-type p53. A common mechanism for cancer cell suppression of wild-type p53 is overexpression of the negative regulators HDM2 and HDMX, which neutralize p53 through protein interaction. Selective HDM2 inhibitor molecules can effectively reactivate p53 in certain cancers, but the co-expression of HDMX can cause resistance, highlighting the need for dual HDM2/HDMX targeting. We previously developed stapled peptides modeled after the p53 transactivation domain to harness the natural propensity of this alpha-helical motif to engage both targets with high affinity and selectivity. Here, we demonstrate that ALRN-7041, a next-generation, clinical-grade stapled peptide, achieves time-dependent cellular uptake and nuclear localization without membrane perturbation, dose-dependently dissociates p53/HDM2 and p53/HDMX complexes as assessed by real-time protein interaction monitoring in live cells, and impairs the viability of cancer cells bearing wild-type p53 by inducing a surge in p53 protein level. Applying an unbiased statistical approach to determine which biophysical parameters dictate the cellular uptake of stapled peptides, we elucidated the design features of ALRN-7041 that confer intracellular access, providing a roadmap for generating cell-permeable stapled peptides with on mechanism cellular activity for clinical translation. We find that cancer cells exhibiting a signature of HDM2, HDMX, and wild-type p53 co-expression are strikingly susceptible to ALRN-7041, highlighting the therapeutic potential of dual HDM2/HDMX inhibition by a cell-penetrant stapled peptide in human cancer.

#2159

A specific 17-beta-hydroxywithanolide (LG-02) sensitizes cancer cells to apoptosis in response to TRAIL and TLR3 ligands.

Poonam Tewary,1 Alan D. Brooks,1 Ya-ming Xu,2 Kithsiri E.M Wijeratne,2 Leslie A. Gunatilaka,2 Thomas J. Sayers1. 1 _Leidos Biomedical Research Inc, Frederick, MD;_ 2 _The University of Arizona, Tucson, AZ, AZ_.

Recent studies have demonstrated a role of toll-like receptor 3 (TLR3) signaling for the initiation of apoptosis in some malignant cells. We have previously shown that, withanolide E (WE), a 17β-hydroxywithanolide (17-BHW) natural product derived from the medicinal plant Physalis peruviana was capable of sensitizing tumor cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis by reducing cellular levels of the anti-apoptotic protein cFLIP. Animal studies also revealed that WE sensitized human renal carcinoma cells to apoptosis at concentrations that did not promote apoptosis in normal cells. Thus we further screened a library of 30 natural and semi-synthetic 17-BHWs for their ability to promote death ligand-mediated cancer cell death. Among the 30 compounds tested, LG-02 (physachenolide C) was found to be 4-5 fold more potent than WE in sensitizing some human renal carcinoma and melanoma cells to apoptotic cell death in response not only to TRAIL but also to the synthetic polynucleotide poly (I:C), which is known to mimic anti-viral responses by activating TLR3 signaling. To date there are no withanolides known to have this dual apoptosis sensitizing activity. LG-02 and Poly (I:C) treatment resulted in increased activation of caspase-8, and apoptosis was blocked by the pan caspase inhibitor zVAD-FMK. Poly (I:C)-driven apoptosis signaling was dependent on endosomal acidification, but independent of IRF 3 and Interferon α/β signaling. Molecular studies suggested a role for changes in the anti-apoptotic proteins cFLIP, IAPs, and Livin on apoptosis signaling in LG-02 treated cells. Loss of cIAP activity is reported to promote spontaneous formation of an intracellular death-inducing protein platform the ripoptosome, that can activate either apoptosis or necroptosis. Immunoprecipitation of either the TRAIL death-inducing signaling complex (DISC) or the Poly (I:C) ripoptosome, demonstrated enhanced levels of FADD and RIP1 and decreased levels of cFLIP in these macromolecular apoptosis signaling complexes in LG-02 treated cells. Intratumor administration of LG-02 and Poly (I:C) in a xenograft M14 melanoma model provided therapeutic benefit leading to complete tumor regression in 90 % of the mice as compared to control mice. Further studies with active 17-BHWs could lead to the identification of more potent analogues, and novel and common therapeutic targets involved in apoptosis signaling in response to both TNF death receptor family members as well as TLR3 ligands.Funded by FNLCR Contract HHSN261200800001E

#2160

Novel sulfonamide derivative inhibits JAK2-STAT3 signaling and induces ROS-mediated apoptosis in colorectal cancer cells.

Rehan Ahmad, Mansoor-Ali Vaali-Mohammed, Omar Al-Obeed, Maha Abdulla. _King Saud Univ. College of Medicine, Riyadh, Saudi Arabia_.

Colorectal cancer is a major worldwide health problem owing to its high prevalence and mortality rate. Developments in screening, prevention, biomarker, personalized therapies and chemotherapy have improved detection and treatment. However, despite these advances many patients with advanced metastatic tumors will still succumb to the disease. New anti-cancer agents are needed for treating advance stage colorectal cancer as most of the deaths occur due to cancer metastasis. A recently developed novel sulphonamide derivative, 4-((2-(4-(Dimethylamino)phenyl)quinazolin-4-yl)amino)benzenesulfonamide (3d) has shown to have potent antitumor effect; however the mechanism underlying the antitumor effect remains unknown. Our study revealed that 3d treatment significantly reduced the viability of human colorectal cancer cells HT-29 and SW620. This is further evidenced by the induction of p53 and Bax, release of cytochrome c, activation of caspase-9, caspase-7 and caspase-3 and cleavage of PARP in 3d treated cells. This compound was found to have significant effect on the inhibition of anti-apoptotic proteins, Bcl2, BclxL and XIAP. The results further demonstrate that 3d inhibited JAK2-STAT3 pathway by decreasing the constitutive and IL-6-induced phosphorylation of JAK2 and STAT3. 3d further decreased JAK2- STAT3 target genes like Cyclin D1 and Survivin. Furthermore 3d treatment induced the generation of reactive oxygen species (ROS) in human colorectal cancer cells. Confirming our observation, NAC significantly inhibited ROS production, induction of apoptosis, cytochrome c release and PARP cleavage. Additionally, treatment with 3d resulted in decreased glutathione (GSH) levels. The results further demonstrate that 3d inhibited cell migration by modulating EMT markers and inhibiting TGFβ-induced phosphorylation of Smad2 and Samd3. Collectively these findings indicate that 3d inhibited JAK2-STAT3 signaling; induced apoptosis via generation of reactive oxygen species and inhibition of cell migration by altering EMT markers and TGFβ-Smad pathway.

#2161

**Pharmacological downregulation of BFL-1 by the BET bromodomain inhibitor CPI203 overcomes ABT-199 resistance in** MYC+/BCL2+ **double hit lymphoma.**

Anna Esteve-Arenys,1 Juan Garcia-Valero,1 David Gonzalez,1 Aránzazu Chamorro-Jorganes,1 Vanina Rodriguez,1 Ivan Dlouhy,2 Itziar Salaverria,1 Elias Campo,2 Dolors Colomer,2 Antonio Martinez,2 Grzegorz Rymkiewicz,3 Patricia Pérez-Galán,1 Armando Lopez-Guillermo,2 Gaël Roué1. 1 _IDIBAPS, Barcelona, Spain;_ 2 _Hospital Clinic, Barcelona, Spain;_ 3 _The Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland_.

Double-hit lymphoma (DHL) is a rare subtype of B-cell lymphoma characterized by MYC and either BCL2 or BCL6 chromosomal rearrangements. DHL cases may have different morphology and are included in the updated 2016 WHO classification as a new category of high grade B-cell lymphoma with rearrangements. DHL patients usually undergo a rapidly progressing clinical course and are poorly responsive to standard chemo-immunotherapy. As a consequence, the prognosis of this entity is particularly poor with a median overall survival of approximately one year.

ABT-199 (venetoclax) is a potent and selective small-molecule antagonist of BCL-2 recently approved for the treatment of some lymphoid malignancies. In this study, we demonstrate that single-agent ABT-199 efficiently displaces pro-apoptotic BAX from BCL-2 complexes but fails to maintain a significant antitumor activity over time in most MYC+/BCL2+ DHL cell lines and primary cultures, as well as in a xenograft mouse model of DHL. We further identify the accumulation of the BCL2-like protein, BFL-1, to be a major mechanism involved in acquired resistance to ABT-199. We show this phenomenon to be manageable by the BET bromodomain inhibitor CPI203, since gene expression profiling identifies BCL2A1 as one of the top apoptosis-related gene modulated by the compound. Upon CPI203 treatment, simultaneous downregulation of MYC and BFL-1 at both mRNA and protein levels further overcomes resistance to ABT-199 both in vitro and in vivo, allowing DHL cultures and tumor xenografts to undergo synergistic caspase-mediated apoptosis.

Together, these findings highlight the relevance of BFL-1 in DHL-associated drug resistance and support the combined use of BCL-2 antagonist and BET inhibitor as a promising therapeutic strategy in patients with aggressive DHL.

#2162

**MEK signaling marks resistance to synthetic lethality induced by PI3K/Akt and BCL-X** L **inhibition in PTEN-mutant cancers.**

Raghav Joshi, Wenying Ren, Paul Mathew. _Molecular Oncology Research Institute, Tufts Medical Ctr., Boston, MA_.

Background: Loss of PTEN represents one of the most common disruptions of tumor suppressor activity in the progression of human neoplasia. There are no established precision medicine approaches defined in PTEN-deficient neoplasms which include prostate, breast, glioblastoma (GBM) and uterine cancers. In PTEN-mutant prostate cancer cells, the PI3K/Akt pathway collaborates with BCL-XL specifically, to regulate the apoptotic threshold (Ren, MCR 2016). Whereas single agent PI3K/Akt inhibitors are ineffective in inducing cell death, combined inhibition of PI3K/Akt and BCL-XL in PTEN-mutant prostate cancer cells results in synergistic apoptosis suggesting a novel therapeutic strategy with translational potential in this important subset of disease.

Hypothesis: We hypothesized that synthetic lethality following combinatorial PI3K/BCL-XL inhibition is conserved across the range of PTEN-mutant cancers.

Methods: PTEN-mutant cell lines (ATCC), validated for PTEN-loss and p-Akt expression, were assessed in apoptosis (cleaved Parp) and cell-viability assays following combined PI3K (Buparlisib, SelleckChem) and BCL-XL (A-1331852, AbbVie) inhibition (1µM each, 24h). Since MEK signaling has been associated with resistance to PI3K pathway inhibition, p-Erk was profiled as a candidate marker of resistance. The contribution of MEK signaling to the apoptotic threshold in p-Erk expressing PTEN-mutant cells was tested with pharmacological MEK inhibition (Trametinib, SelleckChem; 1,3,5 µM 48h) in combination.

Results:

MEK signaling and PI3K/BCL-XL inhibition in PTEN-mutant cancers

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|  | |  | |  | |

|

Source | Cell line | PTEN | p-Akt | p-Erk | Synergistic

Apoptosis with PI3K / BCL-XL

inhibition | Loss of cell viability | p-Erk

suppression with MEK inhibition | Apoptosis with concomitant MEK inhibition

Prostate | PC3 | Absent | Present | Absent | Yes | Yes | N/A | N/A

Prostate | LNCaP | Absent | Present | Absent | Yes | Yes | N/A | N/A

Prostate | C4-2B | Absent | Present | Absent | Yes | Yes | N/A | N/A

Breast | ZR-75-1 | Absent | Present | Weak | Yes | Yes | N/A | N/A

Uterine | SK-UT-1 | Absent | Present | Present | No | No | Yes | Yes

GBM | A-172 | Absent | Present | Present | No | No | Yes | No

Cervix | C33-A | Absent | Present | Present | No | No | - | -

Conclusions: Synthetic lethality in PTEN-mutant tumor cells with PI3K/BCL-XL inhibition was confined to tumor cells without active MEK signaling. MEK inhibition inconsistently overcomes resistance to therapy suggesting heterogeneity of survival pathways in p-erk expressing PTEN-deficient tumors. These data have implications for precision medicine approaches in PTEN-deficient tumors as absence of p-Erk expression may predict for a responsive phenotype.

#2163

Combined targeting of MEK and MCL-1 induces apoptosis and tumor regression of KRAS mutant NSCLC.

Aaron N. Hata,1 Faria M. Siddiqui,1 Maria Gomez-Caraballo,1 Samantha J. Bilton,1 Daria Timonina,1 Varuna Nangia,1 Angela Coxon,2 Sean Caenepeel,2 Paul Hughes2. 1 _Massachusetts General Hospital, Charlestown, MA;_ 2 _Amgen, Inc, Thousand Oaks, CA_.

There are currently no effective targeted therapeutic strategies for KRAS mutant non-small cell lung cancer (NSCLC). Single agent MEK inhibitors have demonstrated showed disappointing clinical activity, partly due to inability to induce a robust apoptotic response. Combining MEK inhibitors with BCL-XL/BCL-2 inhibitors may be effective for a subset of KRAS mutant cancers that are dependent on BCL-XL for survival, however this combination is unlikely to be an effective strategy for cancers dependent on MCL-1. We investigated the effect of combining the MEK inhibitor trametinib with a novel MCL-1 inhibitor (compound A), which possesses potent and selective anti-MCL-1 activity in vitro and in vivo, on KRAS mutant cancers. In contrast to colorectal cancer models, which are largely sensitive to combined MEK + BCL-XL inhibition, a subset of cell line and patient-derived mouse xenograft (PDX) KRAS mutant NSCLC models were significantly more sensitive to MEK + MCL-1 inhibition compared to MEK + BCL-XL. To model potential clinical strategies, we tested intermittent dosing regimens and unexpectedly discovered a method strategy for dramatically sensitizing KRAS mutant NSCLC cells to the MEK + MCL-1 combination. These studies provide rationale for the clinical evaluation of combined MEK + MCL-1 inhibitors for KRAS mutant NSCLC.

#2164

BLID is a novel drug-inducible apoptotic molecule: Identification of an integrative mechanism of chemosensitivity in breast cancer cells.

Sivaramakrishna Yadavalli, Rong Hu, Antonina Rait, Xin Li, Esther Chang, Robert Clarke, Usha Kasid. _Georgetown Lombardi Comp. Cancer Ctr., Washington, DC_.

The triple-negative breast cancer (TNBC) is an aggressive form of breast cancer frequently seen in African American women and BRCA1 mutation carriers. The TNBC tumors often relapse with distant metastases following standard chemotherapy. It is now evident that discoveries of new mechanisms and approaches that explain and target breast cancer biology are urgently needed for durable intervention of metastatic disease. Earlier, we have demonstrated that BLID, BH-3 Like motif containing Inducer of cell Death, is a strong prognostic factor in invasive breast cancer. Frequent lack of BLID has been associated with TNBC, African American ethnicity and younger women. Significant correlations exist between BLID negative breast cancer and declines in overall survival, local relapse-free survival and distant metastasis-free survival. Recently, BLID has been shown to inhibit breast cancer cell growth and metastasis. The purpose of this study was to investigate the role of BLID in response of breast cancer cells to chemotherapeutic drugs. In the dose response and time course studies, BLID mRNA expression was found to be induced by chemotherapeutic drugs. Expression of BLID cDNA nanocomplex (scLBLID) resulted in significant increase in chemosensitivity in SKBr3 and MDA-MB-231 cells, and a comparison of BLID with p53 showed that the chemosensitization effect of BLID was significantly greater than that of p53. Consistently, BLID knockdown led to reversal of drug-induced cytotoxicity. In the ChIP-PCR and ChIP-qPCR assays, drug treatment of breast cancer cells resulted in an increased binding of pro-apoptotic transcription factor FOXO3a to the BLID promoter, and the reversal of drug-induced BLID reporter activity was seen in presence of FOXO3a siRNA. Furthermore, siRNA silencing of FOXO3a was found to be associated with decrease in endogenous BLID mRNA expression. Remarkably, we found that expression of central tumor suppressor microRNA miR34a also resulted in increased BLID mRNA expression and drug toxicity in breast cancer cells. Because lack of BLID expression has been associated with poor prognosis in breast cancer patients, we reasoned that the silencing of BLID may reveal as yet unknown changes in gene expression that may drive breast cancer cell proliferation and therapy resistance. In this context, the mRNA array profiling studies showed that BLID knockdown in MDA-MB-231 cells was associated with increased expression of the oncogenic/anti-apoptotic molecules CYP1B1, BIRC3 and CSF1, and decreased expression of the anti-oncogenic/apoptotic molecules AKAP12, DFNA5 and CHRDL1. Our data suggest that chemotherapeutic drugs induce BLID expression via activation of FOXO3a, and the BLID signaling axis downstream of FOXO3a and miR34a is a novel integrative mechanism of breast cancer response to chemotherapy. SY and RH are equal contributors in this study.

#2165

Deletion of survivin sensitize human hepatocellular carcinoma cells to low dose of doxorubicin and induce apoptosis.

Joseph George, Nobuhiko Hayashi, Takashi Saito, Kazuaki Ozaki, Nobuyuki Toshikuni, Mutsumi Tsuchishima, Mikihiro Tsutsumi. _Kanazawa Medical University, Uchinada, Japan_.

Background and Aims: Hepatocellular carcinoma (HCC) is one of the most common cancers in the world and patients with advanced HCC face a dismal prognosis due to lack of effective therapy. Survivin, a member of the family of inhibitor of apoptosis proteins, is highly upregulated in HCC as well as in experimentally induced intrahepatic tumors. Doxorubicin, the only known chemotherapeutic agent for HCC, is cardiotoxic in addition to several well known side effects.

Methods: Survivin gene deletion was established in HepG2, Hep3B, and PLC/PRF/5 human HCC cells using CRISPR/Cas9 system. All the three HCC cells in culture were treated with doxorubicin at various concentrations before and after survivin gene knockout upto 72 hr. TUNEL assay and FACS analysis were performed to demonstrate the induction of apoptosis after doxorubicin treatment. Western blotting was carried out for cleaved fragments of caspase-9 and caspase-3 as well as major apoptotic executioner molecules.

Results: Cell viability studies depicted around 20% cell death at 24 h, 50% at 48 h, and 80% at 72 h after treatment with doxorubicin at 1 µM (final concentration in the media). Among the three cell lines studied, Hep3B cells were more susceptible to doxorubicin compared to HepG2 and PLC/PRF/5 cells. After deletion of survivin gene, the dosage of doxorubicin could reduce to five fold (200 nM, final concentration in the media) with the same cytotoxic effect before the knockout of survivin gene. HCC cells treated with reduced doses of doxorubicin depicted induction of apoptosis that was proved with TUNEL assay and FACS analysis as wells as increased levels of cleaved caspases and major apoptotic executioner molecules.

Conclusions: Our studies demonstrated that blocking of survivin molecule with effective methods would be a successful approach to treat primary hepatic tumors with low and safe doses of doxorubicin and other anticancer agents.

#2166

Arsenic trioxide targets BCL6 oncoprotein for degradation in BCL6-dependent diffuse large B-cell lymphoma.

Lok Man Yue, David Hau Wing Chau, Wenying Piao, (Eric) Wai Choi Tse, Yok Lam Kwong. _The University of Hong Kong, Hong Kong, Hong Kong_.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma throughout the world. B-cell lymphoma 6 (BCL6) overexpression is frequently observed in DLBCL. Several recent studies have supported that BCL6 is a critical pathogenic oncoprotein in DLBCL. BCL6 represses various downstream genes, including ATR, TP53 and CDKN1A, thereby impairing DNA repair leading to derangement of cellular proliferation. Most importantly, a specific small molecule inhibitor targeting BCL6 successfully suppressed growth of BCL6-dependent DLBCL cell lines and primary human DLBCL cells, further suggesting that BCL6 plays an important pathogenetic role. Arsenic trioxide (As2O3) has been reported to target various oncogenic proteins, including PML-RARA in acute promyelocytic leukemia, cyclin D1 in mantle cell lymphoma, NPMc+ in acute myeloid leukemia and NPM-ALK in anaplastic large cell lymphoma, for degradation through ubiquitin-proteasome pathway. In this study, we investigated the effects of As2O3 on BCL6 in DLBCL.

As2O3 was found to inhibit cell proliferation and induce cell death via apoptosis in DLBCL cells. More interestingly, BCL6-dependent DLBCL cells were found to show higher sensitivity towards As2O3-induced cytotoxicity. BCL6 was found to be degraded by As2O3 at posttranslational level through the ubiquitin-proteasome pathway. Such degradation led to an upregulation of several downstream targets of BCL6, including PRDM1, CD44 and CD69. Moreover, As2O3 synergized with cisplatin to inhibit cell proliferation and enhance apoptosis in BCL6-dependent DLBCL cell lines. Concomitant treatment with As2O3 and cisplatin further enhanced the phosphorylation of Chk1 and γH2AX in these cells.

In conclusion, our data suggest that As2O3 is a potential therapeutic agent for the treatment of BCL6-dependent DLBCL. On top of that, new combination therapies can be developed to expand the therapeutic spectrum of As2O3 to other neoplasms.

#2167

Preclinical validation of an Omomyc cell-penetrating peptide as a viable anti-Myc therapy.

Marie-eve Beaulieu,1 Toni Jauset,2 Daniel Massó-Vallés,2 Peter Rahl,3 Sandra Martinez-Martin,2 Loika Maltais,4 Mariano F. Zacarias-Fluck,2 Silvia Casacuberta,1 Erika Serrano del Pozo,2 Christopher Fiore,3 Laia Foradada,1 Matthew Guenther,3 Eduardo Romero Sanz,5 Marta Oteo Vives,5 Cynthia Tremblay,4 Martin Montagne,4 Miguel Ángel Morcillo Alonso,5 Jonathan R. Whitfield,2 Pierre Lavigne,4 Laura Soucek2. 1 _Peptomyc S.L., Barcelona, Spain;_ 2 _Vall d'Hebron Inst. of Oncology (VHIO), Barcelona, Spain;_ 3 _Syros Pharmaceuticals, Cabridge, MA;_ 4 _University of Sherbrooke, Quebec, Canada;_ 5 _Centre de Investigaciones Energeticas, Medioambientes y Tecnologicas (CIEMAT), Madrid, Spain_.

Deregulation of the MYC oncoprotein promotes tumorigenesis in most, if not all, cancers and is often associated with poor prognosis. However, targeting MYC has long been considered impossible based on the assumption that it would cause catastrophic side effects in normal tissues. Despite this general preconceived notion, we showed that MYC inhibition exerts extraordinary therapeutic impact in various genetic mouse models of cancer, and causes only mild, well-tolerated and reversible side effects. For these studies we employed the systemic and conditional expression of a dominant negative of MYC, called Omomyc, which we designed and validated, and that can inhibit MYC transactivation function both in vitro and in vivo. To date, Omomyc has only been considered a proof of principle, with any potential clinical application limited to gene therapy. Here we actually show that the 11 kDa Omomyc polypeptide spontaneously transduces into cancer cells, demonstrating unexpected cell-penetrating ability. Once inside the nuclei, the polypeptide effectively blocks MYC binding to its

target DNA sites, interfering with MYC transcriptional regulation and halting cell proliferation. Moreover, intranasal (i.n.) administration of the Omomyc polypeptide in mice results in its rapid and persistent distribution to lungs, as well as to other organs (i.e. intestine, liver, kidneys and brain). Importantly, i.n. treatment of mice bearing either Non-Small-Cell-Lung-Cancer (NSCLC) or glioblastoma (GBM) with the Omomyc cell-penetrating peptide (OmomycCPP) significantly reduces tumor burden compared to their control counterparts. Notably, tumor regression is accompanied by significant reprogramming of the tumor microenvironment and tumor immune response. In summary, our data indicate that this novel generation of polypeptides represents a new opportunity to potentially inhibit MYC pharmacologically in a variety of malignant diseases.

#2168

Nucleic acid clamp-mediated recognition and transcriptional modulation of MYC oncogenes through the stabilization of G-quadruplexes.

Taisen Hao, Tracy A. Brooks. _University of Mississippi, University, MS_.

G-quadruplexes (G4s) tend to cluster around biologically important regions such as promoters of DNA, 5'-UTR of mRNA and telomeres. Modulating the stability of G4 as in these regulatory sequences is an emerging approach for cancer treatment. MYC proto-oncogene is overexpressed in over 80% of tumor types, contributing to deregulated cancer cell proliferation. Silencing MYC expression has been demonstrated to be an effective approach to inhibit tumor growth. Among all the silencing strategies, stabilizing MYC promoter G4 that serves as an on-off switch for the transcription of MYC has been explored by the targeting of small molecules. However, the binding specificity of small molecule retarded the drug development process. Exploring novel MYC G4-targeting strategies is necessary to potentiate the pharmacological specificity of MYC G4 modulation. Here, we adopted a nucleic acid clamp based approach to recognize and stabilize the physiologically predominant MYC G4. Previously, a clamp (clamp A) was been demonstrated to recognize the physiologically relevant MYC G4 with high specificity and to downregulate MYC promoter activity. In the current research, we modified and optimized this clamp to allow for flexibility with labeling and monitoring techniques. The original 18 Å polyethylene glycol phosphate linker connecting the 5' and 3' regions of clamp A were substituted with thymine bases of varying lengths; 5 thymines were found to be optimal. The binding specificity and MYC G4 recognizing ability of this optimized clamp A T5 were confirmed by EMSA and ECD; the G4 stabilizing ability of clamp A T5 was demonstrated by DMS footprinting. The cytotoxicity of clamp A T5 was examined by MTS assay on HEK-293 and MCF-7 cells. The intracellular localization of clamp A T5 is being determined microscopically with a 6-FAM-labeled clamp A T5 after being transfected into and incubated with HEK-293 and MCF-7 cells for 48 hr. Nuclear localization is being examined by co-staining with DAPI, and MYC promoter localization is being determined by clustering with a MYC FISH probe. The modification of the clamp is both cost-effective, and enables the detailed study of intracellular functions. This clamp has potential as both a diagnostic tool to inform the use of MYC G4-targeted small molecules, and as a nanotherapeutic.

#2169

**Pharmacological modulation of CXCL12-CXCR4 intracellular trafficking potentiates the** in vitro **and** in vivo **activity of the BET bromodomain inhibitor CPI203 in diffuse large B-cell lymphoma.**

Clara Recasens-Zorzo,1 Teresa Cardesa-Salzmann,2 Laia Ros-Blanco,3 Anna Esteve-Arenys,1 Guillem Clot,1 Martina Guerrero-Hernández,1 Alejandra Valera,2 Alejandra Moros,1 Gonzalo Gutierrez,4 Isolda Casanova,5 Ramón Mangues,5 Alejandra Sanjuan,6 Pablo Menéndez,6 Vanina Rodriguez,1 Antonio Martínez,2 Pedro Jares,2 Dolors Colomer,2 Jordi Teixidó,3 José Ignacio Borrell,3 Elias Campo,2 Armando López-Guillermo,4 Luís Colomo,7 Patricia Pérez-Galán,1 Gaël Roué1. 1 _Institut d'investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain;_ 2 _Hospital Clínic, Barcelona, Spain;_ 3 _Institut Químic de Sarrià, Universitat Ramón Llull, Barcelona, Spain;_ 4 _Hospital Clinic, Barcelona, Spain;_ 5 _Institut d'Investigacions Biomèdiques-Sant Pau (IIB-Sant Pau), Barcelona, Spain;_ 6 _Josep Carreras Leukemia Research Institute, University of Barcelona, Barcelona, Spain;_ 7 _Hospital del Mar, Barcelona, Spain_.

Constitutive activation of CXCR4, a chemokine receptor commonly overexpressed in cancer, is associated with tumor progression, invasion, and chemotherapy resistance. Although the CXCR4 pathway has recently been suggested as an adverse prognostic marker in diffuse large B-cell lymphoma (DLBCL), its biological relevance remains underexplored as the evolution of the malignant clone has been traditionally thought to rely mainly on microenvironment-independent factors, including MYC. To evaluate a possible interplay between CXCR4 and MYC signaling in DLBCL we have analyzed the expression of CXCR4 and its ligand CXCL12 in tissue biopsies of a homogeneous cohort of 86 patients with de novo DLBCL, and correlated it with clinico-pathological characteristics and gene expression profiling. We found that co-expression of CXCL12 and CXCR4 was associated with an inferior overall survival when compared to the sole expression of CXCR4 (p=0.017). An ELISA-based array covering 175 human cytokines further showed that tissue levels of CXCL12 best correlated with high microvessel density (p=0.02) pointing out a role for both CXCR4 and CXCL12 in DLBCL tumor aggressiveness. In support of the established role of nuclear CXCR4 on tumor metastasis, abundant nuclear CXCR4 was detected in DLBCL primary tumors and cell lines compared to normal tonsils. This feature was significantly associated with the migratory potential of the tumor cells, thus suggesting CXCR4 as a potential therapeutic target to hamper lymphoma cell dissemination. In silico and in vitro screening of a CXCR4 inhibitor (CXCR4i) library further identified the tetra-amine IQS-01.01 as a potent inhibitor of CXCR4 nuclear translocation. IQS-01.01 prevented CXCL12-mediated chemotaxis and triggered apoptosis in a panel of 14 DLBCL cell lines and primary cells, with improved pharmacological properties than the standard CXCR4i, AMD3100. Mechanistically, IQS-01.01-mediated blockage of CXCR4 signaling was associated with downregulation of p-AKT, p-ERK1/2 and MYC. The combined use of IQS-01.01 with the BET bromodomain inhibitor CPI203 exerted a synergistic cytotoxic effect in DLBCL cell lines in vitro (mean combination index: 0.6±0.2). In a xenotransplant model of DLBCL the IQS-01.01/CPI203 combination decreased tumor burden (p=0.004) through prevention of CXCR4 nuclear import and MYC downregulation, resulting in a synergistic apoptosis induction. Our results point out an emerging role of nuclear CXCR4 in the pathogenesis of DLBCL and support the simultaneous targeting of CXCR4 and BET bromodomain as a promising, rationale-based strategy for DLBCL.

#2170

Rb1 **suppresses prostate cancer metastasis and lineage plasticity underlying castration resistance.**

Sheng-Yu Ku,1 Spencer Rosario,1 Yanqing Wang,1 Ping Mu,2 Mukund Seshadri,1 Zachary Goodrich,1 Maxwell Goodrich,1 Eduardo Cortez Gomez,1 Jianmin Wang,1 Bo Xu,1 Charles L. Sawyers,2 Leigh Ellis,1 David G. Goodrich1. 1 _Roswell Park Cancer Institute, Buffalo, NY;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Androgen deprivation therapy (ADT) is an effective treatment for metastatic prostate cancer (mPCa), but patients eventually relapse with ADT resistant disease. Well-characterized mechanisms of ADT resistance include AR amplification, intra-tumoral androgen synthesis, AR splice variants, and growth receptor bypass. All of these mechanisms function to maintain sufficient AR signaling for tumor growth and survival. Improved ADT like abiraterone acetate (AA) and enzalutamide (Enza) were developed to combat such resistance mechanisms associated with alterations in androgen receptor or androgen metabolism. While AA and Enza extend survival, clinical benefits are short-lived. A new form of resistance is increasingly appreciated in patients relapsing from AA or Enza, histologic transformation of prostate adenocarcinoma (PADC) to neuroendocrine prostate cancer (NEPC) variants. NEPC is lethal and the survival time is less than a year as effective targeted therapy is unavailable. NEPC typically exhibits reduced AR expression, increased expression of neuroendocrine markers, and visceral metastasis in the absence of rising PSA. Of note, NEPC possesses the similar genome rearrangements with adjacent PADC cells, indicating they share clonal origin. Thus, NEPC may arise by histologic transformation of PADC. Underlying mechanisms of histologic transformation are not understood and experimental models are limited, hindering development of effective remedies. RB1 loss is common in NEPC, but rare in PADC; genetic profiling shows human NEPC exhibit elevated levels of several epigenetic modifiers. We hypothesize that transdifferentiation from PADC to NEPC in the context of RB1 loss is due to epigenetic alterations and can be reversed or blocked by epigenetic targeted therapies. We established several genetically engineered mouse models (GEMMs) to test the role of Rb1, and we find Rb1 loss causes metastatic progression of PADC initiated by Pten deficiency. This Rb1/Pten deficient (DKO) PADC exhibits expression markers for both PADC and NEPC as seen in human patients. Yet, these tumors are sensitive to ADT but relapse with low AR expression and acquired Trp53 mutations. RNA profiling demonstrates the phenotype of DKO tumors is similar to human NEPC. Both human and mouse NEPC is accompanied by increased expression of epigenetic reprogramming factors like Sox2 and Ezh2. Clinically relevant Ezh2 inhibitors GSK126 and EPZ6438 can restore Enza sensitivity by reversing neuroendocrine transformation. This finding has been genetically validated using short-hairpin RNA(shRNA) in vitro. These results uncover genetic mutations driving prostate cancer lineage plasticity and suggest an epigenetic approach for extending the clinical benefits of ADT.

#2171

Multi-CDK inhibition efficiently suppresses AR function and cell growth in prostate cancer.

Brian W. Simons, Maria Ybanez, Emmanuel S. Antonarakis, Jun Luo, Barry D. Nelkin. _Johns Hopkins Univ. School of Medicine, Baltimore, MD_.

Metastatic castration resistant prostate cancer (CRPC) continues to result in over 26,000 deaths per year in the United States. For 70 years, therapy for advanced prostate cancer has relied on AR-directed therapies. Unfortunately, even with the second generation androgen deprivation compounds, treated cancers commonly progress to castration resistance and lethality. This progression to castration resistance remains the central therapeutic roadblock for advanced prostate cancer. Importantly, in the vast majority of CRPC cases, the tumor still depends on AR signaling, but this signaling becomes androgen independent. The most common mechanisms, including mutation, protein truncation, and alternative splicing, affect the C-terminal domain, suggesting that antagonism of AR function via the N-terminal domain may be a promising target for CRPC therapy. Several groups have shown that CDKs 1, 5 and 9 activate AR by phosphorylation of the N-terminal domain. Here, we show that the multi-CDK inhibitor roniciclib (BAY1000394) inhibits activation of AR in a panel of prostate cancer cell lines, including several cell lines which exhibit androgen-independent AR signaling via AR-V7 alternative splicing. In vivo, roniciclib inhibited growth of prostate cancer xenografts. Since AR inhibition in prostate cancer has been shown to induce compensatory PI3K signaling, we also treated xenografts with a combination of roniciclib and the PI3K inhibitor copanlisib (BAY 80-6946). This combination was especially effective, and was well tolerated by the mice. Our results suggest that a combination of a multi-CDK inhibitor and a PI3K inhibitor may be promising for CRPC therapy.

#2172

ON 123300, an orally administered novel CDK4/6 + ARK5 inhibitor, exhibits potent antitumor activity in vivo: comparative studies with Palbociclib.

Shraddha Patel,1 Priya Pancholi,1 Tanvi Visal,1 Amruta Samant,1 Dhvanir Kansara,1 V J. Rajadhyaksha,2 Benjamin S. Hoffman,2 Manoj Maniar,2 Vikas Sehdev1. 1 _Long Island Univ., Brooklyn, NY;_ 2 _Onconova Therapeutics Inc., Newtown, PA_.

Background: The overexpression of cyclin-dependent kinases 4/6 (CDK4/6) is known to cause cell cycle dysregulation in certain cancer types, making these cell cycle kinases attractive targets for pharmacological inhibition. The effectiveness of first-generation non-selective cyclin-dependent kinases, such as roscovitine and flavopiridol, was hampered by toxicities, leading to the development of second-generation compounds like IBRANCE®/Palbociclib that specifically inhibit CDK4 and 6. ON 123300 is a third-generation potent CDK4/6 inhibitor that also inhibits ARK5 with low nanomolar potency and has the potential to improve upon second-generation compounds. Previous studies have demonstrated the inhibitory effect of single-agent ON 123300 in various pre-clinical cancer models of MM and leukemia. In this study, we investigated the comparative therapeutic potential of ON 123300 as an oral anticancer agent and a second-generation inhibitor, Palbociclib, in xenografted Rb+ve mouse models.

Methods: MDA-MB-435S xenografted mice were treated once a day for 21 days with ON 123300 (125mg/kg) or Palbociclib (125mg/kg). Tumor volumes were measured and peripheral blood was gathered to evaluate the effects on hematological parameters. Separately, Western blot analyses were performed to determine the effect of CDK4/6 inhibition on p-Rb following intra-tumoral treatment with ON 123300 (2.5µM) or Palbociclib (2.5µM).

Results: ON 123300 and Palbociclib reduced tumor growth with an equivalent magnitude during the 21-day treatment period, suggesting that the two compounds were equally effective in this model. Both compounds decreased RBC and platelet counts, however Palbociclib had a

more prominent and statistically significant (P≤0.05) inhibitory effect on neutrophil counts when compared to ON 123300 (30.70 ± 3.55 vs. 45.10 ± 2.04). Western blot analysis of tumor tissues demonstrated equivalent effects on p-Rb for both compounds.

Conclusions: Xenograft data indicates that a third-generation CDK4/6 inhibitor, ON 123300, is as effective as Palbociclib in an Rb+ve xenograft model. Moreover, this study also suggests that ON 123300 may have the added advantage of reduced neutropenia compared to Palbociclib. Prior preclinical data suggest that ON 123300 may be efficacious in Rb-ve tumors, where second-generation compounds have diminished single-agent

activity, and our ongoing studies are aimed at further characterizing the in vivo activity of ON 123300 in this setting.

#2173

**Targeting CDK1 and MEK/ERK overcome mutant** BRAF **-mediated apoptosis resistance in human colorectal cancer cells.**

Hisato Kawakami, Shengbing Huang, Frank A. Sinicrope. _Mayo Clinic, Rochester, MN_.

BRAFV600E mutation occurs in ~10% of human colorectal cancers (CRC) where it is associated with treatment resistance and poor prognosis. Data from TCGA and a clinical trial cohort identified a distinct subset of BRAF mutant colon cancers with deregulation of the cell cycle and overexpression of CDK1 (Barras D, et al, Clin Cancer Res. 2016). We tested the hypothesis that CDK1 inhibition can enhance apoptosis in BRAFV600E CRC cells. Since BRAF mutant cells show p-ERK activation, combined inhibition of CDK1 and MEK/ERK was evaluated. Using isogenic colon cancer cells, BRAF mutant alleles were shown to confer resistance to the CDK1 inhibitor, R0-3306, in a gene dose-dependent manner that was associated with reduced cleavage of caspase-3 and downstream PARP, and decreased pH2AX (DNA fragmentation marker). Ectopic BRAFV600E or constitutively active MEK mutant also conferred resistance to R0-3306. CDK1 siRNA was shown to increase cobimetinib-induced apoptosis, including annexin V labeling, and increased pH2AX. Furthermore, ERK siRNA or cobimetinib treatment attenuated CDK1 protein expression and increased R0-3306-induced pH2Ax. Moreover, treatment with R0-3306 plus cobimetinib significantly enhanced a caspase-dependent apoptosis and markedly reduced colony formation vs either drug alone in two CRC cell lines. Caspase-3 cleavage by R0-3306 + cobimetinib was dependent upon caspase-8. Mechanistically, CDK1 inhibition by R0-3306 suppressed the phosphorylation of procaspase-8 at Ser-387 shown by R0-3306 withdrawal which restored p-C8-S387 coincident with expression of the mitotic marker, pH3S10. In conclusion, inhibition of CDK1 can significantly augment apoptosis induction by MEK/ERK inhibition in BRAFV600E CRC cells, suggesting a novel therapeutic strategy to overcome mutant BRAF-mediated resistance.

Apoptosis induction by CDK1 inhibitor (R0-3306) ± MEK/ERK inhibitor cobimetinib

---

|  | Treatment | Treatment | Treatement | Treatment

|

CRC cell lines | DMSO | R0-3306 (5 µM) | cobimetinib (5 µM) | combination

Annexin V+ (%) | RKO | 13.43 ± 1.41 | 32.55 ± 2.93 | 15.04 ± 0.26 | 52.72 ± 3.16

Annexin V+ (%) | HT-29 | 11.22 ± 1.84 | 42.44 ± 2.70 | 25.29 ± 0.16 | 65.62 ± 4.11

#2174

Inhibition of CDK8 kinase with SEL120-34A allows for a personalized approach in AML.

Tomasz Rzymski,1 Michal Mikula,2 Eliza Zylkiewicz,1 Agnieszka Dreas,1 Katarzyna Wiklik,1 Aniela Golas,1 Katarzyna Wojcik,1 Magdalena Masiejczyk,1 Iga Dudzicz,1 Katarzyna Kucwaj,1 Malgorzata Statkiewicz,2 Krzysztof Goryca,2 Aleksandra Grochowska,2 Aleksandra Cabaj,2 Jerzy Ostrowski,2 Urszula Kukliniska,2 Krzysztof Brzozka1. 1 _Selvita S.A., Krakow, Poland;_ 2 _Maria Sklodowska-Curie Memorial Cancer Center, Warsaw, Poland_.

Inhibition of oncogenic transcriptional programs is recognized to be a promising therapeutic strategy. SEL120-34A is a novel inhibitor of Cyclin- dependent kinase 8 (CDK8), which regulates transcription by associating with the Mediator complex. SEL120-34A interacts with the ATP binding site of CDK8 in type I inhibitor fashion and forms several types of interactions, including halogen bonds with the protein's hinge region and hydrophobic complementarities within its front pocket. Although the compound was only modestly active in solid tumor cell lines, it repressed phosphorylation of STAT5 Ser726 and could differentially inhibit viability of AML and ALL cell lines in vitro and in vivo, along with other type I CDK8 inhibitors. Transcriptomic analysis identified major transcriptional programs altered in responder cell lines, which strongly indicated that apart from repression of survival pathways, CDK8 inhibitors could induce differentiation in cell lines with leukemia stem cells characteristics. Further studies on a large panels of responder and non-responder cell lines identified robust biomarkers which could be used with high confidence for stratification and personalized approach in CDK8-dependent AML cases. Favorable pharmacokinetics, confirmed safety and in vivo efficacy in leukemia models provide the rationale for further clinical development of SEL120-34A.

#2175

**Vietnamese medicinal plant compounds show potent anti-pancreatic cancer activity** in vitro **.**

Danielle Bond,1 Phuong Thien Thuong,2 Do Thi Ha,2 Nguyen Minh Khoi,2 Judith Weidenhofer,1 Christopher J. Scarlett1. 1 _The University Of Newcastle, Ourimbah, Australia;_ 2 _National Institute of Medicinal Materials, Viet Nam_.

BACKGROUND: Pancreatic cancer is a devastating disease with a dismal survival rate of less than 7%. This is due to its late diagnosis and aggressive, metastatic disease. Currently treatment typically consists of surgery and/or treatment with gemcitabine; gemcitabine/nab-paclitaxel or FOLFIRINOX - all chemotherapeutic agents that have natural product-derived components that only prolong survival by 5-6 months at best. As traditional Vietnamese medicinal plants have been used to treat a number of diseases for several thousands of years, there is great potential to investigate their importance as sources of novel therapeutic agents for pancreatic cancer.

METHODS: Pancreatic cancer cell lines (BxPC3, MiaPaCa2 and CFPAC1) and the normal pancreatic ductal epithelial (HPDE) cell line were treated with pure compounds isolated from the Vietnamese plants Croton tonkinensis and Salacia cochinchinensis and the effects on cell viability (CCK-8 assay) and apoptosis (caspase-3/-7 activation assay) were assessed to determine their anti-cancer capacity.

RESULTS: Treatment of pancreatic cancer cell lines with Pristimerin (from Salacia cochinchinensis) and CT1 (from Croton tonkinensis) resulted in significant dose-dependent growth inhibition. Pancreatic cancer cell lines showed significantly greater sensitivity to Pristimerin treatment after 24 and 72hrs, with a 1.7 – 2.3 fold lower IC50 value in MiaPaCa2 pancreatic cancer cells compared to normal HPDE cells, respectively (24hrs: 48 vs. 111nM; 72hrs: 63.85 vs. 111nM). Significant cytotoxicity was also seen in other pancreatic cancer cell lines (BxPC3 103 - 210nM; CFPAC1 77nM). Treatment with CT1 also resulted in significant dose-dependent cytotoxicity with an IC50 value that was 1.5 – 1.8 fold lower in MiaPaCa2 cells compared to normal HPDE cells, respectively (24hrs: 116 vs. 177nM; 72hrs: 46.5 vs. 84.5nM). Treatment of BxPC3 and CFPAC1 cells with CT1 also led to significant cytotoxicity with IC50 values of 300nM and 170nM, respectively. Moreover, treatment with Pristimerin and CT1 at concentrations of 625nM and 1μM induced caspase-dependent apoptosis of MiaPaCa2 and HPDE cells after 22hrs of treatment.

CONCLUSIONS: Pure compounds from Vietnamese medicinal plants show significant pancreatic cancer growth inhibition and induce apoptosis in vitro at concentrations that have minimal effect on normal pancreatic cells and therefore show promise as novel anti-pancreatic cancer therapies.

#2176

Thymoquinone synergizes the anticancer properties of cisplatin against head and neck squamous cell carcinoma and protects normal oral epithelial cells.

Abdulwahab Noorwali,1 Omar Aloafi,1 Safia Al-Attas,1 Fatheya Zahran,1 Ahmed M. Al-Abd2. 1 _King Abdulaziz University, Jeddah, Saudi Arabia;_ 2 _National Research Centre of Egypt, Giza, Egypt_.

Cisplatin (CDDP) is potent anticancer agent different tumors such as head and neck cancers. Thymoquinone is natural compound drawing attention as chemotherapeutic and chemomodulator. Herein, we studied the chemomodulatory effect of thymoquinone to CDDP against squamous cell carcinoma cells. CDDP killed head and neck squamous cell carcinoma cells (UMSCC-14C) with IC50's of 5.2±0.2, 2.6±0.2 and 1.6±0.1 µM after treatment for 24h, 48 h and 72 h, respectively. On the other hand, IC50's of CDDP against normal oral epithelial cells were 36.4±1.1, 16.3±0.6 and 6.7±1.1 µM after exposure for 24h, 48 h and 72 h, respectively. Thymoquinone alone exerted considerable cytotoxicity against UMSCC-14C cells with IC50's of 9.0±0.2, 6.9±0.1 and 5.0±0.2 µM after treatment for 24h, 48 h and 72 h, respectively. While significantly weaker killing effect was noticed for thymoquinone against normal oral epithelial cells (OEC) with IC50's of 41.1±0.5, 36.6±1.1and 26.3±1.2µM after exposure for 24h, 48 h and 72 h, respectively. Equitoxic combination of thymoquinone and CDDP showed synergistic interaction against UMSCC-14C cells (Combination index of 0.58) and antagonistic interaction against OEC cells (Combination index of 1.12). Using annexin-V/PI staining, it was found that thymoquinone alone (5 µM) increased apoptotic cell fraction in UMSCC-14C cells, as early as after only 6 hours, from 1.7±0.1% to 24.4±0.6% compared to 2.7±0.04 apoptosis induced by CDDP (5 µM). Combination of CDDP with thymoquinone further increased total apoptosis in UMSCC-14C to 32.9±1.5% after 6 h. prolonged exposure of UMSCC-14C to thymoquinone (5 µM) alone for 24 h resulted in 96.7±1.6% total apoptosis which was increased after combination with CDDP to 99.3±1.2% in UMSCC-14C cells. In contrast to UMSCC-14C cells, thymoquinone did not induce any significant apoptosis in OEC and even decreased apoptosis induced by CDDP alone from 4.5±0.3% to 3.4±0.2% after combination for 24 h. Using western blot analysis, neither thymoquinone nor CDDP managed to increase the expression level of p53 apoptotic protein; while combination of CDDP with thymoquinone significantly increased p53 expression by 4.5 folds. On the other hand, both CDDP and thymoquinone decreased the expression level of the anti-apoptotic protein Bcl-2 to be 60% and 20% of control level, respectively. Combination of both agents further abolished the level of Bcl-2 to be 10% of control level. Caspase-9 expression was similarly induced by 2 folds and 4.5 folds after treatment with CDDP and thymoquinone, respectively. Combination of CDDP and thymoquinone further induced the level of caspase-9 to be 6 folds original basal expression level. None of these finding could be detected in normal OEC cell line. In conclusion, thymoquinone synergize the anticancer properties of CDDP against squamous cell carcinoma cells and protects from its damaging effect against normal epithelial cells

#2177

**The olive biophenol oleuropein selectively induces apoptosis in pancreatic cancer cells** in vitro **.**

Chloe D. Goldsmith,1 Helen Jankowski,1 Danielle Bond,1 Judith Weidenhofer,1 Costas Stathopoulos,2 Paul Roach,1 Christopher Scarlett1. 1 _The University of Newcastle, Ourimbah, Australia;_ 2 _University of Abertay, Dundee, United Kingdom_.

Pancreatic cancer is a devastating disease with a 5-year survival rate of less than 5%. Resistance to conventional treatment options and toxicity of current chemotherapy agents (gemcitabine) makes pancreatic cancer a target for the development of novel therapeutic agents. Oleuropein is the most abundant biophenol found in olive products; it has anti-atherogenic and anti-inflammatory properties as well as activity against cancers of the breast, colon and prostate. However, there has yet to be any investigation into the effects of oleuropein on pancreatic cancer cells. Consequently, this study aimed to assess the anti-pancreatic cancer activity of oleuropein in vitro. Two cell lines were investigated; a pancreatic cancer cell line (MiaPaCa-2) and a normal pancreas cell line (HPDE). The viability of cells after treatment with 0-200μM oleuropein was assessed using the Dojindo CCK-8 viability assay and compared to gemcitabine. The induction of apoptosis was measured by way of caspase 3/7 activation, using a MUSE flow cell analyser, and expression of Bcl-2, Bax and Bac using Western blot. Cell cycle analysis was conducted using the MUSE flow cell analyser. RNA expression was assessed using Affymetrix GeneChip® Whole Transcript (WT) expression arrays. The IC50 values for oleuropein against MiaPaCa-2 cells was148μM. However, importantly, oleuropein did not decrease the viability of HPDE cells within the treatment range. In comparision, 20nM of gemcitabine did not show selectivity; it reduced the viability of MiaPaCa-2 cells to 21% and of HPDE cells to 2%. An increase in the expression of caspase 3/7 was seen in MiaPaCa-2 cells when treated with oleuropein but it had no effect on the HPDE cells. Furthermore, when treated with oleuropein, an increase in the expression of genes involved in the NRF-2 (oxidative stress) pathway was observed in MiaPaCa-2 cells, an effect not observed in HPDE cells.

Conclusion Oleuropein selectively induced apoptosis in the pancreatic cancer cells (MiaPaCa-2), appearing non-toxic to normal pancreas cells (HPDE) within the treatment ranges; this is significant, since gemcitabine was comparatively more toxic to HPDE cells. Furthermore, the link between oleuropein and the NRF-2 pathway in MiaPaCa-2 cells justifies further study into the mechanisms of action of oleuropein and its potential as a novel therapeutic approach for pancreatic cancer.

#2178

**Growth inhibition of the crude extracts of** Musa basjoo **in human colon carcinoma cells.**

Harutoshi Matsumoto,1 Saeko Ando,1 Katsumi Fukamachi,1 Mitsuru Futakuchi,1 Kazunori Kimura,2 Naoki Yoshimi,3 Masumi Suzui1. 1 _Department of Molecular Toxicology, Nagoya City University Graduate School of Medical Sciences and Medical School, Nagoya, Japan;_ 2 _Department of Clinical Pharmacy, Nagoya City University Graduate School of Medical Sciences and Medical School, Nagoya, Japan;_ 3 _Department of Pathology and Oncology, Graduate School of Medicine and Faculty of Medicine, University of the Ryukyus, Okinawa, Japan_.

Musa basjoo (MB) is a tropical evergreen tree growing mainly in subtropical or tropical countries. MB has been used globally as a folk medicine such as antipyretic, diuretic, and hemostatic drugs for centuries but evidence-based biological activities and molecular mechanism of action of MB are unknown. Therefore, in the current study we examined whether the crude extracts of MB exert anticancer activity in HT29 and HCT116 human colon carcinoma cell lines. Dried leaves of MB samples were extracted with acetone or methanol. Crude extracts of MB were then dissolved in dimethylsulfoxide (DMSO) and used for the following experiments. Growth inhibition was determined by colony or MTT assays in these cell lines. Cells were treated with increasing concentrations (12.5 to 200 μg/mL in colony assays, 25 to 400 μg/mL in MTT assays) of acetone/methanol extracts of MB in DMEM/5%FBS. Untreated control cells were treated with DMSO alone. Crude extracts of MB inhibited the growth of cells with IC50 values of 118 µg/mL (acetone extract, HT29), 75 µg/mL (acetone extract, HCT116), >200 µg/mL (methanol extract, HT29), 141 µg/mL (methanol extract, HCT116) in colony assays, and with IC50 values of 137 µg/mL (acetone extract, HT29), 73 µg/mL (acetone extract, HCT116), 240 µg/mL (methanol extract, HT29), 248 µg/mL (methanol extract, HCT116) in MTT assays. Acetone extract was used in the flowcytometry and western blot analyses because it showed stronger growth inhibition than methanol extract in both cell lines. Flowcytometry analysis indicated that when HT29 and HCT116 cells were treated with 100 µg/mL acetone extract of MB for 96h, the percentage of cells in G1 increased by 5.4% and this was associated with a concomitant decrease of cells in the S and G2-M phases of the cell cycle. Acetone extract of MB did not cause subG1 fraction in either HT29 or HCT116 cell lines. The results indicate that acetone extract of MB causes carcinoma cells to arrest in the G1 phase. We then performed western blot analysis to determine whether treatment of carcinoma cells with acetone extract of MB alters cellular levels of the G1 cell cycle control proteins cyclin D1, cdk4 and the cell cycle inhibitor protein p21CIP1. When HT29 and HCT116 cells were treated with 50 and 100 µg/mL acetone extract of MB for 96h, there was a marked decrease in the levels of expression of the cyclin D1 and cdk4 proteins and a marked increase in the levels of expression of the p21CIP1 protein. Thus, a decrease in cyclin D1 may cooperate with the induction of p21CIP1 to arrest cells in G1 and thereby further contribute to MB-induced growth inhibition. Taken together, the crude extracts of MB contain active component(s) that exert growth inhibition of human colon carcinoma cells. The current study is the first systematic examination of the anticancer activity of MB and may provide a novel approach to the chemoprevention and/or chemotherapy of human colon carcinoma. 

## CANCER CHEMISTRY:

### Drug Delivery

#2179

Targeted delivery of nanoparticulate cytochrome c into GL261 glioma cells through the proton-coupled folate transporter.

Yuriy Kucheryavykh,1 Jescelica Ortiz-Rivera,1 Michael Inyushin,1 Luis Cubano,1 Moraima Morales-Cruz,2 Alejandra Cruz-Montañez,2 Kai Griebenow,2 Lilia Kucheryavykh1. 1 _Universidad Central del Caribe, Bayamon, PR;_ 2 _University Of Puerto Rico, Rio Piedras, PR_.

In this study we identified the proton-coupled folate transporter (PCFT) as a route for targeted delivery of drugs to glioma cells. Using the techniques of confocal imaging, western blotting, and siRNA knockdown against the PCFT (encoded by the SLC46A1 gene), we demonstrated that Gl261 glioma cells, but not primary cultured astrocytes, express the PCFT, which provides selective internalization of folic acid (FA)-conjugated cytochrome c-containing nanoparticles (FA-Cyt c NPs), followed by glioma cell death. FA-Cyt c NPs, used at a concentration of 100 µg/ml, had no cytotoxic effects in astrocytes but caused 40% death in GL261 cells. Whole-cell patch clamp recording revealed FA-induced membrane currents, with maximum activity at pH 6.0, which is specific for the PCFT. Currents were reduced by siRNA PCFT knockdown in a similar manner as by application of FA-Cyt c NPs, indicating that the PCFT is a major carrier of FA and a route for internalization of FA-conjugated NPs in GL261 cells. We conclude that the PCFT provides a mechanism for targeted delivery of FA-conjugated nanodrugs to glioma cells with high specificity for GL261 cells, effectively causing apoptosis in these cells, and provides a starting point for the development of efficient methods for treating gliomas. The research was supported by NIH grants SC2GM102040, SC2GM095410, G12MD007583, R25GM110513, and US Department of Education Grant P031S130068.

#2180

Triphenylmethanol conjugates of leuprorelin asanti-cancer prodrugs.

Yousef Ahmadibeni, Kaleh Karim, William Boadi. _Tennessee State University, Nashville, TN_.

Leuprorelin (LEP) is a synthetic analogue of gonadotropin-releasing hormone (GnRH), first was approved by the FDA for treatment of advanced prostate cancer and endometriosis in 1985. The initial effect of administration LEP is to stimulate the pituitary secretion of FSH and LH. After a prolonged stimulation (constant concentration of LEP in the blood) pituitary becomes insensitive to the action of GnRH. This reduces the level of gonadotropin in the blood, resulting in decreased levels of sex hormones to postcastration or menopausal levels. In addition to the usual side effects of this agonist analogs of LH-RH, other reported adverse effects include transient hypertension, dry mouth, excessive salivation, paraesthesia and increased dysuria. Improving the biological activity of LEP by increasing the cellular uptake and retention is a remedy to this end.

In this study, a prodrug strategy has been introduced to optimize the hydrophobicity of LEP by using an appropriate hydrophobic linker attached to tris(4-methoxyphenyl)methanol (TPM) derivatives with the expectation to improve the cellular uptake. In this regard, several LEP conjugates of TPM derivatives with optimized hydrophobicity were synthesized by the reaction of methoxy benzenes (e.g. anisole, 2-fluoroanisole, 2-methylanisole 1,2-dimethoxybenzene or methyl 2-methoxybenzoate and 1,3,5-trioxane, followed by the conjugation with LEP and dodecanedioic acid in the presence of HBTU/DIPEA/DIC in moderate yields.

Comparative antiproliferative assays between TPM-LEP conjugates and the corresponding non-covalent physical mixtures of the TPM derivatives and LEP were performed against human Caucasian prostate adenocarcinoma (PC3), human breast cancer cell line (BT549) and mouse pre-adipocytes (3T3-L1) cells and indicated moderare to high inhibition of the cell proliferation at a concentration of 5-100 µM after 24-72 h of incubation. These data suggest that TPM-LEP derivatives with optimized hydrophobicity can be used to improve the biological activity of LEP.

#2181

Antibody-conjugated nanoparticles for targeting metastatic triple-negative breast cancer.

Vidhi Khanna, Stephen Kalscheuer, Ameya Kirtane, Jayanth Panyam. _University of Minnesota, Minneapolis, MN_.

Early detection and the availability of new treatments have improved the survival rates of patients presenting with local or regional breast cancer to as high as 99% and 85%, respectively. On the contrary, patients with metastatic disease have a dismal 5-year survival rate of 17%. Thus, there is an urgent need for treatment strategies directed towards metastasis. Our lab has developed antibodies (Clone 6 and AM6) capable of recognizing tumor cells that have undergone epithelial-to-mesenchymal transition (EMT), a key step in the generation of circulating tumor cells and metastasis. The goal of the current study was to determine whether we use these antibodies as targeting ligands for directing anticancer drug-loaded polymeric nanoparticles to metastatic triple negative breast cancer cells as a novel therapeutic option. Polymeric PLGA nanoparticles loaded with paclitaxel, a chemotherapeutic agent, were functionalized with the antibodies using thiol-maleimide chemistry. We optimized the conjugation reaction in order to achieve maximal cell uptake of nanoparticles without compromising antibody binding. In vitro studies were carried out in an MDA-MB-231 derivative cell line with enhanced lung metastatic potential as well as a melanoma metastatic cell line M12. Clone 6 nanoparticles and AM6 nanoparticles showed significant improvement in cellular uptake as well as retention. A competition experiment confirmed target-mediated uptake of nanoparticles. Cytotoxicity studies showed improved cell kill using Clone 6 nanoparticles and AM6 nanoparticles. Based on these promising in vitro results, we are currently carrying out in vivo studies in mice. The development of a targeted drug delivery system for the treatment of metastatic triple negative breast cancer can significantly enhance the survival rate for patients who often have a life-expectancy of less than one year.

#2183

Targeted delivery of hyaluronic acid-labeled chitosan nanoparticles against CD44 overexpressed endothelial cell for tumor angiogenesis therapy.

Gahee Kim, Heedong Han. _Konkuk University, Seoul, Republic of Korea_.

Objective: RNA interference (RNAi)-based approaches hold great potential for cancer therapy. Here, we developed siRNA-incorporated chitosan nanoparticles coated with hyaluronic acid (HA-CH-NP/siRNA) to target CD44 receptor on the surface of tumor endothelial cells

Methods: Size and zeta potential of HA-CH-NP were measured by light scattering with a particle size analyzer and Zeta Plus. Morphology of HA-CH-NP was observed by atomic force microscopy (AFM). HA-based selective binding of HA-CH-NP on the CD44 positive cells was assessed by flow cytometry and immunohistochemistry (IHC) analysis. Therapeutic efficacy was examined in orthotopic mouse models of ovarian carcinoma. PLXDC1 mRNA was quantified using real-time reverse transcription-polymerase chain reaction (RT-PCR). Additionally, biological effect on angiogenesis (MCD), cell proliferation (Ki67), and apoptosis (TUNEL) in the tumor tissue were examined by IHC analysis.

Results: HA-CH-NP/siRNA was 185 ± 10 nm in size and zeta potential was 26.4 mV, respectively. The loading efficiency of siRNA into HA-CH-NP/siRNA was up to 60%. We confirmed selective binding efficiency of HA-CH-NP/siRNA with CD44+ tumor endothelial cells was increased 2.1-fold compared to CH-NP/siRNA. PLXDC1 silencing using HA-CH-NP/siRNA significantly inhibited tumor growth in A2780 tumor growth in A2780 tumor-bearing mice compared to control (p < 0.0001), and mRNA expression of PLXDC1 was significantly decreased in HA-CH-NP/siRNA treated group. In addition, treatment with HA-CH-NP/siRNA resulted in significant inhibition of cell proliferation (p < 0.05), microvessel density ( p < 0.05), and increased cell apoptosis (p < 0.05).

Conclusion: This study demonstrated that HA-CH-NP/siRNA was a novel and highly selective delivery system for siRNA with potential for broad applications for tumor angiogenesis therapy.

#2184

Development and testing of nanoformulation of a vascular disrupting agent in rat glioma with MRI monitoring.

Meser M. Ali, Sunalee Jayasundara, James R. Ewing. _Henry Ford Hospital, Detroit, MI_.

Anti-angiogenesis therapy VEGF-VEGF receptors (VEGFRs) axis alone or in combination with other therapeutic agents have demonstrated mixed results, with the majority of reports indicating that glioblastoma multiform (GBM) developed resistance against anti-angiogenesis therapy as well as small molecular receptor tyrosine kinase inhibitors. This result is perhaps not unexpected, because angiogenesis is obviously complex, involving dozen of different growth factors that trigger a cascade of subsequent events. Even if a drug effectively blocks one angiogenic growth factor, such as VEGF, blood vessels may still develop via activating alternative pathways. Yet without a sufficient blood supply, cancerous tumors can't grow larger than the head of a pin and are unlikely to become lethal. Therefore, tumor vascularization is a critical process that determines tumor growth, progression and metastasis. Thus, tumor vasculature has become an emerging target for new chemotherapeutic drugs Vascular disrupting agents (VDAs) for example, combretastatin (CA4), represent a new class of chemotherapeutic agent that targets the newly formed vasculature in solid tumors. Preclinical and early phase trials have demonstrated the promising therapeutic benefits of CA4. Nevertheless, the clinical translation of CA4 has been significantly hampered due to its poor systemic bioavailability and the non-specific distribution of CA4 throughout the body when administered intravenously. Thus, it is reasonable to explore novel formulations of CA4 that overcome the limitations mentioned above. To improve bioavailability combretastatin phosphate (CA4-P) has been synthesized. But, CA4-P showed short blood half life. We have engineered dendrimer-based nano-sized CA4 conjugate which demonstrates high water solubility. Preliminary intravenous (i.v.) delivery of nano-combretastatin in an orthotropic glioma model demonstrated a necrosis at the core of the tumor leaving a rim of viable tissue. The MRI-determined tissue parameters Ktrans, blood flow (CBF), DWI, ADC map, distribution volume and tumor size indicated the effectiveness of nano-combretastatin treatment.

#2185

Cross-disciplinary optimization of nano-drug delivery to ovarian carcinoma and glioma cells.

Rupin Singh,1 Rana Falahat,2 Eva Williams,2 Joseph O. Johnson,3 Norma Alcantar,2 Aleksandra Karolak,3 Katarzyna Rejniak,3 Marzenna Wiranowska1. 1 _University of South Florida Morsani College of Medicine, Tampa, FL;_ 2 _University of South Florida College of Engineering, Tampa, FL;_ 3 _H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL_.

This study reports on the quantitative analysis of the diffusion and localization of a targeted drug delivery system (DDS) consisting of fluorescent labeled 0.01 µM paclitaxel-BODIPY 564/570 encapsulated in non-ionic surfactant vesicles embedded in a thermosensitive cross-linked chitosan hydrogel. This is a multi-tiered DDS that allows for enhanced stability, sustained and controlled delivery of embedded drugs to tumor sites, and reduced toxicity. We showed that cancer cells of epithelial origin, such as human ovarian epithelial carcinoma OV2008 and highly migratory mouse glioma G-26, overexpress MUC1, a mucin surface antigen, which effectively enhances the specific targeting capacity of chitosan. Utilizing this DDS we found that OV2008 carcinoma cells had ~2 times higher fluorescence intensity level than normal ovarian epithelial IMCC3 cells with a statistical significance at 5 min and 24 h incubation times suggesting that this DDS had a higher affinity for tumor cells. Therefore, we hypothesized that there is a difference in PTX diffusion towards epithelial origin tumor cells when compared to normal cells in both distance/location and time. Diffusion and localization of fluorescent labeled PTX was evaluated in vitro using confocal microscopy. The fluorescence intensity captured on the images was quantified with ImageJ software in OV2008 carcinoma cells and compared to IMCC3 normal cells at time intervals 5 min, 1 h, 24 h, 48 h, and 72 h. The fluorescence intensity image data was analyzed in multiple radial line segments separated into three different zones of 24 µm each to represent multiple diffusion distances. Our results showed that fluorescence intensity levels in the zones around the IMCC3 normal cells at 5 min and 1 h were significantly higher (p&lt;0.05) than around OV2008 carcinoma cells correlating reciprocally with our finding of intracellular fluorescence intensity in these two cell lines. Therefore, the data evaluating fluorescence levels in the radial zones outside the cells indicated that the migrating DDS-PTX had already been taken up by the tumor cells. Furthermore, the normal cells which showed significantly lower intracellular levels of fluorescence had higher levels of fluorescence in the measured radial zones outside the cells.

This PTX-BODIPY 564/570 diffusion data from in vitro studies is currently being used for computational modeling in the in vivo intracerebral model of G-26 glioma. It will be integrated with mathematical model simulations that describe the pharmacokinetic and pharmacodynamic (PK/PD) properties of this drug delivery system for the evaluation of its efficacy and to optimize drug delivery in vivo. Computational modelling is being done in Matlab using the treatment scenario of the post-surgical late-stage glioma. The simulation studies will be used to determine optimal drug concentrations, chitosan density, and localization.

#2186

**Peptide-cleavable maytansinoid (ADCs) induce high bystander killing leading to improved anti-tumor activity** in vivo **.**

Wayne C. Widdison, Juliet A. Costoplus, Jose F. Ponte, Leanne Lanieri, Yulius Setiady, Ling Dong, Anna Skaletskaya, Rui Wu, Qifeng Qiu, Yelena Kovtun, Ravi V. Chari. _ImmunoGen Inc., Waltham, MD_.

Antibodies targeting surface antigens on cancer cells typically have progressively lower access to tumor cells that are further removed from blood vessels. Also, the antibody will not bind to cells in the tumor mass that do not express antigen, including stromal cells of the tumor, many of which reportedly aid in the survival or metastasis of cancer cells.

ADCs can bind to antigen positive cancer cells, after which they are internalized and catabolized to release one or more cytotoxic metabolite(s) that can kill the targeted cell. Metabolites that are membrane permeable may also diffuse into and kill neighboring cells, often called bystander cells, that would normally be less accessible.

The goal of this work was to design ADCs that would have increased bystander activity, which could result in greater killing of cancer cells and stromal cells in the tumor environment. We have prepared a new type of peptide-cleavable immolative ADC (PCI-ADC) that efficiently releases membrane permeable cytotoxic maytansinoid metabolites upon cleavage of the peptide linker, followed by immolation. Several PCI-ADCs were prepared that release metabolites having different degrees of hydrophobicity. As the hydrophobicity of the metabolite increased, the PCI-ADCs' bystander activity also increased. The lead PCI-ADC generally displayed a similar degree of in vitro cytotoxicity as maytansinoid ADCs that utilize disulfide linkers, however the PCI-ADC induced significantly more bystander killing. In mice bearing large tumor xenografts (250 mm3) or tumor xenografts that express the target antigen heterogeneously, PCI-ADCs were found to be more efficacious than maytansinoid ADCs that use disulfide linkers, as well as our recently reported peptide-para-anilino maytansinoid ADCs. The nature of the amino acid residues in the peptide linker of the PCI-ADC was also altered so that the tolerability of the ADCs in mice could be increased without impeding efficacy.

In conclusion, we have developed a promising new type of maytansinoid ADC, one that provides a high degree of bystander killing, improved activity in homogeneous and heterogeneous tumor models in vivo, and has a different mechanism of metabolite release than current maytansinoid based ADCs.

#2187

A pro-nanodrug with intratumoral reconstitution and intracellular delivery beyond therapy-resistance molecular pumps.

Rong Xu,1 Mauro Ferrari,2 Haifa Shen2. 1 _Department of Pharmacology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China;_ 2 _Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX_.

Tumor metastases to distant organs pose a serious challenge to cancer treatment. The metastatic tumor lesions are less accessible to cancer drugs and the malignant cells often quickly develop therapy resistance. To address both biological and drug transport concerns in cancer treatment, we have developed a novel composite pro-nanodrug to provide sustained intra-tumoral assembly of nanotherapeutics, and tested its anti-cancer efficacy in murine models of breast cancer metastases to the lung and liver. Specifically, large payloads of polymeric doxorubicin (pDox) pro-drug were packaged as single molecules into a porous silicon microparticles that can be enriched in tumor tissue in the forms of vascular and cellular depots. Once inside the tumor, the pDox molecules self-assemble into nanoparticles that are released into the tissue where they are effectively internalized by tumor cells. The nanoparticulate prodrug is transported through the vesicular system, and doxorubicin is cleaved from the polymer and released in the immediate proximity of the nucleus beyond the reach of the drug efflux pumps. In vivo efficacy studies demonstrated that the pro-nanodrug was effective in treating metastatic tumors with or without expression of the multi-drug resistant genes.

#2188

Targeted delivery of pro-apoptotic peptide using ELP-based carrier for effective cancer therapy in various cancer model.

Sarangthem Vijaya, Rang-Woon Park. _Kyungpook National University, School of Medicine, Daegu, Republic of Korea_.

Therapeutic peptides classified as biopharmaceuticals have gained attention as a new generation of cancer therapeutics with enhanced efficiency and diminished toxicity. They often fail to have sufficient effects due to their unsatisfactory pharmacokinetic profiles, which include poor bioavailability, low stability, short half-life, and poor penetration across the biological membrane. Elastin-like polypeptide (ELP)-based drug delivery has been utilized for various applications including cancer therapies for many years. Genetic incorporation of internalization ligands and cell targeting peptides along with ELP polymer enhanced tumor accumulation and retention time as well as stability and activities of the drug conjugates. Herein, we described a unique delivery system comprised of genetically engineered ELP incorporated with multiple copies of IL-4 receptor targeting peptide (AP1) periodically and proapoptotic peptide (KLAKLAK)2 referred to as AP1-ELP-KLAK. It triggered thermal responsive self-assembly into a nanoparticle-like structure at physiological body temperature and stabilized its helical conformation, which is critical for its membrane disrupting activities. Increased IL-4 receptor specific cellular internalization was associated with the enhanced cytotoxic effect of (KLAKLAK)2 peptide. Additionally, multivalent presentation of targeting ligands by AP1-ELP-KLAK significantly enhanced intratumoral localization and prolonged the retention time compared to ELP-KLAK, non-targeted control. Systemic administration of AP1-ELP-KLAK significantly inhibited tumor growth by provoking cell apoptosis in MDA-MB231 breast cancer and B16F10 melanoma tumor xenograft models without any specific organ toxicity. Thus, our newly designed AP1-ELP-KLAK polymer nanoparticle is a promising candidate for effective cancer therapy and due to the simple preparative procedures of ELPs, this platform can be used as a good carrier for tumor-specific delivery of other therapeutics.

#2189

Adhesion and entry of gold nanoparticles into breast cancer cells: Nanoparticles for localized drug delivery/hyperthermia.

Vanessa O. Uzonwanne,1 John David Obayemi,1 Jingjie Hu,1 Karen Malatesta,1 Nicolas Anuku,2 Winston Soboyejo3. 1 _Princeton University, Princeton, NJ;_ 2 _Bronx Community College, Bronx, NY;_ 3 _Worcester Polytechnic Institute, Worcester, MA_.

The specific targeting of breast cancer cells is important for the early detection and treatment of

cancer cells /tumor tissue, hence the drive for the development of nanoparticles. This paper

presents results of an atomic force microscopy study on the adhesion between triple negative

breast cancer cells (MDA-MB-231 and MDA-MB-468) and biosynthesized gold nanoparticles

(GNP) conjugated with luteinizing hormone releasing hormone (LHRH) which is known to

target LHRH receptors that are over-expressed on the surfaces of breast cancer cells. It also

estimates time scales for Gold nanoparticle entry into cells by means of an analytical model. The

adhesion forces between the LHRH-conjugated gold nanoparticles and the breast cancer cells are

found to be about 4-5 times greater than those between normal breast cells and LHRH-

conjugated gold nanoparticles. The increase in the adhesion of LHRH to breast cancer cells is

shown to be associated with an increase in the LHRH receptor density, which is revealed using

confocal microscopy. The implications of the results are discussed for localized treatment of

breast cancer.

#2190

Theranostical nanoscale metal-organic frameworks-mediated rapid multiplexed microRNA detection and highly therapeutic efficacy in living oral cancer cells.

Pei Yu,1 Zehang Zhuang,1 Guihua Qiu,2 Haolin Chen,1 Yuying Zhao,1 Jinxiang Chen,2 Xiqiang Liu1. 1 _Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangzhou, China;_ 2 _College of Pharmacology, Southern Medical University, Guangzhou, China_.

Oral cancer represents the sixth most frequent cancer in the world, characterized by high poor prognosis and low survival rate due to local relapse and metastasis. MicroRNAs (miRNAs) have been discovered as diagnostic biomarkers and therapeutic targets for oral cancer. However, one of the major challenges of miRNAs-based theranostical approach is to achieve efficient and safe delivery. Thus, a reliable sensing strategy that can detect miRNAs expression levels in living cancer cells is urgently needed. Herein, a novel sensing platform based on nanoscale metal-organic framework (NMOF) conjugated with the fluorophores-labeled single-stranded DNA probe (p-DNA) was fabricated to monitor and inhibit multiplexed miRNAs expression in living oral cancer cells. The flow cytometry and confocal laser scanning microscopy (CLSM) were carried out to evaluate the expression levels of miRNAs in oral cancer cells. The miRNA inhibition efficiency was examined by RT-PCR. The effects of the complexes on tumor cell invasion and migration were assessed by transwell assay and wound healing. The flow cytometry, colony formation assay and CCK8 were used to assess cell apoptosis and proliferation. The results showed that NMOF works as fluorescence quencher of the labeled p-DNA. In presence of the target miRNA (including miR-7, miR-21, and miR-155), p-DNA was able to rapidly hybridize and release from the NMOF leading to the recovery of fluorescence in living cells, which can be detected in real-time. Moreover, RT-PCR results demonstrated that the multiplexed miRNAs could be effectively downregulated after treatment of p-DNA@NMOF, resulting in the simultaneous suppression of oral cancer cell proliferation, anti-apoptosis, migration and invasion in vitro. In summary, our study suggests that p-DNA@NMOF possess great potential for combining early diagnosis and gene therapy of oral cancer.

#2191

Multiple imaging reporter labeled acid-degradable dextran nanopolymer as a COX-2 siRNA carrier for COX-2 specific downregulation.

Zhihang Chen, Balaji Krishnamachary, Marie-France Penet, Zaver M. Bhujwalla. _Johns Hopkins University, SOM, Baltimore, MD_.

Cyclooxygenase-2 (COX-2) as a target has attracted interest for multiple degenerative diseases and cancers. COX-2 siRNA provides specific and effective down-regulation of COX-2. However, because artificial cationic polymers can induce proinflammatory signals that increase COX-2 expression, there are few if any reports about the use of artificial cationic polymers as siRNA carrier to downregulate COX-2 in cancer cells. Here we developed a method to efficiently synthesize a multiple imaging reporter labeled biodegradable dextran to use as an efficient cationic polymer carrier for COX-2 siRNA delivery. Amine function groups were conjugated to the dextran platform through acetal bonds. Acetal bonds were broken at acidic conditions that occur in cancer and endocytosis compartments. Rhodamine was labeled to the amine groups to detect degradation and removal of these groups from the cell, while the dextran scaffold was labeled with Cy5.5. The rapid cleavage and release of amine groups minimized the proinflammatory side effects of the positively charged amine groups.Colorimetric assay of Cy5.5 and rhodamine in different pH buffer indicated that the amine group was cleaved at pH 5.5 buffer but was stable in pH 7.4 buffer. Fluorescence imaging showed that the dextran siRNA nanoplex entered the cells through endocytosis that provided acidic conditions for the breaking of the amine group. For the first time, the intracellular breaking of acetal bonds was clearly visualized by multiple imaging reporters. Because the rapid cleavage and release of amine groups minimized the proinflammatory side-effects, quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay indicated that this dextran COX-2 siRNA nanoplex could downregulate COX-2 expression efficiently. Prostaglandin E2 level decreased significantly with COX-2 siRNA/dextran treatment. In vivo imaging demonstrated that the COX-2 siRNA/dextran nanoplex accumulated in MDA-MB-231 tumors. QRT-PCR and western blot assays of COX-2 levels in tumor tissue demonstrated that this nanoplex significantly downregulated COX-2 expression in vivo efficiently and within 24h. This dextran nanopolymer can be used as a safe, reproducible, and biocompatible siRNA carrier to effectively reduce COX-2 expression in cancer cells and tumors.

#2192

DFP-10825 IP delivery provides a new effective treatment option to peritoneal-disseminated cancers.

Cheng Jin,1 Kenzo Iizuka,1 Kokoro Eshima,1 Masakazu Fukushima,1 Tatsuhiro Ishida,2 Cheng-Long Huang,3 Hiromi Wada,3 Kiyoshi Eshima1. 1 _Delta-Fly Pharma, Inc., Tokushima, Japan;_ 2 _Tokushima University, Tokushima, Japan;_ 3 _Kyoto University, Kyoto, Japan_.

Objective: Peritoneal disseminated ovarian and pancreatic cancers are the most difficult to be treated with conventional cytotoxic or molecular targeted drugs. The treatment option is very limited although an intraperitoneal (IP) paclitaxel has been available and shown to improve a prognosis in patients. Therefore, it is urgent to develop a new IP chemotherapeutic drug regulating the fast DNA synthesis in peritoneal disseminated tumors originated commonly from the ovary, pancreas and stomach. We have developed a unique RNAi molecule consisting of shRNA (55-mer) against TS and a cationic liposome (DFP-10825) and tested its anti-tumor activity and PK profile in peritoneal disseminated ascetic tumor models.

Methods: We developed luciferase gene-transfected ovarian cancer (SKOV3-luc) and pancreatic cancer (PANC-1-luc) models in mice. After IP injection of 5x106 cells, DFP-10825 containing 20 µg TS shRNA (20 mg/mouse) was administered in an IP route (q3d x 4) to the tumor-bearing mice. In combination therapy, paclitaxel (10 mg/kg) was also IP administered to SKOV3-luc model to which the treatment was performed in the same schedule. The anti-tumor effect was assessed by measuring luciferase activity and tumor volume. Furthermore, the TS expression level in both ascetic tumor cells and solid tumors was measured by conventional RT-PCR. For PK study with DFP-10825 (especially TS shRNA), total RNAs were isolated at various time points from ascetic tumor cells and disseminated SKOV3-luc solid tumor models treated with DFP-10825 and TS shRNA levels were determined by Stem-loop RT-PCR.

Results: IP DFP-10825 delivery significantly suppressed the growth of ascetic SKOV3-luc and PANC-1-luc tumor cells and extended the survival time of these tumor-bearing mice compared with that of control group. Combination with the IP paclitaxel augmented the efficacy of DFP-10825. After the IP administration, TS shRNA levels derived from DFP-10825 in ascetic fluid were maintained at nM range (0.7 - 4.3-nM) across 24 hours but not detected (below 5 pM) in the plasma, suggesting that TS shRNA be relatively stable in the peritoneal cavity to be able to exert its anti-tumor activity but not in blood. Also, TS expression (TS mRNA) in ascetic tumor cells was significantly suppressed, supporting the notion that the anti-tumor activity and host survival benefit by DFP-10825 in tumor-bearing mice are through MOA to knock down the TS level in tumors specifically.

Conclusion: IP administration of newly developed DFP-10825, the TS shRNA conjugated with cationic liposome is localized stably in the peritoneal cavity and provides a new effective treatment option to the intractable peritoneal disseminated ovarian and pancreatic cancers without any systemic adverse events.

#2193

Structural modification of the chemotherapeutic anandamide: Designing anti-cancer agents and investigating their COX-2 metabolic products.

Andrew Morris,1 Eman Soliman,2 Rukiyah Van Dross,1 Colin Burns1. 1 _East Carolina University, Greenville, NC;_ 2 _Faculty of Pharmacy, Zagazig University, Zagazig, Egypt_.

Many epithelial cancers have been shown to overexpress the enzyme cyclooxygenase-2 (COX-2), an enzyme responsible for metabolizing anandamide (AEA) to prostamides. AEA has demonstrated cytotoxicity in COX-2 overexpressing cancers via its metabolism to novel J-series prostamides, namely 15d-PMJ2. Fatty acid amide hydrolase (FAAH) degrades AEA into arachidonic acid and ethanolamine (EA), limiting the cytotoxic capability of AEA. Cell lines with high FAAH expression have demonstrated resistance to AEA. By understanding the metabolic characteristics of FAAH, we can design AEA analogs which circumvent FAAH breakdown. To examine the effects of altering polarity, steric bulk, and functional groups on AEA-mediated cytotoxicity, we investigated known AEA derivatives which possess these properties. Arvanil, Arachidonoyl Diethanolamine (ADA), Arachidonoyl Serinol (AS), and R1-methanandamide (m-AEA) add steric bulk to the molecule via aromatic rings, extra EA arms and additional alcohol/methyl functional groups respectively. Arachidonoyl glycine (NAGly) substitutes the terminal EA alcohol with a carboxylic acid increasing polarity. Arachidonoyl-2'-chloroethylamine (AC) substitutes the terminal EA alcohol with a highly soluble chlorine. Furthermore, it is known that Arvanil, ADA, AS, and m-AEA resist degradation of the molecule by FAAH and NAGly is a known substrate of COX-2. Therefore our goal was to determine which structural modifications improve AEA-mediated cytotoxicity. To determine this, JWF2 tumorigenic keratinocytes were exposed to differing concentrations of the AEA analogs for 24 hours and cell viability was measured by conducting MTS assays. Arvanil demonstrated a 90% reduction in cell viability, NAGly demonstrated a 70% reduction in cell viability, and m-AEA demonstrated a 100% reduction in cell viability at 20 µM, the optimal dosage of AEA. Due to the fact that ADA, AS, and AC did not show a significant reduction in cell viability these molecules were not further examined. MTS assays were conducted in other epithelial cancer cell lines with varying expressions of COX-2 and FAAH. Each cell line was exposed to varying concentrations of either NAGly, Arvanil, or both for 24 hours. NAGly demonstrated a 40% reduction in cell viability in HT-29 colon cancer cells (low COX-2, high FAAH). Arvanil demonstrated a 60% reduction in A431 tumorigenic keratinocytes. NAGly demonstrated a 60% reduction in cell viability and Arvanil demonstrated a 100% reduction in cell viability in patient-derived primary melanoma. These findings suggest that modulation and substitute to the core structure of AEA will result in decreased susceptibility to FAAH degradation and enhanced antineoplastic activity.

#2194

Synergistic cytotoxicity of targeted liposomes containing doxorubicin and C6-ceramide against nucleolin-overexpressing ovarian cancer cells is supported by the downregulation of the Akt pathway.

Ana Filipa Cruz,1 Nuno André Fonseca,2 Susana F. Sampaio,2 Vera Moura,3 Ramiro D. Almeida,2 Sérgio Simões,1 João Nuno Moreira1. 1 _CNC - Center for Neuroscience and Cell Biology; FFUC - Faculty of Pharmacy; University of Coimbra, Coimbra, Portugal;_ 2 _CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal;_ 3 _TREAT U, SA, Coimbra, Portugal_.

The acknowledgment that cancer stem cells (CSC) may originate from non-stem cancer cells, interconverting through an EMT-mediated process has turned these distinct cell subpopulations into two relevant therapeutic targets. Moreover, the PI3K/AKT/mTOR pathway is essential to CSC proliferation and survival and it is often over activated in ovarian cancer. One of the strategies to target these different tumor cell populations, relies on the combination of conventional chemotherapeutic drugs (as tumor debulking agents, targeting non-stem cancer cells) with sphingolipids targeting CSC (at the level of PI3K/Akt/mTOR). Nucleolin overexpression has been demonstrated on the surface of both breast CSC and non-stem breast cancer cells (Fonseca NA, Biomaterials 2015) and endothelial cells from tumor blood vessels. The pH-sensitive lipid-based nanoparticle, functionalized with the nucleolin-binding F3 peptide, was recently modified to encapsulate a synergistic combination of a sphingolipid (C6-ceramide) and doxorubicin (DXR). Following the promising results in breast cancer, the aim of this work was to assess the therapeutic potential of this strategy against nucleolin-overexpressing ovarian cancer cell lines, as well as the underlying mechanism of action at the molecular level. A 12.9-fold increase of association of fluorescently-labelled F3 peptide-targeted liposomes ([F3]L), relative to the non-targeted counterparts, into (bulk) ovarian cancer cell lines (SKOV-3, OVCAR-3 and TOV-112D) was observed by flow cytometry. Importantly, a similar pattern of association by putative CSC enriched population (CD44high/EpCAMhigh) was also observed. These results corroborated the marked increase of cytotoxicity (assessed by the resazurin reduction viability assay) enabled by the targeted drug combinations, relative to [F3]L containing only DXR. In fact, the targeted drug combination enabled a 90% death of SKOV-3 cancer cells, following an 8 h incubation, a level of cell killing not achieved by the counterpart containing only DXR. Against the OVCAR-3 and TOV-112D cell lines, the IC90 of the targeted drug combination, following 1 h of incubation, was 2-fold lower than the one resulting from the incubation with [F3]L containing only DXR. In addition, the effect of nucleolin-mediated intracellular delivery of C6-ceramide at the p-Akt protein levels was further discriminated by western blot analysis in SKOV-3 cells. The observed 3.2-fold downregulation of p-Akt levels supported the C6-ceramide-mediated improved cytotoxicity of the drug combination. Overall, these results pointed out the enhanced efficacy of targeted synergistic drug combinations against ovarian cancer, due to the capability to target nucleolin-overexpressing cancer cells and further inhibit the Akt pathway.

#2195

Combined strategy based on pre-activated analogs of oxazaphosphorines for increased therapeutic index and immune modulation.

Julia Delahousse,1 Charles Skarbek,1 Valentine Gauthier,1 M Desbois,2 Emilie Roger,3 C. Pioche-Durieu,4 M. Rivard,5 D. Desmaële,6 T. Martens,5 E. LeCam,4 Jean-Pierre Benoit,3 P. Couvreur,6 Nathalie Chaput-Gras,2 Angelo Paci1. 1 _Gustave Roussy/CNRS-UMR8203, Villejuif, France;_ 2 _Gustave Roussy/CNRS-UMS3655/INSERM-US23, Villejuif, France;_ 3 _INSERM U1066, Angers, France;_ 4 _Signalisations, Noyaux et Innovations en Cancérologie, Villejuif, France;_ 5 _Institut de Chimie et des Matériaux Paris Est Créteil (ICMPE), Thias, France;_ 6 _Institut Galien Paris-Sud, UMR 8612, CNRS, Université Paris-Saclay, Châtenay-Malabry, France_.

Oxazaphosphorines (Oxaza) represented by cyclophosphamide (CPA) and ifosfamide (IFO) are still the corner stone of several polychemotherapy protocols as they are widely indicated in the treatment of numerous cancer from soft tissue sarcomas to lymphomas and immune-related diseases. However, Oxaza are prodrugs requiring cytochrome (CYP) P450 bioactivation responsible of limiting adverse effects. In the case of IFO, bioactivation leads to a low release of 4-OH-IFO (10%), which generates the active nitrogen mustard displaying DNA cross-links. Associated toxicities of IFO due to acrolein, (urotoxicity) and to chloroacetaldehyde (neuro and nephrotoxicity) have been described. Thus, increasing IFO therapeutic index could be of major interest. To circumvent these toxicities, our team has designed new pre-activated IFO analogs to avoid CYP bioactivation (Skarbek et al J Med Chem 2015). Among these analogues some have the ability to self-assemble as nanoassemblies (NAs), the others can be encapsulated within nano-lipid capsules (NLCs). These new drug delivery systems (DDS) can take advantage of passive targeting, as stealthiness of these DDS can be provided by PEGylation by using Cholesterol-polyethylene glycol or the use of surfactant. These DDS can also be functionalized by appropriate monoclonal antibodies leading to multi stage DDS with active targeting properties. Regarding CPA, it has been shown and described in literature that low doses of CPA enhance the immunity by promoting differentiation of CD4+ cell toward Th1. As IFO is isomeric form of CPA, it was assumed that IFO could also have such properties. Studies on immunocompetent MCA205 mouse model, an immunogenic fibrosarcoma mouse model, demonstrate a dose-dependent immunomodulation of IFO towards a modulation of the secretion of IFNy, IL-17A and IL-6 cytokines. The ongoing experiments on mouse model depleted in CD4\+ T cells and CD8\+ T cells show the antitumor efficacy of IFO 150mg/kg on these immune cells in tumor regression. Both strategies could lead to the design of nano-immuno-conjugates (NICs) which could benefit of the immunomodulatory effects of X-Oxaza combined to their antiproliferative properties targeted through immune checkpoint antibodies. These new functionalized DDS may provide a useful strategy to give specificity to active drugs used for many years in clinical practice. Both DDS could be grafted with mAbs which could lead to a new family of DDS aiming to combine antiproliferative and immunomodulatory properties for a dual antitumoral action

#2196

SDF-1/CXCR4 axis-mediated tumor-tropism of monocyte membrane-coated nanoparticles.

Yi-Nan Li, Chien-Wen Chang, Chi-Shiun Chiang. _National Tsing Hua University, Hsinchu City, Taiwan_.

Tumor-associated macrophages are one of the most abundant infiltrating cells in solid tumors. These cells are originated from the bone marrow and circulating in peripheral blood. Through the specific chemoattractant released by the tumor cells or stromal cells, bone marrow-derived monocytes (BMDMs) are able to accumulate at the tumor site by their corresponding receptors. In this study, we utilized the characteristics of tumor-tropism of BMDMs on a nanoparticle-based drug delivery system. We hypothesized that the cellular membrane of BMDMs can provide nanoparticle protection from the mononuclear phagocyte system and the chemokine receptor on the membrane can enhance the tumor-tropism of the nanoparticles. To prove the hypothesis, the cellular membrane was extracted from BMDMs and then coated on the drug-loaded nanoparticles. We also found that the expression of chemokine receptor CXCR4 on the bone BMDMs can be induced following the stimulation of tumor conditioned medium. The tumor targeting effect of the monocyte membrane-coated nanoparticles is diminished against SDF-1 knockdown tumor cells. These results indicate that the SDF-1/CXCR4 axis is contributed to the tumor-tropism of the monocyte membrane-coated nanoparticles toward astrocytoma cell line, ALTS1C1, and this effect can be boosted by the stimulation of tumor conditioned medium. In conclusion, we established a nanoparticle-based drug delivery platform against brain tumor with the cellular membrane coating technology and maximized the effect of tumor-targeting by tumor conditioned medium stimulation.

#2197

Characterization of immunoliposomes as delivery vehicles for the Rac inhibitor Ehop-016 in HER2 type breast cancer.

Michael J. Rivera-Robles,1 Pablo E. Vivas-Mejia,2 Linette Castillo-Pichardo,3 Cornelis Vlaar,4 Eliud Hernández,4 Suranganie Dharmawardhane5. 1 _University of Puerto Rico School of Medicine, San Juan, PR;_ 2 _University of Puerto Rico, San Juan, PR;_ 3 _Universidad Central del Caribe, Bayamon, PR;_ 4 _University of Puerto Rico School of Pharmacy, San Juan, PR;_ 5 _Surangani Dharmawardhane, San Juan, PR_.

Due to the critical need for improved therapeutics against advanced Stage IV breast cancer, we developed and pre-clinically validated the small molecule Ehop-016 as a potential anti-metastatic drug. Ehop-016 is a small molecule inhibitor of the interaction Rac with the guanine nucleotide exchange factor (GEF) Vav2, which consequently decreases invasive actin structures and cellular migration. In a human epidermal growth factor receptor 2 (HER2) type breast cancer model in nude mice, Ehop-016 inhibits mammary tumor growth, angiogenesis, and metastasis. Moreover, Ehop-016 reduces HER2++ cancer cell viability and migration, individually or in combination with anti HER2 or EGFR directed therapeutics. By inhibiting the key signaling intermediate Rac, Ehop-016 has the potential to block signaling downstream of HER2 and EGFRs, as well as signaling via the myriad of receptors that activate Rac, and inhibit cancer progression. Our new results demonstrate that Ehop-016 inhibits both cancer cells and leukocytes in the tumor microenvironment, as well as cytokine levels (IL-6) in mouse plasma. Even though these characteristics are desirable in anticancer agents, systemic therapy with Ehop-016 may adversely affect normal immune system function. Therefore, a strategy was developed for the targeted delivery of Ehop-016 into mammary tumors, using nanoliposomes containing the current breast cancer therapeutic, Trastuzumab, a monoclonal antibody directed at the HER2. We tested various nanoliposome formulations as targeted delivery systems for Ehop-016. First, the encapsulation efficiency of Ehop-016 loaded liposomes was tested using different drug loading methods. To obtain HER2 targeted liposomes, we prepared Trastuzumab-lipid conjugates and incorporated them into the Ehop-016 containing liposomes. The binding, internalization and selectivity of our HER2 targeted delivery system was verified in human metastatic breast cancer cell lines, by using fluorescence microscopy to assess the co-staining of the lipid bilayer and Trastuzumab. In order to evaluate the effectiveness and selectivity of the immunoliposomes in delivering Ehop-016 and inhibiting Rac activity, pull-down assays were performed to detect active Rac-GTP in cells treated with Ehop-016 alone or Trastuzumab-conjugated liposomes with Ehop-016. Results show that the Trastuzumab-conjugated liposomes are more selective at binding to HER2+ breast cancer cells and inhibiting Rac, than treatment with Ehop-016 alone. In summary, our novel delivery system containing Ehop-016 and Trastuzumab has been shown to be effective in breast cancer cells, and needs to be validated in animal models of metastatic breast cancer. The development of this targeted delivery system has implications, not only in the field of breast cancer therapeutics, but may be adapted for the treatment of other diseases, and used to overcome the side effects of current treatments.

#2198

Cancer cell membrane coated biomimetic nanoparticles: Synthesis, characterization, and functionality.

Jiefu Jin, Di Chang, Samit Chatterjee, Balaji Krishnamachary, Yelena Mironchik, Sridhar Nimmagadda, Zaver M. Bhujwalla. _Johns Hopkins Univ. School of Medicine, Baltimore, MD_.

Introduction: Biomimetic nanoparticles (NPs) combining synthetic and biological materials have flexibility and functionality for drug delivery and immunomodulation.1 Red blood cell (RBC) membranes coated poly(lactic-co-glycolic acid) (PLGA) NPs that mimic RBCs and act as nanosponges for toxins were recently described.2-3 Here, we coated cancer cell membranes onto PLGA NPs in a "right-side" out manner, to translocate membrane anchored proteins onto NPs. Cancer cell membrane coated PLGA NPs hold promise for disrupting cancer cell-stromal cell interactions, and for priming the immune system in cancer immunotherapy.

Method: Plasma membrane fractions (MFs) of U87 (low CXCR4) and U87-CXCR4 (high CXCR4) cells were isolated upon homogenization, and sucrose density gradient centrifugation. MFs and PLGA NPs were mixed and physically extruded through a porous membrane to obtain MF-coated PLGA NPs. MFs were probed with antibodies against cell fraction markers, and CXCR4. MFs-coated NPs, MFs and PLGA NPs were characterized on size, morphology, and zeta-potential. The orientation of MFs and MF-coated NPs was investigated by confocal microscopy and flow cytometry. Transwell migration assays were performed to investigate the migration of cancer cells towards human mammary fibroblasts (HMFs). Immune-competent Balb/c mice were immunized with IR700-labeled MFs or MF-coated NPs via subcutaneous injection through hock and imaged in vivo and ex vivo by near-infrared (NIR) fluorescence. Immune responses to MFs or MF-coated NPs were examined on activating CD4+ and CD8+ T lymphocytes in lymph nodes and spleens by flow cytometry.

Results: Plasma membrane purity was confirmed from western blot analysis that showed the significant enrichment of Na+/K+-ATPase, negligible amount of GPR78 or GAPDH in MFs. PLGA NPs, U87-CXCR4 MFs and U87-CXCR4 MFs-coated NPs had average diameters of 50 nm, 200 nm, and 70 nm, respectively. Z-average diameters and zeta-potential of PLGA NPs, U87-CXCR4 MFs, and U87-CXCR4 MFs-coated NPs were 79.8 nm, 336 nm and 168 nm, and -34.3 mV, -24.9 mV and -25.0 mV, respectively. Confocal fluorescence microscopy and flow cytometry detected intense PE fluorescence and higher CXCR4 in U87-CXCR4 MFs and U87-CXCR4 MFs-coated NPs than U87 counterparts, confirming a "right-side" out orientation. When U87 or U87-CXCR4 MFs were added to HMFs in the transwell assay, fewer cancer cells migrated towards HMFs, identifying the unique ability of MFs in disrupting HMF-cancer cell interactions. U87-CXCR4 MFs and MF-coated NPs were observed in the popliteal lymph nodes, and triggered the induction of CD8+ T lymphocytes, identifying a role for MF-coated NPs in providing cancer cell membranes to antigen presenting cells to induce a tumor-specific immune response.

References: 1. Fang, R. et al. Small 2015; 2. Hu, C. et al. PNAS 2011; 3. Hu, C. et al. Nat. Nanotechnol. 2013. Supported by NIH R21 CA198243 and P50 CA103175.

#2199

Magnetoelectric nanoparticles for high-specificity treatment of cancer.

Sakhrat Khizroev,1 Abhi Nagesetti,1 Tiffanie S. Stewart,2 Emmanuel Stimphil,1 Alexa Rodzinski,1 Rakesh Guduru,1 Ping Liang3. 1 _Florida International University, Miami, FL;_ 2 _University of Notre Dame, Notre Dame, IN;_ 3 _Cellular Nanomed, Weston, FL_.

Introduction: Delivering a drug specifically into the tumor cell past its membrane and then releasing the drug into the tumor cells without affecting the normal cells remains a formidable challenge. Unlike any other nanoparticles, magnetoelectric nanoparticles (MENs) display a non-zero magnetoelectric (ME) effect and thus present a unique capability to use external magnetic fields to control intrinsic electric fields associated with cell membranes and the interaction between MENs and therapeutic loads. Because cancer and normal cells of the same type have different electric properties, MENs is used for high-specificity targeted delivery. An a.c. magnetic field is used to trigger drug release off the nanoparticles.

Brief Methods: 30-nm CoFe2O4-BaTiO3 core shell MENs, with a magnetization of 1 emu/g, a coercivity of 300 Oe, and a ME coefficient of 10-100 mV cm-1 Oe-1 were prepared with a coprecipitation process. MENs were coated with fluorescein isothiocyanate to monitor their intra-cellular transport through a high-contrast confocal microscopy. Three cancer cell lines including Skov-3 (Ovarian adenocarcinoma), U87-MG (Glioblastoma), and MCF-7A (Breast adenocarcinoma), and two normal cell lines including brain endothelial cells (Brain EC) and ovarian cells HOMEC were cultured at 37°C. The transport of MENs loaded with drugs, peptides, and RNAs through the cell membranes and the consequent release of the load under different d.c. and a.c. magnetic fields were studied through confocal microscopy and photoabsorption spectroscopy, respectively. Trypan-blue viability count was used to assess cell growth inhibition under different study conditions. Atomic force microscopy of the cell membranes was conducted to understand the interaction between the nanoparticles and the cells.

Summary of new data: Comparison of MENs with purely magnetic iron oxide nanoparticles showed that the penetration through the cancer cell membrane could be achieved only with MENs. It took d.c. fields of 100 Oe and over 1000 Oe to nanoelectroporate the membranes of SKOV-3 and HOMEC cell lines, respectively. An a.c. magnetic field with a strength of 50 Oe and a near-d.c. frequency of 100 Hz was sufficient to enable release of a therapeutic load off MENs. All the cancer cell lines under study showed membrane penetration threshold d.c. fields at least a factor of ten smaller compared to their normal counterparts.

Conclusion: MENs displayed unique capabilities for externally controlled high-specificity targeted anticancer drug delivery and release on demand via application of d.c. and a.c. magnetic fields, respectively. Because MENs rely on a physical mechanism rather than antibody-mediated delivery, they can be used for high specificity delivery and high-efficacy controlled release of a broad range of therapeutic loads including drugs, peptides, and RNAs to treat many different cancers.

#2200

Dual carfilzomib and doxorubicin carrying nanoparticles for synergistic efficacy in multiple myeloma.

Basar Bilgicer, Tanyel Kiziltepe, David Omstead. _University of Notre Dame, Notre Dame, IN_.

We describe the synthesis and analysis of dual carfilzomib and doxorubicin loaded nanoparticles in their ability to deliver both drugs to multiple myeloma tumor cells at their optimal synergistic ratio. First, to identify the optimal synergistic ratio, various molar ratios of carfilzomib to doxorubicin were screened against multiple myeloma cell lines using the Chou-Talalay method. Both therapeutic agents were then incorporated into liposomes at the identified optimal synergistic ratio of 1:1 to achieve dual drug loaded nanoparticles with a narrow size distribution of ~100 nm and with high reproducibility. Our results established that the dual drug loaded nanoparticles exhibited synergy in vitro and were more efficacious in inhibiting tumor growth in vivo than a combination of free drugs, while at the same time reducing systemic toxicity. In conclusion, this study achieved the preclinical evaluation of dual drug loaded liposomes containing carfilzomib and doxorubicin for enhanced therapeutic efficacy to improve patient outcome in multiple myeloma.

#2201

**Downregulation of NF-** k **B by nanoconjugates to overcome drug resistance in ovarian tumors.**

Ajit P. Zambre,1 Sarjukumar Panchal,1 Alyssa Worland,1 Sarah Chapman,2 Matthew Leevy,2 Anandhi Upendran,1 Raghuraman Kannan1. 1 _Univ. of Missouri, Columbia, MO;_ 2 _University of Notre Dame, Notre Dame, IN_.

Ovarian cancer is the fifth leading cause of cancer deaths in women. Globally, around 140k women die from ovarian cancer per year. Recurrence can lead to development of resistance to first line agents such as platinum analogs, taxanes, and doxorubicin. Several studies have shown that decrease of NF-κB expression will inhibit P-glycoprotein maturation and thereby overcome drug resistance. In this context, it is well known that Disulfiram (DSF), a FDA approved drug for anti-alcoholism, have been identified as moderate inhibitor of NF-κB activity. It is also shown that DSF possess mild anti-cancer effects via increased permeability of the outer membrane of mitochondria resulting in leakage of proteins, such as cytochrome C, generation of ROS that simulates the ROS-MAPK pathway leading to apoptosis, and inhibition of P-glycoprotein. As such DSF is not a strong therapeutic agent; however, in combination with copper it showed excellent therapeutic benefit. This effect is mainly attributed to metabolite diethyldithio-carbamate -a proteasome inhibitor that was produced upon complexation of DSF with copper. However, delivery of DSF-Cu complex was not successful in clinical setting. We hypothesized gold nanoparticles can serve as potential delivery vehicle to selectively transfer Cu-DSF to turmor without toxicity. Indeed, cellular studies in drug sensitive (OVCAR) and Adriamycin resistant p-glycoprotein expressing (NCI-ADR/RES) ovarian cancer cells indicated that the gold nanoconjugate exhibit significant cytotoxicity and the IC-50 values was decreased 2.5 fold times compared to the Cu-DSF complex. NF-κB expression was decreased by two folds compared to native Cu-DSF complex. Detailed cellular and mechanistic studies along with the evaluation of [Au(DSF-Cu)NP] in human multi-drug resistant (NCI-ADR/RES) mouse xenografts will be presented.

### Proteomics and Mass Spectrometry 2

#2202

FABP5 in urinary extracellular vesicles is a potential biomarker of high Gleason score prostate cancer.

Kazutoshi Fujita,1 Hideaki Kume,2 Kyosuke Matsuzaki,1 Atsunari Kawashima,1 Takeshi Ujike,1 Akira Nagahara,1 Motohide Uemura,1 Yasushi Miyagawa,1 Takeshi Tomonaga,3 Norio Nonomura1. 1 _Osaka Univ. Graduate School of Medicine, Osaka, Japan;_ 2 _Laboratory of Proteome Research, National Institute of Biomedical Innovation, Health and Nutrition, Ibaraki, Japan;_ 3 _National Institute of Biomedical Innovation, Health and Nutrition, Ibaraki, Japan_.

Urine collected after prostate massage is a promising source of new biomarkers of prostate cancer, and was reported to contain extracellular vesicles (EVs) that are secreted from prostate cancer cells. EVs are microvesicles secreted from various cell types. EVs contain microRNAs, proteins, and mRNAs and play a role in intercellular communications via the mechanisms of exocytosis and endocytosis. We aimed to discover a new biomarker for high Gleason score (GS) prostate cancer (PCa) in urinary EVs via quantitative proteomics.

Material and methods: Urine samples after massage were collected from 18 patients before prostate needle biopsy. Six patients had negative biopsy outcomes, 6 had Gleason score 6 prostate cancer (GS6), and 6 had Gleason score 8 to 9 prostate cancer (GS8-9). Urinary EVs were isolated from urine with ultracentrifuge. Protein extracted from EVs were labeled with 4-plex iTRAQ and liquid chromatography tandem mass spectrometry (LC-MS/MS) was used for proteome analysis. The candidate proteins were quantitated in the independent set by selected reaction monitoring/multiple reaction monitoring analysis (SRM/MRM).

Results: Seven proteins increased in patients with PCa compared to those with negative biopsy (ratio > 2.0, p-value < 0.05). Thirteen proteins were chosen for further analysis and verified in 29 independent urine samples (negative [n = 11], PCa [n = 18]) using selected reaction monitoring/multiple reaction monitoring. Among these candidate markers, fatty acid binding protein 5 (FABP5) was higher in the cancer group than in the negative group (p-value = 0.009) and was significantly associated with GS (p-value for trend = 0.011).

Conclusions: We applied the proteomic analysis to discover biomarkers in EVs in urine collected after prostate massage. FABP5 in urinary EVs could be a potential biomarker of high GS prostate cancer. Additional large-scale studies are warranted to confirm this finding.

#2203

A SRM/MRM based targeted proteomics strategy for absolute quantification of potential biomarkers of TKI sensitivity in EGFR mutated lung adenocarcinoma.

Shivangi Awasthi,1 Xu Zhang,1 Tapan Maity,1 Benjamin L. Oyler,2 David R. Goodlett,3 Udayan Guha1. 1 _Thoracic & Gastrointestinal Oncology Branch, Center for Cancer Research, NCI, Bethesda, MD; _2 _Institute of Marine and Environmental Technology, University of Maryland Center for Environmental Science, Baltimore, MD;_ 3 _School of Pharmacy, University of Maryland, Baltimore, MD_.

Lung cancer remains the leading cause of cancer-related deaths worldwide. Epidermal growth factor receptor(EGFR) is the primary driver oncogene in 10-30% lung adenocarcinoma. Kinase domain mutations in EGFR (L858R and E746-A750 deletion) provide sensitivity to EGFR-specific tyrosine kinase inhibitors (TKIs). Despite good tumor inhibitory effects, after approximately a year, patients acquire resistance to EGFR TKIs. We have carried out a stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative mass spectrometry (MS) on a Thermo Orbitrap Elite to identify and characterize the dynamics of tyrosine phosphorylation upon TKI treatment of human lung adenocarcinoma cell lines. The degree of inhibition of phosphorylation of several phospho-sites correlated with the extent of TKI-sensitivity. Several kinases and adaptor proteins that were significantly differentially phosphorylated include AHNAK-Y160, -Y715, STAT5A-Y694, DAPP1-Y139, ERRFI1-Y394, NEDD9-Y164, NF1-Y2579, INNPL1-Y1135, suggesting that these are a subset of potential biomarkers of TKI response. To corroborate our results, the next logical step is to carry out clinical validation of these phospho-sites in human lung adenocarcinoma patients, however no absolute quantification assay is currently available for these tyrosine phosphorylated peptides in human samples. The MS method most often used to complement shotgun methods is selected/multiple reaction monitoring (SRM/MRM) because of its high sensitivity, speed and dynamic range. We have performed SRM/MRM-MS using a nano-flow high performance liquid chromatography instrument coupled to a nano-electrospray ionization source attached to a Triple Quadrupole MS (Agilent 6495). In this method, digested proteins are chromatographically separated and ionized. The resulting precursor ions are m/z filtered in the first quadrupole(Q1) and collision induced dissociation is carried out in Q2 and the resultant product ions are m/z filtered in Q3. Each precursor-product pair is termed as a transition. For absolute quantification, heavy isotope labelled versions of the target peptides are spiked in biological samples to build a calibration curve. For the robust assay development, we have carried out the initial empirical refinement process using the heavy labelled versions of the target peptides, including selection of optimal transitions, normalizing retention times of peptides, running peptide stability assays and digestion time course experiments. We are currently validating these assays in human lung adenocarcinoma cell lines with known EGFR mutations with or without treatment with 1st and 3rd generation EGFR TKIs. The overall goal of these targeted studies is to develop absolute quantitation assays to precisely and accurately quantify these potential biomarkers in clinical samples.

#2204

A proteomic landscape of diffuse-type gastric cancer.

Sai Ge,1 Xia Xia,2 Chen Ding,3 Bei Zhen,2 Quan Zhou,2 Jinwen Feng,4 Jiajia Yuan,1 Rui Chen,5 Yumei Li,5 Zhongqi Ge,5 Jiafu Ji,1 Lianhai Zhang,1 Jiayuan Wang,1 Zhongwu Li,1 Yumei Lai,1 Ying Hu,1 Yanyan Li,1 Yilin Li,1 Jing Gao,1 Lin Chen,6 Jianming Xu,7 Chunchao Zhang,5 Sung Yun Jung,5 Mingwei Liu,2 Lei Song,2 Wanlin Liu,2 Gaigai Guo,2 Tongqing Gong,2 Yin Huang,2 Yang Qiu,2 Tieliu Shi,4 Weimin Zhu,2 Yi Wang,5 Fuchu He,2 Lin Shen,1 Jun Qin,2 CNHPP. 1 _Peking University Cancer Hospital & Institute, Beijing, China; _2 _National Center for Protein Sciences (The PHOENIX center), Beijing, China;_ 3 _Fudan University, Shanghai, China;_ 4 _East China Normal University, Shanghai, China;_ 5 _Baylor College of Medicine, Houston, TX;_ 6 _General Hospital of Chinese People's Liberation Army, Beijing, China;_ 7 _Affiliated Hospital of Academy of Military Medical Sciences, Beijing, China_.

Gastric cancer is a heterogeneous disease characterized by poor clinical outcomes and limited targeted treatment options. Among them, diffuse-type gastric cancer (DGC) is the subtype with worst prognosis. Here we describe the first proteomic landscape of DGC. We carried out proteome profiling and targeted exome DNA sequencing of 84 DGC samples. We analyzed the 1,008 (168 x 6) raw files together for uniformed quality control and protein identification with 1% global protein false discovery rate (FDR), which resulted in the identification of 11,340 gene products (GPs). A SAM (significance analysis of microarray) analysis identified 1,641 proteins as differentially expressed between T (tumor) and N (nearby) with statistical significance (FDR q value<0.01 by SAM and differential expression ratio>0.5/ < -0.5), including 1,211 up-regulated and 430 down-regulated GPs. Gene Ontology annotation indicated that tumor proteomes were significantly enriched in cell cycle, DNA replication, checkpoint, E2F, WNT, p53 signaling, epithelial mesenchymal transition (EMT), and inflammation/cytokine-receptor interaction pathways, and the proteomes of the nearby tissues are enriched in metabolism pathways, such as fatty acid metabolism, oxidative phosphorylation, and amino acid metabolism. Notably, many gastric makers (ANXA10, VSIG1, CLDN18, CTSE, TFF2, MUC5AC and MUC6) and signature proteins for stomach functions, including digestion, absorption, secretion, and stomach acid generation (PGC, GIF, GAST, and ATP4A), were lost in tumors. Based on proteome profiling alone, DGC can be subtyped into 3 major classes (PX1-3) that exhibit distinct proteome features and correlate with distinct clinical outcomes (Gehan-Breslow-Wilcoxon P = 0.024). PX1 exhibits proteome stability and the best overall survival; PX2 exhibits dysregulation in DNA replication and cell cycle, and is most sensitive to chemotherapy; PX3 features hyper-activated immune response and is not responsive to chemotherapy. We identified seven-marker proteins that can stratify DGC patients into these three subtypes, opening a door for proteome subtyping in clinical application and intervention. Furthermore, we nominated drug target candidates taking into consideration both the altered DGC proteome and association data with patients' overall survival. This study revealed the altered signaling pathways in DGC and demonstrated the advantage of proteomic approach in molecular subtyping of cancer.

#2205

Identification of potential biomarker related to EGFR mutation by functional proteome profiling in primary non-small cell lung cancer.

Yuan-Ling Hsu, Szu-Hua Pan. _National Taiwan Univ., Taipei, Taiwan_.

Non-small-cell lung cancer (NSCLC) accounts for approximately 85% of lung cancers which the leading cause of cancer-related death worldwide. Recently, epidermal growth factor receptor (EGFR) activating mutations have been proved relevant to NSCLC and let treatment of EGFR tyrosine kinase inhibitors (EGFR-TKIs) become an alternative therapy for NSCLC patients. Despite excellent clinical response to EGFR-TKIs, the intrinsic and acquired resistance to EGFR-TKI created challenges in clinical practice. Although several mechanisms may contribute to solve EGFR‐TKIs resistance, there still have 20% to 30% of these patients that how the resistance developed remain unclear. Studies indicated that the increment of membrane efflux may be associated with drug resistance. In this study, we use differential membrane proteomic analysis to identify potential biomarkers for therapeutics of EGFR-TIKs resistance in NSCLC patients.Several primary NSCLC cell lines with different EGFR status were be isolated by our laboratory. Identification of modulators on membrane of primary cancer cells with different EGFR status was analyzed by membrane proteomic assay using LC-MS/ MS. The expression patterns of EGFR mutation cells would compare with that of wild type groups and the potential candidates would be selected by cross the results of interactome profiles of EGFR mutation cell lines. The expression patterns and functional role of these candidates were be examined in NSCLC cells by several molecular, cellular and biochemical analysis; and demonstrated the clinical relevance of these specific targets with EGFR mutation. The expressions of 100 modulators up-regulated in EGFR mutation cells compared with that of wild type group. After cross reacted with the 474 interaction proteins of mutated EGFR, 8 proteins were selected as potential candidates that related to EGFR-TKI resistance, especially the expression of a protein we called SHX in EGFR mutation cells. We demonstrated the real expression levels of SHX in different lung cancer cells, and performed network analysis of SHX and EGFR in translational level.The analysis of differential membrane proteomic found several novelbiomarkers and potential regulation mechanisms that related to the development of EGFR-TKIs resistance in NSCLC. These identification may provide us new direction for exploiting therapeutic strategy for NSCLC treatment in the future.

#2206

Proteome phenotype of stage III metastatic melanoma and response to MEK inhibition.

Christoph Krisp,1 Robert Parker,1 Dana Pascovici,1 James Wilmott,2 John F. Thompson,2 Graham J. Mann,2 Richard A. Scolyer,2 Nicholas K. Hayward,3 Mark P. Molloy1. 1 _Macquarie University, Sydney, Australia;_ 2 _University of Sydney, Sydney, Australia;_ 3 _QIMR Berghofer, Brisbane, Australia_.

Introduction: Melanoma accounts for 5% of all skin cancers, yet is the most common cause of skin cancer-related deaths, and is the commonest lethal malignancy in young people (<40yrs). Patients with BRAF mutant melanoma show survival benefit from MAPK pathway inhibition, although response rates vary. We conducted proteomic screening using mass spectrometry to detect cellular processes that might predict response to MEK inhibition more effectively than genotyping alone.

Methods: Protein extracts from early passages of ten AJCC Stage III local lymph node metastatic melanoma cell lines with known MAPK mutational status (BRAFmut, NRASmut, MAPKwt,) were used. Further, 32 fresh frozen NRASmut or BRAFmut AJCC Stage IIIC metastatic melanoma (lymph node metastases) specimens were obtained from patients with varying survival. Lysed cells and tissue were digested with trypsin and analysed by LC-MS/MS using a TripleTOF 5600/6600 mass spectrometer. Data-independent acquisition (or SWATH-MS) was used to match multiplexed MS/MS spectra against a spectral ion library enabling relative protein quantification. Cell line viability in the presence of 2µM AZD6244 (MEKi) was determined using a PrestoBlue assay.

Results: The SWATH-MS approach enabled quantification of ~2500 proteins across all 10 cell lines. Principal component analysis clearly segregated melanomas based on in vitro sensitivity to MEK inhibition, whereas genotype alone did not. In total, we show 57 proteins whose abundances correlate (r²>0.75) with response to MEKi, revealing roles in cell pigmentation biosynthesis, lipid metabolism, adherence and inter-cell communication. Survival analysis demonstrated that patients whose cell lines were responsive to MEKi had lower mortality than patients whose cells were resistant to MEKi (survival >7.5 years MEKi sensitive versus <1.7 years MEKi resistant, p=0.01). Across the 32 tumour samples, ~2000 proteins were quantifiable. The proteome phenotype associated with MEK inhibition observed in patient cell lines was a dominant signal in fresh frozen stage III metastatic melanoma specimens, suggesting these patients may have profited from MAPK pathway inhibition had these drugs been available. In this patient cohort neither the genotype nor proteome phenotype correlated with survival.

Conclusion: Proteomic profiling by mass spectrometry demonstrated a MEK inhibition phenotype in early passage cell lines and fresh frozen melanoma metastases which may provide utility for selecting patients to treat with MEK drugs.

#2207

HSP90 inhibition leads to a differential proteomics profile in non small cell lung cancer cell lines.

Angela Marrugal,1 Irene Ferrer,1 Maria Dolores Pastor,2 Alvaro Quintanal,1 Antonio Lucena-Cacace,2 Amancio Carnero,2 Luis Paz-Ares,1 Sonia Molina-Pinelo1. 1 _CNIO, Madrid, Spain;_ 2 _IBIS, Sevilla, Spain_.

Background: The cancer chaperone, Heat Shock Protein 90 (HSP90), is known for its ability to regulate the stability of different oncogenic proteins. Thus, its overexpression has been related to unfavorable prognosis in some types of tumors. EGFR and EML4-ALK, two of the most important drivers in non-small cell lung cancer (NSCLC), are HSP90 clients and extremely depend on it. As a consequence, this chaperone is especially important in NSCLC hence HSP90 inhibition shows a lot of possibilities to future treatments in this tumor type. Nevertheless, to obtain a successful clinical development, will be essential supporting evidence of inhibitory efficacy in several molecularly defined subgroups of NSCLC.

Methods: NSCLC cell lines carrying different gene mutations, whose direct (HCC827: EGFR mutated and H3122: EML4-ALK rearrangement) and indirect (A549: KRAS mutated) relationship with HSP90 has been reported, were used. In these cell lines, along with H1781 (EGFR, KRAS, ALK wild type) as control, the activity of the chaperone studied was interrupted. To this end, pharmacological inhibition of HSP90 was achieved through geldanamycin and resorcinol derivates. First, western blotting was carried out to confirm the effect of this inhibition. Later, to identify a proteomic profile associated with HSP90 inhibition, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of selected cell lines were performed.

Results: The expression of the oncogenic HSP90 client proteins studied was decreased by the inhibitors in the NSCLC cell lines. The oncoproteins drivers EGFR and EML4-ALK showed a strong dependence on HSP90 observed through a high sensitivity of the cell lines HCC827 and H3122 to the inhibition. Therefore, these cell lines were selected with the purpose of identify a proteomic signature linked to HSP90 inhibition by 2D-PAGE. The untreated EGFR positive cell line presented 104 protein spots significantly up-regulated, whereas 80 spots were down-regulated compared to inhibited cell line. Meanwhile, the cell line harboring the EML4-ALK translocation showed 16 spots up-regulated and 5 down-regulated in the untreated versus inhibited cell line. In addition HSP70 overexpression, compared to unprocessed cell lines, was detected after treatments. This feedback, previously reported, confirmed HSP90 inhibition in the two cell lines studied.

Conclusions: The evidence of treatment response, in the cell lines studied, was showed through oncogenic client proteins reduction as well as HSP70 induction. The proteomic profiles identified, of HSP90 inhibited and untreated tumor cells, revealed several deregulated pathways involved in the tumorigenesis.

#2208

Peptide-mediated 'miniprep' isolation of extracellular vesicles is suitable for high-throughput proteomics; method evaluation and application in colon cancer.

Meike De Wit,1 Jaco Knol,2 Inge de Reus,2 Tim Tim Schelfhorst,2 Logan Bishop-Currey,2 Nicole van Grieken,2 Sander Piersma,2 Thang V. Pham,2 Remond J. Fijneman,1 Gerrit A. Meijer,1 Henk Verheul,2 Connie R. Jimenez2. 1 _Netherlands Cancer Institute, Amsterdam, Netherlands;_ 2 _VU University Medical Center, Amsterdam, Netherlands_.

Objective:

Extracellular vesicles (EVs) are cell-secreted membrane vesicles enclosed by a lipid bilayer derived from endosomes or from the plasma membrane. Since they are released into body fluids, and their cargo includes tissue-specific and disease-related molecules, EVs represent a rich source for disease biomarkers. However, standard ultracentrifugation methods for EV isolation (UC-EV) are laborious, time-consuming, and require high inputs.

Method:

A recently described isolation method, which can be performed at small 'miniprep' scale, utilizes specific Heat Shock Protein (HSP)-binding peptides to aggregate HSP-decorated EVs (Ghosh et al. (2014), PLoS ONE 9:e110443). The authors showed comparable results for their method (abbreviated HSP-EV here) and UC-EV, but a detailed proteomic comparison was lacking. Therefore, we compared both methods using label-free proteomics of replicate EV isolations from HT-29 cancer cell-conditioned medium. Subsequently we applied this technique on secretomes of fresh human colorectal cancer (CRC) (n=17) and colon adenoma (n=4) tissue as well as patient-matched normal colon tissue.

Results:

Despite a 30-fold different input scale (UC-EV: 60 ml versus HSP-EV: 2 ml), both methods yielded comparable numbers of identified proteins (3115 versus 3085), with reproducible identifications (72.5% versus 75.5%) and spectral count-based quantification (average CV 31% versus 27%). EVs obtained by either method contained established EV markers and proteins linked to vesicle-related gene ontologies. In the EV fraction of the tissue secretomes 6390 proteins were identified, of which 471 proteins were significantly 5-fold more present in CRC samples than in normal tissue EVs. Gene ontology analysis revealed enrichment of nuclear proteins involved in DNA damage response, chromosome organization and RNA processing in the CRC EVs.

Conclusions:

The HSP-EV method provides an advantageous, simple and rapid approach for EV isolation from small amounts of biological samples, enabling high-throughput analysis in a biomarker discovery setting.

#2209

Characterization of FABP5 antibodies in prostate.

Dennis A. Otali,1 Denise K. Oelschlager,1 James Kearns,2 Sandra M. Gaston,2 William E. Grizzle1. 1 _Univ. of Alabama at Birmingham, Birmingham, AL;_ 2 _Tufts Medical Center, Boston, MA_.

FABP5, a member of a family of small proteins (~15 kDa) that transport lipids, is emerging as an important biomarker because it is differentially expressed in prostate cancers (PrCa) compared to uninvolved prostate glands and lipids are important to the progression of prostate cancer. This study was to select an antibody (Ab) for use in immunostaining (IHC) FABP5 in formalin fixed paraffin embedded (FFPE) tissue and also an Ab for use in Western blotting (WB). A mouse monoclonal antibody (mAb) clone A9 to FABP5 from Santa Cruz (SC) Biotechnology, Inc. and a rabbit polyclonal antibody (pAb) to FABP5 from Abcam (AB) (cat. # ab37267) were tested. Six prostate cell lines (PC3, DU145, 22Rv1, MDA PCa 2b, LNCaP, and normal primary prostate epithelial cells (NH Pri Pro) were used to study protein and mRNA levels of FABP5. Protein expression of FABP5 was determined by WB of whole cell lysates as well as cytoplasmic and nuclear lysates. GAPDH, a "housekeeping" gene expressed at similar levels in most types of cells, was used as a loading control for WB. Cells from the six prostate cells lines also were embedded in HistoGel, processed to FFPE and immunostained. The prostate cells were transfected with FABP5 siRNA for WB with aliquots of the transfected prostate cells processed to FFPE. qrtPCR was performed to measure mRNA levels. The expression of whole cell lysates on WB probed with the AB pAb to FABP5 was NH Pri Pro > PC3 > DU145 > 22Rv1 >> MDA PCa 2b > LNCaP compared to NH-Pri Pro > PC3 > DU145 > 22Rv1 when probed with the SC mAb. WB of the cytoplasmic fractions from each cell line has a similar pattern. In HistoGel sections immunostained with AB pAb, the FABP5 cytoplasmic expression was PC3 > DU145 > 22Rv1 > MDA PCa 2b > NH Pri Pro >> LNCaP. These levels of expression among the cell lines followed a similar trend in evaluation of immunostaining of cell membranes, nuclear, and the perinuclear area. In HistoGel sections immunostained with SC mAb, the FABP5 expression was PC3 > DU145 with weak signals in 22Rv1 > MDA PCa 2b > NH Pri Pro >> LNCaP. A recombinant blocking peptide (RBP) to FABP5 strongly inhibited the immunostaining with the antibodies to FABP5 in FFPE sections from archival radical prostatectomy specimens. This RBP is the same sequence as the peptide used to produce the AB antibody. No RBP to FABP5 was available from SC. Incubation with the RBP strongly inhibited the extent of immunostaining with FABP5 SC mAb. Similarly, immunostaining of the 6 cell lines with the AB pAb following incubation with RBP strongly inhibited staining. In analysis of mRNA levels, the expression of FABP5 was PC3 > 22Rv1 > > DU145 > MDA PCa 2b >> LNCaP. We demonstrated by siRNA and the use of a RBP that both these antibodies are specific and sensitive for detecting FABP5 by immunostaining but the AB Ab is more useful for WB. These results are presented with the IHC of FABP5 in normal and uninvolved prostate glands in PrCa.

#2210

Scavenging for lethal prostate cancer biomarkers in FFPE tissue.

Håkon Ramberg,1 Peder R. Braadland,2 Maria E. Stensland,3 Mehrdad Rakaee,4 Ståle Nygård,3 Olov Øgren,1 Viktor Berge,1 Aud Svindland,2 Elin Richardsson,5 Gustavo A. de Souza,3 Sigve Andersen,5 Kristin A. Taskén2. 1 _Oslo University Hospital, Oslo, Norway;_ 2 _Oslo University Hospital and University of Oslo, Oslo, Norway;_ 3 _University of Oslo, Oslo, Norway;_ 4 _University of Tromsø, Tromsø, Norway;_ 5 _University Hospital of North Norway and University of Tromsø, Tromsø, Norway_.

The aim of this study was to identify protein biomarkers that can be used to stratify indolent and aggressive prostate cancer using formalin-fixed paraffin-embedded tissue (FFPE) from radical prostatectomy specimens. The protein expression profiles of benign and malign samples from 23 patients, who had either died of prostate cancer or were alive 10 years after diagnosis, were determined using an Easy nLC1000 Nano-LC system connected to a quadrupole-Orbitrap (Q Exactive) mass spectrometer equipped with a nanoelectrospray ion source. The generated MS raw files were submitted to MaxQuant software for protein identification. Differential expression of LFQ-normalized intensities was tested by Cox proportional hazards modeling and LASSO (least absolute shrinkage and selection operator) regression analysis within the R software environment. The label free Orbitrap analysis identified 3523 unique proteins from the FFPE tissue samples. LASSO of protein expression profiles of indolent and aggressive prostate cancer tissue samples identified ten proteins which could stratify the groups. Seven of the proteins were significantly correlated with time to prostate cancer specific mortality in Cox proportional hazards modeling (p-value < 0.05). The selected proteins were found to be linked to NFkappaB signaling, cell structure, translation and metabolic pathways. Finally, proteins capable of distinguishing benign from malignant prostate tissue were identified by LASSO, and 22 of these were significantly different between the groups (Bonferroni-adjusted p-value < 0.05). Selected candidate biomarkers are currently being validated by immunohistochemical analyses of 535 radical prostatectomy specimens from two independent Norwegian cohorts with long follow-up time.

#2211

Characterization of monoclonal antibodies specific for HPV-positive head and neck cancer.

Hsuan-Chen Liu,1 Falguni Parikh,1 Thomas Kraus,2 Thomas Moran,2 Andrew Sikora1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Icahn School of Medicine at Mount Sinai, New York, NY_.

HPV-driven head and neck squamous cell carcinoma (HNSCC) are among the fastest growing cancers. Although, HPV-HNSCC patients have an overall favorable prognosis, a significant number of patients relapse post-treatment, and currently there is no specific therapeutic approach targeting the unique biology of HPV-driven HNSCC. Our ongoing efforts to target HPV-HNSCC focus on the identification of cell surface antigens that are upregulated by HPV infection. We have developed an "antigen-agnostic" approach for generating HPV-HNSCC-targeting monoclonal antibodies for cancer diagnosis and treatment that does not rely on prior identification of target antigens. We used HPV-HNSCC membrane fractions to immunize recipient mice and generated HPV-specific hybridomas. We then screened five thousand hybridoma colonies by flow cytometry to test the specificity of binding to HPV-positive cancer cell lines (2HNSCC and 2 Cervical Cancer) and HPV-negative cancer cell lines (4 HNSCC and 1 CC). After primary screening, we narrowed down to forty-four clones; among these hybridoma clones, 6D8 and 6B3 bound preferentially to HPV-positive cancer cell lines. The binding targets of 6D8 and 6B3 were identified by immunoprecipitation and mass spectrometry; their targets are integrin alpha6 (ITGA6) and tissue factor (F3) respectively. Future work will validate the biological function of these mAbs in in vitro and in vivo models, and continue identifying more binding target of mAbs. We propose mAbs specifically targeting membrane-expressed antigens on HPV-related cancer cells as a potential approach for early diagnosis and targeted therapy.

#2212

Protein engineering of the decapping enzyme NudT16 enhances its ability to hydrolyze protein-conjugated ADP-ribose for post-translational site identification via mass spectrometry.

Puchong Thirawatananond, Robert L. McPherson, Jasmine K. Malhi, Anthony K. Leung, Sandra B. Gabelli. _Johns Hopkins University, Baltimore, MD_.

During nonhomologous end joining of DNA follow double-stranded breaks, one common signaling response is the adenosine diphosphate ribosylation (ADP-ribosylation) of recruiter scaffold proteins. ADP-ribosylation is a post-translational modification (PTM) that occurs on aspartate, glutamate, lysine, arginine and cysteine on proteins and is mediated chiefly by polyadenosine ribose polymerases (PARP). Using NAD+ as its substrate, PARP transfers an ADP-ribose onto another protein, releasing a nicotinamide ring in the process; this modification presents as a mono-ADP-ribosylation (MARylation) or poly-ADP-ribosylation (PARylation). While site identification of MARylated protein can be achieved through tandem MS/MS and searching for peptides whose spectra have been shifted by 541.0611 Da, a similar approach for PARylated protein is confounded by the variable length and branching of its PTM. To this end, ADP-ribosylated protein is normally incubated with a snake venom phosphodiesterase (SVP) to leave a standardized phosphoribose tag of 212.0086 Da. It has been shown that a recombinantly-expressed Nudix protein NudT16 provides a cost-effective alternative to SVP to process ADP-ribosylated protein. NudT16 in the cell normally decaps small nucleolar RNA. Our studies here sought to utilize the x-ray crystallographic structure of NudT16 in complex with ADP-ribose in guiding rational design mutations to enhance binding of our desired ADP-ribose substrate and to widen the hydrolase's active site to better accommodate a protein-conjugated ADP-ribose. Mutants have demonstrated improved catalytic efficiency for ADP-ribose hydrolysis. Radiolabeled immunoassays of PARylated protein concur with these kinetic experiments by showing a reduction of radiolabeled PARP. Rational design of a ADP-ribose-hydrolyzing NudT16 with comparable catalytic efficiency to SVP eases lead discovery of biological mechanisms that are mediated through ADP-ribosylation such as DNA repair.

#2213

Aberrant glycoprotein expression in recurrent and non-recurrent prostate cancer tissue.

Sarah M. Totten, Cheylene Tanimoto, Abel Bermudez, Amy Hembree, James D. Brooks, Sharon J. Pitteri. _Stanford University School of Medicine, Palo Alto, CA_.

The primary aim of this study is to identify and quantify aberrancies in glycosylation patterns and glycoprotein levels expressed in high grade prostate cancer tissue from men with and without recurrence following radical prostatectomy using multi-dimensional chromatography and tandem mass spectrometry techniques. Glycosylation is a common and highly complex posttranslational modification, and is linked to protein function. Elucidating specific glycan structures is key to understanding the underlying role glycosylation plays in regulating cellular activity, including tumorigenesis, invasion, and metastasis. Aberrant glycosylation is an emerging hallmark of various cancers, demonstrating that alterations in glycosylation disrupt cellular behavior in key pathways. Furthermore, comprehensive glycan characterization can lead to more effective biomarkers with increased clinical utility for distinguishing indolent disease from cancers that are more likely to become metastatic, recur and/or pose higher risk.

In this study, glycoproteomic analysis was performed on prostate cancer tissue and matched normal prostate tissue from ten men with high grade prostate cancer (Gleason 7/8) - five of which experienced recurrence. Proteins were extracted from tissue lysates, denatured, reduced and alkylated. Isotopic labels for quantitation were incorporated during alkylation - normal and cancer tissue was labeled with 12C and 13C acrylamide respectively. Paired normal/cancer tissues were combined. Glycosylated proteins from each pair were separated by multi-lectin chromatography designed to capture sialylated, core-fucosylated, and highly branched complex glycans, fractionating the complex mixture into four discrete fractions containing specific glycoforms. LC-MS/MS was used to analyze the tryptic digest from each fraction for protein identification and quantitation.

This experimental design reveals differences between the glycosylation patterns and protein levels of cancerous and normal tissue in ten men, but also allows for the comparison between those with recurrent cancers. The glycosylation patterns across hundreds of prostate tissue proteins were systematically screened, enabling the detection and relative quantitation of specific glycoforms of proteins that may be dysregulated in prostate cancer. Preliminary results reveal the identification of 6,202 unique proteins, and quantitation of 2,894 proteins, most of which contained sialylated glycoforms. In all patients (regardless of treatment outcome), several hundred proteins had elevated total protein levels in the 13C-labeled cancer tissue compared to the 12C-labeled normal tissue proteins, such as TACSTD2 and LPP, while other proteins demonstrated upregulation only among certain glycoforms. Other glycoproteins were up-regulated in cancer only in recurrent patients, including CPNE1, TXND5, and CASC4.

#2214

Adaptive dynamic artificial polyligand targeting (ADAPT): a method to identify exosomal proteins from a prostate cancer cell line.

Tassilo Hornung, Stephen Logie, Aniket S. Bondre, Varun Maher, Melissa N. Richards, Jelena Zarkovic, Teresa T. Tinder, Heather A. O'Neill, Mark R. Miglarese, David B. Spetzler. _Caris Life Sciences, Phoenix, AZ_.

In the recent years it was demonstrated that a multitude of body fluids contains substantial amounts of exosomes, extracellular vesicles with sizes ranging between 40 and 100 nm. Those vesicles have protein profiles characteristic of their cells of origin. It was shown that exosomes play a role in cell-to-cell communication making them attractive targets to identify early disease stage biomarkers. Cancer heterogeneity is known for a long time to be an important clinical determinant of patient outcome. We developed the highly multiplexed ADAPT platform to capture systems-based biological signatures that may reflect the molecular heterogeneity of various cancer types and help to improve diagnosis of the disease. Exosomes from two prostate cancer cell lines, VCaP and LNCaP, were used to train ssDNA libraries to discriminate them. A highly diverse library of 1012 oligonucleotides (ODNs) was subjected to five rounds of positive and negative selection against exosomes from VCaP and LNCaP prostate cancer cell lines. Individual ODNs that bound preferably to exosomes from VCaP cells were identified by NGS, resynthesized and binding of co-precipitated ODNs to VCaP exosomes was verified by qPCR. LC-MS/MS was used to identify binding partners of ODNs bound to VCaP exosomes. Several of those binding partners (CHMP1b/2a/4b, VPS28, Syntenin-1) were found to be part of the ESCRT machinery, which participates in exosomes biogenesis. It was found that those proteins are overexpressed in human cancers. In addition, we identified the chemokine I-TAC, which is overexpressed in blood and tissue of men with advanced prostate adenocarcinomas. Finally, we found hnRNP-1, a cancer associated splicing factor, and the cold shock proteins RNPL and A18 hnRNP. Knock-down of these cold shock proteins has been shown to enhance chemotherapeutic cell killing of prostate cells. ADAPT is an unbiased profiling platform that identifies cancer associated proteins expressed on exosomes. This platform can be deployed against multiple cancer types and offers broad potential applications in biomarker discovery.

#2215

Detection of novel markers of transitional cell carcinoma of the ovary, the TCC-like variant of high grade serous carcinoma, using proteomics and immunohistochemistry.

Basile Tessier-Cloutier,1 Jamie Magrill,2 Stefan Kommoss,3 Blake C. Gilks,1 David G. Huntsman,2 Dawn R. Cochrane,2 Aline Talhouk,2 Robert Soslow,4 Gregg B. Morin,2 Chris J. Hughes,2 Anthony N. Karnezis,2 Christine Chow,1 Angela S. Cheng,1 Andreas du Bois,5 Jacobus Pfisterer,6 Friedrich Kommoss1. 1 _Vancouver General Hospital, Vancouver, British Columbia, Canada;_ 2 _BC Cancer Agency, Vancouver, British Columbia, Canada;_ 3 _Tübingen University Hospital, Tübingen, Germany;_ 4 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 5 _Kliniken Essen Mitte, Vancouver, Germany;_ 6 _Gynecologic Oncology Center, Kiel, Germany_.

Background: The current WHO classification does not separate transitional carcinoma of the ovary (TCCO) from conventional high grade serous carcinoma of the ovary (HGSC). TCCO has a better prognosis, possibly due to better chemosensitivity or less infiltrative growth pattern. The available immunohistochemical (IHC) markers do not differentiate between the two subtypes. Therefore, we sought to compare the proteomic profiles of conventional HGSC and TCCO to identify surrogate biomarkers of good prognosis from TCCO that could identify conventional HGSC tumors with a better prognosis.

Design: Full proteome analysis of 12 cases of TCCO and 12 cases of HGSC was performed using SP3-clinical proteomics, run on an ThermoFisher Orbitrap Fusion. For validation, tissue microarrays of TCCO (n=89) and HGSC (n=237) were immunostained with antibodies against proteins found to be enriched in TCCO. All cases and immunostains were scored by a gynecologic pathologist. Univariate analysis was performed comparing IHC expression in TCCO vs. HGSC.

Results: We identified 1220 proteins that were significantly enriched in TCCO over HGSC. Claudin 4 and Ubiquitin carboxyl-terminal esterase L1 (UCHL1) were selected as potential markers of TCCO-like biology (p=0.0017, 0.0322). By IHC, Claudin 4 (95% confidence interval (CI) 0.171, 0.430) and UCHL1 (95% CI 0.291, 0.550) showed a significantly higher expression in TCCO as compared to HGSC (see table). | |

---|---|---

|

% of tumors with high immunoreactivity scores

|

Claudin 4 | UCHL1

Pure TCCO | 34/59 (58%) | 26/59 (44%)

Mixed TCCO-HGSC, TCCO component | 14/29 (48%) | 8/29 (28%)

Mixed TCCO-HGSC, HGSC component | 19/28 (68%) | 6/28 (21%)

Conventional HGSC | 33/235 (14%) | 32/237 (14%)

Legend: Mixed TCCO: TCCO with minor component of conventional HGSC

Conclusion: Proteomic analysis showed differing protein profiles for TCCO and HGSC. By IHC, Claudin 4 and UCHL1 were identified as potential markers for TCC-like differentiation of high-grade serous carcinomas. Further studies will focus on the prognostic significance of these and other markers in larger cohorts of HGSC. This study presents a novel approach at identifying potential diagnostic and prognostic biomarkers as well as therapeutic targets.

#2216

A novel MRM-based mass spectrometry assay to quantify HMGB1.

Dawn Zhaohui Chen, Weston R. Spivia, Yang Gao, Jennifer Van Eyk, Shreya Kanodia. _Cedars-Sinai Medical Center, Los Angeles, CA_.

Background: High mobility group box 1 (HMGB1) is a versatile protein with dual roles. Within the cell, this highly conserved chromosomal protein functions as a DNA chaperone. Outside of the cell, it functions as the prototypical damage-associated molecular pattern. There is significant evidence that HMGB1 dysfunction contributes to cancer development, particularly in mesothelioma, where its role in carcinogenesis is better defined.

Goal: To develop a mass spectrometry based immune-multiple reaction monitoring (iMRM) assay to quantify HMGB1 and its disease-associated post-translationally modified forms.

Methods: Mesothelioma cell lines (Ren, Phi and PP-Mill) known to secrete HMGB1 were used. Concentrated cell line supernatants, anti-HMGB1 antibody-loaded magnetic beads were incubated overnight. Beads were extensively washed and the HMGB1 released from the beads was enzymatically digested prior to targeted MRM mass spectrometry. Known quantity of N15 labeled synthetic peptides were spiked in to be used as internal standards to assess the total protein concentrations as well as the acetylated modified peptides that encompass the Lys residue. To ensure tight reproducibility during sample processing, we developed an automated workflow from enrichment to digestion with % coefficient of variance of less than 20%.

Results: Two multiplex MRM assays were developed for HMGB1: 1) total HMGB1 protein assay based on trypsin digested peptides and 2) Acetylated HMGB1 assay based on Endoproteinase GluC (Staphylococcus aureus Protease V8) digestion to obtain the peptides containing hyper-acetylated Lysine residues, which are not located within a suitable tryptic peptide. Four significant milestones were accomplished. i) We optimized the HMGB1 liquid chromatography and mass spectrometry performance through the utilization of signature synthesized peptides representing the common amino acid sequence and potential acetylated regions. The calibration curves for both peptides based on the ratio of light and heavy isotopic labeled internal standards were achieved. LLOD and LLOQ and the inter and intra-day variability of the iMRM assay have been achieved. ii) A robust immuno-capture protocol was established. The final protocol included isolation of HMGB1 using a capture antibody followed by protein release, denaturation, reduction, alkylation and digestion. To date, we have optimized the conditions for HMGB1 attachment to antibody, antibody coupling to magnetic beads, the wash and elution steps. iii) The protocol was applied to quantify HMGB1 in mesothelioma cell lines. HMGB1 was measured in cytoplasm, nucleus and supernatant from the cell lines. iv) Finally, the assay was adapted to work on human plasma samples.

Conclusion: We successfully developed the iMRM workflow for quantification of total HMGB1 and acetylated HMGB1 based on the measurement of the target signature peptides in HMGB1.

#2217

Detection of circulating immune biomarkers of cervical disease using proteome arrays.

Radwa Ewaisha, Ian Meshay, Jack Resnik, Tirinder Bharaj, Karen S. Anderson. _Arizona State University, Tempe, AZ_.

Introduction: More than 260,000 women die of cervical cancer every year. Screening methods have reduced the incidence of cervical cancer in high-income countries, but detection continues to lag in low and middle income countries (LMICs). IgG antibody (Ab) immunity to early (E) HPV antigens (Ags) are potential biomarkers of disease progression. Since HPV16 accounts for only ~50% of invasive cervical cancers, we developed protein microarrays expressing the proteomes of 12 HPV types to detect host IgG Abs to a broad spectrum of viral Ags to detect pre-invasive and invasive cervical disease.

Methods: We developed custom HPV protein microarrays displaying the proteomes of two low-risk HPV types (HPV6 and 11) and ten oncogenic high-risk HPV types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52 and 58). Arrays were probed with serum samples obtained from women with invasive cervical cancer (ICC; n=80), no or low-grade cervical disease (CIN 0/I; n=60), and high-grade cervical dysplasia (CIN II/III; n=60). To identify positive serologic responses, arrays were scanned and the signal intensity of each protein spot was quantified and normalized. Visual examination of diffused signal (ring) around each spot was performed and the Ab response to each protein was scored on a scale from 0 to 5.

Results: To verify array quality and reproducibility, we confirmed high protein expression levels for 98% (96/98) of the antigens printed and high correlation (R ≥ 0.90) of protein expression signals between different randomly selected arrays. Epitope expression was confirmed using four commercial monoclonal Abs raised against HPV16 Ags. Host Abs to at least one early antigen (E1, E2, E4, E6, or E7) were detected in the sera of 31.2% and 43.3% of ICC and CINII/III patients, respectively, compared with 11.7% of women with CIN 0/I. 73.1% of CINII/III cases detected had Abs only to non-HPV16 Ags. Abs to E1, E2, E4, E6, and E7 Ags were detected in 3.8%, 7.7%, 54%, 19%, and 31% of CINII/III versus 16%, 16%, 32%, 32%, and 40% of ICC cases that were positive. The immunodominant Ags in women with CINII/III and ICC were E4 (54%) and E7 (40%), respectively. These results are consistent with the difference in tissue expression levels of these proteins in these two disease stages. This emphasizes the importance of broadening the scope of serological detection to include non-HPV16 proteomes.

Conclusions: This study demonstrates that Abs against high risk HPV16 and non-HPV16 serotypes are potential biomarkers for both the diagnosis of cervical cancer and the differentiation between high grade and low-grade pre-invasive cervical lesions. These results suggest that serology is a potential minimally invasive tool for early detection of cervical cancer. High-throughput immunoprofiling of HPV-associated cancers and pre-invasive lesions may be informative for understanding the diversity of host immunity and viral progression.

#2218

Quantitative multiplex analysis of immune checkpoint protein expression in circulation and in the tumor microenvironment.

Wen-Rong Lie, Jehangir Mistry. _MilliporeSigma, St. Charles, MO_.

Immune checkpoint inhibitors have been proven to be an effective method in improving antitumor immune response. Many immune checkpoint proteins are expressed as soluble forms in circulation and in the tumor and tumor microenvironment. Here we report the development of bead-based Luminex multiplex assays for the quantitative profiling of co-inhibitory and co-stimulating immune checkpoint proteins CTLA-4, PD-1, TIM-3, LAG-3, HVEM. GITRL, BTLA, CD27, CD28, CD40, GITR, PD-L1, B7-1/CD80, B7-2/CD86, and ICOS. In order to explore the use of soluble immune checkpoint proteins as putative cancer biomarkers, we used these multiplex assays to measure checkpoint protein levels in serum samples from breast cancer patients, colon cancer patients, and a corresponding set of normal serum samples. Analysis of the soluble checkpoint protein signatures generated from this multiplex approach revealed a significantly elevated level of soluble TIM-3 protein in the breast cancer serum samples and in the colon cancer serum samples compared to the healthy serum controls (p<0.001). In addition, we analyzed the immune checkpoint protein expression profiles in lysates of tumor and adjacent normal tissues from 3 patients with metastatic breast cancer and 2 patients with colorectal cancer. Differential expression of multiple checkpoint proteins, including BTLA, CD27, TIM-3, HVEM, CD40, GITR, LAG3, CTLA-4, B7-1, PD-L1, or ICOS, were detected in these matched lysates, indicating the roles and complexity of checkpoint proteins in tumor microenvironment. In summary, our results demonstrate that the multiplex assays we developed are useful research tools for the simultaneously quantitation of immune checkpoint proteins, as well as its potential application in cancer biomarker discovery and translational research in cancer immunotherapy.

## PREVENTION RESEARCH:

### Molecular Targets and Cancer Prevention

#2219

Cancer prevention using a novel fatty acid synthase inhibitor.

Neil L. Spector,1 Tim Haystead,1 Sumin Zhao,1 Yazan Alwarawrah,1 David Alcorta,1 William Kim,2 Jose R. Roques,2 Michael Trinkler,2 David B. Darr2. 1 _Duke University Medical Center, Durham, NC;_ 2 _UNC-Chapel Hill, Chapel Hill, NC_.

Tumor growth and survival is dependent upon de novo fatty acid synthesis regulated via preferential upregulation of fatty acid synthase (FASN) in tumor cells. Here a novel FASN inhibitor that has shown antitumor activity in established tumor models (Alwarawrah Y et al., 2016), now shows promise blocking oncogene-induced cell transformation and significantly delaying time to tumor development in a prevention study using a GEMM of triple negative breast cancer.

Non-malignant, immortalized MCF10A human breast epithelial cells were infected with lentivirus encoding for the highly oncogenic 110 kDa truncated form of HER2, herein referred to as p110, under a doxycycline inducible promoter. Lentivirus containing a sham insert served as a control. Forty-eight hours after lentiviral infection, p110 was induced by dox and cells treated with FASN (2 and 4 μM) or vehicle alone for 21 days, and then assessed for cell growth and evidence of senescence. The C3TAg GEMM model of triple negative breast cancer was used to study FASN in the prevention setting. The mice (FVB/N background) express C(3)SV40 T-antigen resulting in the inactivation of p53 and Rb. Eighteen mice were randomized to receive FASN treatment (15mg/kg/ 5 days per week) or control (non-treatment [NT]). The mice were randomized and started treatment at 6 weeks of age, and continued until tumors reached an average size of 87.5mm3. All mice were monitored (5 times/week) for weight loss and time to tumor development.

Expression of p110 led to the transformation of MCF10A cells, as evidenced by the formation of plaques in cell culture. Treatment with FASN blocked transformation in p110 expressing MCF10A cells and instead induced growth arrest, expression of senescence associated β-gal, and morphologic appearance consistent with cell senescence. Analysis of viable p110 expressing MCF10A cells that were maintained in the continuous presence of FASN for 60 days led to the identification of a potential mechanism of therapeutic resistance. In the C3TAg in vivo prevention study, FASN resulted in a significant prolongation in time to tumor development (Log-rank (Mantel-Cox); p <0.0056). The median days of tumor onset between the NT and treated groups were 111 and 145 with a range of 84-130 and 85-163, respectively. No gross toxicity or weight loss was observed in FASN treated animals.

A novel small molecule FASN inhibitor blocked oncogenic cell transformation and induced a state of senescence. Moreover, FASN significantly delayed the onset of mammary tumors in a GEMM model of aggressive triple negative breast cancer. Identification of a mechanism(s) enabling tumor cells to survive long term FASN inhibition will provide rationale combination strategies to induce permanent senescence or apoptosis, and improve the efficacy of FASN inhibitors as preventative agents.

#2220

Repurposing statins for prevention of triple-negative breast cancer.

Anjana Bhardwaj,1 Harpreet Singh,1 Kimal Rajapakshe,2 Cristian Coarfa,2 Isabelle Bedrosian1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Baylor College of Medicine, Houston, TX_.

Introduction: Although triple negative breast cancer (TNBC) accounts for 15% of the 250,000 annual cases of breast cancer, its more aggressive nature, coupled with lack of targeted therapy, results in a disproportionate rate of mortality in women with this disease underscoring the critical need for prevention-based approaches. We hypothesized that identifying miRNA-mRNA functional pairs and their driver pathway(s) deregulated during preneoplastic progression would provide the most informative insights into the molecular drivers of TNBC carcinogenesis. Methods: We performed next generation RNA sequencing of TNBC like MCF10A based model system and focused on detailing the molecular changes preceding the development of in situ carcinoma (DCIS). We used ki67, MTT and colony formation assays to study the growth inhibitory effect of miRNA and pathway targeting. To identify direct gene targets of a miRNA, we cloned the 3'untranslated region containing miRNA-binding sites from its predicted gene targets in a luciferase reporter vector, and studied the effect of miRNA overexpression on the repression of luciferase reporter activity.

Results: In-silico analyses of small RNA seq data showed loss of miRNA-140-3p as one of the top deregulated miRNA relevant to preneoplastic transition from MCF10A parental to MCF 10AT1 preneoplastic cell line. Staining of breast tissue samples also confirmed loss of miRNA-140-3p in tumors compared to normal breast epithelial cells, suggesting a tumor suppressive role. To investigate the functional role of miRNA-140 during TNBC development, we ectopically expressed miRNA-140 in MCF10.AT1 and MCF10.DCIS cells. Interestingly, these studies revealed miRNA-140 replacement to preferentially inhibit colonizing ability and cell proliferation of preneoplastic MCF10.AT1 cells in contrast to a much weaker cell inhibitory effects in DCIS cells. Next, we identified the functional gene target/s of miRNA-140-3p by integromics studies involving next-generation RNA-seq on the MCF10A model employing bioinformatics pipeline, SigTerms. These analyses identified HMGCR and HMGCS1 as top 2 gene targets of miRNA-140-3p that map in cholesterol biosynthesis pathway. Consistently, we found miRNA-140-3p to directly bind in 3'UTR of HMGCR, HMGCS1 and repress their expression as shown by luciferase assays. miRNA-140 binding of these gene targets is also functionally relevant as inhibition of HMGCR activity by an FDA approved statin inhibitor, fluvastatin, preferentially inhibited the cell proliferation and colonizing ability of preneoplastic MCF10AT1 cells. Further studies focused on testing the potential of fluvastatin to prevent progression of breast atypia cells in mouse xenografts are underway.

Conclusions: Our results suggest miRNA-140 and its downstream functional cholesterol biosynthesis pathway targeting, especially by FDA approved Fluvastatin, may be a novel strategy for targeted prevention of TNBC.

#2221

Honokiol stimulates immune reactivity in UV-irradiated skin through DNA demethylation-dependent functional activation of dendritic cells in mice.

Santosh K. Katiyar, Tripti Singh, Harish C. Pal, Ram Prasad. _Univ. of Alabama at Birmingham, Birmingham, AL_.

Solar ultraviolet radiation (UVR), and in particular UVB spectrum (290-320 nm), are considered as a complete carcinogen for cutaneous malignancies. The over-exposure of UVR suppresses the development of allergic contact hypersensitivity (CHS) response in both laboratory animals as well as in humans. CHS response is considered to be a prototypic T-cell mediated immune response. UVR-induced suppression of immune sensitivity has been implicated in skin cancer risk. Therefore, the treatment options which can inhibit UVB-induced suppression of immune sensitivity may be useful in the management of skin cancers, including both melanoma and non-melanoma. Previously, we have shown that topical treatment of honokiol inhibits UVB induced immune suppression, which was associated with the reduction in UVB-induced inflammatory mediators in the mouse skin. Here, we have further determined the underlying mechanism of action of honokiol on UVR-induced immunosuppression including its effect on epigenetic regulators and their relationship with immune sensitivity in mouse skin. Topical treatment of C3H/HeN mice with honokiol (0.5 and 1.0 mg/cm2 skin area) in hydrophilic-cream based topical formulation significantly inhibits UVB-induced suppression of CHS response (P<0.01-0.005), which was associated with reduced levels of DNA methylation as well as Dnmt activity in the mouse skin compared with non-honokiol-treated and UVB-exposed control mice. To characterize the cell population responsible for the honokiol mediated inhibition of UVB-induced immunosuppression, we used an adoptive transfer approach. DNA-methylated (most of cells) dendritic cells (DCs), isolated from the draining lymph nodes of donor mice that had been UVB-exposed and sensitized to 2,4,-dinitrofluorobenzene (DNFB) with and without honokiol (0.5 and 1.0 mg/cm2 skin area) treatment, were transferred into naïve recipient mice. The CHS response of the recipient mice to DNFB was then measured. Honokiol treatment of UVB-exposed donor mice relieved this suppression of the CHS response in naïve mice, while the recipient mice, obtained DNA-methylated DCs from UVB-exposed donor mice that were not treated with honokiol, showed significant suppression in the CHS response. The restoration of CHS response in mice receiving DCs from honokiol treated, UV-exposed donor mice was also associated with enhanced secretion of Th1-type cytokines compared with the Th1-type cytokines from the DCs obtained from non-honokiol-treated and UVB-exposed donor mice. These data suggest that honokiol mediated inhibition of UVB induced suppression of CHS response is associated with functional activation of dendritic cells and that is dependent on DNA demethylation in DCs.

#2222

Sildenafil suppresses inflammation-driven colorectal cancer in mice.

Bianca N. Islam,1 Sarah K. Sharman,1 Yali Hou,1 Allison Bridges,1 Ravindra Kolhe,2 JImena Trillo-Tinoco,1 Nagendra Singh,1 Sangmi Kim,1 Subbaramiah Sridhar,2 Frankin G. Berger,3 Darren D. Browning1. 1 _Augusta University, Georgia Cancer Center, Augusta, GA;_ 2 _Augusta University, Medical College of Georgia, Augusta, GA;_ 3 _University of South Carolina, Columbia, SC_.

Intestinal cyclic guanosine monophosphate (cGMP) signaling regulates epithelial homeostasis, and has been implicated in the suppression of colitis and colorectal cancer (CRC). In this study, we investigated the cGMP-specific phosphodiesterase-5 (PDE5) inhibitor sildenafil to prevent tumorigenesis in the azoxymethane/dextran sulfate sodium (AOM/DSS) inflammation-driven colorectal cancer model. Treatment of mice with sildenafil activated cGMP signaling in the colon mucosa and protected against DSS-induced barrier dysfunction. In mice treated with AOM/DSS, oral administration of sildenafil throughout the disease course reduced polyp multiplicity by 50% compared to untreated controls. Polyps formed in sildenafil-treated mice showed less proliferation and were more differentiated compared to polyps from untreated mice, but apoptosis was unaffected. Polyps in sildenafil-treated mice were also less inflamed; they exhibited reduced myeloid-cell infiltration, and reduced expression of iNOS, IFNγ and IL-6 compared to untreated controls. Most of the protection was observed when sildenafil was added during the initiation stage of carcinogenesis (40% reduction in multiplicity), whereas administration of sildenafil later during the promotion stages did not affect polyp number. While later treatment with sildenafil did not affect multiplicity, it did have a similar effect on polyp phenotype, including increased mucus production, reduced proliferation and inflammation. In summary, our results demonstrate that oral administration of sildenafil suppresses inflammation and polyp formation in mice treated with AOM/DSS. This suggests that PDE5 inhibitors could have therapeutic value for the prevention of CRC in inflammatory bowel disease patients.

#2223

Rapamycin suppresses ErbB2-overexpressing mammary tumors through selective inhibition of luminal progenitor cells and tumor-initiating cells.

Qingxia Zhao,1 Amanda B. Parris,1 Erin W. Howard,1 Ming Zhao,1 Zhiying Guo,1 Ying Xing,2 Zhikun Ma,1 Xiaohe Yang1. 1 _North Carolina Central University, Kannapolis, NC;_ 2 _Basic Medical College of Zhengzhou University, Henan, China_.

Rapamycin is a potent inhibitor of the mTOR pathway and has demonstrated anti-cancer properties in various cancer models. Although rapamycin was reported to inhibit mammary carcinogenesis at multiple stages, including initiation, invasion, and metastasis, the underlying mechanisms remain unclear, especially in individual breast cancer subtypes. Specifically, ErbB2/Her2 amplification occurs in approximately 25% of invasive human breast cancers, and is characterized by Akt-mTOR pathway deregulation. Recent reports also suggest that cancer stem cells (CSCs) play a critical role in tumor development and metastasis. Therefore, we investigated the efficacy of rapamycin in the prevention of mammary tumor development in MMTV-ErbB2 transgenic mice by focusing on its effects on CSCs/tumor-initiating cells (TICs). In this study, MMTV-ErbB2 mice were inoculated with syngeneic 78617 cells, which are derived from MMTV-ErbB2 mammary tumors, followed by rapamycin treatment (1.5 mg/kg/day) for 12 days. We found that rapamycin elicited strong anti-tumorigenic effects on the syngeneic tumors, as indicated by substantially reduced tumor volume and weight. To determine the effect of rapamycin on mammary epithelial cells (MECs) in premalignant tissues, MMTV-ErbB2 mice were treated with low-dose rapamycin (1 mg/kg/day) between weeks 10 and 20 of age. Rapamycin significantly inhibited mammary morphogenesis at 20 weeks of age as compared to the control mice. As such, the branching density, ductal elongation, and proliferative index of the mammary glands from the rapamycin-treated mice were markedly decreased. Flow cytometric analysis of primary MECs demonstrated that rapamycin selectively inhibited the CD61highCD49fhigh subpopulation, which are enriched with luminal progenitor cells and TICs. 3D culture of FACS-sorted MEC subpopulations indicated that CD61highCD49fhigh cells are the major subpopulation that gives rise to 3D colonies. We further demonstrated that rapamycin significantly suppressed colony-forming cell (CFC) number and primary/secondary sphere formation of primary MECs and spontaneous MMTV-ErbB2 tumor cells, indicating rapamycin-induced inhibition of mammary progenitor cells and CSC self-renewal, respectively. Furthermore, molecular analysis of MECs demonstrated that rapamycin inhibited signaling associated with ER, ErbB2, and mTOR. In particular, rapamycin strikingly inhibited Wnt/β-catenin and TGFβ/Smad3 signaling. Taken together, our results demonstrate that rapamycin-mediated prevention of MMTV-ErbB2 mammary tumor development involves selective targeting of mammary progenitor cells and potential TICs in the premalignant mammary tissues through regulation of MEC stemness and multiple signaling pathways, which contributes to its potent anti-tumor effects on ErbB2-overexpressing breast cancer.

#2224

Increased 15-lipoxygenase-1 activity limits tumor development in the azoxymethane mouse model of colon cancer: impact of omega-3-acid ethyl esters.

Xiangsheng Zuo, Jennifer K. Colby, Fuyao Liu, Shen Gao, Ling Wu, Jonathan C. Jaoude, Micheline J. Moussalli, Lin Tan, Peiying Yang, Imad Shureiqi. _University of Texas M.D. Anderson Cancer Center, Houston, TX_.

Pro-inflammatory signaling has been shown to promote colorectal tumorigenesis and is a target for the development of effective chemopreventive approaches. The specialized pro-resolving lipid mediators (SPMs, e.g. resolvins), bioactive metabolites of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), actively terminate inflammation and have been proposed to possibly contribute to the anti-tumorigenic effects of DHA and EPA. The enzyme 15-lipoxygenase-1 (ALOX15) is a key biosynthetic enzyme in generation of resolvins. However, 15-LOX-1 expression is commonly lost during human CRC tumorigenesis starting in premalignant stages via transcriptional mechanisms independent of substrate availability. The impact of ALOX15 on DHA and EPA's effects on tumorigenesis remain unknown. Mice with intestinal epithelium-specific expression of human ALOX15 (15-LOX-1-gut mice) and wild-type FVB controls were injected with azoxymethane (AOM, 7.5mg/kg) once weekly for 6w and followed for 20w. Mice were fed diet with 1% omega-3-acid ethyl esters (O3AEE, a pharmaceutical grade fish oil preparation of EPA and DHA ethyl esters) or control diet starting 3w before initiation with AOM. Colonic tumors developed in 10 of the 13 (77%) wild type (WT) mice fed control diet, 5 of the 10 (50%) WT mice fed O3AEE diet, 5 of the 12 (42%) 15-LOX-1-gut mice fed control diet, and 3 of the 10 (30%) 15-LOX-1-gut mice fed control diet. Lipid mediator levels were measured by liquid chromatography/ tandem mass spectrometry (LC-MS/MS). The SPMs resolvin E1 and D2 (RvE1, RvD2) as well as the pathway intermediates 18-HEPE and 17-HDHE were increased in ALOX15-gut mice on O3AEE (see Table; data are shown as ng/mg protein; mean ± SEM). Our results demonstrate that ALOX15 activity is important to DHA and EPA formation of resolvins and inhibition of colonic tumorigenesis. Supported by grants CPRIT RP150195 and NIH RO1 R01CA195686

SPMs in O3AEE or control diet fed mice

---

|

WT

Control diet | WT

O3AEE diet | 15-LOX-1-gut

Control diet | 15-LOX-1-gut

O3AEE diet

18-HEPE | 0 | 0.46 + 0.5 | 0 | 0.82 + 0.31

17-HDHE | 0.21 + 0.09 | 0.2 + 0.7 | 4.2 + 0.41 | 13.35 + 3.1

RvE1 | 0 | 0 | 0 | 1.98 + 0.77

RvD2 | 0 | 0.024 + 0.024 | 0.17 + 0.022 | 0.3 + 0.077

#2225

Green tea polyphenols suppress tumor growth and invasion by targeting matrix metalloproteinases, RECK and TIMP-3, in a mouse model implanted with prostate tumors.

Eswar Shankar, Natarajan Bhaskaran, Rajnee Kanwal, Sanjay Gupta. _Case Western Reserve University, Cleveland, OH_.

Green tea polyphenols (GTP) and its major constituent, epigallocatechin-3-gallate (EGCG) reactivate epigenetically silenced genes in cancer cells that reduces invasiveness and migration capabilities; however, the mechanisms whereby these effects occur are not well understood. RECK, a novel tumor suppressor and the tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) genes negatively regulates matrix metalloproteinases (MMPs) and inhibits tumor invasion, angiogenesis and metastasis. In the present study, we demonstrate that GTP mediate epigenetic induction of RECK and TIMP-3 thereby playing a key role in suppressing invasiveness and gelatinolytic activity of MMP-2 and MMP-9 in vivo in an orthotopic implantation model of human prostate cancer. Athymic nude mice were implanted with human prostate cancer LNCaP tumor in the ventral prostate for 2 weeks and later treated DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (AZA), histone deacetylase inhibitor, trichostatin A (TSA) and histone methyltransferase inhibitor, 3-Deazaneplanocin A (DZNep) individually at 0.1 mg/kg body weight intraperitoneally at alternate days/week; and combination of AZA+TSA and DZNep+TSA at similar doses and times; whereas GTP was provided peroral by gavage at 7.5 and 15.0 mg/kg body weight freshly prepared in 100µl PBS. Treatment of mice with GTP resulted in marked decrease in tumor growth and its local invasion in dose dependent manner, compared to treatment with epigenetic inhibitors and their combination after 8 weeks of intervention. GTP treatment significantly reduced serum levels of MMP-2, MMP-9 and VEGF, compared to treatment with epigenetic inhibitors alone. Combination of AZA+TSA exhibited similar effect which was equivalent to the lower dose of GTP treatment. Furthermore, GTP treatment significantly reduced EZH2 and H3K27me3 and class I HDAC protein levels in tumors. GTP partially reversed the hypermethylation status of RECK and TIMP3 gene and significantly enhanced their protein expression in the tumor tissue. Inhibition of VEGF, MMP-2 and MMP-9 levels were also noted after GTP treatment in tumor tissue in dose-dependent manner. Our findings suggest that induction of RECK and TIMP-3 are key epigenetic events modulated by GTP that results in suppression of matrix degradation and angiogenesis to delay prostate cancer invasion and its subsequent progression.

#2226

**Sildenafil suppresses tumorigenesis in** ApcMin/+ **mouse model.**

Sarah K. Sharman,1 Bianca N. Islam,1 Yali Hou,1 Allison Bridges,1 Nagendra Singh,1 Subbaramiah Sridhar,2 Frankin G. Berger,3 Darren D. Browning1. 1 _Augusta University, Georgia Cancer Center, Augusta, GA;_ 2 _Augusta University, Medical College of Georgia, Augusta, GA;_ 3 _University of South Carolina, Columbia, SC_.

The cGMP signaling pathway regulates homeostasis in the colon epithelium and has been implicated in the suppression of colorectal cancer. Exisulind is a weak phosphodiesterase-5 (PDE5) inhibitor that was previously shown to reduce polyp formation in familial adenomatous polyposis (FAP) patients. Despite this validation of PDE5 as a therapeutic target, the failure of Exisulind due to toxicity halted further trials of cGMP elevating agents. The present study tested the effect of the PDE5 inhibitor sildenafil, and the receptor guanylyl-cyclase C (GCC) agonist linaclotide on tumorigenesis in the ApcMin/+ mouse model of colon cancer. Administration of either sildenafil or linaclotide to both wild type and ApcMin/+ mice caused dramatic effects on homeostasis that included reduced proliferation and increased goblet cell differentiation. With treatment beginning at 4 weeks, sildenafil and linaclotide caused a 50% and 67% (respectively) reduction in the number of polyps per mouse. Furthermore, the polyps in sildenafil and linaclotide treated animals exhibited a reduced proliferative index and increased mucus density compared to polyps from untreated mice. The mean polyp size was not changed by treatment with either drug. In support of a tumor-suppressive role for cGMP signaling in the intestine, the ApcMin/+ mice showed reduced levels of endogenous GCC agonists in the intestinal mucosa compared to wild type animals. This suggests that part of the tumor preventative effects observed here could be due to compensation for this deficit by augmenting cGMP signaling with PDE5 inhibitors or GCC agonists. While the tumor suppressive mechanism is not fully understood, the results presented here demonstrate in a preclinical model, that increasing intestinal cGMP levels by targeting PDE5 or GCC may be a viable chemoprevention strategy for human FAP patients.

#2227

Fgf2 from visceral adipose tissue stimulates neoplastic transformation of nonmalignant epithelial cells.

Debrup Chakraborty, Vanessa Benham, Elena Y. Demireva, Blair Bullard, Jamie J. Bernard. _Michigan State University, East Lansing, MI_.

Background: Adiposity plays a crucial role in the pathogenesis and prognosis of different types of cancers. Epidemiological evidence suggests visceral adipose tissue (VAT) and high-fat diets (HFD) are associated with increased cancer risk however the mechanism is not understood. The aim of this study was to explore the factors in VAT that stimulate neoplastic transformation.

Methods: We modeled visceral adiposity-stimulated neoplastic transformation using our novel ex vivo system of VAT-condition medium stimulated epithelial cell transformation (measured by growth in soft agar) and our in vivo murine lipectomy model of ultraviolet light B (UVB)-induced, VAT promoted skin tumor formation. FgfR1(-/-) stable cells were generated by using CRISPR-Cas9 technology and were used to investigate the role of fibroblast growth factor-2 (FGF2) and FGFR1 dependent signaling in neoplastic transformation, both in vitro and in vivo.

Results: Only the VAT of obese mice fed a HFD [not VAT from low-fat diet (LFD) fed mice] stimulated neoplastic transformation of skin epithelial cells. Furthermore, human VAT stimulated both skin and mammary epithelial cell transformation. The differences in VAT activity between LFD and HFD fed mice and human donors were associated with the levels of FGF2. Circulating levels of FGF2 were associated with non-melanoma tumor formation in vivo. Human and mouse VAT failed to stimulate transformation in FgfR1(-/-) cells and do not form tumors when injected in Nude mice in vivo.

Conclusion: Collectively, our data show FGF2 released from VAT and its interaction with FGFR1 is a novel and potential direct path of VAT-enhanced tumorigenesis. Blocking the FGFR1 induced signaling in VAT of abdominally obese individuals may be an important cancer prevention strategy as well as an adjuvant therapy for improving outcomes following cancer diagnosis.

#2228

Altering rictor with diosmetin reduces tumor progression in orthotopic prostate cancer model.

Rebecca Pakradooni, Ahmad Khalifa, Riddhi Patel, Sanjeev Shukla. _Case Western Reserve Univ., Cleveland, OH_.

Rapamycin insensitive companion of mTOR (Rictor) is an essential subunit of mTOR complex 2, maintains the integrity of the complex and functions as full activator of Akt. Rictor has been implicated to be involved in growth and progression of malignancies. Reports suggested that overexpression of Rictor in prostate cancer tissues, which may have potential role in prostate cancer progression. We demonstrated silencing Rictor in PC-3 cells decreased p- Akt (Ser-473) expression; conversely p-Akt (Thr-308) and p-PKCα (Ser-657) expression was increasing. Additionally to observe the molecular mechanism of Rictor regulation, we took HAP1 cells (derived from male chronic myelogenous leukemia (CML) cell line KBM-7) were edited and silenced by CRISPR/Cas9 with 2 and 10 base pair Rictor in a coding exon 5. HAP1 haploid human cell line contains a single copy of all chromosomes except for a heterozygous 30-megabase fragment of Chromosome 15; these cells are instrumental for genetic screening, as gene inactivation is greatly facilitated by the absence of a second gene copy. In these cells we observed p-Akt (Ser-473) expression was significantly less with more pronounced in 10 base pair than 2 base pair. Further to determine potential role of diosmetin (5, 7-Trihydroxy-4′-methoxyflavone; a natural flavonoid present in citrus plant, has anti-mutagenic properties) on Rictor silenced 2 base pair and 10 base pair HAP1 cells, we treated these cells in concentration response fashion, and observed decrease in p-Akt (Ser-473) expression than control cells. Next, we determine invivo potential of Rictor silencing, we used luciferase tag PC-3 cells silenced with Rictor gene orthotopically implanted in ventral prostate of mice. After 8 weeks of Rictor silenced cells implanted mice, we observed less luciferase activity in prostate and occasional metastasis than control luciferase PC-3 cells. Moreover, diosmetin fed mice in dose dependent fashion (20 and 50µg/animal/day) represented decrease in tumor luciferase activity with reduced tumor volume and no metastasis than control mice. We observed metastasis only in control and rapamycin (8 mg/kg body weight intraperitoneally every alternate day) treated mice in spine, lung and kidney. Taken together, our studies demonstrate that diosmetin fed animals resulted in growth inhibition and induction of apoptosis by altering Rictor signaling cascade. We are demonstrating that diosmetin modulates molecular targets viz., Rictor and Akt to alter cellular events and elicit anticancer effects in prostate cancer cells.

#2229

Prevention by tolfenamic acid and characterization of the molecular targets in Pirc and FAP colorectal adenomas.

Furkan U. Ertem,1 Wenqian Zhang,2 Kyle Chang,3 Wan-Mohaiza Dashwood,2 Praveen Rajendran,2 Deqiang Sun,2 Ala Abudayyeh,3 Maen Abdulrahim,4 Eduardo Vilar,3 Roderick H. Dashwood2. 1 _University of Pittsburg Medical Center, Pittsburg, PA;_ 2 _Texas A &M Univ. Health Science Ctr., Houston, TX; _3 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 4 _Duke University Medical Center, Durham, NC_.

A critical need exists in sporadic and high-risk colorectal cancer patients for endoscopic screening combined with safe and effective preventive agents. To prioritize molecular targets for prevention studies in vivo, RNA-seq analyses were conducted on colorectal adenomas from familial adenomatous polyposis (FAP) patients, and from the Apc-mutant polyposis in rat colon (Pirc) model. Using a new murine endoscopic polypectomy methodology (F.U. Ertem et al., Gastrointest Endosc 2016;83:1272-6), we evaluated agents that might substitute for the current standard of care, Sulindac, and identified tolfenamic acid (TA) as a promising candidate for further investigation. At clinically-relevant doses in the Pirc model, TA suppressed tumorigenesis significantly both in the small intestine and in the colon. Inhibition by TA of colonic crypt cell proliferation, and the concomitant activation of apoptosis, coincided with reduced expression in target tissues of β-catenin, cyclin D1, Survivin and matrix metalloproteinase 7. Based on the oncogene candidates defined by RNA-seq analysis, endoscopic tracking revealed the time-course of tumor suppression by TA, and the temporal changes in S100a9, Nppb, Aldh1a3 and Mmp7, highlighting their value as potential early biomarkers for prevention in the clinical setting. As an "old drug" repurposed from migraine, TA appears to offer an exciting new therapeutic avenue in FAP and other high-risk colorectal cancer patient populations. This work was supported in part by NIH grants CA090890, CA122959, ES00210, ES023512, the John S. Dunn Foundation, and a Chancellor's Research Initiative.

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#2230

Luteolin selectively inhibits EZH2 and blocks H3K27 methylation in prostate cancer cells.

Rajnee Kanwal,1 Xiaoping Yang,2 Eswar Shankar,1 Sanjay Gupta1. 1 _Case Western Reserve Univ., Cleveland, OH;_ 2 _VA Medical Center, Cleveland, OH_.

The histone methyltransferase enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the polycomb repressive complex (PRC2) and is involved in chromatin remodeling and gene silencing through its methylation of histone H3 on lysine 27 (H3K27). Specifically, trimethylation of H3K27 is associated with gene silencing and plays critical role in cancer initiation and progression. Therefore, blocking EZH2 catalytic activity may present a valid preventive and/or therapeutic strategy for the treatment of cancers with EZH2 overexpression including prostate cancer. Our multistep approach of molecular modeling and direct binding has led to the identification of plant flavone luteolin (3', 4', 5, 7-tetrahydroxyflavone) as a specific inhibitor of EZH2 with preferential blocking of its catalytic site. Here we report luteolin, in micro molar range, inhibits EZH2 catalytic activity, demonstrates anti-proliferative and anti-invasive activities in functional cell based assays. Treatment of human prostate cancer DU145 and 22Rv1 cells, which possess high constitutive EZH2 expression, with 5-20 μM luteolin significantly inhibits EZH2 and SUZ12 protein expression in dose and time dependent manner, although luteolin treatment did not affect protein expression of EED and RbAp46/48 protein, other members of PRC2 complex. Treatment of both cell lines with luteolin also reduced H3K27me3 and H3K27me2 protein and its enzymatic activity in dose and time dependent manner without affecting total H3 protein. In addition, luteolin was also effective in suppressing in vitro methylation performed using recombinant PCR2 complex. These events led to increase in the expression of downstream tumor suppressor genes including E-cadherin, SLIT2 and TIMP3, respectively. Interestingly, treatment of cells with proteasome inhibitor, MG132 together with luteolin did not prevent EZH2 degradation indicating that proteasomal degradation might not contribute to EZH2 inhibition. Taken together, our study suggest that luteolin acts on the catalytic binding site of EZH2 to exhibit downregulation of histone H3 methylation and could be developed as a potential promising agent for the prevention and/or treatment of various human cancers including prostate cancer with EZH2 overexpression.

#2231

**15-Keto prostaglandin E** 2 **inhibits STAT3 signaling in H-** Ras **transformed mammary epithelial cells.**

Eun-Ji Lee,1 Young-Il Hahn,1 Su-Jung Kim,1 Young-Joon Surh,1 Hye-Kyung Na2. 1 _Seoul National University, Seoul, Republic of Korea;_ 2 _Sungshin Women's University, Seoul, Republic of Korea_.

Prostaglandin E2 (PGE2) is a potent lipid mediator that plays a key role in inflammation and carcinogenesis. The intracellular level of PGE2 is regulated by NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). This enzyme catalyzes the oxidation of the 15(S)-hydroxyl group of PGE2 to generate 15-keto PGE2. Down-regulation of 15-PGDH was observed in various malignancies and overexpression of 15-PGDH has been known to suppress the carcinogenic processes. However, mechanisms underlying anti-carcinogenic effects and tumor suppressive functions of 15-keto PGE2 are poorly understood in various cancer. H-Ras transformed mammary epithelial MCF10A-ras cells show enhanced expression of cyclooxygenase-2 and activation of signal transducer and activator of transcription 3 (STAT3) and down-regulation of 15-PGDH compared to normal mammary epithelial MCF10A cells. STAT 3 plays a key role in growth of cancer cells as well as inflammation-associated carcinogenesis. This prompted us to investigate the effect of 15-keto PGE2 on STAT3 activation in MCF10A-ras cells. 15-Keto PGE2 suppressed the phosphorylation of STAT3 in MCF10A-ras cells in concentration- and time-dependent manners as determined by Western blot and immunocytochemical analyses. 15-Keto PGE2 also inhibited the dimerization of STAT3 which led to suppression of nuclear translocation and transcriptional activity of STAT3 as determined by Western blot analysis and the luciferase assay, respectively. 15-keto PGE2 suppressed the expression of cyclin D1. In addition, 15-keto PGE2 inhibited the growth of MCF10A-ras cells as determined by anchorage-independent growth and colony forming assays. However, a non-electrophilic analogue 13,14-dihydro-15-keto PGE2 which lacks the α,β-unsaturated carbonyl moiety failed to inhibit the phosphorylation, dimerization, and nuclear translocation of STAT3. Moreover, 13,14-dihydro-15-keto PGE2 did not affect the growth of MCF10A ras cells. These findings suggest that the α,β-unsaturated carbonyl moiety of 15-keto PGE2 is essential for its suppression of STAT3 signaling. STAT3 has several cysteine residues in its activation sites. We observed that reducing agents N-acetyl-L-cysteine and dithiothreitol abrogated the suppressive effect of 15-keto PGE2 on STAT3 phosphorylation, suggesting that the involvement of the thiol modification. Biotinylated 15-keto PGE2 covalently bound to STAT3 as determined by immunoprecipitation with STAT3 followed by immunoblotted with streptavidin. Molecular docking analysis predicted Cys251 and Cys259 residues of STAT3 as putative binding sites of 15-keto PGE2. In conclusion, 15-keto PGE2 inhibits STAT3 signaling through cysteine thiol modification, thereby suppressing MCF10A-ras cell growth and proliferation. Thus, tumor suppressor function of 15-PGDH is attributable, at least in part, to inactivation of oncogenic STAT3 signaling by its catalytic product 15-keto PGE2.

#2232

Dietary flavonoids, CYP1A1 genetic variants, and the risk of colorectal cancer.

Jeongseon Kim, Young Ae Cho, Jeonghee Lee, Jae Hwan Oh, Dae Kyung Sohn, Hee Jin Chang. _National Cancer Ctr. Korea, Goyang-si, Republic of Korea_.

The role of dietary flavonoid intake in colorectal carcinogenesis might differ according to flavonoid subclasses and individual genetic variants related to carcinogen metabolism. Therefore, we examined whether greater habitual dietary intake of flavonoid subclasses was associated with a lower risk of colorectal cancer and whether CYP1A1 genetic variants altered this association. A semi-quantitative food frequency questionnaire was used to assess the dietary intake of six flavonoid subclasses (flavonols, flavonones, flavanones, flavan-3-ols, anthocyanidins, and isoflavones) in 923 patients with colorectal cancer and 1,846 controls; furthermore, CYP1A1 genetic variants (rs4646903 and rs1048943) were genotyped. A logistic regression was used to investigate dietary flavonoid intake, CYP1A1 genetic variants, and their interaction in relation to colorectal cancer risk. Among the subclasses of flavonoids, flavonols (OR [95% CI] = 0.10 [0.06, 0.16], highest vs. lowest quartile) and flavan-3-ols (OR [95% CI] = 0.54 [0.41, 0.71], highest vs. lowest quartile) were significantly associated with colorectal cancer risk after adjusting for confounders. Homozygous variants of CYP1A1 rs4646903 showed a protective effect on the risk of rectal cancer (OR [95% CI] = 0.60 [0.37, 0.96], CC vs. TT/TC). Carriers of the CC homozygous variant with high flavonol intake showed a greater decrease in colorectal cancer risk compared with T allele carriers with low flavonol intake (P for interaction = 0.02), particularly regarding rectal cancer (P for interaction = 0.005). In conclusion, the effect of dietary flavonoid intake on colorectal cancer risk differs by flavonoid subclasses and CYP1A1 genetic variants.

#2233

The fatty acid amide hydrolase inhibitor URB937 ameliorates radiation-induced lung injury in a mouse model.

Rui Li, Jianxin Xue, You Lu. _West China Hospital, West China School of Clinical Medicine, Chengdu, China_.

Radiation-induced lung injury (RILI) is a common and potentially serious complication from radiotherapy to the thoracic region. In the current study, we examined the potential protective effects of URB937, an inhibitor of fatty acid amide hydrolase in a mouse model of RILI. Here we challenged male C57BL/6 mice with irradiation (16Gy to the thoracic region), and then injected intraperitonealy either URB937 (1 mg/kg) or vehicle three times a week for 30 days. Mice were sacrificed at either the end of treatment or120 days from irradiation to evaluate the extent of RILI and mice survival. Potential effects of URB937 on irradiation induced tumor growth inhibition were also evaluated. We found URB937 increased endocannabinoids in the lungs, attenuated the extent of RILI, prolonged the survival,inhibited mice developed pathological alterations of pneumonitis and lung fibrosis. Irradiation-induced increases of proinflammatory and profibrotic cytokines were decreased, including interleukin-1β, interleukin-6, tumor necrosis factor-α, transforming growth factor-β1 in plasma and lung tissue. Moreover, malondialdehyde in plasma and inflammation cells in bronchoalveolar lavage fluid were lower in mice treated with irradiation plus URB937. URB937 did not affect tumor growth inhibition caused by irradiation. These results suggested that inhibiting fatty acid amide hydrolase through URB937 could ameliorate the RILI, without affecting the efficacy of irradiation on tumor control.

#2234

**Efatutazone reduces mammosphere formation in** Brca1WT/fl11/Cre/p53+/- **and** Brca1fl11/fl11/Cre/p53+/- **mice.**

Sahar J. Alothman, Shan Chao, Weisheng Wang, Priscilla A. Furth. _Georgetown University, Washington, DC_.

Introduction: Efatutazone, a PPAR gamma agonist, may affect tumor growth through the induction of terminal cell differentiation. Thus, we hypothesize that efatutazone could affect progenitor cell number. Here, we evaluate progenitor cell number by measuring mammospheres, which is considered one way to measure potential progenitor cells.

Methods: Two month old Brca1WT/fl11/Cre/p53+/- (n= 14) and Brca1fl11/fl11/Cre/p53+/- (n= 10) C57Bl/6 mice were randomly placed on either control or treated with efatutazone through the diet (30-mg/kg concentration, F3028, rodent diet, grain-based, 1/2-in pellets; Bio-Serv, Frenchtown, NJ) with necropsy at four months and isolation of primary mammary epithelial cells from thoracic glands for studies of mammosphere formation using SCIVAX 96-well low adhesion nanoculture plates (Organogenix, Inc., Japan) using EpiCult-B Mouse Media (Stem Cell Technology, Inc., Vancouver, Canada) with 0,1,5, and 10% fetal bovine serum (FBS) added. Thoracic mammary gland tissue was frozen at -20°C followed by isolation of RNA and evaluation of PPAR gamma pathway gene expression by real-time RT-PCR using TaqMan® Array Mouse Lipid Regulated Genes (Thermo Fisher Scientific, Inc., Waltham MA). One inguinal gland was fixed for mammary gland whole mount and the other inguinal gland was formalin-fixed and paraffin-embedded for histology.

Results: SCIVAX nanoculture plates showed reproducible increases in mammosphere numbers with increasing FBS concentrations (p <0.05 one-way, Kruskal-Wallis). A significant increase in sphere number was seen in Brca1fl11/fl11/Cre/p53+/- compared to Brca1WT/fl11/Cre/p53+/- mice (p<0.05 one-way, Kruskall Wallis). Efatutazone treatment significantly decreased sphere numbers in Brca1WT/fl11/Cre/p53+/- and in Brca1fl11/fl11/Cre/p53+/- mice (p<0.05 one-way, Kruskall Wallis). Expression of PPAR gamma pathway genes were increased at the RNA level with statistically significant increases in Acadvl, Tnf, Alox5 (p<0.05) and Il1B, Srebf2, Hmgcs1, Hmgcr (p<0.01) genes (BootsRatio, http://rht.iconcologia.net/stats/br/several.html) in mice on efatutazone as compared to control diet.

Discussion: SCIVAX nanoculture plates can be used to quantitatively evaluate and compare mammosphere numbers between different genotypes and treatment groups. The higher numbers of mammospheres found with loss of both Brca1 copies as compared to one copy is consistent with previously published literature. The decrease in mammosphere numbers with efatutazone treatment could be secondary to its differentiating impact on mammary epithelial cells.

Conclusion: While efatutazone statistically significantly reduced mammosphere numbers, the absolute reduction was less the 25%.

#2235

Preventive effects of the sodium glucose cotransporter 2 inhibitor tofogliflozin on liver tumorigenesis in obese and diabetic mice.

Yohei Shirakami, Koki Obara, Masaya Ohnishi, Takayasu Ideta, Hiroyasu Sakai, Takuji Tanaka, Masahito Shimizu, Mitsuru Seishima. _Gifu Univ. Graduate School of Medicine, Gifu, Japan_.

Obesity, diabetes mellitus, and their related metabolic abnormalities are associated with increased risk of hepatocellular carcinoma (HCC). Sodium glucose cotransporter (SGLT)-2 inhibitors, recently approved anti-diabetic agents, are expected to ameliorate the abnormalities associated with metabolic syndrome including non-alcoholic fatty liver disease (NAFLD). In this study, we investigated the effects of the SGLT2 inhibitor tofogliflozin on the development of NAFLD-related liver tumorigenesis in C57BL/KsJ-+Leprdb/+Leprdb (db/db) obese and diabetic mice. The direct effects of tofogliflozin on human HCC cell proliferation were also evaluated. Male db/db mice were administered diethylnitrosamine-containing water for two weeks and were treated with tofogliflozin throughout the experiment. Tofogliflozin was kindly provided by Kowa Co., Ltd. and the chemical structure will be disclosed at the time of presentation at the meeting. In mice treated with tofogliflozin, the development of hepatic pre-neoplastic lesions was markedly suppressed, and hepatic steatosis and inflammation significantly reduced, as evaluated using the NAFLD activity score, in comparison to those in the control mice. Serum levels of glucose and free fatty acid and mRNA expression levels of pro-inflammatory markers in the liver were reduced by tofogliflozin treatment. Conversely, the proliferation of SGLT2 protein-expressing HCC cells was not inhibited by this agent. These findings suggest that tofogliflozin suppressed the early phase of obesity- and NAFLD-related liver carcinogenesis by attenuating chronic inflammation and steatosis in the liver, while the agent had no significant direct effect on the proliferation of HCC cells. Therefore, SGLT2 inhibitors may have a chemopreventive effect on obesity-related HCC.

#2236

High-throughput screen for chemopreventative agents that influence adipose-tissue driven epithelial neoplastic transformation.

Vanessa Benham, Debrup Chakraborty, Blair Bullard, Thomas S. Dexheimer, Jamie J. Bernard. _Michigan State University, East Lansing, MI_.

Epidemiological studies have established a link between excess adiposity and cancer risk. Our previous data demonstrate that adipose tissue stimulates the transformation (steps a cell undergoes to become malignant) of both skin and mammary epithelial cells as measured by anchorage-independent growth in soft agar. Therefore, agents that influence growth in soft agar may be used therapeutically for skin and breast cancer prevention. However, soft agar in a 24-well format is unsuitable for high-throughput screens. Our objective was to develop a high-throughput assay to evaluate chemopreventative agents that influence adipose-tissue driven epithelial neoplastic transformation. We demonstrate that growth in low attachment conditions in a 384-well plate format is strongly correlated with growth in soft agar. We utilized this methodology and discovered the ability of several compounds to inhibit adipose tissue-stimulated transformation. These compounds will be used in vivo in our models of adipose tissue- or HFD-promoted tumor formation.

#2237

Novel selenium-containing aspirin molecule AS-10 suppresses androgen receptor signaling and induces apoptosis of LNCap prostate cancer cells.

Deepkamal N. Karelia, Sangyub Kim, Srinivasa Ramisetti, Cheng Jiang, Shantu Amin, Arun K. Sharma, Junxuan Lu. _Penn State College of Medicine, Hershey, PA_.

Aspirin as the best known low cost Non-Steroidal Anti-inflammatory drug (NSAID) has been associated with lowering risk of colorectal cancer, in addition to its cardiovascular health benefit at low dose (baby Aspirin) and over-the-counter pain medication. Through an innovative research program aiming at enhancing the bioactivities of NSAIDs by incorporation of selenium, we have discovered AS-10 as a novel Aspirin-selenium compound with promising anti-cancer drug-like properties in terms of potency enhancement (at least 3 orders of magnitude compared to Aspirin), selective cytotoxicity against cancer cells including majority of NCI-60 panel, while sparing normal moue embryonic fibroblasts in cell culture screening assays. Preliminary toxicology study in mice had shown a wide safety margin. Extensive structure-activity relationship investigation suggested a novel structural basis, instead of releasable selenium, that accounted for the striking potency. To investigate the potential chemopreventive attributes of AS-10 against prostate carcinogenesis, we selected LNCaP cells (wild type p53, functional androgen receptor, AR) to examine the growth suppression and apoptosis responses in cell culture. The rationale for choosing LNCaP cell line as target cells rests in the fact that precancerous prostatic lesions that are the intended targets of chemoprevention as well as early stage prostate cancer retain wild type p53 and they are critically dependent on AR signaling for survival. Our results show that cell viability (MTT) test detected a potent growth inhibition of LNCaP cells in a time and concentration dependent manner, with an EC50 in range of 1.7 to 2.5 μM range compared with Aspirin in the millimolar range. Western blot analysis of AS-10 treated LNCaP cells showed decreased protein level of AR and its best known downstream target prostate specific antigen (PSA) in a concentration-dependent manner. In addition, AS-10 treatment led to increased Annexin V staining, caspase 3/7 activity, and PARP cleavage, all indicators of caspase-mediated apoptosis. Furthermore, AS-10 treatment increased the expression of p53-DNA damage response (DDR) proteins such as p21 (canonical target of p53) and p-H2A.X (a marker for DNA strand breaks). Given that our earlier work with pancreatic cancer cells has indicated a rapid induction of reactive oxygen species (ROS) by AS-10 exposure, we hypothesize that ROS generation may be involved in LNCaP cells to trigger the DDR and apoptosis responses. In summary, our findings suggest AS-10 as a potential chemopreventive agent for prostate carcinogenesis.

#2238

Novel sylibin analogues target ovarian cancer EMT-Wnt/β-catenin resistance pathways.

Haneen A. Amawi,1 Noor Hussein,1 Aubry Fetcenkoa,1 Rawan Alnafisah,1 Karthikeyan Chandrabose,2 Elangovan Manivannan,3 Piyush Trivedi,2 Amit K. Tiwari1. 1 _Univ. of Toledo, Toledo, OH;_ 2 _Rajiv Gandhi Proudyogiki Vishwavidyalaya, Bhopal, India;_ 3 _Devi Ahilya Vishwavidyalaya, Indore, India_.

Ovarian cancer (OC) is one of most lethal malignancies in woman reproductive tract. The OC diagnosed in advanced stage (III-IV) patients is highly aggressive and relapses back in over 80% patient after initial response to chemotherapy. Currently used chemotherapies are limited due to significant adverse/toxic effects, a narrow therapeutic index, and development of multidrug resistance mediated by the epithelial-to-mesenchymal transition (EMT) and cancer cell membrane efflux transporters. It is presumed that an ideal anti-OC drug that has diverse mechanisms, would inhibit several key cell-survival signaling and resistance pathways when combined with conventional chemotherapy (paclitaxel and cisplatin). Silybin, a polyphenolic flavonoid, has previously shown to inhibit metastasis by inhibition of EMT pathways and bypass other drug resistance factors i.e. efflux transporters in ovarian cancer both in vitro and in vivo. However, clinical use of silybin is limited due to their poor absorption and low bioavailability and poor potency. To overcome this, we designed and synthesized 11 silybin derivatives using molecular modeling, computer aided drug design, structure activity relationship, natural product lead optimization and ring disjunction approaches. In our preliminary findings, we found the lead molecule (15k) to have a cytotoxicity (IC50&lt1µM) with potency more than 200-fold compared to silybin (IC50~150µM). The 15k was found to be around 10-fold selective in OC cells (OV2008, A2780) compared to normal ovarian cells. Additionally, compared to other cancer cells belonging to prostate, breast, lung, 15k was (3-6)-fold selective in OC cells (OV2008, A2780). The 15k produced synergistic activity and potentiated the cytotoxic effects of platinum (cisplatin), anthracyclines (doxorubicin) and taxane (paclitaxel) anticancer agents. While 15k also reversed the drug resistance mediated by ABC-efflux transporters to paclitaxel and doxorubicin. Mechanistically, the cytotoxic effect of lead compound 15k, as determined by using molecular biology assays, was found to be due to inhibition of apoptosis, tubulin inhibition, and inhibition of stemness pathways (Wnt-β-catenin-EMT). The 15k had significant anti-metastatic effects as shown by their inhibition of migration and invasion potential on OC cells. In silico findings suggest that pharmacokinetic [PK] profiles of these compounds are favorable compared to silybin. Further in vivo PK/pharmacodynamic studies are underway to establish the clinical use of lead molecule in advanced metastasized, drug resistant OC patients.

#2239

Establishment of lung cancer CTCs competent for lung metastasis.

Zujun Que,1 Bin Luo,2 Qihui Shi,3 Jianhui Tian2. 1 _Shanghai Institute of Traditional Chinese Medicine, Institute of TCM Oncology, Shanghai, China;_ 2 _Longhua Hospital, Shanghai University of Traditional Chinese Medicine, National Clinical Research Centre for Traditional Chinese Medicine and Oncology, Shanghai, China;_ 3 _Systematic Biomedical Research Center of Shanghai Jiao Tong University, Shanghai, China_.

Circulating tumor cells (CTCs) have been implicated as a population of cells that may seed metastasis, which led to the 90% of dead in cancer patients. Though the detection technologies has been developing swiftly, how to culture the cells successfully and established a permanent cell line are the bottleneck in the research of metastasis mechanism. Here, we report, for the first time, the establishment of permanent cell line from CTCs of one Non-small cell lung cancer patient and identifying the biological characterization. The cell line designated CTC-0430 has been cultured for more than one and half years, and the cells have been characterized at the genome, proliferation, drug resistance, the expression of surface marker, cytokine secretion level and the characterization of tumor formation in vivo by immunohistochemisty and immunofluorescence. This thorough analysis showed that CTC-0430 cells display a stable phenotype characterized by an intermediate epithelial/mesenchymal phenotype, stem-cell like properties and the drug sensitivity test indicating stronger drug resistance compared to A549 and 95-D. When compare with A549 and 95-D cells, CTC-0430 cells high expression of ICAM-1 and ICAM-3, low expression of CX3CL1 and CXCL1. Functional studies showed that CTC-0430 cells induced rapidly in vivo tumors after subcutaneous transplantation in immunodeficient mice, and the lung metastasis were found after six months later. Besides, three months after we injected the cells into the caudal vein of NOD/SCID mice, the lung metastasis were formed. And then the metastasis lung tissue were used to make IHC and IF, which indicated that the biological characterizations of this cell line are different from A549. The establishment stable cell line of lung cancer CTCs can provide in vivo research platform for the intervention study of postoperative metastasis of lung cancer.

#2240

"Smart" selenium compound ISC-4 inhibits androgen receptor signaling and induces ROS- and p53-dependent apoptosis in LNCaP prostate cancer cells.

Wei Wu, Kartick Pramanik, Cheng Jiang, Shantu Amin, Arun K. Sharma, Junxuan Lu. _Penn State University College of Medicine, Hershey, PA_.

"Smart" selenium compound phenylbutyl isoselenocyanate (ISC-4) was developed by substituting sulfur in cancer chemopreventive compound phenylbutyl isothiocyanate (PBITC) with selenium, resulting in increased anti-cancer activity in several melanoma and colon cancer cell lines. The in vivo anticancer beneficial effects of ISC-4 have been reported in both melanoma and colon cancer mouse models as well. To explore the potential chemopreventive attributes of ISC-4 against prostate carcinogenesis, we selected LNCaP cells (with wild type p53 and functional androgen receptor (AR) signaling) to examine the growth suppression and apoptosis responses in cell culture and interrogated signaling mechanisms through pharmacological and siRNA knockdown approaches. Results show that when compared with PBITC, ISC-4 was more potent at suppressing cell growth and inducing apoptotic cell death in LNCaP cells. ISC-4 induced caspase-mediated apoptosis (as evidenced by cleavage of PARP) in dose- and time-dependent manners in association with decreased AR and its best known target prostate specific antigen (PSA) protein abundance and upregulation of p53-DNA damage response (DDR) protein expression levels: p53, its canonical target p21, and p-H2AX that marks DNA double-strand break sites. Similar to the rapid induction of reactive oxygen species (ROS) by isothiocyanates, ISC-4 exposure resulted in increased dihydroethidium-detectable ROS in a concentration-dependent manner. Preincubation with the ROS scavenger N-Acetyl-L-cysteine (NAC, 10 µM to 2 mM), markedly reduced ISC-4-induced cell death and attenuated p53, p21 and p-H2AX. Moreover, siRNA knockdown of p53 did not suppress ROS production, but decreased ISC-4-induced Annexin V-positive apoptosis, PARP cleavage and the DDR proteins. Taken together, these data suggest that ISC-4 inhibits LNCaP cell growth and survival with concomitant suppression of AR signaling and induction of ROS-p53-DDR mediated apoptosis. Given the pivotal significance of AR signaling in prostate epithelial survival, cancer genesis and progression, and the preservation of wild type p53 in precancerous lesions and early stage prostate cancer, our findings suggest attractive chemopreventive potential of ISC-4 against prostate carcinogenesis.

#2241

Diosmetin induces apoptosis in human prostate carcinoma cells via the Rictor pathway invitro and inhibits tumor growth in vivo.

Riddhi Patel, Rebecca Pakradooni, Ahmad Khalifa, Natarajan Bhaskaran, Sanjeev Shukla. _Case Western Reserve Univ., Cleveland, OH_.

Cell growth is a fundamental biological process, where mTOR (mammalian target of rapamycin) pathway has its role, regulates cell growth by coordinating energy and nutrient signals with growth factor. mTOR protein kinase have two different complexes; complex-I contains major component raptor; a target of rapamycin and complex II; insensitive to rapamycin, has major component rictor. Rictor is important as it phosphorylates Ser-473 of Akt/PKB, which is essential for full Akt/PKB activation. Reports suggested that overexpression of mTOR in prostate cancer tissues, which may have potential role in prostate cancer progression. The known mTOR inhibitor rapamycin is not specific for mTOR-II complex, as it doesn't inhibit main component Rictor. We need to have effective chemo-preventive agents, which can modulate Rictor signaling cascade to inhibit prostate cancer. Diosmetin (5, 7-Trihydroxy-4′-methoxyflavone), a natural flavonoid present in citrus plant, has anti-mutagenic and anti-allergic properties. Earlier we demonstrated growth factors Insulin-like growth factor-1 and cytokine (IL-6) induces Rictor expression, and phosphorylated Akt at Ser-473. Using diosmetin in concentration dependent fashion (5-40µM) to LNCaP and PC-3 cells down regulated phosphorylation of Rictor (Thr1135), Akt (Ser-473) and PKCα (Ser-657) expressions. This resulted in downregulation of survival molecules; Survivin and X-linked inhibitor of apoptosis protein, which induced apoptosis and inhibited cells growth. Luciferase tag PC-3 cells orthotopic implant in ventral prostate of nude mice represented significant decrease in tumor volume of diosmetin (20 and 50µg/animal/day) fed mice compared to control mice. Similarly bioluminescence imaging of orthotopically implanted mice represented decreased tumor luciferase activity after diosmetin feeding than control mice. Moreover, diosmetin feeding to tumor bearing nude mice reduced the phosphorylation of Rictor, (Thr1135), Akt (Ser-473) and PKCα (Ser-657) expressions and induced apoptotic events to inhibit tumor growth significantly. Based on results we may suggest diosmetin can be developed as an anticancer agent for prostate cancer.

#2242

Vitamin E δ-tocotrienol inhibits NF-κB activation by up-regulating A20 in macrophages and suppresses colitis-promoted colon tumorigenesis in mice.

Chao Yang, Qing Jiang. _Purdue University, West Lafayette, IN_.

Nuclear factor-κB (NF-κB) plays a key role in inflammatory responses and cancer development. Inhibition of NF-κB has been shown to attenuate inflammation and cancer progression. δ-Tocotrienol (δTE) is a natural vitamin E form rich in annatto seeds and has been shown to inhibit NF-κB in pancreatic cells. However, potential effect of δTE on NF-κB activation in immune cells and the underlying mechanism have not been investigated. In the present study, we found that δTE inhibited TNF-α induced activation of NF-κB, as indicated by diminished phosphorylation and degradation of IκBα in the cytosol, and phosphorylation of p65 in the nucleus. Furthermore, δTE potently inhibited TNF-α-induced phosphorylation of TAK1, a key upstream kinase that is required for the activation of NF-κB. Interestingly, δTE significantly increased the expression of A20 and CYLD, both of which are negative regulators of NF-κB. Knockout of A20 partially diminished δTE's anti-NF-κB effect, supporting the notion that induction of A20 plays a key role in δTE's anti-NF-κB effect. To translate mechanistic findings to a whole body environment, we further investigated the effectiveness of δTE and its metabolite, δTE-13'-COOH, against azoxymethane (AOM) induced and colitis-promoted colon tumorigenesis in male BALB/c mice. We found that δTE-rich mixed tocotrienols (δTE /ɣTE ~8/1, at 0.035% diet) inhibited colon tumorigenesis, as indicated by 36% reduction of multiplicity of large adenomatous polyps (>2mm(2), P=0.07) and decreased tumor surface area by 31% (P<0.05). δTE-13'-COOH (at 0.04% diet) significantly suppressed multiplicity of total polyps by 25% (P=0.06) and large polyps by 54% (P<0.01). Our study demonstrates that δTE has potent anti-NF-κB activity and this vitamin E form and its metabolite suppress colitis-promoted colon tumorigenesis in mice.

#2243

Elucidating the mechanism of action of molecular iodine on breast cancer cells.

Zack Xu, Xin Chen, Noymi Yam, kin Chan, Usha Nagavarapu. _Biopharm X, Menlo Park, CA_.

Each year, over 1.7 million women are diagnosed with breast cancer worldwide and about 35% of these diagnosed are expected to die from the disease. A significant barrier to improving treatment of breast cancer lies in our incomplete understanding of breast cancer initiation and metastasis. Regular use of molecular iodine has been shown to reduce the sensitivity of breast cells to the proliferative effects of estrogen, resulting in normalization of breast tissue.The aim of the current study was to evaluate the mechanisms of molecular iodine on two common breast cancer sub-types using well-established breast cancer cell lines. In order to study Luminal A and triple-negative breast cancer in vitro, MCF7 (cells from a luminal A subtype) and MDA-MB231 (cells from a triple-negative subtype), were treated with molecular iodine at various concentrations to measure proliferation and cell death. This was subsequently followed by gene expression analysis of key important molecular markers, which are primarily responsible for cell growth and apoptosis. Primary human mammary epithelial cells derived from healthy female donor were used as an internal control. Data from this study indicated that molecular iodine had potent inhibitory effects on cell growth and showed a dramatic increase in cell death in both breast cancer cell lines. Gene expression analysis using quantitative RT-PCR further confirmed that cell cycle genes controlling G1-S phase transition were largely up-regulated. Changes were not seen in Cyclin B expression levels, which further suggest that cells were arrested before entry into cell division. BCL-2, PPAR-α and PPAR-γ were also up-regulated along with down-regulation of Caspase 3, suggesting molecular iodine induced cell death through activation of a caspase-independent apoptosis pathway. Interestingly, mesenchymal-epithelial transition (MET) occurrence was noticed upon molecular iodine treatment as indicated by sharp increase of GATA3 and E-Cadherin and significant down-regulation of Vimentin in invasive MDA-MB231 cells. Our current study enables us to understand a molecular mechanism controlling tumor cell growth and demonstrates potent cellular effects of molecular iodine on breast cancer cell lines. These results also demonstrate promising effects of molecular iodine for regulation of breast cancer EMT differentiation, which is required for tumor initiation and metastasis. Further studies are underway to determine possible effects of molecular iodine in these breast cancer subtypes using in vitro 3D models.

#2244

Pharmacological TLR4 antagonism using topical resatorvid blocks solar UV-induced skin tumorigenesis in SKH-1 mice.

Sally E. Dickinson,1 Karen Blohm-Mangone,2 Nichole B. Burkett,2 Shekha Tahsin,2 Paul B. Myrdal,1 Kelly L. Karlage,1 Jaroslav Janda,2 Kathylynn Saboda,2 Denise J. Roe,1 Zigang Dong,3 Ann M. Bode,3 Emanuel F. Petricoin,4 Valerie S. Calvert,4 Clara Curiel-Lewandrowski,1 David S. Alberts,1 Georg T. Wondrak1. 1 _University of Arizona, Tucson, AZ;_ 2 _University of Arizona Cancer Center, Tucson, AZ;_ 3 _University of Minnesota, Austin, MN;_ 4 _George Mason University, Manassas, VA_.

An urgent need exists for the development of more efficacious molecular strategies targeting non-melanoma skin cancer (NMSC), the most common malignancy worldwide. Inflammatory signaling downstream of Toll-like receptor 4 (TLR4) is a causative factor in several forms of tumorigenesis, yet its role in solar UV-induced skin carcinogenesis remains undefined. In our recently published work based upon immunohistochemical (IHC) analysis of NMSC tissue microarrays and proteomic analysis of reverse-phase protein microarrays (RPPA) from banked human tissue, we observed increased TLR4 expression in association with tumorigenic progression from normal skin to actinic keratosis and cutaneous squamous cell carcinoma. In immortalized keratinocytes expressing luciferase reporter constructs, resatorvid potently inhibits UV-induced AP-1 and NF-κB signaling, associated with downregulation of inflammatory mediators (IL-6, IL-8, IL-10) and MAP Kinase phosphorylation. In a subsequent acute exposure model in SKH-1 mice, topical resatorvid efficiently antagonized UV-induced stress signaling while potentiating UV-induced epidermal apoptosis. In the current report, we show for the first time that pharmacological inhibition of TLR4 using the specific antagonist resatorvid (TAK-242) blocks UV-induced tumorigenesis in SKH-1 mice. After assessing photostability, efficient cutaneous delivery, and skin residence time of topical resatorvid, we then tested the feasibility of TLR4-directed inhibition of UV-induced tumorigenesis. To this end, SKH-1 mice were split into three groups (n = 20). Each group was exposed to solar-simulated UV (three times a week; 15 weeks duration), followed by another 10 weeks in the absence of UV exposure (sacrifice at week 25). The control group received topical vehicle (acetone) on their backs 1 hour prior to UV exposure (three times a week until sacrifice). The "Prevention" group received topical resatorvid following an analogous dosing regimen. The "Intervention" group received vehicle 1hr prior to each UV treatment but switched to topical resatorvid three times a week only after UV was terminated. In the Prevention model, topical treatment with resatorvid significantly inhibited both tumor area (66% reduction, p = 0.0052) and tumor multiplicity (32% reduction, p = 0.0329; cross sectional Kruskal-Wallis test). In the Intervention model, a marked inhibition of both measures of tumor yield was observed, yet they did not reach statistical significance. Likewise, suppression of UV-induced TLR4-driven signaling was also confirmed in murine skin harvested at week 14 (prior to tumor onset), as assessed by IHC and RPPA analysis. Taken together, these data generated using resatorvid in a murine photocarcinogenesis model suggest that pharmacological TLR4 antagonism may represent a novel molecular strategy for topical prevention of solar UV-induced NMSC.

#2245

Evaluation of new rexinoids for lung cancer.

Di Zhang,1 Ana S. Leal,1 Sarah E. Carapellucci,1 Kayla Zydeck,1 Nicole Chaaban,1 Michael B. Sporn,2 Carl E. Wagner,3 Karen T. Liby1. 1 _Michigan State University, East Lansing, MI;_ 2 _Dartmouth Medical School, Hanover, NH;_ 3 _Arizona State University, Phoenix, AZ_.

Lung cancer remains the leading cause of cancer deaths around the world. It is estimated that there will be over 150,000 deaths in the United States alone from this disease every year. With no significant improvement in 5 year survival rates over the past 30 years, effective prevention or early intervention is a promising approach to reduce the high mortality of lung cancer. Rexinoids are selective ligands for retinoid X receptors (RXR), which regulate the expression of a wide variety of genes. As transcription factors, rexinoids play important roles in proliferation, differentiation, and apoptosis, which are highly relevant in cancer. Bexarotene is the only synthetic rexinoid that has been approved by the FDA and is used for the treatment of refractory cutaneous T-cell lymphoma. It has also been tested in various clinical trials for lung cancer and breast cancer. However, Bexarotene is not potent or selective enough for RXRs, causing limited efficacy and unacceptable toxicities. Therefore, we synthesized a series of new rexinoids and screened them for their ability to inhibit nitric oxide (NO) production in RAW264.7 macrophage-like cells stimulated with LPS. Several of these compounds inhibited NO production at nanomolar concentrations. Based on this screening and other in vitro assays, two new rexinoids were chosen for in vivo testing. Female A/J mice were injected i.p. with vinyl carbamate (16 mg/kg). One week later, the mice were fed control diet or rexinoids in diet (40-80 mg/kg diet) for 16 weeks. Tumor number, size and histopathology were then evaluated. All of the rexinoids reduced the number and size of the lung tumors. However, Bexarotene only reduced the average number of tumors by 8-17%, while a new pyrimidine-analog of Bexarotene—henceforth, pyrimidine-Bexarotene—reduced the number of tumors by 24-28%. The average tumor burden on lung sections was also reduced by pyrimidine-Bexarotene by 59% compared to the controls (from 0.47 ± 0.19 mm3 in the control group to 0.19 ± 0.05 mm3 in the treated group). In contrast, average tumor burden was 0.35 ± 0.08 mm3 in the group treated with Bexarotene, a reduction of 26%. Notably, the percentage of high grade tumors (both histological and nuclear characteristics) were significantly (p<0.05) higher in the mice fed Bexarotene (63%) than in the control group (38%), while the percentage of high grade tumors was only 34% in the pyrimidine-Bexarotene group. Since a major side effect of rexinoids is an elevation in triglyceride levels, we measured total triclycerides in both liver and plasma. Pyrimidine-Bexarotene reduced triclyceride levels in the liver (7.17 ug/kg tissue) compared to Bexarotene (8.23 ug/kg tissue) and plasma (3.6mg/ml vs. 4.8mg/ml, respectively; p<0.05). These studies suggest that new rexinoids can be synthesized that are superior to Bexarotene for prevention of lung cancer. Additional synthesis and testing of new rexinoids will be performed to further enhance the potency and selectivity of this promising class of drugs.

#2246

**Anti-proliferative activities of lipid fraction of extract from the skin of the catfish** Arius Bilineatus, Valenciennes **.**

Peiying Yang,1 Jibin Ding,1 Yong Pan,1 Yan Jiang,1 Mohammad Afzal,2 Bincy M. Paul,2 Sosamma O. George,3 Jassim M. Al-Hassan2. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Kuwait University, Safat, Kuwait;_ 3 _CMS College, Kottayam, Kerala, India_.

The catfish (Arius bilineatus, Val.) secretes a gelatinous substance composed of biochemically active lipids and proteins from its skin upon stress or injury. Preparations from the skin have previously been shown to affect blood clotting and accelerates healing of non-healing diabetic foot ulcers in man. We have reported previously fish skin preparation (CSP) derived from the skin of the Catfish and it's lipid fraction exerted anti-inflammatory activity in the in vitro cells and in vivo animal models. Here anti-proliferative effect of a lipid fraction of fish skin preparation (CSP-L) derived from the skin of the catfish and its plausible mechanism were investigated in human hepatocellular carcinoma (HCC) Hep3B and human pancreatic cancer Panc-1 cells. Cells were treated with CSP-L (0 to 100 μg/ml) for 72 hrs and cell proliferation was measured by the MTT assay. The results showed much stronger inhibition by CSP-L in Panc-1 cells than that of Hep3B cells with IC50 of 5.5 ± 1.4 μg/ml and 19.5 ± 7.7 μg/ml, respectively. Cell cycle analysis with PI staining suggested that CSP-L (25 - 100 μg/ml) led to G1 phase arrest in Hep3B cells whereas S phase arrest was observed in Panc-1 cells, suggesting the differential molecular mechanisms responsible for CSP-L induced cell growth suppression in Hep3B and Panc-1 cells. The alteration of cell cycle was concentration dependent. Additionally, CSP-L concentration dependently suppressed the invasion of Hep3B and Panc-1 cells. The molecular mechanism associated with CSP-L's anti-proliferative effect was examined by Western blotting in both Hep3B and Panc-1 cells. Intriguingly, CSP-L (50 and 100 μg/ml) notably decreased protein levels of cyclin D, Stat3, pRB and pERk in Hep3 B cells in a concentration dependent manner. In contrast, only pRB and pERK protein expression were reduced in CSP-L treated Panc-1 cells. These again suggest that CSP-L inhibited the proliferation of Hep3B and Panc-1 cells by different molecular mechanisms. Collectively, our preliminary data suggest that CSP-L has a great potential to be developed as an anticancer or preventive agent for both HCC or pancreatic cancer and warrants further investigation. This study was support by a grant from Kuwait Foundation for the Advancement of Science No. KFAS 2013-120701A-D, and Kuwait University Research Grant No. SL03/14.

## EPIDEMIOLOGY:

### Biomarkers of Risk and Prognosis

#2247

Associations between total T-lymphocytes and colorectal cancer survival in a prospective cohort study of older women.

Anna E. Prizment,1 Robert A. Vierkant,2 Thomas C. Smyrk,2 Lori S. Tillmans,2 Heather H. Nelson,1 Charles F. Lynch,3 Stephen N. Thibodeau,2 Timothy R. Church,1 James R. Cerhan,2 Kristin E. Anderson,1 Paul J. Limburg2. 1 _University of Minnesota, Minneapolis, MN;_ 2 _Mayo Clinic, Rochester, MN;_ 3 _University of Iowa, IA_.

Background: The increased number of T-lymphocytes residing in the tumors of colorectal cancer (CRC) patients is consistently shown to predict better survival of CRC patients independent of stage. In addition to tumor itself, T-lymphocytes also infiltrate normal tissues adjacent to tumors; however, little is known about how the inter-individual T-cell variability in tumor-adjacent tissues affects CRC prognosis. Our goal was to characterize total T-cells in colorectal tumor and tumor-adjacent tissues and study their associations with all-cause and CRC-specific survival in the Iowa Women's Health Study.

Methods: We constructed tissue microarrays and quantified CD3 antibody staining (Clone F.7.238; Dako), an established marker for total T-cells, in paraffin-embedded tissue samples from 463 women diagnosed with incident CRC from 1986-2002 (mean age at diagnosis was 74 years). Up to 3 tumor and 3 tumor-adjacent cores were immunostained for each person. An experienced pathologist quantified CD3+ T cells in each area using 4 categories: non-detected, mild (1-10 cells per 0.28 mm2); moderate (11-29 cells per 0.28 mm2); and strong infiltration (≥30 cells per 0.28 mm2). The obtained scores were averaged over all quantified cores for each person. Cox regression was used to estimate the hazard ratio (HR) and 95% CI for all-cause and CRC mortality in relation to (1) tumor score; (2) tumor-adjacent score; and (3) the ratio of tumor to tumor-adjacent score, hereafter called Score ratio. All scores were categorized in quartiles. The final model adjusted for age at diagnosis, SEER stage, tumor grade, body mass and smoking history.

Results: During follow-up for a maximum of 25 years, 67% of participants diagnosed with CRC died (~30% died from CRC). There was a weak but significant correlation between CD3 tumor and tumor-adjacent scores (Spearman coefficient: r=0.15, p=0.02). CD3 tumor score was inversely associated with stage and was higher in those with proximal colon cancer, and for MSI-high, CIMP-high and BRAF-mutated tumors. The HRs (95%CI) for the highest versus lowest category of CD3 tumor score were 0.61 (0.44-0.84) (p-trend=0.003) for all-cause mortality and 0.27 (0.15-0.48) (p-trend <0.0001) for CRC-specific mortality. The tumor-adjacent score was not associated with any characteristics or death. The associations between the Score ratio and all-cause and CRC survival mirrored those for the tumor score but were weaker. The HRs (95%CI) for the highest versus lowest quartile of the Score ratio were 0.70 (0.45-1.09) (p-trend=0.09) for all-cause and 0.45 (0.23-0.87) (p-trend=0.003) for CRC-specific mortality.

Conclusions. Based on data from prospectively identified CRC cases in the IWHS cohort, colorectal tumor T-cell infiltration is associated with improved survival. Further investigation is needed to determine the T-cell subtypes that are most predictive of CRC survival outcomes.

#2248

**Common** TDP1 **polymorphisms in relation to survival among small cell lung cancer patients in a multicenter study from the International Lung Cancer Consortium.**

Pawadee Lohavanichbutr,1 Lori C. Sakoda,2 Christopher I. Amos,3 Susanne M. Arnold,4 David C. Christiani,5 Michael P. Davies,6 John K. Field,6 Eric B. Haura,7 Rayjean J. Hung,8 Takashi Kohno,9 Maria Teresa Landi,10 Geoffrey Liu,11 Yi Liu,12 Michael W. Marcus,6 Grainne M. O'Kane,11 Matthew B. Schabath,7 Kouya Shiraishi,9 Stacey A. Slone,4 Adonina Tardón,13 Ping Yang,12 Kazushi Yoshida,9 Ruyang Zhang,5 Xuchen Zong,8 Noel S. Weiss,14 Chu Chen1. 1 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 2 _Kaiser Permanente, Oakland, CA;_ 3 _Geisel School of Medicine at Dartmouth, Lebanon, NH;_ 4 _University of Kentucky Markey Cancer Center, Lexington, KY;_ 5 _Harvard School of Public Health, Boston, MA;_ 6 _University of Liverpool, Liverpool, United Kingdom;_ 7 _Moffitt Cancer Center, Tampa, FL;_ 8 _Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada;_ 9 _National Cancer Research Institute, Tokyo, Japan;_ 10 _National Cancer Institute, Bethesda, MD;_ 11 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada;_ 12 _Mayo Clinic, Rochester, MN;_ 13 _University of Oviedo, Spain;_ 14 _University of Washington, Seattle, WA_.

Introduction: DNA topoisomerase inhibitors (e.g. etoposide, irinotecan, teniposide, topotecan) are commonly used for treating small cell lung cancer (SCLC). Tyrosyl-DNA phosphodiesterase (TDP1) plays a role in repairing DNA damage caused by DNA topoisomerase inhibitors and it is believed to be responsible for resistance to this class of drugs. The purpose of this study was to determine whether common TDP1 polymorphisms are associated with overall survival among SCLC patients.

Method: Two common TDP1 SNPs (rs942190 and rs2401863, r2(European)=0.63 and r2(East Asian)=0.26) were analyzed in 898 patients from 10 studies in the International Lung Cancer Consortium, conducted in the U.S., Canada, Japan, Italy, Spain and the United Kingdom. Genotype data were obtained from either the OncoArray Consortium or other existing genome-wide association studies. Cox proportional hazard regression models with robust standard error were used to evaluate genotype associations for overall survival at 36 months post-diagnosis, adjusting for age, sex, race and tumor stage.

Results: Patients homozygous for the minor allele (GG) of TDP1 SNP rs942190 were associated with poorer survival compared to those carrying both major alleles (AA), with a hazard ratio (HR) of 1.36 (95% confidence interval (CI): 1.07-1.72, p-value=0.01), but there was no association with survival for patients with the heterozygous genotype (HR=1.02, 95% CI: 0.83-1.27, p-value=0.83). For rs2401863, patients carrying both minor alleles (CC) tended to have better survival than patients carrying AA alleles, but this was within the limits of chance given no significant association (HR=0.79, 95% CI: 0.61-1.02, p-value=0.07). In addition, multi-tissue expression quantitative trait loci (eQTL) analyses from the Genotype-Tissue Expression Project indicated a potential effect of rs942190 on lung tissue, with higher TDP1 gene expression for the GG than AG or AA genotypes.

Conclusion: We found that among SCLC patients the TDP1 rs942190 GG genotype was associated with poor overall survival. Further study could determine if this genotype can serve as a predictive marker for the treatment efficacy of DNA topoisomerase inhibitors in SCLC.

#2249

Integrated multi-level omics-based biomarker models for ovarian cancer survival.

Alan Fu,1 Shen-Chih Chang,1 Aileen Baecker,1 Helena R. Chang,2 Zuo-Feng Zhang1. 1 _UCLA Fielding School of Public Health, Los Angeles, CA;_ 2 _David Geffen School of Medicine at UCLA, Los Angeles, CA_.

Background: Although a number of prognostic biomarkers for ovarian cancer have been proposed, the pool of clinically useful biomarkers remains small. While the predictive power of individual biomarkers may be weak, the advent of more affordable high-throughput technologies and readily available "omics" level data point toward the possibility of high performance biomarker panels. In this study, we used tumor-based somatic mutation, mRNA and miRNA expression, and DNA methylation data from The Cancer Genome Atlas (TCGA) to construct integrated biomarker models for overall and progression-free survival.

Methods: The somatic mutations, mRNA expression, and DNA methylation of 451 candidate genes, as well as the expression of the miRNAs targeting them, were individually analyzed for association with overall and progression-free survival in 488 TCGA serous cystadenocarcinoma patients of predominantly Caucasian descent. Following correction for multiple comparisons, molecular variations associated with survival were combined to construct integrated cross-validated prediction models for overall and progression-free survival. The discriminative power of these models was then evaluated for 1-, 3-, and 5-year overall and progression-free survival using ROC analysis.

Results: Our cross-validated integrated molecular models were demonstrated to predict 5-year overall survival in the total patient pool with a ROC AUC of 0.801 and 5-year overall and progression-free survival in Caucasian patients with ROC AUCs of 0.866 and 0.809, respectively.

Conclusions: Our findings suggest the potential utility of our multi-omics-based biomarker models in helping to inform clinical decisions following ovarian cancer diagnosis. Although extensive efforts were made to maximize the predictive potential of our models, further retrospective and prospective validation efforts are necessary before their clinical utility can be accurately assessed.

#2250

Systemic inflammation and sarcopenia predict colorectal cancer survival.

Elizabeth M. (Cespedes) Feliciano,1 Candyce H. Kroenke,1 Jeffrey Meyerhardt,2 Carla M. Prado,3 Patrick T. Bradshaw,4 Marilyn Kwan,1 Jingjie Xiao,3 Stacey Alexeef,1 Erin K. Weltzien,1 Adriemme L. Castillo,1 Bette J. Caan1. 1 _Kaiser Permanente Division of Research, Oakland, CA;_ 2 _Dana Farber Cancer Institute, MA;_ 3 _University of Alberta, Edmonton, Alberta, Canada;_ 4 _University of California, Berkeley, CA_.

Importance: A higher neutrophil-to-lymphocyte ratio (NLR, indicating systemic inflammation), and sarcopenia (reduced skeletal muscle mass) predict morbidity/mortality in a variety of cancers, but no prior research examines associations of pre-diagnostic NLR with at-diagnosis sarcopenia, nor whether NLR and sarcopenia combined identify early-stage patients with poor prognosis in colorectal cancer (CRC).Objective: To evaluate the association between pre-diagnosis NLR and at-diagnosis sarcopenia and of their combination with CRC survival, controlling for age, ethnicity, sex, body mass index, stage, and cancer site.

Design, Setting, and Participants: This observational cohort with prospectively-collected data included 2470 patients diagnosed with stage I-III CRC at Kaiser Permanente from 2006-2011 with computed tomography (CT) scans from clinical care (mean days pre-diagnosis=6).

Exposures: Systemic inflammation measured via average NLR in the 24 months pre-diagnosis (mean count=3 measures, mean months pre-diagnosis=7). The reference value was NLR below 3, indicating low/no inflammation.

Main Outcomes and Measures: Sarcopenia, defined by published cutoffs for skeletal muscle index (CT muscle area in cm2 at the third lumbar vertebra divided by squared height in m2; below 52-cm2/m2 and 38-cm2/m2 for normal/overweight men and women, respectively, and below 54-cm2/m2 and 47-cm2/m2 for obese men and women, respectively), and incident death (overall or CRC-related).

Results: Average age was 63 years; half of patients were female. NLR above 3 and sarcopenia were common (46% and 44%, respectively). Over a median of 6 years, we observed 656 deaths, 357 from CRC. Elevated NLR was associated with sarcopenia in a dose-response manner (compared to NLR below 3, Odds Ratio [OR]=1.35; 95%CI:1.10-1.67 for NLR 3-5; OR=1.47; 95%CI:1.16-1.85 for NLR above 5). NLR above 3 and sarcopenia were also independently associated with survival (Hazard Ratio [HR]=1.65; 95%CI:1.45-1.98 for overall death and HR=1.29; 95%CI:1.09-1.53 for CRC death); patients with both sarcopenia and NLR above 3 (versus neither), had double the risk of death overall (HR=2.53; 95%CI:1.87-3.41) and from CRC (HR=2.19; 95%CI:1.74-2.75).

Conclusions and Relevance: Host inflammatory/immune response shortly prior to diagnosis predicts muscularity at diagnosis. Low muscle combined with systemic inflammation predicts worse CRC prognosis regardless of stage. A better understanding of the crosstalk between inflammatory/immune responses and the onset of changes in skeletal muscle may open new therapeutic avenues to improve cancer outcomes.

#2251

Tumor tissue gene expression in association with survival of triple-negative breast cancer.

Shuyang Wang,1 Qiuyin Cai,1 Hui Cai,1 Pingping Bao,2 Jie Wu,1 Fei Ye,1 Wei Zheng,1 Ying Zheng,2 Xiao-Ou Shu1. 1 _Vanderbilt University School of Medicine, Nashville, TN;_ 2 _Shanghai Municipal Center for Disease Control and Prevention, Shanghai, China_.

Triple-negative breast cancer (TNBC) is an aggressive type of cancer with limited treatment options. Previous studies have shown that gene expression profiles were associated with TNBC prognosis. Information on specific genes that are predictive of TNBC outcome is limited. Using data and samples from a cohort of 469 TNBC cases from Shanghai Breast Cancer Survival Study (SBCSS), we systematically evaluated expressions of 302 genes in tumor tissue with survival of TNBC. Genes included in the study are PAM50 genes, genes encoding drug metabolizing enzymes and, genes implicated in TNBC biology and progression based on literature reviews. Gene expression levels were measured in total RNA isolated from formalin-fixed paraffin-embedded breast cancer tissues using the NanoString nCounter assay. Cox regression was applied to evaluate disease-free survival (DFS) and overall survival (OS) in association with gene expression. Analysis was adjusted for known predictors of TNBC outcome, including age, disease stage, basal like subtype and DUSP4 gene expression. During a median follow-up of 5.3 years (range: 0.7-8.9 years), 100 deaths and 92 recurrences/breast cancer deaths were documented. Expression levels of 17 genes were significantly associated with OS, and 15 genes with DFS (P<0.05). The top 5 most significant genes are EOMES (Hazard Ratio, HR = 0.88; 95% Confidence Interval, 95% CI: 0.82-0.96), GZMK (HR = 0.91, 95% CI: 0.85-0.97), TUBB3 (HR = 1.12, 95% CI: 1.03-1.21), SIT1 (HR = 0.90, 95% CI: 0.83-0.98) and ZNF224 (HR = 0.89, 95% CI: 0.81-0.97) for OS, and TUBB3 (HR = 1.14, 95% CI: 1.05-1.24), KRT14 (HR = 1.08, 95% CI: 1.02-1.14), BAG1 (HR = 1.19, 95% CI: 1.05-1.35), CD44 (HR = 0.73, 95% CI: 0.58-0.92) and EOMES (HR = 0.89, 95% CI: 0.82-0.97) for DFS. Study is on-going to replicate the findings of this study.

#2252

Microbial profiling of the head and neck tumor microenvironment as a biomarker of clinical response to chemoradiation.

Christine M. Pierce Campbell,1 Bo-Young Hong,2 Maria F. Gomez,1 Blake M. Hanson,2 Erica Sodergren,2 Sybil T. Sha,3 Jeffery S. Russell,1 George M. Weinstock2. 1 _Moffitt Cancer Center, Tampa, FL;_ 2 _The Jackson Laboratory for Genomic Medicine, Farmington, CT;_ 3 _Cornell University, Ithaca, NY_.

PURPOSE: Despite advances in cancer treatment, patients with advanced head and neck cancer (HNC) have a median overall survival of <1 year and 30% cure rate. Evidence suggests that microbiota may modulate the response to therapy through a crosstalk with cancer cells, immune cells, and inflammatory mediators. This study was conducted to profile the tumor microbiome of HNC patients to determine whether bacteria and fungi in the tumor microenvironment are associated with the clinical response to chemoradiation.

METHODS: Fresh frozen tumor tissues from 30 newly diagnosed, treatment-naïve patients who received chemoradiation at Moffitt Cancer Center for squamous cell HNC were evaluated. Genomic DNA was extracted from 25mg of tissue. The V1-V3 hypervariable region of the bacterial 16S rRNA gene and fungal internal transcribed spacer (ITS) 1 region were sequenced using a PCR amplicon-mediated workflow on the Illumina MiSeq. 16S sequences were clustered into operational taxonomic units and taxonomic identification was determined using the RDP database and ITS sequences were classified using an in house reference database. Patients' charts were reviewed to ascertain clinical response 3, 6 and 12 months after treatment cessation. Response was dichotomized as complete (no evidence of disease) and incomplete (partial response, stable disease, and progressive disease).

RESULTS: At the time of tumor collection, patients ranged in age from 20-79 years, and were predominantly men (83%) and current smokers (40%). The most common anatomic site was the oral cavity (70%) followed by the larynx (20%) and oropharynx (10%), and most were diagnosed at stage 4 (87%). Three, 6 and 12 months after treatment cessation, 4 (14%), 9 (36%), and 10 (40%) patients had an incomplete response to therapy, respectively. Bacterial sequencing was successful in 29 (97%) samples and fungal in 15 (50%). Clustering analyses of bacterial abundance profiles suggested that 4 types of communities existed, each dominated by Fusobacterium, Prevotella, Streptococcus, or Treponema. HNC patients with incomplete clinical responses (3, 6, or 12 months after treatment) were more likely to have treatment-naïve tumors dominated by Fusobacterium (cluster 1; 56%) than any other genus, whereas patients with complete clinical responses were more likely to have treatment-naïve tumors dominated by Prevotella (cluster 2; 40%) than any other genus. None of the patients with incomplete responses were dominated by Streptococcus (cluster 3) or Treponema (cluster 4). Fungal ITS profiling revealed that the tumor microbiome was largely dominated by the fungi Malassezia.

CONCLUSIONS: The current study demonstrates that intratumoral bacterial profiles dominated by Prevotella may serve as a biomarker of improved prognosis, while profiles dominated by Fusobacterium may indicate a poor prognosis following chemoradiation in HNC patients.

#2253

5 Gene prognostic signature for dedifferentiated liposarcomas.

Deanna Ng, Nicholas Shannon, Johnny Ong, Melissa Teo. _National Cancer Centre Singapore, Singapore, Singapore_.

Introduction: Liposarcomas, despite being rare consist of a number of entities spanning low to high grade sarcomas. De-differentiated liposarcoma (DDLS) is associated with increased frequency of recurrence and lower overall survival. We aimed to identify gene expression changes associated with OS in DDLS. Secondary aims was to postulate a mechanism for DDLS.

Methods: We analysed data from gene expression omnibus (GEO, ID = GSE30929) to identify genes associated with DDLS (n=39). A signature representing genes overexpressed in DDLS was then applied to an independent dataset, utilising data from The Cancer Genome Atlas (TCGA, n=58 DDLS) to segregate samples based on gene expression and groupings compared for survival.

Results: A 5 gene signature was generated consisting of genes with increased expression in DDLS and its relation to OS. The signature was significantly enriched for genes involved in lipid metabolism (AQP7, PRKAR2B, FABP4 p<0.01) and genes involved in the cell cycle and cell proliferation (FBN2, CEBPA p<0.01). Segregation of DDLS in the TCGA cohort on the basis of median gene signature score gave a significant difference in overall survival. High score (stronger DDLS signature) was associated with a lower median survival(33.5 vs 76.4 months, p = 0.027), and poorer 2 year (63.4% vs 89.2%) and 5 year survival (20.6% vs 71.1%). The survival difference remained significant when applied to retroperitoneal DDLS only (n=51, median survival 34.5 vs 54.2 months, p = 0.034) and poorer 2 year survival (. A postulated mechanism is that DDLS have gained deficiency in AQP7, PRKAR2B and FABP4 leading to fatty acid and glycerol accumulation in cells, increasing cancer cell replication. CEBPA inhibits CDK4 and a deficiency of CEBPA in DDLS leads to up regulation of CDK4, increasing cell proliferation. An upregulation of FBN2 in DDLS up regulates TGF-β1, increasing cell proliferation.

Conclusion: Gene expression changes associated with dedifferentiated liposarcomas include changes in cell cycle checkpoint regulation. The signature is associated with poorer overall survival in DDLS and can be used to prognosticate patients.

#2254

Reactive oxygen species modulator 1 (Romo1) predicts unfavorable prognosis in colorectal cancer patients.

Min Jee Jo, Hong Jun Kim, Suk-Young Lee, Dae-Hee Lee, Sang Cheul Oh. _Korea University Guro Hospital, Seoul, Republic of Korea_.

Reactive oxygen species modulator 1 (Romo1) is a novel protein that has been reported to be crucial for cancer cell proliferation and invasion. However, its clinical implications in colorectal cancer (CRC) patients are not well-known. For the first time, we investigated the association between Romo1 and the clinical outcomes of CRC patients. We examined Romo1 expression in resected tumor tissues immunohistochemically and assessed it with histological scores. We conducted survival analyses for patients who had curative resection (n=190) in accordance with clinical parameters including level of Romo1 expression, and we examined the association between Romo1 expression and cell invasion using Matrigel invasion assay in CRC cell lines. We observed significantly longer mean disease-free survival (DFS) in the low Romo1 group compared with the high Romo1 group (161 vs 127.6 months, p=0.035), and the median overall survival (OS) of the low Romo1 group was significantly longer than that of the high Romo1 group (196.9 vs 171.3 months, p=0.036). Cell invasiveness decreased in the Romo1 knockdown CRC cells in contrast to the controlled cells. Romo1 overexpression in tumor tissue was associated with a high lymph node ratio (LNR) between the metastatic and examined lymph nodes (p=0.025). Romo1 overexpression in tumor tissue was significantly associated with survival in curatively resected CRC patients, suggesting Romo1 expression as a potential adverse prognostic marker. Increased Romo1 expression was found to be associated with high LNR. Cancer invasiveness appeared to be a key reason for the poor survival related to highly expressed Romo1.

#2255

SMAD4 loss is associated with poor prognosis in Middle Eastern colorectal cancer.

Maqbool Ahmed, Abdul K. Siraj, Tariq Masoodi, Rong Bu, Fouad Al-Dayel, Khawla S. Al-Kuraya. _King Faisal Specialist Hospital & Res. Ctr., Riyadh, Saudi Arabia_.

Background: Colorectal cancer (CRC) is major cause of morbidity and mortality in cancer patients worldwide and it ranks as the most common cancer in males and the third most common among females in Saudi Arabia. Alteration in TGF-β pathway has been reported in many cancers. So In search for new molecular targets we analyzed the role of SMAD4, critical TGF-β pathway mediator, in Saudi colorectal cancers

Methods: 426 CRC cases were analyzed for SMAD4 mutations by targeted capture sequencing and protein expression status by immunohistochemistry in a tissue microarray format. Results were analyzed for association with any clinico-pathological parameters and for prognostic significance.

Results: SMAD4 mutations were detected in 11.9 %( 50/418) cases of Saudi colorectal cancers. CRC with SMAD4 mutations were significantly associated with NRAS mutations (p=0.0481). SMAD4 complete loss of expression by immunohistochemistry was seen in 12.8% (52/404) cases and this loss was associated with young age (p=0.0236) and inversely with MSI-high tumors (p=0.0005). SMAD4 expression loss by immunohistochemistry and mutation were significantly associated with each other

(p=0.0028). In the multivariate analysis using the Cox proportional hazard model SMAD4 expression loss was an independent marker of poor prognosis (p=0.0119)

Conclusion: SMAD4 alteration was found to be associated with a poor overall survival in the Saudi colorectal cancer patients. With the advent of targeted anticancer therapy, this study confirms the findings of other investigators and highlights a potential target which can be exploited to improve the survival of CRC patients in the Kingdom of Saudi Arabia

#2256

Semaphorin 3C has a possibility of a new prognostic marker in colorectal cancer.

Shingo Kawano,1 Kota Amemiya,1 Yuki Tsuchiya,1 Toshiaki Hagiwara,1 Hirokazu Matsuzawa,1 Yurika Makino,1 Shunsuke Motegi,1 Ryoichi Tsukamoto,1 Ryosuke Ichikawa,1 Kazumasa Kure,1 Kumpei Honjo,1 Hisashi Ro,1 Shingo Ito,1 Yu Okazawa,1 Rina Takahashi,1 Kosuke Mizukoshi,1 Masaya Kawai,1 Shinya Munakata,1 Koichiro Niwa,1 Shun Ishiyama,1 Kiichi Sugimoto,1 Hirohiko Kamiyama,1 Hiromitsu Komiyama,1 Makoto Takahashi,1 Yutaka Kojima,1 Michitoshi Goto,1 Atsushi Okuzawa,1 Yuichi Tomiki,1 Kazuhiro Sakamoto,1 Motohiro Kojima,2 Atsushi Ochiai2. 1 _Juntendo University, Tokyo, Japan;_ 2 _National Cancer Center Hospital East, Chiba, Japan_.

Background: Generally, Semaphorins are secretary or transmembrane-bound molecules that act as axon guidance cues in the nervous system. Recent research showed increased expression of semaphorin 3C correlates with cancers that possess higher invasive and metastatic characteristics. For example, in breast cancer, inhibition of semaphorin 3C reduces adhesion and invasion. The aim of this study was to evaluate a possibility that semaphorin 3C might be a new prognostic marker in colorectal cancer.

Material and methods: We used two cohorts. Cohort 1 was the 192 patients with colorectal cancer resected surgically between 2009 and 2010. We used GSE 14333 dataset as Cohort 2 which included 226 patients with the colorectal cancer. In each cohorts, we divided the patients in to the two groups i.e., high or low semaphorin 3C expression group using receiver operating characteristic (ROC) curve based on the information of the recurrence of colorectal cancer. Disease-free-survival (DFS) rates were calculated using Kaplan-Meier methods. Differences between curves were evaluated with the log-rank test.

Results: In the cohort 1, the DFS in the patients with higher expression was worse than that in the patients with lower expression (P = 0.0467). Semaphorin 3C has two probes (203788_s_at and 203789_s_at) in GSE14333. In the cohort 2, using each two probe of semaphorin 3C, the DFS in the patients with higher expression was worse than that of those patients with lower expression (P = 0.0015 and P = 0.0003).

Conclusions: This study suggested that Semaphorin 3C might be a new prognostic marker in colorectal cancer.

#2257

Pre-diagnostic sex hormone levels and breast cancer survival in the Nurses' Health Study.

Kevin H. Kensler,1 A. Heather Eliassen,2 Bernard A. Rosner,2 Susan E. Hankinson,3 Myles Brown,4 Rulla M. Tamimi2. 1 _Harvard T. H. Chan School of Public Health, Boston, MA;_ 2 _Brigham and Women's Hospital, Boston, MA;_ 3 _University of Massachusetts School of Public Health and Health Sciences, Amherst, MA;_ 4 _Dana-Farber Cancer Institute, Boston, MA_.

Background The role of estrogens has been well established in the development and progression of breast cancers, while an independent function of androgens remains to be fully elucidated. There is consistent evidence that increased levels of circulating sex steroid hormones predict subsequent breast cancer risk. However, few studies to date have evaluated circulating sex hormone levels in relation to breast cancer prognosis and the limited available evidence suggests that higher estrogen and androgen levels are associated with worse prognosis. Identifying the distinct roles of estrogens and androgens and their hormone receptor signaling pathways in breast cancers may inform novel approaches to breast cancer prevention and treatment.

Methods We evaluated the association between pre-diagnostic circulating sex hormones and breast cancer survival among 683 breast cancer cases from the Nurses' Health Study (NHS) cohort. The NHS began in 1976 with the enrollment of U.S. registered nurses between the ages of 30 and 55. The women in this analysis provided a blood sample in 1989-1990, were postmenopausal and not using menopausal hormone therapy at the time of blood draw, and were diagnosed with invasive breast cancer between 1990 and 2010. Levels of estradiol, estrone, estrone sulfate, testosterone, androstenedione, DHEA, DHEAS, and SHBG were measured in plasma. The association between hormone levels and survival was assessed through construction of Kaplan-Meier survival curves and through use of Cox proportional hazards models to estimate hazard ratios and 95% confidence intervals adjusted for patient, tumor, and treatment covariates.

Results Over follow-up through 2014, a total of 219 deaths occurred, including 57 breast cancer deaths. For each hormone, we found no association with breast cancer survival by the log-rank test. Upon adjustment for patient, tumor, and treatment covariates, we observed non-significant 47% increases in breast cancer mortality per unit increase in log estradiol (HR=1.47, 95% CI 0.94-2.31) and estrone sulfate (HR=1.47, 95% CI 0.97-2.24). Log testosterone was not associated with breast cancer survival (HR=0.84, 95% CI 0.46-1.53) and the findings were null for the other hormones. These findings were robust when evaluating overall survival and when stratifying by tumor estrogen receptor expression.

Conclusions We observed no significant associations between pre-diagnostic sex hormone levels and breast cancer survival among postmenopausal women. This is the first analysis to evaluate pre-diagnostic hormone levels in relation to survival. Though limited by sample size, it improves upon past analyses of post-diagnostic hormone levels by reducing the possibility of the tumor affecting hormone levels and by better accounting for tumor and treatment characteristics. The findings should be replicated in a larger population to further assess the prognostic value of circulating sex hormones in breast cancer survival.

#2258

Genome-wide association studies of breast cancer prognosis.

Marjanka K. Schmidt,1 Qi Guo,2 Thilo Dörk,3 Diana Eccles,4 Renske Keeman,1 Jacques Simard,5 Peter Kraft,6 Douglas F. Easton,2 Paul D. Pharoah,2 on behalf of the Breast Cancer Association Consortium2. 1 _Netherlands Cancer Institute, Amsterdam, Netherlands;_ 2 _University of Cambridge, Cambridge, United Kingdom;_ 3 _Hannover Medical School, Hannover, Germany;_ 4 _University of Southampton, Southampton, United Kingdom;_ 5 _Laval University, Québec City, Quebec, Canada;_ 6 _Harvard School of Public Health, Boston, MA_.

Introduction:

The prognosis and response to treatment in women with breast cancer varies considerably after taking into account clinic-pathological variables. Other factors such as host genotype are also likely to be important. We therefore investigated the role of germline genetic variation on survival after breast cancer using data from the Breast Cancer Association Consortium.

Materials and methods:

We included data from ten projects in which breast cancer case cohorts were genotyped using arrays providing genome-wide coverage of common variants. The majority of samples was genotyped using the Illumina custom iCOGS or OncoArray chips. Data were imputed using the 1000 Genome phase 3 as a reference panel using a two-stage procedure. We assessed the association between genotype and 10-year breast cancer specific survival for each SNP using Cox proportional hazards regression adjusted for principle components and stratified by country. Genotype data from 87,877 breast cancer cases, 63,170 ER-positive and 20,297 ER-negative, with 6,511 known breast cancer deaths were included in the analyses.

Results:

Preliminary analyses shows that in all invasive cases, ER-positive and ER-negative disease 123, 110, and 212 variants respectively, were associated with breast cancer-specific mortality at P<5x10-5. A single variant, rs1295683, was associated with higher mortality at genome-wide significance in all patients: HR=1.16 (95%CI:1.10-1.22); P=2.8x10-8 (risk allele frequency: 0.12). We identified four variants to be associated with mortality in ER-negative disease at P<5x10-8. The strongest association was for rs145963877: HR=1.32 (95%CI:1.20-1.46); P=2.3x10-8. No variant reached genome-wide significance for ER-positive disease; the most significant variant was chr7:41400313 with an HR=1.17 (95%CI:1.10-1.24); P=1.8x10-7. In addition, using OncoArray data, we provided independent validation of higher mortality of CHEK2 c.1100delC carriers compared with non-carriers (HR=1.56 (95%CI:1.22-2.01)), previously reported in Weischer et al JCO 2012. Using iCOGS data, we evaluated the association between mortality and the polygenic risk score (PRS) based on 77 known breast cancer susceptibility SNPs. A higher PRS was significantly associated with lower breast cancer-specific mortality: HR=0.87(95%CI:0.81-0.93) (per unit of PRS) with a similar association in ER-negative and ER-positive disease. The association was attenuated after adjusting for tumor grade.

Conclusion:

We have identified a novel set of germline genetic variants that are associated with breast cancer prognosis. The effect sizes are small for each of the variants and unlikely to be of immediate clinical relevance. However, understanding of the biology that underlies the associations may identify novel pathways and targets for therapy. Being at higher risk of breast cancer, as defined by the breast cancer susceptibility PRS, is not associated with an adverse prognosis.

#2259

Evaluation of polymorphisms in myeloid-associated genes and glioma survival.

Daniel I. Jacobs,1 Yanhong Liu,1 Konrad Gabrusiewicz,2 Spiridon Tsavachidis,1 E. Susan Amirian,1 Georgina N. Armstrong,1 Renke Zhou,1 Jun Wei,2 Cristina Ivan,2 George Calin,2 Michael Scheurer,1 Anna Dahlin,3 Terri Rice,4 Paige M. Bracci,4 Helen M. Hansen,4 John K. Wiencke,4 Margaret R. Wrensch,4 Beatrice Melin,3 Amy B. Heimberger,2 Melissa L. Bondy1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _Umeå University, Umeå, Sweden;_ 4 _University of California, San Francisco, San Francisco, CA_.

BACKGROUND: Gliomas are highly infiltrated by immune cells including microglia, macrophages, and myeloid-derived suppressor cells (collectively, glioma-associated myeloid cells). These cells have been shown to be induced by the tumor to be immune-suppressive and tumor-supportive, and are a negative prognosticator for survival in mouse models. Here, we examine whether inherited variants in genes important to the function of glioma-associated myeloid cells are associated with survival following low-grade glioma diagnosis.

METHODS: Subjects for this study were 484 patients with WHO grade II or grade III glioma treated at The University of Texas MD Anderson Cancer Center in Houston, Texas between 1992 and 2008 and followed up for survival through August, 2016. We selected 100 genes for analysis including transcription factors, cytokines and chemokines, receptors, enzymes, and other genes central to the function of glioma-associated myeloid cells. Genotyping was originally performed using the Illumina Human 610-Quad Bead Chip platform and 2,040 tagging SNPs as determined by Haploview Tagger software were selected for analysis with minor allele frequency (AF) ≥ 1%. Associations between selected SNPs and survival were evaluated by Cox regression analysis under an additive allelic model adjusting for age, sex, extent of surgery (biopsy only/partial resection/gross total resection), radiotherapy (yes/no), and chemotherapy (yes/no). Models were examined to ensure that proportional hazards assumptions were not violated.

RESULTS: Median survival among low-grade glioma patients was 6.7 years. Age at diagnosis, extent of surgery, and having received radiotherapy or chemotherapy were each significantly associated with survival. Five SNPs were associated with survival at a significance level of p<0.001, and two remained significantly associated with low-grade glioma survival after adjustment for multiple comparisons (FDR-adjusted p-value (q)<0.10). These results indicated inferior survival for carriers of the C allele (AF=1.4%) at rs147960238 in CD163 (HR=5.47, 95% CI: 2.49-11.99, p=2.23x10-5, q=0.046) and for carriers of the G allele (AF=3.8%) at rs17138945 in MET (HR=2.27, 95% CI: 1.52-3.38, p=5.61x10-5, q=0.057). These SNPs are located in the 10th and 2nd introns of CD163 and MET, respectively.

CONCLUSIONS: Here we provide preliminary evidence of an association between polymorphisms in two genes related to glioma-associated myeloid cell function and low-grade glioma survival. CD163 is a receptor that is highly expressed on macrophages and may play a role in macrophage-mediated anti-inflammatory responses, while MET is a receptor tyrosine kinase and well-studied proto-oncogene that is also involved in the expansion of myeloid-derived suppressor cell populations. Further investigation of these associations is warranted, and validation of these findings is planned in an independent population.

#2260

Haplotypes of chromosome 17q21.31 affect liver metastasis by small intestinal neuroendocrine tumors.

Shinta Kobayashi,1 Edaise da Silva,2 Tanupriya Contractor,3 Laura Tang,2 Chris R. Harris3. 1 _Institute for Advanced Study, Princeton, NJ;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 3 _Raymond and Beverly Sackler Foundation, New Brunswick, NJ_.

Neuroendocrine tumors are the most common tumors of the small intestine. Very little is known about genetic changes that cause SI-NETs, which have a very low frequency of mutations. SI-NETs are highly metastatic to the liver, and patients with liver metastases have a five year survival rate of less than 50%. Caucasian patients are more likely to develop metastasis than patients of other races. We have studied a 900 kB inversion at chromosome 17q21.31, which is common to Caucasians but rare in other races. Within a collection of 117 Caucasian patients with ileal carcinoids, patients with the inversion haplotype were significantly less likely to have liver metastasis. A second Caucasian-specific haplotype in this region, beta2, was also tested for potential association with metastasis of ileal carcinoids. Unlike the inversion haplotype, beta2 associates with increased metastasis of ileal carcinoids. We suspect that the fact that the metastasis-promoting beta2 haplotype is more common among Caucasians than the metastasis-suppressing inversion haplotype (44% incidence vs. 33% incidence) is probably why Caucasians wind up with a higher frequency of metastasis than races that carry neither beta2 nor the inversion.

#2261

Synergistic effect and VEGF/HSP70-hom haplotype analysis: Relationship to prostate cancer risk and clinical outcome.

Sana Sfar,1 Hamadi Saad,2 Faouzi Mosbah,3 Lotfi Chouchane1. 1 _Faculty of Medicine, Monastir, Tunisia;_ 2 _EPS Fattouma Bourguiba, Monastir, Tunisia;_ 3 _EPS Sahloul, Sousse, Tunisia_.

Prostate cancer (PCa) is a complex disorder resulting from the combined effects of multiple environmental and genetic factors. Our previous single-locus analysis showed that VEGF and HSP70-hom polymorphisms were significantly associated with PCa susceptibility and prognosis. Both genes encoding these proteins were located on chromosome 6p21, and combining the neighboring single nucleotide polymorphisms (SNPs) into haplotypes may increase the association with the disease. Three tagging polymorphisms, the HSP70-hom 2437 T/C, the VEGF-1154 G/A, and the VEGF-634 G/C SNPs were genotyped in 101 cases and 80 controls. For the combined analysis of VEGF and HSP70-hom, we found a positive gradient in the odds ratios (ORs) related to the number of high-risk genotypes with a 3.53-fold increase of prostate carcinoma risk (OR= 3.53; p= 0.015). Furthermore, the TAG and CAG haplotypes at positions HSP70-hom, VEGF-1154 and VEGF -634 exhibited a two-fold (OR= 0.46; p= 0.014) and a seven-fold (OR= 0.14; p= 0.00005) reduction in PCa risk, respectively. Regarding PCa prognosis, the TAG haplotype had a negative association with the aggressive phenotype as defined by the histopathological grade (OR= 0.28; p= 0.006). Our findings confirm the role of at-risk haplotype across the HSP70-hom/VEGF gene cluster in determining susceptibility to PCa.

#2262

Correlation between number and sites of metastases and differential pretreated EBV DNA load profiles and their effects on outcomes in patients with metastatic nasopharyngeal carcinoma.

Cheng-Lung Hsu, Hung-Ming Wang, Tung-Liang Lin, Yung-Chia Kuo. _Chang Gung Memorial Hospital, Taipei, Taiwan_.

Differential overall survival of different organ site metastases in nasopharyngeal carcinoma (NPC) had been reported but the underlying mechanism was unclear. We try to find out the possible mechanism from plasma and tumor tissue markers. Total 178 patients of metastatic NPC were enrolled. Their pre-treatment plasma EBV DNA concentrations and cytokines and tissue macrophage, proliferation and apoptosis markers were determined. The overall patient response rate after treatment was 51.7% and median overall survival (OS) was 19 months. Single organ site metastasis had better outcome than multiple organs involved (median OS: 26 months vs. 16 months) with statistical significance. Among single organ site involved, pure lung metastasis had longer survival than bone or liver involved (median OS: 50 months vs. 21 months vs. 18 months) with P<0.001. Pretreatment plasma EBV DNA concentrations were lower in patients with lung metastasis than bone or liver metastasis in single organ involved group. Plasma IP-10 and MCP-1 expression level correlated with the differential single organ site metastasis OS and EBV DNA load. Liver metastatic tissue had higher macrophage infiltration density and higher proliferation index than lung metastatic group. In single organ site metastasis of NPC, lung involved patients had better outcome than bone or liver metastasis. Low pretreatment plasma EBV DNA load, cytokines expression such as IP-10 and MCP-1, tissue macrophage infiltration, and proliferation index may contribute these results.

#2263

Neonatal hormone levels and risk of testicular germ cell tumors (TGCT).

Libby Morimoto,1 David Zava,2 Katherine McGlynn,3 Frank Stanczyk,4 Joseph Wiemels,5 Xiaomei Ma,6 Catherine Metayer1. 1 _UC Berkeley, Berkeley, CA;_ 2 _ZRT Laboratory, Beaverton, OR;_ 3 _National Cancer Institute, Bethesda, MD;_ 4 _University of Southern California, Los Angeles, CA;_ 5 _UCSF, San Francisco, CA;_ 6 _Yale University, New Haven, CT_.

Testicular germ cell tumors (TGCT) are the most commonly occurring cancers in adolescent and young adult males in the U.S. Steroid sex hormones play a central role in the development of the testis. As proposed by the testicular dysgenesis syndrome hypothesis, the origins of TGCT are likely to be in utero or early in life, and to be a manifestation of disturbed prenatal testis development. However, no studies have provided direct, empirical evidence to date. Using an innovative linkage between the California birth records and cancer registry data, we conducted a population-based case-control study of neonatal hormones levels and risk of TGCT diagnosed at 0-19 year of age. We obtained archived neonatal dried blood spot (DBS) specimens from 370 TGCT cases (276 adolescent and young adults [AYA] aged 15-19 yrs at diagnosis; 94 0-4 yrs at diagnosis), and 344 age- and race/ethnicity-matched controls, born between 1982 and 2009. Liquid chromatography with tandem mass spectrometry was used to measure a panel of 17 sex steroids, glucocorticoids, and mineralcorticoids; 12 were present at detectable levels in the newborn DBS samples, including estrone (E1), estradiol (E2), estriol (E3), testosterone (T), dehydroepiandrosterone (DHEA), and androstenedione (A4). Logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI), adjusting for matching factors and age (in hours) of child at blood spot collection. A4, a precursor for T and E1, was positively associated with TGCT (OR: 1.71, 95% CI: 1.09-2.69). Analyses stratified by age group showed that this association was limited to AYA, and was of stronger magnitude in this group (OR: 2.33, 95% CI: 1.37-3.97). A similar, though weaker, trend was observed for T (ORoverall: 1.37, 95% CI: 0.86-2.19; ORAYA: 1.73. 95% CI: 1.00-3.00). There was no significant association of the other measured hormones with risk. In the first case-control study of TGCT with direct measures of neonatal hormone levels, we found that higher levels of T and A4 were associated with increased risk of TCGT, particularly among males diagnosed at 15-19 years of age. These results oppose the dominant theory in TGCT etiology, that TGCT is related to androgen insufficiency in utero, and provides an important link in the etiologic pathway of this increasingly common cancer.

#2264

Telomere biology gene methylation and cancer risk.

Brian T. Joyce,1 Yang Li,1 Yinan Zheng,1 Lei Liu,1 Hushan Yang,2 Chad Achenbach,1 Pantel Vokonas,3 Joel Schwartz,4 Andrea Baccarelli,5 Lifang Hou1. 1 _Northwestern University, Chicago, IL;_ 2 _Thomas Jefferson University, Philadelphia, PA;_ 3 _VA Boston Healthcare System, Boston, MA;_ 4 _Harvard University, Boston, MA;_ 5 _Columbia University, New York, NY_.

Background: Telomeres maintain genomic stability and regulate cellular senescence. Numerous studies have examined blood telomere length (BTL) and cancer risk, including our own which previously identified a dynamic relationship between BTL and cancer risk over time: Relative to subjects who remained cancer-free, those who later developed cancer experienced accelerated telomere shortening until 3-4 years prior to cancer diagnosis/censoring, at which point BTL in the incident cancer group stabilized. Other studies have suggested that cancer cells are able to hijack telomere maintenance mechanisms (e.g., telomerase) in order to circumvent the Hayflick limit, which will otherwise induce senescence and limit cancer cells' ability to multiply. As DNA methylation dysregulation is a known epigenetic hallmark of cancer, our objective was to understand whether DNA methylation of telomere biology genes is associated with cancer risk and BTL.

Methods: We studied 915 blood measurements from 491 subjects in the Normative Aging Study taken between 1999 and 2013, who were cancer free in 1999 and randomly selected for whole-epigenome DNA methylation profiling using the Illumina 450K BeadChip array. Among these subjects, 125 cases developed cancer and 366 controls remained cancer free for our entire follow up (median 10.1 years). Our analysis focused on 2,731 CpG sites on 142 genes related to telomere length maintenance. We used linear models to identify CpGs differentially methylated by cancer status at the first blood draw only, followed by mixed models using our full data to determine the final CpG sites of interest for Cox models of cancer risk, and another set of mixed models to compare trajectories over time of methylation in cancer patients vs. controls. Finally an additional mixed model explored the temporal relationship between methylation measured at the first visit and BTL measured at the second visit.

Results: Our screening identified 167 CpG sites, 10 of which were associated with time-dependent cancer risk at FDR <0.05. Methylation of one CpG on each of CDC73, NSMCE2, and RPA1 was associated with decreased cancer risk. Methylation of one CpG on each of DCLRE1C, DDB1, H2AFY, HNRNPA2B1, and TEP1, as well as two CpGs on TERF2, was associated with increased cancer risk. DNA methylation trajectory at three CpGs was significantly different across cancer status (cg20772347 on DDB1, cg23157637 on TEP1, and cg04818274 on TERF2). One CpG on NSMCE2 and one on TERF2 were associated with BTL in cancer-free subjects, while another CpG on TERF2 was associated with BTL in subjects who later developed cancer.

Conclusion: These findings suggest that methylation changes in telomere maintenance genes may be a mechanism by which cancer cells alter telomere length, thus undermining genomic stability and protecting cancer cells from senescence. Future studies should confirm these findings, and explore these CpG sites and genes as potential early detection biomarkers and therapeutic targets.

#2265

HPV16 serostatus and risk of oropharyngeal carcinoma.

Kristina R. Dahlstrom,1 Karen S. Anderson,2 Matthew S. Field,2 Diego Chowell,2 Guojun Li,1 Erich M. Sturgis1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Arizona State University, Tempe, AZ_.

Background: The current epidemic of human papillomavirus (HPV)-related oropharyngeal cancer (OPC) urgently demands a screening strategy for this disease. Antibodies to HPV type 16 (HPV16) early (E) antigens have been detected in patients with HPV16-related OPC, and are potential biomarkers of HPV-related malignancies. The goal of this study was to evaluate the diagnostic accuracy of a panel of antibodies to HPV16 E antigens.

Methods: This was a case-control study that included 378 patients with OPC, 153 patients with non-oropharyngeal head and neck cancer, and 782 healthy control subjects. All patients had newly diagnosed, previously untreated disease and were matched with controls by age and sex. Tumor HPV status was determined by p16 immunohistochemistry. IgG antibodies to the HPV16 antigens E1, E4, E5, E6, E7, and the N-terminal and C-terminal fragments of E2 (NE2, CE2) were quantified using a custom multiplexed bead array assay. A binary logistic regression classifier based on the combined relative light units of all E antigens on ELISA was pre-defined and applied to the dataset. Odds ratios (OR) with 95% confidence intervals (CI) were calculated using logistic regression analysis.

Results: Of the 378 patients with OPC, 348 had p16-positive OPC. Using the single cutoff values, the frequency of seropositivity for the individual antigens vs. controls was E1 (74% vs 7%); CE2 (68% vs 1%); E6 (42% vs 1%); and E7 (64% vs 1%)(Table 1). Positivity for the binary classifier was associated with an OR of 453 (95% CI, 199-1030) for p16-positive OPC, with a sensitivity of 83% and a specificity of 99% (Table 1).

Conclusions: HPV-specific IgG antibodies to multiple early antigens are detected at the time of diagnosis of HPV-related OPC. A multiparametric algorithm improves the specificity of detection of cases over single antibodies and are potential biomarkers for HPV-associated malignancies that may be advantageous for risk stratification in future screening trials.

Table 1. A blinded study of HPV16 serology of patients with p16-positive OPC (n=348) and controls (n=782). | |  | |  | |  | |

---|---|---|---|---|---|---|---|---

|

Controls | p16+ OPC | |  | |  | |

|

N+ (%) | N+ (%) | OR (95% CI)a | Sensitivity | Specificity | PPV | NPV | AUC

E1 | 52 (6.7) | 256 (73.6) | 43 (28-67) | 74 | 93 | 83 | 89 | .84

NE2 | 14 (1.8) | 128 (36.8) | 43 (20-90) | 37 | 98 | 90 | 78 | .68

CE2 | 7 (0.9) | 235 (67.5) | 290 (114-741) | 68 | 99 | 97 | 87 | .83

E4 | 14 (1.8) | 110 (31.6) | 27 (13-54) | 32 | 98 | 89 | 76 | .65

E5 | 48 (6.1) | 37 (10.6) | 2 (1-4) | 11 | 94 | 44 | 70 | .52

E6 | 9 (1.2) | 146 (42.0) | 80 (34-187) | 42 | 99 | 94 | 79 | .70

E7 | 11 (1.4) | 224 (64.4) | 106 (54-208) | 64 | 99 | 95 | 86 | .82

Classifier | 8 (1.0) | 288 (82.8) | 453 (199-1030) | 83 | 99 | 97 | 93 | .91

aAdjusted for sex, smoking, and alcohol status (never vs. former vs. current)

OR: odds ratio; CI: confidence interval; PPV: Positive Predictive Value; NPV: Negative Predictive Value; AUC: Area Under the Curve

#2266

A prospective study of serum metabolites and glioma risk.

Jiaqi Huang,1 Stephanie J. Weinstein,1 Cari M. Kitahara,2 Edward Karoly,3 Joshua N. Sampson,1 Demetrius Albanes1. 1 _NIH, Bethesda, MD;_ 2 _NIH, bethesda, MD;_ 3 _Metabolon, Inc., Morrisville, NC_.

Malignant glioma is one of the most devastating adult malignancies. Although there exists an urgent need for actionable leads regarding its prevention and early detection, the etiology of glioma remains largely unknown. We conducted a prospective serum metabolomic analysis of glioma based on 64 cases and individually matched controls selected from Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study. Median time from collection of baseline fasting serum to diagnosis of glioma was nine years (interdecile range 3-20 years). We used ultra-high performance LC/MS which identified 730 known metabolites for analysis. Conditional logistic regression models examined odds ratios (ORs) and 95% confidence intervals for one-standard deviation differences in metabolite signals on a log-scale. Forty-three serum metabolites were associated with glioma at P<0.05. 2-Oxoarginine, cysteine, alpha-ketoglutarate, chenodeoxycholate and argininate yielded the strongest metabolite signals and were inversely associated overall risk of glioma (0.0065≤P<0.0083). Three serum acylcarnitines (0.016≤P<0.046), and seven xanthine (i.e., caffeine) metabolites (0.017≤P<0.042) were higher in cases than controls. Findings were mostly similar in high-grade glioma cases, although proportionally more metabolites showed positive risk associations such as N-acetylglutamate, ribonate, and 5-methyluridine (0.04≤P<0.02). Notably, xanthine compounds appeared unrelated to high-grade glioma. Prominent metabolite signals for inverse associations were the bile acids glycocholenate sulfate and 3β-hydroxy-5-cholenoic acid, xenobiotic methyl-4-hydroxybenzoate sulfate, sex steroid 5alpha-pregnan-3beta,20beta-diol monosulfate, and cofactor/vitamin oxalate. We found weaker risk associations for lower-grade glioma, of which none achieved significance at P<0.05. The metabolic sub-pathway analysis revealed associations for primary bile acid, urea cycle/arginine and proline, tocopherol, glycolysis/gluconeogenesis/pyruvate metabolites with overall glioma risk (0.005≤P<0.048); ascorbate and aldarate pathways were associated with high-grade glioma (P=0.02), whereas glycolysis/gluconeogenesis/pyruvate, eicosanoid and glutamate pathways were found to be associated with lower-grade disease (0.01≤P<0.04). These associations did not, however, reach the stringent Bonferroni significance threshold for correction of multiple comparisons. In conclusion, we identified a serum metabolomic profile of glioma, years in advance of clinical diagnoses, characterized by altered molecular signals in the arginine/proline, antioxidant, bile acid, and glycolytic pathways. The observed patterns provide potential new leads regarding the molecular basis relevant to etiologic or sub-clinical biomarkers for glioma. The findings require reexamination in larger and more diverse populations, including through glioma consortia.

#2267

A prospective assessment for telomere length in relation to risk of cancer in the Singapore Chinese Health Study.

Jian-Min Yuan,1 Renwei Wang,1 Kenneth Beckman,2 Aizhen Jin,3 Woon-Puay Koh4. 1 _Univ. of Pittsburgh, Pittsburgh, PA;_ 2 _Univ. of Minnesota, Minneapolis, MN;_ 3 _Health Promotion Board, Singapore, Singapore;_ 4 _Duke-NUS Medical School, Singapore, Singapore_.

Background: Telomeres are crucial in the maintenance of chromosome integrity and genomic stability. A critically short telomere length can trigger cell to enter replicative senescence with a result of cell death; alternatively, cells continue to divide if death does not occur, which results in genomic instability and chromosomal abnormality. A series of epidemiological studies have examined the association between telomere length and the risk of cancers, but the findings remain conflicting.

Methods: Among 63,257 participants of the Singapore Chinese Health Study, a population-based prospective cohort of Chinese men and women aged 45-74 years recruited from 1993 through 1998, 28,219 provided baseline blood samples. We used quantitative polymerase chain reaction (PCR) method to quantify relative telomere length determined by the ratio of telomere repeat copy number (T) to single-copy gene for albumin (S) (i.e., TSR) on all subjects. The present analysis included 24,847 subjects with valid TSR values after excluding samples with insufficient DNA (n = 1,908) and/or patients with prevalent cancer at baseline blood draw (n =1,464). As of December 31, 2015, 3,778 participants developed cancer, including 722 colorectal cancer, 599 lung cancer, and 412 breast cancer. Cox models were used to estimate hazard ratio (HR) and 95% confidence interval (CI) of developing any cancer and these selected specific cancer types for different quintiles of TSR.

Results: Women had a 5.7% higher TSR value than men (P<0.0001). Age, level of education, number of cigarettes/day, years of smoking, and pack-years of smoking were all inversely associated with TSR (P<0.0001). High TSR was associated, in a dose-dependent manner, with significantly increased risk of total cancer and breast, colorectal and lung cancer. Compared with the lowest quintile, the HRs (95% CIs) of total cancer for the 2nd, 3rd, and 4th, and 5th quintile of TSR were 1.03 (0.89, 1.20), 1.08 (0.93-1.26), 1.15 (0.98-1.34), and 1.36 (1.16-1.58), respectively, after adjustment for age, sex, education and smoking (Ptrend=0.001). The corresponding HRs (95% CIs) were 1.21 (0.72-2.03), 1.59 (0.97-2.60), 1.59 (1.04-2.75) and 1.62 (0.99-2.66) for breast cancer (Ptrend=0.023); 1.44 (1.03-2.00), 1.10 (0.76-1.59), 1.32 (0.92-1.90) and 1.88 (1.32-2.66) for colorectal cancer (Ptrend=0.004); and 1.41 (0.98-2.05), 1.37 (0.93-2.02), 1.31 (0.86-1.99) and 1.88 (1.26-2.81) for lung cancer (Ptrend=0.010).

Conclusions: This prospective cohort study demonstrates that longer telomere length is associated with significantly increased risk of total and major cancers in a general population. These results suggest a complex role of telomere in the development of cancer.

#2268

Serum insulin and glucose, indices of insulin resistance, and risk of lung cancer.

Ilona Argirion,1 Stephanie J. Weinstein,2 Satu Männistö,3 Demetrius Albanes,2 Alison M. Mondul1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _National Cancer Institute, Bethesda, MD;_ 3 _National Institute for Health and Welfare, Helsinki, Finland_.

Background: Although insulin is crucial in human growth and development, it also harbors antiapoptotic properties and acts as a growth factor by stimulating mitosis through the Akt pathway, which could lead to tumor growth and promotion. Insulin has been positively associated with several cancer sites, but no studies to date have examined fasting serum insulin concentrations and risk of lung cancer.

Methods: The Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study was a randomized, double-blind intervention trial conducted among 29,133 male smokers in southwest Finland. Participants were randomly assigned to one of four groups in a 2x2 factorial design (α-tocopherol alone, β-carotene alone, both supplements, or placebo). 196 lung cancer cases and 395 subcohort members were sampled from the larger cohort. Fasting serum collected at enrollment (5-12 years prior to diagnosis) was analyzed for insulin using a double-antibody immunochemiluminometric assay, and for glucose using a hexokinase assay. Cox proportional hazards models were used to estimate the hazard ratio (HR) and 95% confidence intervals (CI) of lung cancer risk by quartiles of insulin, glucose, and surrogate indices of insulin resistance (insulin:glucose molar ratio and homeostasis model assessment of insulin resistance [HOMA-IR]). Multivariable models adjusted for age, BMI, family history of lung cancer, and pack-years of smoking.

Results: Insulin was positively associated with risk of lung cancer (Q4 vs. Q1 HR: 2.10, 95% CI: 1.12, 3.94). A similar association was seen with HOMA-IR (Q4 vs. Q1 HR: 1.83, 95% CI: 0.99, 3.38). We observed no statistically significant associations with glucose or the insulin:glucose molar ratio (P-trend=0.55 and P-trend = 0.27, respectively). Insulin and HOMA-IR were associated with lung cancer risk regardless of stage, although the findings were stronger for the lower stage cancers (Q4 vs. Q1 insulin, stage I-II HR=2.85, 95% CI: 1.14, 7.15, P-trend=0.01; stage III-IV HR=0.94, 95% CI: 0.93 - 4.06, P-trend=0.23; HOMA-IR stage I-II HR=2.25, 95% CI 0.89-5.69, P-trend=0.02, stage III-IV HR=1.78, 95% CI=0.87 - 3.61, P-trend=0.34). Findings were similar across histologic subtypes of lung cancer.

Conclusion: Higher fasting serum insulin and insulin resistance appear to be associated with an increased risk of lung cancer. In addition, elevated insulin concentrations may be more strongly associated with lower stage lung malignancies, suggesting a role for insulin in stimulating the growth of early pulmonary cancers.

#2269

Circulating lipids and breast cancer subtypes in a Spanish population.

Manuela Gago-Dominguez,1 Manuel Calaza,2 Victor Muñoz-Garzon,3 Maria Elena Martinez,4 Jose Esteban Castelao5. 1 _Galician Foundation of Genomic Medicine, Servicio Galego de Saude (SERGAS), Instituto de Investigación Sanitaria de Santiago (IDIS), Santiago de Compostela, Spain;_ 2 _University Santiago Compostela. Galician Foundation of Genomic Medicine, Instituto de Investigación Sanitaria de Santiago (IDIS), Santiago de Compostela, Spain;_ 3 _Radiotherapy Department, Instituto de Investigación Galicia Sur CHUVI, Servicio Galego de Saude (SERGAS), Vigo, Spain;_ 4 _Department of Family Medicine and Public Health, Moores Cancer Center, University of California San Diego, San Diego, CA;_ 5 _Oncology and Genetics Unit. Instituto Investigacion Biomedica Galicia Sur, CHUVI, Servicio Galego de Saude (SERGAS), Vigo, Spain_.

Background. Although several studies have looked at the circulating lipids-overall breast cancer relationship, no prior investigation has examined this relationship according to the four major subtypes of breast cancer.

Materials and Methods. The BREOGAN (BREast Oncology GAlicia Network) study is a population-based case-control study conducted in Galicia, Spain between 1997 and 2014, including 1,766 women diagnosed with invasive breast cancer and 1,205 population- and age-matched controls. Data on demographics, breast cancer risk factors, and clinico-pathological characteristics were collected. Three-hundred eighty-five cases and 412 controls had data on fasting blood lipids (total cholesterol, LDL, HDL and triglycerides) before date of diagnosis for cases, and time of interview for controls, and were included in the present study. We examined the blood lipids-breast cancer relationship overall and according to the four major breast cancer subtypes [hormone-receptor-positive, HER2-negative (luminal A); hormone-receptor-positive, HER2-positive (luminal B); hormone-receptor-negative, HER2-negative (TNBC); and hormone-receptor-negative, HER2-positive (HER2 overexpressing)] as well as grade and morphology in Spanish women.

Results. The mean plasma triglyceride levels for breast cancer cases overall and controls were 118.8 (74-142) and 100.7 (68-121) mg/dL, respectively (p < 0.001). Regarding breast cancer subtypes, the mean levels for luminal A breast cancer cases were 123.5 (80-153) mg/dL compared to 100.7 (68-121) mg/dL for controls (p < 0.001). The risk of luminal A breast cancer significantly increased with increasing circulating levels of triglycerides (OR for the highest versus the lowest category = 3.29, 95% CI = 1.66-6.49, p for trend < 0.0001). No associations were detected for any other circulating lipid (total cholesterol, LDL or HDL) or any other breast cancer subtype. No differences were detected by histology (ductal versus lobular) or grade (I, II or III). Results will also be presented on a Mendelian randomization analysis to evaluate associations between circulating triglyceride levels and breast cancer risk using individual-level data from BREOGAN cancer cases and controls genotyped on OncoArray. Variants with genome-wide significant associations with lipid traits will be used to construct polygenic risk scores for analyses of triglycerides in relation to breast cancer risk.

Conclusions. To our knowledge, this is the first study examining the association between circulating lipids and breast cancer according to the four major breast cancer subtypes. Our findings indicate that breast cancer risk increased with increasing triglyceride level among postmenopausal luminal A breast cancer. No differences were detected by histology or grade.

#2270

C-reactive protein and ovarian cancer risk: preliminary results from the Ovarian Cancer Cohort Consortium (OC3).

Elizabeth M. Poole,1 Britton Trabert,2 Renée T. Fortner,3 Nicolas Wentzensen,2 Shelley S. Tworoger1. 1 _Brigham & Women's Hospital, Boston, MA; _2 _National Cancer Institute, Washington, DC;_ 3 _German Cancer Research Center, Germany_.

Background: C-reactive protein (CRP), a general marker of inflammation has been consistently associated with increased ovarian cancer risk. However, few studies have had the ability to evaluate differences in association by ovarian tumor subtype.

Methods: We pooled existing data on CRP and ovarian cancer risk among nested case-control studies conducted within 6 studies participating in the Ovarian Cancer Cohort Consortium (OC3): CLUEII, EPIC, NHS, NHSII, NYUWHS, and PLCO. Cohort-specific tertiles and quartiles were analyzed using conditional logistic regression, as were common cutpoints at <1mg/L, 1-<10mg/L and ≥10mg/L and Odds Ratios (OR) and 95% confidence intervals (CI) were calculated using random-effects meta-analysis. We also evaluated differences in the association between serous and non-serous cancers.

Results: There was no significant association between the top vs. bottom tertile (OR: 1.10; 95% CI: 0.91-1.32) or the top vs. bottom quartile (OR: 1.10; 95% CI: 0.89-1.36). However, there was a strong, positive association between very high CRP (≥10mg/L vs. <1 mg/L: OR: 1.96; 95% CI: 1.36-2.84). Associations of very high (≥10 mg/L vs. < 1 mg/L) did not differ by histologic type: serous OR: 1.63; 95% CI: 1.11-2.43; endometrioid OR: 1.78; 95% CI: 0.63-5.00; mucinous OR: 10.43; 95% CI: 1.27-85.86; clear cell OR: 2.30; 95% CI: 0.39-13.34).

Conclusion: Our results confirm the consistent observation of a positive association between very high CRP levels and ovarian cancer risk. Pooled analyses accounting for potential confounders are ongoing.

#2271

DNMT1 **expression is peripheral mononuclear cells is associated with increased breast cancer risk.**

Lissette Delgado-Cruzata,1 Xinran Ma,2 Yuyan Liao,2 Maya Kappil,2 Regina M. Santella,2 Mary Beth Terry2. 1 _John Jay College, City University of New York, New York, NY;_ 2 _Mailman School of Public Health, Columbia University, New York, NY_.

Loss of global DNA methylation in peripheral tissue has been identified as a biomarker of breast cancer susceptibility in several studies. DNA methyl-transferases or DNMTs are the enzymes in charge of catalyzing the addition of the methyl group to the DNA molecule. Of the different DNMTs described in humans, only DNMT1 has been found to be in charge of adding methyl groups to the newly synthesized DNA strand. Therefore, compromised levels of this enzyme might lead to a loss of global DNA methylation and concomitantly an increase in breast cancer risk. However, to date no study has examined the association between peripheral levels of DNMT1 and breast cancer. To investigate this relation, we conducted a case-control study including sisters in the New York site of the Breast Cancer Family Registry. We extracted mRNA from peripheral mononuclear cells (PMNC) in 196 sister sets, a total of 479 female participants, and assayed DNMT1 gene expression levels using Taqman expression assays. The results were analyzed using generalized estimating equations (GEE). We found that there were no large differences between the mean levels of DNMT1 expression levels of women with breast cancer and controls; mean level in cases was 0.46 ± 0.71 versus 0.63 ± 1.42 in controls (p=0.10). When examining expression levels as a continuous variable, there also were no associations in GEE regression models adjusting for age at blood drawn and smoking status Odds Ratio (OR) = 1.17 (95% confidence interval (CI) = 0.97 - 1.40 per one unit increase in expression levels), p=0.11). However, when results were analyzed by categorizing DNMT1 expression in tertiles, lower DNMT1 expression (≤ 0.17%) was associated with statistically significantly higher odds of breast cancer (OR = 1.47 (95%CI = 1.02 - 2.10), p=0.04). If replicated in other studies, these findings suggest that low DNMT1 expression in PBMC has the potential to be used as a biomarker of breast cancer risk in women at high risk of the disease.

#2272

**A prospective study of urinary prostaglandin E2 metabolite,** Helicobacter pylori **antibodies, and gastric cancer risk.**

Tianyi Wang,1 Hui Cai,2 Wei Zheng,2 Angelika Michel,3 Michael Pawlita,3 Ginger Milne,4 Yong-Bing Xiang,5 Yu-Tang Gao,5 Hong-Lan Li,5 Nathaniel Rothman,6 Qing Lan,6 Xiao-Ou Shu,2 Meira Epplein2. 1 _Peking University Health Science Center, Beijing, China;_ 2 _Division of Epidemiology, Department of Medicine, Vanderbilt Epidemiology Center and Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN;_ 3 _Division of Molecular Diagnostics of Oncogenic Infections, Research Program in Infection, Inflammation, and Cancer, German Cancer Research Center (DFKZ), Heidelberg, Germany;_ 4 _Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN;_ 5 _Department of Epidemiology, Shanghai Cancer Institute, Shanghai, China;_ 6 _Division of Cancer Epidemiology and Genetics, Occupational and Environmental Epidemiology Branch, National Cancer Institute, National Institutes of Health, Rockville, MD_.

Background. Previous studies suggest that a stable end-product of prostaglandin E2, the urinary metabolite PGE-M, is excreted in the urine and can be used as an index of systemic prostaglandin E2 (PGE2) production. In the present study we investigate the PGE-M, Helicobacter pylori (H. pylori), and gastric cancer association.

Methods. The present analysis included 359 prospectively ascertained gastric cancer cases and 700 individually matched controls from two population-based prospective cohort studies, the Shanghai Women's Health Study and Shanghai Men's Health Study. Urinary PGE-M was measured by a liquid chromatography/tandem mass spectrometric method. Sero-positivity to 15 H. pylori recombinantly expressed fusion proteins was detected by H. pylori multiplex serology.

Results. Adjusting for H. pylori, increasing PGE-M was associated with higher risk of gastric cancer (Quartile 4 vs. 1, OR=1.76, 95% CI: 1.17-2.66, Ptrend =0.004). This association remained after excluding those diagnosed within two years from sample collection (OR=1.73, 95% CI: 1.12-2.65, Ptrend =0.007). However it was no longer present among individuals with 10 or more years of follow-up (2-4.9 years, OR=3.15, 95% CI: 1.11-8.91; 5-9.9 years, OR=2.23, 95% CI: 1.22-4.06; ≥10 years, OR=0.73, 95% CI: 0.31-1.70). The association of PGE-M with gastric cancer risk was not modified by H. pylori status, but added predictive ability beyond H. pylori; compared to H. pylori-negative individuals with below-median PGE-M levels, H. pylori-positive individuals with above-median PGE-M levels had a 5-fold increase in the odds ratio of gastric cancer (OR=5.08, 95% CI: 2.47-10.43).

Conclusion. In China, higher PGE-M levels may indicate an increased risk of gastric cancer independent of the risk conferred by H. pylori infection status, particularly for cancers diagnosed within 10 years of sample collection.

#2273

Serum choline, methionine, betaine, dimethylglycine, and trimethylamine-N-oxide in relation to pancreatic cancer risk in two nested case-control studies in Asian populations.

Joyce Huang,1 Lesley Butler,1 Øivind Midttun,2 Renwei Wang,3 Aizhen Jin,4 Yu-Tang Gao,5 Per Ueland,6 Woon-Puay Koh,7 Jian-Min Yuan1. 1 _University of Pittsburgh, Pittsburgh, PA;_ 2 _Bevital A/S, Bergen, Norway;_ 3 _University of Pittsburgh Cancer Institute, Pittsburgh, PA;_ 4 _National Registry of Diseases Office, Health Promotion Board, Singapore, Singapore;_ 5 _Department of Epidemiology, Shanghai Cancer Institute/Shanghai Jiaotong University, Shanghai, China;_ 6 _Department of Clinical Science, University of Bergen; Laboratory of Clinical Biochemistry, Haukeland University Hospital, Bergen, Norway;_ 7 _Duke-NUS Medical School, and Saw Swee Hock School of Public Health, National University of Singapore, Singapore, Singapore_.

Background: Choline, methionine, and betaine are methyl group donors associated with DNA methylation. Diets deficient in choline and methionine have been shown to promote pancreatic carcinogenesis in experimental animals. We have previously reported an inverse association between dietary intake of choline and pancreatic cancer risk in a prospective cohort of Singapore Chinese. In the present study biomarkers of dietary choline and other methyl donor nutrients were evaluated in relation to pancreatic cancer risk.

Method: Two case-control studies were nested within the Shanghai Cohort Study (129 cases and 258 matched controls) and the Singapore Chinese Health Study (58 cases and 104 matched controls). Concentrations of choline, methionine, betaine, dimethylglycine (DMG), and trimethylamine-N-oxide (TMAO) were

measured by LC-MS/MS in pre-diagnostic serum samples. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using conditional logistic regression method with adjustment for potential confounders.

Results: Choline, methionine, and betaine were moderately associated with each other (spearman correlation coefficient: 0.28 ~0.43). In the pooled analysis, serum choline, betaine, and methionine were inversely associated with risk of pancreatic cancer, while TMAO, an oxidative metabolite of choline produced by gut microbiota, was positively associated with risk of pancreatic cancer. Compared with the lowest quartile, ORs (95%CIs) of pancreatic cancer for the highest quartiles of choline, methionine, betaine, and TMAO were 0.37 (0.17-0.80), 0.39 (0.22-0.69), 0.49 (0.28-0.85), and 1.60 (0.94-2.74), respectively (all Ps for trend < 0.05). DMG was not associated with pancreatic cancer risk.

Conclusion: The novel inverse associations of serum choline, methionine, and betaine with risk of pancreatic cancer support the notion that methyl groups related to DNA methylation may modulate the risk of pancreatic cancer development. The positive association between TMAO and pancreatic cancer risk suggested gut microbiota may play an important role in pancreatic carcinogenesis.

#2274

Extreme telomere length was associated with gastric adenocarcinoma risk: The Singapore Chinese Health Study.

Zhensheng Wang,1 Woon-Puay Koh,2 Aizhen Jin,3 Renwei Wang,4 Jian-Min Yuan4. 1 _Univ. of Pittsburgh, Pittsburgh, PA;_ 2 _National University of Singapore, Singapore City, Singapore;_ 3 _National Registry of Diseases Office, Singapore City, Singapore;_ 4 _University of Pittsburgh Cancer Institute, Pittsburgh, PA_.

Background: Evidence regarding extreme telomere length and increased gastric cancer risk has been limited in retrospective case-control study setting which was potentially subjected to selection bias and reverse causality. We conducted a prospective analysis in an Eastern Asian population to examine the hypothesis that extreme telomere length is associated with increased risk of gastric adenocarcinoma.

Methods: Among 63,257 participants of the Singapore Chinese Health Study, a population-based prospective cohort of Chinese men and women aged 45-74 years recruited between 1993 and 1998, 28,219 provided baseline blood samples. We used quantitative polymerase chain reaction (PCR) method to quantify relative telomere length determined by the ratio of telomere repeat copy number (T) to single-copy gene for albumin (S) (i.e., TSR) on all subjects. The present analysis included 24,846 subjects with valid TSR values after excluding samples with insufficient DNA (n = 1,908) and/or patients with prevalent cancer at baseline blood draw (n = 1,465). As of December 31, 2014, 191 study participants who were free of cancer developed gastric adenocarcinoma. The information on demographic and lifestyle characteristics was collected using structured questionnaires at baseline and two follow-up interviews. Cox proportional

hazard regression method was used to estimate hazard ratio (HR) and the corresponding 95% confidence interval (CI) of developing gastric adenocarcinoma for different levels of decile TSR. Restricted cubic spline (RCS) analysis was applied to examine the nonlinear relationship between TSR level and gastric adenocarcinoma risk.

Results: The RCS analysis revealed a U-shaped relationship between TSR and gastric adenocarcinoma risk (p-value for non-linear association=0.009). Compared with the 4th decile, HR (95% CI) for the lowest and highest deciles of TSR 2.27 (1.17-4.40) and 1.98 (0.95-4.13), respectively after adjusting for age, sex, education, interview year, dialect group and smoking status.

Conclusions: This was the first prospective analysis of TSR and risk of developing gastric adenocarcinoma in a general population. The study demonstrates both extreme short and long telomere length to be associated with significantly increased risk of gastric adenocarcinoma. These results suggest a complex relationship between telomere length and gastric cancer risk.

#2275

Serum retinol and risk of overall and site-specific cancer in the Alpha Tocopherol, Beta-Carotene Cancer Prevention Study.

Manila Hada,1 Alison Mondul,1 Stephanie Weinstein,2 Demetrius Albanes2. 1 _University of Michigan, Ann Arbor, MI;_ 2 _National Cancer Institute, Bethesda, MD_.

Retinol, one of the most biologically active forms of vitamin A, influences many biologic pathways potentially related to cancer. However, results of observational studies of serum retinol and cancer risk have been mixed. We prospectively examined serum retinol and risk of overall and site-specific cancer in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study, a randomized, double-blind, placebo controlled trial of α-tocopherol (AT) and β-carotene (BC) supplementation conducted in male smokers in southwestern Finland. At enrollment, overnight fasting blood samples were collected and stored at -70°C protected from light. Serum retinol concentration was measured for all 29,133 participants using reverse-phase high performance liquid chromatography. 10,798 cancer cases occurred through 12/31/2012. Cox proportional hazards models were used to estimate the prospective association between quintiles of baseline serum retinol and overall and site-specific cancer incidence. Known or hypothesized risk factors for cancer were included in the multivariable model (AT and BC supplementation group, cigarettes smoked/day, years smoked, age, BMI, alcohol intake, and serum AT, BC, and cholesterol). Stratified analyses for all subgroups of the covariates (by category or by medians for continuous variables) and follow-up time to diagnosis (<10 vs. ≥10 y) were also performed for overall cancer and for the cancers for which an association with serum retinol was observed. After adjustment for age, higher serum retinol was associated with a lower risk of overall cancer (Q5 vs. Q1: HR=0.93, 95%CI=0.88-0.99, p-trend = 0.04). This finding was attenuated and no longer statistically significant after multivariable adjustment (MV-adj) (Q5 vs. Q1: MV-adj HR= 0.97, 95% CI = 0.91-1.03, p-trend = 0.43). Higher retinol concentrations were associated with increased risk of prostate cancer (Q5 vs. Q1: MV-adj HR=1.28, 95%CI = 1.13-1.45, p-trend < 0.0001) and lower risk of both lung and liver cancers (Q5 vs. Q1: liver MV-adj HR = 0.70, 95%CI = 0.50-0.99,p-trend = 0.03; lung MV-adj HR = 0.80, 95%CI = 0.72-0.88, p-trend < 0.0001). No associations with other cancers were observed. The inverse association between retinol and lung cancer was significantly modified by serum BC with a stronger association among men with higher serum BC (p for interaction = 0.018). No other statistically significant interactions were observed. In this large prospective study, higher serum retinol was associated with increased risk of prostate cancer and lower risk of lung and liver cancers. Understanding the biological mechanisms that underlie these associations may provide insight into retinol's role in cancer prevention.

#2276

Effect modification by isoflavones on the association between salt intake and gastric cancer risk: A case-cohort study within the Korean multi-center cancer cohort.

Jieun Jang, Yunji Hwang, Choonghyun Ahn, Kwang-Pil Ko, Aesun Shin, Keun-Young Yoo, Sue K. Park. _Seoul National University College of Medicine, Seoul, Republic of Korea_.

Backgrounds: Attenuation by genistein, an isoflavone abundant in soybean, of gastric carcinogenesis induced by sodium chloride was reported in an animal study. Up to date, role of isoflavone as an effect modifier on the association between salt intake and gastric cancer risk is not clearly established.

Methods: We randomly selected 640 subcohort subjects without cancer history at enrollment from the Korean multi-center cancer cohort participants. Ninety-nine gastric cancer cases were identified from the inside (N=8) and the outside (N=91) of the subcohort after the 12.2 years of median follow-up time. To estimate individual's salt intake, we measured 24-hour urinary sodium excretion from spot urine using the Tanaka's equation. Three isoflavone plasma concentrations (Genistein, daidzein, and equol) of 167 subjects among case-cohort subjects had been measured using time-resolved fluoroimmunoassay. Cubic spline curve for gastric cancer risk according to urinary sodium level was used to define optimal cut-off points of urinary sodium excretion concentration. Weighted Cox proportional hazard regression was used to calculate hazard ratios (HRs) and their 95% confidence intervals (CIs). Stratified analysis by isoflavone level was conducted to assess effect modification by isoflavone on the association between salt intake and gastric cancer.

Results: Gastric cancer risk in the high salt intake group (urinary sodium excretion ≥6,000mg/day) was significantly increased (HR, 1.69; 95% CI, 0.99-2.90) compared to the reference group (urinary sodium excretion 4500-5,999mg/day). Magnitudes of gastric cancer risk by high salt intake were more strengthened in the group with low plasma isoflavone concentrations (One or no genistein, diadzein or equol levels over the median concentrations) (HR, 6.92; 95% CI, 1.80-26.56). Contrary to the low isoflavone group, no significant differences in gastric cancer risk according to salt intake levels were found in the group with high isoflavone concentration.

Conclusions: High salt intake is associated with gastric cancer risk, but this association can be attenuated by high concentration of isoflavones. We suggest that sufficient intake of soybean products, the rich sources of isoflavone, may give protection against gastric cancer in the population with high salt intake.

### Tobacco, Alcohol, and Other Risk Factors

#2277

Adherence to cancer prevention guidelines and risk of cancer among older white and black adults in the Health ABC Study.

Audrey Y. Jung,1 Christina Gu,1 Iva Milijkovic,2 Susan Rubin,3 Suzanne Satterfield,4 Stephen B. Kritschevsky,5 Heidi Klepin,5 Anne B. Newman,2 Jane Cauley,2 Hilsa Ayonayon,3 Tamara B. Harris,6 Rachel A. Murphy1. 1 _University of British Columbia, Vancouver, British Columbia, Canada;_ 2 _University of Pittsburgh, Pittsburgh, PA;_ 3 _University of California at San Francisco, San Francisco, CA;_ 4 _University of Tennessee Health Science Center, Memphis, TN;_ 5 _Wake Forest School of Medicine, Winston-Salem, NC;_ 6 _National Institute on Aging, Bethesda, MD_.

Background: About one-third of cancers can be prevented by healthy lifestyle behaviors. The American Cancer Society (ACS) have guidelines for reducing the risk of cancer by maintaining a healthy body weight, being physically active, eating well and limiting alcohol. Few studies have examined adherence to cancer preventive behavior and subsequent cancer risk in older populations that are inclusive of men, women and minorities.

Methods: We evaluated adherence to ACS cancer prevention guidelines and subsequent risk of all incident cancer in the Health, Aging and Body Composition Study. The population included 2,124 white and black men and women aged 71-80 years who were initially free of cancer at baseline (1998-1999). Adherence to ACS guidelines was scored from 0 (lowest adherence) to 8 (greatest adherence). Maintenance of healthy body weight was determined from body mass index at ages 25 and 50 (recalled at baseline) and measured height and weight at baseline. Physical activity and alcohol intake were assessed by questionnaire. Diet was assessed by food frequency questionnaire (servings of fruits and vegetables, the ratio of whole grains to refined grains and intake of red meat and processed meats). Incident cancer (all types except non-melanoma skin cancer) was confirmed from pathology reports when available, or medical records and death certificates. Risk of cancer was determined with cox regression adjusted for gender, age and study site. Participants with the lowest adherence to ACS guidelines were the referent.

Results: The lowest adherence category (scores 0-2) had 297 participants (14% of all participants), 1,316 (62%) were in the moderate adherence category (scores 3-5), and 511 (24%) were in the highest adherence category (scores 6-8). Adherence was lower among men (P<0.001), black (P<0.001) and older participants (P<0.01). After a median follow-up of 12.5 years, 517 people developed incident cancer. Participants with moderate adherence had a 23% lower risk of cancer [HR=0.77, 95% CI=0.60-0.97]. Participants with the greatest adherence had a 26% lower risk of cancer [HR=0.74, 95% CI=0.56-0.98].

Conclusions: Only 1 in 4 older adults closely followed healthy lifestyle behaviors outlined by the ACS for cancer prevention. Greater adherence to recommendations was associated with lower likelihood of total cancer in older white and black adults. Public health efforts to increase preventive lifestyle behaviors may be particularly needed among men, black and older individuals.

#2278

Risk factors by molecular subtypes of breast cancer: a pooled analysis of nine cohorts.

Mia M. Gaudet,1 Gretchen Gierach,2 Brian Carter,1 Juhua Luo,3 Roger Milne,4 Elisabete Weiderpass,5 Graham Giles,4 Wendy Chen,6 Rulla Tamimi,6 Heather Eliassen,6 Diane Feskanich,6 Alicja Wolk,7 Hans-Olov Adami,8 Karen Margolis,9 Susan Gapstur,1 Montserrat Garcia-Closas,2 Louise Brinton2. 1 _American Cancer Society, Atlanta, GA;_ 2 _National Cancer Institute, Rockville, MD;_ 3 _Indiana University, IN;_ 4 _Cancer Council Victori, Australia;_ 5 _Karolinska Institutet, Sweden;_ 6 _Brigham and Women's Hospital and Harvard Medical School, Boston, MA;_ 7 _Karolinska Institute, Sweden;_ 8 _Dana Farber/Harvard Cancer Center, MA;_ 9 _HealthPartners, Minneapolis, MN_.

Etiological differences between molecular subtypes of breast cancer, defined by estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), have been examined in previous studies but results are inconsistent, most likely due to small numbers of rarer subtypes. In a recent systematic review of these studies, only six of the possible 44 associations had consistent findings, primarily with the most common luminal A subtype (ER+ or PR+/HER2-). To provide larger numbers from prospective studies for these associations, we utilized a harmonized dataset of nine cohort studies (Cancer Prevention Study-II Nutrition Cohort, Melbourne Cancer Cohort Study, the National Cancer Institute-American Association of Retired Persons cohort study, Nurses' Health Study, Nurses' Health Study-2, Prostate, Lung, Colorectal, and Ovarian Cancer Screening cohort, Swedish Mammographic Cohort, Swedish Women's Lifestyle and Health Study, and Women's Health Initiative) that had case data on ER, PR, and HER2 from medical records or state tumor registry records. Multivariate, joint Cox proportional hazard regression models were used to calculate hazard ratios (HR) and 95% confidence intervals (CI), and to compare associations across subtypes. Among 616,109 study participants, 11,861 invasive breast cancer cases were diagnosed, including 8,777 luminal A, 1,388 luminal B (ER+ or PR+/ HER2+), 532 HER2-enhancing (ER-/PR-/HER2+), and 1,164 triple negative (ER-/PR-/HER2-) subtypes. The mean age at baseline of the study participants was 54.8 (SD=12.2) years for cases and 54.2 (SD=14.4) years for controls, and the majority self-described as white. We found that the associations of race, benign breast disease, parity, number of live births, age at first birth, age at menopause, alcohol consumption, and smoking initiation relative to first birth were statistically-significantly different for at least one of the molecular subtypes compared to the associations for the ER+ or PR+/HER2- subtype (p-value for tumor homogeneity<0.05). For example, women who drank ≥2 drinks/ day were at increased risk of the luminal A-like (HR=1.44, 95% CI 1.28 - 1.63) and luminal B-like subtypes (HR=1.33, 95% CI 0.97 - 1.81), but not HER2-enhancing (HR=0.92, 95% CI 0.52 - 1.62) or triple negative subtypes (HR=0.89, 95% CI 0.61 - 1.30; p-value for homogeneity<0.001). Younger age at menarche, first degree family history of breast cancer, ever use of oral contraceptives, ever use of menopausal hormones, higher education, and postmenopausal body mass index were positively associated with all molecular subtypes. Our results highlight the importance of considering molecular subtype when evaluating breast cancer associations by risk factors, and provide precise estimates of association for the development of molecular subtype-specific risk prediction models.

#2279

Burden of colorectal cancer attributable to lifestyle-related risk factors: a pooled study of seven Australian cohorts.

Claire M. Vajdic,1 Maria Arriaga,1 Peter Hull,1 Karen Canfell,2 Robert MacInnis,3 Emily Banks,4 Graham Giles,3 Paul Mitchell,5 Robert Cumming,5 Barbara-Ann Adelstein,1 Julie Byles,6 Dianna J. Magliano,7 Jonathan Shaw,7 Anne Taylor,8 Kay Price,9 Vasant Hirani,5 Maarit A. Laaksonen1. 1 _University of New South Wales, Sydney, Australia;_ 2 _Cancer Council New South Wales, Sydney, Australia;_ 3 _Cancer Council Victoria, Melbourne, Australia;_ 4 _Australian National University, Canberra, Australia;_ 5 _University of Sydney, Sydney, Australia;_ 6 _University of Newcastle, Newcastle, Australia;_ 7 _Baker IDI Heart and Diabetes Institute, Melbourne, Australia;_ 8 _University of Adelaide, Adelaide, Australia;_ 9 _University of South Australia, Adelaide, Australia_.

Aim: To quantify the burden of colorectal cancer in Australia avoidable by modifications to established lifestyle-related risk factors: smoking, excess body weight, excessive red and processed meat consumption, excessive alcohol consumption and physical inactivity.

Methods: Data on exposure to lifestyle-related risk factors from seven Australian cohort studies (N = 367,772) were harmonized and pooled. The cohorts were linked to the Australian Cancer Database and National Death Index to identify incident cancers and deaths. The strength of the exposure-cancer and exposure-death associations were estimated using a proportional hazards model, adjusting for age, sex and the other lifestyle exposures. Age- and sex-specific exposure prevalence was estimated from the Australian National Health Survey (NHS) 2011-2012 or from the latest 45 and Up cohort study when not available in the NHS. These estimates were then combined to calculate the Population Attributable Fractions (PAFs), i.e. fractions of cancer attributable to the lifestyle-related risk factors, and their 95% confidence intervals (CIs) using an advanced method accounting for competing risk of death.

Results: During the first 10-years follow-up, 3,498 incident colorectal cancers and 20,135 deaths were ascertained as first events. Current or former smoking explained 9% (CI = 5-13%), BMI ≥ 25 kg/m2 9% (CI = 4-14%), red or processed meat consumption 3 or more times/week 6% (CI = 3-8%), and drinking more than 2 alcoholic drinks/day 4% (CI = 2-6%) of the colorectal cancer burden. PAF results for physical inactivity were not significant. The contribution of each factor was more pronounced in men than in women, with most of the burden attributable to excess weight in men (PAF = 15%, CI = 7-22%) and smoking in women (PAF = 7%, CI = 3-12%). Jointly the four significant lifestyle-related risk factors were responsible for 25% (CI = 19-30%) of the colorectal cancer burden, 33% (CI = 25-40%) in men and 15% (CI =7-23%) in women. Given the projected Australian incidence rates, this translates to 49,000 avoidable colorectal cancer cases, 35,000 in men and 14,000 in women, over the next 10 years.

Conclusions: These first Australian and international colorectal cancer PAF estimates based on cohort studies and accounting for competing risk of death show that several modifiable risk factors contribute to the burden of colorectal cancer and their ranking differs by sex. Further analyses may identify subpopulations that would benefit from targeted intervention activities. Due to the high incidence of colorectal cancer, these PAF estimates translate into a significant number of avoidable cases.

#2280

Burden of lung cancer in Australia avoidable by modifications to lifestyle-related risk factors.

Maarit A. Laaksonen,1 Maria Arriaga,1 Peter Hull,1 Karen Canfell,2 Robert MacInnis,3 Emily Banks,4 Graham Giles,3 Paul Mitchell,5 Robert Cumming,5 Barbara-Ann Adelstein,1 Julie Byles,6 Dianna J. Magliano,7 Jonathan Shaw,7 Anne Taylor,8 Kay Price,9 Vasant Hirani,5 Claire M. Vajdic1. 1 _University of New South Wales, Sydney, Australia;_ 2 _Cancer Council New South Wales, Sydney, Australia;_ 3 _Cancer Council Victoria, Melbourne, Australia;_ 4 _Australian National University, Canberra, Australia;_ 5 _University of Sydney, Sydney, Australia;_ 6 _University of Newcastle, Newcastle, Australia;_ 7 _Baker IDI Heart and Diabetes Institute, Melbourne, Australia;_ 8 _University of Adelaide, Adelaide, Australia;_ 9 _University of South Australia, Adelaide, Australia_.

Aim: The cancer burden avoidable through risk factor modification can be quantified using the Population Attributable Fraction (PAF) which combines estimates of the prevalence of the risk factor exposure in the population and the strength of the exposure-cancer association. PAF for risk factors causally related to cancer is most accurately estimated from cohort studies, using analytical approaches that account for death as a competing risk. We provide the first such estimates of the lifestyle-related avoidable lung cancer burden.

Methods: Seven contemporary Australian cohort studies (N = 367,772), with comprehensive data on exposure to lifestyle-related risk factors, were linked to the Australian Cancer Database and National Death Index to identify lung cancers and deaths from any cause. The risk factors were harmonised across the studies and the data pooled. The strength of the exposure-cancer and exposure-death associations were estimated using a proportional hazards model, adjusting for age, sex and the other lifestyle exposures. Age- and sex-specific exposure prevalence was estimated from the Australian National Health Survey 2011-2012. These estimates were then combined to calculate the PAFs and their 95% confidence intervals (CI) using a newly developed method accounting for competing risk of death.

Results: During the first 10-years follow-up, 2,030 incident lung cancers and 20,348 deaths were ascertained as first events. Smoking, physical inactivity and insufficient fruit intake were statistically significantly associated with both lung cancer incidence and death. Most of the lung cancer burden (PAF = 73%, CI = 69-76%) was attributable to current or past smoking, with 31% (CI = 27-35%) of the burden attributable to current smokers. Physical activity below Australian recommendations (< 150 minutes of moderate or 75 minutes of vigorous exercise per week) explained 14% (CI = 6-22%) and fruit intake below Australian recommendations (< 2 daily serves) explained 6% (CI = 0-12%) of the lung cancer burden. Each risk factor contributed more to the lung cancer burden in men than in women. Overall, 78% (CI = 74-81%) of the lung cancer burden was attributable to current or past smoking, physical inactivity and insufficient fruit intake. The combined contribution of modifiable risk factors (i.e. excluding past smoking) was 44% (CI = 37-50%). Based on projected Australian cancer incidence rates, this would translate to 59,000 avoidable lung cancers in the next 10 years.

Conclusions: According to these joint PAF estimates, the current lung cancer burden in Australia could be reduced by up to half in 10 years by quitting smoking and increasing physical activity and fruit intake to the recommended levels. As the combined effect of risk factors is rarely additive, knowing both individual and joint effects of risk factors on cancer burden is essential in planning cancer interventions and public health policies and predicting their potential impact.

#2281

Environmental tobacco smoke exposure and breast cancer risk in the Norwegian women and cancer study cohort.

Inger T. Gram, Tonje Braaten, Eiliv Lund, Idlir Licaj. _UiT The Arctic University of Norway, Tromsø, Norway_.

Background: Today, the majority of middle-aged women in Norway are ever (i.e. either former or current) smokers, who started to smoke in their teens. Many of those who never have smoked themselves were exposed to environmental tobacco smoke (ETS) as children. The purpose of the study, was to examine the effect of ETS in childhood on breast cancer risk in the Norwegian Women and Cancer Study, a nationally representative prospective cohort study.

Material and Methods: We followed 121 662 women, that were aged 34-70 years at enrolment, who completed a baseline questionnaire between 1991 and 2007, through linkages to national registries through December 2014. Questionnaire data included information on lifestyle factors, including lifetime history of smoking. We used Cox proportional hazards models to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) while adjusting for relevant confounders and stratified by birth cohort. We included the following variables in the final multivariate models; age at enrolment, years of education (lt 10, 10-12, 13-16, 17+), age at menarche (lt 13,13-14, 15+), ever oral contraceptives use (yes, no), a variable including nulliparous and a combination of total number of births, (1, 2, 3+) and of age at first childbirth (age lt 20, 20-24, 25-29, 30+) for a total of 13 categories, family history of breast cancer in the mother (yes, no), body mass index (calculated from current height and weight (lt 20, 20-24.9, 25-29.9, 30+) kg/m 2, , menopausal status (yes, no), postmenopausal hormone therapy use (never, former, current) and average alcohol consumption, based on the content of pure alcohol in different sorts of beverages and portion sizes, as grams of alcohol per day (0, 0.1-1.40, 4.1-10, >10)). Women who reported to be teetotalers and those answering 'seldom or never' had their alcohol consumption set to 0.

Results: During a mean follow-up of 15 years, 4501 women developed invasive breast cancer confirmed by histology. Altogether, 23.7% of the women reported to be exposed to ETS during childhood. The women reported that the active smoker in 7.7% was the father, in 0.66 % the mother, in 3.57% both parents, and in 11.8 % the information about the active smoker was missing. Compared with never smokers, the multivariable adjusted HR estimate for ETS during childhood where the father was the smoker was statistically significantly increased with 23% (HR=1.23; 95% CI 1.07-1.42). The corresponding figures for those being exposed to ETS by the mother was a HR of 1.22 (95% CI 0.88-1.69), for those being exposed by both parents was a HR of 1.06 (95% CI 0.88-1.26) and for those with no information about the active smoker the HR was 1.03 (95% CI 0.89-1.20). In conclusion, we found that exposure to environmental tobacco smoke during childhood was associated with a higher risk of breast cancer for women who reported that the father was the active smoker.

#2282

Smoking and alcohol and the risk of myelodysplastic syndrome: The JPHC study.

Tomotaka Ugai,1 Keitaro Matsuo,1 Norie Sawada,2 Motoki Iwasaki,2 Taiki Yamaji,2 Taichi Shimazu,2 Shizuka Sasazuki,2 Manami Inoue,2 Yoshinobu Kanda,3 Shoichiro Tsugane2. 1 _Aichi Cancer Center Research Institute, Nagoya, Japan;_ 2 _National Cancer Center, Tokyo, Japan;_ 3 _aitama Medical Center, Jichi Medical University, Omiya, Japan_.

Background. Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal disorders of hematopoietic stem cells that are characterized by ineffective hematopoiesis, multilineage dysplasia, peripheral cytopenias, and susceptibility to leukemic transformation. Even without progression to leukemia, there is substantial morbidity and mortality. Therefore, a better understanding of the etiology of this disease can lead to a significant reduction in the incidence and the mortality as a consequence, but it remains largely unknown. Smoking and alcohol are important modifiable risk factors for human cancers. However, only a few epidemiological studies have investigated their association with the risk of MDS. Here, we evaluated the association of smoking and alcohol consumption and the risk of MDS in a large-scale population-based cohort study in Japan.

Methods. We included 95,950 Japanese subjects (45,451 men and 50,059 women; age 40-69 years at baseline) of the Japan Public Health Center-based Prospective Study who completed a questionnaire about their smoking and alcohol habits. During 18.3 years of follow-up, we identified 70 MDS cases (50 men and 20 women). We calculated hazard ratios (HRs) and their 95% confidence intervals (95% CI) using the Cox proportional hazards model to describe the relative risk of MDS associated with the smoking categories at baseline (never smokers, former smokers, current smokers, ever smokers of <30 and ≥30 pack-years) and the drinking categories at baseline (non-drinkers, occasional drinkers, and regular drinkers of 1-299 or ≥300 g/week of ethanol) after adjustment for potential confounders.

Results. Smoking was marginally associated with an increased risk of MDS among men, with a HR for current smokers relative to never smokers of 2.11 (95% CI: 0.91-4.89). This risk increase was also observed in ever smokers with more than 30 pack-years compared to never smokers (HR=2.22, 0.95-5.19). A linear increase in HR with increasing pack-years was also seen, albeit without statistical significance (p-trend=0.083). In contrast, alcohol consumption was associated with a dose-dependent decrease in the risk of MDS among men (nondrinkers: reference, occasional drinkers: HR=0.48, 0.16-1.43; 0-299 g/week: HR=0.37, 0.19-0.74; ≥300 g/week: HR=0.45, 0.20-0.99, p for trend=0.008). All types of alcohol beverages (sake, distilled spirit and beer) consistently showed a protective effect. We also explored a possible interaction between smoking and alcohol consumption, but no obvious interaction was observed.

Conclusions. This study showed that alcohol has a significant protective effect on the risk of MDS among Japanese men. In addition, this study also provides evidence that smoking increases the risk of MDS among Asian population, as it does among Western populations. Previous findings on the association between alcohol and the risk of MDS have been inconsistent, and further investigation across multiple populations is needed.

#2283

Population-level relevance of lifestyle-related risk factors for pancreatic cancer in Australia.

Maria E. Arriaga,1 Maarit A. Laaksonen,1 Karen Canfell,2 Robert MacInnis,3 Emily Banks,4 Graham Giles,3 Paul Mitchell,5 Robert Cumming,5 Barbra-Ann Adelstein,1 Julie Byles,6 Dianna J. Magliano,7 Jonathan Shaw,7 Anne Taylor,8 Kay Price,9 Vasant Hirani,5 Claire M. Vajdic1. 1 _University of New South Wales (UNSW), Sydney, Australia;_ 2 _Cancer Council, Sydney, NSW, Australia;_ 3 _Cancer Council, Melbourne, Victoria, Australia;_ 4 _Australian National University, Canberra, Australia;_ 5 _University of Sydney, Sydney, Australia;_ 6 _University of Newcastle, Newcastle, Australia;_ 7 _Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia;_ 8 _University of Adelaide, Adelaide, South Australia, Australia;_ 9 _University of South Australia, Adelaide, South Australia, Australia_.

Aim: To estimate the proportion of exocrine pancreatic cancers in Australia that can be attributed to the established risk factors, smoking and excess body weight, and the suggested risk factors, excessive alcohol consumption and excessive red and processed meat consumption.

Methods: The proportion of pancreatic cancers attributable to lifestyle-related risk factors was quantified using the Population Attributable Fraction (PAF) which combines estimates of the strength of the exposure-cancer association and the exposure prevalence in the population. The study population included seven Australian cohort studies (N=367,772) with comprehensive data on exposure to lifestyle-related risk factors and long-term follow-up. Pancreatic cancer incidence and all-cause mortality were identified through linkage with the Australian Cancer Database and National Death Index, respectively. A proportional hazards model was used to estimate the strength of exposure-cancer and exposure-death associations adjusting for age, sex and other lifestyle exposures. The age- and sex-specific exposure prevalence was calculated from the Australian National Health Survey (NHS) 2011-2012 except for red and processed meat consumption which was not available and was calculated from the latest 45 and Up study. A newly developed method that takes into account the competing risk of death was used to produce PAF estimates and their 95% confidence intervals (CIs).

Results: During the first 10-years follow-up to incidence of pancreatic cancer, death or end of follow-up, there were 21,541 deaths and 613 incident exocrine pancreatic cancers. 10% (CI = 1-17%) of pancreatic cancers in Australia could be attributed to current or past smoking. Current smoking explained 8% (CI = 4-13%) of pancreatic cancers. Using cancer incidence projections estimated by the Australian Institute of Health and Welfare, a PAF of 8% translates to 2,700 potentially preventable pancreatic cancers over the next 10 years. The PAF results for excess body weight (≥ 25 kg/m2), excessive alcohol consumption (> 2 drinks/day) and excessive red and processed meat consumption (≥ 4 times/week) were not statistically significant.

Conclusion: According to the first pancreatic cancer PAF results from cohort studies, and accounting for competing risk of death, smoking control could reduce the number of pancreatic cancers by one tenth. Further PAF studies should include confidence intervals for the estimates to allow an evaluation of their significance.

#2284

Effects of smoking on breast tumor pathology.

Nicholas Costantino,1 Craig D. Shriver,2 Rachel E. Ellsworth3. 1 _Chan Soon-Shiong Institute of Molecular Medicine at Windber, Windber, PA;_ 2 _Murtha Cancer Center, Bethesda, MD;_ 3 _Murtha Cancer Center, Windber, PA_.

Background Although smoking is not a well-established breast cancer risk factor, proximity of lungs to the breasts and findings that tobacco smoke constituents serve as mammary carcinogens in animal models suggests that smoking may alter the breast microenvironment and affect breast tumorigenesis. To determine how smoking influences tumor pathogenesis, the relationships between smoking and pathological characteristics and outcome were evaluated in 1,707 women with invasive breast cancer.

Methods Patients enrolled in the Clinical Breast Care Project 2001-2013 filled out a questionnaire that included answers to current smoking status, current and previous pack use/day, number of years smoked, and length since last smoked. Patients were classified as never, former or current smokers. Pack years was calculated as years smoked x pack use/day. Logistic regression was performed to determine whether smoking was associated with any characteristics. Significance was defined as P<0.05

Results 55.7% of women never smoked, 31.5% were former smokers and 12.8% were current smokers. The average number or pack years was 14.5 in former smokers and 25.5 in current smokers. Current smoking status was associated with lymph node metastasis (OR 1.44; 95% CI 1.03-2.01), positive HER2 status (OR 1.54, 95% CI 1.04-2.25), larger tumor size (OR 1.55; 95% CI 1.14-2.11) and higher tumor stage (OR 1.54, 95% CI 1.16-2.04) when compared to never smokers. Former smokers were less likely to be diagnosed with later stage tumors (OR 0.77, 95% CI 0.62-0.96) compared to never smokers. No significant associations were detected when pack years were considered as a continuous variable; when categorized as 0, <20, 20-34 or >35 pack years, >35 pack years was associated with positive HER2 status (OR 1.80, 95% CI 1.06-3.03) and tumors of the ER+HER2+ subtype (OR 1.99, 95% CI 1.07-3.7). Overall survival was significantly lower for former and current smokers compared to never smokers (P=0.014); however, breast cancer specific survival did not differ significantly between groups (P=0.226).

Conclusions Current smokers are at increased risk for having larger and later stage tumors with positive lymph node and HER2 status. Tumor size, stage and lymph node status are temporal factors, thus suggest that current smokers may have delayed diagnosis compared to never and former smokers. In contrast, HER2 status reflects a biological phenomenon and the risk of HER2 positivity increases with pack years, suggesting that carcinogens found in tobacco smoke may promote overexpression of HER2.

#2285

Menopausal hormone therapy (MHT) use and breast cancer risk by receptor subtypes: results from the New South Wales Cancer Lifestyle and EvAluation of Risk (CLEAR) study.

Usha G. Salagame,1 Emily Banks,2 Dianne O'Connell,3 Sam Egger,3 Karen Canfell3. 1 _Cancer Council NSW, NSW 2011, Australia;_ 2 _Australian National University, Australian Capital Territory, Australia;_ 3 _Cancer Council NSW, Woolloomoolloo, NSW 2011, Australia_.

Background: Prior observational studies have identified an elevated breast cancer risk associated with current MHT use for ER+ (Estrogen Receptor positive), and for ER+ / PR+ (Estrogen and Progesterone Receptor positive) breast cancers than for ER- and ER-/PR- subtypes respectively. We have previously reported, from a large case-control study for all cancer types (the NSW CLEAR study) that current MHT use was associated with a doubling of the odds of breast cancer. Here, we describe further analyses investigating the MHT-breast cancer association for the breast cancer tumor receptor subtypes defined by ER expression, by ER and PR expression and by the joint expression of ER, PR, and HER-2 (Human Epidermal growth factor Receptor-2).

Methods: Analyses were carried out for a subset of registry-verified CLEAR breast cancer cases with hormone receptor status data (n=410) and CLEAR (cancer-free) controls recruited over the same period (n=324). We used a multinomial logistic regression model to estimate Odds Ratios (ORs) adjusted for other breast cancer risk factors and 95% Confidence Intervals (CI) for current and past MHT use in subgroups defined by tumor receptor subtypes. Never users comprised the reference group.

Findings: In a multinomial model, current MHT use was associated with an elevated risk of ER+ breast cancer (aOR= 2.04, 95%CI: 1.28 -3.24). When breast cancers were categorised by ER and PR status, current use was associated with an elevated risk of developing ER+PR+ breast cancer (aOR= 2.29, 1.41-3.72). Current MHT use was associated with the surrogate luminal A breast cancer characterized by ER+/PR+/HER2- phenotype (aOR= 2.30, 1.42-3.73). None of the other subtypes of breast cancer (ER+/PR+/HER2+, ER-/PR-/HER2+, and ER-/PR-/HER2-) were significantly associated with current MHT use. A significant difference in the odds of developing breast cancer for current MHT users was detected between the surrogate luminal A and luminal B (ER+/PR+/HER2+) subtypes only (aOR= 0.28, 0.09-0.88, p=0.029). None of the other groups were significantly differently associated with MHT use, although this may be due to lack of power. Past MHT use was not associated with an increased risk of breast cancer for any breast cancer subtype.

Conclusion: The findings from this contemporary Australian study are consistent with findings from other studies that current, but not past, use of MHT is associated with increased risk of breast cancer, with higher risks reported for ER+, ER+ and PR+ and ER+/PR+/HER2- (surrogate luminal A) subtypes. Our findings are consistent with the hypothesis that breast cancers induced by MHT may occur through receptor-mediated mechanisms.

#2286

Atopic allergic conditions and risk of pancreatic cancer: the Multiethnic Cohort.

Brian Z. Huang,1 Loic Le Marchand,2 Christopher A. Haiman,3 Kristine Monroe,3 Lynne Wilkens,2 Zuo-Feng Zhang,1 Veronica Wendy Setiawan3. 1 _UCLA School of Public Health, Los Angeles, CA;_ 2 _University of Hawaii Cancer Center, Honolulu, HI;_ 3 _University of Southern California, Los Angeles, CA_.

Background: Previous studies, mostly case-control studies, have suggested that atopic allergic conditions (AACs) are associated with a decreased risk of pancreatic cancer. Scarce data, however, are available from prospective, US multiethnic populations. In this largest prospective study to date, we investigated the association between AACs (asthma, hay fever, or allergy) and risk of developing pancreatic cancer in the US Multiethnic Cohort Study (MEC).

Methods: AACs were assessed via a baseline questionnaire when participants joined the MEC in 1993-1996. Hazard ratios (HRs) and 95% confidence intervals (CIs) for pancreatic cancer incidence by AACs status were calculated using Cox regression, adjusting for age, sex, race/ethnicity, education, smoking status, family history of pancreatic cancer, body mass index, diabetes, and alcohol intake.

Results: During an average of 16.2-year follow-up, 1,455 incident cases of pancreatic cancer were identified among 187,226 white, African-American, Native Hawaiian, Japanese-American, and Latino men and women. AACs were not associated with pancreatic cancer incidence (HR=1.00; 95% CI: 0.88, 1.12). The null association was observed in men and women and across racial/ethnic groups, smoking status, BMI groups, diabetes status, and family history of pancreatic cancer.

Conclusions: Based on these results, AACs is unlikely to be associated with risk of pancreatic cancer.

#2287

Number of risky lifestyle behaviors and breast cancer subtypes in a large nested case-control study from Norway.

Merete Ellingjord-Dale,1 Linda Vos,1 Steinar Tretli,1 Solveig Hofvind,1 Anette Hjartåker,2 Hege Russnes,3 Isabel dos-Santos-Silva,4 Giske Ursin1. 1 _Cancer Registry of Norway, Oslo, Norway;_ 2 _University of Oslo, Oslo, Norway;_ 3 _Oslo University Hospital, Oslo, Norway;_ 4 _London School of Hygiene and Tropical Medicine, London, United Kingdom_.

Background: Alcohol, smoking, physical inactivity, high body mass index (BMI) and menopausal hormone therapy have been shown to be associated with increased risk of breast cancer. However, it is unclear how these risk factors combined influence overall breast cancer risk, and whether they are associated with only certain subtypes.

Methods: We conducted a case-control study nested within a cohort of 457,036 women who participated in the Norwegian Breast Cancer Screening Program (NBCSP) in 2006-2014, and who completed a questionnaire at baseline screening. In all, 4 686 breast cancer cases with information on risk factors and hormone receptor status (i.e. estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor 2 (HER2) occurred during the follow-up. The following surrogate definitions of breast cancer subtypes were used: ER+PR+HER2- ("luminal A-like"), ER+PR-HER2- ("luminal B-like, HER2 negative"), ER+ PR+/PR-HER2+ ("luminal B-like, HER2 positive"), ER-PR-HER2+ ("HER2 positive") and ER-PR-HER2- ("triple negative"). We defined risky lifestyle behaviors as ever smoking, weekly consumption of more than 2 glasses of alcoholic beverage, less than 3 hours leisure time physical activity per week, ever use of menopausal hormone (estrogen or estrogen and progesterone) therapy and BMI (kg/cm²) >25. We used conditional logistic regression to estimate odds ratios (ORs), with 95% confidence intervals (CIs), adjusted for age, education, age at menarche, number of pregnancies and menopausal status.

Results: Number of risky lifestyle behaviors was associated with an increased risk of breast cancer overall (p-trend<0.0001). Compared to women with no risky lifestyle behaviors, women with 1 (OR=1.28, 95% CI 0.87-1.88), 2 (OR=1.63, 95% CI 1.13-2.36), 3 (OR=1.85, 95% CI 1.28-2.68), 4 (OR=2.27, 95% CI 1.56-3.29) and 5 (OR=2.38, 95% CI 1.58-3.59) risky lifestyle behaviors had increased risk of the luminal A-like subtype (p-trend<0.0001). However, number of risky lifestyle behaviors was not associated with an increased risk of the triple negative subtype (p-trend=0.27).

Conclusions: This study showed that number of risky lifestyle behaviors was positively associated with a marked increased risk for luminal A-like breast cancer. These findings suggest that healthy lifestyle choices may play an important role in the prevention of the commonest form of this cancer.

#2288

Effects of tobacco smoking and alcohol consumption on risks of CYP1B1 polymorphisms for prostate cancer.

Taku Kato, Yutaka Hashimoto, Shigekatsu Maekawa, Marisa Shiina, Mitsuho Imai-Sumida, Pritha Dasgupta, Priyanka Kulkarni, Soichiro Yamamura, Shahana Majid, Sharanjot Saini, Varahram Sharryari, Guoren Deng, Rajvir Dahiya, Yuichiro Tanaka. _University of California San Francisco and Veterans Affairs Medical Center San Francisco, San Francisco, CA_.

Cytochrome P450 1B1 (CYP1B1) converts xenobiotics to carcinogens and polymorphic variants have been shown to increase activity levels. Lifestyle choices such as tobacco smoking and alcohol consumption are known to enhance the carcinogenesis process and in this study, how these factors may interact with CYP1B1 polymorphisms and affect prostate cancer risk was assessed. Blood genomic DNA from a Caucasian population consisting of 405 healthy men and 400 prostate cancer patients were obtained. Of these, 507 were current or former smokers and 407 were alcohol drinkers. Eight polymorphic sites of the promoter region of CYP1B1 (rs2551188 G to A, rs2567206 G to A, rs2567207 A to G, rs162556 A to G, rs10175368 C to T, rs163090 T to A, rs162330 T to G, and rs162331 A to G) were analyzed in samples using Taqman genotyping assays and real-time PCR. Lifestyle factors and its influence on CYP1B1 polymorphisms toward cancer risks were also evaluated. Overall, both alcohol (P=0.006) and smoker (P=0.069) status were associated with prostate cancer. CYP1B1 variants were also risks for cancer at rs2551188 (P=0.043), rs2567206 (P=0.008), and rs10175368 (P=0.001). Evaluation of linkage disequilibrium show rs2551188, rs2567206, rs2567207, and rs10175368 to be linked and interestingly, the G-G-A-C haplotype (wildtype at respective sites) was significantly reduced in cancer (P=0.0282). When classified by lifestyle factors, no associations for CYP1B1 variants were found for cancer among non-smokers with rs10175368 (P=0.051) being a risk among non-drinkers. On the other hand, variants at both rs2567206 and rs10175368 showed increased cancer risk among smokers (P=0.032 and 0.002, respectively) as well as drinkers (P=0.044 and 0.019, respectively). No genotyping differences were observed when analyzing cancers by pathological grades. These results demonstrate smoker and alcoholic drinker status to modify the risks of CYP1B1 polymorphisms for prostate cancer and this is of importance in understanding their role in the pathogenesis of this disease.

#2289

Β-carotene supplementation and lung cancer risk in the ATBC Study: the role of tar and nicotine.

Pooja Middha,1 Stephanie J. Weinstein,2 Satu Männistö,3 Demetrius Albanes,2 Alison M. Mondul1. 1 _University of Michigan School of Public Health, Ann Arbor, MI;_ 2 _National Cancer Institute, Bethesda, MD;_ 3 _National Institute for Health and Welfare, Helsinki, Finland_.

Background: Globally, lung cancer is the most common cancer and the leading cause of cancer death. Early observational studies reported a protective association between intake of β-carotene rich vegetables and lung cancer risk, which led to interest in β-carotene supplementation as a potential chemopreventive strategy, particularly in high-risk populations such as cigarette smokers. A large randomized controlled trial, the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study, demonstrated that β-carotene supplementation increased lung cancer incidence by 18% in smokers, and led to an 8% excess in overall mortality. It is also well-documented that cigarettes with higher (versus lower) tar and nicotine content are associated with a higher risk of lung cancer. To our knowledge, however, no studies have examined whether the increased risk associated with β-carotene supplementation in smokers varies by cigarette tar or nicotine content.

Methods: The ATBC Study was a randomized, double-blind vitamin supplementation trial conducted among 29,133 male smokers in southwest Finland. Participants were randomly assigned to one of four groups in a 2x2 factorial design (α-tocopherol alone, β-carotene alone, both supplements, or placebo). A detailed smoking history was collected at baseline, including brands of cigarettes smoked (for which machine measured tar and nicotine content was available). Cox proportional hazards models were used to estimate the hazard ratio (HR) and 95% confidence intervals (CI) of lung cancer risk by trial Β-carotene assignment stratified by a priori categories of cigarette tar (ultra-light ≤7 mg, light 8-14 mg, medium/regular 15-20 mg, high/non-filtered/self-made >20 mg) and nicotine content (ventilated filtered ≤0.8 µg, unventilated filtered 0.8-1.3 µg, non-filtered >1.3 µg). Statistical interaction was estimated using the likelihood ratio test.

Results: The β-carotene supplemented arm had a statistically significantly higher risk of developing lung cancer in all categories of tar content [yes vs. no β-carotene supplementation - ultra-light: HR=1.31 95% CI=0.91-1.89; non-filtered: HR=1.22 95% CI=0.91-1.64; p-interaction=0.91]. Similarly, there was no apparent interaction with cigarette nicotine content [yes vs. no β-carotene supplementation - ventilated cigarettes: HR=1.23, 95% CI=0.98-1.54; non-filtered cigarettes: HR=1.22, 95% CI=0.91-1.64; p-interaction=0.83].

Conclusion: These findings indicate that supplementation with β-carotene increases lung cancer incidence in smokers regardless of the tar or nicotine content of cigarettes smoked. Our data suggest that all smokers, regardless of the type of cigarette smoked, should continue to avoid β-carotene supplementation.

#2290

Perceived stressfulness of life events impacts breast cancer risk.

Avital Fischer, Argios Ziogas, Hoda Anton-Culver. _UCI, Irvine, CA_.

Background: Breast cancer is the most prevalent malignancy and the second most common cause of cancer mortality among women. Approximately 60% of variability in breast cancer risk remains unexplained. It is imperative to perform further research focusing on identifying preventable risk factors for breast cancer. The relationship between psychological stress and breast cancer risk is unclear. Psychological stress arises when environmental demands are appraised as going beyond one's coping abilities. Higher perceived stress has been linked to increased cortisol levels and HPA perturbations. The purpose of this study is to understand the relationship between stressfulness appraisal of salient life events and breast cancer risk.

Methods: A case-control design was used and included 664 female cases identified through the Cancer Surveillance Program of Orange County, CA and 203 female population-based controls. Participants completed a risk factor questionnaire that included a life event section. Participants were asked to determine if events occurred and if they were considered to be significantly stressful or not. Multivariate unconditional logistic regression was used to calculate ORs while adjusting for known breast cancer covariates.

Results: Cumulative adverse life events perceived as stressful were associated with increased breast cancer risk in a dose response fashion (OR=1.63, 95% CI=1.00-2.66, Ptrend=0.045). Conversely, cumulative adverse life events not reported as stressful did not have a significant impact on breast cancer risk (OR=2.04, 95% CI=0.77-5.43). History of personal illness was directly related to increased breast cancer risk, when perceived as stressful (OR=2.84, 95% CI=1.96-4.11) or non-stressful (OR=3.47, 95% CI=1.34-8.94). Interestingly, abortion and relocation were observed to have a protective effect on breast cancer risk only when reported as being stressful (OR=0.54, 95% CI=0.32-0.92; OR=0.63, 95% CI=0.43-0.93 respectively).

Conclusions: This study broadens our understanding of the relationship between major life events and breast cancer risk and underscores the importance of stressfulness appraisal. Our results indicate the importance of incorporating assessments of perceived stressfulness in future epidemiological investigations focusing on the assessment of life events and breast cancer risk.

#2292

Lung function and lung cancer risk: a Mendelian randomization study of UK Biobank cohort and the International Lung Cancer Consortium.

Linda Kachuri,1 Mattias Johansson,2 Paul Brennan,2 Phillip Haycock,3 Geoffrey Liu,4 Maria Teresa Landi,5 David C. Christiani,6 Neil E. Caporaso,5 Xifeng Wu,7 Melinda C. Aldrich,8 Demetrius Albanes,5 Adonina Tardón,9 Gad Rennert,10 Chu Chen,11 Gary E. Goodman,11 Jennifer A. Doherty,12 Heike Bickeböller,13 Dawn Teare,14 Lambertus A. Kiemeney,15 Stig E. Bojesen,16 John K. Field,17 Aage Haugen,18 Stephen Lam,19 Loic Le Marchand,20 Matthew B. Schabath,21 Angeline S. Andrew,12 Jonas Manjer,22 Philip Lazarus,23 Susanne M. Arnold,24 Valérie Gaborieau,2 Richard Martin,3 Caroline Relton,3 George Davey Smith,3 Christopher I. Amos,12 James D. McKay,2 Rayjean J. Hung1. 1 _Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada;_ 2 _International Agency for Research on Cancer, Lyon, France;_ 3 _University of Bristol, Bristol, United Kingdom;_ 4 _Princess Margaret Hospital, Ontario Cancer Institute, Toronto, Ontario, Canada;_ 5 _National Cancer Institute, MD;_ 6 _Harvard T.H. Chan School Of Public Health, MA;_ 7 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 8 _Vanderbilt University Medical Center, TN;_ 9 _University of Oviedo, University Institute of Oncology, Oviedo, Spain;_ 10 _Technion Faculty of Medicine and Carmel Medical Center, Israel;_ 11 _Fred Hutchinson Cancer Research Center, WA;_ 12 _Geisel School of Medicine, Dartmouth College, NH;_ 13 _University Medical Centre Göttingen, Göttingen, Germany;_ 14 _School of Health and Related Research, University of Sheffield, Sheffield, United Kingdom;_ 15 _Radboud University Medical Centre, Nijmegen, Netherlands;_ 16 _Herlev and Gentofte Hospital, Copenhagen University Hospital, Herlev, Denmark;_ 17 _University of Liverpool, Liverpool, United Kingdom;_ 18 _National Institute of Occupational Health, Oslo, Norway;_ 19 _BC Cancer Agency, Vancouver, British Columbia, Canada;_ 20 _University of Hawaii Cancer Center, HI;_ 21 _Moffitt Cancer Center, FL;_ 22 _Skåne University Hospital, Lund University, Lund, Sweden;_ 23 _College of Pharmacy, Washington State University, WA;_ 24 _Markey Cancer Center, University of Kentucky, KY_.

Background: Impaired lung function (LF) is strongly associated with increased lung cancer risk. However, since airflow obstruction is a diagnostic criterion for obstructive lung disease, and a consequence of tobacco smoking, isolating the causal relationship between LF and lung cancer has remained a challenge.

Methods: We investigated 3 standardized (mean=0, standard deviation=1) LF metrics: forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC), and FEV1/FVC. To evaluate the causal relevance of LF in lung cancer etiology we conducted: i) survival analyses in the UK Biobank cohort (UKB); and ii) Mendelian Randomization (MR) analyses using genetic instrumental variables (IVs) developed in UKB and tested using individual-level data from the OncoArray, a genome-wide array with in-depth coverage for common cancers. Results: 702 incident lung cancers were diagnosed in 484,194 UKB participants during follow-up. Cox regression was used to estimate hazard ratios (HR) and 95% confidence intervals (CI), adjusted for age, sex, smoking status, socioeconomic status, and assessment center. Adjustment for other smoking metrics yielded similar results. Lung cancer risk increased per 1 unit decrease in FEV1 (HR=1.80, 95% CI: 1.64-1.98, p=3.3×10-34), FVC (HR=1.45, 1.30-1.60, p=2.3×10-12), and FEV1/FVC (HR=1.39, 1.33-1.46, p=1.3×10-38). This pattern was observed for adenocarcinoma (n=300): FEV1 (HR=1.77, p=6.0×10-12), FVC (HR=1.48, p=1.4×10-5), FEV1/FVC (HR=1.34, p=8.3×10-11); and squamous cell carcinoma (n=166): FEV1 (HR=1.97, p=9.9×10-10), FVC (HR=1.60, p=1.0×10-4), FEV1/FVC (HR=1.38, p=5.9×10-8). Next, a genome-wide association study of 67,708 UKB participants and 12.6 million variants was carried out to develop genetic IVs for LF.

Results were filtered to retain independent variants (R2<0.2) associated with each LF phenotype (p<5×10-8). The following IVs were developed: FEV1 (n=28 variants, 0.72% of variation explained), FVC (n=44, 1.08%), and FEV1/FVC (n=45, 1.85%). Odds ratios (OR) for each IV and lung cancer were estimated for 18,686 cases 15,190 controls (>80% European ancestry) from 23 studies. Effect estimates were combined using maximum-likelihood MR models to estimate causal ORs. MR results indicate that genetic scores associated with improved airflow are unrelated to lung cancer risk: FEV1 (OR=1.00, 95% CI: 0.96-1.03, p=0.86), FVC (OR=1.00, 0.97-1.03, p=0.93) and FEV1/FVC (OR=1.00, 0.91-1.10, p=0.95). The null association observed for the genetic determinants of FEV1, FVC and FEV1/FVC was not modified by tumor histology or smoking status.

Conclusions: LF is a robust predictor of lung cancer risk, however, our findings do not support the existence of causal pathways that are independent of obstructive lung disease or smoking. This apparent lack of a causal relationship should be interpreted with caution since pleiotropic effects of LF loci cannot be ruled out.

#2293

Oligomenorrhea, polycystic ovary syndrome, and risk of ovarian cancer histotypes, evidence from the Ovarian Cancer Association Consortium.

Holly R. Harris,1 Susan Jordan,2 Harvey Risch,3 Mary Anne Rossing,1 Marc T. Goodman,4 Francesmary Modugno,5 Susanne Krüger Kjær,6 Joellen M. Schildkraut,7 Elisa V. Bandera,8 Nicolas Wentzensen,9 Catherine Phelan,10 Hoda Anton-Culver,11 Anna H. Wu,12 Kathryn L. Terry13. 1 _Fred Hutchinson Cancer Research Ctr., Seattle, WA;_ 2 _QIMR Berghofer Medical Research Institute, Herston, Australia;_ 3 _Yale School of Public Health, New Haven, CT;_ 4 _Cedars-Sinai Medical Center, Los Angeles, CA;_ 5 _University of Pittsburgh School of Medicine, Pittsburgh, PA;_ 6 _Danish Cancer Society Research Center, Copenhagen, Denmark;_ 7 _The University of Virginia, Charlottesville, VA;_ 8 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ;_ 9 _National Cancer Institute, Bethesda, MD;_ 10 _Moffitt Cancer Center, Tampa, FL;_ 11 _University of California Irvine, Irvine, CA;_ 12 _University of Southern California Norris Comprehensive Cancer Center, Los Angeles, CA;_ 13 _Brigham and Women's Hospital, Boston, MA_.

Background: Oligomenorrhea, defined as infrequent or irregular periods has been shown to be associated with epithelial ovarian cancer risk in some but not all studies. Polycystic ovary syndrome (PCOS), which is characterized by oligomenorrhea and abnormal hormone levels including hyperandrogenism, hyperinsulinemia, and gonadotropin imbalance, has been suggested to increase ovarian cancer risk. However, these associations have been rarely examined by histologic subtype. We sought to examine these associations among 14 studies participating in the Ovarian Cancer Association Consortium.

Methods: Participants included 16,594 patients with invasive (n=13,719) or borderline (n=2,875) ovarian cancers and 17,718 controls who had answered questions regarding menstrual cycle irregularity, menstrual cycle length, and/or PCOS. Study specific odds ratios (ORs; 95% confidence intervals [95% CI]), adjusted for ovarian cancer risk factors, were calculated and then combined using a random-effects meta-analysis. Pooled histologic subtype specific ORs (95% CI) were calculated using polytomous logistic regression.

Results: Women reporting menstrual cycle length >35 days had decreased risk of invasive ovarian cancer compared to women reporting cycle length ≤35 days (OR=0.70; 95% CI=0.58-0.84). Decreased risk of invasive ovarian cancer was also observed among women who reported irregular menstrual cycles compared to women with regular cycles (OR=0.83; 95% CI=0.76-0.89). No significant association was observed between self-reported PCOS and invasive ovarian cancer risk (OR=0.87; 95% CI=0.65-1.15). For menstrual cycle length >35 days, decreased risk was observed for all invasive histotypes: low grade serous (OR=0.48; 95% CI=0.25-0.92), high grade serous (OR=0.62; 95% CI=0.48-0.80), mucinous (OR=0.38; 95% CI=0.19-0.76), endometrioid (OR=0.75; 95% CI=0.54-1.06), and clear cell (OR=0.70; 95% CI=0.43-1.13) but not for serous borderline tumors (OR=1.18; 95% CI=0.87-1.59) (pheterogeneity=0.006). An increased risk of serous borderline disease was observed among women reporting irregular cycles (OR=1.34; 95% CI=1.16-1.55) (pheterogeneity<0.0001). No statistically significant differences by histologic subtype were observed for self-reported PCOS.

Conclusion: In this large consortium analysis we observed a decreased risk of most invasive epithelial ovarian cancer histotypes among women reporting longer or irregular menstrual cycles. In contrast, such cycles were associated with increased risk of the serous borderline tumors.

#2294

Atypical nevi and risk of incident skin cancer in US men: a prospective study.

WEN-QING LI,1 Eunyoung Cho,1 Alisa Goldstein,2 Yen-Tsung Huang,1 Martin Weinstock,1 Abrar Qureshi1. 1 _Brown University, Providence, RI;_ 2 _National Cancer Institute, Bethesda, MD_.

Background and Aims: Atypical nevi (AN) are a strong risk factor as well as a precursor of melanoma. However, most previous studies on the associations between atypical nevi (AN) and the risk of melanoma have been conducted in retrospective case-control settings and the few available prospective studies have been based on small sample sizes. Further, few studies have examined the associations between AN and risk of keratinocyte carcinomas (formerly called non-melanoma skin cancer). We prospectively examined the risk of incident melanoma, squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) associated with AN.

Methods: In the Health Professionals Follow-up Study, information on AN was collected by self-report. Only those confirmed cases of AN by a supplementary questionnaire, the Atypical Nevus Screening Questionnaire (ANQ), were included in the analyses. Diagnosis of skin cancers was reported biennially and information on melanoma and SCC was pathologically confirmed. A total of 50193 men were included in our study, with 1239 confirmed cases of AN with information on diagnosis year. Cases of AN that were not confirmed or did not respond to the ANQ were excluded from the analyses. Time-dependent Cox-regression analyses were conducted for the associations between AN and risk of incident melanoma, SCC, and BCC respectively.

Results: We identified 545 melanoma cases, 1721 SCC cases, and 13498 BCC cases during the follow-up (1986-2012). A history of AN was significantly associated with increased risk of incident melanoma (hazard ratio [HR]=3.50, 95% confidence interval [CI]: 2.13-5.76) overall and at different body sites (head and neck, trunk, or limbs). The risk of melanoma was particularly augmented with the increasing number of AN that were surgically removed (HR=1.26, 95% CI: 1.16-1.37 per AN) and for cases of AN with tissue re-excision from the nevus removal sites (HR=4.59, 95% CI: 1.70-12.41). A history of AN was also significantly associated with increased risk of BCC (HR=1.84, 95% CI: 1.60-2.10) and was only associated with risk of SCC at the trunk sites (HR=3.09, 95% CI: 1.24-7.72).

Conclusions: A history of AN significantly increased the risk of subsequent melanoma in a prospective study of men. A history of AN may also be associated with increased risk of BCC overall and trunk SCC, which requires further studies to replicate.

#2295

Association between metformin use and the risk of colorectal adenomas: A systematic review and meta-analysis.

Sheetal Hardikar,1 Jessica S. Li,2 Qianchuan He,1 Jonathan Kocarnik,1 Edward J. Boyko,2 Polly A. Newcomb1. 1 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 2 _University of Washington, Seattle, WA_.

Metformin, the most commonly prescribed drug in type-2 diabetes, has been evaluated for its potential cancer chemopreventive role. While evidence from meta-analyses suggests a reduced risk for colorectal cancer in association with metformin use, recent European studies have reported no association between metformin and colorectal cancer risk. For precursors to colorectal cancers such as colorectal adenomas, however, literature evaluating their relationship with metformin use is sparse. We performed a systematic review and meta-analysis of available studies to quantitatively assess the association of metformin use with risk of colorectal adenomas. In accordance with PRISMA guidelines and using relevant MeSH terms, we searched the PubMed, EMBASE, and Web of Science databases through September 30, 2016 to identify full-text articles investigating the risk for colorectal adenomas associated with the use of metformin. Overall risk ratios (RR) and 95% confidence intervals (CI) for colorectal adenomas were calculated using a fixed-effects model. Heterogeneity between studies was evaluated using the Cochrane's Q- test and the I2\- statistic. Potential publication bias was assessed by constructing a funnel plot. Sub-group analyses were performed to examine whether the association with metformin use differed by country/region. Of the 609 articles identified through searching the three databases, 6 studies were eligible for our meta-analysis (1 randomized trial, 2 case-control, and 3 cohort studies; 4 Asian studies and 2 US-based studies) and contributed a total of 9613 patients. The meta-analysis suggested that the risk for colorectal adenomas was significantly lower among metformin users as compared to non-users [overall RR (95% CI): 0.69 (0.62-0.77), test for overall effect Z =18.52, P-value <0.0001]. This protective risk applied both for studies within the US and Asian regions [pooled RRs (95% CI): 0.72 (0.64-0.81) and 0.62 (0.47-0.76), respectively]. There was no heterogeneity observed between studies (Q-test p= 0.09, I2-statistic 47.3%) and no evidence of any publication bias on funnel plot visualization. This meta-analysis indicates that the use of metformin is significantly associated with a lower risk of colorectal adenomas. As metformin is an affordable and well-tolerated drug, it has the potential to be an effective agent for polyp chemoprevention. Further long-term randomized controlled trials are needed to confirm the anti-neoplastic effects of metformin in reducing the risk of colorectal polyps, and eventually colorectal cancer, in the general population.

#2296

Metformin reduces colon cancer risk in patients with type 2 diabetes: a retrospective cohort analysis.

Chin-Hsiao Tseng. _National Taiwan Univ. College of Medicine, Taipei, Taiwan_.

Background: The effect of metformin on colon cancer is rarely investigated in Asian populations.

Methods: This is a retrospective cohort study. Patients with newly diagnosed type 2 diabetes during 1999-2005 were recruited from Taiwan's National Health Insurance. A 1:1 propensity score matched-pair sample of 16314 ever users and 16314 never users of metformin were followed until December 31, 2011. Cox regression incorporated with the inverse probability of treatment weighting using propensity score was used to estimate hazard ratios.

Results: The incidence of colon cancer was 272.04 and 392.44 per 100,000 person-years, respectively, in ever and never users. The overall hazard ratio (0.691, 95% confidence interval: 0.578-0.826) suggested a significantly lower risk in metformin users. While compared to never users, the hazard ratio (95% confidence interval) for the first (<20.00 months), second (20.00-45.20 months) and third (>45.20 months) tertile of cumulative duration of metformin therapy was 1.455 (1.165-1.816), 0.683 (0.526-0.885) and 0.224 (0.152-0.331), respectively. The protective effect of metformin on colon cancer was not affected by the use of aspirin and statin or the infection of Helicobacter pylori in additional analyses.

Conclusions: Metformin reduces colon cancer risk in patients with type 2 diabetes. The protective effect is more remarkable when the cumulative duration is more than 20 months.

#2297

Household stove improvement and risk of chronic obstructive pulmonary disease in Xuanwei, China: a cohort study.

Jinming Zhang,1 Bryan A. Bassig,1 Roel Vermeulen,2 Wei Jie Seow,1 Jason Y.Y. Wong,1 Wei Hu,1 Bofu Ning,3 George S. Downward,2 Hormuzd A. Katki,1 Bu-Tian Ji,1 Nathaniel Rothman,1 Robert S. Chapman,4 Qing Lan1. 1 _National Cancer Insitute, Rockville, MD;_ 2 _Utrecht University, Utrecht, Netherlands;_ 3 _Xuanwei Center of Disease Control, Xuanwei, China;_ 4 _Chulalongkorn University, Bangkok, Thailand_.

Background: In Xuanwei County, China, chronic obstructive pulmonary disease (COPD) rates are over twice the national average. Patients with COPD have higher risks of both lung cancer and poor treatment outcomes after lung cancer diagnosis. In a prior retrospective cohort study (1976-1992), household stove improvement was found to be associated with decreased COPD incidence among lifetime smoky (bituminous) coal users. However, whether the protective effects persist over time is unclear. Our objective was to evaluate the associations between risk of COPD, and vented and portable stove use with extended follow-up (1976-2011).

Methods: The study population comprised 22,833 residents who were born between 1917 and 1951 and lived in Xuanwei as of January 1, 1976. Participants were followed retrospectively from 1976 to 1992, and then followed prospectively through 2011. We conducted two surveys in 1992 and 2011. During each survey, we collected comprehensive information on household stove type, fuel use, family size and number of rooms in each residence during the lifetime, smoking, education, age started cooking food and time spent indoor from each subject. Participants were also asked to report any medically diagnosed chronic bronchitis or emphysema, age and place of diagnosis, which we combined into a single category of COPD. COPD cases diagnosed before January 1, 1976 were excluded. Sex-specific time-dependent Cox regression models were used to estimate the hazard ratio (HR) of stove improvement for COPD incidence among lifetime smoky coal users.

Results: The analysis was restricted to lifetime smoky coal users. A significant reduction in COPD incidence was found after change to vented stoves (Men's model: HR=0.66, 95% confidence interval(CI): 0.59-0.75; Women's model: HR=0.63, 95% CI: 0.55-0.72) and portable stoves (Men's model: HR=0.77, 95% CI: 0.67-0.88; Women's model: HR=0.63, 95% CI: 0.54-0.74). There was a downward trend of COPD incidence over time after change to vented stove or portable stove (p-trend<0.01). Similar findings were noted in both men and women.

Conclusion: Consistent with prior research, our findings suggest that household stove improvement was associated with a substantially decreased risk of COPD, a strong risk factor for lung cancer. Domestic smoky coal use and unvented coal combustion are important risk factors for COPD. Our findings support the need for replacing smoky coal with less carcinogenic fuels in areas where domestic coal use is common for cooking and heating, especially in the developing countries.

#2298

Antibiotic use and breast cancer risk: results from the Sister Study.

Sandra L. Deming-Halverson,1 M. Elizabeth Hodgson,1 Aimee D'Aloisio,1 David Shore,2 Dale Sandler3. 1 _Social & Scientific Systems, Inc., Durham, NC; _2 _Westat, Durham, NC;_ 3 _NIEHS, Durham, NC_.

Background: Regular antibiotic use is hypothesized to influence breast cancer risk by a number of mechanisms including disruption of intestinal microflora which plays a role in the conversion of food-based phytochemicals into bioactive substances suggested to be protective against cancer, and by influences on the immune system and inflammatory response. Previous results have been mixed, and given the sparse and conflicting data in epidemiologic literature, we analyzed the association between regular antibiotic use and breast cancer risk, overall and by different classes of antibiotics.

Methods: Regular antibiotic use, as characterized by class type and number, duration, and indication for use, was evaluated for its association with breast cancer risk among participants of the NIEHS Sister Study, a prospective cohort of 50,884 US women aged 35-74 without breast cancer who have had a sister diagnosed with breast cancer.

Results: 5,312 (10.4%) of women in the Sister Study reported ever having regular antibiotic use (at least 3 times per week for 3 months in a row or longer) with a median duration of 1.50 years (0.25 – 53.8). 34% of regular uses reported use greater than 2 years. Skin conditions including acne and rosacea were the most common indications for use (6.02%). The hazard ratio (HR) was 1.10 (0.99-1.27) for ever regular antibiotic use and 1.40 (1.16-1.69) for greater than 2 years of regular use. Results are suggestive of an increased risk among regular users who have only ever used one class rather than more than one class of antibiotics. Adjusted hazard ratios were 1.40 (0.99-1.99) for tetracyclines only, 1.45 (0.95-2.20) for penicillins only, 1.42 (0.94-2.15) for macrolides alone. However, there does not appear to be an association between ever regular use of more than one class of antibiotics and breast cancer. Furthermore, there was no association between indications for ever regular use and breast cancer risk, among all women reporting regular use of antibiotics, regardless of class.

Conclusion: Overall, there is a suggestion that ever regular use of a single class of antibiotics, but not use of more than one class, may be associated with increased breast cancer risk. However, given that most individuals reported only a single episode of regular antibiotic use for a duration of less than 2 years; further exploration is warranted to rule out effects of underlying indication for use or explore other possible class specific-confounders.

#2299

Toxic metals in private well water and bladder and kidney cancer incidence in North Carolina.

Deepika Shrestha,1 Selin Cronkrite,1 Alison Sanders,2 Rebecca Fry,3 Katie M. Applebaum1. 1 _George Washington University, Milken Institute School of Public Health, Washington, DC;_ 2 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 3 _University of North Carolina, Gillings School of Global Public Health, Chapel Hill, NC_.

Introduction: There are a limited number of studies conducted in the US that have related metals in well water with kidney and bladder cancer. We investigated levels of arsenic (As), cadmium (Cd), manganese (Mn), and lead (Pb) in private well water and the association with incidence of bladder and kidney cancer in 100 counties in North Carolina, a state that has among the highest proportion of well water consumers in the country.

Methods: Between 1998-2010, private wells across 100 NC counties were analyzed for levels of As, Cd, Mn and Pb (n=63,836, n=22,915, n=70,675 and n=65,535 measurements, respectively) and geocoded. Bladder and kidney cancer incidence for each county between 1990 and 2011 were obtained from the North Carolina Department of Health and Human Services. County-level data on potential confounders were obtained from US Census Bureau data (2010) and the Behavioral risk factor surveillance study (2003, 2005). For each county, the mean concentration of each metal and the incidence rate of kidney and bladder cancer were calculated separately. Using counties as the unit of analysis, we used Poisson regression to estimate incidence rate ratios (IRRs) and 95% confidence intervals (CIs) between mean concentration of the four metals and kidney and bladder cancer incidence, adjusting for age, gender, race, education, physical activity, smoking, diabetes, hypertension and residential well water supply. County mean metal levels were analyzed in tertiles. Counties with less than 10 cases or fewer than 10 well measurements for any metal were excluded from the analysis.

Results: Ninety-eight counties were included in the final analysis. In the fully adjusted model, elevated IRRs for kidney cancer were observed in counties with the highest tertile of As (3rd tertile: 0.99-11.44 ppb, IRR=1.05 (95% CI 1.00, 1.11)). Similarly, elevated IRRs for kidney cancer were observed for both the 2nd and 3rd tertiles of Cd (3rd tertile 0.61-2.98 ppb: IRR=1.05 (95% CI 1.00, 1.10)) and the 2nd tertile of Pb (3.88-5.05 ppb, IRR=1.05 (95% CI 1.00, 1.10)). A dose response was observed for kidney cancer incidence and Mn (2nd tertile: 37.33-51.74 pbb; IRR =1.06 (95% CI 1.02, 1.11); 3rd tertile: 52.95-146.60 ppb: IRR=1.13 (95% CI 1.08, 1.19)). For bladder cancer, no relationship was observed with As but IRRs were elevated for the other metals including potential dose-response trend with Cd and Mn (Pb 2nd tertile 3.88-5.05 ppb: IRR=1.03 (95% CI 1.00, 1.07); Cd 3rd tertile 0.61-2.98 ppb: IRR=1.10 (95% CI 1.06, 1.15); Mn 3rd tertile 52.95-146.60 ppb: IRR=1.12 (95% CI 1.08, 1.16)).

Conclusion: In North Carolina, counties with higher mean private well metal levels had elevated kidney and bladder cancer incidence rates. The conclusions are limited by the ecological approach, but the consistency of the associations suggests that the influence of metals in private well water in North Carolina on health outcome deserves additional investigation.

#2300

Pesticides and primary liver cancer: A systematic review and meta-analysis.

Hamdi Abdi, Jennifer Lee, Gary Ellison, Gabriel Lai, Tram Lam. _National Cancer Institute, Rockville, MD_.

Background: Primary liver cancer is the sixth most occurring cancer worldwide and the second most common cause of cancer mortality. Hepatocellular carcinoma (HCC) is the most prevalent histologic type of liver cancer, with well-established risk factors (e.g., chronic infections with hepatitis B virus or hepatitis C virus, and alcohol-related liver disease). Exposure to pesticides may also contribute to the etiology of liver cancer. The use of pesticides has increased significantly in both developed and developing countries within the last few decades. The association between pesticides and liver cancer is unclear. We conducted a systematic review and meta-analysis to investigate the relationship between exposure to pesticides (e.g.; organophosphates, organochlorines, carbamates, dichlorodiphenyltrichloroethane, dichlorodiphenyldichloroethylene, agent orange) and liver cancer incidence and mortality.

Methods: We searched PubMed and EMBASE databases using a combination of medical subject heading terms and key words for articles published before June 2016. Articles were identified as relevant if they were original studies and met the following criteria: 1) primary liver cancer was an identified outcome for risk or mortality, 2) pesticides were the main exposure of interest, 3) exposure to pesticides was assessed histologically (e.g.; blood serum) or through questionnaires, and 4) the estimates for the association between liver cancer risk/mortality and pesticide exposure were reported. Titles and abstracts meeting search criteria were reviewed and relevant full-text articles were read and analyzed independently by two researchers. We calculated the summary estimates [Odd Ratios (ORs) or Relative Risks (RR)] and 95% confidence intervals (CIs) using random-effects models. All analyses were conducted using STATA 14.0.

Results: Of 1644 articles reviewed, a total of 34 observational studies were identified as relevant and 20 were included in the meta-analysis for liver cancer risk (5 case-controls and 15 cohorts). All of the identified studies investigated HCC as the outcome; none investigated other types of liver cancer. The results showed that pesticide exposure was associated with a 73% increased risk of HCC (RRsummary:1.73, 95% CI 1.40-2.13; I2=85.9%, P=<0.001). Stratified analyses by study design showed similar results (case-control: 1.69, 95% CI 1.31-2.18; cohorts: 1.47, 95% CI 1.25-1.72). Furthermore, in analysis stratified by sex and type of pesticide similar elevated risks were observed. Analysis of articles that reported on liver cancer mortality (n=7) showed an excess in mortality from HCC for individuals exposed to pesticides.

Conclusion: The existing epidemiological data supports the hypothesis that exposure to pesticides is associated with a significant increased risk of HCC and death from liver cancer.

#2301

Genital use of talc and risk of ovarian cancer: A meta-analysis.

Paolo Boffetta,1 Wera Berge2. 1 _Mt. Sinai Icahn School of Medicine, New York, NY;_ 2 _University of Dresden, Dresden, Germany_.

Some epidemiological studies suggest an association between genital use of talc powders and increased risk of ovarian cancer, but the evidence is not consistent. We performed a meta-analysis of epidemiologic studies to formally evaluate this suspected association. A systematic search was conducted in Medline, Embase and Scopus, leading to the identification of 24 case-control studies and 3 cohort studies, including 302,705 women with ovarian cancer. In the meta-analysis, we used a random-effect model to calculate summary estimates of the association between genital use of talc and occurrence of ovarian cancer. We assessed potential sources of between-study heterogeneity and presence of publication bias. The summary relative risk (RR) for ever use of genital talc and ovarian cancer was 1.22 (95% confidence interval [CI] 1.13-1.30). The RR for case-control studies was 1.26 (95% CI 1.17-1.35) and for cohort studies was 1.02 (95% CI 0.85-1.20, p heterogeneity=0.007). Serous carcinoma was the only histologic type for which an association was detected (RR 1.24; 95% CI 1.15-1.34). There was no trend in RR with either duration or frequency of genital talc use. This meta-analysis resulted in a weak but statistically significant association between genital use of talc and ovarian cancer, which appears to be limited to serous carcinoma. The heterogeneity of results by study design and the lack of a trend for duration and frequency of use, however, detract from a causal interpretation of this association.

#2302

Differences in risk factor-colorectal adenoma associations according to non-steroidal anti-inflammatory drug use.

Sobia Mujtaba, Roberd M. Bostick. _Emory University, Atlanta, GA_.

Multiple observational studies and large, randomized controlled trials indicate that non-steroidal anti-inflammatory drugs (NSAIDs) strongly reduce risk for colorectal neoplasms. The strengths of these findings suggest that NSAIDs may so mask various risk factor-colorectal neoplasm associations, that they may be undetectable among NSAID users. Temporal changes and between-population differences in regular NSAID use prevalence may account for some of the inconsistencies in reported findings for risk factor-colorectal neoplasm associations. We investigated whether associations of known colorectal cancer risk factors with colorectal adenoma differed by non-aspirin NSAID use using pooled data from 3 colonoscopy-based case-control studies of incident, sporadic colorectal adenoma conducted in Minnesota, North Carolina, and South Carolina between 1991 and 2002. Participants (n=789 cases, 2,035 polyp-free controls) provided risk factor data prior to colonoscopy. The multivariable-adjusted odds ratios (OR) (95% confidence intervals [CI]) for those in the highest relative to the lowest quartiles of exposure, by regular non-aspirin NSAID non-use/use, respectively, were 1.57 (CI 0.96, 2.55) vs. 1.22 (CI 0.40, 3.70) for total fat, 1.37 (CI 0.86, 2.18) vs. 0.78 (CI 0.26, 2.35) for saturated fat, 0.92 (CI 0.67, 1.26) vs. 1.31 (CI 0.62, 2.76) for total calcium, 0.88 (CI 0.64, 1.22) vs. 1.38 (CI 0.65, 2.93) for total fruits and vegetables, 1.05 (CI 0.73, 1.50) vs. 0.73 (CI 0.33, 1.62) for red and processed meats, and 0.84 (CI 0.65, 1.10) vs. 0.97 (CI 0.54,1.75) for physical activity. For current smokers relative to never smokers, the ORs (CIs) among regular non-NSAID users/non-users were 2.88 (CI 2.20, 3.77) vs. 1.64 (CI 0.85, 3.17), respectively, and for those who were obese relative to those who were normal weight, they were 1.65 (CI 1.27, 2.16) vs. 1.20 (CI 0.70, 2.04). The associations of age, height, and intakes of alcohol, dietary fiber, and total folate with adenoma did not substantially differ according to NSAID use.

These findings suggest that regular non-aspirin NSAID use may mask, beyond simple confounding, associations of various risk factors with colorectal adenoma, suggesting that differential proportions of regular NSAID users between study populations may explain some inconsistencies in reported risk factor-colorectal neoplasm associations, and support routinely assessing such associations stratified by regular non-aspirin NSAID use.

#2303

Entacapone and prostate cancer in patients with Parkinson's disease: A large Veterans Health Administration study.

Jacqueline M. Major,1 Francesca Cunningham,2 Diane Dong,2 Kunthel By,3 Kwan Hur,2 David C. Shih,1 Simone P. Pinheiro,1 Gerald D. Podskalny,4 David J. Graham1. 1 _US Food and Drug Administration/CDER/OSE, Silver Spring, MD;_ 2 _US Department of Veterans Affairs, Hines, IL;_ 3 _US Food and Drug Administration/CDER/OB, Silver Spring, MD;_ 4 _US Food and Drug Administration/CDER/OND, Silver Spring, MD_.

Background: An increased risk of prostate cancer was observed in Parkinson's disease (PD) patients treated with entacapone during a pre-approval randomized clinical trial.

Objective: To investigate a potential association between entacapone use and prostate cancer in an ambulatory setting.

Methods: Using data from the Department of Veterans Affairs (VA) healthcare system, new-user cohorts were created of PD patients treated with add-on entacapone or add-on dopamine agonist/monoamine oxidase B inhibitors between January 2000 and December 2014. Patients were followed on-treatment for occurrence of prostate cancer, identified via VA cancer registry linkage. Cox proportional hazards regression with time-dependent exposure was used to calculate hazard ratios (HR) and 95% confidence intervals (CI). Confounding was controlled by using inverse probability treatment weighting calculated from propensity scores.

Results: Mean follow-up time was 3.1 and 4.0 years in the entacapone and control cohort, respectively. There were 17,666 subjects meeting study criteria (mean age, 74 (SD 8.6) years); the group treated with entacapone comprised 5,257 subjects. Twenty-three prostate cancers occurred in the entacapone cohort and 97 in the control cohort. The overall incidence of prostate cancer was 1.8 per 1000 person-years of risk. There was no difference in risk of prostate cancer between the cohorts for increased duration of entacapone intake (adjusted HR: 1.08; 95% confidence interval: 0.46-2.51 for cumulative exposure of ≥2 years). Time since starting drug therapy and cumulative dose (mg) were also examined.

Conclusion: In this cohort of PD patients, extended duration of entacapone use was not associated with an increased incidence of prostate cancer.

#2304

Environmental pollutants and breast cancer: 2006-2016 epidemiological studies designed to evaluate biological hypotheses provide evidence of risk for certain pesticides, organic solvents, and products of combustion.

Kathryn M. Rodgers, Julia O. Udesky, Ruthann A. Rudel, Julia G. Brody. _Silent Spring Institute, Newton, MA_.

Common environmental chemicals have plausible links to breast cancer because they are mammary gland carcinogens in animal studies or are known to activate relevant hormonal pathways or enhance susceptibility of the mammary gland to carcinogenesis. An institute of Medicine report prioritized human studies of these exposures. Yet the long latency and multifactorial etiology of breast cancer make evaluation of these chemicals in humans challenging, adding to the importance of study designs that are well-suited to capture hypothesized breast-cancer mechanisms. With this in mind, we updated Brody et al's 2007 review in Cancer to evaluate the strength of the epidemiologic evidence for several classes of chemicals with widespread exposure.

We conducted a systematic search of the PubMed database for articles published in 2006-2016 using 147 terms and the inclusion/exclusion criteria defined in Brody et al's 2007 review. We critically reviewed articles identified by our search, including whether study designs were suited to the biological evidence.

We identified 158 new articles since 2006. In the past 10 years, the strength of evidence for an association between breast cancer and banned but persistent endocrine disrupting chemicals polychlorinated biphenyls (PCBs) and organochlorine pesticides has increased, based on a unique 50-year cohort study that captured exposure during potentially critical windows for breast development (in utero, adolescence, pregnancy) and at the time when the chemicals were still in use. Studies that considered early life exposure to organic solvents and/or exposure of long duration also added to evidence for an association with breast cancer. Solvents are used in dry cleaning, electronics manufacturing, leather and fur processing, and metal, auto, textile, and some military work. Other important reports included the U.S. Agricultural Health Study, which provided the first evidence of an association between organophosphate pesticides and breast cancer. Analyses from the Long Island Breast Cancer Study Project showed higher risk associated with polycyclic aromatic hydrocarbons (PAHs, products of combustion) in women with certain genetic variations. Consumer product chemicals, including BPA, phthalates, per fluorinated compounds, and hair dyes were evaluated in a few studies with inconsistent results; many of these studies had serious methodologic limitations.

Animal and mechanistic evidence suggest that timing could be crucial to carcinogenic actions of many environmental chemicals, however we found that most epidemiologic studies in the past 10 years did not evaluate a specific timeframe of exposure prior to diagnosis. Those that did enhanced our understanding of relevant windows for biological activity of certain classes of chemicals.

#2305

Residential airborne heavy metal concentrations and breast cancer characteristics.

Jacob K. Kresovich, Serap Erdal, Maria Argos, Hua Yun Chen, Peter H. Gann, Garth H. Rauscher. _University of Illinois-Chicago, Chicago, IL_.

Background: Ambient air metal concentrations have recently been implicated in the etiology of breast cancer. Previous studies have shown airborne concentrations of arsenic and cadmium are associated with the development of estrogen receptor-negative tumors. This study aims to replicate these findings and examine the role of other toxic and essential heavy metals.

Methods: Participants were women who were diagnosed with breast cancer in Chicago between 2005 and 2008. We examined estrogen and progesterone receptor-negative (ER-/PR-) and high-grade tumors as markers of aggressive breast cancer, and estimated 15-year markers of exposure of 11 heavy metals. Exposures were calculated using census tract-level airborne concentrations from the National-scale Air Toxics Assessment and participants' residential histories. We adjusted all models for socioeconomic status and reproductive factors.

Results: We found that prevalent ER-/PR- tumors were associated with increased residential exposure to airborne concentrations of antimony (Q4 vs Q1: OR= 1.81; 95% CI: 0.95, 3.44; Ptrend= 0.04), cobalt (Q4 vs Q1: OR= 2.37; 95% CI: 1.26, 4.45; Ptrend < 0.01), manganese (Q4 vs Q1: OR= 2.55; 95% CI: 1.24, 5.24; Ptrend= 0.04), and selenium (Q4 vs Q1: OR= 1.85; 95% CI: 1.03, 3.29; Ptrend= 0.05), and also identified marginally significant trends for arsenic (Ptrend= 0.06), chromium (Ptrend= 0.08), lead (Ptrend= 0.08), and mercury (Ptrend= 0.07). We did not identify any overall associations with high-grade tumors, however when stratifying by menopausal status we found that antimony (Q4 vs Q1: OR= 6.97, 95% CI: 1.61-30.19) and arsenic (Q4 vs Q1: OR= 6.97, 95% CI: 1.61-30.19) were associated with prevalent high-grade tumors in premenopausal women only.

Discussion: This study found further support for a role of airborne arsenic concentrations, and novel evidence implicating other airborne estrogen-pathway disrupting metal concentrations, in the development of aggressive breast cancer subtypes. Additionally, this is the first study to implicate heavy metal exposure in the etiology of high-grade tumors. These results suggest that long-term, low-dose exposures to certain heavy metals play a role in the etiology of aggressive breast cancer characteristics. Airborne exposures have the ability to affect large populations and findings from this and similar studies have large public health implications.

#2306

Sleep duration and risk of fatal prostate cancer in CPS-I and CPS-II.

Susan M. Gapstur, Victoria L. Stevens, Brian D. Carter, Ying Wang, Eric J. Jacobs. _American Cancer Society, Atlanta, GA_.

This abstract has been withheld from publication due to its inclusion in the AACR Annual Meeting 2017 Official Press Program. It will be posted online following its presentation. 

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

### Apoptosis and Other Regulated Cell Death in Cancer

#2307

The p53/p21 complex is a functional unit that regulates cancer cell invasion and apoptosis by targeting Bcl-2 family proteins.

Eun Mi Kim, Hong-Duck Um. _Korea Inst. of Radiological & Medical Sci., Seoul, Republic of Korea_.

Although well known as a transcription factor, the p53 tumor suppressor also exists in the cytoplasm (including the mitochondria) of normal and cancer cells. Moreover, nuclear p53 is translocated to the mitochondria upon apoptotic stimulation of cells. The cytoplasmic/mitochondrial p53 can bind pro-survival Bcl-2 family proteins (e.g., Bcl-2, Bcl-XL, and Bcl-w), liberating Bax therefrom. Bax then exerts its pro-apoptotic or anti-invasive function depending on experimental conditions. Given the ability of p53 to interact with p21, we investigated the possible involvement of p21 in these functions. Here, we show that whereas p53 can bind to Bcl-w alone, it requires p21 to liberate Bax from Bcl-w and suppress/promote cell invasion/death, respectively. p21 consistently binds Bcl-w, forming a p53/p21/Bcl-w complex. Only this trifold complex, but not any of the pairings, facilitates the release of Bax from Bcl-w. Accordingly, a p53 derivative incapable of binding p21 fails to mediate radiotherapy-induced tumor retardation and cell death in mice. We further show that Bcl-2 and Bcl-XL also serves as a target of p53/p21 cooperative action. Overall, our findings indicate that the p53/p21 complex rather than p53 itself regulates cell invasion and death by targeting Bcl-2 proteins. We propose that the p53/p21 complex is a functional unit that acts on multiple types of cellular components, providing a new foundation for understanding the tumor-suppressing functions of p53 and p21.

#2308

Sigma-2 receptor ligands induce apoptosis and inhibit proliferation in breast cancer cell line mda-mb-231.

Gladys Asong, Felix Amissah, Olufisayo Salako, Rosemary Poku, Elizabeth Ntantie, Augustine Nkembo, Nazarius Lamango, Seth Ablordeppey. _Florida A &M University, Tallahassee, FL_.

Introduction: Despite the reduction in mortality from breast cancer achieved in the last 30 years due to more effective targeted therapies, the survival rate for patients with triple negative breast cancer (TNBC) is poor and has virtually plateaued. Due to the absence of estrogen, progesterone, and human epidermal growth factor receptors 2, no targeted treatment options are currently available for TNBC. Moreover, the current standard systemic treatments are known to cause tremendous side effects. Thus, there is an urgent need to identify novel strategies for treating TNBC. Several reports have revealed that sigma-2 receptors are over-expressed in all solid tumors including TNBC, making it a plausible biomarker to explore for TNBC treatment. In this study, we investigated the effects of novel sigma-2 receptor ligands (XYZ-XI-14 and XYZ-VII-69) synthesized in our lab on the viability and survival of the TNBC, MDA-MB-231.

Methods: MDA-MB-231 cells were treated for 48 h with XYZ-XI-14 and XYZ-VII-69, cell viability and proliferation were assessed using resazurin cell titre assay and cell count methods respectively. The effects of the ligands on spheroids formation, cell cycle, and mode of cell death were also investigated.

Results: XYZ-XI-14 and XYZ-VII-69 decreased cell viability of MDA-MB-231 cells in a concentration-dependent manner. The EC50 for XYZ-XI-14 and XYZ-VII-69 were 12 and 13 µM respectively; and at 5 µM, the ligands inhibited cell proliferation, induced apoptosis, and arrested MDA-MB-231 cells at the G0/G1 phase of cell cycle. Additionally, concentrations of the ligand as low as 1 µM prevented the formation of spheroids as evidenced by the lack of compact spheroids, and caused the disintegration of preformed spheroids.

Conclusion: This study indicates that targeting sigma-2 receptors with novel sigma-2 ligands (XYZ-XII-14 and XYZ-VII-69) effectively inhibits TNBC cancer cell growth by inducing apoptosis and cell cycle arrest, thus presenting a unique and effective pathway for treating TNBC. Additionally, the sigma-2 receptor ligands (XYZ-XI-14 and XYZ-VII-69) have the potential to halt tumor growth and prevent tumor relapse, as seen in our 3D culture assays. Thus, the sigma-2 receptor has the potential to be a valuable target for the development of novel agents for the treatment of TNBC.

#2309

Acetylation of survivin represses transactivation of STAT3 to induce sodium butyrate-induced apoptosis in human mucoepidermoid carcinoma cell lines.

Sung-Dae Cho. _Seoul National University, School of Dentistry, Seoul, Republic of Korea_.

Sodium butyrate (NaBu) is one of histone deacetylase inhibitors and possesses anticancer activity. Here, the aim of this study is to determine the molecular mechanism by which NaBu controls apoptosis in MC3 and YD15 human mucoepidermoid carcinoma (MEC) cell lines. NaBu caused growth inhibition and induced apoptosis in both cell lines accompanied by the acetylation of histone proteins H2A and H3. NaBu clearly increased survivin and cleaved caspase 3 evidenced by human apoptosis array and it regulated survivin molecule at a post-transcriptional level. NaBu caused nuclear translocation of survivin protein and increased survivin acetylation in both cell lines. In addition, STAT3 and survivin were co-localized in nucleus by NaBu resulting in the decrease in the expression level of Bcl-XL mRNA which is one of STAT3 downstream molecules. In addition, NaBu induced caspase-dependent apoptosis in human MEC cells. Taken together, these results suggest that NaBu is a potent apoptosis inducer in human MEC cell lines through survivin-STAT3 signaling cascades and these findings provide the basis of its clinical application for the treatment of human MEC.

This abstract is supported by SNU Invitation Program for Distinguished Scholar and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2014R1A12055874)

#2310

Molecular dynamics simulation of permeation pathway of cytochrome C through Bax pore.

Mingzhen Zhang,1 Jie Zheng,1 Ruth Nussinov,2 Buyong Ma2. 1 _University of Akron, Akron, OH;_ 2 _Leidos Biomedical Research, NCI-Frederick, Frederick, MD_.

How cytochrome C is released from the mitochondria to the cytosol via Bax oligomeric pores, a process which is required for apoptosis, is still a mystery. Based on the residue-residue distances detected experimentally for Bax and its homologous protein (Bak), we recently computationally solve the first atomic model for Bax oligomeric pores at the membranes. Next, we investigate the mechanism at the microsecond time- and nanometer space- scale using coarse-grained replica exchange and all-atom MD simulations. Our free energy landscape depicts a low barrier for the permeation of cytochrome C into the Bax C-terminal mouth, with the pathway proceeding to the inner cavity and exiting via the N-terminal mouth. Release is guided by organized charged/hydrophilic surfaces. The hydrophilicity and negative charge of the pore surface gradually increase along the release pathway from the pore entry to the exit opening. Rather than inert passing of cytochrome C through a rigid pore, the flexible pore may selectively aid the cytochrome C passage. The energy barrier is under 4 kcal/mol. Thus, once the Bax pore is formed in the membrane, the release of cytochrome C may be readily achieved through energy fluctuations. Collectively, our work provides mechanistic insight and atomic detail into the release of cytochrome C through Bax oligomeric pores.

#2311

A peptide derived from a photosynthetic cupredoxin protein, auracyanin, induces a p53-independent apoptosis in cancer cells.

Samer A. Naffouje, Tohru Yamada, Tapas K. Das Gupta. _University of Illinois at Chicago, Chicago, IL_.

Cupredoxins are a family of low-molecular weight, water soluble, copper-containing redox proteins involved mainly in the electron transfer chain in prokaryotes. Azurin is a member of the cupredoxin family secreted by Pseudomonas aeruginosa, of which a 28-amino acid sequence (p28) was identified to acquire the capability of preferential entry into cancer cells of various histogenesis. Upon entry, it exerts a cytostatic effect in these cancer cells via a p53-mediated pathway. Our aim was to study whether the other members of the cupredoxin family possess a similar function to that of azurin in the eukaryotic cancer cells. Based on structural and sequence analyses, helical regions of auracyanin A & B were shown to resemble p28 closely. Thus, we studied the biologic function of two 28-amino-acid peptides derived from auracyanin A (aurA; pI 4.5, MW 2.6 kD), and auracyanin B (aurB; pI 3.4, MW 2.7 kD). Cancer growth and MTT assays demonstrated that aurB, but not aurA, induced cytotoxicity in various cancer cell lines in a dose-dependent (range 0.1uM-100uM) and time-dependent (~72 hours) pattern, including p53-null (prostate cancer PC-3) and dn-p53 (breast cancer MDD-2 and ovarian cancer SKOV-3) in which p28 did not demonstrate a significant growth inhibition. Annexin-V assays showed a dose-dependent apoptosis in prostate cancer cell lines of various p53 statuses (wt-p53 LNCaP, mut-p53 DU-145, p53-null PC-3) following treatment with aurB at 5-50uM for 48 hours, whereas p28 did not demonstrate an apoptotic activity in p53-null cell lines, suggesting an aurB-induced apoptosis via a p53-indepdnent pathway. In contrast, aurA did not appear to induce apoptosis in any of the above cell lines. Induction of apoptosis was mediated through depolarization of the mitochondrial membrane and caspase cascade activation in cancer cells. Moreover, fluorescence microscopy demonstrated that aurB, unlike p28, does not acquire a similar pattern of preferential entry into cancer cells. This proposes that aurB acquires the potential as an anticancer cell penetrating peptide, and differs from p28 in its mode of entry and p53-mediated action. Our studies pertaining to azurin and auracyanin, suggest that cupredoxins, as a family of prokaryotic metalloproteins, could represent a fountainhead of numerous derived peptides that hold promise as novel and efficient antineoplastic agents against various cancer cell lines.

#2312

Feasibility study of a fractionation method in the treatment of tumor treating fields.

Yunhui Jo,1 Jiwon Sung,1 Hyo Sook Song,1 Eun Ho Kim,2 Myonggeun Yoon1. 1 _Korea University, Seoul, Republic of Korea;_ 2 _Korea Institute of Radiological and Medical Sciences, Seoul, Republic of Korea_.

Tumor treating fields (TTFs) treatment has been reported to have great potential to treat various cancers. Inspired by a similarity between TTFs and radiotherapy in electromagnetic property, TTFs using a fractionated treatment scheme originally designed for radiotherapy were applied to cancer (Human glioblastoma, U373) and normal cells (intestinal epithelial, IEC-6) for three days with durations of 3, 6, 9, or 24 hours/day. As the treatment time of TTFs increases from 3 to 24 hours/day, the relative cancer cell growth rates in both cell lines were reduced. While the difference in cancer cell growth rate compared to control was ~25% (decreased from 100% to ~75%) for the treatment time of 6 hours/day, the growth rates of cells treated for additional 6 hours (i.e., 12 hours/day) and additional 18 hours (i.e., 24 hours/day) differed only ~5% and ~8% from that of cells treated for 6 hours/day, respectively. Our results suggested that the dependence on treatment time in cancer cell inhibition is weakened distinctly at treatment times over 6 hours/day. For normal cells, the relative growth rates corresponding to the treatment time of TTFs was not decreased much for the treatment time of 3, 6, 12 hours/day revealing 93.3%, 90.0%, 89.3% of cell growth rates compared to control, respectively, but it suddenly reduced to ~73% for 24 hours/day of treatment. The experimental results indicated that the effects of TTFs on growth rates of cancer cells are higher than the effects of TTFs on growth rates of normal cells for treatment times of 3-12 hours/day, but the difference became minimal for treatment times of 24 hours/day. Our results suggest that the fractionated scheme in the treatment of tumor treating fields may reduce treatment time while maintaining efficacy, suggesting that this method can increase patients' quality of life.

#2313

Promoting caspase-8-dependent apoptosis signaling using 17-beta-hydroxywithanolides.

Alan D. Brooks,1 Ya-ming Xu,2 E. M. Kithsiri Wijeratne,2 Curtis J. Henrich,1 Poonam Tewary,1 Leslie Gunatilaka,2 Thomas J. Sayers1. 1 _Frederick National Laboratory for Cancer Research, Frederick, MD;_ 2 _The University of Arizona, Tucson, AZ_.

We have previously reported that withanolide E (WE), a steroidal lactone from Physalis peruviana, was highly active in sensitizing various human carcinoma cell lines to TRAIL-mediated apoptosis. Therefore, over 100 natural and semi-synthetic withanolides were evaluated for their ability to promote caspase-8-dependent cancer cell death. Our studies identified several withanolides that were 4-8 fold more potent than WE in sensitizing the renal carcinoma cells and melanoma cells to caspase-8-dependent apoptosis in response to either TRAIL or the TLR3 ligand poly (I:C). All active withanolides were 17-beta-hydroxywithanolides (17-BHW). The highly active 17-BHWs were more efficient than withanolide E at reducing cellular levels of both cFLIPL and cFLIPS and enhancing caspase-8 activation. Furthermore, immunoprecipitation of the TRAIL death-inducing signaling complex (DISC), or the related ripoptosome, demonstrated enhanced levels of both FADD and RIP1 in these macromolecular apoptosis signaling complexes following treatment with active 17-BHWs. The 17-BHWs used in this work were obtained by the application of an efficient method of plant biomass production involving our innovative and patented soil-less aeroponic cultivation of P. crassifolia and P. peruviana and by chemical modification of natural withanolides produced by these plants. Preliminary structure activity relationship (SAR) studies suggested that the enone moiety in ring A was essential for activity. In addition, acetoxylation at C-18, an alpha orientation of the side-chain lactone group and the double bond at C-24(25) of the lactone ring played important roles in determining the activity of 17-BHWs as apoptosis sensitizers. This suggests that the 17-BHW scaffold is amenable to optimization by a medicinal chemistry approach, which could lead to the identification of highly active natural product-based sensitizers of cancer cells to caspase-8-dependent apoptosis. The cellular molecular target(s) of active 17-BHWs are currently under further investigation. Funded by FNLCR Contract HHSN261200800001E.

#2314

Novel BH3 mimetic based therapeutic strategies for the treatment of breast cancer.

Zachary Kratche, MacKenzie Adams, Robert Wilson, Stephen Ethier, Stephen Guest. _Medical Univ. of South Carolina, Charleston, SC_.

Bcl-2 homology domain 3 (BH3) mimetics are a new class of targeted anti-cancer compounds that function by inducing cancer cell apoptosis. Testing in a variety of pre-clinical tumor models has shown that BH3 mimetics are capable of inducing remarkable tumor regressions. Recently, a clinical trial of the BH3 mimetic venetoclax in Chronic Lymphocytic Leukemia (CLL) resulted in an impressive response rate of 80% leading to FDA approval. Using unbiased genomic and functional genomic approaches, we identified gene amplification of the BCL2L1 gene, which encodes the anti-apoptotic protein BCL-XL, as a novel driver event in breast cancer. We identified focal amplification of the BCL2L1 gene in the SUM-185 breast cancer cell line and a genome-wide RNAi-based screen identified BCL2L1 as one of the top genes necessary for viability of this cell line (ranked 5th out of 15,256 genes). Treatment of SUM-185 cells with navitoclax showed that these cells are intrinsically sensitive to this drug (IC50 = 100nM). This sensitivity was 1-2 logs greater than what was observed for breast cancer cell lines that do not harbor BCL2L1 gene amplification. To expand the relevance of these findings beyond the SUM-185 cell line, we used the cancer cell line encyclopedia (CCLE) database to identify an additional breast cancer cell line, HCC38, that also harbors a focal BCL2L1 gene amplification. Treatment of HCC38 cells with navitoclax revealed a high level of sensitivity that was equivalent to that of SUM-185 cells. These findings demonstrated that a subset of breast cancers that harbor BCL2L1 gene amplification are highly sensitive to a BH3 mimetic that targets BCL2L1 suggesting that BH3 mimetics could be used as a single agent to effectively treat these breast cancers. We also examined the effectiveness of BH3 mimetics when used as part of a combination therapy approach. In addition to a focal amplification of the BCL2L1 gene, the SUM-185 breast cancer cell line also harbors a focal amplification of the fibroblast growth factor receptor 3 (FGFR3) gene as well as an activating mutation in the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene. Treatment of SUM-185 cells with low doses of navitoclax, a FGFR inhibitor, or a PIK3CA inhibitor had little effect on colony forming efficiency. At these same low doses, combined treatment with navitoclax and the FGFR inhibitor or the PIK3CA inhibitor resulted in a strikingly synergistic effect with near complete loss of colony forming ability (less than 10% of controls). We further showed that this loss of clonogenicity was the result of a rapid induction of apoptosis by the combination therapies. These results suggest that combining BH3 mimetics with inhibitors of PIK3CA or FGFR is a promising novel therapeutic strategy for a subset of breast cancer patients.

#2315

Overcoming treatment resistance in cisplatin-resistant ovarian carcinoma cells by tissue-specific miRNAs.

Michael Kleemann,1 Jeremias Bereuther,1 Simon Fischer,2 Kim Marquart,1 Simon Hänle,1 Kristian Unger,3 Verena Jendrossek,4 Christian U. Riedel,5 René Handrick,1 Kerstin Otte1. 1 _University of Applied Sciences Biberach, Biberach, Germany;_ 2 _Boehringer Ingelheim, Biberach, Germany;_ 3 _Helmholtz Zentrum München, München, Germany;_ 4 _University of Essen, Essen, Germany;_ 5 _University of Ulm, Ulm, Germany_.

Ovarian cancer is a common human cancer with a poor outcome and a high risk of death. Due to resistances to current standard therapies (carboplatin and paclitaxel for 3 to 6 cycles) additional therapeutic approaches are required and novel specific biomarkers for ovarian cancer patients are necessary. MicroRNAs (miRNA) are conserved, small noncoding RNAs regulating gene expression by binding to their target mRNA which have been shown to regulate biological processes including apoptosis. Apoptosis is a conserved, irreversible process that allows cells to undergo a highly regulated form of cell death. However, the final role of miRNAs in apoptotic signaling has not yet been fully determined. Modulating the expression of key molecular components of the cell death machinery is an attractive strategy for cancer therapy overcoming chemo drug resistance. To extend the pool of potential miRNA as anticancer agents and biomarkers, we transferred a high content miRNA screening for pro-apoptotic miRNAs to human cancer cell lines. Based on the high recovery rate of apoptosis inducing miRNAs in SKOV3 cells analyses were extended to multiple ovarian carcinoma cell lines with different genetic background. After transfection of miR-1912-5p the apoptosis rate increased strongly in cisplatin-resistant cells. In combination treatment with carboplatin the apoptotic effect increased after transfection of miR-1912-5p in the cisplatin-resistant A2780 cells. Furthermore, miR-1912-5p, miR-147b and miR-3073a showed significant apoptosis induction in the various ovarian cancer cell lines alone and in combined therapy with carboplatin. Western Blot analysis revealed an enhanced expression of the pro-apoptotic proteins Bak1 and Bax and a decrease inBcl2 and Bcl-xL. We characterized the miRNAs in regard to their endogenous expression and detected an enhanced expression after apoptosis induction by chemotherapeutic drugs. Finally, analysis of The Cancer Genome Atlas data showed an influence on the median survival of ovarian cancer patients (> 69 years). Our data indicate a potential benefit of tissue specific pro-apoptotic miRNAs in regard to cisplatin-resistant cells. Furthermore the study identified novel pro-apoptotic miRNAs with their potential use as novel therapeutic entities or prognostic biomarkers for ovarian cancer.

#2316

Elevated H-Ras suppresses death receptor-mediated apoptosis in cancer cells.

Su-Ryun Kim. _FDA, SIlver Spring, MD_.

TNF-related apoptosis inducing ligand (TRAIL) induces apoptosis through its death receptors (DRs) 4 and/or 5 expressed on the surface of target cells. Despite its selectivity in killing cancer cells over most normal cells, recombinant human TRAIL or its receptor agonists (monoclonal antibodies against DR4 or DR5) encountered resistance in many tumor cells while the underlying mechanisms remain partially understood. Here, we show that wild-type H-Ras GTPase is systemically upregulated in TRAIL-resistant cells compared to TRAIL-sensitive cells. The elevated H-Ras expression correlated with a deficiency of DR4 and DR5 on plasma membrane in TRAIL-resistant cell lines. Notably, knockdown of H-Ras in TRAIL-resistant cells successfully restored the surface expression of DR4 and DR5, thereby sensitizing the cells to TRAIL-induced apoptosis. Consistently, ectopic expression of H-Ras in TRAIL-sensitive cells reduced surface DR4 and DR5 which was associated with a loss of TRAIL sensitivity. By contrast, the status of K-Ras or its mutations was not casually linked to TRAIL receptor expression or TRAIL sensitivity across the panel of cancer cell lines tested. These data suggest that H-Ras may play a distinct role to negatively regulate TRAIL receptors and apoptosis. The upregulated H-Ras could be a predictor of tumor resistance to DR-targeted agents and a potential therapeutic target for combinational therapy to achieve better treatment outcomes.

#2317

Prostanoid EP4 receptor induces cleavage of HSP90 via ROS generation in human colon cancer cells.

In Gyeong Chae, Mi-hee Yu, Kyung-Soo Chun. _Keimyung Univ., Daegu, Republic of Korea_.

Prostaglandin E2 (PGE2) has been reported to play critical roles in cell fate decision by interacting with four types of G protein-coupled membrane receptors such as EP1, EP2, EP3 and EP4. We previously reported that EP4 stimulation by treatment with its agonist CAY10598 induced apoptosis via reactive oxygen species (ROS) formation in colon cancer HCT116 cells. Moreover, treatment with CAY10598 diminished the phosphorylation of JAK2 and induced degradation of this oncoprotein, leading to the attenuation of STAT3 activation in HCT116 cells. In the present study, we attempted to delineate the molecular mechanisms underlying the degradation of JAK2 by activation of EP4. HSP90, a member of the heat shock protein family, is a molecular chaperone that supports stability of client proteins, such as, EGFR, MET, Akt and JAK2. HSP90-mediated stabilization/activation of these client proteins contributes to the acquisition of cancer cell hallmarks, including proliferation, survival, angiogenesis and invasion. It has been recently reported that the chaperoning function of HSP90 may be disrupted by post-translational modification induced by oxidative stress. Treatment of human colon cancer HCT116 cells with CAY10598 down-regulated expression of HSP90 client proteins in a concentration- and time-dependent manner and the down-regulation was restored by pretreatment with ROS scavenger N-acetyl cysteine (NAC) or proteasome inhibitor MG132. However, cotreatment with cycloheximide, protein synthesis inhibitor, accelerated the CAY10598-induced degradation of HSP90 client proteins. These data suggest that CAY10598-induced HSP90 client protein degradation may be caused by ROS generation. In renal carcinoma Caki cells, CAY10598 also down-regulated expression of HSP90 client proteins, suggesting that EP4 stimulation may regulate HSP90 activity in colon cancer cells as well as renal cancer cells. We found that HSP90α was cleaved to 40 or 55 kDa, while HSP90β cleaved to 25 kDa by CAY10598 treatment and the cleavage of HSP90α and β was blocked by NAC treatment. Furthermore, EP4 inhibition by treatment with antagonist GW627368x attenuated not only degradation of HSP90 client proteins but also cleavage of HSP90 in CAY105998-treated HCT116 cells. In conclusions, EP4 agonist CAY10598 induces degradation of HSP90 client proteins via ROS-dependent HSP90 cleavage, leading to apoptosis in HCT116 cells. This is a novel mechanism by which EP4 activation induces apoptosis of cancer cells that involved inhibiting HSP90 function.

#2318

Cyclopamine sensitizes endoplasmic reticulum stress-mediated increase of death receptor 5 and survivin degradation in trail-resistant gastric cancer cells.

Yoo Jin Na, Dae-Hee Lee, Jung Lim Kim, Bo Ram Kim, Seong Hye Park, Min Jee Jo, Yoon A Jeong, Sang Cheul Oh. _Korea University, Seoul, Republic of Korea_.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has been known as one of the most effective cancer treatment owing to its selective killing effect of cancer but not normal cells. However it has been reported that several gastric cancer cells showed resistance to TRAIL because of scarcity of death receptor 5 (DR5) expressed on the cell surface. In this study, we elucidated cyclopamine sensitizes to TRAIL-induced apoptosis by elevating the expression level of DR5 in gastric cancer cells. Interestingly, survivin hampers existence of DR5 protein under normal condition. Cyclopamine decreases expression level of survivin. It is a reason for cyclopamine to act as a TRAIL sensitizer. Mechanistically, cyclopamine induces endoplasmic reticulum (ER) stress by Reactive Oxygen species (ROS), and CHOP, which as a last protein of ER stress pathway regulates stability of survivin protein by proteasome degradation. Taken together, our results indicate that cyclopamine can be used for the purpose of combination therapy in TRAIL-resistant gastric cancer cells.

#2319

Deficiency of protease-activated receptor 2 signaling sensitizes EGFR-TKI-induced apoptosis in colorectal cancer.

Weiwei Li, Yiming Ma, Longmei He, Hongying Wang. _State Key Laboratory of Molecular Oncology, National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China_.

Colorectal carcinoma (CRC) patients show resistance to EGFR-TKI (epidermal growth factor receptor -tyrosine kinase inhibitor) treatment. Protease-activated receptor 2 (PAR2) has been shown to transactivate EGFR. We aim to investigate whether PAR2 sensitizes EGFR-TKI-induced apoptosis in CRC. Firstly, inhibition of PAR2 with shRNA or ENMD-1068 (a selective antagonist of PAR2) significantly sensitized EGFR-TKI (gefitinib or AG1478)-induced apoptosis, which was measured with Annexin V/ PI staining and activation of caspase-3 in different CRC cell lines. In xenograft tumor model, gefitinib treatment dramatically reduced the tumor volume in HT-29-shPAR2 compared with HT-29-vector. Mechanism study showed that PAR2 knockdown significantly reduced bcl-xL expression at both mRNA and protein level. Consistently, activation of PAR2 upregulated bcl-xL. Furthermore, inhibition of protein phosphatase (PP) with okadaic acid significantly downregulated bcl-xL. Moreover, knockdown of PP1 with siRNA blocked PAR2-induced accumulation of bcl-xL. In summary, our findings suggest that inhibition of PAR2 sensitizes EGFR-TKI-induced apoptosis via downregulation of bcl-xL in colorectal cancer. Considering the extensive expression of PAR2 and PAR2-activating proteases in colon, it strongly indicates that inhibition of PAR2 may be a potential avenue to overcome EGFR-TKI resistance in patients with CRC.

#2320

Cucurbitacin D induces cell cycle arrest and apoptosis by inhibiting STAT3 and NF-κB signaling in doxorubicin-resistant human breast carcinoma (MCF7/ADR) cells.

Jin Mo Ku, Se Hyang Hong, Myeong-Sun Kim, Hyo In Kim, Soo-Yeon Kang, Kangwook Lee, Yu-Jeong Choi, Chunhoo Cheon, CHING WEN HUANG, Youme Ko, Yui Sasaki, Sohyeon Kang, Ji Hye Kim, Hye Sook Seo, Tai Young Kim, Yong Cheol Shin, Seong-Gyu Ko. _Kyung Hee University, Seoul, Republic of Korea_.

Objective: We examined whether cucurbitacin D affects doxorubicin resistance of MCF7/ADR breast cancer cells.

Methods: Cell viability was measured by MTT assay. Levels of p-STAT3, p-NF-κB, IκB, and caspases were measured by western blot analysis. Nuclear staining of Stat3 and NF-κB was measured by immunocytochemistry. STAT3 and NF-κB transcriptional activity was detected by STAT3 and NF-κB luciferase reporter gene assays. Analysis of cell cycle arrest was performed by flow cytometry. Induction of apoptosis by cucurbitacin D was measured by annexin VFITC/PI assay.

Results: More than 90% of MCF7/ADR cells lived upon treatment with doxorubicin for 24 h. However, upon treatment with cucurbitacin D, cell death was more than 60%. Co-administration of cucurbitacin D and doxorubicin induced apoptosis, G2/M cell cycle arrest, and inhibited upregulated Stat3 by doxorubicin on MCF7/ADR cells. Additionally, cucurbitacin D led to an increase in the IκBα level in the cytosol and a decrease in the p-NF-κB level in the nucleus. Finally, cucurbitacin D inhibited translocation of Stat3 and NF-κB and decreased transcriptional activity in the nucleus.

Conclusion: Cucurbitacin D decreases cell proliferation and induces apoptosis by inhibiting Stat3 and NF-κB signaling in doxorubicin-resistant breast cancer cells. Cucurbitacin D could be used as a useful compound to treat Adriamycin-resistant patients.

#2321

Reactive oxygen species dictate the apoptotic response of melanoma cells to TH588.

jiayu wang, Jin Lei, Xu Guang yan, Simonne Sherwin, Margaret Farrelly, Yuan Yuan Zhang, Fen Liu, Chun Yan Wang, Su Tang Guo, Hamed Yari, Ting La, Jennifer McFarlane, Fu Xi Lei, Hessam Tabatabaee, Jie Zhong chen, Amanda Croft, chen chen Jiang, Xu Dong Zhang. _life sciences building, Newcastle, Australia_.

Cancer cells commonly contain elevated levels of reactive oxygen species (ROS) resulting from oncogenic stimulation. On one hand, ROS promote cancer cell survival, proliferation, and metastasis. On the other, high levels of ROS suppress tumour growth through inhibition of proliferation and induction of apoptosis and senescence via damage to DNA. Incorporation of oxidized dNTPs such as 8-oxo-deoxy-guanine (8-oxo-dGTP) and 2-OH-deoxy-adenosine (2-OH-dATP) into genomic DNA plays an important role in apoptosis induced by ROS. Human MutT homolog 1(MTH1) is an enzyme that sanitizes oxidized dNTP pools through converting 8-oxo-dGTP and 2-OH-dATP into monophosphates, thus preventing their incorporation into genomic DNA. Inhibition of MTH1 by small molecule inhibitors has been suggested to be a promising approach in cancer treatment. However, we have found that while silencing of MTH1 does not affect survival of melanoma cell, TH588, one of the first-in-class MTH1 inhibitors, kills melanoma cells through apoptosis independently of its inhibitory effect on MTH1. Induction of apoptosis by TH588 was not alleviated by MTH1 overexpression or introduction of the bacterial homologue of MTH1 that has 8-oxodGTPase activity but cannot be inhibited by TH588, indicating that MTH1 inhibition is not the cause of TH588-induced killing of melanoma cells. Although knockdown of MTH1 did not impinge on the viability of melanoma cells, it rendered melanoma cells sensitive to apoptosis induced by the oxidative stress inducer elesclomol. Of note, treatment with elesclomol also enhanced TH588-induced apoptosis, whereas a ROS scavenger or an antioxidant attenuated apoptosis triggered by TH588. Indeed, the sensitivity of melanoma cells to TH588 was correlated with endogenous levels of ROS. Collectively, these results suggest that: 1) TH588-induced apoptosis of melanoma cells is not associated with its inhibitory effect on MTH1; 2) TH588 remains a promising candidate for the treatment of melanoma; 3) MTH1 inhibition in combination with oxidative stress inducers may be a useful approach in melanoma treatment; and 4) the endogenous levels of ROS are a potential biomarker for prediction of the response of melanomas to TH588 and MTH1 inhibition in combination with oxidative stress inducers.

#2322

Antitumor activity of spicatoside A, a steroidal saponin, via induction of switch from autophagy to apoptotic cell death.

Won Kyung Kim, Yuna Pyee, Hyen Joo Park, Ji-Young Hong, Sang Kook Lee. _Seoul National University, Seoul, Republic of Korea_.

The antitumor activity of spicatoside A (SA), a steroidal saponin isolated from the tuber of Liriope platyphylla (Liliaceae), and its underlying mechanisms were investigated in HCT116 human colorectal cancer cells. SA induced autophagy and apoptotic cell death and inhibited tumor growth in a nude mouse xenograft model implanted with HCT116 cells. Treatment with SA for 24 h enhanced the formation of acidic vesicular organelles in the cytoplasm, indicating the induction of the onset of autophagy. This event was associated with the regulation of autophagic markers including microtubule-associated protein 1 light chain 3 (LC3)-II, p62, beclin 1, lysosomal-associated membrane protein 1, and cathepsin D by inhibiting the PI3K/Akt/mTOR signaling pathway, regulating mitogen-activated protein kinase signaling, and increasing p53 levels. However, a prolonged exposure to SA resulted in apoptosis characterized by the accumulation of a sub-G1 cell population and an annexin V/propidium iodide-positive cell population. Apoptosis induced by SA was associated with the regulation of apoptotic proteins including Bcl-2, Bax, and Bid, the release of cytochrome c into the cytosol, and the accumulation of cleaved poly ADP-ribose polymerase (PARP). Further study revealed that cleavage of beclin 1 by caspase-8 plays a critical role in the SA-mediated switch from autophagy to apoptosis. Taken together, these findings highlight the significance of SA in the modulation of crosstalk between autophagy and apoptosis, as well as the potential use of SA as a novel candidate in the treatment of human colorectal cancer cells. Acknowledgement: This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean Government (MEST) (No. 2009-0083533)

#2323

**The inhibitory role of Kaempferol-3-** O- **rutinoside induced AMPK activation on the growth of human breast cancer cell lines.**

Chea Ha Kim. _Hallym UNIV., Chuncheon, Republic of Korea_.

Solidago virga-aurea, herbaceous perennial plant of the family Asteraceae, has astringent, diuretic, and antiseptic assets. In our current study, we found that kaempferol-3- O -rutinoside (KR) from Solidago virga-aurea extract prompted cellular apoptosis in breast cancer cells. The present study was conducted to investigate its effects on MCF-7 cells with respect to the induction of inhibitory effect on cell viability against MDA-MB-231 and MCF-7 breast cancer cell lines. In addition, apoptotic protein, especially Bim, was also increased in KR treated MDA-MB-231 and MCF-7 cell line. However, an anti-apoptotic protein, Bcl-xL, was not changed by KR. To find out positive regulator of Bim

expression, FOXO (forkhead-box transcription factor, group O) 3a and AMPK (5' AMP-activated protein kinase) protein expressions were examined in KR treated cells. Immunoblot analysis showed dramatically increased expressions of FOXO3a and AMPK in KR treated MDA-MB-231 and MCF-7 cells. In addition, cell cycle analysis indicated that G0/G1 arrest was intensely increased in KR treated MDA-MB-231 and MCF-7 cells. Moreover, phospho-AMPK (pT172) and phospho-FOXO3a (S472) expression were also increased in KR treated MDA-MB-231 and MCF-7 cells. Our results suggest that KR induces apoptosis by AMPK-FOXO3a mediated Bim expression. The mechanisms of KR

mediated AMPK and FOXO3a regulation is not fully elucidated yet, but further studies about these molecular mechanisms between two proteins will give some ideas for cancer therapies for certain breast cancer patients.

#2324

The role of SERCA pump in cell death and autophagy.

Paula Szalai,1 Morten Luhr,1 Frank Sætre,1 Søren B. Christensen,2 Jesper V. Møller,3 Poul Nissen,3 Nikolai Engedal1. 1 _University of Oslo, Oslo, Norway;_ 2 _University of Copenhagen, Kopenhagen, Denmark;_ 3 _University of Aarhus, Aarhus, Denmark_.

The purpose of the study is to elucidate structural and molecular determinants of SERCA inhibition by Thapsigargin (Tg) and related analogs for their effects on intracellular calcium homeostasis, ER stress, cell death and autophagy. Tg specifically binds and blocks the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), which pumps Ca2+ from the cytosol to the ER. Sustained SERCA inhibition leads to calcium depletion from the ER causing ER stress and ultimately cell death. Recently we showed that Tg-induced calcium perturbation potently blocks autophagy (1). Tg is an attractive potential anti-tumor drug because it effectively kills both slow and fast proliferating cancer cells. However, since Tg is toxic also to normal cells, it must be targeted towards the cancer cells. Replacing a side chain with a linker connecting the Tg core to a peptide prevents Tg from entering cells. Two different linker-peptide sequences have been introduced in clinically tested Tg prodrugs; one is cleaved by PSA, secreted by prostate cancer cells, and the other is cleaved by PSMA, which is secreted by neovascular tissues of a broad range of tumors. The Tg analogs unmasked by the cleavage are able to enter cells and exert their toxic effects. Interestingly, however, in vitro experiments indicate that Tg analogs have different potencies and cellular effects depending on the terminal amino acid residue (2). Exploring why this is the case may lead not only to better Tg prodrug formulations, but also to a deeper understanding of the biological functions of SERCA pump activity. We compare a broad panel of Tg analogs in various cell types for their biological effects. Methods used so far are western blotting, real-time RT-PCR, live-cell imaging, flow cytometry, and assays that measure autophagic sequestration and degradation activity. Our unpublished data indicate the ER-stress sensors PERK, ATF4, CHOP, and IRE1 but not XBP1 and ATF6 to be involved in cell death signaling in LNCaP prostate cancer cells. Moreover, Tg-induced cell death required death receptor 5 and caspase-8. All Tg analogs tested displayed exactly the same molecular requirements for induction of cell death, although some required 5 to 10 times higher doses than Tg to evoke the same strength of death signal. Like Tg (1) the analogs inhibited autophagy before the closure of phagophores, but at higher doses and/or with slower kinetics. These results indicate that thapsigargin and its analogs evoke similar anti-autophagic and death signaling pathways. Further investigations are aimed at exploring the causes for differential potencies, and include measurements of cytosolic and compartmentalized calcium, as well as solving crystal structures of selected analog:SERCA complexes complemented with biophysical studies of analog:SERCA interactions.

References 1. N. Engedal et al., Autophagy 9, 1475 (2013). 2. C. Dubois et al., Febs j 280, 5430 (2013).

#2325

Destruction of circulating tumor cells by fluid shear stresses generated in a microfluidic system.

Sagar Regmi,1 Afu Fu,1 Sierin Lim,1 Kathy Qian Luo2. 1 _Nanyang Technological University, Singapore, Singapore;_ 2 _University of Macau, Taipa, Macau, China, Macao_.

Circulating tumor cells (CTCs) are mainly responsible for the cause of cancer metastasis. Although most CTCs can be destroyed by the bloodstream, some of them can still manage to withstand hemodynamic shear stress in blood stream. To study the effects of fluidic micro-environment on CTCs, we designed a microfluidic system that can produce various levels of shear stress which can be generated in human artery under resting or exercise condition. We also generated three human breast cancer cell lines with increased ability to form lung metastases in nude mice (i.e. 213-M1A>231-M1>231-C3). All three cell lines can produce a fluorescence resonance energy transfer (FRET)-based sensor which can reveal apoptosis in real-time by changing its color from green to blue. Besides these, we also investigated the effects of shear stress on multiple types of cancer cells including lung cancer (A549), ovarian cancer (2008), breast cancer (UACC-893) and leukemia (K562). For all the cell lines, except the non-attached K562 cells, cells were cultured on petri-dishes, detached by trypsinization and re-suspended in a normal culture medium to a cell density of 2x105 cells/ml. One milliliter of this cell suspensions was circulated in our microfluidic system under the shear stresses of 15, 30, 45 and 60 dyne/cm2 for 2-18 h. The effects of shear stress on morphology and viability of CTCs were determined by FRET imaging microscopy and MTT assay. And the impact of shear stress on causing necrosis in CTCs were examined by propidium iodide (PI) staining and LDH assay. Our findings are summarized below: 1) High shear stress (SS60, 4 h) is more potent to destroy CTCs than low shear stress (SS15, 4 h). 2) High shear stress can destroy most of the CTCs via necrosis. 3) This high level of shear stress is theoretically achievable via physical exercise. 4) Highly metastatic 231-M1A cells are more resistant to high shear stress compared to less metastatic 231-C3 cells. 5) Leukemia K562 cells representing the white blood cells showed stronger resistance to high shear stress compared to the cancer cells tested in this study. We think these findings can help people to understand how metastatic CTCs resist shear force-induced cell death which are useful for designing more effective therapies against metastatic CTCs. Additionally, the finding that high shear stress is more effective to kill CTCs may inspire cancer patients to consider exercise as a way to increase their hemodynamic shear stress, consequently destroy CTCs and prevent cancer metastasis.

#2326

A high-throughput screen identified cancer selective small molecules that kill independent of mitochondrial apoptosis.

Catriona M. Dowling,1 Micheal Hemann,2 James Bradner,3 Anthony Letai,4 Triona Ni Chonghaile1. 1 _Royal College of Surgeons, Dublin, Ireland;_ 2 _Koch Institute for Integrative Cancer Research at MIT, Massachusetts Institute of Technology, MA;_ 3 _Dana-Farber Cancer Institute, Harvard Medical School, Department of Medical Oncology, MA;_ 4 _Dana-Farber Cancer Institute, Boston, MA_.

Triple negative breast cancer (TNBC) lacks expression of oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor 2 (HER2). Importantly, there have been fewer advances in the treatment of TNBC with the mainstay of treatment being cytotoxic chemotherapy. All too often though tumor responds poorly to chemotherapy, or relapse and resistance follow an initial response. We previously found in a range of hematological and solid tumors that chemoresistant cancers are far from the mitochondrial apoptotic threshold. To identify novel therapeutics for the treatment of chemoresistant breast cancer, we performed a high-throughput screen to identify small molecules that are cancer selective but can kill independent of the mitochondrial pathway of apoptosis. To perform this screen we used TNBC line MDA-MB-231 in which the pro-death proteins BAX/BAK were knocked down. BAX/BAK are essential for mitochondrial apoptosis. To enrich for compounds that were not non-specifically toxic, we simultaneously counter-screened against the non-transformed breast epithelial cell line MCF10a. We screened a total of 30,000 compounds in duplicate across the two cell lines. There was a hit rate of 0.3% in the screen and 85 compounds were retested in the validation cherry pick screen. From the cherry pick screen 18 compounds were further validated with low-throughput assessment for mitochondrial independent killing and selectivity for cancer cells. To identify the mechanism of action of the lead compound we used a genetic approach to generate an RNAi signature for the compound. This signature enables classification of the compound into known or novel class of compounds. The lead compound signature was most like that of histone deacetylase inhibitors (HDAC). Using an in vitro HDAC inhibitor screen, we identified that the compound selectively inhibited HDAC6. Importantly, we validated that the lead compound caused an increase in acetylated tubulin in cells, a target of HDAC6. In conclusion, we have identified a novel HDAC6 specific inhibitor that selectively kills cancer cells independent of mitochondrial apoptosis.

#2327

Apoptosis of melanoma induced by non-thermal atmospheric pressure bio-compatible plasma activated media.

Ihn Han, Ying Li, Eun Ha Choi. _Kwangwoon University, Seoul, Republic of Korea_.

Melanoma is the most dangerous type of skin cancer, which mostly related with the exposure of ultraviolet (UV), and especially happened to those who have low skin pigment levels. Epidemiological studies showed Europeans and North Americans have a high incidence of melanoma, while it is less common in Asia and Africa. Therefore, the new and more effective methods to treat melanoma are still necessarily needed.

Non-thermal atmospheric pressure bio-compatible plasma (NBP) is defined as a partially ionized gas with electrically charged particle, which has been reported have cytotoxicity on various cancer cells induce DNA damage and apoptosis other than normal cells. Reactive oxygen and/or nitrogen species (RONS) were proved to play the most important role during this process, by inducing oxidative stress and depolarization of mitochondria membrane potential with the consequence of various cancer cells apoptosis.

The plasma activated media (PAM), which the media treated directly by NBP with different time, containing mostly long-live secondary species such as hydrogen peroxide (H2O2) which have been confirmed anti-microorganism and cytotoxic activity. Researches already investigated that PAM also effective in cancer therapy via inducing apoptosis. However, the underlying molecular mechanisms of apoptosis are remaining elusive.

The purpose of this study was to evaluate the potential of PAM as an effective tool to induce apoptosis in melanoma cells. Our results showed that PAM has a killing effect to melanoma cells in a time-dependent manner, for 3minutes treatment could induce almost 40% cells entry death, after treated by 5 minutes more than 60%, and 10 minutes treatment reduced the cell viability to approximately 30%. Annexin-V/PI staining demonstrated that PAM kills these cells via apoptosis pathway. We also found that with a manner of treatment time PAM significantly increased the concentration of intracellular NO and H2O2, reflecting an influx of extracellular RONS may result in melanoma cells apoptosis. Besides, western blot assay showed that P53 and caspase 3 increased after PAM treatment. Taken together, PAM is effective to induce the apoptosis in melanoma cells in vitro, further in vivo experiments will be performed to investigate the functional effects in the animals.

#2328

Race specific hyper-activation of CCR9-mediated survival signals and its impact on efficacy of docetaxel in prostate cancer.

Neeraj Kapur,1 Hina Mir,1 Guru Sonpavde,2 Shailesh Singh1. 1 _Morehouse School of Medicine, Atlanta, GA;_ 2 _UAB Comprehensive Cancer Center, Birmingham, AL_.

Current approaches to treat and manage prostate cancer (PCa) have failed to reduce racial disparity primarily due to undefined molecular mechanism. We have shown higher expression of chemokine receptor-9 in PCa cells and CCR9 expression was higher in PCa cells (MDA-PCa-2b) derived from African American (AA) patient compared to PCa cells derived form European American (EA) patients (LNCaP and PC3). Using antibody microarray we observed hyper activation of survival molecules and down-regulation of apoptotic molecules in AA PCa cells compared to EA PCa cell lines, following CCL25 stimulation. Comparative heat map analysis showed significant increase (~2-3 fold) in phosphorylation of pro-survival proteins in AA cells compared to EA cells treated with CCL25, which was further confirmed by western blot analysis. Furthermore, CCL25 treated PCa cells showed decreased expression of pro-apoptotic proteins (Bim, Bid, Bak), which was significantly abrogated by CCR9 shRNA. This effect was more pronounced in AA cells compared to EA cells. Efficacy of docetaxel (DTX) was higher after CCR9 blockade in presence of CCL25 compared to cells treated with CCL25 without blocking CCR9. Improvement in efficacy of DTX was due to inhibition of cell survival and activation of pro-apoptotic signals following CCR9 blockade. These observations suggest potential involvement of CCR9 mediated molecular pathways in disparity associated with outcome of PCa.

#2329

TMEM33 induces apoptosis via UPR signaling and autophagy in breast cancer cells.

Rong Hu, Xiyuan Zhang, Leena Hilakivi-Clarke, Usha Kasid, Robert Clarke. _Georgetown Lombardi Comp. Cancer Center, Washington, DC_.

TMEM33 is a novel transmembrane protein that resides in the endoplasmic reticulum (EnR). It has been shown to activate the PERK and IRE1α branches of the unfolded protein response (UPR). However, the underlying mechanism of action of this EnR resident protein TMEM33 and the cellular functions that it regulates remain largely unknown. In this study, we show that overexpression of TMEM33 induces robust cell death in breast cancer cells. Inhibition of the PERK/eIF2alpha pathway with eIF2alpha inhibitor ISRIB blocks the TMEM33 induced cell death. TMEM33 overexpression strongly activates UPR associated pro-death JNK-p53 signaling. We also observed a significant inhibition of the downstream survivin, which blocks cell death activation by binding to caspases and inhibiting their activation. We further show that the blockage of JNK activation with either an inhibitor or overexpression of survivin, protects cells against TMEM33 induced apoptosis. In addition, we show that TMEM33 overexpression induces autophagy in breast cancer cells. Inhibition of autophagy with using either the inhibitor chloroquine or knockdown of the Atg5 gene, further sensitizes breast cancer cells to the effects of TMEM33 overexpression. Cell death induced by TMEM33 is also decreased by overexpression of the autophagy gene Beclin 1. The findings in this study demonstrate that the novel EnR resident protein TMEM33 induces cell death by activating IRE1α-JNK-p53-survivin signaling in breast cancer cells. Concurrently, autophagy is also activated by TMEM33, and functions as a pro-survival mechanism. Cell fate reflects the balance between the pro-death and pro-survival activities as regulated by TMEM33.

#2330

BI5: a novel SMAC mimetic that triggers tumor cell death and potentiates PD-1 mediated cancer therapy.

Markus Reschke, Maria Antonietta Impagnatiello, Ulrich Reiser, Dirk Scharn, Walter Spevak, Alexander Savchenko, Andreas Wernitznig, Martina Sykora, Rebecca Langlois, Elisabeth Zier, Daniel Zach, Sabine Kallenda, Pilar Garin-Chesa, Jens Quant, Mark Pearson, Darryl McConnell, Norbert Kraut, Juergen Moll. _Boehringer Ingelheim, Vienna, Austria_.

Background: Inhibitors of apoptosis proteins (IAPs) regulate cellular apoptosis by interfering with the proteolytic activities of caspases. IAP inhibitors (SMAC mimetics) have been developed to restore the defective apoptosis that characterizes many tumour cells. Emerging evidence demonstrates that IAPs are critical components of immune-modulatory pathways that control innate and adaptive immunity. Accordingly, SMAC mimetics hold the promise of both inducing tumour cell killing and stimulating the immune system to recognize and eliminate dying tumour cells. Here we show that BI5 primes immune components and synergises with PD-1 checkpoint inhibitors to promote eradication of syngeneic tumors.

Methods: Here we report the efficacy and modulation of the immune response by a potent and selective SMAC mimetic, BI5. We characterised the effect of BI5 on tumor growth inhibition as a single agent and in combination with an anti-PD-1 antibody in syngeneic mouse tumor models. A detailed 17-colour multi-color flow cytometry analysis was used to investigate the mechanisms by which the SMAC mimetic interacts with anti-PD-1 therapy in vivo.

Results: Treatment of the syngeneic mouse tumor models MBT-2 and EMT-6 with the SMAC mimetic in combination with an anti-PD-1 antibody results in remarkable tumor regressions in vivo. Importantly, the combined effect of the SMAC mimetic and anti-PD-1 on tumor growth was dependent on the adaptive immune system in vivo. Mechanistic studies show that degradation of IAP triggers tumor cell death, which leads to a potent activation of dendritic cells in the draining lymph nodes and a subsequent influx of T and NK cells into the tumor microenvironment. Interestingly, in the presence of the SMAC mimetic alone, an induction of PD-1 expression on tumor-infiltrating CD8+ T cells was observed, which in turn resulted in the exhaustion of these cells and tumor outgrowth. In the presence of the anti-PD-1 antibody, T cells are reactivated leading to potent and long term tumor eradication.

Conclusion: We show that our SMAC mimetic leads to a potent induction of immunogenic cell death and sets up a "virtuous cycle" by potentiating dendritic cell and T cell mediated immune responses that further promote induction of cell death. These effects are potentiated by checkpoint inhibitors, leading to long term tumor control. Tumours with minimal T-cell infiltration are poorly responsive to PD-1 monotherapy. These studies indicate that SMAC mimetics, such as BI5, represent promising and tolerated combination partners for checkpoint inhibitors in patients that lack a strong immune inflammatory signature.

#2331

FAM3B/PANDER inhibits cell death and increases tumor growth by modulating the expression of Bcl-2 and Bcl-XL cell survival genes.

Izabela Caldeira,1 Paula Maciel-Silva,1 Flavia Ramos Siqueira,1 Anna Carla Goldberg,2 Viviane Abreu Nunes,1 Jose Ernesto Belizario,1 Humberto Miguel Garay-Malpartida1. 1 _University of Sao Paulo, São Paulo, Brazil;_ 2 _Hospital Israelita Albert Einstein, São Paulo, Brazil_.

FAM3B/PANDER is a novel cytokine-like protein that induces apoptosis in insulin-secreting beta-cells. Since in silico data revealed that FAM3B can be expressed by prostate and breast tumors, we evaluated the putative role of this cytokine in prostate and breast tumor progression. The FAM3B expression was compared by quantitative PCR in LnCAP, PC-3 and DU145 prostate tumor cell lines and MCF-7, ZR-75 and MDA-MB-231 breast tumor cell lines. After treatment with either recombinant FAM3B protein or secreted FAM3B obtained from conditioned media (CM) derived from FAM3B-overexpressing 293T cells , the cell death and viability of DU145 and MDA-MB-231 cell lines were evaluated. DU145 and MDA-MB-231 cells overexpressing FAM3B protein were produced by lentiviral-mediated transduction of full-length FAM3B cDNA. Cell viability and apoptosis were analyzed in DU145-FAM3B and MDA-231-FAM3B cells after treatment with several cell death inducers. Anchorage-independent growth and scratch wound assays were used to evaluate in vitro tumorigenicity and cell migration, respectively. In vivo tumorigenicity and invasiveness were evaluated by tumor xenograft growth in nude mice. We observed that FAM3B was highly expressed by hormone responsive cells (LnCAP and MCF-7) and low expressed in unresponsive hormone cells (PC-3, DU145 and MDA-MB-231). Cell viability and survival of DU145 and MDA-MB-231 cells increased after exogenous treatment with recombinant FAM3B protein or CM containing FAM3B-secreted protein. Overexpression of FAM3B in DU145 and MDA-MB-231 cells promoted an inhibition of apoptosis triggered by TNF-alpha, staurosporine and serum-deprived conditions. Cell death inhibition was accompanied by increased gene expression of the anti-apoptotic Bcl-2 and Bcl-xL genes, and by slight decrease in expression of pro-apoptotic gene Bax and diminished caspases-3, -8 and -9 proteolytic activities. When compared to control, cells overexpressing FAM3B displayed a decreased anchorage independent growth and increased motility in vitro, as well as an increased tumor growth in xenografted nude mice. The immunohistochemistry analysis from tumor xenografts revealed similar anti-apoptotic phenotype displayed by FAM3B-overexpressing tumor cells. Taken together, by activating pro-survival mechanisms, FAM3B overexpression contribute to increased cell death resistance and tumor growth in nude mice, highlighting a putative role for this cytokine in prostate and breast cancer progression.

#2332

Integral modulation of nuclear factor-kappa B activation by C/EBPβ and the endoplasmic reticulum stress sensor PERK to mediate estrogen-induced apoptosis in estrogen-deprived breast cancer cells.

Ping Fan,1 Amit K. Tyagi,1 Fadeke A. Agboke,2 Niranjana Pokharel,1 V. Craig Jordan1. 1 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Georgetown University, Washington, DC_.

Stress responses are critical for estrogen (E2) to induce apoptosis in E2-deprived breast cancer cells. Nuclear factor-kappa B (NF-κB) is well known as a therapeutic target to prevent stress responses in chronic inflammatory diseases including cancer. However, whether E2 activates NF-κB to participate in stress-associated apoptosis in E2-deprived breast cancer cells is unclear. We demonstrated that E2 differentially modulates NF-κB activity in E2-deprived breast cancer cells according to the treatment time. Because E2 initially has significant potential to down modulate the NF-κB activation, it completely suppresses the tumor necrosis factor alpha (TNFα)-induced NF-κB activation. We found that E2 preferentially and constantly enhances the expression of transcription factor CCAAT/enhancer binding protein beta (C/EBPβ) which is responsible for suppression of NF-κB activation by E2 in MCF-7:5C cells. The mTOR signaling pathway promotes repression of NF-κB by C/EBPβ which is confirmed by the evidence that inhibition of mTOR is synergistic with E2 to upregulate NF-κB-dependent genes, such as TNFα. Interestingly, NF-κB p65 activity is upregulated when E2-treatment is administered for 48 hours, leading to induction of TNFα. Blocking the nuclear translocation of NF-κB completely prevents E2 from induction of TNFα and apoptosis. Importantly, protein kinase RNA-like endoplasmic reticulum kinase (PERK), a stress sensor of unfolded protein response, is activated by E2 and plays an essential role in increasing NF-κB p65 DNA binding through the activation of STAT3, independently of canonical IκBα signal pathway. Thus, inhibition of PERK kinase activity completely blocks nuclear activation of NF-κB and NF-κB-dependent induction of TNFα, thereby preventing E2-induced apoptosis. All of these findings illustrate a crucial role for the novel PERK/NF-κB/TNFα axis in E2-induced apoptosis which is integrally modulated by the stress responsive transcription factor C/EBPβ and endoplasmic reticulum stress. This study provides an important rationale for exercising caution in clinical trials when considering targeting PERK or NF-κB following the development of acquired resistance to aromatase inhibitors whereas mTOR may be a target to enhance the therapeutic effects of E2 in antihormone resistant breast cancer.

#2333

Modulation of Bax and mTOR for cancer therapeutics.

Rui Li,1 Chunyong Ding,2 Jun Zhang,1 Maohua Xie,1 Dongkyoo Park,1 Ye Ding,3 Guo Chen,1 Guojing Zhang,1 Melissa Gilbert-Ross,1 Wei Zhou,1 Adam Marcus,1 Shi-Yong Sun,1 Zhuo Chen,1 Gabriel Sica,1 Suresh Ramalingam,1 Andrew Magis,4 Haian Fu,1 Fadlo Khuri,1 Walter Curran,1 Taofeek Owonikoko,1 Dong Shin,1 Jia Zhou,2 Xingming Deng1. 1 _Emory Univ., Atlanta, GA;_ 2 _University of Texas Medical Branch, Galveston, TX;_ 3 _University of Texas Medical Branch, Atlanta, TX;_ 4 _Institute for Systems Biology, Seattle, WA_.

Pharmacologic manipulation of the serine (S)184 phosphorylation site of Bax protein to functionally regulate its proapoptotic activity is an attractive anticancer strategy. We recently identified three small molecule Bax agonists. SMBA1 was selected as the lead compound based on its chemical structure and its drug-like properties, from which a more effective analog, CYD-2-11, was generated. CYD-2-11 targets the structural pocket around S184 in the C-terminal tail of Bax, directly activating its proapoptotic activity via 6A7 conformational change and formation of Bax homo-oligomers in mitochondrial membranes. CYD-2-11 suppresses tumor growth in SCLC, NSCLC and patient-derived lung cancer xenografts as well as the genetically engineered mutant KRAS-driven lung cancer model with no significant normal tissue toxicity. Inhibition of mTOR by RAD001 enhances S184 Bax phosphorylation in lung cancer cell lines and tumor tissues from lung cancer patients treated with RAD001, which inactivates the propaoptotic function of Bax, contributing to rapalog-resistance. Combined CYD-2-11 and RAD001 treatment not only displays strong synergistic activity against lung cancer but also overcomes rapalog-resistance in vitro and in vivo. Therefore, a mechanism-driven combination of Bax agonist and mTOR inhibitor represents a highly attractive therapeutic strategy to improve lung cancer patient outcomes.

#2334

**Examining the contribution of autophagy to TRAIL resistance in an** in vitro **model of circulating breast cancer cells.**

Julianne D. Twomey, Baolin Zhang. _Food and Drug Administration, Silver Spring, MD_.

Circulating tumor cells (CTCs) are cancer cells which dissociate from the primary tumor and circulate within the peripheral blood, initiating metastasis at a distant location. CTCs present a "non-invasive, liquid biopsy", making them attractive targets for the development of cancer biomarkers and therapies. In order to form metastases, these cells must undergo a phenotypic shift acquiring the ability to survive anoikis within the blood stream and evade immune surveillance involving death events mediated by death receptors (DRs) expressed on the surface of cancer cells. Under physiological conditions, these DRs can be activated by circulating cytokines such as TNF, Fas ligand, and TNF-related apoptosis inducing ligand (TRAIL), triggering cell death. In vitro studies have demonstrated that environmental stress caused by non-adherent suspension culture conditions, similar to circulation, will increase autophagy in cancer cell lines aiding anoikis survival. Our lab has previously identified a link between increased autophagic rates and decreased DR surface expression. Based on this we were interested in determining whether increased autophagy due to non-adherent conditions caused breast cancer cell lines to decrease death receptor expression, evading immune surveillance and resisting death-ligand induced apoptosis. To this end, breast cancer cell (BCC) lines were cultured in monolayer or a non-adherent suspension condition for 7 days. Following 7 days of culture, death receptor expression and autophagy initiation were measured using immunoblot and flow cytometry microscopy. BCCs were treated with TRAIL and analyzed over 24 hours for apoptosis and caspase activation. We found that the BCC lines decreased total and surface expression of DR5 and total expression of TNFR1 over the 7 days of suspension condition. The reduced expression of DR5 in suspension cultured BCCs resulted in a delayed response to TRAIL mediated apoptosis, with delayed caspase activation and PARP cleavage compared to monolayer cultured parental cells. Under suspension condition, autophagy was initiated by increased LC3-II/ LC3-I turnover and p62 degradation. This initiation of autophagy is potentially due to increased expression of the mitochondrial related proteins BNIP3 and BNIP3L, which have been implicated in anoikis survival. This work warrants additional studies investigating the therapeutic potential of targeting CTCs within the blood to limit metastatic spread and disease progression in breast cancer. Our findings suggest the potential to sensitize CTCs to anoikis and circulating death ligands through autophagy inhibition.

#2335

Targeting the DNA repair as well as survival functions of MCL-1 enhances cancer cell killing.

Abid Mattoo,1 J Milburn Milburn Jessup,1 Tej K. Pandita2. 1 _Inova Schar Cancer Institute, Falls Church, VA;_ 2 _The Houston Methodist Research Institute, Houston, TX_.

MCL-1 is a a pro-survival BCL2 protein family member which is over-expressed in drug resistant cancer cells. Our purpose was to assess whether MCL-1 increases genomic instability along with its anti-apoptotic function in cancer cells. Depletion of MCL-1 by treating human small cell lung cancer (SCLC) lines with immunotoxin sensitizes these cells to increased killing by BH3 mimetic - Navitoclax (ABT-263) both in vitro and in mice xenograft models. These SCLC cell lines are resistant to both single agent Immunotoxin or ABT-263. In addition, inhibition of NANOG or NANOGP8 by lentivrus delivered shRNA depletes MCL-1 in human colorectal cancer cells and sensitizes these cells to increased cell killing by venetaclox (ABT-199). This increased killing depended on loss of MCL-1, was caspase-dependent and reversed by re-expression of MCL-1. Depletion of MCL-1 in cancer cells , increases cell sensitivity to ionizing radiation (IR) induced death, which was reversed by expression of MCL-1. In response to IR, MCL-1 depleted cells showed reduced survival even at radiation doses less than 6 Gy, however an increase in caspases 3/7 activity was observed at 6 Gy and above, suggesting that apoptosis is activated only when MCL-1 depleted cells are irradiated with higher IR doses. In order to explain the decreased survival post- irradiation the genomic instability and DNA repair pathways were analyzed in MCL-1 depleted cells. Post-irradiation MCL-1 depleted cells exhibited increased genomic instability as measured by increase in chromosome aberrations at different phases of the cell cycle. The increase in aberrations were significantly higher in G2 and S- phases of the cell cycle suggesting defect in Homologous recombination repair pathway (HR). Moreover, the MCL-1 depleted cells show decreased gamma-H2AX foci at earlier time points (30 and 90 minutes) post-irradiation indicating defect in DNA Damage response (DDR) and higher residual foci at 360 minutes, indicating defective DNA repair. These observations were further confirmed by decreased phosphorylation of ATR suggesting defect in DNA Damage response and higher levels of residual 53BP1 and RIF1 foci in MCL-1 depleted cells , confirming DNA DSB repair by homologous recombination (HR) was compromised. Consistent with this model, MCL-1 depleted cells exhibited a reduced frequency of IR-induced MRE11, BRCA1, RPA and Rad51 foci formation, decreased DNA end resection and decreased HR repair in the DR-GFP DSB repair model. Similarly, after Hydoxyurea (HU) induction of stalled replication forks in MCL-1 depleted cells there was a decreased ability to subsequently restart DNA synthesis, which is normally dependent upon HR mediated resolution of collapsed forks. In summary, MCL-1 is an important therapeutic target in cancer cells and its depletion increases cell killing by either increase in apoptosis or suppression of HR and increased replication stress.

#2336

Novel regulatory mechanisms for Bcl2-related Ovarian Killer (BOK) expression in breast cancer.

Benjamin Chidi Onyeagucha,1 Panneerdoss Subbarayalu,1 Subapriya‎ Rajamanickam,1 Nourhan Abdelfattah,1 Santosh Timilsina,1 Rosa M. Guzman,2 Carla Zeballos,1 Vijay Eedunuri,1 Sanjay Bansal,1 Hima Bansal,1 Tabrez A. Mohammad,1 Yidong Chen,1 Manjeet K. Rao1. 1 _University of Texas Health Science Center San Antonio, San Antonio, TX;_ 2 _St Mary Unversity, San Antonio, TX_.

Deregulation of apoptosis is central to cancer progression and a major obstacle to effective treatment. The Bcl-2 gene family members play important roles in the regulation of apoptosis and are frequently altered in cancers. One such member is Bcl-2-related Ovarian Killer (BOK), which is a pro-apoptotic protein. Despite its critical role in apoptosis, the regulation of BOK expression is poorly understood in cancers. Here, we discovered that miR-296-5p, regulates BOK expression by binding to its 3'UTR in breast cancers. Furthermore, we show that depletion of BOK by either miR-296-5p or siRNA against BOK protected breast cancer cells from undergoing paclitaxel-induced apoptosis. Interestingly, miR-296-5p also regulates the expression of Mcl-1, which is an anti-apoptotic protein and is highly expressed in breast cancers. Our results reveal that Mcl-1 is important for suppression of BOK function as ectopic BOK expression induced Mcl-1, while silencing of BOK resulted in reduced Mcl-1 levels in breast cancer cells. In addition, we show that specific silencing of Mcl-1 reduced the long-term growth of breast cancer cells, whereas BOK inhibition didn't have any effect on the growth of breast cancer cells. Surprisingly, silencing of both Mcl-1 and BOK rescued the effect of Mcl-1 silencing on breast cancer cell growth, suggesting that BOK is important for attenuating cell growth in the absence of Mcl-1, and also showing a tight feedback regulatory loop between BOK and Mcl-1 in breast cancer cells. Furthermore, we demonstrated that BOK protein level is regulated post-translationally by GSK3α and to some extent GSK3β as GSK3 inhibitor (CHIR99021) or silencing of GSK3 significantly increased BOK protein levels in breast cancer cells. Notably, we found that Mcl-1 interacts with GSK3α/β and silencing of Mcl-1 using siRNA significantly attenuated endogenous GSK3α/β levels in breast cancer cells. Taken together, our results suggest that fine tuning (either post-transcriptionally by miR-296-5p or post-translationally by GSK3) of the levels of pro-apoptotic protein BOK and anti-apoptotic protein Mcl-1 decide the fate of cancer cells to either undergo Apoptosis or proliferation.

### CDKs and CDK Inhibitors

#2337

miR-6883 and family miRNAs induce G1-arrest in colon cancer cells by targeting CDK4/6.

Amriti R. Lulla, Margret B. Einarson, Yan Zhou, Michael Slifker, David T. Dicker, Wafik S. El-Deiry. _Fox Chase Cancer Ctr., Philadelphia, PA_.

Dysregulation of the CDK4/6-Rb pathway is a hallmark of various tumor types. Cancer cells subvert different cell cycle checkpoints to continue unchecked growth and proliferation. Some common mechanisms of overcoming cell cycle checkpoints involve loss of tumor-suppressor proteins such as p16 INK4A or Retinoblastoma (Rb). Alternately, cancer cells can also increase expression of oncogenes like cyclin D1, other CDKs by gene amplification or gene translocation. Thus, targeting different oncogenes in the cell cycle pathway, particularly CDKs has been considered an important therapeutic strategy. Events of overexpression of cell cycle oncogenes and suppression of tumor suppressors are mutually exclusive and tumor-type specific. In colorectal cancer (CRC), it has been shown that there is overexpression of CDK4/6 . We conducted TCGA analysis of 50 matched tumor and normal CRC tumors, and validated that CDK4 and CDK6 are significantly overexpressed in patient tumor samples by RNA-seq (p= 1.55 e-09 and p= 4.439 e-05). Thus, ongoing clinical trials for CRC patients with advanced disease are currently evaluating the efficacy of CDK4/6 inhibitors as single agents and in combination with chemotherapy (J Clin Oncol 33, 2015 ,suppl 3; abstr 626; Zhang 2016). We evaluated the efficacy of miRNAs targeting CDK4/6 as novel therapeutics in CRC. We performed in silico analysis using TargetScan to identify miRNAs that can target the 3'UTR regions of CDK4/6. We have identified a novel family of miRNAs (based on seed sequence similarity) that can potently target CDK4/6 mRNA and thereby reduce both the RNA and protein levels of CDK4/6 in cancer cells. Of the four miRNAs in the family, we chose to characterize miR-6883-5p and miR-149* in a panel of CRC, melanoma and pancreatic cancer cell lines. Our preliminary TCGA analysis in 11 matched T/N samples shows miR-149* expression is significantly lost (p= 0.0049) in CRC samples and this further positively correlates with the stage of the tumor. Ongoing in vitro studies using miRNA mimics in CRC cell lines indicate that both miR-6883-5p and miR-149* have anti-proliferative effects as assayed by CellTiterGlo (CTG) and MTT. Further, both miRNAs induce G1-arrest as a consequence of downregulation of CDK4 and CDK6 in all cell lines and we also observe induction of apoptosis in a subset of CRC cell lines. Knockdown of CDK4 and CDK6 mimics the phenotypes observed with restoring expression of both miR-6883 and miR-149* in CRC cell lines. Combination of miR-6883 with FDA-approved drugs Irinotecan and 5-FU showed strong synergy and led to apoptosis as assayed by CTG and PARP cleavage. Our ongoing work is further looking into the mechanisms of synergy and identifying additional target genes in CRC cell lines. Thus, our novel miRNA based strategy to target CDK4 has potential to translate as both a single agent/combinatorial therapy and to identify biomarkers of response, which is critical for understanding the clinical results seen with CDK inhibitors.

#2338

CDK4/6 and autophagy inhibitors synergize to induce senescence in cancers with an intact G1/S checkpoint.

Smruthi Vijayaraghavan,1 Cansu Karakas,1 Xian Chen,1 Iman Doostan,1 Akshara S. Raghavendra,1 Min Yi,1 Ravi Amaravadi,2 Kelly Hunt,1 Debu Tripathy,1 Khandan Keyomarsi1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _U Penn School of Medicine, PA_.

Deregulation of the cell cycle machinery is a hallmark of most cancers. The crucial role of the CDK4/6-CyclinD pathway in tumorigenesis has led to the successful development and FDA approval (palbociclib) of CDK4/6 inhibitors for the treatment of advanced estrogen receptor positive breast cancer. However, two major clinical challenges remain: i) lack of a reliable biomarker to predict treatment response and ii) adverse events leading to interruption or discontinuation of treatment which possibly thus curtailing therapeutic benefit.

We found that treatment of ER+ breast cancer cell lines with the CDK4/6 inhibitor palbociclib resulted in a dose-dependent sustained growth inhibition and senescence. Interestingly, breast cancer cells activate autophagy in response to palbociclib, a stress response process that promotes cancer cell survival. Genetic ablation of crucial autophagic genes or pharmacological inhibition of autophagy increases the sensitivity of ER+ breast cancer cells to palbociclib and the other CDK4/6 inhibitors, ribociclib and abemaciclib. This was confirmed in vivo, where the combination of palbociclib and autophagy inhibitor, hydroxychloroquine (HCQ) resulted in a significantly improved and a sustained tumor shrinkage. To identify biomarkers that predict response to CDK4/6 inhibition, we examined the G1 checkpoint proteins and found that knockdown of Rb or overexpression of the oncogenic low molecular weight isoforms of Cyclin-E (LMW-E) mediates resistance to palbociclib and its combination with autophagy inhibitor. More significantly, immunohistochemically staining of pre-treatment biopsies from palbociclib treated patients strengthened the correlation between palbociclib efficacy and an intact G1/S checkpoint (Rb+ve /LMWE-ve), resulting in a significantly longer progression free survival compared to the other patient groups; thus solidifying Rb and LMW-E as reliable prognostic biomarkers for palbociclib treatment. Finally, we examined the biomarker driven synergy between CDK4/6 and autophagy inhibition in several other cancer cell lines. Several solid tumors (ovarian, lung, pancreatic, colon, prostate) and triple negative breast cancer (TNBC) cell lines exhibited a synergistic response to palbociclib/HCQ combination treatment dependent upon an intact G1/S transition (Rb+/LMWE-). This was also verified in a TNBC patient derived xenograft (PDX) model.

Thus, this study addresses the aforementioned limitations and provides a novel and promising biomarker-driven combination therapeutic strategy to treat breast and other solid tumors. We predict that this combination of CDK4/6 and autophagy inhibitors would be more beneficial than standard dose palbociclib in patients, allowing us to lower the dose, minimize palbociclib mediated toxicities and potentially improve overall patient survival - a goal that has not yet been met with currently approved treatment combinations.

#2339

Downregulation of cell cycle genes in HNSCC by erufosine.

Shariq S. Ansari,1 Ashwini K. Sharma,1 Michael Zepp,1 Frank Bergmann,2 Rainer König,3 Martin R. Berger1. 1 _The German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 2 _University Clinic Heidelberg, Heidelberg, Germany;_ 3 _Leibniz Institute for Natural Products Research and Infection Biology, Jena, Germany_.

Head and neck cancer ranks amongst the sixth most prevalent cancers worldwide and oral squamous cell carcinomas (OSCC) constitute 90% thereof. Erufosine is an ether-lipid-derived synthetic compound belonging to alkylphosphocholines (APCs), which has been shown to inhibit the proliferation of OSCC cells. It simultaneously induces apoptosis and autophagy by modulating the Akt-mTOR signaling pathway, however, its exact mechanism of action is not fully understood. Here, we describe the activity of erufosine on the expression of cell cycle related genes in OSCC cells.

The anti-proliferative effect of erufosine in cells of two human OSCC cell lines, HN-5 and SCC-61, was determined by MTT assay after 24h, 48h and 72h exposure. Based on these results, HN-5 cells were exposed to erufosine at concentrations corresponding to IC25, IC50 and IC75 concentrations, and then their mRNA was isolated and analyzed by Illumina Chip array. Gene expression modulation was confirmed qRT-PCR and Western blot. Gene set enrichment analysis was performed to identify core KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways and GO (Gene Ontology) terms. In addition, erufosine's effect on cell cycle distribution and colony formation was determined as well as its antineoplastic effect on respective xenografts in nude mice.

As shown from TCGA data, cell cycle deregulation in HNSCC ranks amongst the top three out of 24 different types of cancers. In line with this, the median expression of cell cycle genes was higher than that of other genes within HNSCC. We also looked at the median gene expression of all CDKs and cyclins in 519 HNSCC patient samples from TCGA and found that the expression levels were higher than the global gene expression. This was in agreement with IHC staining of CCND1 in tumors from 30 South East Asian HNSCC patients. Furthermore, increased CCND1 and CDK6 expression levels from TCGA data had negative implication on patient survival. In our experimental results erufosine caused a dose dependent growth inhibition of OSCC cell lines and negative enrichment of genes related to

the cell cycle process. Our microarray findings revealed that cyclins and CDKs were downregulated in a dose dependent manner in response to erufosine exposure. These findings were verified at both, mRNA and protein levels. Erufosine not only caused a massive G2M block but also inhibition of colony formation thus preparing the OSCC cells to undergo apoptosis. We are the first to show that erufosine inhibited tumor growth in vivo in a HNSCC xenograft model and caused downregulation of cyclinD1, CDK4 and CDK6 in lesions of the animals.

These findings collectively show the potential of erufosine to be used as a cell cycle inhibitor in HNSCC progression and support the future evaluation of erufosine as a therapeutic approach in cancer treatment alone, or in combination.

#2340

Thymidine kinase activity as a response marker for CDK 4/6 inhibition.

Geoffrey Shapiro,1 Magnus Neumuller,2 Sara Lööf,3 Smaranda Bacanu,3 Takahiro Seki,4 John Hilton,1 Khan Do,1 Nicole Chau,1 Leena Gandhi,1 Joseph W. Gibson,1 Robert Distel,1 Pawel Niekrasz,2 Edward M. Suh,2 Mattias Bergqvist2. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Biovica International AB, Uppsala, Sweden;_ 3 _Karolinska University Hospital, Stockholm, Sweden;_ 4 _Karolinska Institute, Stockholm, Sweden_.

Selective CDK4/6 inhibition is now part of standard treatment for HR+ breast cancer. There is a pressing need for a practical biomarker that can provide early indication of the biologic activity of these agents and correlate with clinical outcome. Expression of thymidine kinase (TK) occurs in actively proliferating cells, is E2F-dependent and is downregulated after CDK4/6 inhibitor-mediated G1 arrest. Here, we have investigated TK activity (TKA) as a pharmacodynamic marker for CDK4/6 inhibition in both preclinical and clinical contexts and demonstrate the potential of this assay as CDK4/6 inhibitors are further developed. TKA in response to the CDK 4/6 inhibitor palbociclib (palbo) was studied in cell culture, mouse models and samples from clinical studies. TKA was determined by the DiviTum Assay (Biovica, Sweden). In culture, the intracellular TKA and TK release in response to palbo were determined by analysis of cellular extracts and tissue culture media. In vivo, mice bearing human xenografts were treated with vehicle or palbo to determine effects on TKA in serum and in tumor extracts. Finally, serum TKA (s-TKA) was assayed in patient samples procured before and after palbo treatment in a Phase 1 study of palbo and the MEK inhibitor PD0325901 (NCT02022982). Palbo was administered on 3/4 (21 of every 28 days) or 4/4-week (continuous) schedules. In K562 cells, intracellular TKA levels exhibited a clear dose response to palbo. Reductions in TKA were seen at lower drug concentrations than those affecting cell viability. Similar results were observed in MCF-7 xenografts, where lower TKA in serum and tumor was observed after palbo treatment, whereas in E0771 xenografts, no change in TKA was observed. Clinically, serum samples were obtained before and at various time points after palbo exposure in 20 cancer patients. One patient, who discontinued study treatment at the end of cycle 1, demonstrated an increase in s-TKA. In contrast, for the other 19 patients, there was a marked decrease in s-TKA at day 21. Pre-treatment s-TKA values were higher than C1D21 (paired t-test, p<.0001). In 11 patients there was a reduction in s-TKA to below the assay detection limit. Five patients exhibited a reduction in s-TKA within the working range, with mean residual activity at day 21 of 32%. In the 6 patients receiving continuous palbo, s-TKA remained low after day 21. Of the 14 patients on the 3/4-week schedule, 9 exhibited an increasing level of s-TKA between day 21 and day 1 of cycle 2, consistent with reversible effects of palbo on cell cycle progression after cessation of exposure. These data suggest that s-TKA reflects MOA response to the selective CDK4/6 inhibitor palbo. The DiviTum assay may provide a practical, non-invasive tool for monitoring the effects of CDK4/6 inhibitors in both preclinical models and patients. We plan inclusion of the assay into additional studies to determine whether the assay can predict clinical outcome or portend development of resistance.

#2341

In situ **labelling identifies a novel landscape of nuclear CDK2 substrates.**

Bruce E. Clurman, Yong Chi. _Fred Hutchinson Cancer Research Ctr., Seattle, WA_.

Cellular signal transduction pathways rely heavily on protein kinase-mediated phosphorylation events. Comprehensive identification of the physiologic targets of protein kinases is key to understanding their functions and elucidating cellular signalling pathways. Various direct and indirect approaches, combined with advanced mass spectrometry technologies, have made it possible to identify many candidate targets of a given kinase, but have also yielded many false-positive identifications, largely due to technical limitations. Identifying direct and physiological kinase targets thus remains a difficult task. We developed a mass spectrometry-based in situ approach that utilizes substrate thiophosphorylation and ATP analog-sensitive CDK2 (AS-CDK2) to identify over one hundred potential cyclin-dependent kinase 2 (CDK2) substrate in intact nuclei. More than one-third of these candidates are known CDK substrates, indicating significant enrichment for physiologic CDK2 substrates, and we also we identified and validated new CDK2 substrates. Importantly, many of these new CDK2 substrates are chromatin-associated proteins with roles in histone modification, DNA replication, and DNA repair, which likely reflects our use of AS-CDK2 within its normal nuclear context, and regulated by endogenous cyclins. These methods should be broadly applicable to the study of other kinases with complex substrate networks.

#2342

TNFAIP8 promotes prostate cancer cell survival by modulating autophagy.

Suresh Niture,1 Malathi Ramalinga,2 Habib Kedir,3 Deepak Kumar1. 1 _North Carolina Central University, Durham, NC;_ 2 _University of District of Columbia, Washington, DC;_ 3 _North Carolina Central University and UDC, Durham, NC_.

TNF-α inducible protein (TNFAIP8) is an antiapoptotic protein with roles in tumor cell growth and survival. Mechanisms of cell survival by TNFAIP8 remains elusive. In the current study, we investigated the role and molecular mechanism related to TNFAIP8 in the modulation of cell/cycle, autophagy and drug resistance/cell survival in prostate cancer cells. Microarray data from PC3 prostate cancer cells ectopically expressing TNFAIP8 demonstrated modulation of cell cycle related genes such as CDKs, CDCs and PCNA. Immunoblotting data from overexpression of TNFAIP8 in PC3 cells shows upregulation of cell cycle proteins cyclin A, cyclin B1, Myt1 and Chk1. However, no change in cell cycle was found. Phosphorylation of Histone-S10, CDC2-Tyr15 and Wee1-S642] was also observed. Autophagy plays an important role in tumor cell survival. We evaluated the effects of TNFAIP8 in modulating autophagy. Overexpression of TNFAIP8 leads to induction of autophagy. TNFAIP8 positively modulates the expression/stabilization of autophagy markers and effectors such as LC3β I/II, Beclin1, 4EBP1, oncogene p62 and SIRT1. Knockdown of TNFAIP8 inhibits autophagy induced by nutrient starvation in PC3 prostate and MCF7 breast cancer cells. We also evaluated cell growth and survival in PC3 cells. We demonstrate that TNFAIP8 promotes cell growth and proliferation in PC3, LNCaP and C4-2 prostate cancer cells. Furthermore, we have found that TNFAIP8 increase resistance against anticancer drugs docetaxel and doxorubicin. These data collectively suggest that by the creation of cellular autophagy events TNFAIP8 promotes cell survival and drug resistance in prostate cancer cells.

#2343

The novel potential of palbociclib (CDK4/6 inhibitor) in the treatment of triple-negative breast cancer.

Shinichiro Kashiwagi, Yuka Asano, Wataru Goto, Koji Takada, Tsutomu Takashima, Tamami Morisaki, Satoru Noda, Naoyoshi Onoda, Kosei Hirakawa, Masaichi Ohira. _Osaka City University Graduate School of Medicine, Osaka, Japan_.

Background: The effectiveness of palbociclib (CDK4/6 inhibitor) for estrogen receptor positive breast cancer has been demonstrated by large-scale clinical studies, with the drug garnering attention as a key drug for breast cancer subtypes with endocrine sensitivity in the future. According to PALOMA-3 trial, palbociclib has been demonstrated to contribute to the extension of progression-free survival in patients with advanced hormone receptor-positive and HER2-negative metastatic breast cancer after endocrine therapy. On the other hand, in the case of triple-negative breast cancer (TNBC), luminal AR (LAR) related to androgen signaling is believed to have endocrine activity. Previous clinical data revealed that palbociclib shows high sensitivity in luminal breast cancer cell lines with endocrine activity, with effectiveness also expected in LAR. In this study, we created TNBC cell lines that forcibly express AR and examined the effectiveness of palbociclib for TNBC.

Materials and Methods: MCF-7 and T-47D were used as luminal breast cancer cell lines, while MDA-MB-231 and BT-549 were used as TNBC cell lines. In addition, we created TNBC cell lines that forcibly express AR, called AR-MDA-MB-231, by the transfection of pEGFP-C1-AR Plasmid Vector using Lipofectamine® 3000 Reagent. We confirmed the expression of AR by qRT-PCR as well as Western blotting and examined the impact of palbociclib on proliferation as well as apoptosis of breast cancer cell lines.

Results: AR was found to have been expressed only in luminal breast cancer cell lines but not TNBC cell lines. It was confirmed that AR was expressed in AR-MDA-MB-231 which are stable cell lines with the properties of LAR. In a CCK assay, palbociclib showed high sensitivity in AR-MDA-MB-231 as in luminal breast cancer cell lines. Furthermore, in an apoptosis assay using FACS and cell cycle assay, apoptosis was induced in AR-MDA-MB-231 and cell cycle arrest at the G1S check point was confirmed.

Conclusion: palbociclib (CDK4/6 inhibitor) showed effectiveness for TNBC cell lines that compulsively express AR, suggesting it may be one treatment option for TNBC in the future.

#2344

Beyond RB1: the pocket protein p130 mediates the effects of CDK4/6 inhibition in RB1-deficient urothelial carcinoma.

Bishoy M. Faltas, Ethan Shelkey, Rebecca Meyer, Nathan Young, Mark A. Rubin. _Weill Cornell Medical College, New York, NY_.

Introduction: Urothelial carcinoma is characterized by a high incidence of molecular alterations in the CDKN2A-RB-E2F axis. Loss of the cyclin-dependent kinase inhibitor p16 encoded by CDKN2A or amplification of CDK4/6 is associated with sensitivity to CDK4/6 inhibitors in various cancers. Functional RB1 is believed to be canonically required for mediating the effects of CDK4/6 inhibition in this setting. However, muscle-invasive urothelial carcinomas harbor RB mutations and copy-number losses in 21% of patients. To extend the activity spectrum of CDK4/6 inhibitors to these RB1-deficient cancers, we investigated the role of pocket protein p130 encoded by the Retinoblastoma-Like 2 (RBL2) gene, in mediating the downstream effects of CDK4/6 inhibition.

Methods: CRISPR-Cas9 was used for CDKN2A knockout in the 5637-urothelial cancer cell line with an RB-mutant background and in TCCSUP cell line with wild-type RB. CDKN2A- cells were subsequently treated with CDK4/6 inhibitor palbociclib. Levels of RB1, phospho-RB1 (at serine 807/ serine 811), p130 and phospho-p130 (specifically, the CDK4/6-dependent serine-672 phosphorylation) proteins were quantified using SDS-PAGE. Cell-cycle phase analysis was performed using flow cytometric measurement of BrdU and PI staining.

Results: CRISPR-induced disruption of CDKN2A was validated using genomic PCR and targeted Miseq sequencing. p16 protein knockdown was validated using SDS-PAGE. CRISPR-induced CDKN2A knockdown resulted in increased cell proliferation and faster G1/S transition even in the absence of functional RB in the 5637-urothelial cancer cell line. A significant increase in the levels of phospho-p130 was observed after p16 knockdown without changes in the levels of unphosphorylated p130. TCCSUP cell line with functional RB demonstrated a similar increase in proliferation, faster G1/S transition and an increase in phospho-p130. Palbociclib was effective in reversing these changes in all the tested cell lines.

Conclusions: the pocket protein p130 plays an important role in mediating downstream effects of CDK4/6 inhibition in urothelial cancer cell lines with both wild-type and more importantly, deficient RB. CDK4/6-dependent phosphorylation of p130 can effectively compensate for the functional loss of RB activating E2F family of transcription factors that control G1/S checkpoint. These findings potentially explain the lack of correlation between RB and response to CDK4/6 inhibitors in clinical trials. These results provide a mechanistic rationale for extending clinical trials of CDK4/6 inhibitors to patients with RB1-deficient tumors.

#2345

CDK4 inhibition enhances anti-melanoma effects of BRAF/MEK inhibition.

Yanping Zhang, Eddy C. Hsueh. _Saint Louis Univ., St. Louis, MO_.

Introduction: Targeted therapy against BRAF-mutated melanoma has shown clinical efficacy. However, resistance to therapy often occurs. CDK4 (INK4)-retinoblastoma (RB) pathway controls cell cycle progression by regulating the G1-S checkpoint. Defect in the p16INK4A: cycling D-CDK4/6: RB pathway is also detected in melanoma. We study the effect of combining CDK4-RB pathway inhibition with BRAF/MEK inhibition as a possible strategy to overcome resistance to therapy.

Methods: Three BRAF wild-type and three BRAF-mutated melanoma cell lines were selected. CDK4/6 inhibitor (PD0332991; PD), BRAF inhibitor (PLX4032; PLX), MEK inhibitor (AZD6244; AZD), and PI3K/AKT inhibitor (XL765; XL) were used alone or in combination. Cell proliferation assay was performed using Cell Titer Blue assay. Cell migration assay was performed by artificial wounding of melanoma cell monolayer and observing migration of cells into the wound up to 48 hours. Western blotting was performed for expression of pERK/ERK, pRB/RB, Cyclin D1, CDK4, and GAPDH. Data were presented as means ± SD for triplicate experiments. For comparison between groups, the student's t test was used and p<0.05 was considered to be statically significant.

Results: Variable inhibition of cell proliferation and migration was observed in a dose- and time-dependent fashion. Enhanced inhibition of melanoma proliferation and migration was observed with PD+AZD and PD+PLX combination compared with single agent AZD or PLX. Differential enhancement was observed in BRAF wild-type cell lines with PD+AZD and in BRAF mutated cell lines with PD+PLX combination. Minimal change in tumor proliferation and migration inhibition was noted with PD+XL combination compared with PD or PLX alone. No correlation was observed between expression of CDK4 and cyclin D1 and sensitivity to singe agent or the combination. Inhibition of phosphorylated RB1 and ERK expression was observed with exposure to single agent PD, PLX, XL, AZD. Enhancement of pRB1 and pERK inhibition was noted with PD+PLX and PD+AZD combination but not PD+XL.

Conclusion: CDK4 inhibition can enhance anti-proliferation and migration effects of BRAF/ MEK inhibition in melanoma cells. The mechanism is via augmented down-regulation of pRB expression.

#2346

Preclinical selectivity profile of the CDK4/6 inhibitor ribociclib (LEE011) compared with that of palbociclib and abemaciclib.

Ralph Tiedt,1 Scott Delach,2 Steven Kovats,2 Thomas Horn,2 Michael Acker,2 Barbara Schacher Engstler,1 Giordano Caponigro2. 1 _Novartis, Basel, Switzerland;_ 2 _Novartis, Cambridge, MA_.

A hallmark of cancer is unchecked cell division. Retinoblastoma protein (Rb) is a human tumor suppressor that guards a cell's entry into S phase by binding E2F transcription factors and keeping them inactive. Many growth-promoting stimuli increase expression of D-type cyclins, which bind to and activate cyclin-dependent kinases 4 and 6 (CDK4/6). The cyclin D–bound CDK4/6 holoenzymes phosphorylate Rb, resulting in release of E2F, which in turn activates genes required for S phase entry and DNA replication. Numerous oncogenic aberrations converge at the CDK4/6–Rb pathway, providing a strong rationale for developing CDK4/6 inhibitors as cancer therapeutics.

Ribociclib (LEE011) is a selective CDK4/6 inhibitor that has received FDA breakthrough therapy and priority review designations for treatment of hormone receptor–positive breast cancer in combination with letrozole and is being tested in additional clinical trials. Here we describe the preclinical selectivity profile of ribociclib in biochemical and cellular assays. Ribociclib inhibits both CDK4–cyclin D1 and CDK6–cyclin D3 kinase activity with nanomolar IC50s in biochemical assays. To comprehensively address the selectivity of ribociclib in direct comparison with 2 other clinical CDK4/6 inhibitors, palbociclib and abemaciclib, we made use of the KINOMEscan assay consisting of >450 kinase active site–directed competition-binding assays. We adjusted the test concentrations in the kinase-selectivity panel per the binding constants for CDK4 and CDK6 to account for the higher potency of abemaciclib. Data showed that both ribociclib and palbociclib have high selectivity for CDK4 (CDK6 was not covered in the panel), with very few distinct additional binding events detected. In contrast, abemaciclib is a much more promiscuous kinase inhibitor.

Next, we sought to determine the relative potencies of the 3 inhibitors against CDK4 vs CDK6 in cellular assays. When testing different routinely used readouts of cellular viability, we found that assays that measured metabolic activity (eg, CTG) tended to underestimate the effects of CDK4/6 inhibition; thus, assays that either directly or indirectly assessed cell number were used instead. We first identified cancer cell lines primarily dependent on either CDK4 or CDK6 as judged by combined RNA expression analysis and shRNA or CRISPR-based functional assays. When determining IC50s of the 3 CDK4/6 inhibitors in these cell lines, we found that ribociclib and abemaciclib demonstrated greater activity in CDK4-dependent cells vs CDK6-dependent cells, whereas palbociclib was similarly active in both cell types.

The high degree of CDK4 selectivity of ribociclib suggests that off-target kinase inhibition is an unlikely complication in patients. Moreover, the apparent preference for CDK4 over CDK6 could be an advantage in certain cancer types that are primarily dependent on CDK4.

#2347

MicroRNA mediated CDK4/6 inhibitor resistance via extracellular signaling.

Liam Cornell, Geoffrey I. Shapiro. _Dana Farber Cancer Institute, Boston, MA_.

Multiple potent and highly selective inhibitors of the cell cycle kinases - CDK4 and CDK6 are in development. One such inhibitor, palbociclib, was recently approved for use in combination with letrozole for the treatment of estrogen receptor positive (ER+) breast cancer. Cyclin D-dependent kinase activity is a driving factor for ER+ breast carcinogenesis, irrespective of CCND1 amplification, making CDK4/6 inhibition a promising approach for this breast cancer subset. However, as with all cancer treatments, resistance will be a major issue limiting the efficacy of this approach. To date, mechanisms of palbociclib resistance have not been extensively investigated.

Through interrogation of the generated CDK4/6 inhibition resistant cells we discovered overexpression of CDK6, specifically, mediates resistance. CDK6 depletion reversed resistance and overexpression caused resistance, whereas the same was not true when CDK4 or D cyclin protein levels were manipulated. Interestingly, when generating and analyzing resistant cell populations, we observed a "bystander effect", by which resistance was transmissible between cells. The "resistant bystander" cells display all characteristics of the drug exposure induced resistant cells, however became resistant in just 48 hours as oppose to 14 weeks. Analysis of conditioned medium revealed that the transmission of resistance is dependent on exosomes, but not protein or DNA. We identified a specific miRNA, present in the exosomes of resistant cells, by microarray which caused resistance when excreted. Additionally, overexpression and inhibition of the miRNA confirmed that it is responsible for causing resistance. miRNA overexpressing cells exhibited the same phenotype as drug induced resistant cells, and furthermore, could cause resistance in neighboring cell populations by exosome mediated signaling. Using Biotin labelled miRNA-mRNA pulldown followed by RNA-seq, we identified the TGFβ pathway as the miRNA target. Downregulation of the TGFβ caused a decrease in the CDK inhibitors, p15 and p21, resulting in an increased CDK6 protein level and palbociclib resistance. We subsequently confirmed these data in patient samples by comparing before treatment and post relapse biopsies.

These findings highlight a novel mechanism of conferred drug resistance as well as new insights into acquired CDK4/6 inhibitor resistance.

#2348

Targeting the p27kip1/cdk4/cdk2/Rb axis in breast cancer using a peptidomimetic of Brk's SH3 domain.

Stacy W. Blain,1 Jason Quinones,1 Priyank Patel,1 Vladislav Tsiperson,1 Susan Gottesman,1 Jonathan Somma,1 Yun Wu2. 1 _State University of New York, Downstate Medical Center, Brooklyn, NY;_ 2 _State University of New York, University of Buffalo, Buffalo, NY_.

Purpose: Cyclin D-cdk4 (DK4) has been a highly sought after therapeutic target because it drives cancer proliferation in a majority of human tumors. We have explored the clinical utility of a recently discovered mechanism of cell cycle control exerted on DK4 by p27Kip1 and its activator, the Breast tumor Related Kinase (Brk), in predicting responsiveness to therapy and as a new target for treatment. Although known as a DK4 assembly factor and cdk2 inhibitor, p27 also acts as a DK4 ON/OFF "switch." Tyrosine (Y) phosphorylation of p27 (pY) by Brk gatekeeps both ATP binding and CAK phosphorylation of cdk4's T loop, essential for DK4 activation. This function is restricted to cdk4: p27's association with cdk2, whether Y phosphorylated or not, appears to be inhibitory. However, in vivo Y phosphorylated p27 is a target for cdk2-dependent ubiquitin-mediated degradation, reducing p27's association with cdk2, indirectly activating this complex. We showed that blocking p27 pY inactivates cdk4 directly AND cdk2 indirectly, and thus represents a novel way to block cancer cell proliferation. pY also serves as a predictive biomarker of cdk4 activity and tumor response.

Methods: We used a small peptide, ALT, which contains a portion of Brk's SH3 domain. ALT binds to p27, blocks Brk's association and ability to phosphorylate p27, inhibiting cdk4 and increasing p27's ability to inhibit cdk2. We engineered a lipid-based nanoparticle delivery vehicle (NP-ALT), permitting us to test ALT as a first generation therapeutic in breast cancer cell lines that were both responsive and non-responsive to cdk4i therapy. ALT was also used with Palbociclib to determine if combination therapy reduced drug resistance. We developed a dual IHC assay for p27 and pY, which we used to analyze paraffin-embedded, archival human tumor samples, to determine whether we could pinpoint patients who would have responded to cdk4 inhibition therapy.

Results: NP-ALT blocks pY, cdk4 and cdk2 activity, and proliferation in both Palbociclib sensitive and resistant cell lines. As a dual therapy, ALT treatment synergized with Palbociclib to arrest cells for >30 days, increased senescence, and in animal models caused tumor regression instead of just slowing tumor growth as seen with Palbociclib alone. Analysis of human cancer, obtained from archival sources, demonstrated that pY is never detected in quiescent benign mammary tissue, but is detected in about half of the advanced ER/PR+/Her2- tumors analyzed, and using explant culture techniques, we were able to stratify pY with Palbociclib response.

Conclusion: Use of an Brk SH3 based peptide (NP-ALT) has proven effective in blocking p27 pY, inhibiting both cdk2 and cdk4, inducing senescence and increased durability. pY levels correlate with Palbociclib sensitivity in low, moderate and non-responders, suggesting that this may be a biomarker highlighting responsiveness to cdk4i therapy.

#2349

Sensitivity and resistance to cell cycle and IGF-1R inhibitors in rhabdomyosarcoma.

Justin Montoya, David W. Lee, Eiman Aleem. _Institute of Molecular Medicine at Phoenix Children's Hospital, Phoenix, AZ_.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and young adults. The five-year survival rate for RMS has hardly improved over the last three decades despite intensive and toxic chemotherapy, radiotherapy with surgery. Therefore, novel treatment approaches are required to change these outcomes. RMS has two major subtypes, embryonal RMS (ERMS) and alveolar RMS (ARMS). ARMS, the more aggressive subtype, is characterized by translocations that fuse two transcription factor-encoding genes; creating novel PAX3/PAX7-FOXO1 fusion proteins. The PAX3-FOXO1 is highly expressed in the G2 phase of the cell cycle, allowing the cell to divide following a sustained checkpoint arrest despite DNA damage induced by chemotherapy, suggesting that PAX3-FOXO1 may enhance the survival of tumor cells in response to chemotherapy. Many cell cycle regulators are altered in RMS, including CDK2, CDK4 and p53. Furthermore, RMS cells are highly dependent on the insulin-like growth factor -1 receptor (IGF-1R) signaling, however, IGF-1R targeting was not successful in the clinic. Therefore, targeting key cell cycle regulators individually or in combination with IGF-1R inhibition may expand the available therapeutic options for RMS.

Purpose: The goal of the present study was to investigate the cytotoxicity of 15 small molecule inhibitors targeting the IGF-1R and cell cycle regulators in RMS cell lines and to determine potential mechanisms of drug sensitivity or resistance.

Methods and Results: Seven RMS cell lines including ERMS and ARMS were studied. The IC50 values were determined for the following targeting compounds: linsitinib, BMS-754807 and picropodophyllin (PPP) (IGF-1R), riobociclib and palbociclib (CDK4/6), dinaciclib and flavopiridol (pan CDK inhibitors), BS-181HCL (CDK7), MK-1775 (WEE-1), MK-8776 (CHK1), alisertib (AURKA) and volasertib (PLK1). The most potent compounds with IC50< 10 nM were dinaciclib and volasertib. The ARMS cell lines were resistant to alisertib in comparison to the ERMS cell lines. Most cell lines were sensitive to flavopiridol, MK-1775, BMS-754807 and PPP with IC50< 100 nM, and relatively resistant to BS-181 HCL, linsitinib, R0-3306 and MK-8776 with IC50 (1-70 μM). Palbociclib and BMS-754807 showed a synergistic effect in some RMS cell lines. Ongoing studies are focusing on determining the mechanisms of interaction of these two compounds through studying cell cycle, apoptosis, and mRNA and protein expression of key regulators in the IGF-1R and RB pathways.

Conclusion: These data demonstrate that dinaciclib, volasertib, flavopiridol, MK-1775, BMS-754807 and PPP are highly cytotoxic in RMS cell lines. The CDK4/6 inhibitor palbociclib may sensitize selected RMS cell lines to IGF-1R inhibitors. Targeting selected cell cycle regulators individually, or in combination with IGF-1R inhibitors may thus provide an efficacious treatment approach to be further validated in RMS patients with poor outcome.

#2350

In vivo E2F reporting on efficacious dosing schedules of MEK plus CDK4/6 inhibition in melanoma.

Jessica Teh,1 Neda Nikbakht,1 Timothy Purwin,1 Inna Chervoneva,1 Prem Patel,1 Michael Davies,2 Andrew Aplin1. 1 _Thomas Jefferson University, Philadelphia, PA;_ 2 _MD Anderson University of Texas Houston, Houston, TX_.

Pharmacological targeting of cyclin dependent kinases 4 and 6 (CDK4/6) could represent a viable therapeutic option in combination with BRAF and/or MEK inhibitors in different genetic subsets of melanoma. Indeed, continuous and concurrent dosing of MEK inhibitor (MEKi) plus a CDK4/6 inhibitor (CDK4/6i) leads to melanoma regressions in in vivo models and delays the onset of MEKi resistance. Current scheduling in breast cancer patients for the CDK4/6i, palbociclib, is intermittent due to adverse effects resulting in neutropenia. It is unclear what the most efficacious schedules are of palbociclib plus a MEKi. Utilizing an E2F reporter system, we sought to analyze the efficacy of different CDK4/6i plus MEKi schedules in a quantitative and temporal manner. Intermittent dosing (3 weeks on/1 week off) of both CDK4/6i and MEKi combination therapy resulted in tolerant tumors and rapid reactivation of E2F activity during drug holiday. Continuous MEKi with intermittent CDK4/6i led to more complete responses as compared to continuous CDK4/6i with intermittent MEKi. Weight loss of mice was also evident in the continuous CDK4/6i plus intermittent MEKi arm suggesting adverse events related to continuous CDK inhibition. Importantly, functional proteomic analysis revealed distinct mechanisms of acquired resistance/drug tolerance that arose from the three scheduling arms. Here, we report that upregulation of S6 phosphorylation is associated with acquired resistance to MEK plus CDK4/6 inhibition and can be overcome with an mTOR inhibitor. Taken together, in vivo reporting allows for quantitative measurement of pathway activity associated with inhibitor resistance and can be utilized to optimize combination schedules to improve the therapeutic index in patients.

#2351

The novel potential of palbociclib (CDK4/6 inhibitor) in the treatment of triple-negative breast cancer.

Yuka Asano, Shinichiro Kashiwagi, Wataru Goto, Katsuyuki Takahashi, Tsutomu Takashima, Satoru Noda, Naoyoshi Onoda, Shuhei Tomita, Kosei Hirakawa, Masaichi Ohira. _Osaka City University Graduate School of Medicine, Osaka, Japan_.

Background: The effectiveness of palbociclib (CDK4/6 inhibitor) for estrogen receptor positive breast cancer has been demonstrated by large-scale clinical studies, with the drug garnering attention as a key drug for breast cancer subtypes with endocrine sensitivity in the future. According to PALOMA-3 trial, palbociclib has been demonstrated to contribute to the extension of progression-free survival in patients with advanced hormone receptor-positive and HER2-negative metastatic breast cancer after endocrine therapy. On the other hand, in the case of triple-negative breast cancer (TNBC), luminal AR (LAR) related to androgen signaling is believed to have endocrine activity. Previous clinical data revealed that palbociclib shows high sensitivity in luminal breast cancer cell lines with endocrine activity, with effectiveness also expected in LAR. In this study, we created TNBC cell lines that forcibly express AR and examined the effectiveness of palbociclib for TNBC.

Materials and Methods: MCF-7 and T-47D were used as luminal breast cancer cell lines, while MDA-MB-231 and BT-549 were used as TNBC cell lines. In addition, we created TNBC cell lines that forcibly express AR, called AR-MDA-MB-231, by the transfection of pEGFP-C1-AR Plasmid Vector using Lipofectamine® 3000 Reagent. We confirmed the expression of AR by qRT-PCR as well as Western blotting and examined the impact of palbociclib on proliferation as well as apoptosis of breast cancer cell lines.

Results: AR was found to have been expressed only in luminal breast cancer cell lines but not TNBC cell lines. It was confirmed that AR was expressed in AR-MDA-MB-231 which are stable cell lines with the properties of LAR. In a CCK assay, palbociclib showed high sensitivity in AR-MDA-MB-231 as in luminal breast cancer cell lines. Furthermore, in an apoptosis assay using FACS and cell cycle assay, apoptosis was induced in AR-MDA-MB-231 and cell cycle arrest at the G1S check point was confirmed.

Conclusion: palbociclib (CDK4/6 inhibitor) showed effectiveness for TNBC cell lines that compulsively express AR, suggesting it may be one treatment option for TNBC in the future.

#2352

Effect of inhibition of cell cycle versus transcription cyclin-dependent kinases (CDKs) in ovarian cancer cells.

Rosaria Chilà, Nicolò Panini, Eugenio Erba, Giovanna Damia, Massimo Broggini. _IRCCS - Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy_.

Background. Cell cycle is regulated by cyclin-dependent kinases (CDKs) activity, whose deregulation can lead to uncontrolled proliferation and cancer. Other CDKs are engaged in the regulation of transcription and post-transcriptional mRNA processing through the phosphorylation of the C-terminal domain of RNA polymerase II, such as CDK9 and CDK12. Inhibitors of cell cycle CDKs have been developed as anticancer agents and some of them are under clinical validation (e.g. palbociclib). While recent data would suggest that inhibition of CDK9 is feasible and has antitumor effect, the data on the therapeutic role of CDK12 inhibition are very scanty.

Methods. Ovarian cancer cell lines were maintained in RPMI medium supplemented with 5% glutamine and 10% FBS. Cells were treated with different drug concentrations and after 72 hours cell survival was evaluated by MTS assay (Promega). IC50 values were calculated by interpolation method. Cell cycle analysis and apoptosis were performed with standard flow cytometric methods. A2780 and SKOV3 ovarian cancer cell lines knocked out for CDK12 were generated with CRISPR/CAS9 genome editing tool.

Results. The cytotoxicity of palbociclib (a CDK4/6 inhibitor) and LDC000067 (a CDK9 inhibitor) was tested in a panel of ovarian cancer cell lines (A2780, SKOV3, OVCAR3, OVCAR5, OVCAR8, OVCA432, OVCA433, IGROV1, EFO27). Sensitivity of cells was similar for palbociclib and LDC000067, ranging from 10 to 33 and from 8 to 60 μM, respectively. A preferential G1 block was observed with palbociclib, while LDC000067 caused a S-G2 block. A higher induction of apoptosis was observed after LDC000067 than after palbociclib treatment in both A2780 and SKOV3. The palbociclib-induced G1 block was associated with decreased Rb phosphorylation, while no modulation of the Ser2 in the carboxyterminal domain of RNA polymerase II was observed after LDC000067 treatment. We generated CDK12 knocked out cells transfecting CRISPR/CAS9 engineered plasmid in both A2780 and SKOV3 ovarian cancer lines. The biological and pharmacological characterization of these clones is under study.

Conclusions. Palbociclib and LDC000067 showed a dose dependent cytotoxic effect in the panel of ovarian cancer cell lines tested and were active in the μM range. Preliminary data of treatment induced cell cycle perturbation and apoptosis suggest that the two drugs behave in a different manner and have distinct molecular effects on cells.

#2353

**CDKI-15, a novel and highly selective CDK4/6 inhibitor: discovery,** in vitro **and** in vivo **anticancer efficacy.**

Solomon Tadesse, Laychiluh Bantie, Khamis Tomusange, Saiful Islam, Muhammed H. Rahaman, Benjamin Noll, Frankie Lam, Mingfeng Yu, Shudong Wang. _University of South Australia, Adelaide, Australia_.

Cyclin D dependent kinases CDK4 & CDK6 are crucial regulators of the G1 to S phase transition of the cell cycle. The facts that myriad cancer types show aberrance in INK4-CDK4/6-cyclin D-Rb-E2F pathway, & the rapidly emerging positive clinical data of pharmacological inhibitors have validated CDK4/6 as anticancer drug targets. As the first commercialized CDK inhibitor, palbociclib in combination with letrozole or fulvestrant has received regulatory approval for the treatment of breast cancer. This represents an important scientific advance in the field. However, the limited structural diversity & undesired side effects due to broader kinase interactions of existing inhibitors mean that the hunt for new & highly selective CDK4/6 inhibitor drug candidates continues. Using our advanced medicinal chemistry, targeted biochemical & cell-based assays, & animal pharmacology, we synthesized & evaluated a novel series of inhibitors. Many compounds were highly potent & selective against CDK4/6, & exhibited low nanomolar potency against a range of human cancer cell lines. Notably, inhibition of CDK4D1 by compound CDKI-15 (Ki = 4 nM) was over 3 orders of magnitude greater than CDK1B, CDK2A, CDK7H & CDK9T1. Interrogation of a panel of 369 protein kinases revealed CDKI-15 to be highly selective for CDK4/6 with only 3 other kinases inhibited potently. CDKI-15 reduced the level of Rb phosphorylation & induced G1 cell cycle arrest, confirming cellular inhibition of CDK4/6 in cancer cells. Moreover, CDKI-15 possesses superior pharmacokinetic profile with oral bioavailability of 100% in mice. Treatment of nude BALB/c mice bearing human MV4-11 acute myeloid leukemia cells with CDKI-15 at daily dose of 100 mg/kg by oral administration resulted in a robust inhibition of tumor growth compared to vehicle treated animals (T/C = 30%, p < 0.00001). Strikingly, CDKI-15 caused a complete & sustained tumor regression in one-third of the animals. No detectable toxicity was observed in the animals during & post treatment. Taken together, our data provide a rationale for CDKI-15 to be developed towards clinic for cancer therapy.

#2354

Identification of CDK1 as an aspirin and salicylic acid binding protein: a potential role in chemoprevention.

Jayarama B. Gunaje,1 D.Ramesh Kumar,2 Siddharth Kesharwani,1 Eduardo Callegari,3 Hemachand Tummala,1 Rakesh Dachineni1. 1 _South Dakota State Univ. College of Pharmacy, Brookings, SD;_ 2 _University of Kentucky, Lexington, KY;_ 3 _University of South Dakota, Vermillion, SD_.

Data emerging from the past 10 years have consolidated the rationale for investigating the use of aspirin for chemoprevention; however, the mechanisms leading to its anti-cancer effects are still being elucidated. We hypothesized that aspirin's ability to exert chemopreventive effects may involve altering the levels and activity of cell cycle regulatory proteins. In the present study, using HT-29 colon cancer cells and other cancer cells, we demonstrated that both aspirin and its primary metabolite, salicylic acid, downregulated the protein and mRNA levels of cyclin B1 and cyclin dependent kinase-1 (CDK1). Lactacystin, a 26S proteosomal inhibitor, prevented aspirin and salicylic acid mediated degradation of cyclin B1, but not CDK1. Decrease in protein levels of cyclin B1/CDK1 was correlated with a corresponding decrease in CDK1 kinase activity. Molecular docking studies showed that aspirin and salicylic acid independently can dock on CDK1 through interactions with Leucine 83. Incubation of recombinant CDK1 with aspirin resulted in acetylation at lysine residues, this was also observed in cell culture experiments. Pre-incubation of CDK1 with salicylic acid dose dependently prevented aspirin's ability to acetylate CDK1 in purified preparations confirming the data obtained from molecular docking studies. Our results show that CDK1 is a salicylic acid binding protein (SABP) and the chemopreventive actions of aspirin may involve modulation of levels and activity of cyclin B1 and CDK1.

#2355

Palbociclib enhances the antitumor activity of taxanes by abrogating cell cycle checkpoints and alleviating hypoxia in squamous cell lung cancer.

Joan Cao,1 Zhou Zhu,1 Hui Wang,1 Tim Nichols,1 Edward Rosfjord,2 Christine Hopf,2 Erik Upeslacis,2 Paul Rejto,1 Scott Weinrich,1 Todd Vanarsdale,1 James Hardwick,1 Ping Wei1. 1 _Pfizer, Inc., San Diego, CA;_ 2 _Pfizer, Inc., Pearl River, NY_.

Lung cancer remains one of the leading causes of cancer-related mortality. Squamous cell lung cancer (SqCLC) is the second most common subtype of non-small cell lung cancer (NSCLC) and is responsible for ~100,000 deaths in the US and EU. Most SqCLC patients receive chemotherapy as 1st line treatments and have a high un-met medical need for new therapies. Therapeutic approaches that enhance the efficacy of chemotherapy would therefore improve clinical outcomes for this patient population. CDK inhibitors comprise a class of drugs that target the dysregulated cell cycle in malignant cells. Treatment of tumor cells with the CDK4/6 inhibitor palbociclib inhibits tumor growth by decreasing retinoblastoma (RB) protein phosphorylation and inducing cell cycle arrest at the G1/S phase transition. Based on promising clinical trial results, palbociclib in combination with letrozole was granted accelerated approval by the US FDA for the treatment of postmenopausal women with ER-positive, HER2-negative advanced breast cancer. Like hormone receptor positive breast cancer patients, the vast majority of SqCLC patients harbor wild type RB and thus may also benefit from palbociclib treatment. Previously, we reported robust cytotoxicity and antitumor effects of palbociclib plus taxanes, including nanoparticle albumin-bound paclitaxel (Nab-PTX) or docetaxel (DTX), in several preclinical models of SqCLC. In the present study, we extended our efficacy studies of this combination to additional RB+ SqCLC models with diverse molecular genetic backgrounds. In search of mechanisms of action underlying the observed combinatorial effects, we identified several novel mechanisms, including cell cycle checkpoint abrogation as well as reduction of hypoxia-inducible factor 1 alpha (HIF-1α). Decrease in HIF-1α protein led to strong modulation of downstream genes involved in angiogenesis, resulting in reduced blood vessel size in tumor vasculature. Our results suggest that palbociclib enhances the antitumor activity of taxanes by abrogating cell cycle checkpoints and alleviating hypoxia in SqCLC.

#2356

**Functional characterization of** CDKN1A **loss in bladder cancer and effects on cisplatin sensitivity.**

Philip H. Abbosh, Rahmat K. Sikder, Wafik S. El-Deiry. _Fox Chase Cancer Ctr., Philadelphia, PA_.

Introduction

Muscle-invasive bladder cancer (MIBC) is most optimally treated with cisplatin-containing combination chemotherapy, and if clinically localized, bladder removal. With recent discoveries delineating aberrant driver genes involved in MIBC, it is now possible to personalize treatment approaches. Development of a therapeutic rationale for personalized therapies which is based on mutational landscape or other characteristic of each tumor may prospectively identify patients prone to chemoresponse, thus maximizing therapeutic index. Inactivating mutations in CDKN1A, the gene encoding cyclin dependent kinase inhibitor p21, occur in about 14% of MIBCs, with a majority of these truncating the peptide. We hypothesized that after DNA-damaging events, cells deficient in p21 would be unable to halt the cell cycle to repair the damage, and subsequently proceed down an apoptotic pathway, potentially serving as a therapeutic vulnerability.

Methods

RT4 and SW780 cell lines were chosen for study based on chemoresistance to cisplatin and WT status of CDKN1A and TP53. Cell-cycle arrest and apoptotic pathways were evaluated with Western blot after treatment with IC50 doses. CRISPR sgRNAs were generated to introduce frameshifts 5' to the regions encoding the cyclin binding domain (CDI), within the CDI, and 5' to the PCNA binding domains. Knock-outs were confirmed by immunoblot. MTT was used to assess cipslatin response.

Results

p53 increased in both cell lines 24 hours after a 3h pulse of cisplatin exposure. However, p53 peaked at 24 hours and then diminished in RT4 but continued to rise in SW780. p21 was basally expressed in both cell lines, decreased at 24 hours, then increasing at 72 hours. In RT4, peak Rb phosphorylation (pRb) occurred at 24 hours, indicating cell cycle arrest, and gradually decreased at 72 hours. In SW780 pRb remained unchanged during the course of the treatment. In RT4, PARP cleavage was observed at 24 hours and increased at 48 hours indicating apoptosis. RT4 cells harbored extensive DNA damage as indicated by persistent phosphorylated γH2AX. In contrast a smaller fraction of SW780 cells underwent PARP cleavage at 48 and 72 hours and there was less phosphorylated γH2AX. Single cell CDKN1A knockout clones were generated and confirmed by immunoblotting. sgRNAs targeting amino acids 12 and 59 completely abolished p21 detection, and an sgRNA targeting amino acid 109 generated a truncated peptide, confirming p21's apparent stability. CDKN1A disruption at any of the three sites did not reverse chemorensitance to cisplatin in either cell line.

Conclusion

Cisplatin resistant cell lines use a combination of arrest and repair to survive an apoptotic insult. Although p21 loss did not reverse cisplatin resistance, it may

impact sensitivity to other agents. Other mechanisms of chemo- and radio-sensitization are being explored.

#2357

CDK4/6 inhibition in early stage triple negative breast cancer.

William B. Kietzman, Virginie Ory, Fransisco Saenz, Ghada Sharif, Anton Wellstein, Anna T. Riegel. _Georgetown University, Washington, DC_.

Palbociclib (PD), a CDK4/6 inhibitor, has recently been approved for use in advanced ER positive breast cancer, demonstrating significant benefit in combination with endocrine therapies. Ductal carcinoma in situ (DCIS) is an inobligate precursor to invasive ductal carcinoma with anywhere from 14-53% of cases progressing. Progression is unpredictable and driven by poorly understood mechanisms. In this study, we assess the efficacy of PD in the context of this early stage disease using a triple negative DCIS model(MCF10DCIS), not only in inhibiting cell cycle but also in hindering the progression from in situ to invasive lesion. We demonstrate similar efficacy of PD in 2D on MCF10DCIS and immortal mammary epithelial cells (MCF10As) in preventing proliferation, inhibiting phosphorylation of RB and stopping cell cycle progression. Yet in 3D, MCF10DCIS cells form smaller, more organized spheres in the presence of drug while MCF10As appear unaffected, highlighting potential disease selectivity in vivo. Interestingly, when a cDNA array is performed on MCF10DCIS cells grown in 3D +/- PD, the most regulated genes are not well characterized cell cycle genes as one might predict based on previously published data and 2D response to treatment, suggesting alternative CDK4/6 targets may be responsible for the phenotype observed in 3D. We also demonstrate in a nude mouse xenograft model of MCF10DCIS that PD is able to significantly reduce overall tumor burden while simultaneously delaying the progression of lesions to invasive disease. Though the growth of PD treated MCF10DCIS lesions is delayed we demonstrate that proliferation is unaffected in vivo by the presence of the drug, based on equivalent Ki67 signal within the lesions. This reiterates the possibility that in 3D and in vivo contexts PD targets mechanisms outside of cell cycle regulation in order to slow tumor growth and delay invasive transition. We believe that investigation into alternative targets of CDK4/6 altered by PD in 3D and in vivo may provide insights on important mechanisms of DCIS progression in DCIS models and human patients.

#2358

Impact of MDM2 inhibition on cell cycle regulation through Aurora Kinase B - CDK1 axis in prostate cancer cells.

Thanigaivelan Kanagasabai,1 Khalid Alhazzani,2 Thiagarajan Venkatesan,1 Sivanesan Dhandayuthapani,1 Ali Alaseem,2 Appu Rathinavelu1. 1 _Rumbaugh Goodwin Institute for Cancer Research, Nova Southeastern University, Fort Lauderdale, FL;_ 2 _College of Pharmacy, Nova Southeastern University, Fort Lauderdale, FL_.

Cyclin-dependent kinases (CDKs) are critical regulators of cell cycle progression and therefore overexpression of CDKs contributes to the proliferation of cancer cells. A variety of genetic and epigenetic events cause over activity of the CDKs in human cancers, and their inhibition can lead to cell cycle arrest and apoptosis. In malignant cells, both elevated expression of CDKs and their modulators including cyclins, and loss of expression of CDK inhibitors result in deregulated CDK activity, providing a selective growth advantage for cancer progression. In this respect, the multifunctional mouse double minute 2 homolog (MDM2) oncoprotein has been gaining a significant amount of attention towards better understanding the cell cycle regulatory mechanisms. It is well established that MDM2 gene amplification can occur in diverse human malignancies including prostate cancer. Also, MDM2 oncoprotein has been shown to exert both p53-dependent and p53-independent roles in oncogenesis. It has been well established in several laboratories, including ours, that overexpression of MDM2, can eventually lead to the inactivation of cell cycle control and loss of apoptotic ability in many tumors. However, the mechanisms underlying the regulation of CDK1 by MDM2 in cancer cells have not been fully identified. Therefore, the main objective of our study was to understand the impact of MDM2 overexpression on CDK1 that regulates cell cycle progression and apoptosis. Our preliminary data from human cell cycle PCR array experiments revealed the expression profile of genes that are involved in different phases of cell cycle regulation in LNCaP-MST cells with and without nutlin-3 treatment. Our study clearly demonstrated a significant increase in the expression level of Aurora Kinase B (AURKB), CDC25C and CDK1 in MDM2 transfected LNCaP-MST cells as compared with non-transfected LNCaP cells. However, after treating the cells with 20 µM of MDM2 specific inhibitor nutlin-3, for 24 h, the expression levels of the above mentioned proteins were significantly altered when compared to untreated controls. In addition, inhibition of MDM2 with nutlin-3 leads to increased expression of pro-apoptotic proteins p53, p21, and Bax. Our results offer significant evidence towards the effectiveness of MDM2 inhibition in causing cell cycle arrest via blocking the transmission of signals through AURKB-CDK1 axis and inducing apoptosis in cancer cells. It is clearly evident from our data that MDM2 overexpression probably is the primary cause for CDK1 up-regulation in the LNCaP-MST cells, which might have occurred possibly through activation of AURKB. However, further studies in this direction should shed more light on the intracellular mechanisms involved in the regulation of CDK1 in MDM2 positive cancers. (This project was supported by The Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida).

### Cell Growth Signaling Pathways 5

#2359

Inhibition of the ATR kinase enhances therapeutic efficacy of cisplatin in ATM low uterine carcinosarcoma cells.

Emily R. Penick,1 Paulette Mhawech-Fauceglia,2 Nicholas Bateman,1 Kelly Conrads,1 Tracy Litzi,1 Chunqiao Tian,1 Chad A. Hamilton,1 Kathleen Darcy,1 George Maxwell,3 Thomas Conrads3. 1 _Gynecologic Cancer Center of Excellence, Murtha Cancer Center, Walter Reed National Military Medical Center, Department of Obstetrics and Gynecology, Uniformed Services University of the Health Sciences, Bethesda, MD;_ 2 _University of Southern California, Los Angeles, CA;_ 3 _Inova Schar Cancer Institute, Inova Center for Personalized Health, Annandale, VA_.

Objective: Uterine carcinosarcoma (UCS) is an aggressive malignancy, making up less than 5% of uterine cancers. Pharmacologic inhibition of ATR kinase synergistically enhances the ability of cisplatin to kill carcinoma cells of many solid tumor types and is further enhanced in ATM low carcinoma cells. It is unknown whether ATR inhibition (ATRi) offers a therapeutic opportunity in UCS. The goal of this study was to evaluate ATM expression in UCS tumors and determine the response of an ATM low UCS model to ATRi + cisplatin combination.

Methods: ATM expression was evaluated in full tissue sections from 74 UCS tumors by immunohistochemistry (IHC) with an ATM specific monoclonal antibody (clone Y170, Abcam), and was categorized as negative vs any positive staining in the carcinomatous (C) vs sarcomatous (S) components. In vitro models of UCS: SK-UT-1, SK-UT-1B, KLE, and RL95-2 cell lines were treated with an ATRi (AZD6738), cisplatin, and ATRi + cisplatin. ATM low models were generated in RL95-2 cells using short hairpin RNA (shRNA) lentivirus targeting ATM and a non-target control. Cells were subjected to cisplatin and ATRi dose response analyses.

Results: Negative IHC expression of ATM protein was observed in 24% of the C vs 31% of the S components of the 74 UCS cases (Table 1). ATRi increased the sensitivity of uterine carcinosarcoma cell line models to cisplatin. Western blot confirmed a 57% knockdown in ATM in RL95-2 cells infected with shRNA for ATM and treatment with ATRi +cisplatin therapy showed ~20% increase in cell death in ATM low vs control cells lines, p=0.02.

Conclusions: IHC analyses of UCS tumors showed an average of ~27% of tumors have loss of ATM in C and S components. Preliminary evidence shows that ATRi increases the sensitivity of UCS cell models to cisplatin therapy, which is further increased in ATM low uterine carcinoma cells. These findings suggest a novel therapeutic opportunity for ATRi + cisplatin therapy in UCS patients with low ATM expressing tumors.

Immunohistochemistry-based quantification of ATM expression in uterine carcinosarcoma tumors

---

ATM | |

Uterine Carcinosarcoma

Intensity | Carcinomatous Component | Sarcomatous component

0-1 | 31 (41.9%) | 33 (44.6%)

|

0 | 18 (24.3%) | 23 (31.1%)

|

1 | 13 (17.6%) | 10 (13.5%)

2-3 | 43 (58.1%) | 41 (55.4%)

|

2 | 11 (14.9%) | 10 (13.5%)

|

3 | 32 (43.2%) | 31 (41.9%)

TOTAL | 74 | 74

#2360

Regulation of the MAPK phosphatase MKP-1 by the E3 ligase GP78 in cancer cells.

Dhonghyo Kho, Gen Sheng Wu. _Wayne State Univ. School of Medicine, Detroit, MI_.

Mitogen-activated protein kinase (MAPK) phosphatase 1 (DUSP1/MKP-1) is a member of the threonine-tyrosine dual-specificity phosphatase family that is endogenous inhibitor for extracellular signal-regulated protein kinases (ERKs), p38 MAPKs, and JNKs signaling. MKP-1 plays a key role in determining the magnitude and duration of MAPK activation in response to extracellular stimuli. In human cancers, abnormal expression of DUSP1 is associated with tumor development, chemoresistance and prognosis. However, the mechanism by which MKP-1 is regulated is not fully understood. Here, we show that GP78, an E3 ligase, plays an important role in regulation of MKP-1 protein levels. Specifically, we found that MKP-1 interacts with GP78 both in vitro and in vivo. Their interaction occurs between the N-terminal, Rhodanese domain of MKP-1 and C-terminal E3 ligase domain of GP78. We also found that K230, 280, and 289 residues on MKP-1 are responsible for its ubiquitination. Importantly, this interaction causes MKP-1 ubiquitination and subsequent degradation. Furthermore, we showed by overexpression and knockdown studies that GP78 downregulates the level of MKP-1 protein in cancer cells. Therefore, we identify a novel mechanism by which GP78 controls MKP-1 protein levels and suggest that this regulatory mechanism may play an important in cancer.

#2361

Pim kinase inhibition alters mRNA splicing in AML cell lines.

Tejashree A. Joglekar, Xiang Li, Charles J. Bieberich. _University of Maryland, Baltimore County, Baltimore, MD_.

Oncogenic kinase activity is a common feature of nearly all cancers and kinases are major targets for therapeutic intervention. Pim kinases are deregulated in hematopoietic cancers including Acute Myeloid Leukemia (AML), as well as prostate cancer. While cancer cells can become dependent on Pim activity to sustain proliferation, in normal adult tissues, Pim kinase activity appears to be dispensable. These features make Pim kinases an attractive target for cancer therapy, however, their physiological roles have not been fully characterized. Using the reverse in-gel kinase assay (RIKA), we identified a battery of novel Pim substrates that are involved in mRNA splicing regulation. We hypothesized that Pim family kinases regulate mRNA splicing through phosphorylation of splicing factors. Microarray analysis revealed more than 10,000 splicing changes in AML cells treated with the highly selective small molecule Pim kinase inhibitor AZD1208. Using RT-PCR analysis, multiple AZD1208-induced splicing changes were validated. To discern the mechanisms whereby Pim kinases regulate splicing, we determined whether Pim inhibition alters phosphorylation of serine/arginine-rich (SR) proteins. Inhibition of serine arginine protein kinase (SRPK) activity using a small molecular inhibitor (SRPIN340) reduced SR protein phosphorylation, whereas Pim kinase inhibition did not, suggesting that Pim kinases regulate splicing through an SRPK independent pathway. Comparison of splicing changes in AZD1208 and SRPIN340 treated AML cells demonstrated distinct patterns, providing further evidence of SRPK-independent splicing regulation by Pim kinases. Biomarkers of kinase inhibitor efficacy are critical components of clinical trials. These data suggest that changes in mRNA splicing may serve as a biomarker for assessing patient responsiveness to Pim inhibition therapy.

#2362

Mechanistic insights into phosphoinositide 3-kinase α.

Sweta Maheshwari. _Johns Hopkins University School of Medicine, Baltimore, MD_.

Phosphoinositide 3-kinases (PI3Ks) are at the center of mTOR pathway that plays key roles in regulating cell survival, proliferation, protein synthesis and vesicle trafficking. Dysregulation of the mTOR pathway has been implicated in various diseases such as cancer, diabetes, arthritis, inflammation and respiratory illnesses. PI3Ks have dual kinase specificity with a lipid kinase activity that phosphorylates the 3'-hydroxyl of phosphoinositides, and a protein kinase activity including auto-phosphorylation. In this study, we explored the roles of residues of the catalytic domain and regulatory subunit of human PI3Kα in the lipid and protein phosphorylation. We used site-directed mutagenesis and kinetic assays to precisely map the molecular basis of substrate recognition and catalysis in PI3Kα. Kinetic analysis of K776, located in the P-loop of PI3Kα, reveals that a lysine at this position is essential for the recognition of the lipid and ATP substrates. This same residue plays an important role in the auto-phosphorylation of the enzyme. Replacement of histidine residues–H936 and H917 in the activation and catalytic loops respectively with alanine residues results in a dramatic change in the kinetics of PI3Kα. While H936A inactivates the lipid kinase activity without affecting auto-phosphorylation, H917A abolishes both the lipid and protein kinase activities of PI3Kα. On the basis of kinetic and structural analysis, we proposed possible mechanistic roles of these critical residues in catalysis.

#2363

**Targeting Src-family kinases to combat acquired inhibitor resistance in FLT3-ITD** + **AML.**

Ravi K. Patel, Mark Weir, Sabine Hellwig, Heather Dorman, Thomas E. Smithgall. _University of Pittsburgh, Pittsburgh, PA_.

Activating internal tandem duplication (ITD) mutations in the FMS-like receptor tyrosine kinase 3 (Flt3) are found in approximately 30% of acute myeloid leukemia (AML) patients. Flt3-ITD expression is associated with aggressive, chemotherapy-resistant disease and decreased overall survival. Multiple selective Flt3 kinase inhibitors have been developed as targeted therapy for Flt3-ITD+ AML. However, the clinical utility of these kinase inhibitors has been limited by the rapid recurrence of drug-resistant disease that often results from Flt3 kinase domain mutations that prevent inhibitor action. Recent studies revealed that multiple non-receptor tyrosine kinases are over-expressed in AML, including the myeloid Src-family kinases Hck, Lyn and Fgr, and may cooperate with Flt3 to drive disease. These observations suggest that kinase inhibitors with specificity profiles targeting Flt3 and these AML-associated cytoplasmic kinases may be less prone to acquired resistance. In the present study, we determined the contributions of Hck and Fgr kinase activities to Flt3-ITD\+ AML cell growth using a chemical genetics approach. While RNAi-mediated knockdown of each of these kinases reduces proliferation and increases apoptosis in primary AML cells, the impact of pharmacologic inhibition of their kinase activities on AML cell growth is not known. Because no selective Hck or Fgr inhibitors exist, we developed kinase domain mutants that desensitize each kinase to the broad-spectrum SRC-family kinase inhibitor, A-419259. Examination of a previous crystal structure of Hck with A-419259 bound to the active site suggested a role for the 'gatekeeper' residue in compound binding. Substitution of the gatekeeper threonine of Hck and Fgr with methionine conferred 3-fold resistance to A-419259 in vitro without diminishing kinase activity. When expressed in human TF-1 myeloid cells transformed with Flt3-ITD, these inhibitor-resistant Hck and Fgr mutants conferred resistance to growth inhibition by A-419259 to a similar extent, while expression of the corresponding wild-type kinases was without effect. The sensitivity of the TF-1/Flt3-ITD cell lines to A-419259 correlated with inhibition of mutant Hck and Fgr kinase activity in the cells. Our findings suggest that suppression of AML cell growth by A-419259 is due in part to inhibition of Hck and Fgr, and identify these AML-associated kinases as therapeutic targets. Ongoing studies are addressing the role of Hck and Fgr as inhibitor targets in Flt3-ITD+ AML cell lines and patient samples as well as the propensity of A-419259 and other multi-targeted inhibitors of AML-associated kinases for acquired drug resistance.

#2364

Src-like adaptor protein 2 negatively regulates FLT3 and KIT downstream signaling.

Sausan A. Moharram, Lars Rönnstrand, Julhash U. Kazi. _Lund University, Lund, Sweden_.

Type III receptor tyrosine kinases (RTKs) including FLT3 and KIT play a major role in cell differentiation, proliferation, and survival of hematopoietic stem cells. FLT3-ITD and KIT-D816V mutations are the most common oncogenic mutations in FLT3 and KIT found in hematological cancers. These mutations lead to constitutive activation of proliferative and survival signals. Tyrosine kinase inhibitors (TKIs) in combination with chemotherapy display promising results in a clinical setting, but patients develop resistant disease after short-term treatment,. Hence, proteins that regulate the activity of RTKs can be alternative targets for patients carrying these mutations. Activation of FLT3 and KIT results in phosphorylation on several tyrosine residues that recruit SH2 domain-containing signaling proteins. In this study we identified Src-like adaptor protein 2 (SLAP2) as a potent FLT3 and KIT interacting protein. The interaction requires an intact SH2 domain of SLAP2 as well as phosphorylation of the receptor. Overexpression of SLAP2 in murine proB Ba/F3 cells inhibited oncogenic FLT3-ITD-mediated cell proliferation and colony formation. SLAP2 displayed a similar inhibitory potential in cells expressing KIT-D816V. SLAP2 partially blocked phosphorylation of several FLT3 and KIT downstream signaling proteins such as AKT, ERK and p38. Moreover, SLAP2 expression inhibited FLT3-ITD-mediated STAT5 phosphorylation and KIT-D816V-mediated STAT3 phosphorylation. SLAP2 expression significantly accelerated ubiquitination-mediated degradation of FLT3 and KIT. Collectively, these data suggest that SLAP2 negatively regulates FLT3 and KIT signaling and therefore, modulation of SLAP2 expression levels may become a potential target for anticancer therapy.

#2365

Dual inhibition of the PI3k/Akt and MEK5/ERK5 pathways in tamoxifen resistant breast cancer.

Thomas D. Wright, Mahmud Hasan, Paula Witt-Enderby, Jane Cavanaugh. _Duquesne Univ. School of Pharmacy, Pittsburgh, PA_.

Approximately 70% of breast cancers are considered estrogen receptor alpha (ER) and/or progesterone receptor (PR) positive, and their growth is primarily driven by the hormone estrogen. Initially these cancers are responsive to endocrine therapies such as tamoxifen. However, over time patients become resistant to conventional endocrine therapy and treatment becomes more difficult. Tamoxifen resistance occurs due to several mechanisms: loss of ER expression, gain of function mutations, pharmacological tolerance, alterations of co-activators, and ligand independent activation of ER by various kinase cascades. Aberrations in the Phosphoinositide-3-kinase (PI3K) and Mitogen Activated Protein Kinase (MAPK) pathways have been linked to increased breast cancer proliferation and survival. It has been proposed that these survival characteristics are enhanced through compensatory signaling and crosstalk mechanisms. The crosstalk between PI3K/Akt and MEK1/2/ERK1/2 has been characterized in several systems. However, new evidence suggests that MEK5/ERK5, a member of the MAPK family, is a key component in the proliferation and survival of therapy resistant cancers. MEK5/ERK5 has been shown to promote ER alpha driven transcription in ER+ breast cancers and actin reorganization and metastasis in ER- breast cancers. Furthermore, MEK5/ERK5 promotes hormone independent tumorigenesis in breast cancer. Our lab has previously investigated these pathways in hormone independent breast cancers, TNBC (triple negative breast cancer). Our previous results indicate that combinations of PI3k/Akt and MEK5/ERK5 blockade are promising because they inhibit both the pro-proliferative and pro-metastatic pathways in TNBC. Additionally, PI3k/Akt and MEK5/ERK5 inhibition was shown synergistically reduce viability in a diverse panel of cell lines (TNBC, ER+, and Triple Positive). Based on these initial results we hypothesize that PI3k/Akt and MEK5/ERK5 inhibition decreases viability via a hormone independent mechanism: perhaps by reducing the cytosolic sequestration of Bad. In this study we investigate the utility PI3k/Akt and MEK5/ERK5 inhibition in a MCF-7 TamR cell line. The goal of our study is to elucidate the roles of PI3k/Akt/mTOR and MEK5/ERK5 in endocrine resistant breast cancer and broaden the scope of a dual inhibition strategy.

#2366

Targeting PI3 pathway in ibrutinib resistant diffuse large B-cell lymphoma.

Neeraj Jain,1 Lalit Sehgal,1 Stephen Joseph Shuttleworth,2 Felipe Samaniego1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Karus, Abingdon, United Kingdom_.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL) and approximately 30% of patients with DLBCL develop relapsed/refractory disease that becomes a major cause of mortality and morbidity. The Bruton kinase (BTK) inhibitor ibrutinib successfully blocks B-cell receptor signaling and shows clinical benefit in leukemia and lymphomas, including mantle cell lymphoma (MCL) and DLBCL. Ibrutinib elicits overall desirable response rate with relapsed/refractory MCL and ABC DLBCL. However, in spite of these encouraging results, responses are variable and generally incomplete, acquired resistance is common, and recurrence and persistence of lymphoma is a problem. We undertook a study of factors underlying acquired ibrutinib resistance (IR) in initially ibrutinib-sensitive DLBCL cell lines. In this study, IR DLBCL cell lines were generated by continuous culturing of parental (PT) cell lines in increasing concentrations of ibrutinib. Once established, IR cell lines were removed from ibrutinib, expanded, and cultured under the same conditions as the PT cell lines for further experiments. Of 5 ABC DLBCL cell lines tested, two (OCI-LY3, U2932) were initially resistant to ibrutinib (IC50>10µM).Three (TMD8, OCI-LY10, HBL1) were sensitive (IC50 < 10 nM). In comparison to PT versions of these cell lines, IR cells did not form clumps in suspension cultures, displayed irregular cell morphology, showed elevated colony formation ability in methylcellulose matrix, and had a higher proliferation rate. Western blots and gene expression profile analysis showed increased expression by IR cell lines of inhibitors of apoptosis (IAP) family members, survivin, cIAP2, and oncogenic BCL2 and BCL6. The DNA damage repair pathway was found to be elevated with enhanced expression of CHK1 kinase. IR cell lines had reduced BTK expression without acquiring mutations, and downstream enhanced PI3K-Akt signaling. Analysis of PI3K isoforms revealed up-regulation of PI3Kα and PI3Kβ with decreased expression of PI3Kδ and PTEN (PI3K negative regulator). Given the enhanced PI3K isoform expression with IR, we treated IR cell lines with the PI3Kβ/δ isoform-targeting drug KA2237 and observed reduced metabolic activity (survival) of PT cell lines and further reduction of surviving IR cell lines. In conclusion, this study highlights how changes in a regulator (PTEN) and mediator (p110β) of PI3K/AKT signaling have important roles in the development of ibrutinib resistance in DLBCL. Treatment with KA2237 may provide a better outcome for ibrutinib-resistant DLBCL.

#2367

Identification of ALK alternative transcription initiation in BRAF-negative metastatic melanoma patients.

Shan Zhong,1 Christine Lovly,2 Christine Malboeuf,1 Kristina Brennan,1 Douglas Johnson,2 Kelsie Riemenschneider,2 Catherine Meador,2 Emily White,1 Geoff A. Otto,1 Doron Lipson,1 Philip Stephens,1 Vincent Miller,1 Jeffrey Ross,1 Jie He1. 1 _Foundation Medicine, Inc, Cambridge, MA;_ 2 _Vanderbilt University, Nashville, TN_.

Background:

Genomic alterations in the ALK tyrosine kinase, such as copy number gains, point mutations, and ALK gene fusions, have been described in a broad spectrum of malignancies. Recently a novel mechanism of ALK activation in melanoma was discovered in which ALK transcription was initiated from a de novo alternative transcription initiation (ATI) site in ALK intron 19 (ALK-ATI) [1]. In this study, we used whole transcriptome sequencing (WTS) to interrogate a cohort of clinical melanoma specimens in order to identify those that express ALK-ATI.

Method:

RNA-seq libraries of 33 BRAF-negative clinical metastatic melanoma specimens were prepared using the KAPA Stranded RNA-Seq Kit with RiboErase with 200ng RNA as input and sequenced on Illumina HiSeq 4000 at 2x75bp. The specimens were sequenced to an average of 69.4m total read pairs with 11.3m on-target distinct read pairs. Sequencing reads were mapped to the human genome and transcriptome, gene- and exon-level expressions were quantified. An algorithm to identify the ALK-ATI was developed based on assessing 1) transcription of intron 19 putative ATI region, 2) ALK relative RNA expression and 3) ALK 5' to 3' differential expression. In addition, immunohistochemistry (IHC) staining for ALK (using D5F3 antibody) was done on 10 of the 33 samples with adequate tumor material.

Results:

High coverage (median 30x) of intron 19 ATI region and high ALK relative expression (log ratio > 2) were observed in 4 of the 33 specimens tested, in which the ATI site is consistent with what had been reported in [1]. Among the 4 potential ALK-ATI positive cases, 3 have shown significant 3' to 5' overexpression of ALK. IHC staining were performed on the 4 putative ALK-ATI positive cases and 6 putative negative cases, and has confirmed ALK-ATI calls detected by WTS assay. Two of the ALK IHC positive cases harbor the highest ALK expression, and the other two ALK-ATI likely cases are ALK IHC weak positive. ALK-ATI occurred in cutaneous, mucosal, and unknown primary melanoma, and co-occurred with other driver mutations (NRAS, NF1, KIT). None of the above specimens had ALK rearrangements detected from DNAseq (by FoundationOne) or WTS.

Conclusions:

Our findings confirmed previously reported ALK activation through ALK-ATI in an independent melanoma cohort, and provided direct evidence that such events can be detected using whole transcriptome sequencing and align with IHC results in clinical FFPE tumor specimens. This confirmation together with future functional validation and clinical evidence may provide new therapeutic opportunities for BRAF-negative metastatic melanoma patients.

1. Wiesner, T. et al. Alternative transcription initiation leads to expression of a novel ALK isoform in cancer. Nature 526, 453-457 (2015).

#2368

**Differential role of** Pik3caH1047R **and** Pten-/- **mutations in thyroid cancer development.**

Michela Ranieri, Antonio Di Cristofano. _Albert Einstein College of Medicine, Bronx, NY_.

Anaplastic thyroid cancer (ATC) is one of the most lethal malignancies, with a median survival of less than 6 months from time of diagnosis.

Molecular changes that characterize ATC involve most often p53 loss or inactivation (up to 80%), activation of the PI3K cascade through PTEN loss (12%) and PIK3CA activating mutations (20%) or gene amplification (30-60%), RAS family members activation (6-50%), and BRAF activation (25-50%).

Studies performed in endometrial and breast cancer suggest that although PI3K activating mutations and PTEN loss of function both enhance PI3K signaling, these mutations are not equivalent.

We have tested whether Pik3caH1047R and Pten-/- mutations have the same impact on thyroid cancer development and if these two mutations differ in their ability to cooperate with additional genetic alterations, such as p53 deletion and the KrasG12D activating mutation.

We have generated a series of mouse models and cell lines carrying thyroid-restricted p53-/-, KrasG12D, Pten-/-, and Pik3caH1047R alleles.

Analysis of single and compound mutants has revealed important and striking differences between Pten and PI3K abilities to control the PI3K signaling pathway.

Tumor development analysis in Pten-/- and Pik3caH1047R mice has revealed that Pik3ca activation is less potent in inducing thyroid hyperplasia and subsequent transformation than Pten loss, even though the two mutations appear to have similar ability to induce AKT activation. This differential activity correlates with lower levels of PIP3 found in the thyroids of Pik3caH1047R mice, compared to that of Pten-/- mice.

We also observed that while Pten loss synergizes with Trp53 loss to induce anaplastic thyroid cancer, activation of Pik3ca leads to the development of a smaller and well differentiated tumors.

On the other hand, Pten loss and Pik3ca activation are equivalent in inducing poorly differentiated tumors in the presence of a Kras activating mutation.

However, KrasG12D, Pik3caH1047R cell lines are more sensitive to PI3K, MEK and combined PI3K/MEK inhibition than KrasG12D, Pten-/- cell lines.

Accordingly, tumors developing in mice carrying the KrasG12D, Pik3caH1047R alleles are more sensitive than KrasG12D, Pten-/- mice to a dual PI3K/mTor inhibitor as well as a MEK inhibitor.

All together these results suggest that Pten loss has a stronger impact than Pik3ca activation to thyroid cancer development and resistance to small molecule inhibitors.

#2369

Transcription activators that regulate PKC- ἱ expression and are downstream targets of PKC- ἱ.

Andre H. Apostolatos, Wishrawana S. Ratnayake, Tracess Smalley, Anisul Islam, Mildred Acevedo-Duncan. _University of South Florida, Tampa, FL_.

Protein Kinase C-iota (PKC-ἱ) is an anti-apoptotic oncogene over-expressed in multiple cancers including prostate, ovarian, and glioma. PKC-ἱ is part of a cycle that helps cancer cell avoid senescence by releasing the transcription factor NFkB and promoting apoptotic resistance. PKC-ἱ is activated externally by factors like loss of PTEN (Paget 2012). However, while under the effect PKC-ἱ specific inhibitors, expression levels decreased, suggesting PKC-ἱ plays a role in regulating its own expression. A previous study showed the ELK1 transcription factor to be a regulator of PKC-ἱ (Gustafson 2003). Other transcription factors including Jun, ISGF3, PAX3, EGR1, and FOXO1 bind on or near the promoter sequence of the gene and their role in PKC-ἱ regulation was analyzed. Each transcription factor was systematically silenced with its own siRNA. Western Blotting revealed expression of PKC-ἱ in the transcription factor silenced cells determining which transcription factors are key players in regulation of PKC-ἱ. qPCR and microarray were performed to analyze the transcriptome of treated cells to match protein levels with mRNA levels. Targets both up and downstream of PKC-ἱ were analyzed to find the pathway that PKC-ἱ uses to help regulate itself.

#2370

Depletion of p21-activated kinase 4, PAK4, inhibits cellular proliferation, motility and clonogenicity in oral squamous cell carcinoma.

Felicia F. Chung,1 Si-Hoey Tan,1 Vijay J. Raja,2 Pei Yuen Ng,3 Ian C. Paterson,4 Chye Ling Tan,5 Chee-Onn Leong1. 1 _International Medical University, Kuala Lumpur, Malaysia;_ 2 _Weill Cornell Medical College, New York, NY;_ 3 _The National University of Malaysia, Kuala Lumpur, Malaysia;_ 4 _University of Malaya, Kuala Lumpur, Malaysia;_ 5 _Agency for Science, Technology and Research (A*STAR), Singapore, Singapore_.

Oral squamous cell carcinoma (OSCC) accounts for 90% of all malignancies in the oral cavity, and is often associated with low survival and poor prognosis. While considerable progress in OSCC treatment has been made, options for targeted therapy remain limited. Thus, it is imperative that new druggable targets are identified and characterized. p21-activated kinase 4 (PAK4) is a member of the PAK family of serine/threonine kinases, which are known modulators of a diverse range of oncogenic pathways including cell proliferation, escape from apoptosis, and anchorage-independent growth. Previous reports have indicated that PAK4 is overexpressed in a subset of invasive OSCC, and is highly associated with poor prognosis. However, the specific role of PAK4 in OSCC has yet to be elucidated. Here, we show that RNAi-mediated depletion of PAK4 conferred reduced clonogenicity, cell motility, and cell proliferation in OSCC cell lines in a cell line-dependent manner. Global proteomic profiling of PAK4-depleted H103 and H357 cells was conducted to identify the molecules and signaling pathways responsive to PAK4 depletion. In addition to reported targets of PAK4 such as CFL1, ACTB, and TUBB6, we observed that p16INK4a, pre-mRNA splicing factors (SRSF2, SRSF5, SRSF6, SLU7), and proteasome activator subunits (PSME1-4) were dysregulated as a result of PAK4 depletion. These results indicate that PAK4 plays a role in promoting OSCC progression by regulating multiple signaling pathways. Of note, there have yet to be any reports describing interactions between PAK4 signaling and mRNA splicing or proteasome activation. Further investigation is thus warranted to further delineate the potential role of PAK4 in regulating these targets.

#2371

Oncogenic transformation of FLT3-ITD is partially dependent on tyrosine 842 in the activation loop.

Julhash U. Kazi, Lars Rönnstrand. _Lund University, Lund, Sweden_.

Acute myeloid leukemia (AML) is a heterogeneous disease of the blood. About 30 % of AML patients carry an oncogenic mutant of the type III receptor tyrosine kinase FLT3. Among the various mutations in FLT3, the internal tandem duplication (ITD) mutations in the juxtamembrane domain are the most common type of mutation, while point mutations in the kinase domain also occur. Although oncogenic FLT3 mutants are constitutively active, the wild-type receptor needs its ligand (FLT3 ligand, FL) for activation. Ligand binding to the receptor induces dimerization, auto-phosphorylation, and activation of the receptor. Tyrosine phosphorylation sites on FLT3 constitute docking sites for interacting proteins that transduce pro-survival and proliferative signals. The so-called activation loop tyrosine residue (in FLT3, Y842) is well conserved in all receptor tyrosine kinases and long been known essential for the activity of some but not all receptor tyrosine kinases. Recently we have shown that activation loop tyrosine residue in KIT is not essential for its activation but plays an important role in receptor stabilization and downstream signaling. However, so far the role of FLT3 activation loop tyrosine residue remains unknown. We generated 32D cell lines carrying a Y-to-F mutant (Y842F) of FLT3 in either the wild-type or ITD background to study the role of this tyrosine residue. We show that the Y842 residue is not necessary for FLT3 activation or stability but is required for oncogenic transformation. We observed that Y842F mutation results in reduced cell viability, proliferation and in vitro colony formation in semi-solid medium. In addition, cells expressing the Y842F mutant in FLT3-ITD background display a significant reduction in tumor volume and weight in a xenograft model in nude mice. Gene set enrichment analysis (GESA) shows that mutation of Y842 causes suppression of anti-apoptotic genes. Furthermore, we demonstrated that phosphorylated Y842 is one of the binding sites of the protein tyrosine phosphataseSHP2, which is required for activation of signaling through the RAS/ERK pathway. Taken together, our data suggest that activation loop tyrosine residue in FLT3 plays an important role in the cellular transformation which is regulated by RAS/ERK signaling.

#2372

Xkr5 negatively regulates KIT/D816V signaling.

Jianmin Sun,1 Tine Thingholm,2 Tianfeng Li,3 Julhash U. Kazi,2 Hui Zhao,3 Lars Rönnstrand2. 1 _Ningxia Medical University, Yinchuan, China;_ 2 _Lund University, Lund, Sweden;_ 3 _the Chinese University of Hong Kong, Hong Kong, Hong Kong_.

D816V is the most often occurred mutation of type III receptor tyrosine kinase KIT in mastocytosis and CBF-AML. In attempting to identify specific downstream signaling pathways of KIT/D816V, we found that KIT/D816V but not wild-type KIT can bind to Xkr5 that was not studied previously. In addition to the association with KIT/D816V, Xkr5 is phosphorylated at Tyr 369, Tyr487 and Tyr 543 by KIT/D816V, and the phosphorylation is mediated by only mutations of Asp 816 in KIT but not by other KIT mutations that occurred in gastrointestinal stromal tumors and melanoma. Furthermore, the tyrosine phosphorylation of Xkr5 is not dependent on Src family kinases that play a crucial role in the activation of wild-type KIT. In KIT/D816V expressing mast cells, phosphorylation of Xkr5 inhibits KIT/D816V downstream signaling molecules Akt, Erk and p38. As a consequence, cell proliferation and colony formation are inhibited by the tyrosine phosphorylation of Xkr5 as well, indicating that Xkr5 is a negative regulator of KIT/D816V signaling.

#2373

Analysis of the distinct activities of the C1a and C1b domains of protein kinase C theta.

Agnes Czikora,1 Satyabrata Pany,2 Youngki You,2 Amandeep Saini,1 Noemi Kedei,1 Nancy E. Lewin,1 Adelle Abramovitz,1 Joydip Das,2 Peter M. Blumberg1. 1 _National Cancer Institute, LCBG, Bethesda, MD;_ 2 _Department of Pharmacological and Pharmaceutical Sciences, Houston, TX_.

PKC isozymes, the major receptors for tumor-promoting phorbol esters, play a central role in cellular signal transduction downstream of the second messenger diacylglycerol. The PKCθ isozyme has been extensively studied particularly in the context of hematopoiesis and immunity, where is required for the development and maturation of specific T cell subsets. PKCθ also plays a role in Notch-driven T cell leukemia and in a subset of gastrointestinal stromal tumors, melanoma and breast cancers. The C1 domains represent the regulatory motif in the PKCs responsible for their recognition of diacylglycerol / phorbol esters. PKCθ is unique among the conventional and novel PKC isoforms in that one of the two C1 domains of PKCθ, the C1a domain, has been reported to show little or no affinity for diacylglycerol or phorbol ester, unlike the C1b domain. Likewise, the PKCθ C1b domain has been reported to play the predominant role in the membrane translocation and activation process of PKCθ. We observed that the C1a domain in fact has appreciable binding activity for [3H]phorbol 12,13-dibutyrate (PDBu), with a Kd of 254 nM, albeit much less than that of the C1b domain, 1.58 nM. Replacing individually in the C1a domain ten of the amino acid residues that differed between the C1a and C1b domains, using the corresponding residue from the C1b domain, we found that only the P9K replacement restored [3H]phorbol 12,13-dibutyrate binding activity to the C1a domain, yielding a Kd of 3.60 nM, close to that of the C1b domain. Strikingly, the reciprocal replacement in the C1b domain of K9P still preserved appreciable binding affinity, 11.7 nM. We conclude that other features in the C1a domain contribute to the extent to which the P9 residue negatively influences binding. We further explored the role of the C1 domains of PKCθ on translocation using confocal microscopy with a variety of full length constructs in which the C1 domains were manipulated. Consistent with the binding studies, we showed that the C1b domain was the main contributor to translocation, that the full length PKCθ with the C1b K9P retained response, albeit more weakly, and that deletion of the C1a domain or its replacement with a second C1b domain enhanced the translocation to the plasma membrane. Exploitation of the structural differences between the C1 domains in PKCθ and those of other PKC isoforms, both at the level of the direct ligand interactions and the more complicated interplay between C1 domain, ligand, and phospholipid bilayer, may help to afford specific ligands for this member of the PKC family.

#2374

Mixed-Lineage Kinase 3 signals through the FRA1-MMP1 axis to drive triple-negative breast cancer invasion and transendothelial migration.

Chotirat Rattanasinchai, Brandon Llewellyn, Susan E. Conrad, Kathleen A. Gallo. _Michigan State Univ., East Lansing, MI_.

Mixed-lineage kinase 3 (MLK3), a mitogen-activated kinase kinase kinase (MAP3K), contributes to metastasis of triple-negative breast cancer (TNBC), in part through its regulation of paxillin phosphorylation and focal adhesion turnover. However the signaling mechanisms through which MLK3 promotes invasion and metastasis are not fully understood. We found that in non-metastatic, estrogen receptor positive (ER+) breast cancer cells, induced MLK3 expression robustly upregulates the oncogenic transcription factor, FOS-related antigen-1 (FRA-1), as well as matrix metalloproteinases (MMPs), MMP-1 and MMP-9. MLK3-induced MMP-1 induction is abrogated by FRA-1 silencing, demonstrating that MLK3 signals through FRA-1 to control MMP-1. In complementary experiments performed in metastatic TNBC models, high levels of FRA-1 are significantly reduced upon gene silencing of MLK3 or by MLK3 disruption using the CRISPR/Cas9n approach. Furthermore, both MMP-1 and MMP-9 are decreased upon ablation of MLK3 or MLK inhibitor treatment. Reduced endothelial permeability and transendothelial migration is observed in experiments with MLK3-depleted TNBC cells compared with parental counterparts. In addition, MLK inhibitor treatment or MLK3 depletion renders TNBC cells defective in Matrigel invasion. Furthermore, circulating tumor cells (CTCs) derived from a TNBC-bearing mouse which show an intermediate epithelial-mesenchymal phenotype display increased levels of MLK3, FRA-1, and MMP-1 compared with parental cells, supporting a role for the MLK3-FRA-1-MMP-1 signaling axis in tumor cell invasion and vascular intravasation. Consistent with the role of MMP-1 in multiple steps of metastasis, high levels of MMP-1 in breast cancer patient tumor samples are associated with increased distant metastases and poorer overall survival, particularly in TNBC. Our results demonstrating the requirement for MLK3 in controlling the FRA-1/MMP-1 axis suggest that MLK3 is a potentially effective therapeutic target for TNBC.

#2375

Functional role of the tumor suppressor protein phosphatase, PP2A-B55α, in breast cancer.

Nikita Panicker,1 Abdul Mannan,1 Lauren F. Watt,1 Ben Copeland,1 Matt D. Dun,1 Simon King,2 Megan Clarke,3 Kathryn Skelding,1 Severine Roselli,1 Nicole M. Verrills1. 1 _Univ. of Newcastle, Callaghan, Australia;_ 2 _Pathology North, New Lambton, Australia;_ 3 _Hunter Cancer Research Alliance, New Lambton, Australia_.

Breast cancer is the most common cancer in women and a leading cause of death. Dysregulation of cellular signalling pathways controlling proliferation, survival and migration, such as the PI3K/Akt and Ras/MAPK pathways, are key features of breast cancer. Protein phosphatase 2A (PP2A) negatively regulates many components of these pathways. PP2A is a family of trimeric serine/threonine phosphatases, each consisting of a structural, a catalytic and a regulatory subunit of which there are multiple isoforms. The addition of specific regulatory subunits provides subcellular targeting and substrate specificity to the enzyme. While PP2A is generally considered a tumor suppressor, a specific role for individual PP2A subunits in breast cancer has not been described. To address this, we first examined PP2A subunit expression in human breast tumors. Immunohistochemical analysis revealed significantly lower expression of the structural subunit, PP2A-A, and regulatory subunits PP2A-B55α and PP2A-B56α, in primary tumors and metastases, compared to adjacent normal mammary tissue. We further found an association of low PP2A-B55α with aggressive breast cancer subtypes, and with worse disease-free and overall survival. Functionally, shRNA-mediated knockdown of PP2A-B55α in normal mammary epithelial 3D cultures induced a tumorigenic phenotype, characterised by increased proliferation and enlarged multi-lobular acini. In contrast, overexpression of PP2A-B55α in breast cancer cells inhibited proliferation. Thus PP2A inactivation, in particular loss of B55α, is functionally important in breast tumorigenesis. PP2A-B55α complexes play an important role in DNA damage repair, and we found B55α knockdown impaired DNA damage repair. Thus low PP2A-B55α may contribute to genomic instability. To examine the functional role of PP2A-B55α in vivo, we have generated the first PP2A-B55α (Ppp2r2a) knockout mouse. Constitutive knockout of Ppp2r2a is embryonic lethal, with embryos dying during late development, post 14.5 days p.c. Heterozygous Pppr2ra mice (Ppp2r2a+/-) are viable, despite expressing only ~10% of PP2A-B55α protein levels compared to WT mice. Interestingly, Ppp2r2a+/- mice have significantly reduced branching in the developing mammary gland, similar to that observed in mice with mammary-specific loss of the breast tumor susceptibility gene, Brca1. Analysis of breast tumor formation in these mice, either alone or when crossed with MMTV-Neu animals, is underway. Finally, we show that pharmacological activation of PP2A, using FTY720, or the non-phosphorylatable analogue, AAL(S), inhibits tumor growth and metastases in an orthotopic xenograft model of aggressive, triple negative breast cancer (MDA-MB-231). Together this work demonstrates the importance of PP2A as a tumor suppressor in breast cancer, and suggests that targeting PP2A is a potential therapeutic strategy for poor outcome patients.

#2376

Novel insights into the regulation of ERK3's kinase activity and its ability to promote cancer cell invasiveness.

Lobna Elkhadragy, Hadel Alsaran, Weiwen Long. _Wright State University, Dayton, OH_.

In comparison with the well-studied conventional MAPKs, such as extracellular signal-regulated kinase (ERK)1/2, much less is known about the regulation and downstream targets of ERK3, an atypical MAPK. Whereas dual phosphorylation of the TXY activation motif in ERK1/2 is critical for their activation, it is unclear if phosphorylation of the single phospho-acceptor site (S189) in the SEG activation motif of ERK3 is critical for its kinase activity or function. In addition, little is known about the functions of the structurally distinct elongated C-terminus extension of ERK3. Recent studies have revealed important roles for ERK3 in promoting cancer progression. Of note, our previous study showed that ERK3 increases cancer cell invasiveness by phosphorylating the oncogenic protein steroid receptor co-activator 3 (SRC-3) and upregulating SRC-3-mediated matrix metalloproteinase gene expression. Here we aimed to elucidate the importance of the phospho-site S189 and the C-terminus in regulating ERK3 kinase activity and its functions in cancer cells. By performing in vitro kinase assays, we found that as compared to the wild type (WT) ERK3 protein, ERK3-S189A has remarkably reduced kinase activity towards auto-phosphorylation and phosphorylating SRC-3. In line with the critical role of S189 in ERK3 kinase activity, mutation of S189 to alanine greatly reduced ERK3's activity in promoting the migration and invasion of lung cancer cells. To study the regulatory role of the C-terminus in ERK3 kinase activity, we expressed and purified the N-terminal ERK3 (aa 1-340) that retains the kinase domain but has deletion of the C-terminus. While it showed higher auto-phosphorylation ability than the full length ERK3 protein, ERK3 (aa 1-340) had greatly decreased ability to phosphorylate SRC-3. Interestingly, ERK3-S189A (aa 1-340) exhibited kinase activity equivalent to that of ERK3 (aa 1-340). These results suggest that on one hand, the C-terminus may play an auto-inhibitory role on ERK3 kinase activation through intramolecular interaction and that the phospho-S189 is required for relieving this auto-inhibition; on the other hand, C-terminus plays a critical role for ERK3 to recruit substrates, such as SRC-3. In agreement with its inability to phosphorylate SRC-3, ERK3 (aa 1-340) had decreased ability to stimulate migration and invasion of lung cancer cells as compared to full length ERK3. Taken together, our study unravels the importance of ERK3 S189 residue as well as the C-terminus for the intramolecular regulation of ERK3 kinase activity and invasiveness-promoting ability in cancer cells.

#2377

The AML linked Src family kinase Fgr is uncoupled from SH2 and SH3 domain regulation and drives oncogenic transformation.

Kexin Shen,1 Heather R. Dorman,1 Haibin Shi,1 Ravi K. Patel,1 Jamie A. Moroco,2 John R. Engen,2 Thomas E. Smithgall1. 1 _Univ. of Pittsburgh School of Medicine, Pittsburgh, PA;_ 2 _Northeastern University, Boston, MA_.

Fgr is a member of the Src family of non-receptor protein-tyrosine kinases, which are over-expressed and constitutively active in many human cancers. Fgr expression is restricted to myeloid hematopoietic cells and strongly upregulated in a subset of primary human AML bone marrow samples, suggesting that Fgr kinase activity may contribute to AML pathogenesis. To investigate the oncogenic potential of Fgr, we expressed the wild-type kinase in Rat-2 fibroblasts and scored transformation in soft-agar colony-forming assays. Remarkably, Fgr expression resulted in strong transforming activity, suggesting that over-expression of the kinase is sufficient to overcome regulatory control by its SH2 and SH3 domains. To test this idea, we mutated the negative regulatory tail tyrosine of Fgr. Phosphorylation of the homologous tyrosines in c-Src, Hck and other Src-family members by the regulatory kinase Csk causes intramolecular engagement of the SH2 domain to suppress kinase activity. Substitution of the Fgr tail tyrosine with phenylalanine did not further enhance the transforming or kinase activity of Fgr in Rat-2 cells, suggesting that its kinase domain is uncoupled from regulation by its non-catalytic SH3-SH2 region. To explore the regulatory mechanism further, we expressed near-full-length Fgr in insect cells, purified it to homogeneity, and confirmed that it was singly phosphorylated on its negative regulatory tail (and not the activation loop). Hydrogen-deuterium exchange mass spectrometry demonstrated that the SH3 and SH2 domains are protected from deuterium uptake as observed previously for recombinant, downregulated Hck, suggesting that they are packed against the back of the kinase domain. We then tested recombinant Fgr for sensitivity to activation in vitro using peptide ligands for the SH3 and SH2 domains, as well as a peptide that engages both domains simultaneously. Fgr kinase activity was unaffected by these peptides, providing further evidence that the kinase domain is not allosterically coupled to SH3 and SH2 control. In contrast, Hck and c-Src were both stimulated by these peptides, consistent with a domain displacement mechanism of activation described previously. Control experiments using surface plasmon resonance spectroscopy confirmed that the SH3 and SH2 domains of both Hck and Fgr bind to each of the peptide ligands with similar kinetics and affinity. Taken together, our data show that Fgr is a unique Src family member in that its kinase domain is not subject to regulatory domain control despite evidence for intramolecular interactions with the SH3 and SH2 domains. As a consequence, simple over-expression of Fgr is sufficient to induce transformation of rodent fibroblasts, unlike Hck or other Src family members. By extension, over-expression of Fgr may contribute to AML development and selective targeting of its kinase activity may be of therapeutic benefit.

#2378

A kinome analysis of the molecular pharmacodynamics of PT-112 in a human cancer cell line.

Diego Tosi,1 Esther Perez-Gracia,1 Philippe Pourquier,1 Tyler D. Ames,2 Richard A. Wing,2 José Jimeno,2 Céline Gongora1. 1 _Institut de Recherche en Cancérologie de Montpellier, Montpellier, France;_ 2 _Phosplatin Therapeutics, New York, NY_.

Background: To better understand the mechanism of action of PT-112, we investigated the effects of PT-112 on kinase activity within HCT116 human colorectal cancer cells. PT-112 is a pyrophosphate-conjugated platinum (Pt) containing new chemical entity under clinical development in the US and Taiwan. Designed to reduce certain toxicities associated with Pt agents and drug resistance from DNA-repair pathway activity, PT-112 differs in significant ways from traditional Pt therapies. PT-112 results in potent anti-proliferation effects with lower Pt accumulation in whole cell and DNA extracts. Upregulation of p16 and p21 and, consistently, inhibition of the cell cycle at the G1/S phase transition is observed with PT-112 use, a finding not commonly associated with Pt agents. Further, sensitivity to PT-112 is not significantly affected by the functionality of DNA repair pathways. Finally, the induction of immunogenic cell death by PT-112 has been indicated via release of HMGB1, cell surface localization of calreticulin, and suppression of STAT3.

Methods: A broad analysis of the phosphorylation pattern of kinase targets was achieved by utilizing the Tyrosine Kinase and Serine / Threonine Kinase PamChip® Arrays, each spotted with 144 peptides harboring kinase phosphorylation sites. Data were collected after 1, 6, and 24hr of exposure to 10 μM PT-112, a concentration achievable in human patient plasma at sub-toxic doses, and the degree of phosphorylation of these peptide targets was measured by the PamStation®.

Results: A set of kinase targets was identified as differentially phosphorylated relative to untreated control samples. In total, 4/15/30 targets were more phosphorylated than controls at the 1/6/24hr time points, respectively, while 1/0/10 targets were less phosphorylated. Several of these targets connect to key cancer relevant pathways, including anti-angiogenesis (VGFR1), apoptosis (BAD), cell cycle regulation (CDC2, Rb, RBL2), cell growth (EGFR, MK07, RBL2), and cancer immunology (STAT4, CSF1R).

Conclusions: A narrow set of initial effects on kinase activity from PT-112 exposure, which broaden over time, was observed. While we cannot discriminate direct from indirect effects, these data are consistent with PT-112 having mechanistic targets independent of nuclear DNA. Several observations were consistent with findings from previous experiments (e.g. evidence of impacts on apoptotic, cell cycle, and immunological pathways), while others were novel (e.g. impact on VGFR1, EGFR, MK07). In conclusion, PT-112 at pharmacologically achievable concentrations leads to a unique pattern of kinase disruption in vitro. Further work will be conducted to validate these findings, identify the driver kinases, and connect these observations into a coherent network of kinases and targets affected by PT-112 treatment, and to understand the clinical impact of the unique pharmacodynamic profile of PT-112.

#2379

Nuclear lemur tyrosine kinase-2 regulates RNA polymerase II dependent transcription in prostate cancer.

Kalpit Shah,1 Justin Foley,2 Michael L. Nickerson,1 Michael Dean,1 Neil Bradbury2. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Rosalind Franklin University of Medicine & Sciences, North Chicago, IL_.

The Androgen Receptor (AR), a DNA-binding transcription factor plays a key role in the development and maintenance of prostate epithelia by modulating expression of growth-promoting genes. AR has been proposed to regulate gene expression by enhancing the efficiency of RNA polymerase II (RNAPII) dependent transcription elongation. Dysregulation of this AR-dependent transcriptional activity has been implicated not only in prostate cancer but also androgen-independent prostate cancer. Recently, we showed that protein expression of Lemur Tyrosine Kianse-2 (LMTK2), a serine-threonine kinase is downregulated in prostate cancer. Importantly, our study ascribed a novel role for LMTK2, as a negative regulator of AR-dependent transcriptional activity in prostate epithelial cells. However, the mechanism through which LMTK2 is able to regulate AR remains to be determined. Here, we show that LMTK2 is not only present in cytoplasmic fraction but also, within the nucleus of mammalian cells where it co-localizes with AR, newly transcribed mRNA and with RNA polymerase II (RNAPII). Interestingly, our data reveals that LMTK2 colocalizes with elongating RNAPII, phosphorylated on serine 2 of the carboxyl-terminal domain. This presents an interesting possibility that LMTK2 might be able to regulate transcription of AR-dependent genes by interacting with the elongating RNAPII and transcription & epigenetic factors. As such, our study potentially identifies LMTK2 as an important player regulating the AR-RNAPII mediated transcription machinery in mammalian cells.

#2380

Insertion mutations in the tyrosine kinase domain of FLT3 display a higher oncogenic potential than the D835Y mutation in acute myeloid leukemia.

Alissa Marhäll,1 Thomas Fischer,2 Florian Heidel,3 Julhash U. Kazi,1 Lars Rönnstrand1. 1 _Translational Cancer Research, Lund University, Lund, Sweden;_ 2 _Otto-von-Guericke University Medical Center, Magdeburg, Germany;_ 3 _University Hospital Jena, Jena, Germany_.

Acute myeloid leukemia (AML) remains the most common form of acute leukemia among adults and accounts for a large number of deaths. Mutation in FMS-like tyrosine kinase 3 (FLT3) is one of the most prevalent factor in this heterogeneous disease. The major mutations in FLT3 can be categories as internal tandem duplications (ITD) and point mutations. Recent studies suggest that ITDs are subdivided in to two groups depending on their location: the juxtamembrane domain (ITD-JM) and the tyrosine kinase domain (ITD-TDK). Although, ITD-JM has been characterized well the ITD-TKD has not yet been studied well due to its recent discovery. For this reason, we compared ITD mutations in TKD and JM, as well as the most frequently occurring point mutation located in the TKD, D835Y. The purpose of this study was to understand whether it is the mutation's nature or location that plays the driving role in leukemogenesis. We used a cytokine-dependent mouse pro-B cell line, BaF3, to overexpress different FLT3 mutants. We first examined the colony formation capacity in semisolid cytokine- and serum-free medium. The assay resulted in indistinguishable number and size of colonies for both ITD-JM and ITD-TKD, while D835Y-TKD transfected cells failed to form colonies suggesting that the ITD-TKD mutations have stronger transforming potential than other TKD mutations. In addition to colony formation assays, cell proliferation and survival was significantly higher in ITD-TKD expressing cells compared to cells expressing D835Y-TKD. Finally, we showed that phosphorylation of STAT5 and AKT is increased in ITD-TKD, while other FLT3 downstream signaling remained unaffected. All together, our data suggest that ITD-TKD displays higher oncogenic potential than other TKD mutants.

#2381

The effect of ERK5 inhibition in clear cell renal cell carcinoma.

Hidenori Kanno, Sei Naito, Osamu Ichiyanagi, Norihiko Tsuchiya. _Yamagata University, Yamagata City, Japan_.

Introduction Clear-cell renal cell carcinoma (ccRCC) is the most prevalent kidney cancer and is characterized by von Hippel-Lindau tumor suppressor gene (VHL) mutation. VHL protein acts in a ubiquitin ligase complex, mediating the proteasomal degradation of ubiquitinized proteins, including hypoxia-inducible factor (HIF). Therefore, HIF accumulates in VHL-defective cell lines. A recent study revealed that extracellular signal-regulated kinase 5 (ERK5) is degraded through the ubiquitin-proteasome system, in a process mediated by VHL and HIF. Our objectives were to examine the effects of ERK5 in ccRCC and to investigate ERK5 as a potential therapeutic target. Methods ERK5 expression was investigated in surgically resected ccRCC specimens using immunohistochemistry. To confirm that ERK5 is degraded by the VHL pathway, ERK5 levels were examined by western blotting in Caki-1 VHL wildtype renal cell carcinoma (RCC) cells and in A498 and A704 VHL-mutant RCC cells with or without the MG132 proteasome inhibitor. Furthermore, VHL-mutant cell lines were examined, with or without siRNA and XMD8-92-mediated ERK5 inhibition, to investigate the role of ERK5 in RCC cells using flow cytometry, MTS assays, and western blotting . Results In surgical specimens, RCC cells showed higher levels of ERK5 expression than did normal cells. In addition, MG132 enhanced ERK5 expression in wildtype cell lines, but not in VHL-mutant cell lines. Therefore, ERK5 degradation involves the VHL pathway. Furthermore, ERK5 inhibition resulted in increase of the sub-G1 population as observed by flow cytometry and cleaved RARP expression as seen by western blotting, indicating an increase in apoptotic cells. ERK5 inhibition also suppressed cell viability and downregulated p16 and Bcl-2 expression. Conclusion Our results show that ERK5 inhibition contributes to the downregulation of anti-apoptotic and cell cycle proteins and suggest that ERK5 is a promising therapeutic target for ccRCC.

#2382

RACK1 regulates PKA activity to drive tumorigenesis in colon cancer.

Sheri L. Hayes, Catríona M. Dowling, John C. Coffey, Patrick A. Kiely. _University of Limerick, Limerick, Ireland_.

RACK1 (Receptor for Activated C Kinases) is a scaffolding protein with 7 WD repeats that interacts with the Insulin-like Growth Factor I receptor (IGF-IR), integrins, and other signalling proteins. RACK1 functions as a scaffolding protein and regulator of many key biological processes and it is highly expressed in most tissue. We recently reported that RACK1 is essential for IGF-I-mediated regulation of PP2A activity and AGAP2 activity at focal adhesions. Downstream of IGF-1R signaling in cancer cells, the scaffolding properties of RACK1 are altered providing distinct migratory advantages to the cell and suggesting that RACK1 is an important regulator of IGF-I signalling in cancer progression. It is important to further characterise the role of RACK1 in cancer and to delineate the molecular mechanisms by which it regulates key signalling pathways. We have identified Protein Kinase A (PKA) as RACK1 interacting protein. PKA is a complex, multicomponent enzyme which is a fundamental protein that functions in cell survival, proliferation, and cytoskeletal remodelling among others. Our hypothesis is that RACK1 regulates the PKA axis in cancer cells leading to Akt and MAP Kinase activation downstream of IGF-1R signalling. We have demonstrated that RACK1 is required to mediate PKA activity in colon cancer cells. Although we show that PKA expression is not altered in colon cancer tissue, we believe that PKA activity is altered significantly in the disease state. Using PKA inhibitors, we have shown that PKA regulates IGF-I-mediated MAP Kinase activation in colon cancer cell lines. In conclusion, this research project will help elucidate the role of PKA in colon cancer. Inhibiting or promoting specific protein interactions with RACK1 will provide very novel therapeutic opportunities and anti-cancer drug targets.

#2383

Nicotinamide N-methyltransferase inhibits Protein Phosphatase 2A activity by promoting Y307 phosphorylation.

John R. Jacob, Arnab Chakravarti, Kamalakannan Palanichamy. _Ohio State Univ. College of Medicine and Comprehensive Cancer Center, Columbus, OH_.

Recent evidence indicates that suppression of protein phosphatase 2A (PP2A) activity plays an essential role in malignant transformation and cancer cell maintenance. Despite the importance of PP2A as a tumor suppressor in cancer, the mechanisms by which other enzymes regulate PP2A remain largely unknown. We recently identified Nicotinamide N-methyltransferase (NNMT) as a novel regulator of PP2A activity in glioblastoma (GBM). NNMT is a cytosolic N-methyl transferase which utilizes methyl groups from S-Adenosyl methionine (SAM), forming the by-product S-Adenosyl Homocysteine (SAH). PP2A becomes activated following the trimer formation of the three PP2A subunits, which requires the methylation of the catalytic subunit of PP2A (PP2A C) at L309.In cancer, NNMT has been reported to decrease the cellular methylation potential (SAM:SAH) and correspond with decreased PP2A activity. It was hypothesized that the decrease in methylation potential accounts for the decrease in PP2A activity by kinetically disfavoring the methylation of PP2A C. Interestingly, when we overexpressed NNMT in GBM cell lines and used LC-MS/MS (LC-MS Quadrupole Time-of-Flight and LC-MS Triple Quad) to monitor the methylation potential, we discovered that overexpression of NNMT increased methylation potential, however, still lead to a decrease in PP2A activity. As a result, we hypothesized that NNMT suppresses PP2A activity in a methylation-independent manner. In an effort to identify an alternative mechanism by which NNMT decreases PP2A activity, we found that NNMT expression was required for the phosphorylation of PP2A C at residue T307, which inhibits PP2A trimer formation and activation. Further, we found that NNMT expression leads to the constitutive activation of the PP2A target serine/threonine kinases (STKs): Akt, MAPK, and SAPK/JNK. NNMT silencing resulted in the deactivation of these STKs and radiation-induced cellular stress was not sufficient to rescue their activation. This suggests that NNMT-induced PP2A suppression is essential for the activation of these kinases. Overall, this study demonstrates a novel mechanism independent of methylation by which NNMT represses the activity of tumor suppressor PP2A.

#2384

Aberrant expression of PDZ binding kinase modulates human pancreatic cancer cell invasion.

Charles Hinzman,1 Jose Trevino,2 Partha P. Banerjee1. 1 _Georgetown Univ. Medical Ctr., Washington, DC;_ 2 _University of Florida Medical Center, Gainesville, FL_.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths and projected to be the second leading cause of cancer deaths by 2030. Incidence to mortality ratio for PDAC is almost one suggesting its aggressive phenotype. The poor survival rate of PDAC patients is due to metastasis to distant organs, and therefore, recent focus has been aimed towards the identification of molecular targets that drives metastatic disease. We are investigating an under-studied kinase, PDZ-domain binding kinase (PBK) as a target for pancreatic cancer (PanCa) therapy. PBK is a dual-specificity serine/threonine kinase involved in mitosis and invasive behavior of cancer cells. It is expressed physiologically in testis, but aberrant expression results in an aggressive phenotype in a variety of cancers. Our results demonstrate that PBK expression varies across a panel of human PanCa cell lines, and directly correlates with their invasive ability. Similarly, a panel of patient-derived primary PanCa shows higher PBK levels associated with aggressive behavior and invasion. To assure specificity of PBK to this invasive phenotype, we measured the effect of PBK overexpression or knockdown on the PanCa lines' invasive ability. Overexpression of PBK in low PBK expressing cells increased PanCa invasion ~10-fold, while PBK knockdown reduced invasive ability ~5-fold. Immunohistochemical analyses of resected human PanCa specimens revealed that PBK is present in the majority of human PanCa tissue samples tested but not in the adjacent normal pancreatic tissue. Interestingly, aberrant PBK localization was found in even early stages of PanCa development and was sustained throughout its progression. In search of molecular effectors of invasion regulated by the PBK, we examined various genes/proteins that are involved in cancer cell invasion and metastasis. RelB a well-known metastatic inducer in various cancers was found to be a major payer in PanCa and regulated by PBK. These data indicate a novel molecular pathway of PBK where its presence plays a role in invasion of human PanCa cells. Therefore, the role of PBK in promoting cancer cell invasion, combined with its general lack of expression in normal cells, nominates PBK as a potentially important therapeutic target for pancreatic cancer.

#2385

Systematic characterization of kinase inhibitors reveals heterogeneity in responses by class and cell line.

Caitlin E. Mills,1 Marc Hafner,1 Dejan Juric,2 Peter K. Sorger1. 1 _Harvard Medical School, Boston, MA;_ 2 _Massachusetts General Hospital, Boston, MA_.

Several publications have addressed concerns surrounding drug response screens by pointing out sources of variability and by presenting recommendations for better experimental methods and more robust analytical approaches. In the presented profiling effort, we integrated the latest advances in drug response measurement and focused on data diversity and quality rather than on breadth. We selected 32 breast cancer cell lines with a strong bias towards triple negative lines as well as 4 cell lines established from relevant patient-derived xenografts. We evaluated a panel of clinically relevant kinase inhibitors using a microscopy-based drug response assay to measure drug potency, and to quantify drug efficacy in terms of growth inhibition (GR metrics) and cell death. The use of the GR metrics to quantify drug sensitivity enabled us to identify and study differences between cytostatic and cytotoxic responses. This systematic dose response dataset is complemented by measurements of baseline mRNA expression levels by RNAseq and endogenous protein levels by shotgun mass spectrometry across all cell lines. The completeness and controlled conditions under which these data sets were collected provide confidence in their integration. The baseline RNA and protein expression levels were used to build predictors of the measured drug responses with the goal of identifying the biological processes responsible for the differences in sensitivity across drugs and cell lines. Differences in the phenotypic responses of cell lines to kinase inhibitors with the same nominal targets have been investigated, and associated with variable inhibitor polypharmacology.

#2386

HDAC6 regulates ERK1's kinase activity via deacetylation.

Jheng-Yu Wu,1 Xiaohong Zhang2. 1 _Univ. of South Florida College of Medicine, Tampa, FL;_ 2 _Karmanos Cancer Institute, Wayne State University, Detroit, MI_.

Extracellular signal-regulated kinases 1/2 (ERK1/2) are important kinases regulating cell proliferation and cell migration, and have been established as therapeutic targets for cancer treatment. Previously, we found that ERK1 phosphorylates histone deacetylase 6 (HDAC6), a class IIb HDAC, to regulate its enzymatic activity. HDAC6 plays an important role in several types of cancers. HDAC6 governs cancer cells' migration by cooperating with cytoskeleton proteins and their associated proteins, and HDAC6 is arising as a determinant for cisplatin-resistance in non-small cell lung cancer (NSCLC) and ovarian cancer. Here, we have shown that HDAC6 manipulates ERK1's kinase activity via deacetylation. We demonstrated that both ERK1 and ERK2 interact with HDAC6 physically via GST pull-down assays. To examine whether ERK1/2 are acetylated, we transfected GST-ERK1 or GST-ERK2 into HEK293T cells, and treated the cells with a pan-HDAC inhibitor, Trichostatin A. We showed that the level of acetylated GST-ERK1/2 increased in a dose- and time-dependent manner. Furthermore, when GST-ERK1 or GST-ERK2-expressed cells were treated with an HDAC6-specific inhibitor, ACY-1215, the level of acetylated GST-ERK1/2 increased. These results suggest that HDAC6 is a deacetylase for ERK1/2. In addition, we determined that acetyltransferases CBP and p300 acetylate ERK1/2. We found a novel acetylation site located in ERK1 N-terminus by mass-spectrometry analysis. We then mutated this lysine (K) site with either Glutamic acid (Q) or Arginine (R) to mimic the acetylation or deacetylation status, and performed kinase assays using ELK1 as a substrate. The acetylation mimicking mutant exhibits a decreased kinase activity toward ELK1, while the deacetylation mimicking mutant exhibits a similar level of kinase activity as the wild-type ERK1 does, suggesting that acetylation/deacetylation alters ERK1 enzymatic activities. Therefore, HDAC6 may regulate ERK1's kinase activity via deacetylation of this site. Furthermore, we have shown that HDAC6 is overexpressed in lung cancer tissue samples and plays an important role in chemo-sensitivity in lung cancer. We predict that inhibition of the HDAC6-ERK1 pathway may provide clinical benefit for lung cancer patients. 

### Epigenetics 2

#2387

Overexpression of NSUN2 by DNA hypomethylation is associated with metastatic progression in human breast cancer.

Jie Yi,1 Yu Chen,1 Ran Gao,1 Zhuo Yang,1 Jinhua Wang,2 Yingchun Xu1. 1 _Peking Union Medical College, Beijing, China;_ 2 _Chinese Academy of Medical Science and Peking Union Medical College, Beijing, China_.

NSUN2 is a RNA methyltransferase that has been shown to be implicated in development of human cancer. However, the functional role of NSUN2, mechanism of NSUN2 overexpression and its association with clinicopathologic features in breast cancer remain unclear. To investigate alterations in the expression and functional role of NSUN2 in breast cancer, NSUN2 expression was assessed in breast cancer cells and tissues obtained from cancers at different American Joint Committee on Cancer (AJCC) stages, and its functions were investigated using breast cancer cells. NSUN2 expression was shown to be significantly higher in breast cancer cells and tissues than in normal breast epithelial cells and tissues, at both mRNA and protein levels. Overexpression of NSUN2 was shown to promote cell proliferation, migration, and invasion while NSUN2 knockdown inhibited these processes in vitro and in vivo. NSUN2 expression level was associated with the methylation level of its promoter. Our results demonstrated that the overall expression of NSUN2 significantly correlated with clinical stage (P=0.027), tumor classification (P=0.012), pathological differentiation (P=0.023), as well as with the expression levels of estrogen receptor (P<0.001), progesterone receptor (P=0.001), and Ki-67 (P<0.001). Our findings provide a unique insight into the roles and effects of NSUN2 overexpression in breast cancer cells, and highlight the necessity of the investigation of novel therapeutic targets, such as NSUN2, for the improvement of breast cancer treatments.

#2388

Low grade gliomas exhibit distinct patterns of epigenetic aging associated with prognostic molecular subtype.

Peter Liao, Jill Barnholtz-Sloan. _Case Western Reserve University, Cleveland, OH_.

Background: Low grade gliomas make up a significant proportion of malignant brain tumors and possess a high degree of heterogeneity, highlighting a need for clinically useful markers for prognosis and further biologic investigation. We used an existing model of biological age based upon DNA methylation to characterize epigenetic age in low grade gliomas (LGG) according to existing molecular classifications and assessed the prognostic utility of epigenetic age.

Methods: We analyzed the full TCGA LGG cohort consisting of 516 patients; 216 grade 2 and 241 grade 3 tumors. Age at diagnosis ranged from 14-87 years with median age 43. We calculated "DNA methylation age" (DNAm age) based upon 353 CpG probes according to the epigenetic clock developed by Steve Horvath, and calculated age acceleration according to the DNAm age of tumor tissue relative to reported chronological age of patient at diagnosis. DNAm age was assessed for prognostic value using Cox proportional hazards regression. Age acceleration differences were assessed across LGG molecular classification methods using the Wilcoxon Ranked Sum test.

Results: DNAm age remained highly correlated with chronological age in LGG tumor tissue (cor=0.58, p<0.001), with an average age acceleration of 33.5y. DNAm age was weakly positively correlated with mutational burden (cor=0.12, p=0.01). Higher DNAm age was positively associated with survival independent of chronologic age and Karnofsky Performance Score (p=0.003), but not when IDH mutation status was included into the Cox proportional hazards model. Further investigation showed that DNAm age acceleration was associated with IDH mutation status and was significantly increased in IDH-mutant compared to IDH-wt tumors (95% CI= 18.6-28.8y) as well as MGMT-promoter methylated versus unmethylated cases (95%CI= 18.1- 28.9y). No significant DNAm changes were observed across histological subtype or grade.

Conclusions: Although the regulation of DNAm age in cancers remains poorly understood, significant association of DNAm age acceleration with prognostically useful molecular subtypes in LGGs underscores biological differences that cannot be discerned in LGGs based upon histological classification. This association suggests that LGGs may provide an avenue for investigating DNAm age, molecular subtype, and their relation to tumor behavior and clinical prognosis.

#2389

LINE-1 tumor hypomethylation is associated with shorter recurrence-free survival in localized clear-cell renal cell carcinoma.

Gabriel G. Malouf, Roger Mouawad, Frederick Allanic, Eva Compérat, Morgan Roupret, David Khayat, Jean-Philippe Spano. _Salpetriere Hospital, Paris, France_.

Background: Cancer cells are characterized by alterations of DNA methylation patterns involving global DNA hypomethylation and site-specific CpG island promoter hypermethylation. Global DNA hypomethylation is thought to play a role in genetic instability and tumor aggressiveness. Long interspersed nucleotide element-1 (L1/LINE-1) repetitive elements represent 40% of the genome and their methylation is a good indicator of the global DNA methylation level. Although LINE-1 methylation has been previously shown to be associated with prognosis of patients with cancer, the value of LINE-1 methylation in predicting recurrence of patients with localized clear-cell renal cell carcinomas remains unknown.

Material and Methods: We quantified the LINE-1 methylation using bisulfite pyrosequencing in cohort of 200 patients with resected clear-cell renal cell carcinomas (AJCC stage I-III). LINE-1 methylation of adjacent normal kidney was also assessed in 128 cases. Threshold of tumor LINE-1 hypomethylation was defined as LINE-1 methylation in normal samples minus three standard deviation.

Results: Median methylation of tumor samples was 59.01% versus 61.61% for normal adjacent kidney samples (p<10-5). LINE-1 methylation level of normal adjacent kidney was associated with tumor size (Spearman R=0.21, P=0.02) but not with age (p=0.24) or Leibovitch score (p=0.05). Tumor LINE-1 hypomethylation was observed in 20 samples (n=10%). Strikingly, those were enriched for tumors occurring in female (60% versus 27.7%) (p=0.005); of note, no association was found between LINE-1 tumor hypomethylation and tumor grade (p=0.31), stage (p=1), age (p=0.24) and Leibovitch score (p=0.82). In multivariate analysis, only Leibovitch score and LINE-1 hypomethylation were independently associated with poor recurrence-free survival.

Conclusion: LINE-1 hypomethylation is associated with shorter recurrence-free survival in patients with resected clear-cell renal cell carcinomas, suggesting the possibility of using it as predictive biomarker of recurrence.

#2390

Clinical validation of an epigenetic field of susceptibility to detect significant prostate cancer from 2 non-tumor biopsies.

Bing Yang,1 Johnathon McCormick,1 Adam Schultz,1 Glen Leverson,1 Geoffrey Sonn,2 Cristina Magi-Galluzi,3 Eric A. Klein,3 Michael Fumo,4 David F. Jarrard5. 1 _Univ. of Wisconsin-Madison, Madison, WI;_ 2 _Stanford University, Palo Alto, CA;_ 3 _Glickman Urological and Kidney Institute, Cleveland, OH;_ 4 _Rockford Urological Associates, LTD, Rockford, WI;_ 5 _Univ. of Wisconsin-Madison Carbone Cancer Center, Madison, WI_.

Background: An epigenetic field of cancer susceptibility exists for prostate cancer that gives rise to multifocal disease in the peripheral zone. In a previous studies (Neoplasia 2013, J Urol 2014) we identified using methylation arrays over 50 altered regions in normal prostate tissue of men with prostate cancer. In the current multicenter study, a validation was performed to determine the predictive strength of this approach.

Methods: We evaluated 2 archived, cancer negative prostate biopsy core tissue from 129 subjects from 4 urological centers. All negative cases (controls) underwent 2 or more repeat negative biopsies within 24 mo with a central review of all histopathology. Cancer cases were negative biopsies selected from patients who ultimately went prostate removal to confirm a final grade >Gleason Score 7. Biopsies were analyzed using pyrosequencing for DNA methylation changes at multiple CpGs surrounding the genes EVX1, CAV1, PLA2G16, FGF1, SPAG4 and NCR2. Analyses used multiplex logistic regression modeling with backward elimination.

Results: Patients diagnosed with GS>7 cancer (77) and the control group (52) were similarly matched except for PSA (7 vs 5.8; p<0.03) and prostate size (46 vs 36; p<0.02). We observed robust methylation differences for all genes (p<0.05) (Table). A multiplex regression model for biomarker performance incorporating a gene combination of EVX1, NCR2, PLAG2 and SPAG4 discriminated non-tumor from tumor-associated tissues (AUC 0.744, p<0.0001). A multiplex model incorporating 3 genes (EVX1, PLA2G16, NCR2) and clinical information (PSA, age) identified patients with GS>7 prostate cancers (AUC 0.808, p=0.001).

Conclusions: A widespread epigenetic field defect can be used to detect GS>7 prostate cancers in patients with histologically negative biopsies. This assay is sensitive in requiring only 2 biopsies and detects alterations in nontumor cells at distance from the cancer. This has the potential to decrease the need for repeat prostate biopsies, a procedure associated with cost and complications.

Support: UW Ignitor; Gen X foundation

#2391

Integrated analysis of methylation and expression profile of telomere-related genes in breast cancer patients.

Jianfu Heng,1 Xinwu Guo,2 Lili Tang,3 Fan Zhang,1 Limin Peng,2 Ming Chen,2 Xipeng Luo,2 Xunxun Xu,2 Shouman Wang,3 Jun Wang1. 1 _Central South University, Changsha, China;_ 2 _Sanway Gene Technology Inc., Changsha, China;_ 3 _Xiangya Hospital, Central South University, Changsha, China_.

Telomeres at the ends of chromosomes are critical in maintaining the integrity and stability of the genome. Aberrant telomere or telomerase dysfunction participates in tumorgenesis. A majority of human cancers exhibit critically aberrant telomere length, suggesting that tumors can arise from genetically instable cells with dysfunctional telomeres. Many genes are involved in the complex regulatory mechanisms of telomere length and telomerase activity. In addition, the methylation and expression pattern for most of telomere related genes in breast cancer are still unknown. Using microfluidic-PCR based target enrichment and next-generation bisulfite sequencing technology, we explored the promoter methylation profile of 29 telomere-related genes in 184 breast cancer patients with paired tumor and matched normal tissues. The average methylation level was significantly higher in tumor (8.13%) than that in matched normal tissues (7.08%) (P = 4.30E-21). Four genes showed significant hyper-methylation in the breast tumor tissues. All of these 4 genes are annotated with potential TFBSs in the promoter regions. In subtype analysis, RAD51D showed significant methylation difference among four subtypes. In analysis of the association between methylation and clinicopathologic characteristics, TERC showed significant difference between ER+/ER- tumors. The expression profile of the 4 significant hyper-methylated genes was explored in the same cohort using qPCR method. All of them showed significantly lower expression in breast tumor tissues compared with the matched normal tissues. Two genes showed significant and negative cis correlation between methylation and gene expression. These results were also validated in the TCGA database. The 4 genes panel showed a good performance in predicting breast cancer with ROC analysis. In summary, our results revealed the methylation pattern of telomere related genes in breast cancer and illustrated the epigenetic regulatory mechanism on expression of aberrant methylated genes. Our study provides a novel panel of telomere related genes which may be a valuable diagnostic biomarker for breast cancer prediction.

#2392

A multiplex histone H3 PTM assay for epigenetic biomarker discovery in tissue biopsy and archived clinical samples.

Mary Anne Jelinek, Melissa Ritland, AJ Westergren. _Active Motif, Inc., Carlsbad, CA_.

Dysregulation of epigenetic mechanisms is known to play an important role in the development and progression of cancer. One such mechanism is manifested as altered levels of histone modifications involved in regulating gene transcription. N-terminal histone tails can have a variety of modifications, such as phosphorylation, methylation and acetylation at specific amino acid residues which are conserved throughout eukaryotes and function by altering chromatin structure and creating binding sites for chromatin readers, writers or erasers.

Numerous studies have reported aberrant global levels of several histone H3 and H4 post-translational modifications (PTMs) in a wide range of solid tumor types. These changes have been shown to be predictive of clinical outcome, raising the possibility that histone modifications have potential as epigenetic biomarkers. Clinical samples have immense potential for biomarker identification since they are often accompanied with valuable information pertaining to patient history, treatment courses and disease outcome. The preferred method for clinical sample preservation is formalin-fixation followed by paraffin embedding (FFPE). While extraction and downstream analysis of DNA and RNA from FFPE samples is now routine, proteomic studies of FFPE samples is hampered by extensive protein cross-linking generated by formalin fixation. Analysis of histone PTMs in patient archival samples is limited to low-throughput immunohistochemical staining, and is not an ideal approach for mining large sample cohorts for biomarker identification. Western blot or ELISA methods, which have large sample requirements, are simply not an option for FFPE samples.

We have developed a multiplex bead-based ELISA assay which enables simultaneous interrogation of thirteen histone H3 PTMs. The assay is performed in 96-well plates and is ideally suited for profiling histone modification levels in limiting or small samples. We will present the application of this technology in a variety of sample types including frozen tissue, formaldehyde-cross linked cells and tissues, and in small molecule inhibitor screens with as few as 2,000 cells per well. The ability to simultaneous detect up to thirteen histone H3 PTMs provides a unique feature that enables determination of for on-target and off-target effects within the same sample.

#2393

Decreased expression of 5-hydroxymethylcytosine is associated with grade of malignancy in lung cancer.

Yoshiaki Tominaga,1 Ken-ichi Ito,1 Jun Nakayama2. 1 _Division of Breast, Endocrine and Respiratory Surgery, Department of Surgery(II), Shinshu University School of Medicine, Matsumoto, Japan;_ 2 _Department of Molecular Pathology, Shinshu University Graduate School of Medicine, Matsumoto, Japan_.

Purpose: The ten-eleven translocation (TET) proteins are critical regulators in epigenetic modification. It is well known that TET proteins catalyze the oxidation of 5-methylcytosine (5-mC) to generate 5-hydroxymethylcytosine (5-hmC), and 5-hmC levels reflect diverse functions of TET proteins. It has been reported that the expression of 5-hmC in cancer tissues is decreased compared with that in normal tissues in several organs. However, 5-hmC expression in lung cancer has not been fully analyzed. To examine whether or not 5-hmC was associated with phenotypes or progression of lung cancer, we evaluated 5-hmC expression in a variety of histological types of lung cancers.

Materials and methods: The expression of 5-hmC was analyzed by immunohistochemistry in 109 paired specimens of lung cancer and adjacent normal tissues obtained from the patients underwent operation at Shinshu University Hospital. Eighty-nine adenocarcinomas, 17 squamous cell carcinomas, two large cell carcinomas, and one small cell carcinoma were analyzed in the study.

Results: 5-hmC expression in female was significantly lower than that in male, and was lower in smokers than non-smokers. 5-hmC expression was positive in 34.1±22.0% of lung cancer tissues and 69.4±18.8% of normal tissues. The expression of 5-hmC was significantly decreased in the lung cancer tissues compared with that in the adjacent normal tissues (p<0.001). With regard to the histological types, the positive ratios of 5-hmC expression were 36.0±21.7% in adenocarcinoma, 24.0±20.3% in squamous cell carcinoma, 8.0±7.3% in large cell carcinoma, and 13.0% in small cell carcinoma. Thus, the frequency of positive expression of 5-hmC was decreased in accordance with the increase of grade of malignancy of lung cancer, and a negative correlation between 5-hmC expression and the grade of malignancy of lung cancer was observed. On the other hand, no difference of the expression of 5-mC was observed between lung cancer tissues and normal tissues.

Conclusions: Our results indicate a possibility that 5-hmC may be related to carcinogenesis and progression or differentiation of lung cancer.

#2394

Subpopulation of infiltrating lymphocyte and MGMT methylation in brain tumor prognosis.

Carlos Castaneda, Miluska Castillo, Carolina Belmar-Lopez, Sandro Casavilca, Luis Angel Bernabe, Tatiana Vidaurre. _Instituto Nacional de Enfermedades Neoplasicas, Lima, Peru_.

Background: Clinicopathological features including MGMT methylation have been correlated to prognosis in Glioblastoma (GBM). Tumor infiltrating lymphocytes (TILs) are predictive of clinical outcome in different malignancies. We evaluated association between clinicopathological features including MGMT methylation and IHC infiltrating immune cell subpopulations in 43 resected GBM cases who came from 2008 to 2013.

Methods: A prospective evaluation of TIL (CD3, CD4, CD8 and CD20) and macrophages (CD68 and CD163) through H&E was performed by a pathologist and evaluated Immune cell subpopulations was evaluated through scanning, and software analysis (BX63 Olympus and TissueMorph-Visionpharm) with pathologist support. MGMT evaluation was prospectively performed by bisulphyte PCR test in paraffin samples.

Results: Median age was 47.9 y, men (51.2%), Karnofsky scale 90% (62.8%), median tumor size of 5.1 cm, and partial/ complete tumor resection (22/21). Methylation MGMT test was performed in 31 cases and 17 showed un- methylated, 4 partial methylated and 10 methylated. TILs evaluation by HE showed low- intensity (29 cases), middle- intensity (1) and high- intensity level (9). Pattern of distribution was focal (9 cases), multi- focal (13) and diffuse (17). Median CD3, CD4, CD8, CD20, CD68 and CD163 were 1.6%, 0.032%, 1.6%, 0.03%, 9.1% and 2.2% (regarding total cells), respectively. Higher CD163 level was associated to male (p=0.03) and larger size (p=0.05). Higher levels of CD8 was associated to larger size (p=0.05). Higher CD8 and CD20 was associated to unmethylated MGMT (p=0.041 and 0.013). Median follow- up was 14 months. Only total resection (p<0.05) and MGMT methylation (p<0.05) was associated to outcome. Lymphocyte infiltration (H&E) was not associated to outcome: Lymphocyte intensity (p=0.703), distribution (p=0.61). Longer OS was associated to lower CD4 levels (p=0.01).

Conclusion: Tumor Infiltrating immune cell are not common in GBM, CD8 and CD20 was associated to unmethylation, and CD4 and MGMT methylation were associated to prognosis.

#2395

**Differentiation of carcinoma ex pleomorphic adenoma from pleomorphic adenoma by** TERT **promoter hypermethylation and elevated** TERT **mRNA expression.**

Seungjae Lee,1 Sumit Borah,1 Kurt Patton,2 Armita Bahrami1. 1 _St. Jude Children's Research Hospital, Memphis, TN;_ 2 _Trumbull Laboratories, Memphis, TN_.

The purpose of this study was to determine whether i) genetic or ii) epigenetic aberrations of the TERT promoter, iii) elevated TERT mRNA level or iv) abnormal p53 expression pattern effectively distinguishes cases of carcinoma ex pleomorphic adenoma (CA ex PA) from pleomorphic adenoma (PA). PA is the most common salivary gland tumor which in rare cases progresses into an epithelial malignancy, termed CA ex PA. Fusion genes involving PLAG1, or less commonly HMGA2, are common in both PA and CA ex PA and are thought to be early genetic drivers in their development. Although the key molecular events which occur during malignant transformation of PA are still unknown, some genetic and epigenetic changes proposed to play a role include amplification of PLAG1, HMGA2, MYC, ERBB2, or chromosome 12q and aberrant promoter hypermethylation of RASSF1, especially in combination with promoter hypermethylation of p16, TERT and WT1. The importance of TERT promoter hypermethylation for malignant transformation in PA may be underappreciated since hypermethylation of that region most critical for malignancy in several cancers, termed the upstream of transcriptional start site (UTSS), has not previously been assayed in PA or CA ex PA. Point mutations or genomic rearrangement in the TERT promoter and elevated TERT mRNA levels are also characteristic of many cancers. We used a panel of 10 PA and 6 CA ex PA clinical samples to determine whether these alterations of TERT, or aberrant expression of TERT mRNA or p53 protein, could effectively distinguish CA ex PA from PA. UTSS hypermethylation in each sample was determined by treatment of genomic DNA with sodium bisulfite followed by MassARRAY and Sanger sequencing. Mutation and arrangement of TERT were screened for by Sanger sequencing and fluorescence in situ hybridization, respectively. p53 expression pattern was visualized by immunohistochemistry and TERT mRNA levels were measured by RT-qPCR. Mutation or rearrangements of the TERT promoter were not observed. However, all 6 CA ex PA samples harbored hypermethylated TERT promoter and had much higher TERT mRNA levels than the PA samples. Two of these samples also had aberrant p53 expression patterns, suggesting the presence of a TP53 missense mutation. None of the PA samples had aberrant p53 expression patterns, although 2 had hypermethylated TERT promoter and elevated TERT mRNA levels as compared with the other PA samples. The significance of this for long term outcome in these patients was unclear. Overall, these results suggest that TERT is commonly upregulated in CA ex PA and that this upregulation is often mediated through hypermethylation of the TERT promoter UTSS. Future large scale studies are warranted to determine the usefulness of TERT promoter hypermethylation for differentiating benign PA from CA ex PA, or the PAs at risk of malignant transformation.

#2396

Methylation of subtelomeric repeat D4Z4 in peripheral blood leukocytes is associated with biochemical recurrence in localized prostate cancer patients.

Yuyan Han, Jeri Kim, Xifeng Wu, Jian Gu. _UT MD Anderson, Houston, TX_.

Background: Global DNA methylation may affect chromosome structure and genomic stability and is involved in carcinogenesis. In this study, we aimed to investigate whether methylation of pericentromeric repeat (NBL2) and subtelomeric repeat (D4Z4) in peripheral blood leukocytes was associated with the aggressiveness of prostate cancer (PCa).

Methods: We measured the methylation status of different CpG sites of NBL2 and D4Z4 in 900 PCa patients via pyrosequencing after bisulfite treatment. We then used multivariate logistic regression to estimate the odds ratio (ORs) and 95% confidence intervals (CIs) for analyzing the association of NBL2 and D4Z4 methylation with the aggressiveness of PCa at diagnosis. We then analyzed the association of CpG sites with the risk of biochemical recurrence (BCR) in patients receiving radical prostatectomy or radiotherapy using a multivariate Cox proportional hazards model adjusting for age, BMI, smoking status, pack year, D'Amico risk groups, and treatments. In addition, we used the Kaplan-Meier survival function and log-rank tests to assess BCR-free survival associated with these markers.

Results: There was no significant differences in the methylation level of NBL2 and D4Z4 between clinically defined aggressive and non-aggressive PCa at diagnosis. However, the methylation of subtelomeric region D4Z4 was associated with BCR, while the methylation of NBL2 was not associated with BCR. In tertile analysis, we found that patients with higher methylation of D4Z4 exhibited an increased risk of BCR in localized patients receiving definitive therapy. Because of the relatively low rate of BCR among patients, we combined the highest and second tertiles to increase statistical power. Patients in the second and third (highest) tertiles had an increased risk of BCR (HR=1.74, 95% CI, 1.14-2.65; p= 0.0096) compared to patients in the lowest tertile after adjustment of age, BMI, smoking status, pack year, D'Amico risk groups, and treatments. We assayed 4 CpG sites in this region. In detailed analysis of each CpG site, the association was mostly attributable to the methylation of the 2nd methylation site of D4Z4 (HR=1.80, 95% CI, 1.19 - 2.72).

Conclusion: These data suggest that methylation in the subtelomeric region D4Z4 may be able to predict worse prognosis of localized PCa patients.

#2397

**Epigenetic silencing in clear renal cell carcinoma:** KEAP1 **promoter hypermethylation.**

Federico Pio Fabrizio,1 Manuela Costantini,2 Massimiliano Copetti,1 Anna Maria la Torre,1 Angelo Sparaneo,1 Andrea Fontana,1 Maria Luana Poeta,3 Michele Gallucci,2 Steno Sentinelli,2 Paolo Graziano,1 Paola Parente,1 Vincenzo Pompeo,2 Laura De Salvo,2 Giuseppe Simone,2 Rocco Papalia,2 Francesco Picardo,4 Teresa Balsamo,1 Gerardo Paolo Flammia,5 Domenico Trombetta,1 Angela Pantalone,4 Klaas Kok,6 Paranita Ferronika,6 Lucia Anna Muscarella,1 Vito Michele Fazio1. 1 _IRCCS Casa Sollievo Della Sofferenza, San Giovanni Rotondo, Italy;_ 2 _Regina Elena National Cancer Institute, Rome, Italy;_ 3 _University of Bari, Bari, Italy;_ 4 _University Campus Bio-Medico, Rome, Italy;_ 5 _Campus Bio-Medico University Hospital, Rome, Italy;_ 6 _University Medical Center Groningen, Groningen, Netherlands_.

The Keap1/Nrf2 pathway is a master regulator of the cellular redox state through the induction of several antioxidant defense genes implicated in chemotherapeutic drugs resistance of tumor cells. An increasing body of evidence supports a key role for Keap1/Nrf2 pathway in kidney diseases and renal cell carcinoma, but data concerning the molecular basis and the clinical effect of its deregulation remain incomplete.

Here we performed a comprehensive genetic and epigenetic analysis of the KEAP1 gene in 37 tumor/normal paired tissues of clear cell Renal Carcinoma (ccRCCs). Promoter methylation analysis was performed by using a quantitative methylation specific PCR assay in real time, whereas mutation scanning was performed on FFPE tissues by direct sanger sequencing of the exons 4-7 codifying for the DGR domain of the Keap1 protein.

A tumor-specific DNA methylation of the KEAP1 gene promoter region was found in 18 out of 37 ccRCCs (48,6%) and a direct effects on the modulation of Keap1 mRNA levels was confirmed by in vitro 5-azacytidine treatment on three different ccRCCs cell lines. Analysis of an independent TGCA data set corroborate the epigenetic findings and reveals a significant correlation in multivariate analysis of epigenetic KEAP1 silencing with Overall Survival in ccRCCs.

Our results further suggest that epigenetic deregulation of the Nrf2/Keap1 system could play a pivotal role in the cancerogenesis of ccRCCs. In addition identifying patients with KEAP1 epigenetic abnormalities may contribute to disease progression prediction and response to therapy in ccRCC affected patients.

#2398

Molecular analysis of histone methyltransferases SUV420H1 and SUV420H2 and their potential as prognostic markers in head and neck cancer.

Doralina do Amaral Rabello Ramos, Beatriz Itai Haupt Ribeiro, Tércia Maria Mendes Lousa de Castro, Vívian D'Afonseca da Silva Ferreira, Gustavo Henrique Soares Takano, Eliza Carla Barroso Duarte, Fabiana Pirani Carneiro, Fábio Pittella Silva. _University of Brasilia, Brasilia, Brazil_.

The incidence of head and neck cancer as well as its mortality rates continues to increase, evidencing the need for diagnostic and prognostic markers that can improve clinical management of these patients. Histone Methyltransferases (HMT) are responsible for the methylation of histone tails, a mechanism important for the regulation of gene transcription, cell differentiation and proliferation. Although alterations on these enzymes have been associated with many types of cancer, their relationship with head and neck carcinogenesis is not yet understood. SUV420H1 and SUV420H2 are members of the SUV family of HMTs that contain the conserved SET domain, which catalyzes the addition of methyl groups to specific lysine residues, leading to chromatin compaction and gene repression. In the present study, we investigated the tissue expression profile of SUV420H1 and SUV420H2 using immunohistochemistry in 10 formalin-fixed paraffin-embedded human oral cancer samples and adjacent non-tumor tissues. We also investigated the alterations of SUV420H1 and SUV420H2 genes in head and neck cancer with in silico analyses from public databases. Immunohistochemistry revealed moderate to strong SUV420H1 and SUV420H2 expression in the majority of oral cancer cells, mainly with cytoplasmic staining in the invading tumor cells. In silico analyses using data from 530 head and neck squamous cell carcinoma samples (The Cancer Genome Atlas - TCGA, via cBioPortal) revealed that amplification is the most frequent genetic alteration in SUV420H1. Considering all known lysine methyltransferases, SUV420H1 was among the top three with the highest frequency of amplification. For SUV420H2, the frequency for amplification and deletion alterations was similar. The gene expression level of these methyltransferases was also analyzed. SUV420H1 showed a high expression of mRNA in head and neck cancer samples, with a moderate correlation (R: 0.588) between genetic copy number alteration and mRNA level. SUV420H2 did not show significant altered expression. In addition, SUV420H1 mRNA overexpression was associated with decreased overall survival in head and neck cancer patients (p: 0.0233). Since these enzymes are important in chromatin compaction and gene transcription repression, an increase in their expression level, as detected in this study, could lead to changes in histone methylation pattern, resulting in aberrant silencing of genes essential to maintain normal cellular function, such as tumor suppressor genes. Taken together, our data indicates a possible role of SUV420H1 in head and neck carcinogenesis, with the potential to be used as a prognostic marker for the disease.

#2399

Metabolic and epigenetic targets in multiple myeloma therapy.

Laure Maneix, Fu-Yuan Shih, Polina Iakova, Joanne I. Hsu, Andre Catic. _Baylor College of Medicine, Houston, TX_.

The dynamic interaction of transcription factors (TFs) with promoters and enhancers allows cells to continuously adjust gene expression. Whereas the composition and binding of TFs at genomic sites is the focus of a widespread research effort, relatively little is known about how these complexes are being removed. We previously devised a method to detect genomic locations that are associated with nuclear protein turnover mediated by the ubiquitin-proteasome system (UPS).

Multiple myeloma, the second most common hematopoietic malignancy, has become a model disease for drugs that interfere with the UPS through either blocking or facilitating protein elimination. The proteasome inhibitor Velcade, for instance, has become first-line treatment in myeloma. Yet, our knowledge of how myeloma cells are killed by this drug is incomplete. Our research is focused on defining how proteolysis regulates transcriptional dynamics in this disease and how this impacts Velcade sensitivity.

Following proteasome inhibition, we are utilizing next generation sequencing to identify sites of nuclear protein turnover and evaluate epigenetic changes. Our aim is to identify TFs and epigenetic marks that may qualify as more specific targets for treatment compared to blunt proteasome inhibition. Our findings reveal that certain TFs, including metabolic and cell growth regulators, are particularly sensitive to Velcade. Among these transcriptional regulators, the co-repressor NCoR1 plays a key role in controlling cell metabolism by adjusting mitochondrial gene expression. We are currently validating the mechanism of NCoR1 degradation, with a particular emphasis on the new role of its E3-ubiquitin ligase Seven in absentia 2 (Siah2) in mitochondrial regulation, to better understand how this degradation pathway impacts myeloma proliferation and metabolism.

This research project will contribute to our understanding of epigenetic and transcriptional dynamics in multiple myeloma. With the focus on gene programs that are continuously adapting or changing, we seek to unlock new attractive targets for molecular therapy.

#2400

Identification of histone modifying enzymes associated with tamoxifen resistance in MCF7 breast cancer cells.

Min-Ho Lee, Mi-Ock Lee. _Seoul National Univ. College of Pharmacy, Seoul, Republic of Korea_.

Tamoxifen is widely used to treat estrogen receptor-alpha positive breast cancer. However, resistance to tamoxifen often prevent the success of endocrine therapy. Epigenetic change in tumors are associated with cancer development and progression. Although altered histone modification has been implicated in tumorigenesis, their role in endocrine resistance was little known. To identified tamoxifen-resistance associated histone methylation modifying enzyme, we examined the global histone H3 methylation patterns in tamoxifen-resistance MCF7 breast cancer cells. Our results show that, the levels of methylated histone H3 lysine 4 (H3K4) and histone H3 lysine 9 (H3K9) was upregulated in tamoxifen resistance MCF7 breast cancer cells. Next, we analysis expression levels of H3K4 and H3K9 methylation modifying enzyme in tamoxifen resistance breast cancer cells. qRT-PCR results show that H3K4 methyltransferases (KMT2A and KMT2C) is upregulated in tamoxifen resistance breast cancer cells. Moreover, we observed that si-RNA mediated knockdown of KMT2C was more susceptible to tamoxifen-induced growth inhibition. Finally, Analyses of publicly available patient data sets indicated that KMT2A and KMT2C levels are higher in patients after endocrine therapy failure (recurrence), compared to those of responders (non-recurrence). Our study indicates that elevated levels of KMT2A and KMT2C in the tamoxifen-resistance breast cancers may contributed tamoxifen resistance.

#2401

Connecting oxidative DNA damage to epigenetic alterations during inflammation-driven tumorigenesis.

Ashley Maiuri, Ning Ding, Heather M. O'Hagan. _Indiana University, Bloomington, IN_.

Inflammation contributes to the development of a diverse array of diseases, including cancer. At sites of chronic inflammation epithelial cells are exposed to high levels of reactive oxygen species and undergo cancer-associated DNA methylation changes, suggesting that inflammation initiates epigenetic alterations. Cancer cells are globally DNA hypomethylated, but have aberrant gains in promoter DNA methylation that transcriptionally silence tumor suppressor genes (TSGs), linking DNA methylation directly to tumorigenesis. However, the mechanisms of targeting and initiation for these stable disease-specific epigenetic marks are unknown. We have linked inflammation and oxidative DNA damage to acute changes in the interaction of epigenetic silencing proteins with each other and the chromatin. Recently, we demonstrated that the mismatch repair (MMR) protein heterodimer MSH2-MSH6 participates in the oxidative damage-induced recruitment of DNA methyltransferase 1 (DNMT1) to chromatin and provided evidence that the role of DNMT1 at sites of oxidative damage is to reduce transcription. To connect these findings to epigenetic changes in tumors we use a model of inflammation-driven tumorigenesis where we infect mice with the human commensal bacterium enterotoxigenic Bacteriodes fragilis (ETBF). ETBF infection causes colon inflammation that drives tumorigenesis in Multiple intestinal neoplasia (Min) mice. The central hypothesis for this work is that oxidative damage at sites of inflammation causes acute genome-wide changes in the binding of epigenetic silencing proteins that result in the permanent epigenetic silencing of genes in tumors that form at the sites of exposure. Using genome-wide DNA methylation techniques we identified TSGs with increased promoter DNA methylation and decreased expression in tumors from ETBF infected Min mice as compared to normal tissue. In Min mice lacking expression of Msh2 in their intestinal epithelium (Msh2lox/loxVCMin) there is a loss of the inflammation-induced change in binding of epigenetic proteins that occurs in Min mice even though the cytokine response to infection is normal. Because they lack functional MMR, ETBF infection of Msh2lox/loxVCMin mice causes an increase in colon tumor number and these tumors are positive for microsatellite instability, unlike tumors from ETBF infected Min mice. Importantly, DNA hypo- and hypermethylation changes in tumors from Msh2lox/loxVCMin ETBF infected mice are reduced both globally and at promoter CpG islands of candidate TSGs as compared to tumors from Min ETBF infected mice. Altogether, this work suggests a novel mechanism by which oxidative damage induces acute epigenetic changes through the interaction of DNMT1 with MMR proteins and that this acute change drives DNA methylation alterations during tumorigenesis.

#2403

A novel mechanism for regulation of breast cancer metastasis.

Kenneth Jiao, Yin Peng, Lizhong Wang, Runhua Liu. _University of Alabama at Birmingham, Birmingham,, AL_.

Our major goal is to determine the role of Chromodomain-helicase-DNA-binding protein 7 during breast cancer (BC) metastasis. This protein is an ATP-dependent nucleosome remodeling factor and is critical for embryonic development in both animal models and human patients. Our most recent studies provide direct evidence suggesting its novel role in repressing BC metastasis. We found that this protein, but not the mRNA, is highly expressed in a low-metastatic breast cancer cell line (Mcf-7), while its expression is much reduced in metastatic cell lines (MDA-MB-468 and MDA-MB-231). Furthermore, this protein is predominantly localized in the cytoplasm of 468 and 231 cells, in contrast to its nuclear localization in Mcf-7 cells. In the cytoplasm of 468 and 231 cells, this protein is colocalized with a lysosome marker, suggesting that in high metastatic BC cells, this protein is exported to the cytoplasm to be degraded in lysosomes. Blocking the activity of lysosomes increased expression of this protein in high metastatic BC cell lines. This result suggests a mechanism accounting for the reduced expression of this protein in high metastatic BC cell lines. To further support the potential clinic relevance of our results, this protein is localized in the nucleus of almost all duct epithelial cells in normal breast tissues; however, in a large portion of epithelial cells of breast tumors, this protein is localized in the cytoplasm. Through reporter and mutagenesis analysis, we identified the nuclear exporting signal (NES) sequence responsible for exporting this protein into cytoplasm. In the next set of experiments, we attempted to determine the function of this gene in BC metastasis. We found that knocking down expression of this gene in 468 cell significantly increased their migration and invasion. We next applied the CRISPR genome editing technique to mutate the critical amino acids within the NES region in the endogenous gene locus and found that the amount of protein in the nucleus is significantly increased compared to wild type cells. Migration/invasion of these cells was also dramatically decreased. At the molecular level, we found that forced expression of this protein in nucleus reduced expression of epithelial markers and increased expression of epithelial-mesenchymal-transition markers. We are now applying ChIP-Seq and RNA-Seq approaches to determine direct regulatory network of this protein in 231 cells. We will further test whether this protein acts through modulating the epigenetic status of the enhancers/promoters to regulate expression of its target genes to repress BC metastasis. In summary, our data collectively suggest that Chromodomain-helicase-DNA-binding protein 7 is a novel epigenetic regulator to repress BC metastasis. To the best of our knowledge, our study represents the first to address the activity of this protein in BC.

#2404

Paternal intake of an obesity-inducing diet before conception modulates the risk of pancreatic cancer in offspring in a mouse model.

Raquel Santana Da Cruz, Johan Clarke, Ali Baird, Hong Cao, Carlos Benitez, M. Idalia Cruz, Sonia De Assis. _Georgetown University Medical Center, Washington, DC_.

Pancreatic cancer is a mostly untreatable malignancy, with 5-year survival rates of about 5%. Life-style and dietary factors have been associated with pancreatic cancer risk. Particularly, the consumption of high-fat diets and obesity (and underlying metabolic dysfunction) have been linked to increased susceptibility to this cancer. Recent studies have shown that the paternal diet and life-style can have a significant influence on offspring's health via epigenetic information transmitted in the germ-line. Paternal overweight before conception has been shown to increase offspring's susceptibility of developing metabolic diseases and some types of cancer. Here, we evaluated the effects of paternal overweight in the susceptibility of pancreatic cancer in offspring using the P48Cre/+ /KrasG120/+ mouse model of pancreatic cancer. LSL-KrasG120/+ and P48Cre/+ male mice were fed either an obesity-inducing (OID) or control (CO) diet for 8 weeks from weaning to sexual maturity. After this period, OID-fed and CO-fed male mice were housed together with 7 week-old female mice, with free access to CO diet, for 3 days. Pregnant dams were kept on the CO diet during pregnancy and after giving birth. Pups were weaned from mothers at 21 days of age, fed a standard chow diet for the extent of the study and weighed weekly. The offspring of CO or OID fathers were used to study body weight, metabolic parameters and pancreatic cancer development. Fathers fed an OID gained significantly more weight (CO 15.7±1.0g; OID 19.7±1.3g, p=0.02) and had higher leptin levels (p=0.04), compare to CO group fathers. Body weight analyses of OID and CO offspring, showed gender-specific effects. While the OID female offspring had higher weight at birth (p=0.005) and at weaning (p=0.02), compare to CO group, no significant differences were observed between the CO and OID male offspring. Those gender-specific differences were also observed in metabolic parameters such GTT with the OID male, but not female, offspring showing impaired glucose tolerance (p=0.0004) compared to CO. While the monitoring period is still ongoing, 8-week old OID offspring present gender-specific differences in susceptibility to pancreatic cancer: Males OID offspring have higher number/area (481±25; 2.6±0.3) of pancreatic intraepithelial neoplasia (PanIn), compared to CO (359±61; 2.0±0.5). On the other hand, female OID offspring have similar levels of PanIn, but have higher incidence of PDACs compare to CO. In conclusion, an ancestral history of overweight through the paternal lineage may be associated with an increased susceptibility to pancreatic cancer development in adulthood. The mechanisms mediating this effect remain to be elucidated.

#2405

The 8p11 amplicon oncogenes ASH2L and NSD3 alter the epigenomic landscape and provide the foundation for novel application of epigenetic therapy in luminal B breast cancers.

Jamie N. Mills, Stephen P. Ethier. _Medical University of South Carolina, Charleston, SC_.

The 8p11-p12 genomic region is amplified in 15% of breast cancers and is associated with poorer prognosis, especially in the luminal B (ER+) subtype. This region harbors several oncogenes, three of which are epigenetic modifiers of chromatin: NSD3 (WHSC1L1) and ASH2L, which are histone methyltransferases, and KAT6A, a histone acetyltransferase. NSD3 preferentially di-methylates histone 3 lysine 36 (H3K36me2) in gene bodies to facilitate elongation, and ASH2L primarily tri-methylates histone 3 lysine 4 (H3K4me3) in promoter regions, which is also associated with active transcription. Previous work in the amplicon-bearing cell line SUM-44 implicated NSD3 in growth, survival, and overexpression and estrogen-independent activation of the estrogen receptor. Current work is focused on elucidating the mechanism behind these transforming features and exploring the epigenomic interactions of 8p11 oncogenes. Chromatin immunoprecipitation and high throughput sequencing (ChIP-seq) using an NSD3 antibody confirmed the presence of this factor in the gene body of ESR1, as well as several other important genes. As NSD3 lacks the ability to bind chromatin directly, it relies on adaptor proteins such as BRD4, a member of the BET bromodomain family that binds acetylated histones such as those generated by KAT6A. Indeed, we have shown that BRD4 binds NSD3 in SUM-44 cells. Similar to our previous findings with NSD3, knockdown of BRD4 and ASH2L reduced cell proliferation and clonogenic potential. To examine the influence of ASH2L on histone methylation in gene promotors, we performed ChIP-seq using an H3K4me3 antibody. The data showed a dramatic decrease in the number of peaks called following knockdown of ASH2L and peaks in the NSD3 promotor disappeared. This finding solidifies the connection between these oncogenes and their cooperative regulation of the epigenome, and therefore the transcriptome, in this cell line, explaining the effects of NSD3 on cell growth and behavior. Amplification and overexpression of oncogenes in the 8p11 region is most prevalent in ER+ breast cancer, treated clinically with endocrine therapies. Patients with poor response to these therapies have few alternatives and often succumb to their disease. This study and continued investigation of 8p11 amplicon oncogenes opens an important new treatment opportunity for these patients: epigenetic therapy. Previous work identifying these patients as candidates for novel anti-estrogen SERD compounds suggests that combination therapy with a BET bromodomain inhibitor could have major therapeutic benefit in a patient group currently without treatment options, thereby improving patient outcomes by a novel mechanism in luminal B breast cancer.

#2406

LSD1 promotes castration-resistant prostate cancer cell survival independently of the androgen receptor and of histone demethylation.

Archana Sehrawat,1 Lina Gao,1 Junior Tayou,1 Armand Bankhead,1 Laura M. Heiser,1 Carly J. King,1 Yuliang Wang,1 Jacob Schwartzman,1 Joshua Urrutia,1 Daniel J. Coleman,1 Sheila Weinmann,2 Bhaskar V. Kallakury,3 Deborah L. Berry,3 Reina Haque,4 Stephen K. Van Den Eeden,5 Tomasz M. Beer,1 George V. Thomas,1 Shannon McWeeney,1 Joshi J. Alumkal1. 1 _OHSU Knight Cancer Institute, Portland, OR;_ 2 _Kaiser Permanente NW, Portland, OR;_ 3 _Georgetown University Medical Center, Washington, DC;_ 4 _Southern California Kaiser Permanente Medical Group, Pasadena, CA;_ 5 _Kaiser Permanente Northern California, Oakland, CA_.

Background: Androgen deprivation therapy (ADT) or interference with androgen receptor (AR) function is the principal treatment for advanced prostate cancer. However, progression is universal, and therapies following the emergence of castration resistance do not offer durable control of the disease. Lysine specific demethylase 1 (LSD1) is a histone demethylase and a key regulator of gene expression in cancer. Prior work demonstrates that LSD1 may act as a cofactor of the AR in androgen-dependent prostate cancer cells. In this report, we describe a distinct role of LSD1 as a driver of proliferation and survival of castration-resistant prostate cancer (CRPC) cells independently of the AR and independently of histone demethylation.

Methods: We used gain and loss of function studies to determine the importance of LSD1 for survival of prostate cancer cells. To identify transcriptional networks that contribute to cell survival, we suppressed LSD1 with RNAi and measured gene expression changes with microarrays. To determine the importance of histone demethylation in regulation of these gene networks, we suppressed LSD1 and measured levels of LSD1 canonical histone substrates (H3K4me2 and H3K9me2) genome-wide with chromatin immunoprecipitation-sequencing.

Results: Cell viability assays demonstrated that LSD1 is important for proliferation and survival of CRPC cells independently of the AR. Microarray studies demonstrated that LSD1 activates androgen-independent genes that comprise cell cycle and embryonic stem cell maintenance gene sets that are enriched in lethal human tumors. Importantly, our global epigenomic studies after LSD1 suppression demonstrated that LSD1 activates these gene sets independently of demethylation of its canonical histone substrates.

Conclusions: Our results demonstrate that LSD1 promotes CRPC cell survival independently of the AR and suggest that LSD1 regulates key pathways in CRPC through demethylation of non-histone substrates or via a scaffold function―mechanisms we are currently investigating. In summary, LSD1 contributes to CRPC cell survival through non-canonical mechanisms and represents an attractive therapeutic target in lethal prostate cancer.

#2407

Inhibition of gene expression during non-alcoholic steatohepatitis (NASH)-related hepatocarcinogenesis is mediated by histone H4 lysine 16 deacetylation.

Kostiantyn Dreval,1 Aline de Conti,1 Orish Ebere Orisakwe,2 Frederick A. Beland,1 Igor P. Pogribny1. 1 _FDA-National Center for Toxicological Research, Jefferson, AR;_ 2 _University of Port Harcourt, Rivers State, Nigeria_.

Hepatocellular carcinoma (HCC) is one of the most aggressive human cancers and the incidence is steadily increasing worldwide. The molecular mechanisms leading to the development of HCC consist of genetic and epigenetic aberrations, including changes in global and gene-specific DNA methylation and altered expression of several classes of non-coding RNAs. While changes in DNA methylation and microRNA expression during the development of HCC are well-studied, the role of aberrant post-translational histone modifications remains unexplored. Using a mouse Stelic Animal Model (STAM) of NASH-associated liver carcinogenesis, we have investigated alterations in hepatic histone modifications at steatotic (6 weeks), fibrotic (12 weeks), and full-fledged HCC (20 weeks) stages of the HCC development. NASH-related liver carcinogenesis was characterized by a progressive decrease in the levels of histone H4 lysine 20 trimethylation (H4K20me3) and histone H4 lysine 16 acetylation (H4K16ac), with the greatest decrease occurring in full-fledged tumors. Mechanistically, the deacetylation of H4K16ac was associated with nuclear protein 1 (Nupr1)-mediated inhibition of histone lysine acetyltransferase KAT8. In addition to a global loss of hepatic H4K16ac, there was a significant decrease of gene-specific H4K16ac, as indicated by a reduced level of H4K16ac in 16 out of 25 critical cancer-related genes. More importantly, the changes in the extent of gene-specific H4K16 deacetylation were positively correlated with the level of gene transcription (r = 0.965, p < 0.01). These results indicate that a reduction of global and gene-specific of H4K16ac is a key pathophysiological mechanism contributing to the development of NAFLD-derived HCC.

#2408

Epigenomic characterization of MSC from myeloid malignancies.

Manuel Rodríguez-Paredes,1 Stefanie Geyh,2 Mahshid Gazorpak,1 Julian Gutekunst,1 Felix Bormann,1 Rainer Haas,2 Thomas Schröder,2 Frank Lyko1. 1 _German Cancer Research Center, Heidelberg, Germany;_ 2 _University of Düsseldorf Medical Faculty, Düsseldorf, Germany_.

Ineffective hematopoiesis is a hallmark of myeloid malignancies such as Myelodysplastic Syndromes (MDS) or Acute Myeloid Leukemia (AML). Our previous research has shown that mesenchymal stromal cells (MSC), one of the main cellular regulatory components of the bone marrow niche and essential for the differentiation of new blood cells from Hematopoietic Stem Cells (HSC), are molecularly and functionally impaired in MDS and AML patients. More specifically, these cells display reduced proliferative and differentiation capacities, an increased cellular senescence, or the altered expression of key hematopoietic factors, defects that can provide an explanation for the cytopenia associated with these diseases. But most interestingly, we have also discovered that MDS- and AML-derived MSC present aberrant methylomes. Our data, based on 450k DNA methylation microarrays, show that these abnormalities are shared by the MSC from both malignancies and affect key transcription factors like TBX15, PITX2 or HOXB6, formerly linked to differentiation and development. This results can account for some of the defects observed in the disease-related MSC and represent an important paradigm for cancer-induced epigenetic changes in normal cells. Aiming to further explore the importance of this epigenetic deregulation, we performed RNAseq experiments using MSC from healthy donors as well as from MDS and AML patients. This approach not only suggests a profound impact of the epigenetic aberrancies on gene expression, but also allowed us to identify TGFβ as the soluble factor that, released by the malignant cells in the bone marrow, appears to trigger the above-mentioned defects in the MDS- and AML-derived MSC. Indeed, our results show that, when treated with TGFβ, healthy MSC undergo the deregulation of TBX15, PITX2 or HOXB6 and develop some of the observed aberrant phenotypes. Conversely, the treatment of MDS- and AML-derived MSC with a TGFβ antagonist like SD208 can abrogate these effects. Finally, our preliminary DNA methylation data reveal a very similar deregulation in the methylome of MSC from Multiple Myeloma (MM) patients, strongly suggesting that the same scenario could also be common to other hematological malignancies from the lymphoid branch.

#2409

**Loss of** L1TD1 **suppresses Notch1 induced** LINE1 **methylation and genomic stability.**

Chia-Chen Chiu,1 Wei-Chen Huang,1 Kuan-Der Lee,2 Chih-Cheng Chen,2 Chia-Chen Hsu,2 Mei-Ling Kang,1 Yu-Wei Leu,1 Shu-Huei Hsiao1. 1 _National Chung Cheng Univ., Chiayi County, Taiwan;_ 2 _Chang Gung Memorial Hospital, Chiayi County, Taiwan_.

The silencing of endogenous retrotransposon like LINE1 is critical for the maintenance of genomic stability and the LINE-1 type transposase domain containing 1 (L1TD1) gene possesses the repeated, putative LINE-1 RNA-binding domains and was hypothesized to regulate the activity of LINE-1 through DNA methylation. To verify if L1TD1 is responsible for LINE-1 silencing and the maintenance of somatic genome stability, CRSPR/Cas9 system was used to knockout (KO) L1TD1 locus in gastric, colon and breast cancer cells so as in the mesenchymal stem cells (MSCs). We observed that L1TD1 KO blocked the entrance of SUV39H1, histone methyl transferase, into cell nuclear which suppressed the tri-methylation of histone 3 at lysine 9 (H3k9me3) and LINE-1 methylation. A global demethylation and reduced HP1α recruitment were also observed. The global distribution of CTCF binding loci was also distorted as well as the bivalent histone marks. Further, L1TD1 KO distorted normal distribution of RassF1A expression, cytoskeleton conformation and therefore cell stiffness. The MSC-to-neuron differentiation was also blocked by the KO. Further, we found that overexpressed Notch-1 increased DNA methylation within LINE1 promoter, and this increase was attenuated by L1TD1 KO. Therefore, we conclude that the external signals like Notch1 affect L1NE1 methylation and genome stability through possible LINE1 interacting L1TD1. Since L1TD1 hypermethylation was observed in colon (n=100), gastric (n=19) and breast (n=79) cancers, L1TD1 abnormality is then a candidate for cancer biomarker. (Supported by: MOST-105-2320-B-194-004, MOST-105-2314-B-182A-134, MOST Taiwan and CMRPG6F0092, Chang Gung Memorial Hospital, Chia-Yi, Taiwan)

#2410

Toward mutation analysis of regulatory elements: Epigenetic profiling of primary breast tumors.

Samah El Ghamrasni, Paul Guilhamon, Rene Quevedo, Cindy Yang, Mathieu Lupien, Trevor Pugh. _Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada_.

Non-coding mutations found in regulatory elements can function as driver mutations in breast cancer by changing the binding affinity of transcription factors for DNA, thereby resulting in direct change of expression of genes that promote cancer development. Identifying such additional driver mutations can reveal the molecular mechanisms favorable to breast cancer development and progression, as well as reveal new biomarkers to better tailor personalized/precision cancer medicine. In this study we have collected 20 primary luminal breast tumors and optimized experimental workflow to dissociate solid tumors and map open chromatin using ATAC-seq. In our initial experiments using ATAC-seq profiling of bulk tumor tissues, we were able to call an average of 15x103 peaks. Subsequently, flow cytometry analysis showed the presence of 15-25% of immune cells in our primary tumors. Therefore, we have optimized a workflow to eliminate immune cells and focus mainly on epithelial tumor cells. Primary breast tumors were digested using collagenase and further dissociated with dispase. Cells were sorted into two populations (mammary epithelial and immune cells) using anti-CD45, anti-CD49f and anti-EpCAM antibodies. Sorted mammary epithelial cells were then used for ATAC- and RNA-seq library preparation as well as for generation of patient derived organoids. Our new workflow resulted in an increased number of called peaks (40x103 vs 15x103), as well as a significant increase in the percentage of unique peaks compared to bulk sequencing (45% vs 15%). By refining our workflow to enrich for tumour content, we will continue our ongoing effort to profile these open chromatin regions and contextualize the mutations within in a large cohort of luminal breast cancers using targeted sequencing. These data will be compared with large-scale whole genome data generated by our group and made publicly available by others.

#2411

The role of A-to-I RNA editing in human cancer.

Han Liang. _UT MD Anderson Cancer Ctr., Houston, TX_.

Recent studies show that A-to-I RNA editing introduces a large number of specific nucleotide changes in cancer transcriptomes. Using the genomic/transcriptomic data from The Cancer Genome Atlas, we systematically characterized the landscape of RNA editing in a variety of cancer types and revealed many sites with clinically relevant patterns. We further integrated proteomic data from the Clinical Proteomic Tumor Analysis Consortium and found that mutations and RNA editing introduced similar numbers of amino acids changes per tumor sample in breast and ovarian cancers. The amino acid changes engendered by these RNA editing events may significantly alter protein stability. Some of the identified RNA editing events show critical consequent changes driving tumor growth and altering therapeutic activity. We also identified the RNA editing hotspots in miRNAs across cancer types, and some of RNA editing events modify the miRNA target genes. They could function as potential therapeutic targets and therapeutics. Our study suggests a large contribution of A-to-I RNA editing to protein diversity and gene regulation, and highlights its translational potential as both biomarkers and therapeutic targets.

#2412

Monitoring small molecules and macromolecules methyltransferase activities using homogenous luminescent assay.

Said A. Goueli, Kevin Hsiao. _Promega Corp., Madison, WI_.

It is well recognized that methylation/demethylation of not only DNA, RNA, Proteins but also methylation of small molecules play major roles in modulation of the epigenome, transcriptome, neurotransmitter uptake and metabolic regulation. Recent biochemicval and biological data suggest that the activity of these enzymes and their expression level are under very strict regulation and any abnormal alteration in either one or both results in a wide varieties of pathogenic conditions such as cancer, inflammation, and neurodegenerative diseases. In addition to chromatin modulation, altered methylation of DNA and more recently mRNA have been recognized to regulate the transcriptome and the rate of message translation and stability. Furthermore, methylation of small molecules such as catechols and nicotinamide play critical roles in neurotransmitters uptake and function, and metabolic regulation, respectively. Thus, pharmacological modulation of these enzymes by small molecules will be beneficial in developing novel therapeutics for multiple unmet medical needs. Towards this goal of searching for activators/inhibitors of these enzymes for the development of next generation of drugs, screening assays for these modulators are urgently needed. To address these unmet needs, we have developed a novel assay that monitors the activities of these enzymes and their modulation by small molecules. The assay is bioluminescent based, HTS formatted and highly sensitive. A unique feature of this assay is its universality since it is based on monitoring the formation of the universal product S-adenosylhomocysteine (SAH), i.e., capable of detecting changes in activity of a broad range of methyltransferases such as DNA, RNA, protein, and small molecules. In addition, the assay has been validated for all classes of protein methyltransferases (Lysine and Arginine), and with different types of substrates (small peptides, large proteins, or even nucleosomes). This enables determining the specificity of these enzymes and their substrate requirements. The assay has high signal to background, low C.V., robust (Z' value > 0.7), and has been validated using various plate densities such as 96-, 384, and 1536-well plates. A strong feature of this assay is its utility with broad range of substrates concentrations or the composition of the substrates (short vs. long peptides), thus enabling the generation of kinetic data and determining the mechanism of action of various modulators of methyltransferases of interest.

#2413

**SIRT1 regulates** Mxd1 **throughout melanoma progression.**

Fabiana M. Meliso,1 Danilo Micali,1 Camila T. Silva,1 Thais S. Sabedot,2 Simon G. Coetzee,2 Adrian Koch,3 Fabian B. Fahlbusch,3 Houtan Noushmehr,2 Regine Schneider-Stock,3 Miriam G. Jasiulionis1. 1 _Universidade Federal de São Paulo, São Paulo, Brazil;_ 2 _Universidade de São Paulo, Ribeirão Preto, Brazil;_ 3 _Friedrich-Alexander University, Erlangen, Germany_.

In a murine melanoma model of malignant transformation promoted by sustained stress, increased levels of reactive oxygen species were found to result in DNA damage and were related to massive epigenetic alterations. Since the chromatin modifier Sirtuin-1 (SIRT1) is a protein attracted to double-stranded DNA break (DSB) sites and can recruit other components of the epigenetic machinery, we aimed to define the role of SIRT1 in melanomagenesis through our melanoma model. The DNA damage marker, γH2AX was found increased in melanocytes after 24 hours of deadhesion, accompanied by increased SIRT1 expression and decreased levels of its target, H4K16ac. Moreover, SIRT1 started to be associated to DNMT3B during the stress condition, and this complex was maintained along malignant progression. Mxd1 was identified by ChIP-seq among the DNA sequences differentially associated with SIRT1 during deadhesion and was shown to be a common target of both, SIRT1 and DNMT3B. In addition, Mxd1 was found down-regulated from pre-malignant melanocytes to metastatic melanoma cells. Treatment with DNMT inhibitor 5AzaCdR reversed the Mxd1 expression. Sirt1 stable silencing increased Mxd1 mRNA expression and led to up-regulation of MYC targets, such as Cdkn1a, Bcl2 and Psen2, the upregulation of which is associated with human melanoma aggressiveness and poor prognosis. We demonstrated a novel role of the stress responsive protein SIRT1 in malignant transformation of melanocytes associated with deadhesion. Mxd1 was identified as a new SIRT1 target gene. SIRT1 promoted Mxd1 silencing, which led to increased activity of MYC oncogene contributing to melanoma progression.

### Genome-Wide Analysis of Transcriptional Regulation

#2414

Fibromodulin expression in human prostate cancer tissue: Implications for prostate cancer progression.

Estebana Ines Benedetti Padron,1 Angelo De Marzo,2 Javier Baena Del Valle,2 Jan Geliebter,3 Niradiz Reyes1. 1 _Universidad de Cartagena, Cartagena, Colombia;_ 2 _Johns Hopkins University, Baltimore, MD;_ 3 _New York Medical College, Valhalla, NY_.

Background: Fibromodulin is a small leucine-rich proteoglycan important for extracellular matrix organization and essential for tissue repair in multiple organs. Recently, differential expression of fibromodulin has been shown in human prostate cancer cell lines with different cancer phenotypes and in human prostate cancer tissue compared to normal/benign tissue.

Methods: The protein expression level of fibromodulin was analyzed by immunohistochemistry using a prostate tissue microarray (TMA), constructed from archival radical prostatectomy and trans-urethral prostatectomy tissue specimens, from patients with localized prostate cancer (PCa). Stained sections from the donor blocks were evaluated, and a representative area from PCa, high grade prostatic intraepithelial neoplasia (HGPIN), proliferative inflammatory atrophy (PIA), and benign prostate tissue (BPT) was included into the TMA. Staining for each lesion was scored, using H-score, by microscopic assessment of the percentage of epithelial cells with positive staining. H-score values were compared between groups by ANOVA test, and p < 0.05 was considered statistically significant. The Ethical Review Board of the academic institution approved this study.

Results: Fibromodulin expression at protein level was evaluated in 378 cores representing PCa (143 cores), HGPIN (58), PIA (102), and BPT (78). Mean fibromodulin expression in cores with PCa, HGPIN, and PIA was significantly higher than mean of fibromodulin expression in normal glands (p<0.0001). There was a higher mean staining score in PCa (156, 95% CI:144–167), compared to HGPIN (131.4, 95% CI:109-153), PIA (128, 95% CI:117-138), and BPT (33.3, 95% CI:27-38). Thus, fibromodulin expression increased across the PCa progression model.

Conclusions: The expression pattern of fibromodulin showed a significant increase from benign prostate tissue through pre-cancerous lesions to PCa. These findings suggest that this proteoglycan may be involved in the process of prostate cancer progression, with low levels in pre-cancerous lesions and higher levels in cancer tissue. Additional studies are required to determine the biological role and potential clinical implications of fibromodulin in prostate cancer.

#2415

Genome-wide alterations in gene expression of prostate cancer (PC) cells surviving neo-adjuvant androgen deprivation therapy.

Anna C. Ferrari,1 Hatem Sabaawy,1 Mark Stein,1 David Foran,1 Ying Chen,1 Srinivasan Yegnasubramanian2. 1 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ;_ 2 _Johns Hopkins University Sidney Kimmel Cancer Center, Baltimore, MD_.

Background: Although androgen deprivation therapy initially decreases PC tumor burden, resistance to further androgen receptor (AR)-directed treatments or chemotherapy is inevitable once CRPC is established. We postulated that the stress of ADT triggers widespread alterations in expression that renders a metastable physiologic state conditioned by epigenetic changes that might be initially reversible by targeting non-androgen pathways. We conducted a pilot study to explore genome-wide expression alterations in PC foci surviving 3 months ADT (eADT).

Methods: mRNA from 7 frozen microdissected PC foci and normal counterparts (NC) were processed for RNA-seq. RNA-seq changes in eADT specimens were compared first with NC and the untreated PC in the TCGA PRAD (TCGA) database to castrate resistant (mCRPC) specimens in the dbGAP study phs000915.v1.p1database. The raw data (fastq files) was quantified using kallisto, normalized by TMM using R package edgeR, batch effects corrected using R package SVA. Analysis of differential gene expression by R package sleuth. Pathway and gene set by GSEA, GAGE/pathview packages for Gene Ontology (GO) and KEGG.

Results: TMPRSS2-ERG+, 5/7. Highest DEG in eADT vs. TCGA vs mCRPC were non-coding RNA's. Among 17431 differentially regulated paths; GSEA of eADT vs TCGA or mCRPC: 341 (1.95%) and 1366 (7.84%) up- vs 46 (0.26%) and 59 (0.34%) down-regulated. KEGG paths, eADT vs. TCGA or mCRPC, 11 and 53 up vs. 2 and 3 down- respectively. Highly down- path in eADT vs TCGA (log q<10-17) was ribosomal vs. cell cycle and DNA replication in mCRPC. Six paths significantly up- in eADT vs TCGA or mCRPC: Wnt, adherence junction, steroid biosynthesis, unsaturated fatty acids, citrate cycle, ErbB. Calcium, MAPK, insulin, GnRH and Hedgehog were also up- in eADT vs mCRPC. AR full-length was marginally higher in eADT than TCGA and lower than mCRPC, no differences in gene targets.

Conclusions: This pilot data shows that ADT triggers a wide range of gene expression alterations that support PC cell survival and may be vulnerable to therapeutic targeting in addition to the androgen pathway. Validation of these findings is planned in a larger set of samples from the same bank.

#2416

Transcriptome and cluster analysis of infant acute lymphoblastic leukemia cases with and without MLL (KMT2A) rearrangement.

Byunggil Yoo,1 Midhat S. Farooqi,1 Rumen Kostadinov,2 Neil Miller,1 Jeff Johnston,1 Emily Farrow,1 Shannon Kelley,2 Margaret Gibson,1 Patrick Brown,2 Erin Guest1. 1 _Children's Mercy Hospital and Clinics, Kansas City, MO;_ 2 _Johns Hopkins University, Baltimore, MD_.

Background: Infant acute lymphoblastic leukemia (ALL) is a high-risk cancer with poor survival due to treatment failure and relapse. The majority of infant ALL cases possess an MLL (KMT2A) translocation (MLL-R), where MLL joins with AF4 (AFF1), ENL (MLLT1), or another partner gene to create a novel fusion gene. A subset of infant ALL cases do not involve an MLL rearrangement (non-MLL-R). Gene expression profiling delineates MLL-R from non-MLL-R cases. Here, we describe transcriptome profiling and cluster analysis of MLL-R and non-MLL-R cases and compare between partner genes.

Methods: We performed RNA sequencing (RNA-seq) on diagnostic blood or bone marrow samples from 40 infants with ALL. Cohort A included 16 patients with MLL-R ALL known to have relapsed; cohort B, 10 patients with MLL-R ALL not known to have relapsed; and cohort C, 14 patients with non-MLL-R ALL whom to date have also not relapsed. RNA-seq was performed to an average of 8.8Gb and analyzed using Bowtie2, RSEM, and limma-voom. Multiple different groups of cases (e.g. cohort A versus B) were compared for differential gene expression; the resulting genes were ranked by adjusted P value with a significance cutoff of 0.01 and further evaluated for pathway analysis with Ingenuity software. Hierarchical clustering of samples was conducted using distance measures computed from normalized gene expression.

Results: Transcriptome analysis of MLL-R versus non-MLL-R cases found 2,334 differentially expressed genes. Ingenuity pathway analysis of these genes showed enrichment in specific gene sets, such as those involved in PTEN, EGF, and NF-κB signaling. Ingenuity predicted increased PTEN signaling and diminished EGF and NF-κB signaling in MLL-R cases relative to non-MLL-R cases. Transcriptome analysis of relapsed versus non-relapsed MLL-R cases found no differentially expressed genes meeting significance criteria, while analysis of MLL fusion partners identified 46 differentially expressed genes including BMI1 and MYC. Cluster analysis demonstrated a distinct grouping of non-MLL-R cases relative to MLL-R cases. Additionally, MLL-R samples sub-clustered according to their MLL fusion gene partners. However, no difference in clustering was appreciated between MLL-R cases that later went on to relapse versus those that did not.

Conclusions: Our findings show that MLL-R infant ALL has a distinct gene expression signature at diagnosis that correlates with the type of MLL translocation present. This is important because it suggests treatment strategies could be tailored to the MLL partner gene. Risk of relapse in MLL-R cases could not be predicted by this signature due to a paucity of differentially expressed genes between relapsed and non-relapsed groups at diagnosis. Similar to prior profiling studies, we find that non-MLL-R cases independently cluster together and have a gene expression signature distinct from MLL-R cases.

#2417

Large scale integrated transcriptomic and epigenetic profiling defines the molecular hallmarks of HGSOC and disease origins.

Kate Lawrenson,1 Marcos Abraao,2 Felipe Segato,2 Janet M. Lee,1 Simon Coetzee,1 Ji-Heui Seo,3 Matthew L. Freedman,3 Dennis Hazelett,1 Simon Gayther,1 Houtan Noushmehr4. 1 _Cedars-Sinai Medical Center, Los Angeles, CA;_ 2 _Ribeirão Preto Medical School, Sao Paulo, Brazil;_ 3 _Dana-Faber Cancer Center, Boston, MA;_ 4 _Henry Ford Hospital, Detroit, MI_.

High-grade serous ovarian cancers (HGSOCs) are the most common subtype of epithelial ovarian cancer. Pathways driving HGSOC development are poorly understood, and the cellular origins are debated in the literature. Previously, ovarian surface epithelial cells (OSECs) were thought to be cellular precursors of HGSOC, but there is now strong evidence that at least half of all HGSOCs arise from fallopian tube secretory epithelial cells (FTSECs). We took a large-scale integrated transcriptomic-epigenomic profiling approach to defining the molecular hallmarks of HGSOC and to explore the disease cell-of-origin. RNA sequencing was performed on 121 OSEC cultures and 84 FTSEC cultures and integrated with 394 HGSOC transcriptomes profiled by TCGA. We identified around 100 genes that differentiate OSECs and FTSECs, and pathway analysis of this gene list identified ovarian cancer genes as highly enriched (p=2.2x10-8), with ZEB1 and estrogen the most significant predicted upstream regulators (P<3.7x10-7). We integrated gene expression data with clinical metadata to identify associations between gene expression signatures and clinical variables and found that in patients diagnosed with ovarian cancer, OSEC and FTSEC profiles converged, suggestive of a field cancerization phenotype occurring in the normal OSECs on the unaffected ovary contralateral to a clinically diagnosed ovarian carcinoma. Transcriptomic signatures for OSECs, FTSECs and HGSOCs were then merged with superenhancers defined by performing chromatin immunoprecipitation data for acetylated H3K27 profiled in primary HGSOC tissue specimens. We detected many more superenhancer-target gene relationships in the normal cells than in the tumor cells, suggesting superenhancer fingerprints define the cell of origin rather than determining upregulation of oncogenes driving cancer. To explore this further we determined the similarities of HGSOCs to OSEC and FTSEC using a machine learning model. Using a pre-defined gene signature of OSECs and FTSECs, our model (high specificity and sensitivity; 95% CI = 0.77-1, p-value=6.1-5) was applied to HGSOCs and we observed that the majority of HGSOCs were classified as FTSEC-like. Interestingly, the mesenchymal subtypes were enriched as FTSEC-like. Moreover, a subset of HGSOCs were characterized as OSEC-like, and these tumors were associated with poorer outcomes. Our data describe molecular subgroups within HGSOC precursor tissues as well as defining the transcriptional networks that define HGSOC precursor tissues, drive HGSOC development; furthermore we describe the interplay between the epigenetic landscape and the transcriptome in these cell types. In closing, these large-scale integrative analyses support a predominantly FTSEC origin for HGSOCs, but reinforce conclusions that OSECs cannot be excluded as origins of HGSOC, and may represent a subset of tumors with a different biology.

#2418

Characterization of the PAX8 regulatory network in epithelial ovarian cancer.

Rosario I. Corona,1 Emily Adler,2 Janet M. Lee,1 Norma Rodriguez-Malave,1 Paulette Mhawech-Fauceglia,2 Heidi Sowter,3 Dennis J. Hazelett,1 Kate Lawrenson,1 Simon A. Gather1. 1 _Cedars-Sinai Medical Center, Los Angeles, CA;_ 2 _University of Southern California/Keck School of Medicine, Los Angeles, CA;_ 3 _University of Derby, Derby, United Kingdom_.

PAX8 is a lineage-restricted transcription factor expressed in a large proportion of epithelial ovarian cancers (EOCs). PAX8 is commonly upregulated in EOCs relative to precursor tissues, suggesting it functions as an oncogene. However, the biological role of PAX8 during cancer initiation and development is poorly understood, and the genome-wide transcriptional targets have yet to be comprehensively catalogued. Using stable models of PAX8 knockdown in HEYA8 and IGROV1 EOC cell lines we show that PAX8 knockdown reduces cell proliferation in vitro and tumor growth in vivo. To understand how PAX8 regulates neoplastic phenotypes in cancer cells we performed RNA expression profiling by microarray in HEYA8 and IGROV1 before and after PAX8 knockdown. We also performed chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) for PAX8 and a marker of active chromatin (H3K27ac) in both cell lines. De novo motif discovery in the ChIP-seq profiles identified a PAX-like binding motif and several potential PAX8 cofactors in IGROV1 that are not enriched in HEYA8. These cofactors include members of the TEAD and Sp/KLF families of transcription factors. We divided the genes from our differential expression (DE) analysis in PAX8 knockdown into three non-overlapping categories: (a) direct regulatory targets (RTs), i.e. DE genes with PAX8 binding sites in their promoter regions; (b) putative enhancer RTs, where DE genes and PAX8 binding sites reside within the same topological association domain (TAD) defined from embryonic stem cells; and (c) indirect RTs. Our findings reveal that PAX8 can function as either an activator or a repressor, and that PAX8 regulates a very different set of genes in each cell line, as only a small fraction RTs (~50) is shared between HEYA8 and IGROV1. Nonetheless, independent pathway enrichment analysis reveals four pathways downstream of PAX8 that are common to both cell lines: DNA replication, cellular response to lipopolysaccharides, TNFA signaling via NFKB and anatomical structure morphogenesis. Individually, we discovered enrichment for G2M checkpoint and EMT signaling in HEYA8, and tissue morphogenesis and mesenchymal cell development in IGROV1. Collectively, our results suggest that PAX8 regulates cell proliferation and cancer promoting processes via highly tissue-specific regulation of common oncogenic pathways.

#2419

Genome-wide profiling of PAK4 DNA-binding sites and transcriptome reveals its potential transcriptional control on DNA repair-related genes in ovarian cancer cells.

Ivy Tsz-Lo Wong,1 Oscar Gee-Wan Wong,1 Yiming Qin,1 Junwen Wang,2 Annie Nga-Yin Cheung1. 1 _The University of Hong Kong, Hong Kong;_ 2 _Mayo Clinic Arizona, Scottsdale, AZ_.

Oncogenic role of p21-activated protein 4 (PAK4) in relation to cell growth and migration through various signalling cascades has been well studied in multiple cancer types. Such role might be contributed largely by cytoplasmic PAK4 as most of the known PAK4 signalling events occur in the cytoplasm. However, recent studies have demonstrated that PAK4 is capable of shuttling between nucleus and cytoplasm. The precise function of nuclear PAK4 (nPAK4) remains substantially unexplored. We have previously shown that PAK4 can act as a transcription factor. Herein, we aim to identify nPAK4-regulated genes by delineating the PAK4-binding landscape in ovarian cancer genome and mapping the PAK4 transcriptome in ovarian cancer cells.

PAK4-enriched genomic regions were identified by chromatin immunoprecipitation with a PAK4 antibody followed by next-generation sequencing (ChIP-seq) in OVCAR3 cells. PAK4 transcriptome data was generated using RNA extracted from SKOV3 cells stably expressing PAK4 in the Affymetrix GeneChIP® Human Transcriptome Array 2.0 system. Gene lists generated from both ChIP-seq and microarray dataset were subjected to pathway enrichment analysis by Partek® PathwayTM. Among the 20 most significantly enriched pathways from microarray studies, several pathways in which PAK4 has no known reported function by far have been identified. Notably, DNA repair pathways that are known to influence drug chemosensitivity in ovarian cancer, such as the nucleotide excision repair (p=0.008), homologous recombination (p=0.012), mismatch repair (p=0.018) and DNA replication (p=0.018) pathways were significantly enriched. In concordance with the findings, the nucleotide excision repair pathway was also significantly enriched (p<0.006) in the gene list derived from promoter-restricted PAK4 binding sites in the ovarian cancer genome. Among the many candidates that were deregulated by PAK4, cyclin-dependent kinase 7 (CDK7), a core component in the CAK subcomplex crucial to nucleotide excision repair pathway, was further validated. The transcription start site (TSS) of cyclin-dependent kinase 7 (CDK7) was bound by PAK4. CDK7 mRNA expression was upregulated upon PAK4 overexpression. Quantitative-PCR showed downregulated CDK7 mRNA expression upon siRNA-mediated knockdown of PAK4 in OVCAR3 and TUOS3. This study points to the possible role of PAK4 in regulating gene transcription as PAK4 is mapped to several core promoter/TSS regions in the ovarian cancer genome. Our findings also shed light into the potential role of PAK4 in regulating drug chemosensitivity via its transcriptional control on genes implicated in DNA repair pathways. We postulate CDK7 as a direct transcriptional target of PAK4 and thereby contributes to chemoresistance in ovarian cancer cells.

#2420

Integrative analyses of gene expression, DNA methylation, genotype and copy number alterations characterize X-chromosome inactivation in ovarian cancer.

Stacey J. Winham,1 Nicholas B. Larson,1 Sebastian M. Armasu,1 Zachary C. Fogarty,1 Melissa C. Larson,1 Kimberly R. Kalli,1 Kate Lawrenson,2 Simon Gayther,2 Brooke L. Fridley,3 Ellen L. Goode1. 1 _Mayo Clinic, Rochester, MN;_ 2 _Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA;_ 3 _University of Kansas Medical Center, Kansas City, KS_.

Introduction: In females, X-chromosome inactivation (XCI) epigenetically silences transcription of one copy of the X chromosome. Which chromosome is silenced is randomly selected, and is tissue- and cell-specific. While some genes are known to escape XCI under normal conditions, aberrant XCI patterns are thought to occur in female-specific cancers, although the role of XCI in ovarian tumorigenesis and progression is largely unknown. The process of XCI is complex, and integration of gene expression, DNA methylation, and copy number data can inform the XCI status of individual genes and chromosome-wide XCI patterns for individual patients.

Methods: We evaluated gene- and chromosome-level patterns of XCI by integrating RNA sequence, copy number alteration, genotype, and DNA methylation data to study XCI escape patterns in tumor samples from 99 ovarian cancer patients. We measured allele-specific expression (ASE) for 397 X-linked genes to identify the active alleles for each tumor. Combining ASE data with knowledge of copy number status, we used a Bayesian beta-binomial mixture model to estimate which genes escaped XCI for each patient, and validated our findings using DNA methylation data. To assess global XCI patterns, we performed cluster analyses on the ASE and methylation data, after adjusting for loss of heterozygosity. We examined the relationship between the clusters and clinical factors, including overall survival and time to recurrence.

Results: DNA promoter methylation demonstrated inverse regional correlations with ASE. Cluster analyses using ASE and methylation data demonstrated evidence of two tumor clusters, representing normal XCI and global XCI dysregulation. The dysregulated XCI cluster (N=52) was associated with lower X-inactive specific transcript expression as expected (p<0.01). Patients with XCI dysregulated tumors were higher grade, stage, serous histology and were sub-optimally debulked (p<0.05). These patients also had shorter overall survival time (HR=1.87, p=0.02) and time to recurrence (HR=2.34, p<0.01), although associations were attenuated after covariate adjustment. In 45 tumor samples with sufficient data, we observed escape patterns largely consistent with previous reports of multiple tissue types. When comparing tumor to normal ovarian tissue, eight genes (CXorf23, CXorf36, BRWD3, ELF4, SLITRK4, GABRE, CLCN4, SH3BGRL) showed putative escape in the tumor and two genes (RBBP7, OFD1) showed discrepant tumor inactivation.

Conclusions: We identified discrepant gene-level XCI tumor classifications compared to normal tissue and identified a group of patients with chromosome-wide XCI dysregulation associated with worse clinical prognosis. This provides evidence of the role of XCI in ovarian cancer and highlights the need to integrate multiple genomic data types to study XCI.

#2421

The role of p53 mutational status and SOX9 suppression in chemotherapy response of ovarian cancer.

Noelle L. Cutter, Matthew Lucito, Ryan Frank, Kimberly Doyle. _Molloy College, Rockville Centre, NY_.

Ovarian cancers are highly heterogeneous where platinum based chemotherapy which induces DNA crosslinking resulting in apoptosis of the cell is the preferred treatment. However, many patients are intrinsically resistant or quickly develop resistance. The Sox factors are a large family of transcription factors that play important roles in tumor development and progression in a variety of human malignancies and diverse developmental processes, but their impact in clinical tumorigenesis is still unclear. An analysis of genomic changes in ovarian cancer has provided the most comprehensive and integrated view of cancer genes for any cancer type to date. Ovarian serous adenocarcinoma tumors from 500 patients were examined by The Cancer Genome Atlas (TCGA) Research Network and analyses are reported in a recent issue of Nature. This evidence suggests that epigenetic deregulation, such as methylation, may be a key factor in the onset and maintenance of chemoresistance. Previous microarray analysis results in our lab correctly identified a subset of about 300 genes that when methylated altered the chemoresistance of the ovarian epithelium cells in culture. Of the genes identified in the analysis we further set out to characterize oncogenes and tumor suppressor genes that interact with the guardian of the genome, TP53, to determine if we could elucidate the mechanism by which it increased resistance. Using several in-vitro assays, we determined that the loss of p53 in conjunction with SOX9 decreased the level of apoptosis in response to carboplatin. Furthermore, in cells with mutated p53/SOX9 show an increase in tumorigenesis. Regulation of several pathways with p53 mutations in ovarian cancer might represent a therapy response prediction and could be a future therapeutic target for ovarian cancer. In addition, the crosstalk between p53/SOX9 and epigenetic regulators may present a valid treatment option for increasing carboplatin sensitivity in resistant patients.

#2422

Simultaneous detection of activating somatic DNA mutations and expressed fusion transcripts from lung tumor FFPE samples.

A. McGarry Houghton,1 Gavin Meredith,2 Julia Kargl,1 Jill McKay-Fleisch,2 P. Martin Ross,2 Anisha Kharkia,2 Afshin Mashadi-Hossein,2 Dae Kim,2 Joseph Beechem2. 1 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 2 _NanoString Technologies, Inc., Seattle, WA_.

Worldwide, lung cancer is the most commonly diagnosed form of cancer with a survival rate among the lowest. Combined, somatic mutations (in the form of SNVs and InDels) and gene fusions, account for the majority of interpretable and actionable genomic alterations. Importantly, this typically requires the analysis of DNA and RNA from limited amounts of FFPE-preserved specimens. Currently, these analyses typically require complex sample pre-processing for assay on separate platforms or separate complex library preparation methods for assessment by high throughput sequencing. To provide a unified and simpler alternative, NanoString's molecular barcoding technology has been modularized to permit simultaneous digital measurement of cancer-relevant targets that span these two analyte classes. Novel 'SNV' probes enable sensitive and specific identification of DNA mutant allele sequences down to a level of detection of ≤ 5% from 5 ng of FFPE-extracted genomic DNA. Fusion transcripts are detected with 5'/3' imbalance probes and toehold-mediated junction probes. This dual analyte workflow requires just a single 5-10 micron section of FFPE tissue and provides to sample-to-answer results with approximately 5 minutes of hands-on time per sample after nucleic acid extraction.

To demonstrate utility, 37 lung cancer samples were assayed simultaneously with an SNV panel that targets >100 solid tumor somatic mutations and a lung cancer fusion gene panel that provides general evidence of ALK, RET, and ROS1 gene fusion events along with specific detection of 35 unique fusion transcripts that correspond to known break-points. In this particular cohort, 16 samples were positive for activating KRAS SNVs (one of which was also positive for an activating STK11 variant), 3 were positive for activating EGFR mutations including two SNVs and an 18-base InDel and one was positive for an activating KIF5B16:RET12 fusion transcript. Positive mutation calls obtained with the SNV panel could only be confirmed by whole-exome sequencing (average depth of 100X) for 13 of 20 variants detected; however, ultra-deep (average depth of 4400X) targeted sequencing revealed that the 7 additional panel-detected mutations were, in fact, present. Measured against the sequencing datasets, the SNV panel provided 100% sensitivity, specificity, accuracy and precision for all variants present at 5% or greater allele frequency. The KIF5B16:RET12 fusion event was also confirmed by sequencing. Combined, these results show that these two important classes of activating mutations can be readily and efficiently assayed together on a NanoString nCounter® system (for research use only).

#2423

Bronchial airway gene expression signatures in mouse lung squamous cell carcinoma and their modulation by cancer chemopreventive agents.

Donghai Xiong,1 Jing Pan,1 Qi Zhang,1 Eva Szabo,2 Mark S. Miller,3 Ronald A. Lubet,3 Ming You,1 Yian Wang1. 1 _Medical College of Wisconsin, Milwaukee, WI;_ 2 _National Cancer Institute, Bethesda, MD;_ 3 _Division of Cancer Prevention, National Cancer Institute, Bethesda, MD_.

Due to exposure to environmental toxicants, a "field cancerization" effect occurs in the lung resulting in the development of a field of initiated but morphologically normal appearing cells in the damaged epithelium of bronchial airways with dysregulated gene expression patterns. Using a mouse model of lung squamous cell carcinoma (SCC), we performed transcriptome sequencing (RNA-Seq) to profile bronchial airway gene expression and found activation of the PI3k and Myc signaling networks in cytologically normal bronchial airway epithelial cells of mice with preneopastic lung SCC lesions, which was reversed by treatment with the PI3K Inhibitor XL-147 and pioglitazone, respectively. Activated MYC signaling was also present in premalignant and tumor tissues from human lung SCC patients. In addition, we identified a key microRNA, mmu-miR-449c-5p, whose suppression significantly up-regulated Myc expression in the normal bronchial airway epithelial cells of mice with early stage SCC lesions. We developed a novel bronchial genomic classifier in mice and validated it in humans. In the classifier, Ppbp (pro-platelet basic protein) was overexpressed 115 fold in the bronchial airways of mice with preneoplastic lung SCC lesions. This is the first report that demonstrates Ppbp as a novel biomarker in the bronchial airway for lung cancer diagnosis.

#2424

The in-parallel whole-genome ChIP-Seq analysis of primary tissues, patient derived xenografts, and cancer cell lines from HPV-relative HNSCC samples.

Dylan Z. Kelley,1 Emily L. Flam,1 Hildegard A. Wulf,1 Theresa Guo,1 Evgeny Izumchenko,1 Dzov A. Singman,1 Ludmila V. Danilova,1 Elena D. Stavrovskaya,2 Michael Considine,1 Justin A. Bishop,1 William H. Westra,1 Zubair Khan,1 Wayne M. Koch,1 David Sidransky,1 Sarah Wheelan,1 Joseph A. Califano,3 Alexander V. Favorov,1 Elana J. Fertig,1 Daria A. Gaykalova1. 1 _Johns Hopkins Univ., Baltimore, MD;_ 2 _Moscow State University, Moscow, Russian Federation;_ 3 _Univ. of California, San Diego, La Jolla, CA_.

This project develops a novel experimental technique to perform ChIP-Seq (chromatin immunoprecipitation with massively parallel DNA sequencing) analysis of chromatin structure in primary tumor tissues from high risk HPV-related head and neck squamous cell carcinomas (HPV+ HNSCC). Recent data suggest that chromatin structure is the central regulator and predictor of cancer-specific expression and mutagenesis landscape of diseased cells. Genome-wide gene expression dysregulation in many tumors, including HPV+ HNSCC, are incompletely described by current knowledge. Methods for study of chromatin structure in primary tumor tissue are needed to better understand the role global epigenetic changes may play in these tumors. However, ChIP-Seq, which is the state-of-the-art method of elucidating chromatin structure, until now, has not been reliably performed on any HNSCC samples. Because chromatin structure is disrupted at room temperature, ChIP-Seq is especially complicated for primary patient tissues, which are primarily obtained as surgical waste after pathology review. Snap freezing of leftover waste surgical tissues and further tissue thawing for the analysis decreases chromatin structure integrity necessary for highly sensitive ChIP-Seq methodology, especially for tumor samples with chromatin structure deformed during carcinogenesis. To improve the chromatin structure integrity in tumor sample we added a xenografting step and minimized the exposure of cancer tissue to room temperature conditions after mouse surgery. We also minimized patient non-cancer tissue preservation at ambient temperature after patient surgery. We successfully performed ChIP-Seq for H3K4me3, H3K9me3, and H3K9ac on frozen uvulopalatopharyngoplasty (UPPP) primary tissues, frozen patient derived xenograft tissues, and freshly-cultured head and neck squamous cell carcinoma cell lines, revealing comparable success rates between tissue type and sample preservation techniques. ChIP-Seq techniques were performed and cross validated using tried and true qRT-PCR methods to demonstrate data reproducibility. The biological relevance of the ChIP-Seq data was confirmed through massive RNA-Seq analysis of 47 HPV+ HNSCC samples and 25 non-cancer controls. Analysis revealed that most H3K9ac and H3K9me3 enrichment is similar in primary tissues, regardless of disease status. Only small portion of them showed differential histone enrichment, which correlated with differential expression of corresponding genes. On the other hand, H3K4me3 showed strong tissue specificity and were found differentially enriched especially in tumor samples. The proposed experimental pipeline demonstrates high reproducibility between biological replicates, diversity of tissue models, and low dependence of ChIP-Seq analysis on tissue preservation techniques.

#2425

Delving into molecular phenomena of methylation, polyadenylation and mutation suggests their importance in regulating gene expression in GBSCC.

Richa Singh,1 Esita Chattopadhyay,1 Roshni Roy,2 Bidyut Roy1. 1 _Indian Statistical Institute, Kolkata, India;_ 2 _Baylor Scott and White Health, TX_.

Background: Gingivo-buccal squamous cell carcinoma (GBSCC) is one of the most prevalent types of oral cancers in India. Although whole exome studies in oral cancer have reported somatic mutational driver genes, studies on methylation, mutations and alternative polyadenylation (APA) to identify differentially expressed transcripts and exons, respectively, are rare. The aim of our study is to investigate possible causes of expression deregulation due to methylation, APA or mutations.

Methods: Whole transcriptome data was generated from 12 tumor-normal paired GBSCC tissues and analyzed for differential gene expression, differential exon usage and predicting APA site. Whole exome sequencing was performed with same sample set and the data was analyzed to identify somatic mutations. Reduced representation of bisulphate sequencing (RRBS) was also done for some of these samples to inquire about the methylation status of deregulated genes.

Results: A total of 465 genes were found to have differential exon usage in tumor leading to expression of different isoforms and change in overall gene expression. About 194 genes were predicted to utilize alternate polyadenylation site in tumors. Amongst these, 11 genes including ERBB3, CDC25B and LTBP4 were shown to have differential exon usage in tumor with altered gene expression hinting upon the influence of polyadenylation in expression change. From the exome sequencing study, 180 SNVs and 11 indel somatic mutations per sample were observed. Apart from other non-silent exonic mutations at TP53, CASP8, DIS3 and DLG1, splice site mutations were also found in 37 and 4 genes due to point and indel mutations respectively. Genes such as TPM3, NBN, CXCL12 along with others showed differential exon usage and were found to harbor somatic mutations and APA site. This suggests for coupled regulation by APA and mutation in altering expression of those genes. Apart from this, a subset of deregulated genes (n=54) were observed to have differential methylation in tumors. Expression change of TAP1, SLN, ELN etc. corroborated with their methylation status suggesting the influence of methylation in regulating expression of these genes.

Conclusion: Differential exon usage suggested different transcript formation leading to overall change in gene expression in tumor. Upon excavating into the regulatory causes, a subset of genes showed change in methylation pattern while few others showed change in APA site with/without somatic mutation effect. These molecular phenomena explain how different mechanisms are involved in expression deregulation of genes observed in GBSCC which would help in further functional research for cure of the disease.

#2426

Oncopig soft-tissue sarcomas recapitulate key transcriptional features of human sarcomas.

Kyle M. Schachtschneider,1 Yingkai Liu,2 Suvi Makelainen,3 Ole Madsen,3 Laurie A. Rund,2 Martien A. Groenen,3 Regina M. Schwind,1 Ron C. Gaba,1 Schook B. Lawrence2. 1 _University of Illinois at Chicago, Chicago, IL;_ 2 _University of Illinois, Urbana, IL;_ 3 _Wageningen University, Wageningen, Netherlands_.

Human soft-tissue sarcomas (STS) are rare, aggressive mesenchymal tumors with a late stage 5-year survival rate (50-60%) that has for decades remained unchanged. Research into STS treatment is hampered by the limited human STS cell line availability and the large number of STS subtypes. Therefore, there is a need to develop STS cell lines and animal models representative of diverse human STS subtypes. Pigs represent ideal human disease models due to their similar size, anatomy, metabolism, genetics, and epigenetics compared to humans. In this regard, porcine cancer models provide the opportunity to produce STS cell lines and in vivo tumors similar to those clinically observed in humans. The Oncopig encodes Cre recombinase inducible porcine transgenes encoding KRASG12D and TP53R167H, allowing the Oncopig to model a number of human sarcomas in an inducible and temporal manner. However, comparative analysis is required to determine to what extend the Oncopig STS model mimics human STS on a molecular level. The purpose of this study was to identify similarities between Oncopig and human STS transcriptional profiles to validate the Oncopig model as a viable model for human STS. Towards this end, Oncopig fibroblasts were isolated from ear notches of 4 Oncopigs and cultured in vitro. Following confirmation of their mesenchymal origin (positive vimentin immunostaining), Oncopig fibroblasts were transformed via Cre recombinase exposure, resulting in the formation of Oncopig STS cell lines. Oncopig STS tumors were produced in vivo through intramuscular injection of adenovirus encoding Cre in 2 Oncopigs (2 sites/Oncopig), resulting in the formation of 4 tumors detectable by 10 days post injection. The mesenchymal origin of the resulting tumors was confirmed through histological characterization. Genome-wide expression of Oncopig STS cell lines and tumors was profiled via RNA-seq. Reproducible Oncopig STS cell line and tumor expression profiles were observed, and Oncopig STS cell lines also displayed high temporal reproducibility. Differential expression analysis was performed by comparing Oncopig STS cell lines and tumors to untransformed fibroblasts and skeletal muscle, respectively. A total of 3,360 and 7,652 differentially expressed genes were identified in the Oncopig STS cell lines and tumors, respectively. Commonly identified alterations in human STS gene expression and pathway regulation were identified in Oncopig STS, including altered TP53 signaling, activation of Wnt signaling, and evidence of epigenetic reprogramming. Furthermore, master regulators of Oncopig STS gene expression were identified, including FOSL1, which was previously identified as a potential therapeutic target for human STS. These results demonstrate the Oncopig STS model's ability to mimic human STS on a transcriptomic level, making the Oncopig a valuable resource for sarcoma research and cell line development.

#2427

Alterations in gene expression after exposure of MDA-MB-231 cells to isobutylparaben.

Christine Strelchuk,1 Holly Jones Taggart2. 1 _University of Toronto, Toronto, Ontario, Canada;_ 2 _University of Ontario Institute of Technology, Oshawa, Ontario, Canada_.

Parabens are a group of chemical compounds extensively used as preservatives in consumer products such as cosmetics, pharmaceuticals and processed food. Parabens have been shown to have endocrine disrupting properties, to accumulate in breast tissue and to increase the proliferation of hormone receptor positive breast cancer cell lines through competitive binding to the estrogen receptor. Studies have shown that isobutylparaben has the highest binding affinity for the aryl hydrocarbon receptor (AhR), reported to bind environmental toxins. This study examined the alterations in gene expression correlated to exposure of the hormone receptor negative breast cancer cell line MDA-MB-231 to isobutylparaben over a 72 hour time frame. Exposure of the cells to concentrations of isobutylparabens commonly found in consumer products alters the genetic expression profile of the cells. In our study, detectable expression levels of AhR mRNA decreased, while cytochrome P450 (CYP1A1) and receptor activator of NFκB ligand (RANKL) mRNA levels increased, as analyzed by qPCR. The downregulation of AhR and altered expression levels of invasion related genes is suggestive of an increased metastatic phenotype with exposure to isobutylparaben.

#2428

The cytidine deaminase APOBEC3B is a regulator of gene expression in breast cancer.

Manikandan Periyasamy. _Imperial College London, Du Cane Road, London, United Kingdom_.

The APOBEC3B (A3B) cytidine deaminase gene has been implicated driving the mutational landscape in breast and other cancer types. We have discovered that A3B is a co-activator of estrogen receptor (ER) mediated gene expression in breast cancer. Mechanistic studies established that A3B regulates gene expression by promoting C-to-U deamination at ER binding regions. We show that these C-to-U changes lead to the generation of DNA strand breaks through activation of base excision repair (BER) and to repair by non-homologous end-joining (NHEJ) pathways. We have performed RNA-seq and A3B ChIP-seq, in multiple ER+ and ER- breast cancer cell lines. Remarkably, these studies demonstrate that A3B regulates the expression of many genes in inflammatory and interferon signaling pathways, in addition to the regulation of ER target genes. These findings, together with other data to be presented, provide a clear role for A3B in transcription regulation. These findings also raise the possibility that transcription factor mediated recruitment of A3B to DNA may facilitate acquisition of A3B driven cancer mutations.

#2429

Generation of a transcriptome of oncogenic mutant p53 in triple-negative breast cancer.

Rebecca E. Steele, Simon S. McDade, Paul B. Mullan. _Queen's University Belfast, Belfast, United Kingdom_.

The purpose of this study was to generate a transcriptional profile specific for mutant p53 in triple-negative breast cancer (TNBC), representing potential novel therapeutic targets. The highly heterogeneous TNBC tumor subgroup is established as having a poor clinical outcome. There are currently no targeted therapies for women with TNBC; consequently, there is an unmet clinical need to identify targetable pathways. Approximately 75% of these tumors are known to harbour a mutation in Tp53 (mutp53). Chromatin Immunoprecipitation Sequencing (ChIP-Seq) for mutp53 and ETS-1 (a known mutp53 interactor) was carried out using the D0-1 (sc-126) and sc-20 antibodies respectively, in the MDA-MB-468 TNBC cell line. The aim of these ChIP-seq experiments was to identify sites bound by endogenous R273H mutp53 and ETS-1 and to confirm that target genes were co-regulated. We identified 282 novel mutp53 specific genomic binding sites and 37 mutant p53 and ETS1 shared binding sites. In-house data analysis and peak calling generated a list of potential transcriptionally co-regulated genes adjacent to these genomic sites. PDGFβ, TRIB3, BTRC, TNFSF18 & PI3 were successfully validated as being transcriptionally regulated by R273H mutp53 and not wild-type p53 (as shown by MCF7 ChIP-seq comparisons). A number of these genes are known to be aberrantly expressed in TNBC patient tumors and harbor interesting immune biology, providing potential opportunities for novel therapeutic strategies. In addition, DNA damaging chemotherapy-resistant MDA-MB-468 derivatives express elevated levels of mutp53 protein and altered expression of our validated transcriptional targets, suggesting that they may also have roles in promoting resistance to current chemotherapy treatments, downstream of mutp53. In conclusion, we have identified a cohort of 'mutp53 specific' transcriptional targets which may have roles in modulating immune responses to TNBCs, driving resistance to current chemotherapies and could provide opportunities for the development of novel treatments for TNBCs.

#2430

The expression of BCL6 and ZEB family transcription factors and their correlation in breast cancers.

Min Zhao, Lin Ang, Li Zheng, Jin Wang, Jin Huang, Hongguang Hu, Qiang Zou. _the Second People's Hospital of Hefei, Hefei, China_.

The proto-oncogene B-cell lymphoma 6 (BCL6) is an evolutionarily conserved zinc finger protein and a potent transcriptional repressor. It is also the master regulator of B-lymphocyte development. Emerging evidence indicates that BCL6 may play an important role in breast cancer progression. In this study, we investigated the expression of BCL6 and tis correlation with the expression of epithelial-mesenchymal transition (EMT)-associated transcription factors zinc finger E-box-binding homeobox 1 (ZEB1) and ZEB2 in invasive breast cancers. The mRNA and protein expression of BCL6, ZEB1 and ZEB2 were examined using in situ hybridization and immunohistochemistry, respectively, in 228 breast cancer patients and 80 patients with breast benign diseases. The association of BCL6, ZEB1 and ZEB2 expression with clinicopathological parameters and patient survival were analyzed. We found that the expression of BCL6, ZEB1 and ZEB2 protein and mRNA were significantly higher in breast cancer tissues than in breast benign disease tissues (P<0.05). The expression levels of BCL6, ZEB1 and ZEB2 were all positively correlated with the tumor size, lymph node metastasis and higher tumor stages (P<0.05). Furthermore, patients with BCL6, ZEB1 and ZEB2 protein-positive primary tumors were associated with a significant lower overall survival (OS) rate (P=0.001, 0.002 and 0.001, respectively) and post-operative relapse-free survival (RFS) rate (P=0.002, 0.001 and 0.003 for the expression of BCL6, ZEB1 and ZEB2, respectively). The mRNA expression of ZEB1 and ZEB2 were positively correlated with BCL6 mRNA expression (rs=0.326, P<0.001; and rs=0.382, P<0.001, respectively); the protein expression of ZEB1 and ZEB2 were also positively correlated with BCL6 protein expression (rs=0.449, P<0.001; and rs=0.669, P<0.001, respectively). These results indicate that BCL6, ZEB1 and ZEB2 may be as potential biomarkers for predicting invasion, metastasis and prognosis of breast cancer and that BCL6 may be a regulator of ZEB family transcription factors (ZEB1 and ZEB2).

#2431

Single cell gene expression profiling in breast cancer cells with the Her2/neu gene knockout by CRISPR-Cas9.

Xiaoyang Wang,1 Chip Lomas,1 Michael A. Tycon,1 Craig Betts,2 Suzanne Weaver2. 1 _BD Biosciences, San Jose, CA;_ 2 _BD Genomics, Menlo Park, CA_.

The Her2/neu gene is amplified and overexpressed in 15%-30% of breast cancers. The overexpression of this oncogene is strongly correlated with decreased survival, increased cancer relapse, and poor prognosis. Although therapy has been successfully developed to target this oncogene, a better understanding of this oncogene will provide further insight for breast cancer biology and future drug development. In recent years, the CRISPR-Cas9 system has emerged as an efficient method for genetic engineering, enabling targeted gene knockout with minimal off-target effects.

In this study, we employed a plasmid-based CRISPR-Cas9 system to knock out the Her2/neu gene in breast cancer cells. Puromycin selected, GFP-positive (two indicators of the incorporation of the CRISPR-Cas9 system), Her2-negative individual cells were index-sorted using a BD FACSMelody™ system into individual wells of a 96-well BD™ Precise plate with sample barcoding and molecular indexing. A whole transcriptome amplification (WTA) assay was performed to obtain a gene expression profile for each of the Her2/neu knockout cells. Her2 gene knockout was performed on two cell lines: T47D, a ductal carcinoma-derived breast cancer cell line with no Her2 amplification that shows low to intermediate Her2/neu expression; and SKBR3, an adenocarcinoma-derived breast cancer cell line known for Her2/neu gene amplification and overexpression. For comparison, WTA assays were also carried out on parental cells without gene editing. Comparison of the gene expression profiles reveals downstream changes in gene expression correlated to the Her2 knockout.

The profiling of gene expression in Her2/neu CRISPR-Cas9 knockout cells on a single cell level promises to provide insight into the mechanisms in these aggressive cancers that could help future drug discovery.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.

23-19341-00

#2432

GPER1 expression is modulation by D-glucose concentration in estrogen-responsive cancer and tumor cells.

Yan Zheng, Kevin D. Houston. _New Mexico State Univ. - Las Cruces, Las Cruces, NM_.

G protein-coupled estrogen receptor 1 (GPER1, aka GPR30), is a 7-transmembrane receptor that mediates rapid cell signaling events stimulated by 17β-estradiol (E2) in cancer and tumor cells. In breast cancer cells, GPER1 mediates E2-induced CTGF-dependent cell migration and cyclin E expression resulting in cell cycle progression. GPER1 has also been shown to mediate antiproliferative cell signaling such as p53-dependent inhibition of cell cycle progression in breast cancer cells treated with the GPER1-specific agonist G1 and the inhibition of IGF-1R signaling in tamoxifen-treated breast cancer cells. In addition to E2, both natural and synthetic compounds have been shown to activate GPER1 in breast cancer cells. GPER1 has also been shown to regulate cell signaling in cells that lack ERα. For example, GPER1 is antiproliferative in triple-negative breast cancer cells. While the importance of GPER1 in the modulation of E2-responsive tumor and cancer cells as been demonstrated, there is very little information regarding the regulation of GPER1 expression in these cell types. The work presented here is aimed at determining the molecular mechanisms that regulate GPER1 expression. To determine if GPER1 expression is sensitive to D-glucose concentration in breast cancer cells, MCF-7 and T47D human breast cancer cells were cultured in media containing increasing concentrations of D-glucose and GPER1 expression was measured using real-time PCR and immunoblot. Data from these experiments showed that GPER1 expression was significantly increased in breast cancer cells cultured in media containing low D-glucose concentrations (0 and 2.5mM) and significantly reduced in media containing high D-glucose (25 mM). Since low D-glucose concentration is known to activate the energy-sensing AMP kinase (AMPK), the observed GPER1 induction in low D-glucose conditions was determined after pretreatment with the AMPK inhibitor compound C. GPER1 expression was inhibited in both MCF-7 and T47D breast cancer cells cultured in low D-glucose media when pretreated with compound C. Additionally, the AMPK activator metformin induced GPER1 expression in MCF-7 and T47D breast cancer cells cultured in high D-glucose (25mM) conditions. These data suggest that AMPK mediates GPER1 expression in cells cultured in low D-glucose. The D-glucose sensitivity of GPER1 expression was also determined in the Eker rat-derived uterine leiomyoma cells (ELT-3 and ELT-6) which are characterized by hyperactive AMPK. In both ELT-3 and ELT-6 cells, GPER1 expression was not sensitive to D-glucose concentration nor was the activation state of AMPK sensitive to D-glucose concentrations. However, the inhibition of AMPK by compound C in both ELT-3 and ELT-6 cells resulted in decreased GPER1 expression. These findings reveal a previously unknown mechanism that regulates GPER1 expression in E2-responsive tumor and cancer cells.

#2433

Altering nuclear size impacts cancer cell characteristics in melanoma cell lines.

Lidija D. Vukovic,1 Bradley A. Stohr,2 Dan L. Levy1. 1 _University of Wyoming, Laramie, WY;_ 2 _University of California, San Francisco, CA_.

Nuclear size is altered in cancer cells, a change used by pathologists to distinguish cancer from normal cells. Previous studies in Xenopus identified two nuclear transport proteins, importin α and NTF2, as regulators of nuclear size. In general, importin α and NTF2 levels positively and negatively affect nuclear size, respectively. Increased importin α expression is used as a biomarker in non-small cell lung carcinoma and breast cancer and correlates with increased nuclear size. We identified decreased NTF2 protein expression as a potential biomarker in melanoma, consistent with NTF2 levels negatively regulating nuclear size. Following up on this result, our current studies focus on manipulating nuclear size in melanoma cells to assess the impact on cancer cell characteristics, including proliferation rate, migration potential, and apoptosis.

First, we measured nuclear size and NTF2 expression levels in different stage melanoma cell lines. Compared to a normal melanocyte cell line, we consistently observed larger nuclei and lower NTF2 protein levels in all melanoma cell lines examined. Next, we performed transient transfections in HeLa, MRC5, and melanoma cell lines, demonstrating that ectopic NTF2 expression leads to reduced nuclear size. The most dramatic effects were observed in primary melanoma cell line WM3211 where higher NTF2 expression caused a 40% reduction in nuclear cross-sectional area.

To obtain more precise control over NTF2 expression levels, we generated a stably transfected metastatic melanoma cell line (WM983B) in which NTF2 expression can be titrated by varying the concentration of doxycycline in the growth media. Higher doxycycline levels lead to increased NTF2 expression and smaller nuclei. In particular, 20 ng/ml doxycycline induced a 10% reduction in nuclear cross-sectional area. In a wound healing assay, doxycycline-treated cells exhibited a 40% reduction in cell motility compared to non-treated cells or the parent cell line treated with doxycycline. Furthermore, the doxycycline-treated cells exhibited an increased rate of apoptosis. We propose that reduced nuclear size in the doxycycline-treated cells causes changes in chromatin organization and gene expression, thus giving rise to observed effects on cell migration and apoptosis. To test this idea, we are currently performing a global transcriptomics analysis of our cell lines to identify genes whose expression is altered by nuclear size. Using this information, we will use DNA FISH to map the intranuclear position of differentially-expressed genes as a function of nuclear size. Also in vivo experiments are ongoing in which we have subcutaneously injected NTF2-inducible melanoma cells into NSG mice in order to examine tumor formation and metastatic capacity of cells with differently-sized nuclei.

Regarding that in most cancer cells nuclear size is enlarged, understanding the causes and effects of these changes can help us to better understand cancer biology.

#2434

LEF1 negatively regulates differentiation of HCC by activation of Notch signaling pathway.

Shuo FANG,1 Lei LI,2 MIng LIU,3 Janice Tsang,4 Xinyuan GUAN5. 1 _The university of Hong Kong, Hong Kong, China;_ 2 _SUN YAT-SEN UNIVERSITY CANCER CENTER, Guangzhou, China;_ 3 _Guangzhou Medical University, Guangzhou, China;_ 4 _The university of Hong Kong, Queen Mary Hospital, Hong Kong, China;_ 5 _The university of Hong Kong, Department of Clinical Oncology, Hong Kong, China_.

Lymphoid enhancer binding factor-1 (LEF1), an essential regulatory protein, plays a crucial role on self-renewal and differentiation properties of several cancers, contributing to tumor malignancy degree and prognosis. However, its molecular mechanisms to promote tumor differentiation still keep unknown, especially in hepatocellular carcinoma (HCC). In this study, by analyzing the transcriptome datasets and our qPCR result of clinical HCC samples, we found that LEF is frequently overexpressed in HCCs and significantly associated with HCC differentiation (p=0.015). In addition, functional study showed that LEF1 could enhance the cell proliferation, matrigel invasion, transwell migration, as well as self-renewal abilities both in vivo and in vitro experiments. We also demonstrated LEF1 is closely related to Notch intracellular domain (NICD), and revealed several putative sites as the targets for LEF1 activating Notch signaling pathway. LEF1 could induce the Notch signaling via transcriptionally up-regulate Notch expression through direct DNA binding. The expressions of several downstream members in Notch signaling pathway including Notch1 and Notch2 were subsequently increased. Moreover, LEF1 could down-regulate various differentiation markers and over-express tumor stemness related genes. Together, LEF1 is a novel oncogene that show a crucial role in the tumorigenesis and progression of HCC via activating Notch signaling pathway and inhibiting the differentiation and stemness of HCC, resulting in poor pathologic differentiation and leads to high degree of malignant. All the findings suggest a novel treatment possibility and a new target for liver stem cell eradication. LEF1 could be investigated for clinical early diagnosis and prognosis prediction of HCC patients and improving the development of new therapy against HCC.

#2435

Smoothened a new perspective for uterine leiomyoma treatment.

Natalia Garcia,1 Laura Gonzalez Anjos,2 Giovana De Nardo Maffazioli,1 Nilo Bozzini,2 Edmund Chada Baracat,2 Katia Candido Carvalho2. 1 _Univ. of São Paulo Faculty of Medicine, Mogi das Cruzes, Brazil;_ 2 _Univ. of São Paulo Faculty of Medicine, São Paulo, Brazil_.

Background: Uterine leiomyomas (LM) are the most common gynecologic tumors occurring in women of reproductive age. Patients may present clinical complications such as bleeding, pain and infertility. Additionally, some researchers believe that a leiomyosarcoma can arise from a degenerated LM. Efficient treatments for LM are still limited. Identification of gene markers can help to elucidate these tumors pathogenesis and to reveal molecules that can be used as therapeutic targets.

Objective: To assess gene and miRNAs expression, and DNA methylation profiles of genes related to the Sonic Hedgehog (SHH) signaling pathway in uterine leiomyoma samples in order to look for potential therapeutic targets.

Methods: 80 samples of LM and 20 myometrium (MM) were obtained in the Department of Obstetrics and Gynecology at HCFMUSP/Sao Paulo - Brazil. Expression profile of 106 genes and 84 miRNAs sequences related to SHH regulation, and the percentage of methylation in the promoter region of the SHH, PTCH1, SMO, SUFU, GLI and GLI3 genes were performed by quantitative Real Time PCR. Results: Among 106 genes evaluated, 16 showed hyper- and 11 showed hypoexpression in LM samples, compared to MM. Only SMOOTHENED (SMO) was found hyperegulated in LM. miRNAs analysis showed 16 sequences with different regulation profiles between LM and MM. None of them is involved in the regulation of SMO expression. DNA methylation profile showed a higher percentage of methylation in LM samples, but this difference did not exceed 2% of methylation for the PTCH1, SMO, GLI1 and GLI3 genes. Conclusion: Our results suggest an activation of SHH pathway in LMs by hyperexpression of SMO. These results are very interesting because there are SMO specific drugs approved by FDA for other tumors types.

#2436

Non canonical activation of Sonic Hedgehog pathway in uterine leiomyosarcoma.

Natalia Garcia, Bianca C. Oliveira, Kelly P. Ferreira, Edmund C. Baracat, Katia C. Carvalho. _Univ. of São Paulo Faculty of Medicine, São Paulo, Brazil_.

Background: Uterine leiomyoma (ULM) and leiomyosarcoma (ULMS) are smooth muscle tumors with distinct clinical and biological behavior. They can be a cause of infertility and even death. Little is known about the factors that can to influence these tumors behavior and biology. Sonic Hedgehog (SHH) pathway components were previously implicated in the ULMS malignancy risk; however its activation mechanism is unknown. In this study, we investigated the gene expression profile of Sonic Hedgehog Signaling and pathway upstream genes in myometrium (MM), ULM and LMS cell lines.

Methods: Total RNA obtained from cell lines were submitted to cDNA synthesis and quantitative real time PCR (qRT-PCR), using 7500 System (Life Technologies, USA). Gene expression of SHH, PTCH1, SMO, SUFU, GLI 1-3, CCND1, BCL-2 and BMP4 were assessed.

Results: Gene expression data showed an upregulation of SMO, SUFU, GLI1, BCL-2 and BMP4 in LMS cells when compared to MM. GLI1 (RQ=11) and BCL-2 (RQ=20) genes had higher expression. In the other side, SHH, PTCH1, GLI2 and GLI3 showed upregulation in MM cells.

Conclusion: Our results showed that the Sonic hedgehog pathway is activated in LMS cells but by SHH independent form (non-canonical pathway). This could be explained by the downexpression of SHH ligand and the PTCH1 receptor, and by the upexpression of SMO, GLI1 and pathway target genes as BCL-2 and BMP4 in uterine LMS cells compared to LM and MM. These results suggest a new therapeutic perspective for LMS by target drugs to inhibit SMO and GLI1 molecules.

#2437

Overexpression of MYL5 promotes cervical cancer cell metastasis through SLUG and HIF-1α signaling.

Lan Zhang. _Sun Yat-sen University Cancer Center, Guangzhou, China_.

Myosin light chains (MLC) serve important regulatory functions in a wide range of cellular and physiological processes. Previous research found that MLC are also chromatin-associated nuclear proteins which regulate gene transcription. In this study, we show that the MLC member myosin regulatory light chain 5 (MYL5) controls metastasis in cervical cancer. We uncover this role of MYL5 through clinical cervical cancer samples and various in vitro and in vivo models of metastasis. MYL5 promotes metastasis by enhancing the transcription of SLUG and hypoxia inducible factor-1α (HIF-1α). Moreover, we reveal a bidirectional regulation between MYL5 and HIF-1α. HIF-1α specifically binds to the MYL5 promoter region, and hypoxia leads to increased levels of MYL5 in cervical cancers. Overexpression of MYL5 sustained HIF-1α expression in normoxic condition. Clinical data confirmed a positive correlation between MYL5 and HIF-1α. In summary, our observations suggest a potential application of MYL5 in prognosis prediction and cancer treatment.

#2438

**Analysis of IL-13 signaling through IL-13Rα2 in human brain tumor specimens** in situ **.**

Rukmini Bhardwaj, Akiko Suzuki, Pamela Leland, Bharat H. Joshi, Raj K. Puri. _FDA-CBER, Silver Spring, MD_.

Previously, we have demonstrated that IL-13 receptor alpha2 (IL-13Rα2), a high affinity receptor for Th2 cytokine IL-13, is overexpressed in most glioblastoma multiforme (GBM) cell lines and approximately 78% of the patient-derived samples. We have also demonstrated that IL-13Rα2 can be targeted by a number of immunotherapeutic agents including chimeric antigen receptor modified T (CAR-T) cells, targeted lentivirus and adenovirus vectors, and a chimeric fusion immunotoxin consisting of IL-13 and truncated Pseudomonas exotoxin (IL-13-PE). However, the signal transduction initiated by IL-13 in GBM tumor is not known to occur through the IL13Rα2, which has a high affinity for IL-13. We have recently observed that IL-13 can signal through IL-13Rα2 by activating AP-1 transcription factors in human brain tumor cell lines. In this study, we have examined IL-13Rα2 expression in human brain tumor and normal brain specimens, and the subsequent signaling through the AP-1 pathway in situ. Using six human glioblastoma and three astrocytoma specimens, we evaluated the expression of AP-1 transcription factors by immunohistochemistry (IHC) and compared the extent of immunostaining and percent positive fields with three normal brain specimens. Six GBM specimens examined showed high degree of immunostaining for c-Fos, c-Jun, Jun D and Fra-1 (AP-1 family of transcription factors) and a high percentage of positive fields. These specimens also showed strong immunostaining for IL-13Rα2 (4+) in >70% fields (P<0.001 compared to normal brain). Three astrocytoma specimens showed staining for IL-13Rα2 (>2+ and 32% fields, P<0.01 compared to normal brain), but the extent of staining was lower compared to GBM. Similar to IL-13Rα2 expression, the extent of staining and percentage of positive fields for AP-1 transcription factors were highly statistically significant between tumors and normal brain (P<0.001 for GBM compared to normal brain and P<0.01 for astrocytoma compared to normal brain). The extent of immunostaining in GBM was highest for c-Fos (4+, 78% fields) followed by c-Jun (3+, 57% fields), Fra-1 (2+, 70% fields) and Jun-D (2+, 28% fields). Jun-B expression was the lowest among the AP-1 transcription factors (<1+, 7% fields) in GBM specimens. Astrocytoma specimens showed lesser extent of immunostaining for AP-1 members compared to GBM; c-Fos showed 2+ staining and 42% positive fields followed by c-Jun (2+, 12% fields), Fra-1 (2+, 48% fields) and Jun-D (<1+, 18% fields). Jun-B staining intensity was <1+ in only 6% fields. Normal brain specimens showed no immunostaining for AP-1 family members. Our results generally corroborate with data obtained from GBM cell lines and confirm that IL-13 can signal in IL-13Rα2 positive GBM tumors in-situ through the AP-1 pathway, thus indicating that this pathway may be an important target for therapeutic intervention of GBM in addition to targeting IL-13Rα2.

#2439

Establishment of six human colorectal cancer cell lines: identification of genes that have metastatic potential.

Soon-Chan Kim, Chang-Won Hong, Sang-Geun Jang, Seung-Yong Jeong, Jae-Gahb Park, Ja-Lok Ku. _Seoul National University, Medical Campus, Seoul, Republic of Korea_.

A number of genes have been studied in accordance with their metastatic potential. Nevertheless, widely used metastatic colorectal cancer cell lines such as SW-620 or IS3 only show the influence of metastatic genes within limited mutational landscape. Here, three pairs of primary CRC (SNU-2335A, SNU-2335D, SNU-2404A, SNU-2404B, SNU-2414A, and SNU-2414B) and corresponding metastasis cell lines were established and analyzed by whole exome sequencing and microarray.

Three pairs of primary CRC (SNU-2335A, SNU-2404A, and SNU-2414A) and corresponding matched metastasis cell lines (SNU-2335D, SNU-2404B, and SNU-2414B) were analyzed for gene expression by microarray and whole exome sequencing. Selected target genes from microarray were analyzed again by real-time PCR in order to confirm the mRNA expression level in the paired CRC cell lines.

The genes selected for examination were calponin 3, acidic (CNN3); sorbin and SH3 domain containing 1(SORBS1); epithelial stromal interaction 1 (EPSTI1); bone marrow stromal cell antigen 2(BST2); kelch-like 5 (KLHL5); trypsinogen C (TRY6); and synaptotagmin-like 5 (SYTL5). We calculated the intersection of all three metastatic CRC cell lines. Five genes (BST2, SORBS1, CNN3, EPSTI1, and KLHL5) were up-regulated in metastatic cell lines compared to primary cell lines. Two genes (TRY6 and SYTL5) were down-regulated in metastatic cell lines compared to primary cell lines. Then, we ranked 15 genes that were up-regulated and 15 genes that were down-regulated in omental metastasis regardless of the 2-folds threshold. Of the 30 genes that were differently expressed, we selected three genes (CNN3, SORBS1, and TRY6) that showed the highest fold changes and are found in 2-fold change analysis.

We identified 7 genes that were differently expressed in metastatic CRC cell lines compared to primary CRC cell lines. These genes may serve as diagnostic markers and therapeutic targets for patients with metastatic CRC.

#2440

Cross-tissue interactome analyses unravel novel oncogenic roles of SOX2.

Thorsten Schaefer,1 Silvia Candido,1 Hui Wang,1 Thomas Bock,2 Alexander Schmidt,2 Claudia Lengerke1. 1 _University of Basel and University Hospital Basel, Basel, Switzerland;_ 2 _Biozentrum, University of Basel, Basel, Switzerland_.

First recognized for its significance in early embryogenesis, SOX2 (sex determining region Y box 2) more recently received major attention as a (i) transcriptional master regulator of stemness driving the re-programming of terminally differentiated somatic cells back into a pluripotent stem cell state and (ii) powerful oncogene of the cancer stem cell (CSC) compartment. We and others have shown that SOX2 expression relies on canonical PI3K/AKT signaling involving direct physical contact and phospho-modification by AKT. Interestingly, we observed that these interactions are highly tissue specific which prompted us to perform a systematic proteomic analysis of co-factors regulating SOX2 biology.

The SOX2 interactome was analyzed in human breast carcinoma and glioblastoma cell lines by co-immunoprecipitation (co-IP) and HPLC/MS revealing >900 individual and >70 conserved proteinaceous co-factors, thus underscoring a hitherto underestimated scaffolding function of SOX2. As expected, a major proportion of binding partners cluster in functional categories implicated in DNA binding and/or nucleotide modification (e.g. helicases, transferases, and ligases). However, a near equal share of SOX2 associated factors fall into functional classes formally unrelated to transcription modulation such as cell adhesion, cytoskeletal organization, or metabolic adaptation. Using SOX2 knock-down and overexpression cells generated by lentiviral technology, we confirm co-regulation of SOX2 with selected binding partners. Moreover, we observe co-depletion of SOX2 and protein co-factors in cells of stalled AKT activity, and a molecular restoration of both upon proteasomal inhibition.

We conclude that, besides its known significance in DNA activity control, the SOX2 protein engages in various interactions seemingly unrelated to transcription modulation. These newly discovered interactions are likely to synergize with canonical SOX2 functions (i.e. the induction and maintenance of stemness), but involve regulation at post-translational level and of extra-nuclear components. Our efforts thus add an exciting new facet to SOX2 biology and eventually, may open new therapeutic avenues for targeted anti-CSC therapy.

#2441

NanoString 3D Biology™ technology: simultaneous digital counting of DNA, RNA and protein.

Chris Lausted,1 Yong Zhou,1 Jinho Lee,2 Christopher Vellano,2 Karina A. Eterovic,2 Ping Song,2 Lin-ya Tang,2 Gloria Fawcett,2 Tae-Beom Kim,2 Ken Chen,2 Gary Geiss,3 Gavin Meredith,3 Qian Mei,3 Gokhan Demirkan,3 Dwayne Dunaway,3 Dae Kim,3 P. Martin Ross,3 Elizabeth Manrao,3 Nathan Elliott,3 Sarah Warren,3 Christina Bailey,3 Chung-Ying Huang,3 Joseph Beechem,3 Gordon Mills,2 Leroy Hood1. 1 _Institute for Systems Biology, Seattle, WA;_ 2 _MD Anderson Cancer Center, Houston, TX;_ 3 _Nanostring Technologies, Inc., Seattle, WA_.

Introduction: Development of improved cancer diagnostics and therapeutics requires detailed understanding of the genomic, transcriptomic, and proteomic profiles in the tumor microenvironment. Current technologies can excel at measuring a single analyte, but it remains challenging to simultaneously collect high-throughput DNA, RNA, and protein data from small samples. We have developed an approach that uses optical barcodes to simultaneously profile DNA, RNA, and protein from as little as 5ng DNA, 25ng RNA, and 250ng protein or just 2 5µm FFPE slides, and simplifies data analysis by generating digital counts for each analyte.

Methods: The approach uses paired capture and reporter oligonucleotide probes and optical barcodes to enumerate up to 800 targets. The platform was initially developed to measure RNA, and we have adapted it to measure DNA single nucleotide variants (SNVs), proteins, and phospho-proteins. SNVs are detected by direct hybridization of sequence discriminating probes to the wild-type and mutant sequence of interest. Proteins are detected via binding of oligonucleotide-conjugated antibodies.

Results: Combinations of DNA, RNA, and protein in biological and experimental contexts. SNV probes are able to detect variant alleles down to 5% abundance within a wild type population and can discriminate variants within mutation hotspots. It was >96% accurate at identifying variants from samples displaying a range of allele frequencies and DNA integrity when benchmarked against next-generation sequencing. Protein detection has been developed for cell surface, cytosolic, and nuclear proteins, as well as phospho-proteins. It was validated against flow cytometry, western blot, and mass spectrometry using cell lines with ectopic target expression and primary cells. To demonstrate concurrent measurement of DNA, RNA, and protein from a single system, BRAFWT or BRAFV600E cell lines were treated with the BRAFV600E inhibitor vemurafenib and the MEK inhibitor trametinib. We measured the allele usage at the BRAFV600 locus, as well as BRAFV600E dependent changes in mRNA expression, protein expression and protein phosphorylation in a single experiment.

Conclusions: 3D Biology has several advantages over other analytical approaches. Direct, single-molecule digital counting allows detection over a broad dynamic range with high reproducibility, often over 98% concordance between technical replicates. The simultaneous interrogation of DNA, RNA, and protein maximizes the amount of data obtained from precious samples and minimizes instrumentation demands by leveraging a single detection platform. The 3D Biology approach allows holistic, digital analysis of biological samples with high specificity and precision. This technology is currently available for research use, but may also have clinical application in the future.

#2442

Simplified sequencing of full-length isoforms in cancer on the PacBio Sequel System.

Meredith Ashby, Ting Hon, Elizabeth Tseng, Aparna Vedula, Tyson A. Clark. _Pacific Biosciences, Menlo Park, CA_.

Tremendous flexibility is maintained in the human proteome via alternative splicing, and cancer genomes often subvert this flexibility to promote survival. Identification and annotation of cancer-specific mRNA isoforms is critical to understanding how mutations in the genome affect the biology of cancer cells. While microarrays and other NGS-based methods have become useful for studying transcriptomes, these technologies yield short, fragmented transcripts that remain a challenge for accurate, complete reconstruction of splice variants. In cancer proteomics studies, the identification of biomarkers from mass spectroscopy data is often limited by incomplete gene isoform expression information to support protein to transcript mapping.

The Iso-Seq™ protocol developed at PacBio offers the only solution for direct sequencing of full-length, single-molecule cDNA sequences needed to discover biomarkers for early detection and cancer stratification, to fully characterize gene fusion events, and to elucidate drug resistance mechanisms. Knowledge of the complete isoform repertoire is also key for accurate quantification of isoform abundance. As most transcripts range from 1 - 10 kb, fully intact RNA molecules can be sequenced using SMRT® Sequencing without requiring fragmentation or post-sequencing assembly. However, some cancer research applications have presented a challenge for the Iso-Seq protocol, due to the combination of limited sample input and the need to deeply sequence heterogenous samples.

Here we report the optimization of the Iso-Seq library preparation protocol for the PacBio Sequel platform and its application to cancer cell lines and tumor samples. We demonstrate how loading enhancements on the higher-throughput Sequel instrument have decreased the need for size fractionation steps, reducing sample input requirements while simultaneously simplifying the sample preparation workflow and increasing the number of full-length transcripts per SMRT Cell. The results highlight the potential for broader application of the Iso-Seq method to more comprehensively characterize alternative splicing in cancer.

#2443

Massively parallel single-cell RNA-Seq identifies diverse subpopulations displaying EMT and stem-like features.

Billy Lau, Jiamin Chen, Hanlee P. Ji. _Stanford School of Medicine, Stanford, CA_.

Single-cell transcriptome analysis enables a new paradigm for studying complex systems in cancer. As opposed to bulk sequencing, which averages genomic signals across thousands or millions of cells and obscures the presence of rare subtypes, single cell sequencing enables the interrogation of individual cells. In cancer, intratumoral heterogeneity is observed at both genomic and epigenomic levels, and its analysis enables the discovery of new actionable targets and treatment modalities tailored to individual subpopulations. As an example, many cancer cell lines and those derived from patients contain subpopulations marked by distinct patterns of surface markers such as CD44, and are linked to drug resistant and tumor initiating phenotypes. A complete characterization of such cellular populations ideally requires marker-free sampling, followed by clustering into distinct subgroups. In this study, we demonstrate the significant advantages of such an approach; we utilize a high-throughput single-cell RNA-Seq method to characterize the transcriptomic profiles of cellular populations.

We performed single-cell RNA-Seq on thousands of cells in the matched SW480 (primary) and SW620 (metastatic) colorectal cell lines using a microfluidic droplet barcoding technology that enables the tracking of single cells during library preparation. By focusing on genes with high inter-cell variability, we discovered a small subpopulation of cells that displayed a distinct gene expression signature from the major subpopulation. Differential gene expression analysis of this subpopulation yielded genes virtually all enriched in the epithelial-to-mesenchymal transition (EMT) pathway. These cells showed significant increases in canonical mesenchymal marker genes such as VIM, CD44, and SOX9. Gene expression profiles of these subpopulations also correlated with established EMT signatures. Remarkably, this subpopulation did not display mutual exclusivity in gene expression with the epithelial marker EPCAM, which possibly indicates an intermediate mesenchymal phenotype. We also observed in the major population cluster a small subset of cells totaling less than 1% of the population that were significantly enriched for LGR5 expression, a common stem-like marker in colorectal cancer. Overall, we demonstrate the use of single-cell RNA-Seq to discover and characterize a diversity of cellular states that would otherwise be impossible from bulk analysis.

### Genomic Landscape of Lymphoma, Leukemia, and Lung, Bladder, and Other Cancers

#2444

Recurrent mutations and clinical outcomes in patients with follicular lymphoma.

Kilannin Krysiak,1 Cody Ramirez,1 Felicia Gomez,1 Christopher A. Miller,1 Robert S. Fulton,1 Friederike Kreisel,1 Amanda F. Cashen,1 Nancy L. Bartlett,1 Ana Ruano,2 Eric D. Hsi,2 Malachi Griffith,1 Obi L. Griffith,1 Todd A. Fehniger1. 1 _Washington University in Saint Louis, Saint Louis, MO;_ 2 _Cleveland Clinic, Cleveland, OH_.

Follicular lymphoma (FL), the most common indolent non-Hodgkin lymphoma (NHL), is associated with a highly heterogeneous clinical course. The genetic landscape of FL is less understood than other B-cell NHLs such as chronic lymphocytic leukemia and diffuse large B-cell lymphoma. With ongoing clinical trials using therapies targeting B cell receptor signaling (e.g., ibrutinib) in FL, recurrent mutations could impact the interpretation of these trial outcomes and the identification of specific patient subpopulations that have increased or decreased clinical benefit.

To identify novel recurrent mutations in FL, we designed a large custom capture lymphoma-related gene panel (WUSM-LP) and queried 113 predominantly treatment-naïve FL samples. To design the WUSM-LP reagent (NimbleGen), we targeted the coding regions of 1716 genes identified in exome sequencing data from a cohort of 24 patients (28 samples) with FL combined with genes recurrently mutated in B-cell non-Hodgkin lymphoma in the literature (Krysiak K et al., Blood, 2016). Previously, we applied the WUSM-LP to the discovery cohort and an additional 81, primarily treatment-naïve, FL samples. Using mutations identified in these 105 individuals, we identified 39 significantly mutated genes (SMGs). These included well-characterized genes in FL (e.g., KMT2D, EZH2, TNFRSF14), and identified novel mutations (BTK, HVCN1, and others) thereby expanding the repertoire of mutations affecting genes in the BCR signaling pathway, SWI/SNF complex and histone family. Including novel BTK mutations, we identified 23 (22%) individuals harboring mutations that likely up-regulate signaling downstream of BTK, potentially affecting response rates to BTK-targeting therapies at the point of FL diagnosis.

We also focused on the subset FL patients (N=59) that were sequenced within 1 year of front-line therapy in order to identify clinical associations between recurrent mutations and clinical outcomes, comparing mutation status of SMGs to progression-free survival (PFS) rates. Improved PFS was observed in patients harboring HVCN1 mutations (p<0.05) in the treated cohort. CREBBP mutations were associated with significantly reduced PFS (p<0.05).

Since our initial analysis, we have completed sequencing of an additional 174 FL samples with clinical data available. Confirming our earlier observations with an independent cohort, we observed 9 TNFAIP3 truncating, 18 CARD11 coiled-coil domain, 4 BCL10 truncating and 8 BTK mutations in these samples. These mutations affect 18.4% of FL patients in this cohort, are likely to up-regulate NFkappaB signaling and may impact ibrutinib sensitivity. With the additional power of 8 HVCN1 and 110 CREBBP mutations we have identified, we will evaluate the previously identified associations. The analyses of recurrent mutations and clinical correlations in this combined cohort of nearly 300 FL patients are ongoing and will be presented.

#2445

Integrative genetic analysis identifies therapeutic relevance of cell of origin-specific genetic alterations in diffuse large B-cell lymphoma.

Daisuke Ennishi,1 Ali Bashashati,2 Saeed Saberi,2 Anja Mottok,1 Barbara Meissner,1 Merrill Boyle,1 Susana Ben-Neriah,1 Robert Kridel,1 Kerry J. Savage,1 Laurie H. Sehn,1 Joseph M. Connors,1 Ryan D. Morin,3 Marco A. Marra,3 Sohrab P. Shah,2 Christian Steidl,1 David W. Scott,1 Randy D. Gascoyne1. 1 _Centre for Lymphoid Cancer, British Columbia Cancer Agency, Vancouver, British Columbia, Canada;_ 2 _Molecular Oncology, British Columbia Cancer Agency, Vancouver, British Columbia, Canada;_ 3 _Genome Science Centre, British Columbia Cancer Agency, Vancouver, British Columbia, Canada_.

Introduction: Diffuse large B-cell lymphoma (DLBCL) comprises two distinct cell-of-origin (COO) subtypes: the germinal center B-cell-like (GCB) and the activated B-cell-like (ABC) subtypes. Several sequencing studies have informed on DLBCL biology and identified numerous candidate genes implicated in pathogenesis. However, comprehensive analysis of recurrent genetic alterations, and related prognostic significance according to COO subtypes has not yet been performed. To understand the molecular distinctions and their therapeutic relevance specific to COO subtype in DLBCL, we performed a comprehensive genetic analysis on a population registry-based cohort of 347 patients with de novo DLBCL uniformly treated with R-CHOP.

Methods: Tissue biopsies from 347 patients were analyzed using deep amplicon sequencing of 58 genes, Affymetrix SNP 6.0 arrays, and RNAseq. Immunohistochemical staining was performed on tissue microarrays. COO subtype was assigned by Lymph2Cx assay.

Results: We detected COO-specific mutations, copy number alterations (CNAs) and gene fusions. Furthermore, integrative analysis showed COO-specific trans gene expression effects on CNAs, demonstrating that CNAs in GCB-DLBCL are tightly linked to the deregulation of networks associated with PI3K-AKT signaling.

We also identified the significant prognostic impact of recurrent genetic alterations within each COO subtype, including deletions of MIR15a/16-1 and TP53 and NFATC1 gain in ABC-DLBCL, TP53 and CREBBP mutations, PRAME deletions and MYC gain in GCB-DLBCL. In addition, deletions of PTEN and INPP4B, which are negative regulators of PI3K-AKT signaling, were individually associated with poor outcome in GCB-DLBCL (p=0.01 and p=0.045, respectively). Furthermore, patients whose tumors harbored both genetic alterations had an even worse prognosis (p=0.004), suggesting the clinical importance of PI3K-AKT signaling in GCB-DLBCL.

Based on these genetic data, we found that the PI3K-AKT signaling pathway was more commonly altered in GCB-DLBCL than in ABC-DLBCL (64% vs 23%, p<0.0001). We also confirmed frequent genetic alterations involved in the BCR-NFκB signaling pathway in ABC-DLBCL (80%), while this pathway was less frequently altered in GCB-DLBCL (54%; p<0.0001). In addition, epigenetic modification and immune recognition pathways were more commonly altered in GCB-DLBCL compared to ABC-DLBCL (70% vs 44%; p<0.0001, 71% vs 56%; p=0.015, respectively). Finally, we demonstrated that the majority of DLBCL cases are altered for at least two of the four pathways mentioned above (86% of ABC-DLBCL and 87% GCB-DLBCL).

Conclusion: we describe the landscape of common genetic aberrations in a population-based uniformly-treated DLBCL cohort, broadly representative of patients treated with curative intent, providing the genetic foundation for implementing precision medicine in this disease.

#2446

Integrated analysis of somatic mutations in subcutaneous panniculitis-like T-cell lymphoma by whole-exome and -transcriptome sequencing.

Hyojin Song,1 Youngil Koh,2 Daeyoon Kim,1 Hongseok Yun,3 Choong-Hyun Sun,3 Hogune Im,3 Dong-Yeop Shin,2 Sung-Soo Yoon2. 1 _Seoul National University College of Medicine, Seoul, Republic of Korea;_ 2 _Seoul National University Hospital, Seoul, Republic of Korea;_ 3 _Samsung SDS, Seoul, Republic of Korea_.

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rarely differentiated form of non-Hodgkin lymphoma. This uncommon disease is triggered by preferential lymphoma infiltration into subcutaneous adipose tissue, and nodule formation in the fatty tissue. Consequently, the early stage symptoms of SPTCL is found as multiple subcutaneous nodules beneath the skin. Due to the low incidence rate and indolent progression of the disease, the genetic alterations associated with SPTCL still remains poorly characterized. Genetic profiling of SPTCL by integrated whole-exome and -transcriptome sequencing will augment the comprehensive characterization of somatic mutations in SPTCL. To investigate the genetic alterations associated with the incidence of SPTCL, we performed whole-exome sequencing of both biopsy sample collected from the patient's left shoulder lesion, and matched saliva sample to 200X and 100X read depth of coverage, respectively. Whole-transcriptome sequencing of the tumor sample was also performed for expression level analysis; gene fusions are also explored by TopHat-fusion-post. Somatic mutations were precisely detected by our internally developed somatic variant caller, which is an adjusted version of VarScan2 with additional filters based on thresholds of p-value and odds ratio. Overall, we discovered a total of 158 somatic variants: 139 SNVs and 19 indels, by using somatic variant detection algorithm of our own. By applying the additional filters to these variants, two genes were remarkably identified: SQSTM1 and BAGE3. SQSTM1 (sequestosome 1), also known as ubiquitin-binding protein p62, encodes proteins that regulate ubiquitination, autophagy, and activation of NFkB1. Through cBioPortal database search, SQSTM1 found to be highly amplified across the various cancer types, such as neuroendocrine prostate cancer (NEPC), kidney renal clear cell carcinoma (RCC), and pancreatic cancer. On the other hand, BAGE3, B melanoma antigen 3, was reported as a candidate gene encoding tumor antigens. Gene fusions were identified by comparing the number of spanning reads and mate pairs, and fusion of NOL7 and RANBP9 on chromosome 6 showed the highest fusion score of 604.45. Comprehensive characterization of the initiation, progression, and relapse of subcutaneous panniculitis-like T-cell lymphoma is yet thoroughly understood. Hence, establishing the genetic basis and profiling the genomic landscape of SPTCL will broaden our understanding of SPTCL, and enhance the therapeutic effectiveness in the pre-diagnosis and treatment of the disease, as well as targeted gene therapies. Furthermore, to strengthen the statistical power of the somatic mutation analysis, we are planning to expand the cohort and conduct functional validation study of selected genes by using secured paraffin blocks of SPTCL patients.

#2447

The mutational landscape of chemo-refractory Burkitt lymphoma.

Claudia M. Wever,1 Maryse Lemaire,1 Donald C. Vinh,1 Josée Hébert,2 Yasser Riazalhosseini,1 Ryan Morin,3 Nathalie A. Johnson1. 1 _McGill University, Montreal, Quebec, Canada;_ 2 _Université de Montréal, Montreal, Quebec, Canada;_ 3 _Simon Fraser University, British Columbia, Canada_.

Background: Burkitt Lymphoma (BL) is characterized by rapid cellular proliferation driven by the canonical IG-MYC translocation. BL can evade apoptosis by silencing the tumor suppressor TP53 or by mutating MYC. Other mutations contribute to the pathogenesis of BL, including TCF3 or ID3, which stimulate PI3K/MAPK/MTOR signaling, or mutations in CCND3, which accelerate cell cycle progression. While BL is curable in 90% of patients, the prognosis for relapsed BL is dismal. The mutational landscape of relapsed BL (rBL) is not well characterized because it is rarely re-biopsied at the time of relapse. Two young men at our institution experienced a BL relapse within 3 months of completing intensive chemotherapy. We hypothesized that mutations implicated in therapeutic resistance would be enriched in the tumor cells taken at the time of relapse. Our aim was to identify these mutations and develop cell lines from these patients to study these tumours in more detail.

Methods: Both patients had whole exome sequencing (WES) performed on tumor cells obtained at the time of diagnosis (T1) and relapse (T2), using unaffected peripheral blood to exclude germline variants. The novel cell lines (CL) derived from T2 were also sequenced and karyotyped.

Results: The total number of coding mutations in patient 509 was: T1 = 78, T2 = 84 and CL= 63, and in patient 533: T1= 43, T2 = 66 and CL= 70. All samples had multiple missense mutations (5 to 7) in MYC and had evidence of clonal heterogeneity. Patient 509 had a TP53 (R248) mutation that was clearly selected for during chemotherapy, where the variant allele fraction (VAF) increased from 2% at T1 to 93% at T2. 19 genes had acquired missense mutations at T2 including: CD14, GSTM3, BCR and ELMO3. The following genomic rearrangements were observed in the 509CL by karyotype:-Y,t(8;14)(q24.21;q32),der(10)t(10;22)(p1?3;q11.2),t(13;17)(q3?2;q11.2), del(17)(p11.2) and -22. Patient 533 had multiple frameshift mutations that occurred in all three samples (T1, T2 and CL), including in CCND3, BAX, ARID1A, ARID1B, DDX5, DETD1B, TSFM, SP3 and TFAP4. T2 and the CL had additional acquired missense or nonsense mutations in ZFP36L2, CCL7, FAM13C and IKBKB. The 533CL had 3 dominant clones by karytope, The rearrangements observed were: del(2)(p21p23), t(8;14)(q24.21;q32) ,t(1;12)(q12;q24) and +7. The frameshift mutations in ZFP36L2 and BAX were homozygous in the 533CL.

Conclusions: Multiple genes are implicated in the pathogenesis of chemotherapy-resistant BL, many of which had not been appreciated in studies focused on sequencing de novo BL (e.g. BAX, ARID1A). To our knowledge, these are the first BL cell lines that have been well-characterized with respect to the serial acquisition of mutations after exposure to chemotherapy and with knowledge of germline variants. These are useful resources to study oncogene cooperation, clonal evolution, chromatin remodelling (ARID1A), RNA processing (DDX5, ZFP36L2) and apoptosis (P53 and BAX null) in BL.

#2448

Deep exome sequencing reveals recurrent somatic mutations in Hodgkin's lymphoma.

Felicia Gomez, Kilannin Krysiak, Cody Ramirez, Nancy Bartlett, Amanda Cashen, Friederike Kreisel, Eric Duncavage, Malachi Griffith, Todd Fehniger, Obi Griffith. _Washington University, St. Louis, MO_.

High-throughput sequencing provides insights into cancer pathogenesis. The application of genomics technologies to cancers characterized by rare cell populations is a challenge. DNA extracted from bulk tumor samples is a mixture of malignant and non-malignant cells. This is a problem in cancers defined by rare cell populations because it complicates the task of identifying true somatic mutations. Hodgkin lymphoma (HL) exemplifies this challenge. The pathologic hallmark of HL is Hodgkin-Reed-Sternberg (HRS) cells. HRS cells have a low abundance within an affected lymph node (1-30% of tumor microenvironment). Because HRS cells are rare, a limited number of studies have described characteristic genomic events. These studies have examined HL cell lines, and, recently, a small number of flow-sorted HRS-cells (Reichel et. al. 2015). We hypothesized that recurrent somatic mutations can be identified in HRS cells using bulk HL biopsies and ultra deep exome sequencing.

The Illumina HiSeq X,which can generate> 1.6 Tb of sequence data per run, is a applicable tool for the discovery of somatic mutations in HL. Frozen tumor (lymph node)/normal (skin) pairs from 31 patients with HL were examined. Three KAPA libraries were constructed per sample. The libraries were pooled, size selected, and captured using an IDT xGen capture reagent. The pools were sequenced across eight lanes of a HiSeq X. A total of 7.04 x 1012 bases were sequenced across all samples with an average of 1.10 x 1011 bases per sample. The mean depth of coverage achieved across all samples was 783.4x (425.9x - 1091.9x). Given the low abundance of HRS cells we anticipated most somatic sites would have a VAF of 1% -5%. Given the level of coverage we anticipate that most somatic variants will have >5 reads of support.

Instrument data were processed using the McDonnell Genome Institute somatic variant calling pipeline that includes 5 SNV callers and 3 indel callers. We used these steps to filter variants: min. 50x coverage, max. 5000x coverage, min. 1% tumor VAF, max. 5%, normal VAF, and min. 5 variant reads in the tumor. Here we report preliminary data on mutations in five pilot samples. The remaining 26 will be presented at the meeting. We confirmed 7 previously identified recurrent mutations in HL (Reichel et al. 2015). Within our pilot data 13 recurrently mutated genes were found. The most interesting of our recurrent genes is TNFAIP3, which was the second most frequently mutated gene in Reichel et al. 2015. These data suggest that ultra deep sequencing of tumor samples containing rare HRS cells can identify recurrent somatic mutations. Thus, ultra deep exome sequencing may be a useful discovery tool for rare tumor populations, and will improve our understanding of HL.

#2449

Transcriptome and whole-genome sequencing analysis of a novel murine model of chronic lymphocytic leukemia.

Lili Wang,1 Jing Sun,1 Amaro Taylor-Weiner,2 Jaegil Kim,2 Zachary J. Cartun,1 Angela N. Brooks,3 Donna Neuberg,1 Mark D. Fleming,4 Benjamin L. Ebert,4 Gad Getz,2 Ruben Carrasco,1 Catherine J. Wu1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Broad Institute, Cambridge, MA;_ 3 _University of California, Santa Cruz, CA;_ 4 _Boston Children's Hospital, Boston, MA_.

Large-scale cancer sequencing of primary chronic lymphocytic leukemia (CLL) has identified SF3B1, an RNA splicing factor, as one of the most frequently mutated CLL genes. SF3B1 mutations localize to a hotspot (>50% at K700E site) and highly co-occur with mutations in ATM or deletion of chromosome 11q (minimally deleted region contains ATM). How this splicing factor mutation alone or in cooperation with ATM deletion contributes to CLL remains elusive. Genetically engineered mice are powerful tools in understanding genetic lesions and cancer phenotypes. We therefore generated a mouse line that conditionally expresses heterozygous Sf3b1-K700E mutation. We modeled the effects of the combined alterations by crossing mice with conditional knockout of Atm and mice with Sf3b1-K700E. By breeding these mice with homozygous CD19-Cre transgenic mice, we achieved B cell-restricted expression of heterozygous Sf3b1 mutation and Atm deletion. B cell co-expression of these two mutations in vivo led to clonal expansion of CD19+CD5+ B cells in blood, marrow and spleen in aged mice (18 to 24-month old) at low penetrance. No leukemia cells were found in the Sf3b1-K700E mice (up to 24-month old). The CLL cells from the double mutant mice could be engrafted in both immunocompetent and immunodeficient mice, with detectable disease within 2-4 weeks following transfer, thus making this mouse line amenable to drug discovery and biologic investigations. To investigate how Sf3b1 mutation and Atm deletion synergistically contribute to CLL, we asked if there are RNA and DNA level changes in the double mutant mice with CLL. First, we performed transcriptome sequencing of splenic B cell RNA collected from age-matched mice that either express wild-type, singly mutant alleles of Sf3b1 or Atm, or doubly mutant alleles with or without CLL-like disease. Using the tool JuncBASE, we classified and quantified splice variants associated with the different genetic alterations. Consistent with prior findings in human CLL, we observed that splice variants in Sf3b1-mutant mice alone were highly enriched at 3' splice sites. Sf3b1 and Atm doubly mutant B cells displayed a splicing pattern similar to that in Sf3b1 singly mutant cells. Moreover, we identified unique CLL splice variants in genes (Setdb2, Phf11c) previously shown to be associated with CLL. Next, we examined the mutation rate in DNA derived from splenic B cells collected from mice with a singly mutated allele of Sf3b1 or Atm, or with doubly mutated alleles with and without CLL-like disease through comparison against matched germline DNA from kidney by whole-genome sequencing. We have observed that co-expression of Sf3b1 mutation and deletion of Atm results in a higher mutation rate compared to singly mutant cells. Our analysis has revealed that altered RNA splicing and genomic instability all contribute to CLL leukemogenesis. We will further dissect how the two lesions contribute to CLL functionally using this model.

#2450

Mutational profiling of MLL-PTD acute myeloid leukemia.

Lingwen Ding,1 Qiaoyang Sun,1 Kar-Tong Tan,1 Wenwen Chien,1 Anand Mayakonda,1 Dechen Lin,1 Xinyi Loh,1 Jinfen Xiao,1 Manja Meggendorfer,2 Tamara Alpermann,2 Manoj Garg,1 Su-Lin Lim,1 Vikas Madan,1 Norimichi Hattori,1 Yasunobu Nagata,3 Satoru Miyano,4 Allen Yeoh Eng Juh,1 Hsin-An Hou,5 Yan-Yi Jiang,1 Yan-Yi Jiang,1 Sumiko Takao,1 Li-Zhen Liu,1 Siew-Zhuan Tan,1 Siew-Zhuan Tan,1 Michael Lill,6 Mutsumi Hayashi,7 Akitoshi Kinoshita,7 Hagop M. Kantarjian,8 Steven M. Kornblau,8 Seishi Ogawa,3 Torsten Haferlach,2 Henry Yang,1 H. Phillip Koeffler1. 1 _Cancer Science Institute of Singapore, Singapore, Singapore;_ 2 _MLL Munich Leukemia Laboratory, Munich, Germany;_ 3 _Department of Pathology and Tumor Biology, Kyoto, Japan;_ 4 _The University of Tokyo, Tokyo, Japan;_ 5 _National Taiwan University Hospital, Taiwan, Taiwan;_ 6 _Cedars-Sinai Medical Center, Los Angeles, CA;_ 7 _Keio University School of Medicine, Tokyo, Japan;_ 8 _MD Anderson Cancer Center, Houston, TX_.

In this study, we performed whole-exome and targeted sequencing on 85 MLL-PTD AML patients. These AMLs have oncogenic tandem duplication of the MLL gene. At least one well-known oncogenic driver mutation was identified in over 90% of the MLL-PTD patients. In line with earlier sequencing studies of other AML subtypes and the TCGA-AML-sequencing project, DNMT3A was the most often mutated epigenetic regulator (25%); IDH1/2 hotspot mutations were identified in 31% of patients. TET family was the third most prominently mutated epigenetic regulator (TET1 (5%), TET2 (16.3%). Mutations of epigenetic regulators also occurred in polycomb-associated proteins (EZH2, ASXL family members), chromatin remodelers (ARID2, ARID1A), genes associated with histone acetylation (CREBBP, EP300, KAT6A, KAT6B) and histone methylation (MLL2, MLL3).

Proliferation-related pathway was extensively mutated, with 54 of 80 MLL-PTD patients (67.5%) carrying at least one mutation of proliferative genes. Specifically, FLT3 mutations were found in 46% of patient samples. Notably, some FLT3-ITD patients had more than one type of internal tandem duplication (ITD) insertion, probably reflecting existence of multiple subclones in these leukemias.

We found highly prevalent mutations of cohesin genes: STAG2 (16%), SMC1A (6%), SMC3 (1%), RAD21 (1%) and CTCF (6%). Cohesin pathway is more frequently mutated in MLL-PTD patients (26%) than the AML samples from either TCGA (13%) or a meta-analysis of 1000 AML (9.1%). Remarkably, an extremely high proportion of the mutations had a strong tendency to disrupt the coding sequence in STAG2, emphasizing their crucial tumor-suppressor role in this AML subtype (16% in MLL-PTD vs 3% in TCGA-AML.

RNA processing pathway was also strikingly altered in MLL-PTD patients. The most prominently mutated genes within this category were the splicing factors. They included U2AF1 (13%, S34F/Y), SRSF2 (3%), SF3A1 (5%), ZRSR2 (3%), DHX15 (1%) and CWC22 (1%).

Multiple mutations co-occur with MLL-PTD which are usually acquired in a sequential manner. A potential ordering for acquisition of many mutations include IDH2/DNMT3A/U2AF1/TET2→MLL-PTD→RAS-receptor tyrosine kinase based on the following reasons: #1, real-time-PCR showed that MLL-PTD was absent in remission while mutations of IDH2, DNMT3A, TET2 and U2AF1 were still retained with a high VAF. This suggests that MLL-PTD was acquired after mutations of IDH2, DNMT3A, TET2 and U2AF1; #2, MLL-PTD is highly stable during disease progression as compared with mutations of the RAS-RTK. On the other hand, RAS-RTK mutations frequently exist as subclonal mutations and tend to be unstable during disease progression. These observations support a notion that MLL-PTD was acquired prior to RAS-RTK. Taken together, MLL-PTD is acquired after those remission-persisting, initiating mutations (IDH2, DNMT3A, TET2 and U2AF1), but prior to lesions of the proliferation-related drivers.

#2451

Genomic wide microarray analysis identifies novel copy number alterations in adult acute myeloid leukemia.

Maria Chiara Fontana, Giovanni Marconi, Cristina Papayannidis, Eugenio Fonzi, Giorgia Simonetti, Antonella Padella, Anna Ferrari, Emanuela Ottaviani, Silvia Lo Monaco, Stefania Paolini, Simona Soverini, Giovanni Martinelli. _Department of Experimental, Diagnostic and Specialty Medicine. University of Bologna, Bologna, Italy_.

Introduction: Novel array-based technique as SNP microarray can detect losses or gains of chromosomic material, which could be predictive of response and can help define therapeutic strategies. The aim of this study is to improve conventional cytogenetic analysis and identify new genetic alterations relevant to leukemogenesis, by a SNP array-based genotyping approach.

Materials and Methods: We performed SNP 6.0 or Cytoscan HD (Affymetrix) in 235 Acute Myeloid Leukemia (AML) patients at diagnosis. Seventy-eight/235 samples were also performed by Whole Exome Sequencing, WES (HiSeq,Illumina). SNP Array data were analyzed by Nexus Copy Number v8.0 (BioDiscovery) and R Core Team.

Results: Copy Number Alterations (CNAs) were scattered across all chromosomes and all pts showed CNA events. SNP array analysis showed that several genes were preferentially deleted, including MRPS5 (14.8%), PHF6 (9.3%), SCAPER (7.2%), CASK (5.9%), WNK (4.6%), STAG2 (4.2%), LRRK1 (3.4%), PALB2 (3.4%), while the genes preferentially amplified were RABL2B (16.1%), NF2 (10.2%), NBPF9 (7.6%), JAK2 (6.8%), RB1, NF1 and KMT2A (4.2%), PTEN (3.4%), TP73 and SMAD2 (2.5%). Single-copy losses and deletions were enriched (p<.001) for genes mapping into the following pathways: aberrant PD-1 signaling, loss of function of SMAD4 in cancer and SMAD4 MH2 Domain mutants in cancer. The functional pathways significantly (p<.001) deregulated in our cohort with single copy gain and homozygous amplification were: regulation of transcription and nucleic acid, negative regulation of metabolic processes, constitutive signaling by aberrant PI3K in cancer and PI3K/AKT network. Finally, in order to define driver alterations, we correlate deletions and losses with mutational data. Interestingly, we found losses which are also targeted by mutations (BRCA2, LRRK1). Moreover, some deleted genes, as CASK, CDK6 and MAPT, were involved in pathways affected by genomic mutations (CASK deletion and MPP6 mutation, CDK6 deletion and PPM1D mutation, MAPT deletion and SPAG5 mutation).

Conclusion: We have identified new CNAs and pathways involving novel potential leukemia-related genes. Our results suggest that the comparison between SNP and WES data could provide important findings on prognosis of AML patients. Minimal deleted regions of genes implicated in deregulated pathways deserve further investigation in order to identify new candidate genes which could be relevant AML biomarkers. Acknowledgements: ELN,AIL,AIRC,progetto Regione-Università 2010-12 (L. Bolondi),FP7 NGS-PTL project,HARMONY.

#2452

Detailed genomic characterization of CNL/aCML/MPN-U/CMML reveals disease subgroups that may benefit from rationally-designed combination therapies.

Haijiao Zhang,1 Beth Wilmot,1 Daniel Bottomly,1 Libbey White,1 Erik Segerdell,1 Shannon K. McWeeney,1 Vishesh Khanna,1 Angela Rofelty,1 Sophie Means,1 Brian Junio,1 Samantha Savage,1 Emily Stevens,2 Kim-Hien Dao,1 Julia E. Maxson,1 Jeffrey W. Tyner1. 1 _Oregon Health & Science University Knight Cancer Institute, Portland, OR; _2 _Fred Hutchinson Cancer Research Center, Seattle, WA_.

Purpose: Chronic neutrophilic leukemia (CNL), chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia (aCML), and unclassified myeloproliferative neoplasms (MPN-U) are a group of heterogeneous disorders belonging to rare entities of myeloproliferative or myelodysplastic/myeloproliferative (MDS/MPN) syndromes. Due to lack of specific molecular markers and the limited understanding of pathogenesis, the treatment of these diseases remains empirical, resulting in poor outcomes. Recently, recurrent mutations in ASXL1, TET2, SRSF2 and cell signaling genes have been identified in these diseases. In this study we aim to analyze the co-occurrence patterns of these gene mutations, as well as the association of different mutations with disease subtypes and treatment outcomes.

Methods and results: We performed whole exome sequence and RNA-Seq on primary patient samples. Consistent with previous studies, high frequencies of ASXL1, SRSF2, TET2, SETBP1 and signaling pathway mutations were observed; whereas mutations of MPL, CEBPa, IDH1/2, and TP53 were rare. Further variant allelic frequency analysis demonstrated that mutations of the chromatin modifiers, epigenetic markers and splicing factors are mostly present in the major clones indicating early acquisition of these mutations. In addition, ASXL1/2, splicing factor and signaling pathway mutations co-occur in around 45% of all patients with these mutations, indicating that they drive these diseases in a cooperative manner.

RNA-seq analysis demonstrated two major gene expression clusters with high levels of either RAS or JAK-STAT signaling alterations respectively, indicating a potential need for distinct therapeutic targeting of specific subgroups of these diseases. In addition, we observed that 17% of patients have more than one signaling pathway mutation, possibly in distinct subclones, providing rationale for drug combination treatment.

Furthermore, we observed that mutations related to RAS signaling pathway are prevalent in CMML; CSF3R and JAK2 mutations are enriched in CNL; whereas, RAS and JAK-STAT pathway double mutations are more frequent in aCML. Similar incidence of ASXL1, splicing factor and epidemics mutations are observed across all these diseases.

Conclusions: CNL/aCML/MPN-U/CMML is a group of heterogeneous diseases associated with chromatin modifier, epigenetic, splicing factor and signaling pathway mutations in concomitant manner. We propose future studies of rational drug combinations with agents, targeting epigenetic and splicing factors, together with the appropriate signaling pathway inhibitors. Our study represents a large-scale comprehensive genomic analysis of these diseases, which reveals novel insight into patterns of mutation co-occurrence which could translate into new treatment paradigms for these difficult to treat hematologic malignancies.

#2453

Chromatin remodeling genes are frequently somatically altered in cervical carcinomas.

Akinyemi Ojesina, Aishwarya Sundaresan, Vinodh Srinivasasainagendra, Hemant Tiwari, Warner Huh. _University of Alabama at Birmingham, Birmingham, AL_.

Introduction: Cervical cancer is responsible for 15% of all deaths due to cancer in women worldwide. There is a need to identify somatic mutations driving this cancer in order to facilitate development of novel therapeutic strategies.

Experimental procedures: We analyzed whole exome sequencing data from 430 cervical carcinomas. The data were derived from a combination of our previously published work (Ojesina et al. Nature 2014; Chung et al, Int J Cancer 2015), and publicly available data generated by the Cancer Genome Atlas (TCGA). The Mutect2.0 and MutSig2CV algorithms were used to identify somatic mutations and significantly mutated genes (SMGs) respectively. Copy number alterations were analyzed using the GISTIC algorithm.

Findings: Based on MutSig2CV false discovery rate threshold of 0.1, we identified MLL3 (19%) and MLL2 (13%) as novel significantly mutated genes (SMGs) in cervical cancer. In addition, other chromatin modifying genes including EP300 (12%) and ARID1A (7%) were confirmed as SMGs. Further investigation of genes that did not meet the threshold revealed that 228 of 430 (53%) of cervical tumors harbored at least one somatic mutation in a chromatin modifying gene. These include genes in the myeloid/lymphoid or mixed-lineage leukemia (MLL) family (28%), lysine (K)-specific demethylase (KDM) family (23%), AT rich interactive domain (ARID) family (13%), SWI/SNF related, matrix associated, actin dependent regulator of chromatin (SMARC) family (8%) and histone cluster 1 (HIST1) family (4%).

Conclusions: We have demonstrated that a majority of cervical cancers harbor somatic mutations in chromatin remodeling genes. This novel finding was facilitated by the larger sample size derived from combining multiple datasets, and may have diagnostic and therapeutic implications in cervical cancer.

#2454

Genetic landscape of glioma reveals defective neuroactive ligand receptor interaction pathway as a poor prognosticator in glioblastoma patients.

Jagriti Pal,1 Vikas Patil,1 Anupam Kumar,2 Kavneet Kaur,2 Chitra Sarkar,2 Kumaravel Somasundaram1. 1 _Indian Institute of Science, Bangalore, India;_ 2 _All India Institute of Medical Science, New Delhi, India_.

Glioblastoma (GBM; grade IV), is highly proliferative, infiltrative and treatment refractory. Hence, understanding the complete genetic alteration profile of GBM would help us in identifying molecules or pathways that have strong implications in GBM pathogenesis, thus opening up avenues for targeted therapy. Recent large scale studies suggest that three pathways - receptor-tyrosine kinase, TP53 and RB, are significantly altered in GBM. However, even with the tremendous increase in our understanding of the tumor, advancement in therapeutics is minimal and the median survival still remains at 15 months. Hence, we need to elucidate novel altered molecules and pathways in GBM progression such that more effective therapeutic options can be explored.

Here, we have carried out whole exome sequencing of grade II, grade III and GBM samples which revealed the mutation spectrum of glioma from our patient set. Further, we performed integrative analysis of mutated genes from our patient cohort as well as TCGA cohort (The Cancer Genome Atlas) to find out mutated pathways that predict survival in GBM patients. The most significant pathway - neuroactive ligand-receptor interaction pathway was explored further. Patients with mutations in one or more genes of this pathway had poor survival. The pathway comprises of G-protein coupled receptors, ion channels and ligands which functions in modulation of neural plasticity, memory processes, behavior etc. Of the enriched genes belonging to this pathway, Calcitonin Receptor (CALCR), which was highest mutated in GBM (2.75%), was taken up for further investigation. CALCR was found to be downregulated in GBM and mutation or downregulation of the gene was found to predict poor survival in patients. Functional studies through cell-line based experiments revealed CALCR is a tumor suppressor in GBM. The peptide hormone calcitonin (CT), a high affinity CALCR ligand, inhibited proliferation, migration and anchorage-independent growth of glioma cells expressing CALCR with a concomitant decrease in the phosphorylation levels of ERK, AKT and JNK signaling

molecules. However, CT failed to do these functions in CALCR silenced glioma cells. Exogenous overexpression of CALCR in glioma cells expressing low levels of the receptor was found to inhibit proliferation, migration and anchorage independent growth and this effect was further augmented when CT was added. Further, introduction of tumor-derived mutations in CALCR led to the abrogation of its tumor suppressor function. Studies are ongoing to demonstrate the tumor suppressive nature of CALCR using in vitro astrocyte transformation and intracranial orthotopic mouse glioma model. Thus, our study finds CT-CALCR signaling axis is an important tumor suppressor pathway in glioma development and underscores the importance of using CT as a novel therapeutic molecule for GBM treatment.

#2455

Human glioblastoma arises from the distant subventricular zone normal appearing but harboring tumor-initiating mutations.

Joo Ho Lee,1 Jeong Eun Lee,2 Jee Ye Kahng,1 Junseong Park,3 Seon Jin Yoon,3 Se Hoon Kim,3 Jong Hee Chang,3 Seok-Gu Kang,3 Jeong Ho Lee1. 1 _Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea;_ 2 _Chungnam National University Hospital, Daejeon, Republic of Korea;_ 3 _Severance Hospital, Yonsei University College of Medicine, Seoul, Republic of Korea_.

Glioblastoma multiforme (GBM) is the most devastating and incurable brain tumor. Although the identification of cells with tumor initiating mutations or their location can provide the fundamental basis for understanding disease progression, the origin of GBM remains controversial due to the lack of direct evidence in human GBM patients. Here, we performed ultra-deep sequencing of triple-matched tissues of i) radiologically and pathologically normal subventricular zone (SVZ), which is distant from tumor, ii) GBM tumor, and iii) blood (or normal cortical tissues) from patients with GBM (IDH-wildtype), compared to those with other type of brain tumors such as GBM (IDH-mutant), meningioma, oligodendrgolioma, metastatic brain tumor, and GBM (IDH-wildtype) with SVZ-invasion. Surprisingly, we found that in 55.5% of IDH-WT GBM patients (5 of 9), normal appearing and distant SVZ already contained the low level of GBM mutations such as TP53, EGFR, RB1, PDGRF or TERT variations observed in the matched tumor. Single cell sequencing of GBM tumors and laser capture microdissection analysis of the SVZ show that mutations are enriched in the astrocyte ribbon area, which clonally evolved from the SVZ to the distant GBM tumor. Furthermore, using CRISPR-Cas9 system in the postnatal mouse brain, we showed that neural stem cells with TP53, PTEN, EGFR mutations migrated away from the mutated SVZ site and then formed the high grade malignant glioma in the distant cortical region. Taken together, this study provides the direct evidence that human glioblastoma arises from the distant SVZ that is normal-appearing but harboring tumor-initiating mutations.

#2456

Distinct genomic landscape of upper urinary tract urothelial carcinoma.

Yoichi Fujii,1 Yusuke Sato,2 Hiromichi Suzuki,1 Tetsuichi Yoshizato,1 Yusuke Shiozawa,2 Kenichi Yoshida,1 Yuichi Shiraishi,3 Kenichi Chiba,3 Hiroko Tanaka,3 Tohru Nakagawa,2 Haruki Kume,2 Hiroaki Nishimatsu,4 Toshikazu Okaneya,5 Masashi Sanada,6 Hideki Makishima,1 Satoru Miyano,3 Yukio Homma,2 Seishi Ogawa1. 1 _Graduate School of Medicine Kyoto University, Kyotoshi, Japan;_ 2 _The University of Tokyo Hospital, Bunkyoku, Japan;_ 3 _Institute of Medical Science, The University of Tokyo, Minatoku, Japan;_ 4 _The Fraternity Memorial Hospital, Sumidaku, Japan;_ 5 _Toranomon Hospital, Minatoku, Japan;_ 6 _Clinical Research Center, Nagoya Medical Center, Nagoya, Japan_.

Backgrounds:

Upper urinary tract urothelial carcinoma (UTUC) is relatively rare, accounting for 5-10% of urothelial malignancies with frequent multifocal development. To clarify distinct characteristics of UTUC, we comprehensively investigated the genetic alterations of this disease.

Materials & methods:

Surgical specimens of UTUC and matched normal samples were obtained from 99 patients with various stages who underwent nephroureterectomy, and subjected to whole exome and RNA sequencing. We compared our results in UTUC with datasets previously reported in bladder urothelial carcinoma (BUC). Mutations in apparently normal urothelial epithelia in 5 cases were also interrogated.

Results:

Genetic alterations were most frequently observed in TERT promoter (51% of cases), followed by KMT2D (48%), FGFR3 (44%), CDKN2A (42%), TP53 (31%), and RAS pathway (HRAS/KRAS/NRAS, 21%). More than 95% of cases harbored either TP53/MDM2, FGFR3, or RAS pathway mutations in an almost mutually exclusive manner, based on which UTUCs are classified into 3 distinct subgroups with unique molecular and clinical features; FGFR3-mutated tumors showed a significantly better postoperative overall survival than those with TP53/MDM2 (p<0.001) and RAS pathway (p=0.010) lesions. KMT2D mutations were common in patients with TP53, MDM2, and FGFR3 alterations, but rare in those with RAS pathway alterations.

Mutation patterns were compared between different urothelial cancers with regard to their location. Despite common genes affected, their mutation frequencies were substantially different; KMT2D mutations were more frequent in UTUC, while RB1 alterations were more prevalent in BUC. In addition, KMT2D mutations were significantly more common in UTUCs in the ureter than those in renal pelvis (85% vs. 35%, p<0.0001). UTUC showed 4 predominant mutation signatures; age-related, APOBEC, Transcriptional coupled repair (TCR), and mismatch repair (MMR) associated ones. Among these, TCR and MMR signatures were more specific to UTUC, although the latter is only detected in 4 hypermutated cases with biallelic MMR-gene deficiencies.

In the analysis of normal ureter tissues (N=25 from 5 patients), driver mutations were identified in 6 samples from 2 patients. In one case, tumor and normal samples shared 10-42 mutations, indicating that the cancer evolved within a background of clonal precancerous proliferation in apparently normal epithelia. By contrast, in the other case, none of the mutations were shared between tumors and normal epithelia, suggesting the presence of a field effect on urothelial carcinogenesis.

Conclusions:

UTUC tumors are classified into 3 molecularly and clinically distinct subtypes based on the status of mutations in TP53/MDM2, FGFR3, and RAS pathway. Depending on their location, urothlial cancers have different genetic backgrounds, where a field effect unique to urothelial epithelia might contribute to multifocal occurrence of UTUC.

#2457

Investigating the functional significance of novel, recurrent noncoding mutations of TBC1D12 in bladder cancer.

Angela S. Li, Jason A. Reuter, Can Cenik, Michael P. Synder. _Stanford University School of Medicine, Stanford, CA_.

Somatic noncoding mutations remain widely unexplored despite examples of critical importance such as driver mutations in the promoter of the TERT gene. We recently identified novel somatic mutations in the regulatory Kozak sequence (just upstream of the translation start site) of TBC1D12 in ~15% of bladder cancer patients, making it one of the most frequently mutated noncoding regions in any cancer (Araya et. al 2016). TBC1D12 was not previously implicated in cancer, and little is currently known about its function. It is a member of the TBC1 superfamily, suggesting a role in intracellular trafficking as a RAB-specific GTPase-activating protein (GAP). To investigate the functional implications of TBC1D12 mutations in bladder cancer, we undertook a combined computational and experimental approach. We analyzed clinical correlates, identified downstream effector candidates, and assessed the impact of these mutations on gene expression.

To determine the clinical relevance of the TBC1D12 mutation, we did correlation and survival analyses of clinical data of bladder cancer patients from TCGA. We found that TBC1D12 mutations confer slightly worse survival but were not linked to other clinical correlates. To identify potential downstream effectors, we used reverse phase protein array (RPPA) data from 127 patients to search for proteins that were differentially expressed or phosphorylated between patients with or without TBC1D12 mutations. We found differential expression of GSK3 and differential phosphorylation of p90RSK and c-Jun, suggesting three potential downstream effector candidates. Hierarchical clustering of RPPA data for these three proteins revealed 4 subgroups with distinct molecular phenotypes. Furthermore, most of the TBC1D12 mutants were clustered within one molecular subtype. To experimentally test these downstream effector candidates and identify new ones, we modulated the levels of TBC1D12 in bladder cancer cell line Ku-19-19 using overexpression plasmids or siRNAs. After validating that these transfections were effective in changing TBC1D12 expression, we performed RNA-Seq experiments. Results from this study are expected to be presented at the meeting.

In summary, these studies suggest that the novel and recurrent noncoding mutations in TBC1D12 may affect patient survival and alter the expression or post-translational modification of downstream targets. Potential molecular mechanisms associated with the presence of TBC1D12 mutations will be discussed, and a series of follow-up studies are underway. These studies also demonstrate that applying both computational and experimental approaches can help develop and test evidence-based hypotheses, contributing to an understanding of the clinical and functional impact of TBC1D12.

#2458

Spatial genomic heterogeneity of multifocal bladder cancer.

Rachel Goldberg, Maria Del Carmen Rodriguez, Leslie Cope, Aline C. Tregnago, Evgeny Izumchenko, George J. Netto, David Sidransky, Mohammad O. Hoque. _Johns Hopkins University, Baltimore, MD_.

Urothelial cancer (UC) is a well-known multifocal disease with frequent recurrences. The clonal origin of spatially separated UC foci is controversial. Here we propose to elucidate the multifocal UC genome by next generation sequencing (NGS) and thereby identify novel clonal heterogeneity among the lesions of a single bladder. Until now, all experiments aimed at understanding the molecular heterogeneity were generated mostly by candidate gene approach and older technologies. There is a lack of comprehensive molecular information in these multifocal lesions partly due to inadequate sample size for comprehensive molecular studies. Identification of mutational status at the gene level in different multifocal lesions will allow us to identify markers for prognostic classification, and predictive classification of response to UC therapies, as well as identify potential therapeutic targets.

To understand the molecular heterogeneity among the lesions of a single bladder, we used The Ion AmpliSeq™ Comprehensive Cancer Panel (CCP) (ThermoFisher). The Ion AmpliSeq™ CCP was designed to target all exons of 409 key tumor suppressor genes (TSGs) and oncogenes most frequently cited and mutated in cancer. We tested 41 lesions from 16 cystectomized bladders. Among these lesions, we performed an initial analysis of a total of 21 lesions and 8 germ line controls from 8 patients. We further technically validated selected mutational events found by NGS by a complementary approach, namely droplet digital PCR (ddPCR). We then compared the intra- and inter-tumoral mutation profiles. Two separate lesions were used for analysis in 3 patients and 3 lesions were taken from each of the remaining 5 patients. Our initial analysis showed mutational heterogeneity among the lesions of most of the samples. Briefly, two of the patients had one clone found at all sites, while the rest of the patients showed more variation amongst their lesions. For instance, ARID2, TRRAP, PDGFB, and FBXW7 were mutated in two out of three lesions in one of the patients, indicating hotspot for heterogeneous mutational events. Three mutational events were confirmed by ddPCR, denoting the accuracy of our NGS data analysis.

Our analysis demonstrates that targeted next generation sequencing is a sensitive and reliable method for interrogating the molecular landscape of bladder cancer, and indicates that both clonal and spontaneous events can account for multifocal bladder cancer.

Our data suggests that sequencing of multiple lesions from an individual patient is necessary to accurately elucidate the mutation profile and tailor appropriate targeted treatment. Further analysis in an extended number of samples is necessary to fully understand the molecular heterogeneity at the mutational level in urothelial cancer.

#2460

Copy number alterations in NOTCH2 and PTP4A3 are associated with prognosis and support novel therapeutic strategies for malignant pleural mesothelioma.

Leticia G. Leon,1 Maria Gemelli,2 Paolo A. Zucali,2 Christos Zoumadakis,3 Elisa Giovannetti1. 1 _University of Pisa, Pisa, Italy;_ 2 _Humanitas Clinical and Research Center, Rozzano, Italy;_ 3 _University of La Laguna, La Laguna, Spain_.

Malignant pleural mesothelioma(MPM) is an aggressive cancer, which incidence has constantly increased over the past two decades and is expected to peak in 2020. The overall prognosis is poor, and predictive biomarkers of drug activity are missing. Given the heterogeneous and complex nature of MPM, it is likely that genomic aberrations changing the expression of several genes, might affect therapeutic response. Therefore, the aim of the present study was to identify genes whose copy number alterations might predict the MPM prognosis.

Recurrent copy number alterations of genes were analyzed by high-resolution whole-genome sequencing in DNA obtained from a "discovery cohort" of 26 resected MPM patients treated with pemetrexed-based chemotherapy (8 with progressive disease, vs. 10 with stable disease and 8 with partial response). Prognostic markers identified by Copy Number Variation analysis with Nexus, Control-FREEC and ReadDepth software were validated by PCR gene copy number and gene expression analyses both in the "discovery" and in two "validation" cohorts of pemetrexed-treated and untreated patients (N=45 and 40). The role of emerging genes was evaluated through siRNA and pharmacological studies using proliferation, migration and apoptosis assays in MPM cells.

As reported previously we observed copy number loss of CDKN2A (15.4%) and BAP1 (7.7%). Interestingly, copy number gain of NOTCH2 was observed in 50% of samples of the patients who underwent progression, whereas losses of PTP4A3 were associated with clinical benefit (SD+PR). The prognostic relevance of NOTCH2 was confirmed by PCR analysis. NOTCH-2 silencing reduced MPM cell migration and enhanced apoptosis induction by pemetrexed, while a PTP4A3 inhibitor overcame pemetrexed resistance in MPM cells characterized by high NOTCH2 and PTP4A3 expression.

These results support the role of NOTCH2 as a novel prognostic/predictive biomarker for MPM, prompting prospective randomized trials for its validation. Moreover, preclinical data suggest that NOTCH2 and PTP4A3 are oncogenes suitable for effective therapeutic targeting in pemetrexed-resistant MPM cells.

#2461

Molecular classification and clinical characterization of a large uterine leiomyoma patient cohort.

Netta Mäkinen,1 Hanna-Riikka Heinonen,1 Annukka Pasanen,2 Jaana Tolvanen,1 Simona Bramante,1 Miika Mehine,1 Jari Sjöberg,3 Oskari Heikinheimo,3 Ralf Bützow,2 Lauri A. Aaltonen1. 1 _Genome-Scale Biology Research Program and Department of Medical and Clinical Genetics, University of Helsinki, Helsinki, Finland;_ 2 _Department of Pathology and Laboratory of Helsinki University Hospital (HUSLAB), Helsinki University Hospital, University of Helsinki, Helsinki, Finland;_ 3 _Department of Obstetrics and Gynecology, Helsinki University Hospital, Helsinki, Finland_.

Uterine leiomyomas (ULs), benign smooth muscle tumors, represent one of the most common neoplasms in women with an estimated prevalence varying from 20% to over 70% during the reproductive years. Approximately every fourth woman with ULs has clinically relevant lesions, which cause morbidity and thus require treatment. Still today, hysterectomy is the primary treatment option for ULs worldwide, and remarkably, their annual societal costs exceed those of colon and breast cancer combined. Our previous findings, derived from the use of high-throughput technologies, suggest that there are at least three distinct molecular UL subclasses, each displaying a characteristic genetic driver aberration and unique global gene expression profile: MED12 (mediator complex subunit 12) mutation-positive, HMGA2 (high mobility group AT-hook 2)-overexpressing, and FH (fumarate hydratase)-deficient ULs. The aim of this study is to examine the molecular subclasses, their respective proportions, and clinical characteristics in a large UL patient cohort. The study material consists of 1026 ULs and corresponding normal myometrium tissue from 322 patients, who had ultrasound-diagnosed ULs and underwent hysterectomy in Helsinki University Hospital, Finland between October 2013 and June 2016. From each uterus, we harvested all feasible ULs ≥1 cm in diameter and a piece of the corresponding normal myometrium tissue. The location of the collected samples in the uterus, their size, and any observed special characteristics were carefully documented at the time of sample removal. In addition, comprehensive clinical information of the patients was obtained from medical and pathology reports, as well as from a questionnaire, and the histopathology of all lesions were characterized according to the WHO 2014 criteria. All collected ULs have been systematically screened for MED12 mutations by Sanger sequencing. Overexpression of HMGA2 typically arises from a chromosomal translocation, and deletions at FH locus suggest the presence of potential biallelic FH inactivation. Therefore, HMGA2-overexpressing and FH-deficient ULs have tentatively been identified using a high-density customizable Infinium® HumanCore-24+ BeadChip with over 305,000 markers. Currently, the data analyses are ongoing. Sufficient numbers of well-documented high-quality samples are a prerequisite for successful research. We hypothesize that ULs can emerge through several distinct molecular mechanisms that contribute to different biological and clinicopathological features and response to treatment. Their molecular classification and further characterization will lead to increased knowledge on UL genesis as well as improved management of the disease.

#2462

Whole-exome sequencing reveals genetic variants in ERC1 and KCNG4 in complete hydatidiform moles from Chinese Han women.

Yan Yu,1 Bingjian Lu,1 Weiguo Lu,1 Xinyu Wang,1 Pengyuan Liu,2 Yan Lu,2 Xiaodong Cheng,1 Xing Xie1. 1 _Women's Hospital School of Medicine Zhejiang UNIV., Hangzhou, China;_ 2 _Institute for Translational Medicine School of Medicine, Zhejiang University, Hangzhou, China_.

Complete hydatidiform mole (CHM) is an uncommon pregnancy-related disease with an invasive potential. The genetic background of the sporadic CHM has not been addressed previously despite the possible mechanisms of maternal genetic variants to the development of this disease with biparental origin. We performed the whole-exome sequencing analysis on 51 CHM patients and 47 normal healthy women, then the second screening of the probable mutations by mass spectrometry in 199 CHM patients and 400 normal healthy women. Finally the candidate genetic polymorphisms were validated by direct Sanger sequencing in 247 cases and 599 controls with another 205 new controls. We eventually found that two SNPs c.G48C(p.Q16H) in ERC1 and c.G1114A(p.G372S) in KCNG4, were associated with an increased risk for CHM (p<0.05). These genetic variants will facilitate our understanding on the pathogenesis of CHM and related tumors as well as the oogenesis and embryonic implantation. They will provide beneficial information for the pregnant women both in physiology and psychology. Further multiple-disciplinary collaborations should be encouraged to clarify the accurate pathogenesis of CHM.

#2463

Tumor profiling of separated carcinomatous and sarcomatous components from uterine carcinosarcoma biopsies provides insights into their development.

Yihua Liu,1 Zachary Weber,2 F. Anthony San Lucas,3 Aditya Deshpande,4 Raed Sulaiman,5 Mary Fagerness,5 Natasha Flier,5 Joseph Sulaiman,5 Christel M. Davis,2 Jerry Fowler,3 Gareth E. Davies,2 David Starks,5 Luis Rojas-Espaillat,5 Paul Scheet,3 Erik A. Ehli2. 1 _Graduate School of Biomedical Science, University of Texas at Houston Health Science Center, Houston, TX;_ 2 _Avera Institute for Human Genetics, Sioux Falls, SD;_ 3 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 4 _Weill Cornell Graduate School of Medical Sciences, New York, NY;_ 5 _Avera Cancer Institute, Sioux Falls, SD_.

Uterine carcinosarcoma (UCS) is a rare and aggressive form of uterine cancer. It is bi-phasic, exhibiting histological features of both malignant epithelial (carcinomatous) and mesenchymal (sarcomatous) elements. Studies have indicated that UCS arises from sarcomatous differentiation of high-grade carcinoma while others have suggested a bi-clonal nature. Given these differences, we sought to separate the carcinoma and sarcoma elements of UCS to try to understand their molecular differences and gain further insights into how these tumors develop. We macrodissected carcinomatous, sarcomatous, and normal cells from formalin fixed paraffin embedded (FFPE) uterine samples of 10 UCS patients. DNA and RNA were isolated and extracted using the Qiagen AllPrep DNA/RNA FFPE kit. Whole-genome SNP microarrays and deep sequencing of 26 cancer genes was performed, using the Illumina Infinium OmniExpressExome array and the TruSight Tumor panel respectively. Illumina HiSeq mRNA sequencing was also performed to quantify gene expression. The genomic allelic imbalance (AI) profiling, called from the SNP data by hapLOH, showed that sarcoma samples were more aberrant than their carcinoma counterparts (abstract 131, AACR 2016). From the targeted sequencing, the Illumina Amplicon-DS Somatic Variant Caller was employed to call somatic mutations. Mutations were identified in TP53 in both the sarcoma and carcinoma samples of all 10 patients. Frequently mutated genes included APC, EGFR, MET and MSH6 which were found in 60-80% of the patients. Genes mutated in less than 50% of the patients included PTEN, KRAS, KIT, FBXW7, PIK3CA, FGFR2, and CTNNB1. Current results showed no association of a mutated gene to either the sarcoma or carcinoma component of UCS. RSEM, STAR and EBSeq were applied to the RNA-seq data for gene expression quantification. Approximately 2500 genes were identified as being differentially expressed (DE) between normal and carcinoma samples. Just over 4000 genes were identified as being DE between normal and sarcoma samples. 75% of the DE genes in the carcinoma were also identified in the sarcoma. Using DAVID functional annotation tool, we characterized these gene sets with KEGG pathways. Deregulated pathways identified in both carcinoma and sarcoma include: cell cycle, transcriptional regulation, Ras and p53 signaling. Some additional pathways are putatively associated with sarcoma only, including MAPK and PI3K-Akt signaling. We report here the differences between sarcoma and carcinoma components of UCS from multiple molecular perspectives. From the genomic AI and DE analysis, the carcinoma aberrations appear to be mostly a subset of the sarcoma tumor profiles, where sarcoma samples appear to be more highly aberrant compared to the carcinoma samples. One possible inference from this is that the sarcoma originated and evolved from the carcinoma cells.

#2464

Papillary renal cell carcinoma, proposal of a new classification system based on integrated molecular, histological and clinical analysis.

Rola Saleeb,1 Mina Farag,1 Fadi Brimo,2 Fabio Rotondo,1 Pamela Plant,1 George Yousef1. 1 _Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada;_ 2 _McGill University Health Center, Montreal, Quebec, Canada_.

Background:

Papillary Renal Cell Carcinoma (PRCC) is divided into histological subtypes 1 and 2. Type 2 is known to have worse clinical behavior. A number of PRCC cases (~ 50%), fail to meet all reported morphological criteria for either type, hence are best characterized as PRCC not otherwise specified (NOS). There are yet no reliable markers to resolve the PRCC NOS category. That in turn reflects the clinical dilemma of how to manage these patients.

Experimental Design:

PRCC patient cohort of 115 cases was selected for the study. Cases were subtyped histologically into PRCC types 1, 2 and NOS. Potentially distinguishing markers ABCC2, CA9, SAll4, and BCL2 selected from our previous genomic analysis, were assessed by immunohistochemistry (IHC). A total of 24 cases were further selected for molecular analysis using miRNA expression and copy number variation (CNV). Univariate and multivariate survival analysis were performed using Log rank test and cox proportionate hazards.

Results:

Markers ABCC2, CA9 exhibited distinct staining patterns between the two classic PRCC subtypes; and successfully classified many of the PRCC NOS (45%) cases. Moreover, immunomarkers revealed a third distinct subtype of PRCC (35% of the PRCC cohort). Molecular

testing using miRNA expression and CNV analysis confirmed the presence of three distinct molecular signatures corresponding to the 3 subtypes. On univariate analysis DFS was significantly enhanced in the type1 versus 2& 3 (p value 0.047). PRCC subtyping retained

significance on multivariate analysis (p value 0.025, HR:6, 95% CI 1.25 to 32.2) .

Conclusion:

We propose a new classification system of PRCC integrating morphological, immunophenotypical, and molecular analysis. Our classification reveals a 3rd PRCC subtype that was not previously described. This subtype has overlapping morphology of with PRCC types 1 and 2, hence would be subtyped as PRCC NOS in the current classification. Molecularly PRCC type 3 has a distinct signature and clinically it behaves similar to PRCC type 2. The new classification stratifies PRCC patients into clinically relevant subgroups and has significant future implications on the management of PRCC.

#2466

Identifying confidently measured genes in single pediatric cancer patient samples using RNA sequencing.

Holly Beale,1 Du Linh Lam,1 John Vivian,1 Yulia Newton,1 Avanthi Tayi Shah,2 Isabel Bjork,1 Ted Goldstein,1 Angela N. Brooks,1 Josh Stuart,1 Sofie Salama,1 E. Alejandro Sweet-Cordero,2 David Haussler1,1 Olena Morozova1. 1 _UCSC Genomics Institute, Santa Cruz, CA;_ 2 _Stanford University, Stanford, CA_.

In the UC Santa Cruz Treehouse Childhood Cancer Initiative (treehousegenomics.soe.ucsc.edu), we are exploring the utility of using RNA-Seq analysis of tumor samples from children to identify potential novel therapeutic options for each individual. Within a single RNA-Seq data set, the gene expression measurements are not equally accurate. The identification of activated, druggable pathways requires accurate gene-level expression measurements.

We receive samples from a variety of clinical and research settings, and the quantity and complexity of the available input material and the depth of sequencing differ. These factors inspired us to develop a tool that will allow us to identify accurate measurements in most RNA-Seq samples we receive.

First, we characterized the relationship between depth of sequencing and the accuracy of the gene expression measurement. We analyzed subsets of reads in samples with more than 50 million Uniquely Mapped, Exonic, Non-duplicate (UMEND) reads. UMEND reads typically constitute over 80% of the reads in a high quality experiment with sufficient starting material. We compared gene expression across the subsets of reads to calculate how many UMEND reads are required to produce consistent measurements. We found that, on average, genes expressed at 1-5 TPM in our data require 30 million reads to be accurately measured. For this calculation, we define accuracy as the condition in which 75% of genes are measured to within 25% of the true value.

Secondly, we use these known relationships to identify genes that have been accurately measured in our tumor RNA-Seq samples. For a sample with 15 million UMEND reads, we find that genes expressed above 5 TPM can be accurately measured and are retained. In the first twelve samples analyzed, samples with more than 10 million UMEND reads retained at least 46% of the genes expressed above zero. We exclude as references those samples with fewer than 10 million UMEND reads due to the marked gene loss after thresholding for this group.

Using accurately measured genes allows us to more confidently assess similarity to other samples, identify enriched pathways, and confirm the expression of drug targets and related molecules under consideration. For example, we reconsidered the CDK4 inhibitor Palbociclib in one patient because the expression of RB1, downstream effector required for Palbociclib-mediated tumor cell death, was under our accuracy threshold. Accuracy thresholds can also be used in experiment planning.

Accuracy thresholding allows us to better assess the value of an RNA-Seq data set and, if necessary, identify the subset of genes whose expression can be confidently considered in a clinical setting. Our experience points to the importance of careful quality control in this process.

#2467

Mutation analysis using next-generation sequencing in histologically heterogeneous of primary lung cancers.

Hayato Koba, Hideharu Kimura, Kazuo Kasahara. _Kanazawa University Hospital, Kanazawa, Japan_.

Rare cases of primary lung cancer are known to have different histological types. Recently, small cell carcinoma was reported to be developed in adenocarcinoma patients with epidermal growth factor receptor (EGFR) mutations after EGFR tyrosine kinase inhibitor (EGFR-TKI) treatment. The purposes of this study are to evaluate mutational status in two different histological types separately, and to further understand the molecular pathogenesis. Twelve tumor samples from 6 patients were collected in our institution. 4 patients had primary adenocarcinoma and small cell carcinoma transformed after EGFR-TKI. One patient had primary adenocarcinoma and squamous cell carcinoma transformed after EGFR-TKI. The other one patient had carcinosarcoma in a surgically resected tumor. Two samples per patient were collected from histologically different areas within a resected tumor or the 2 distinct metastatic sites. DNA was extracted from formalin-fixed, paraffin-embedded tumor tissues. Next-generation sequencings in the 12 samples were performed on 160 cancer-related genes using GeneRead™ DNAseq Targeted Panels V2 Human comprehensive Cancer Panel (QIAGEN). In a carcinosarcoma case with deletional mutation in EGFR gene, a total 504 in adenocarcinoma and 595 in sarcoma variants in 160 genes were detected with high confidence. The gene of identical alterations, as truncal alterations, were EGFR, CBLB, TP53 and MEN1 and the number of that as an unique alteration corresponding to each histology is 25 for adenocarcinoma and 85 for sarcoma. We show relations between the histological types and the genomic alterations, identical alterations and unique alterations, in each patient. Furthermore, we raise essential factors for the histological transformation.

#2468

Gene aberrations for precision medicine against lung adenocarcinoma.

Motonobu Saito,1 Koji Kono,2 Takashi Kohno1. 1 _National Cancer Ctr. Japan, Tokyo, Japan;_ 2 _Fukushima Medical University, Japan_.

Lung adenocarcinoma (LADC) is often triggered by an aberration in a driver oncogene. LADC harboring EGFR mutation and ALK fusion can be treated with anti-cancer drugs that target the aberrant gene products. These molecularly targeted drugs showed significant positive effectiveness for the LADC patients with EGFR mutations and ALK fusions. Additional oncogene aberrations, including RET, ROS1, and NRG1 fusions, skipping of exon 14 of MET, and mutations in BRAF, HER2, NF1, and MEK1, were recently added to the list of such "druggable" driver oncogene aberrations, and their responses to targeted therapies are currently being evaluated in several clinical trials. On the other hand, about 30% and 50% of LADCs in patients in Japan and Europe/USA, respectively, lack the driver oncogene aberrations listed above. Therefore, novel therapeutic strategies, such as those that exploit the vulnerabilities of cancer cells with non-oncogene aberrations, are urgently required. In this presentation, we show gene mutation profiles according to driver oncogenes and summarize the frequencies of those aberrations. We also compare driver aberrations between Japan and USA according to smoking status and sex. This will provide the current status of research on precision medicine against LADC and enumerates the research priorities for the near future.

#2469

The B-cell lymphoma specific aptamer C10.36 binds a ribonucleoprotein complex on the cell surface of cancer cells.

Sonal S. Tonapi, Janet E. Duncan, Matthew Rosenow, Melissa Richards, Teresa L. Tinder, Heather A. O'Neill, Mark R. Miglarese, David Spetzler, Michael Famulok, Günter Mayer. _Caris Life Sciences, Phoenix, AZ_.

Aptamers have recently gained prominence for their diagnostic and therapeutic potential. The DNA aptamer C10.36 forms a G-quadruplex and has been shown to bind the Ramos Burkitt's lymphoma cell line. However, its binding partner on the cell surface remains unknown. Here we report on the identification of the molecular target of C10.36, which suggests its application in the therapy of B-cell lymphoma and leukaemia. Aptamer-affinity purification, followed by LC-MS/MS revealed unique proteins pulled down with C10.36 associated with Ramos cells but not Jurkat, a T-cell lymphocyte cell line. The majority of the identified target molecules were found to be associated within ribonucleoprotein complexes, of which the abundant and consistent ones belong to the nucleolin complex including nucleolin (NCL) itself and its interacting partners, i.e. nucleophosmin (NPM1), heterogeneous nuclear ribonucleoprotein (HNRNP) family members such as HNRNP C1C2 and U, rRNA 2'-O-methyltransferase fibrillarin (FBL), actin (ACTB), nucleolar RNA helicase 2 (DDX21), and proline- and glutamine-rich splicing factors (SFPQ). All proteins identified in the above ribonucleoprotein complex are aberrantly expressed on the surface of several disparate cancer cell types and have been shown to play an oncogenic role in cancer. Another G-rich anti-NCL aptamer, AS1411, has been shown to induce cell death in >100 cancer cell lines. Therefore, we tested the effect of C10.36 on viability of Ramos and other non-Hodgkin B-cell lymphoma (NHL) cancer cell lines. Our results indicate that C10.36 treatment causes specific cell death of certain lymphoma cell lines such as Ramos but not Jurkat cells. The present study identifies C10.36 as a novel anti-B cell lymphoma aptamer and underscores its potential for the development of a new targeted therapy to treat B-cell lymphomas. 

### Homologous Recombination and DNA Double-Strand Break Repair

#2470

Compromised BRCA1-PALB2 interaction is associated with breast cancer risk.

Tzeh Keong Foo,1 Marc Tischkowitz,2 Srilatha Simhadri,1 Talia Boshari,3 Kathleen A. Burke,4 Samuel H. Berman,4 Nadia Zayed,3 Yuan Chun Ding,5 Susan L. Neuhausen,5 Britta Weigelt,4 Jorge S. Reis-Filho,4 William D. Foulkes,3 Bing Xia1. 1 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ;_ 2 _University of Cambridge, Cambridge, United Kingdom;_ 3 _Jewish General Hospital, McGill University, Montreal, Quebec, Canada;_ 4 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 5 _Beckman Research Institute at the City of Hope, Duarte, CA_.

The major breast cancer suppressor proteins BRCA1 and BRCA2 play essential roles in homologous recombination (HR)-mediated DNA repair, which is thought to be critical for tumor suppression. The two BRCA proteins are physically and functionally linked by a third tumor suppressor, PALB2 (partner and localizer of BRCA2), in the HR pathway. Heterozygous PALB2 mutation carriers have increased risk of breast, ovarian and pancreatic cancer. While truncating mutations in BRCA genes are generally pathogenic, interpretations of missense variants remains a challenge. To date, patient-derived missense variants that disrupt PALB2 binding have been identified in BRCA1 and BRCA2; however, there has not been sufficient evidence to establish their pathogenicity in humans. Variants in PALB2 that disrupt either its BRCA1 or BRCA2 binding have not been reported. Here, we report on the identification of a novel PALB2 variant, c.104T>C [p.L35P], that segregated in a family with a strong history of breast cancer. Functional analyses showed that L35P abrogates the PALB2-BRCA1 interaction, resulting in impaired HR and sensitivity to platinum salts and PARP inhibitors. Whole-exome sequencing of breast tumor from a c.104T>C carrier revealed a somatic, truncating mutation in the second allele of PALB2, with the tumor displays hallmark genomic features of tumors with BRCA mutations and HR defects. Using a combination of traditional clinical genetics, tumor whole-exome sequencing and in-depth functional analyses, we have provided direct evidence to cement the pathogenicity of L35P. Parallel analyses of other germline variants in the PALB2 N-terminal BRCA1-binding domain also identified multiple variants that affect HR function to varying degrees, suggesting their possible contribution to cancer development. Our findings establish p.L35P as the first pathogenic missense mutation in PALB2 identified to date and directly demonstrate the requirement of the PALB2-BRCA1 interaction for breast cancer suppression.

#2471

Role of PALB2-BRCA1 interaction in tumor suppression.

Amar H. Mahdi, YanYing Huo, Bing Xia. _CINJ, New Brunswick, NJ_.

Homologous recombination (HR) is the only error-free pathway for the repair of DNA double strand breaks (DSBs). BRCA1 and BRCA2, the two major breast cancer suppressor proteins, play essential roles in HR-mediated repair of DSBs by promoting the recruitment of RAD51, the recombination enzyme, to DNA damage sites for the initiation of HR. PALB2 (partner and localizer of BRCA2) plays a key role in this pathway by acting as a chromatin adaptor for BRCA2 and a linker between BRCA1 and BRCA2. Like BRCA1 and BRCA2, PALB2 is a tumor suppressor gene itself. Germline, heterozygous mutations in the gene increase the risk of breast, ovarian and pancreatic cancers. However, its mechanism is not fully understood. To investigate the in vivo role of the PALB2-BRCA1 interaction, we previously generated a Palb2 knockin mouse strain which contains a mutation that disrupts BRCA1 binding. This mouse model also allows us to bypass the embryonic lethality of the Palb2 KO mice. In this study, we hypothesized that the direct communication between the two proteins is critical for proper DNA damage repair and response in vivo and for suppression of tumorigenesis. Indeed, both immunohistochemistry (IHC) and immunofluorescence (IF) demonstrated that different tissues of the mutant mice have higher levels of endogenous DSBs (γH2AX foci) and slower DSB repair kinetics after ionizing radiation (IR). Yet, mutant cells were more resistant to cell death. When aged under normal conditions, mutant mice showed increased tumor incidence in multiple tissues, particularly in the liver. When challenged by IR, mutant mice quickly developed thymic lymphoma and later in other tissues including liver and ovary, etc. Interestingly, when crossed with Trp53 mutant mice, the resulting Palb2m/m;Trp53+/- mice showed greatly accelerated development of thymic lymphoma and osteosarcoma, which are typically associated with Trp53 but not Palb2 mutations. Exome sequencing revealed focal deletion of the wild-type allele of Trp53 in the majority of the tumors, suggesting that disruption of BRCA1-PALB2/BRCA2 axis promotes regional genomic deletions that may lead to loss of other tumor suppressors such as p53. Our results underscore the importance of the BRCA1-PALB2/BRCA2 pathway for tumor suppression and suggest a potentially novel mechanism for BRCA1/PALB2-mediated tumor suppression, which is by preventing Trp53/TP53 loss of heterozygosity (LOH), which allows for tumor development.

#2472

Defining the role of the XAB2 complex during homologous recombination.

David O. Onyango, Jeremy Stark. _City of Hope, Duarte, CA_.

Several factors associated with RNA processing are important for homologous recombination (HR), but the mechanistic links between these processes remain poorly understood. To this end, we have examined the function of the tetratricopeptide repeat factor XAB2 in HR, since this factor has a conserved function in RNA processing. We have found that XAB2 is important for chromosomal double strand break (DSB) repair via two pathways of HR that require end resection as an intermediate step, end resection of camptothecin (Cpt) induced DNA damage, and RAD51 recruitment to ionizing radiation induced foci (IRIF). Furthermore, XAB2 mediates CtIP hyperphosphorylation induced by Cpt and BRCA1 IRIF, as well as histone acetylation events linked to HR proficiency. The capacity for XAB2 to promote HR correlates with its ability to form a complex with ISY1 and PRP19, which show a similar influence as XAB2 on HR. Our recent efforts include examining other members of the XAB2 complex, as well as the transcription unit aberrations caused by loss of this complex, which may contribute to the defects in HR.

#2473

Breast cancer whole genomes link homologous recombination deficiency (HRD) with therapeutic outcomes.

Eric Y. Zhao,1 Yaoqing Shen,1 Erin Pleasance,1 Katayoon Kasaian,1 Martin R. Jones,1 Carolyn Ch'ng,1 Caralyn Reisle,1 Peter Eirew,2 Karen Mungall,1 Nina Thiessen,1 Yussanne Ma,1 Alexandra Fok,1 Andrew J. Mungall,1 Yongjun Zhao,1 Richard Moore,1 Diego Villa,2 Tamara Shenkier,2 Caroline Lohrisch,2 Stephen Chia,2 Stephen Yip,3 Karen Gelmon,2 Howard Lim,2 Sophie Sun,2 Kasmintan A. Schrader,2 Sean Young,3 Aly Karsan,1 Robyn Roscoe,1 Janessa Laskin,2 Marco A. Marra,1 Steven J. Jones1. 1 _Canada's Michael Smith Genome Sciences Centre, Vancouver, British Columbia, Canada;_ 2 _British Columbia Cancer Agency, Vancouver, British Columbia, Canada;_ 3 _The University of British Columbia, Vancouver, British Columbia, Canada_.

Background: Homologous recombination deficiency (HRD) is common in cancer - germline BRCA1 & BRCA2 mutations account for 5-10% of breast cancers and confer 85% lifetime risk. HRD cancers exhibit genomic instability and sensitivity to platinum-based therapy and PARP inhibitors. While not all causes of HRD are known, recent sequencing efforts have revealed genome-wide somatic mutation signatures that characterize the HRD genomic instability phenotype, also known as "BRCA-ness". This provides a promising new assay to predict sensitivity to platinum-based therapy. Here, we integrate two whole-genome sequencing metrics to assess their association with therapeutic outcomes in a breast cancer cohort.

Methods: Whole-genome sequencing of 47 breast cancer tumors (100x coverage) and matched normals (60x) was performed on an Illumina HiSeq. Alignment, assembly, SNV calling, and loss of heterozygosity (LOH) detection were performed with BWA, ABySS, Strelka, and APOLLOH respectively. SNV signatures were deciphered by non-negative matrix factorization with Monte Carlo resampling. An HRD score comprised of LOH, telomeric allelic imbalance (TAI), and large scale transition (LST) counts was computed. Clinical endpoints were obtained by retrospective review of treatment and imaging reports. Analysis is ongoing in an independent validation cohort of 62 sequenced cases.

Results: The HRD-linked SNV signature was significantly associated with radiographic clinical response (CR) to platinum-based therapy (p=0.015). Logistic regression demonstrated a 59% improved odds of CR to platinum-based therapy per 1000 somatic SNVs attributed to HRD (odds ratio 1.16-2.50). Tumors carried up to 10,246 such SNVs and all patients with CR were among the top quartile. The LOH-TAI-LST score was correlated with SNV signature (r=0.6, p=7×10-6) and associated with CR (p=0.025). Notably, elevated HRD signatures associated with CR were identified in tumors with wild-type BRCA1/BRCA2 or variants of unknown significance. Tumors with above median HRD signatures were associated with a 69-day longer time to treatment failure and an 18% daily decreased probability of treatment failure per 1000 HRD-attributed SNVs (hazard ratio 0.71-0.95, p = 0.007).

Discussion: We found that HRD mutation signatures are associated with clinical response and longer time to treatment failure with platinum-based therapy. While similar benefits were observed in patients with somatic bi-allelic loss of BRCA1/BRCA2, such cases are less common (8% of our cohort) compared to those with elevated HRD signature. Thus, mutation signature methods may identify patients who stand to benefit from platinum-based therapy missed by BRCA screening alone.

#2474

The mechanistic role of polyamines in DNA double-strand break repair.

Chih-Ying Lee,1 Guan-Chin Su,1 Min-Yu Ko,1 Wen-Yen Huang,2 Geen-Dong Chang,1 Sung-Jan Lin,2 Peter Chi1. 1 _Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan;_ 2 _Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan_.

Polyamines are essential organic cations for cell growth, proliferation and tissue remodeling. The rate-limiting step in the biosynthesis of polyamines is catalyzed by ornithine decarboxylase (ODC). The activity of ODC and the level of intracellular polyamines significantly increase in many cancers, indicating the critical role of polyamines in tumorigenesis. Depletion of polyamines sensitizes cells to DNA damage agents that lead to DNA double-strand breaks (DSBs), suggesting that endogenous polyamines may participate in DNA repair process. However, the mechanistic role of polyamines in DSB repair remains largely unknown. Here, we wish to ascertain the role of polyamines in DSBs repair pathway and to delineate the underlying mechanism by combining biochemical analyses, cell-based and animal approaches. Our cell-based experiments demonstrated that polyamines indeed participate in DNA double-strand break repair. Specifically, the level of intracellular polyamines regulates homologous recombination (HR) repair pathway rather than non-homologous end joining (NHEJ). Furthermore, our in vitro reconstitution system and functional analyses demonstrated that polyamines significantly enhance RAD51-mediated DNA strand exchange reaction. Importantly, we reveal that the stimulatory effect of polyamines by RAD51 stems from the enhancement of duplex DNA captures in the DNA exchange reaction. Finally, the physiological role of polyamines in DSBs repair was examined by using mouse hair follicle as a model system. The animal model clearly highlights the significant contribution of polyamines to the DNA repair in vivo. Our findings thus furnish valuable insight into the mechanistic basis of polyamines in DNA repair.

#2475

In vitro **and** in vivo **assessment of the mechanism of action of the PARP inhibitor rucaparib.**

Liliane Robillard, Minh Nguyen, Thomas C. Harding, Andrew D. Simmons. _Clovis Oncology, Inc., Boulder, CO_.

Background: Rucaparib (CO-338) is an oral small molecule inhibitor of poly (ADP-ribose) polymerase (PARP) that is being developed for patients with BRCA1 and BRCA2 mutated and homologous recombination deficient ovarian cancer (Swisher et al., 2016; Lancet Oncol. Epub). The mechanism of action (MOA) of rucaparib was characterized in the studies reported here.

Methods: Enzymatic and cellular reporter assay profiling were performed by BPS Biosciences. Rucaparib potency was assessed using 6-day cell viability assays (CellTiter-Glo). In vivo PK/PD and efficacy studies in the MDA-MB-436 (BRCA1 mutant) and the HBCx-17 (BRCA2 mutant) TNBC models were performed at Crown Biosciences and XenTech, respectively.

Results: In biochemical assays rucaparib inhibited PARP-1, PARP-2 and PARP-3, with IC50 values of 0.8, 0.5, and 28 nM, respectively. Rucaparib weakly inhibited PARP5a (tankyrase 1[TNKS1]) and PARP5b (tankyrase 2[TNKS2]) with an IC50 of 796 and 486 nM; however rucaparib did not inhibit TNKS1/2 in a cellular reporter assay evaluating Wnt pathway signaling (IC50 >10 µM). Rucaparib cytotoxicity was evaluated in the BRCA1 mutant (UWB1.289) and wild-type (UWB1.289+BRCA1) isogenic pair, and a panel (n=26) of breast and ovarian cancer cell lines. The UWB1.289 cell line was more sensitive to rucaparib (IC50 = 375 nM) than the UWB1.289+BRCA1 cell line (IC50 = 5430 nM). Rucaparib also decreased the cell viability of several BRCA1 and BRCA2 mutant ovarian and breast cancer cell lines. Mechanistic analyses in the UWB1.289 cell line demonstrated a dose dependent decrease in poly (ADP) ribosylation (PAR; IC50 = 2.8 nM), and an increase in DNA damage and apoptosis. Taken together, these results are consistent with the concept of synthetic lethality, where simultaneous inhibition of multiple DNA repair pathways results in irreparable DNA damage and cell death. In vivo PK/PD and efficacy studies assessed the MOA and activity of rucaparib in the MDA-MD-436 (BRCA1 mutant) xenograft model. In a PK/PD study, dose dependent inhibition of PAR was observed in the tumors of mice treated with 15, 50 and 150 mg/kg BID rucaparib, resulting in statistically significant (p < 0.05) inhibition of 45%, 86% and 96%, respectively. An inverse and dose dependent correlation between PAR and rucaparib levels in the plasma and tumor was observed, and the levels of rucaparib in the tumor were consistently higher (> 2-fold) than the levels of rucaparib measured in plasma. Dose dependent and statistically significant tumor growth inhibition was observed in both subcutaneous and orthotopic xenograft studies evaluating the anti-tumor activity of rucaparib in the MDA-MB-436 model. Rucaparib also demonstrated potent activity in the BRCA2 mutant HBCx-17 patient-derived xenograft model.

Conclusion: These results demonstrate that rucaparib is a potent and selective inhibitor of PARP1, PARP-2, and PARP-3 with in vitro and in vivo activity in BRCA1/2 deficient tumors.

#2476

Preclinical assessment of the PARP inhibitor rucaparib in homologous recombination deficient prostate cancer models.

Minh Nguyen, Andrew D. Simmons, Thomas C. Harding. _Clovis Oncology, Inc., Boulder, CO_.

Rucaparib (CO-338) is an oral small molecule inhibitor of the poly(ADP-ribose) polymerase (PARP) enzymes PARP-1, PARP-2 and PARP-3 that is being developed for cancer patients with evidence of homologous recombination (HR) deficiency, including BRCA1 and BRCA2 mutations. PARP inhibitors have demonstrated clinical activity in BRCA1, BRCA2, and ATM mutant prostate cancer patients (Mateo et al. N Engl J Med. 2015;373:1697-708); however, limited preclinical validation of PARP inhibitors in prostate cancer has been reported to date, since no BRCA mutant nor HR deficient prostate cell lines have been identified. In this study, we modelled the functional inactivation of DNA repair genes using siRNA and CRISPR/Cas9, and examined the impact on PARP inhibition, in the DU145 and PC-3 prostate cancer cell lines. These cell lines were chosen based on transfection efficiency, lack of obvious genetic defects in DNA repair pathways, and baseline insensitivity to rucaparib (IC50 > 10 µM). A panel of 26 DNA repair genes were individually knocked down by siRNA, and changes to rucaparib potency were examined in a 7-day cell viability assay. The knockdown of BRCA1 or BRCA2 increased rucaparib cytotoxicity in both prostate cell lines. For example, the IC50 of rucaparib in DU145 and PC-3 cells with reduced BRCA2 levels was 275 and 297 nM, representing a 36- and 37-fold increase in rucaparib sensitivity, respectively. In addition to BRCA1 and BRCA2, the knockdown of several other genes including BARD1, CDK12, FANCA, PALB2, RAD51, RAD51C, RAD51D, and RAD54L increased rucaparib efficacy ≥ 2-fold in at least 1 cell line examined. To further investigate HR deficiency in prostate cancer, BRCA2 was knocked out in the human 22Rv1 prostate cancer cell line using CRISPR/Cas9. Clone 1-50 displayed biallelic BRCA2 inactivation due to the introduction of a deleterious premature stop codon, and immunoblot analysis verified lack of the full-length BRCA2 protein. The IC50 of rucaparib in the parental and clone 1-50 cells was 9,504 and 287 nM, respectively, demonstrating a 33-fold increase in rucaparib cytotoxicity in this BRCA2 deficient model. Androgen receptor (AR) signaling and proliferation were also examined in clone 1-50. In contrast to published reports (Schiewer et al. Cancer Discov. 2012;2:1134-49), no impact of PARP inhibition on dihydrotestosterone (DHT) induced gene expression was observed in the parental 22Rv1 and BRCA2 deficient clone 1-50 cell lines. In vivo model development and efficacy studies with the 22Rv1 and clone 1-50 cell lines are ongoing. Taken together, these findings support the hypothesis that deficiencies in BRCA1 and BRCA2, as well as additional HR genes, may sensitize prostate cancer cells to rucaparib. A phase 2 study investigating rucaparib in patients with metastatic castration-resistant prostate cancer and HR gene deficiency (TRITON2) is ongoing (NCT02952534).

#2477

Genomic instability in BRCA1-deficient cells is a result of the anti-recombinogenic activity of BLM helicase.

Dharm S. Patel, Sarah M. Misenko, Samuel F. Bunting. _Rutgers University, Piscataway, NJ_.

Mutations in BRCA1 are responsible for approximately 5% of cases of breast cancer, and there are few treatment options that substantially alter the probability of disease onset in individuals with BRCA1 mutations. BRCA1-deficient cells exhibit increased genomic instability following DNA damaging treatments due to a defect in the homologous recombination (HR) DNA repair pathway. Presently, Olaparib, a PARP inhibitor, is approved as a targeted monotherapy for germline BRCA1 mutated advanced ovarian cancers. As use of this treatment has expanded, data suggests that patients exhibit a further relapse and resistance to PARP inhibitors via mechanisms that include the development of secondary BRCA1 reversion mutations, enhanced drug efflux relating to P-glycoprotein, and mutations in other DNA repair proteins that restore HR. Here, we show that the RECQ helicase, BLM, mediates the genomic instability observed in BRCA1-deficient cells by a mechanism that depends in part on its anti-recombinogenic activity. We have generated conditional knockout mice with single and combined deficiencies in BRCA1, BLM, and 53BP1 in the B lymphocyte cell population. Ablation of BLM in BRCA1-deficient cells allows the HR repair pathway to be restored, leading to a partial rescue of the genomic instability that is present in BRCA1-deficient cells. The stable accumulation of RAD51, a marker for HR, at DNA double-strand break sites is inhibited by BLM in BRCA1-deficient B cells. However, DNA end resection is not impacted by single or co-deletion of BRCA1 and BLM. Furthermore, cells co-deficient in BRCA1 and BLM display limited sensitivity to PARP inhibition. The rescue in HR and PARPi sensitivity phenotypes following deletion of BLM is only present in hypomorphic BRCA1Δ11/Δ11 cells and not the RING domain deficient BRCA1Δ2/Δ2. These results demonstrate that the anti-recombinogenic activity of BLM is of potentially great significance for regulating the balance of HR versus other error-prone repair pathways. Mutation of BLM in BRCA1-deficient tumors is therefore a potential pathway leading to resistance to PARPi and other DNA damaging agents.

#2478

PARP trapping by PARP inhibitors have distinct effects on HR and ALT NHEJ DSB repair, potentially impacting its therapeutic efficacy in breast cancers.

Bryan M. Pelkey. _University of MD, Baltimore, Baltimore, MD_.

PARP inhibitors (PARPi) have exhibited clinical success in inherited breast cancers with mutations in homologous recombination (HR) DNA double strand break (DSB) repair genes BRCA 1/2 by synthetic lethality. Novel PARPi have been shown to function not only by catalytic inhibition of PARP 1/2 activity, but also by "trapping" of PARP to sites of DNA damage. Trapping of PARP is a response generated by PARPi that is independent of the inhibition of PARP's catalytic activity, and leads to lethal unrepaired DSBs during replication. Importantly, PARP trapping ability of PARPi correlates with its cytotoxic effect. In this study, we investigated whether PARP trapping has distinct effects on regulation of DNA DSB repair in breast cancer cells. Major DSB repair pathways in mammalian cells include HR and non-homologous end joining (c-NHEJ). Alternative NHEJ (ALT-NHEJ) is a recently discovered and highly error-prone pathway which requires DNA resection like HR, and utilizes PARP1. Our hypothesis is that PARP1 catalytic inhibition vs 'trapping' leads to distinct outcomes on DSB repair, highlighting inhibitor efficacy. To test our hypothesis, we utilized MCF10A (non-tumorigenic), MDA-MB-231 (TNBC/BRCA+), and SUM149PT (TNBC/BRCA-) breast cancer cell lines with stably integrated GFP-reporter plasmids designed to assess interchromosomal activities of the specific DSB repair pathways. For our analysis, we used varying concentrations of Talazoparib (MDV3800), which is a potent PARP trapping agent when used in combination with DNA damaging agents such as methyl methanesulfonate (MMS). First, we determined optimal conditions to induce PARP trapping in cells without leading to significant toxicity. As observed by others, combination treatments with of MMS/MDV3800 led to a significant increase in PARP trapping relative to treatments with individual drugs. Studies were also performed with a potent inhibitor of coenzyme NAD+ (Daporinad/FK866) that blocks poly-ADP ribosylation activity in combination of MMS. We observed significant PARP trapping with FK866/MMS combination treatment, indicating that PARP depletion under the conditions of DNA damage also leads to potent PARP trapping. Next, we investigated the effects of these inhibitors on DSB repair. Surprisingly, combination MDV3800/MMS lead to an overall decrease in HR and increase in ALT NHEJ activity. These results were observed in both our BRCA+/- lines. Similar results are observed between the repair activities when using FK866/MMS treatments. Overall, our results suggest that PARP inhibitors with potent PARP trapping activity increases ALT NHEJ, which may potentially compensate for lack of HR. Studies are ongoing to determine whether targeting ALT NHEJ may increase cytotoxicity in these cells.

#2479

**A functionally null** RAD51D **missense mutation is strongly associated with ovarian carcinoma.**

Barbara Rivera. _McGill University, Montreal, Quebec, Canada_.

Background and goal: RAD51D is a key player in DNA repair by homologous recombination (HR) and carriers of truncating RAD51D mutations have an increased risk for ovarian cancer (OC). However, the contribution of non-truncating RAD51D variants to cancer predisposition remains uncertain. We sought to fully characterize the previously described missense RAD51D variant c.620C>T;p.S207L in order to elucidate its role in OC.

Methods: A clinical panel screening was used to identify the RAD51D variant c.620C>T;p.S207L in two French Canadian (FC) kindred affected with familial High Grade Serous Cancer (HGSC) of the ovary or endometrium. High resolution melting, TaqMan genotyping and Sanger sequencing were used to genotype the p.S207L variant in a series of unselected cases of HGSC of the ovary and endometrium, breast, pancreas and colorectal cancer and healthy controls, all of a FC origin. Whole exome sequencing (WES) was performed to study the genetic signature characterizing RAD51D associated tumors. RAD51 foci formation and CRISPR-Cas9-stimulated and HR-mediated gene targeting assays were used to assess HR activity of RAD51D-S207L mutated CHO cells. HR activity in RAD51D-S207L mutated human cells was tested by a DR-GFP assay. The effect of RAD51D p.S207L on RAD51D-XRCC2 interactions was analyzed by co-immunoprecipitation and quantified in-vivo in a single cell colocalization assay. Sensitivity to PARP inhibitors (PARPi) was evaluated in a cell survival assay.

Results: Using deep sequencing and case-control genotyping studies, we showed that the missense RAD51D variant c.620C>T;p.S207L is over-represented in the French Canadian population affected by HGSC of the ovary (3.8% cases vs 0.002% controls; p < 0.0001).The frequency of the p.S207L variant did not differ from that of controls in breast, endometrial, pancreas and colorectal adenocarcinomas. A common haplotype shared by all the carriers suggested a founder origin for c.620C>T;p.S207L mutation. WES analysis of RAD51D tumor profiles revealed the presence of signature 3 which is known to be associated with HR defects. Functionally, we show that this mutation impairs HR by disrupting the RAD51D-XRCC2 interaction and confers PARP-inhibitor sensitivity to CHO cells.

Conclusions: This work identifies RAD51D p.S207L as the first bona fide pathogenic missense susceptibility allele for HGSC of the ovary and supports the use of targeted PARPi therapies in OC patients carrying missense RAD51D mutations.

#2480

**Suppression of homology-dependent DNA double-strand break repair induces PARP inhibitor sensitivity in** VHL **-deficient human renal cell carcinoma.**

Susan E. Scanlon, Parker L. Sulkowski, Peter M. Glazer. _Yale School of Medicine, New Haven, CT_.

The von Hippel-Lindau (VHL) tumor suppressor gene is inactivated in the vast majority of human clear cell renal carcinomas. The pathogenesis of VHL loss is currently best understood to occur through stabilization of the hypoxia-inducible factors, activation of hypoxia-induced signaling pathways, and transcriptional reprogramming towards a pro-angiogenic and pro-growth state. However, hypoxia also drives other pro-tumorigenic processes, including the development of genomic instability via down-regulation of DNA repair gene expression. Here, we find that DNA repair genes involved in double-strand break repair by homologous recombination (HR) and in mismatch repair, which are down-regulated by hypoxic stress, are decreased in VHL-deficient renal cancer cells relative to wild type VHL-complemented cells. Non-homologous end joining (NHEJ) genes are not decreased in VHL-deficient cells, demonstrating specificity of the down-regulation and similarity to hypoxia. Functionally, this repression of HR genes is associated with impaired DNA double-strand break repair in VHL-deficient cells, as determined by the persistence of ionizing radiation-induced DNA double-strand breaks and reduced activity in a homology-dependent plasmid reactivation assay. Furthermore, VHL deficiency conferred increased sensitivity to the DNA crosslinking agent mitomycin C and PARP inhibitors, analogous to the synthetic lethality observed between hypoxia and these chemotherapeutic agents. Finally, we discovered a correlation between VHL inactivation and reduced HR, but not NHEJ, gene expression in a large panel of human renal carcinoma samples. Together, our data elucidate a novel connection between VHL-deficient renal carcinoma and hypoxia-induced down-regulation of DNA repair, and identify potential opportunities for targeting DNA repair defects in human renal cell carcinoma.

#2481

RAD51 and AICDA define a new synthetic lethal interaction that is targetable in multiple tumor types.

Kevin D. Mills,1 Marly I. Roche,1 Amber Cyr,1 Tyler Maclay,1 Muneer G. Hasham,2 Kristin Lamont,2 Jane Branca,2 Francesca Cavallo,3 Francesca Cavallo,3 Maria Jasin,3 Alvin Stern,4 Youngho Kwon,5 Weixing Zhao,5 Patrick Sung5. 1 _Cyteir Therapeutics, Inc, Cambridge, MA;_ 2 _The Jackson Laboratory, Bar Harbor, ME;_ 3 _Memorial Sloan-Kettering Cancer Center, New York, NY;_ 4 _Cepter BioPartners, Hoboken, NJ;_ 5 _Yale University School of Medicine, New Haven, CT_.

Antigen stimulated B-cells transiently express Activation Induced Cytidine Deaminase (AID) which initiates point mutations and DNA double strand breaks (DSB) in immunoglobulin genes to promote B-cell maturation. However, AID is capable of broadly damaging the B-cell genome. We have shown that AID-induced DSBs require the homologous recombination factor RAD51 for their repair. Using genetic approaches, we discovered that reduced HR function is cytotoxic to AID-expressing B-cells. We subsequently demonstrated the feasibility of small molecule inhibition of RAD51 to sensitize AID-overexpressing tumor cells. Here we build on those observations, finding that AID-RAD51 synthetic lethality occurs via mitotic catastrophe, involving the mTOR pathway. We have now developed potent and selective RAD51 modulatory small molecules that preferentially kill AID-expressing tumor cells. Using mouse models of lymphoma and leukemia we provide in vivo efficacy data demonstrating the potential therapeutic feasibility of RAD51 modulation. These studies provide evidence for a novel "synthetic lethal" approach for treating AID-expressing malignancies, via the induction of mitotic catastrophe.

#2482

Inhibition of RAD51 with a cell penetrating antibody, 3E10.

Peter M. Glazer, Audrey Turchick. _Yale University, New Haven, CT_.

Many human malignancies arise in the setting of defective DNA repair pathways, leading to genetic instability, carcinogenesis and tumor progression. However, DNA repair deficiency can provide for a therapeutic window that can be exploited with targeted agents. One such anti-cancer agent is 3E10, a unique cell penetrating, anti-DNA autoantibody. 3E10 by itself is considered non-toxic to normal cells but has been shown to have deleterious effects on cells with DNA repair deficiencies. Specifically, 3E10 has been shown to reduce the efficiency of homologous recombination (HR). This has been attributed to its ability to preferentially binding to ssDNA tails, thereby inhibiting Rad51 mediated strand exchange. Prior work has shown that treatment with 3E10 alone can selectively kill cells with genetic mutations that lead to abnormal DNA repair. For example, 3E10 has been shown to be synthetically lethal in BRCA2 deficient human cancer cells. Additionally, 3E10 sensitizes cells to radiation and other DNA damaging therapies. The goal of this project is to characterize the specific mechanism by which 3E10 inhibits DNA repair pathways. Previously, the mechanism by which 3E10 inhibits HR was attributed to 3E10's DNA binding ability. Through the biochemical evaluation of 3E10 variants, new evidence now suggests that 3E10 inhibits HR independently of 3E10's ability to bind DNA. We have found that this HR inhibition is a result of a physical interaction between 3E10 and Rad51. Biochemical analysis revealed that 3E10 binds to the N-terminal domain of Rad51, the domain that plays a role in Rad51 homo-oligomerization and filament formation. This 3E10-Rad51 interaction impairs Rad51's ability to localize to the nucleus and form repair foci. Taken together, this data suggests that 3E10 inhibits HR by physically interacting with Rad51. Overall, 3E10 holds promise as a novel therapeutic agent for certain subsets of cancer.

#2483

Molecular mechanisms of dianhydrogalactitol (VAL-083) in overcoming chemoresistance in glioblastoma.

Beibei Zhai,1 Anna Gobielewska,2 Anne Steino,3 Jeffrey A. Bacha,3 Dennis M. Brown,4 Simone Niclou,2 Mads Daugaard1. 1 _University of British Columbia, Vancouver, British Columbia, Canada;_ 2 _Luxembourg Institute of Health, Luxembourg, Luxembourg;_ 3 _DelMar Pharmaceuticals, Inc., Vancouver, British Columbia, Canada;_ 4 _DelMar Pharmaceuticals, Inc., Menlo Park, CA_.

Standard treatments for glioblastoma (GBM) include surgery, radiation and chemotherapy with temozolomide (TMZ). Nearly all tumors recur and 5-year survival is less than 3%, largely due to chemoresistance. Evidence shows that cancer cells utilize DNA damage repair pathways to overcome cytotoxic effects of chemotherapy. GBM tumors expressing O6-methylguanine-DNA-methyltransferase (MGMT) display intrinsic chemoresistance to TMZ and nitrosoureas, while a deficient DNA mismatch repair system (MMR) confers chemoresistance to TMZ and platinum agents. Alterations in p53, particularly gain-of-function mutations, are correlated with increased MGMT-expression and poor prognoses in GBM. Second line anti-angiogenic treatment with bevacizumab has not improved overall survival and has been shown to induce intratumor hypoxia and increased chemoresistance. VAL-083 is a bi-functional alkylating agent that readily crosses the blood-brain barrier, accumulates in brain tumor tissue and has demonstrated activity against GBM in prior NCI-sponsored clinical trials. VAL-083 induces interstrand cross-links at guanine-N7 causing DNA double-strand breaks and cancer cell death. VAL-083 is equiactive against GBM cancer stem cells (CSCs) and non-CSCs independent of MGMT and p53 status, in vitro. We recently showed that VAL-083 leads to irreversible S/G2-phase cell cycle arrest, proposing synergy with S-phase specific chemotherapeutics, including topoisomerase and PARP inhibitors. VAL-083 further showed persistent activation of the homologous recombination (HR) DNA repair pathway and its potency was increased when HR was impaired, demonstrating that VAL-083-induced lesions are repaired via HR suggesting increased VAL-083 potency in HR-impaired tumors. Further, hypoxic cancer cells are known to downregulate their HR pathway, proposing increased VAL-083 potency in hypoxic tumors. Bevacizumab treatment increases hypoxia in tumor cells, presumably impairing HR, proposing VAL-083 as a treatment option in HR-deficient or hypoxic cancers following, or as part of a combination treatment with, bevacizumab. Here, VAL-083 cytotoxicity and DNA damage response was evaluated by crystal violet assays, western blot and flow cytometry. VAL-083's ability to overcome MMR-related chemoresistance was investigated using lentiviral MLH1 and MSH2 vectors in HCT116 and LoVo cancer cells. We report synergy between VAL-083 and etoposide or camptothecin in A549 and PC3 cancer cell lines. We also investigated the potency of VAL-083 in GBM under hypoxia either in vitro or in vivo as part of a combination treatment with bevacizumab. Our results demonstrate a distinct anti-cancer mechanism for VAL-083, resulting in the ability to overcome resistance to TMZ and nitrosoureas, increased activity in cancers with impaired HR and synergy with etoposide or camptothecin.

#2485

Development and validation of Predi-HRD liquid biopsy test for PARP inhibitor clinical trials.

Zhixin Zhao, Toni Wiegers, Tak Cheung, Pan Du, Shidong Jia. _Predicine, Inc, CA_.

Response to DNA-damaging agents, such as platinum and recently emerged PARP inhibitors, is associated with homologous recombination deficiency (HRD) pathway. In fact, as reported by multiple clinical trials, patients with advanced breast, prostate, and ovarian cancers who have harmful mutations in BRCA1 and BRCA2 have demonstrated a great benefit from treatment of PARP inhibitors including olaparib, niraparib, and talazoparib. Current companion diagnostic assays that mainly examine germline defects from blood or somatic alterations from tumor tissues have shown several practical challenges: germline test does not capture somatic alterations of a tumor, initial biopsies from a tumor may not reflect the current status of the tumor's genetic profile over time, and tissue biopsy may be of insufficient quantity or unavailable/unobtainable for downstream molecular profiling.

Here we introduce the Predi-HRD blood test, a non-invasive, next-generation sequencing (NGS)-based multiplex assay that enables real-time monitoring of temporal change of tumor heterogeneity and highly sensitive detection of defects in HR genes including BRCA1, BRCA2, ATM, CHEK2, among others. Technical validation was performed to evaluate assay sensitivity, specificity and accuracy using reference samples with known genetic profiling and further validated on a variety of orthogonal platforms such as digital PCR, allele-specific PCR and Sanger sequencing. Mutation prevalence of HR genes was examined in clinical plasma samples from patients with advanced ovarian cancer after systemic chemotherapy. The successful development of Predi-HRD liquid biopsy test offers an efficient solution to stratify and monitor cancer patients who may benefit from HRD-targeted therapeutics such as PARP inhibitors.

#2486

Tumor hypoxia induces DNA repair vulnerabilities through contextual loss of heterozygosity.

Osman Mahamud,1 Melvin Chua,2 Winnie Lo,2 Gaetano Zafarana,3 Robert Bristow2. 1 _University of Toronto, Toronto, Ontario, Canada;_ 2 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada;_ 3 _University Health Network, Toronto, Ontario, Canada_.

Introduction: Intratumoural hypoxia leads to decreased expression in DNA damage response (DDR) and repair pathways. Given the 'two-hit' model for loss of gene function, we hypothesize that hypoxia-mediated down-regulation of gene expression and function, coupled with an already inactive allele contributes to an exploitable contextual 'loss-of- heterozygosity' phenotype.

Method: To interrogate this relationship, isogenic DLD-1 cells heterozygous and homozygous null for BRCA2, were placed under normoxic (21%) or hypoxic (0.2%) conditions for 72 hours. Hypoxia-mediated changes in DDR response and DNA repair were evaluated by cell proliferation, cell cycle analysis, western blots, qPCR, immunofluorescence, clonogenic assays and functional repair assays.

Results: No differences in proliferation and cell survival were observed between oxic and hypoxic cells (72 hours - 0.2% O2). Under chronic hypoxic conditions, confirmed by the up-regulation of VEGF and HIF1a, mRNA and protein expression of key homologous recombination (HR) genes (BRCA1, BRCA2, RAD51) were down-regulated. Functionally, BRCA2-/- null cells proved unable to recruit Rad51 foci and were profoundly sensitivity to PARP inhibition. Conversely, heterozygote BRCA2+/- cells retained the ability to recruit Rad51 foci under both oxic and hypoxic conditions. However, exposure to chronic hypoxia resulted in a reduction of foci formation. Chronically hypoxic BRCA2+/- cells exhibited a 30-40% increase in sensitivity to PARP inhibition compared to their oxic counterparts. Preliminary data shows a similar synthetically lethal relationship in genetic (BRCA2-/- ) and tumor microenvironment induced (BRCA2+/-) HR deficient cells, when challenged with DNA damage response (DDR) kinase inhibitors ATRi and DNAPKi.

Conclusions: Herein, we demonstrate a novel mechanism of contextual 'loss-of heterozygosity', which marries the tumour microenvironment and innate genetic alterations. The resultant increased sensitivity to DDR kinase inhibitors and PARPi highlights the therapeutic significance of this phenomenon.

#2487

Functional characterization of the BRCA2 variant, K3326X.

Srilatha R. Simhadri, Sonia C. Dolfi, Atul Kulkarni, Bing Xia, Shridar Ganesan, Kim M. Hirshfield. _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ_.

Individuals with germline mutations in the breast cancer susceptibility gene, BRCA2, have an approximate 70% risk of developing breast cancer, a 30% risk of developing ovarian cancer, a 20-fold increased risk of developing prostate cancer, and a 10-fold risk of developing pancreatic cancer during their lifetime. Loss of function germline mutations in BRCA2 affect its role in the homologous recombination (HR) DNA repair pathway leading to significant genomic instability. In addition to deleterious truncating mutations, several sequence variants, collectively called Variants of Unknown Significance (VUS), have been identified and are distributed along its length. One such variant is rs11571833, a nonsense mutation in the last exon (c.9976A>T, K3326X), resulting in the loss of the C-terminal 93 amino acid residues in BRCA2. This truncated variant has been previously described as a polymorphism that does not increase susceptibility to breast and ovarian cancers, and as a neutral unclassified variant non-deleterious to its function. However, recent studies identified K3326X to be enriched in breast cancer cases and to increase the risk for lung, pancreatic, ovarian, and upper aero-digestive tract cancers. Preliminary data, obtained from the Rutgers Cancer Institute of New Jersey breast cancer case-control study, identified K3326X enrichment in 1.25% of cases compared to 0.7% of controls. Several of the carriers had second primaries and displayed a trend toward increased number of family members diagnosed with colon cancer. Notably, K3326X was also identified in 1.38% (11 of 796) of our histologically-diverse cohort of genomically-profiled tumors that included cancers of the breast, ovarian/fallopian tube, lung, vulvar, cancer of unknown primary, and one breast cancer case having prolonged response to platinum-based therapy. Thus, K3326X may represent a functional loss of wild type BRCA2 function, as we observe concomitant loss of heterozygosity at this locus. In a preliminary study, we evaluated the K3326X variant, in vitro, in a functional DR-GFP-based reporter assay measuring HR. Our data reveal the BRCA2 K3326X variant to be impaired in the HR pathway indicating a loss of wild-type protein function. We will also evaluate cell viability of the K3326X variant in the presence of DNA damaging drugs like cisplatin, poly-ADP ribose polymerase inhibitors and mitomycin C. Future studies will also incorporate a retrospective evaluation of tumor specimens that have undergone comprehensive genomic profiling. These data would indicate that BRCA2 K3326X represents a functional hypomorphic variant that may have implications in therapeutic approaches and cancer risk evaluations across a spectrum of tumor types.

#2488

Tumor necrosis factor-alpha signaling determines cytotoxicity induced by BRCA2 deficiency.

Anne M. Heijink, Francien G. Talens, Anouk Baars, Marcel A. van Vugt. _University Medical Center Groningen, Groningen, Netherlands_.

Introduction

Breast cancer 2, early onset (BRCA2) is involved in DNA-damage repair via homologous recombination (HR). Defective HR leads to severe genomic instability and homozygous deletion of BRCA2 is therefore deleterious for survival of normal cells. In apparent contrast, inherited heterozygous mutations in BRCA2 increase the risk to develop cancer, which goes along with somatic loss of the wild type allele. So paradoxically, BRCA2 loss is lethal in normal cells, but can be tolerated in cancer cells. It is currently unclear how tumor cells can survive without BRCA2. To address this question, we performed a genome-wide functional genetic screen and identify Tumor Necrosis Factor-alpha (TNFα) signaling, via TNF receptor 1 (TNF-R1) and Src-associated-substrate-during-mitosis-of-68kDa (Sam68), as an important determinant of cell death induction upon BRCA2 inactivation.

Material & Methods

A genetic screen was performed in human near-haploid KMB-7 cells. Two monoclonal KMB-7 lines were engineered to express independent doxycycline-inducible BRCA2 short hairpin RNAs (shRNAs). Cells were randomly mutagenized using retrovirus carrying a strong splice-acceptor, resulting in gene knock-outs. Deep sequencing of surviving colonies revealed gene mutations that prevented shBRCA2-mediated cell death. Validation of TNF-R1 and Sam68 was done by shRNAs in multiple breast- and leukemic cell lines. Cell viability was determined by MTT and clonogenic assays. Competition assays with vectors expressing shRNA including IRES-driven mCherry were analyzed with flow cytometry. Biochemical analyses were assessed by western blotting and ELISA. Immunoprecipitation with FLAG-tagged TNFα was performed to study TNF-R1 complex formation.

Results

From our genetic screens, loss-of-function mutations involved in TNFα signaling were found to significantly rescue cell death in BRCA2-depleted cells. To validate these findings, BRCA2 was stably depleted in a panel of cancer cell lines, which decreased viability. Subsequent co-depletion of TNF-R1 or Sam68 rescued survival. Notably, BRCA2 depletion resulted in the production of TNFα. In line with this finding, the majority of tested cancer cell lines were found to be sensitive to recombinant TNFα upon BRCA2 depletion. This phenomenon was not restricted to BRCA2 inactivation, since depletion of related DNA repair genes (BRCA1, FANCD2) or hydroxyurea-induced replication stress also sensitized cells to recombinant TNFα. Mechanistically, BRCA2 depletion was induced to observe differential complex formation of TNF-R1 with downstream effector proteins upon TNFα activation.

Conclusion

Our data reveal that down-regulation of TNF-R1 or its downstream signaling component Sam68 rescued cytotoxicity upon BRCA2 inactivation. These findings and mechanistic follow-up experiments describe a novel mechanism by which autocrine TNFα signaling, induced by loss of BRCA2, limits tumor cell viability.

#2489

Mre11-CDK2 interaction during the DNA double-strand break response.

Mary J. Morgan, Todd A. Festerling, Jeffrey Buis, David O. Ferguson. _University of Michigan, Ann Arbor, MI_.

The MRN (Mre11/Rad50/NBS1) complex is a group of highly conserved proteins central to the detection and repair of DNA double-strand breaks (DSBs). Inherited deficiencies in DNA DSB signaling result in a spectrum of disorders featuring cancer predisposition, neurodegeneration, and immunodeficiency. MRN directly binds DNA DSB ends and facilitates activation of ataxia-telangiectasia mutated (ATM) kinase, which initiates cellular responses including DNA repair, cell cycle checkpoints, and in some circumstances, apoptosis. Cellular responses to DNA DSBs require rapid communication between DNA repair complexes and the cell cycle machinery, but much about this relationship remains unclear. Our lab recently discovered an interaction between Mre11 and Cyclin-dependent kinase 2 (CDK2), a key component of the cell cycle machinery (Buis et al., NSMB, 2012). CDK2, when bound to its regulatory partner Cyclin A, promotes cell cycle progression through S phase. We demonstrated that the Mre11-CDK2/Cyclin A interaction is important in regulating the capacity for DNA repair by homologous recombination in normally dividing cells. Current work is focused on understanding the role of this interaction during DNA damage responses. We hypothesize that Mre11 interaction with CDK2 is a component of a molecular switch between the normal cell cycle and the DNA damage response. To test our hypothesis, we take advantage of murine alleles of Mre11 previously generated in the lab. We performed immunoprecipitation studies to investigate the protein-protein interaction in the presence of DNA damage. We also utilized kinase inhibitors to investigate the role of apical kinases in regulating the interaction. Finally, we are currently optimizing in vitro kinase assays to further understand a relationship between Mre11 and CDK2 enzymatic activity.Unpublished findings reveal that the Mre11-CDK2/Cyclin A interaction is altered upon DNA damage, a process which appears to be important for cell cycle checkpoint regulation. In conclusion, we demonstrate that the Mre11-CDK2 interaction plays important roles both in normally dividing cells and in the DNA damage response. Taken together, our data suggests that Mre11 may act to bridge the gap between DNA repair complexes and the cell cycle machinery.

#2490

Rad51 suppresses innate immune response by blocking MRE11-mediated degradation of newly replicated genome.

Kalayarasan Srinivasan, Souparno Bhattacharya, Salim Abdisalaam, Shibani Mukherjee, Asaithamby Aroumougame. _UT Southwestern Medical Ctr., Dallas, TX_.

Purpose of the study: Eukaryotic cells accrue DNA damage as a result of endogenous metabolic activities such as DNA replication, recombination errors or environmental exposures such as ionizing radiation, ultra-violet light and chemical mutagens. Unrepaired DNA damage leads to tumorigenesis. Rad51 is a multifunctional protein that plays a central role in DNA replication and homologous recombination repair. It is known that defects in Rad51 function can cause cancer. The goal of this study is to identify a novel role for Rad51 outside of its known functions in DSB repair and replication fork processing.

Methods: Since Rad51 knockout is lethal to cells, we generated an inducible system in which we can down-regulate Rad51 expression in HT1080 cells after Doxycycline treatment. To determine the effect of Rad51-knockdown in global gene expression pattern, we carried out unbiased microarray gene expression analysis and after induction of DNA damage and replication stress by radiation. ssDNA and dsDNA in the cytosolic fractions were quantified using Quant-iT OliGreen and PicoGreen Assay Kits. For cytoplasmic BrdU detection, exponentially grown cells were labeled with BrdU for 18-20 h and then immunostained with anti-BrdU antibody. Additionally, we measured the expression and post-translational modification of proteins involved in innate immune signaling by western blotting. We also employed DNA fiber assay to determine the role of Rad51 in replication fork processing.

Results: We found that defects in Rad51 lead to the accumulation of self-DNA in the cytoplasm, triggering a STING-mediated innate immune response after replication stress and DNA damage. Mechanistically, the unprotected newly replicated genome in the absence of Rad51 is degraded by the exonuclease activity of Mre11, and the fragmented nascent DNA accumulates in the cytosol, initiating an innate immune response. Our data revealed that in addition to playing roles in homologous recombination-mediated DNA double-strand break repair and replication fork processing, Rad51 is also implicated in the suppression of innate immunity.

Conclusion: Rad51 plays a novel role in immunity outside its known functions in DSB repair and replication fork processing. We discovered that the lack of Rad51 leads to the upregulation of innate immune response pathway genes upon DNA damage and replication induced by irradiation. We found that in the absence of Rad51 the newly replicated genome is degraded by the exonuclease activity of Mre11. We also showed that these degraded nascent DNA fragments are exported to the cytoplasm, triggering innate immune response signaling. Our study reveals a previously unidentified role for Rad51 in triggering an innate immune response, and places Rad51 at the hub of new interconnections between DNA replication, DNA repair, and immunity. Funding: This work was supported by NIH R01AG053341 grants.

#2491

Beclin1 promotes DNA double-strand breaks repair.

Fei Xu,1 Lili Yan,1 Jianrong Wang,1 Jinming Yang2. 1 _Soochow University, China, Suzhou, China;_ 2 _Penn State Univ., PA_.

Beclin1 is a well-established core mammalian autophagy protein that has been presumed to suppress oncogenesis via an autophagy-mediated mechanism. Since its discovery, studies have also shown that Beclin1 interacts with an increasing number of cofactors. However, the tumor suppressive roles of Beclin1 remain a major research focus, and the exact mechanism by which Beclin1 acts as a tumor suppressor has remained largely obscure. Our confocal analysis showed that in embryonic and neonatal mice, Beclin1 is primarily located in the cytoplasm in hepatocytes, with a very small portion located in the nucleus. When the mice were 15d, roughly half of the total Beclin1 was located in the nucleus. Thereafter, Beclin1 progressively redistributed from cytoplasma to nucleus within a few weeks after birth. This pattern, was similarly sustained not only in hepatocytes but also in other tissues. And we found both 0-50 and 254-278 residues are required for Beclin1 nuclear localization. Furthermore, loss of Beclin1 resulted in reduced DNA damage repair. We found that the beclin1-/- cells showed a marked increased γ-H2AX foci that are considered as a biomarker of DNA DSBs. Moreover, beclin1-/- cells showed approximately about 60-fold and 30-fold reductions in DSB repair by NHEJ and HR pathways. A series of key DSBs repair proteins were significantly reduced in beclin1-/- cells. Interestingly, Beclin1 regulates DSB repair independent of autophagy. Autophagic flux was maintained in the beclin1-/- cells. Perhaps an alternative autophagy may be activated when beclin1 is deleted as ULK1 was upregulated. We further found that Beclin1 promotes DSB repair via a direct interaction with DNA topoisomerase IIβ (Top2b). Pull down results showed that Top2b is one of the major Beclin1-interacting proteins in the nucleus. And we found that this interaction was intensified after exposure to IR. Top2b and Beclin 1 were found to be colocalized to the DSB sites, after exposure to IR. Strikingly, depletion of Top2b by gene silencing completely prohibited Beclin1 localized to the DNA break sites. We constructed a beclin1-/- cell line using CRISPR/Cas9 system. The spot counts of protein foci were measured by imaging flow cytometry after exposure to 6Gy IR. HR and NHEJ reporter assays were used to detect the activity of DSBs repair pathways. We pulled down nuclear proteins with ectopically expressed Flag-tagged Beclin1. The identities of the Beclin1-interacting proteins were determined using mass spectrometry. Our work establishes that Beclin1 plays an autophagy-independent role in maintaining genomic integrity by promoting DNA damage repair. The finding challenge the long-standing paradigm of the mechanisms that are involved in the autophagy-mediated tumor suppressive role of Beclin1. This finding suggests that when manipulating cytoplasmic Beclin1 that is involved in the autophagy pathway in order to explore targeted tumor therapies, the non-autophagy cytoprotective role of Beclin 1 in the nucleus should also be considered.

#2492

DNA polymerase beta participates in DNA end-joining.

Sreerupa Ray, Michelle DeVeaux, Gregory Breuer, Ranjit Bindra, Daniel Zelterman, Joann Sweasy. _Yale University, New Haven, CT_.

DNA double strand breaks (DSBs) are one of the most deleterious lesions. If left unrepaired, DSBs lead to genomic instability and carcinogenesis. The cells combat DSBs by two classical pathways that include homologous recombination (HR) which requires the sister chromatid for sequence homology, and non-homologous end-joining (NHEJ), wherein the two DNA ends are re-joined. Recently a back-up NHEJ pathway has been reported and is referred non-canonical NHEJ which has been given many names, such as alternative NHEJ or microhomology-mediated end joining (MMEJ). The enzymatic mechanisms of non-canonical NHEJ are not well defined. NHEJ requires processing enzymes including nucleases and polymerases, although the roles of these enzymes in the pathway are poorly understood. Recent studies have implicated a role for DNA polymerase theta (Pol θ), an A-family polymerase is essential for non-canonical NHEJ pathway. Emerging evidence indicates X-family polymerases, mammalian DNA polymerases lambda (λ) and mu (μ) and yeast Pol 4 promote DNA end-joining. Our laboratory has recently provided evidence for a role for DNA polymerase beta (Pol β), another X-family polymerase, in V(D)J recombination, a process that requires end-joining. Here, using a recently developed fluorescence based assay that monitors non-canonical NHEJ and HR, we provide evidence that Pol β plays a role in the non-canonical NHEJ process. DNA sequencing at the break point junctions revealed Pol β-depleted cells have fewer small deletions than control cells, but significantly greater numbers of insertions and large deletions. We further demonstrate that Pol β-depleted cells have increased sensitivity to DNA damaging agents that induce double-strand breaks and that there is persistent accumulation of DSBs in these cells. In combination, our results suggest that Pol β is critical for double-strand break repair.

#2493

Identification of the mutant p53-PARP-MCM chromatin axis as a triple negative breast cancer replication stress target.

Wei-Gang Qiu,1 Alla Polotskaia,2 Gu Xiao,2 Lia Di,2 Yuhan Zhao,3 Wenwei Hu,3 John Philip,4 Ronald Hendrickson,4 Jill Bargonetti1. 1 _City Univ. of New York at Hunter College and The Graduate Center, New York, NY;_ 2 _City Univ. of New York at Hunter College, New York, NY;_ 3 _Rutgers Cancer Institute of New Jersey, Rutgers, NJ;_ 4 _Memorial Sloan-Kettering Cancer Center, New York, NY_.

Approximately 15% of all breast cancer is triple-negative and of these about 80% are found to have mutations in the gene for the tumor suppressor p53 (TP53). Many TP53 mutations encode gain-of-function oncogenic mutant p53 (GOF mtp53) protein. We used inducible knockdown of endogenous GOF mtp53 in MDA-MB-468 cells in conjunction with stable isotope labeling with amino acids in cell culture (SILAC) and subcellular fractionation. We sequenced over 70,000 total peptides for chromatin and cytoplasmic reciprocal data sets and were able to identify 3,010 unique cytoplasmic fraction proteins and 3,403 unique chromatin fraction proteins. We found that the heterohexomeric minichromosome maintenance (MCM) complex (MCM 2-7) along with PARP are high mtp53-chromatin associated pathways. When we depleted R273H mtp53 we found a large reduction of the amount of MCM complex and PARP proteins on the chromatin. Furthermore a direct mtp53-MCM2 interaction was detected. Overexpressed mtp53, but not wild type p53, showed a protein-protein interaction with MCM2 and MCM4. We treated cells with the PARP inhibitor talazoparib and the alkylating agent temozolomide and detected synergistic activation of apoptosis only in the presence of functional MCM2-7 and mtp53. The mtp53-PARP-MCM axis has potential use as a therapeutic and diagnostic target.

#2494

ATR inhibitor AZD6738 as monotherapy and in combination with olaparib or chemotherapy: defining pre-clinical dose-schedules and efficacy modelling.

Alan Y. Lau,1 James Yates,1 Zena Wilson,2 Lucy A. Young,1 Adina M. Hughes,1 Alienor Berges,1 Amy Cheung,1 Rajesh Odedra,2 Elaine Brown,2 Mark J. O'Connor,1 Simon Hollingsworth1. 1 _AstraZeneca, Cambridge, United Kingdom;_ 2 _AstraZeneca, Macclesfield, United Kingdom_.

Introduction: AZD6738 is a potent and selective oral inhibitor of the ataxia telangiectasia and rad3 related (ATR) protein kinase. ATR has a key role in the DNA replication stress response (RSR) pathway of DNA repairby facilitating the recovery and repair of potentially cytotoxic persistent, stalled DNA replication forks. Inhibition of ATR leads to the inability to resolve replication associated damage and the accumulation of DNA strand breaks, which if remains unrepaired leads to cell death. AZD6738 is currently in Phase I/II clinical trials being evaluated as a monotherapy and in combination with novel agents olaparib / Lynparza (PARP DNA damage response inhibitor), durvalumab (PD-L1 immune checkpoint inhibitor) and DNA-damaging agents such as carboplatin and ionising radiation. Critical in helping to guide the clinical usage of AZD6738 and maximise patient benefit, pre-clinical studies were performed to determine optimal doses and schedules as monotherapy and in combination with olaparib and carboplatin.

Experimental procedures: Human cancer cell lines, xenograft and patient-derived explant (PDX) models of non-small cell lung cancer (NSCLC), head & neck squamous cell carcinoma (HNSCC), and triple-negative breast cancer (TNBC) were tested comparing once daily versus twice daily dosing, the number of consecutive days dosing (3 days/week, 5 days/week, continuous) and co-dosing versus sequential or intermittent dosing with AZD6738 alone or in combination with olaparib or carboplatin. The magnitude and duration of anti-tumour responses were then compared with AZD6738 mouse pharmacokinetic (PK), pharmacodynamic (PD) and in vitro target (IC) / growth inhibition (GI) profiles.

Results: A mathematical model was derived which adequately described the AZD6738 PK/PD-efficacy relationship. This modelling confirms that duration of cover (time) above cellular ATR target inhibition thresholds (IC90 pCHK1 / GI90) per day, rather than Cmax or exposures per se, is the major determinant of anti-tumour responses. As monotherapy, in sensitive ATM-deficient models, it is necessary to inhibit ATR continuously to give tumour stabilisation, which can be achieved through repeat daily of AZD6738 over several weeks. Co-dosing AZD6738 in combination with olaparib or carboplatin gives best efficacy compared to sequential dosing and PK cover over the first 48-72 hours is necessary to give tumour regressions. The models predict that extending the duration of ATR cover, achieved through repeat daily dosing, further increases efficacy. These pre-clinical dose-schedules were compared to human free plasma AZD6738 PK data and predicted efficacious exposures found to be clinically achievable.

Conclusions: Together these data further support the clinical evaluation of AZD6738 and suggest optimal dosing schedules for ATR inhibitors.

### Metabolomics and Metabolic Regulation

#2495

The metabolic secretome of cachexia inducing pancreatic ductal adenocarcinoma.

Santosh K. Bharti,1 Paul T. Winnard,1 Louis Dore-Savard,2 Yelena Mironchik,1 Marie-France Penet,1 Zaver M. Bhujwalla1. 1 _The Johns Hopkins University School of Medicine, Baltimore, MD;_ 2 _McGill University Health Center, Montreal, Quebec, Canada_.

Cachexia is an underexplored and yet devastating consequence of cancer that is the cause of 20% of all cancer related deaths1. Cachexia inducing tumors cause a 'wasting away' of the body. The condition is associated with poor treatment outcome2, fatigue, and extremely poor quality of life2,3. Because of the multi-factorial characteristics of this condition, it has been difficult to understand the mechanisms driving the impact of the tumor on body organs and the sequence of events that leads to this lethal condition. Here we have used 1H MRS to characterize the metabolic profile of tumor interstitial fluid (TIF) obtained from noncachexia (Panc1) and cachexia inducing (Pa04C) tumors to further understand the impact of the deranged metabolism of cachexia-inducing tumors on the tumor metabolic secretome.

The human pancreatic cancer cell line, Panc1, was obtained from ATCC. The human pancreatic cancer cell line, Pa04C, was provided by Dr. Maitra4. Six to 8 week old male SCID mice were inoculated in both the right and left flank with cancer cells (5×106, Panc1 N=2, Pa04C N=2). We created a collection chamber to collect TIF. The chamber was implanted together with small tumor pieces harvested from the subcutaneous flank tumors, into the subcutaneous flank space of SCID mice (Panc1 N=8 and Pa04C N=6) until the tumor encompassed the chamber (4-5 weeks). The tumor was then removed and the tumor tissue and TIF were collected. To obtain control TIF, an empty chamber was implanted in the subcutaneous flank space of healthy mice. Dual phase solvent extraction was performed on tumor tissue. The water phase was separated, freeze dried, reconstituted in D2O PBS for spectral acquisition. All 1H MR spectra were acquired on an Avance III 750 MHz (17.6T) Bruker NMR spectrometer equipped with a 5 mm broad band inverse (BBI) probe. Spectral acquisition, processing and quantification were performed using TOPSPIN 2.1 software.

Notable differences between Pa04C compared to Panc1 TIF or normal interstitial fluid were a significant decrease of polyunsaturated fatty acids (PUFA) and lipids, and formate, pyruvate, glutamine, and glucose. Lactate, glutamate, succinate, glycine and acetone significantly increased in Pa04C TIF compared to Panc1 TIF or normal interstitial fluid. These differences in TIF cannot be explained solely by the differences in the tumor metabolic profile. Our data provide new insights into changes in the metabolic secretome with induction of cachexia that may shed new light on the cachexia cascade, and identify metabolic strategies to reverse the syndrome.

References:

(1) Argiles, J. M., et al. Nature reviews. Cancer 2014, 14, 754-762.

(2) Ozola Zalite, I., et al. Pancreatology 2015, 15, 19-24.

(3) Fearon, K. C., et al. HPB (Oxford) 2010, 12, 323-324.

(4) Penet, M. F., et al. Clinical Cancer Research 2015, 21, 386-395.

Acknowledgment: This work was supported by NIH R01 CA193365, NIH P50CA013175 and NIH P30CA06973.

#2496

Targeting brain tumor stem cells by interfering with choline metabolism: Evidence for an EMT-choline oncometabolic network.

Katharina Koch,1 Rudolf Hartmann,2 Abigail K. Suwala,1 Dayana Herrera Rios,1 Ulf D. Kahlert,1 Jaroslaw Maciaczyk1. 1 _University Hospital Duesseldorf, Duesseldorf, Germany;_ 2 _Forschungszentrum Juelich, Juelich, Germany_.

Glioblastoma (GBM) is the most lethal primary malignant brain tumor with a median survival of less than two years. High levels of therapy resistance, strong cellular invasiveness and rapid cell growth demand aggressive multimodal therapies involving resection as well as radio-chemotherapy. Recent evidence has pointed to the existence of brain tumor stem cells (BTSCs), a subpopulation of human brain tumors which is thought to be responsible for tumor dissemination, relapse and chemo resistance. BTSCs have been associated with the expression of mesenchymal features as a result of epithelial-mesenchymal transition (EMT). Using high resolution proton nuclear magnetic resonance spectroscopy (1H NMR) we compared the intracellular metabolic composition of GBM cells after induction vs. inhibition of EMT as well as under stem cell or differentiated conditions. We identified that both EMT and enrichment for stemness induces the cholinic phenotype which is characterized by high intracellular levels of phosphocholine and total choline derivatives. Furthermore, interference with choline metabolism by targeting choline kinase alpha (CHKα) reversed EMT in GBM cells as we observed reduced invasiveness, clonogenicity, and expression of EMT associated genes. Taken together, interfering with choline metabolism is a powerful strategy to suppress EMT and thus target BTSCs. Moreover, the newly identified BTSC-oncometabolic network could be used to non-invasively monitor the invasive properties of glioblastomas and the success of anti-BTSC therapy.

#2497

Differential requirement of amino acids on cell survival of ovarian cancer cells.

Akiko Furusawa,1 Jun Inoue,1 Hitoshi Tsuda,2 Naoyuki Miyasaka,1 Johji Inazawa1. 1 _Tokyo Medical and Dental University, Tokyo, Japan;_ 2 _National Defense Medical College Hospital, Tokorozawa, Japan_.

Amino acids (AAs) are traditionally classified into nutritionally essential AAs (EAAs) or nonessential AAs (NEAAs) for animals and humans. Recent studies have demonstrated that not only EAAs but also NEAAs play important roles on energy metabolisms in cancer cells. However, which AAs are indispensable for human cancer cell survival has not been fully elucidated. The purposes of this study were 1) to determine the indispensable AAs for ovarian cancer (OVC) cells and 2) to investigate the relationship between the AAs synthetase and cancer cell survival.

Firstly, OVC cell lines were cultured with different medium in which each AA was depleted and cancer cell survival was examined. Among 17 OVC cell lines, EAAs were required in 12 cell lines, whereas others did not require EAAs for cell survival. Interestingly, cancer cell lines which required EAA also required at least one NEAAs, including arginine, cystine, glutamine, serine and tyrosine. Although extracellular glutamine has been known to be a critical factor for cancer cell survival and growth, 3 OVC cell lines did not require extracellular glutamine. Hence, we next examined glutamine synthetase (GS) gene, which is a critical enzyme for production of endogenous glutamine from glutamate. In OVC cell lines which did not require extracellular glutamine, GS expression level was positively correlated with cell survival rate under glutamine-depleted condition. Furthermore, we found that the expression of GS protein was negative in 38 of 645 primary OV tumors (5.9 %). Taken together, these results suggest that GS-down-regulated OVC cells require extracellular glutamine for their survival. Therefore, uptake inhibition of extracellular glutamine or reduction of extracellular glutamine level may be a novel therapeutic strategy for patients with ovarian cancer with "GS down-regulation", as well as development of an AA-based precision medicine of OVC.

#2498

Amine and lipid metabolites are enriched in advanced prostate cancer.

Lorelei A. Mucci,1 Ericka Ebot,1 Clary Clish,2 Sarah Markt,1 Renea Taylor,3 Matthew Watt,3 Philip Kantoff,4 Edward Giovanucci,1 Matthew VAnder Heiden,5 Kathryn Wilson1. 1 _Harvard School of Public Health, Boston, MA;_ 2 _Broad Institute of MIT/Harvard, Cambridge, MA;_ 3 _Monash University, Australia;_ 4 _Memorial Sloan Kettering Cancer Center, NY;_ 5 _Masachusetts Institute of Technology, Cambridge, MA_.

Background: Obesity is an established risk factor for advanced prostate cancer. We performed whole body metabolomics profiling in pre-diagnostic blood specimens to identify metabolites associated with risk of advanced prostate cancer, and assess the extent to which obesity modifies this association.

Methods: Our study was nested within the prospective US Health Professionals Follow-up Study during the study period 1993 and 2014. We selected 212 advanced prostate cancer (≥ stage T3b, distant metastasis or cancer death during follow-up) patients and 212 matched controls for whom pre-diagnostic bloods were collected with a median 5.5 years prior to diagnosis. Metabolomics profiling was performed at the Broad Institute using liquid chromatography-tandem mass spectrometry to assay 295 known metabolites. We estimated odds ratios and 95% confidence intervals using multivariable conditional logistic regression to examine associations of individual metabolites and cancer risk. We also performed pathway analyses using a method adapted from gene set enrichment analysis to identify metabolite classes associated with advanced prostate cancer.

Results: We identified 29 circulating metabolites in pre-diagnostic bloods that were significantly associated with advanced prostate cancer risk. These metabolites included carnitines and phospholipids, diacylglycerols (DAG) and triacylglycerols (TAG). Using pathway analyses, we found metabolites in DAG and TAG pathways were highly significantly (FDR<10-8) enriched in men with advanced prostate cancer. 166 of the 295 metabolites were significantly (p<0.001) associated with obesity. When we stratified by a man's obesity status, we observed unique metabolomics profiles associated with advanced prostate cancer. Circulating amines (including carnitines) were associated with advanced prostate cancer in overweight men while lipid metabolites (including DAGs and TAGs) were associated with advanced cancer in the healthy weight men.

Conclusion: Altered levels of TAG and DAG metabolites in prediagnostic bloods are strongly associated with an increased risk of advanced prostate cancer, independent of obesity. The finding that circulating lipids were specifically associated with advanced prostate cancer in healthy weight patients suggests there are men with normal body mass index but who are metabolically obese. The specific lipid metabolite classes enriched in advanced prostate cancer support the hypothesis that obesity acts through dysregulation of liver fatty acid metabolism rather than through metabolic actions of the adipose tissue itself.

#2499

Arginine deprivation-induced autophagy renders ASS1-deficient sarcomas dependent on serine metabolism.

Jeff Kremer, Bethany Prudner, Brian Van Tine. _Washington University in St Louis, St. Louis, MO_.

Objective: Argininosuccinate Synthetase 1 (ASS1) is silenced in ~90% of sarcomas. Upon arginine starvation, ASS1 (-) sarcoma cells undergo autophagy and growth arrest, but not cell death. In order to identify potential synthetic lethal targets arising from the arginine deprivation, mass spectroscopy was performed to determine the metabolic alterations caused by short and long term arginine deprivation resulting from treatment with pegylated arginine deiminase (ADI-PEG20). With recent studies showing the importance of serine biology in cancer, as well as recent generation of a small molecule inhibitor to the rate limiting enzyme in serine biosynthesis, this newly identified synthetic lethality proves to be an exploitable therapeutic option for ASS1 deficient sarcomas.

Methods: The SKLMS1 cell line was cultured in normal culture conditions, as well as with ADI-PEG20 for three days and subjected to metabolite extraction and capillary electrophoresis mass spectrometry (CE-MS). Similarly, additional samples were cultured for 2 days, with or without ADI-PEG20, and subsequently treated with UC13 labeled glucose for an additional 24 hours before being subjected to metabolite extraction. CE-MS was performed on the samples to determine the abundance of 54 metabolites, and all respective isotopomers. Cell death was measured by propidium iodide fluorescent activated cell sorting after inhibition of serine metabolism by the small molecule inhibitor of PHGDH (CBR-5884) with and without ADI-PEG20.

Results: Upon treatment with ADI-PEG20, the stable isotope labeled glucose derived component of a vast majority of metabolites decreased significantly. The pathway with the largest increase in metabolic flux was serine biosynthesis, including subsequent conversion into glycine, rendering LTAT lines significantly more susceptible to antifolate metabolites. Significant changes in the enzyme responsible for the rate limiting step of serine biosynthesis, phosphoglycerate dehydrogenase (PHGDH) were observed upon short term arginine deprivation, with further upregulation observed upon long term treatment. Cell death levels were significantly higher in samples when inhibition of PHGDH was paired with ADI-PEG20 treatment, with LTAT lines having even further susceptibility to cell death induction upon PHGDH inhibition than short term ADI-PEG20 treated samples.

Conclusion: By determining the alterations in the fate of glucose in the SKLMS1 cell line upon short and long term treatment with ADI-PEG20, we were able to illustrate an increase in the level of serine biosynthesis. Inhibition of this escape pathway by inhibition of PHGDH induced cell death. LTAT lines were the most sensitive to treatment with the PHGDH inhibitor as well as the commonly used antifolate metabolite, methotrexate.

#2500

Urinary metabolites are diagnostic biomarkers of liver cancer.

Majda Haznadar,1 Kristopher W. Krausz,1 Christopher M. Diehl,1 Elise D. Bowman,1 Anuradha S. Budhu,1 Jittiporn Chaisaingmongkol,2 Siritida Rabibhadana,2 Marshonna Forgues,1 Takahiro Oike,1 Mathuros Ruchirawat,2 Frank J. Gonzalez,1 Xin W. Wang,1 Curtis C. Harris1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Chulabhorn Research Institute, Bangkok, Thailand_.

Liver cancer is third leading cause of cancer-related deaths worldwide. A tremendous burden of this disease is evident in that there is an estimated rise in liver cancer incidence from 39,239 in 2016 to 95,374 by 2020. This bleak statistic serves as a motivation to search for early diagnosis biomarkers, especially considering that the 5-year relative survival rate for people with localized disease is ~30.5%, and for regional stage is only 10.7%. Hepatocellular Carcinoma (HCC) is the most common primary malignant tumor of the liver, and is considered to be the third leading cause of all cancer-related deaths and fifth common cancer worldwide. Hepatitis virus B and C infections, fatty and alcoholic liver disease are established risk factors for developing HCC. Cholangiocarcinoma (CCA) is the second most common primary hepatic malignancy. Liver fluke, a chronic parasitic disease of the bile ducts, is a risk factor for developing CCA; otherwise not enough is known about the etiology of this form of liver cancer. We initially conducted a pilot study in the NCI-MD cohort comprising 98 HCC cases, 101 high risk subjects and 95 controls, to evaluate whether previously identified urinary metabolite biomarkers of lung cancer were also predictive of liver cancer, given preliminary evidence that they were universally elevated in several cancer types. Thus, we leveraged ultraperformance liquid chromatography coupled to tandem mass-spectrometry (UPLC-MS/MS) for quantitation of creatine riboside, N-acetylneuraminic acid, cortisol sulfate, and a lipid molecule designated as 561+. This study resulted in findings that all four aforementioned metabolites are significantly increased in HCC cases compared to both, population controls and, more importantly, high risk subjects. We next evaluated whether our previously described findings from the NCI-MD liver case-control study validate in the TIGER-LC cohort (n=370 cases, 471 high risk subjects, 251 controls), where CCA is highly prevalent. We confirmed that indeed the four metabolites were significantly increased in HCC cases compared to both, controls and high risk subjects in TIGER-LC. Most importantly, we also showed that these metabolites are also deregulated in CCA, as their levels are significantly elevated in CCA cases when compared to both, controls and high risk subjects. Furthermore, we showed in the Receiver Operating Characteristic (ROC) analysis a robust ability of the four-metabolite profile to classify both, HCC (AUC=0.75) and CCA (AUC=0.81) compared to high risk subjects. Additionally, the four-metabolite profile performed better in classifying CCA than a clinically utilized CCA tumor marker, CA19-9, and their combination led to a significantly improved classifier (AUC=0.88, P=9.4E-7). Currently, we are investigating a number of pesticides and herbicides used in the areas of Thailand from where TIGER-LC subjects were recruited, to investigate whether these putative carcinogens participate in the etiology of CCA.

#2501

Assessing metabolic activity of patient-derived models of non-small cell lung cancer using stable isotope resolved metabolomics (SIRM).

Connor J. Kinslow,1 Jinlian Tan,2 Yihua Cai,2 Jun Yan,2 Richard M. Higashi,3 Andrew N. Lane,3 Teresa W. Fan3. 1 _Columbia University College of Physicians & Surgeons, NEW YORK, NY; _2 _University of Louisville, Louisville, KY;_ 3 _University of Kentucky, Lexington, KY_.

Stable Isotope-Resolved Metabolomics (SIRM) has been a useful tool in interrogating metabolic reprogramming in cancer. We have recently published several protocols using SIRM in patient-derived models of non-small cell lung cancer (NSCLC)1. In this case study, we compared central metabolic activity in three different model systems, all derived from the same patient. Slices from the human tumor (Warburg slices 2,3), slices from an NSG mouse bearing the human tumor 4, and the patient-derived cell culture were incubated in [U-13C]-glucose or [U-13C,15N]-glutamine for 24 hours. Samples were then harvested and analyzed by gas-chromatography-mass spectrometry and NMR. All three models displayed similar levels of fractional enrichment after 24 hours, making metabolic comparison possible, despite their very different 3D architectures. Glucose metabolism in particular, showed consistent labeling patterns of metabolites in glycolysis and the Krebs cycle. Glutamine metabolism, however, was distinct in the cell culture, with a pattern that indicated upregulation of anaplerotic glutaminolysis. Activation of glutaminolysis may be an important transformative step in the establishment of a viable cell culture. We are also in the process of validating the present finding by comparing multiple patient-derived models of NSCLC and tracking the evolution of their metabolic activity.

1 Lane, A. N., Higashi, R. M. & Fan, T. W.-M. Preclinical models for interrogating drug action in human cancers using Stable Isotope Resolved Metabolomics (SIRM) Metabolomics 12, 1-15 (2016).

2 Fan, T. W.-M. et al. Distinctly perturbed metabolic networks underlie differential tumor tissue damages induced by immune modulator β-glucan in a two-case ex vivo non-small cell lung cancer study. CSH Molec. Case Studies J. 2, a00083, (2016).

3 Fan, T. W.-M., Lane, A. N. & Higashi, R. M. Stable Isotope Resolved Metabolomics Studies in ex vivo Tissue Slices. Bio-protocol 6, e1730 (2016).

4 Lane, A. N., Yan, J. & Fan, T. W.-M. 13C Tracer Studies of Metabolism in Mouse Tumor Xenografts. Bio-protocol 5, e1650 (2015).

#2502

Liquid diet introduction of tracers into mice for stable isotope-resolved metabolomics (SIRM) investigations.

Ramon C. Sun, Marc O. Warmoes, Ye Yang, Pan Deng, Qiushi Sun, Andrew N. Lane, Richard M. Higashi, Teresa W-M Fan. _University of Kentucky, Lexington, KY_.

Tracer-based mapping of metabolic networks in vivo is a powerful approach for revealing metabolic reprogramming in human cancer. However, current in vivo labeling techniques for model animals face important challenges including insufficient depth of pathway coverage (e.g. inability to detect labeled nucleotides, proteins, and lipids) and stress-related artifacts. Here, we report stress-free administration of 13C6-glucose via liquid diet into mice. 13C enrichment was observed in metabolites of glycolysis, the Krebs cycle, the pentose phosphate pathway, nucleobases, UDP-sugars, as well as macromolecules glycogen, lipids, and proteins from major organs in NSG mice. We have applied the liquid diet method to map the glucose metabolic networks in NSCLC tumors in a patient-derived xenograft (PDX) model. We observed a high enrichment in the metabolites of glycolysis, Krebs cycle, and PPP as well as de novo synthesized nucleotides and amino acids by IC-UHR-FTMS analysis. Lung PDX displayed unexpected metabolic complexity, such as the use of pyruvate to fuel anaplerosis as well as gluconeogenesis. We also found high 13C enrichment in both tumor and plasma glutamine, which implies that glutamine in the PDX tumors largely came from other organs via the blood rather than being synthesized in situ. Our data showed that liquid diet is an effective and noninvasive means for comprehensive analysis of glucose-associated metabolic networks in human tumor xenografts, which can also be extended to SIRM studies with other fuel sources.

Acknowledgements: This work was supported in part by grants: 1R01ES022191-01 (to TWMF and RMH), 1P01CA163223-01A1 (to ANL and TWMF), and 1U24DK097215-01A1 (to RMH, TWMF, and ANL)

R.C. Sun was supported by a T32 training grant to M. Vore (5T32ES007266-25)

#2503

Metabolomics identifies a caveolin-1-associated plasma glycosphingolipid and sphingomyelin signature that differentiates aggressive from indolent prostate cancer in an active surveillance cohort.

Johannes Fahrmann, Jody Vykoukal, Spyridon Basourakos, Jianxiang Wang, Jennifer Dennison, Eunice Murage, Jeri Kim, Timothy C. Thompson, Samir Hanash. _UT MD Anderson Cancer Center, Houston, TX_.

Purpose: To identify a plasma signature that can distinguish aggressive from indolent prostate cancer in patients on active surveillance based on metabolomics profiling.

Introduction: The slow growth rate and low likelihood of disease progression in low-risk prostate cancer patients have led to the widely implemented active surveillance (AS) strategy. Given tumor heterogeneity, a clinical challenge is how to expand the pool of patients eligible for AS safely. Consequently, additional markers able to identify patients at increased risk of disease progression are needed. We previously identified plasma caveolin-1 (CAV1) as predictive of aggressive prostate cancer in an AS cohort. Complementary in vitro and in vivo studies demonstrated that CAV1 mediates major reprogramming of lipid metabolism, collectively suggesting that dysregulated lipid biogenesis may be associated with aggressive prostate cancer.

Experimental Procedures: An integrated approach was utilized which included multi-assay untargeted metabolomics to characterize alterations in the metabolome and lipidome of conditioned media from prostate cancer cell lines following overexpression of CAV1 (LNCaP) or CAV1 siRNA knockdown (PC3M). In parallel, sera from PBCre+PTENloxp/loxp;PBCav-1\+ and PBCre+PTENloxp/loxp;PBCav-1\- mice were collected for metabolomics analysis to further delineate signatures related to CAV1-associated aggressive prostate cancer. We further conducted untargeted metabolomic analysis on plasma samples (n=16 per group) prospectively collected from patients with early stage prostate cancer undergoing AS who exhibited early progression (defined as upgrading of Gleason score (GS) and/or increased tumor volume on surveillance biopsy within 18 months after start of AS) or indolent disease (did not progress for a minimum of 5 years after start of AS). Patients were matched with respect to age, clinical stage, prostate-specific antigen, and GS on baseline biopsy at start of AS. We evaluated plasma samples collected at baseline (start of AS) and after 12 months.

Results: Analysis of conditioned media identified a metabolomics signature with major alterations in lipid composition highlighted by CAV1-mediated elevations in lipid-raft associated sphingomyelins and glycosphingolipids. This signature was observed in patients who had early progression but not in patients with indolent disease. Importantly, this signature was observed at baseline and at least 18 months prior to disease progression on surveillance biopsy.

Conclusions: We have identified a plasma-derived lipid signature that differentiates aggressive versus indolent prostate cancer in an active surveillance cohort. Our findings highlight the potential utility of altered lipid profiles as additional risk markers for aggressive prostate cancer.

#2504

Melatonin decreases plasma arginine, its precursors and acylcarnitines in breast cancer xenograft model at specific time point during circadian rhythm.

Debora Zuccari,1 Rubens Paula Junior,1 Nathália Martins Sonehara,1 Roger Chammas,2 Florence Raynaud3. 1 _Faculdade de Medicina de São José do Rio Preto - FAMERP, São José do Rio Preto, Brazil;_ 2 _Instituto do Câncer do Estado de São Paulo - ICESP, São Paulo, Brazil;_ 3 _The Institute of Cancer Research, Sutton, London, United Kingdom_.

The circadian rhythm regulates the whole metabolism modulating the concentration of various circulating substances through the clear and dark variation. Melatonin is the main mediator of this flow and breast tumors development is linked to imbalance of this rhythm. This hormone has several anti-cancer effects extensively proved by our group. Currently metabolomics analysis have been used in cancer to provide a comprehensive profile of the metabolic changes. Important tumor features are regulated by amino acids likewise fatty acids metabolism. Therefore the comprehension of specific metabolic pathways modulated by melatonin is essencial to verify its useful as adjuvant treatment. The aim of this study was to evaluate the metabolic profile in breast cancer xenograft model treated with melatonin and according the circadian cycle. Were used 160 female Balb/c nude athimic mice divided into four groups: Tumor / Melatonin, Tumor / Vehicle, Non-tumor / Melatonin and Non-tumor / Vehicle. The tumor-bearing animals were developed by implantation of 3x106 triple negative human breast cancer cells (MDA-MB-231) in the mammary fat pad. All the animals received 100 ul of solution by intraperitoneal injection (IP) for five days a week during 21 days. The treated groups received 40 mg/kg of melatonina. Blood sample were collected every 3 hours during 24 hours (eight time points) and the plasma was extracted for analysis by liquid chromatography-mass spectrometry (LC-MS) using the Absolute IDQ p180 Kit (BioCrates). Orthogonal Partial Least Squares (OPLS) analysis showed evident separation of the animals metabolic profile not only because the circadian cycle, but also because the melatonin treatment. We found a significant increasing in arginine and precursors concentration in blood of the tumor-bearing animals specificaly at 6am and 9am. Beside these data we also found alterations in the acylcarnitines levels. The concentrations of medium and long-chain acylcarnitines increased significantly at 3pm. Tumorbearing animals which have been treated with melatonin showed reduced levels of these amino acids and acylcarnitines in blood achieving the same levels of non-tumour bearing animals. In summary ourt work reveals a strong relationship between the metabolic profile and the entire circadian variation as well as the exogenous melatonin can regulates the metabolism. This pre-clinical trial so far unpublished has shown an increase of arginine, its precursors and acylcarnitines at specific time points in the plasma of breast tumor-bearing animals. The treatment with melatonin is capable to deacrese the concentration of these amino acids and acylcarnitines achievning basal levels. Our results corroborate the literature which characterizes these metabolites as biomarkers for cancer and increasingly allow the use of melatonin as an adjuvant therapy for breast cancer.

#2505

High-resolution MRS characterization of malignant ascites in two models of ovarian cancer.

Santosh Bharti,1 Flonne Wildes,1 Chien-Fu Hung,2 TC Wu,2 Zaver Bhujwalla,1 Marie-France Penet1. 1 _JHU ICMIC Program, Division of Cancer Imaging Research, Department of Radiology, Johns Hopkins Univ. School of Medicine, Baltimore, MD;_ 2 _Department of Pathology, Johns Hopkins Univ. School of Medicine, Baltimore, MD_.

Build-up of malignant ascites occurs in more than one third of ovarian cancer patients and significantly contributes to poor quality of life and mortality. Advances in understanding malignant ascites formation and finding new therapeutic options are urgently needed. High-resolution 1H MRS has been described as extremely useful in detecting endogenous metabolites to diagnose cancer, by providing a detailed overview of metabolic pathways in a single measurement. In the present study, we are using two ovarian cancer cell lines, the murine ID8-VEGF-Defb29 cell line and the human OVCAR3 cell line. Implanted orthotopically, these two ovarian cancer models are characterized by different profile of ascites formation. The mouse cell line ID8-VEGF-Defb29 induces large volumes of ascites, often more than 10 mL, while the human OVCAR3 cell line induces ascites less frequently and at smaller volumes, usually less than 0.2 mL. We applied high-resolution 1H MRS to compare the metabolic composition of both ascitic fluids. To better understand the differences observed, we characterized the metabolism of these ovarian cancer cells in culture by analyzing cell lysates and conditioned culture media with 1H MRS to advance our understanding of cancer cell metabolic reprogramming in malignant ascites formation and the role of the tumor microenvironment in ascites formation and composition. The two tumor models used in this study induced different ascitic profiles. While OVCAR3 tumor bearing mice developed small viscous volume of ascites, ID8-VEGF-Defb29 induced higher volumes. ID8-VEGF-Defb29 ascitic fluids were characterized by higher levels of glutamine, glucose, poly-unsaturated fatty acids and pyruvate compared to the OVCAR3 fluids, while all the other metabolites, including glutamate, lactate, myo-inositol, choline and acetate, were lower. To determine if the differences observed in the ascitic fluids were only due to a different metabolism of the cancer cells, we investigated their metabolism in vitro. We analyzed the metabolites present in the conditioned cell culture media, and in the cells, and observed differences in OVCAR3 and ID8-VEGF-Defb29 cells metabolism in vitro, without replicating the differences observed in vitro. A Venn diagram of the different metabolites present in the cells, media and ascites showed differences in the metabolites present in those 3 compartments, and highlighted the ones in common. Beta-hydroxybutyrate, lipids, maleic and citrate were found in both ascites, and not in the cells or media. Ascites MRS derived biomarkers could help in ovarian cancer diagnosis, and enhance our understanding of the biochemical and metabolic changes associated with ovarian cancer, and with ascites formation.

Supported by Tina's Wish Foundation, NIH P50CA013175 and P30CA06973.

#2506

Untargeted and targeted multiplatform metabolomic and lipidomic approaches for monitoring biological effects in serum from total body irradiated humans.

Evagelia C. Laiakis,1 Evan L. Pannkuk,1 Siddheshwar Chauthe,1 Yi-Wen Wang,1 Ming Lian,2 Tytus D. Mak,3 Christopher A. Barker,2 Giuseppe Astarita,1 Albert J. Fornace1. 1 _Georgetown University, Washington, DC;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 3 _National Institute of Standards and Technology, Gaithersburg, MD_.

Investigation of responses to radiation therapy or exposure to radiation in an intentional or accidental manner have identified significant biomarkers for biodosimetry and monitoring of individuals in urine through metabolomics (1). We extend the investigation in serum by combining targeted and untargeted methodologies with a multiplatform approach. Serum was collected from patients exposed to total body irradiation (TBI) prior to hematopoietic stem cell transplantation at Memorial Sloan Kettering Cancer Center. The underlying diseases (chronic myelogenous leukemia, acute lymphocytic leukemia, acute myeloid leukemia, non-Hodgkin lymphoma, or myelodysplastic syndrome) were in remission and the cohort included both males and females. Serum was collected prior to irradiation, at 3-8 h after a single dose of 1.25 Gy, and at 24h after three fractions of 1.25 Gy each. Untargeted metabolomic and lipidomic approaches with liquid chromatography (LC) mass spectrometry (MS) and gas chromatography (GC) MS demonstrated significant perturbations at the later time point and higher total dose. Alterations in circulating levels were observed in lipids from monoacylglycerol, triacylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylserine, and phosphatidic acid lipid classes. Fatty acids were some of the most dysregulated lipids, with increased levels linked to pro-inflammatory processes. To further investigate the presence of low abundance pro-inflammatory metabolites, we utilized a targeted and highly quantitative approach measuring downstream molecules from fatty acids, such as thromboxanes, prostaglandins, and leukotrienes that can act as signaling molecules and mediators of systemic inflammation. Results identified a rapid response within the first 3-8 hours primarily mediated through the enzymatic action of lipoxygenase (LOX) and cytochrome P450 (CYP450), although some dissipated at the later time point. Less significant alterations were observed in the anti-inflammatory pathway, although the fatty acid precursor α-linolenic acid remained elevated in both time points. Finally, as observed in our previous published study of a similar cohort (1), sex-dependent differences were analyzed and unlike the urinary results, females appeared to be more responsive to radiation effects as measured in serum with lipidomics and metabolomics. No sex differences were observed with the targeted approach. The ability to identify and quantify small molecules in blood following radiation exposure can therefore be utilized to not only monitor systemic effects of radiotherapy but also to generate signatures that can be utilized to identify accidentally exposed individuals that may require medical intervention. 1. Laiakis EC et al. Radiat Res 2014; 181: 350-361.

#2507

**Using** 1 **H NMR metabolomics to study breast cancer and endothelial cell metabolic interactions.**

Suehelay Acevedo-Acevedo, Sean P. Palecek. _University of Wisconsin-Madison, Madison, WI_.

Breast cancer is the second most commonly diagnosed cancer among women worldwide. Patient death is typically caused by metastasis development rather than the primary tumor. Metastasis in breast cancer has been shown to occur via blood and lymphatic vessels. Research shows that breast cancer cells 'educate' lymphatic and blood endothelial cells to support tumor growth by stimulating growth factor secretion; specifically VEGF, PDGF-BB and EGF secretion. In addition, cell metabolism is altered during malignant transformation. Cancer cells have increased energy and macromolecule biosynthesis requirements to sustain rapid proliferation. Increased angiogenesis observed in tumors points to a need for an increased nutrient supply. However, it remains unclear how tumor endothelial cell metabolism is altered and how metabolism regulates tumor angiogenesis. Therefore, this study aims to use 1H NMR metabolomics to identify breast cancer-endothelial cell metabolic interactions in a high-throughput manner. Firstly, we studied if in vitro co-culture with breast cancer altered endothelial cell metabolism. We identified distinct metabolic profiles for HUVECs grown in monoculture or co-culture with three different breast cancer cell lines. Metabolism of HUVECs co-cultured with MCF7 and SKBR3 breast cancer cell lines was significantly altered compared to mono-culture control. The most impacted metabolic pathways by co-culture were amino acid metabolism, energy metabolism and lipid biosynthesis pathways. Culturing HUVECs with MDA-MB-231 triple negative breast cancer cells did not have a significant impact on HUVEC metabolism, however. Together, these results indicate that culturing HUVECs with certain breast cancer lines causes significant changes in endothelial metabolism. Alterations in endothelial cell metabolism in response to breast cancer co-culture can further our understanding of tumor-vascular interactions and may lead to identification of metabolic biomarkers or therapeutic targets that can disrupt tumor angiogenesis.

#2508

Profiling the metabolic effects of AGI-5198 treatment on IDH1-mutated gliomas.

Victor Ruiz Rodado, Adrian Lita, Tyrone Dowdy, Mark R. Gilbert, Mioara Larion. _National Institutes of Health, Bethesda, MD_.

Background: AGI-5198 (a specific IDH1mut inhibitor) inhibits the formation of D-2-hydroxyglutarate (D-2HG) typically produced in millimolar quantities by the R132H mutation of isocitrate dehydrogenase I (IDH1). AGI-5198 was found to delay growth, to promote differentiation in cells and to decrease tumor volume in IDH1-mutant (IDH1mut) mice xenographs, and as a result, has been advanced to clinical trials in humans with IDH1mut glioblastomas. However, no metabolic study of this drug has been reported to date. Here, we investigated the metabolic changes induced by AGI-5198 treatment in order to determine if the drug restores the normal metabolic status once D-2HG concentration is decreased. Methods: We treated glioma cells carrying an IDH1 mutation with 10 μM AGI-5198 for 72 h. Metabolic changes were explored using an untargeted metabolomics approach. Cell viability analysis verified that the concentration of drug does not lead to significant cell death. NMR and MS-linked metabolomics together with in vivo cell imaging using RAMAN were used. Principal component analysis was employed to explore the differences in the metabolic profiles comparing treated and untreated glioma cells. Results and Conclusions: We confirmed that AGI5198 treatment reduced D-2HG levels in the cell lines investigated, validating previous reports. Moreover, we observed a decrease in phospholipid levels upon AGI-5198 treatment, which suggests a decreased proliferative potential for these cells; of particular importance for clinical application. Our preliminary analysis also suggests that metabolites involved in one carbon metabolism are altered upon AGI-5198 treatment. This pathway provides the methyl group for the DNA methylation by DNA methylases producing a hypermethylated phenotype. Our findings suggest that combining the AGI-5198 treatment with a DNA methylase inhibitor may improve outcomes in IDH1-mutated gliomas.

#2509

**High-resolution** 1 **H <MRS> human plasma profiling of pancreatic ductal adenocarcinoma.**

Santosh K. Bharti, Michael Goggins, Zaver M. Bhujwalla. _The Johns Hopkins University School of Medicine, Baltimore, MD_.

Early detection of pancreatic ductal carcinoma (PDAC) is critically important because by the time PDAC is detected almost 80% of patients are surgically unresectable1. While imaging with CT, MRI and ultrasound is making significant inroads in PDAC detection, the costs associated with imaging impose a barrier for routine screening. Plasma based detection of PDAC would provide a relatively inexpensive and easy method for routine screening for the purpose of early detection. Metabolic profiling of plasma samples using high resolution 1H MRS provides an opportunity to assist in the detection of PDAC. Here we characterized the plasma metabolome of normal subjects, subjects with benign pancreatic disease, and subjects with PDAC to evaluate the ability of 1H MRS to identify metabolic changes in plasma associated with PDAC.

Plasma from patients with PDAC (n=4), patients with benign pancreatic disease (n=2) and from healthy control subjects (n=4) were included in this study. Final diagnosis was established by histopathological evaluation of surgical specimens. Three hundred micro liter of D2O phosphate buffer saline (NaCl 0.9% in 90% D2O) was mixed with 300 μL of plasma and transferred to 5 mm NMR tubes. High-resolution 1H MRS was performed on an Avance 750 MHz Bruker MR spectrometer. The Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence with water suppression [PRESET-90°-(d-180-d)n-Aq] was performed to remove short T2 components arising due to the presence of macromolecules2.

The metabolites were identified and quantified from 1H MRS spectra from the three groups. Even with a small sample size clear differences were identified in metabolites such as lactate, pyruvate, β-hydroxybutyrate, acetate and acetoacetate in plasma from PDAC subjects compared to normal subjects. The increase of lactate and pyruvate detected in plasma may reflect altered glucose metabolism occurring in PDAC. β-hydroxybutyrate, acetate and acetoacetate are involved in ketone body synthesis and may reflect altered ketone body metabolism occurring in PDAC.

In a recent study, higher levels of ketone bodies and lactate were detected in the serum of rats with 7,12-dimethylben(a)anthracen (DMBA)-induced pancreatic intraepithelial neoplasia (PanIN) compared to plasma. Interestingly, with progression to PDAC several of these metabolites decreased3. Our data support further investigation of 1H MRS of human plasma to detect PDAC in combination with techniques such as circulating tumor cell phenotyping

References:

(1) Kotteas, E., et al. J Cancer Res Clin Oncol 2016, 142, 1795-1805.

(2) Van, Q. N., et al. Biochem Biophys Res Commun 2003, 301, 952-959.

(3) Lin, X., et al. Mol Biosyst 2016, 12, 2883-2892.

This work was supported by NIH R01 CA193365.

#2510

Interrogating liver metabolic stress due to cancer-induced cachexia.

Santosh K. Bharti,1 Paul T. Winnard,1 Yelena Mironchik,1 Anirban Maitra,2 Zaver M. Bhujwalla1. 1 _The Johns Hopkins University School of Medicine, Baltimore, MD;_ 2 _The Uuniversity of Texas MD Anderson Cancer Center, Dallas, TX_.

Cancer-induced cachexia accounts for approximately 20% of all cancer deaths 1. In pancreatic cancer, the syndrome affects nearly 80% of patients 2,3. Cachectic patients experience a wide range of symptoms affecting the function of several organs such as muscle, liver, brain, and heart, that decrease quality of life and worsen prognosis. A major characteristic of cachexia is the accelerated skeletal muscle and fat storage wasting causing nutrient mobilization both directly as lipid and amino acids, and indirectly as glucose derived from the exploitation of liver gluconeogenesis that reaches the tumor through the bloodstream4. Patients with cachexia develop a wide range of metabolic stress from increased proteins and fat tissue burning resulting in increased energy expenditure. Here, for the first time, we have performed high-resolution quantitative 1H magnetic resonance spectroscopy (MRS) of liver tissue obtained from normal, noncachectic and cachectic mice bearing PDAC that are cachectic (Pa04C) and noncachectic (Panc1). A significant reduction in liver weight and significant changes in 1H MRS derived metabolite profiles were detected with cachexia.

Human pancreatic cancer cell lines, Panc1 and Pa04C, were used for the study. Six to 8 week old male severe combined immunodeficient mice were inoculated in the right flank with cancer cells (5×106) and in the right hind leg muscle with reporter myoblasts (2×106) to monitor the development of cachexia in mice5. Live animal optical imaging was done using a Xenogen IVIS® Spectrum (PerkinElmer) optical scanner. Dual phase solvent extraction was performed on liver tissue to extract water soluble metabolites. All 1H MR spectra were acquired on an Avance 750 MHz Bruker NMR spectrometer.

We found, for the first time, that the liver in Pa04C tumor bearing mice underwent a profound weight loss; although Panc1 tumor bearing mice showed some liver weight loss this was not as profound as observed with Pa04C tumors. Significant decreases in lactate, glucose and glutathione were observed in cachectic mouse liver compared to noncachectic mouse liver, and the liver from healthy control mice. The significant decrease of these metabolites in cachectic livers may reflect increased utilization of glucose, lactate and glutathione by the tumor or other organs during the cachexia cascade4. These results provide new insights into changes in liver metabolism during cachexia, and support investigating metabolic strategies such as supplementing glutathione or glucose to reduce cachexia associated morbidity.

References: (1) Argiles, J. M., et al. Nature reviews. Cancer 2014, 14, 754-762. (2) Fearon, K. C., et al. HPB (Oxford) 2010, 12, 323-324. (3) Ozola Zalite, I., et al. Pancreatology 2015, 15, 19-24. (4) Porporato, P. E. Oncogenesis 2016, 5, e200. (5) Winnard, P. T., et al. Cancer Research 2015.

Acknowledgment: This work was supported by NIH R01 CA193365. We thank Dr. Marie-France Penet for useful discussions.

#2511

The role of lipids metabolism in bladder cancer.

Gang Wang, Rui Cao, Qiangqiang Ge, Yu Xiao, Xinghuan Wang. _Zhongnan Hospital of Wuhan Univesity, Wuhan, China_.

Bladder cancer (BCa) is one of the most common malignancy of the urinary tract. Many risk factors for BCa has been demonstrated, including aging, smoking, exposure to chemicals, dietary total cholesterol intake, dietary fatty acids intake, etc. In order to investigate the role of lipids metabolism in bladder cancer, we collected several human BCa tissues and normal bladder tissues to generate an affected pathway network by microarray analysis, and it revealed that glycerolipid metabolism, fatty acid degradation and fatty acid biosynthesis have a close correlation with bladder cancer. Therefore, Simvastatin, one of the 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase inhibitors, have been well-known regarding its effects on rate-limiting step in cholesterol synthesis, was used to treat with BCa cells. We observed that simvastatin could reduce BCa cell proliferation, induce cell cycle arrest at G0/G1 phase and inhibit cell metastasis. Then, the potential alterations of lipids metabolism in bladder cancer cells after simvastatin treatment was investigate by lipidomics analysis. These foundings demonstrate that lipids metabolism plays important role in bladder cancer and some lipids or related enzymes may be the novel targets for bladder cancer treatment.

#2513

Multi-omics-based approach to colorectal cancer metabolism.

Kiyotoshi Satoh, Tomoyoshi Soga. _Institute for Advanced Biosciences, Keio University, Tsuruoka, Japan_.

Cancer cells exhibit many metabolic alterations to acquire energy and cellular components required for their proliferation and thus, cancer specific metabolic pathways are attractive targets for cancer therapy. However, the regulation mechanisms underlying metabolic reprogramming are poorly understood. Here, we performed multi-omics-based analyses of paired normal and tumor tissues from patients with colorectal cancer (CRC) to understand the regulation mechanisms of metabolism in colorectal cancer. First, we carried out capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS)-based metabolome profiling of tumor and normal tissue obtained from 275 patients with CRC. S-adenosylmethionine (SAM), a principal methyl group donor produced by one-carbon metabolism, was the most increased metabolite in tumor tissue. Glucose was the second most decreased metabolite in tumor tissue, whereas lactate was accumulated, indicating activation of glycolysis, commonly known as the Warburg effect. Every amino acid except glutamine, the starting metabolite in glutaminolysis, was significantly accumulated in tumor tissue. Interestingly, most of the changes in metabolite levels were occurred in the adenoma stage. In addition, next-generation and Sanger sequencing analyses of genomic DNA obtained from tumor tissue revealed that metabolic alterations occurred independently of mutations in oncogenes and tumor suppressor genes, such as KRAS, APC, and TP53. Furthermore, DNA microarray analysis of paired normal and tumor tissues from CRC patients demonstrated that expression profiles of many metabolic genes were also altered at the adenoma stage. Most of genes regulating purine/pyrimidine synthesis, one-carbon metabolism and fatty acid synthesis were highly expressed, whereas those in fatty acid oxidation were down-regulated in tumor tissue. We will discuss the raised questions, which molecules contribute to the regulation of metabolic reprogramming of colorectal cancer metabolism.

#2514

**Tetra-O-methyl nordihydroguaiaretic acid (M** 4 **N) broadly suppresses cancer metabolism and synergistically induces strong anticancer activity in combination with etoposide, rapamycin and UCN-01.**

Kotohiko Kimura, Ru Chih C. Huang. _Johns Hopkins Univ., Baltimore, MD_.

The ability of Tetra-O-methyl nordihydroguaiaretic acid (M4N) to induce rapid cell death in combination with Etoposide, Rapamycin, or UCN-01 was examined in LNCaP cells, both in cell culture and animal experiments. Mice treated with M4N drug combinations with either Etoposide or Rapamycin showed no evidence of tumor and had a 100% survival rate 100 days after tumor implantation. By comparison all other vehicles or single drug treated mice failed to survive longer than 30 days after implantation. This synergistic improvement of anticancer effect was also confirmed in more than 20 cancer cell lines. In LNCaP cells, M4N was found to reduce cellular ATP content, and suppress NDUFS1 expression while inducing hyperpolarization of mitochondrial membrane potential. M4N-treated cells lacked autophagy with reduced expression of BNIP3 and ATG5. To understand the mechanisms of this anticancer activity of M4N, the effect of this drug on three cancer cell lines (LNCaP, AsPC-1, and L428 cells) was further examined via transcriptome and metabolomics analyses. Metabolomic results showed that there were reductions of 26 metabolites essential for energy generation and/or production of cellular components in common with these three cell lines following 8 hours of M4N treatment. For instance, among the amino acids, the content of aspartate, a key metabolite in electron transport system in the mitochondria, was specifically suppressed by M4N. Deep RNA sequencing analysis demonstrated that there were sixteen genes whose expressions were found to be modulated following 6 hours of M4N treatment similarly in these three cell lines. Six out of these 16 genes were functionally related to the 26 metabolites described above. One of these up-regulated genes encodes for CHAC1, a key enzyme affecting the stress pathways through its degradation of glutathione. In fact M4N was found to suppress glutathione content and induce reactive oxygen species production. The data overall indicate that M4N has profound specific negative impacts on a wide range of cancer metabolisms supporting the use of M4N combination for cancer treatments.

#2515

Understanding metabolic dysfunction in retinoblastoma development.

Nilanjan Guha,1 Carolina Livi,2 Vishnu Suresh Babu,3 Lisa Winer,2 Deepak SA,1 Syed Lateef,1 Seetaraman Gundimeda,1 Arunkumar Padmanabhan,1 Brian Dranka,2 Arkasubhra Ghosh3. 1 _Agilent Technologies, Bangalore, India;_ 2 _Agilent Technologies, Santa Clara, CA;_ 3 _Narayana Nethralaya, India_.

Metabolic dysfunction or reprogramming is the underlying cause of many diseases including cancer. In this study, we looked into the transcriptomic and metabolomic profile of retinoblastoma (Rb), a pediatric eye cancer, from tumor and normal tissue, aqueous humor, vitreous humor and tear. The samples were collected from enucleated eyes of 9 subjects and 2 deceased controls, whose cause of death is not due to any eye related disease. The study was corroborated with cell line models of retinoblastoma, used for metabolic phenotyping. Combined pathway analysis of transcriptomics data from subject tissue samples and retinoblastoma cell line WERI-Rb-1 revealed a significant overlap in the differentially regulated genes belonging to both glycolysis and OXPHOS pathways. The results were correlated with Rb1 overexpressed and its respective control also. To better understand the implications of these two major pathways in the progression of retinoblastoma, metabolic study using Seahorse analyzer enables measurement of extracellular flux (oxygen consumption rate and extracellular acidification rate) to provide insight into mitochondrial function and glycolytic activity. Using a pharmacological approach, the contribution of glucose, glutamine and fatty acid oxidation pathways can be assessed. Overall the study demonstrates the value of using a multi-omics approach with functional metabolic profiling to understand the extent of metabolic dysfunction in retinoblastoma and also to identify implications of various biological pathways in Rb.

#2516

Pokemon functions as a metabolic oncoprotein integrating glucose & lipid metabolism in hepatocellular carcinoma.

Yibao Jin,1 Shangfu Li,2 Deliang Cao,3 Dan Gao,2 Hongxia Liu,2 Feng Liu,2 Tiejie Wang,1 Yuyang Jiang4. 1 _Shenzhen Institute for Drug Control, Shenzhen, China;_ 2 _The State Key Laboratory Breeding Base-Shenzhen Key Laboratory of Chemical Biology, Tsinghua University, Shenzhen, China;_ 3 _Southern Illinois University School of Medicine, Springfield, IL;_ 4 _Department of Pharmacology and Pharmaceutical Sciences, School of Medicine, Tsinghua University, Shenzhen, China_.

Pokemon (also known as FBI-1 or LRF) is a proto-oncogene encoded by ZBTB7A gene. It functions as a transcription factor, playing important roles in embryonic development, cell differentiation and neoplasia. Pokemon is abnormally expressed in hepatocellular carcinoma (HCC) and may contribute to the metabolic homeostasis of HCC cells, but the mechanism of action remains to be fully understood. This study demonstrated that Pokemon may integrate glycolysis and lipid metabolism in hepatocellular carcinogenesis. Our data showed that Pokemon was upregulated in HCC cell lines and clinical samples. Ectopic expression of Pokemon stimulated HCC cell growth and proliferation in culture and tumorigenesis in nude mice. Lipid metabolomic analysis by LC/MS exhibited that in HCC cells lipogenesis was positively correlated with Pokemon expression levels, and targeted expression of Pokemon in HCC cells promoted the flux of radiolabeled glucose to lipids, including membrane phospholipids, through the aerobic glycolysis-acetyl-CoA metabolic stream. Mechanistic studies revealed that Pokemon stimulated glycolysis through activation of PKA and its mediated Raf/c-Myc pathway. In the tested HCC cells, Pokemon activated fatty acid synthase (FASN) and suppressed AMPK, an inhibitor of acetyl-CoA carboxylase α (ACC-α), stimulating fatty acid and lipid synthesis. Together our results suggest that Pokemon correlates glycolysis and lipid synthesis and thus promotes lipogenesis and hepatocellular tumorigenesis.

#2517

Prognostic biomarkers of PDAC - a cross-validation study.

Hung-Jen Wu,1 Smrithi S. Menon,1 Keith Unger,2 Massimo S. Fiandaca,3 Yassi Fallah,1 Mark Mapstone,3 Howard J. Federoff,3 Amrita K. Cheema1. 1 _Georgetown University, Washington, DC;_ 2 _Med-Star Georgetown University Hospital, Washington, DC;_ 3 _UCI, Irvine, CA_.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) represents 90% of pancreatic neoplasms and the fourth leading cause of cancer death in the United States. Recently, several research groups have focused on conducting metabolomics based clinical investigations to identify metabolite markers of PDAC. Since a central biomarker repository for PA is lacking, however, it remains challenging to delineate the total number of metabolites reported, and importantly, which bio-signatures overlap between the disparate studies.

Approach:

We performed an extensive literature search to delineate PDAC case-control studies reporting dysregulated metabolite biomarkers for PDAC utilizing three main sources: PubMed, EDRN and GDOC. We refined our search to 36 publications that reported blood based metabolomics/lipidomic biomarkers. Data from these studies was compiled based on up/down-regulation of specific metabolites in PDAC, study sample size, and other clinical determinants. The final list consisted of 12 metabolites (including amino acids, fatty acids and small organic acids), that were reported by two or more research groups as being dysregulated in PDAC. Of note, these 36 disparate studies were carried out using different methodologies, analyzed on diverse analytical platforms, and utilized different statistical and bioinformatics methodologies. Resultant common features, therefore, were deemed significant as candidate biomarkers for cross-validation.

Next, we used stable isotope dilution multiple-reaction monitoring mass spectrometry (SID-MRM-MS) for targeted quantitation of these 12 metabolite markers in plasma samples obtained from patients that were diagnosed with PDAC and compared the same profiles to matched normal controls.

Results: We delineated of a sub-set of the original 12 metabolites that showed concordance with those reported in the literature. Statistical analyses to determine the sensitivity and specificity of the resultant panel, and any influence provided by age, gender, and type 2 diabetes, on the predictive performance of the biomarker panel are ongoing. These data will be presented at the AACR meeting.

Conclusion: Developing a specific and sensitive panel of blood-derived biomarkers offers a practical approach towards screening and allows increasing overall survival rates for PDAC with early diagnosis. Such disease-specific bio-signatures allow identification of molecular targets for therapeutic development; improve early treatment strategies and thereby clinical outcomes. Creating a compendium of existing biomarker data and performing cross-validation studies represent the first steps for developing clinical assays for the diagnosis and prognosis determination of PDAC. The integration of data generated from multiple analytic platforms and diverse subject cohorts is likely to help identify robust bio-signatures that would then be ready for large scale validation studies.

Supported by American Cancer Society

#2518

Accumulation of a liver- and microbiome-derived metabolite in human breast tumors is associated with patient survival.

Wei Tang,1 Tiffany Dorsey,1 Vasanta Putluri,2 Nagireddy Putluri,2 Stefan Ambs1. 1 _NCI-CCR, Bethesda, MD;_ 2 _Baylor College of Medicine, Houston, TX_.

Breast cancer is the most common cancer in women in the United States. Gene expression profiling studies of breast tumors led to the discovery of disease subtypes with different biologies. These studies also described novel biomarkers for therapy response and disease survival. However, it remains a challenge to define breast cancer biology solely based on gene expression. Recently, metabolomics emerged as a new discovery tool with the promise of identifying prognostic biomarkers and targetable metabolic dependencies of cancer cells. We previously measured the abundance of 536 metabolites in 67 breast tumor and their tumor adjacent noncancerous tissue by untargeted mass spectrometry. In the current study, we explored the prognostic power of the metabolome and conducted an integrated analysis comprising of the metabolome, transcriptome, and proteome to explore the association of metabolites with cell systems, tumor biology, and disease outcome. We built a predictive model based on multivariable Cox proportional hazards using the L1 penalized log partial likelihood (LASSO) method after pre-selecting prognostic metabolites following cross-validation with 1000 iterations. The median C-index was 0.73, indicating a certain robustness of metabolites as classifiers of breast cancer outcome. The models identified five metabolites including a bile acid-related metabolite, glycochenodeoxycholate (GCDC), as the most frequently selected features and outcome markers among these metabolites. An increased GCDC tumor content was associated with improved patient survival. We corroborated the presence of GCDC and three other bile acids in the human breast tissues using absolute measurements and confirmed their occurrence in breast tumors. Because additional large-scale transcriptome and proteome data were available for the same tissue samples, we further characterized the tumors based on their GCDC abundance, which showed that cell cycle-related pathways were enriched for differently expressed genes and tumors with a high GCDC content tended to have a low cell proliferation score, indicating that accumulation of GCDC leads to growth inhibition. We also conducted a correlation analysis for the relationship between GCDC abundance with other metabolites and identified 51 metabolites that were significantly correlated with GCDC. In this analysis, metabolite abundance in the sterol/steroid pathway associated most strongly with the GCDC tissue content. Lastly, we evaluated the effect of bile acids on breast cancer cell lines. The data showed a strong reduction of cancer cell proliferation under bile acid treatment, consistent with the tumor data, and distinct changes in gene expression. In conclusion, we propose that integrated metabolomics can provide powerful prognostic information with GCDC being a novel prognostic marker in breast cancer because accumulation of GCDC reduces cancer cell proliferation.

#2519

Analysis of gene expression patterns and metabolomics correlated to obesity, diabetes, and outcomes in patients with epithelial ovarian cancer.

Allison M. Montgomery,1 Angelina I. Londono,1 Eric R. Craig,1 Cindy Tawfik,1 Haller J. Smith,1 Charles A. Leath,1 Ashwini A. Katre,1 Sara J. Cooper,2 Rebecca C. Arend1. 1 _University of Alabama at Birmingham, Birmingham, AL;_ 2 _HudsonAlpha Institute for Biotechnology, Huntsville, AL_.

Objective: Diabetes and obesity have been associated with a poor prognosis in ovarian cancer (OVCA); the exact mechanism has yet to be determined. Data suggests that obesity diminishes normal immunological response and better prognoses in non-obese patients may be attributable to an intact immune response. The objective of this study was to analyze the correlation between gene expression pattern/metabolomics and obesity/diabetes in OVCA patients..

Methods: Following IRB approval, UAB patients with suspected OVCA undergoing surgery were consented. Tissue was collected during cytoreduction and 35 samples were analyzed using RNAseq technology and mass spectrometry-based metabolomics. DESeq2 was used for RNAseq analysis to identify gene expression differences between diabetics and non-diabetics stratified by BMI: obese (BMI ≥30) and non-obese (BMI <30). Gene set enrichment analysis was conducted to determine whether there was an over-representation of immune pathways among altered genes. Profiles were normalized in the analysis of the metabolite profiles using ChromaTOF.

Results: 76 genes (p-value <0.05) were differentially expressed in the tumor samples from patients with BMI ≥30 to those <30. These genes were highly enriched for immune-related genes, including 34 immunoglobulin genes, and complement activation. The list of identified genes also included 3 HLA genes (HLA-G(down), HLA-H(up) and HLA-DMA(down). Furthermore, when analyzing tumor from diabetics (n=7), there were 18 genes differentially expressed compared to controls. These genes are not statistically enriched for any functional class. Additionally, genes associated with response to platinum-based therapy, differentiating patients with BMI≥30 v. <30 were analyzed. No genes met genome-wide significance; however, there were 14 genes with a genome-wide p-value<0.1. When diabetes status as a covariate was controlled for, this number was reduced to 9.

Conclusion: By evaluating the transcriptomic profiles generated through RNAseq analysis, a significant number of differences in RNA expression were identified in comparing obese to non-obese OVCA patients. Due to the small sample size, no genes were identied as being associated with the presence or absence of diabetes.

#2520

Differential metabolite requirements in breast cancer cells correlate with ER/PR status and doubling time.

Vijesh J. Bhute, Kylie R. Knutson, Yan Ma, Sean P. Palecek. _University of Wisconsin-Madison, Madison, WI_.

Metabolism in cancer cells is significantly altered as compared to their normal counterparts. Metabolic rewiring can assist cancer cells to meet their requirements for uncontrolled proliferation and survival. In addition to their role in protein synthesis and energy generation, metabolites also act as signaling molecules, antioxidants and perform osmoregulatory functions. Differences in genetic makeup and phenotype of cells can lead to differences in these metabolic needs. A detailed analysis of metabolic signatures of different cancer cells can therefore prove useful in understanding metabolic interactions with the phenotype in cancer cells. This study aims at identifying these metabolic differences among distinct breast cancer cell lines and gaining mechanistic insights into possible reasons for differential abundance of metabolites. We used nuclear magnetic resonance (NMR) spectroscopy to quantify intracellular metabolites in five breast cancer cell lines having distinct genetic and phenotypic composition. We show that the intracellular abundance of essential amino acids depends on cell type and medium composition. We also show that myo-inositol abundance was significantly higher in ER-/PR- than ER+/PR+ breast cancer cells due to increased expression of inositol-3-phosphate synthase 1 expression. Myo-inositol is an osmolyte and also a precursor of inositol phosphates which participate in PI3K and calcium signaling pathways. Although the reason ER-/PR- cells contain greater concentrations of myo-inositol than ER+/PR+ cells is not yet known, increased myo-inositol abundance could potentially serve as a marker of ER-/PR- breast cancer cells. We also observed significant changes in phospholipid metabolism, specifically, glycerophosohocholine (GPC) concentration correlated with doubling time. We also found that reducing serum concentration in the growth medium reduced the growth rate of MCF7 cells, which led to increased GPC abundance in the cells. GPC is also an intracellular osmolyte and is a degradation product of phosphatidylcholine (PtdCho). Phospholipid metabolism has been shown to be significantly altered in breast cancer cells and has been used as prognostic marker in breast cancer cells. A positive correlation of doubling time with GPC irrespective of ER/PR status may prove useful in deciding therapeutic options. Our results highlight the differences in metabolic requirements in different breast cancer cell lines and suggest potential for gaining mechanistic understanding of these differences using metabolomics.

#2521

Inhibition of metabolic reprogramming by zerumbone alters the tumorigenic potential of hepatocellular cancer.

Nissar A. Wani, Bo Zhang, Kun-yu Teng, Juan Barajas, Kalpana Ghoshal, Rafael Brüschweiler, Samson T. Jacob. _Ohio State Univ., Columbus, OH_.

Incidence of hepatocellular carcinoma (HCC) that represents ~90% of all cases of primary liver cancer is on the rise worldwide. Sorafenib, the only approved targeted therapy, prolongs median survival only by ∼3 months. Thus, new therapeutic strategies are urgently needed. In this study, we investigated therapeutic potential of zerumbone, a sesquiterpene from an edible ginger, against HCC. Zerumbone inhibited proliferation and clonogenic survival of HCC cells by blocking glycolysis and pentose phosphate pathway, arresting cells at G2/M phase of cell cycle and inducing apoptosis. To uncover the underlying molecular mechanisms of its action on HCC cells, we employed several unbiased approaches. First, phosphokinase array showed significant inhibition PI3K/AKT/mTOR and STAT3 signaling in zerumbone-treated HCC cells. Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) of the microarray data revealed that zerumbone-deregulated genes showed positive correlation with PI3/AKT/mTOR and STAT3 pathways and those involved in metabolism, apoptosis and cell cycle. Notably, the alteration in expression of genes regulated by PI3/AKT/mTOR or STAT3 pathways and downregulated by zerumbone in HCCs is associated with poor patient survival. These results implicate that treatment with this dietary sesquiterpene might lead to a better outcome in HCC patients. Indeed, zerumbone treatment significantly inhibited growth and lung metastasis of orthotopic HCC xenografts in NSG mice. These findings reveal novel therapeutic strategies targeting cancer metabolism. Collectively, these observations underscore the therapeutic potential of zerumbone in HCC. 

### MicroRNAs and Other Noncoding RNAs as Tumor Suppressors or Oncogenes 1

#2522

MiRNA-652 induces neuroendocrine-like differentiation in LNCaP prostate cancer cells through decreased PP2A function.

Tania Benatar, Yutaka Amemiya, Christopher J. Wallis, Linda Sugar, Christopher Sherman, Robert Nam, Arun K. Seth. _Sunnybrook Research Institute, Toronto, Ontario, Canada_.

Background: MicroRNA (miRNA) dysregulation has been shown to contribute to prostate cancer progression. Recently, we identified a panel of five miRNAs associated with prostate cancer recurrence and metastasis using next generation miRNA sequencing. Here, we examine the clinical implications of dysregulation of miR-652 and its biological mechanism.

Methods: Using a cohort of 585 patients treated with radical prostatectomy, we examined the prognostic significance of miR-652 using the Kaplan Meier method and Cox proportional hazard models. We examined miR-652 expression in prostate cancer cells and created cell lines that overexpressed miR-652 for functional assays. Finally, we examined pathways through which miR-652 may function using prediction algorithms, and confirmed by Western blotting, IHC and luciferase reporter assays.

Results: Patients overexpressing miR-652 had significantly increased rates of biochemical recurrence (p<0.0001). In multivariable models adjusting for known clinical prognostic factors, patients with high miR-652 expression had an increased risk of biochemical recurrence (HR 1.47, 95% CI 1.09-1.98). Overexpression of miR-652 in PC3 and LNCaP cells resulted in increased growth, migration and invasion. Prostate cancer cell xenografts overexpressing miR-652 had increased tumorigenicity and metastases. Using the miRNA target prediction program miRanda, we identified the B" regulatory subunit, PPP2R3A, of the tumor suppressor PP2A as a potential target of miR-652. PC3 and LNCaP cells transfected with miR-652 mimic dowregulated the PR72 isoform of PPP2R3A. Mutation of the miR-652 binding site in the PPP2R3A gene negated this effect. Western blotting demonstrated that mir-652 induced PPR2R3A inhibition induced epithelial-mesenchymal transition (EMT) in PC3 cells. Prolonged miR-652 expression in LNCaP cells induced a neurite-like morphology, suggesting neurodocrine-like differentiation (NED). Western blotting of LNCaP cells after prolonged miR-652 exposure, resulted in increased phospho-AKT and phospho-β-catenin (phosphorylated at Serine 552), reduced AR expression, and upregulation of the NED marker, neuron specific enolase (NSE), consistent with NED. Expression of NED markers, chromogranin A, NSE, and synaptophysin were also observed in a xenografted tumor derived from LNCaP-652 overexpressing cells.

Conclusion: These observations suggest that increased levels of miR-652 found in prostate cancer may contribute to tumor progression by promoting NED, tumor cell survival and cell migration/invasion through decreased PP2A function which may provide an opportunity for novel therapy in prostate cancer.

#2523

Key cluster for prostate cancer diagnosis and therapy; miR-888 cluster.

Tsuyoshi Hasegawa, Aurora Kerscher. _Eastern Virginia Medical School, Norfolk, VA_.

Prostate cancer (PCa) is the 2nd leading cause of cancer death among men in the U.S. No accurate biomarkers exist and therapeutic options are limited for advanced PCa. MicroRNAs (miRNAs), small non-coding RNAs, act as negative regulators of gene expression. MiRNAs have recently emerged as promising therapeutic tools for cancers. It is poorly understood how they function to promote cancer progression. We found miR-888 was involved with PCa progression. miR-888 belongs to a genomic cluster on human chromosome Xq27.3, linked to hereditary PCa. I hypothesized additional members of this cluster (miR-892c, -890, -892a, -892b, -891b, -891a) function to promote PCa progression. Our lab profiled miR-888 cluster expression in paired syngenic human PCa cell lines (RWPE1 & WPE1-NB26, LNCaP & C4-2, PC3-N & PC3-ML) differing in metastatic status and androgen response. We noted that the miR-888 cluster was preferentially elevated in metastatic PC3-ML and under-expressed in non-aggressive PC3-N cells. I examined miR-888 cluster function in vitro and found that forced expression (via miRNA mimics) of certain miR-888 cluster members, notably miR-888 and miR-891a, promoted cell proliferation, colony formation, migration and invasion in PC3-N cells. Conversely, other cluster members inhibited these activities. Experiments with hormone sensitive LNCaP cells showed similar results in these assays. PC3-ML transfected with miRNA inhibitors exhibited reciprocal effects. In mouse xenograft studies, I used lentiviral vectors to individually overexpress miR-888 and miR-891a in PC3-N cells and injected them subcutaneously into flanks of NOD/SCID mice. The miRNAs accelerated tumor formation and increased tumor size compared to controls. miR-888 may promote tumorigenesis by suppressing the tumor suppressors TIMP2 and SMAD4, which our lab validated as miR-888 targets using luciferase reporter assays. I confirmed by western blot that xenograft tumors overexpressing miR-888 had decreased protein levels for these targets. Studies reveal miRNAs exist in exosomes, small membrane bound vesicles involved in cell-to-cell communication. I postulated that aggressive tumors secrete exosomes with the miR-888 cluster as cargo and promote PCa progression. Indeed, I noted that miR-888 cluster expression is elevated in exosomes isolated from aggressive PC3-ML compared to PC3-N cells as well as in EPS urine exosomes from high-grade PCa patients versus those with low-grade disease. My work indicates miR-888 and miR-891a are novel oncogenic factors in the prostate and may use exosomes to exert their effects. This is the first report to explore the functional role of the miR-888 cluster in any tissue and may lead to promising clinical strategies for PCa.

#2524

**MiR-124 suppresses p62 and p65/NFkB to regulate autophagy, inflammation and cell death in** KRAS **mutant mesenchymal NSCLC cells.**

Anita K. Mehta,1 Kevin Hua,1 William Whipple,1 Mihn-Thuy Nguyen,1 Rushika M. Perera,2 Johanns Haybaeck,3 Joanne Weidhass,4 Jeffrey Settleman,5 Anurag Singh1. 1 _Boston University School of Medicine, Boston, MA;_ 2 _University of California at San Francisco, San Francisco, CA;_ 3 _Medical University of Graz, Graz, Austria;_ 4 _University of California at Los Angeles School of Medicine, Los Angeles, CA;_ 5 _Calico Life Sciences, South San Francisco, CA_.

Background KRAS mutant non-small cell lung cancers (NSCLC) are molecularly and histologically diverse. Epithelial-like cells are more KRAS dependent, whereas mesenchymal-like cells are less KRAS dependent. These two subtypes are designated KE (epithelial) and KM (mesenchymal), respectively. A KE versus KM subtype transcriptional signature reveals specific modes of KRAS dependent survival signaling in the KE subtype. This KRAS dependency signature is significantly enriched with predicted microRNA (miRNA) target genes of miR-205 and miR-34b/c. MiRNAs can function as tumor suppressors by coordinately regulating multiple oncogenic signaling pathways. The role of deregulated miRNA function in mediating the survival of KM versus KE NSCLC cells has not been investigated to date.

Methods Differential miRNA expression in KRAS mutant cell lines was determined using Taqman low-density qPCR arrays (TLDA). Functional miRNA reconstitution experiments of downregulated miRNAs were performed in a panel of KM cell lines. Effects on apoptosis and autophagy were performed by Western blotting, immunofluorescence and live cell microscopy and caspase assays. The molecular targets of miR-124 were computationally identified by TargetScan or miRWalk and experimentally verified using 3'UTR luciferase-based assays. Functional rescue of miRNA-dependent cell viability defects was determined by ectopic predicted target gene expression.

Results Comparison of KE to KM cells yielded a KE-KM miRNA subtype classifier/signature. This signature revealed a number of silenced or suppressed miRNAs in KM cell lines, including members of the miR-200 family. MiR-200 and miR-205 reconstitution in KM cells modulated epithelial plasticity by Zeb1 protein suppression and increased E-cadherin levels. Reconstitution of miR-124, miR-625 and miR-518-3p in KM cells caused pronounced loss of cell viability. Furthermore, miR-124 caused autolysosome maturation defects. We identified SQSTM1/p62, TRAF6 and RELA/p65 as key predicted targets of miR-124. MiR-124 reconstitution in KM cells caused decreased p62, TRAF6 and p65 protein levels. The effect of miR-124 on p62 expression was verified using a Luciferase-p62-3'UTR reporter construct. Overexpression of p62 in KM cells rescued the cell viability defects caused by miR-124.

Conclusion These studies implicate miR-124 as a context-dependent tumor suppressor miRNA in KM subtype cells. MiR-124 directly suppresses expression of SQSTM1/p62 to promote defects in autolysosome maturation. In parallel, miR-124 suppresses RELA/ p65 and in some cases, TRAF6, to alter expression levels of several cytokines. Thus, miR-124 coordinately regulates autophagy and inflammation to disrupt the finely-tuned balance between pro and anti-inflammatory signals, resulting in cytotoxic effects in a specific subtype of mesenchymal-like KRAS mutant NSCLC cells.

#2525

Targeting myeloma cell-derived runx2 by miRNAs suppresses multiple myeloma growth and progression.

Pramod S. Gowda, Mohammad Q. Hassan, Timothy N. Trotter, Yang Yang. _University of Alabama at Birmingham, Birmingham, AL_.

In Multiple myeloma (MM), abnormal plasma cells accumulate in the bone marrow and spread to new bone sites. However, the mechanisms underlying this spread of MM cells remain unclear. Runx2 is a bone specific transcription factor upregulated in various human tumors, including MM. Our studies have previously demonstrated that Runx2 is a major driver of MM progression in bone. In the present study, our goal was to identify miRNAs targeting Runx2 to reduce tumor growth and dissemination of MM to new bone sites and asses their therapeutic potential. Expression analysis of a panel of miRNA's regulating Runx2 revealed an inverse relationship between Runx2 expression and two miRNAs: miR-342 and miR-363. miRNA expression analysis using the Gene Expression Omnibus database showed that miR-342 and miR-363 are highly expressed in plasma cells of normal donors (n= 3), while Runx2 is detected at very low levels. In contrast, MM cells of patients with newly diagnosed (n=23) and relapsed MM (n=17) expressed very little miR-342 and miR-363 but showed high levels of Runx2 expression. Reconstituting CAG human MM cells with synthetic miR-342, miR-363 or miR-342+miR-363 in combination reduced the expression of Runx2 and the metastasis-promoting Runx2 target genes RANKL, DKK1 and DMP1. Tumor cell viability and migration were also decreased compared to control in vitro. We then transfected miR-342, miR-363 or miR-342+miR-363 in mouse 5TGM1 MM cells expressing high levels of Runx2 and tested bone-homing and growth in syngenic C57Bl/KaLwRij mice by intravenous injection. Mouse sera were collected at week 2 and 4 after tumor cell injection and the levels of IgG2bκ, a marker of 5TGM1 cells, were measured by ELISA. The results showed significantly decreased IgG2bκ levels in mice bearing miR-342 or miR-363 transfected tumors compared to mice bearing control miR-scramble tumors. Additionally, miR-342+miR-363 co-transfection produced a synergistic effect on reducing tumor growth. Mechanistic studies demonstrated that miR-342 and miR-363 induced Runx2 suppression, results in inhibition of Runx2-downstream signaling pathways Akt/β-catenin/survivin, which are required for MM tumor progression. In conclusion, we have identified two novel miRNAs, miR-342 and miR-363, that negatively regulate Runx2 expression in MM cells. We further demonstrated that enhanced expression of miR-342 or miR-363 in MM cells inhibits MM growth in vivo. Thus, miR-342 and miR-363 are potential novel markers of MM prognosis and can be developed as new therapeutic drugs for MM treatment.

#2526

**MicroRNA expression signature of patients with tyrosine kinase inhibitors failure:** miR-10a-5p **inhibits cancer cell aggressiveness in renal cell carcinoma.**

Takayuki Arai,1 Atsushi Okato,1 Akira Kurozumi,1 Mayuko Kato,1 Yusuke Goto,1 Keiichi Koshizuka,1 Satoko Kojima,2 Yukio Naya,2 Tomohiko Ichikawa,1 Naohiko Seki1. 1 _Graduate School of Medicine, Chiba University, Chiba, Japan;_ 2 _Teikyo University Chiba Medical Center, Ichihara, Japan_.

Renal cell carcinoma (RCC) is a disease in which cells undergo oncogenic transformation in the kidney tubules. The five-year survival rate of advanced stage RCC is poor (5-10%) due to recurrence or distant metastasis. Recently, anti-angiogenic multi-tyrosine kinase inhibitors (TKIs) have been developed and have been used as first and second line treatments for RCC. However, these treatments extend progression-free survival only slightly, and relapse and metastasis eventually develop in most patients. The molecular mechanisms of RCC recurrence, metastasis and drug resistance are not yet fully understood. Therefore, analysis of the molecular mechanisms underlying RCC development and progression and studies of novel oncogenic pathways based on current genome-based approaches could significantly improve diagnosis, therapy, and prevention of the disease. miRNAs (miRNAs) are small noncoding RNAs that function to fine tune the expression of protein coding/noncoding RNAs by repressing translation or cleaving RNA transcripts in a sequence-depending manner. The unique characteristic function of miRNAs is to regulate RNA transcripts in human cells. Therefore, dysregulated expression of miRNAs can disrupt tightly regulated RNA networks in cancer cells. In this study, we constructed a miRNA expression signature to identify pathways activated by TKI treatment using autopsy specimens from patients with RCC. We have sequentially identified tumor-suppressive miRNA and its regulated RCC pathways based on the signature. The aim of this study was to investigate the functional significance of miR-10a-5p and to identify the molecular targets and pathways mediated by miR-10a-5p in RCC cells. The expression levels of miR-10a-5p were significantly reduced in RCC clinical specimens and RCC cell lines compared with non-cancerous kidney tissues (P < 0.001). TCGA data showed that the overall survival of low miR-10a-5p expression group was significantly shorter than that of high expression group (P = 0.00408). Restoration of miR-10a-5p significantly inhibited cancer cell migration and invasion in RCC cell lines (P < 0.0001). Spindle and kinetochore associated complex subunit 1 (SKA1) was identified as a direct target gene of miR-10a-5p by genome-wide gene expression analysis and in silico analysis. Overexpression of SKA1 was observed in RCC clinical specimens. Moreover, the overall survival of high SKA1 expression group was significantly shorter than that of low expression group by TCGA analysis (P = 1.44E-07). Tumor-suppressive miR-10a-5p was identified by using miRNA signature of patients with TKI failure. Overexpression of SKA1 might be involved in RCC aggressiveness, metastasis and drug resistance. Elucidation of tumor-suppressive miRNAs regulated molecular pathways and targets could provide new information on potential therapeutic strategies in the disease.

#2527

The role of miR-601 in prostate cancer progression.

Jessica L. Fleming, Erica H. Bell, Kathryn Andrews, Arnab Chakravarti. _The Ohio State University, Columbus, OH_.

Prostate cancer (PCa) is the second most common cancer among men worldwide. In order to advance treatment options for these men, it is crucial to understand the molecular underpinnings behind this cancer. Previously, our group identified miR-601 as a biomarker of significance in PCa. Few studies to date have functionally validated molecular biomarkers and currently little is known about the function of miR-601 in PCa. Based on our previous publication, we hypothesized that miR-601 plays a role in PCa progression and radiation response. To test this hypothesis, miR-601 was over-expressed and knocked down in DU145 and PC3 cell lines and assays were performed evaluating cell proliferation, colony formation, apoptosis, and cell cycle progression. Additionally, to provide more information regarding miR-601-associated pathways, we identified putative gene targets of miR-601 using in silico prediction programs, microrna.org and TargetScan.org and evaluated top gene targets in vitro. To date, our data suggest that miR-601 may be playing a role as a tumor suppressor. Over-expression of miR-601 in cell lines resulted in a significant reduction in cell viability. This was confirmed both by MTS as well as colony formation assays. We looked into the mechanism behind the reduction in cell viability by testing the effect of miR-601 on apoptosis and cell cycle progression. We found that cells over-expressing miR-601 had slightly higher levels of apoptosis, however miR-601 did not induce a substantial effect on cell cycle progression. Additionally, we found that miR-601 enhances radiosensitization in DU145 and PC-3 cell lines. Similar results have been observed in breast cancer 1 as well as in a large miRNA screen performed in the LNCaP cell line2. Two putative gene targets of miR-601 were identified and investigated in vitro, SIRT1, a histone deacetylase known to be both an oncogene and tumor suppressor, and BCL2L2, an anti-apoptotic gene known to be an oncogene. SIRT1 and BCL2L2 had strong scores on both online prediction programs as likely targets of miR-601. Our in vitro results confirmed this. We saw reduced mRNA and protein expression of these targets in cells over-expressing miR-601. Our data thus far suggest that miR-601 is acting as a tumor suppressor in PCa. Targeted therapies for miR-601 and/or its targets may be promising in the treatment of PCa, however additional work is needed to validate this. Future work will focus on the role of miR-601 in cell migration and invasion as well on identifying the direct molecular mechanism(s) by which miR-601 is reducing cell viability and conveying radiation sensitivity.

Funding: R01CA108633 (To AC), 1RC2CA148190 (To AC) U10CA180850-01 (To AC), 1R01CA169368 (To AC) from the NCI, Brain Tumor Funders Collaborative Grant (To AC), OSUCCC Award (To AC).

1. Hu JY et al. (2016) Biomedicine and Pharmacotherapy. 79: 247-53

2. Hatano K. et al. (2015) Nucleic Acids Research. 43: 4075-86

#2528

**miR-452 inhibits migration and invasion of prostate cancer cells by targeting E3 ubiquitin ligase-1 (** WWP1 **).**

Satoko Kojima,1 Yusuke Goto,2 Akira Kurozumi,2 Mayuko Kato,2 Atsushi Okato,2 Takayuki Arai,2 Tomohiko Ichikawa,2 Yukio Naya,1 Naohiko Seki2. 1 _Teikyo University Chiba Medical Center, Ichihara, Japan;_ 2 _Chiba University Graduate School of Medicine, Chiba, Japan_.

Introduction: Most prostate cancer (PCa) patients initially respond to androgen-deprivation therapy (ADT), but eventually acquire resistance to ADT and progress to castration-resistant prostate cancer (CRPC), resulting in metastasis causing PCa death. We have developed the microRNA (miRNA) expression signature of PCa using clinical specimens and determined the target genes of the tumor suppressive miRNAs, whose expressions are significantly decreased in PCa specimens. Based on the signature, our previous study revealed that miR-224 functions as a tumor suppressor, especially contributes to cancer cell metastasis directly targeting miR-224-TPD52 signaling. In human genome, miR-452 is located nearby tumor-suppressive miR-224, forming clustered miRNAs on chromosome Xq28 region. The aim of the study was to investigate the functional significance of miR-452 and to identify novel miR-452-mediated cancer pathways and responsible genes in PCa cells.

Material and methods: Clinical prostate specimens were obtained from patients admitted to the Teikyo University Chiba Medical Center from 2008 to 2013. Ninety-two patients with elevated PSA levels underwent transrectal prostate needle biopsy, and three patients who died of CRPC underwent autopsies. PCa tissues (n=54), noncancerous prostate tissues (non-PCa, n=36), were used for analysis of expression levels of miRNAs. Functional studies of differentially expressed miRNAs were analyzed using PC3 and DU145 cells. The influence on CRPC-free survival of expression levels of miRNA was estimated using the Kaplan-Meier methods. In silico database and genome-wide gene expression analyses were performed to identify molecular targets regulated by the miRNAs.

Results: (The miRNA expression signature of PCa specimens showed that the cluster miRNAs miR-224 was significantly downregulated, suggesting that this miRNA may act as tumor suppressor.) Clinical data using advanced PCa showed that low expression of miR-452 predicted a short duration of progression to CRPC. Restoration of miR-452 in PC3 and DU145 cells revealed significant inhibition of cancer cell migration and invasion. WW domain-containing E3 ubiquitin protein ligase-1 (WWP1) was confirmed as a direct target of miR-452 by in vitro transfection of miR-452 and Luciferase assay. Knockdown of the expression of WWP1 using siRNA showed inhibiting cell migration and invasion in PC3 and DU145 cells. Immunohistochemistry showed overexpressed WWP1 in advanced PCa.

Conclusions: Loss of the tumor suppressive miR-452 enhanced migration and invasion in PCa cells. Regulation of the miR-452-WWP1 axis contributed to PCa cell migration and invasion, and elucidation of downstream signaling of this axis will provide new insights into the mechanisms of (progression to CRPC) metastasis of PCa.

#2529

TTF-1/NKX2-1 induced miR-532-5p targets KRAS and MKL2 oncogenes and causes apoptosis in lung adenocarcinoma cells.

Sebastian Griesing,1 Taisuke Kajino,1 Mei Chee Tai,1 Zhuoran Liu,1 Masahiro Nakatochi,2 Motoshi Suzuki,1 Takashi Takahashi1. 1 _Nagoya University Graduate School of Medicine, Nagoya, Japan;_ 2 _Nagoya University Hospital, Nagoya, Japan_.

Our group and others have previously identified TTF-1/NKX2-1 as a lineage survival oncogene in lung adenocarcinoma. Subsequent studies revealed double-edged sword characteristics of TTF-1 in the development of lung adenocarcinoma. TTF-1 elicits lineage-survival signaling by inducing transcriptional targets such as ROR1, while TTF-1 also inhibits tumor progression by decreasing cell motility through direct activation of genes such as MYBPH. It is thus clear that a more comprehensive picture of this still enigmatic lineage-survival oncogene needs to be elucidated. Whereas most of the previous studies on TTF-1 have focused on its regulation of protein-coding genes, very little is known about regulation of microRNAs by TTF-1. In the present study, we combined in vivo patient data with in vitro data from lung adenocarcinoma cell lines, aiming at identifying miRNAs, which are transcriptionally regulated by TTF-1.

Our integrative approach identified miR-532-5p as a microRNA regulated by TTF-1. Expression of miR-532-5p decreased, when cell lines with a high endogenous level of TTF-1 were transfected with siRNA against TTF-1, while it was increased by overexpression of exogenous TTF-1 in TTF-1-negative cell lines. By ChIP and luciferase assays, TTF-1 was found to induce miR-532-5p expression through its binding to the MIR532 promoter region, identifying miR-532-5p as a direct target of TTF-1. We next employed microarray analysis and target prediction using TARGETSCAN to search for potential targets of miR-532-5p. We consequently identified KRAS and MKL2 as candidates and confirmed this by qRT-PCR, Western blot and luciferase analyses. Overexpression of miR-532-5p induced apoptosis and inhibited colony formation in lung adenocarcinoma cell lines, while depletion of MKL2 phenocopied the effects of miR-532-5p introduction, similarly exhibiting occurrence of marked apoptosis. In addition, miR-532-5p significantly decreased tumor formations in vivo in a mouse xenograft model. It was also of note that miR-532-5p inhibited lung adenocarcinoma cell lines regardless of the presence or absence of KRAS mutations.

In summary, we have found that TTF-1 transactivates miR-532-5p, which potently induces apoptosis in lung adenocarcinoma cells. MiR-532-5p directly targets MKL2, which we have also uncovered to be crucially involved in lung adenocarcinoma survival. In addition, while previous reports have shown that TTF-1 inhibits KRAS-driven tumorigenesis, this study identifies KRAS as a target of miR-532-5p, providing their potential mechanistic link. Our results thus shed light on how TTF-1 plays a tumor-suppressive role in the process of lung carcinogenesis, which might ultimately lead to development of a novel therapeutic strategy for this devastating disease.

#2530

Validation and pathway analysis of a metastasis-specific microRNA signature in primary colon cancer.

Robert R. Coebergh van den Braak, Anieta S. Sieuwerts, Zarina S. Lalmahomed, Marcel Smid, Vanja de Weerd, Michelle van der Vlugt - Daane, Anne van Galen, Shanshan Xiang, Katharina Biermann, John A. Foekens, John W. Martens, Jan N. IJzermans. _Erasmus University Medical Center, Rotterdam, Netherlands_.

Introduction: In lymph node negative (LNN) colon cancer 20% of the patients develops disease recurrence. Identification of these patients is needed. MicroRNAs (MiRNAs), a group of short non-coding RNAs, can function as tumor suppressors or oncogenes. Hur et al recently found 6 differentially expressed MiRNAs when comparing primary colorectal cancer and matched liver metastasis (MiR-320, MiR-221, MiR-30b, MiR-10b, MiR-885-5p, Let-7i). The expression of 2 MiRNAs was significantly correlated with distant metastasis (low Let-7i expression and high MiR-10b) in primary colorectal cancers. When split at the median and combined into a signature (Let-7i high and MiR-10b low [n=22] vs. Let-7i low and/or MiR-10b high[n=122]), the first group showed 100% metastasis free survival (MFS). We assessed the prognostic value of these MiRNAs and the signature in a clinically well-defined cohort of primary colon cancers.

Methods: Expression of the 6 MiRNAs were measured using RT-qPCR in a cohort of 232 colon cancer patients (n=155 untreated LNN and n=77 adjuvant treated lymph node positive [LNP] patients) selected from the MATCH-cohort. Expression levels were related to microsatellite instability (MSI), and MFS, hepatic metastasis free (HFS) and overall survival (OS).

Results: MiR-221, Mir-30b, Mir-10b and MiR-885-5p levels were significantly associated with MSI. In univariate Cox regression analysis, MiR-30b was significantly associated with MFS (HR=1.23 p=0.004), and MiR-30b and Let-7i were associated with HFS (HR=2.52 p=0.005 and HR=0.40 p<0.001, respectively) in the total group. In the LNN group results were similar for MiR-30b (MFS HR=2.07 p=0.008 and HFS HR=2.94 p=0.005) and Let-7i (HFS HR=0.28 p<0.001), but did not reach significance in the LNP group. These associations remained significant when correcting for MSI. Pathway analysis for MiR-30b and Let-7i with the GSEA hallmark gene sets between the 50 samples with the highest and 50 samples with lowest expression revealed a significantly higher expression of the TGF-beta pathway in the MiR-30b high group, and a significantly higher expression of the EMT pathway in the Let-7i high group. MiR-30b and Let-7i expression was split at the median level and combined into two groups ('Let-7i high and MiR-30b low' vs. 'Let-7i low and/or MiR-30b high'). The 'Let-7i high and MiR-30b low' group in the total group (n=74) had a significantly better 5-yr HFS (100% vs 87.4% p=0.002), and had a significantly better 5-yr MFS (91.7% vs 78% p=0.036) and HFS (100% vs 87.8% p=0.01) in the LNN group (n=52).

Conclusion: in our cohort and more specifically the LNN group, we confirmed Let-7i and identified MiR-30b as a prognostic factor for MFS and HFS. We did not confirm the prognostic value of Mir-10b. The combination of Let-7i and MiR-30b identified a group with a 100% HFS. Pathway analysis showed higher expression of the TGF-beta pathway in the MiR-30b high group, and higher expression of the EMT pathway in the Let-7i high group.

#2531

Regulation of androgen signaling axis and tumor suppressive function of miR-149-5p in prostate cancer.

Savita Singh,1 Jey Sabith Ebron,1 Eswar Shankar,2 Sanjay Gupta,2 Daniel Lindner,3 Girish Shukla1. 1 _Cleveland State University, Cleveland, OH;_ 2 _Case Western Reserve University & University Hospitals Case Medical Center, Cleveland, OH; _3 _Taussig Cancer Center, Learner Research Institute, Cleveland, OH_.

Prostate cancer growth and proliferation depends on androgen signaling mediated by transactivation of Androgen Receptor (AR). Androgen ablation remains the mainstay therapy for treatment of the disease. However, despite androgen ablation, the disease relapses to more aggressive form known as castration-resistant prostate cancer (CRPC). Androgen Signaling Inhibitor such as Abiraterone Acetate and Enzalutamide are the most effective treatment methods currently being used to treat CRPC. However, more than one-third of CRPC patients develop resistance to these treatments, mostly due to the gain of function in the AR protein and increase in intratumoral dihydrotestosterone (DHT) synthesis. Intratumoral DHT synthesis from steroid precursors in tumors is augmented by up-regulation of enzymes of cholestrogenesis and steroidogenesis, such as HMG-CoA reductase, SCARB1, and 17βHSD respectively. Tumor-specific downregulation of microRNAs which regulate the AR and steroid biosynthesis has been implicated in tumor growth and resistance to therapeutics in CRPC. We are focusing on tumor suppressive role of miR-149-5p in PCa. We discovered that miR-149-5p expression was significantly lower in prostate cancer tissues compared to normal tissues. The mechanistic investigation revealed that miR-149-5p downregulates wild type and alternatively spliced variant of AR. It also down regulates the expression of SCARB1, HMGCS and HMG-CoA reductase, the proteins known to facilitate intratumoral DHT synthesis. Ectopic expression of miR-149-5p negatively regulated the expression of prostate-specific antigen, a downstream target of androgen signaling and it also inhibited invasion and proliferation of CRPC cells. Our findings indicate a significant role of miR-149-5p in regulating androgen signaling and possibly DHT synthesis in CRPC. This provides a strong rationale for further investigating the significance of miR-149-5p for generation of new therapeutic for CRPC.

#2532

High expression of MiR-432-3p is associated with the chemoresistance by NRF2 stabilization via directly targeting KEAP1.

Burak Akdemir,1 Jun Inoue,1 Tatsuyuki Kawano,2 Johji Inazawa1. 1 _Department of Molecular Cytogenetics, Tokyo Medical and Dental University, Tokyo, Japan;_ 2 _Department of Esophageal and General Surgery, Tokyo Medical and Dental University, Tokyo, Japan_.

NF-E2-related factor 2 (NRF2) is a master transcriptional regulator that integrates cellular stress responses and is negatively regulated by Kelch-like ECH-associated protein 1 (KEAP1) at the post-translational level. In human cancers, aberrantly stabilized NRF2, either by mutation of NRF2 or KEAP1, plays a vital role in chemoresistance through the activation of target genes, suggesting that targeted inhibition of NRF2 is a potential therapy for NRF2-stabilized tumors. MicroRNAs (miRNA) are endogenous small noncoding RNAs that can negatively regulate gene expression by interfering with the translation or stability of target transcripts. Previously, we identified miRNAs negatively regulating NRF2 pathway via directly targeting NRF2 itself by a reporter-coupled miRNA library screening and demonstrated the potential of miRNA-based therapy against NRF2-stabilized tumors in esophageal squamous cell carcinoma (ESCC) (Mol. Cancer Res 2014, Cancer Res 2015).

In the present study, we further identified miR-432-3p as a miRNA positively regulating NRF2 pathway. MiR-432-3p could directly targeted KEAP1 via binding to its seed sequence within the cording region. Overexpression of miR-432-3p resulted in enhanced resistance to cisplatin via activation of NRF2 in ESCC cell lines. Furthermore, miR-432-3p was overexpressed in 55 of 84 primary ESCC tumors, compared with that of corresponding non-cancerous esophageal mucosa (65.5%). Importantly, we showed the negative correlation between the expression level of KEAP1 and miR-432-3p in primary ESCC tumors. Taken together, these findings suggest that the high expression of miR-432-3p may contribute to the chemoresistance by NRF2 stabilization via down-regulation of KEAP1 expression in ESCC.

#2533

Sox2 promotes cell proliferation of esophageal squamous cell carcinoma through the miR-17-92 cluster ,PTEN,AKT,mTORC1 pathway.

Kei Terasaki. _Kyoto Prefectural University of Medicine, Kyoto, Japan_.

SOX2, a member of the SOX family of transcription factors, play an important role of differentiation and morphogenesis of the esophagus. We previously revealed that SOX2 is the target of gene amplification at 3q26 in esophageal squamous cell carcinoma (ESCC) (Cancer Genet Cytogenet. 2010), and that SOX2 promotes ESCC cell proliferation in vitro and in vivo through the activation of the AKT/mTORC1 signaling pathway (Cancer Sci. 2013). However, the mechanisms by which SOX2 promotes AKT/mTORC1 signaling remain unknown. It is known that PTEN is a negative regulator of AKT and the PTEN gene is a target of the miR-17-92 cluster. In the present study, we showed that SOX2 up-regulated the expression of miR-17-92 cluster and repressed PTEN expression using RNA interference (RNAi)-mediated knockdown of SOX2 and enforced expression of SOX2. In summary, SOX2 promotes cell proliferation of ESCC through the miR-17-92 cluster /PTEN/AKT/mTORC1 pathway.

#2534

miRNA expression as potential biomarker for synovial sarcoma.

Laura Pazzaglia, Serena Pollino, Mattia Vitale, Amalia Conti, Piero Picci, Maria Serena Benassi. _Istituto Ortopedico Rizzoli, Bologna, Italy_.

Synovial sarcoma (SS) is a rare tumor, with dismal survival when metastatic. SS contains a characteristic translocation (X;18)(p11;q11), representing the fusion of SYT on chromosome 18 with either SSX1, SSX2, or rarely SSX4 on chromosome X The resulting fusion genes appear to be mutually exclusive and concordant in primary and metastatic tumours. New prognostic and predictive factors are needed. Chemokine receptor 4 (CXCR4) is a seven-transmembrane G protein-coupled chemokine receptor and it is the chemokine receptor most commonly expressed in tumour cells, involved in cell migration and invasion, as well as angiogenesis. microRNAs (miRNAs) are involved in post-transcriptional gene expression regulation and control important physiological processes like development, cell differentiation and cell signaling. Altered expression of miRNAs is strongly correlated with the malignant phenotype and there is data reporting a strong association between microRNA expression, patient age and STS prognosis. By three different databases (miRBase, TargetScan , miRanda) and by literature data we identified two miRNA regulators of CXCR4, miR-133b and miR-494. The expression of these miRNAs was evaluated by RT-PCR in 42 SS primary samples stored at Rizzoli biobank. 20 tissues from non oncologic patients were used as control. Our results showed a significant lower expression of miR-133b and miR-494 (respectively p=0.0005 and p=0.001) when compared to non tumor tissue. In vitro study on SW982 cell line with miR-133b precursor show a CXCR4 downregulation and a decrease of cell proliferation. Our preliminary data confirmed a miR-133b and miR-494 downregulation in SS. Correlation with clinical data, with CXCR4 expression and in vitro studies also with miR-494 in several SS cell lines are on-going to better investigate their role as potential prognostic and therapeutic markers.

#2535

MicroRNA-518c-5p promotes the metastasis of oral cancer.

Makoto Kinouchi, Daisuke Uchida, Nobuyuki Kuribayashi, Yuske Komiyama, Shuji Tsuchida, Hitoshi Kawamata. _Dokkyo University School of Medicine, Tochigi, Japan_.

We have demonstrated that CXCR4 system is involved in the metastatic process of oral cancer. Recently, we identified the miR-518c-5p as a downstream target of the CXCR4 system. In this study, we examined the function of miR-518c-5p on the metastasis of oral cancer. We transfected miR-518c expression vector into oral cancer cells, B88 and CAL27, and isolated stable transfectants, B88-518c and CAL27-518c, respectively. The growth and migration of both cells were significantly enhanced in compared with those of mock cells. LNA-based miR-518c-5p inhibitor significantly impaired the enhanced cell growth and migration of these cells, indicating that these phenomena were dependent on the expression of miR-518c-5p, not on that of miR-518c-3p. Next, we examined the function of miR-518c-5p in vivo. miR-518c or mock transfectants were inoculated into the masseter muscle or the blood vessels of nude mice. Tumor volume, lymph nodes metastasis, and lung metastasis were significantly increased in the mice inoculated with miR-518c transfectants. Furthermore, we examined the mRNA expression induced by miR-518c-5p using B88 cells and B88-518c cells by cDNA microarray analysis. Consequently, some of the genes involved in the cell growth and metastasis were upregulated in the B88-518c cells.

These results indicate that CXCR4 system regulates the metastases of oral cancer via induction of miR-518c-5p.

#2536

miR-187-5p and miR-219a-1-3p: potential biomarkers associated with chemotherapeutic response and prognosis in ovarian cancer.

Chia-Yen Huang,1 Hsien-Da Huang2. 1 _Cathay General Hospital, Taipei, Taiwan;_ 2 _National Chiao Tung University, Hsinchu, Taiwan_.

Background: Epithelial ovarian cancer (EOC) is the fifth-most common cause of female cancer death and is associated with high recurrence and mortality despite recent advancements in therapeutic strategies. Chemoresistance remains the main cause of treatment failure in EOC patients. The aim of this study is to identify specific miRNA markers closely associated with the chemoresponse in EOC patients.

Methods: We analyzed 374 serous ovarian cancer patients who receiving combination chemotherapy from The Cancer Genome Atlas (TCGA) and identified the most significantly altered miRNAs between different groups patients of chemotherapy response. We then used Kaplan-Meier and log-rank methods to analyze the relationship between these miRNAs and progression-free and overall survival in TCGA (n = 460) and Bagnoli (n = 130) datasets. In vitro study for response to chemotherapy agents were performed in human ovarian cancer cell lines and their transfectants. The biological relevance of putative miRNA targets was also analyzed using bioinformatics.

Results: MiR-187-5p and miR-219a-1-3p were associated with better response to chemotherapy and longer progression-free and overall survival in two independent epithelial ovarian cancer patient cohorts (all p <0.05). These two miRNAs also sensitized ovarian cancer cells to chemotherapy, thus recapitulating the clinical observation.

Conclusions: This study showed that miR-187-5p and miR-219a-1-3p could be useful biomarker for prediction of chemotherapy response and survival in serous ovarian cancers. MiR-187-5p and miR-219a-1-3p may be potential molecules in the treatment of EOC for sensitizing cancer cells to chemotherapy.

#2537

MicroRNA targeting anti-apoptotic and G2/M pathways as therapeutic targets for castration resistant prostate cancer.

Pheruza Tarapore,1 Sarah To,2 Bin Ouyang,1 Yuet-Kin Leung,1 Ana Cheong,1 Shuk-mei Ho1. 1 _University of Cincinnati, Cincinnati, OH;_ 2 _Hudson Institute of Medical Research, Melbourne, Australia_.

Prostate cancer (PC) is one of the most common cancers in men. Unfortunately, limited treatment options currently exist for those who have developed advanced castration-resistant PC (CRPC). G-1 is a GPER1/GPR30 agonist, and a promising candidate for CRPC therapy. G-1 is effective in halting the growth of CR tumors but not those grown in intact hosts. These findings suggest GPER1 is a therapeutic target for CRPC. We found that G-1, through activation of GPER1, inhibited growth of CRPC cells via cell-cycle arrest at the G2-M phase, probably leading to mitotic catastrophy. However, the exact mode of action of G-1 was not known. To better understand the pathways involved in G-1 action, we conducted a genome-wide mRNA-seq and miRNA-seq study on G-1 treated xenografts. We identified a panel of novel G-1-associated tumor suppressive miRNAs and genes. Moreover, Ingenuity Pathway Analysis revealed that the G-1 differentially regulated genes are involved in "Cellular Growth and Proliferation", "endocrine function" and "Cancer" pathways. We validated the expression of the key miRNA (miR-34c, miR-10b, miR-138 and miR218) and genes in G-1 treated castration resistant xenografts and cell lines (C4-2 and 22Rv1). Furthermore, we have shown the tumor suppressor function of these miRNA in cell survival, migration, and invasion. The predicted target genes were next examined for decreased expression by qRT-PCR. We confirmed decreased expression of some of the downstream targets, which consist of genes involved in cell cycle regulation (CCNA and CCND, CDK1, CDK4, PLK1), cell survival (Bcl2, Survivin), cell migration/F-actin formation (LASP, PCDH7, ITGA9, ROBO1, SLIT1) and G2/M checkpoint (CDK1, PLK1, BCL-2 Survivin). Thus these miRNAs - miR-34c, miR-10b, miR-138 and miR218 - are ideal candidates for therapy of CRPC.

#2538

MiR-638 promotes autophagy and malignant phenotypes of cancer cells via directly suppressing DACT3.

Ming Yang. _Shandong Cancer Hospital and Institute, Jinan, China_.

Dyregulation of autophagy is implicated in human cancers and the mechanism details remains largely unclear. Herein we report the regulatory role of miR-638 in autophagy of esophageal squamous cell carcinoma (ESCC) and breast cancer cells. We found that miR-638 overexpression promotes starvation- and rapamycin-induced autophagy. In ESCC and breast cancer cells, miR-638 acts as an oncogene and promote cell proliferation, migration, as well as invasion in vitro and in vivo. In accordance with this, we observed significantly higher miR-638 expression in ESCC and breast cancer tissues compared to normal tissues. To further elucidate regulatory mechanisms of miR-638 in autophagy, we performed a computational nomination of its target genes through intersecting the results of multiple prediction algorithms. DACT3, a key regulator of Wnt/β-catenin signaling, was predicted to be regulated by miR-638 by all programs and confirmed by experimental results. Depletion of DACT3 phenocopied effects of miR-638 overexpression, demonstrating its importance in autophagy. These results elucidate that the miR-638-DACT3 axis might be an important molecular pathway in controlling and autophagy and tumorigenesis. Our data in clinical tissue samples highlight miR-638 and DACT3 with histological marker for cancer detection and potentially therapeutic implications.

#2539

MiR-96-5p functions as an oncogenic miRNA by inhibiting apoptosis in hepatocellular carcinoma.

Naoto Iwai, Kohichiroh Yasui, Akira Tomie, Kei Teasaki, Tomoko Kitaichi, Osamu Dohi, Yasuyuki Gen, Yoshito Ito. _Kyoto Prefectural University of Medicine, Kyoto, Japan_.

Hepatocellular carcinoma (HCC) is one of the most aggressive cancers with high mortality worldwide. MicroRNAs (miRNAs) are small non-coding RNAs that have been used as cancer-related biomarkers and expected to be therapeutic agents. We performed genome-wide miRNA expression profiling of paired HCC tumors and non-tumorous liver tissues from patients with primary HCCs using the miRNA microarray (Agilent). We found that miR-96-5p was most significantly up-regulated in HCC tumors compared to non-tumor tissues. Although miR-96-5p is suggested to be an oncogenic miRNA, the function of miR-96-5p remains largely unknown. We identified the caspase-9 gene (CASP9) as a novel target of miR-96-5p, in addition to the forkhead box O1 gene (FOXO1) which is the known target of it. Caspase-9 protein is thought to play a central role in apoptosis and to be a tumor suppressor. Overexpression of miR-96-5p decreased caspase-9 protein expression and resulted in resistance to apoptosis induced by doxorubicin and UV in HCC cells. Our results suggested that miR-96-5p functions as an oncogenic miRNA by inhibiting apoptosis through decreasing caspase-9 expression in HCC.

#2540

miR-196b as a potential biomarker for human esophageal cancer.

Xiaohui Tan,1 Tao Chen,2 Robert Siegel,1 Sidney Fu1. 1 _George Washington Univ., Washington, DC;_ 2 _Chengdu Military General Hospital, Chengdu, China_.

Esophageal cancer is the sixth most common cause of cancer related death. Although multiple genetic and epigenetic alterations have been detected in esophageal cancer, molecular markers for early diagnosis and prediction of prognosis or treatment responses are quite limited. microRNA (miRNA) is a class of small-regulatory non-coding RNA, acting as either a tumor suppressor or oncogene by regulating gene expression through pairing with complementary seed of the targeted messenger RNAs (mRNA). A number of miRNA expression profiling studies have been conducted in esophageal. In our current study, we analyzed the most recent studies on miRNAs expression profiles in esophageal cancer. Consistently dysregulated miRNAs have been identified in esophageal cancer, including upregulation of miR-196b, miR-135a, and downregulation of miR-141, miR-200a-5p, miR-200b-3p, miR-27b, miR-210. We first assessed expression of miRNAs in esophageal cancer cell lines and primary esophageal cancer tissues by real-time reverse transcriptase-polymerase chain reaction (qRT-PCR). Significantly overexpressed miR-196b was observed in human esophageal squamous cell carcinoma KYSE-70 and KYSE-180 compared with normal esophageal squamous cell line, HET-1A. Furthermore, overexpression of miR-196b was detected in 12 out of 14 (86%) cancer tissues compared to the matched normal tissues. Forced expression of miR-196b promotes the proliferation and invasion in esophageal cancer cell lines. TargetScanS and miRanda were used to identify the target genes of miR-196b. A list of targets genes were predicted, including GATA6, ARHGAP28, EPS15 and EPHA7. Forced expression of miR-196b resulted in significant downregulated EPS15 and EPHA7 expression in esophageal cancer cell lines. Our present work suggested that miR-196b acts as an oncomiR by regulating EPS15 and EPHA7. Therefore, miR-196b could be used as a biomarker for esophageal cancer.

#2541

Two miR-34 loci, miR-34a and miR-34b/c, have differential anticancer effects in lung adenocarcinomas.

Jeong Seon Kim, Young-Ho Ahn. _Ewha Womans University College of Medicine, Seoul, Republic of Korea_.

Three miR-34 family members (miR-34a, miR-34b, and miR-34c) are clustered on two different chromosomal loci, miR-34a and miR-34b/c, and have identical seed-sequences predicted to target the same set of genes; however, miR-34a and miR-34c have different sets of negatively correlated genes in TCGA data of human lung adenocarcinomas. Therefore, we hypothesized that miR-34 family members, which are tumor suppressive miRNAs, might have differential effects on lung tumorigenesis. To prove this, we overexpressed each miR-34 cluster, miR-34a and miR-34b/c, in murine lung cancer cells. miR-34b/c enhanced cancer cell attachment and suppressed cell growth and invasion compared with miR-34a. In a syngeneic mouse model, both miR-34a and miR-34b/c blocked lung metastasis; whereas, miR-34b/c suppressed tumor growth more than miR-34a. miR-34b/c also decreased mesenchymal markers (Cdh2 and Fn1), and increased epithelial markers (Inadl, Crb3, Ocln, and Dsp) and miR-200 family members better than miR-34a, implying that miR-34b and miR-34c partially inhibit EMT. Furthermore, miR-34b and miR-34c are better prognostic markers than miR-34a in human lung adenocarcinoma patients. Collectively, we suggest that miR-34b and miR-34c are more effective tumor suppressors than miR-34a.

#2542

MSI2 binds LncRNAs and promotes self-renewal and oncogenesis through MYC expression.

Hifzur Siddique, Padmani Narayan, Vasu Punj, Douglas E. Feldman, Keigo Machida. _University of Southern California Keck School of Medicine, Los Angeles, CA_.

Backgrounds and Aims: RNA-binding protein MSI2 is elevated in several cancers and is linked to poor prognosis. Here, we sought to elucidate the role of MSI2 in regulating the expression of proto-oncogenes or tumor suppressor genes in hepatocellular carcinoma (HCC).

Methods: We performed RIP-seq using anti-MSI2 antibody in tumor-initiating stem-like cells (TICs) and subsequent validation by qPCR analysis.

Results: Among the MSI2-bound RNAs, MYC mRNA and long noncoding RNA (LncRNA) miR22 host gene (MIR22HG) and MARAT1 were identified by RIP-qPCR analysis. MSI2-MARAT1 binding dereppresses polycomb reppressor to recruit E2F1 in MYC promoter regions. It is known that the 5'-untranslated region of MYC contains an internal ribosome entry sequence (IRES) which allows IRES-dependent translation initiation along with canonical cap-dependent mechanisms. Our data show that MSI2 acts as an IRES trans-acting factors for MYC mRNA translation. MSI2 does not have any effect on the steady state level of MYC mRNA but significantly increases MYC protein levels, indicating that MSI2 regulates MYC at the post-transcriptional level. Further, we observed that MSI2 reduced the level of mature miR22 derived from MIR22HG processing. We showed that miR22 suppresses the expression of MYC thorough a 3'-UTR dependent process. Overexpression of MSI2 promotes TIC self-renewal and tumor initiation property in xenograft tumor mouse models, whereas silencing MSI2 reduces this occurrence.

Conclusion: we demonstrate that MSI2 promotes liver tumorigenesis by maintenance of MYC expression through dereppression of polycomb repressor and an IRES-dependent translation mechanism and possibly by inhibition of miR22 processing. Thus, MSI2 may be a useful prognostic factor for HCC and MSI2 targeted therapy and thus may be beneficial in the treatment of HCC patients.

#2543

The novel long non-coding RNA TALNEC2, regulates tumor cell growth and the stemness and radiation response of glioma stem cells.

Wei Jiang,1 Shlomit Brodie,2 Simona Cazacu,1 Cunli Xiang,1 Laila Poisson,1 Steve Kalkanis,1 Doron Ginsberg,2 Chaya Brodie1. 1 _Henry Ford Hospital, Detroit, MI;_ 2 _Bar-Ilan University, Ramat Gan, Israel_.

Despite advances in novel therapeutic approaches for the treatment of glioblastoma (GBM), the median survival of 12-14 months has not changed significantly. Therefore, there is an imperative need to identify molecular mechanisms that play a role in patient survival. Here, we analyzed the expression and functions of a novel lncRNA, TALNEC2 that was identified using RNA seq of E2F1-regulated lncRNAs. TALNEC2 was localized to the cytosol and its expression was E2F1-regulated and cell-cycle dependent. TALNEC2 was highly expressed in GBM with poor prognosis, in GBM specimens derived from short-term survivors and in glioma cells and glioma stem cells (GSCs). Silencing of TALNEC2 inhibited cell proliferation and arrested the cells in the G1\S phase of the cell cycle in various cancer cell lines. In addition, silencing of TALNEC2 decreased the self-renewal and mesenchymal transformation of GSCs, increased sensitivity of these cells to radiation and prolonged survival of mice bearing GSC-derived xenografts. Using miRNA array analysis, we identified specific miRNAs that were altered in the silenced cells that were associated with cell-cycle progression, proliferation and mesenchymal transformation. Two of the downregulated miRNAs, miR-21 and miR-191, mediated some of TALNEC2 effects on the stemness and mesenchymal transformation of GSCs. In conclusion, we identified a novel E2F1-regulated lncRNA that is highly expressed in GBM and in tumors from patients of short-term survival. The expression of TALNEC2 is associated with the increased tumorigenic potential of GSCs and their resistance to radiation. We conclude that TALNEC2 is an attractive therapeutic target for the treatment of GBM.

#2544

The lncRNA Meg3 acts as an epigenetic determinant of oncogenic signaling in multiple endocrine neoplasia type 1.

Sucharitha Iyer, Sunita Agarwal. _National Institutes of Health, Bethesda, MD_.

Loss of tumor suppressors and gain of oncogenic properties is a hallmark of cancer. But the pathways leading to tumor formation are poorly understood. A good example is the multiple endocrine neoplasia type 1 (MEN1) syndrome, in which patients inherit germline mutations in the MEN1 gene, predisposing to tissue-specific loss of the encoded tumor suppressor protein menin. Consequently, tumors develop in multiple endocrine organs - the pituitary, the parathyroids, and the duodeno-pancreatic tissues, including pancreatic neuroendocrine tumors (PNETs). Insulinomas are the most common type of functioning PNETs that also occur in MEN1 patients. We investigated the molecular pathways associated with insulinomas. We previously showed that menin loss downregulated Maternally Expressed Gene 3 (MEG3), a long non-coding RNA (LncRNA), by eliciting promoter hypermethylation along with the loss of Histone H3 lysine 4 trimethylation (H3K4me3). Gene expression microarray analyses and RT-PCR, of mouse insulinoma MIN6 cells stably transfected with Meg3, showed a 5-fold decrease in the proto-oncogenic signaling receptor c-Met, suggesting a tumor suppressor function for Meg3. We also found MEG3 downregulation with concurrent upregulation of c-MET in PNETs. However, the molecular underpinnings of MEG3 governed c-MET repression remain elusive. To identify the mechanisms by which MEG3 inhibits oncogenic c-MET signaling to suppress tumorigenesis, we examined the effect of various Meg3 isoforms, explored Meg3 association with the epigenetic regulatory machinery, and RNA-DNA triplex formation. We provide direct evidence for the first time that ectopically expressed Meg3 isoforms could attenuate the highly abundant c-Met transcript in MIN6 insulinoma cells. Meg3 also interacted with components of the epigenetic machinery, such as the Polycomb Repressive Complex 2 (PRC2) protein conglomerate, to regulate c-Met RNA expression. Additionally, analyses of triplex forming oligos (TFOs) in MIN6 cells revealed that triple helix formation between Meg3 and dsDNA could potentially disrupt c-Met transcription. Combined, these data offer mechanistic insight into the dysregulation of c-MET repression in PNETs, and support the conclusion that MEG3 acts as an important determinant of oncogenic signaling in MEN1-associated endocrine tumor cells (insulinoma). These findings warrant further investigation into the tumorigenic pathways that may result from the loss of tumor suppressor MEG3 and gain of oncogenic c-MET signaling in other MEN1-associated endocrine tumors.

#2545

Upregulated lncRNA-HNGA1, a target of miR-378a, contributes to aerobic glycolysis of head and neck squamous cell carcinoma through increasing levels of the C-C chemokine receptor type 7 (CCR7).

Yun Wang. _Sun Yat-sen University, Guangzhou, China_.

Introduction: Long non-coding RNAs (lncRNAs) have been regarded as key regulators in aerobic glycolysis of human cancer. However, the role and function of lncRNAs in Head and Neck Squamous Cell Carcinoma (HNSCC) aerobic glycolysis remain unclear. Here, we report a novel lncRNA, HNGA1, which could promote HNSCC aerobic glycolysis and malignancy by competing for miR-378a binding to regulate CCR7.

Materials and methods: Microarrays were performed to explore the lncRNA/miRNA profiles in tissues samples. qRT-PCR and functional analysis were used to confirm the expression and role of lncRNA/miRNA. Bioinformatics approach and luciferase assay were used to verify the miRNA target gene and the interation between lncRNA and miRNA. Nude mouse model was utilized to observe the effect of lncRNA/miRNA in vivo. Tissue array was performed to explore the association between lncRNA and postoperative survival.

Results: 1. LncRNA HNSCC glycolysis-associated 1 (HNGA1) was up-regulated in tumor tissues, while miR-378a was down-regulated significantly. These observations were confirmed in 60 pairs of HNSCC tissues/non-tumor tissues samples and 7 cohorts of HNSCC cell lines. 2. Silencing of HNGA1 inhibited HNSCC cells proliferation and glycolysis, while overexpression of HNGA1 had the opposite effect. 3. Ectopic expression of miR-378a repressed HNSCC cells proliferation and glycolysis, whereas miR-378a inhibition resulted in the opposite effect. MiR-378a could repress the CCR7 expression by binding to the 3'-UTR region of CCR7 directly. 4. There was an inverse correlation between HNGA1 and miR-378a in HNSCC specimens. Moreover, miR-378a suppressed HNGA1's expression and function by directly binding to HNGA1. In addition, HNGA1 could reverse the inhibitory effect of miR-378a on HNSCC cells, which might act as an endogenous 'sponge' by competing for miR-378a binding to regulate CCR7. 5. The xenograft mouse model unveiled the suppressive effects of miR-378a on HNSCC tumor growth and glycolysis, while HNGA1 could accelerate this process. 6. The clinicopathological findings suggested that the up-regulation of HNGA1 in HNSCC patients was associated with the poorly differentiated degree and more metastasis. Moreover, the results of tissue array showed that HNGA1 was correlated with postoperative survival.

Conclusion: Taken together, our data highlights the pivotal role of HNGA1 in HNSCC aerobic glycolysis. More importantly, we elucidate a novel lncRNA-miRNA-mRNA regulatory network that is HNGA1-miR-378a-CCR7 axis in HNSCC malignancy and progression.

#2546

The long noncoding RNA SChLAP1 inhibits the SWI/SNF complex, revealing a therapeutic opportunity in prostate cancer.

Julia Dancourt,1 Anirban Sahu,2 John R. Prensner,2 Benjamin Chandler,2 Qi Cao,2 Nithin Edara,2 Udit Singhal,2 Matthew K. Lyer,2 Rohit Malik,2 Xuhong Cao,2 Saravana M. Dhanasekaran,2 Yashar S. Niknafs,2 Shuang Zhao,2 Corey Speers,2 Arul M. Chinnaiyan,2 Felix Y. Feng1. 1 _University of California San Francisco, San Francisco, CA;_ 2 _University of Michigan, Ann Arbor, MI_.

Background: Recently, we identified the long noncoding RNA SChLAP1 that is expressed highly in 20-30% of prostate cancers and significantly associates with metastatic and lethal disease. SChLAP1 enhances cell invasiveness in part by interacting with and abrogating genome-wide binding of the tumor-suppressive SWI/SNF complex. Approximately 20% of all cancers harbor a mutation in SWI/SNF, and several recent studies have identified therapeutic opportunities arising from SWI/SNF inactivation. We hereby investigate the relationship between SChLAP1 and SWI/SNF and its potential therapeutic implications.

Methods: In silico data mining was used to correlate the SWI/SNF complex mutation frequency with SChLAP1 expression in prostate cancer samples. A systematic tiling deletion approach was then used to map important domains on the SChLAP1 gene. In vitro migration and invasion assays as well as cell proliferation assays were used to quantify phenotypes associated with over-expression of SChLAP1 or its deletion mutants. Moreover, CHIP was used to study the influence of SChLAP1 on the genomic binding of SWI/SNF.

Results: In prostate cancer samples, SWI/SNF mutations correlate with low SChLAP1 expression while high SChLAP1 expression may represent a mutation-independent modality of SWI/SNF inhibition. We identify a 250 nucleotide region necessary for SChLAP1 to bind to SWI/SNF and to confer invasiveness to a prostate epithelial cell line. Further studies suggest that SChLAP1 inhibition is selective for the BRG1-containing SWI/SNF sub-complex, inferring potential genomic specificity. Moreover, we show that SChLAP1 over-expression is associated with enhanced sensitivity to BRM knockdown, in accordance with the BRG1/BRM synthetic lethality previously described in BRG1-deficient cancers.

Conclusions: Our findings indicate that SChLAP1-mediated SWI/SNF inhibition may function similarly to SWI/SNF mutation, exposing similar therapeutic opportunities.

#2547

Discovery and characterization of late-stage breast cancer estrogen receptor alpha 1 bound long non-coding RNAs.

Jessica Monique Silva-Fisher,1 Abdallah M. Eteleeb,1 Torsten Nielsen,2 Charles M. Perou,3 Jorge S. Reis-Filho,4 Mathew J. Ellis,5 Elaine R. Mardis,6 Christopher A. Maher1. 1 _Washington University School of Medicine, St. Louis, MO;_ 2 _Vancouver Hospital & Health Sciences Centre, British Columbia, Canada; _3 _Univeristy of North Carolina, NC;_ 4 _Memorial Sloan Kettering, NY;_ 5 _Baylor College of Medicine, TX;_ 6 _Nationwide Childrens Hospital, OH_.

Breast cancer (BC) is the second most common newly diagnosed cancer and the second leading cause of cancer death among women in the United States. Despite the proven benefits of adjuvant endocrine therapy in women with hormone receptor positive BC, relapses still occur even after initial treatment with endocrine therapy for 5 years, referred to as late-stage relapse. Long non-coding RNAs (lncRNAs) have been shown to be dysregulated in breast cancer. Recent studies have also shown lncRNAs to function by interfacing with corresponding RNA binding proteins to play critical regulatory roles of diverse cellular processes. Therefore, we hypothesize that lncRNAs may interact with ER to regulate genes promoting late-stage relapse. To address this, we aimed to identify lncRNAs bound to the estrogen receptor alpha 1 protein (ESR1) that promote late-stage relapse breast cancer. We first used transcriptome sequencing to identify altered expression levels of lncRNAs between 72 primary tumors and 24 late-stage relapse breast cancer patients. We detected 1192 altered lncRNAs when comparing the metastatic to the primary samples (FDR <0.05). Next, to identify ESR1 bound lncRNAs associated with late-stage BC, we conducted RNA Immunoprecipitation Sequencing of all transcripts bound to ESR1 as compared to an IgG control in the ER+ T47D cell line. We identified 217 lncRNAs bound to ESR1 of which 50 were up-regulated in late-stage BC, termed Late-Stage Relapse BC ESR1-bound lncRNAs (LASERs). Next, we focused on characterizing the most up-regulated differentially expressed lncRNA, LASER1. We found that LASER1 has increased expression in ER+ breast cancer cell lines. Further, elevated expression of LASER1 was also detected in MCF7 long-term estrogen deprived cells lines that have amplified ER+, suggesting an ER dependent mechanism. Preliminary functional studies indicate LASER1 to promote proliferation and invasion in BC cell lines. Ongoing studies will decipher how LASERs interact with ESR1 to promote late-stage relapse. Overall, this is the first study to discover ESR1 bound lncRNAs that may be contributing to late-stage relapse in BC patients.

#2548

Oncogenic function and molecular mechanism of H19 noncoding RNA in colorectal cancer.

Hui Ling,1 Masahisa Ohtsuka,1 Cristina Ivan,1 Martin Pichler,1 Meng Chen,1 Ondrej Slaby,2 Ajay Goel,3 Milan Radovich,4 George Calin1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Masaryk University, Czech Republic;_ 3 _Baylor University Medical Center, TX;_ 4 _Indiana University School of Medicine, TX_.

The clinical significance of long noncoding RNAs (lncRNAs) in colorectal cancer (CRC) remains largely unknown. We analyzed a large panel of cancer-associated lncRNAs in 483 TCGA CRC cases, and identified that H19 correlates most significantly with overall survival and disease-free survival, independent of stage and other clinical parameters. We validated the TCGA findings with two additional CRC cohorts. As one of the "oldest" lncRNAs, diverse activities of H19 including interaction with epigenetic regulators, regulation of microRNAs as competing endogenous RNA (ceRNA), and production of miR-675 as a primary transcript have been reported. However, an unbiased study to clarify the essential mechanisms of H19 is lacking. To decipher the functional involvement of H19 in CRC, we used an integrative approach combining unbiased microarray analysis, experimental validation, and bioinformatic analysis of clinical data. With such approach, we revealed not only previously reported mechanisms such as let-7 and MYC regulation by H19, but also several novel mechanisms that have not been appreciated before. We discovered a mechanism of H19 on regulating RB-E2F signaling and accordingly controlling cell cycle progression. We also identified for the first time that H19 regulates CDK8, an oncogenic driver in CRC pathogenesis, and consequently affects β-catenin activity (Firestein et al., Nature, 2008). We further identified an interaction of H19 with macroH2A, a histone variant reported to repress CDK8 transcription (Kapoor et al., Nature, 2010). Importantly, expression correlation analysis with clinical samples strongly supported the above-identified mechanisms. Combined analysis of H19 with its targets showed strong independent prediction power for CRC survival. These data indicate the clinical relevance of Rb-E2F and CDK8/β-catenin signaling in mediating H19's oncogenic activity in CRC.

#2549

A long non-coding RNA regulates the androgen receptor and mediates prostate cancer progression.

Sethuramasundaram Pitchiaya, Rohit Malik, Marcin Cieslik, Yajia Zhang, Xia Jiang, Arul M. Chinnaiyan. _Univ. of Michigan Comp. Cancer Ctr., Ann Arbor, MI_.

The pervasive expression of long non-coding RNAs (lncRNAs) and their roles in a plethora of cellular processes has revolutionized our understanding of functional genetic elements. Emerging evidence suggest that lncRNAs are promising cancer biomarkers and dysregulation of lncRNAs results in attenuated or accelerated oncogenic phenotypes. In support of these findings, recent reports from the Chinnaiyan lab suggest that lncRNAs can potentially drive and act as independent predictors of aggressive prostate cancer (PCa). Central to PCa progression is the androgen receptor (AR), a nuclear hormone receptor, and the dysregulated transcription program it mediates. Consequently, androgen deprivation therapy (ADT) supplemented with anti-androgens is the initial standard-of-care in treating advanced PCa. Yet, the disease often manifests as a lethal, hormone-refractory castration-resistant prostate cancer (CRPC) after initial ADT and this phenomenon is linked to the resumption of AR activity. Therefore, we sought to identify novel and therapeutically actionable targets of AR that can detect the onset of PCa or stratify PCa variants. To this end, our lab recently characterized the transcriptional landscape of cancer and discovered > 50,000 novel transcripts, a significant fraction of which were tissue- and cancer-specific lncRNAs. Using this updated transcriptome as a reference and performing RNA-seq on PCa cells stimulated with dihydrotestosterone (an androgen), we identified Androgen Receptor regulated lncRNA-1 (ARlnc1) as an important PCa-specific, AR-regulated lncRNA that reciprocally regulates AR. By employing single-molecule fluorescence in situ hybridization (smFISH), we find that ARlnc1 and AR transcripts colocalize within the nucleus of PCa cells and that ARlnc1 promotes transcription of AR. Moreover, we found that ARlnc1 interacts with anti-apoptotic, stress-granule related proteins and regulates PCa cell proliferation in vitro and in mouse xenografts. We have additionally found that anti-sense oligonucleotides (ASOs) that target this lncRNA can reduce tumor growth in xenograft models. Taken together, our data suggests that ARlnc1 and AR reciprocally and positively modulate each other to promote PCa progression and that ARlnc1 may serve as an enhancer RNA that regulates AR transcription. Our data suggests that ARlnc1 can be therapeutically targeted to orthogonally modulate the AR signaling axis for PCa treatment.

#2550

Anti-MALAT1 synthetic oligonucleotides target the proteasome and exert anti-multiple myeloma activity.

Maria Angelica Stamato,1 Nicola Amodio,1 Eugenio Morelli,1 Giada Juli,1 Martina Manzoni,2 Maria Eugenia Gallo Cantafio,1 Lavinia Biamonte,1 Elisa Taiana,2 Annamaria Gullà,1 Emanuela Altomare,1 Francesca Scionti,1 Daniele Caracciolo,1 Cirino Botta,1 Maria Cucè,1 Mariamena Arbitrio,1 Maria Teresa Di Martino,1 Marco Rossi,1 Antonino Neri,2 Pierosandro Tagliaferri,1 Pierfrancesco Tassone1. 1 _University Magna Grecia of Catanzaro, Catanzaro, Italy;_ 2 _Universita' Degli Studi di Milano, Milano, Italy_.

Dysregulation of non-coding RNAs features prominently in the biology of multiple myeloma (MM). However, if short non-coding RNAs have been extensively studied in this malignancy, the role of long non-coding RNAs remains to be elucidated. A custom annotation pipeline of microarray data investigating lncRNA expression in plasma cells from 20 MGUS, 33 smoldering MM, 170 MM, 36 plasma cell leukemia patients, and 9 healthy donors, indicates overexpression of the lncRNA MALAT1 during MM progression. On this finding, we sought to investigate the functional significance of MALAT1 expression in MM by the use of 16-mer LNA-gapmeR synthetic oligonucleotides, that trigger specific RNAse H-dependent degradation of MALAT1. Transfected or gymnotic (naked) anti-MALAT1 gapmeRs decreased MM patients (n=3) and cell lines growth (n=7), triggering ER stress and apoptosis, while spared healthy peripheral blood mononuclear cells. Microarray gene profiling indicates that MALAT1 depletion was associated with modulation of various gene sets, and specifically had a negative impact on genes encoding for 20S proteasome β subunits, such as PSMβ2/4/5 and 7, whose overexpression is linked to proteasome impairment in cancer cells. Down-modulation of PSMβs after anti-MALAT1 gapmeRs exposure was confirmed at protein level and was associated to reduced trypsine, chymotrypsine and caspase-like proteasomal activities and to the accumulation of poly-ubiquitylated species. Consistently, anti-MALAT1 gapmeRs enhanced the in vitro anti-MM activity of the proteasome inhibitor bortezomib in bortezomib-sensitive and resistant MM cells. In vivo, i.p. administration of naked anti-MALAT1 gapmeRs (10 and 25 mg/kg) inhibited the growth of bortezomib-resistant MM xenografts, with no evidence of organ toxicity. Mechanistically, anti-MALAT1-induced PSMβs down-modulation could be ascribed to the inhibition of the transcription factors NRF1-2, which act as positive regulators of PSMSβs transcription. On the other hand, siRNA or pharmacologic inhibition of NRF1 by all-trans retinoic acid decreased promoter activity and expression of MALAT1, indicating the occurrence of a feedback loop involving MALAT1 and NRF1. Collectively, our data indicate that MALAT1-targeting represents a promising strategy to dampen aberrant proteasome activity in MM cells, and provide the preclinical rationale to use oligonucleotide therapeutics as anti-MALAT1 gapmeRs for MM treatment.

#2551

**Long noncoding RNA** HOXA11 antisense **promotes tumor progression via the Notch pathway in serous ovarian cancer.**

Hee Jung Kim,1 Ga Won Yim,2 Kyung Jin Eoh,1 Lee Kyung Kim,1 Jung-Yun Lee,1 Eun Ji Nam,1 Sang Wun Kim,1 Young Tae Kim1. 1 _Institute of Women's Life Medical Science, Yonsei University College of Medicine, Seoul, Republic of Korea;_ 2 _Department of Obstetrics and Gynecology, National Medical Center, Seoul, Republic of Korea_.

The biological function of long noncoding RNAs (lncRNAs) is only partially understood. We investigated the expression of the novel HOXA11 antisense (HOXA11as) lncRNA and its oncogenic role in serous ovarian cancer (SOC). HOXA11as expression was examined in 129 serous ovarian cancer tissue samples by real time RT-PCR. Clinicopathologic factors and patient survival were compared between high (N = 27) and low HOXA11as expression group (N = 102). To investigate the role of HOXA11as in cell proliferation, invasion, and migration, HOXA11as expression in ovarian cancer cells was knocked down using RNA interference. HOXA11as expression in cancer tissue was 77-fold higher than that of noncancerous tissue (P < 0.05). Higher HOXA11as expression significantly correlated with histologic grade (P = 0.017) and preoperative CA125 (P = 0.048). HOXA11as overexpression in SOC cells led to increased cell proliferation, invasion, and migration. In addition, HOXA11as was associated with the expression of genes that involve in cell proliferation, invasion, and migration. Moreover, Knockdown of HOXA11as decreased the expression of vascular endothelial growth factor, matrix metalloproteinase-9 and epithelial-mesenchymal transition (EMT), which are important for cell motility and metastasis. Mechanistic investigation revealed that Notch1, Hes1 and p300 proteins could be inhibited by HOXA11as depletion. In multivariate analysis, HOXA11as was a prognostic factor of progressive disease and mortality (HR = 1.730, 2.170 and P = 0.043, 0.033, respectively). Progression-free and overall survival were significantly shorter in patients with high HOXA11as expression. These findings highlight the clinical significance of HOXA11as in predicting the prognosis of SOC patients and suggest its potential in promoting tumor aggressiveness by regulation of the Notch signaling pathway and EMT-related mechanisms.

### Nuclear Oncogenes and Tumor Suppressors

#2552

MDM2 induces cell fusion in breast cancer and osteosarcoma.

David J. Olivos, Mikhail Chtcherbinine, Eric R. Wolf, Ciarán McAtarsney, Lindsey D. Mayo. _Indiana University School of Medicine, Indianapolis, IN_.

Cell fusion is a physiological process utilized by a variety of biological systems during fertilization, embryogenesis, and cellular differentiation and morphogenesis. During the malignant transformation of breast and bone tissues, normal and malignant cells become fused to form hybrid structures. These enhanced phenotypes acquire genetic characteristics which support initiation, progression, and oncogenesis. In breast cancer and osteosarcoma cell lines, cell fusion generates chromosomal instability, aneuploidy, and DNA damage while maintaining the characteristics of enhanced migration, invasion, and chemoresistance. We have found that mouse double minute 2 (Mdm2) significantly induces cell fusion and the generation of large multinucleated cells in MDA468, MDA231, MDA157, T47D breast cancer and MG63 osteosarcoma cell lines under normoxia when compared to silenced Mdm2 controls (P<0.0005). Interestingly, silencing mutant p53 in the MDA468 cell line resulted in significantly higher fusion events (P<0.0004) suggesting mutant p53 may play a regulatory role in reducing Mdm2-associated cell fusion. Silenced Mdm2 MDA231 cells showed a three-fold increase under hypoxia whereas MG63 cells showed similar levels of cell fusion events as in normoxic conditions suggesting cell specific dependency to environmental stimulus. The variation in proportions of cell fusion among each cell type suggests this phenomenon is dependent on Mdm2 expression and cell type. CD133+ MDA468 cells have increased fractions of polyploidal cell events suggesting stemness may influence the polyploidal giant cancer cell (PGCC) dynamics. The mechanism by which cell fusion occurs in breast and osteosarcoma is poorly understood. Collectively these data suggest the interplay of Mdm2, p53, and stemness in supporting the formation of fused polypoidal cells and may serve as relevant targets for cancer therapy.

#2553

High-density array-CGH with targeted NGS unmask multiple non-contiguous minute deletions on chromosome 3p21 in mesothelioma.

Michele Carbone,1 Yoshie Yoshikawa,2 Mitsuru Emi,2 Tomoko Hashimoto-Tamaoki,2 Masaki Ohmuraya,2 Ayuko Sato,2 Tohru Tsujimura,2 Seiki Hasegawa,2 Takashi Nakano,2 Masaki Nasu,1 Sandra Pastorino,1 Agata Szymiczek,1 Angela Bononi,1 Harvey I. Pass,3 Haining Yang1. 1 _Univ. of Hawaii Cancer Ctr., Honolulu, HI;_ 2 _Hyogo College of Medicine, Hyogo, Japan;_ 3 _New York University, New York, NY_.

We used a custom-made comparative genomic hybridization-array (aCGH; average probe interval 254 bp) to screen 33 malignant mesothelioma (MM) biopsies for somatic copy number loss throughout the 3p21 region (10.7 Mb) that harbors 251 genes, including BRCA1-associated protein 1 (BAP1), the most commonly mutated gene in MM. We identified frequent minute biallelic deletions (<3 Kb) in 46/251 genes: four were cancer associated genes: SETD2 (7/33), BAP1 (8/33), PBRM1 (3/33) and SMARCC1 (2/33). These four genes were further investigated by targeted Next Generation Sequencing (tNGS), which revealed sequence-level mutations causing biallelic inactivation. Combined high-density aCGH and tNGS revealed biallelic gene inactivation in SETD2 (9/33, 27%), BAP1 (16/33, 48%), PBRM1 (5/33, 15%) and SMARCC1 (2/33, 6%). The incidence of genetic alterations detected is much higher than reported in the literature because minute deletions are not detected by NGS or commercial aCGH. Many of these minute deletions were not contiguous but rather they alternated with segments showing oscillating copy number changes along the 3p21 region. Functional assays revealed that SETD2, PBRM1 and SMARCC1 act as tumor suppressor in MM. In summary, we found that in MM: 1) multiple minute simultaneous biallelic deletions are frequent in chromosome 3p21, where they occur as distinct events involving multiple genes; 2) in addition to BAP1, mutations of SETD2, PBRM1 and SMARCC1 are frequent in MM; 3) our results suggest that high-density aCGH combined with tNGS provide a more precise estimate of the frequency and types of genes inactivated in human cancer, than approaches based exclusively on NGS strategy.

#2554

PRR14, a novel regulator of Ras signaling pathway.

Mei Yang,1 Meijun Long,2 Xiaoliu Shi,1 Zhi-Min Yuan,3 Hang Su4. 1 _The Second Xiangya Hospital of Central South University, Changsha, China;_ 2 _The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China;_ 3 _Harvard T.H. Chan School of Public Health, Boston, MA;_ 4 _The University of Texas Health Science Center at San Antonio, San Antonio, TX_.

PRR14, a component of nuclear lamina, has recently been identified as a novel oncogene in lung cancer. Frequently upregulated in lung cancers, PRR14 promotes tumorigenesis through activating the PI3-kinase/Akt/mTOR signal pathway. The regulatory effect is mediated by direct interaction with GRB2, a docking protein upstream of both PI3K and Ras. Indeed, PRR14 is commonly elevated in many cancer types including lung cancer and breast cancer, but our understanding of the underlying molecular mechanism is still very elusive. Given the complicated interplay between PI3K and Ras signal pathways and their importance in carcinogenesis and optimal treatment, we further explored the effect of PRR14 on Ras activation. We used MCF10A human mammary epithelial cells and its K-RasV12-induced cancerous counterparts to establish isogenic cell lines that express different levels of PRR14. Colony formation and growth curves were monitored simultaneously. Western blot and Ras pull-down activation assay were employed to examine protein level and Ras activity. Cell fractionation was performed to detect protein location. We found that the expression level of PRR14 positively correlated with both Ras protein level and its activity, resulting in consequent changes in cell proliferation and colony formation. Remarkably, elevated PRR14 promoted translocation of Ras from cytoplasm into the cell nucleus and its stabilization. In conclusion, PRR14 sequesters, stabilizes and activates Ras in cell nuclei. When upregulated, PRR14 promotes tumorigenesis, and Ras signaling pathway presents one of the complex underlying mechanisms.

#2555

ZC3H8 associates with PML bodies and influences aggressive tumor cell behavior.

John A. Schmidt, Emily Duffner, Gerard Walker, Keith G. Danielson, Janice E. Knepper. _Villanova Univ., Villanova, PA_.

The ZC3H8 gene encodes a protein with three zinc finger motifs in the C-terminal region, suggesting a role as an RNA binding protein, but whose specific function remains unclear. ZC3H8 protein localizes to both PML bodies and Cajal bodies within the nucleus, as confocal microscopy demonstrates colocalization of ZC3H8 with both PML and coilin proteins. ZC3H8 has a casein kinase 2 phosphorylation site in the N-terminal region, and treatment with a casein kinase 2 inhibitor causes the numerous PML bodies and their associated ZC3H8 to coalesce to a few larger bodies. Removal of the inhibitor restores PML bodies to their original state. We constructed a mutant of ZC3H8 that removed the predicted CK2 phosphorylation site by replacing the serine with an alanine residue. Cells transfected with this mutant had reduced numbers of PML bodies, similar to cells treated with the CK2 inhibitor. In contrast, a mutant constructed with a glutamic acid in place of the phosphorylatable serine was predicted to behave as a constitutive mutant. This mutant did not exhibit differences from wild type localization. These experiments suggest that ZC3H8 integrity is key to maintenance of PML bodies. Other studies have identified ZC3H8 as a component of the LEC (little elongation complex), and we found that another identified component, ICE2 (Narg) also localizes to PML bodies. The ZC3H8 gene is overexpressed in a number of human and mouse breast cancer cell lines, and elevated mRNA levels are associated with a poorer prognosis for women with breast cancer. We have used RNA silencing to decrease levels of expression in two independent mouse mammary tumor cell lines. Cells with lower ZC3H8 expression have dramatic phenotypic changes, showing decreased rates of proliferation, slower migration in wound healing assays, formation of fewer and smaller colonies in soft agar assays, and decreased ability to invade through a basement membrane in transwell assays. Further, ZC3H8-depleted cells have decreased rates of growth as spheroids and decreased growth in vivo in syngeneic mice. We developed an inducible vector driving expression of ZC3H8. Upon treatment with the inducer, doxycycline, cells assume a more aggressive phenotype. We introduced a synonymous mutant of ZC3H8 unable to be silenced by the RNAi into the knockdown cells, and rescued the aggressive phenotype of the original tumor cells. Overexpression of ZC3H8 in non-tumorigenic COMMA-D cells leads to a less pronounced, but opposite, increase in proliferation, motility, and invasion. We have engineered the synonymous ZC3H8 with disrupted zinc finger motifs, and preliminary results suggest that disruption of the first zinc finger is not sufficient to cause loss of the aggressive phenotype. We suggest that ZC3H8 is an oncogene with substantial effects on the cancer cell phenotype. These effects are hypothesized to be associated with ZC3H8 function in PML bodies.

#2556

Loss of progesterone receptor through epigenetic regulation is associated with poor prognosis in multiple solid tumors.

Yiyang Li, Tamar Kavlashvili, Cheng Huang, Yuping Zhang, Xiangbing Meng, Kristina Thiel, Kimberly Leslie, Shujie Yang. _Univ. of Iowa, Iowa City, IA_.

Introduction: As a tumor suppressor in the endometrium, progesterone and its synthesized analogue progestin have a long history as a treatment for endometrial cancer. Loss of progesterone receptor (PR) in endometrial cancer leads to therapeutic failure, and our group has identified several mechanisms underlying PR loss, most notably epigenetic silencing of PR transcription. Recently, a Finnish group demonstrated that progestin therapy in premenopausal women is associated with a lower incidence of not only endometrial cancer, but also ovarian, pancreatic and lung cancers. This unexpected protective function of progestin in organs outside of the reproductive system led us to hypothesize that tumor progression effects in endometrial, ovarian, pancreatic and lung cancer is occurs due to the loss of progesterone's protective effects.

Methods and results: Supporting a potential protective role of progesterone, we discovered that PR is downregulated in multiple tumors, including breast, endometrial, ovarian, cervical, pancreatic and lung cancers, and loss of PR was consistently associated with a poor prognosis based on analysis of the Oncomine database. Similarly, PR expression was low in multiple ovarian, pancreatic and lung cancer cells. Mechanistic studies revealed that PR downregulation was mediated by the polycomb-repressor complex and DNA methylation, though the precise mechanism differed among cell lines and cancer types. Treatment with epigenetic modulators, which are FDA-approved for multiple myeloma, restored functional PR expression at both the mRNA and protein level and promoted marked cell death through induction of apoptosis. . Moreover, overexpression of PR or treatment with epigenetic modulators in combination with progesterone enhanced this effect, indicating a key protective role for progesterone signaling through PR.

Conclusion: These data suggest loss of PR may be a general theme for tumorigenesis or disease progression in multiple tumor types beyond the classically studied endometrial cancer. In addition, the epigenetic mechanisms contributing to PR downregulation in endometrial tumors were recapitulated in other tumor types, including ovarian, pancreatic and lung tumors. These studies collectively set the stage for use of progestin therapy in combination with epigenetic modulators, a concept we term "molecularly enhanced progestin therapy," as a novel therapeutic approach for many cancers. This combinatorial strategy has the power to revitalize the use of epigenetic modulators, which have proved disappointing in solid tumors, as an approach to resensitize cancer cells to a tumor suppressor.

#2557

Role of microRNAs in breast cancer.

Hesham Abbas El-Mahdy,1 Ossama Abdelmotaal Mansour,1 Mohamed Mohamed Badr,1 Reham Aly Elshimy2. 1 _Faculty of pharmacy, Cairo, Egypt;_ 2 _National Cancer Institute, Cairo, Egypt_.

Background: Breast cancer (BC) is one of the most common cancer in women around the world and the second leading cause of death worldwide. MicroRNAs (miRNAs) expression participates in breast cancer.

Objectives: The purpose of this study is to investigate the expression of miR-133a and miR-155 in breast cancer serum and study their correlation with tumor suppressor protein (p53), carcinoembryonic antigen (CEA) and cancer antigen-15.3 (CA-15.3) concentrations in serum of breast cancer patients and also study their correlations with clinicopathological features.

Material and methods: In this study the expression of miR-133a and miR-155 in serum were measured using quantitative real-time polymerase chain reaction (qRT-PCR), P53 concentration was measured by enzyme-linked immunosorbent assays (ELISA), CEA and CA-15.3 concentration were measured using ARCHITECT immunoassay in women with breast cancer (n=60) and controls (n = 80).

Results: In this study miRNA-155 was significantly overexpressed (P<0.001) while miR-133a had significant down expression (P<0.001) in the serum of breast cancer patients compared to control serum. P53 had no significant correlations with any of the studied miRNAs. Carcinoembryonic antigen and CA-15.3 have significant higher concentration in the serum of breast cancer patients compared to control serum. A significant association was observed between miR-133a with tumor grade (P<0.05) and miR-155 with lymph node involvement (P<0.05). A significant correlation between miR-155 and CEA (P<0.05). No correlations between miR-133a and P53, CEA, CA-15.3. By using receiver operating characteristic (ROC) curve, the two miRNAs showed higher sensitivity, specificity, positive predictive value and negative predictive value than routine markers.

Our Conclusion: These miRNAs have a significant signature in the pathogenesis of breast cancer and can be used as non-invasive potential biomarkers for breast cancer detection.

#2558

MYC-dependent transformation model of triple-negative breast cancer in vivo.

Corey Lourenco, Manpreet Kalkat, Dharmesh Dingar, Jason De Melo, Rosemary Yu, Linda Penn. _Princess Margaret Cancer Centre, Toronto, Ontario, Canada_.

Recent comprehensive breast cancer studies examining mutations and genomic alterations have determined that deregulation of MYC and the PI3K pathway occur frequently during breast cancer progression and may be useful targets for therapy. As a result, there have been large efforts to develop PI3K, AKT and mTOR inhibitors (PAM inhibitors) for clinical use, however clinical trial data demonstrates that many patients treated with PAM inhibitors develop resistant disease. An alternative strategy would be to target Myc, though a lack of effective and specific inhibitors makes this difficult. To identify the core vulnerabilities in these cancers we developed an in vivo xenograft model of triple-negative breast cancer driven by deregulated PI3K signaling and MYC. We hypothesize that determining how these pathways co-operate to transform normal human breast cells into breast carcinomas will reveal a tumor progression signature and highlight new therapeutic opportunities. We developed our model using the spontaneously immortalized, basal, triple-negative MCF10A cell line. By expressing the hotspot PIK3caH1047R protein alone in MCF10A cells (MCF10.H) in addition to MYC (MCF10.HM), we can model normal/early breast cancer and invasive ductal carcinoma respectively. This is the first in vivo human model of breast cancer dependent on MYC for transformation. When injected into female NOD-SCID mice, MCF10A.H cells form organized acinar ducts embedded in extracellular matrix. MCF10A.H ducts form with hollow lumen and a single layer of myoepithelial cells, recapitulating normal human breast histology. Alternatively, MCF10A.HM cells grow as high-grade carcinomas indicative of invasive disease. MCF10A.H benign growths and MCF10A.HM tumors remain basal-like and triple-negative by immunohistochemistry. Importantly, MCF10A.HM tumors are sensitive to MYC repression and therefore may be a suitable model to evaluate direct and indirect anti-MYC therapies. Having relevant human xenograft samples representing both normal and IDC tissue, we performed RNA-seq to identify a MYC-signature driving breast cancer transformation. Our current work will involve targeting the resulting MYC-driven pathways identified by RNA-seq to therapeutically target MYC in breast cancer.

#2559

FoxF1 is a potential oncogene in prostate cancer.

Carolin Bossmann,1 Constanze Merz,1 Yuri Tolkach,1 Jessica Carlsson,2 Sven Perner,3 Ove Andrén,2 Glen Kristiansen,1 Michael Nowak1. 1 _University Hospital Bonn, Bonn, Germany;_ 2 _University Hospital of Örebro, Örebro, Sweden;_ 3 _University Medical Center Schleswig-Holstein, Lübeck, Germany_.

Background: FoxF1 belongs to the family of forkhead box transcription factors. Many forkhead box proteins are linked to cancer development and progression. However, the regulation and exact function of FoxF1 remains unclear. The aim of this study was to elucidate how the transcriptional activity of FoxF1 is controlled and to assess the role of FoxF1 within prostate cancer.

Design: For assessment of FoxF1 functions tumor cell lines were stably transfected with wild type FoxF1 or mutant FoxF1 constructs lacking putative phosphorylation sites. Protein expression and localization of FoxF1 were analyzed by immunprecipitation and Western Blot. Using tissue microarrays of prostate cancer cohorts we analyzed the protein expression and intracellular localization of FoxF1 in tumors compared to benign tissue.

Results: Primary tumors and distant metastases exhibited a significantly higher FoxF1 expression compared to benign prostate tissue. In tumors and metastases nuclear localized FoxF1 was more abundant compared to benign tissue.

Transfection of tumor cell lines with FoxF1 followed by immunprecipitation and Western Blot showed that nuclear FoxF1 but not cytoplasmic FoxF1 is phosphorylated in tyrosine residues, conceivably tyrosine-39, as suggested by literature data. We further transfected tumor cell lines with different amino acid exchange mutants of putative phosphorylation sites and analyzed the transcription of the known FoxF1 target gene Pecam-1, revealing a complex pattern of regulation, dependent on the site of phosphorylation.

Overexpression of FoxF1 in tumor cell lines resulted in epithelial-mesenchymal transition, as observed by loss of E-cadherin expression, gain of Vimentin expression, the up-regulation of Snail and Twist and changes to a spindle-like morphology. Furthermore, overexpression of FoxF1 led to anchorage-independent growth in soft agar as well as increased migration rates in vitro without affecting the proliferation rate.

Transfection of tumor cell lines with FoxF1 followed by immunprecipitation and Western Blot showed that nuclear FoxF1 but not cytoplasmic FoxF1 is phosphorylated in tyrosine residues, conceivably tyrosine-39, as suggested by literature data. We further transfected tumor cell lines with different amino acid exchange mutants of putative phosphorylation sites and analyzed the transcription of the known FoxF1 target gene Pecam-1, revealing a complex pattern of regulation.

Conclusion: In summary, our results point to a role of FoxF1 as a potential oncogene in prostate cancer whose nuclear localization and activity is regulated by phosphorylation.

#2560

Identification and characterization of EWS-FLI1 binding partners in Ewing sarcoma cell lines.

Matthew L. Rotondi, Peter J. Houghton. _Greehey Children's Cancer Research Institute, San Antonio, TX_.

Ewing sarcoma (ES) is characterized by a reciprocal translocation t(11;22) that results in a fusion of the EWSR1 and FLI1 genes (EWS-FLI). The objective of this study is to identify protein binding partners of the purportedly undruggable chimeric transcription factor EWS-FLI1 to serve as alternative pharmacological targets for potential ES-selective drug therapies. The literature identifies the orphan nuclear receptor DAX1 (encoded by NR0B1) as a binding partner of EWS-FLI1 the expression of which is restricted outside ES, thereby providing a target for a potential ES selective drug. Yeast two-hybrid (Y2H) screening using EWS-FLI1 as the bait was employed to identify other potential targets. The importance of the binding partners identified by the Y2H screen was initially assessed by a series of proliferation assays involving the ES cell lines EW8, ES7 and ES6 as well as rhabdomyosarcoma cell line Rh30 (Non-Ewing control). Prior to measuring proliferation, cells were transfected a siRNA designed to target the putative binding partner of EWS-FLI1. These cells were then grown alongside cells transfected with a control siRNA over a period of 96 hr. The change in cell confluence in the wells was measured by a live-cell real-time measurement (Incucyte). Gene knockdown was confirmed by RT-PCR and western blot analysis. The proliferation assays showed that the knockdown of DAX1 produces a notable reduction in cell proliferation compared to siRNA control proliferation in all three of the Ewing cell lines (10% to 25%). No significant DAX1 siRNA based inhibition of cell proliferation was detected in the Rh30 non-Ewing control cell line. Three of the proteins identified by the Y2H screen showed significant reductions in ES cell proliferation. Methyl-CpG-Binding Domain Protein 1 (MBD1) knockdown caused the proliferation of the ES cell lines to level off at 60% to 80% within 2 to 4 days. Proliferation of MBD1 siRNA and control siRNA transfected Rh30 cells was similar for the full 4 days of the assay. Mannosidase-α-class 2B-member 2 (MAN2B2) knockdown caused the proliferation of the ES and Rh30 cell lines to level off at 25% to 65% within 1 to 2 days. Mixed-Lineage Leukemia Protein 3 (MLL3) knockdown caused the proliferation of the EW8 and ES7 cell lines to level off at 52% and 70% respectively. MLL3 knockdown did not inhibit the proliferation of Rh30 cells when compared to siRNA control cells. In contrast, ES6 cell proliferation was also not inhibited by MLL3 knockdown when compared to their siRNA control cells. The knockdowns of DAX1, MLL3 and MBD1 suggest that these EWS-FLI1 binding partners would likely provide potential targets for ES drug development. The effect of knocking down Y2H-identified EWS-FLI interactors on colony formation, migration and tumorigenicity is ongoing.

#2561

Wip1 constrains p53 activity during embryogenesis to permit normal neuronal development.

Sharlyn J. Mazur, Yantenew Gete, Aamir Akbarali, Charles Halsey, R Mark Simpson, Ettore Appella. _National Cancer Institute, Bethesda, MD_.

The oncoproteins MDM2 and Wip1 (PPM1D) each negatively regulate tumor suppressor p53 activity and overexpression of either promotes tumorigenesis. Both MDM2 and PPM1D genes are characteristically induced by p53 following exposure to DNA damage and both negatively regulate p53, but by different mechanisms. In mice, homozygous deletion of Mdm2 is embryonically lethal (E5.5-E6.5) resulting from excess p53-dependent apoptosis, whereas Ppm1d-/- embryos exhibit partial mid-gestation lethality. To investigate the role of p53 in the partial lethality of Ppm1d-/- embryos, we generated an endogenous Ppm1d promoter β-galactosidase reporter allele, Ppm1dΔ3(LacZ), and a new Ppm1d knockout allele, Ppm1dΔ3. We also generated Trp53-/-Ppm1dΔ3/Δ3 double knockout embryos and mice. From Ppm1d+/Δ3 × Ppm1d+/Δ3 crosses, approximately 40% of Ppm1dΔ3/Δ3 embryos die between E10.5 and E12.5 and an additional 40% die shortly after birth, leading to the survival of only approximately 20% of Ppm1dΔ3/Δ3 pups to adulthood. Sections of Ppm1d+/Δ3(LacZ) embryos show dispersed LacZ staining activity with regions of higher activity, especially in specific regions of developing neuronal tissues. The developing neural tissues of Ppm1dΔ3/Δ3 embryos are characterized by distorted cellular architecture, cell dropout, and dark, pyknotic nuclei. Although the frequency of apoptotic cells was nearly three-fold higher in Ppm1dΔ3/Δ3 compared with Ppm1d+/+ embryos (p < 0.005), the number of apoptotic cells were less than 1%, even in the knockout. Interestingly, the numbers of pHH3 positive cells (M phase) in ventricular and sub-ventricular regions of neural tissue were reduced in E10.5 (p < 0.01) and sharply reduced in E11.5 (p < 0.05) Ppm1dΔ3/Δ3 embryos compared with Ppm1d+/+ embryos. Intriguingly, the numbers of γ-H2AX foci per cell observed in Ppm1dΔ3/Δ3 sections were approximately three-fold higher than in Ppm1d+/+ sections (p < 0.002). The specific phenotypes of excess mid-gestation embryonic lethality, excess neuronal apoptosis, and deficient ventricular and sub-ventricular pHH3 cell numbers, but not excess in γ-H2AX foci numbers, were rescued by loss of p53 in Trp53-/-Ppm1dΔ3/Δ3 embryos. These results support a specific function of Wip1 in regulating p53 activity to ensure normal development of the brain and spinal cord during embryogenesis.

#2562

Stromal antigen 1 (SA1) as a potential pro-neoplastic factor in non-small-cell lung cancer (NSCLC).

Michelle Zhang, Mart Dela Cruz, Navneet Momi, Sanjib Chowdhury, Hemant Roy, Adam Lerner. _Boston Medical Center, Boston, MA_.

Introduction:

SA1, encoded by the STAG1 gene, is a subunit of the higher-order chromatin remodeler cohesin. SA1 deficiency has been implicated as a driver of aneuploidy and tumorigenesis (Remeseiro et al, EMBO J 2012). Furthermore, our lab has recently shown that SA1 functions as a tumor suppressor protein in early colon cancer (Wali et al, Cancer Prev Res 2016). The aim of this project was to investigate the role of SA1 in NSCLC. Combining data from The Cancer Genome Atlas (TCGA), human lung tissue, and in vitro gene knockdown, we have surprisingly identified SA1 as a potential pro-neoplastic factor in NSCLC.

Methods:

Data was extracted from TCGA and plotted to characterize STAG1 gene alteration in NSCLC. Immunohistochemistry (IHC) was performed on human lung tissue microarray assay (TMA) to assess SA1 protein expression. Transient knockdowns were performed on A549 human lung adenocarcinoma cells using STAG1 siRNA (Dharmacon) with an incubation time of 48 hours. Following this, quantitative real-time polymerase chain reaction (qPCR, Life Technologies) and WST-1 cell proliferation assays (Promega) were performed as per protocol.

Results:

STAG1 alterations occur in about 3% of lung adenocarcinoma and 17% of lung squamous cell carcinoma. Of these alterations, the majority were amplifications. Furthermore, although not reaching statistical significance, there was a trend towards decreased survival with STAG1 alteration in adenocarcinoma patients (11.6 months vs. 46 months). IHC demonstrated a 1.7-fold increase in SA1 protein expression in NSCLC when compared with non-malignant lung tissue (p<0.001). Transfected A549 cells showed a 78% decrease in SA1 RNA expression (qPCR, p=0.0002) and a 50% decrease in cell proliferation (WST-1, p<0.0001). Proliferating cell nuclear antigen (PCNA), a marker of cell proliferation, was also decreased by 37% in transfected cells (p<0.001).

Conclusions:

Using TCGA data, we found that STAG1 alteration occurs with some frequency in NSCLC and that many of these alterations are amplifications of the gene. Furthermore, there is a trend towards decreased survival with a STAG1 gene alteration. Our IHC data shows robust over-expression of SA1 in NSCLC when compared with non-malignant lung tissue. Our qPCR data showed a profound decrease in SA1 mRNA expression following transfection; accordingly, our WST-1 proliferation data suggests that knockdown of the STAG1 gene significantly decreases proliferation of A549 lung adenocarcinoma cells. Decreased PCNA confirms a true anti-proliferative effect, rather than a pro-apoptotic effect. This novel data suggests a link between SA1 and NSCLC, of which there are no prior published accounts. Even more compelling is the implication that in NSCLC, SA1 may be pro-neoplastic rather than anti-neoplastic, which introduces a new potential target for future gene therapy.

#2563

LSD1 maintains differentiation and survival of mammary luminal cells and suppresses invasion of luminal breast cancer cells via GATA3.

Xin Hu,1 Dongxi Xiang,2 Luwei Tao,2 Ying Xie,2 Yue Jin,1 Yidi Guo,1 Luca Pinello,3 Guo-Cheng Yuan,3 Zhe Li2. 1 _Jilin University, Changchun, China;_ 2 _Brigham & Women's Hospital, Boston, MA; _3 _Dana-Farber Cancer Institute, Boston, MA_.

LSD1 (also known as KDM1A) is a histone demethylase that has been shown to play both oncogenic and tumor suppressor roles in breast cancer. However, the exact context under which it functions as a tumor suppressor or oncoprotein remains largely elusive. To address this, we characterized its role in normal mammary epithelial cells (MECs) and found MEC-specific ablation of Lsd1 largely phenocopied that of Gata3, which encodes a master regulatory transcription factor for luminal MECs. In the luminal lineage, LSD1-loss led to reduction in both mature ductal and alveolar luminal subpopulations. In human luminal breast cancer cells, we found LSD1 interacts with GATA3 and their common target genes are highly related to breast cancer. In particular, we found LSD1 might be recruited by GATA3 to the GATA3 promoter region, to positively regulate GATA3 expression via demethylation of H3K9me2. Knockdown of LSD1 led to increased invasion of luminal breast cancer cells, in part via downregulation of GATA3 and multiple cell junction genes (e.g., CDH1, VCL, CTNNA1), as well as upregulation of mammary stem/progenitor cell genes (e.g., ELF5, ITGB1). However, LSD1 knockdown also led to reduced proliferation of luminal breast cancer cells, in part via downregulation of RBBP4 (encoding a Histone-binding protein) and upregulation of CDKN1A (encoding p21). Collectively, our data suggest that LSD1 is required for maintaining luminal MEC differentiation and survival and for suppressing luminal breast cancer cell invasion, in part via GATA3. As inhibitors of LSD1 are currently under clinical trials (e.g., in leukemia), our data suggest that although treatment of luminal breast cancer by the LSD1 inhibitor may lead to reduced tumor cell proliferation (and thus tumor shrinkage), the treatment may also lead to increased invasion of surviving breast cancer cells. Thus, our study is expected to have important implications for developing better epigenetic therapy in an individualized, context-dependent fashion.

#2565

VCP/p97 segregase-mediated regulation of chromatin-bound PTEN stability.

Jinshan He, Gulzar Wani, Qianzheng Zhu, Altaf A Wani. _The Ohio State University, Columbus, OH_.

Phosphatase and Tensin homolog (PTEN) tumor suppressor protein is associated with many human cancers. PTEN has been shown to accumulate in the nucleus and control DNA repair in response to genotoxic stress. PTEN down-regulation impairs the capacity of global genomic nucleotide excision repair by suppressing the early DNA damage sensor, Xeroderma pigmentosum C (XPC), protein. Our recent work has demonstrated that the Valosin-containing protein (VCP)/p97 segregase participates in proteolytic processing of XPC and Cockayne syndrome B (CSB) proteins. Here, we show that (i) VCP/p97 segregase, in conjunction with its adaptor proteins, specifically extracts PTEN from damaged chromatin for targeted proteasomal degradation, (ii) inhibition of VCP/p97 activity induces the accumulation of PTEN on chromatin, and (iii) VCP/p97 physically interacts with PTEN. Depletion of VCP/p97, in p97-knockout cells or siRNA-mediated ablation in normal cells, affects the stability of PTEN in response to cellular UV irradiation. Results show that compromising the proteasome and VCP/p97 functions causes the accumulation of PTEN accompanied by an increase of UBXD7, proteasomal RPN2 and Sug1 within the chromatin compartment. To our knowledge, this is the first report to reveal that VCP/p97 segregase extracts tumor suppressor PTEN from damaged chromatin. (This work was supported by NIH grants ES002388 and ES012991 to AAW).

#2566

Positive cross-regulatory loop links ZBTB7A to estrogen receptor alpha expression in breast cancer.

Mary Ellen Molloy, Monika Lewinska, ThanhThao Nguyen, Zhi-Min Yuan. _Harvard TH Chan School of Public Health, Boston, MA_.

In breast development and tumorigenesis the biological functions of estrogen are mediated by estrogen receptor alpha (ERα). Approximately 70% of breast cancers are classified as ERα positive and are therefore candidates to receive various forms of endocrine therapy. De novo or acquired resistance to endocrine therapies limits their clinical effectiveness. Identification of novel targets is critical to combat disease progression. Our lab has recently discovered that ZBTB7A suppresses glycolysis by transcriptionally repressing key genes within the glycolytic pathway in various cancer types (Liu, X.S., et al. 2014). Currently, ZBTB7A is proposed to be an oncogene in breast cancer (Zu, X., et al. 2011), however its function in ERα positive breast cancers has yet to be fully addressed. Using publically available datasets, we found that in luminal breast cancers relapse free survival was improved for patients whose tumors expressed high levels of ZBTB7A. Gene expression analysis of TCGA datasets revealed that ZBTB7A and ERα are highly correlated in human breast tumors prompting us to evaluate their potential cross regulation. ERα mRNA and protein levels were significantly decreased upon ZBTB7A silencing both in MCF-7 and T47D breast cancer cell lines. Upon estradiol stimulation, ZBTB7A silencing reduced ERα target gene expression and estrogen response element driven luciferase reporter activity. Additionally, silencing of ZBTB7A inhibited estradiol-induced proliferation and decreased sensitivity to tamoxifen in MCF-7 and T47D cells. These data suggest that ZBTB7A may be required to mediate the pro-proliferative signal of estradiol through its regulation of ERα expression in breast cancer cell lines. To evaluate the potential regulation of ERα on ZBTB7A, ERα was silenced and ZBTB7A mRNA and protein expression were determined. ERα silencing had no effect on ZBTB7A mRNA levels, but led to decreased ZBTB7A protein expression. These effects on ZBTB7A protein expression were attenuated by the presence of the proteasomal inhibitor MG132. Together, these data suggest a model in which ZBTB7A promotes ERα expression at the transcript level while ERα stabilizes ZBTB7A post-transcriptionally conferring a positive feedback loop and sensitivity to estradiol and endocrine therapies. ZBTB7A may contribute to the transcriptional program maintaining a hormone and endocrine therapy responsive phenotype in breast cancer.

#2567

CYP24A1-induced vitamin D insufficiency promotes breast cancer growth.

Makoto Osanai, Akira Takasawa, Kumi Takasawa, Masaki Murata, Norimasa Sawada. _Sapporo Medical Univ., Sapporo, Japan_.

Vitamin D plays a critical role in the maintenance of tissue homeostasis by regulating the expression of genes affecting cell proliferation, differentiation, and apoptosis. The vitamin D 24-hydroxylase CYP24A1 functions in vitamin D target tissues to degrade the hormonal form of vitamin D. Existing knowledge regarding dysregulated CYP24A1 expression supports its candidacy as a putative oncogene. In our presentation, we demonstrate that the suppression of constitutive CYP24A1 expression by CYP24A1-specidic shRNA conferred target cells with increased susceptibility to apoptosis and consequently inhibited anchorage-independent growth in breast carcinoma cells. In addition, suppression of vitamin D metabolism following knockdown of CYP24A1 significantly reduced tumor growth in vivo. Since these data provide substantial evidence for a pro-survival and stimulatory oncogenic effect of CYP24A1 in breast carcinoma cells, we will discuss the potential feasibility of CYP24A1-inhibitory cancer therapies.

#2568

Mutant p53 regulates HSP70 expression and nuclear localization.

Kishore Polireddy, Kanchan Singh, Melissa Pruski, Wasim Dar, John S. Bynon, Jennifer M. Bailey. _University of Texas Health Science Center, Houston, TX_.

Pancreatic cancer is the 3rd leading cause of cancer related deaths in the US with a median survival of less than one year. No effective therapies or early detection tests are available for these patients. Scientists have been studying cellular origins of pancreatic cancer to aid in the development of novel treatments and biomarkers. Several studies have demonstrated that acinar cells have a high propensity to undergo acinar to ductal metaplasia and form precursor lesions of pancreatic ductal adenocarcinoma. We have recently shown that simultaneous expression of Kras and mutant TP53 can generate invasive ductal adenocarcinoma from ductal cells. We hypothesized specific mutations in TP53 have different mechanisms of transforming ductal cells. In order to understand the role of mutant TP53 in transforming pancreatic ductal cells into invasive ductal adenocarcinoma, we used a lentiviral system to express mutant TP53R175H and TP53R273H, two of the most frequently mutated TP53 alleles in pancreatic cancer patients, in immortalized pancreatic ductal epithelial cells carrying a Kras mutation (HPNE-KrasG12D). Trypan blue dye exclusion assay and Spheroid formation assays were used to study cellular proliferation and cancer stem cell (CSC) populations. Mutant TP53 over expression enhanced CSC populations without altering cellular proliferation in HPNE-KrasG12D cells. Reverse phase protein array (RPPA) was carried out to detect gene expression changes in HPNE-KrasG12D cells upon mutant TP53 over expression. RPPA assay results suggested that TP53R175H uniquely induced HSP70 expression in HPNE-KrasG12D cells, as cells expressing either vector control or TP53R273H failed to do so. HSP70 expression was further validated by transiently overexpressing TP53R175H and TP53R273H. Surprisingly, TP53R175H specifically promoted nuclear localization of HSP70 without altering the expression of a recently identified HSP70 nuclear transporter, Hikeshi. Future studies will determine 1) whether HSP70 is required for p53-mediated stemness in HPNE-KrasG12D cells and 2) the function of nuclear HSP70. In summary, over expression of mutant p53 enhanced cancer stem cell properties of HPNE-KrasG12D cells, through upregulation HSP70. The exact mechanism behind HSP70 nuclear localization and increased cancer stem cell properties is being more rigorously explored.

#2569

BCL6 promotes glioma and serves as a novel therapeutic target.

Liang Xu,1 Ye CHEN,1 Marina Dutra-Clarke,2 Anand Mayakonda,1 Masaharu Hazawa,1 Steve E. Savinoff,2 Ngan Doan,3 Jonathan W. Said,3 William H. Yong,3 Henry Yang,1 Ling-Wen Ding,1 Yan-Yi Jiang,1 Jeffrey W. Tyner,4 Jianhong Ching,5 Jean-Paul Kovalik,5 Markus Müschen,6 Joshua J. Breunig,3 De-Chen Lin,1 Phillip Koeffler1. 1 _National University of Singapore, Singapore, Singapore;_ 2 _Cedars-Sinai Medical Center, UCLA, Los Angeles, CA;_ 3 _UCLA, Los Angeles, CA;_ 4 _Oregon Health & Science University, OR; _5 _Duke-NUS Graduate Medical School, Singapore, Singapore;_ 6 _University of California San Francisco, CA_.

ZBTB transcription factors orchestrate gene transcription during tissue development. However, their roles in glioblastoma multiforme (GBM) remain unexplored. Here, through a functional screening of ZBTB genes, we identify that BCL6 is required for GBM cell proliferation and is overexpressed in GBM samples. Using a somatic transgenic mouse model, Bcl6 confers the proliferative and invasive stimuli to glioma cells in vivo. Moreover, we discover AXL as a novel downstream target of BCL6. Through AXL, BCL6 enhances both MEK-ERK and S6K-RPS6 axes. Pharmacological inhibition of BCL6 activity effectively blocks GBM growth and inhibits AXL expression. Together, these findings uncover a novel glioma-promoting role of BCL6, and provide the rationale of targeting BCL6 as a potential therapeutic approach.

#2570

Induction of mild oxidative stress as a strategy for reactivation of mutant p53 proteins: KSS72, a small molecule derived from ethacrynic acid restores the biological functions of R248W/Q mutant.

Surendra R. Punganuru, Hanumantha Rao Madala, Kalkunte S. Srivenugopal. _Texas Tech Univ. Health Sciences Ctr., Amarillo, TX_.

p53 gene mutations in 50% of human cancers drive the emergence of oncogenic genomes and confer resistance to anticancer drugs & apoptosis. Therefore, a pharmacological reactivation of p53 mutant proteins to their wild-type forms has emerged as an intense research area. We have shown that induction of oxidative protein modifications primarily underlie the mechanistic conversion of mutant to wt or wt-like p53 forms (Eur J Medchem 107:233, 2016; Int J Oncol 48:1426, 2016). R248W/Q DNA contact mutations in the p53 core are highly frequent and cause functional inactivation. No small molecules reactivating this p53 mutation are available. Here, we describe the design and synthesis of a small molecule KSS72 using a cell-based screening and its potency in reactivating the R248 p53 mutants. KSS72 is a non-diuretic hydrophobic analog [1-(2,3-dichloro-4-methoxyphenyl)-2-methylenebutan-1-one] of ethacrynic acid (Edecrin) obtained by removing the carboxylate side chain. KSS-72 retained the ability to bind with GSH, inhibit GSTs and induce marked redox-imbalance in tumor cells. KSS72 displayed preferential cytotoxicity (3-4 fold more cell killing) against cancer cells expressing the allele specific R248W mutation (MiaPaca-2) in comparison with the wt p53 or p53-null cancer cell lines. Further, we developed isogenic cell lines expressing the R248W and R248Q mutants in the p53-null H1299 lung cancer cells and evaluated their sensitivity towards KSS72; these models again verified the greater drug efficacy. To prove the hypothesis the higher efficacy of KSS72 stems from the reactivation of mutant p53, the conformation-specific antibodies (Pab1620 for wt and Pab420 for mutant) were employed. Immunoprecipitation and immunostaining using confocal microscopy showed that KSS72 treatment of R248W or R248Q p53-cells results in a marked reciprocal loss of mutant protein and gradual increase of wt-like protein. EMSA revealed a time-dependent restoration of DNA-binding of the mutant p53, which was accompanied by a distinct upregulation of pro-apoptotic signaling pathways mediated by PUMA, Bax, p21cip1 and MDM2 proteins. A strong G2/M arrest and significant apoptosis were observed. Other data showed that KSS72 restores the wt structure and function by covalently binding the p53R248 protein. Pharmacokinetics showed that KSS72 has excellent oral bioavailability with a half-life of 3.5 h. In MiaPaca-2 xenografts developed in nude mice, there was a marked tumor growth suppression with KSS72 as a single drug. In conclusion, KSS72 is an attractive lead compound for further development because p53 reactivation occurred without discernible organ toxicities after oral doses and a wide therapeutic window. The redox-sensitive cysteines involved in the p53 reactivation are being identified by mass spectrometry (supported by CPRIT grants RP130266 & RP170207 to KSS).

#2571

DBC1, a novel CBP-interacting protein, promotes p53 stability by regulating CBP-dependent p53 polyubiquitination.

Oluwatoyin E. Akande,1 Priyadarshan K. Damle,1 Nicholas E. Sherman,2 Steven R. Grossman1. 1 _Virginia Commonwealth University, Richmond, VA;_ 2 _University of Virginia, Charlottesville, VA_.

The acetyltransferase CBP, in conjunction with the E3 ubiquitin ligase enzyme, Mdm2, maintains physiologic levels of the tumor suppressor protein p53 in the absence of cellular stress, via a cytoplasmic, but not nuclear, p53-directed E4 polyubiquitin activity. CBP also possesses cytoplasmic, but not nuclear, E3 ubiquitin ligase, autoubiquitination activity. To understand the mechanism by which the ubiquitin ligase activities of CBP are compartmentalized in the cell, we have employed Multidimensional Protein Identification Technology (MudPIT) to identify cytoplasmic and nuclear CBP interacting proteins. MudPIT analysis revealed that Cell Cycle and Apoptosis Regulator protein (CCAR2), also known as Deleted in Breast Cancer 1 protein (DBC1), is a novel CBP- interacting protein in both the nucleus and cytoplasm. The N-terminus of DBC1 bound to multiple CBP domains, including the putative E3/E4 domain of CBP located at the N-terminus. Interaction between DBC1 and CBP suppressed the in vitro E3 autoubiquitination activity of CBP. Functional studies demonstrated that DBC1 directly regulates the cellular compartmentalization of CBP E3 and p53-directed E4 ubiquitin ligase activities. Knockdown of DBC1 in U2OS cells stimulated normally absent nuclear p53 polyubiquitination, and also caused a decrease in p53-dependent and p53-independent apoptosis, rescued by CBP/DBC1 double knockdown. Further, over-expression of DBC1 rescued the increase in nuclear p53 polyubiquitination and decrease in p53 half-life observed in DBC1 depleted cells. Together, these results identify DBC1 as a novel binding partner of CBP and a critical regulator of CBP ubiquitin ligase activities. Based on our data that DBC1 loss inactivates p53 function through destabilization, we queried the TCGA database for co-occurrence of DBC1 loss with maintenance of wild-type p53 status which showed that 33% of breast, 40% of lung, and 96% of prostate cancer cases with DBC1 alterations maintained wild-type p53 status. Therefore, by restoring DBC1 function, this work could lead to reactivation of p53 tumor suppressor activity in tumors with DBC1 alterations maintaining wild-type p53 status.

#2572

The novel long non-coding RNA PANCR is an intracellular p53 activator.

Yu Cao. _Southern illinois University, Springfield, IL_.

Long non-coding RNAs (LncRNAs) function as oncogenes or tumor suppressors in development and progression of cancer. Chromosome 16q22.1 region is frequently deleted in breast cancer, which may contribute to breast carcinogenesis by inactivation of tumor suppressor genes. This study characterized a new LncRNA tumor suppressor, named p53 activating non-coding RNA (PANCR), located in this Chromosome 16q22.1 region. This PANCR LncRNA consists of 1.5kb in length.Our data showed that PANCR was downregulated in breast cancer cell lines and tissues. In the breast cancer cell lines, PANCR expression appeared reversely correlated with cell malignancy, and in breast cancer tissues, PANCR was downregulated over 2 times in 31(62.0%) of 50 cases when compared to adjacent normal breast tissues. In breast cancer cells MCF7 and immortalized human mammary epithelial cells MCF10A, ectopic expression of PANCR induced marked apoptosis, suppressing cell proliferation in culture and tumor growth in xenografts, but in contrast, shRNA-mediated silencing of PANCR promoted cell growth and proliferation. Mechanistic approaches revealed that in both MCF7 and MCF10A cell, PANCR activated p53 and upregulated pro-apoptotic proteins bid and bim and cell cycle inhibitors p21waf/cip1 and p27Kip1. We further explored the mechanism of action that PANCR activates p53, and our results from RNA immunoprecipitation (RIP), RNA pulldown and immunoprecipitation (Co-IP) demonstrated that the PANCR can bind to p53, dissociate p53-MDM2 complex and thus activate p53.Together our data suggest that the novel LncRNA PANCR located in the deleted Chromosome 16q22.1 region is an intracellular p53activator, functioning as a tumor suppressor by activating p53-mediated apoptosis.

#2573

TP73's regulation and expression in human cancer cell lines.

Zhixing Yao, David R. Yao, William Yu, Hassan Brim, Hassan Ashktorab, Zaki A. Sherif. _Howard Univ., Washington, DC_.

TP73 is the homologue of the master tumor suppressor TP53. It is involved in cellular responses to stress and development. The TP73 gene encodes two different proteins, TAp73 and ΔNp73 and maps to a region on chromosome 1p36 that is frequently deleted in neuroblastoma and other tumors, and thought to contain multiple tumor suppressor genes. However, the analysis of p73-knockout mice yielded conflicting results with respect to tumor suppression. World-wide efforts in sequencing the TP73 gene in patient tumor samples have not provided evidence for genetic alterations as a common cause of p73 inactivation in human cancer. The role of TP73 in tumorigenesis has remained elusive to date. In this study, we isolated two stem cell lines from normal young and old human liver tissues and determined TP73 expression in human breast cancer cell lines (MCF7 & MDA321), human hepatocellular carcinoma (HCC) cell lines (HepG2, SNU398, SNU449 & SNU475), neuroblastoma cell lines (IMR32 & SK-N-SH), normal liver stem cell, non-cancerous Li-Fraumeni Syndrome (LFS) skin fibroblasts cells and normal skin fibroblasts. Results show that TAp73 only expresses in cancer cell lines. Moreover, when HepG2 and MDA231 were treated with paclitaxel, a cytoskeletal drug that targets tubulin, TAp73 expression was drastically down-regulated in HepG2 cells and abrogated in MDA231 cells at 1 to 5 µmol/L concentrations. Furthermore, ChIP assay results demonstrated that TP53 and CTCF (a chromosomal networking protein CCCTC binding factor) bind to TP73 promoter and regulate TP73 expression. Our observation may prove significant for the development of future therapeutic and diagnostic applications.

*These authors contributed equally to this work.

#2574

The PICT-1 is required for USP7 to effectively stabilize p53 protein, and codon 389 polymorphism is a risk factor for ovarian cancer.

Masafumi Yoshimoto, Aoi Tokuda, Yuji Yaginuma. _Kumamoto Univ. Graduate School of Health Sci., Kumamoto, Japan_.

The ovarian cancers causes the deaths of more than 100,000 women per year worldwide, and it is a complex disease, with multiple both histological subtypes and molecular characteristics recognized; therefore, overall survival rates have improved little in the last 30 years. We have to precisely identify genetic and molecular events which could serve as markers for early diagnosis or targets for new therapeutics for ovarian cancers.

The PICT-1 (also named GLTSCR2), one of the nucleolar proteins, has several tumor suppressor functions, such as the stabilization of PTEN and p53. Some studies reported that the loss of or low PICT-1 expression is the common alteration in many cancers, such as brain, skin, lung, esophageal, prostate, kidney, breast, and cervix, but its association with ovarian carcinogenesis has been unknown. Therefore, we investigated its alteration, regulation mechanism, and the effect for p53 stabilization in ovarian cancer.

We found that polymorphism on codon 389 (rs184994) was a statistically significant association with the risk of ovarian cancers. Furthermore, ectopic expression of PICT-1 was required for USP7 to effectively stabilize p53 protein. Because PICT-1 was reported to bind both p53 and MDM2 that enabled to form USP7-p53-MDM2 complex, we investigated whether PICT-1 was regulated by USP7-p53-MDM2 complex. Our results suggested that this complex was the important regulator for PICT-1. In the investigation of the role of PICT-1 that played in the ovarian carcinogenesis using immunohistochemical stainig, we found that PICT-1 protein levels decreased in ovarian cancer tissues compared to normal ovary, and there was a positive correlation between PICT-1 and USP7 protein expression. Our findings suggested that PICT-1 involved in exactly p53 regulation along with MDM2 and USP7, and PICT-1 should be considered as a potential target for therapeutic human cancers.

#2575

**Concurrent mutation of** CDKN2A **and** TP53 **loci in primary keratinocytes.**

Bin Li, Edmund A. Mroz, James W. Rocco. _The Ohio State University Comprehensive Cancer Center, Columbus, OH_.

Concurrent inactivation of the p16Ink4a and p53 tumor suppressors, often through mutation of CDKN2A and TP53, is frequent in early development of head and neck squamous cell carcinoma (HNSCC) and other cancers. The role of this early combined inactivation, and the oncogenic drivers that subsequently lead to tumor development, are poorly defined. To characterize the precancerous role of concurrently mutated CDKN2A and TP53, we combined a number of recent advances to disrupt both loci in primary cells. First, we grew primary keratinocytes in medium conditioned by irradiated J2-3T3 cells and containing an inhibitor of ROCK kinases, conditions that greatly extend their normally limited proliferative potential. Second, we transfected primary keratinocytes with plasmid vectors expressing both Cas9 and sets of guide RNAs targeting specific regions of CDKN2A and TP53. We targeted either or both of the p14Arf and p16Ink4a protein products of CDKN2A for knockout (KO), together with either TP53 KO or a TP53 point mutation (R248Q protein product), two major categories of genetic alterations of p53 in HNSCC. Third, we evaluated use of nutlin-3, an inhibitor of p53-MDM2 interaction, to try to select against cells that still contained wild-type p53. We assessed KO efficiency by PCR of genomic DNA; the TP53 point mutation introduced a SauA3i site into TP53. Two weeks of nutlin-3 treatment led to a 3 fold increase in p53 KO efficiency versus untreated cells (50% versus 14%). p16Ink4a KO efficiencies were enhanced 2 to 14 fold after nutlin-3 treatment, depending on the specific Cas9/guide-RNA constructs used. In cells transfected to produce p53 R248Q, 14% of PCR products amplifying the mutated region after nutlin-3 exposure showed Sau3Ai digestion, whereas no Sau3Ai digestion was detected in untreated cells. This result suggests that up to 14% of nutlin-3 treated keratinocytes had integrated the mutated donor oligo sequence. We similarly enrich for mutated keratinocytes having p14Arf-specific or combined CDKN2A KO along with TP53 KO or mutation. Combined transfection to generate concurrent CDKN2A and TP53 mutations under conditions that allow extended proliferation in culture, followed by selection for successful TP53 mutation with nutlin-3, will facilitate identification of keratinocyte clones with bi-allelic knockout genotypes. This panel of keratinocytes with concurrent CDKN2A and TP53 mutations will help delineate the roles of these genes in early cancer development and will allow screening for the oncogenic drivers required for tumor development and metastasis, with emphasis on discovering more effective and specific druggable targets for improving treatment.

#2576

Benzyl isothiocyanate potentiates p53 signaling and antitumor effects against breast cancer through activation of p53-LKB1 and p73-LKB1 axes.

Arumugam Nagalingam,1 Bei Xie,1 Panjamurthy Kuppusamy,2 Nethaji Muniraj,1 Peter Langford,1 Balázs Győrffy,3 Neeraj Saxena,2 Dipali Sharma1. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _University of Maryland, Baltimore, MD;_ 3 _Hungarian Academy of Sciences,, Hungary_.

Functional reactivation of p53 pathway, although arduous, can potentially provide a broad-based strategy for cancer therapy owing to frequent p53 inactivation in human cancer. Using a phosphoprotein-screening array, we found that Benzyl Isothiocynate, (BITC) increases p53 phosphorylation in breast cancer cells and reveal an important role of ERK and PRAS40/MDM2 in BITC-mediated p53 activation. We show that BITC rescues and activates p53-signaling network and inhibits growth of p53-mutant cells. Mechanistically, BITC induces p73 expression in p53-mutant cells, disrupts the interaction of p73 and mutant-p53, thereby releasing p73 from sequestration and allowing it to be transcriptionally active. Furthermore, BITC-induced p53 and p73 axes converge on tumor-suppressor LKB1 which is transcriptionally upregulated by p53 and p73 in p53-wild-type and p53-mutant cells respectively; and in a feed-forward mechanism, LKB1 tethers with p53 and p73 to get recruited to p53-responsive promoters. Analyses of BITC-treated xenografts using LKB1-null cells corroborate in vitro mechanistic findings and establish LKB1 as the key node whereby BITC potentiates as well as rescues p53-pathway in p53-wild-type as well as p53-mutant cells. These data provide first in vitro and in vivo evidence of the integral role of previously unrecognized crosstalk between BITC, p53/LKB1 and p73/LKB1 axes in breast tumor growth-inhibition.

#2577

Expression of P53/PTEN/c-Myc in peripheral blood leukocytes from patients with breast cancer and its clinical significance.

Guangcheng Huang,1 Qiang Ma,2 Jiang Zou,1 Ru Sun,1 Lihua Yao,1 Lei Xu,1 Dongsheng Wang,2 Xiaolan Guo2. 1 _North Sichuan Medical College, Nanchong, China;_ 2 _Affiliated Hospital of North Sichuan Medical College, Nanchong, China_.

Breast cancer remains the most frequently diagnosed malignancy and leading cause of cancer deaths in women worldwide. Current data showed that the morbidity of breast cancer has been rising shifting with younger age in the last decades, therefore early detection is urgently needed to improve the management of breast cancer as what has been working on many other malignancies. Dysfunction of tumor suppressor gene and/or oncogene might play vital roles in tumorigenesis and cancer progression, such as P53, PTEN and c-Myc. In this study we aim to detect the expression of P53/PTEN/c-Myc mRNA in peripheral blood leukocytes from patients with breast cancer and benign breast diseases, and investigate its correlation with the clinicopathologic characteristics, further to explore the potential clinical application significance.

In this study, fluorescent quantitation Real-time Polymerase Chain Reaction (FQ-RT-PCR) was employed to detect the expression of P53/PTEN/c-Myc mRNA in peripheral blood leukocytes from 304 cases, including 105 patients with breast cancer, 107 cases of benign breast diseases and 92 healthy women as control. 2-ΔΔCT was used to calculate the relative quantitative gene expression and Mann-whitney U test was carried to distinguish the differences among the independent cohorts. The results showed that P53 and PTEN mRNA were significantly reduced in both breast cancer group and benign breast diseases group, respectively(P<0.01), moreover, the P53 mRNA in benign breast diseases was even lower than that in breast cancer(P<0.01). However, c-Myc didn't show any obvious difference among the three groups. Further investigation the genes expression with clinicopathologic characteristics, we found that PTEN in patients with breast cancer was correlated with lymph nodes metastasis and clinical stage as well. Among those breast cancer patients without lymph node metastasis, the level of PTEN mRNA in peripheral blood leukocytes was obviously lower than that in patients with metastatic lymph node positive (P<0.05), on the other hand, breast cancer patients with ductal carcinoma in situ and earlier stages(0+I+II) showed lower PTEN mRNA than those in later or advanced stages (III+IV) (P=0.01).

Above results indicated that P53 and PTEN mRNA in peripheral blood leukocytes displayed apparent difference among patients with breast cancer, benign breast diseases and healthy control, and dysregulation of P53/PTEN was found in patients with benign breast diseases, even more obvious than that in breast cancer patients. Moreover, down-regulation of PTEN might predict and/or display the early events of breast cancer carcinogenesis and lymph nodes metastasis. Our findings suggest that detection of tumor related genes in peripheral blood leukocytes would serve as a unique potential approach in early and noninvasive diagnosis of tumors, also would be very promising in clinical applications as well.

#2578

Investigating the role of mutant p53 in esophageal squamous cell carcinoma.

Ashley Lento,1 Apple Long,1 Veronique Giroux,1 Qiaosi Tang,1 Morgan Sammons,1 Andres Klein-Szanto,2 Shelly Berger,1 Anil Rustgi1. 1 _The University of Pennsylvania, Philadelphia, PA;_ 2 _Fox Chase Cancer Center, Philadelphia, PA_.

Esophageal squamous cell carcinoma (ESCC) is a highly aggressive cancer characterized by a high rate of metastasis, limited therapeutic options, and a poor prognosis. However, there is limited information regarding the molecular mechanisms underlying the metastatic properties of ESCC. p53 is one of the most commonly mutated genes in ESCC, and our group has shown that esophageal cells lines expressing a mutation in human p53 shows signs of malignancy and increased invasion in 3D organotypic culture. A mouse model of mutant p53 (R172H) in ESCC is lacking in the field. To elucidate the role of mutant p53 in ESCC we developed a novel mouse model utilizing a genetic and carcinogenic approach. L2cre;p53-/- and p53R172H/- mice were generated and treated with 4NQO in their drinking water for 16 weeks, which resulted in the development of ESCC. Compared to wildtype mice, p53R172H/- mice and p53-/- mice exhibited a decreased tumor latency time, increased tumor frequency and a more severe tumor diagnosis. However, p53R172H/- mice and p53-/- mice displayed similar tumorigenic properties. RNA-seq was performed on cell lines established from wildtype and p53 mouse models and reveled different gene expression profiles between wildtype, p53R172H/-, and p53-/- cells. p53R172H/- cells displayed an increase in mesenchymal and decrease in epithelial marker expression, supporting the idea that they are undergoing EMT. In addition, several endocytic recycling related genes, including Rab11-fip1, Rab25, and Myo5b were downregulated in mutant and null p53 compared to wildtype cells. Further examination of the differing genetic profiles in our mouse models can provide novel insight into the role of mutant p53 in ESCC tumorigenesis and lead to the identification of new therapeutic targets.

#2579

Identifying pathways that regulate the turnover of gain-of-function p53 mutant proteins in ovarian cancer cells.

Achuth Padmanabhan,1 Nicholes Candelaria,1 Kwong-Kwok Wong,2 Bryan C. Nikolai,1 David Lonard,1 Bert W. O'Malley,1 JoAnne S. Richards1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _M D Anderson Cancer Center, Houston, TX_.

Ovarian cancer is the fifth leading cause for cancer-associated deaths among women in the US. High-grade serous ovarian carcinoma is the most common histological subtype and is characterized by frequent (>90%) mutations in the TP53 (p53) gene. Gain-of-function (GOF) p53 mutant proteins form highly stable aggregates that accumulate in ovarian cancer cells and have been shown to have oncogenic activity. Increasing evidence indicates the stabilization of GOF p53 mutants to be important for tumor progression and drug resistance. Therefore, selective degradation of the GOF p53 mutant proteins is a highly attractive therapeutic strategy to specifically target ovarian cancer cells. We identified that a small molecule named MCB-613, originally discovered as a stimulator of steroid receptor coactivator-3 (SRC-3), causes rapid degradation of the usually stable p53R175H conformational mutant aggregates leading to catastrophic cell death in human ovarian cancer cell lines. Our results indicate this effect is independent of SRC-3 mediated gene expression, but rather by post-translational modification of p53 mutant protein. Upon MCB-613 treatment, the p53R175H mutant is rapidly exported from the nucleus, ubiquitinated and degraded through a lysosomal pathway. Interestingly, in contrast to its effect on the p53R175H mutant, MCB-613 causes a slight stabilization of wild type (WT) p53 but had less of an effect on the turnover of other frequently observed p53 mutants (R248Q and R273H) in human ovarian cancer cell lines. MCB-613 also causes a rapid decrease in MDM2 protein but not mRNA. Further, the effect of MCB-613 on p53R175H is phenocopied by multiple deubiquitinase (DUB) inhibitors, confirming the role of ubiquitination in the selective turnover of p53R175H mutant aggregates. Taken together, these results show that different pathways regulate the turnover of WT p53 and the specific GOF p53 mutant proteins. We are currently investigating the precise mechanism by which MCB-613 and the different DUB inhibitors induce selective turnover of p53R175H mutant protein. We anticipate the proposed work to significantly advance our understanding of pathways important in ovarian cancer progression and pave way for the development of new targeted therapies for ovarian cancer patients with specific p53 mutations. This work is funded by NIH-CA-181808, NIH-HD-07857 and NIH-HD-076596.

#2580

Ceramide-rubusoside nano-micelles restores p53-mediated tumor suppression in p53R248W mutant ovarian cancer cells.

sachin K. khiste,1 Zhijun Liu,2 Yong-Yu Liu1. 1 _University of Louisiana at Monroe, Monroe, LA;_ 2 _Louisiana State University, Monroe, LA_.

Ceramide (Cer), a sphingolipid metabolite, has crucial functions in anti-proliferation, cell differentiation and apoptosis. Cellular Cer levels highly correlates to therapeutic efficacy of anticancer drugs, however, the hydrophobicity Cer has limits to develop it as a therapeutic agent. Here, we report that Cer-rubusoside (Cer-RUB) nanomicelles enhanced Cer bioavailability and restored p53-mediated tumor suppression. These nanomicelles significantly increased the levels of NBD-C6-Cer by more than 50-fold in serum, 40-fold in tumor and 70-fold in lung of tumor-bearing mice (1 mg/kg, i.p., 24 hr), respectively. Cer-RUB (3 µM) significantly increased OVCAR-3 (p53 R248W) cell sensitivity to cisplatin, and the IC50 values decreased by 3-fold, even though these values were no significant changes in A2780 (wt p53) cells. Furthermore, Cer-RUB treatments (1 mg/kg, i.p. every 3 days for 24 days) significantly increased cisplatin efficacy in tumor growth-arrest in OVCAR-3 tumor-mice, and the tumor volumes reduced by 52% (1653 vs. 805 mm3) in combination with cisplatin, compared to cisplatin alone. We found that ovarian cancer stem cells (CD44+/CD133+) decreased by 10-fold (0.85 vs. 0.08% of total cells) in OVCAR-3 tumors treated with Cer-RUB combined with cisplatin, as compared to cisplatin alone, respectively. Western blotting showed that Cer-RUB treatments selectively increased the expression levels of phosphorylated p53 (Ser15), BAX and p21 in OVCAR-3 cancer cells and tumors, respectively. These studies indicates that the gain-of-function (GOF) of p53 R248W leads OVCAR-3 cell resistance to cisplatin and tumor growth. Cer-RUB can restore p53 and eliminates the GOF of p53 R248W in cancers.

#2581

VGLL4 suppresses the human breast cancer progression through the inhibition of YAP oncogenic function.

Jianmin Zhang. _Roswell Park Cancer Inst., Buffalo, NY_.

VGLL4 is a Vestigial-like (VGLL) protein that is deregulated in various types of cancer. However, the functions of VGLL4 in breast cancer remain largely unknown. Here, we demonstrate that VGLL4 expression positively correlates with survival for breast cancer patients. The overexpression of VGLL4 in breast cancer cells reduced cell proliferation, colony formation, cell migration and invasion in vitro and inhibited tumor growth in vivo. We found that the 2nd TEAD-interacting domain (TDU2) of VGLL4 is involved in interactions between VGLL4 and the transcription factor TEAD and that the deletion of this domain completely abolished the phenotype induced by VGLL4 overexpression. Interestingly, RNAseq analysis indicated that VGLL4 overexpression not only inhibits YAP targets but also activates the TGF-β and Notch signaling pathways. Our study provides evidence of the tumor-suppressive effects of VGLL4 on breast cancer progression and suggests that VGLL4 and its TDU2 domain may become promising and efficient therapeutic targets for delaying the development of breast cancer and improving patient survival. 

## BIOINFORMATICS AND SYSTEMS BIOLOGY:

### Computational Tools and Databases

#2582

KIRnome: KIR genotyping for whole genome/exome sequencing data.

Daeyoon Kim,1 Sung-Soo Yoon,1 Youngil Koh,1 Su Yeon Lee,2 Hongseok Yun,2 Sunghoon Cho,3 Hyung-Lae Kim4. 1 _Seoul National Univ. Cancer Research Inst., Seoul, Republic of Korea;_ 2 _Development Group, Healthcare Analytics Team, Samsung SDS, Seoul, Republic of Korea;_ 3 _Functional Genome Institute, PDXen Biosystems Inc, Seoul, Republic of Korea;_ 4 _Department of Biochemistry, Ewha Womens University College of Medicine, Seoul, Republic of Korea_.

In this study, we tried to develop valid KIR calling algorithm from WES/WGS data. As a consequence, here we suggest KIRnome (KIR typing for whole genome and exome sequencing) which is a KIR typing method for applying sequencing data such as WES/WGS. A total of 71 sequencing data (18 WES and 53 WGS data, respectively) and matched experimentally validated KIR genotype data are used to train and validate. Before developing this method, two types of allele references (genomic and coding, here after called gSeq and cSeq) are constructed by allele sequence of IPD/KIR. KIRnome consists of two part, first part is calculating normalized depth (here after called depth) of 16 KIR genes and considering allele reference length and second is estimating KIR type based on depth. In general, we get higher normalized depth when using genomic references for WGS data and using coding references for WES data. This coincides with scheme KIRnome and the characteristics of WGS and WES data. Given 71 samples of 3 data sets, we evaluated KIR genotyping performance of KIRnome per each KIR gene. For all 16 KIR genes, KIRnome shows >=98.5% accuracy. When KIRnome performance was tested only in 13 WES samples, the accuracy slightly drops to 94%. We assume this is attributable to relatively small number of the samples. In conclusion, we developed a novel and unique method named KIRnome for KIR typing from NGS data. KIRnome could determine KIR genotype accurately from WES and WGS data respectively. We expect KIRnome would facilitate revealation of immunogenetic facts in various disease. Moreover, future generation of KIR specific NGS data and improved reference sequence information of KIR would enable KIRnome to type KIR at allele level in a near future.

#2583

A model for capturing and integrating complex molecular alterations related to clinically relevant efficacy evidence.

Cara M. Statz, Sara E. Patterson, Taofei Yin, Susan M. Mockus. _The Jackson Laboratory for Genomic Medicine, Farmington, CT_.

The ability to capture data relevant to different types of oncology-related variations, including single nucleotide variations, deletions, insertions, copy number variations, and fusions in a single system is crucial to a comprehensive understanding of cancer biology. However, collectively linking these varied types of molecular alterations to capture their compound impact to clinically relevant efficacy evidence within a database can prove challenging. Thus, we have built the JAX Clinical Knowledgebase (JAX-CKB), a flexible relational database that allows curation of complex molecular profiles and provides the ability to associate these profiles with documented efficacy evidence, thereby providing a more detailed overview of therapeutic relevance. To demonstrate the utility of creating complex molecular signatures in relation to efficacy evidence, the JAX-CKB was queried to first determine the overall degree of efficacy evidence content related to complex profiles. Additionally, two specific types of complex molecular profiles were queried, which included EML4-ALK plus a missense mutation(s) and BRAF V600E plus any type of molecular alteration(s). Within the JAX-CKB, there are 1,383 unique efficacy evidence lines linked to complex molecular profiles. The complex molecular profiles consisting of EML4-ALK and one or more additional missense mutations were associated with 174 unique efficacy evidence lines. Of the 174 lines, 69 were annotated with a resistant response type, while 68 were annotated with a sensitive response type. The combination of BRAF V600E with one or more molecular alterations was linked to 229 unique efficacy evidence lines. The majority of the lines, 118, were specific to a resistant response type and 83 were associated with sensitivity. Comprehensive genomic profiling of cancer patient samples can often reveal complex molecular signatures. The JAX-CKB is an inclusive knowledgebase that allows one to interpret these complex signatures and rapidly identify appropriate targeted therapies, which could be critical in a clinical setting.

#2584

The UCSC Xena system for cancer genomics data visualization and interpretation.

Mary Goldman, Brian Craft, Jingchun Zhu, David Haussler. _UC Santa Cruz, Santa Cruz, CA_.

The UCSC Xena platform (http://xena.ucsc.edu/) allows biologists and bioinformaticians to securely analyze and visualize their private functional genomics data in the context of public genomic and clinical data sets. The Xena platform consists of a set of federated data hubs and the Xena browser, which integrates across hubs, providing one location to analyze and visualize all data. Our expanding public Xena Data Hubs currently hosts 1400+ data sets from more than 35 cancer types, as well as Pan-Cancer data sets. Our public data hubs serve seminal cancer genomics and functional genomics data set to the scientific community, including the latest TCGA, TARGET, ICGC, and GTEx data sets. We support most data types including somatic and germline SNPs, INDELs, large structural variants, CNV, gene-, transcript-, exon- protein-, miRNA-expression, DNA methylation, phenotypes, clinical data, subtype classifications and genomic biomarkers. Additionally, investigators' own functional genomics data can be hosted on private hubs running on their laptop or behind the firewall. Data is integrated on the UCSC Xena Browser, allowing biologists to view and interpretation of their genomic data in the context of a large collection of cancer genomics data sets such as TCGA. The lightweight Xena data hubs are straightforward to install on Windows, Mac and Linux operating systems and loading data is easy using either our application or command line interface. This system of the browser and hubs helps researchers combine new or preliminary results from their laptops or internal servers, or even data from a new paper, securely with vetted data from the public sphere.

Visualizations and analyses include dynamic Kaplan-Meier survival analysis to assess survival stratification by any information in addition to our visual spreadsheet, scatter plots and bar graphs. We seek feedback at our poster on new visualizations and functionalities.

#2585

viGEN: An open source bioinformatics pipeline for viral RNA detection and quantification in human tumor samples.

Krithika Bhuvaneshwar, Lei Song, Yuriy Gusev. _Georgetown University, Washington, DC_.

We present a novel pipeline for viral RNA detection and quantification in human RNA-seq data. Our pipeline has been tested on the TCGA liver cancer cohort, can not only detect the presence of a viral species, but also provide gene level read counts for individual viral species and extract viral-variants.

Introduction Approximately 20% of human cancer types are associated with viral infection that is routinely detected in blood samples. However the extent and biological significance of viral presence/infection in actual tumor samples is generally unknown but could be measured using existing Human RNA-seq data from tumor samples. We have developed a bioinformatics pipeline viGEN combining existing and novel RNAseq tools that allows for detection and quantification of viral RNA in human RNAseq data.

Methods The pipeline includes 4 major modules: The first module allows to align and filter out human RNA sequences; second module maps and count (remaining un-aligned) reads against reference genomes of all known and sequenced human viruses; the third module calculates quantitate read counts at the individual viral genes level thus allowing for downstream differential expression analysis of viral genes between experimental and controls groups. The fourth module calls variants in these viruses. To the best of our knowledge there are no publicly available pipelines or packages that would provide this type of complete analysis in one package. Customized solutions have been reported in the literature however were not made public.

Results We used this pipeline to examine viruses present in RNA-seq data from 75 liver cancer patients in the TCGA data collection. Our pipeline allows conducting quantitative analysis at the gene level for visualization and detection of statistically significant differentially expressed viral genes between groups of patients known to be infected with both HBV and various subtypes of HCV. Once the viral genomes are detected at the genome level, we examine the differences between "Dead" and "Alive" samples at the viral-transcript level, and at the viral-variant level.

Conclusion Our results show that it is possible to detect viral sequences from whole-transcriptome (RNA-seq) data in humans. We were able to not only quantify them at a viral-gene/CDS level, but also extract variants from the 75-sample dataset from TCGA. The results presented here are in correspondence with published literature and are a proof of concept of our pipeline. This pipeline can be used on cancer and non-cancer human RNA-seq or other NGS data to provide additional insights into the biological significance of viral infection in complex diseases, tumorigeneses and cancer immunology.

#2586

CellMinerCDB: Enabling cross-database exploration of molecular pharmacology data and response determinant discovery in cancer cell lines.

Vinodh N. Rajapakse,1 Augustin Luna,2 Chris Sander,2 William C. Reinhold,1 Yves Pommier1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Dana-Farber Cancer Institute, Boston, MA_.

Cancer cell line panels are widely used for evaluating drug response across diverse tissue types. A growing set of molecular profiling data complements measurements of chemosensitivity, providing novel avenues for response determinant discovery and clinical translation. Accessing and inter-relating data from different sources is essential for evaluating such determinants, but remains challenging. To enable wider access to cell line pharmacogenomic data, we have developed CellMinerCDB (CellMiner Cross-Database, discover.nci.nih.gov/cellminercdb), a web application integrating data from several widely studied cancer cell line panels, including the NCI-60 (NIH), GDSC (Sanger/MGH), and CCLE/CTRP (Broad). All together, our database spans over 1300 distinct cell lines, 400 clinically relevant cancer drugs, 20,000 experimental compounds, and molecular profiling data, such as gene/protein expression, DNA copy, methylation, and mutational status. Cell line and tested drug overlaps allow cross-database validation of genomic and drug data, and CellMinerCDB simplifies this by transparently matching differently named entities between sources. Data exploration can be additionally restricted to particular tissue types, with individual cell lines annotated to the OncoTree ontology for consistent treatment across sources.

A range of analysis tools support interactive data exploration, from 2D plots of drug response and molecular profiling features to exhaustive correlation analyses and multivariate predictive models. We illustrate the power and utility of CellMinerCDB with examples of response determinant discovery and predictive modeling for Top1 and PARP inhibitors. Beginning with established individual determinants, such as SLFN11 mRNA expression, we show how both unbiased and biological knowledge network-based feature selection methods enable iterative refinement of a multivariate genomic signature of drug response. For Top 1 inhibitors, additional predictive features include expression of chromatin remodeling factors and genes modulating apoptosis capacity, while complementary PARP inhibitor response determinants include PARP1 and drug efflux pump expression. Pathway and process-based gene annotations allow biological interpretation of response predictive features. CellMinerCDB also includes ongoing algorithmic work to improve the construction of multivariate predictive models using constraints from biological networks. These approaches bridge a limiting gap in existing methods, which either ignore biological knowledge altogether or are limited to exploration within known pathways and processes.

#2587

VCF2CNA: a tool for efficiently detecting copy number alteration using VCF genotype data.

Daniel K. Putnam, Xiaotu Ma, Stephen V. Rice, Yu Liu, Jinghui Zhang, Xiang Chen. _St. Jude Children's Research Hospital, Memphis, TN_.

Whole genome sequencing (WGS) is increasingly used in both research and clinical settings. The Variant Call Format (VCF) specification is a widely adopted file format for genetic variation data exchange partially due to its smaller file size compared to raw WGS BAMs. Each variant in a typical VCF file contains its chromosome position, reference/alternative alleles and corresponding allele counts. This makes it possible to identify copy number alterations (CNAs). To this end, we developed VCF2CNA (http://vcf2cna.stjude.org), a web interface tool for CNA analysis from VCF files.

A user of VCF2CNA, uploads a VCF file via the provided web interface. The entire analysis runs remotely with an average run time of 23 minutes. Results are emailed to the user as either a downloadable link or file attachments. VCF2CNA also accepts input in the Mutation Annotation Format (MAF) and the variant file format produced by the Bambino program.

We analyzed 22 TCGA glioblastoma tumor/normal pairs by Illumina technology to evaluate VCF2CNA's performance. It achieved high consistency (average F1-score: 0.952 ± 0.082) with CONSERTING, a tool that incorporated read-depth and SV data from raw BAMs for CNA detection. A segment-by-segment comparison between results from CONSERTING and VCF2CNA indicated that the latter was less sensitive to focal CNAs. This is expected because there is less information in the VCF input than in raw BAMs. Further analysis using samples with a "fractured genome" pattern revealed that VCF2CNA was more robust to library artifacts and produced relatively clean CNA profiles (on average 76.2-fold reduction compared to the number of segments reported by CONSERTING).

Finally, we analyzed 137 pediatric neuroblastoma samples from the TARGET project, sequenced by Complete Genomics, Inc. (CGI) technology. MYCN amplification has been clinically validated in 33 samples. VCF2CNA identified high amplitude MYCN gains in 32 samples and the remaining sample carried a low-level broad gain covering MYCN. For comparison, CGI's HMM-based method reported MYCN gains in only 15 out of the 33 samples. VCF2CNA further identified two additional MYCN amplifications among the remaining samples. Collectively, our analysis suggests that VCF2CNA is a platform-independent, efficient, robust and accurate tool for general WGS-based CNA analysis. It further complements CONSERTING, which produces more accurate result in focal CNAs at the cost of significantly higher computational burden.

#2588

GenePattern Notebook: an environment for reproducible cancer research.

Michael M. Reich,1 Thorin T. Tabor,1 Ted Liefeld,1 Barbara Hill,2 Helga Thorvaldsdottir,2 Jill P. Mesirov1. 1 _University of California, San Diego, La Jolla, CA;_ 2 _Broad Institute, Cambridge, MA_.

As the availability of genetic and genomic data and analysis tools from large-scale cancer initiatives continues to increase, the need has become more urgent for a software environment that supports the entire "idea to dissemination" cycle of an integrative cancer genomics analysis. Such a system would need to provide access to a large number of analysis tools without the need for programming, be sufficiently flexible to accommodate the practices of non-programming biologists as well as experienced bioinformaticians, and would provide a way for researchers to encapsulate their work into a single "executable document" including not only the analytical workflow but also the associated descriptive text, graphics, and supporting research. To address these needs, we have developed GenePattern Notebook, based on the GenePattern environment for integrative genomics and the Jupyter Notebook system. GenePattern Notebook unites the phases of in silico research – experiment design, collaborative analysis, and publication – into a single interface.

GenePattern Notebook presents a familiar lab-notebook format that allows researchers to build a record of their work by creating "cells" containing text, graphics, or executable analyses. Researchers add, delete, and modify cells as the research evolves, supporting the initial research phases of prototyping and collaborative analysis. When an analysis is ready for publication, the same document that was used in the design and analysis phases becomes a research narrative that interleaves text, graphics, data, and executable analyses, serving as the complete, reproducible, in silico methods section for a publication.

GenePattern Notebook features are designed to make it easy for nonprogramming users to create and adapt notebooks. We have developed new cell types that allow users to choose analyses, specify input parameters and datasets, navigate results, send result files to new analyses, and create richly formatted text, all without the need for programming.

We have released a freely available online GenePattern Notebook workspace, http://notebook.genepattern.org, where researchers can develop and publish notebook documents. We have provided a collection of template notebooks that walk users through various machine learning analyses, and are collaborating with cancer research laboratories to create integrative cancer genomics notebooks as well. Notebook topics in development include characterization of intratumoral heterogeneity from single cell RNA-Seq data, effective clinical interpretation of comprehensive genomic profiling from whole exome sequencing of a patient's tumor and germ line samples, and identification of master regulators/transcription factors associated with the downstream transcriptional effects associated with the activation of an oncogene.

#2589

Identifying cancer susceptibility variants and genes using whole exome and genome sequencing: a literature review.

Melissa Rotunno,1 Mindy Clyne,1 Tram Lam,1 Leah Mechanic,1 Danielle Daee,1 Stefanie Nelson,1 Elizabeth Gillanders,1 Alisa Goldstein2. 1 _NCI-DCCPS, Rockville, MD;_ 2 _NCI-DCEG, Rockville, MD_.

The application of Next Generation Sequencing (NGS) technologies in cancer research enabled the discovery of novel somatic mutations at an unprecedented speed through efficient high-throughput profiling of DNA variation in tumor samples. In contrast, the identification through NGS studies of germline variation associated with cancer has been more difficult and with limited consensus around best practices for study design and analytical techniques. We performed a review of the 2005-2016 PubMed English literature for genome-wide germline sequencing studies that were aimed at identifying new cancer susceptibility genes or variants. We extracted and summarized the experimental and analytical designs, methods and tools implemented in the selected literature together with their degree of success and limitations in identifying cancer susceptibility variants. About 80 in-scope publications were identified and examined in depth. The most studied cancer types were colorectal, breast, blood and lung and were investigated predominantly via whole exome sequencing (primarily using Illumina HiSeq) in familial cases of Caucasian ethnicity. Only 10 studies used whole genome sequencing and 15 adopted a case control study design. A large fraction of the familial studies was limited to 1 or 2 families and sequenced 2 or less cases per family. Reporting of key data quality information (e.g., human sequence coverage) was inconsistent throughout the publications. While the methods for sequence alignment and variant calling converged towards those implemented in established bioinformatic tools (e.g., BWA, GATK), there were no standard approaches for filtering or prioritizing of variants. Most studies included some degree of technical validation, often restricted to the top few variants or genes. About 60% of the studies included replication in an independent group of cancer patients and 55% functionally validated their findings to some extent. Our work provides valuable data and insights to inform future sequencing studies of cancer genetic susceptibility, and points to the need for improving standardization and completeness in reporting of quality control metrics, study design rationale and interpretation of findings.

#2590

Analysis of APOBEC3A and APOBEC3B mutational signatures using next-generation sequencing data from cancer cell lines.

Suleyman Vural, Julia Krushkal, Richard Simon. _National Cancer Institute, Rockville, MD_.

The APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) gene family of cytidine deaminases includes evolutionarily conserved genes that play important roles in DNA repair and mRNA editing. Activity of at least two APOBEC family members, APOBEC3A and APOBEC3B, can lead to kataegis, a mutagenic process in cancer cells that generates clusters of closely spaced, single strand specific C->T DNA substitutions. APOBEC mutagenesis has a characteristic signature, most commonly represented by the 5'-Tp(C->T)pW-3' sequence motif, with additional substitutions also reported. This hypermutation signature and high mRNA expression of APOBEC3A and APOBEC3B have been associated with several cancer types. Most previous studies of APOBEC signatures have examined tumor sequence data from clinical samples, for which limited or no information about drug response was available. We investigated the presence of the mutational signature and mRNA expression patterns of the APOBEC3A and APOBEC3B genes in extensively characterized cell lines, in order to identify those cell lines that carry mutations generated by kataegis, with the aim of establishing associations between the APOBEC mutational signature, individual cancer types, and the patterns of sensitivity to antitumor agents. For this purpose, we analyzed whole exome sequencing (WES) data and mRNA expression of the APOBEC3A and APOBEC3B genes in two resources with extensive drug response data: the NCI-60 cell line panel, which includes 59 human cancer cell lines representing 9 cancer types and drug response information for thousands of anticancer agents, and the Cancer Cell Line Encyclopedia (CCLE), which provides WES, whole genome, and RNA-seq sequence information on hundreds of cancer cell lines and drug response data to over 200 agents. We analyzed WES data of 325 CCLE cell lines and 59 NCI-60 cell lines, with variants identified using GATK pipeline and Varscan2 software. The variants in each cell line were filtered to remove common polymorphisms in dbSNP and 1000 Genome Project databases. We searched the discovered variants for the presence of APOBEC signatures, 5'-Tp(C->AGT)pN-3', 5'-Tp(C->AGT)pD-3', and 5'-Tp(C->AGT)pW-3' in closely spaced (1000 and 10,000 bp) windows that appeared on the same DNA strand. We will discuss the use of optimal filters for detecting APOBEC mutational signatures and will present the analyses of associations between APOBEC signatures, mutational load of the tumor cell lines, APOBEC gene expression, and chemosensitivity to treatment. These results contribute to additional characterization of available cell lines by providing information about specific mutational signatures in different categories of cancer. Our findings may assist with identifying antitumor agents that would be appropriate for treatment of cancer cells with specific signature patterns generated by APOBEC mutagenesis.

#2591

A guide to filtering TARGET Complete Genomics germline variants.

James P. Evans,1 Rajesh Patidar,2 Zalman Vaksman,1 Sivasish Sindiri,2 Douglas R. Stewart,2 Javed Khan,2 Jun S. Wei,2 Sharon J. Diskin1. 1 _Children's Hospital of Philadelphia, Philadelphia, PA;_ 2 _National Cancer Institute, Bethesda, MD_.

With the public release of TARGET's pediatric cancer data, researchers are eager to investigate and integrate variants from both tumor and germline sequencing data, but caution should be used when interpreting TARGET variant calls from Complete Genomics data, as they unlike the Illumina based calls for which most of our databases and tools are customized. Here, we use germline samples from 55 neuroblastoma cases with both whole genome Complete Genomics variants and whole exome Illumina variants from GATK, Pindel, Platypus, and bam2mpg to investigate differences between germline variant calls from Complete Genomics and Illumina platforms. Our study of ~160,000 single nucleotide polymorphisms (SNPS) and ~19,000 insertions or deletions (indels) reveals that while SNP sets largely agree between Complete Genomics and Illumina, Complete Genomics data have many indels not present in Illumina calls, making it difficult to screen Complete Genomics indels against popular Illumina based variant databases like those provided by the Exome Aggregation Consortium and the National Heart, Lung, and Blood Institute Exome Sequencing Project. Further probing of Complete Genomics indels uncovered inconsistent indel formatting resulting a loss of recurrent variants, and miscalculated read depths in earlier versions of the Complete Genomics platform. To solve these issues and make Complete Genomics variant calls comparable to Illumina calls, we introduce a Snakemake pipeline that reformats Complete Genomics variant files and creates variant features for use in decision tree or random forest classifiers trained to recognize Complete Genomics variants that will be present in Illumina variant call sets. We determine that the most important features for locating Complete Genomics variants likely to be found in Illumina germline variant call sets are the presence of reads from both directions, average variant allele fraction across the cohort, a variant call in the matched tumor, the variant fraction of reads, and read depth. Using ten fold cross validation with our decision tree, we obtain >95% recall and >98% precision for SNPs, and >90% recall and >85% precision for indels. We advocate the use of our pipeline and filtering scheme for making Complete Genomics variant calls comparable to Illumina based calls.

#2593

Accelerating pediatric brain tumor research through team science solutions.

Amanda Christini,1 Angela J. Waanders,2 Joost B. Wagenaar,1 Alex S. Felmeister,2 Mariarita Santi,2 Nitin R. Wadhwani,3 Jennifer L. Mason,2 Mateusz P. Koptyra,2 Jena V. Lilly,2 Jeffrey W. Pennington,2 Rishi R. Lulla,3 Adam C. Resnick2. 1 _Blackfynn, Incorporated, Philadelphia, PA;_ 2 _The Children's Hospital of Philadelphia, Philadelphia, PA;_ 3 _Ann & Robert H. Lurie Children's Hospital of Chicago, Chicago, IL_.

Introduction: The Children's Brain Tumor Tissue Consortium (CBTTC), an international repository of genomic and phenotypic data, has partnered with Blackfynn, Inc., to create a cloud-based data management platform to facilitate team-science across disciplines.

Background: The CBTTC through the CHOP Department of Biomedical and Health Informatics (DBHi) has developed a network of informatics and data applications for researchers across the globe to work together and perform real-time analyses on existing clinical, phenotypic, and genomic data. Historically, rare disease datasets are siloed, locked in proprietary formats, segregated by data types, and hidden from the view of experts in the field. This has been a significant barrier to finding effective therapeutics for children with pediatric brain tumors. Blackfynn was founded by a group of multidisciplinary experts in neuroscience, neurology, medicine, software development, engineering, computer science and business with the goal to empower researchers to cure neurologic disease and provide solutions to these challenges.

Description of Methods: The CBTTC and Blackfynn teamed up to provide a cloud-based, team-focused data management and analytics platform. The platform provides a commercial grade, scalable approach to upload, view, and integrate digital pathology images with relevant subject data such as MRIs, pathology reports and genomic information. Stakeholders can search integrated data without requiring users to change their current workflow or conform to imposed data standards. This platform is a simple, intuitive, end-to-end software platform for teams of scientists and pathologists to review, annotate and discuss cases, enabling rapid diagnostic consensus, quality control, and empowered discovery.

Summary of Unpublished Results: The CBTTC/Blackfynn data platform enabled CBTTC members to engage in a cross-institutional collaboration to reach consensus on digital pathology data in ways that were previously not possible. We demonstrated that this solution removes existing barriers to collaborative efforts and provides a rich analytic and discovery platform bridging imaging with genomics and other data formats. The platform provides a new model for the scientific community to facilitate translation towards improved treatments for children diagnosed with brain tumors.

Discussion and Future Direction: This pilot project will be scaled to other CBTTC sites for centralized review of pathology images to enable the research community to collaborative on specific projects. The next phase of platform development will include further integration CBTTC platforms fully integrating genomics data, and side-by-side viewing and analyses of MRI, pathology and clincal data to facilitate specific project work around large and complex research data types in a cloud environment.

#2594

Optimizing the replication of cancer genomics workflows: case studies.

Jerry Fowler,1 F. Anthony San Lucas,1 Smruthy Sivakumar,1 Aditya Deshpande,2 Humam Kadara,3 Paul A. Scheet1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Weill Cornell Graduate School of Medical Sciences, New York City, NY;_ 3 _American University of Beirut, Houston, TX_.

Reproducing results is a major issue in cancer biology, whose "work bench" is dynamic and complex, with frequently updated algorithms and software. The better to manage our work in this environment we have developed SyQADA, a System for Quality-Assured Data Analysis -- a workflow automation system designed to simplify common sequential analysis processes on the same or different data. SyQADA manages many of the details of procedural bookkeeping involved in bioinformatics workflows: What samples are we using? Where are the raw data? Were all the samples processed? Did every job complete satisfactorily? Is there as much output as expected? Where are the input files for the next step? How long does a typical job take to run? Which program versions did we use? Can we easily compare these results with the output of a different version of a program, or with different input data?

Using SyQADA, we have found ourselves better able to reproduce results while at the same time reducing the human effort required to manage our upstream data analyses. Here, we briefly describe how our lung cancer studies have benefitted from the use of SyQADA.

To understand the effect of different variant callers for Ion Torrent deep sequencing data in a lung cancer genomics study, we created a work protocol that allowed us to compare the different sets of variants called on 34 distinct somatic DNA samples from 4 patients. This complex processing framework involved running multiple variant callers, annotating variants, filtering germline variants using quality control metrics, and collating results across samples and callers. With SyQADA, we were able to re-run individual processes changing parameters with trivial changes to our configuration, yielding improved output. We then applied that unmodified protocol to the 500 samples from 48 individuals in our study, and rapidly produced data from which we could perform biological analysis. We then applied the protocol to a study of pre-malignant lesions in 25 lung cancer patients. In both studies, our workflow allowed us to generate comparable results in a matter of hours rather than days.

SyQADA has been used by individuals with backgrounds ranging from expert programmer to Unix novice, to perform and repeat dozens of diverse analytical workflows. Projects to which SyQADA has been applied include allelic imbalance studies of TCGA samples for cancers of the breast, pancreas, lung, and colon, processing roughly 6000 samples through a dozen steps.

A zipfile containing the SyQADA executable source code, documentation, tutorial examples, and workflows used in our lab will be available.

#2595

Enabling petabyte-scale cancer genomics with the NCI Cancer Cloud Pilots.

Gaurav Kaushik, Yilong Li, Erik Lehnert, Zeynep Onder, Devin Locke, Brandi N. Davis-Dusenbery, Deniz Kural. _Seven Bridges, Cambridge, MA_.

The advent of next generation sequencing has accelerated the generation of genomic data and created a need for methodologies to organize, share, and analyze large volumes of data. To date, petabytes of multi-dimensional information from thousands of patients have been collected. Access and analysis of this information becomes increasingly challenging as the amount of data grows. This difficulty is exemplified when we consider data generated by the efforts of The Cancer Genomics Atlas (TCGA) network, which encompasses more than 2.5 petabytes. Historically, downloading the complete TCGA repository can require several weeks with a highly optimized network connection and access to large institutional compute clusters to perform integrated analysis, which is out of reach for many researchers. The Cancer Cloud Pilot project seeks to directly address these challenges by co-localizing data with the computational resources to analyze it where researchers can access it securely and easily. The project was born out of the recognition that conducting biological research is increasingly computationally-intensive and new approaches are required to support effective data discovery, storage, computation, and collaboration. Funded by the National Cancer Institute, the Cancer Genomics Cloud (CGC) enables researchers to leverage the power of cloud computing to gain actionable insights about cancer biology and human genetics from massive public datasets including TCGA and the Cancer Cell Line Encyclopedia on the CGC. Our approach to create a cancer cloud platform includes collaborative tools, security permissions, data harmonization, and making the data easier to query through the use of metadata curation, resource description frameworks, and visual tools. Additionally, we implemented the Common Workflow Language, an emerging standard for describing computational workflows, to support computational reproducibility. To date, more than 1200 researchers have accessed and analyzed TCGA and analyzed more than 50000 samples on the CGC since its launch in February 2016. In addition to the motivation, inception, and development of the CGC, we will present a case study on the application of unsupervised learning methods to identify individual cell types within tumors using RNA Sequencing data from TCGA cohorts. We will demonstrate how these computationally-intensive methods are benefited by the cloud and how researchers can apply open pipelines to interrogate cancer subtypes and mixed cell populations from TCGA data on their own data.

#2596

MediSapiens Explorer Technology Platform - Integrated Solution for big biomedical data.

Henrik Edgren, MediSapiens bioinformatics and development teams. _MediSapiens Ltd, Helsinki, Finland_.

During recent years, the increasing throughput and decreasing cost of technologies for genomic, transcriptomic, proteomic and other high throughput profiling of samples has enabled collection of very rich data sets on large sample cohorts. As methods have been developed to analyze e.g. large human germline sequencing studies, it has simultaneously become clear that the potential of the data can be fully and effectively leveraged only if technical solutions are developed to curate, store, harmonize and jointly use these omics data sets together with clinical and phenotypic data on the samples.

In the field of modern data-driven research, heterogeneous data is increasingly available through many dispersed resources, and in many cases, data from public sources is combined with proprietary data for analysis. Thus, it is not only important to have effective solutions for data storing and data management, but for data curation and data analysis and visualization. Data curation ensures that data from various sources are harmonized, standardized and cross-comparable. Powerful data analytics and visualization is pivotal in transforming data into information, new insights and added value.

To address these problems, MediSapiens has developed the Explorer™ Technology Platform solution, based on its extensive experience in both industrial software development and academic biomedical research. The Explorer™ Technology Platform has been designed for all of the three major parts of the data research workflow: For curating data, storing and managing data, and analyzing and visualizing data. This web browser based, integrated solution provides functionalities to import, curate, ontology map and store rich clinical data, as well as very large data sets of gene expression, copy number, mutation and other profiling data for both bulk and single-cell analyses (e.g. single-cell RNA-seq), and perform various analyses and visualization on the data.

Existing software components in the Explorer™ Technology Platform can quickly and efficiently be used to construct custom UI tools to query, analyze and interpret data according to individual customer needs. Further, the platform provides APIs which enable retrieval of data for integration with other systems, or development of custom analyses using e.g. R.

Here, we will illustrate the application of Explorer Technology Platform concept to solve a variety of problems in preclinical cancer research and personalized medicine, starting from efficient automated curation of clinical data through to integration of clinical and multi-omics data.

#2597

Celecoxib fixed dose combinations - patient level data analyses.

Sanjive Qazi,1 Lynne Murphy,2 Anshuma Mehta,2 Wen Wang,2 Mihir Munsif,2 Zachary Yim,2 Vuong Trieu2. 1 _Autotelic Inc, Costa Mesa, CA;_ 2 _Marina Biotech, Agoura Hills, CA_.

Background: Celecoxib is a highly effective anti-inflammatory drug. However, its use is associated with adverse events including edema and hypertension (HTN). High dose celecoxib has been shown to be effective against familial adenomatous polyposis (FAP). However, it was discontinued because of safety concerns. Here we examined the impact of celecoxib intake on HTN and edema. The concomitant intake of celecoxib and one of the antihypertensives (AH) was also evaluated as means to suppress the side effects of celecoxib.

Methods: This is a retrospective study that uses two patient level data (PLD) sources: (1) anonymous PLD from Symphony from Jan 2012 to Dec 2014 (3 years), and (2) Pinnacle registry data from American College of Cardiology (ACC) over the same time period with detailed clinical data such as blood pressure (BP) readings, peripheral edema flags, etc. In Symphony dataset there are 162 million (M) unique patients, 4.3M patients on celecoxib, 16.3M patients with osteoarthritis (OA)(15.4M only OA) ,and 2.3M patients with rheumatoid arthritis (RA) (1.4M only RA). In ACC dataset, there are 1.58M unique patients with BP readings, 870K patients with edema flag.

Results: There was no impact of celecoxib consumed on the change in BP readings. There were as many increases as decreases, regardless of the amount of celecoxib consumed between BP readings. A breakdown by baseline BP readings did not reveal any trends. Concomitant use of AH with celecoxib did not impact BP. Onset of edema was not correlated with total dose of celecoxib. Less than 10% of the observations indicated a change in edema status. However, unlike BP, increasing % of edema was observed for increment in doses and days of treatment with celecoxib plus any other drug combination, suggesting drug induced edema. Edema was baselined with concomitant consumption of angiotensin receptor blocker (ARB) or angiotensin converting enzyme (ACE) or hydrochlorothiazide. It was further aggravated by non-thiazide diuretics and minimally impacted by calcium channel blocker.

Conclusion: Previous analysis has shown that dose increment of celecoxib to more than 200mg/day did not improve treatment efficacy, but significantly increased the edema rate but not BP in OA patients. Here we confirmed our meta-analysis using PLD data. Celecoxib with ARB exhibited the lowest incidence of edema and with non-thiazide diuretics the highest incidence (Odds ratio = 3.34 (95% CI = 2.85-3.94), p<0.0001). ARB with celecoxib displayed a significant reduction in edema incidence compared to celecoxib alone (Odds ratio = 0.626 (95% CI = 0.539-0.724), p<0.0001). The celecoxib-ARB and celecoxib-ACE FDCs are being investigated further as therapy for FAP and more broadly for colorectal cancers.

#2598

Paradigm to ensure curation transparency and consistency of a cancer genomic database - the JAX Clinical Knowledgebase (CKB).

Taofei Yin, Sara E. Patterson, Cara M. Statz, Anuradha Lakshminarayana, Daniel Durkin, Susan M. Mockus. _The Jackson Laboratory for Genomic Medicine, Farmington, CT_.

In the era of personalized medicine, comprehensive cancer genomic databases represent critical tools for interpreting gene variants and their relevant therapies. As the number of cancer genomic databases grow, it is challenging for users to compare and assess the data integrity and validity of such databases. The lack of disclosure of curation processes makes it difficult to cross reference different databases. The JAX Clinical Knowledgebase (CKB) is a comprehensive relational database providing evidence-based information on gene variants, targeted therapies, efficacy evidence, and clinical trials. At the Jackson Laboratory for Genomic Medicine, we have developed a curation paradigm to ensure curation transparency and consistency of the JAX-CKB. First, to ensure standardization of nomenclature, we have integrated standard nomenclature and ontologies, including HGNC approved nomenclatures for genes, HGVS guidelines for variants, and the Disease Ontology for indications/tumor types. Second, we implemented decision matrices to maintain uniformity of our evaluation of scientific and clinical data. For example, evaluation of the effect of gene variants on protein function is solely based on changes in the intrinsic activity of the protein instead of downstream pathway activation or the effects on pathogenesis; the response type of gene variants to therapies are classified based on the evidence of targeting specificity; and the efficacy evidence are further classified as actionable, diagnostic, prognostic, risk factor, emerging, or not active centered on the response type and the strength of the evidence. Third, to provide convenient evaluation of the clinical relevance of efficacy data, we further developed a tier ranking system based on emerging consensus guidelines, which takes response type, evidence type, and approval status into consideration. Finally, all decision matrices are clearly outlined on the JAX-CKB open access website for users' reference. In summary, we have curated a highly structured and semantically consistent cancer genomic database following a set of specific curation guidelines. As a result, JAX-CKB open access has become a valuable resource for both the clinical and research communities, and is utilized by numerous independent groups for variant interpretation.

#2599

COSMIC Cancer Gene Census: expert descriptions across genes in oncogenesis.

Zbyslaw Sondka, Sally Bamford, Charlotte G. Cole, Elisabeth Dawson, Laura Ponting, Raymund Stefancsik, Sari A. Ward, John Tate, Peter J. Campbell, Simon A. Forbes. _Wellcome Trust Sanger Institute, Cambridge, United Kingdom_.

The Cancer Gene Census is an ongoing effort to catalogue genes for which somatic mutations have been causally implicated in cancer. The Census comprises manually curated summaries of the most relevant information for cancer-driving genes and their somatic mutations and brings together the expertise of a dedicated curation team, cancer scientists and the comprehensive resources of the COSMIC database. Current research focuses on characterising the participation of 609 census genes in hallmarks of cancer and identification of additional genes involved in these biological traits primarily via altered expression, CNA or epigenetic changes. New overviews of cancer gene function focused on hallmarks of cancer pull together manually curated information on the function of proteins coded by cancer genes and summarises the data in simple graphical form. It presents a condensed overview of most relevant facts with quick access to the literature source, aiming to provide summary characteristics of a cancer gene, rather than a full monography, to avoid information overload. This functional characterisation enables the creation of lists of genes of interest focused on the particular role they play in the development of cancer, as well as aiming to identify the cellular functions affected by mutations in particular tumours, and help to choose right targets for targeted therapy or synthetic lethality experiments. The Census is available from the COSMIC website for online use or download at: http://cancer.sanger.ac.uk/census.

#2600

Analysis of drug resistance mechanisms and strategies for overcoming resistance in cancer therapy using a curated clinical knowledgebase.

Sara E. Patterson, Cara M. Statz, Taofei Yin, Susan M. Mockus. _The Jackson Laboratory for Genomic Medicine, Farmington, CT_.

Emergence of resistance to targeted therapies is a critical problem in cancer therapy, and understanding both the mechanisms of resistance and strategies for overcoming resistance are crucial to effective treatment of cancer patients. The JAX Clinical Knowledgebase (JAX-CKB), which incorporates data on therapeutic efficacy in the context of molecular alterations, enables rapid analysis of known therapy resistance mechanisms and current data surrounding strategies for overcoming resistance, both in the preclinical and clinical setting. Using the JAX-CKB, we have identified over 1250 lines of evidence corresponding to therapy resistance across tumor types. Of those lines, 147 correspond to therapy resistance in lung cancer. The JAX-CKB contains 22 variants in ALK associated with ALK therapy resistance. Within ALK fusion-positive lung cancer, evidence lines corresponding to ALK inhibitor resistance are associated primarily with complex molecular profiles containing secondary ALK mutations, and mechanisms for overcoming resistance in this setting were associated with use of novel agents. Within EGFR mutation positive lung cancer, lines associated with resistance to EGFR inhibitor therapy included copy number alterations, primary resistance mutations, secondary resistance mutations, and expression level changes. Strategies for overcoming resistance in these settings include novel agents and/or various combination therapies. The JAX-CKB provides a unique global view into current data on resistance to targeted therapies in oncology, which may enable more rapid assessment of effective therapy options and expose opportunities for additional research into strategies for overcoming resistance.

#2601

COSMIC-3D: exploring cancer mutations in three dimensions for drug design and discovery.

Harry C. Jubb,1 Harpreet Saini,2 Marcel Verdonk,2 Simon Forbes1. 1 _Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom;_ 2 _Astex Pharmaceuticals, Cambridge, United Kingdom_.

Explosions in the availability of cancer genomic data and protein structure data give us the potential to explore cancer molecular biology at an unprecedented scale, and in atomic detail. We present COSMIC-3D, which combines COSMIC, the most comprehensive cancer mutation database available (http://cancer.sanger.ac.uk), with the wealth of publicly available 3D protein structure data, to create a resource with which the protein-structural nature of cancer can be probed.

COSMIC-3D will help us understand which cancer mutations drive the progression of cancers, by identifying which are in functional sites that have an effect on driving cell growth and proliferation, and where they are clustered in protein structures across the proteome, for specific cancer types. Through understanding the effects of cancer mutations on known and predicted drug binding sites, we aim to predict potential new drug targets in cancers, and improve the specificity and efficacy of new or existing drugs, by using protein structure with cancer mutation data to guide mutation-specific drug design.

COSMIC-3D is available as a web interface at http://cancer.sanger.ac.uk/cosmic3d, and enables interactive exploration of the cancer mutome in a 3D peptide environment, showing all forms of exonic point mutation. Individual mutant locations can be highlighted as molecular surfaces, while recurrence across nonsynonymous substitutions is visualized as 3D heat-maps. Cancer mutations can also be visualised in combination with precalculated small-molecule "druggable" binding sites, providing a powerful visual approach for development of hypotheses across structural, functional, and drug-resistance impacts of cancer variants. COSMIC-3D shows, for example, the steric occlusion of the binding site of ATP-competitive small-molecule inhibitors in the EGFR kinase domain by the mutation of L858 to arginine; these kinds of insights can be applied to novel cancer targets for any protein-structural mutation of interest.

The human cancer structural proteome comprising COSMIC-3D extends to nearly 8,500 human genes; 1/3rd of genes in COSMIC. Over 30,000 human protein structures are available across these genes, to which over 345,000 cancer mutations are mapped. From structure based druggability prediction, over 6,500 proteins in COSMIC are predicted to have small-molecule druggable binding sites, making COSMIC-3D a powerful and exciting resource for the exploration and guidance of drug design and discovery in oncology.

#2602

The ICGC data portal and its underlying open source software architecture.

Junjun Zhang, Bob Tiernay, Dusan Andric, Phuong-My Do, Sid Joshi, Vitalii Slobodianyk, Chang Wang, Shane Wilson, Andy Yang, Vincent Ferretti. _Ontario Institute for Cancer Research, Toronto, Ontario, Canada_.

The goal of the International Cancer Genome Consortium (ICGC) is to analyze the cancer genomes of at least 500 tumour samples with matched controls from 50 different cancer types and subtypes, building a comprehensive catalogue of somatic abnormalities for the benefit of the research community. The amount of data ICGC members will generate is close to that of 50,000 human genome projects and, to date, has received commitments for 107 projects to study more than 27,000 tumor genomes.

The ICGC Data Coordination Center (DCC) is responsible for collecting, curating, aggregating, and disseminating the data generated by the consortium's member projects. Given the size and the complexity of the ICGC data, these tasks represent significant scientific and technological challenges that require a performant, robust software infrastructure. Key to this infrastructure is the ability to scale as data grows.

Using state-of-the-art Big Data, bioinformatics and cloud computing technologies, we developed a suite of web-based applications and microservices that enable member projects to first submit their data and validate their submissions according to the rules defined in the submission specification. Following validation, the data is processed, annotated and loaded into the data portal using a modular Extract-Transform-Load (ETL) pipeline. Submission, ETL and portal systems are built using scalable and distributed technologies such as Hadoop, Spark, MongoDB and ElasticSearch. Spark is used to validate, join, index, and harmonize annotations on submitted variants while ElasticSearch powers our variant query engine, API and portal displays.

Here we present the ICGC Data Portal and describe both the current features and capabilities accessible to users along with the architecture of the underlying infrastructure. The portal provides scientists with powerful and unique tools for exploring and visualizing the millions of variants and annotations available. These include sophisticated, faceted search capabilities making data exploration extremely fast and easy, a suite of interactive Javascript components for in-depth analysis and visualization of specific genomic features, embedded genome and pathway browsers, synthetic cohorts comparisons and a streaming data download service. The portal integrates a large variety of annotations such as variant consequences and frequencies, functional impact factors and druggability. The portal also offers cloud-based tools for searching a catalog of raw ICGC data files stored in worldwide repositories and compute clouds.

All source code is open to the community under the GPLv3 license.

#2603

A patient driven cancer database to collect information, analyze data, and predict outcomes.

Lori Marx-Rubiner,1 AnneMarie Ciccarella,2 Vincent An,3 Jared Bass,3 Will Berman,3 Jackson Berry,3 Chloe Chan,3 Christopher Han,3 Louis Harboe,3 Joshua Lurie,3 Sara Ma,3 Parker Malachowsky,3 Naylee Nagda,3 Simon Schneider,4 E. Bircan Çopur,4 Jorge J. Nieva,5 Jan Liphardt,6 Peter Kuhn,7 Jeremy M. Mason7. 1 _Independent Research Advocate and Metastatic Breast Cancer Patient, Los Angeles, CA;_ 2 _Independent Research Advocate and Breast Cancer Patient, Glen Head, NY;_ 3 _University of Southern California, Iovine and Young Academy, Los Angeles, CA;_ 4 _Zyncd Ltd, Munich, Germany;_ 5 _Norris Comprehensive Cancer Center, Los Angeles, CA;_ 6 _Stanford University, CA;_ 7 _University of Southern California, Los Angeles, CA_.

It is widely held that major breakthroughs in cancer treatment will result from properly amassing, analyzing and utilizing existing and emerging "big data." To date there is no single vehicle that integrates the data available, and of those being developed, none that put patient needs and outcomes as primary foci.

CancerBase was released as part of the Cancer Moonshot launch in June 2016. It is a global, real-time data collection tool designed by and for patients. Patients were recruited through social media to share their information, including but not limited to, demographic details, personal cancer data, and history of treatment. Combining open text boxes and drop down preselected response boxes allow for the capture of information in a way that patients understand. In addition to cancer data, Facebook comments demonstrated immediate engagement with the tool and captured the organic community of patients who eagerly shared their stories with others. Personal interviews and a communal Twitter chat have recorded the interests and priorities from patients who were not necessarily familiar with CancerBase. In addition to medical data, general information and questions CancerBase participants wanted to add to the system have been collected.

Incorporation of patient-driven deliverables is unique in the space of big data and cancer in that other tools, both existing and still in development, are geared toward the needs of researchers, payors, policy-makers and clinicians. CancerBase is unique in that it adapts to growth. Patient feedback drives improvements via ongoing communication between developers, PIs and participants. Wherever possible, participant recommendations and questions are rapidly incorporated into the website design. The remaining patient recommendations are cataloged and will be incorporated in the forthcoming releases of CancerBase.

The initial launch of CancerBase demonstrated patient willingness to share personal, anonymized data and to recruit others. Patients were eager to engage and manipulate the limited data collected in the first release. Moving forward, patient priorities include: How do my treatment decisions compare with others and what can I learn from those who have come before me; What is the likelihood that I will have a recurrence; and, in the case of metastatic patients, how long might I have between progressions, to which organ(s) is progression most likely, and what are my likely survival outcomes. CancerBase is a database tool that resonates with the patient community and is driven by patient needs and interests. As the tool becomes increasingly robust it will grow to support the decision-making needs of clinicians and guide the investigations of researchers. The relaunch of CancerBase in Spring 2017 will address emerging patient concerns, integrate collected data, and utilize existing forecasting databases to add value to patients.

#2604

The Georgetown Database of Cancer (G-DOC): A web-based data sharing platform for precision medicine.

Krithika Bhuvaneshwar, Anas Belouali, Shruti Rao, Adil Alaoui, Yuriy Gusev, Robert Clarke, Louis M. Weiner, Subha Madhavan. _Georgetown University, Washington, DC_.

Introduction An overarching goal of biomedical research is to improve the use and dissemination of rapidly growing biomedical datasets to support precision medicine. Individualized molecular profiling and the identification of predictive biomarkers can powerfully inform the choice of therapies for cancer patients. However, both require integration of extensive molecular, clinical, and pharmacological data, often from disparate and diverse sources. The Georgetown Database of Cancer (G-DOC) was designed and engineered to be a unique multi-omics data analysis platform to enable translational research and precision medicine.

Methods G-DOC is home to 61 datasets that contain data from over 10,000 patients across 14 diseases (10 cancers and 4 non-cancers). 1700+ researchers from over 48 different countries worldwide currently use the platform. The data and tools in the G-DOC system have enabled over 40 research publications. G-DOC has the largest public collection of brain cancer patients from NCI Rembrandt dataset (671 patients).G-DOC integrates clinical, transcriptomic, metabolomic, microRNA, next generation sequencing (NGS) data, and MRI medical images with systems-level analysis tools into a single, user-friendly platform. The "Variant Search" feature in G-DOC currently enables exploratory analysis of mutations based on genes, chromosomes, and functional location. A researcher can use this feature to 1) identify clinically actionable mutations in their dataset 2) identify pathways that may be affected by these mutations, and 3) identify novel mutations in their dataset and explore their potential impact on protein function.

Results and Conclusion We are currently working on developing features to support the import, integration, search, and retrieval of CLIA/CAP-certified cancer molecular diagnostic (molDx) data. This will enhance G-DOC's interoperability with clinical and patient molecular profiling data that may be already stored in other databases. Our vision is to continuously improve and expand G-DOC with the long-term vision of supporting integration of informatics techniques into everyday research and practice.

#2605

Improving data quality in oncology immunotherapy clinical research by big data analytics and data visualization.

Chengsen Xue, Joanne Cuomo, Walter Meyers, Thomas W. Mc Closkey. _ICON Laboratory Services, Farmingdale, NY_.

Flow cytometric assessment of cellular and intracellular biomarkers facilitates the development of new classes of cancer immunotherapy therapeutics. This powerful tool has multiple applications including 1) detecting PD1, PDL-1 and caspase-3 for apoptosis, 2) isolating, defining, and quantifying dendritic cells and their intracellular cytokine profile, 3) immunophenotyping subpopulations of B, T, NK, regulatory T, and helper T cells, and, 4) using tetramer analysis to detect and monitor the frequency of antigen-specific cytotoxic T lymphocytes (CTL). However, lack of knowledge of normal distributions and standard ranges for the esoteric reportables generated by flow cytometry is one of the most challenging problems in this promising field.

Here we describe a new method to improve data quality in clinical research by big data analytics and data visualization. Each data point was tagged. By applying a simple statistical analysis, a self-defined range allowed all of the outlier numbers to be identified. By repeating this process, clear patterns emerged, and all questionable data points were marked. In addition, the system can differentiate biodiversity in subjects from a more homogenous population, which can be exploited further for the purpose of personal and precision medicine. Other benefits of the new system are to predict the outcome of the clinical study and to generate new clues in the understanding of the underlying biological mechanisms.

Big data analytics and data visualization show unparalleled advantages in oncology studies through simultaneously monitoring unlimited data points related to surface and intracellular biomarkers. It opens a new avenue in cost saving for long, time-consuming clinical trials by using all available data to detect a status change of the immune system. With the improvement of data quality, big data analytics has become an essential tool and an integrated part of cancer immunotherapy and the development of cancer vaccines.

#2606

Accelerating clinical research using cloud technology.

Arun Apte,1 Shonali Paul,1 Aditi Gade,1 Carolyn Compton2. 1 _CloudLIMS.com, Wilmington, DE;_ 2 _Arizona State University, AZ_.

Introduction: Existing protocols for clinical trials are typically slow and error-prone due to lack of informatics tools for sharing and accessing clinical data in real time. There is an increasing pressure on clinical trial laboratories to change the way clinical research is performed so as to minimize costs and speed up R&D, which in turn will produce better and faster outcomes. Cloud-based technologies are emerging as one of the most viable options for streamlining the heavily regulated clinical research processes.

Description: Cloud-based software tools such as laboratory information management systems (LIMS) are much more capable than traditional paper-based or desktop tools for accelerating a clinical trial life cycle. Cloud-based LIMS enhance collaboration between sponsors and investigators, enabling more timely and accurate sharing of information with pharmaceutical companies.

Summary: Many sponsors are now adopting cloud-based LIMS for conducting clinical trials. The onus is on service providers to keep pace with the demands of the industry, comply with the regulatory framework and at the same time deliver value to CROs and testing labs by reducing costs and maintaining efficiency.

Conclusion: CloudLIMS.com has developed a cloud based software called CloudLIMS Lite for managing clinical research workflows to provide a cost effective solution to the clinical research industry. Being on the cloud, CloudLIMS Lite facilitates users to access and share data in real time, across multiple laboratories in different geographical locations and adhere to the regulatory framework. This in turn improves their operational efficiency. CloudLIMS Lite uses 256-bit AES encryption SSL protocol, thereby enabling secure data transmission. CloudLIMS Lite deploys a separate database layer for each of its customers making it impossible for any third party to view or access data.

#2607

The cBioPortal for Cancer Genomics: an open source platform for accessing and interpreting complex cancer genomics data in the era of precision medicine.

Jianjiong Gao,1 Ersin Ciftci,2 Pichai Raman,3 Pieter Lukasse,4 Istemi Bahceci,5 Adam Abeshouse,1 Hsiao-Wei Chen,1 Ino de Bruijn,1 Benjamin Gross,1 Zachary Heins,1 Ritika Kundra,1 Aaron Lisman,1 Angelica Ochoa,1 Robert Sheridan,1 Onur Sumer,1 Yichao Sun,1 Jiaojiao Wang,1 Manda Wilson,1 Hongxin Zhang,1 James Xu,1 Andy Dufilie,2 Priti Kumari,2 James Lindsay,2 Anthony Cros,2 Karthik Kalletla,3 Fedde Schaeffer,4 Sander Tan,4 Sjoerd van Hagen,4 Jorge Reis-Filho,1 Kees van Bochove,4 Ugur Dogrusoz,5 Trevor Pugh,6 Adam Resnick,3 Chris Sander,2 Ethan Cerami,2 Nikolaus Schultz1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _Children's Hospital of Philadelphia, Philadelphia, PA;_ 4 _The Hyve, Netherlands;_ 5 _Bilkent University, Turkey;_ 6 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada_.

The cBioPortal for Cancer Genomics is an open-access portal (http://cbioportal.org) that enables interactive, exploratory analysis of large-scale cancer genomics data. It integrates genomic and clinical data, and provides a suite of visualization and analysis options, including cohort and patient-level visualization, mutation visualization, survival analysis, enrichment analysis, and network analysis. The user interface is user-friendly, responsive, and makes genomic data easily accessible to translational scientists, biologists, and clinicians.

The cBioPortal is a fully open source platform. All code is available on GitHub (https://github.com/cBioPortal/) under GNU Affero GPL license. The code base is maintained by multiple groups, including Memorial Sloan Kettering Cancer Center, Dana-Farber Cancer Institute, Children's Hospital of Philadelphia, Princess Margaret Cancer Centre, and The Hyve, an open source bioinformatics company based in the Netherlands. More than 30 academic centers as well as multiple pharmaceutical and biotech companies maintain private instances of the cBioPortal. This includes the recently launched cBioPortal instance at the NCI Genomic Data Commons (https://cbioportal.gdc.nci.nih.gov/), and two large cBioPortal instances hosting genomic and clinical data at MSK and DFCI, supporting the MSK-IMPACT and DFCI Profile projects, two of the largest clinical sequencing efforts in the world.

Our multi-institutional software team has accelerated the progress of evolving the core architectural technologies and developing new features to keep pace with the rapidly advancing fields of cancer genomics and precision cancer medicine. For example, we have integrated multi-platform genomics data with extensive clinical data including patient demographics, treatment history, and survival data. We have also developed a patient-centric view that visualizes both clinical and genomic data with annotation from OncoKB knowledge base. In the next few years, the development team will focus on the following areas:

(1) Implementing major architectural changes to ensure future scalability and performance.

(2) New features to support precision medicine, including (i) improved integration of knowledge base annotation, (ii) enhanced visualization of patient timeline, drug response, and tumor evolution, (iii) new patient similarity metrics, (iv) improved support for immunogenomics and immunotherapy, and (v) new visualization and analysis features for understanding response to therapy.

(3) New analysis and target discovery features for large cohorts, including (i) supporting user-defined virtual cohort by selecting samples from multiple studies, and (ii) comparison of genomic or clinical characteristics of two or more selected cohorts.

(4) Expanding community outreach, user support and training, and documentation.

#2608

Global integration of knowledgebases for clinical interpretation of cancer variants.

Obi L. Griffith,1 Malachi Griffith,1 David Tamborero,2 Alex H. Wagner,1 Kilannin Krysiak,1 Catherine Del Vecchio Fitz,3 Debyani Chakravarty,4 Ethan Cerami,3 Olivier Elemento,5 Nikolaus Schultz,4 Adam Margolin,6 Nuria Lopez-Bigas2. 1 _Washington University School of Medicine, Saint Louis, MO;_ 2 _Institute for Research in Biomedicine, Barcelona, Spain;_ 3 _Dana-Farber Cancer Institute, Boston, MA;_ 4 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 5 _Weill Cornell Medical College, New York, NY;_ 6 _Oregon Health & Science University, Portland, OR_.

The paradigm of precision medicine envisions a future in which a cancer patient's molecular information can be interpreted in the context of accumulated knowledge to inform diagnosis, prognosis and treatment options most likely to benefit that individual patient. Accordingly, many groups have created knowledgebases to annotate cancer genomic mutations associated with evidence of pathogenicity or relevant treatment options. However, in real-life practice, clinicians and researchers are unable to utilize the accumulated knowledge derived from such efforts. Integration of the available knowledge is currently infeasible because each group (often redundantly) curates their own knowledgebase without adherence to any interoperability standards. Therefore, there is a clear need to standardize and coordinate clinical-genomics curation efforts, and create a public community resource able to query the aggregated information. To this end we have formed the Variant Interpretation for Cancer Consortium (VICC) as part of the Global Alliance for Genomic Health (GA4GH) to bring together the leading institutions that are independently developing comprehensive cancer variant interpretation databases. VICC participants share a desire to coordinate efforts and thus enhance the value of each independent effort. Each participant has agreed to: (1) sharing at least a minimal set of required data elements for cancer variant interpretations; (2) Protecting patient privacy by focusing on only clinical interpretations of variants derived from published findings, not individual patient/variant-level observations, thus avoiding the possibility of linking variants to individuals; (3) Sharing all or a significant proportion of interpretations accumulated by their ongoing curation efforts; (4) Releasing content under a permissive license (free and non-exclusive for research use); (5) Releasing software in public repositories with open source licenses; (6) Making data available through publicly accessible and documented APIs and as cross-knowlegebase bulk downloads; and (7) Using, the existing schemas, APIs and demonstration implementations developed by GA4GH. To date a large number of academic and commercial groups have agreed to participate including those at Washington University (CIViC), MSKCC (OncoKB), Weill Cornell (PMKB), Oregon Health and Science University, Dana-Farber, Institute for Research in Biomedicine, Illumina Inc, and others. Additional participants are welcome (http://ga4gh.org/#/vicc). We will present progress by the VICC to create a federated query service able to interrogate associations between cancer gene alterations and clinical actions, for each cancer disease setting, based on evidence amassed from all participating institutions world-wide.

#2609

Columbia University's Center for Cancer Systems Therapeutics (CaST) 2017 Scholars Program: A synergistic partnership with students from Brooklyn College, CUNY.

Rahimah Ahmad,1 Tasnim Azad,1 Carlos Barreto,1 Kamrun Begum,1 Mahlaqa Butt,1 Daniel Gruffat,1 Aulon Jerliu,1 Jonathan Kwiat,1 Mikaela Murph,1 Katherine A. Rivera Gómez,1 Barry Honig,2 Andrea Califano,2 Shaneen Singh,1 Diana Murray2. 1 _CUNY Brooklyn College, Brooklyn, NY;_ 2 _Columbia University, New York, NY_.

The Cancer Systems Therapeutics (CaST) Outreach helps foster community building throughout the NCI Cancer Systems Biology Consortium (CSBC). The overall mission of the CaST Outreach Core is to advance progress in cancer systems biology by tapping into and making connections across research talent at all stages of educational and professional attainment. A unique component is our partnership with motivated undergraduate, post-baccalaureate, and graduate students from Brooklyn College of the City University of New York (CUNY). These students comprise the first cohort of the CaST Scholars Program and are immersed in the cutting-edge biological and biomedical science underlying Cancer Systems Biology. In subsequent years, CaST Outreach will partner with students from other CUNY Senior Colleges while continuing interactions with our Brooklyn College Scholars. This integrative approach will create a network of CaST Scholars throughout the CUNY system. Because Systems Biology is highly interdisciplinary, participants in the program can come from many backgrounds, including biology, computer science, physics, chemistry, genomics, engineering, and other fields.

The program has no fixed curriculum and each of nine Brooklyn College students has developed an individualized CSBC educational plan tailored to their interests. The CaST Scholars Program opportunities include 1) tuition-free attendance at a master's level Systems Biology course taught by a CaST investigator and offered through Columbia University's School of Professional Studies; 2) supported attendance at the AACR annual meeting; 3) monthly informal discussion meetings with CaST investigators; 4) weekly formal tutorials in the design and applications of CaST computational algorithms; 5) research internships with CaST investigators; 6) participation in a wide-range of Center activities; 7) a scholarship to support participation; and 8) ongoing mentorship from CaST investigators.

This poster provides summaries of the collaborative, synergistic experiences and achievements of each Scholar and of the group as a whole. The CaST Scholars Program offers a unique opportunity for early-stage scientists to learn how Systems Biology is transforming cancer research and precision medicine at Columbia University Medical Center. Regardless of their chosen professional path, CaST Scholars will obtain a solid foundation in the methods and potential of Cancer Systems Biology.

#2610

A database for evaluating methylation biomarkers in bladder cancer.

Meaghan M. Kennedy, Garrett M. Dancik. _Eastern Connecticut State University, CT_.

Bladder cancer is the fourth-most common cancer in men, one of the most expensive types of cancer to treat, and recurs frequently. While the genomic pathways associated with high-grade, muscle invasive (MI) tumors and low-grade, non-muscle invasive (NMI) tumors are well-established, recent studies have also found that MI and NMI tumors have unique methylation patterns, and that methylation may play a role in the development of early-stage bladder cancer. Interestingly, bladder cancer patients have frequent mutations in chromatin remodeling genes that alter known histone markers of DNA methylation. Since methylation marks are pharmacologically reversible, the identification of methylation biomarkers is a promising avenue for therapeutic treatment. Indeed, several candidate prognostic methylation biomarkers have been identified, but await prospective evaluation.

The Bladder Cancer Biomarker Evaluation Tool (BC-BET) is a web-based resource for rapidly evaluating biomarkers in publicly available genomic datasets. Initially developed for gene expression biomarkers, BC-BET includes 1451 patients across 13 cohorts, and 40 bladder cancer cell lines, with gene expression profiles. In this work, we describe the addition of methylation data to BC-BET, so that candidate methylation biomarkers can be rapidly evaluated across multiple patient cohorts.

BC-BET now contains methylation profiles from four patient cohorts (250 patients) and includes the available bladder cancer data from the Gene Expression Omnibus and The Cancer Genome Atlas. In "evaluation" mode, a user selects a gene of interest, and BC-BET evaluates whether the selected gene is differentially methylated between tumor vs. normal samples, NMI vs. MI tumors, and low- vs. high-grade tumors. Alternatively, the "biomarker discovery" mode allows a user to identify all differentially methylated genes at a desired false discovery rate (FDR). In both cases, graphical summaries of the results are displayed and statistical results can be downloaded to an Excel spreadsheet. The methylation module of BC-BET is implemented using shiny, a web application framework for R for developing interactive web pages.

The addition of methylation data to BC-BET makes it a valuable resource for evaluating methylation biomarkers in bladder cancer. BC-BET is available from the following link: https://gdancik.github.io/bioinformatics/BCBET

#2611

A crowdsourcing-based clinical trial information curation and searching system.

Pengfei Yu, Qingxuan Song, Yang Han, Guanghui Hu. _Admera Health, South Plainfield, NJ_.

Targeted cancer therapies based on actionable mutations identified are the future of cancer treatment. Besides those approved by the Food and Drug Administration (FDA) for different types of cancers, many other target therapy drugs are in development under different phases of clinical trials. Access to information about these clinical trials is important to health care professionals and patients. Public clinical trials database (www.clinicaltrials.gov) provides a very comprehensive resource for clinicians and researches to search for relevant clinical trials based on different kinds of searching criteria. However, due to the complexity of wording and phasing upon submission, some of the searching results returned by native search engine did not perfectly match the search criteria. Many undesired results are presented with false positives and false negatives when users are searching for clinical trials targeting specific actionable mutations. To tackle this problem, we developed a crowd-sourcing based clinical trial curation and searching system on top of the information extracted from public database. We divide the information extracted from clinicaltrials.gov into three different levels: standard, easy-to-clean and unstructured. With the crowdsourcing approach, our online system can harness the brainpower of biological scientists to translate the most unstructured information such as cancer types, gene mutations into standard format that is easy to be queried. In the searching phase, we combine the aspects of actionable mutation, cancer type, location, and clinical trial phase to select the best matches between patient conditions and clinical trials in our curated database. With the same searching criteria, our system can provide more relevant clinical trials compared with those provided by clinicaltrials.gov. The curation system is currently open to reviewers with eligibility check. And the search engine is publicly available at: https://agis.admerahealth.com/AGIS/ClinTrials/.

## IMMUNOLOGY:

### Checkpoints 2: Small-Molecule Inhibitors

#2612

Anti-Tigit induces T cell mediated anti-tumor immune response and combines with immune checkpoint inhibitors to enhance strong and long term anti-tumor immunity.

Minu K. Srivastava, Rui Yun, Erin Mayes, Janice Yu, Hyun-Bae Jie, Fumiko Axelrod, Ming-Hong Xie, Jorge Monteon, Andrew Lam, May Ji, Yuwang Liu, John Lewicki, Tim Hoey, Austin Gurney, Angie Inkyung Park. _OncoMed Pharmaceuticals, Inc., Redwood City, CA_.

TIGIT (T cell immunoreceptor with Ig and ITIM domains) has been recently described as an inhibitory receptor which blocks CD8 T cell-mediated anti-tumor immune responses. We have generated an anti-mouse TIGIT antibody (313R12) to evaluate drug efficacy and mechanism of action in pre-clinical tumor models. Anti-TIGIT as a single agent promoted an anti-tumor immune response in multiple syngeneic mouse tumor models. Anti-TIGIT enhanced tumor specific T cell responses, particularly of the Th1 type and reduced Th2 type responses and also increased the function of cytotoxic T cells. Furthermore, anti-TIGIT displayed combination activity with immune checkpoint inhibitors anti-PD1 and anti-PDL1 in inhibiting tumor growth, promoting complete tumor rejection and significantly increasing mouse survival in the murine CT26 colon carcinoma model as compared to controls and single agents alone. Mice "cured" with anti-TIGIT/anti-PDL1 or anti-TIGIT/anti-PD1 combination treatments did not form tumors upon subsequent re-challenges with increasing number of CT26 tumor cells, suggesting the existence of immunologic memory. IL2 and tumor-specific IFN-γ production by splenic T cells were increased in mice who responded to combination treatment compared to controls. Additionally, both effector and memory CD8+ T cell frequencies were increased within the total CD8+ T cell population in responding mice. We also demonstrated a systemic increase in tumor-specific CD8 T cells after anti-TIGIT/anti-PDL1 combination treatment compared to controls. Therefore, these results suggest that co-targeting of TIGIT and PD1 or PDL1 may be an effective and durable cancer therapy by increasing T cell-mediated anti-tumor immune responses and promoting long-term immunological memory.

#2613

Combination agonist and antagonist antibody therapy enhances vaccine induced T cell responses in non-immunogenic cancers.

Hayley S. Ma, Evanthia Roussos Torres, Brian Christmas, Blake Scott, Tara Robinson, Todd Armstrong, Elizabeth Jaffee. _Johns Hopkins University School of Medicine, Baltimore, MD_.

Immune tolerance by multiple mechanisms is a major obstacle in the implementation of successful immunotherapy. T regulatory cells (Tregs) and myeloid derived suppressor cells (MDSCs) suppress the immune response, and the presence of extensive stromal networks within the tumor microenvironment (TME) can prevent T cell infiltration in many tumor types. Furthermore, tumor antigen-specific T cell tolerance limits the efficacy of therapeutic cancer vaccines. CD40 is expressed on antigen presenting cells, and CD40 signaling is critical to the decision of whether cytotoxic T lymphocytes become primed or tolerized. Administration of monoclonal CD40 agonistic antibody (Ab) has been shown to promote CD8 activation in vivo, and likely alters the myeloid component of the TME. Our study asks the question of whether combining a T cell inducing vaccine and PD-1 inhibition with CD40 agonistic Ab can induce T cell priming and tumor infiltrating lymphocyte (TIL) activation in non-immunogenic solid malignancies. We utilized an orthotopic transplant model in which HER2/neu-expressing breast tumor cells were implanted into the mammary fat pad of syngeneic neu-N mice to assess the effects of drug combinations on intratumoral immune responses. Tumor-bearing mice were treated with a granulocyte macrophage colony-stimulation factor secreting vaccine (GVAX) + anti-PD-1 Ab alone or in combination with CD40 agonistic Ab, and monitored for tumor clearance and survival. A separate cohort of mice were analyzed by IHC and multi-color flow cytometry to assess T cell infiltration and activation, myeloid maturation, and MDSCs after one week of treatment. We demonstrated delayed tumor progression and increased median survival in mice treated with GVAX + anti-PD-1 Ab + CD40 Ab relative to CD40 Ab alone, although endogenous T cell infiltration remained low across treatment groups. However, adoptive transfer of neu-specific TCR transgenic cells revealed 100% tumor clearance in the combination-treated mice, along with significant T cell infiltration and activation as measured by IFNγ, Granzyme B and TNFα-secreting CD8+ T cells. In contrast, only 20% of mice treated with GVAX + anti-PD-1 Ab were able to clear tumor, and none of the mice receiving vehicle or CD40 Ab alone had long-term survival. Trends were also observed in monocytic and dendritic cell infiltration and maturation in the tumors of combination-treated mice, and warrant further investigation. In conclusion, GVAX, anti-PD-1 Ab and CD40 agonist Ab have potential synergy in modulating anti-tumor immunity in breast cancer. These results support further studies more broadly in non-immunogenic solid tumors.

#2614

Decrease of benefit from immune checkpoint inhibitors in women with non-small cell lung cancer.

Joseph A. Pinto,1 Luis A. Mas,1 Carlos S. Vallejos,1 Jhajaira Araujo,1 Leny Bravo,2 Alfredo Aguilar,1 Zaida Morante,1 Denisse Bretel,1 Henry L. Gomez,1 Christian Rolfo3. 1 _Oncosalud, Lima, Peru;_ 2 _Universidad Privada San Juan Bautista, Lima, Peru;_ 3 _Antwerp University, Lima, Peru_.

Introduction: In a previous work we found differences in immune gene sets enrichment in NSCLC between genders, regardless their smoking status, where women had higher expression of gene sets associated with immune processes. Based in this fact, we hypothesize fewer benefits from immune checkpoint inhibitors in women patients compared than men patients.

Objectives: Evaluate the benefit (in terms of progression-free survival) of women patients from immune checkpoint inhibitors nivolumab and pembrolizumab in published randomized phase III trials.

Methods: We evaluated by meta-analysis four randomized phase III studies, two of prembrolizumab (Herbst et al., 2015 and Reck et al., 2016) and two of nivolumab (Borghaei et al., 2015 and Brahmer et al., 2015). We analyzed TCGA data for lung adenocarcinoma to evaluate differences in the expression of PDCD1, CD274 genes (encoding for PD-1 and PD-L1, respectively).

Results: For all studies, it was observed an overall HR=0.79 (CI95%:0.71-0.87;P<0.00001), although was observed a significant heterogeneity between studies (P=0.002). In male patients, was observed an overall HR=0.71 (CI95%:0.62-0.80;P<0.00001) with significant heterogeneity between studies (P=0.002). For female patients, there was not seen a clear benefit from nivolumab or pembrolizumab (overall HR=0.97, CI95%:0.82-1.14;P=0.67). There was not heterogeneity between the cohorts (P=0.45). There were not differences in the expression of PDCD1 and CD274 genes according to gender in the TCGA data (P=0.371 and 0.144, respectively).

Conclusion: Although there are not differences between genders in the expression of PDCD1 and CD274 genes , such as is previously described for expression for PD-1 and PDL-1 proteins, in our meta-analysis we shown that women have modest benefit from anti immune checkpoint inhibitors. Blockade of PD1 and PD-L1 is a promising therapy in lung cancer; however, a better stratification of patients should be done.

Meta-analysis of four phase III randomized studies

---

Study | All patients | |

Men | |

Women

|

|

Weight | Hazard Ratio IV, Fixed, 95% CI | Weight | Hazard Ratio IV, Fixed, 95% CI | Weight | IV, Fixed, 95% CI

Reck et al., 2016 | 10.6% | 0.50 [0.37, 0.68] | 10.4% | 0.39 [0.26, 0.581] | 8.0% | 0.71[0.40, 1.26

Brahmer et al., 2015 | 13.7% | 0.63 [0.48, 0.82 | 17.8% | 0.63 [0.46, 0.86] | 11.2% | 0.75[0.46, 1.22]

Borghaei et al., 2015 | 30.3% | 0.91 [0.76, 1.09 | 26.6% | 0.81 [0.63, 1.04] | 37.8% | 1.02[0.78, 1.33]

Herbst et al., 2015 | 45.4 | 0.85 [0.73, 0.98] | 45.2% | 0.78 [0.64, 0.95] | 43.0% | 1.04[0.81, 1.33]

Total (95% Cl) | 100.0% | 0.79 [0.71, 0.87] | 100.0% | 0.71 [0.62, 0.80 | 100.0% | 0.97[0.82, 1.14]

|

Heterogeneity: Chi2=14.73,df=3 (P=0.002); I2= 80% Test for overall effect: Z= 4.73 (P <0.00001) | |

Heterogeneity: Chi2=11.12, df=3 (P=0.01); I2=73% Test for overall effect: Z= 5.28 (P<0.00001) | |

Heterogeneity: Ch2=2.67, df=3 (P= 0.45); I2=0% Test for overall effect: Z= 0.42 (P= 0.67)

|

#2615

Preclinical evaluation and mechanistic characterization of M7824 (MSB0011359C), a novel bifunctional fusion protein targeting the PD-L1 and TGFβ pathways.

Yan Lan, Dong Zhang, Chunxiao Xu, Bo Marelli, Jin Qi, Huakui Qi, Guozhong Qin, Xiaomei Xu, Hong Wang, Aroop Sircar, Beatrice Brunkhorst, Eric Austin, Laszlo Radvanyi, Kin-Ming Lo. _EMD Serono Research and Development Institute, Inc., Billerica, MA_.

Dual targeting of the programmed death ligand 1 (PD-L1) and transforming growth factor β (TGFβ) pathways, 2 key mediators of tumor immune evasion, may provide synergistic antitumor activity. The novel bifunctional fusion protein M7824 (MSB0011359C) is a fully human IgG1 monoclonal antibody against PD-L1, fused via a glycine-serine linker at the CH3-C terminus to the extracellular domain of human TGFβ receptor II, which functions as a TGFβ "trap." Thus, M7824 is designed to exert independent and complementary anti-immunosuppressive functions by simultaneously blocking the PD-L1 and TGFβ pathways. The purpose of this study was to evaluate M7824 preclinically. M7824 was efficacious in multiple syngeneic tumor models (EMT-6, MC38, PANC02, 4T1, CT26, and GL261). In orthotopic EMT-6 and intramuscular MC38 models, M7824 showed superior additive antitumor effects compared to anti-PD-L1 antibody or TGFβ trap monotherapy. M7824 also inhibited spontaneous metastases in EMT-6 and 4T1 models. Furthermore, M7824 extended survival and conferred long-term protective antitumor immunity, as demonstrated by protection of cured mice against tumor cell rechallenge. Mechanistically, the dual anti-immunosuppressive activity of M7824 in the MC38 model resulted in unique activation of both the adaptive and innate immune systems, including increased tumor infiltration, proliferation, and cytotoxicity of CD8+ T cells; increased prevalence of splenic tumor antigen-specific CD8+ T cells; increased CD8+ TEM infiltration and CD8+ TEM/naïve ratio; increased tumor-infiltrated natural killer (NK) cells; increased ratio of M1/M2 macrophages; and decreased infiltration of tumor-associated neutrophils and myeloid-derived suppressor cells. Similar immunophenotypic changes were observed in the EMT-6 model. Activation of both the adaptive and innate immune systems contributed to the antitumor activity of M7824 in the MC38 model, as depletion of CD8+ T cells and NK cells led to a substantial decrease in M7824-mediated tumor regression; CD4+ T cells and antibody-dependent cellular cytotoxicity, however, appeared dispensable. Toxicologic evaluation in cynomolgus monkeys indicated M7824 was well tolerated, with only minor hematologic effects observed. Although M7824 is a bifunctional molecule, its antitumor activity can be further improved by combination with standard-of-care therapies: in the MC38 model, combining M7824 with radiotherapy or chemotherapy resulted in more effective inhibition of tumor growth and an increased frequency of tumor antigen-specific IFN-γ-producing CD8+ T cells vs either monotherapy. Collectively, our preclinical data suggest that M7824 elicits potent and synergistic antitumor activity via the simultaneous blockade of the PD-L1 and TGFβ pathways. Phase 1 clinical trials with M7824 are under way in patients with solid tumors.

#2616

Upregulation of program death ligand 1 expression after a single cycle of chemotherapy is associated with inferior survival in patients with local advanced esophageal squamous cell carcinoma treated with preoperative chemoradiotherapy.

Ta-Chen Huang,1 Cher-Wei Liang,1 Chia-Chi Lin,1 Ya-Jhen Chen,2 Kuan-Ling Lin,1 Ann-Lii Cheng,1 Chin-Hung Hsu1. 1 _National Taiwan University Hospital, Taipei, Taiwan;_ 2 _National Taiwan University, Taipei, Taiwan_.

Introduction

Previous studies have demonstrated that the expression of program death-ligand 1 (PD-L1), a ligand of an immune checkpoint, on cancer cells or on immune cells may have prognostic significance in multiple cancer types including esophageal squamous cell carcinoma (ESCC). We evaluated the expression of PD-L1 and its dynamic changes following one cycle of chemotherapy before standard preoperative chemoradiotherapy (CRT) in a cohort of locally advanced ESCC patients.

Patients and Methods

A cohort of 66 patients of locally advanced ESCC have been enrolled to a phase II clinical trial of a single-cycle chemotherapy (a combination of weekly paclitaxel, cisplatin and 24 hour infusional 5-fluorouracil, 2 week on plus one week off, followed by paclitaxel/cisplatin (TP) -based preoperative CRT and radical surgery. Paired tumor tissues from endoscopic biopsy before and after the single-cycle chemotherapy were available in 21 patients. Immunohistochemical stain for PD-L1 expression was performed using the Clone SP142 antibody (Spring Bioscience, Pleasanton, CA, USA). The expression of PD-L1 was scored as previously reported. Tumor cells were scored according to percentage of positively-stained cells: TC3≥50%, TC2≥5% and <50%, TC1≥1% and <5%, and TC0<1%; Immune cells: IC3≥10%, IC2≥5% and <10%, IC1≥1% and <5%, and IC0<1%.

Results

In 21 baseline ESCC tissues, 7 of 19 (37%) exhibited positive PD-L1 expression on tumor cells, and 10 of 21 (48%) exhibited positive expression on immune cells. In 21 ESCC tissues obtained a median of 18 days after initiating chemotherapy, 9 of 18 (50%) and 15 of 21 (71%) demonstrated positive PD-L1 expression on tumor cells and immune cells, respectively. The expression of PD-L1 was significantly increased in post-chemotherapy ESCC tissues compared with baseline tissues (p=0.010); and the increased PD-L1 expression between paired tissues was significant on immune cells (p=0.0056) but did not reach statistical significance on tumor cells (p=0.11). The positive PD-L1 expression in baseline ESCC tissues was correlated with better survival (HR: 0.37, p=0.023). Besides, patients with more increase of PD-L1 expression after chemotherapy had significantly worse survival than those with less (median survival: 19.4 vs 71.20 months, HR: 4.07, p=0.0089).

Conclusion

PD-L1 expression of ESCC can be up-regulated by chemotherapy. The

up-regulation of PD-L1 is associated with poor outcome for locally advanced ESCC patients. (The study was supported by the grant NTUH 104-M2891 and the grant MOST 105-2314-B-002-186-MY3)

#2617

Inducing neoantigens in therapeutic and prophylactic cancer immunotherapy.

Greta Garrido Hidalgo, Agata Levay, Alexey Berezhnoy, Brett Schrand, Eli Gilboa. _University of Miami, Miami, FL_.

Correlation between clonal neoantigen burden (neoantigens generated early in tumorigenesis and therefore represented at high frequency in all tumor lesions) and responsiveness to checkpoint blockade has underscored the relevance of neoantigens in promoting tumor immunogenicity. Yet, the most of patients do not express, or express low numbers of, clonal neoantigens, and consequently will be less likely to benefit from checkpoint blockade. We describe strategies to generate de novo neoantigens in the patient' disseminated tumors and show that in mouse tumor models they potentiate checkpoint blockade. We previously showed that tumor inhibition of the Nonsense-mediated mRNA decay (NMD) results in the neoantigens' induction and reduces tumor growth (Pastor et al., 2010). We now demonstrate that tumor-targeted NMD inhibition potentiates PD-1 blockade. A general concern and potential limitation of this approach is that a significant proportion of the induced neoepitopes come from mutated products and hence will not be shared by all tumor lesions. To that end, we are exploring alternative strategies by targeting key components of antigen presentation pathways, specifically the TAP transporter, ERAAP peptidase, and Invariant chain. Studies show that downregulation of these products, not only reduces the canonical antigenic presentation but also upregulates alternative pathways that present new, otherwise silent or subdominant, epitopes. Since such epitopes are not generated by random events they are more likely to represent clonal neoepitopes. We are developing approaches to inhibit the aforementioned mediators using corresponding siRNAs targeted to tumor cells by conjugation to a nucleolin-binding aptamer. Nucleolin, a nucleolar product, is translocated to the surface the majority of tumors and thereby serves as a broad target to deliver therapeutic cargo to the disseminated tumors. We show that nucleolin aptamer-targeted downregulation of TAP, ERAAP or Invariant chain inhibits tumor growth and potentiates PD-1 blockade. Recent studies suggest that tumor neoantigen-specific T cells are dysfunctional due to constant antigenic exposure. Transiently expressed siRNA inhibition-induced neoantigens are not expected to be defective. Ongoing studies explore the combinatorial use of neoantigen induction methods with others immune potentiating strategies. Prophylactic cancer vaccination obviates the limitations of therapeutic vaccination. A major barrier for developing this modality is the choice of antigens that will appear in the future cancer. The ability to induce tumor neoantigens can serve the basis for developing a new approach to prophylactic, though not preventative, cancer vaccination whereby neoantigens are induced in the healthy individual (at risk for cancer), and if or when a cancer develops induce the same antigens in the patient' tumor by the methods described above. Preliminary studies in mice show that the approach has merit.

#2618

Agonist antibodies targeting OX40 and GITR enhance the activity of the IDO1-selective inhibitor epacadostat in preclinical models.

Holly K. Koblish,1 Brendan Horton,2 Michael Hansbury,1 Sybil O'Connor,1 Kerri Lasky,1 Christina Stevens,1 Thomas Condamine,1 Leslie Hall,1 Liang-Chuan Wang,1 Yue Zhang,1 Horacio Nastri,1 Gregory Hollis,1 Reid Huber,1 Thomas Gajewski,2 Peggy Scherle1. 1 _Incyte Corporation, Wilmington, DE;_ 2 _University of Chicago, Chicago, IL_.

The majority of immunotherapeutic agents developed thus far either attempt to stimulate a more productive anti-tumor immune response or to inhibit key proteins in the immunosuppressive tumor milieu. PD-1/PD-L1 axis blockade, CTLA-4 blockade and IDO1 inhibition are examples of the latter approach and have been utilized to reverse the suppressive tumor microenvironment, resulting in clinical benefit for cancer patients. Recent clinical and preclinical data have also demonstrated that combining these approaches results in enhanced therapeutic benefit. Notably, the IDO1-selective inhibitor epacadostat has been shown to increase the efficacy of two checkpoint inhibitors, the anti-CTLA-4 antibody ipilimumab and the anti-PD-1 antibody pembrolizumab, in patients with melanoma. Because both checkpoint receptors and IDO1 serve as negative regulators of the immune response, we also explored the ability of IDO1 inhibition to combine with agents that directly activate T cells through costimulatory receptors of the tumor necrosis factor receptor (TNFR) superfamily. Rodent active surrogate agonist antibodies to 4-1BB, OX40 and GITR were tested with epacadostat in multiple preclinical models. In the B16-SIY melanoma model that does not express IDO1 in tumor cells, both epacadostat and anti-OX40 had little effect, but the combination resulted in enhanced efficacy. This was associated with increased infiltrates of CD8+ T cells and decreased numbers of FoxP3+ TILs. Increased numbers of SIY-reactive T cells were found in both the tumor and the TDLN post-therapy. In contrast, epacadostat did not provide any enhancement to the activity seen with 4-1BB. Clear combinatorial effects were seen with anti-GITR and epacadostat in the more inflamed, IDO1-expressing PAN02 pancreatic cancer model. These data suggest that IDO1 inhibition can be effective in combination with agents that agonize T cell costimulatory receptors as well as with agents that block coinhibitory receptors.

#2619

Combination of ECP1014 and anti-PD-L1 reduces tumor growth in the CT26 murine colon carcinoma model of a cold tumor.

Bobby W. Sandage,1 John J. Talley,1 Eduardo J. Martinez,2 Maryland R. Franklin,3 Mary Anne Meade,3 Dan Saims,3 Matt Thayer,3 Scott Wise,3 David Draper,3 Wilber Leopold3. 1 _Euclises Pharmaceuticals, Inc., St. Louis, MO;_ 2 _Rgenix, Philadelphia, PA;_ 3 _MI Bioresearch, Ann Arbor, MI_.

Objective: The objective of this study was to evaluate the effect of ECP1014, a new selective COX2 inhibitor, in combination with a PDL1 inhibitor on tumor growth in a CT26 murine colon carcinoma model. Colon cancer has been described as a "cold tumor" and antiPDL1monotherapy has shown limited or no benefit. In addition, many colon cancer cell lines produce high levels of PGE2, which may in turn dampen the immune system. The hypothesis was that combining ECP1014, which we have shown potently decreases PGE2 in CT26, with a checkpoint inhibitor would increase immune response to the tumor, turning it "hot" and producing superior tumor control versus either agent alone.

Methods: Balb/C mice were implanted with CT26 cells and randomly assigned to 7 treatment groups; vehicle (G1), 10mg/kg rat IgG2b control (G2), 10mg/kg antiPDL1 (G3), 10mg/kg ECP1014 (G4), 10mg/kg ECP1014+10mg/kg rat IgG2b (G5), 10mg/kg ECP1014+10mg/kg antiPDL1 (G6), 1mg/kg ECP1014+10mg/kg antiPDL1(G7). ECP1014 was given daily by oral gavage; rat IgG2b and antiPDL1 (clone 10F.9G2) were administered IP. Treatment was started on day 3 post implant and mice were followed for 16 days. Tumor measurements were taken every 2 to 3 days, and a nonparametric approach was taken to assess the pairwise differences among groups using day 16 tumor volumes. In addition, a responder analysis was used. Tumor infiltrating lymphocytes (TIL) were also assessed by flow cytometry on day 16.

Results: All animals developed tumors. G4 to 7 demonstrated statistically significantly (p<0.05) slowing of tumor growth when compared to vehicle (G1). In particular, G7 produced an 87% suppression of tumor growth compared to vehicle. Three animals in G7 showed regression of tumor growth and one additional animal had no detectable growth until day 16. G6 produced 65% suppression of tumor growth compared to G1. G2 to 5 produced 30, 54, 73 and 50% suppression of tumor growth respectively when compared to G1. G7 had the highest number of responders 75% vs. 62.5% for G6 and G4, with all other groups having < 25% responders. TIL analysis from G4, G6 and G7 also displayed the highest levels of CD8+ Tcells (6.3%, 6.8%, and 6.7% respectively, versus 3.8% for antiPDL1 and 2.5% for vehicle groups).

Summary: ECP1014 alone was superior to antiPDL1 in slowing tumor growth. However, the combination of ECP1014+antiPDL1showed the greatest tumor response and growth suppression and markedly increased CD8+ Tcells infiltrating into the tumor vs. single agent anti-PDL1 treatment.

#2620

**Selecting the right chemotherapy partner for checkpoint inhibitors: an** in vivo **comparison of different drugs and dosages.**

Stefania Orecchioni, Giovanna Talarico, Valentina Labanca, Patrizia Mancuso, Francesco Bertolini. _European Inst. of Oncology, Milan, Italy_.

Anti-PD-1 and anti-PD-L1 monoclonal antibodies (checkpoint inhibitors, CIs) have shown a remarkable clinical activity in a variety of types of solid cancers and hematological malignancies. However, complete and/or long-lasting clinical responses have been observed only in a minority of patients. The clinical response to CIs depends on a complex balance between tumor-associated and circulating CD3+CD4+ and CD3+CD8+ T, regulatory, B, NK and myeloid/suppressor cell subsets. We have used immunocompetent mice to generate orthotopic models of breast cancer (BC, injecting 4T1 triple negative murine cells) and of non-Hodgkin's lymphoma (NHL, injecting A20 murine malignant B cells). In these models and in cancer-free mice we studied by 10-color flow cytometry the effect of different doses of 3 widely-used, orally-active chemotherapy drugs (cyclophosphamide, CTX; capecitabine, 5FU; and vinorelbine, VNR) over a variety of immune cell subsets including CD3+CD4+ and CD3+CD8+ T cells, CD56+ NKs, CD19+ B cells, CD3+CD4+CD25++CD127low/neg Tregs, GR1-CD11b+CD11c+ monocytes, SSClowCD11b+GR1+ total Myeloid-Derived Suppressor Cells (t-MDSC, monocytic and polymorphonuclear), SSChighCD11b+GR1+ granulocytes and CD11c+CD11b-GR1- antigen-presenting cells (APCs). Chemotherapy drugs were given alone or in combination with murine anti-PD-1 or anti PD-L1 monoclonal antibodies (Bioxcell clones J43 and 10F.9G2, respectively). For every type of chemotherapy drugs, we selected 3 dosages representative of clinical low-dose metronomic (LDM), medium (M), or maximum tolerable (MT) dosages. Mice were treated for 3 weeks, blood cell subsets were measured weekly. CTX and 5FU, at M and MT dosages, reduced significantly the number of T, B, and NK cells and of t-MDSC. CTX, but not 5FU, reduced Tregs and APCs. 5FU, but not CTX, reduced t-MDSC. At variance, VNR at LDM, M and MT doses did not reduce T, NK and t-MDSC. At M and MT doses, VNR reduced Tregs, B and SSChighCD11b+GR1+ granulocytes. Considering these data, we designed preclinical protocols in both BC and NHL models associating CTX, 5FU and VNR with CIs. Cumulative and unbearable toxicities were observed in mice treated with 2 or more chemotherapy drugs at MT doses plus CIs. In both models, anti-PD-L1 plus M or LDM combinatory doses of CTX and VNR were the most effective therapies for reducing local and metastatic neoplastic growth. Our data indicate that chemotherapy drugs have relevant effects on immune cell subsets involved in the anti-neoplastic activity of CIs, and that the type and the dosage of chemotherapy are crucial to obtain synergy with CIs. Further studies are ongoing to find the right chemotherapy/CI balance to a) maintain an adequate T, APC and NK cell activity, b) reduce Tregs and myeloid suppressors, c) avoid toxicity, and d) target cancer cells.

#2621

Microsatellite instability is a potential biomarker for immune checkpoint inhibitor in endometrial cancer.

Hitomi Yamashita, Kentaro Nakayama, Noriyoshi Ishikawa, Toshiko Minamoto, Tomoka Ishibashi, Kohei Nakamura, Kaori Sanuki, Ruriko Ono, Masako Ishikawa, Takeshi Isobe, Satoru Kyo. _Shimane University Faculty of Medicine, Izumo city, Japan_.

[Introduction] In recent years, tumor cells have immune escape mechanism and immune checkpoint inhibitor therapy (anti PD-1/ PD-L1 antibody) has shown benefit in various cancers. Somatic mutations have the potential to encode ''non-self''immunogenic antigens and lymphocytes infiltrate tumor cells in Microsatellite-instable (MSI) endometrial cancers. Therefore, immune checkpoint inhibitor therapy might be effective in MSI endometrial cancers.

[Method] Mismatch repair protein (MLH1, PMS2, MSH2, and MSH6), tumor-infiltrating lymphocytes (CD8), and PD-1/PD-L1 expression were assessed by immunohistochemistry in 60 patients with endometrial cancer. We examined whether MSI status have enhanced immune microenvironment and become the therapeutic effect predictor of PD-1/PD-L1 immunotherapy in endometrial cancer.

[Results] Loss of mismatch repair protein (MSI group) was identified in 8 (13.3 %) of 60 patients with endometrial cancer. Expression of tumor-infiltrating lymphocytes (CD8) and PD-L1/PD-1 were significantly higher in MSI group compared to MSS group (p=0.001, p=0.044 and p=0.013).

[Conclusion]These results suggested that immune checkpoint inhibitor (anti PD-1/PD-L1 antibody) is effective in endometrial cancers with MSI. MSI testing is likely to be a biomarker for PD-1/PD-L1 immunotherapy in endometrial cancer.

#2622

Two new TLR9 agonists for cancer immunotherapy: Combination with checkpoint inhibitors.

Kerstin Kapp,1 Barbara Volz,1 Detlef Oswald,1 Burghardt Wittig,2 Manuel Schmidt1. 1 _MOLOGEN AG, Berlin, Germany;_ 2 _Foundation Institute Molecular Biology and Bioinformatics, Freie Universitaet Berlin, Berlin, Germany_.

Introduction

TLR9 agonists are developed as anti-cancer therapies based on their broad activation of the innate and adaptive immune system. Single-stranded oligodeoxynucleotides (ODN) containing non-methylated CG-motifs activate TLR9. Previously, chemical modification was used to prevent their degradation by exonucleases. To avoid the off-target effects observed with chemical modifications, new TLR9 agonists containing only natural DNA were stabilized by structural components. The dSLIM® family of TLR9 agonists is protected from exonucleolytic degradation by its covalently-closed dumbbell-shaped structure. It contains an immunomodulatory sequence with CG-motifs in its loops. The linear single-stranded EnanDIM® family of TLR9 agonists utilizes L-deoxyribonucleotides (natural enantiomers of D-deoxyribonucleotides) at their 3'-ends to prevent degradation. Since the mode-of-action of TLR9 agonists starts upstream of the targets of checkpoint inhibitors anti-PD-1/anti-PD-L1 a combinatory approach may support synergistic immune activation and thus enhanced anti-tumor effects.

Methods

The impact of dSLIM2006 and EnanDIM-1 on T cell responses was analyzed employing an in vitro assay using human peripheral blood mononuclear cells (PBMC). PBMC were treated with peptides selected from HLA class I-restricted T-cell epitopes of recall-antigens (CMV, EBV, Flu = CEF), TLR9 agonists and anti-PD-1 as checkpoint inhibitor. In addition, in vivo studies were used to investigate the anti-tumor effect of dSLIM2006 and EnanDIM-1 in combination with anti-PD-1 in a syngeneic murine CT26 tumor model.

Results

The IFN-gamma secretion of human PBMC after stimulation of CEF peptides was roughly 5-fold increased by EnanDIM-1 and dSLIM2006, whereas treatment with anti-PD-1 resulted barely in a two-fold increase. The combination of the TLR9 agonists and anti-PD-1 further enforced IFN-gamma secretion by about 7-fold. In the murine colon cancer model CT26 the subcutaneous injection of EnanDIM-1 or intraperitoneal injection of anti-PD-1 had a moderate effect on the tumor growth when used in monotherapy (28.3% or 57.0% tumor growth inhibition, TGI). Notably, a combination of EnanDIM-1 and anti-PD-1 further reduced tumor growth (74.7% TGI). Intratumoral injection of dSLIM2006 in combination with intraperitoneal injection of anti-PD-1 reduced tumor growth (54.2% TGI) whereas the single components had more limited effects (dSLIM2006: 18.7%, anti-PD-1: no inhibition). Combined treatment with TLR9 agonists and anti-PD-1 prolonged survival of the mice in comparison to single treatments.

Conclusions

The TLR9 agonists and immune surveillance reactivators (ISR) EnanDIM-1 and dSLIM2006 enhance T cell responses and anti-tumor effects of the anti-PD-1 checkpoint inhibitor. These data show their promising potential not only for monotherapeutic but also combinatory approaches.

#2623

**Identification of neo-antigens driving melanoma response to immune checkpoint blockers via** in vivo **screening.**

Chi-Ping Day,1 Eva Perez-Guijarro,1 Rajaa El Meskini,2 Zoe Weaver Ohler,2 Maxwell Lee,1 Howard Yang,1 Suman Vodnala,1 Shyam Sharan,3 Glenn Merlino1. 1 _National Cancer Institute, NIH, Bethesda, MD;_ 2 _Frederick National Laboratory for Cancer Research, Frederick, MD;_ 3 _National Cancer Institute, NIH, Frederick, MD_.

Immune checkpoint blockers (ICBs) have rendered unprecedented, durable responses in metastatic melanoma, but the heterogeneous response among patients continues to be the major obstacle for their therapeutic development. It is generally hypothesized that neoantigens derived from mutated genes are involved in tumor response to ICBs, since the latter is correlated witt mutational loads of tumors. However, direct experimental evidence showing that threshold quantity or specific properties of neoantigens drive ICB response are mostly lacking. To identify and characterize neoantigens implicated in ICB response, we have generated two UV-induced melanoma models based on the BrafV600E/Pten-knockout (Braf/PKO) and Hgf-transgenic (Hgf-tg) mouse, which displayed intrinsic resistance and high sensitivity to an anti-CTLA-4 antibody, respectively. Exome sequencing identified 216 and 291 non-synonymous mutations in the Braf/PKO and UV-Hgf melanoma cell lines, respectively. By RNA sequencing, 74 (34%) and 121 (42%) of these mutated genes were found to be expressed in each model, respectively, and there were no overlapping mutations between them. The mutations found in the "sensitive" UV-Hgf melanoma were analyzed in silico for their binding affinity to MHC-I and/or MHC-II, thus characterizing putative neoantigens. A "neo-epitope" library was generated by cloning the DNA sequences flanking non-synonymous mutations in frame with the eGFP gene in a lentiviral vector. We further showed that such eGFP-fused epitopes can be presented by the cells to induce specific T cell responses. The library will be transduced into the "resistant" Braf/PKO melanoma, which will be treated with anti-CTLA-4 in mice to identify the neoantigens required for the response. To prevent immunity against eGFP expressed by tumors, the library-transduced melanoma cells will be transplanted into the eGFP-tolerant "glowing head mice". The results will be used to determine if one, or more, of our candidate neo-epitopes can induce a response to anti-CTLA-4. We will also analyze if the response to this ICB is an epitope-specific reaction or require multiple epitopes, which will help to identify resistance mechanisms. We anticipate that our results will provide insight into the role of neoantigens in ICB response. Moreover, our models will serve as a platform to study the specific contribution and predictive value of neoantigens for melanoma response to immunotherapy, which could help improve therapeutic strategies involving ICBs.

#2624

An FKBP5-based immunophenotype for assessment of the immunosuppression status and possible prediction of immunotherapy response in melanoma patients.

Simona Romano,1 Ester Simeone,2 Anna D'Angelillo,1 Paolo D'Arrigo,1 Mario Capasso,1 Vito Alessandro Lasorsa,1 Martina Tufano,1 Anna Rea,1 Michele Russo,1 Nicola Zambrano,1 Paolo Antonio Ascierto,3 Maria Fiammetta Romano1. 1 _Univ. of Naples Federico II, Napoli, Italy;_ 2 _National Cancer Institute "G Pascale", Napoli, Italy;_ 3 _National Cancer Institute, Napoli, Italy_.

Background and aim: FKBP51 is an immunophilin encoded by FKBP5 gene on chromosome 6. It is a protein resident in lymphocytes and involved in immune response. Recently, our group demonstrated that the inhibitory checkpoint PD-L1/PD1 promoted the alternative splicing of FKBP5 gene, resulting in increased expression of its variant 4, in PBMC of melanoma patients. The aim of this study was to address whether such a molecular signature could help in identifying an immune profile associated with increased probability of immunotherapy response.

Experimental Design: The splicing FKBP51 isoform or FKBP51s was measured in peripheral blood T lymphocytes subsets (CD3/CD4, CD3/CD8, CD25 and PD-L1) and CD14 monocytes from a cohort of 118 patients and 77 age- and sex-matched healthy controls, by flow cytometry. Blood samples were collected before patients underwent ipilimumab treatment. Furthermore, in 64 out of 118 patients, expression of FKBP51s was also assessed in regulatory T cells.

Results: Physiologically, each PBMC subset analyzed contained a fraction of an FKBP51spos component, which resulted expanded in melanoma patients. We also measured an increase in CD3/CD8 and PD-L1 lymphocytes in patients. CD4 T lymphocytes showed the FKBP51sneg fraction significantly impaired, which might reflect the condition of impaired T cell help. Treg count was increased, in accordance with previous studies. The count of FKBP51sposTregs defined a subgroup of nonresponder patients to ipilimumab, by 92.6%. A 2D hierarchical partitioning of data from FKBP51s-immunophenotype (heatmap) revealed 3 main clusters: C1 (33 pts, 51,5%), C2 (14 pts, 22%) and C3 (17 pts, 26,5%). FKBP51spos Treg subset appeared globally increased in all clusters. In C1, values of effector T cells and monocytes were not different from normal donors. C2 showed a significant increase of FKBP51spos PD-L1 monocytes and a significant probability of not responding (Chi-square=5.46; p=0.019). This finding is consistent with the hypothesis that response to ipilimumab is prevented by accessory cells exerting a negative immune regulatory control. C3 showed a significant increase of FKBP51s in overall lymphocyte subsets analyzed. Patients in this cluster showed reduced overall survival. This finding suggests that a prevalence of activation (CD25) and co-inhibitory (PD-L1) markers together with the expansion of FKBP51spos effector T cells might reflect a condition of chronic lymphocyte stimulation in some advanced melanoma patients, contributing to T cell-exhaustion.

Conclusions: FKBP51s-based immunophenotype of melanoma patients revealed several profiles virtually related to a negative immune regulatory control and highlights an impairment of a Treg subset endowed with increased suppressive potential. Such FKBP51sTreg subset is likely to be associated with immunotherapy response (Chi-square=9.916, p=0.002) .

#2625

Clinicopathological features of programmed cell death ligand 1 (PD-L1) expression in resected non-small cell lung cancers.

Yoshihito Ohhara,1 Ichiro Kinoshita,1 Utano Tomaru,1 Kanako C. Hatanaka,2 Yutaka Hatanaka,2 Rio Honma,1 Satoshi Takeuchi,1 Yasushi Shimizu,1 Kichizo Kaga,1 Yoshihiro Matsuno,2 Hirotoshi Dosaka-Akita1. 1 _Hokkaido University Graduate School of Medicine, Sapporo, Japan;_ 2 _Hokkaido University Hospital, Sapporo, Japan_.

Background: Antibodies against programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) have recently demonstrated a survival benefit in several types of cancer, including melanoma and non-small cell lung cancer (NSCLC). PD-L1 expression has provided a predictive biomarker for anti-PD-1/PD-L1 therapy. However, the relationship between PD-L1 expression and clinical and clinicopathological characteristics remains unclear in NSCLCs. The aim of this study was to investigate the relevance of PD-L1 expression to clinical and clinicopathological factors in resected NSCLCs. Materials and Methods: PD-L1 expression was evaluated in 154 surgically resected NSCLC specimens by immunohistochemistry (IHC) using the SP142 antibody. Before the IHC of clinical samples, we performed a concordance study of Western blot analysis and IHC in 17 lung cancer cell lines. We evaluated the relationship between PD-L1 expression and clinical and clinicopathological factors by univariate and multivariate analyses. Survival curves were estimated by the Kaplan-Meier methods, and differences in survival distributions were evaluated by the log-rank test.

Results: PD-L1 expression was well concordantly observed in Western blot analysis and IHC in the lung cancer cell lines analyzed. PD-L1 expression was observed in 51 of 154 (33%) surgically resected NSCLCs. PD-L1 expression was significantly correlated with male, heavy smoking history, squamous cell carcinoma, and moderate/poor differentiation of tumors by univariate analysis. In multivariate analysis, tumor differentiation had a strong relationship with PD-L1 expression (odds ratio: 6.32, 95% CI: 1.70-23.49, P = 0.006). The survival was not statistically different between patients with high and low PD-L1 expression (5-year survival rate: 44.1% versus 58.1%, P = 0.22). PD-L1 expression was not an independent prognostic factor in univariate and multivariate analyses.

Conclusion: PD-L1 expression was strongly associated with tumor differentiation in resected NSCLCs. PD-L1 expression was not a prognostic factor in this cohort of resected NSCLCs.

#2626

Expression of human poliovirus receptor, an immune checkpoint biomarker in lung cancer.

Camilla Koczara,1 Hui Yu,1 Kim Ellison,1 Christopher Rivard,1 Leslie Rozeboom,1 Daniel Chan,1 Kenichi Suda,1 Kristine Brovsky,1 Rafal Dziadziuszko,2 Fred Hirsch1. 1 _University of Colorado Anschutz Medical Campus, Aurora, CO;_ 2 _Medical University of Gdansk, Gdansk, Poland_.

Background: Recently, immune therapy has shown notable efficacy in melanoma, non-small-cell lung cancer (NSCLC), among others, indicating that the targeting of immune checkpoints may be a promising treatment for malignant tumors. The human Poliovirus Receptor (CD155), an immune checkpoint, has been found to be overexpressed on the membranes of various tumor cells including colorectal and ovarian carcinoma. Examining PVR's expression in NSCLC could shine light on PVR's potential role and its possible correlation to the prognosis and outcomes for NSCLC patients.

Material and Methods: Gene expression data for lung cancer cell lines and tumor tissues was derived from the Cancer Cell Line Encyclopedia (CCLE [www.broadinstitute.org/ccle]) and the TCGA database (www.cbioportal.org), respectively. Immunohistochemistry (IHC) was performed on a Ventana Benchmark XT platform using an anti-PVR antibody (Cell Signaling #D3G7H at a 1:100 dilution). IHC staining was evaluated for a NSCLC Cell Line Tissue MicroArray (TMA) and a NSCLC patient cohort TMA. The H-score system was used to generate a semi-quantitative score, ranging from 0 to 300, that evaluates the intensity and prevalence of the staining on tumor cells. Patient data was analysed to uncover correlations between outcome and PVR expression in the NSCLC TMA cohort. Western blot analysis was performed to confirm antibody specificity and PVR expression levels in three NSCLC cell lines.

Results: Analysis of mRNA expression data from CCLE and TCGA databases identified that PVR is highly expressed in lung cancer. Three lung cancer cell lines were chosen based on mRNA expression for this study: NCI-H211 (high), NCI-H1650 (moderate) and NCI-H187 (negative). Cell line data revealed that PVR demonstrated higher expression levels in lung cancer than other immune checkpoints, such as PD-L1, PD-L2, and Gal-9 among others. In addition, PVR expression was not correlated with other negative immune checkpoints including PD-L1 and Gal-9. Western blot analysis confirmed that the Cell Signaling antibody was specific to the PVR protein in NSCLC cell lines. IHC staining of NSCLC cell lines revealed that non-squamous NSCLC cell lines express higher levels of PVR than do squamous cell cell lines (p<0.0001, Fisher test). The prevalence of PVR in the NSCLC patient cohort was 68% (68/100) using a H-score cutoff of ≥ 7. Analysis revealed a lower positivity of 61.7% (37/60) in squamous carcinoma as compared to 77.5% (31/40) in non-squamous NSCLC. Analysis of the patient outcome data demonstrated that higher levels of PVR protein expression is correlated with poorer outcomes in patients with both squamous lung cancer and adenocarcinoma.

Conclusions: PVR protein expression could potentially serve as a new immune checkpoint biomarker in lung cancer. This study demonstrates that high PVR protein levels predict poorer outcomes in lung cancer patients; suggesting that PVR may be an important target for future immune therapy.

#2627

**A study of dynamic changes in PD-L1 expression in** KRAS **mutant adenocarcinoma of the lung exposed to signal transduction inhibitors.**

Anna R. Minchom,1 Parames Thavasu,1 Zai Ahmad,1 Adam Stewart,1 Alexandros Georgiou,1 Mary ER O'Brien,2 Sanjay Popat,3 Jaishree Bhosle,2 Timothy A. Yap,1 Johann de Bono,1 Udai Banerji1. 1 _Institute of Cancer Research, Sutton, United Kingdom;_ 2 _Royal Marsden Hospital, Sutton, United Kingdom;_ 3 _Royal Marsden Hospital, Chelsea, United Kingdom_.

Aim

MEK and AKT inhibitors inhibit signaling down steam of KRAS and are being evaluated as single agents or in combination with other anticancer drugs for the treatment of KRAS mutant adenocarcinoma of the lung (adeno-NSCLC). We investigated whether increased PD-L1 expression with functional T cell inhibitory consequences were a common mechanism of resistance to these drugs in this setting.

Material and Methods

A panel of 10 KRAS mutant adeno-NSCLC cell lines were studied. Cells were exposed to GI50 concentrations of a MEK (trametinib) and AKT (AZD5363) inhibitor for 6hrs, 24hrs and 3 weeks. PD-L1 expression on cell lines was studied by immunofluorescence. Immunofluorescence at various time points were expressed as a ratio of values in drug treated cells compared to untreated control. Functional consequences of PD-L1 expression were studied using a T cell cancer cell line (Jurkat) transfected with a luciferase reporter where co-culture of cancer cells expressing PD-L1 led to reduction in luminescence. Luminescence at various time points were expressed as ratio of luminescence values of drug treated cells compared to untreated controls.

Results

Following characterization of expression of PD-L1 in 10 KRAS mutant adeno-NSCLC cell lines, 5 cell lines with the highest expression of PD-L1 was chosen for functional assays (H441, H2291, H23, H2030 and A549). When exposed to trametinib for 3 weeks, 3/5 cell lines (H23, H2030 and A549) showed an statistically significant increase in expression of PD-L1 (range 1.1-2.4) however significant reduction of luciferase activity was only observed in H23 and H2030, 0.93 and 0.75, p= 0.018 and p=0.01 respectively. When exposed to AZD5363 for 3 weeks, 3/5 cell lines (H441, H23 and H2030) showed a statistically significant increase in PD-L1 expression (range 1.3-1.9) however significant reduction in luciferase reduction was seen in only H441 and H23, 0.86 and 0.76, p= 0.03 and p= 0.04 respectively.

Conclusion

Chronic exposure of KRAS mutant cell lines to MEK and AKT inhibitors cause minor increases in PD-L1 expression but this does not result in functional inhibition of T cells in all instances. Consequently, a functional increase in PD-L1 expression is unlikely to be a common mechanism of resistance to MEK and AKT inhibitors in KRAS mutant adeno-NSCLC.

1

#2628

BGB-A425: a humanized anti-human Tim-3 antibody that exhibits strong immune cell activation.

Tong Zhang, Liu Xue, Jing Zhang, Qi Liu, Jie Ma, Yi Zhang, Yingdi Shi, Hongjia Hou, Hao Peng, Ning Liu, Yilu Zhang, Xiaomin Song, Yucheng Li, Jing Song, Lai Wang, Min Wei, Kang Li. _BeiGene, Beijing, China_.

Background: Tim-3 (T-cell immunoglobin and mucin-domian containing-3) is a "checkpoint" inhibitory receptor, which is primarily expressed in activated or "exhausted" T cells, NK cells, macrophages and DCs. Engagement of Tim-3 receptor by its ligand phosphatidylserine (PtdSer) or galectin-9 leads to activation of negative regulatory signaling in T-cells, promoting functional exhaustion of tumor-infiltrating T-lymphocytes. BGB-A425 is a novel humanized IgG1 (variant) anti-Tim-3 antibody under preclinical development. The immunomodulatory activity of BGB-A425 was evaluated both in vitro and in vivo.

Materials and methods: BGB-A425 was generated through hybridoma fusion, humanized by CDR grafting and structural simulation. The Fc region (IgG1) of BGB-A425 was engineered to remove Fc gamma receptor (FcγR) binding. The binding affinity and specificity were studied by ELISA, FACS and SPR (Biacore). The immunomodulatory functions of BGB-A425 were evaluated using primary immune cells as well as cell lines.

Results: BGB-A425 binds to the extracellular domain of human Tim-3 with high affinity (KD = 0.36 nM) and specificity. In a competition assay, BGB-A425 efficiently blocks the interactions between Tim-3 and PtdSer. In vitro, BGB-A425 significantly enhances IFN-γ production of primary T cells and NK-mediated cytotoxicity against tumor cells. In a MLR assay, BGB-A425 augments T-cell response to allogeneic antigens either alone or in combination with an anti-PD-1 antibody BGB-A317. Besides blocking Tim-3, BGB-A425 can also induce the internalization of Tim-3 receptor on cell surface. In vivo, BGB-A425 in combination with BGB-A317 inhibits tumor growth in a mouse xenograft cancer model.

Conclusions: BGB-A425 demonstrates potent activity to stimulate immune cell function both in vitro and in vivo, supporting its clinical development for the treatment of human cancers.

#2629

The synergy between BXCL701, a DPP inhibitor, and immune checkpoint inhibitors discovered using AI and Big Data analytics.

Luca Rastelli, Snigdha Gupta, Akhil Dahiya, Zeenia Jagga, Krishnan Nandabalan, Sanatan Upmanyu. _BioXcel, Branford, CT_.

Using the proprietary Big Data PharmGPS® Discovery platform, BioXcel has created a comprehensive relationship map between immune-evasion and immune-activation pathways, comprising interacting genes and all overlapping pharmacological agents and tumors.

This map was used to identify clinically validated compounds that would act synergistically in combination with immune-checkpoint inhibitors (ICI) by remodelling the tumor micro-enviroment and transforming cold, non-inflamed tumors into hot immune-sensitive tumors.

One of the several compounds thus identified is BXCL701, previously known as Talabostat/PT-100, a DPP inhibitor that by inducing a wide panel of cytokines and chemokines stimulates both the innate and acquired immune system.

BXCL701 has a dual immuno-oncology related MOA. Via the Fibroblast Activator Protein (FAP) target, it inhibits the activation of immuno-suppressive fibroblasts and through an angiogenic related effect, it increases immune cell extravasation into the tumor tissue. Via the DPP8/9 targets, it depresses the immuno-suppressive activity of MDSCs by inducing a granulocytic differentiation while it stimulates the priming, migration and cytotoxicity of T-cells and NK cells and the formation of memory T-cells.

The hypothesis that BXCL701 immune-mediated MOA would complement the action of ICIs was validated in-vivo in the syngeneic MC38 mouse model of colon adenocarcinoma.

Co-administration of BXCL701 combined with anti-PD1 showed a synergistic inhibition of tumor growth as well as synergistic up-regulation of immuno-stimulatory cytokines, IL-2, IL12 and GM-CSF. The effects of the combination on the immune-phenotyping of the circulating and tumor infiltrated immune cells will also be presented.

The findings support BXCL701 ability to transform the immune-suppressive tumour microenvironment to an immuno-permissive milieu sensitive to immune-checkpoint inhibitors. Further supporting the therapeutic potential of BXCL701, an analysis of genomic alterations in FAP, DPP8 and DPP9 across a wide range of tumors singled out castration-resistant prostate cancer with a high level of DPP9 amplification (14%) and overexpression of DPP8 in 50% of the patients which could make this patient population uniquely sensitive to the combination as shown by in-vitro and in-vivo experiments.

This study provides further evidence of the capability of Big data analytics to generate in-silico hypothesis of synergistic combination effects that can be converted in validated therapeutic opportunities to benefit patients non- responsive to ICI therapy.

#2630

MEN1309, a novel antibody drug conjugate (ADC) targeting Ly75 antigen, induces complete responses in several xenografts of solid tumors.

Mario Bigioni,1 Giuseppe Merlino,1 Cristina Bernadó Morales,2 Rossana Bugianesi,1 Attilio Crea,1 Rosanna Manno,3 Joaquin Arribas,2 Rachel Dusek,4 Nickolas Attanasio,4 Keith Wilson,4 Christian Rohlff,5 Monica Binaschi1. 1 _Menarini Ricerche S.p.A., Pomezia (RM), Italy;_ 2 _Vall D'Hebron, Institute of Oncology, Barcelona, Spain;_ 3 _Research Toxicology Center, Pomezia (RM), Italy;_ 4 _Oxford BioTherapeutics, Inc., San Jose, CA, USA, San Jose, CA;_ 5 _Oxford BioTherapeutics, Ltd., Abingdon, UK, Abingdon, United Kingdom_.

The cell surface antigen Lymphocyte antigen 75 (LY75, CD205, DEC-205) is over-expressed in several tumor histotypes. It is a type I C-type lectin receptor (CLR), normally expressed on various APC subsets, characterized by a cytoplasmic domain containing protein motifs crucial for endocytosis and internalization upon ligation. These features make the antigen ideal to be exploited as a target for a novel ADC. MEN1309 is a humanized IgG1 antibody directed against the cell surface antigen Ly75, conjugated through a cleavable linker to a potent maytansinoid microtubule disruptor, DM4. In this study, we evaluated the in vitro and in vivo (xenografts and PDX) efficacy of MEN1309 in different tumor histotypes. A PK/PD relationship was also investigated in tumor-bearing mice. IHC demonstrated high prevalence of Ly75 in human pancreatic, triple negative breast, and bladder cancers, as well as in diffuse large B-cell lymphoma. In vitro experiments showed that cytotoxic activity of MEN1309 was in nM/sub nM range against several lymphoma, pancreatic, bladder and triple-negative breast cancer (TNBC) cell lines. Moreover, MEN1309 exhibited high cell-killing ability against cells having either strong as well as low to moderate antigen expression. In vivo, MEN1309 at 2.5-5 mg/kg (schedule varying from single dose, q7dx3, or q21dx3) showed an impressive antitumor activity, resulting in complete and long lasting responses in most of the xenograft models representing lymphoma, TNBC, bladder and pancreatic cancers, expressing the antigen at high but also at low levels. No treatment related toxicity in terms of change of body weight and death events were detected. Moreover, the administration of (i) isotype control-DM4, (ii) the non-conjugate antibody IgG1 and (iii) the free toxin DM4 (at a dosage corresponding to the equimolar concentration linked at 10 mg/kg ADC) showed little to no therapeutic efficacy on tumor growth. In TNBC patient-derived xenograft (PDX) model (coming from a heavily pre-treated patient and expressing high level of the antigen Ly75), MEN1309 (5 mg/kg q21dx3) showed a complete tumor regression. Finally, in the pancreatic adenocarcinoma xenograft model HPAFII, the pharmacokinetics profile in serum of MEN1309 at 5 mg/kg was characterized and it was qualitatively correlated, using immunofluorescence, with the occurrence of phosphorylation of Serine 10 of H3 Histone in cancer cells, as a pharmacodynamic (PD) marker of DM4 activity on microtubules. Initial ADC exposure was noteworthy and was followed by a relatively fast decline. In parallel with the decay of the serum ADC concentrations there was a progressive increase in the number of positive cells showing the PD marker for mitotic arrest. Overall, our data suggest that MEN1309 is a selective and potent novel antitumoral ADC and it deserves to enter into aPhase I study for a variety of Ly75 positive tumor histotypes.

#2631

**Enhanced anti-tumor effects by a combination approach of interferon-γ producing recombinant** Bifidobacterium **and anti-mPD-1 antibody in syngeneic mouse model.**

Yuji Seki,1 Koichiro Shioya,1 Satoshi Kobayashi,1 Yuko Shimatani,1 Minoru Fujimori,2 Shun'ichiro Taniguchi3. 1 _Anaeropharma Science, Inc., Matsumoto, Japan;_ 2 _Department of Breast Surgery, Tokyo Medical University Ibaraki Medical Center, Ibaraki, Japan;_ 3 _Department of Comprehensive Cancer Therapy, Shinshu University School of Medicine, Matsumoto, Japan_.

Cancer immunotherapies like antibodies targeting immune checkpoint molecules have resulted in an extraordinary success in cancer treatment. Still, large fraction of cancer patients doesn't respond to the antibody immunotherapy and lots of patients are suffered from immune-related adverse events (irAEs) which sometimes cause discontinuation of the treatment. The above problems highlight the needs in the cancer immunotherapy for attempting various approaches like synergistic combination therapy to minimize adverse events and enhance efficacy.

Interferon-γ is a cytokine having antitumor activity and has been developed as an anticancer drug in multiple cancer indications but failed due to its limited efficacy and severe adverse events. In several publications, IFN-γ has been clarified to induce PD-L1 expression in tumors [1,2]. Upreguration of PD-L1 renders tumor resistance to cytotoxic T cell and it may cause the limited efficacy of IFN-γ therapy. Our preliminary experiments suggested that an intratumoral injection of purified IFN-γ and following addition with anti-murine PD-1 antibody showed significant tumor suppression, implying a combination effects of regional IFN-γ and immune checkpoint blockers.

Here in the present report, in an aim of decreasing adverse events, we have created recombinant Bifidobacterium, which is a non-pathogenic anaerobic bacterium to secrete IFN-γ specifically inside solid tumor, and then demonstrated the combination of IFN-γ producing Bifidobacterium and anti-murine PD-1 antibody significantly suppress tumor growth whereas each single treatment moderately contributes to tumor suppression in a CT26 bearing syngeneic mouse model. In addition, ELISA assay indicated that there are substantial IFN-γ detected in tumor tissue but none of them are detectable in blood on the Day 4 of treatment. All in together, combination treatment with IFN-γ producing Bifidobacterium and anti-murine PD-1 antibody offers a promising anti-tumor approach.

Reference:

1. Abiko K., Br J Cancer, 2015 Apr 28;112(9):1501-9. IFN-γ from lymphocytes induces PD-L1 expression and promotes progression of ovarian cancer.

2. Mimura K., Cancer Sci., 2014 Oct;105(10):1236-44. Inhibition of mitogen-activated protein kinase pathway can induce upregulation of human leukocyte antigen class I without PD-L1-upregulation in contrast to interferon-γ treatment.

#2632

4SC-202 induces inflamed tumor microenvironment, strongly enhances tumor infiltration with cytotoxic T cells and primes tumors for anti-PD1/PD-L1 therapy.

Tanja Wulff, Kerstin Kronthaler, Sabine Schrepfer, Ulrike Parnitzke, Anne Catherine Bretz, Roland Baumgartner, Svetlana Hamm. _4SC, Martinsried, Germany_.

Various HDAC inhibitors were described as beneficially affecting anti-tumoral immune response. Although different HDAC inhibitors were investigated in syngeneic tumor models, the mode of anti-tumoral action is not yet fully understood. Here, we analyzed the anti-tumoral mode-of-action (MOA) of 4SC-202, an orally available clinical stage epigenetic small molecule inhibitor specifically targeting histone deacetylases HDAC class I as well as the lysine-specific demethylase LSD1. To ensure that the conclusions would be relevant for the clinical situation we used a clinically equivalent dose and schedule.

Anti-tumoral efficacy and the impact of 4SC-202 alone or in combination with an anti-PD-L1 antibody on the tumor microenvironment were analyzed in the syngeneic colorectal CT26 model in immunocompetent BALB/c or in nude/irradiated BALB/c mice. A broad spectrum HDAC inhibitor was used for comparison. Transcriptome analysis was performed by RNA-Seq, and the composition of immune cell subpopulations in the tumor was determined by flow cytometry.

4SC-202 treatment at clinically relevant dosage regimen significantly inhibited growth of CT26 tumors. A competent immune system was apparently necessary for the anti-tumoral effect of 4SC-202 since its tumor-reducing effect was lost in immunocompromised mice. 4SC-202 treatment led to an increase of MHC class II molecules and enhanced expression of inflammatory markers like IFN-γ and various chemokines in the tumors. Detailed analysis of the tumor microenvironment revealed that 4SC-202 strongly increased the number of tumoricidal cytotoxic T cells (CTL). In contrast, a broad-spectrum HDAC inhibitor tested in the same model demonstrated anti-tumoral efficacy but did not affect the number of CTLs in tumors demonstrating that HDAC inhibitors employ different MOAs for their anti-tumoral response and that the effect on CTLs is not attributed to HDAC inhibition in general. Since the T cell abundance is pre-requisite for the efficacy of PD1/PD-L1 blockade, we tested the combination of clinically relevant dosage regiment of 4SC-202 with an anti-PD-L1 antibody. The combined treatment was more efficacious than monotherapies and resulted in significantly longer survival.

4SC-202 already demonstrated a favorable safety profile in a phase I clinical trial with relapsed or refractory hematological malignancies with two objective responses (1 CR, 1 PR) and disease stabilizations in several patients. 4SC-202's immune priming capacity offers further options for clinical development of 4SC-202 in combination with various cancer immunotherapy approaches.

#2633

Identification of novel T cell co-inhibitory and co-stimulatory receptors from screening a comprehensive library of extracellular proteins.

Arthur Brace, Nathan Sallee, Justin Chou, Lindsay Garrenton, Diana Chen, Ryan Liang, Greg Kemper, David Yang, Marina Jaquez, Janine Powers, Mikayel Mkrtichyan, Thomas Brennan, Luis Borges. _Five Prime Therapeutics, South San Francisco, CA_.

Antibody blockade of immune checkpoint regulators such as PD-1 and CTLA-4 has been shown to be an effective cancer treatment strategy; however, a large percentage of patients still do not respond to existing therapies. Discovery of additional immune checkpoints and co-stimulatory receptors and development of antibody therapeutics against them are likely critical to address this unmet patient need. We generated a comprehensive library of essentially all human extracellular proteins and have used this library to screen for new agents that modulate the function of various immune cell subsets both in vitro and in vivo. Using primary T effector and regulatory T cells, we have identified new targets that modulate the activities of these T cell populations in vitro and are currently evaluating these proteins as possible targets for cancer immunotherapy. In addition, we have expressed a subset of the library in three different models of tumor-bearing mice and have identified several proteins that modulate anti-tumor immunity in vivo. Several of the proteins identified in these screens have activity across multiple primary cell and in vivo tumor model screens and we are rapidly evaluating them as possible targets for novel immuno-oncology therapeutics. Taken together, we believe that we have developed robust in vitro and in vivo platforms that will allow us to discover novel cancer immuno-therapeutics that will help address the needs of cancer patients that fail to respond to current immunomodulatory therapies.

#2634

**EVT801: Standalone cancer immunotherapy in VEGFR3** + **tumors and combination with immune checkpoint therapies in VEGFR3** - **tumors.**

Michael Esquerré,1 Pierre Fons,1 Gaëlle Badet,1 Pauline Barron,1 Jérémy Kagan,1 Antoine Alam,2 Jérôme Meneyrol,1 Isabelle Blanc,2 Roselyne Broussy,1 Florence Gaujarengues,1 Joanna Lisztwan,1 Michael Paillasse,1 Mark Whittaker,1 Françoise Bono1. 1 _EVOTEC, Toulouse, France;_ 2 _Sanofi, Lyon, France_.

In collaboration with Sanofi, we previously showed that inhibition of the VEGFR3 pathway by SAR131675 leads to reduction of tumor growth by acting on the tumor microenvironment. We have identified a new drug candidate, EVT801, and evaluated its activity on tumor models expressing VEGFR3. For proof-of-concept, we transfected the mouse BNL hepatoma cell line with VEGFR3. EVT801 displayed a strong therapeutic activity by acting on both VEGFR3+ tumor cells and on the Tumor Microenvironment (TME). By deciphering the mechanism of action of the compounds we showed that EVT801 strongly decreased tumor-associated immunosuppression by decreasing MDSCs (Myeloid Derived Suppressor cells) and CD4+ regulatory T cells and by increasing macrophages with a M1 phenotype inside the tumor. As a consequence the T-cell:MDSC ratios were increased in the TME and also in peripheral blood. In addition to its immunomodulatory properties, EVT801 decreased angiogenesis without increasing hypoxia. We have evaluated EVT801 therapeutic activity in syngeneic tumor mouse models which are not expressing VEGFR3 such as 4T1 mammary carcinoma and CT26 colon carcinoma models. As expected, we observed an intermediate therapeutic activity of the compound on both tumor models. Positive modulation of the TME was equivalent to what we observed with the VEGFR3+ tumor model. With this unique mechanism of action of EVT801 on the TME, we evaluated its ability to increase therapeutic activity of standard Immune Checkpoint Therapies (ICT) such as anti-CTLA-4 and anti-PD-1 mAbs. Strong additive therapeutic activities were observed with EVT801 in combination with these ICT as illustrated by the development of long-term tumor-specific memory CD8+ T cell responses.

Toxicological data show that EVT801 has a favorable pharmacological profile consistent with its entry into pre-clinical development. Taken together, these results indicate that EVT801 represents an innovative drug for cancer Immunotherapy which provide a favorable microenvironment to promote tumor regression. In addition, EVT801 may improve the frequency of response to ICT.

#2635

Baseline myeloid-derived suppressor cell and eosinophil cell counts predict clinical efficacy in patients with non-small cell lung cancer (NSCLC) treated with nivolumab in second line.

Patrizia Mancuso, Antonio Passaro, Valentina Labanca, Sara Gandini, Gianluca Spitaleri, Elena Guerini, Massimo Barberis, Cristina Noberasco, Ester del Signore, Alessia Pochesci, Chiara Catania, Filippo De Marinis, Francesco Bertolini. _European Inst. of Oncology, Milan, Italy_.

The anti-PD-1 monoclonal antibody nivolumab is clinically active in a variety of tumor types including squamous (sq) and non-squamous (non-sq) NSCLC in second-line, where randomized phase III trials have shown a survival benefit. However, no predictive/prognostic or dynamic biomarkers have been found so far to correlate with clinical benefit in patients treated with anti-PD-1 antibodies. The aim of the present study is to investigate the potential role of baseline peripheral blood cell counts in relation to survival and response rate in NSCLC patients treated with nivolumab in a second-line setting. From July to May 2016 we evaluated 45 patients with Sq (n = 10) and non-Sq (n = 35) NSCLC, previously treated with first-line platinum-based chemotherapy, who received nivolumab 3 mg/kg IV on day 1 of each 2 week treatment cycle. Clinical characteristics (T-Stage, lymph nodes involvement, M-Stage) were assessed. Total numbers of white blood cells, myeloid-derived suppressor cells (MDSCs, including both monocytic [Mo-MDSC]) and polymorphonuclear [PMN-MDSC] types), regulatory T cells (T-regs), and serum lactate dehydrogenase (LDH) were assessed. Endpoints were correlations with objective response rate (RR), progression-free survival (PFS, categorized as ≤ 3 or > 3 months) and overall survival (OS). Tumor response was assessed using RECIST criteria, version 1.1, at week 9 and every 6 weeks thereafter until disease progression. Statistical investigations were based on univariate analyses by the Wilcoxon rank test. The median PFS of the overall study population was 3 months. Data about PMN-MDSCs (identified by flow cytometry as Lin-CD15+CD14-CD11b+HLA-DRlow/-), Mo-MDSCs (Lin-CD14-CD11b+HLA-DRlow/-) and absolute eosinophil count (AEC) were available in 37/45 patients (82% of treated patients). Baseline absolute numbers of PMN-MDSCs, Mo-MDSCs and AEC were greater in patients with a good prognosis (PFS > 3 months) and a better RR. In particular, among patients with shorter PFS and lower RR, the median numbers of PMN-MDSCs, Mo-MDSCs and AEC were significantly lower than those detected in patients with longer PFS (4 vs 13 cell/µl, p=0.01; 4 vs 21 cell/µl, p=0.06; 55 vs 155 cell/µl; p=0.02, respectively). Our data suggest that a baseline blood signature characterized by low levels of PMN-MDSCs, Mo-MDSCs and AEC is associated with a poor clinical outcome (median PFS ≤ of 3 months and low RR) in 67.6% of patients treated with nivolumab. In contrast, patients with high levels of these three biomarkers showed a median PFS significantly longer than 3 months and a higher RR. The OS analysis is ongoing, and further studies have been planned to understand whether this signature has a biomarker potential also in chemotherapy-naïve, first line NSCLC patients.

#2636

Targeted therapies to ERBB receptors downregulate expression of PD-L1: implications in combination therapies.

Christopher A. Hamm,1 Sumin Zhao,2 Cesar A. Santa-Maria,3 Massimo Cristofanilli,3 Neil L. Spector,2 Sarah Bacus1. 1 _American Molecular Laboratories, Vernon Hills, IL;_ 2 _Duke University Medical Center, Durham, NC;_ 3 _Northwestern University Feinberg School of Medicine, Chicago, IL_.

Background: Inflammatory breast cancer (IBC) is the most aggressive and lethal form of primary breast cancer. Inflammatory signaling pathways are active in IBC, but the function of the immune response remains elusive. ErbB receptors play a role in IBC. ERBB2 is amplified in 50% of IBCs, and ERBB3 is also mutated in IBC. Currently, Lapatinib, a dual ErbB inhibitor, is used in IBC patients with ERBB2 amplification.

Although immune cell inflammatory signaling may promote IBC tumor growth and metastasis, the presence of cytotoxic tumor-associated lymphocytes has also been associated with a more favorable breast cancer prognosis. The contradictory nature of the immune data highlights the need to elucidate the relationship between the immune response and subsequent treatments. Given the active IBC immune component and the recent clinical benefit of immune checkpoint inhibitors in cancer treatment, IBC's may represent a clinically unique breast cancer population that may benefit from immunomodulating agents.

Interestingly, Myc is a downstream effector of ErbB signaling, and Myc is thought to regulate the expression of immune checkpoint proteins CD47 and PD-L1. The presence of PD-L1-positive IBC immune infiltrate suggests that IBC's may benefit from therapies that disrupt PD-L1 signaling together with ErbB inhibitors.

METHODS: IHC was used to examine immune checkpoint signaling and characterize the tumor-immune infiltrate. Tumor tissues were also characterized using an RNA-seq panel that examined the expression of 377 immune-related genes. Cell lines (BT474, SKR3, AU565, and SUM225) were treated with Lapatinib and Neratinib to examine the relationship between growth factor receptors and downstream immune signaling pathways.

RESULTS: RNA-seq revealed the expression of specific immunosuppressive signaling pathways in tumors. Treatment of breast cancer cells with ErbB inhibitors resulted in a decrease in the levels of PD-L1. Treatment with Lapatinib and Neratinib diminished PD-L1 in all cell lines. Levels of Phospho-Stat3 decreased in BT474 and Sum225 but not in SKBR3 and AU565, implying that PD-L1 is regulated by another mechanism (ERK-MYC).

CONCLUSION: Our results provide mechanistic insight into ErbB receptor activation and the expression of downstream signaling molecules (Stat3, Myc, PD-L1, and others). In addition, our unique RNA-seq immune signature reveals the expression of several genes that may serve as biomarkers of inhibitory immune signaling pathways. Our laboratory is currently examining the correlation between PD-L1 expression and the activation of ErbB2 in model systems and clinical trials using ErbB inhibitors in combination with PD-L1 inhibitors. Our immune panel gene signature may serve as a useful diagnostic test that, in conjunction with traditional ErbB testing, can identify patients that will benefit from combination therapy of an ErbB inhibitor and an immune checkpoint inhibitor.

#2637

Anti-tumor activity of cisplatin is enhanced by PD-1 blockade in preclinical models of head and neck squamous cell carcinoma.

Linda Tran,1 Clint T. Allen,2 So-Jin Park,1 Roy Xiao,1 Carter Van Waes,1 Nicole C. Schmitt2. 1 _NIDCD, Bethesda, MD;_ 2 _Johns Hopkins University and NIDCD, Bethesda, MD_.

Rationale: Cisplatin, which remains the most commonly used systemic drug for head and neck squamous cell carcinoma (HNSCC), reduces numbers of circulating immune cells. However, recent studies suggest that cisplatin may enhance some aspects of anti-tumor immunity in the tumor microenvironment. We previously found that cisplatin increases expression of major histocompatibility (MHC) class I and programmed death ligand 1 (PD-L1) in HNSCC cell lines in vitro.

Methods: In the current study, we investigated other components of the antigen processing machinery (APM) by flow cytometry following treatment with cisplatin. Using the syngeneic mouse oral cancer 1 (MOC1) model of HNSCC, we then investigated the effects of cisplatin and anti-PD-1 antibody, alone or in combination, on tumor growth and survival.

Results: Using three different HNSCC cell lines, we found that levels of the APM components LMP2, ERp57 and calreticulin increased in all cell lines treated with cisplatin, whereas TAP1/2 increased dramatically only in one cisplatin-treated cell line with wildtype TP53 (UMSCC-74A). In MOC1 tumor-bearing mice, tumor growth was partially delayed by treatment with cisplatin and anti-PD-1 antibody alone and substantially delayed when these agents were given in combination, with resultant increased survival. Comparison of different treatment schedules suggested that concurrent administration is more effective than giving cisplatin one day prior to anti-PD-1 or giving anti-PD-1 one day prior to cisplatin.

Conclusions: Despite upregulating PD-L1 on tumor cells and decreasing levels of circulating immune cells, cisplatin may enhance some aspects of anti-tumor immunity in the tumor microenvironment. While no mice were cured with concurrent cisplatin and anti-PD-1 antibody, tumor growth was substantially delayed. These results suggest a rationale for combining cisplatin and anti-PD-1 monoclonal antibodies, perhaps with radiation or other therapies, in additional preclinical studies and clinical trials of HNSCC.

Supported by NIDCD intramural project ZIA-DC-DC000090.

#2638

Clinical characteristics and PD-L1 expression of pulmonary sarcomatoid carcinoma.

Sung Yong Lee, Jae Kyeom Shim, Jee Youn Oh, Kyung Hoon Min, Gyu Young Hur, Jae Jeong Shim, Kyung Ho Kang, Bong Kyung Shin, Young Seok Lee. _Korea University Guro Hospital, Seoul, Republic of Korea_.

Introduction: Pulmonary sarcomatoid carcinoma (PSC) is a poorly differentiated non-small cell lung cancer (NSCLC) containing a component of spindle cell or giant cell. Because the prevalence of PSC is very rare, there is insufficient data about clinical feature, therapeutic strategy and prognosis.

Method: We reviewed the medical records of 26 patients diagnosed with PSC from January 2009 to June 2015, and analyzed the clincopathological characteristics, treatment modality and their outcome, and risk factors of overall survival (OS). Programmed death ligand 1 (PD-L1) expression was tested in 13 patients by using archival tissues (PD-L1 IHC 22C3 pharmDx assay).

Results: The median age of PSC patients was 69.5 years. 23 patients were male. 25 patients were current or former heavy smokers. Pleomorphic carcinoma was the most common subtype, and epidermal growth receptor mutation (EGFR) was positive in 2 of 11 patients who were tested. Among 13 patients tested for programmed death ligand 1 (PD-L1), eight showed PD-L1 high expression (tumor proportion score, TPS ≥ 50%). Four patients showed low expression (1% ≤ TPS ≤ 49%), and one patient showed no expression (TPS < 1%). The median OS of all patients was 286 days. Age and the time from symptom onset to diagnosis were significantly associated with OS. PD-L1 expression showed the trend of positive prognostic value on OS in univariate analysis but was insignificant in multivariate analysis. 12 patients were treated with chemotherapy: 9 with platinum-based doublet therapy, 2 with tyrosine kinase inhibitor and 1 with docetaxel. 7 patients showed partial response or stable disease. The median OS and progression free survival of patients receiving chemotherapy were 260 days and 85 days, respectively.

Conclusions: It has poor prognosis compared with ordinary NSCLC. However, PSC patients showed comparatively fair response to chemotherapy and generally showed higher PD-L1 expression. Active treatment with cytotoxic chemotherapy, EGFR TKIs or immune checkpoint inhibitors are necessary to achieve a better results of PSC management.

#2639

Development of small molecule selective inhibitors of GCN2 as an immunotherapy aimed at preventing immune escape of tumor cells.

Michał Gałęzowski, Kamil Sitarz, Eliza Majewska, Stefan Chmielewski, Kinga Michalik, Magdalena Masiejczyk, Agnieszka Adamus, Arkadiusz Białas, Joanna Fogt, Marcin Bień, Mateusz Świrski, Maciej Mikulski, Julian Zachmann, Krzysztof Brzózka. _Selvita S.A., Kraków, Poland_.

GCN2 is a protein kinase capable of sensing amino acid (AA) shortage. It gets activated by binding of unloaded tRNAs and subsequently phosphorylates translation initiation factor 2 alpha (eIF2α) eventually leading to induction of ATF4-mediated integrated stress response (ISR). Elevated catabolism of one of the essential amino acids - tryptophan (Trp), driven by overexpression of critical enzymes in Trp metabolism - IDO and TDO, leads to immunosuppressive microenvironment in many types of cancer. GCN2 is a key effector signaling component for IDO/TDO and is considered as a metabolic checkpoint of highly Trp-dependent T-cells. GCN2 activation through accumulation of unloaded tRNAs leads to inhibition of CD8+ effector T-cells and increase in generation and activation of regulatory T-cells. According to these findings, selective inhibition of GCN2 may be an effective strategy for targeting Trp-dependent immunosurveillance of tumor cells. In this study, we report the results for a series of novel small molecule GCN2 kinase inhibitors that have been developed at Selvita and, to our best knowledge, are the most potent GCN2 inhibitors reported so far. Newly synthesized compounds exert low nanomolar potency for GCN2, good selectivity as well as ADME profiles. Profound assessment of the compounds potency and efficacy using a selection of in vitro activity/binding assays will be presented.

#2640

**Small-molecule inhibitors of CD73, CD39 and A** 2a **R: Three anti-cancer targets in the ATP/adenosine signaling pathway.**

Ulrike Schindler, Joanne B. Tan, Matt Walters, Annette Becker, Fangfang Yin, Ada Chen, Yu Chen, Wan Hsin Lim, Steve Young, Manmohan Leleti, Jay Powers, Juan C. Jaen. _Arcus Biosciences Inc., Hayward, CA_.

INTRODUCTION: In the tumor micro-environment (TME) Adenosine (ADO) dampens the immune response towards cancer cells by inhibiting the cytotoxic activity of effector cells and promoting the proliferation of immunosuppressive cells. Extracellular ADO is generated by the two ecto-nucleotidases CD39 (ATP→AMP) and CD73 (AMP→ADO). In immune cells ADO signals primarily through the G-protein coupled receptor A2aR. Inhibition of ADO generation and signaling have been shown to be promising therapeutic approaches for the treatment of cancer.

METHODS: Potent and highly selective small molecule inhibitors for A2aR, CD73 and CD39 have been designed and synthesized by the Medicinal Chemistry Department at Arcus Biosciences. Enzymatic assays: CD39 and CD73 activity was assayed by quantitating hydrolysis of ATP or AMP, respectively. Inorganic phosphate was detected using a colorimetric (malachite green) protocol. ADO receptor assays: The potency of A2aR antagonists and selectivity towards related receptors was determined by measuring cAMP elevation in stably-expressing CHO cells following NECA (ADO mimic) stimulation. CD8+ T cell assays: Activation, proliferation and effector function of CD8+ T cells were quantified following compound treatment in the presence and absence of ATP. Mixed Lymphocyte Reaction (MLR): Cytokine levels were determined in an MLR assay after compound treatment in the presence and absence of ATP. Tumor models: CT26 and B16F10 syngeneic tumor models were used to assess the therapeutic effect of the inhibitors.

RESULTS: Potent and highly selective small molecules have been generated to block either ADO generation or ADO signaling. The therapeutic potential of these molecules was assessed in CD8+ T cell assays, MLR assays and in various tumor models. CD73 inhibition blocked the conversion of AMP to ADO. CD39 inhibition blocked the conversion of ATP to AMP and A2aR inhibition abolished the increases in intracellular cAMP following ADO stimulation. The compounds are highly selective relative to related enzymes/receptors, as well as a large panel of unrelated targets. Inhibition of any one of the three targets robustly reversed adenosine-mediated inhibition of proliferation, CD25 expression, and IFN-γ and granzyme B production by human CD8+ T-cells. Robust inhibition of tumor growth in combination with anti-PD1 antibody was observed for several of these compounds.

CONCLUSIONS: Highly potent and selective inhibitors of each of the 3 molecular targets involved in the ATP/adenosine pathway have been identified. Their ability to interfere with the extracellular generation of ADO and the immune-suppressive effects of ADO, as well as their effects on experimental tumor biology, have been demonstrated in various in vitro and in vivo models.

#2641

**AZD4635 A** 2A **receptor occupancy in cynomolgus monkey using PET and its application to an oncology clinical development program.**

Peter Johnström,1 Pablo Morentin Gutierrez,2 Katarina Varnäs,3 Magnus Schou,1 Akihiro Takano,3 Lorraine Jones,4 Ganesh Mugundu,5 Patricia McCoon,5 Paul Lyne,5 Jeffrey Infante,6 Gerald Falchook,6 Manish Patel,6 Janet Karlix,6 Melinda Merchant,5 James Clarke,2 Alan Cross,5 Nicholas Seneca,1 Lars Farde,1 Miles Congreve,7 Jon S. Mason,7 Fiona H. Marshall7. 1 _AstraZeneca, Stockholm, Sweden;_ 2 _AstraZeneca, Cambridge, United Kingdom;_ 3 _Karolinska Institutet, Stockholm, Sweden;_ 4 _AstraZeneca, Macclesfield, United Kingdom;_ 5 _AstraZeneca, Waltham, MA;_ 6 _Sarah Cannon, Nashville, TN;_ 7 _Heptares, Welwyn Garden City, United Kingdom_.

Introduction

AZD4635 is an A2A receptor antagonist currently being tested as monotherapy and in combination with durvalumab in patients with advanced solid cancers. High adenosine levels found in tumors are immune suppressive and therefore AZD4635 could potentiate immune checkpoint inhibitors such as durvalumab (anti-PDL1). Predictions of A2A receptor engagement in patients at different doses and at different time points may enable better interpretation of clinical biomarker data measuring effects on immune modulation. A quantitative assessment of the receptor occupancy in the brain of non-human primates was conducted for AZD4635 with PET imaging and the resulting PK/PD model was applied to predict occupancy in humans in tumors.

Methods

PET measurements of A2AR occupancy in brain was performed using the radioligand [18F]MNI-444 in three anesthetized cynomologus monkeys. PET data acquisition was performed for 120 min following IV-administration of [18F]MNI-444 at baseline and following pretreatment of AZD4635. Sampling for AZD4635 plasma exposure determination was performed. As part of PK/PD analysis of the occupancy data, a novel modification of the non Invasive-LOGAN data analysis of the PET data was performed to obtain a time course of occupancy for each dose. A bio-phase PK/PD mathematical model was then used to describe the relationship of occupancy with circulating concentrations of AZD4635. In parallel, a PK model for AZD4635 in humans was developed using data from cohort 1 (Clinical trial NCT02740985) after 125 mg and used for PK predictions for alternative doses of AZD4635 in the clinic.

Results

A clear Exposure-Effect relationship was observed for AZD4635-driven A2AR occupancy in cyno brain when dosed 30 min prior to PET measurement. The PK/PD analysis of cyno PET-determined receptor occupancy provided an Occ50 that is in line with the in vitro potency for the compound under physiological concentrations of adenosine in the brain. The resulting PK/PD model has then been applied to predict the level of occupancy in human tumours at other clinically relevant doses. Different simulations were done varying the amount of endogenous adenosine levels.

Conclusions

AZD4635 was shown to occupy A2AR in cyno brain in an exposure dependent manner. The resulting PK/PD model built using this dataset was used to run simulations of expected tumor receptor occupancy in man and aid clinical dose selection for AZD4635. Sensitivity analysis has shown that prediction of human occupancy in the tumour is highly dependent on adenosine concentrations in the tumour. Simulations with tumor adenosine concentrations of 1 μM indicate that AZD4635 is predicted to provide ~90% receptor occupancy over the whole dosing interval at a clinically relevant dose.

### Immune Response to Hematopoietic Tumors: New Development in Tumor Immunology

#2642

Preclinical efficacy of daratumumab in acute lymphoblastic leukemia.

Karen Lee Bride,1 Tiffaney Vincent,1 Soo-Yeon L. Im,1 Tori Fuller,1 Theresa Ryan,1 David M. Barrett,1 Shannon L. Maude,1 Mignon L. Loh,2 Michelle L. Hermiston,2 Stephan A. Grupp,1 Brent L. Wood,3 David T. Teachey1. 1 _CHOP, Philadelphia, PA;_ 2 _UCSF Medical Center - Mission Bay, San Francisco, CA;_ 3 _Seattle Children's Hospital, Seattle, WA_.

Targeted immunotherapy has become critical for the successful treatment of many forms of cancer, particularly therapeutic antibodies with cytotoxic abilities. No safe and effective immunotherapies have been developed for T-cell acute lymphoblastic leukemia (T-ALL). CD38 is a transmembrane glycoprotein found on the cell surface of activated T cells, terminally differentiated B cells, but relatively low levels on normal lymphoid and myeloid cells. Daratumumab (dara) (Darzalex, Janssen Biotech, Inc.) is a human IgG1κ monoclonal antibody that binds to a unique CD38 epitope and was recently FDA approved for the treatment of refractory multiple myeloma. In order to ensure CD38 is a relevant target in T-ALL and surface expression does not change with chemotherapy, we evaluated CD38 expression by flow cytometry (FACS) from 21 patients with T-ALL (10 early T-cell precursor (ETP) and 11 non-ETP) at diagnosis and after one month of induction chemotherapy. All of the samples had detectable CD38 expression which did not change significantly after induction (mean CD38 MFI at diagnosis vs end-induction: 3.27 log vs 3.19 (S.I.; p = 0.25). Therefore we hypothesized that targeting CD38 with dara would be effective against T-ALL. In order to test this hypothesis, we xenografted primary ALL blasts from 15 different patients, including 7 with ETP-ALL and 8 with non-ETP T-ALL. Mice were randomized to dara (200 μg /mouse intraperitoneally (IP) weekly) vs control (isotype control at 200 μg/mouse IP weekly; 5 mice per arm for each sample) after they developed >1% peripheral blood (pb) blasts by FACS. Disease burden was assessed by FACS enumeration of pb blasts weekly and splenic blasts at sacrifice. We demonstrate striking efficacy of dara monotherapy in 6 of 7 ETP-ALL samples with reduction of pb and splenic blasts. Mice treated with high disease burden from 5 of the 8 non-ETP samples were moribund immediately after dara injection, possibly from aggregates of antibody bound to tumor cells leading to pulmonary embolism or tumor lysis, and were therefore inevaluable. We have since repeated the experiments for 3 of the samples, treating the mice after injection but before detectable engraftment of peripheral blasts. Dara was effective in all 3 samples. Experiments are ongoing for the other 2 samples. Overall, we have found Dara was effective in 4 of 6 evaluable non-ETP T-ALL samples. In summary, we found dara is a highly effective novel monotherapy for T-ALL in preclinical models. Based on these results, we are developing an early phase trial for children and young adults with relapsed/refractory T-ALL.

#2643

Thymic precursor cells generate acute myeloid leukemia in NP23-NHD13 double transgenic mice.

Subhadip Kundu,1 Eun Sil Park,2 Yang Jo Chung,3 Trevor Stephen Barlowe,4 Peter D. Aplan3. 1 _NIH/NCI, Rockville, MD;_ 2 _Gyeong-Sang Natl Univ.Hosp., Jinju, 660-702, Republic of Korea;_ 3 _NIH/NCI/CCR, Bethesda, MD;_ 4 _Darthmouth Geisel School of Medicine, Hanover, NH_.

Oncogenic fusion genes have been identified in a large number of hematologic malignancies. We previously generated mice that expressed either a NUP98-PHF23 (NP23) or NUP98-HOXD13 (NHD13) fusion in the hematopoietic compartment. Both NP23 and NHD13 mice develop a wide variety of leukemias, including myeloid, erythroid, megakaryocytic and lymphoid, at 9-14 months of age. NP23-NHD13 double transgenic mice were generated by interbreeding NP23 and NHD13 mice. Surprisingly, 100% of the NP23-NHD13 double transgenic mice showed rapid onset of acute myeloid leukemia (AML) within 3 months. The leukemias were characterized by extraordinarily high WBC and replacement of the thymus with Mac1+/Gr1+ myeloblasts; the percent of malignant myeloid cells in the thymus was often higher than the bone marrow (BM). It led to the intriguing hypothesis that the AML generated in NP23-NHD13 mice arose in the thymus, as opposed to the BM. We transplanted unfractionated cells from the thymus of a NP23-NHD13 mouse, invaded by Mac1+/Gr1+ AML cells or residual CD4-/CD8- double negative (DN) thymocytes into sub-lethally irradiated recipients. All mice (transplanted with either unfractionated leukemic cells or DN cells from same leukemic mouse) developed Mac1+/Gr1+ AML within 26 days, indicating that the AML was aggressive and transplantable, and could be transmitted by DN thymocytes. The observation that the AML was transmitted by the DN thymocytes or Mac1+/Gr1+ AML suggested that the DN thymocytes may be the cell of origin for this AML. To rule out the possibility that the leukemia in this experiment was transmitted by rare, contaminant Mac1+/Gr1+ cells, we repeated the experiment, twice, using DN thymocytes from 4-5 wk old mice with no signs of leukemia. DN thymocytes again transmitted a Mac1+/Gr1+ AML. We further fractioned DN thymocytes into DN1, DN2, DN3 and DN4 populations; the recipients of DN1 and DN2 populations developed a Mac1+/Gr1+ AML, but DN3 and DN4 recipients were healthy with no signs of engraftment in the peripheral blood. DN thymocytes from non-leukemic NP23-NHD13 mice were cultured on an OP9 stromal layer, which has been shown to support myeloid differentiation in vitro. DN thymocytes from non-leukemic NP23-NHD13 mice cultured on a OP9 stromal layer showed a markedly enhanced ability to differentiate into Mac1+/Gr1+ myeloid lineage cells compared to WT (56% vs 1.4 % at day 10). The NP23-NHD13 cells lost expression of Mac1 and Gr1 after 26 days; with immunophenotype now CD4-, CD8-, CD25-, CD44+, Thy-1.2+ and cKit+, consistent with a self-renewing DN1 thymocyte. These cells were transplanted; all recipients were anemic, and demonstrated engraftment of NP23-NHD13 myeloid cells as well as a less prominent (7.85%-38.5%) population of CD71+/Ter119+ erythroid cells in the BM and spleen. Taken together, these results indicate that NP23-NHD13 thymic progenitors are potently leukemogenic, and retain myeloid and erythroid differentiation potential.

#2644

SY-1425, a selective RARα agonist, induces high levels of CD38 expression in RARA-high AML tumors creating a susceptibility to anti-CD38 therapeutic antibody treatment.

Kathryn Austgen, Michael R. McKeown, Darren Smith, Emily Lee, Chris Fiore, Matthew L. Eaton, Christian Fritz, Tracey Lodie, Eric Olson. _Syros Pharmaceuticals, Cambridge, MA_.

CD38 is a cell surface protein expressed primarily on white blood cells and considered a marker of differentiation initiation. CD38 is involved in the immune system by engaging cross-talk with T and B cells as well as activation of NK cells. In multiple myeloma (MM), a subset of tumor cells have high CD38 expression (CD38hi), which has led to the development of effective anti-CD38 therapeutic antibodies, such as daratumumab (DARA). Thus, cancer cells that express CD38 can be selectively targeted for elimination by the immune system using these therapeutic antibodies. In multiple myeloma, DARA is most effective in patients whose tumor cells are CD38hi. In contrast, CD38 expression in AML tumors is generally observed to be negative (CD38neg) or dim (CD38dim), and DARA has not shown activity in preclinical AML models. We previously reported that SY-1425, a clinical stage RARα agonist with improved pharmacokinetics, potency, and selectivity over pan-retinoic acid agonists, induces differentiation in non-APL AML cell lines and primary patient samples with a RARA super-enhancer associated biomarker (RARA-high). Since CD38 was found to be among the most differentially expressed genes in response to SY-1425, we hypothesized that SY-1425 mediated CD38 induction to levels comparable to MM may sensitize RARA-high AML cells to anti-CD38 therapy. We demonstrate that SY-1425 treatment of four RARA-high AML cell lines and four RARA-high primary AML patient PBMCs induces the CD38hi phenotype, as measured by flow cytometry, similar to that found in the DARA sensitive MM cells. In contrast, we see no induction in RARA-low cell lines. We then demonstrated the activity of the SY-1425 and DARA combination in an ex vivo NK cell co-culture assay. Two RARA-high AML cell lines treated with SY-1425 and DARA were co-cultured with NK cells and monitored for both antibody dependent cell-mediated cytotoxicity (ADCC) and NK cell activation by interferon gamma production. The combination of SY-1425 and DARA led to a six-fold increase in tumor cell death relative to the single agent controls, and 5-10 fold increases in NK cell activation is observed only in the SY-1425 and DARA combination treatment of RARA-high AML cell lines. Neither single agent, when administered alone, resulted in ADCC. Furthermore, a RARA-low AML cell line does not respond in the ADCC assay following the combination treatment due to the lack of CD38 induction in this SY-1425 insensitive line. In summary, we have identified a novel and rational combination treatment approach for a subset of patients with RARA-high AML. By inducing the expression of CD38, SY-1425 in combination with DARA elicits tumor cell death and NK cell activation. Based on these findings, a phase 2 clinical study with SY-1425 in combination with an anti-CD38 antibody is planned in AML using an RARA biomarker patient selection strategy.

#2645

Development of a first in class APRIL fully blocking antibody BION-1301 for the treatment of multiple myeloma.

John Dulos,1 Lilian Driessen,1 Marc Snippert,1 Marco Guadagnoli,1 Astrid Bertens,1 David Lutje Hulsik,1 Tai Yu Tzu,2 Kenneth Anderson,3 Jan Paul Medema,4 Kate Cameron,4 Hans Eenennaam,1 Andrea Elsas1. 1 _Aduro Biotech Europe, Oss, Netherlands;_ 2 _Jerome Lipper Multiple Myeloma Center, Harvard Medical School, Boston, MA;_ 3 _LeBow Institute for Myeloma Therapeutics and Jerome Lipper Multiple Myeloma Center, Dana Farber Cancer Institute, Harvard Medical School, Oss, MA;_ 4 _University of Amsterdam, Amsterdam, Netherlands_.

APRIL, or tumor necrosis factor super family member 13 (TNFSF13), is a ligand for the receptors B-cell maturation antigen (BCMA) and transmembrane activator calcium modulator and cyclophilin ligand (CAML) interactor (TACI). APRIL serum levels are enhanced in patients diagnosed with multiple myeloma (MM), chronic lymphocytic leukemia (CLL) and colorectal carcinoma and are correlated with poor prognosis.

As a paracrine factor produced by osteoclasts, macrophages and other cells in the bone marrow niche, APRIL binds to BCMA to drive proliferation and survival of human Multiple Myeloma (MM) cells and induces resistance to several standard of care agents. Using a mouse anti-human APRIL blocking antibody 01A1 originally discovered using Aduro's B-Select platform, we demonstrated that osteoclast-induced MM cell in vitro proliferation and survival is dependent on APRIL. In these co-cultures, cytolytic activity of lenalidomide and bortezomib is significantly enhanced by 01A in a dose-dependent fashion2. Importantly, APRIL drives and 01A inhibits expression of an immunosuppressive gene set including the immune checkpoint programmed death ligand-1 (PD-L1), interleukin-10, vascular endothelial growth factor, and transforming growth factor beta.

The human APRIL antagonist antibody 01A was humanized and designated BION-1301. The antibody binds an epitope overlapping the BCMA and TACI binding sites potently and fully blocks BCMA and TACI binding (IC50 <1 nM). Biophysical and functional experiments indicated that BION-1301 recapitulated all characteristics of 01A. In vivo, BION-1301 was shown to suppress T cell-independent B cell responses to NP-Ficoll. Furthermore, APRIL blockade demonstrated single agent anti-multiple myeloma activity in a humanized SCID model2 confirming its activity in vivo, and potentially indicating that BION-1301 is active targeting multiple myeloma cells in a tumor-protective bone marrow microenvironment.

To our knowledge, BION-1301 is a first-in-class humanized APRIL antagonist demonstrated in to inhibit multiple myeloma survival, drug resistance and an immune suppressive phenotype preclinical. These data suggest a rationale to develop BION-1301 as a single agent, and in combination with lenalidomide, bortezomib, or possibly checkpoint inhibitors such as anti-PD-1. BION-1301 is expected to enter Phase 1 in 2017.

1) Guadagnoli at al. Blood. 2011 Jun 23;117(25):6856-65

2) Yu-Tzu et al. Blood. 2016 Jun 23;127(25):3225-36

#2646

Intratumoral delivery of TTI-621 (SIRPαFc), a CD47-blocking immunotherapeutic, inhibits tumor growth and prolongs animal survival in a subcutaneous B cell lymphoma model.

Gloria H. Lin, Marilyse Charbonneau, Vien Chai, Alison M. O'Connor, Bolette Bossen, Hui Chen, Mark Wong, Natasja N. Viller, Emma Linderoth, Lisa D. Johnson, Xinli Pang, Jeffery Winston, Penka S. Petrova, Robert A. Uger. _Trillium Therapeutics Inc., Mississauga, Ontario, Canada_.

Tumor cells often evade macrophage-mediated destruction by increasing cell surface expression of CD47, which delivers an anti-phagocytic ("do-not-eat") signal by binding the inhibitory signal-regulatory protein α (SIRPα) receptor on macrophages. We have previously shown that blockade of the CD47-SIRPα pathway using TTI-621, a soluble SIRPα-IgG1 Fc fusion protein, triggers macrophage phagocytosis of tumor cells in vitro as well as inhibits tumor growth in vivo when delivered systemically. In the current study, the efficacy of intratumoral delivery of TTI-621 was evaluated in a subcutaneous diffuse large B-cell lymphoma (Toledo) xenograft model.

Tumor bearing mice were randomized into treatment groups when tumor volumes reached approximately 120 mm3. Weekly intratumoral administration of TTI-621 at 10, 1 and 0.1 mg/kg dose levels resulted in statistically significant decreases in tumor growth and improved survival relative to vehicle control treatment. Notably, at day 50 post-tumor implantation 100% survival was achieved at the highest dose level (vs. 0% survival with vehicle control treatment). Moreover, weekly intratumoral administration of TTI-621 was efficacious even at a high tumor load setting in which the pre-dose volumes were approximately 300 mm3. Flow cytometry analysis of the dissociated tumor samples demonstrated no significant change in the numbers of M1 and M2 tumor-associated macrophages (TAMs) following intratumoral administration of TTI-621. Nevertheless, TTI-621 dramatically increased the phagocytosis of Toledo cells by both M1 and M2 TAMs to a similar extent ex vivo, suggesting that TTI-621 is efficacious in increasing the phagocytosis of tumor cells by a heterogeneous population of TAMs.

Collectively, these results demonstrate that TTI-621 is efficacious when delivered intratumorally and can increase the phagocytosis of tumor cells by both M1 and M2 TAM populations. These data support the evaluation of intratumoral administration of TTI-621 in cancer patients, and a Phase I study of intratumorally delivered TTI-621 in patients with percutaneously accessible solid tumors and mycosis fungoides is ongoing (NCT02890368).

#2647

TGF-β type I receptor inhibitor (TEW-7197) diminishes myeloma progression by multiple immunomodulatory mechanisms in combination with ixazomib.

Byung-Gyu Kim,1 Olga Sergeeva,1 George Luo,1 Sung Hee Choi,1 Zhenghong Lee,1 Seong-Jin Kim,2 John Letterio,1 Ehsan Malek1. 1 _Case Western Reserve University, Cleveland, OH;_ 2 _Seoul National University, Republic of Korea_.

Background: Multiple myeloma (MM) is an incurable cancer of plasma cells. MM thrives in the bone marrow tumor microenvironment (TME) where several factors sustain MM growth and viability. TGF-β is a multi-functional cytokine elaborated by MM cells and by cells in the bone marrow (BM) TME. TGF-β stimulates MM progression through promotion of catabolic bone remodeling, IL-6 secretion and Th17 T cell development, which act in concert to induce osteolytic bone disease, immune suppression and myeloma progression. Therefore, we evaluated the anti-MM therapeutic potential of a small molecule inhibitor of the TGF-β type I receptor kinase (TβRI), TEW-7197, in combination with the proteasome inhibitor, ixazomib. Currently, TEW-7197 is being evaluated in phase I clinical trials in patients with solid tumors, and exposure to this agent is associated with an acceptable tolerability profile.

Methods: The preclinical immunocompetent 5T33MM model (C57BL/KaLwRij) was used to characterize the role of TGF-β signaling in the BM TME. Mice bearing 5T33MM cells expressing luciferase were treated with TEW-7197, ixazomib and the combination of TEW-7197 plus ixazomib daily for 3 weeks, and evaluated for MM growth by bioluminescence imaging (BLI). Cellular and molecular assays were performed in human RPMI8226 and U266 as well as murine 5T33MM cells via apoptosis, real-time PCR and Western blotting. Immunological assays were performed via immunohistochemistry and FACS analyses. Peripheral blood monoclonal protein concentration, M-spike, was measured by ELISA.

Results: TEW-7197 attenuated the growth and viability of human and murine MM cells by inducing apoptosis and it inhibited TGF-β-induced activation of Smad2/3 in MM cells in vitro. In our 5T33MM preclinical model, oral administeration of TEW-7197 as single agent inhibited MM progression as measured by peripheral blood monoclonal protein concentration and BLI before and after treatment. TEW-7197 also induced a decrease in mortality and an increase in body weight of mice bearing MM. TEW-7197 alone or combination with ixazomib also attenuated TGF-β activation of Smad2/3, reduced the expansion of CD11b+Gr-1+ myeloid derived suppressor cells (MDSCs) in BM TME, and diminished the population of Foxp3+ regulatory T cells (Treg) in the spleen. Combination therapy of TEW-7197 plus ixazomib prolonged survival and exhibited a synergistic anti-tumor effect when compared to either TEW-7197 or ixazomib alone by significant a reduction in both the M-spike and BLI.

Conclusion: Our data demonstrate that the small molecule inhibitor of the TβRI, TEW-7197, effectively modulate the MM TME and is associated with a potent anti-myeloma effect in an immunocompetent murine model of MM. These data provide a rationale for clinical evaluation of the combination therapy of ixazomib and TEW-7197 as a potential therapeutic strategy to improve outcomes in patients with MM.

#2648

CD47 antibody-induced engulfment of human T-cell leukemia cells by bone marrow-derived macrophages.

Gillian Lovell,1 Clare Szybut,1 Kalpana Patel,1 Hinnah Campwala,1 Nicola Bevan,1 Dan Appledorn,2 Tim James Dale,1 Derek John Trezise1. 1 _Essen BioScience Ltd, Welwyn Garden City, United Kingdom;_ 2 _Essen BioScience Inc, Ann Arbor, MI_.

CD47 is a trans-membrane "don't-eat-me" signaling protein that enables tumor cells to evade clearance by neighboring phagocytes. Blocking CD47 allows phagocytes to identify and clear tumor cells and is a promising new approach for cancer immunotherapy. In this study, we characterized anti-CD47 antibody-mediated engulfment of living tumor cells (CCRF-CEM) by mouse bone-marrow derived macrophages (BMDMs) or immortalized mouse macrophages (J774A.1). Phagocytosis was quantified using a pH-sensitive cell-labeling fluorescent probe, pHrodo, and automated kinetic live-cell analysis (IncuCyte). CCRF-CEM cells were first labeled using pHrodo (250ng ml-1 for 1h), washed and then treated with antibody for 1 h. Target cells were then added to BMDMs or J774A.1 that had been seeded overnight on 96-well plates. Phase- and fluorescence images were captured and quantified every 15min. Anti-CD47 antibody (B6H12.2, 0.04-5μg ml-1), but not IgG-control, produced time- and concentration-dependent engulfment of CCRF-CEMs by BMDMs (30'-4h), as evidenced by an increase in intracellular fluorescence as the label accumulates in the acidic phagosome. After 4h the red fluorescence area was increased by 25-fold (1975 ± 391 μm2 vs 80 ± 41 μm2). From close inspection of the time-lapse images cellular engulfment could be clearly observed, coincident with the appearance of the fluorescent signal. Similar observations were made with J774A.1 as the effector cell. Interestingly, the rate and degree of engulfment appeared effector cell-dependent. The mechanism of engulfment was not via induction of target cell apoptosis since anti-CD47 did not induce PS externalization (Annexin V) or activate caspase 3/7. Anti-CD47 had no direct effect on CCRF-CEM proliferation for the first 4 h but upon longer exposures (>8 h) cell growth was attenuated. Our experimental findings substantiate the known pro-phagocytic effects of anti-CD47 antibodies, and provide a model system and method for quantitative functional analysis and mechanistic insight of CD47 modulators as cancer therapeutics.

#2649

Fc-effector function activity of the CXCR4 IgG1 antibody PF-06747143: a novel clinical candidate for the treatment of hematologic malignancies.

Flavia Pernasetti, Shu-Hui Liu, Gu Yin, Bernadette Pascual, Zhengming Yan, Max Hallin, Rolla Yafawi, Cathy Zhang, Connie Fang, Wenlian Wang, Justine Lam, Mary E. Spilker, Eileen Blasi, Brett Simmons, Nanni Huser, Wei-Hsien Ho, Kevin Lindquist, Thomas-Toan Tran, Jyothirmayee Kudaravalli, Jing-Tyan Ma, Gretchen Jimenez, Ishita Barman, Colleen Brown, Sherman-Michael Chin, Maria Costa, David Shelton, Tod Smeal, Valeria R. Fantin. _Pfizer, CA_.

The chemokine receptor CXCR4 triggers signaling pathways that control cell migration to tissues where its ligand, CXCL12, is highly expressed, including the bone marrow (BM). In hematologic cancers, CXCR4 expression is associated with poor prognosis. CXCR4-driven homing of malignant cells to the BM protective niche is a key mechanism of chemotherapy resistance. PF-06747143 is a novel humanized IgG1 therapeutic antibody that binds to CXCR4 and inhibits CXCL12-driven pathways. Human IgG1 antibodies can induce strong cytotoxicity mediated by the antibody Fc-region, including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-driven cytotoxicity, while human IgG4 antibodies show minimal or no Fc-driven cytotoxicity. Here we generated an IgG4 version of PF-06747143 (m15-IgG4), which has similar binding to CXCR4 as the IgG1 antibody. We then characterized the role of Fc-driven cytotoxic function, comparing both antibodies in efficacy and safety studies. In an ADCC assay, PF-06747143 showed strong cytotoxicity of non-Hodgkin's lymphoma (NHL) and acute myeloid leukemia (AML), while m15-IgG4 had no significant cytotoxicity. In a NHL mouse tumor model, the IgG1 CXCR4 antibody resulted in superior tumor growth inhibition, with 50% of mice exhibiting complete tumor regressions, compared to the m15-IgG4 antibody, which had limited activity, with no tumor regressions (p<0.01). The two antibodies had comparable exposure, suggesting that the difference in efficacy is due to the IgG1 antibody Fc-driven cytotoxic function. Furthermore, CXCR4 inhibition was previously shown to induce leukocyte mobilization from the BM; however, prolonged cell mobilization may lead to safety related issues, including hyperleukocytosis. In a study in non-human primates, we show that the IgG1 CXCR4 antibody elicited short leukocyte mobilization, lasting 16-24 hrs, while m15-IgG4 induced prolonged cell mobilization, lasting > 4 days. Since both antibodies had comparable exposures, the different mobilization duration is likely due to the ability of the IgG1 CXCR4 antibody to reduce the number of mobilized cells via Fc-driven cytotoxic function. Finally, CXCR4 has been shown to play a key role in chemotherapy resistance. In a chemo-resistant PDX AML mouse model, in which the standard of care agents daunorubicin and cytarabine had limited activity, resulting in 30% of tumor cells remaining in the BM post-treatment, we show that combination of PF-06747143 with these chemo agents led to synergistic activity, with tumor burden reduced to 0.3% tumor cells in the BM. In conclusion, PF-06747143 attributes offer potential efficacy- and safety-related advantages over other CXCR4 antagonists currently in development, which do not have Fc-driven cytotoxic activity. PF-06747143 is now being evaluated in a Phase 1 clinical trial in relapsed and refractory AML (NCTID 02954653).

#2650

Radioimmunotherapy for acute myeloid leukemia targeting human aspartyl (asparaginyl) β-hydroxylase.

Michael S. Lebowitz,1 Ekaterina Revskaya,2 Kanam Malhotra,1 Amir Shahlaee,3 Steven Fuller,1 Maria R. Baer,4 Noa G. Holtzman,4 Ashkan Emadi,4 Ekaterina Dadachova,5 Hossein A. Ghanbari1. 1 _Panacea Pharmaceuticals, Inc., Gaithersburg, MD;_ 2 _Albert Einstein College of Medicine, Bronx, NY;_ 3 _Institute for Asthma and Allergy P.C., Chevy Chase, MD;_ 4 _University of Maryland School of Medicine, Baltimore, MD;_ 5 _University of Saskatchewan, Saskatoon, Saskatchewan, Canada_.

Treatment options for acute myeloid leukemia (AML) are limited and have modest impact on 5-year survival rates in patients <60 years of age (~35-40%) and on median survival of older patients (< 1 year). The incidence of AML has been rising, with an anticipated 19,950 new cases in 2016. Thus, the need for novel, more efficacious and better tolerated therapy for AML is urgent. Human aspartyl (asparaginyl) β-hydroxylase (HAAH) is an embryonic/developmental protein, which is down-regulated in normal cells after birth but overexpressed on the surface of many malignant cells, where it has been demonstrated to be sufficient to induce cancer cell proliferation, motility and invasiveness. We hypothesized that HAAH is selectively overexpressed on leukemia cells and thus targeting it with a fully human monoclonal antibody (mAb) conjugated to the short half-life α-emitter 213Bi could provide a novel radioimmunotherapy (RIT) treatment option with a reasonable therapeutic index. Using immunocytochemistry and flow cytometry with anti-HAAH mouse mAbs as well as a fully human mAb, PAN-622, we investigated the expression of HAAH on a variety of cytogenetically- and mutationally heterogeneous leukemia cell lines, including MV4-11, MOLM-14, HL-60, KG1α, Kasumi-1, K562, J45.1, and CCRF-CEM, as well as on normal human leukocytes. PAN-622 was further modified with DTPA-maleimide to allow chelation of radionuclides, specifically 213Bi, to the antibody. The 213Bi-DTPA-PAN-622 was then employed in killing assays to measure selective dose-dependent killing of leukemic cells. HAAH was highly expressed, as detected by flow cytometry, on all tested leukemia cell lines with KG1α and HL-60 demonstrating the highest levels of expression amongst the AML lines. Binding was observed by both PAN-622 and the modified DTPA-PAN-622 conjugate. Interestingly, expression of HAAH was not detected on CD45\+ normal human leukocytes, including in subpopulations of monocytes (CD14+), B-cells (CD19+), T-cells (CD3+) or hematopoietic stem cells (CD34+). To determine whether PAN-622 could selectively target leukemia cells in a background of normal human blood, flow cytometry was performed on leukemia cells spiked into whole blood. Only leukemia cells were stained with the PAN-622 antibody as indicated by simultaneous staining for surface markers specific to the various leukocyte subpopulations. Selective killing of leukemia cell lines was demonstrated following incubation with 213Bi-DTPA-PAN-622 which induced dose dependent specific killing over a 6.7-670 pM concentration range. No killing was detected when using 213Bi conjugated to an isotype-matched control antibody at a similar specific activity and dose range. Thus, HAAH is a novel oncogenic target expressed on leukemia cells, which can be selectively targeted for RIT via the fully-human PAN-622 mAb. Experiments targeting primary leukemia cells in patient-derived bone marrow aspirates are ongoing.

#2651

A novel CD19 targeting antibody-drug conjugate, huB4-DGN462, shows promising in vitro and in vivo activity in CD19-positive lymphoma models.

Eugenio Gaudio,1 Chiara Tarantelli,1 Alberto J. Arribas,1 Roberta Pittau Bordone,2 Andrea Rinaldi,1 Georg Stussi,2 Emanuele Zucca,2 Davide Rossi,1 Anastasios Stathis,2 Min Li,3 Alan Wilhem,3 Kate Lai,3 Qifeng Qiu,3 Stuart Hicks,3 Callum Sloss,3 Francesco Bertoni1. 1 _Institute of Oncology Research - IOR, Bellinzona, Switzerland;_ 2 _Oncology Institute of Southern Switzerland - IOSI, Bellinzona, Switzerland;_ 3 _Immunogen Inc., Waltham, MA_.

Background. CD19 is a cell surface membrane protein expressed in most mature and immature B cell neoplasms, making it a promising target for antibody-drug conjugate (ADC) therapy for B cell malignancies. Here we describe the preclinical activity of a novel CD19-targeting ADC, based on the potent indolinobenzodiazepine DNA-alkylating payload DGN462.

Methods. The humanized anti-CD19 antibody, huB4, was conjugated to DGN462 via a cleavable disulfide linker, sulfo-SPDB. In vitro activity of the huB4-DGN462 ADC or the unconjugated DGN462 toxin was evaluated in 54 lymphoma cell lines [27 diffuse large B cell lymphomas (DLBCL); 10 mantle cell lymphomas; 6 marginal zone lymphomas; 5 anaplastic large T-cell lymphomas; 6 others]. Cell proliferation/viability after 72h of exposure was measured using a MTT assay. Apoptosis activation was defined by at least 1.5-fold increase in caspase 3/7 signal activation in respect to controls using the Promega ApoTox-Glo Triplex Assay. Gene expression profiling (GEP) was performed with the Illumina HumanHT-12 Expression BeadChips on untreated cell lines followed by GSEA (NES > |2|, P<0.05, FDR<0.25) and limma t-test (FC> |1.2|; P< 0.05; top 200 up and top 200 down). In vivo efficacy of huB4-DGN462 was evaluated in CD19-expressing xenograft tumor models.

Results. huB4-DGN462 was cytotoxic against a broad panel of 48 B cell lymphoma cell lines (median IC50 100 pM; 95%CI, 38-214). The cytotoxic activity was not limited by P53, BCL2, MYC or CDKN2A status, or associated with DLBCL cell of origin. Consistent with overall lower CD19 expression, huB4-DGN462 was significantly (p-value=0.007) less active in eight T cell-derived lymphomas (median IC50 of 1.75 nM (95%CI, 0.5-5.75)) than in B cell lymphomas. huB4-DGN462 induced caspase 3/7 activation in 48/54 cell lines (89%) consistent with an apoptotic mechanism of action. huB4-DGN462 demonstrated compelling anti-tumor activity after a single intravenous dose in two diffuse large B-cell lymphoma cell line xenograft models: DoHH2 (a subcutaneous model) and Farage (a disseminated model). In the DoHH2 model, huB4-DGN462 resulted in a significant, dose-dependent tumor growth delay and survival benefit at 1.7 mg Ab/kg compared to a non-targeted control DGN462 ADC. In the disseminated Farage model, a significant dose-dependent increase in survival was observed in mice treated with as low as 0.17 mg Ab/kg of huB4-DGN462. At 1.7 mg Ab/kg, the life span was increased >400% compared to untreated mice.

Conclusions. The novel ADC huB4-DGN462 presented strong preclinical anti-lymphoma activity, which provides evidence for further study.

#2652

Favorable outcome of primary mediastinal large B-cell lymphoma treated by Dose Adjusted EPOCH-Rituximab regimen.

Fatiha Grifi, Soraya Bougherira. _university of Annaba, Annaba, Algeria_.

Background Primary Mediastinal Large B-Cell Lymphoma (PMLBCL) is a unique type of B-cell lymphoma probably arising from a putative thymic medulla B-cell. One to 2% of NHL, 7% of all diffuse large B-cell lymphomas (DLBCL), occurring more often in young females. Often presents with superior vena cava syndrome and airway compromise. Treatment is with chemotherapy combined with Rituximab, followed by involved field radiation therapy to mediastinum. PMLBCL has better outcome than others DLBCL. We aimed to develop a more dose-dense/intense regimens that improves the rate of cure with an attempt to avoid mediastinal radiation. Patients and methods A prospective study was conducted in our department from January to December 2015, in 6 patients with untreated primary mediastinal B-cell lymphoma. An infusional dose-adjusted etoposide, doxorubicin, and cyclophosphamide with vincristine, prednisone, and rituximab (DA-EPOCH-R) has been administred according to the protocol Day 1 : Rituximab 375 mg/m2 IV Day 1-4 : Etoposide 50 mg/m2 IV, Doxorubicin 10 mg/m2 AND Vincristine 0.4 mg/m2 Day 5 : Cyclophosphamide 750 mg/m2 Days 1-5 : Prednisone 60 mg/m2 PO BID. Administer G-CSF 5 mcg/kg SQ on day 6 until ANC exceeds nadir Cycles were repeated every 3 weeks for a toal of 6 cycles Results The median age of the patients was 32 years, the male to female ratio : 2:4. Systemic symptoms mainly fever and weight loss, were present in all cases; increased LDH levels were also observed in 50% of cases. Majority of the patients (83%) presented with stage I/II disease, bulky mass with a median tumor diameter 15 cm. Most had features of local extension like pleuro-pericardial effusion. Absence of infradiaphragmatic lymph nodes and no bone marrow involvement at presentation. All patients required biopsy from the mediastinal mass by image guided core biopsy. The patients were treated by RDA-EPOCH, they received 4 to 6 courses, without involved field radiotherapy (only one patient underwent radiotherapy for residual mass). No late morbidity or cardiac toxic effects were found in any patients. After 24 months of follow-up, one patient still received treatment and was not evaluable. 5 patients achieved complete remission and were disease-free at the last visit. Conclusion PMBCL is a specific lymphoma entity seen in the young with good survival. It has a distinct clinical behavior and prognosis, and therefore require a different therapeutic approach. The R/DA-EPOCH regimen need to be confirmed in further prospective study for a large cohort.

#2653

The anti-myeloma activity of TTI-621 (SIRPαFc), a CD47-blocking immunotherapeutic, is enhanced when combined with a proteasome inhibitor.

Emma Linderoth, Simone Helke, Vivian Lee, Tapfuma Mutukura, Mark Wong, Gloria H. Lin, Lisa D. Johnson, Xinli Pang, Jeff Winston, Penka S. Petrova, Robert A. Uger, Natasja N. Viller. _Trillium Therapeutics Inc., Mississauga, Ontario, Canada_.

CD47 is transmembrane glycoprotein that delivers an anti-phagocytic ("do not eat") signal by binding to its receptor, signal-regulatory protein α (SIRPα), on the surface of macrophages. Many tumors, including multiple myeloma (MM), express high levels of CD47 as a means to exploit the CD47-SIRPα pathway and escape macrophage-mediated immune surveillance. TTI-621 (SIRPαFc) is a soluble recombinant fusion protein consisting of the CD47-binding domain of human SIRPα linked to the Fc region of human IgG1. It is designed to promote anti-tumor responses by blocking the CD47 "do not eat" signal and engaging activating Fcγ receptors on macrophages. We have previously shown that TTI-621 enhances phagocytosis of malignant cells in vitro and exhibits anti-tumor activity in acute myeloid leukemia and B lymphoma xenograft models. In this study we investigated the anti-myeloma activity of TTI-621 as both a single agent and in combination with bortezomib or carfilzomib, two proteasome inhibitors that are approved for use in MM patients. The ability of TTI-621 to trigger macrophage phagocytosis of MM cells was assessed using a flow cytometry-based phagocytosis assay. Blockade of CD47 using TTI-621 effectively triggered macrophage-mediated phagocytosis of MM cells, and this anti-tumor effect was significantly enhanced by pre-treatment of MM cells with bortezomib or carfilzomib. In order to understand the molecular mechanism behind this enhanced phagocytic effect, we performed immunophenotyping of the tumor cells and observed an upregulation of pro-phagocytic "eat" signals on the tumor cell surface that potentially augment the phagocytic response. To investigate whether CD47 blockade in the context of proteasome inhibition translates to enhanced anti-tumor activity in vivo, we employed a MM xenograft model. NOD.SCID mice were subcutaneously engrafted with human MM cells followed by treatment with TTI-621, in the absence or presence of concurrent administration of a proteasome inhibitor. As monotherapies, both TTI-621 and the proteasome inhibitor reduced tumor burden relative to vehicle controls. Moreover, the combination of CD47 blockade and proteasome inhibition resulted in a greater reduction in tumor growth as compared to the two drugs alone. In conclusion, these data demonstrate that TTI-621 exhibits anti-myeloma activity that is further enhanced by combination with bortezomib or carfilzomib. TTI-621 monotherapy is currently being evaluated in a Phase 1b study in patients with MM and other hematological malignancies (NCT02663518). These data provide a rationale to evaluate a combination study of TTI-621 and a proteasome inhibitor in MM patients.

#2654

Resveratrol and piceatannol synergistically induce PDL1 expression.

Justin P. Lucas,1 Joseph M. Wu,2 TzeChen Hsieh,2 Zbigniew Darzynkiewicz,2 Halina Dorota Halicka-Ambroziak2. 1 _Pfizer/New York Medical College, Pearl River, NY;_ 2 _New York Medical College, Valhalla, NY_.

The interaction of programmed cell death-1 (PD-1) and its ligand programed cell death-1 ligand (PD-L1) is a major focus of recent immune oncology therapy efforts. The expression of PD-1 on T lymphocytes and its subsequent interaction with PD-L1, either from antigen presenting or tumor cells, will result in apoptosis-dependent inactivation of the T lymphocytes. This interaction plays an integral role in tumor immunology, specifically augmenting immune evasion. Relatively little is known about the regulation of PD-L1 in either the tumor or normal environment. We investigated the ability of natural dietary compounds to induce PD-L1 expression on normal epithelial cells and various cancer cell lines. These molecules have been evolutionary selected to control inflammation and cancer cell transformation and progression, understanding their mechanism of action could be important to understanding how this system works. Using flow cytometry and immunohistochemistry, we focused our studies on resveratrol and its metabolite piceatannol, key phytochemicals extracted from grapes, after an initial screen of various natural products. Experimental data showed that both compounds can individually up-regulate the expression of PD-L1 on tumor cell lines and normal epithelial cells, by an IFN-γ-independent mechanism. PD-L1 induction by both of these compounds was higher in tumor than in normal epithelium, suggesting greater significance in tumor regulation than inflammation. Additionally, the combination of resveratrol and piceatannol acted synergistically, leading to a significantly greater induction of PD-L1 expression across multiple tumor indications. Understanding whether resveratrol and piceatannol use a common signaling pathway to induce PD-L1 expression in tumor cells, was critical to understand the synergistic induction observed. Studies utilizing specific inhibition of IKK phosphorylation were accompanied by a significant reduction in the ability to induce PD-L1 expression on tumor cells, either as single agents or in combination. These results are consistent with the hypothesis that induction of PD-L1 by resveratrol or piceatannol, or their combination involves molecular determinants down-stream of NF-kB signaling.

#2655

**Drug resistant B-cell tumors eliminated by novel therapeutic antibodies** in vivo **.**

Hong Qin,1 Guowei Wei,1 Ippei Sakamaki,2 Zhenyuan Dong,1 Wesley A. Cheng,1 Diane L. Smith,1 Feng Wen,3 Han Sun,1 Soung-chul Cha,4 Sattva S. Neelapu,4 Larry W. Kwak1. 1 _City of Hope, Duarte, CA;_ 2 _University of Fukui, Awara, Japan;_ 3 _Sichuan University, China;_ 4 _University of Texas MD Anderson Cancer Center, Houston, TX_.

B-cell malignancies have been successfully targeted in the clinic by therapies such as anti-CD20 antibody rituximab or Bruton's tyrosine kinase inhibitor ibrutinib. However, leukemias and lymphomas remain incurable due to primary or acquired resistance, ultimately leaving patients without an effective treatment option. We sought to circumvent this drug resistance by pursuing an alternative target known as B cell activating factor receptor (BAFF-R). Despite past limited success, BAFF-R remains a prime target for B-cell lymphoma and leukemia therapeutic antibody development due to its key role in B-cell proliferation and development.

We report the development of two novel monoclonal antibodies (mAbs) against human (h) BAFF-R. The mAbs were generated by immunizing mice with (h)BAFF-R-expressing mouse fibroblast cells presenting a natively folded, cell-surface immunogen. The two mAbs presented unique complementarity determining regions that specifically bound (h)BAFF-R-expressing mouse fibroblast cells but not the parental counterpart. Furthermore, the antibodies were specific to B-cell containing organs such as tonsil and spleen, by immunohistochemical staining and without detectable reactivity in heart, lung, brain, liver, and kidney tissues. To tailor the antibodies for clinical application, a human IgG1 Fc capable of eliciting an immune response was substituted, creating chimeric versions. We showed that both chimeric mAbs bound with high affinity to human B-cell lymphoma cell lines including JeKo-1 (mantle cell lymphoma; MCL), SU-DHL-6 (diffuse large B cell lymphoma; DLBCL), Raji (Burkitt lymphoma), and RL (follicular lymphoma). The chimeric antibodies also elicited antibody-dependent cell-mediated cytotoxicity (ADCC) with primary human natural killer (NK) cells in vitro against these tumor lines as well as primary lymphoma samples (n=5) from patients who progressed after rituximab exposure. Most notably, the antibodies demonstrated efficacy in two in vivo drug resistant lymphoma models we developed, a rituximab-resistant CD20 genomic knockout variant of JeKo-1 and the naturally ibrutinib-resistant Z-138. Using these lymphomas lines for xenogeneic tumor models in NOD scid gamma (NSG) mice, we found our antibodies significantly inhibited tumor growth, conferring long-term and tumor free survival on the mice.

Our in vitro and in vivo results robustly demonstrate the high specificity and significant anti-tumor effects of our anti-BAFF-R antibodies against a broad variety of B-cell malignancies, especially against cases of rituximab and ibrutinib resistance. This successful development of novel anti-BAFF-R therapeutic antibodies warrants support for further translational development for clinical use in light of current resistance cases.

#2656

**Polatuzumab vedotin significantly enhances** in vitro **cell death and overall survival against CD79b+ Burkitt lymphoma (BL)/primary mediastinal large B-cell lymphoma (PMBL) NSG xenograft mice.**

Aradhana A. Tiwari, Janet Ayello, Christeen Azmy, Carmella van de Ven, Mitchell S. Cairo. _New York Medical College, Valhalla, NY_.

Background: Mature B-NHL, including Burkitt lymphoma and primary mediastinal large B cell lymphoma express CD79b+ and have an excellent prognosis with chemo-immunotherapy (Cairo et al Blood, 2007, Gerrard/Cairo et al. Blood, 2013). However, a subset of patients with relapsed/refractory mature B-NHL has chemoimmunotherapy resistant disease a dismal prognosis (≤ 10% 5 years, EFS) (Cairo et al. JCO, 2012). The antibody drug conjugates (Polatuzumab Vedotin, PV) has demonstrated significant preclinical activity against indolent CD79b+NHL (Polson et al.Can. Res. 2009). More recently PV has been safe and well tolerated in adult with CD79b refractory CLL (Palanca-Wessels et al. Lancet Oncol, 2014) but its preclinical activity against mature B-NHL (BL/PMBL) is unknown.

Objective: To determine the efficacy of the PV against CD79b+ PMBL and rituximab (RTX) sensitive/resistant BL tumor cell lines in-vitro and in-vivo.

Design/Methods: Raji/Raji4RH (BL, provided by M. Barth, Roswell Park Cancer Institute) and Karpas1106P and MedB-1(PMBL) were cultured in 10-20% RPMI. Tumor cells were incubated with hu anti-CD79b-vc-MMAE, and/or anti-CD79b, MMAE or huIgG1 (generously supplied by Genentech Inc.) for 24 hrs. Cell death was evaluated by staining with annexin V/7AAD and analyzed by flow cytometry, n=3. Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), were divided into 5 groups: PBS only (control), isotype control (IgG), PV (5mg/kg), anti-CD79b mAb (5 mg/kg) and MMAE (5 mg/kg). Mice were xenografted with intravenous injections of Luc+ Raji/Raji4RH and Karpas1106P cells as we have previously demonstrated (Awasthi/Cairo et al, BJH, 2015). Mice were treated twice a week for 6 weeks. Tumor burden was monitored by IVIS spectrum system.

Results: Anti-CD79b-vc-MMAE compared to anti-CD79b Ab or IgG1 Ab alone (10µg/ml, 24hrs), significantly enhanced cell death in Raji, 47.2±1.3% vs 29.1±6.0% vs. 28.2±4.3%, (p=0.0008 and p=0.00006), Raji4RH, 29.8±9.1% vs 25.4±3.9% vs. 18.0±8.2% (p=NS and p=0.03), Karpas1106P, 46.8±5.3% vs 33.8±3.5% vs. 26.2±0.4% (p=0.02 and 0.006) and MedB-1, 47.4±2.2% vs 27.6±2.4% vs. 23.9±1.7% (p=0.002 and 0.0001), respectively. Further, median survival time in mice receiving 5 mg/kg of PV was significantly increased when compared to mice receiving 5 mg/kg of anti-CD79b Ab or isotype control in Raji, Raji4RH and Karpas1106P (35.5 vs.17 vs. 19.5 days, p=0.0001, 0.0003, 50 vs. 18 vs. 18.5 days and 150 vs 89 vs 64 days, p=0.03 ,0.003, respectively)

Conclusions: Our preliminary data indicates that PV significantly enhances cell death in RTX sensitive/ resistant BL and PMBL compared to CD79b Ab or isotype control. Furthermore, PV significantly increased survival in BL and PMBL NSG xenografts

#2657

PD-L1 blockade enhances T cell cytotoxicity against primary mediastinal B cell lymphoma.

Tishi Shah, Wen Luo, Aradhana Awasthi, Janet Ayello, Jessica Hochberg, Mitchell Cairo. _New York Medical College, Valhalla, NY_.

Background:

Primary Mediastinal B cell Lymphoma (PMBL) represents 2-4% of Non-Hodgkin Lymphomas (NHL) in adolescents and young adults (AYA) (Gerrard/Cairo et al., Blood, 2013). Disease progression, relapse and long-term toxicity remain a concern for patients treated on current chemo-immunotherapy and mediastinal radiotherapy. Alternative therapeutic regimens are urgently needed, especially in patients without an early response to therapy. Programmed Death 1 (PD-1) is a negative co-stimulatory receptor critical for suppression of T-cell activation, with binding of PD-1 and Programmed Death Ligand 1 (PD-L1) resulting in T cell exhaustion (Postow/Wolchok et al., J Clin Oncol, 2015). Gain in 9p associated with amplification of PD-L1 has been reported in up to 60% of PMBL specimens, leading to overexpression of PD-L1 and potential immune cell evasion of PMBL (Rosenwald/Staudt et al., J Exp Med, 2003; Twa/Steidl et al., Blood, 2014). Blockade of PD-1/PD-L1 interaction, therefore, constitute a potentially promising alternative for treatment of resistant PMBL.

Objective:

In the current study, we sought to investigate whether PD-L1 blockade enhances T cell responses in PD-L1 expressing PMBL.

Methods:

PD-L1 expression on Karpas 1106p PMBL treated with or without IFN γ was investigated by western blotting and flow cytometry analyses. T cells were isolated from human PBMC followed by activation and expansion using anti-CD2, anti-CD3 and anti-CD28 Biotinylated MACSiBead particles. T cell activation was confirmed by CD25 and CD69 expression using flow cytometry. Activated T cells were incubated with or without anti-PD-L1 (Clone 6E11, Genentech) at a dose of 10 ug/ml together with Karpas 1106p cells treated with or without IFN γ. Cell proliferation was assessed with MTS assays after incubation for 24 hours.

Results:

We demonstrated that Karpas 1106p cells express a low level of PD-L1. However, following IFN γ treatment (48 hours) there was a significant increase in PD-L1 expression. Anti-PD-L1 had no significant effect on T cell mediated inhibition of cell proliferation in Karpas 1106p cells with low PD-L1 expression. However, anti-PD-L1 and T cell treatment significantly inhibited cell proliferation in IFN γ treated Karpas 1106p cells when compared to T cell treatment alone (32±18% vs. 3±3% at E:T=5:1, p=0.048).

Conclusion:

PD-L1 blockade enhances T cell cytotoxicity against PMBL, which is dependent on PD-L1 expression level on PMBL cells. Future in-vivo NSG xenograft studies are ongoing.

#2659

MT-4019: a de-immunized engineered toxin body targeting CD38 for multiple myeloma.

Garrett L. Robinson, Sangeetha Rajagopalan, Brigitte Brieschke, Jane Neill, Jennifer Erdman, Rodney Flores, Jensing Liu, Jack P. Higgins, Erin K. Willert. _Molecular Templates Inc., Austin, TX_.

Molecular Templates is developing engineered toxin bodies (ETBs), potent recombinant immunotoxins that combine the specificity of an antibody fragment with the powerful direct cytotoxicity of the Shiga-like toxin A subunit (SLTA). ETBs can induce their own internalization, route through the cell in a predictable manner, enzymatically and irreversibly destroy ribosomes to shutdown protein synthesis and induce apoptosis of tumor cells. This mechanism of action is distinct from that of other therapeutics, making ETBs an attractive treatment for patients who have become resistant to chemotherapy and other treatments. Safety and efficacy in refractory non-Hodgkin's lymphoma patients has been observed in a phase I study with Molecular Template's first-generation CD20-targeting compound, MT-3724.

CD38 is a surface receptor that is highly expressed on malignant plasma cells. CD38 is a clinically validated target of monoclonal antibodies for treatment of multiple myeloma and is known to persist after failure of antibody treatment. MT-4019 is a CD38-targeted next-generation ETB, utilizing a modified SLTA. The SLTA subunit of the next-generation ETB scaffold has been modified through proprietary genetic engineering to systematically and comprehensively reduce B cell and CD4+T cell epitopes, as well as to dampen the innate response by decreasing binding to TLR-4. This de-immunized next-generation scaffold retains the potency and specificity of an ETB containing an unmodified SLTA. Pre-clinical studies in rodents and non-human primate models have demonstrated the tolerability of MT-4019, along with a decreased anti-drug antibody response. Additionally, in the non-human primate model, MT-4019 showed reduced neutrophil and monocyte activation as compared to an ETB with an unmodified SLTA subunit, indicating that SLTA modification also exhibits a diminished innate immune response.

Molecular Template's ETB technology has resulted in potent, targeted therapeutic agents that have a unique mechanism of action in the field of oncology. MT-3724, a first-generation CD20-targeted ETB, has shown promising clinical results in the refractory setting for non-Hodgkin's lymphoma. The next-generation scaffold, as exemplified in the CD38-targeted MT-4019, retains potent and specific direct cell kill activities, and additionally reduces the innate and adaptive immune response to the therapeutic. MT-4019 is a promising lead and is under further development to enable clinical studies in multiple myeloma.

#2660

PD-1 blockade in combination with zoledronic acid to enhance the antitumor efficacy in the breast cancer mouse model.

Yuan Li,1 Yang Du,2 Ting Sun,1 Huadan Xue,1 Jie Tian,2 Zhengyu Jin1. 1 _Peking Union Medical College, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing, China;_ 2 _Key Laboratory of Molecular Imaging of Chinese Academy of Sciences, Institute of Automation, Chinese Academy of Sciences, Beijing, China_.

Purpose: Blockade of PD-1 receptor may provide proof of concepts for the activity of an immune-modulation approach for the treatment of a breast cancer (BC). Zoledronic acid (ZA) has been proven to inhibit angiogenesis, invasion, and adhesion of tumor cells. The aim of this study was to investigate the potential of antibody treatment towards the T-cell checkpoint inhibitor PD-1 alone or in combination with ZA in BC mouse model.

Experimental Design: The 4T1-Luc mouse mammary tumor model was used in this study. The anti-tumor efficacy of anti-PD-1 antibody alone or in combination with ZA was monitored by measuring tumor volume and bioluminescence imaging (BLI). At the end of study, the tumor immunohistochemistry was performed to confirm the in vivo observation and flow cytometry was used to determine the immune cell population in tumors. The survival rate was also calculated to evaluate the treatment efficacy.

Results: The results showed that mice treated with the combination therapy exhibited the best antitumor response compared to untreated controls or single therapy. Furthermore, Combination therapy inhibited tumor regrowth as determined by BLI imaging, and thus significantly improved the median overall survival compared to other groups.

Conclusions: Our study provides evidence for enhanced clinical BC treatment benefit from targeting co-signal molecules by combining PD-1 blockade with ZA treatment.

#2661

A broad approach for the selection of therapeutic mAbs to TNFR-superfamily members for use in combination with an anti-PD-1 mAb.

Cheng Chen, Xiaoniu Miao, Yao Yan, Liang Tang, Bingliang Chen, Junjian Liu, Xiaolin Liu, Michael Yu, Andy Tsun. _Innovent Biologics Co. Ltd., Suzhou, China_.

The rise of therapeutic antibodies directed towards T cell checkpoint inhibitors has paved the way to a novel and exciting frontier of cancer treatments. Spearheaded by anti-CTLA-4 and PD-1/PD-L1 inhibitors, many investigators have strived to uncover combinatory therapies that could synergize with CTLA-4 and PD-1 inhibitors to improve the overall survival of cancer patients. The TNFR-superfamily has been seen as an ideal combinatory target in providing co-stimulatory T cell signals in sync with the unleashing of T cells from their CTLA-4 and PD-1 brakes. The ideal outcome of these combinations is the induction of T cell memory and effector function towards tumour cells. Current clinical-stage agents against TNFR-superfamily targets include conventional or Fc-engineered therapeutic antibodies and multimeric ligands that aim to cluster and activate targets such as OX40, 4-1BB, GITR, and CD27. For OX40 and GITR, wildtype IgG1 variants offer target cell depletion of OX40/GITR-high expressing Treg cells, and also provide agonism through FcgR-mediated crosslinking. We have taken a broad approach of screening IgG1, IgG2, Fc-engineered and tetravalent antibodies to select for potent therapeutic antibody candidates to TNFR-superfamily targets, and conducted humanized mouse studies as a screen for candidate potency.

#2662

Statins modulate MMP-9 and beta catenin in epithelial cells.

Nadeem Fazal, Walid Al-Ghoul. _Chicago State University, Chicago, IL_.

The effect of statins (Simvastatins) mediated mechanisms that affect inflammatory or non-inflammatory pathways causing formation and progression of cancer need to be investigated. Currently, sufficient data are lacking to support the use of statins for the prevention of cancer and further research is clearly warranted. We studied these antineoplastic mechanisms in rodent inflammatory model, which might be instrumental in drug discovery research for the development of future cancer therapeutics. MMP9 is a member of the family of zinc-containing endopeptidases, which degrade various components of the extracellular matrix, thereby regulating matrix remodeling. Since matrix remodeling plays an important role during growth and progression of cancer and considering the fact that, tumor cells switch to aerobic glycolysis as its major energy source, this study was designed to analyze the effect of Statins on MMP9 and epithelial cell integrity at gastrointestinal gut barrier. We used an established model of thermal injury (TI) to study the effects of simvastatins. We found that the percentage of stained MMP-9 in the control tissue and TI+SMV tissue was lower than the percentage in thermal injury tissue (P< 0.05). Moreover, fluorescent stained sections of intestinal epithelial from control, thermal injury and thermal injury+SMV. β-catenin determination was performed with fluorescence microscope. β-catenin levels were significantly (p<0.05) decreased in thermal injury tissue, which was reversed by statins. Our studies conclude that statins have anti-neoplastic effects and affect metallopeptidases, β-catenin and e-cadherins.

#2663

Characterization of the pharmacodynamic immune response to a novel immunotherapeutic agent, ALKS 4230, in mice and non-human primates.

Jared E. Lopes, Heather C. Losey, Reginald L. Dean, Heather L. Flick, Michael R. Huff, Rosemarie A. Moroso, Lei Sun, Juan C. Alvarez. _Alkermes, Inc, Waltham, MA_.

ALKS 4230 is a selective agonist of the intermediate-affinity IL-2 receptor (IL-2R). A phase 1 study is ongoing to evaluate the safety and tolerability of ALKS 4230 in the treatment of patients with refractory solid tumors. The selectivity of ALKS 4230 is achieved through the stable fusion of circularly permuted IL-2 to the extracellular portion of the IL-2Rα chain, CD25. The resulting fusion protein is sterically prevented from binding to the high-affinity IL-2R complex, comprised of IL-2Rα, IL-2Rβ, and common gamma chain, expressed preferentially on CD4+ FOXP3+ regulatory T cells (CD4+ Tregs) yet retains full ability to signal through the intermediate-affinity IL-2R complex, comprised of IL-2Rβ and common gamma chain, expressed on memory CD8+ T cells and NK cells. Repeated dosing of ALKS 4230 drives the significant expansion of various CD8\+ T cell and NK cell populations without activation and minimal expansion of CD4+ Tregs in mice and non-human primates (NHP). The kinetics of the immunological responses in mice and NHP demonstrate that the pharmacodynamic effects persist beyond systemic exposure of ALKS 4230. Data demonstrating the effects of ALKS 4230 on tumor-infiltrating lymphocyte populations in syngeneic tumor models will also be presented.

#2665

Green tea catechin, EGCG, enhances antitumor immunity by down-regulation of PD-L1 expression in non-small human lung cancer cell lines.

Anchalee Rawangkan,1 Keisuke Iida,1 Ryo Sakai,1 Hirota Fujiki,2 Masami Suganuma1. 1 _Saitama University, Saitama, Japan;_ 2 _Saga University, Saga, Japan_.

(-)-Epigallocatechin gallate (EGCG) is now widely accepted as a non-toxic, effective cancer preventive compound. EGCG also acts as a synergist with anti-cancer drugs for cancer treatment. Considering the wide beneficial effects of EGCG, we think EGCG might have antitumor immunity. Programmed death-ligand 1 (PD-L1) expression on tumor cells is involved in PD-L1/PD-1 pathway. PD-L1 protein levels varied among 6 non-small human lung cancer cell lines (NSCLCs): LC-AI and Lu99 cell lines showed the highest PD-L1 expression, A549 and H322 cell lines were medium, and H1703 and H1299 were very low, independent on cancer cell lines. Since PD-L1 expression is induced by various cytokines and growth factors, we studied the effects of IFN-γ and EGF on cell-surface PD-L1 level using flow cytometry, and its gene expression using RT-PCR. Treatment with IFN-γ dose-dependently stimulated PD-L1 expression in both A549 and H1299 cells, and not effective on Lu99 cells, whereas treatment with EGF stimulated PD-L1 in Lu99 cells only.

Pretreatment with EGCG for 3 h, dose-dependently inhibited INF-γ-induced PD-L1 gene expression and protein level on cell-surface in both A549 and H1299. It is important to note that EGCG also inhibited phosphorylation of STAT1. In addition, EGCG inhibited EGF-induced PD-L1 gene expression and protein level in Lu99 cells via inhibition of phosphorylation of Akt. In order to study the effects of EGCG in between rodent carcinogenesis experiment and antitumor immunity, green tea extract (GTE) was given to NNK-induced lung tumors in A/J mice, which showed significant reduction of PD-L1 protein level, determined by immunohistochemical analysis using anti-PD-L1 antibody. Furthermore, the group treated with NNK + GTE significantly showed reduced number of tumors per mouse from 3.2 to 2.2, and decreased tumor size. Treatment with Wortmannin, a PI3K inhibitor, as a control, did not inhibit PD-L1 expression in A549 and H1299 cells. The results strongly suggest that EGCG enhances antitumor immunity via inhibition of PD-L1 expression in cancer cells, resulting in significant cancer preventive activity.

#2666

Using "omics" to select immunotherapy and conventional therapy combinations.

Sarit Schwartz,1 Robert Heaton,1 Yuan Tian,1 Zack Sanborn,2 Shankar Sellappan,1 Kerry Scott,1 Fabiola Cecchi,1 Steve Benz,2 Todd Hembrough1. 1 _NantOmics, Rockville, MD;_ 2 _NantOmics, Santa Cruz, CA_.

Background: As only a subset of patients respond to immunotherapies, therapeutic biomarkers are needed to predict which patients will benefit and which should be spared from potentially toxic treatment. Proteins expressed by tumor cells and by the immune microenvironment may predict response to immune checkpoint inhibitors. In non-inflamed tumors, chemotherapy and targeted therapy may stimulate an immune response thereby affecting the relationship between tumor and immune system. Combinations of immunotherapies and conventional therapies are the subject of ongoing clinical trials. We hypothesized that genomic and proteomic evaluation of multiple immune biomarkers in tumor tissue and in lymphocytes would identify a signature that could (a) differentiate between responders and non-responders to checkpoint inhibitors and (b) identify candidates for combination therapy.

Methods: A pathologist marked areas of tumor and tumor-associated lymphocytes on archived tissue sections (N=2) of non-small cell lung cancer (NSCLC) and melanoma. The marked areas were microdissected and solubilized to tryptic peptides. In each liquefied tumor sample, 110 protein biomarkers, including 60 immunomarkers were quantified with a mass spectrometry-based proteomic assay. The genomic material was analyzed by whole genome sequencing (WGS) and RNA-seq.

Results: Tumor and lymphocytes expressed several immunomarker proteins (eg, PDL1, IDO1, B7H3, B7-2, STAT1, GBP1). The melanoma patient's tumor and lymphocytes expressed protein markers for response to immunotherapy (PDL1 and IDO1), and the tumor had high mutational burden as identified by WGS. Furthermore, biomarker analysis suggested sensitivity to BRAF inhibitors and resistance to platinum and taxane. The NSCLC patient's tumor and lymphocytes expressed IDO1 protein, but genomic analysis suggested a non-inflamed tumor. Based on expression levels of ERCC1 and hENT1, one possible regimen for this patient is platinum plus gemcitabine followed by immunotherapy. In one patient, comparison of protein expression in tumor-associated lymphocytes versus distal lymphocytes (>1 mm from marked tumor areas) showed variation < 10% for 11 proteins and < 30% for 14 proteins.

Conclusions: Both lymphocytes and tumor expressed immunoproteins that may be used to select patients for approved immunotherapies or clinical trials. Proteomic and genomic profiling also characterized biomarkers that inform selection of targeted therapies and chemotherapies. Ongoing clinical trials of immunotherapy and combination therapies could benefit from precise, quantitative molecular stratification of patients. Protein level cutoffs that correlate with response to immunotherapy are in development.

#2667

In vitro properties and pre-clinical activity of PF-06801591, a high-affinity engineered anti-human PD-1.

Sawsan Youssef,1 Yasmina Abdiche,2 HoangKim Nguyen,1 Joyce Chou,1 Sherman Michael Chin,1 Cris Kamperschroer,1 Patricia A. Schneider,1 Eugenia Kraynov,1 Heike I. Krupka,1 Arvind Rajpal,3 John Lin1. 1 _Pfizer Inc., South San Francisco, CA;_ 2 _Wasatch Microfluidics, Salt Lake City, UT;_ 3 _BMS, Redwood City, CA_.

Monoclonal-antibody-based therapies targeting the immune checkpoint receptors have become the new standard of care in many cancers. Antibodies that specifically target programmed death receptor-1 (PD-1) or its cognate ligand, programmed death receptor ligand-1 (PD-L1), alone or in combination, have yielded clinical benefits and durable responses in patient subsets with various cancers (including metastatic melanoma, NSCLC, RCC, urothelial cancer, cHL and others). Here we report on the biophysical characteristics and non-clinical antagonistic activities of PF-06801591. PF-06801591 is a humanized anti-PD-1 antibody of human IgG4 isotype, that binds selectively and with similar potency to human and cynomolgus monkey PD-1 receptor (EC50 ~50 pM) and blocks its interaction with its cognate ligands PD-L1 and PD-L2 (IC50 < 1 nM) with no detectable Fc effector function. The interaction of PF-06801591 to PD-1 rescues T cell suppression and exhaustion that translates into NFAT activation, IL-2 and IFN-gamma secretion and T cell proliferation both in vitro cultures and in vivo using an acute xeno GvHD model with human PBMC transfer. Binding of PF-068001591 to human and cynomolgus PD-1 is characterized by the formation of a very stable complex (T1/2 = 2 h) as measured by SPR at 37 C, resulting in high affinity (KD ~20 pM) as measured in solution by KinExA at 23 C. In addition, we explored therapeutic potential of anti-PD-1 in combination with other immunotherapy agents using surrogate antibodies in non-clinical tumor models. The data presented here support future development of PF-06801591 as a single agent or in combination with other immunotherapies.

#2668

Efficacy study of immuno-checkpoint antibodies in humanized CDX and PDX models.

Feifei Zhang, Yang Yang, Hongkui Chen, Lijun Jia, Kedong Ouyang, Danyi Wen, Taiping Chen. _LIDE Biotech Ltd, Shanghai, China_.

Immune therapeutic intervention has been accepted widely for many hematopoietic malignancies and solid malignancies in past several years. Preclinical animal models with reconstitution of immune cells and target-expressing tumor cells were urgently needed for prove of concept studies demonstrating stimulatory immune system activation. Here we describe two established models of human PBMC co-transplantation of CDX and PDX. The study design was based on co-inoculation of human PBMC and PD-L1 expressing cell in NOG mice for reconstitution of immuno-checkpoint blockage. A therapeutic anti-PD1 antibody was applied for immune system activation and tumor growth inhibition measurement. After drugs were administrated i.p. for three weeks, 95 % TGI for A375 CDX model were observed. At the termination of the efficacy study at 28 days post therapeutic intervention, tumors of vehicle (tumor only, PMBC only), PD1 Ab treated groups were collected for immunostaining of CD45. Infiltrating T-cell with CD45+ label was found within the tumors when co-inoculated with human PBMC. PBMC humanized PDX models of ovarian cancer was implanted into mice which reconstituted with human PBMC for therapeutic intervention. Results showed that 85% TGI for PBMC humanized ovarian PDX models was observed. In conclusion, the models established here are feasible for immunotherapeutic evaluation and further additional immune therapeutic test articles can be explored with similar approaches.

#2669

Antitumor activity of the CMP-001 (TLR9 agonist) alone or combined with immune modulators in syngeneic tumor models.

Francis Bichat,1 Sylvie Maubant,1 Jean-François Mirjolet,1 Philippe Slos,1 Arthur M. Krieg,2 Aaron Morris2. 1 _Oncodesign S.A., Dijon, France;_ 2 _Checkmate Pharmaceuticals, Cambridge, MA_.

Targeted blockade of checkpoint inhibitors such as CTLA-4 or PD-1 with antagonist monoclonal antibodies (mAbs) has shown impressive and durable clinical responses in patients with advanced cancer. An alternative strategy to boost anti-tumor immunity is to promote T cell activation through co-stimulatory receptors such as OX40 and 4-1BB. OX40 is of particular interest as treatment with an activating anti-OX40 mAb augments T cell differentiation and cytolytic function leading to enhanced anti-tumor immunity. However, each of these agents benefits only a subset of patients, highlighting the critical need for more effective combinatorial therapeutic strategies.

Toll-like receptor 9 (TLR9) agonist CpG oligodeoxynucleotides (ODN) are candidates to promote an anti-tumor immune response. CMP-001, a CpG-A ODN formulated within a virus-like particle, is designed to activate TLR9 (the receptor for CpG-A) in tumor-associated plasmacytoid dendritic cells (pDC) within the tumor or tumor-draining lymph nodes. Resting or immature pDC promote tumor growth, but when activated by CpG-A, the resulting mature pDC promote a robust anti-tumor immune response. Activation of pDC causes secretion of very large quantities of type I interferons, increased expression of costimulatory molecules, and recruitment and activation of other DC subsets to enhance tumor antigen presentation to T cells, culminating in the generation of effective anti-tumor T cell responses.

The preclinical efficacy of intratumorally administered CMP-001 alone or in combination with an intraperitoneally administered PD-1 antagonist and/or an OX40 agonist was examined and assessed in a variety of syngeneic tumor models: CT-26 colon tumor model, MBT-2 bladder tumor model, RenCa kidney tumor model, 4T1 breast tumor model and LLC-1 lung tumor model. Tumors were implanted into left and right flanks while only one tumor was injected with CMP-001. In addition to body weight and overall survival, tumor volume was monitored on both flanks to assess direct and abscopal/systemic anti-tumor activity.

Some discrepancies were observed between evaluated syngeneic tumor models with non-responders (LLC-1, 4T1) and responders (CT-26, MBT-2, RenCa). The most efficacious results were registered in the CT-26 model. Each therapeutic yielded weak activity as a single agent, which improved when combined with another treatment modality. The best therapeutic efficacy was obtained with the combination of all three agents resulting in cures of both treated and untreated CT-26 tumors in 40% of the mice. The median survival time was increased for these animals compared to those treated with only vehicle or one or two immune modulators (50 vs 18 vs 21 or 23-28 days, respectively). Similar results were generated in the MBT-2 model (and to a less extent in RenCa model) though no complete response was recorded. These data support the clinical investigation of these combinations in cancer patients

#2670

Combination of anti-HER2 ADC XMT-1522 and checkpoint inhibitor pembrolizumab for treatment of NSCLC in preclinical models.

Natalya Bodyak, Marina Protopopova, Qingxiu Zhang, Alex Yurkovetskiy, Mao Yin, LiuLiang Qin, Laura L. Poling, Rebecca Mosher, Donald A. Bergstrom, Timothy B. Lowinger. _Mersana Therapeutics, Inc., Cambridge, MA_.

The combination of antibody-drug conjugates (ADCs) and immunomodulatory cancer therapies is emerging as a powerful strategy for cancer treatment. Tumor-targeted delivery of a cytotoxic payload capable of inducing immunogenic cell death (ICD) can trigger both an innate and an adaptive immune response, whereby increased recruitment of effector T-cells to the tumor and formation of tumor specific immunological memory can result in a durable treatment response. The ADC XMT-1522 consists of a novel human IgG1 anti-HER2 monoclonal antibody and a novel, auristatin-based cytotoxic payload (Auristatin F-hydroxypropylamide, AF-HPA). An average DAR of 12 AF-HPA molecules is achieved via a biodegradable polymer conjugation platform. We have characterized the ability of both the free payload AF-HPA and the ADC XMT-1522 to induce ICD in vitro in multiple cell lines (NCI-N87, HT-29, SKBR3), as measured by the cell surface expression of the ICD marker calreticulin (CRT) by microscopy and flow cytometry (FACS). CRT, usually contained in the lumen of the endoplasmic reticulum, translocated to the cell surface within a few hours after treatment with AF-HPA or XMT-1522. XMT-1522 as a single agent induced significant tumor regressions in two patient-derived xenograft (PDX) models of HER2-espressing non-small cell lung cancer (NSCLC) at a dose of 3 mg/kg weekly for 3 doses. The combination of XMT-1522 in combination with the checkpoint inhibitor pembrolizumab was tested in one of these patient-derived HER2-expressing PDX models in a mouse with a humanized immune system. Expression of huPD-L1 in the tumor was confirmed by FACS and immunohistochemistry (IHC). Lymphocyte sub-populations were quantified in whole blood and in tumor by FACS and IHC. XMT-1522 treatment alone induced tumor growth delay after 3 weekly doses of 1 mg/kg. Pembrolizumab as a single agent administered every 5 days for 6 doses (q5dx6) at a dose of 2.5 mg/kg led to less tumor growth delay than XMT-1522 treatment. The combination of these two treatment regimens resulted in a better response than either of the two monotherapies. These data provide a rationale for XMT-1522 to be tested clinically as a single agent in HER2-expressing NSCLC, as well as a rationale for combination of XMT-1522 and immunomodulatory therapies in NSCLC.

#2671

Antitumor activity of NKTR-214 in combination with pmel-1 ACT in an aggressive murine melanoma model.

Giulia Parisi,1 Justin Saco,1 Siwen Hu-Lieskovan,1 Ruixue Zhang,1 Paige Krystofinski,1 Cristina Puig Saus,1 Deborah H. Charych,2 Antoni Ribas1. 1 _UCLA, Los Angeles, CA;_ 2 _Nektar Therapeutics, San Francisco, CA_.

The adoptive cell transfer (ACT) of genetically engineered T cells expressing cancer-specific T-cell receptors (TCR) has been shown to induce effective anti-tumor response. However, tumors frequently relapse after an initial response. Another strategy towards stimulating the immune system is the use of high-dose interleukin-2 (IL-2) to target the IL-2 receptor (IL2R), leading to immune cell expansion. However, clinically approved IL-2 expands both tumor-killing CD8+ effector T cells (CD8T) as well as regulatory T cells (Tregs) through binding the IL-2Rβγ and IL-2Rαβγ complexes, respectively. Tregs in the tumor lead to immune suppression, which hampers the antitumoral response. NKTR-214 is a CD122-biased cytokine agonist conjugated with multiple releasable chains of polyethylene glycol and designed to provide sustained signaling through the heterodimeric IL-2 receptor pathway (IL-2Rβγ) to preferentially activate and expand effector CD8+ T and NK cells over Tregs. We used the pmel-1 ACT/B16 melanoma tumor model to test the anti-tumor activity of NKTR-214 and evaluate its effects on tumor-specific TCR transgenic T cells. On Day 0 (D0) C57BL/6 mice were implanted with B16-F10 mouse melanoma cells and lymphodepleted with 500 cGy on D6. On D7, mice were treated with either the combination of ACT (T lymphocytes activated in vitro with 1 μg/ml gp100) plus NKTR-214 (0.8 mg/kg, q9dx3, i.v.) or with C57/B6 T cells plus PBS (vehicle control). The tumors of the vehicle control mice (n=12) rapidly grew to the 1500mm3 endpoint in 12 days post-treatment, versus 35 days for the NKTR-214 group (n=12) with only 1 out of 12 mice reaching endpoint. Bioluminescence imaging was used to visualize the in vivo distribution and tumor-homing of antigen-specific T cells. Interestingly, the reporter T cells were retained in the spleen until D7 and could be seen migrating to the tumor at D9 reaching peak of bioluminescence at day 12, a delayed time point compared to the 5 days usually observed in mice treated with standard IL-2. The signal persisted in the NKTR-214 + ACT group until D20 versus D7 in the vehicle-control animals. These data suggest that NKTR-214 + ACT is well tolerated and provides a robust anti-tumor response in the aggressive B16F10 model. Treatment with NKTR-214 + ACT robustly mobilizes T cells into the tumor where they durably persist. The robust and long-lasting effect of NKTR214 supports its potential use in combination with cell-based therapeutics. 

### Inflammation and Cancer

#2672

Response to anti-PD-1 based therapy in metastatic melanoma patients is associated with the diversity and composition of the gut microbiome.

Vancheswaran Gopalakrishnan,1 Christine Spencer,1 Alexandre Reuben,1 Peter Prieto,1 Diego Vicente,1 Tatiana V. Karpinets,1 Courtney W. Hudgens,1 Diane S. Hutchinson,2 Michael Tetzlaff,1 Alexander Lazar,1 Michael A. Davies,1 Jeffrey E. Gershenwald,1 Robert Jenq,1 Patrick Hwu,1 Padmanee Sharma,1 James Allison,1 Andrew Futreal,1 Nadim Ajami,2 Joseph Petrosino,2 Carrie Daniel-MacDougall,1 Jennifer A. Wargo1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Baylor College of Medicine, Houston, TX_.

This abstract has been withheld from publication due to its inclusion in the AACR Annual Meeting 2017 Official Press Program. It will be posted online following its presentation.

#2673

Divergent therapeutic responses to CD40L blockade or CD40 activation in Ras-driven skin cancers determined by origin of tumor initiating cell.

Adam B. Glick,1 Jacob T. Bailey,1 Andrew Gunderson,2 Kyle Breech,1 Michael Podolsky1. 1 _Pennsylvania State University, University Park, PA;_ 2 _Earle A. Chiles Research Institute, Portland, OR_.

Heterogeneity in tumor immune responses is a poorly understood yet critical parameter for successful immunotherapy. We used doxycycline-inducible epidermal squamous cancer models driven by oncogenic Ras to test whether the origin of the tumor-initiating cell influenced the tumor immune response. Threshold doxycycline induced expression of H-RasG12V in either the basal/stem cell layer with a Keratin14-rtTA transgene (K14Ras), or committed progenitor/suprabasal cells with an Involucrin-tTA transgene (InvRas), caused distinct immune responses despite similar levels of Ras, tumor latency, and tumor numbers. End stage K14Ras tumors had an immunosuppressed microenvironment with abundant Tregs and Bregs which evolved from an initially Th1 dominant environment in the earliest detectable lesions. In contrast InvRas tumors had a Th2 pro-inflammatory microenvironment, with significantly fewer Tregs or Bregs at any time point. This difference in immune microenvironment between the tumor models was also found in hyperplastic skin after 5 days of maximal Ras expression, indicating that it was not simply a property of the end stage tumor. Surprisingly, adaptive immunity had opposite roles in tumor development. InvRasRag1-/- mice developed fewer and smaller tumors that regressed while K14RasRag1-/- mice developed more tumors with shorter latency than Rag1+/+ controls. In both models adoptive transfer and depletion studies showed that cooperation between B and CD4 T cells drove the opposing tumor responses, lymphocyte polarization, and tumor immune phenotype. Co-culture of tumor-conditioned but not splenic B cells from each model with naive CD4 T cells showed that direct contact and CD40-CD40L ligation was required for opposite polarization towards either a Th2 or Treg phenotype. Importantly, we found that anti-CD40L mAb caused regression of preexisting InvRas tumors but enhanced growth of K14Ras tumors. In contrast, CD40 agonist mAb enhanced growth of preexisting InvRas tumors, and suppressed growth of K14Ras tumors. Thus in an in vivo setting the type of tumor immune microenvironment and opposing role of CD40-CD40L in tumor development generates distinct responses to therapeutic antibodies. Together these data show that the position of a tumor initiating cell within the stem cell hierarchy in a stratified squamous epithelia has important consequences for the type of tumor immune microenvironment and response to checkpoint therapy.

#2674

Fusobacterium nucleatum **subspecies** animalis **influences pro-inflammatory cytokine expression and monocyte activation in human colorectal tumors.**

Xiangcang Ye,1 Rui Wang,1 Rajat Bhattacharya,1 Delphine R. Boulbes,1 Fan Fan,1 Ling Xia,1 Adoni Harish,1 Nadim J. Ajami,2 Matthew C. Wong,2 Daniel P. Smith,2 Joseph F. Petrosino,2 Susan Venable,2 Wei Qiao,1 Veera Baladandayuthapani,1 Dipen Maru,1 Lee M. Ellis1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Baylor College of Medicine, Houston, TX_.

Chronic infection and associated inflammation have long been suspected to promote human carcinogenesis. Recently, certain gut bacteria, including some in the Fusobacterium genus, have been implicated in playing a role in human colorectal cancer (CRC) development. However, the Fusobacterium species and subspecies involved and their oncogenic mechanisms remain to be determined. We sought to identify the specific Fusobacterium spp. and ssp. in clinical CRC specimens by targeted sequencing of Fusobacterium 16S ribosomal RNA gene. Five Fusobacterium spp. were identified in clinical CRC specimens. Additional analyses confirmed that Fusobacterium nucleatum ssp. animalis was the most prevalent F. nucleatum subspecies in human CRCs. We also assessed inflammatory cytokines in CRC specimens using immunoassays and found that expression of the cytokines interleukin-17A and tumor necrosis factor-alpha was markedly increased but interleukin-21 decreased in the colorectal tumors. Furthermore, the chemokine (C-C motif) ligand 20 was differentially expressed in colorectal tumors at all stages. In in vitro co-culture assays, F. nucleatum ssp. animalis induced CCL20 expression in CRC cells and monocytes. It also stimulated the monocyte/macrophage activation and migration. Our observations suggested that infection with F. nucleatum ssp. animalis in colorectal tissue could induce inflammatory response and promote CRC development. Further studies are warranted to determine if F. nucleatum ssp. animalis could be a novel target for CRC prevention and treatment.

#2675

MMP9 modulates ROS levels and DNA repair pathway to maintain genomic stability in colitis associated cancer.

Lewins Walter, Adani Pujada, Brandon Canup, Hamed Laroui, Pallavi Garg. _Georgia State University, Atlanta, GA_.

Introduction: Chronic inflammation predisposes tissues to oncogenic, and in the colon this condition is termed as colitis associated cancer (CAC). CAC is the inflammation-dysplasia-carcinoma pathway which is significantly different compared to the adenoma-carcinoma pathway of sporadic colon cancer (CRC). In CAC, chronic inflammation causes accumulation of reactive oxygen species (ROS) resulting in DNA damage that nurtures tumor microenvironment and accelerates cancer cell growth. Gut microbiota is important in regulating ROS levels, and DNA repair pathways are critical in restoring the genomic instability. Matrix Metalloproteinases (MMPs) are a family of zinc dependent endopeptidases which mediate inflammation, tissue remodeling, and tumorigenesis. MMP9 is the most unique MMP because it is undetectable in healthy tissue but highly upregulated during inflammation and cancer. We have previously shown that MMP9 plays a protective role in CAC which is opposite to its conventional role of mediator in acute inflammation and cancer.

Aim: In this study, we examined that if MMP9 acts as a tumor suppressor by modulating gut microbiota and mismatch repair (MMR) genes to reinstate genomic stability in CAC.

Methods: We used C57/B6 transgenic MMP9 mice which can express MMP9 under villin promoter (TgM9) and their wild type (WT) littermates. CAC was induced by azoxymethane (AOM) and dextran sodium sulfate (DSS, inflammation inducer) to assess the microbiota population. As a proof of principal model, we used MMP9 small interfering RNA (siRNA) encoded within nanoparticles and gavaged WT mice to inhibit MMP9 in colonic epithelium.

Results: QPCR data showed that mRNA levels of total microbiota population were more among TgM9 mice compared to WTs in CAC. QPCR analysis also indicated increased mRNA levels for the phylum Bacteroidetes sp., Akkermansia muciniphila but decreased mRNA levels for the phylum Firmicutes sp. TgM9 mice had lower levels of ROS compared to WTs in CAC. WB data showed decrease in protein expressions of γH2AX and MDC1 while an increase in protein expressions of MLH1 and pCNA compared to WTs in CAC. WT mice treated with MMP9 siRNA loaded nanoparticles showed worsened CAC condition compared to WT mice given scrambled siRNA loaded nanoparticles as reflected by significant bodyweight loss and higher clinical score. We also observed that WT mice treated with MMP9 siRNA loaded nanoparticles showed increase in protein expression of γH2AX while decrease in MLH1 protein levels compared to control group.

Conclusion: Our study has two clinically relevant outcomes that MMP9 expression in CAC: i) promotes the beneficial microbiota population and controls the ROS production, ii) restores the DNA damage via activation of the MMR pathway. This implies the contradiction of the use of targeted MMP9 inhibitors in CAC treatment therapies by showing that MMP9 expression may be a natural way to suppress CAC.

#2676

Role of the RON kinase in the regulation of the antitumor immune response in pancreatic cancer.

Alex Cazes, Michele L. Babicky, Jeffery Chakedis, Divya Sood, Dawn Jaquish, Andrew M. Lowy. _University of California San Diego, La Jolla, CA_.

RON is a cell-surface receptor for the macrophage-stimulating protein expressed in tissue-resident macrophages and overexpressed in multiple epithelial neoplasms including pancreatic cancer. Our laboratory recently found that RON serves to mediate pancreatic duct carcinogenesis initiated by KRAS. We demonstrated that RON overexpression in the pancreas results in more rapid local and metastatic progression ultimately associated with decreased survival. We also discovered profound changes in the immunophenotype within the tumor microenvironment associated with RON expression. We notably established that RON expression drives the polarization of tumor-associated macrophages into an alternative, pro-tumorigenic state. We hypothesize that these unique activities help to shape the immunosuppressive milieu of the pancreatic cancer microenvironment and that RON directed therapy has the potential to serve as a novel immunomodulatory strategy in this disease. The aim of this work is to define how RON signaling contributes to shaping the pancreatic cancer microenvironment. In order to further interrogate the role of RON during pancreatic carcinogenesis, we bred kinase-dead RON mice (KD) to Pdx1-cre/LSL-KRASG12D mice. We observed a significant delay in the onset of pancreatic neoplasia and a significant decrease in tumor weight. The absence of functional RON kinase markedly reduced trafficking of CD11b+ cells to the pancreas and immunophenotyping in tumors showed a clear reduction of alternatively polarized macrophages when compared to RON overexpression or KRAS mutation alone. Because RON is expressed in both epithelial and stromal cell populations, we further sought to dissect the relevance of RON function to tumor growth within these cellular compartments. We performed orthotopic injections of organoïd-3D cells derived from tumors of KPC mice (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre). These cells were injected in wild type (WT) or RON KD mice. Preliminary results show that tumor growth is impaired in KD mice, suggesting that RON activity in the host microenvironment influence tumor growth. In order to identify all immune populations regulated by RON, we will conduct an exhaustive immunophenotyping of murine pancreatic tumors. A transcriptomic analysis will then be conducted on selected cell populations to better define the molecular mechanisms associated with the RON regulation. Finally, given the effect of RON signaling on macrophage polarization, we will also dissect the effects of RON signaling from bone marrow derived myeloid cells versus resident tissue macrophages. Preliminary results and future experiments will significantly narrow the gap in our understanding of how RON signaling contributes to shaping the pancreatic cancer chimera and therefore how RON-directed therapies could be most successfully utilized to the benefit of those afflicted with this most deadly disease

#2677

Role of CLEC4D in inflammation-driven liver carcinogenesis.

Elena Riboldi,1 Luca Di Tommaso,2 Nausicaa Clemente,3 Chiara Raggi,2 Elisa Forti,2 Simone Merlin,3 Fabio Pasqualini,2 Antonia Follenzi,3 Antonio Sica1. 1 _Università del Piemonte Orientale, Dept. of Pharmaceutical Sciences, Novara, Italy;_ 2 _Humanitas Clinical and Research Center, Rozzano, Italy;_ 3 _Università del Piemonte Orientale, Dept. of Medical Sciences, Novara, Italy_.

Hepatocellular carcinoma (HCC) accounts for approximately 90% of all primary liver tumors and is the second most deadly cancer in the world. HCC commonly arises in a chronically damaged liver that contains large amounts of inflammation and fibrosis. Indeed, interactions among hepatocytes, stromal, and inflammatory cells create a complex microenvironment permissive to tumor development. Myeloid cells are crucial players in cancer-related inflammation. From transcriptional profiling of liver myeloid cells, we identified the gene encoding for the C-type lectin receptor CLEC4D as one of the genes upregulated during the inflammatory response that precedes cancer development in a spontaneous model of murine HCC. We hypothesized that CLEC4D may be involved in the molecular mechanisms that drive HCC pathogenesis. We used the murine model of diethylnitrosamine (DEN)-induced liver carcinogenesis and evaluated tumor growth in genetically modified mice lacking CLEC4D (CLEC4D KO mice), as compared to C57BL/6 wild type (WT) mice. We observed that tumor burden (number of tumors per mouse and tumor dimensions) was reduced in CLEC4D KO mice compared to WT mice. Gene expression analysis of tumor lesions showed that the tumor microenvironment of CLEC4D KO mice was less inflammatory. CLEC4D expression was then evaluated in surgical specimens and tissue microarrays from HCV+ HCC patients. CLEC4D-positive inflammatory cells (macrophages and granulocytes) were present both in the peritumor and in the tumor areas. The percentage of intratumor CLEC4D-positive cells inversely correlated with the degree of the lesion: CLEC4D expression was higher in dysplastic nodules compared to high grade HCCs. Our results indicate that the CLEC4D-dependent pathway contributes to the initiation and the progression of HCC. By targeting CLEC4D, we could modulate immune responses and provide an environment less favorable to tumor growth. An intervention at this level could represent a chemopreventive strategy to arrest the development of HCC in a cirrhotic liver.This work has been supported by Fondazione Cariplo, grant n° 2014-0962.

#2678

Novel host-pathogen interactions of Helicobacter pylori and their implications to gastric cancer.

Emine Guven Maiorov, Ruth Nussinov, Chung-Jung Tsai. _National Cancer Institute, Leidos Biomed. Research, Inc., Frederick, MD_.

About 20% of the cancer incidences worldwide have been estimated to be associated with infections. There is a strong correlation of some pathogens with various cancer types. Although presence of commensal microbiome helps chemotherapy to be more effective, pathogenic microbiota increases the cancer risk. Invading pathogens interact with the host mainly through proteins. To subvert host defense, pathogens hijack host pathways by mimicking the binding surfaces (interfaces) of host proteins. This similarity in interfaces permits the pathogenic protein to compete with host proteins to bind to a target protein, alter the physiological signaling and cause persistent infections as well as cancer. Detection of host-pathogen interactions (HPIs) and mapping the re-wired HPI network - along with its structural details - is critical for in-depth understanding of the underlying pathogenesis mechanisms of infections and pathogen-triggered cancers, and developing efficient therapeutics. Here, we developed a novel computational approach to identify novel HPIs by employing "interface mimicry", We applied this approach to Helicobacter pylori, dominant species in gastric microbiome that greatly increases the gastric cancer risk in order to understand how they modulate host immunity and lead to tumorigenesis. We found that its proteins interfere with the functioning of host apoptosis pathway, cytokine and chemokine pathways, and also cell-cell adhesions. Our results shed light on the molecular mechanisms of resistance to apoptosis, immune evasion and loss of cell junctions that are seen in Helicobacter pylori-infected host cells. In conclusion, HPIs can help us unravel which human pathways are targeted by the pathogenic proteins and how they contribute to pathogenesis of infections and pathogen-triggered cancer. With a better grasp on immunomodulatory strategies of pathogens, we can develop better therapies against them.

#2679

A promoting role for the epithelial MyD88/IRAK4/NF-kB signaling in K-ras mutant lung tumorigenesis.

Susana Castro,1 Soudabeh Daliri,1 Maria Miguelina De La Garza,1 Amber M. Cumpian,1 Misha Umer,1 Diana Del Bosque,2 Sabah Akbani,2 Scott E. Evans,1 Seyed Javad Moghaddam1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _University of Houston, Houston, TX_.

K-ras mutation is the most common oncogenic alterations associated with lung cancer development. Unfortunately, all attempts to develop therapies directly targeting K-ras have been failed thus far, clearly stating the need for new strategies targeting downstream effectors and/or cooperating pathways of K-ras to overcome lung cancer displaying such a molecular profile. Using a conditional K-ras mutated lung cancer mouse model, CC-LR (CCSPcre/LSL-K-rasG12D) we previously showed that K-ras mutated lung tumorigenesis is associated with lung inflammation due to activation of NF-κB pathway and increased expression of its downstream targets in the lung. Here we have shown that lack of NF-κB activity in the airway epithelium by selectively targeting IKKβ, which is required for NF-kB activation, significantly reduces lung tumor burden (3.4-fold) and changes the inflammatory cells and mediators in the bronchoalveolar lavage fluid (BALF) of CC-LR mice. Immunohistochemically staining of lung tissues with specific markers, Ki-67 and CD-31, demonstrated significantly lower tumor cell proliferation and angiogenesis in CC-LR mice with lack of epithelial NF-kB activity. To further dissect the role of NF-κB pathway in this process, CC-LR mice were crossed with MyD88f/f mice to develop a mouse with lack of MyD88 (an adaptor protein upstream to IKKβ) in the airway epithelial cells (CC-LR/MyD88Δ/Δ mice). As we had hypothesized, the resulting tumor numbers in the lungs were significantly lower (1.9-fold) in CC-LR mice with lack of MyD88 in the airway epithelial cells compared to control CC-LR mice. Tumor reduction in CC-LR-MyD88Δ/Δ mice was also associated with decreased tumor cell proliferation and angiogenesis compared to control CC-LR mice. Surprisingly, unlike to lack of epithelial NF-kB activity, absence of MyD88 in the airway epithelium did not change the BALF inflammatory cell component of CC-LR mice. We then targeted another upstream signaling molecule to NF-kB, IRAK4, which is down stream of MyD88 by crossing CC-LR mice to IRAK4 knock out mice. Similar to lack of MyD88, we found a significant reduction in lung tumor number (1.8-fold) with no changes in BALF inflammatory cell component in CC-LR mice with lack of IRAK4 compared to the control CC-LR mice. Taken these together, we conclude that there is an essential role for MyD88/IRAK4/NF-kB pathway activation in promotion of K-ras mutant lung cancer.

#2680

A novel pro-tumorigenic role for IDO1 in inflammatory neovascularization.

Arpita Mondal,1 Erika Sutanto-Ward,1 James B. DuHadaway,1 Arturo Bravo-Nuevo,2 Sunil Thomas,1 George C. Prendergast,1 Alexander J. Muller1. 1 _Lankenau Inst. for Medical Research, Wynnewood, PA;_ 2 _ArtDem Therapeutics, Holmes, PA_.

Small molecule inhibitors of the tryptophan catabolizing enzyme IDO1 (indoleamine 2,3-dioxygenase 1) have shown early promise in clinical trials as immuno-oncology agents. While the tolerogenic activity of IDO1 has been well established, we have recently identified an additional role for IDO1 in supporting neovascularization at the regulatory interface between the inflammatory cytokines IFNγ (interferon γ) and IL6 (interleukin 6). IFNγ is a primary inducer of IDO1, but is also a key mediator of immune-based tumor suppression, which studies have associated with its anti-angiogenic activity. Conversely, genetic studies in mice have clearly established the tumor-promoting role of IDO1, suggesting that it may act in a negative feedback capacity. Targeted disruption of the Ido1 gene in mice resulted in enhanced resistance to lung tumor and metastasis development. This corresponded with attenuated induction of the pro-angiogenic cytokine IL6, which, when provided through ectopic expression, was able to restore pulmonary metastasis susceptibility to Ido1-/- mice. These initial findings led us to hypothesize that IDO1 might contribute to cancer promotion by countering the anti-neovascular effect of IFNγ, possibly through IDO1-potentiated elevation of IL6. Testing this hypothesis in mouse models of oxygen-induced retinopathy and pulmonary metastasis, we determined that loss of IDO1 did indeed result in reduced neovascularization in conjunction with impaired metastasis outgrowth, effects that were completely reversed by the concurrent elimination of IFNγ. Loss of IL6 was likewise associated with IFNγ-dependent reductions in neovascularization and impaired metastasis outgrowth, as predicted. Having established a novel role for IDO1 in inflammatory neovascularization, current investigations are focused on the underlying molecular and cellular mechanisms involved. At the molecular level, one of the consequences of tryptophan catabolism by IDO1 can be to trigger the ISR (integrated stress response) through activation of the GCN2/CHOP pathway that has previously been linked to the downstream induction of IL6. Thus far, data collected in both the oxygen-induced retinopathy and pulmonary metastasis models are consistent with the ISR being the relevant downstream signaling pathway from IDO1 in this biological context. At the cellular level, we have detected the incorporation of non-endothelial, IDO1-positive cells into the vessels that comprise the neovascular tufts, implicating IDO1 in the process of vasculogenesis. These insights into this unrecognized aspect of IDO1 biology are likely to have important ramifications for IDO1 inhibitor development, not only in cancer where clinical trials are currently ongoing, but in other disease indications involving neovascularization as well.

#2681

Oral micro biome enhances stemness in oral cancer cells by activating Toll like receptor signaling.

Joyeeta Talukdar,1 Rashmi Bhuyan,2 Bidisha Pal,2 Sorra Sandhya,1 Hong Li,2 Seema Bhuyan,1 Sukanya Garhyan,1 Debabrat Baishya,3 Anupam Sarma,4 Jyotirmoy Phukan,1 Amal Kataki,4 Bikul Das2. 1 _KaviKrishna Laboratory, Guwahati Biotech Park, IIT, Guwahati, India;_ 2 _Forsyth Institute, Cambridge, MA;_ 3 _Gauhati University, Guwahati, India;_ 4 _B Borooah Cancer Institue, Guwahati, India_.

Background: The resistance and progression of cancers after chemotherapy to invasive and metastatic stages accounts for the overwhelming majority of cancer deaths. Recent studies suggest, microbiomes can induce a cascade of host events to either support or inhibit tumor growth. Specially, in oral cancer, chemotherapy treatment may alter the oral microbial flora, which may favor or inhibit tumor growth. Hence, it is importantl to develop novel experimental approaches to study the role of oral microbial flora in oral cancer stemness (self-renewal and undifferntiated state of cancer stem cells). Importantly, patients in developing area, including Assam, where KaviKrishna laboratory is located, may have distinct oral microbial flora that could favor oral cancer growth. Hence, it is important to include patients from developing countries for such studies. Our previous research showed that chemotherapy ehances stemenss in many cancer cell types, including oral squamous cell carcinoma cell line SSC-25. The stemness switch is characterized by enhanced expression of stemness associated genes including Nanog, Lin28A/B, Oct-4, MYC, HIF-2alpha and inflammation associated genes including Toll like receptor (TLR) 2/4. Here we investigated the role of oral microbiomes in the TLR mediated stemness switch of oral cancer cells.

Methods: SCC-25 oral cancer cell line was treated with bacterial product lipopoly saccharide (LPS), and the stemness switch evaluated by isolation of ABCG2+ cells and expression of stemness associated genes by these cells. Capacity of interaction of tumor stromal cells with mesenchymal stem cells was also evaluated. Additionally, we obtained sputum from oral cancer subjects undergoing chemotherapy. The patients were from the Kamrup district of Assam, where KaviKrishna laboratory is located. The sputum was processed and then added to the culture medium of SCC-25 cells. These post-sputum treated SCC-25 cells were subjected to phenotypic stemness switch analysis.

Results: We found, LPS and sputum treatment led to the enhanced stemness of ABCG2+ cells, including the high expression of TLR2/4, MYC, Nanog, Sox-2, and HIF-2alpha. Importantly, sputum derived from oral cancer subjects under remission showed inhibitory activity on ABCG2+ cell self-renewal. In contrast, sputum obtained from oral cancer subjects with relapse showed enhanced stemness of ABCG2+ cells, and also increased tumorigenic potential. The post-sputum treated ABCG2+ cells exhibited high expression of TLR2/4 and associated increase of HIF-2alpha and MYC transcriptional activity. The sputum treated with broad spectrum antibiotic ciprofloxacin did not enhance the stemness and TLR2/4 signaling of SCC-25 cells.

Conclusion: These results indicate that oral microbiomes may differentially influence the stemness of oral cancer cells. We also conclude that live bacteria present in the sputum may be required to enhance stemness in a TLR2/4 dependent manner.

#2682

IL-6/STAT3 activation in hepatocytes drives the formation of a pro-metastatic niche in the liver during pancreatic tumorigenesis.

Jae W. Lee, Paige M. Porrett, Chad A. Komar, Whitney L. Gladney, Gregory L. Beatty. _University of Pennsylvania, Philadelphia, PA_.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth-leading cause of cancer-related deaths in the United States with metastasis to the liver as the major cause of mortality. While the propensity of PDAC to spread to the liver may reflect mechanical trapping of tumor cells that enter the portal circulation, primary tumor cells have also been suggested to secrete factors that may promote recruitment of myeloid cells to establish a pro-metastatic niche. In this study, we used the LSL-KrasG12D/+;LSL-Trp53R127H/+;Pdx-1-Cre (KPC) mouse model of PDAC to investigate the impact of PDAC development on the formation of a pro-metastatic niche in the liver. We found that KPC mice (compared to age- and gender-matched control mice) demonstrated an increased susceptibility to tumor seeding in the liver even prior to development of invasive PDAC. Examination of the liver of KPC mice revealed diffuse activation of Signal Transducer and Activator of Transcription 3 (STAT3) signaling, particularly in hepatocytes. Although hepatocytes are recognized as important regulators of inflammation, their role in establishing a pro-metastatic niche is unknown. To define changes in the liver associated with development of a pro-metastatic niche, we performed QuantSeq analysis on RNA isolated from the liver of KPC versus control PC mice. Our results showed increased transcriptional levels of myeloid chemoattractants, particularly serum amyloid A proteins that are predominantly produced by hepatocytes. Consistent with this finding, we observed an accumulation of F4/80+ and Ly6G+ myeloid cells in the liver of KPC mice by immunofluorescence microscopy. We next determined the role of tumor cells in driving cellular activation seen in the liver by establishing intraperitoneal and orthotopic models of PDAC. Using these models, we found that implantation of pancreatic tumor cells induced STAT3 activation in hepatocytes and stimulated F4/80+ and Ly6G+ myeloid cell recruitment to the liver. To determine whether cellular activation in the liver was associated with systemic release of soluble factors, we performed parabiotic joining of tumor-implanted mice and control wild type mice, and we found evidence of STAT3 activation and myeloid recruitment to the liver in parabiotic pairs. As interleukin-6 (IL-6) is a key inflammatory cytokine that can activate STAT3 signaling, we hypothesized a role for IL-6 directed STAT3 activation in hepatocytes for development of a pro-metastatic niche in the liver. Consistent with this hypothesis, we found that IL-6 receptor blocking antibodies administered after tumor implantation reduced STAT3 activation in hepatocytes and decreased transcriptional levels of hepatocyte-derived chemoattractants. Together, our findings support a role for IL-6/STAT3 signaling in hepatocytes in driving a pro-metastatic niche in the liver during PDAC development.

#2683

The gastrointestinal microbiome and its composition are critical for antitumor efficacy of immune checkpoint inhibition by anti-PD-L1.

Benjamin G. Cuiffo, Caitlin S. Parello, Chelsea Ritchie, Katie Pedrick, Alexandra Kury, Catarina Costa, Brett Van Dam, Jonathan Jung, Gregory D. Lyng, Stephen T. Sonis. _Biomodels, Watertown, MA_.

The intestinal microbiome has become increasingly appreciated as a significant mediator of systemic antitumor immunity/response in both naïve and treatment contexts. In naïve contexts, an intact intestinal microbiome has been demonstrated to enhance tumorigenesis, and its composition to mediate primary tumor growth kinetics. In the context of cancer treatment, antibiotic depletion of the intestinal microbiota has been reported to inhibit the efficacy of cyclophosphamide and that of the immune checkpoint inhibitor αCTLA4. Compositional modulation of the intestinal microbiota has been found to be sufficient to enhance the antitumor efficacy of αPD-L1. Here, we assessed the relative importance of the intestinal microbiota in mediating αPD-L1 antitumor efficacy in a B16.F10.SIY murine model of melanoma, by performing parallel efficacy studies in C57BL/6 germ-free (Taconic) or specific pathogen free (Taconic or Jackson) mice. We observed that αPD-L1 treatment provided significant antitumor efficacy of in Taconic mice carrying an intact microbiome; however, this efficacy was abolished in germ-free Taconic mice. Furthermore, we observed that tumors of Jackson mice carrying an intact but compositionally different microbiome did not respond to αPD-L1 treatment. Phenotyping of local tumor and systemic immune responses, as well as characterization of the intestinal microbiome in responder vs nonresponder animals provided mechanistic insights. Taken together, these observations suggest that rational modulation of the microbiome may enhance response to immune checkpoint inhibition, and indicate that the gastrointestinal microbiome and its composition are critical for the antitumor efficacy of αPD-L1.

#2684

Multiplex immunofluorescence profiling of tumor infiltrating immune subsets in HNSCC biopsies provides a powerful tool when combined with patient outcome data.

Matt Levin, Mark Lingen, David Schwartz, Helen Snyder. _Cell IDx, San Diego, CA_.

Head and neck squamous cell carcinoma (HNSCC) is one of the growing number of tumors for which an anti-tumor immune response is implicated to play a role in the course of the disease. Profiling of the tumor microenvironment including specific infiltrating lymphocyte subsets, antigen presenting cells, expression of regulatory molecules on both tumor and immune cells and spatial relationships between cells could lead to predictive signatures for responsiveness to both conventional and immune modulatory therapies. While flow cytometry results in multi-parametric analysis of immune populations within the tumor, their context i.e. the spatial relationship of these cells to tumor and to other immune cells, is lost. In this current study we use Cell IDx's novel UltraPlex technology, which is based on a simple two-step, autostainer compatible protocol using hapten labeled primary antibody cocktails followed by fluor labeled anti-hapten secondary cocktails to simultaneously detect 4 cellular markers on the same cell in tumor biopsies. Image registration of serial sections increases multiplexing to 12-16 markers. This technology, which allows generation of full immune subset profiling, quantitative marker expression and relative cellular localization, was applied to a HNSCC tumor microarray as a test system. Data are presented showing the potential of this technology for correlation of tumor "immune signature" with patient outcome.

#2685

Modulating tumor microenvironments through inflammasome and IL-1 pathways.

Beichu Guo, Jinyu Zhang, Shun-Jun Fu. _Medical University of South Carolina (MUSC), Charleston, SC_.

Chronic Inflammation has been shown to play important roles at all stages of tumor development including initiation, growth, invasion and metastasis. The inflammasome is an important innate immune pathway critical for the production of active IL-1β, a potent inflammatory cytokine. While extensive evidence indicates that inflammasomes are involved in infections and autoimmune diseases, the role of inflammasomes in tumor development remains controversial. To dissect the roles of the inflammasome and IL-1 pathway in tumor development, we utilized the MMTV-PyMT transgenic model, which develops mammary gland tumors with a high incidence of lung metastasis. Our results have demonstrated that inflammasome and IL-1β play a critical role in promoting tumor growth and metastasis. We found that tumor growth was associated with inflammasome activation and elevated levels of IL-1β in tumor microenvironments in mouse mammary tumor models and in human breast cancer tissues. Mice deficient for inflammasome components or IL-1R signaling exhibit significantly reduced lung metastasis. Our data also show that inflammasome activation led to the accumulation of myeloid cells, such as such as myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) in tumor microenvironments. Furthermore, blocking IL-1R signaling with IL-1R antagonist (IL-Ra) or anti-IL-1R antibody inhibited tumor growth and metastasis. Our results suggest that targeting the tumor microenvironment through inflammasome and IL-1 blockade may provide a novel approach for the treatment of breast cancer.

#2686

Dual inhibition of BCR and TLR signaling has therapeutic potential in chronic lymphocytic leukemia.

Eman L. Dadashian, Sarah Herman, Adrian Wiestner. _NIH/NHLBI, Bethesda, MD_.

Introduction: The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib is clinically active in lymphoproliferative diseases driven by B-cell receptor (BCR) and Toll-like receptor (TLR) signaling, including chronic lymphocytic leukemia (CLL) and Waldenstrom macroglobulinemia (WM), respectively. However, deep remissions are uncommon and resistance to single agent has been described. The hallmark of WM is an activating mutation in MYD88 in the TLR signaling pathway. While mutations in MYD88 are uncommon in CLL, our previous study identified gene signatures indicative of active BCR and TLR signaling in CLL cells residing in lymphoid tissues (Herishanu, Blood 2011). Further, TLR9 activating CpG oligonucleotides induce proliferation and extend CLL cell survival in vitro. These observations suggest that BCR and TLR signaling may cooperate to activate CLL cells in the tissue microenvironment. Here, we tested the hypothesis that targeting both BCR and TLR signaling could improve therapy for CLL.

Methods: CLL PBMCs were treated with ibrutinib and/or an IRAK1/4 inhibitor (Calbiochem) for 1h and then stimulated with soluble αIgM, CpG, or both. We quantified changes in phosphorylation of BTK, PLCγ2 and ERK (BCR pathway) and STAT3 and STAT1, as well as total IRAK1 (TLR pathway).

Results: As expected, ibrutinib inhibited phosphorylation of BTK, PLCγ2 and ERK (P<.05) and decreased the survival of CLL cells stimulated with αIgM (P=.001). CpG stimulated TLR signaling degrades IRAK1 and the stimulates cytokine secretion that can, in autocrine fashion, activate STAT phopsphorylation. The IRAK1/4 inhibitor effectively inhibited TLR signaling resulting in stabilization of IRAK1 (P=.002), decreased phosphorylation of STAT1/3 (P=.04) and decreased viability compared to CpG stimulated but not IRAK inhibitor treated cells. Ibrutinib had no effect on CpG-induced IRAK1 degradation, but was comparable to IRAK1/4 inhibition in reducing STAT phosphorylation, suggesting that inhibition of BTK can antagonize downstream effects of TLR activation but not upstream IRAK dependent steps. In contrast, IRAK1/4 inhibition had no effect on αIgM-induced BCR activation. Next we evaluated the effect of dual BCR and TLR activation, modelling co-operative activation of both pathways in the tumor microenvironment. Under these in vitro conditions, ibrutinib prevented BCR activation and partial TLR activation, while IRAK1/4 affected only the TLR pathway. When ibrutinib and the IRAK1/4 inhibitor were combined, activation of both BCR and TLR signaling was prevented resulting in a significant reduction in CLL cell viability (P=.01) compared to co-activation of both pathways.

Conclusion: While ibrutinib partially inhibited TLR signaling, an IRAK1/4 inhibitor was required for full inhibition of the pathway. The combination of BTK and IRAK1/4 inhibition for the treatment of lymphoproliferative diseases warrants further investigation.

#2687

Toll like receptors mediated inflammatory signals mediate promotion of K-ras mutant lung cancer by chronic obstructive pulmonary disease.

Nasim Khosravi,1 Nelly Torres-Garza,2 Soudabeh Daliri,1 Maria Miguelina De La Garza,1 Amber Cumpian,1 Evelyn Beltran,1 Misha Umer,1 Diana Del Bosque,1 Saba Akbani,1 Scott Evans,1 Seyed Javad Moghaddam1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Tecnológico de Monterrey School of Medicine, Monterrey, Mexico_.

Lung cancer is the leading cause of cancer death worldwide, and cigarette smoking is its main cause. However, smoker with chronic obstructive pulmonary disorder (COPD), an inflammatory disease of the lung, have an increased risk of lung cancer (3 to 10 fold) compared to smokers without COPD. Importantly, lung inflammation persists and lung function continues to deteriorate as does the increased risk of lung cancer even after cessation of cigarette smoking among former smokers. These facts suggest a strong link between COPD-related airway inflammation and lung cancer promotion, however, the precise mechanistic link is not known. We have previously developed a COPD-like mouse model of airway inflammation through repeated aerosol challenge to a lysate of nontypeable (i.e., unencapsulated) Haemophilus influenzae (NTHi). NTHi is the most common colonizing bacteria in the lower respiratory tract of patients with COPD and could be a potential cause of perpetuating and promoting airway injury and inflammation in these patients. We then showed that this type of airway inflammation promotes lung cancer in a K-ras mutant mouse model of lung cancer (CC-LR), which was associated with the activation of MyD88/NF-κB pathway and increased expression of its downstream targets in the lung. We have further shown that lack of NF-kB or MyD88 (an adaptor protein upstream to NF-κB) in the airway epithelium of CC-LR mice changes the bronchoalveolar lavage fluid cellular component of CC-LR mice and inhibits the promoting effect of COPD-like airway inflammation on lung tumorigensis. Upstream to MyD88 and NF-κB and downstream to bacterial stimuli is the toll-like receptors family (TLRs), which play critical role in the innate immune response. Among TLRs, TLR-2, TLR4, and TLR9 play critical roles in mediating inflammatory responses in lung and are required for primary epithelial response to inflammatory stimuli and activation of MyD88/NF-kB pathway in the airway epithelium. Accordingly, we further hypothesized that TLR-2, 4, and 9 mediate promoting effect of inflammtion on lung tumorigenesis in an MyD88/NF-kB dependent manner. Therefore, CC-LR mice were separately crossed to TLR-2, TLR-4 and TLR-9 knock out mice in order to test this hypothesis. We found that genetic ablation of these TLRs in CC-LR mice, causes significant reduction in lung surface tumor numbers compared to age and sex matched control CC-LR mice in the presence of COPD-like airway inflammation. This tumor reduction was associated with significant reduction in the numbers of inflammatory cells in bronchoalveolar lavage fluid of mice with lack of these TLRs. Taken these together, we conclude that promoting effect of COPD on lung cancer is mediated through TLR2, 4, 9-mediated activation of epithelial MyD88/NF-kB pathway.

#2688

Multiplex IHC detection of immune checkpoint receptors in the tumor microenvironment.

Jennifer E. Ziello, Sarah R. Klein, Emily Alonzo, Herbert Haack. _Cell Signaling Technology, Inc, Danvers, MA_.

As immune checkpoint blockade has been shown to partially reverse the exhausted T cell phenotype and consequently lead to a decrease in tumor burden, there is a need for an understanding of this T cell type. Using recently developed, highly validated antibodies, we have developed a fluorescent multiplex, TSA-based assay in order to examine the interaction of PD-L1 with PD-1-expressing exhausted T cells in various tumor types. Here, we construct a seven-color multiplex panel in order to simultaneously visualize cytokeratin (the tumor mask), DAPI (nuclear counterstain), CD8 (cytotoxic T cell marker), the exhausted T cell markers PD-1, Tim-3 and Lag-3, and the ligand for PD-1, PD-L1. This technique not only enabled the concurrent detection of these markers, but also provided high-resolution visualization of interactions between PD-1-expressing CD8+ T cells and PD-L1-expressing CD68+ macrophages in the tumor microenvironment. The seven-plex panel was applied to FFPE tumor microarrays (TMAs) consisting of breast, lung and ovarian tumor tissue and each core was subsequently analyzed for the distribution, co-localization, frequency and proximity of these targets in relation to one another. While we often visualized co-expression of Lag-3 and PD-1 on T cells, Tim-3 was frequently observed on PD-L1+ macrophages in several tumor types. This data provides valuable insight into the co-expression profiles of these markers in multiple tumor types and has implications for the use of combination therapies that aim to target both the innate and adaptive immune systems.

#2689

The protumorigenic, proinflammatory effects of obesity are reversed by weight loss via bariatric surgery, but not a low-fat diet.

Laura W. Bowers,1 Emily L. Rossi,1 Subreen A. Khatib,1 Steven Doerstling,1 Alfor Lewis,2 Randy J. Seeley,2 Stephen D. Hursting1. 1 _Univ. of North Carolina, Chapel Hill, NC;_ 2 _Univ. of Michigan, Ann Arbor, MI_.

Background: Obesity negatively impacts basal-like breast cancer (BLBC) prognosis, but the reversibility of these pro-cancer effects via weight loss remains unclear. However, there is consistent evidence suggesting that weight loss via bariatric surgery reduces breast cancer risk. This may be related to the reductions in metabolic perturbations and inflammation that follow bariatric surgery, effects that could be mediated by epigenetic reprogramming and/or changes in the gut microbiome.

Purpose: We previously demonstrated that mammary tumor growth and inflammation remain elevated in formerly obese mice, in concordance with aberrant methylation of inflammation-related genes. Here we aim to determine the differential effects of surgical versus non-surgical weight loss on inflammation, DNA methylation, the gut microbiome, and tumor burden in a mouse model of BLBC.

Methods: Mice were fed a low fat control (n=25) or high fat diet-induced obesity (DIO, n=75) regimen for 15 weeks to model chronic obesity. DIO mice were then randomized to remain on DIO (Obese) or receive either a surgical (sleeve gastrectomy) or dietary (low fat control diet) weight loss intervention, resulting in formerly obese (FOb)-Surg or FOb-Diet mice, respectively. The Control mice were maintained on the low fat diet throughout study. Four weeks after weight stabilization in the FOb mice, all mice were orthotopically injected with E0771 mammary tumor cells, which model BLBC. Stool samples were collected at baseline and prior to tumor cell injection.

Results: The average weight and percent body fat of the FOb-Surg and FOb-Diet mice were equivalent to Control and significantly lower than Obese mice at study endpoint. Average tumor weight in FOb-Surg mice was statistically equivalent to Control mice, but tumor weight in FOb-Diet mice was significantly greater than Control mice and statistically equivalent to Obese mice. Furthermore, FOb-Surg mice had significantly lower serum tumor necrosis factor alpha, mammary gland interleukin-6 expression, and tumor-infiltrating adipocyte area in comparison to FOb-Diet. To further define the effects of surgical versus non-surgical weight loss, characterization of the gut microbiota as well as global mammary tissue gene expression and DNA methylation via paired RNA sequencing and reduced representation bisulfide sequencing is in progress.

Conclusions: Our results suggest that the strong anti-cancer benefits seen with bariatric surgery may be related to a significant reduction in systemic and local inflammation, which did not occur with non-surgical weight loss. Identification of the mechanisms mediating the protective effects of bariatric surgery against breast cancer could help identify new targets and strategies for breaking the obesity-cancer link.

#2690

MK2 pathway blockade inhibits inflammatory cytokine production and colorectal cancer growth and invasion.

Anita L. Ray, Amanda S. Peretti, Wade Johnson, Gregory Gan, Ellen J. Beswick. _The Univ. of New Mexico, Albuquerque, NM_.

Introduction: Colorectal cancer is the third most common malignancy diagnosed for both men and women in the United States of America. High levels of inflammatory cytokines in colorectal tumors cause increased growth and invasion and carry a higher risk of metastasis. Identifying new targets to control inflammation is important for developing improved treatment approaches. Mitogen-activated protein kinase-activated protein kinase 2 (MK2) is a regulator of pro-inflammatory cytokines that may promote colorectal tumor progression. MK2 signaling is known to induce IL-1, IL-6, and TNF-α production. These pro-inflammatory cytokines are associated with CRC development, invasion, and metastasis. We hypothesized that the MK2 pathway could be an important component of CRC growth, invasion and tumor regrowth.

Methods: To investigate this pathway, CT26 CRC cells were examined. Cells were flank-injected into Balb/c mice with and without MK2 inhibitor treatment. At day 19 after tumor injection, tumors were harvested and measured using calipers. Cytokines were quantitated by multiplex bead array in organ culture supernatants and in supernatants from tumor cells plated in fibronectin coated wells. In culture, tumor cell invasion was measured in scratch wound assays containing matrigel.

Results: Treating CT26 cells with MK2 inhibitors markedly reduced tumor growth in mice by a mean of 60% compared to vehicle control treated cells. Inflammatory cytokines were also dramatically decreased with MK2 inhibition compared to controls by up to 80%. Cytokines affected included both known MK2 downstream cytokines (IL-1, IL-6, and TNF-α) and also chemokines such as MIP-1α and MCP-1 in both supernatants from cultured tumor cells and in organ culture supernatants. MK2 inhibition also decreased invasion of tumor cells in a cytokine dependent manner.

Conclusions: The MK2 pathway regulates production of multiple pro-inflammatory cytokines in colorectal tumors, including several previously unreported chemokines. Inhibition of this pathway markedly decreases tumor growth and invasion. Thus, MK2 may be a promising tumor target to prevent colorectal cancer progression.

#2691

Cigarette smoke induced upregulation of endothelin axis in the initiation of pancreatic cancer.

Suprit Gupta, Satyanarayana Rachagani, Sushil Kumar, Surinder Kumar Batra, Maneesh Jain. _University of Nebraska Medical Center, Omaha, NE_.

Background: Cigarette smoke is an established risk factor for pancreatic ductal adenocarcinoma (PDAC). Smoke induced inflammation accelerates the progression of PDAC in presence of constitutively active K-Ras mutation. Endothelin (ET) axis comprising of endothelin converting enzymes (ECE-1, ECE-2, and ECE-3), endothelin isoforms (ET-1, ET-2 and ET-3) and two receptors A (ETAR) and B (ETBR), is linked to pathobiology of pancreatitis and its components exhibit aberrant overexpression in PDAC. However, the expression of ET-axis in the oncogene-associated early Pancreatic Intraepithelial neoplastic (PanINs) lesions following exposure to cigarette smoke is unknown. We hypothesize that smoke induced alterations in the ET axis facilitate acinar to ductal metaplasia (ADM) in presence of oncogenic K-Ras. Thus, we characterized the impact of cigarette smoke exposure on ET axis components in the pancreas of mice harboring mutant K-Ras.

Methods: Expression of ECE-1, ET-1, ETAR and ETBR was analyzed by IHC and RT-PCR first in the murine model of preneoplastic lesions [KC model: (Pdx1-Cre, KrasG12D)). To determine the changes in ET axis after smoke exposure, wild type (WT) and KC mutant mice were exposed to cigarette smoke for 20 weeks and expression was analyzed in the pancreas. The impact of smoking on ET-axis was also studied on murine acinar and tumor cell lines derived from KC mice (UN-KC6141) and KPC (Pdx1-Cre, p53 (R172H) KrasG12D) mice (UN-KPC-961) by western blot and RT-PCR analysis.

Results: In contrast to the normal ducts, the expression of ECE-1, ET-1, ETAR and ETBR was upregulated in the early PaNIN lesions (20-30 weeks). In the advanced lesions (50 weeks), significant overexpression of all four molecules was noticeable in the tumor cells and stromal compartment. Smoke exposure resulted in significant increase in the transcripts of ET-1, ETAR and ETBR (p values = 0.04, 0.03 and 0.01 respectively) in the KC mice while the increase in WT mice was not significant. The changes in the expression were also corroborated by tissue IHC. Expression of ET axis components was predominantly seen in the islet cells in WT mice, while low immunoreactivity was observed in the acinar compartment. Smoke exposure of KC mice resulted in accelerated progression of PanIN lesions along with concomitant increase in the expression of ET axis components both in the ductal and stromal cells. In vitro exposure of cigarette smoke extract (CSE) to UN-KC6141 and UN-KPC-961 cells for 24 hours elicited a dose dependent upregulation of ECE 1 and ET-1.

Conclusions: In presence of mutated KrasG12D, smoking-mediated inflammatory insult promotes PanIN progression and tumorigenesis. The sustained increase and activation of ET axis with increasing dysplasia in the ductal compartment during this progression in KC mice and its further upregulation following smoke exposure suggests its possible role in promoting inflammation-associated pancreatic tumor progression.

#2692

Inflammatory patterns exhibited by African American colon tumor-derived cell lines.

Jenny E. Paredes Sanchez,1 Ping Ji,2 Maria Munoz-Sagastibelza,1 Laura Martello-Rooney,1 Jennie Williams2. 1 _SUNY Downstate Medical Ctr., Brooklyn, NY;_ 2 _Stony Brook University, Stony Brook, NY_.

Despite progress in closing the gap, health disparities still persist among African American (AA) colon cancer patients both in incidence and death rates. Previous studies from our group reported that colon tumors from AAs displayed hypermethylation of DNA regions in inflammatory genes such as NELL1, GDF1, ARHGEF4, and ITGA4 when compared to Caucasian Americans (CA). To assess potential differences in the inflammatory response, we utilized two AA colon cancer cell lines generated in our laboratory and compared them with the commercially available CAs colon cancer cell lines, Caco-2 and HT-29. Recent experiments by our group with the anti-inflammatory drugs Ibuprofen, Sulindac and Aspirin, showed significantly higher IC50 values in proliferation assays for AA cell lines compared to CA cell lines. Therefore, we evaluated the anti-inflammatory effects of these drugs by determining secretion of inflammatory cytokines and MAPKs activation in response to the pro-inflammatory cytokine TNF-alpha. The same parameters were tested for the anti-inflammatory cytokine IL-10. As hypothesized, our results in the CA cell lines demonstrated down-regulation of MAPKs activation in response to TNF-alpha after pre-treatment with Ibuprofen. On the contrary, Ibuprofen concentrations as high as the IC50 values for the CA cell lines were not able to induce down-regulation of MAPKs in the AA cell lines. Interestingly, IL-10 treatment was more effective in the AA compared to the CA cell lines, suggesting that the main anti-inflammatory cytokine of the colon will be more effective in controlling inflammation in AA colon tumors. Further studies are needed to elucidate the major differences in inflammatory responses between AA and CA colon cancer cell lines and their potential role in CRC health disparities. As it has been documented that AA colon cancer patients are less responsive to the chemotherapeutics Fluorouracil and Capecitabine, in future studies we will evaluate the effect of these therapeutic agents on AA and CA cell lines in terms of inflammation, cell viability, apoptosis and invasion.

#2693

Inflammatory signalling in the colon tumour microenvironment enhances stromal cell mediated suppression of anti-tumour immune responses.

Grace O'Malley, Kevin Lynch, Serika Naicker, Paul Lohan, Athina Rigalou, Thomas Ritter, Laurence J. Egan, Aideen E. Ryan. _National University of Ireland, Galway, Galway, Ireland_.

The colon tumour microenvironment (TME) comprises many cell types including endothelial cells, stromal cells and immune cells. Recent evidence suggests that high tumour stromal cell density correlates with a poor prognosis for colon cancer patients. The majority of these stromal cells are of mesenchymal origin (MSCs) and are known contributors to tumour angiogenesis and invasiveness. Little is known about the role of their immunosuppressive potential in the colon TME. We investigated the molecular regulation of the induced immunosuppressive, tumour-promoting phenotype of tumour-associated MSCs, and the effect of inflammation on this process. Balb/c bone marrow derived MSCs were treated with conditioned medium from untreated CT26 tumour cells (MSCTCM) or TNF-α treated CT26 cells (MSCTNF-TCM). Cell surface analysis of MSCs after 72h revealed an increased expression of TCR ligands MHC-I, MHC-II and PD-L1 compared to MSCControl. This was further potentiated by TNF-α induced tumour cell inflammation. MSCTCM co-cultured with syngeneic activated T cells displayed an enhanced ability to suppress CD8+ T cell proliferation, which was further potentiated by inflammatory activation of CT26 (MSCTNF-TCM). This effect was dependent on induced PD-L1 expression on MSCs as PD-1 blockade restored CD8+ T cell proliferation, activation and granzyme B secretion. In an immunocompetent Balb/c syngeneic model, we assessed tumour growth and anti-tumour immune responses following sub-cutaneous injection of CT26 cells alone or co-injection with MSCControl/MSCTNF-TCM. Co-injection of MSCControl significantly promoted tumour growth, and this was further potentiated by the co-injection of MSCTNF-TCM. This effect was associated with significantly reduced tumour infiltration of CD8 Granzyme B secreting T cells. We showed that this stromal cell mediated tumour promotion could be reversed by administration of a PD-1 blocking antibody, via restoration of granzyme B secreting CD8+ T cells. We show for the first time that stromal cells in the inflammatory TME directly modulate anti-tumour immune responses via PD-L1. This data could lead to better stratification of patients for immunotherapeutic regimens resulting in more targeted and durable responses to overcome stromal mediated tumour immunosuppression in colon cancer.

#2694

Target obesity-associated inflammation to decrease murine basal-like mammary tumor burden.

Emily L. Rossi,1 Subreen A. Khatib,1 Laura W. Bowers,1 Steven S. Doerstling,1 Andrew J. Dannenberg,2 Stephen D. Hursting1. 1 _Univ. of North Carolina at Chapel Hill, Chapel Hill, NC;_ 2 _Weill Cornell Medical College, New York, NY_.

Background: Adipose tissue dysregulation, a hallmark of obesity, contributes to a chronic state of low-grade inflammation that promotes cancer growth through multiple signaling pathways. We previously showed that inflammation and basal-like breast cancer (BLBC) growth are increased in chronically obese mice and persist following weight normalization.

Purpose: We tested the hypothesis that targeting inflammation in obese mice by treating them with the nonsteroidal anti-inflammatory drug (NSAID) Sulindac would offset the procancer effects of obesity in a mouse model of BLBC.

Methods: Mice were administered a control diet (10 kcal % fat; n=34) or diet-induced obesity regimen (DIO, 60 kcal % fat; n=34). After 15 weeks on control or DIO diets, mice were randomized to either receive Sulindac supplementation at 160 ppm in the diet (n=17/diet) or no supplementation (n=17/diet). Twelve weeks later, all mice were orthopically injected with E0771 cells, a model of basal-like breast cancer. Five mice/group were killed at a 4-week interim time-point after injection, and their tissue collected and stored. The remaining 12 mice/group continued in a survival study; these mice were killed when tumor size reached 1.2 cm in diameter in any direction.

Results: Sulindac supplementation in DIO mice significantly reduced serum insulin and leptin to levels statistically equivalent to control mice, but had no effect on body weight, body fat percentage, or ex vivo visceral white adipose weight. Sulindac supplementation in DIO mice (but not control) significantly reduced mean tumor volume in the interim tumor study and significantly increased tumor latency in the survival study. Analysis of H&E stained tumor demonstrated that DIO mice had significantly increased adipocytes infiltrating into the tumor (relative to control), but Sulindac supplementation in DIO mice decreased adipocyte infiltration to levels observed in control.

Conclusions: Sulindac supplementation significantly reduced insulin and leptin in DIO mice, and increased tumor latency in DIO mice, but had no effects on body weight or fat depots, suggesting that Sulindac offsets some of the pro-tumorigeneic effects of obesity rather than targeting obesity directly. Preliminary analyses of inflammatory surrogates, including circulating cytokines and prostaglandins, mammary gland crown-like structures and cyclooxygenase-2 levels, suggests Sulindac's effects in obese mice are mediated through its eicosanoid-depressing effects.

#2695

Role of tumor generated acidity in immune stromal interactions during prostate carcinogenesis.

Asmaa El-Kenawi, Jasreman Dhillon, Arig Ibrahim-Hashim, Dominique Abrahams, Shari Pilon-Thomas, Brian Ruffell, Robert Gatenby, Robert Gillies. _Moffitt Cancer Ctr., Tampa, FL_.

Insufficiency in tumor perfusion and high rate glycolysis combine to reduce the pH of tumor microenvironment. In a TRAMP model of prostate cancer, we had shown that carcinogenesis is associated with increasing acidification of the microenvironment and that neutralization of this acidity can prevent cancer emergence or metastases. Carcinogenesis in the TRAMP model is also associated with increased fibrosis and immune cells infiltration. We thus sought to determine if fibrosis drives immune infiltration in early tumorigenesis or vice-versa; and whether this dynamics is affected by tumor acidity. To investigate this, we harvested prostates from TRAMP mice or their matching non-transgenic controls at different time points and stained serial prostate tissue sections with F4/80 (macrophages), SMA (cancer-associated fibroblasts, CAFs), and Masson's Trichome (collagen). Quantitative image analysis reveals that increase in fibrosis occur prior to macrophage infiltration and that both events preceded tumor development. However, the relative amount of collagen fibers was unchanged across all time points. Notably, neither fibrosis nor macrophage infiltration occurred in mice treated with buffer, suggesting an involvement of acidity in this immune stromal interactions. Interestingly, macrophages isolated from latter time points in the untreated group as well as macrophages co-cultured with prostate tumor cells at acidic pH, possessed an M2-like phenotype by expressing immunosuppressive genes (e.g. Arginase 1, Arg1) and a range of scavenging receptors (e.g. mannose receptor, Cd206), as well as releasing more angiogenic factors (e.g. VEGF and MMPs). Similar results were recapitulated when M2 macrophages were stimulated at acidic pH by showing enhanced Cd206 and Arg1 expression. On the functional level, macrophages activated at acidic pH had a higher ability to uptake fluorescently labelled ovalbumin and collagen, as examples of mannosylated ligands that prevail the fibrotic microenvironment. In summary, these results suggest that tumor acidity may promote fibrosis, with subsequent macrophage infiltration and phenotypic switching, leading to increased collagen turnover. It is suspected that this extracellular matrix remodeling may be permissive for tumor progression.

#2696

PD-1 antibody treatment induces gut microbiota changes in CT-26 colon cancer syngeneic model.

Jian Ding, Binchen Mao, Qian Shi. _CrownBio, Taicang, Jiangsu Province, China_.

The gut microbiota plays an important role in shaping systemic immune response. One novel frontier in cancer research is investigating how the gut microbiota change immune response and influence the efficacy of anticancer immunotherapy drugs; this has been reported in CTLA4 and PD-L1 antibody treatment, both in laboratory and clinical settings. One recent study reported that composition of patient`s gut microbes appears to be a determining factor for immunotherapy, at least in melanoma patients. These findings shed light on the potential use of gut microbial as a biomarker for responders in cancer immunotherapy and even as a novel treatment.

Here, we evaluated the gut microbiota profiling upon PD-1 antibody (RMP1-14) treatment in CT-26 colon cancer syngeneic mouse model. Fresh feces were collected at different time points before and after treatment, snap frozen and sent for 16s RNA sequencing for microbiome profiling. Our results identified top 25 abundant taxa in genus level in CT-26 feces. Significant abundance changes were observed in several genera after PD-1 antibody treatment, especially in Akkermansia. These findings indicate that immunotherapy like PD-1 antibody can induce the profile changes of gut microbiota in CT-26 model.

Furthermore, we also investigated the correlation of gut microbiome with immune- microenvironment and the efficacy upon IO therapy, as well as the possibility to utilize microbiota profiling as a quality control step that may help to explain the variability observed in many syngeneic studies.

#2697

A role for the membrane type-1 matrix metalloproteinase in the transcriptional regulation of carcinogen-induced inflammasome components.

Samuel Sheehy, Borhane Annabi. _Université du Québec à Montréal, Montréal, Quebec, Canada_.

BACKGROUND : Signal transducing functions driven by the cytoplasmic domain of membrane type-1 matrix metalloproteinase (MT1-MMP) are believed to regulate many inflammation-mediated cancer cell functions including migration, proliferation, and survival. Besides the upregulation of the inflammation biomarker cyclooxygenase (COX)-2 expression, MT1-MMP's role in relaying the signals triggered from pro-inflammatory cues remain poorly understood.

METHODS : Here, we treated HT1080 fibrosarcoma cells with phorbol-12-myristate-13-acetate (PMA), a well-known carcinogen and inducer of COX-2 and of MT1-MMP. In order to assess the global transcriptional regulatory role that MT1-MMP may exert on inflammation biomarkers, we combined gene array screens to transiant MT1-MMP gene silencing strategy.

RESULTS : We found that MT1-MMP expression exerted both stimulatory and repressive transcriptional control of several inflammasome-related biomarkers such as IL-1B, IL-6, IL-12A, and IL-33, as well as of transcription factors such as EGR1, ELK1, and ETS1/2 in PMA-treated cells. Among the signal transducing pathways explored, silencing of MT1-MMP prevented PMA from phosphorylating Erk, IκB, and p105 KF-κB intermediates. We also highlight a signaling axis linking MT1-MMP to MMP-9 transcriptional regulation.

CONCLUSIONS : Altogether, our data evidence an important involvement of MT1-MMP in the transcriptional regulation of inflammatory biomarkers consolidating its contribution in signal transducing functions, in addition to its classical hydrolytic activity.

#2698

IL-17A alone or in combination with IL-4/IFN-γ induces Duox2/DuoxA2 expression and reactive oxygen production in human colon and pancreatic cancer cells.

James H. Doroshow,1 Yongzhong Wu,2 Jiamo Lu,2 Smitha Antony,1 Jennifer Meitzler,2 Agnes Juhasz,2 Guojian Jiang,2 Iris Dahan,1 Mariam Konate,1 Krishnendu Roy1. 1 _NCI-DCTD, Bethesda, MD;_ 2 _NCI-DTP, Bethesda, MD_.

NADPH oxidase (NOX)-derived reactive oxygen species (ROS) contribute significantly to inflammation-associated carcinogenesis. Expression of dual oxidase 2 (Duox2), one of seven members of the NOX gene family, is up-regulated in inflammatory bowel disease, chronic pancreatitis, and in many human malignancies including carcinomas of the colon, pancreas, prostate, lung, and breast. Previously, we demonstrated that Stat1 and/or NF-kB play a critical role in modulating the enhanced expression of Duox2, and its cognate maturation factor DuoxA2, by the pro-inflammatory mediators IFN-γ and lipopolysaccharide in human pancreatic cancer cells. This cytokine-mediated increase in Duox2 expression was responsible for a significant increase in tumor cell H2O2 production, DNA damage, and HIF-1α and VEGFA upregulation in pancreatic cancer cell lines. Using the T84 human colon cancer line, and BxPC-3, AsPC-1, and HTB134 pancreatic cancer cell lines, we now report that the pro-inflammatory cytokine IL-17A, pivotal in the adaptive immune response against various pathogens, and in the promotion of colon and pancreatic cancer progression, strongly induces Duox2/DuoxA2 mRNA and protein expression (alone or in combination with IL-4 or IFN-γ). In BxPC-3 cells, IL-17A enhances the expression of Duox2/DuoxA2 in a time- and concentration-dependent manner, which leads to a significant increase in the generation of extracellular H2O2. When IL-17A is combined with IL-4 in either T-84 or AsPc-1 lines, Duox2-mediated ROS formation is sufficient to produce a DNA damage response, indicated by the strong induction of γH2AX expression; moreover, in AsPc-1 cells, Duox2-specific siRNA significantly attenuated cytokine-induced ROS production and γH2AX phosphorylation. IL-17A in combination with IL-4 also significantly increases Stat3 phosphorylation and promotes proliferation in AsPC-1 cells. Ongoing cellular experiments are focused on whether the cytokine-mediated upregulation of Duox2/DuoxA2 is responsible for Stat3 activation and cell growth promotion in the K-ras mutated AsPC-1 cells. Bio-informatic analysis with the TCGA data set, as well as RT-PCR using surgically-resected human colon cancer samples compared to adjacent, uninvolved colonic epithelium revealed that Duox2 was significantly upregulated in human colon cancers. These studies, in concert with our previous data demonstrating high-level Duox protein expression in pancreatitis, pancreatic intra-epithelial neoplasia (PanIN), and frank pancreatic cancer, strongly suggest that ROS, produced as a consequence of cytokine-related Duox2 up-regulation, may be associated with the development of inflammation-related gastrointestinal malignancies.

#2699

NFATc2 in non-small cell lung cancer mediates inhibition of peripheral blood mononuclear cell proliferation.

Ian K. Lam, Zhi-Jie Xiao, Vicky P. Tin, Fang Ping Huang, Maria P. Wong. _The University of Hong Kong, Hong Kong, Hong Kong_.

Lung cancer is the leading cause of cancer death worldwide. While genetic mutations that provide growth advantages to cancer cells are fundamental to malignant transformation, ability to evade the immune system is also crucial in tumorigenesis. We have previously shown that human lung cancers with high expression of nuclear factor of activated T cells, cytoplasmic 2, (NFATc2) are associated with reduced recurrence-free and overall survival. NFATc2 is expressed in most immune cells and has been extensively studied in immune cells. However, its function in cancer development is not completely understood. In this study, the role of NFATc2 in lung cancer on immune escape is explored. Lung cancer cell lines with high NFATc2 expression were co-cultured with peripheral blood mononuclear cells (PBMC) and a significant inhibition of PBMC proliferation was observed compared to PBMC only control. However, the inhibitory effect of PBMC proliferation was reversed in co-culture with NFATc2 knockdown cancer cells. When cancer cells and PBMC were separated by a membrane using Transwell inserts, the anti-proliferative effect on PBMC was not observed suggesting that cell-cell contact was required for the NFATc2-mediated inhibition. Moreover, interleukin-2 (IL-2) was produced by PBMC irrespective of the presence or absence of lung cancer cells. These results showed that the inhibition of proliferation was independent of the activation of PBMC. Taken together, we have shown NFATc2 in lung cancer plays a role in immune modulation by inhibiting PBMC proliferation and this could be a novel mechanism of immune evasion in lung cancer.

#2700

Intestinal Vitamin D receptor deficiency increases susceptibility for the DSS-induced colon disease.

Jung-Hwan Kim,1 Satoshi Yamaori,2 Frank J. Gonzalez2. 1 _Gyeongsang National University, Jinju, Republic of Korea;_ 2 _National Cancer Institute, Bethesda, MD_.

To investigate the function of the intestinal Vdr gene in inflammatory bowel disease (IBD), in conjunction with the discovery of possible metabolic markers for IBD using intestine-specific Vdr knockout mice. VdrΔIEpC mice were generated, phenotyped and treated with a time-course of 3% dextran sulfate sodium (DSS) to induce colitis. Colitis was diagnosed by evaluating clinical symptoms and intestinal histopathology. Gene expression analysis was carried out. In addition, metabolic markers of IBD were explored by metabolomics. VdrΔIEpC mice showed abnormal body size, colon structures and feces color. Calcium, collagen, and intestinal proliferation-related gene expression were all decreased, and serum alkaline phosphatase was highly increased. In the acute model which was treated with 3% DSS for six days, VdrΔIEpC mice showed a high score of IBD symptoms; enlarged mucosal layer and damaged muscularis layer. In the recovery experiment model, where mice were treated with 3% DSS for four days and water for three days, VdrΔIEpC mice showed a high score of IBD symptoms; severe damage of mucosal layer and increased expression of genes encoding proinflammatory cytokines. Feces metabolomics revealed decreased concentrations of taurine, taurocholic acid, taurodeoxycholic acid and cholic acid in Vdr ΔIEpC mice. In conclusion, disruption of the intestinal Vdr gene showed phenotypical changes. These factors combined may exacerbate IBD. Taken together, the results suggest that VDR may play an important role in IBD.

#2700A

Impact of radiation and EGFR inhibitor treatment on the expression of immune susceptibility markers in human head and neck squamous cell carcinoma.

Lauryn R. Werner, Lindsey K. Werner, Joseph G. Kern, Shyhmin Huang, Jasdeep Kler, Clinton Heinze, Zachary S. Morris, Paul M. Harari. _Univ. of Wisconsin-Madison, Madison, WI_.

Treatment with radiation (RT) and/or molecular targeted therapies may enhance tumor susceptibility to immune response. We previously demonstrated a cooperative interaction between RT and various immunotherapies in vivo. We subsequently demonstrated that there are delayed, transcriptionally-mediated effects of RT on the expression of tumor markers of immune susceptibility in murine and human melanoma. To expand these findings and explore applications in other tumor types, we investigated the effects of RT on immune susceptibility markers in human head and neck squamous cell carcinoma (HNSCC) cell lines. In addition, we examined whether treatment with the anti-EGFR monoclonal antibody cetuximab could further enhance these effects.

We used quantitative polymerase chain reaction (qPCR) to measure RT-induced changes in gene expression of a variety of immune susceptibility markers over time in HSCCC cell lines. We observed a time-dependent increase in the expression of specific death receptors (Fas, DR5), as well as T cell co-stimulatory/co-repressor ligands (PD-L1, CD40). The timing of these changes correlated with tumor susceptibility to ADCC immune response in vivo. Western blots confirmed RT-induced changes in protein expression of selected markers consistent with the observed changes in gene expression. To assess the effect of cetuximab on the expression of immune susceptibility markers, we performed western blots and qPCR to measure the effect of treatment with radiation, cetuximab, or combination treatment on protein and gene expression of immune susceptibility markers in HNSCC cells. We found that cetuximab treatment enhanced RT-induced changes in gene expression of several markers in both SCC6 and SCC1 cells, including MHC I-A, CD40, and OX40-L. Similar enhancement of RT-induced changes in protein expression was confirmed via western blotting. To validate that the effects of combination treatment were specific to EGFR inhibition, we examined the effects of RT on gene and protein expression of selected markers in cells in which EGFR had been transiently knocked down via siRNA transfection. Similar enhancement of RT-induced changes was observed with EGFR-knockdown and treatment with cetuximab.

This study highlights the impact that treatment with RT and/or molecular targeted therapies may have on tumor cell immunogenicity and suggests opportunities to enhance anti-tumor response by combining immunotherapeutic agents with RT or RT and EGFR inhibitors. These findings suggest opportunities to further optimize clinical treatment regimens by combining RT, molecular targeted therapies, and immunotherapies in the treatment of patients with HNSCC.

## CLINICAL RESEARCH:

### Clinical Molecular Genetics and Laboratory Correlates

#2701

**Large-scale ex vivo generation of human neutrophils from cord blood CD34** + **cells.**

Zhenwang Jie,1 Yu Zhang,1 Chen Wang,2 Bin Shen,1 Xin Guan,1 Zhihua Ren,1 Xinxin Ding,3 Wei Dai,4 Yongping Jiang1. 1 _Biopharmaceutical R &D Center, Peking Union Medical College of Tsinghua University, Suzhou, China; _2 _Biopharmagen corp., Suzhou, China;_ 3 _College of Nanoscale Science and Engineering, SUNY Polytechnic Institute, Albany, NY;_ 4 _Environmental Medicine, NYU Langone Medical Center, Tuxedo, NY_.

Ex vivo expansion of hematopoietic stem cell and subsequent differentiation into mature neutrophils remains a challenge. Here, we have developed a three-stage culture system to produce efficiently functional neutrophils derived from cord blood CD34+ cells. A procedure of ex vivo expansion and differentiation in a large-scale was developed in a modified IMDM basal medium supplemented with transferrin, insulin, fetal bovine serum, and some other nutrients with selected cytokine combination that contained stem cell factor (SCF), Flt-3 ligand (FL), granulocyte-colony stimulating factor(G-CSF), granulocyte macrophage colony-stimulating factor(GM-CSF) and thrombopoietin (TPO) in stage I (days 0~6); SCF, FL, G-CSF, interleukin 3 (IL-3), and GM-CSF in stage II (days 6~9); SCF, FL, and G-CSF in stage III (days 9~18), respectively. Enriched CD34+ cells were firstly cultured and expanded in 25-T flasks. After 6 day-culture, the cells were transferred to a 2-L bottle with 500 ml of medium in the bottle-turning device system. During the differentiation process, neutrophil marker CD66b was evaluated by flow cytometry. Ex vivo generated neutrophils or medium only were incubated with E.coli overnight for its bacteria killing assay, and then the E.coli colony-forming units were counted separately. Matured neutrophils or vehicle were transplanted into NOD/SCID mice intravenously for chemotactic activity in vivo. The cells that accumulated in the pouch with chemoattractant were collected and subjected to flow cytometric analysis for human CD66b antigen. The ex vivo generated neutrophils/progenitors were then injected into sub-lethal irradiated NOD/SCID mice to monitor the viability and maturation in vivo. After the three-stage culture, proliferation fold of total cell reached 30013 ± 286.5 with 60.2% ± 2.4% for CD66b+ neutrophils. The calculated yield of matured neutrophils from each CD34+ cell was ranged from 1.8 × 104 to 1.87 × 104 for 18-day culture. There was no E.coli colony formed after incubation with neutrophils in bacteria killing assay, indicating that the generated neutrophils was functional. For in vivo chemotaxis assay, the neutrophils collected from 18-day culture were injected into mice and detected at 1.08% ±0.16% for human CD66b+ cells and no any CD66b+ cell observed for negative control group. In addition, the CD66b+ cells were extended for 4 days from a 15-day cultured neutrophil group in mouse peripheral blood (PB), while only for 2 days from a freshly isolated human PB neutrophils post injection, indicating that the ex vivo generated neutrophils/progenitors could further matured in vivo. Taken together, we have established a pilot-scale culture system to produce functional human neutrophils ex vivo. Considering that one neutrophil transfusion unit (100ml) contains 2×1010 cells, the CD34+ cells from one CB unit (80 ml) would generate 1.6×1011 neutrophils, which are equivalent to 8 unit doses of neutrophils in the clinical application.

#2702

**Mutations of** SPOP **,** FOXA1 **and** IDH1 **are associated with prostate cancer metastasis and biochemical outcomes.**

Jinlu Ma,1 Suxia Han,1 Xin Sui,1 Wenyan Lu,2 Yonghe Li,2 Bo Xu2. 1 _The First Hospital, Xi'an Jiaotong University, Xi'an, China;_ 2 _Southern Research Inst., Birmingham, AL_.

Recent genomic studies have shed light on cataloging genomic alterations in prostate cancer. Mechanistic studies allow the use of this information to subclassify the heterogeneous prostate tumors for optimized treatment planning. Speckle-Type POZ Protein (SPOP), Forkhead Box Protein A1 (FOXA1) and Isocitrate Dehydrogenase 1 (IDH1) are among the genes that show frequent point mutations in primary prostate cancers. However, the clinical significance of these gene mutations in cancer patients is less clear. To determine the whether SPOP, FOXA1 and IDH1 mutations are associated with clinical outcomes in prostate cancer, we pyro-sequenced the three genes in prostate cancer patients and evaluated the clinical and prognostic significance. 198 patients from the first hospital of Xi'An Jiaotong University (China) were analyzed. All patients were newly diagnosed with prostate cancer from January 2010 to December 2015. No patient was lost to follow-up. The average follow-up time after the primary diagnosis was 27 months. The Fisher's exact tests were used to compare categorical data. The risk of metastasis among patients with SPOP, FOXA1 and IDH1 mutations was evaluated using univariate and multivariate logistic regression models. The prognostic significance of clinical and gene mutations was determined using multivariate Cox regression analysis. SPOP mutations were found in 16 patients (8.1%), with all of the mutations clustered in the substrate-binding MATH domain. FOXA1 mutationswere found in nine patients (4.5%), and were clustered in the Forkhead domain. IDH1 mutations were detected in three patients (1.5%). Mutation frequencies of SPOP, FOXA1 and IDH1 were associated with prostate-specific antigen (PSA), a biochemical marker of prostate tumor presence/risk which is decided by Gleason Score, T stage and PSA level, along with prostate cancer PSA failure and metastasis. Among patients with SPOP mutation, 56.3% showed metastasis at the time of diagnosis of primary cancer, compared to only 11.5% of patients with wild-type SPOP. Similarly, the probabilities of FOXA1 and IDH1 mutations with metastasis were

approximately 44.4% and 66.7% versus 13.8% and 14.4%, respectively, comparing to their wild-type counterparts. The metastasis risk for patients with SPOP mutation was 1.27 times of the SPOP wild-type patients (P = 0.003). We also found that patients with SPOP (P < 0.0001) and FOXA1 (P = 0.0403) mutations showed higher risk of PSA failure compared with wild-type. The PSA failure risk for patients with SPOP mutation was 35.49 times of the SPOP wild-type patients. In contrast, IDH1 mutation was not associated with PSA failure (P = 0.054). Multivariate analyses demonstrate that SPOP mutation is an independent factor that is associated with prostate cancer metastasis and PSA failure recurrence. These findings underscore the clinical significance for profiling SPOP, FOXA1 and IDH1.

#2703

Extraskeletal myxoid chondrosarcoma: a clinicopathologic and molecular study reveals novel genetic aberrations by targeted next-generation sequencing.

Marcel Trautmann,1 Magdalene Cyra,1 Ilka Isfort,1 Inga Grünewald,1 Konrad Steinestel,1 Sebastian Huss,1 Reinhard Büttner,2 Eva Wardelmann,1 Wolfgang Hartmann1. 1 _University Hospital Münster, Gerhard-Domagk-Institute of Pathology, Münster, Germany;_ 2 _University Hospital Cologne, Institute of Pathology, Cologne, Germany_.

Background: Extraskeletal myxoid chondrosarcomas (EMCs) are rare mesenchymal neoplasms comprising approx. 3% of all soft tissue tumors. EMCs arise mainly from the deep soft tissues of the extremities, accompanied with high rates of recurrence and metastases. The molecular hallmarks of EMCs are various cytogenetic NR4A3 rearrangements, generating chimeric -NR4A3 proteins. The most common reciprocal translocation t(9;22)(q22;q12), results in a fusion of the EWS RNA-binding protein 1 gene (EWSR1) to the nuclear receptor subfamily 4, group A, member 3 gene (NR4A3 or TEC; approximately 75% of cases). Further cytogenetic t(9;17)(q22;q11) rearrangements involve TAF15 RNA polymerase II, TATA box binding protein (TBP)-associated factor (TAF15; approximately 15% of cases). The less frequent reciprocal translocations t(9;15)(q22;q21) and t(9;3)(q22;q12), result in transcription factor 12 (TCF12)-NR4A3 and TRK-fused gene (TFG)-NR4A3 fusion proteins. Although the oncogenic -NR4A3 fusion transcripts seem to have a crucial role in EMC tumorigenesis and progression, the specific biological function and the mechanism of action remain to be defined.

Methods: We characterized the cytogenetic rearrangements of 25 comprehensive EMC tumors by RT-PCR and/or fluorescence in situ hybridization (FISH). Next generation sequencing (NGS) was performed (Illumina MiSeq platform) to reveal additional genetic alterations besides the known chromosomal translocation. Therefore, a comprehensive cancer panel was designed, comprising 27 cancer-related genes known to be frequently mutated across various malignancies.

Results: Overall, fusion transcripts were detected in 22 of 25 samples (88%). Sixteen were positive for the EWSR1-NR4A3 and six for the TAF15-NR4A3 fusion gene. The t(9;15) and t(9;3) translocations, resulting in TCF12-NR4A3 and TFG-NR4A3 fusion proteins were not identified in any EMC case. In Addition, several known oncogenic mutations were detected which have not been previously reported in EMC.

Conclusions: The combination of RT-PCR and FISH on paraffin-embedded tissue is a sensitive and specific method for the molecular detection of the pathogenic translocations to be applied in the differential diagnosis of extraskeletal myxoid chondrosarcomas. Our results emphasize that cytogenetic NR4A3 rearrangements are the initiating events in the pathogenesis of EMC. Furthermore, our results indicate the occurrence of additional genetic aberrations providing a rational base for novel targeted therapeutic approaches.

#2704

Pathogenic mutations and variants of unknown significance (VUS) in cancer predisposition genes are associated with over 10% of pediatric rhabdomyosarcoma: a report from the Children's Oncology Group.

Erin L. Young,1 Luke Maese,1 Rosann Robinson,1 Lance Pflieger,1 Barry Moore,1 Shawn Rynearson,1 Trent Fowler,1 Sean V. Tavtigian,1 Mark Yandell,1 Clinton C. Mason,1 Douglas S. Hawkins,2 Philip J. Lupo,3 Joshua D. Schiffman1. 1 _University of Utah, Salt Lake City, UT;_ 2 _University of Washington, Fred Hutchinson Cancer Research Center, Seattle, WA;_ 3 _Baylor College of Medicine, Houston, TX_.

Approximately 5% of rhabdomyosarcoma (RMS) cases are due to known cancer predisposition syndromes (e.g., Li-Fraumeni syndrome, Neurofibromatosis-1), but these estimates have not been confirmed in large-scale studies. Furthermore, no recommended germline testing protocols exist for RMS. We tested the hypothesis that germline mutation burden is greater than previously reported and present several new predisposition genes as potential drivers of pediatric RMS. We sequenced 59 cancer susceptibility genes in 213 children with RMS enrolled on one clinical trial (COG ARST0531), unselected for family history of cancer. Validation was performed with Sanger sequencing. Our analysis included determining the incidence of pathogenic variants in known RMS predisposition genes, followed by other cancer predisposition genes on our panel but not previously associated with RMS. In our cohort, 3.9% of unselected RMS cases harbor a pathogenic variant in a known predisposition gene (Table 1). Using VAAST, we next identified the top 10 genes possibly associated with RMS. Variants of uncertain significance (VUS) were restricted to rare variants (<0.1%) with severe CADD and Polyphen2 scores, which added 16 additional patients (7.4%). To distinguish incidental findings from true correlations, we compared our allele frequencies to those in the GnomAD database (126,216 exome sequences and 15,136 whole-genome sequences). Including both pathogenic mutations and rare VUS, 10.8% of RMS cases carry a variant with potentially important clinical implications, suggesting that newly diagnosed RMS would benefit from multigene genetic testing. Future directions include linkage to clinical data such as family history, age at diagnosis, tumor stage/location/histology, and clinical outcome, as well as expanding both the RMS and control cohorts.

Table 1. List of genes with numbers of carriers identified in 213 rhabdomyosarcoma cases

---

Gene | Carriers | % | % of carriers in GnomAD | SIR | 95% CI | P=value

Truncating Variants and Known Pathogenic Missense

ATM | 0 | |

0.42% | - | - | -

BAP1 | 0 | 0.87% | 0.05% | - | - | -

BRCA2 | 2 | 0.87% | 0.85% | 1.01 | (0.12-3.66) | 1.1736

DICER1 | 1 | 0.43% | 0.06% | 7.75 | (0.20-43.19) | 0.2421

MSH2 | 1 | 0.43% | 0.15% | 2.93 | (0.07-16.30) | 0.5790

NF1 | 2 | 0.87% | 0.88% | 0.98 | (0.12-3.54) | 1.2088

PMS2 | 0 | 0.87% | 0.18% | - | - | -

PTCH1 | 0 | |

0.15% | - | - | -

TP53 | 3 | 1.30% | 0.13% | 10.10 | (2.08-29.51) | 0.0070

WT1 | 0 | 1.30% | 0.02% | - | - | -

Total | 9 | 3.90% | 3.42% | 1.14 | (0.52-2.16) | 0.7882

Combined Analysis of Pathogenic Variants plus Variants of Uncertain Significance (VUS)

ATM | 2 | 0.87% | 0.78% | 1.11 | (0.13-4.02) | 1.0734

BAP1 | 2 | 0.87% | 0.22% | 3.89 | (0.47-14.05) | 0.1891

BRCA2 | 2 | 0.87% | 1.52% | 0.57 | (0.07-2.06) | 1.7286

DICER1 | 7 | 3.03% | 0.86% | 3.52 | (1.41-7.24) | 0.0089

MSH2 | 1 | 0.43% | 0.78% | 0.56 | (0.01-3.10) | 1.6690

NF1 | 2 | 0.87% | 1.70% | 0.51 | (0.06-1.84) | 1.8062

PMS2 | 1 | 0.43% | 0.64% | 0.68 | (0.02-3.78) | 1.5425

PTCH1 | 3 | 1.30% | 0.86% | 1.52 | (0.31-4.43) | 0.6348

TP53 | 4 | 1.73% | 0.25% | 6.93 | (1.89-17.76) | 0.0058

WT1 | 1 | 0.43% | 0.31% | 1.40 | (0.04-7.81) | 1.0197

Total | 25 | 10.82% | 9.94% | 1.09 | (0.70-1.61) | 0.7232

#2706

KRT16 **germline mutation associated with familial syndrome of tylosis with esophageal cancer (TOC).**

Erin L. Young,1 Lance Pflieger,1 Luke Maese,1 Trent Fowler,1 Kinley Garfield,2 N. Jewel Samadder,1 Bella Johnson,1 Clinton C. Mason,1 Barry Moore,1 Shawn Ryanearson,1 Mark Yandell,1 Wendy Kohlmann,2 Joshua D. Schiffman1. 1 _University of Utah, Salt Lake City, UT;_ 2 _Huntsman Cancer Institute, Salt Lake City, UT_.

Tylosis (palmoplantar keratoderma) with esophageal cancer (TOC) also known as Howel-Evans syndrome has been associated with pathogenic mutations in RHBDF2. A potential TOC family was referred for clinical evaluation at the Family Cancer Assessment Clinic (FCAC) at Huntsman Cancer Institute, Salt Lake City, Utah. Multiple relatives of the proband had hyperkeratosis on the areas of skin associated with pressure and friction, especially the feet, as well as oral leukoplakia. The proband's father, paternal aunt, paternal grandfather, and paternal great grandfather had been diagnosed with esophageal cancer. Clinical testing was unable to identify a germline mutation in RHBDFR2 that explained the observed phenotype and inheritance pattern. As part of Heritage 1K Project (University of Utah), Pediatric & Adult Cancer Section, we performed whole genome sequencing (WGS) on 5 family members, 4 that were affected with the hyperkeratosis, and 1 unaffected family member, to identify other potential genetic causes for the observed TOC phenotype. We prioritized variants via VAAST (Variant Annotation, Analysis and Search Tool). Reducing our genes of interest to those involved in palmoplantar keratoderma with PHEVOR (Phenotype Driven Variant Ontological Re-ranking Tool), we identified a pathogenic mutation: KRT16 c.379C>T p.Arg127Cys. This mutation is reported in a large palmoplantar keratoderma family (without esophageal cancer) and is listed as pathogenic in Clinvar (www.ncbi.nlm.nih.gov/clinvar). KRT16 c.379C>T p.Arg127Cys was present in each of the affected family members, but not in the unaffected relative. Our analysis is the first of its kind to suggest carriers of pathogenic variants in KRT16 are at-risk for esophageal cancer, and may benefit from esophageal surveillance. Additionally, patients presenting with a family history of esophageal cancer should be considered for germline testing for KRT16 mutations along with RHBDF2 mutations.

#2707

Targeted RNA sequencing reveals thus far unknown diagnostically relevant fusion partners confirming its diagnostic potential.

Niroshan Nadarajah, Manja Meggendorfer, Torsten Haferlach, Wolfgang Kern, Claudia Haferlach. _Munich Leukemia Laboratory, Munich, Germany_.

Introduction: The genomic landscape of hematological malignancies has been resolved mainly based on whole exome and whole genome sequencing, primarily targeting gene mutations. In addition to mutations, gene fusions have also been identified as therapeutic targets, impressively shown e.g. for BCR-ABL1 and ETV6-PDGFRB. Even though fluorescence in situ hybridization (FISH) is the current gold standard in fusion detection, it is by concept limited to the selected genes it is applied to. In contrast, targeted RNA sequencing is a valuable hypothesis-free approach to discover all possible fusion junctions in a single reaction.

Aim: Explore the value of targeted RNA sequencing in a routine diagnostic work up.

Patients and Methods: We sequenced 134 cases in parallel to our routine diagnostics workflow using chromosome banding analysis (CBA), FISH and real-time quantitative (RQ-PCR). Targeted RNA sequencing was performed on the NextSeq 500 using the TruSight RNA Fusion panel (Illumina, San Diego, CA) consisting of 7690 probes covering 507 genes known to be involved in gene fusions. Analysis was performed with the RNA-Seq Alignment App v1.2.0 (BaseSpace Sequence Hub) using Star for Alignment and Manta for gene fusion calling with default parameters (Illumina, San Diego, CA).

Results: In 127 of 134 (95%) cases the results of FISH, subsequently confirmed with RT-PCR were also picked up by RNA Seq. This included diagnostically highly relevant fusions like BCR-ABL1 (n=8), KMT2A rearrangements (n=7), PML-RARA (n=4), and ETV6 and NPM1 rearrangements. In addition to the confirmation of orthogonal results, we were able to identify novel rare gene fusions, which we subsequently confirmed by RQ-PCR. This included immediately targetable fusions like TNIP1-PDGFRB and ETV6-EFL1, ETV6-FOXO1, IRF2PB1-RARA, RARA-SAE1).

Conclusion: 1) In the vast majority of instances targeted RNA sequencing confirmed results obtained by FISH/RT-PCR and in addition discovered new rare gene fusions 2) Targetable genetic aberrations were identified, which were not identifiable by chromosome banding analysis but would now lead to more individualized treatment. 3) Thus, targeted RNA sequencing may be a valuable tool in routine diagnostics and for patients with rearrangements unresolved by standard techniques, also paving the way to precision medicine in a considerable number of patients.

#2708

A custom gene expression panel for consensus molecular subtype classification of archival primary and metastatic colorectal cancers.

Ling-Yuh Huw, Robert Piskol, Felipe de Sosa e Melo, Doris Kim, Xueping Qu, Hartmut Koeppen, Mark Lackner, Garret Hampton, Omar Kabbarah, Rachel Tam. _Genentech, Inc., South San Francisco, CA_.

Stratification of Colorectal Cancer (CRC) into actionable molecular subtypes has tremendous clinical value. Recently, a consolidated classifier identified four molecularly distinct CRC subtypes (CMS1-4) that were associated with unique biology and clinical outcomes based on global transcriptional analysis of frozen tissues. Here, we developed and applied a novel CRC panel that is ideally suited for transcriptional classification of archival clinical samples. Findings from in silico analysis demonstrated that the 800 genes on our panel could accurately classify CRC samples from external public datasets into the correct CMS subtypes. We applied our panel in the analysis of a novel cohort of 312 formalin-fixed paraffin-embedded (FFPE) tissues from 205 patients, and were able to detect all 4 CMS subtypes in primary CRCs and in metastases. When we examined the CMS subtypes of primary tumors and matched metastases from 50 patients we found 70% of cases to be concordant, as were key biologies, such as WNT/MYC pathway activation in CMS2 and EMT features in tumors of the CMS4 subtypes. This was confirmed by in situ hybridization (ISH) using the markers ASCL2 for CMS2 and SPARC for CMS4, respectively. Discordance in the CMS subtypes between primary tumors and matched metastases were observed in 30% of cases and may reflect tumor heterogeneity. Our findings suggest that our CRC-focused panel many have clinical utility for CMS classification of FFPE samples, and point to potential risks of using CMS subtypes of primary tumors to inform clinical decision-making at the metastatic stage in a subset of patients.

#2709

Evaluation of CD37 expression and binding of AGS67E, an antibody-drug conjugate (ADC) against CD37, on white blood cells (WBCs) collected from phase I non-Hodgkin lymphoma (NHL) patients.

Sher Karki,1 Hector Avina,1 Jacqueline Lackey,1 Ahmed Sawas,2 Kerry J. Savage,3 Raymond Perez,4 Ranjana Advani,5 Jasmine Zain,6 Owen A. O'Connor,2 Sara Gulesserian,1 Hui Zhao,1 Peng Yang,1 Karen Morrison,1 Leonard Reyno,1 Fernando Donate1. 1 _Agensys Inc., an affiliate of Astellas Pharma, Santa Monica, CA;_ 2 _Columbia University Medical Center, New York, NY;_ 3 _British Columbia Cancer Center, British Columbia, Canada;_ 4 _University of Kansas, Kansas City, KS;_ 5 _Stanford University, Stanford, CA;_ 6 _City of Hope, Duarte, CA_.

AGS67E is an antibody drug conjugate (ADC) against CD37 conjugated to monomethyl auristatin E (MMAE). CD37 is expressed on normal WBCs, but is also highly expressed in NHL, CLL and AML (Pereira et al., 2015). A phase I study is currently evaluating the safety, PK and anti-cancer activity of AGS67E with or without growth factor (GF) in subjects with relapsed/refractory NHL. To assess CD37 expression on WBCs, binding of AGS67E, and potential pharmacodynamic effects, samples from subjects were collected at pre-dose, D2, D8, and D15 and analyzed by flow cytometry. CD37 expression on subject tumor samples was also evaluated by immunohistochemistry (IHC). Our results demonstrated that CD37 was highly expressed in tumor samples and that AGS67E binds to WBCs causing down-regulation of CD37, achieving saturation of binding at 24 hours post-treatment (earliest time measured) at or above 0.9 mg/kg. A dose-dependent decrease in the number of all cell types examined was observed with a nadir occurring at D8, with partial or full recovery at D15, except for neutrophils. NK and T cell counts appeared to be least impacted while neutrophils were most affected. B cell counts were extremely low pre-dose for some patients, presumably from prior therapies. In patients treated at 0.9 mg/kg and higher without GF, recovery of neutrophils was delayed beyond D15. At doses of 1.2 mg/kg and higher, use of GF resulted in a significant recovery of neutrophils by D15. The extent of cell count decreases did not correlate to the proportion of cells expressing CD37. For example, decreases in NK cells, monocytes, and, in some cases, T cells, were much greater than the proportion of cells expressing CD37. Furthermore, mature WBCs are unlikely to be affected by AGS67E. This raises the possibility that the main effect of AGS67E may be on rapidly growing precursor cells and that cells with low, or no, CD37 expression may be impacted by the membrane permeable MMAE through a by-stander effect. The effect of AGS67E on neutrophils was investigated in an in vitro assay where hematopoietic stem cells were differentiated into neutrophils. Using this method, we showed that when AGS67C antibody was conjugated to a non-cleavable, membrane impermeable auristatin (mcMMAF) less cytotoxicity to differentiating neutrophils was observed compared to AGS67E. Previously, we have shown that neutrophils secrete proteases that can liberate MMAE from ADCs (Zhao et al, 2016). These results suggest that AGS67E contributes to neutropenia through a by-stander effect, in addition to the CD37-mediated internalization of the ADC. In conclusion, the results showed that AGS67E bound to its target CD37, modulated its expression, achieved saturation of binding at doses at or above 0.9 mg/kg, and reversibly depleted WBCs, with the exception of neutrophils for which GF administration appeared to significantly improve recovery rate.

#2710

Molecular and clinical characteristics of IGHV4-34 expressing classic and variant HCL.

Evgeny Arons, Katherine Potocka, Maryalice Stetler-Stevenson, Hong Zhou, Mark Raffeld, Mark Sokolsky, Sarah Davies, Robert J. Kreitman. _National Cancer Inst., Bethesda, MD_.

Classic hairy cell leukemia (HCLc) is a B-cell malignancy with distinctive immunophenotype, typically expressing CD20, CD22, CD25, CD11c, CD103, CD123, annexin A1, tartrate-resistant acid phosphatase (TRAP), and BRAF V600E mutation. Purine analog therapy is highly effective with complete remission rates of approximately 85% in first line. HCL variant (HCLv) is recognized as a distinct entity, lacking CD25 and usually lacking annexin A1, tartrate-resistant acid phosphatase expression, CD123, and also lacks the BRAF V600E mutation. Patients respond poorly to purine analogs, with partial response in less than 50% and relatively poor overall survival from diagnosis. HCLc expressing B-cell receptor (BCR) with the IGHV4-34 immunoglobulin rearrangement has a poor prognosis like HCLv, whether immunophenotypically consistent with HCLv or HCLc, and also lacks BRAF V600E. We previously reported a series of 17 patients expressing IGHV4-34, immunophenotypically resembling HCLc in 7 and HCLv in 10 cases. Using routine deep sequencing of patient samples for immunoglobulin rearrangements, we have been able to accumulate 42 IGHV4-34 expressing cases including 23 classic HCL and 19 HCLv. Six (26%) of 23 HCLc vs 12 (63%) of 19 HCLv were truly unmutated, with VH gene germline identity (GI) 100%. Eleven (48%) of 23 HCLc and 7 (37%) of 19 HCLv were borderline-mutated (97≤GI<100), and 6 (26%) of 23 HCLc were significantly mutated (97<GI). There was no significant difference between the 5 groups with respect to either IGHD or IGHJ genes usage, or CDR3 length. However, we found a significant restriction of IGHD genes between 12 HCLv truly unmutated and 6 HCLc truly unmutated cases, in that there was no overlap of any IGHD genes between these 2 groups (p<0.0001). Of 28 cases test for BRAF V600E, only 5 were positive, all HCLc with GI 93.18-97.49%. The 23 BRAF wild-type (WT) cases included one with only one with GI=93.64%, but GI was >98.7% for the remaining 22 cases (p<0.0001). Of the 23 BRAF WT cases, 9 patients died of disease, 9 have active disease, and only 2 are in remission. Patients commonly had highly aggressive courses, with infiltration of HCLc/HCLv into spinal cord and cranial nerves, cardiac muscle, lungs, and cervical lymph nodes. Complete remission was achieved only through either recombinant immunotoxin therapy of combination purine analog and rituximab, but not by purine analogs alone. Additional clinical and genetic studies are ongoing to better characterize this poor-prognosis syndrome and determine if therapy can be optimized.

#2711

Whole exome sequencing analyses of colorectal cancers associated with liver metastasis.

Yanlei Ma,1 Wenhao Weng,2 Yongzhi Yang,1 Qing Wei,3 Sanjun Cai1. 1 _Fudan University Shanghai Cancer Center, Shanghai, China;_ 2 _Tongji University, Shanghai, China;_ 3 _Shanghai Tenth People's Hospital, Shanghai, China_.

Purpose: Liver metastasis is one of the major causes of death in colorectal cancer (CRC) patients. But the process is still unknown. Here, we analyzed genetic differences between primary tumors associated with liver metastasis and those without liver metastasis and predicted clinical significance.

Experimental design: Eight primary tumors associated with liver metastasis and twelve without liver metastasis of CRC were analyzed by whole-exome sequencing. The functional characterisation for the role of metastatic-related genes was investigated through in vitro migration and invasion assays. For clinical relevance, TMAs were used for immunohistochemistry assay.

Results: The mutation spectrum was similar to that of previous studies. TP53, APC and KRAS were the three most frequently mutated genes, with no significant difference of mutation frequency between primary tumors from patients with liver metastasis and those from other patients. Notably, four recurrently mutated genes (FBXW7, EDIL3, KCNK9 and PCDHGA7) were identified exclusively in the tumors associated with liver metastasis. Furthermore, in vitro analyses showed that EDIL3 and KCNK9 overexpression resulted in enhanced cell migration and invasion, while CRISPR-mediated deletion of FBXW7 and PCDHGA7 exhibited enhanced migratory and invasive properties as well. For clinical relevance, highly expressed EDIL3 and KCNK9 was significantly associated with liver metastasis, whereas the expression of FBXW7 and PCDHGA7 negatively correlated with it in our cohorts. Pathway enrichment analysis also indicated a role for cell adhesion signaling in liver metastasis of CRC patients.

Conclusion: Our study identified candidate genes, which might response to the initiation of metastasis, exclusively mutated in tumors associated with liver metastasis. These findings also provided potential therapeutic targets for personalized treatment.

#2712

Relationship between CLDN18-ARHGAP fusion gene and clinicopathological features of gastric cancer in young adult.

Izuma Nakayama, Eiji Shinozaki, Seiji Sakata, Noriko Yamamoto, Satoko Baba, Kensei Yamaguchi, Shunji Takahashi, Kengo Takeuchi, Tetsuo Noda. Japanese Foundation for Cancer Research, 江東区, Japan.

Introduction: Gastric cancer (GC) in young adults comprises a small part of this disease population having the distinctive clinicopathological features, such as diffusive tumor growth and peritoneal dissemination. Genetic alterations underlying these features and the development of cancer in early ages remain unclear. Comprehensive molecular analyses by TCGA have suggested 4 molecular subtyping of GC and revealed enrichment of CLDN18-ARHGAP fusion gene in Genomically stable (GS) type which mainly included diffuse type. In this study, we examined the relationship between CLDN18-ARHGAP and clinicopathological features of GC affecting young adults.

Method: Patients (pts) diagnosed at the age younger than 40 years-old were defined as the GC in young adults, and underwent surgery at our institute between Jan 2006 and Dec 2015 were enrolled to this study. Tissue microarrays were prepared from formalin-fixed and paraffin-embedded surgical specimen. DNA probes for CLDN18, ARHGAP-26 and -6 were created using bacterial artificial chromosomes and fusion genes were detected by a FISH method.

Results: Out of 155 surgical specimens available for analysis, we found CLDN18-ARHGAP fusion gene in 21 pts (13.5%). Among these, 19 cases were CLDN18-ARHGAP26 fusion and the others were CLDN18-ARHGAP6. Cancers with CLDN18-ARHGAP fusion genes were all diagnosed as diffuse type and included more large-sized, multiple lymph node metastases and advanced stages tumors (table 1). The proportion of detected fusion genes in this study was almost equivalent to that of GS type in TCGA cohort. Analysis of variations of breakpoint is planned to confirm by RT-PCR and sequencing. Additionally, we are going to study the association of CLDN18-ARHGAP with RHOA or CDH-1.

Conclusion: CLDN18-ARHGAP fusion gene might account for the characteristic of a part of diffuse type GC. Further comprehensive molecular analysis comparing to general GC population is warranted.

Patient demographics

---

|

positive (n=21) | negative (n=134) | p-value

Age | |  | 0.646

<30 / 30-39 | 2 / 19 | 9 / 125

|

Sex | |  | 0.482

man / woman | 8 / 13 | 65 / 69

|

Tumor location | |  | 0.507

Upper /Middle /Lower | 4 / 10 / 7 | 43/ 53 / 38

|

Tumor size (mm) | 75.6 | 64.7 | 0.117

≤80 / 80< | 9 / 12 | 34 / 100

|

Borrmann | |  | 0.648

Type2 / 3 / 4 / others | 0 / 8 / 3 / 10 | 10 / 38 / 20 / 62

|

Lauren | |  | NS

intestinal / diffuse / mixed | 0 / 21 / 0 | 5 / 124 / 5

|

T (TNM) | |  | 0.479

T1 / T2 / T3 / T4 | 6 / 1 / 4 / 10 | 50 / 18 / 18 / 48

|

N (TNM) | |  | 0.044

N1 / N2 / N3 | 6 / 2 / 2 / 11 | 71 / 12 / 20 / 31

|

Stage (TNM) | |  | 0.032

I / 2 / 3 / 4 | 7 / 1 / 5 / 8 | 57 / 28 / 31 / 18

|

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#2713

The mutational landscape of recurrent and nonrecurrent human papillomavirus-associated head and neck squamous cell carcinoma.

Richard A. Harbison,1 Mark Kubik,2 Eric Q. Konnick,1 Seok-Geun Lee,3 Michael Kao,1 Michael Mason,4 Thomas Yu,4 Chang Xu,3 Daniel Faden,2 Colin C. Pritchard,1 Cristina P. Rodriguez,1 Chu Chen,3 Justin Guinney,4 Umamaheswar Duvvuri,2 Eduardo Mendez1. 1 _University of Washington, Seattle, WA;_ 2 _University of Pittsburgh, Pittsburgh, PA;_ 3 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 4 _Sage Bionetworks, Seattle, WA_.

Background: Despite the better prognosis for human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC) compared to HPV-negative HNSCC, 10% to 25% of HPV-associated cases recur within 3 years of completing therapy. We sought to compare the mutational profiles of primary tumors among recurrent and non-recurrent HPV-associated HNSCC. We hypothesized that the mutational profile of primary tumors from recurrent cases resembles that of HPV-negative HNSCC.

Methods: Sequencing was performed on DNA extracted from the primary tumors of p16-positive recurrent and non-recurrent HNSCC cases utilizing a high-coverage (>500X), CLIA-certified, 262 multiplexed gene sequencing panel (i.e., UW OncoPlex). Primary tumors from 11 recurrent and 13 non-recurrent cases were included in our sample. Mutational differences between the primary tumors of recurrent and non-recurrent tumors were evaluated.

Results: 88% of patients were male with a mean (standard deviation; SD) age of 60.3 (11) years. Among the recurrent cases, 100% arose from the oropharynx. Among the non-recurrent cases, 77% arose from the oropharynx with the remainder originating from the oral cavity, nasal cavity, and larynx. 83% of patients presented with stage III or higher disease, and the median survival time was 21 months and 19 months among the recurrent and non-recurrent cases, respectively. The mean (SD) number of somatic nucleotide variants per tumor among the recurrent cases was 16.4 (12.2) compared to 4.3 (1.5) among non-recurrent cases (p < 0.05). The ratio of nonsynonymous to synonymous variants among primary tumors of recurrent cases was 2 vs 5 among the non-recurrent cases (p = 0.224). Among primary tumors of recurrent cases, 17% of variants included indels, splice site, and nonsense mutations vs 14% of variants among the non-recurrent cases. The mean (SD) number of tumor suppressor mutations per tumor was 1.2 (1.3) among recurrent cases vs 1.0 (1.0) among non-recurrent cases (p = 0.81). Interestingly, the mean (SD) number of mutations in DNA damage response (DDR) genes among the primary tumors of recurrent cases was 2.7 (2.4) compared to 0.7 (1.2) mutations per tumor among non-recurrent cases (p = 0.07).

Conclusion: We observed a greater mutational burden among the primary tumors of recurrent HPV-associated HNSCC compared to non-recurrent HNSCC. Moreover, among the primary tumors of recurrent cases, there were more mutations in DNA damage response genes coupled and relatively fewer deleterious mutations compared to non-recurrent cases. Dysregulation of DDR genes may select for advantageous mutations via genomic instability. Alternatively, gain of function of DDR genes may promote treatment resistance. Future work aims to further characterize mutational differences between the primary tumors of HPV-associated recurrent and non-recurrent HNSCC and evaluate mechanisms promoting treatment resistance.

#2714

Analytical validation of clinical whole genome and transcriptome sequencing of patient derived tumors: clinical application of whole genome sequencing for reporting targetable variants in cancer.

Kazimierz O. Wrzeszczynski,1 Avinash Abhyankar,1 Vanessa Felice,1 Esra Dikoglu,1 Lukasz Kozon,1 Nicolas Robine,1 Anne-Katrin Emde,1 Olca Basturk,2 Umesh K. Bhanot,2 Alex Kentsis,2 Mahesh Mansukhani,3 Govind Bhagat,3 Vaidehi Jobanputra1. 1 _New York Genome Center, New York, NY;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 3 _Columbia University Medical Center, New York, NY_.

Next Generation DNA Sequencing (NGS) technologies are currently being applied in the clinical setting for the treatment of disease. The goal is to use high-throughput sequencing to identify specific variants within each tumor and recommend personalized treatment approaches or clinical trials tailored to the individual's disease and genomic profile. These assays are comprised of either predefined sequencing panels, where a handpicked set of clinically significant genes are examined within each patient, or are cancer type specific targeted sequencing protocols or whole exome platforms covering only the coding region of the patient's genome. Whole genome sequencing allows hypothesis-free interrogation of both coding and non-coding regions of the genome revealing more potential therapeutic options than examining a small set of genes or genomic loci. The protocol eliminates sequence capture related bias observed in whole exome or panel sequencing. The New York Genome Center therefore has performed analytical validation of whole genome and transcriptome sequencing (WGTS) of patient derived tumors and matched normals for the purposes of clinical testing and have devised a clinical reporting strategy of significant driver and therapeutic associated mutations. Many clinical NGS guidelines are directed toward targeted panel or exome sequencing validation. Here, we expanded on New York State's Department of Health NGS guidelines developing them into novel standards applicable to WGTS for the purposes of clinical test validation. We first sequenced a virtual tumor at very high coverage (300x) and downsampled to determine the optimum depth of sequencing necessary for high confidence somatic variant calling across the entire genome. We then validated whole genome sequencing laboratory protocols for DNA and RNA sequencing on a total of 50 specimens derived from fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) tumor samples. We performed a series of experiments to assess the accuracy and reliability of the results based on our laboratory and bioinformatics protocols. We performed our validation on the 50 tumor normal pairs, a subset of which had known genomic profiles. Comparisons were also made for variant calling concordance and reproducibility between matched FF and FFPE tumors. Here, we present our validation results and clinical WGTS standards for depth of sequencing, reproducibility, sensitivity, and present limit of detection analysis for SNV calling, copy number identification and structural variants. RNA sequencing is performed to call fusion or exon skipping events and to confirm the DNA variants. The New York Genome Center WGTS clinical assay is intended to provide a more comprehensive patient variant discovery approach suitable for directed oncological therapeutic applications.

#2715

Precision-designed, rapid and low-cost single molecule detection of mutations from circulating tumor DNA.

Christina M. Wood-Bouwens,1 Christine Handy,2 Billy Lau,2 Hanlee Ji1. 1 _Stanford University, Stanford, CA;_ 2 _Stanford Genome Technology Center, Stanford, CA_.

Detecting and quantifying mutations from circulating tumor DNA (ctDNA) is a new approach for longitudinally monitoring cancer patients throughout the course of treatment. However, general limitations to the clinical utility of ctDNA include sample concentration, overall assay cost, and result turnaround time. We have developed a novel molecular assay that utilize single color digital droplet PCR (ddPCR) to both genotype and quantify the number of tumor derived DNA molecules in a given sample. Our assay routinely detects as few as three mutant DNA molecules per a reaction volume, can be tested efficiently for fewer than ten dollars per reaction, and generates useable mutation information within four hours. Additionally, the assay can be rapidly configured to detect different mutations specifically identified in any cancer patient. To demonstrate the single molecule sensitivity and specificity for clinically relevant hotspot mutations, we have validated the assay using multiple input sources including high quality cell line DNA, formalin-fixed paraffin embedded tissue (FFPET) DNA, and ctDNA. Our ddPCR assay utilizes a novel primer design that is not rely on fluorescent probes. The overall simplicity of assay design enables one to detect nearly any coding mutation; practically, this means that any cancer or DNA sample can be tested efficiently. We have created customized precision mutation assays for two individual cancer patients. After extracting ctDNA from 500µL of plasma we prepare a controlled mixed wild type and mutant standard curves which contain between 150-3 detectable mutant molecules of interest per reaction. We then assay the patient ctDNA sample in parallel to the controlled standard curve and generate clustering confidence intervals. We have designed precision mutation assays targeting KRAS A146V, AKT1 E17K, and TP53 R175H for Patient A as well as KRAS G12V and PIK3CA H1047R for Patient B. The results of our single-color precision mutation quantification assays support the original mutation findings from the primary tumors. In one case, from 1µL of un-amplified ctDNA equivalent to less than 0.5ng of DNA, we identified 4 mutant molecules per ddPCR reaction volume. We have verified the presence of hotspot mutations from un-amplified patient derived ctDNA and quantified the number of mutant molecules present in each ctDNA sample. This assay has many features that make it amenable to automation. We are currently developing a massively high throughput process to design assays for tens of thousands of mutations in a matter of weeks. Overall this is an extremely low cost, highly sensitive and scalable diagnostic technology. We anticipate that this technology will be a valuable tool for rapidly monitoring ctDNA longitudinally in cancer patients.

#2716

A multi-parameter assay for the detection of rare malignant cells in classical Hodgkin lymphoma.

Lance U'Ren,1 David Wu,2 Jonathan R. Fromm,2 Jackie Stilwell,1 Eric Kaldjian1. 1 _RareCyte Inc., Seattle, WA;_ 2 _University of Washington, Seattle, WA_.

Background: Classical Hodgkin Lymphoma (CHL) ranks among the most frequently occurring lymphomas and is marked by Hodgkin and Reed-Sternberg (HRS) cells, frequently multi-nucleated neoplastic cells that are derived from germinal center B cells. The genetic alterations involved in the pathogenesis of this lymphoma have been difficult to study because HRS cells are rare and are surrounded by robust, non-neoplastic infiltrate, therefore they frequently represent less than 0.1% of cells within involved lymph nodes. Consequently, methods that isolate these rare cells are required to understand pathobiology of this lymphoma. We used the AccuCyte® \- CyteFinder® system (RareCyte) to develop a method for detection and isolation of HRS cells.

Methods: A 6-parameter immunofluorescence assay was designed to identify HRS cells that included HRS markers (CD40, CD30, CD15), CD3, combined CD20/CD14 and a cell membrane permeability viability dye. The assay was tested using the KMH2 Hodgkin lymphoma cell line spiked into PBMC and transferred to well slides for imaging by CyteFinder. This instrument utilizes 6-color scanning microscopy coupled with predefined algorithms to identify rare cell populations. The assay was then applied to a frozen fine needle aspirate (FNA) sample taken from the lymph node of a patient with histologically confirmed CHL. The FNA sample was thawed, prepared into a suspension, stained and analyzed as above. Cells identified with HRS phenotype were individually retrieved using the integrated CytePicker module. PCR of framework regions of the immunoglobulin heavy chain gene (IGH) was performed on single HRS cells (InvivoScribe) to assess clonality.

Results: Of ~200 KMH2 cells spiked into blood, the assay identified 180 cells (90%) with the correct HRS phenotype. HRS phenotype cells were identified in the patient lymph node FNA sample. Two individual cells analyzed by IGH framework region PCR demonstrated identical size PCR products, indicating clonality of the B cell receptor rearrangement.

Conclusion: A multi-parameter assay was successfully developed for identifying rare HRS cells with the CyteFinder system. Cells with HRS phenotype in a lymph node FNA sample were demonstrated to have clonal IGH rearrangements, demonstrating that actual HRS cells were identified. This method may be a useful tool for the diagnosis of CHL by identification of rare HRS cells from FNA specimens, as well as to investigate the molecular genomics of CHL.

#2717

Identification of three distinct molecular subtypes in meningioma samples using microarrays for copy-number variants.

Josef Srovnal,1 Vladimir Balik,2 Magdalena Houdova Megova,3 Jiri Ehrmann,3 Miroslav Vaverka,2 Lumir Hrabalek,2 Radek Trojanec,3 Katerina Staffova,3 Jana Vrbkova,3 Marian Hajduch3. 1 _Palacky University, Olomouc, Czech Republic;_ 2 _University Hospital Olomouc, Olomouc, Czech Republic;_ 3 _Palacky University, Faculty of Medicine and Dentistry, Olomouc, Czech Republic_.

Background: Meningioma represents one of the most common intracranial tumors. They are generally thought to progress from low to high-grade lesions. However, the molecular mechanisms underlying their pathogenesis remain still uncertain. Identification of meningioma molecular subgroups may have significant potential to improve clinical management, through molecular disease risk stratification strategies and the identification of patients who could benefit from targeted molecular therapeutics.

Methods and patients: Formalin-fixed paraffin embedded tumor samples were obtained from 45 meningioma patients and 5 healthy controls (dura mater). Comprehensive clinical-pathological data were mined. There were 15 males and 30 females; median age was 54 years, range 28 - 99 years. Total DNA was purified from FFPE samples after pathological verification using proteinase K treatment followed by QIAmp DNA FFPE Tissue Kit (Qiagen). Microarray analysis was performed using the OncoScan FFPE Assay Kit (Affymetrix), raw data were obtained using Chromosome Analysis Suite (Affymetrix) in default manner. The data were analyzed using "R" software, version 3.2.3.

Results: Our results confirm that chromosome 22 deletion and del(1p) are the most common (55%, resp. 47% of cases) deletions in meningioma. We revealed chromosomal gains not as rare as was published previously while the dup(3q) was present in 31% of cases. Three meningioma molecular patterns were identified based on CNV profiling - normal-like profile, deletion profile and complex profile. Deletion profile was characterized by loss of 1p and monosomy of chromosomes 6, 10, 14, 18 and 22. The complex profiles combined gains (3q, 8q, chromosomes 12, 15 and 20) and losses (1p, chromosomes 13, 21 and 22). The primary tumor samples of patients with meningioma recurrence (20 patients) had different CNV profile in comparison with patients without recurrence (25 patients). Recurrent tumors had statistically significant higher frequencies of losses on chromosomes 1, 9, 10, 11, 13 and 21 and gains on chromosomes 12, 19 and 20. Moreover, we analyzed CNV profiles of paired tumor samples of 15 recurrent meningioma patients (primary tumor and recurrent tumor in each patient). After extraction, it was found, that the most common alteration in recurrent tumors is chromosome 7q31 and 1p36 gains and chromosome 15q21 losses.

Conclusion: We have identified three distinct meningioma molecular patterns - normal-like profile, deletion profile and complex profile. Chromosome 22 deletion, del(1p) and dup(3q) are the most common CNV (55%, 47%, resp. 31% of cases) in meningioma. Potential CNV changes in recurrent meningioma were identified. However, it will require further validation using FISH. Acknowledgment: This work was financially supported by Ministry of Health of the Czech Republic, grant nr. 15-29021A, IGA UP LF 2016_010 and NPU LO1304.

#2718

**Molecular Tumor Board treatment predictions on rare** EGFR **exon 20 mutations.**

Anthonie J. van der Wekken,1 Matthew R. Groves,2 Arja ter Elst,1 Nils A. 't Hart,1 Lucie B. Hijmering-Kappelle,1 Thijo J. Hiltermann,1 Anke van den Berg,1 Wim Timens,1 Ed Schuuring,1 Harry J. Groen1. 1 _University of Groningen, University Medical Center Groningen, Groningen, Netherlands;_ 2 _University of Groningen, Groningen, Netherlands_.

Purpose:

To evaluate treatment response following the Multidisciplinary Molecular Tumor Board (MTB) decisions which focused on EGFR exon 20 mutations in lung adenocarcinoma.

Methods:

Molecular studies were routinely performed using the Ion Torrent sequencing platform for histologically or cytologically diagnosed lung adenocarcinoma. Since October 2014 patients with rare (prevalence <1%) or combinations of rare mutations were discussed at the MTB of the University Medical Center Groningen. More common, known sensitive EGFR, BRAF V600E, as well as KRAS, and PIK3CA mutations were excluded. To predict the best therapy for a subset of patients with EGFR exon 20 insertion mutations the literature is reviewed and molecular models were built using SWISS-MODEL, is used to predict protein tertiary structure (https://swissmodel.expasy.org/interactive/wcurbt/models/).

Summary of data:

Over a period of 2 years 1389 samples were tested. A total of 170 (12.2%) rare mutations were detected in lung cancer. Among these rare mutations we observed 16 EGFR exon 20 insertions and other mutations like e.g: p.T790M, p.D761N, p.D770delinsGY, p.S768_D770dup, p.V769_D770insSFL, p.N771_H773dup, p.T790S. Using an in silico modeling of protein responses with TKI were predicted to be likely in EGFR T790M mutations and p.S768_V769delinsIL, but not in p. D770delinsGY, p.S768_D770dup, p.V769_D770insSFL, and p.N771_H773dup mutations. Eight patients with an exon 20 mutation were treated with an EGFR TKI. One patient (p.D761N) had a partial response on erlotinib 300mg daily. On afatinib monotherapy 2 out of 3 pts had stable disease (PFS 3-11 months). On afatinib/cetuximab treatment 1 out of 2 pts (p.D770delinsGY) had a partial response and the other patient had stable disease (p.S768_D770dup) (PFS 11 and 4 months, resp.). Two evaluable patients were treated with osimertinib: the first patient initially progressed on afatinib (p.V769_D770insSFL). The protein model predicted no response, since we predicted hindrance of osimertinib. The model predicted no response on osimertinib in the second patient as well, and during treatment a stable disease for 4 months was observed (p.N771_H773dup).

This molecular information from the weekly MTB meeting was delivered within 2 weeks to the treating physician. Feedback on treatment outcome helped to further improve treatment predictions.

Conclusion:

The Molecular Tumor Board is an effective multidisciplinary team to discuss rare mutations. Our pilot data show that the evaluation of the use and effectiveness of a theoretical model concerning protein structures is not possible yet, however, did provide a clear insight in protein structures in a mutated EGFR receptor supporting decision making of treatment options.

#2719

**Accelerated** BRAF **mutation analysis using a fully automated PCR platform improves the management of patients with metastatic melanoma :** MELFAST **trial.**

Alexandre Harlé, Delphine Serre, Julia Salleron, Marie Husson, Agnès Leroux, Lionnel Geoffrois, Jean-Louis Merlin. _Institut de Cancérologie de Lorraine, Vandoeuvre Les Nancy, France_.

Background : Molecular diagnosis has become a standard of care in many cancers and BRAF mutations analysis in FFPE tumor specimens is needed to initiate personalized therapy using BRAF tyrosine kinase inhibitor (vemurafenib, dabrafenib) in BRAF-mutated metastatic melanoma. Accelerated BRAF mutation analysis is achievable using CE-IVD fully automated (FA) PCR-based platform (Idylla, Biocartis) and enables the determination of BRAF mutational status in less than 2 hours including sample preparation and proved suitable for routine molecular diagnosis of metastatic melanoma (Harlé et al. PloS ONE 11(4); e0153576).

Patients and methods : MELFAST trial is an observational monocentric study aiming at evaluating the clinical impact of reducing BRAF status determination delay in patients with metastatic melanoma. 40 patients (pts) were included in MELFAST trial, 31 pts were included retrospectively with BRAF mutation analysis being performed according to standard operating procedures (SOP) using conventional PCR, 10 pts were included prospectively with BRAF mutation analysis being performed using FA-PCR.

Results : Among the 40 pts included, 3 pts were excluded because of violation of inclusion criteria. 37 pts that were analyzed (29 pts with retrospective inclusion and 8 pts with prospective inclusion). BRAF mutational status was not known at the time of treatment decision in 11/29 (38%) pts included retrospectively and in all the pts included prospectively. Using FA-PCR enables to provide BRAF mutational status within the same day for most of the samples and the reporting delay was significantly reduced using FA-PCR as compared to SOP (0 vs 7 days, p<0.001) and the delay of initiation of anti-BRAF therapy was subsequently reduced (16 vs 26 days, p=0.035). This reduced delay was found to be consistent with that observed when BRAF mutational status had been anticipated and was already available at the time of treatment line decision (20 days, p=0.798).

Conclusion: As compared to conventional SOP, using FA-PCR accelerates BRAF mutation analysis reporting and significantly reduces the delay before initiation of personalized therapy in pts with metastatic melanoma. This warrants the investigation of the impact on the patients outcome i.e. progression free and overall survival.

#2720

Association between five common polymorphisms of plasminogen activator inhibitor-1 gene and colorectal cancer risk in Korean population.

Jisu Oh. _CHA Medical Center, CHA University, Seongnam, Republic of Korea_.

Background: The plasminogen activator inhibitor-1 (PAI-1) is expressed in many cancer cell types and allows the modulation of cancer growth, invasion, and angiogenesis. The present study investigated the association between five polymorphisms in PAI-1 gene and colorectal cancer (CRC) risk.

Material and Methods: Genotyping of five PAI-1 polymorphisms (-844G>A [rs2227631], -675 4G/5G [rs1799889], +43G>A [rs6092], +9785G>A [rs2227694], and +11053T>G [rs7242]) was assessed by polymerase chain reaction-restriction fragment length polymorphism assay in 459 CRC cases and 416 controls.

Results: The increased risk of CRC was found for the PAI-1 -675 5G/5G polymorphism compared with 4G/4G (AOR=1.556; 95% CI, 1.012 to 2.391, p=0.044). In contrast, for the PAI-1 +11053 polymorphism, we found a reduced risk of CRC for GG genotype (AOR=0.620; 95% CI, 0.413 to 0.932, p=0.022) than TT genotype, as well as for recessive carriers (TT+TG vs. GG, AOR= 0.662; 95% CI, 0.469 to 0.933, p=0.019). For patients with age < 61 years (AOR= 1.942; 95% CI, 1.247 to 3.024, p=0.003), without HTN (AOR= 1.694; 95% CI, 1.093 to 2.627, p=0.019), without DM (AOR= 1.464; 95% CI, 1.051 to 2.039, p=0.024), with folate ≥ 3.8ng/mL (AOR= 1.449; 95% CI, 1.030 to 2.039, p=0.033), with homocysteine ≤ 13.2umol/L (AOR= 1.630; 95% CI, 1.163 to 2.285, p=0.005), PAI-I -675 5G/5G genotype was shown increased risk of CRC. For patients with age < 61 years (AOR=0.563 ; 95% CI, 0.333 to 0.950, p=0.031), with HTN (AOR=0.618 ; 95% CI, 0.382 to 1.001, p=0.05), without DM (AOR=0.633 ; 95% CI, 0.425 to 0.942, p=0.024), with folate ≥ 3.8ng/mL (AOR=0.669 ; 95% CI, 0.448 to 0.999, p=0.049), with BMI ≥ 25 (AOR=0.420 ; 95% CI, 0.192 to 0.920, p=0.03), however, PAI-1 +11053 TG+GG genotype was related to protective effect about CRC. In multivariate Cox proportional analysis, compared with the +43 GG genotype, +43 AA genotype was associated with poor OS (HR=8.551; 95% CI, 1.833 to 39.89; p=0.006) and RFS (HR=12.71; 95% CI, 1.330 to 121.3; p=0.027). Moreover, OS (HR=9.330; 95% CI, 2.043 to 42.61, p=0.004) and RFS (HR=11.97; 95% CI, 1.433 to 100; p=0.022) were shown in recessive genotype (GG+GA vs. AA).

Conclusion: Our results support the relationship between CRC susceptibility and PAI-1 gene polymorphisms. Our study suggests that the -675 5G/5G genotype is associated with an increasing CRC development but +11053 GG genotype is vice versa. Additionally, we found evidence that the +43 AA genotype may be related to unfavorable CRC prognosis. This is the first study to identify an association between five PAI-1 polymorphisms and CRC incidence in the world. Further research into the association between fibrinolysis and cancer could lead to the discovery of novel drug targets for cancer prevention or treatment.

#2721

Prospective whole exome and whole transcriptome sequencing of hematologic cancers for biomarker-driven treatment : Preliminary results of a randomized pragmatic clinical trial.

Sanga Kim,1 Ryul Kim,1 Youngil Koh,1 Choong Hyun Sun,2 Hogune Im,2 Hong Seok Yun,2 Dong-Yeop Shin,1 Inho Kim,1 Sung-soo Yoon1. 1 _Seoul national university, Seoul, Republic of Korea;_ 2 _Samsung SDS, Republic of Korea_.

Introduction: Biomarker-driven early clinical trials of molecularly targeted agents have reported improvement of the response rate in various malignancies. In this pragmatic clinical trial, we aimed to assess whether biomarker-driven treatment strategy could improve outcomes of incurable hematologic cancer patients.

Methods: This is an open-label, randomized, pragmatic trial in adult patients with hematologic cancer. Patients with any kind of hematologic cancer refractory to standard-of-care are eligible. We randomly assigned these patients to receive a matched molecularly targeted agent (experimental group) or treatment at physician's choice (control group). Randomizations are determined by central block randomization in 2:1 ratio. Clinicians and patients are not masked to allocation. Whole exome and whole transcriptome sequencing (WES/WTS) are applied for detecting all classes of genomic alterations: single nucleotide variation (SNV), insertions and deletions (INDEL), copy number alterations, Fms-Related Tyrosine Kinase 3 (FLT3) internal tandem duplication (ITD), Mixed-lineage leukemia partial tandem duplication and gene rearrangements. The primary endpoint is response rate in the intention-to-treat population.

Results: Twenty-three patients with median age of 53 years and male to female ratio of 11:12 were enrolled between December 2015 and October 2016; 15 patients in experimental group, 8 patients in control group. Actionable mutations were identified in four patients in experimental arm. A 78-year-old female with acute myelogenous leukemia (AML) had FLT3 ITD at exon 14, which was identified as a long insertion mutation in SNV/INDEL calling. She received ASP2215 (FLT3 inhibitor), and showed a dramatic reduction in blast counts of peripheral blood. A 75-year-old female with myelodysplastic syndrome had TSC1P39A mutation. Because mTOR exists in downstream signaling pathway of TSC1, everolimus was administered and regression of disease activity, reflected by reduction of C-reactive protein level, was observed. NOTCH2 c.17′ and KITD815V mutation were identified in two patient; 69-year-old female with multiple myeloma and 42-year-old male with AML, respectively. Among 8 patients in control arm, seven patients received cytotoxic chemotherapy. One undergone another comprehensive genomic profiling called FoundationOne Heme, however no actionable mutations was identified.

Conclusions: Among patients with incurable hematologic malignancy, actionable mutations could be identified in one fourth of patients for whom WES/WTS were performed. They showed favorable initial response to molecularly targeted agents. This randomized pragmatic clinical trial is currently ongoing(NCT02758080).

#2722

Next generation targeted sequencing reveals four unknown mutations in the Mexican population with Gorlin syndrome.

Fragoso Veronica,1 Natalia Espinosa,2 Julieta Dominguez,1 Felipe Vaca-Paniagua,1 Luis A. Herrera,1 Rosa M. Alvarez1. 1 _Instituto Nacional de Cancerología, Mexico City, Mexico;_ 2 _Centro Dermatológico Pascua, Mexico City, Mexico_.

Aim: Nevoid Basal Cell Carcinoma Syndrome (NBCCS), an autosomal dominant disorder, is characterized by the presence of a variety of developmental anomalies, and predisposition to multiple basal cell carcinomas (BCCs), and other neoplasias. NBCCS mainly results from germinal mutations in the PTCH1 gene, that encodes a transmembrane glycoprotein, component of the hedgehog signalling pathway. The incidence of NBCCS in general population is estimated at 1 in 150000. Data related to NBCCS in Mexican patients is reduced to clinical case reports, without molecular testing. Therefore, we conducted next-generation sequencing (NGS) of PTCH1, in 9 Mexican patients with diagnosis of NBCCS.

Material and methods: NGS of PTCH1 was performed, in the context of a hereditary cancer gene panel. The PTCH1 regions with insufficient coverage (<30× read depth), were complemented by Sanger sequencing, to ensure 100% coverage of the gene. In-silico analysis was perfomed for missenses mutations. The identified pathogenic mutations were verified by capillary sequencing.

Results: Targeted analysis of PTCH1 gene, looking for deletions, insertions, splice-site alterations, nonsense and missense mutations, was done. Four PTCH1 mutations (4/9) were identified: a deletion (c.1044delG); one insertion (c.631dupA); a non-sense mutation (c.3406G>T), and a missense mutation (c.1488C>A). None of the mutations had been previously reported.

Conclusions : NGS of PTCH1 gene in 9 Mexican patients with clinical criteria for NBCCS allowed the identification of four novel pathogenic mutations (44% frequency). This is the first molecular overview of PTCH1 in NBCCS patients of our population. The perspectives of the current study lie in the molecular testing of other genes involved in NBCCS (PTCH2, SUFU), and other mechanisms (whole gene deletion), as well as the review of possible phenotype-genotype correlations.

#2723

MET exon 14 skipping mutations in advanced non-small cell lung cancer (NSCLC) are not associated with MET amplification and overexpression.

Ana Gimenez-Capitan,1 Cristina Teixidó,1 Cristina Aguado,1 Sonia Rodríguez,1 Jordi Bertran-Alamillo,1 Josep Castellví,1 Zaira Yeste,1 Ana Pérez,1 Rafael Rosell,2 Miguel Angel Molina-Vila1. 1 _Pangaea Oncology, Barcelona, Spain;_ 2 _Institut Català d' Oncologia, Badalona, Spain_.

Background: Activating alterations of the mesenchymal epithelial transition (MET) oncogene in NSCLC are potentially actionable with targeted MET inhibitors. MET exon 14 skipping mutations have been described in 3% of patients (p) in NSCLC,.Fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC) are currently used for detecting MET amplification and overexpression, respectively, but are not useful to detect MET splicing variants. On the other hand, the reverse transcription-polymerase chain reaction (RT-PCR) technique has the potential to detect this actionable alteration.

Methods: We designed and validated a custom set of 5´and 3´ primers to detect the MET exon 14 splicing variant by RT-PCR. RNA isolation from FFPE samples was performed with Roche High Pure FFPET RNA isolation kit and M-MLV Reverse Transcriptase enzyme was used in the RT-PCR. A panel of cell lines was initially employed to assess the performance of the technique. Subsequently, a total of 232 formalin-fixed paraffin-embedded (FFPE) samples from advanced NSCLC patients were analyzed. Of them, 15 were positive by RT-PCR (n=209) for the MET exon 14 variant. The bands corresponding to the splicing variant were submitted to Sanger sequencing.

Results were compared with FISH (ZytoVision Dual Color probe Z-2087-200) and IHC (Ventana CONFIRM anti-Total c-MET (SP44)). Results: A total of 232 EGFR-wt advanced NSCLC p were analyzed by IHC and 42 (18.1%) were considered as positive (cut-off 3+≥ 50%). Regarding FISH, MET amplification was detected in 13 out of 58 p (22.4%) evaluable while the MET exon 14 skipping variant was detected in 15 (7.2%) out of 209 p . Unexpectedly, only three (21.4%) of the positive MET exon 14 skipping p by RT-PCR were positive for IHC. Finally, of the 15 MET exon 14 positive p, 5 were evaluable for FISH and none of them were positive for MET amplification.

Conclusion: In our cohort of 232 EGFR-wt, advanced NSCLC p, the MET exon 14 skipping mutation had an incidence of 7.2% No correlation was found between the presence of the MET exon 14 variant by RT-PCR and MET overexpression or amplification. Detection of MET exon 14 alterations poses a challenge for diagnostic testing.

#2724

Brush biopsy miRNA based OSCC detection and diagnosis.

Guy R. Adami,1 Joel L. Schwartz,1 Yalu Zhou,1 Jessica L. Tang,1 Michael Markiewicz,1 Gina D. Jefferson,1 Joel Epstein,2 Antonia Kolokythas3. 1 _Univ. of Illinois at Chicago, Chicago, IL;_ 2 _Cedars-Sinai Medical Center, Los Angeles, CA;_ 3 _University of Rochester Medical Center, Chicago, IL_.

RNA based diagnosis and prognosis of squamous cell carcinoma has been slow to come to the clinic. Improvements in RNA measurement, statistical evaluation, and sample preservation still have not made these methods accurate enough to allow truly independent external validation. We propose that in the case of squamous cell carcinoma of the oral cavity that a chief source of variability is sample dissection which leads to variable amounts of stroma mixed in with tumor epithelium. We have used brush biopsy of suspicious lesions to characterize miRNA expression in OSCC epithelium. We used it to show an 11 miRNA signature for OSCC that can differentiate OSCC in both smokers and never smokers from normal tissue. We will report on efforts to optimize this classifier to distinguish OSCC from benign oral lesions such as lichen planus and leukoplakia that can be mistaken for OSCC. We also provide evidence that body fluid samples show similar changes in RNAs with OSCC that are seen in brush biopsy samples -suggesting much of the RNA is coming from the same source tumor epithelium. Finally, using a variation of mirPath designed to eliminate false positive, we have identified the neurotrophin signaling pathway as disregulated in most OSCC epithelium, and for the first time highlight the miRNAs that may control this pathway in OSCC, miR-486-5p, miR-7-5p, miR-146-5p, miR-101-3p, miR-18b-5p, miR-10b-5p, miR-21-5p, miR-20b-5p, miR-126-3p and miR-31-5p.

#2725

Circulating CD9-GFAP-survivin exosomes during active specific immunotherapy, a potential biomarker for glioma.

Michael J. Ciesielski, Phillip Galbo, Sheila Figel, Laura Wiltsie, Cheryl Frank, Jingxin Qiu, Robert A. Fenstermaker. _Roswell Park Cancer Inst., Buffalo, NY_.

Background: We evaluated circulating exosomes isolated from the serum of malignant glioma patients enrolled in a completed phase I clinical trial of an anti-survivin vaccine (SurVaxM). Exosomes are microvesicular bodies with potential mechanisms for cell-cell communication. Identifying circulating exosomes from cancer patients as potential indicators of disease status and response to therapy is of interest. The inhibitor of apoptosis protein (IAP) survivin (SVN) promotes cancer cell proliferation and resistance to chemotherapy. Survivin is expressed in many cancer types including malignant gliomas and is a potential target for active immunotherapy. In the above mentioned clinical trial, five patients experienced early tumor progression with a mean of 2.8 months (1.9-5.4 months) from study entry, while three patients had either late (20.5-22.5 months) or no tumor progression (no evident disease at 48 months in one patient).

Methods: To determine whether changes in CD9+/GFAP+/SVN+ exosomes correlated with disease progression, patient serum was evaluated prior to treatment (study entry), 8-10 weeks after initial vaccination, and at the time of MRI-defined tumor progression. By employing ImageStream flow cytometry technology we were able to analyze multiple markers on individual exosomes, identifying highly specific populations of survivin+ exosomes which co-expressed the brain/tumor marker protein GFAP. The combination of these markers made it possible to differentiate patient exosomes from those of healthy individuals.

Results: The levels of CD9+/GFAP+, CD9+/SVN+, and CD9+/GFAP+/SVN+ (SVN+) exosomes were significantly increased in patients. Serum from three patients who experienced the longest progression-free intervals, showed 98-99% decreases in SVN+ exosomes after treatment with SurVaxM, and maintained similarly low exosome levels over several months. Five early progressing patients experienced a detectable persistence of, or increase in SVN+ exosomes at 9 weeks following initial vaccination. One patient had rising SVN+ exosomes detectable 16 weeks prior to the discovery of tumor progression by brain MRI scan. One patient with no tumor progression sustained a 99% reduction in serum SVN+ exosomes which was sustained over 48+ months.

Conclusion: This study demonstrates that increased numbers of CD9+/GFAP+/SVN+ specific exosomes appear to be associated with early tumor progression in recurrent malignant glioma patients. Larger studies are needed to determine whether the correlation between SVN+ exosome levels and disease progression is specific to SurVaxM treatment, and whether rises in SVN+ exosomes levels are predictive of generalized glioma progression. Our study suggests that levels of circulating SVN+ exosomes may be altered during anti-survivin immunotherapy and could serve as a potential biomarker for clinical trials and tumor monitoring.

#2726

Guidance by molecular selection improves the outcome of early phase treatment for gynecological (GYN) cancers.

Rowan E. Miller,1 Michael John Devlin,1 Nicholas Brown,1 Kin Woo,1 Tami Grunewald,2 Arran Speirs,1 Miriam Mitchison,2 Michelle Lockley,2 Mary McCormack,2 Jonathan Ledermann,2 Martin Forster,1 Tim Meyer,1 Rebecca Kristeleit1. 1 _NIHR Clinical Research Facility, University College London Hospitals, London, United Kingdom;_ 2 _University College London Hospitals, London, United Kingdom_.

Background: Patients (pts) with advanced gynecological (GYN) cancers have limited therapeutic options and the prognosis is poor. Early phase trials may be a suitable option for pts with good performance status. Increasingly, molecular characterisation guides pt selection for early phase trials. We sought to determine the outcome of GYN pts treated in a phase 1 unit and examined the role of molecular selection to inform therapeutic decision making.

Methods: Medical records of all pts with a GYN malignancy treated within an early phase trial between 2010 and 2016 were reviewed. Data comprising patient and tumor characteristics, prior treatment, trial therapy and outcome were analysed.

Results: 81 pts with a median age of 60 years (range 20-75) with a diagnosis of ovarian (OC, 54), endometrial (EC, 15) or cervical/vulval (CC, 12) cancer were identified. The median number of prior therapies for advanced disease was 3 (OC) and 2 (EC and CC) (overall range 1-6). 9 pts (11%) entered a second and 1 pt a third phase 1 study on disease progression. Next Generation Sequencing (NGS) using a targeted panel was performed in 32 pts (40%) with an actionable mutation identified in 9 including; KRAS (3pts), PIK3CA (2pts) and EGFR (2pts). Germline BRCA (gBRCA) testing was performed in 35 OC pts (65%) with 24 gBRCA mutations identified.

Pts were allocated, in order of priority, where available, to (1) a trial selected on the basis of NGS or gBRCA ('genomic' 35%), (2) a 'tumor specific' cohort within an early phase trial (15%) or (3) a 'generic' study (51%). For the whole cohort there was an overall response rate (ORR) of 18% with 41% stable disease (SD) and median progression free survival (PFS) and overall survival (OS) of 13 and 46 weeks respectively. Outcomes were best for pts in the genomic group. Both PFS and OS were significantly longer with genomic selection (p < 0.01 for both, Mantel-cox test) with median PFS of 29.7, 14.2, 8.0 weeks and OS of 84.1, 69.7, 33.6 weeks for genomics, tumor specific and generic studies respectively. The ORR was also greatest for the genomic cohort (32%) compared to the tumour specific (7%) and generic (11%) groups.

Within the heavily pre-treated EC and CC cohorts there was an OS of 30 and 42 weeks respectively. 24% of EC pts had an ORR with a further 24% with stable disease (SD). There was only 1 response (9%) in the CC cohort, however SD was seen in 64%. The OS for the OC was 55 weeks with an ORR of 20% and 46% SD.

Conclusions: Early phase trials represent a good option for pts with advanced GYN malignancies. Whilst applicable to all GYN cancers, this is particularly relevant for EC and CC pts as standard treatment options are limited. For OC patients (median 3 prior lines of chemotherapy in this cohort) where standard treatment options exist, early access to phase 1 genomic trials may result in improved response rates and allow further standard options to be given subsequently. NGS is feasible in real time and may have a positive impact on outcome.

#2727

Clinical significance of PTEN in UTUC.

Liang-Chen Li,1 Hung-Ying Chiang,1 Ying-Tzu Chen,1 See-Tong Pang,2 Wen Hui Weng*1. 1 _National Taipei University of Technology, Taipei, Taiwan;_ 2 _Chang Gung Medical Foundation, Taoyuan, Taiwan_.

Upper tract urothelial carcinoma (UTUC) is a rare malignancy accounts for about 5% of all urothelial tumours. The cancer cells start in the layer of tissue urothelium, and found in the renal pelvis, renal calyces or ureters. When compare the UTUC to the bladder urothelial carcinoma, the pathological and clinical findings are often look and act alike. Previous studies indicated that deletion of phosphatase and tensin homologue (PTEN) gene is common to be found in bladder cancer. As known tumor suppressor gene PTEN is located on chromosome ten and considered as an important negative regulator for the PIP3⁄Akt signaling pathway to promote cell proliferation and inhibit apoptosis. Thus, this study we aimed to determine clinical significance of PTEN in UTUC. We collected sixty-eight formalin-fixed paraffin-embedded (FFPE) UTUC samples, the tumor stage was according to American Joint Committee on Cancer (AJCC) with Ta (n=15), T1 (n=11), T2 (n=10), T3 (n=18), T4 (n=7) and seven patients with unknown; in which male no.32, female no.36; the median age is 68 y/o (range 40-85 y/o). Fluorescence in situ hybridization (FISH) was performed to exam the PTEN gene copy number or gene structure alterations of UTUC; besides, immunohistochemistry (IHC) analysis were used to observe the PTEN protein expression in the tumor. Our results showed PTEN mutations were observed in twenty-two tumors with heterozygous PTEN deletion; one tumor with homozygous PTEN deletion, and three tumors with PTEN monosomy. Further, heterozygous PTEN deletion was shown significantly associated with stage of pTa, pT1 and pT2 (P=0.048) and history of smoking (P=0.030). The IHC results showed lower expression level was common to be observed in the tumor area. Our findings suggest loss of PTEN is associated with the low-risk group of UTUC but not the high-risk group, the unknown mechanisms between these two subtypes might be existed. Besides, smoking could also as a factor highly emerge from genetic alterations in UTUC. In conclusion, PTEN genomic loss might act a predictive indicator of classification of UTUC, however, the clinical treatment could be different due to the underlying diversity of the molecular activities.

#2728

Frequency and prognostic value of PI3KCA mutations in early stage of colorectal cancer in Mexican patients.

Erika Ruiz-Garcia,1 Alette Ortega,1 Antonio Bahena,1 Edith Fernandez,1 German Calderillo,1 Cesar Lopez Camarillo,2 Laurence Marchat,3 Jorge Guadarrama,1 Abelardo Meneses-Garcia,1 Horacio Astudillo-de la Vega4. 1 _National Cancer Institute, Mexico City, Mexico;_ 2 _Universidad Autónoma de la Ciudad de México, Mexico City, Mexico;_ 3 _Escuela Nacional de Medicina y Homeopatía, Instituto Politécnico Nacional, Mexico City, Mexico;_ 4 _Lab Investigacion Traslacional en Cancer, HOCMN SXXI, IMSS, Mexico City, Mexico_.

Background: There are molecular associations between mutated profiles and microsatelital instability, both related with tumor growth and oncogenic development by activating tyrosine receptors families in colorectal cancer (CRC). It is known that exon 9 and exon 20 (H1047R) mutations in PIK3CA increased PI3K activity. Moreover, PIK3CA mutations are significantly associated with tumor recurrence and poor overall survival. The frequency of PIK3CA mutations in CCR occurs in 15-20%, being the catalytic subunit more frequently affected. The aim of our study was to investigate the frequencies and clinical implications of these genetic alterations in advanced CRC.

Methods: Retrospectively, we analyzed the initial paraffin embedded tissue block of 71 Mexican patients with CCR stage II. We evaluated mutations frequency of PIK3CA exon 9 (E542K, E545K) and exon 20 (H1047R) using qBiomarker Somatic Mutation PCR for real-time PCR-based. These methodology increases specificity, multiplex PCR and allelic discrimination. The data analysis was done using Ct values and Analysis Webportal.

Results: We found in our population a frequency of PIK3CA mutations of 26%. Of those, double mutants were present in 7% with a higher frequency for E545K/H1047R. Clinical implication that we found is that any active mutation of PIK3CA is associated to an increase of recurrence risk (HR 1.86;CI 95% =0.46-7.5), but most important is for E545K mutation (HR 4.17; CI 95% 0.85-20.39), meanwhile comparing no mutated with H1074R mutated samples there were no differences in HR (0.53 vs. 0.60, respectively).

Conclusions: We found a higher number of PI3KCA mutations that literature reports and that E545K mutation have a worst prognostic implication but we need to increase our sample looking for statistical significance results. Data from H1047R mutation raise de hypothesis that it could exist a compensatory mechanism related to structural interactions between both helicoidal and catalitic domains.

#2729

A significant differential sensitivity to AKT inhibitors GSK690693 and MK-2206 2HI was identified in primary cells established from a low-grade mucoepidermoid carcinoma.

Ahmad M. Alamri, Priscilla A. Furth. _Georgetown University, Washington, DC_.

The AKT pathway is frequently activated in cancer cells and is a defined target for therapeutic intervention.

Methods: RNA expression levels of candidate AKT pathway genes (AREG, EGFR, AKT, mTOR, GSK3α/β) were defined following RNAseq of replicate primary cell cultures (GUMC220 and GUMC221), which were established independently from two areas of the same low-grade salivary mucoepidermoid carcinoma (MEC). Protein expression and phosphorylation levels of the corresponding gene products were characterized using immunohistochemistry on FFPE sections from the original patient tumor, cell pellets, and cell line generated patient-derived xenografts and through western blotting of proteins extracted from cells grown under different culture conditions before and after treatment with AKT inhibitors GSK690693 (ATP-competitive) and MK-2206 2HCl (allosteric inhibitor). The effects of GSK690693 and MK-2206 2HCl on cell viability were tested at different concentrations ranging from 1.2-40μM in 2D and 3D conditions with three different culture media (Conditional media+ ROCK kinase inhibitor Y27632, conditional media without Y27632, and EpiCult-C BasalTM) using CellTiter-GloTM. Doxorubicin was used as a comparative control for evaluating the impact of a chemotherapeutic on cell viability and DMSO as a negative vehicle control. MDA-MB-453 cells were tested as a positive control for MK-2206 2HCl and GSK690693 sensitivity.

Results: Replicate primary cell cultures showed the same chemosensitivity results. MK-2206 2HCl induced a significantly greater reduction in cell viability as compared to GSK690693 in the MEC primary cells. This differential response was replicated in all three 2D culture medias tested (GUMC220_CMY p= 0.0017, GUMC220_CM p=0.0004, GUMC220_EpiC p<0.0001, GUMC221_CMY p=0.0010, GUMC221_CM p<0.0001, and GUMC221_EpiC p=0.0041, One-way ANOVA) with the same pattern reproduced in 3D cultures. Expected changes in levels of p-AKT(Ser473), GSK3α/β, p-GSK3α/β, and p-mTOR were found following treatment with both drugs as compared to vehicle controls. In MK-2206 2HCl treated cells, expression levels were all reduced whereas in GSK690693 treated cells, p-AKT(Ser473), p-AKT(Thr308) were increased and GSK3α/β, p-GSK3α/β, p-mTOR reduced.

Conclusion: Characterization of primary cell cultures using RNAseq led to the identification of a potentially druggable pathway. Direct testing revealed a differential response to the two AKT inhibitors evaluated. Although the two primary cell cultures were established from two geographically distinct areas of the same tumor, response to drug testing was the same and reproducible across different culture modalities. Studies establish the feasibility of deriving primary cells for drug testing and personalized therapy, even from small tumors.

#2730

Image-based single cell-sorting to separate and recover distinct cell populations from complex heterogeneous mixed tissue: precise sample preparation upstream of FISH.

Amanda Gerber,1 Aditi Khurana,2 Lisa Koenig,3 Lindsay Strotoman,4 Lori Millner,4 Valeria Sero,1 Chiara Bolognesi,5 Sabine Kasimir-bauer,3 Gianni Medoro,5 Matthew Moore,2 Philip Cotter,2 Nicolo Manaresi,5 Farideh Bischoff1. 1 _Menarini Silicon Biosystems Inc, San Diego, CA;_ 2 _Research Dx, Irvine, CA;_ 3 _Universitätsklinikum Essen, Essen, Germany;_ 4 _PGXL Laboratories, Louisville, KY;_ 5 _Menarini Silicon Biosystems SpA, Bologna, Italy_.

Fluorescent in Situ Hybridization (FISH) is commonly used for assessment of chromosomal alterations. Guidelines for determining FISH-based classification of clinical biomarkers exist but are based on pre-analytical factors, including fixation/sectioning/thickness/age, that can greatly influence biomarker status determination. Here, we use single-cell image-based cell sorting by DEPArrayTM for the separation and recovery of pure distinct cell populations prior to FISH.

Methods: A multi-center study to evaluate HER2-FISH based analysis on FFPE with and without DEPArrayTM pre-processing was conducted using breast tumors classified as infiltrating ductal carcinoma (n=12), metastatic (n=1) and ductal carcinoma (n=1). From each block, four 50-micron thick curls were sectioned. One curl from each sample was sent to each of four centers (3 US; 1 EU). Each site performed disassociation of curls to generate a single cell suspension. Cells were then stained and sorted using the DEPArrayTM platform for recovery of tumor (cytokeratin+/vimentin-/DAPI+) and stromal (cytokeratin-/vimentin+/DAPI+) cells. Dual-probe FISH for HER2 and centromere 17 was performed on the sorted cells and compared with conventional tissue section FISH.

Results: Overall, ≥ 90% concordance between the sorted tumor cells and the conventional HER2 FISH result was observed. Among the 7 HER2+ cases, HER2 ratio scores for the sorted tumor cells ranged slightly higher, from 2.60 to 8.95, as compared to the conventional method (from 2.10 to 5.14). In all cases in which stromal cells were also recovered, an expected normal ratio was observed, thus verifying that the populations were efficiently separated. Discordance can be attributed to intra-tumoral heterogeneity and the fact that conventional FISH on FFPE requires only a 4-micron section for analysis.

Conclusion: Today, a percentage of patients are likely misclassified for the biomarker of interest as result of pre-analytical factors. We demonstrate here the ability to overcome these pre-analytic factors and ultimately improve the accuracy in determining biomarker status using the DEPArrayTM

### Liquid Biopsies 2: cfDNA

#2731

Identifying circulating tumor DNA in patients with osteosarcoma.

David M. Barris, Michael Fremed, Robert Durbin, Xusheng Zhang, Sajida Piperdi, Wendong Zhang, Shahina Maqbool, Jonathan Gill, Michael Roth, Bang Hoang, David Geller, Richard Gorlick, Daniel Weiser. _Albert Einstein College of Medicine, Bronx, NY_.

Introduction:

Osteosarcoma (OS) is the most common primary malignant bone tumor and primarily affects children and adolescents. During and after treatment there is no non-invasive test to assess disease response and early relapse. We hypothesize that circulating tumor DNA (ctDNA) can be used to assess tumor burden, evaluate response to treatment, and monitor for recurrence in OS. We effectively tested this hypothesis on a mouse model and have utilized our methods on a first cohort of human patients.

Materials and Methods:

Twenty blood samples with matched primary tumor material were obtained from 10 patients with OS who were at various stages of treatment or post treatment. Cell free DNA was isolated from blood collected in Cell-Free DNA blood collection tubes using the QIamp Circulating Nucleic Acid kit. We performed massively parallel sequencing (MPS) with the Illumina HiSeq 2500 in the Epigenomics Shared Facility (ESF) to a depth of at least 800x using custom designed probes (Roche NimbleGen SeqCap EZ) capturing genes commonly altered in OS including TP53, RB1, ATRX, DLG2, MET, PTEN, and SLC19A1. We aligned the fastq files with BWA, called the variants using Varscan and GATK HaplotypeCaller, and annotated the variants with SnpEff.

Summary of Data:

Using an initial threshold of 2% of variants, ctDNA was identified in two of the patients in our study. The first patient's sample, collected at the completion of therapy, contained a missense mutation in the coding exon of DLG2 at position chr11 84027990 (2.07% of reads). This patient has no radiographic evidence of recurrent disease 12 months after completion of treatment. In a second patient with 4 samples drawn, a mutation was found in the intron region of RB1 at position chr13 48986157. The variant was 0.17% of reads upon completion of therapy, undectable at 3 months, 2.16% of reads at 6 months, and 1.5% of reads at 9 months. The patient is at high-risk for recurrence but is currently disease-free. Sequencing and analysis of serially collected patient samples, including from 3 patients with recurrent disease, and primary tumor is ongoing.

Conclusions:

We have identified pathogenic variants in the cell free DNA of patients with no radiologic evidence of OS but who are at high risk for relapse. This is the first step to utilizing a non-invasive test to assess tumor burden, response to treatment, and likelihood of recurrence. As part of future work, we will lower the threshold for calling variants and quantify ctDNA abundance in serially collected samples from treatment and during surveillance, and correlate the findings with clinical outcome.

#2732

Novel methods for enrichment of mutations and differentially methylated sequences from liquid biopsy genomes.

Chen Song, Yibin Liu, Mariana Fitarelli-Kiehl, Ioannis Ladas, Harvey Mamon, Mike G. Makrigiorgos. _Dana-Farber Cancer Inst., Boston, MA_.

Presence of excess unaltered, wild-type DNA providing no information of biological or clinical value may often mask rare alterations containing diagnostic or therapeutic clues for cancer. With the surge of high-throughput technologies there is a growing demand for removing unaltered DNA over large pools-of-sequences. Here we present nuclease-assisted minor-allele enrichment with probe-overlap (NaME-PrO), a single-step approach with broad genome-coverage that removes WT-DNA prior to DNA-amplification, following which current genomic analysis processes remain unchanged. NaME-PrO employs a double-strand-DNA-specific nuclease and overlapping oligonucleotide-probes interrogating multiple DNA targets. Following genomic-DNA denaturation, or upon partial denaturation, the probes form transient double-stranded regions with their targets, thereby guiding nuclease digestion to selected sites. Mutations create mismatches that inhibit digestion, thus subsequent amplification yields DNA with alterations enhanced at multiple targets. In this manner, WT DNA at hundreds or thousands of DNA regions can be digested simultaneously. We demonstrate several-hundred-fold multiplexed mutation-enrichment in diverse human samples on multiple clinically-relevant targets in 50-247-plex reactions. By adjusting conditions it is possible to sequence mutations down to 0.00003% abundance, essentially isolating mutated-DNA. Application with targeted re-sequencing of tumor-circulating DNA (cfDNA) demonstrated detection of mutations at the 0.01-0.1% levels with few sequence reads.

In another adaptation of the same approach in the field of methylation detection, we developed methylation-sensitive nuclease-assisted minor allele enrichment, MS-NaME. Aberrant methylation changes, often present in a minor allelic fraction in clinical samples such as cfDNA, are potentially powerful prognostic and predictive cancer biomarkers. During MS-NaME, oligonucleotide probes targeting unmethylated sequences in bisulfite-treated DNA generate local double stranded regions resulting to digestion of unmethylated targets, and leaving methylated targets intact; and vice versa. We validated MS-NaME by demonstrating enrichment of promoters from cancer-related genes like RARb2, ATM, MGMT and GSTP1 in multiplexed 177-plex reactions using dilutions of methylated/unmethylated DNA as well as clinical lung cancer samples and matched normal tissue. NaME-PrO and MS-NaME introduce simple, low-cost, broad genomic coverage processes that combine seamlessly with next

generation sequencing. This reveals clinically useful DNA alterations that normally would stay undetectable or require very high read-depth to be detected. Applications in liquid biopsy-based diagnostics using circulating DNA will be presented.

#2733

Comparison of copy number aberrations (CNAs) between plasma cell free DNA (cfDNA) and tissue DNA in metastatic castrate resistant prostate cancer (mCRPC).

Chiang-Ching Huang,1 Meijun Du,2 Liguo Wang,3 Yen-chen Lin,1 Hua Huang,1 Liewei Wang,3 Liang Wang,2 Manish Kohli3. 1 _University of Wisconsin, Milwaukee, Milwaukee, WI;_ 2 _Medical College of Wisconsin, Milwaukee, WI;_ 3 _Mayo Clinic, Rochester, MN_.

Background: Circulating cell-free DNA (cfDNA) analysis is emerging as a less invasive approach to assessing tumor genomic alterations in cancer patients. Although high concordance has been reported between tumor tissue NGS and cfDNA in studies investigating specific genetic alterations, the fidelity of cfDNA to tumor tissue DNA in the global genomic scale is largely unknown. In a correlative study of a prospective clinical trial (NCT# 01953640) conducted in mCRPC stage patients treated with abiraterone acetate/prednisone (A/P), we evaluated correlation of genomic CNA in tumor DNA obtained from biopsy of metastatic lesions and matched plasma cfDNA.

Methods: mCRPC patients (pts) underwent two image-guided core biopsies of metastases prior to initiation of A/P (visit 1, V1) and 12 weeks after treatment (visit 2, V2). At both time points plasma was obtained at the time of the core biopsies and cfDNA was extracted using DNA Blood Mini Kit (Qiagen, Valencia, CA). High coverage (for tumor tissue) and low coverage (for cfDNA) whole genome sequencing reads were first mapped to the human genome hg19. Read counts (RC) from the mapped sequence files were then binned into 1Mb windows. The RC ratio in each genomic bin was calculated by comparing tumor tissue DNA to lymphocyte gDNA derived from the same patient, and was further log2 transformed, corrected for GC content, and normalized by CGHnormaliter. The fully normalized log2 ratios data was subjected to segmentation using DNAcopy algorithm.

Results: Between 05/2013 and 09/2015, 92 patients (pts) were enrolled of which tissue and plasma NGS data both visits was available for 18 pts. The correlation of CNAs between tumor tissue and its cfDNA counterpart ranges from 0.013 to 0.83 for V1 samples, and -0.05 to 0.9 for V2 samples. The decreased correlation in some pairs of samples is largely due to low tumor content and heterogeneity in the cfDNA. Although there is a wide range of correlation, commonly shared CNAs were identified in multiple chromosomal regions, including loss in 8p, gain in 8q and chromosome 5 and X. On the other hand, several genomic regions show inconsistent CNAs between tumor tissue and cfDNA among 15 pairs of samples in which cfDNA have large tumor content. These include: loss in 6q, 1p, 2p, 9q, 11q, 13p, 15p, 20p and gain in 8q and 10p in cfDNA but not in tumor tissue; marked loss in chromosome 22 and gain in p arm of X chromosome in tumor tissue but much less evident in cfDNA.

Conclusion: High concordance of CNAs between cfDNA and tumor tissue DNA can be achieved given sufficient tumor content of cfDNA. However, more CNAs can be identified in cfDNA than its tumor tissue counterpart. Our result suggests that cfDNA NGS is a useful tool to investigate clonal evolution associated with cancer progression.

#2734

Impact of circulating and tissue biomarkers in patients with metastatic colorectal cancer treated with first-line FOLFOX-aflibercept therapy. Results of the GERCOR VELVET Phase II Study.

Annemilai Tijeras-Raballand,1 Armand de Gramont,1 Marielle Chiron,2 Jean-Baptiste Bachet,3 Thierry Andre,4 Dominique Auby,5 Jerome Desramé,6 Baba-Hamed Nabil,7 Lecaille Cedric,8 Valérie Lebrun,9 Christophe Louvet,10 Annette Larsen,11 Christophe Tournigand,12 Sylvie Benner,1 Malika Attia,13 Aimery de Gramont,14 Franck Bonnetain,15 Benoist Chibaudel14. 1 _AFR Oncology, Paris, France;_ 2 _Sanofi France, Vitry-sur-Seine, France;_ 3 _Salpetriere University Hospital, Paris, France;_ 4 _Saint-Antoine University Hospital, Paris, France;_ 5 _Mont de Marsan Hospital, Paris, France;_ 6 _Jean Mermoz Hospital, Paris, France;_ 7 _Saint-Joseph Hospital, Paris, France;_ 8 _Clinique Bordeaux Nord, Paris, France;_ 9 _Limoges University Hospital, Paris, France;_ 10 _Institut Mutualiste Montsouris, Paris, France;_ 11 _Inserm U938-Institut Universitaire de Cancérologie/UPMC, Paris, France;_ 12 _Henri Mondor Univeristy Hospital, Paris, France;_ 13 _GERCOR group, Paris, France;_ 14 _Institut Hospitalier Franco-britannique, Paris, France;_ 15 _Inserm UMR 1098- Besançon University Hospital, Paris, France_.

Background: The combination of aflibercept to OPTIMOX (VELVET study) was evaluated in patients with previously untreated advanced colorectal cancer. A biomarker program was set-up to explore the expression of several tissue biomarkers at baseline and circulating biomarkers upon treatment cycles to identify the best monitoring biomarkers of the treatment strategy.

Methods: VELVET was a prospective, single arm phase II trial. Patient's plasma samples were collected at baseline, and during induction therapy at day 1 of the first 6 cycles. Circulating biomarkers were analyzed using multiplexing immunoassays (31 biomarkers, 3 panels). Assays were conducted on a Biorad Bioplex platform, expect for PlGF, VEGF-A and Neuropillin-1 which were assessed using ELISA assays. Tissue biomarker were analyzed at baseline by IHC, using a BOND-Max autostainer (Leica biosystems). EGFR mutation status were analyzed using next generation sequencing.

Results: Among 49 patients included in the VELVET study from May 2013 to May 2014, 44 (90%) patients were evaluable for circulating biomarkers expression. The proportion of patients with tumor response (CR or PR) was higher in patients with high baseline levels of sVEGFR2, sEGFR, G-CSF, Prolactin and low baseline levels of VEGFA and MIF. Progression-free survival (PFS) was higher in patients with low baseline levels of Osteopontin (HR: 0.53; P=0.045) and in patients with high baseline levels of VEGF-C (HR: 0.45; P=0.014) and sVEGFR3 (HR: 0.50; P=0.045). Overall survival was higher in patients with high sVEGFR3 (HR: 0.36; P=0.030) levels. In both responders and non-responders, sVEGFR-1 and PlGF dramatically increased upon exposure to aflibercept and remained overexpressed for the all course of induction therapy. Circulation biomarkers modulations will be analyzed upon the 6 cycles. Tumor tissue biomarker assessment at baseline using IHC and theEGFR pathway mutations status will be added.

Conclusions: Exposure to aflibercept is associated with an increase of sVEGFR-1 and PlGF at cycle 1. Elevated baseline expression levels of on-target sVEGFR3 predict favorable outcome in patients treated with aflibercept.

#2735

Targeted molecular profiling of tumor and matched circulating tumor DNA in patients with early stage endometrioid type endometrial adenocarcinoma.

Ana M. Bolivar, Rajyalakshmi Luthra, Meenakshi Mehrotra, Wei Chen, Bedia A. Barkoh, Peter Hu, Rajesh R. Singh, Russell R. Broaddus. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Endometrioid-type endometrial adenocarcinoma (EEA) is the most common type of gynecological cancer in the USA and the fourth most common cancer in women. Contrary to other cancer types, its incidence is rising, likely due to its tight association with obesity. A major clinical problem for EEA patients presenting with early stage disease is identifying those at risk of recurrence, as extra-vaginal recurrence is incurable. If these at-risk patients could be identified up-front, they could receive more aggressive surgical treatment and adjuvant radiation therapy/chemotherapy/targeted therapy to help prevent the development of incurable recurrences. The long-term goal of our work is to develop a blood-based molecular diagnostics platform that can help to identify women with early stage EEA who are at highest risk for tumor recurrence. To achieve this goal, we designed a proof-of-principal study to determine if it was possible to detect common EEA associated mutations in circulating tumor DNA (ctDNA) of early stage EEA patients. From our previous work, we have shown that 98% (43/44) of EEA patient's tumors have somatic mutations in CTNNB1, KRAS, PTEN, and/or PIK3CA. Using a custom panel designed by Swift Biosciences targeting 30 amplicons in these 4 genes, MiSeq NGS analysis was performed on ctDNA extracted from 3mL of frozen plasma using QIAamp circulating nucleic acid kit and on the matching tumor DNA (tDNA) extracted from FFPE slides using an Arcturus Pico Pure DNA extraction kit from 33 stage I and II EEA patients (stages in which tumor is confined to the uterus). NGS data were analyzed using a custom bioinformatics pipeline. Using a cutoff of 0.5% allelic frequency (AF) in the ctDNA and 5% in the tDNA, at least one EEA associated mutation was detected in both ctDNA and tDNA in 5/33 (15%) of cases. Mutations with more than 5% AF in the tumor and 0.02-0.49% AF in the cfDNA (total coverage >250X and variant coverage >25) were also detected in another 6/33 patients. Each of these 11 cases was re-sequenced at a higher depth (>20,000X), with comparable results. For the remaining 22 patients at least one EEA associated mutation was detected only in the tDNA, with no mutation detected in ctDNA. Primary tumor size may play a role in detection of mutations in ctDNA, as mean tumor size in the group with mutations in both tDNA and ctDNA was significantly larger (mean 10.4 cm) compared to the group in which mutations were only present in tDNA (mean 3.6 cm; p=0.005). To improve the analytical sensitivity in the ctDNA and confirm the current findings, all cases will be analyzed using digital droplet PCR. Molecular barcodes will also be incorporated into our custom panel. In this proof-of-principal study, we have demonstrated that relevant mutations can be detected in the plasma of a subset of patients when endometrial carcinomas are confined to the uterus.

#2736

A digital droplet PCR assay for the quantitation of androgen receptor and splice variant expression in CTCs from metastatic castration resistant prostate cancer patients.

Ada Gjyrezi,1 Giuseppe Galletti,1 Areti Strati,2 Seaho Kim,1 Evi Lianidou,2 David M. Nanus,1 Jun Luo,3 Emmanuel Antonarakis,3 Scott T. Tagawa,1 Andrew Armstrong,4 Paraskevi Giannakakou1. 1 _Weill Cornell Medicine, New York, NY;_ 2 _University of Athens, Athens, Greece;_ 3 _John Hopkins Medicine, Baltimore, MD;_ 4 _Duke University School of Medicine, Durham, NC_.

Prostate cancer (PC) is the second leading cause of cancer death in men in the US. The aberrant functioning of androgen receptor signaling is the central driving force behind prostatic tumorigenesis and its transition into metastatic castration resistant disease. Hence, androgen deprivation therapy (ADT) is the first line of treatment for PC patients. However, many patients progress becoming resistant to ADT therapy, due to the expression of AR splice variants (AR-Vs), which lack the ligand binding domain and are constitutively active in the nucleus. Expression of the AR splice variant, AR-v7, in circulating tumor cells (CTCs) isolated from the blood of PC patients was correlated with resistance to enzalutamide and abiraterone, which are the next generation AR signaling inhibitors in CRPC. Further, there is evidence that AR-Vs may convey cross-resistance, not only to enzalutamide and abiraterone, but also to taxanes, highlighting that their assessment in the clinic may have clinical utility.

We developed a novel, specific and highly sensitive assay to measure mRNA expression of the AR full length (AR-FL) and the splice variants ARv7 and ARv567es, by using Droplet Digital PCR in CTCs isolated from CRPC patients. The analytical specificity of the assay was determined by transfecting cells with plasmids encoding AR-FL, AR-v7 and AR-v567 and showed that each probe detected signal only in cells expressing the respective transcript. No signal was detected against genomic DNA, indicating lack of non-specific binding. Also, the assay detected endogenous expression of AR-FL and AR-v7 in VCAP or 22RV1 cells, while no variant expression was detected in healthy donor blood. The analytical sensitivity of the assay was determined in a series of serial dilution experiments that showed sensitivity down to single cell.

We then used this assay to determine the clinical prevalence and expression pattern of each of these variants in CTCs from about 200 mCRPC patient samples and blood from 40 healthy donors. CTCs were enriched by EpCAM- or PSMA-based positive selection or CD45 negative depletion in an antigen-agnostic manner. AR-FL was detected in ~80% of mCRPC samples irrespective of CTC-enrichment technology. AR-v7 was expressed in 65% of the samples in which in CTCs were enriched either by PSMA-positive selection or by negative depletion. In contrast, EpCAM-based CTC enrichment showed lower AR-v7 expression both in terms of expression levels and prevalence. In addition, CTC enrichment following negative depletion showed that 30% of the samples had higher AR-v7 expression levels as compared to AR-FL. This expression pattern was not observed in the samples using EpCAM-based selection. Collectively, these data suggest distinct CTC subpopulations are present in CRPC patient samples, with differential expression of AR-Vs that could have important predictive and prognostic implications.

#2737

Development of a clinically actionable, ultra-sensitive multi-oncogene panel compatible with ctDNA testing in urine and plasma.

Karena A. Kosco,1 Matthew Wiggin,2 Peter J. Croucher,1 David Broemeling,2 Shiloh Guerrero,1 Laura Mai,2 Kurt Krummel,1 Victoria M. Raymond,1 Mark G. Erlander,1 Andre Marziali,2 Karsten Schmidt1. 1 _Trovagene, Inc, San Diego, CA;_ 2 _Boreal Genomics, Vancouver, British Columbia, Canada_.

Background: Circulating tumor DNA (ctDNA) is rapidly emerging as a viable alternative to tissue molecular diagnostics for the detection of actionable oncogenic mutations. In non-small cell lung cancer, identifying the emergence of EGFR T790M in patients receiving first line EGFR tyrosine-kinase inhibitors (TKIs) is a clinical need. Obtaining a tissue biopsy to determine T790M status is associated with significant cost and patient morbidity. In a retrospective analysis of the TIGER-X trial, patients had similar therapeutic response to a 3rd generation TKI, independent of the sample type positive for T790M; tissue, plasma, or urine. This indicates that non-invasive urine liquid biopsy is a viable diagnostic option (Wakelee et al, ASCO, 2016). We report on the expansion of single mutation assays to a multiplex-panel of clinically actionable mutations having analytical properties with similar ultra-sensitive detection.

Methods: We previously developed two next generation sequencing methodologies with high sensitivity and specificity for ctDNA detection: 1) A series of individual ultrasensitive ctDNA hotspot assays capable of detecting ultra-short DNA fragments (31-45 base pairs) and a 0.01% lower limit of detection (LLoD). The short sized amplicons coupled with mutant allele enrichment enabled high clinical sensitivity for plasma and urine ctDNA (Reckamp et al, J Thorac Oncol., 2016) 2) A multiplex panel using sequence-specific synchronous coefficient of drag alteration (SCODA) technology that enriches for mutant ctDNA to obtain a LL0D of 0.001%-0.03%, (Pel et al, PNAS, 2009; Kidess et al, Oncotarget, 2015). By combining a multiplex of short amplicons with SCODA enrichment capabilities, we developed an assay enriched for over 200 clinically actionable driver and resistance mutations within 7 oncogenes.

Results: An iterative process was used to design and optimize primer and enrichment probe length and placement to produce a panel where mean coverage across hotspot regions was within 2 to 3-fold (read uniformity) and greater than 1,000-fold enrichment of mutant DNA prior to sequencing. Technical validation was performed utilizing in-house and commercially available model systems for plasma and urine demonstrating mutation detection from inputs ranging from hundreds to fewer than 10 copies and LLoD as low as 0.001%. Clinical performance evaluation of the newly developed multiplex assay is ongoing using urine and plasma ctDNA from patients with advanced cancer.

Conclusions: High sensitivity is critical when identifying targetable driver mutations as well as emergence of resistance mutations which can inform therapeutic decision making. By combining short amplicon and mutation enrichment technology, we developed an ultra-sensitive ctDNA assay for use in sample types with highly degraded and fragmented DNA.

#2738

A commutable circulating tumor DNA (ctDNA) reference material.

Yves Konigshofer,1 Farol L. Tomson,1 Matthew Ryder,1 Russell Garlick,2 Bharathi Anekella1. 1 _SeraCare Life Sciences, Inc., Gaithersburg, MD;_ 2 _SeraCare Life Sciences, Inc., Milford, MA_.

We developed and assessed a novel ctDNA reference material that was designed with an emphasis on commutability. Possessing qualities that are well-matched with clinical specimens is critical for a reference material, however many existing methods of preparing ctDNA analogues result in material that is not commutable for certain assays. For example, sonication of genomic DNA yields fragments across a broad range of sizes that must be used at greater input amounts relative to native cell-free DNA (cfDNA) - even after enzymatic end-repair to reduce damage. This can affect the utility of the reference material for assays that depend on the ligation of adapters to double-stranded DNA, such as many hybrid/capture-based Next Generation Sequencing (NGS) assays. If higher input amounts are not used, then the LOD and precision of such an assay may appear substantially poorer than expected. At the same time, sonicated DNA has been shown to be acceptable for amplicon-based ctDNA assays.

We created commutable ctDNA reference material by starting with genomic DNA from GM24385 cells and mixing with synthetic variant-containing DNA sequences at defined ratios. The use of GM24385 cells allows for the comparison of detected variants against the NIST RM 8391 high confidence variants. The use of synthetic DNA permits addition of large numbers of different cancer-associated variants at defined allele frequencies. Fragmentation was followed by size-selection in order to obtain ctDNA-like sizes. Additional steps were performed to increase the amount of output material from the size-selection step. The general method of preparing the reference material is also compatible with native cfDNA, such as that isolated from cancer patients.

The reference material was submitted to several testing laboratories along with similar sonicated material. When tested using hybrid/capture-based assays, variant detection was superior in the novel reference material compared to sonicated DNA when similar amounts of material were used as the input. We therefore conclude that our new reference material has greatly improved commutability compared to existing materials composed only of sonicated DNA.

#2739

Low-pass whole genome sequencing detects copy number variations in circulating tumor DNA.

Xiaoji Chen, Jill M. Spoerke, Kathryn Yoh, Walter C. Darbonne, Ling-Yuh Huw, Steven Gendreau, Shih-Min A. Huang, Mark R. Lackner. _Genentech, South San Francisco, CA_.

Circulating tumor DNA (ctDNA) is released from necrotic/apoptotic tumor cells into the bloodstream. Recent studies have demonstrated the value of using ctDNA as biomarkers in cancer diagnosis, prognosis, and drug resistance. Unlike local tissue biopsy, ctDNA collection and analysis is non-invasive, allows continuous monitoring of clonal evolution, and provides an overview of tumor heterogeneity. Copy number variations (CNVs) play an important role in cancer biology. However, traditional CNV analyses of ctDNA using droplet digital PCR (ddPCR) and SNP arrays can only assess a small number of genes due to the low abundance of ctDNA in the majority of patient samples. Next generation sequencing (NGS) offers a more efficient and high-throughput way to study CNVs in ctDNA.

Here, we evaluated the use of low-pass whole genome sequencing (WGS) in determining CNVs in ctDNA. In this work, cell-free DNA was isolated from 1-5 ml of plasma from phase II clinical trial patients with metastatic breast cancer and non-small cell lung cancer (NSCLC) using the QIAamp Circulating Nucleic Acid Kit (QIAGEN). DNA yield was determined by ddPCR, with a range from 2-4000 ng. Sequencing libraries were prepared using 2-10 ng DNA by the ThruPLEX Plasma-Seq Kit (Rubicon Genomics). WGS at 0.1x, 0.25x, 0.5x, and 1x coverage was performed on Illumina NextSeq, and data was analyzed using Nexus Copy Number software (BioDiscovery). As a reference, we sequenced DNA from peripheral blood mononuclear cells (PBMCs) of 20 healthy donors at the same coverage. Low-pass WGS was also performed on a subset of matched tumor tissue samples as comparisons.

Our results showed that WGS of ctDNA at 0.5x coverage was efficient to identify CNVs. CNVs were detected in ctDNA from about half of the patients analyzed, and in general CNVs identified in ctDNA matched the ones found in tumor tissue from the same patient. We also found that CNV patterns from different time points of the same patients clustered together. With this promising system, we will present CNV analysis in ctDNA from breast cancer and NSCLC patients enrolled in phase II clinical trials of the PI3K inhibitor pictilisib. We will evaluate the ability of the method to classify patients into different subgroups, monitor tumor progression, and identify drug resistance mechanisms.

#2740

High-throughput and sensitive quantification of circulating tumor DNA.

Yinghui Guan, Oleg Mayba, Shan Lu, Thomas Sandmann, Younjeong Choi, Walter Darbonne, Vincent Leveque, Lisa Ryner, Eric Humke, Nga W. Tam, Sundari Sujathasarma, Anna Cheung, Richard Bourgon, Yulei Wang. _Genentech, A Member of the Roche Group, South San Francisco, CA_.

Circulating tumor DNA (ctDNA) has a great potential to serve as non-invasive biomarkers for monitoring disease progression and treatment response. However, its broad clinical applications have been hindered by sensitivity, throughput and complexity of existing ctDNA detection methods. Here we report an adaptation of a multiplex PCR pre-amplification (PreAmp) into the microfluidic multiplex PCR (MMP-Seq) technology for high-throughput and sensitive quantitation of ctDNA. We have demonstrated that PreAmp MMP-Seq enabled sensitive (down to 0.6% allele frequency), reproducible (R2=0.97), accurate and quantitative mutation detection from as low as 1.6 ng DNA input. An Empirical Bayesian Model has been established to estimate background error rates for every base of amplicons in the ctDNA panel using data from 43 normal plasmas. 0.1-0.3% of median error rates were observed across all positions. 149 longitudinal plasma samples from 12 ovarian and 10 pancreatic cancer patients of PhI DMUC5754A anti-MUC16 ADC trials were then profiled using PreAmp MMP-Seq. 79% (11/14) driver mutations found in tissue samples were also identified in matching baseline plasma samples at frequencies well above estimated error rates. Moreover, we found that changes of corresponding ctDNA concentration in post-treatment plasma samples were correlated with changes in tumor burdens and circulating protein marker levels. In conclusion, the MMP-seq PreAmp workflow provides an inexpensive, reproducible and flexible solution for mutation detection in plasma DNA samples.

#2741

Assessing the utility of circulating tumor DNA as a surveillance tool for sarcomas and Li-Fraumeni syndrome using a pre-clinical model.

Sangeetha Paramathas, Nathan Lewis, Tanya Guha, Zainab Motala, David Malkin. _The Hospital for Sick Children, Toronto, Ontario, Canada_.

Li-Fraumeni Syndrome (LFS) is a hereditary cancer predisposition syndrome commonly characterized by the presence of inherited mutations in the tumor suppressor gene TP53. This leads to an early onset of a wide spectrum of tumors in multiple organ systems. Current surveillance protocols for early tumor detection include biochemical screening, MRI and ultrasound scans, colonoscopy and mammography (for adults). While early detection is associated with improved survival, the complexity of testing, potential to 'misdiagnose' tumors (false positive/negative) and requirement for multiple imaging modalities makes clinical surveillance challenging to implement and interpret. Liquid biopsies are a recently described diagnostic and prognostic tool that takes advantage of analyzing circulating tumor DNA - fragmented genomic material that are released into the blood from dying tumor cells.

In this proof-of-principle study, we use mouse xenograft tumor models to assess the dynamic relationship between tumor burden and ctDNA concentrations, as well as resolve the sensitivity of capturing the presence of lesions in their earliest stages of growth. Longitudinal analyses of ctDNA from serially collected blood were performed on mice with xenografts of rhabdomyosarcoma (Rh4 and Rh30), osteosarcoma (HOS) and non-small cell lung carcinoma (H1975) of both localized and simulated metastatic tumors. We characterized ctDNA using droplet digital PCR (ddPCR) for known gene mutations that were specific for the cancer cell lines used. We observe obvious increases in ctDNA with increased tumor burden and a complete clearance of ctDNA after tumor resection. In addition, using the metastatic model, we have conducted synchronized imaging and blood-based biopsies to determine the smallest lesion able to be detected in the blood by ctDNA.

These studies provide the foundation for early tumor detection with ctDNA in Trp53 mutant mice that develop spontaneous tumors analogous to LFS. This model will help further our understanding on the utility of ctDNA as a surveillance and diagnostic tool for LFS as well as assess its potential for clinical use.

#2742

**Cross-platform comparison of four leading technologies for detecting** EGFR **mutations in circulating tumor DNA from plasma of patients with non-small cell lung carcinoma.**

Ting Xu,1 Xiaozheng Kang,1 Xiaofang You,1 Dai Liang,1 Dequan Tian,1 Wanpu Yan,1 Yongbo Yang,1 Hongchao Xiong,1 Zhen Liang,1 Grace Q. Zhao,2 Shengrong Lin,2 Ke-Neng Chen,1 Guobing Xu1. 1 _Peking University Cancer Hospital and Institute, Beijing, China;_ 2 _AccuraGen, Menlo Park, CA_.

Analysis of circulating tumor DNA (ctDNA) is emerging as a powerful tool for guiding targeted therapy and monitoring tumor evolution in patients with non-small cell lung cancer (NSCLC), particularly when fresh tissue biopsy is not available. This study compared the ability of four leading technology platforms to detect epidermal growth factor receptor (EGFR) mutations (L858R, exon 19 deletion, T790M and G719X) in ctDNA from NSCLC patients. The platforms included two amplification refractory mutation systems (cobas-ARMS and ADx-ARMS), a droplet digital polymerase chain reaction platform (ddPCR) and a next-generation sequencing platform (Firefly NGS). Fifteen EGFR mutations across twenty NSCLC patients were identified. We observed superior sensitivity and specificity of cobas-ARMS, ddPCR and Firefly NGS platforms, while ADx-ARMS was only suitable for the qualitative detection of EGFR mutations with allele frequency higher than 1% in plasma samples. We observed high concordance between the plasma and tissue EGFR mutational profiles for three driver mutations that are known targets of the first generation EGFR-TKI therapy (L858R, E19-dels, and G719X). Discrepancies between plasma and tissue EGFR mutational profiling could be attributed to spatial and temporal tumor heterogeneity. This pilot study illustrates the promise of ctDNA analysis in the context of treatment evaluation and drug resistance detection, and results will be validated in follow-up studies.

#2743

Comparison of enhanced Tagged-Amplicon Sequencing and digital PCR for circulating tumor DNA analysis in advanced breast cancer.

Isaac Garcia-Murillas,1 Matthew Beanney,1 Michael Epstein,2 Karen Howarth,2 Andrew Lawson,2 Sarah Hrebien,1 Emma Green,2 Nitzan Rosenfeld,2 Nick Turner1. 1 _The Institute of Cancer Research, London, United Kingdom;_ 2 _Inivata, Cambridge, United Kingdom_.

Background Circulating tumor DNA (ctDNA) analysis allows non-invasive detection of tumor mutations and amplifications in advanced breast cancer. Multiple technologies have been developed to analyse ctDNA and here we compared two leading ctDNA detection technologies, enhanced Tagged-Amplicon Sequencing™ (eTAm-Seq™) and digital PCR (dPCR) assays, in advanced breast cancer.

Methods We recruited a cohort of 35 women with advanced breast cancer, of whom 23 had two separate blood samples taken in a standard EDTA tube processed immediately or in preservative Streck tubes processed up to 120 hours after venipuncture. Digital PCR was conducted with assays for hotspot actionable mutations in 3 known drivers in breast cancer: PIK3CA exon 9 and 20, ESR1 ligand binding domain and AKT1 (c.49G>A; p.E17K), and ctDNA sequencing was conducted with eTAm-Seq method using a 35-gene panel including cancer hotspots, entire coding regions and copy number variants (CNVs).

Results Across both assays, 37 mutations were detected in 35 patients, with PIK3CA mutation in 13 patients (37%), ESR1 mutations in 10 patients (29%), and no AKT1 mutations. ESR1 mutations were polyclonal in 8 patients, with ctDNA eTAm-Seq method revealing substantially more diversity in mutations, with up to 8 individual mutations detected in a patient. There was 96.15% agreement for PIK3CA mutation detection between assays (Kappa 0.89, 95% CI 0.743 to 1.000), 100% agreement for ESR1 mutations (Kappa 1.00, 95% CI 1.000 to 1.000). There was very high correlation in mutation allele frequency between eTAm-Seq and dPCR (r=0.93, 95%CI 0.86 to 0.96, p<0.0001). The sensitivity and specificity for HER2 amplification detection by eTAm-Seq was 100% compared to tumor HER2 status. Comparison of immediate processing and Streck tubes revealed 97.92% agreement (Kappa 0.95, 95% CI 0.868 to 1.000) for mutation calling.

Conclusions This study demonstrates that ctDNA analysis using eTAmSeq and dPCR have very high agreement in mutation detection in patients with advanced breast cancer patients. Streck tubes present a robust alternative to immediate processing of samples. eTAm-Seq and digital PCR have high clinical validity in mutation detection.

#2744

Inter-site comparison of performance of an ultra-sensitive sequencing technology for circulating tumor DNA.

Stephanie Yaung,1 Alex Lovejoy,1 Dan Klass,1 Fergal Casey,1 Aruna Arcot,1 Melissa Loyzer,1 William Chiu,2 Nabil Azhar,2 Sophie Beckert,3 Maria Lange,3 Sandra Siemann,3 Sebastian Froehler,3 Shelly Gunn,2 John F. Palma1. 1 _Roche Sequencing Solutions, Pleasanton, CA;_ 2 _ResearchDx, Irvine, CA;_ 3 _Roche Sequencing Solutions, Potsdam, Germany_.

The combination of liquid biopsies and ultra-sensitive next-generation sequencing (NGS) holds tremendous promise in oncology for the detection and surveillance of somatic mutations that correlate with diagnosis, prognosis, response prediction, resistance monitoring and tumor burden. We have developed a capture-based NGS workflow that provides ultra-sensitive detection of 4 mutation classes - single nucleotide variants (SNVs), insertions/deletions, copy number amplifications (CNAs) and fusions - from low input cell-free DNA isolated from plasma. Sensitivity and specificity of 2 different AVENIO ctDNA Analysis Kits (for Research Use Only) across 3 sites for 48 samples that included contrived DNA blends with known mutations validated by digital PCR and normal reference plasma samples were assessed. The AVENIO ctDNA Targeted Kit (Targeted) was designed to detect mutations in 17 genes, across 80kb, included in NCCN Guidelines for solid tumors plus additional variants relevant for therapy selection. The AVENIO ctDNA Expanded Kit (Expanded) was designed to detect mutations in 77 genes, across 200kb, associated with solid tumor including variants relevant for clinical trial research. Testing sites achieved 100% sensitivity for SNVs and fusions at 0.5% and 1% allelic frequency, respectively, from 30 ng of input DNA for both kits. Median coverage of 4,200-6,400X was greater with the Targeted kit relative to that of 2,700-3,800X with the Expanded kit. Specificity was 100% for the Targeted kit either by sample or variant. The Expanded kit had a specificity of 91.7-100% by sample and 99.7-100% by variant. At an expected 5X or 8X copy number amplification for multiple genes, both the Targeted and Expanded kits were 100% sensitive and 93.1-98.4% specific per gene. The specificity for fusion detection was 100% for both kits down to 1% mutant allele frequency. The AVENIO ctDNA Analysis Kits for plasma and NGS applications provide exquisite sensitivity and specificity for the simultaneous detection of 4 different mutation classes.

#2745

Evaluation of a new system for collection, stabilization, and purification of circulating tumor DNA.

Michael Fleischhacker,1 Bernd Schmidt,2 Rohan Baker,3 Susanne Pedersen,3 Natasha Cant,4 Maryam Zahedi-Nejad,4 Thorsten Voss,5 Andrea Ullius,5 Daniel Groelz5. 1 _University of Halle, Germany;_ 2 _DRK-Kliniken Berlin-Mitte, Germany;_ 3 _Clinical Genomics, North Ryde, Australia;_ 4 _QIAGEN Ltd., Manchester, United Kingdom;_ 5 _PreAnalytiX GmbH, Hilden, Germany_.

Introduction:

The current preanalytical workflows for circulating tumor DNA (ctDNA) analysis have limitations that affect the accurate detection and quantification of these plasma cancer biomarkers. Release of genomic DNA (gDNA) from white blood cells (WBCs) due to cell lysis or apoptosis during whole blood storage in EDTA tubes creates higher gDNA background levels, affecting the sensitivity of ctDNA assays. Current tubes that stabilize WBCs often contain crosslinking reagents, which have negative effects on sensitive downstream assays, including methylation-based assays.

Using ctDNA assays, the new PAXgene® Blood ccfDNA System*, consisting of a blood collection tube with unique, non-crosslinking chemistry and an automated circulating cell-free DNA (ccfDNA) extraction kit, was evaluated in three research studies.

Methods:

Blood samples were collected into paired PAXgene and EDTA tubes and stored for 7 days at room temperature (RT). ccfDNA was isolated from plasma using the PAXgene kit or the QIAGEN QIAamp® Circulating Nucleic Acid Kit.

Study 1: The ccfDNA from lung cancer patients was quantified by real-time PCR for the amount of the ERV sequence (as a measure of the total plasma DNA quantity) and after bisulfte treatment for mSHOX2 as a marker for ctDNA.

Study 2: Blood from healthy donors was spiked with fragmented, fully-methylated CpGenome DNA. During storage, tubes were intermittently inverted to simulate tubes in transit. Subsequent to bisulfite conversion, PCR assays targeting ACTB and methylated BCAT1 and IKZF1 DNA were used to determine the yields of ccfDNA and ctDNA, respectively.

Study 3: Restriction enzyme treated EGFR DNA containing exon 19 deletions and exon 20 and 21 substitutions (T790M, L858R) were spiked into healthy donors' blood. ccfDNA was tested with the QIAGEN therascreen® EGFR Plasma RGQ PCR Kit*.

Results:

Both study 1 and 2 demonstrated constant levels of the methylation ctDNA markers, SHOX2, BCAT1 and IKZF1, over the investigated time course. There was no significant release of gDNA in the PAXgene tube whereas a significant release of gDNA was detected in EDTA samples. Likewise, study 3 showed constant EGFR Ct values in the PAXgene system with reliable mutation detection, whereas the high DNA concentration from the EDTA system resulted in false-positive callings.

Conclusions:

The new system allows researchers to accurately detect and quantify plasma cancer biomarkers from blood samples that have been stored in the tube for up to 7 days at RT. This includes challenging assays based on methylated ctDNA. The system provides the required assay sensitivity to allow the correct assay interpretation beyond the typical 3–6 hour storage limit for EDTA tubes.

*For Research Use Only. Not for use in diagnostic procedures.

#2746

Detection of tumor-specific mutations in plasma DNA: A potential esophageal adenocarcinoma biomarker.

Matthew Egyud,1 Jennifer Jackson,1 Emiko Yamada,1 Anders Ståhlberg,2 Paul Krzyzanowski,3 Virginia Litle,1 Lincoln Stein,3 Tony Godfrey1. 1 _Boston Medical Center, Boston, MA;_ 2 _University of Gothenburg, Gothenburg, Sweden;_ 3 _Ontario Institute for Cancer Research, Ontario, Canada_.

BACKGROUND: Recent studies have shown that tumor-specific DNA from multiple types of tumors can be detected circulating in plasma and this has raised the possibility of "liquid biopsies" using mutated tumor DNA as a potential diagnostic and prognostic biomarker. Detection of mutations with allele frequencies below 0.1% remains challenging however given that circulating cell-free DNA is highly degraded and in low abundance. Detection of multiple different mutations in the same sample presents an additional challenge particularly when the mutation panel may change from patient to patient. We have developed a novel approach, called SimSen-Seq, to introduce molecular barcodes into sequencing libraries with DNA inputs as low as 5ng. Barcodes enable differentiation of true mutants from background noise introduced by Taq polymerase errors and permits detection of variant alleles with frequencies below 0.1%. The barcodes are protected from mis-priming using a hairpin structure which permits a high degree of multiplexing and flexibility for detection of multiple mutations from one plasma sample. We are using this technology to test the utility of liquid biopsy as a biomarker for esophageal adenocarcinoma (EAC) diagnosis and disease monitoring.

METHODS: Blood samples were obtained at a single time point from patients with various stages of EAC and longitudinal blood samples were also collected from patients undergoing neoadjuvant therapy followed by surgery. Tumor samples were obtained from biopsy or resection specimens. Tumor DNA was sequenced using a targeted EAC panel to identify mutations in each tumor. SimSen-Seq assays were designed to identify these mutations in plasma, and hairpin barcodes were attached. Sequencing libraries were generated from circulating DNA, sequenced and analyzed using the barcodes to reduce background noise.

RESULTS: Mutations were identified in tumor samples from 37 patients. To date, 29 patients have had plasma analyzed; 5 stage I, 6 stage II, 13 stage III, and 5 stage IV. Of these 29, the same mutations have been identified in 15 plasma DNA samples (20% stage I, 50% stage II, 54% stage III, 80% stage IV. Six patients demonstrated multiple mutant alleles in plasma DNA. One patient with detectable pre-treatment ctDNA underwent serial blood draws during their treatment course, and post-operative detection of tumor-specific markers preceded physical evidence of disease recurrence.

DISCUSSION: SimSen-Seq shows promise as a novel ultra-sensitive, highly multiplexed sequencing method for identifying rare circulating mutations. Possible applications include prognostication in early stage patients and rapid monitoring of therapeutic response and recurrence. Further work is to evaluate this is ongoing.

#2747

Plasma DNA methylation marker and hepatocellular carcinoma risk prediction model for the general population.

Hui-Chen Wu,1 Hwai-I Yang,2 Qiao Wang,1 Chien-Jen Chen,2 Regina M. Santella1. 1 _Columbia Univ. School of Public Health, New York, NY;_ 2 _Genomics Research Center, Academia Sinica, Taipei, Taiwan_.

Metastases in the later stages of hepatocellular carcinoma (HCC) cause the majority of deaths associated with the disease, making early detection crucial to patient survival. Risk models that assess HCC risk in the general population can be used for risk stratification for further HCC surveillance, however, none have been validated externally. Methylation of circulating DNA shows potential for noninvasive and early diagnosis of HCC. Most evidence, however, comes from cross-sectional studies raising concerns about temporality. We conducted a prospective case-control study nested within a community-based cohort. We measured methylation levels in of 6 genes (CDKN2A, RASSF1A, STEAP4, TBX2, VIM, and ZNF154) using pre-diagnostic plasma DNA from 237 HCC cases and 257 matched controls. We found TBX2 hypermethylation was associated with increased HCC risk, with ORs (95%CI) of 3.7 (1.9-7.2). The associations were mainly among high-risk subjects; among subjects infected with HBV/HCV, the OR (95%CI) of TBX2 methylation was 5.3 (2.2-12.7). Among subjects with high risk, the ORs (95%CIs) were 8.9 (1.0-73.9) for Wen-HCC score ≥16, 5.6 (2.1-14.6) for Hung-HCC score ≥15, and 7.2 (2.1-24.4) for Michikawa-HCC ≥8. Adding information on TBX2 methylation improved the accuracy of risk models for a high risk population, with the area under the curve (AUC) of 74% for TBX2 methylation and Wen-HCC score compared with 64% with Wen-HCC score alone. The AUCs were 63% for Hung-HCC score and TBX2 methylation, 53% for Hung-HCC score alone, 65% for Michikawa-HCC score and TBX2 methylation and 59% for Michikawa-HCC risk alone. Our findings suggest the potential increase in risk assessment discrimination and accuracy from incorporation of DNA methylation.

#2748

Liquid biopsy using extracellular vesicular DNA for EGFR mutation testing in the patients with NSCLC.

Jae Young Hur,1 Hee Jung Kim,1 Chang-Min Choi,2 Jae Cheol Lee,2 Kye Young Lee1. 1 _Konkuk University Medical Center, Seoul, Republic of Korea;_ 2 _Asan University School of Medicine, Seoul, Republic of Korea_.

Background Tumor-derived extracelluar vesicles (EV) have been proved to carry double-stranded DNA including oncogenic mutants and expected to be a novel biomarker in cancer detection. Liquid biopsy using cell-free (cf) DNA for EGFR mutation testing is being actively investigated. In this study, we investigated whether liquid biopsy using EV DNA is superior to cf DNA for EGFR mutation testing in the patients with NSCLC.

Methods EV was isolated using ultracentrifugation and confirmed by transmission electron microscopy and zetasizer in 20 plasma samples (all tissue-proven EGFR mutated) and 32 bronchoalveolar lavage fluid (BALF) samples (23 EGFR-mutated including 14 TKI-naïve and 9 TKI resistant, and 9 wild-type). EV and cf DNA was separately extracted from each samples and EGFR mutation testing was performed by peptide nucleic acid (PNA)-clamping method.

Results In 20 plasma samples, the concordance rate of EV DNA for tissue EGFR genotyping was 55% (11/20), while 30% (6/20) in cf DNA. In 32 BALF samples, however, the sensitivity, specificity and concordance rate of EV DNA for activating EGFR mutations was 100% (23/23), 100% (9/9), and 100% (32/32), while cf DNA showed 73.9% (17/23), 100% (9/9) and 81.3% (26/32), respectively. The kappa value (p-value) of EV DNA was 1.0 (<0.001), while it was 0.714 (p,0.001) in the case of cf DNA. As for 9 re-biopsy cases for T790M detection in which only 3 patients (33%) was proved as positive T790M, BALF EV DNA identified 2 more T790M patients (total 5 among 9 patients, 56%) without non-matching with tissue typing, while BALF cf DNA found 1 more T790M positive patient with missing 1 tissue proven case (total 3 among 9 patients, 33%)

Conclusions Liquid biopsy using EV DNA is significantly superior to cf DNA in both plasma and BALF. A little low sensitivity in plasma EV DNA is supposed to be improved with much more sensitive method such as ddPCR or BEAMing. Liquid biopsy using BALF EV DNA showed almost 100% matching with tissue EGFR typing and seems to be highly promising for T790M detection. A large number of clinical trials should be prompted.

#2749

Liquid biopsy testing allows highly-sensitive detection of plasma cfDNA mutations in 87 breast cancer-related genes.

Maruja E. Lira, Tao Xie, Shibing Deng, Jennifer Kinong, Jingjin Gao, Zhou Zhu, Nathan Lee, Paul Rejto, Jadwiga Bienkowska, James Hardwick, Kai Wang, Stephen Huang. _Pfizer, Inc., San Diego, CA_.

Liquid biopsies have the potential to revolutionize the way physicians select personalized anti-cancer therapies, monitor patient responses to treatment, and characterize acquired resistance to cancer drugs. New tests that use a simple peripheral blood draw offer snapshots of a patient's total tumor DNA mutation profile and are attractive because of their minimally-invasive modality and because they integrate information from both primary and metastatic disease. Currently, most plasma cell-free DNA (cfDNA) mutation detection tests used in clinical research detect known hotspot mutations in a limited number of genes. Technologies that interrogate multi-gene panels in cfDNA are advancing, but commercially-available options suitable for clinical use are limited, come at a high cost, and are not customizable. We designed and developed a customized, next generation sequencing-based, liquid biopsy assay capable of detecting somatic mutations in 87 breast cancer genes involved in cell cycle and estrogen receptor signaling. Targeted regions (147 Kb) were enriched using hybrid capture resulting in an average capture specificity and uniformity of 65.93% and 96.38%, respectively. When tested on cfDNA from healthy donors (n=14), we demonstrated a level of specificity >99.99%. Analytical sensitivity of 0.1% was established on HapMap and reference mutant cell line DNA. Using a pool of HapMap genomic DNA (n=10), 96% (48/50) of SNPs with expected allele frequency of 0.1% were detected. In reference mutant cell line DNA with 1% or 0.1% mutant allele frequencies, we were able to reliably detect all mutations present at 1% and mutations at 0.1% in 50% of the cases. Assay validation on plasma cfDNA with matching tumor from ER+, HER2- breast cancer patients will be presented. In conclusion, we developed a highly sensitive and specific liquid biopsy assay to interrogate 87 breast cancer-related genes. The high level of specificity and sensitivity makes the test ideal not only for detecting known cancer gene hotspot mutations but also for detection of novel gene mutations that may arise during treatment as a result of acquired drug resistance.

#2750

Detecting resistance mechanisms in patients on EGFR TKI treatment by liquid biopsy.

Laure Sorber,1 Karen Zwaenepoel,1 An Wouters,2 Janssens Annelies,3 Birgitta Hiddinga,3 Jan Van Meerbeeck,3 Filip Lardon,2 Christian Rolfo,3 Patrick Pauwels1. 1 _Antwerp University (UA) / Antwerp University Hospital (UZA), Wilrijk, Belgium;_ 2 _Antwerp University (UA), Wilrijk, Belgium;_ 3 _Antwerp University Hospital (UZA), Edegem, Belgium_.

The rise of liquid biopsy, including the molecular analysis of circulating cell-free tumor DNA (ctDNA), has enabled real-time follow up of cancer patients who receive targeted therapy.

Here, we report the case of a lung cancer patient (adenocarcinoma), initially harboring the activating EGFR L858R mutation. Blood and tissue samples were collected during treatment with the EGFR tyrosine kinase inhibitor (TKI) gefitinib, and during the appearance of resistance. Analysis of the tissue biopsies consisted of EGFR mutational analysis by high-resolution melting analysis (HRMA) (all tissue samples), immunohistochemistry (IHC) of cMET (sample 2); FISH analysis of the cMET gene (sample 1 and 3); and next-generation sequencing (NGS) (sample 3). We performed digital droplet PCR (ddPCR) to detect EGFR mutations and cMET amplification in both tissue and plasma samples. To the best of our knowledge, this is the first time that ddPCR is used to screen for cMET amplification in ctDNA.

The HRMA analysis of the biopsy during progression under EGFR TKI treatment revealed the EGFR T790M resistance mutation. Progression was also seen by radiological examination and this resulted in discontinuation of gefitinib treatment. As a third generation EGFR TKI was not available by that time, the patient was retreated with carboplatin plus pemetrexed, which resulted in a mixed response. In the second biopsy, only the EGFR activating L858R mutation was detected by HRMA. IHC analysis revealed a strong membrane positivity for cMET in 100% of the tumor cells. After progression upon rechallenging the patient with erlotinib, treatment with osimertinib (third generation EGFR TKI) was initiated. This resulted in only a partial response, with some strongly progressing lesions. NGS analysis of the third tissue biopsy indicated the presence of both the EGFR L858R and T790M mutations, while ddPCR analysis of the plasma sample only detected the EGFR L858R mutation. Due to the fast progression, it was opted to also screen for cMET amplification. DdPCR cMET analysis of the tissue and plasma resulted in a copy number variation of 10.0 and 3.7, respectively. FISH analysis confirmed the cMET amplification with a ratio of 3.1 and an average of 8.4 cMET-signals per nucleus.

The results of the ddPCR EGFR and cMET analysis of both the plasma and tissue samples are now being processed to determine the course of the mutational load and the best course of treatment.

As such, real-time follow-up of cancer patients is crucial to detect the underlying mechanisms of resistance to targeted therapies. Correlation of molecular analysis with the disease course is key. Our case study highlights the potential of ctDNA analysis for detection of both the EGFR T790M resistance mutation and cMET amplification. Hence, tissue biopsies can be supplemented by liquid biopsies in the screening for resistance mechanisms.

#2751

**Automatic DNA extraction system can improve the** EGFR **point mutation detection rate of liquid biopsy.**

Chiho Nakashima,1 Akemi Sato,1 Tomonori Abe,1 Tomomi Nakamura,1 Kazutoshi Komiya,1 Eisaburo Sueoka,1 Shinya Kimura,1 Naoko Sueoka-Aragane,1 Junichi Kato,2 Mitsuharu Hirai2. 1 _Saga University, Saga city, Japan;_ 2 _ARKRAY Inc., Kyoto, Japan_.

The usefulness of liquid biopsy to detect mutations from cancer patients has been well recognized today. However, because the mutation detection rates from plasma DNA were relatively lower than those of tissue re-biopsy, its clinical utility has not been confirmed yet. As previously we reported, we have developed fully automatic high-sensitive point mutation detecting system named mutation-biased PCR and quenched probe (MBP-QP) system for liquid biopsy. Recently, the importance of pre-analytical procedures for plasma DNA anazysis has been highlighted. In this study, we examined whether the automatic DNA extraction system can improve the mutation detection rate in our MBP-QP system. Sixty-one plasma samples were obtained from advanced non-small cell lung cancer patients, and plasma DNA extraction was performed from 200μl plasma by manually (200-M), and 200μl (200-A), 1000μl (1000-A) plasma by automatically. We used silica membrane spin column system for manual DNA extraction, and magnet beads system for automatic DNA extraction procedure. The median DNA concentrations quantified by quantitative real-time PCR of 200-M, 200-A, 1000-A were 4.92, 6.00, 20.1 ng/mL plasma, respectively. In terms of the epidermal growth factor receptor (EGFR) L858R point mutation detection, the sensitivity of 200-M, 200-A, 1000-A were 36.6%, 58.5%, 77.5%, that of the specificity were 93.3%, 100%, 96.7%, and the concordance rates were 60.6%, 76.1%, 85.7%, respectively. The size distribution of automatically extracted plasma DNA represented two peaks characteristics at 170 bp and 5 kb. In this study, we indicate the automatic DNA extraction can improve mutation detection rates in plasma DNA.

#2752

**Liquid biopsy analysis of FGFR3 and PIK3CA** **hotspot mutations for disease surveillance in bladder cancer.**

Emil Christensen,1 Karin Birkenkamp-Demtröder,1 Iver Nordentoft,1 Søren Høyer,2 Kirstin Van Der Keur,3 Kim Van Kessel,3 Ellen Zwarthoff,3 Mads Agerbæk,2 Torben Falck Ørntoft,1 Jørgen Bjerggaard Jensen,1 Lars Dyrskjøt1. 1 _Aarhus University Hospital, Aarhus N, Denmark;_ 2 _Aarhus University Hospital, Aarhus C, Denmark;_ 3 _Erasmus MC Cancer Institute, Rotterdam, Netherlands_.

Background: Disease surveillance in patients with bladder cancer is important for early diagnosis of progression and metastasis and for optimal therapeutic treatment.

Experimental procedures: Droplet digital PCR (ddPCR) assays were developed and tumour DNA from two patient cohorts was screened for FGFR3 and PIK3CA hotspot mutations. One cohort (NMIBC cohort) included 363 patients with non-muscle invasive bladder cancer (NMIBC). Another cohort (Cx cohort) included 468 patients with bladder cancer undergoing radical cystectomy. Urine supernatants (NMIBC cohort: n=216, Cx cohort: n=27) and plasma samples (NMIBC cohort: n=39, Cx cohort: n=27) from patients harbouring mutations were subsequently screened using ddPCR assays. Liquid biopsies were collected from 2003-2015 with up to 11.3 years of follow-up for the NMIBC cohort and from 1995-2009 with up to 13.5 years of follow-up for the Cx cohort.

Results and limitations: In total, 36% of the patients in the NMIBC cohort (129/363) and 11% of the patients in the Cx cohort (44/403) harboured at least one hotspot mutation in FGFR3 or PIK3CA. Screening of DNA from serial urine supernatants from the NMIBC cohort showed high levels of tumour DNA (tDNA) to be significantly associated with later disease progression (p=0.003). Furthermore, high levels of tDNA in preoperative urine supernatants and plasma samples were associated with recurrence in the Cx cohort (p=0.068 and p=0.016 respectively). A positive correlation between the levels of tDNA in urine and plasma was observed (corr.=0.6). The retrospective study design and the low volumes of plasma available for analysis were limitations of the study.

Conclusions: Increased levels of FGFR3 and PIK3CA mutated DNA in urine and plasma is indicative of later progression and metastasis in bladder cancer.

#2753

Mutational profiles from targeted NGS combine with miRNA-based liquid biopsy to predict survival in LDCT screening-detected lung cancers.

Mattia Boeri,1 Carla Verri,1 Cristina Borzi,1 Todd Holscher,2 Matteo Dugo,1 Andrea Devecchi,1 Elisa Romeo,2 Stefano Sestini,1 Paola Suatoni,1 Ugo Pastorino,1 Gabriella Sozzi1. 1 _Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy;_ 2 _Gensignia Life Sciences, Inc, San Diego, CA_.

In order to increase cost-effectiveness of low-dose computed tomography (LDCT) lung cancer screening programs, a change in the management of screening-detected lung tumors should be considered. The higher percentage and the better 5-year overall survival of early stage tumors in LDCT screening series than in clinical practice argue in favor of screening programs. On the other hand, the overall limited reduction of mortality observed in screening trials with an observational control arm suggests that some of the screening-detected early stage tumors might be over-diagnosed. In our Institution we are trying to address this issue by the development of complementary biomarkers able to improve detection of aggressive disease. Targeted next-generation sequencing was performed in 94 LDCT screening-detected lung tumors resected from subjects participating in 3 screening trials enrolling 9,248 volunteers. Mutation profile was associated with subjects' clinicopathologic features and with the risk profile of a plasma microRNA signature classifier (MSC). Using data available through The Cancer Genome Atlas database (TCGA), we compared the mutations of a selected set of non-small cell lung cancer (NSCLC) cases detected in standard clinical practice to LDCT screening-detected NSCLC cases. The 5-year overall survival (OS) of screening patients with and without mutations in the tumors was 64% and 100%, respectively (p=0.019). By combining the mutational status with the MSC, patients were stratified into 3 groups with 5-year OS ranging from 41% to 96% (p<0.0001) and the prognostic value was significant even when controlling for stage (p=0.017). The comparison with TCGA data revealed a higher number of non-mutated NSCLC among screening patients (21% vs. 13%), despite the similar spectrum and frequency of mutations. In addition, the difference in 5-year OS between subjects with and without mutations was exclusively detected in screening patients. The mutation profile of screening-detected tumors, while similar to that of clinically detected tumors, was a strong predictor of OS. The combination of tumor mutational status and a circulating miRNA-based risk classifier predicts tumor aggressiveness and clinical outcome and may find rapid application in LDCT screening programs.

#2754

A novel liquid biopsy method for development of aptamer libraries that bind blood plasma exosomes from breast cancer patients.

Valeriy Domenyuk, Symon Levenberg, Adam Stark, Mark Miglarese, David Spetzler. _Caris Life Sciences, Phoenix, AZ_.

Improved technologies capable of characterizing system-wide changes associated with complex diseases will be required to be able to detect millions of proteins and their isoforms as well as multi-molecular complexes. We present a method for developing aptamer libraries using blood plasma exosomes that provides unprecedented system-wide coverage of native exosomal complexes. To train a naïve aptamer library toward cancer samples (positive selection), the library (~1013 biotinylated ssODN species) was incubated with plasma from individual cancer patients and aptamer-bound exosomes were isolated using polymer-based precipitation. Negative selection was performed by contacting the aptamer library with exosomes from donors without breast cancer and recovering unbound aptamers from the supernatant. In all, 12 libraries trained toward 12 individual breast cancer patients were used to probe additional samples. Exosome-bound aptamers were identified and quantified by Next Generation Sequencing (NGS) to build highly accurate signatures for cancer/healthy donor classification. Using these signatures, cancer patients' binding profiles were easily distinguishable from controls. Interestingly, cancer-trained libraries did not distinguish any of the negative control samples from each other, indicating that the selection pressure for cancer was high and noise due to inherent inter-healthy donor heterogeneity was minimal. Full validation studies are ongoing. Aptamer libraries may ultimately be deployed as a minimally-invasive diagnostic adjunct in breast and other cancers.

#2755

Comparison of blood based liquid biopsy methodologies for improved risk assessment of prostate cancer (PCa).

Yashoda Rajpurohit, Shibu Thomas, Jaymala Patel, Dong Shen, Michael Gormley, Denis Smirnov, Deborah Ricci. _Janssen R &D, Spring House, PA_.

Approximately 28,000 men die from prostate cancer in the US each year. Predictive biomarkers can provide patient risk assessment to enable therapeutic decision making. Compared to tumor, blood based liquid biopsies offer greater flexibility for noninvasive sample collection and allows for continuous monitoring of treatment response. While various liquid biopsy sample types and methodologies are currently available, it is critical to identify which method offers the highest sensitivity and specificity for clinical decision making. In this study we systematically compared specificity and sensitivity of prostate cancer detection using a pre-selected panel of PCa specific mRNA transcripts using four different methodologies. In this prospective laboratory study, we collected five blood tubes from a cohort of 40 metastatic castrate resistant prostate cancer (mCRPC) patients and 20 age matched healthy volunteers (HV). Circulating tumor cells (CTCs) were enumerated and blood samples enriched for CTCs were isolated for mRNA evaluation. Two tubes of plasma samples were utilized to isolate exosomes using ExoRNAeasy columns and CellSearch EpCAM capture respectively, while RNA was extracted directly from whole blood collected in PAXgene tubes. RNA isolated from blood samples enriched for CTCs, two exosome preps and whole blood samples were tested using qPCR (Fluidigm) for a panel of 48 prostate specific mRNA transcripts and three endogenous controls previously shown to be detected in whole blood. Receiver operator analysis (ROC) was performed to compare expression of mRNA transcripts across all four methods. Results showed PCa specific genes were detected in whole blood, Exo-52 exosome prep and samples enriched for CTCs, while exosomes isolated using CellSearch EpCAM capture failed to detect any PCa transcripts. ROC showed ExoRNAeasy exosome prep detected PCa specific mRNA transcripts with significantly higher sensitivity and specificity compared to three other methodologies. Area under curve (AUC) for ExoRNA prep 0.65 to 0.75 (average of z score at 3.27 p= 0.0023), compared to AUC for PAXgene is 0.55 to 0.83, (average of z score at 2.64 and p=0.05) and CTC samples is 0.52 to 0.67, (average of z score at 1.63 and p= 0.19). For CellSearch exosome method, the AUC is 0.47 to 0.52, average of z score at 0.66 and p= 0.32). These early results support the utility of exosome derived mRNA assay as a viable alternative to whole blood and CTC based assays. Further testing for expression of markers associated with prognosis and treatment response is necessary to warrant its prognostic or predictive clinical utility.

#2756

Multiplexing done differently: flexible, sensitive, and accurate detection of miRNA and protein analytes using Firefly particle technology.

Elnaz Atabakhsh, Graeme Doran, Conor Rafferty, Jennifer Heath, Sarah Albertson, Peter Gillis, Jonathan Pinney, Russell Neuner, Michael Chipchase, James Murray, Daniel Pregibon. _Abcam, Inc., Cambridge, MA_.

Detection of molecular biomarkers in biological fluids is used as an indicator of normal biological processes, pathogenic processes, and response to therapeutic intervention1,2. Using a combined signature of multiple biomarkers can better account for patient and epidemiological heterogeneity, and provide a more accurate indication of patient health2. This has necessitated the development of technologies that can be used for multiplex biomarker profiling directly from biofluids such as plasma, serum, and urine. Conventional assays consume excessive quantities of precious clinical samples, time and budget when used for discovery and validation of multi-analyte biomarker signatures. To address this need, we developed the Firefly Technology platform, which allows for sensitive and accurate detection of up to 75 protein analytes or 68 miRNAs directly from a biological sample. The Firefly platform uses patented Firefly® hydrogel particles and a three-region encoding design that allows for true, in-well multiplexing, providing flexibility over which analytes can be quantified in the same well and allowing for custom panel design. For the detection of protein analytes, the Firefly immunoassays use high-performance matched antibody pairs that reduce cross reactivity between individual analytes, provide up to 5 logs dynamic range, and typically demonstrate single-digit pg/ml sensitivity, while requiring only 12.5 µl biofluid input. Similarly, the Firefly microRNA assay can reliably detect as few as 1000 microRNA copies per sample with a linear dynamic range of ~5 logs, and without the need of prior RNA purification. This assay utilizes single step RT-PCR signal amplification using universal primers, thus leveraging PCR sensitivity while eliminating the need for separate reverse transcription reactions and mitigating amplification biases introduced by target-specific qPCR. The Firefly assays have been validated in a wide range of biological samples including plasma, serum, urine and cell culture supernatant, providing experimental flexibility. The 96well plate assay format enables high-throughput screening of samples, with readout conducted on standard flow cytometers, thereby omitting the need for complex and expensive dedicated instrumentation. Finally, the integrated Firefly Analysis Workbench software enables easy and rapid data analysis, visualization, and export in under ten minutes, and includes key features such as standard curve analysis and publication-quality heatmaps and graphs. Here we present data from several studies investigating cytokine profiling in human and rodent samples, and circulating and tumor microRNA profiles, using the Firefly Platform. Together, this novel combination of bioinformatics tools and multiplex, high-sensitivity assays enables rapid discovery and validation of biomarker signatures from fluid samples.

#2757

Using liquid biopsies and NGS as tools to analyze mutation burden and copy number variation in the blood of a patient with triple negative breast cancer to better inform therapeutic targets.

Kellie Howard,1 Kimberly Kruse,1 Brianna Greenwood,1 Elliott Swanson,1 Mathias Ehrich,2 Christopher K. Ellison,2 Taylor Jensen,2 Sharon Austin,1 Arturo Ramirez,3 Debbie Boles,4 John Pruitt,4 Elisabeth Mahen,1 Jackie L. Stilwell,3 Eric P. Kaldjian,3 Michael Dorschner,5 Sibel Blau,6 Marcia Eisenberg,4 Steve Anderson,7 Anup Madan1. 1 _Covance, Redmond, WA;_ 2 _Sequenom Laboratories, San Diego, CA;_ 3 _RareCyte, Inc., Seattle, WA;_ 4 _Laboratory Corporation of America® Holdings, Research Triangle Park, NC;_ 5 _University of Washington, Seattle, WA;_ 6 _Northwest Medical Specialties, Puyallup, WA;_ 7 _Covance, Durham, NC_.

The ability to characterize molecular features of cancer from liquid biopsies is resulting in the development of innovative health care for patients. Longitudinal changes in the mutational profiles of DNA isolated from liquid biopsies are being used to better understand and monitor the development, progression, and evolution of therapy resistance in cancer patients. To define changes in the mutational landscape and predict drug susceptibilities in Triple Negative Breast Cancer (TNBC) patients, we used whole exome analysis to profile circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) from eight selected time points of a patient enrolled in the Intensive Trial of OMics in Cancer clinical Trial (ITOMIC-001). The patient initially received weekly cisplatin infusions followed by additional targeted therapy. Peripheral blood samples were collected at specific time points over a period of 272 days following enrollment in the clinical trial. Our data indicates that the identified mutations in genomic DNA isolated from CTCs and ctDNA can be used to understand and mitigate the impact of tumor heterogeneity in addition to identifying clinically relevant mutations at these selected time points. To further increase the resolution of our analysis, we profiled ctDNA from these samples to a higher depth targeting only clinically relevant genes. These analyses increased the sensitivity of detection and identified additional targets that could have been used for therapeutic intervention. In addition to sequence variants, copy number variations (CNVs) have also been significantly associated with the development of metastasis and changes in CNVs have been used to monitor disease progression. We performed a bioinformatics analysis of genomic instability and CNVs across 32 different time points from ctDNA from the same patient throughout the treatment period. The genomic instability number (GIN) calculated for each of the 32 time points seems to mirror the overall CTC burden in the patient at each time point tested. CNV analysis is ongoing and these data sets are being further analyzed in combination with TCGA data to define possible cancer driver genes for the functional prediction of significant TNBC candidate alterations and the results of these analyses will be presented.

#2758

Comparison of cell-free DNA blood collection tubes.

Cecille D. Browne, Margrith E. Mattmann, Marc J. Wycoco, Sheila N. Chen, Rajeswari Ravichandran, Joel Desharnais, Laura J. Varela, Jonathan D. Browne, Vasco Liberal, Florence Lee. _Biomatrica, Inc., San Diego, CA_.

Critical biomarkers such as cell-free DNA (cfDNA) derived from tumors and circulating tumor cells (CTCs) can be detected and analyzed from a simple blood draw. These analytes are fragile, prone to degradation, and present in extremely low quantities. Therefore, proper preservation of these analytes is necessary to ensure accuracy of test results. Several blood collection tubes are commercially available for cfDNA applications, and selecting the ideal blood collection tube for cfDNA impacts test results, sample collection logistics and costs. However, no comprehensive and systematic evaluation of performance among these tubes is available. This study evaluates five commercial blood collection tubes: LBgard™ Blood Tube (Biomatrica), Streck cfDNA BCT® (Streck), PAXgene Blood ccfDNA Tube (PreAnalytiX), Cell-Free DNA Collection Tube (Roche), and EDTA (BD). Healthy donor blood samples were collected in each tube type and incubated over several days at different temperatures. Total plasma DNA was subsequently isolated, and the yield, fold increase versus time 0 and quality of purified DNA were compared. cfDNA controls were also spiked into blood samples collected in each tube type and measured by droplet digital PCR to determine the mutant allele frequencies over time. Finally, the yield and quality of cfDNA isolated from stage IV colorectal cancer blood collected in LBgard Blood Tube, Streck cfDNA BCT and EDTA were compared. Additionally, the inhibition of hemolysis and CTC stabilization were also assessed. Our results show that LBgard Blood Tube, Streck cfDNA BCT, PAXgene ccfDNA Tube and Cell-Free DNA Collection Tube are superior to EDTA tubes in maintaining cfDNA yield and quality over 7 days at ambient temperature. LBgard Blood Tube out-performs all tested blood tubes in inhibiting genomic DNA release for the longest duration (14 days) and across the widest temperature range (4°C, 25°C and 37°C). LBgard Blood Tube shows equivalent inhibition of genomic DNA release to Streck cfDNA BCT for clinical samples, and both tubes out-perform EDTA blood tubes. LBgard Blood Tube also consistently shows better CTC stabilization and inhibition of hemolysis over Streck cfDNA BCT. This comprehensive and systematic study of blood collection tube performance provides a quantitative and exhaustive assessment of blood sample stability, allowing researchers to make informed decisions based on their sample stabilization needs.

#2759

Technical limitations of capture-based, targeted deep sequencing for the detection of somatic variations in cell-free DNA.

Gahee Park. _Seoul National University/ Samsung Genome Institute, Seoul, Republic of Korea_.

Despite widespread applications of capture-based targeted deep sequencing, its limitations on detecting circulating tumor DNA (ctDNA) have not been systematically elucidated. Here, we analyze and characterize parameters critically determining its detection limits; depth of coverage and background errors. The depth increase by using more cell-free DNA (cfDNA) was saturated at around ~3000x with ~50 ng of cfDNA primarily due to a deduplication artifact, which limited the detection sensitivity of the method. Next, characterizing background errors, we uncovered that a majority of errors were not derived from biological origins but technical artifacts, especially during sample preparation and library construction rather than sequencing. Thus, it is important to use an appropriate protocol for those steps minimizing background errors. Considering the depth of coverage and the background error rate, we estimated the detection limit of the method approximately to be 0.4% variant allele frequency at 95% confidence level. Applying the method to pancreatic cancer patients, we found that the levels of ctDNAs measured by cfDNA sequencing well correlated with clinical responses to therapy and/or disease progression, but biopsy-free profiling of somatic variants was limited by the moderate sensitivity of 72% and PPV of 74% at the variant level. Our results clarify technical limitations of targeted deep sequencing and provide a guideline for applying the technique to analyze cfDNA.

#2760

Proof of principle studies for detection of circulating renal cancer cells from blood samples using diverse technologies.

Yvonne Maertens, Verena Humberg, Julie Steinestel, Martin Boegemann, Andres J. Schrader, Christof Bernemann. _University Hospital Muenster, Muenster, Germany_.

Background: During the past years, much progress has been made in detection and analysis of circulating tumor cells (CTCs) in several tumor entities, including prostate cancer, breast cancer or lung cancer. However, little is known about circulating tumor cells in patients suffering from clear cell renal cell carcinomas. The majority of technologies detecting CTCs is based on expression of epithelial markers on the surface of these cells, e.g. EpCAM. Additionally, biophysical approaches have been invented to detect CTCs based on size, invasive capacity or density. In order to be able to detect CTCs in patient samples, in vitro establishment of the most accurate isolation procedure followed by precise detection techniques has to be performed. Aim of our studies was to build a stable in vitro fundament of isolation and subsequent detection of CTCs in ccRCC patients. Methods: We made use of 4 different technologies, all of which have been approved for detection of CTCs in distinctive tumor entities. We used EpCAM based positive enrichment of CTCs, Ficoll densitiy centrifugation followed by CD45-positive cell depletion, rosette formation followed by CD45 positive cell depletion as well as size and deformability based enrichment technologies by using the Parsortix system. Furthermore, by using 4 phenotypically distinct ccRCC cell lines, we tried to detect markers unique for tumor cells in demarcation to blood cells. Results: By performing spiking experiments of renal cancer cells, we found the highest recovery rates by using the size based Parsortix system. Interestingly, the most established technique of EpCAM based isolation failed in three out of four cell lines to recover more than 40%. Expression of well-established markers for ccRCC, like carboanhydrase (CA)-9, could be detected in renal cancer cell lines. However, expression was also found in blood samples of healthy donors. Another marker used for immunohistochemical diagnosis of ccRCC, PAX8, showed weak to absent expression in established renal cancer cell lines. The highest specificity to detect renal cancer cells in blood samples was found when analyzing KRT8 or KRT 19 expression. Conclusion: Our results demonstrate that firstly, using the EpCAM based CTC enrichment, which is the basis of the CellSearch system, which up to now is the only methodology approved by the FDA, the majority of renal cancer cells will presumably not be detected in blood samples. This seems largely due to low or absent expression of EpCAM on renal cancer cells. The usage of the size based Parsortix system showed the highest recovery rates and should therefore be analyzed in more detail on samples of ccRCC patients. Secondly, an exclusive marker for defining a renal CTC is still missing. Some well-established ccRCC markers, like CA-9, failed to specifically detect renal cancer cells in blood samples, as they were either present also in healthy blood samples or absent in renal cancer cell lines.

### Outcomes in Patients and Survivors

#2761

Adjuvant chemotherapy outcomes of node negative, T1a,T1b,T1c hormone receptor-negative HER2-positive breast cancer patients.

Anu Paul, Runhua Shi, Prakash Peddi, Gary Burton. _LSU Health, Shreveport, LA_.

Background: Patients (pts) with early stage HER2neu-positive breast cancers have a higher risk of recurrence and death compared to hormone receptor positive patients. NCCN guidelines recommends adjuvant chemotherapy for Stage II and above HER2-positive breast cancer patients. It is unclear what benefit patients with Stage I HER2-positive breast cancer receive from adjuvant chemotherapy. The current study evaluates the effects of adjuvant chemotherapy on the survival of early stage ER/PR negative, HER2-positive breast cancer patients registered to the National Cancer Data base (NCDB) from 2010-2012.

Patients and Methods: 3416 women from the NCDB were identified with Stage I ER, PR negative, HER2-positive breast cancer diagnosed between 2010-2012. The primary measured outcome was overall survival. Pts were grouped by tumor size (T1a,T1b,T1c) and adjuvant chemotherapy or no adjuvant chemotherapy. Additionally, patients were also grouped into lumpectomy with radiation and mastectomy with or without radiation. Adjusted variables included age, race, Charlson Comorbidity Index (CCI), payer status, income, education, distance travelled, diagnosing/treating facility and treatment delay. Multivariate Cox regression was used to investigate the effect of adjuvant chemotherapy on overall survival while adjusting for other factors.

Results: There were a total of 3416 patients, mean age at diagnosis of 57.9 (range 22-90). The mean age was 56.2 years for the chemotherapy group and 61.8 for the no chemotherapy group. Pathological stage distribution was T1a (24.9%), T1b (24.7%) and T1c (50.2%). Adjuvant chemotherapy was received by 49.5% of T1a, 83.5% of T1b and 95% of T1c pts. Mean patient follow up was 2.7 years (range 0.05-5 years)

In univariate analysis, the hazard ratio (HR) of death for chemotherapy vs no chemotherapy in each T stage was: T1a; 1.22 (0.46-3.28), T1b; 0.221 (0.095-0.512), T1c; 0.217 (0.094-0.498).

In multivariate analysis, adjusting for other factors in the model, HR of death was 0.51 (95%CI: 0.26-1.03) for chemotherapy vs no chemotherapy. The HR of death was 1.92 (95%CI: 1.11-3.32) for pts who received mastectomy without radiation compared to lumpectomy with radiation. In addition, we observed a HR of 5.16 for CCI score of 2 or above as compared to score of 0. There was no difference in outcome based on payer status or race.

Conclusion: Patients with early stage T1 a-c HER2-positive breast cancer had a marginal improvement in overall survival with the addition of chemotherapy. Patients who underwent lumpectomy with radiation were also found to have better overall survival compared to mastectomy without radiation. However, the relatively short follow up and the lack of cancer recurrence information preclude making definitive conclusions relative to adjuvant therapy for pts with these tumors. Longer follow-up and prospective controlled trials will be needed to quantify these potential benefits.

#2762

The comparison of allogeneic stem cell transplantation outcomes between haploidentical donor and international donor: A retrospective multi-institutional study in Korea.

Hyunkyung Park,1 Yoo Jin Lee,2 Sang-Jin Shin,3 Jayoun Lee,3 Inho Kim,1 Sung-Soo Yoon,1 Silvia Park,4 Hyewon Lee,5 Joonho Moon,2 Jun Ho Jang,4 Youngil Koh1. 1 _Seoul National University, Seoul, Republic of Korea;_ 2 _Kyungpook National University Hospital, Dague, Republic of Korea;_ 3 _National Evidence-based Healthcare Collaborating Agency, Seoul, Republic of Korea;_ 4 _Samsung Medical Center, Seoul, Republic of Korea;_ 5 _National Cancer Center, Goyang, Republic of Korea_.

Introduction: The successful rate of hematopoietic stem cell transplantation (HSCT) from HLA-mismatched donor has been increased according to the development in management of complications including graft versus host disease (GVHD) and infections. Especially, haploidentical HSCT provides an opportunity for all patients who do not have HLA-matched sibling donor.

Methods: In this study, we compared HSCT outcomes between haploidentical familiar donor and international donor (donors from Japan, China, Germany, Unites States of America and Taiwan) in acute leukemia patients. We reviewed the overall survival (OS), relapse free survival (RFS) and complications.

Results: Total 142 acute leukemia patients performed HSCT from 2000 to 2016; 98 patients underwent haploidentical donor transplantation and 44 patients underwent international donor transplantation. Major variables such as age, sex, disease status and the number of CD34 stem cells were not statistically different between two groups. In survival analysis, there was no significant difference according to the donor-type. 1-year OS rate for haploidentical transplantation group was 44.1% and 52.3% for international transplantation group (p=0.345). 1-year RFS rate was 36.6% versus 40.9%, respectively (p=0.362). In addition, the incidence of complication events was similar between two groups. The acute GVHD in haploidentical group and international group were 42.9% and 43.2% (P=0.97), and the infection event within 30days after transplantation were 49.5% in haploidentical group and 34.1% in international group (p=0.09). The cumulative incidence of chronic GVHD was no significantly different (P=0.34), respectively.

Conclusion: These data suggest that HSCT from haploidentital donor shows similar outcomes including survival outcomes and complications with international donor transplantation. Therefore, haploidentitial donor transplantation can be good choice for acute leukemia patients who have no HLA_matched sibling donor.

#2763

Health-related quality of life among long-term prostate cancer survivors by primary treatment: A systematic review.

Salome Adam,1 Anita Feller,2 Sabine Rohrmann,1 Volker Arndt2. 1 _Epidemiology, Biostatistics and Prevention Institute, University of Zurich, Zurich, Switzerland;_ 2 _National Institute for Cancer Epidemiology and Registration, Zurich, Switzerland_.

Introduction: Due to improving prognosis for prostate cancer survivors and increased

awareness of late- or long-term treatment effects, monitoring long-term well-being among

prostate cancer survivors has gained increasing attention. In this systematic review, we

identified and synthesized studies comparing health-related quality of life (HRQoL) among

long-term prostate cancer (PC) survivors (i.e. ≥5 years past diagnosis) by primary treatment.

Methods: In order to summarize current literature research results, we searched multiple

databases, including Pubmed, Medline, Embase, PsychInfo, Cinahl, Web of Science and

Cochrane Central Register of Controlled Trials to identify all relevant articles. Studies had to

assess at least overall/ general HRQoL plus one HRQoL domain or a minimum of two HRQoL

domains. Two independent reviewers systematically extracted the content of the included

studies.

Results: A total of twelve studies were identified (2 from US, 3 from Japan, 6 from Europe, 1

from US/UK); the majority were observational prospective cohort studies (n=7), and two

were RCTs. HRQoL was assessed using the SF-36 (n=7) or EORTC QLQ-C30 (n=5). Instruments

to assess symptoms included various questionnaires (e.g., EPIC, IPSS, UCLA and EORTC PR

25). The sample size (all arms combined) was below 100 in 6 studies, between 100 and 200

in 5 studies, and 780 in one study. Comparison groups differed across studies.

Although, long-term prostate cancer survivors reported comparable HRQoL to the general

population, survivors treated using external beam radiation therapy (EBRT) reported poorer

role, social and role physical functioning and a higher burden of diarrhea, pain, sleep

disturbance and nausea/vomiting.

Results for comparisons of different therapies were not consistent. Whereas studies using

the EORTC QLQ-c30 questionnaire did not reveal any statistically or clinically significant

results, studies using the SF-36 showed that survivors treated with a radical prostatectomy

reported better physical and role functioning as well as higher vitality than survivors treated

with AS/WW, ADT and EBRT. However, the latter results were only based on one study. No

clear associations were seen between EBRT and OM.

Conclusion: This review suggests that HRQol among long-term prostate cancer survivors

varies according to primary treatment. However, it remains unclear which treatment options

are superior with respect to HRQoL since studies yielded conflicting results, used different

comparison groups, or did not have enough power to draw any firm conclusions.

#2764

Creating a review process of a digital photo database collected on NRG NSABP B39/RTOG 0413 phase III clinical trial for evaluation of cosmetic results from breast conserving therapy (BCT).

Ashley Sekhon,1 Ruiqi Zhao,1 Yan Wang,1 Debora Grant,2 Kathryn A. Winter,2 Jennifer Moughan,2 Douglas W. Arthur,3 Stephen J. Chmura,4 Atif J. Khan,5 Simona F. Shaitelman,6 Aleix M. Martinez,1 Frank A. Vicini,7 Julia R. White1. 1 _Ohio State University, Columbus, OH;_ 2 _NRG Oncology, Philadelphia, PA;_ 3 _Virginia Commonwealth University, Richmond, VA;_ 4 _University of Chicago, Chicago, IL;_ 5 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ;_ 6 _MD Anderson Cancer Center, Houston, TX;_ 7 _21st Century Oncology, Pontiac, MI_.

Introduction:

After local control and overall survival, acceptable cosmesis is the next most important goal of BCT. Digital photos (DP) are commonly used for assessing cosmesis on clinical trials. On NRG NSABP B39/RTOG 0413, DP at baseline, 1, and 3 years post-radiation were submitted for review by a panel of breast cancer specialists. Given no current standards for central DP review, this project seeks to develop a standard for subjective DP evaluation. The initial standardization of the photo database and reliability of the panel review is described.

Methods:

De-identified DP submitted and stored at NRG SDMC were downloaded to a secure FTP site. Cases with only 1 time point, no baseline, or photos from different patients were excluded. A minimum photo quality was set, including pre-specified criteria for contrast/light, breast/torso size, and cropping to exclude anatomy above the clavicle and below the umbilicus. DP were edited with MATLAB™ and saved in a Reformatted Digital Photo Database (RDPD). A training set was created defining 4 global cosmetic scores, and each individually assessed criteria of breast symmetry, shape, skin color, nipple, and scar appearance. A 14 case calibration set followed the training set. The process for panel review was adapted from the methods reported in the START and RAPID trials. The review panel consisted of 6 breast-specific radiation oncologists, who reviewed in groups of 3. For each session, the training set was reviewed and calibration set scored. A separate 30 case test set randomly selected from the RDPD was reviewed by 2 groups of 3. The calibration and test sets were evaluated for agreement of categorized scores (excellent/good vs. fair/poor).

Results:

There were 2064 DP from 892 patients on NRG NSABP B39/RTOG 0413 documenting the anterior view of both breasts. After exclusions, 1,717 reformatted DP were saved in the RDPD. Five 1-3 hour review sessions resulted in scoring of 777 DP (~ 155 per session). Only 2/777 (0.1%) DP were deemed poor quality and not able to be scored. There was full agreement in categorized scores for 12/14 cases (86%; kappa=0.82) across the 5 calibration sets; and 27/30 (90%; kappa=0.71) for the 2 test set reviews.

Conclusions:

Cosmesis DP prospectively collected on a large phase III clinical trial can be digitally formatted in a systematic fashion for consistent photo quality, allowing for cosmesis consensus panel review without photo quality bias. High agreement in categorized scores allowed reviews to be completed by multiple 3-person review groups. Future work will focus on creating an updated, semi-automatic analysis tool of these photos for objective evaluation of cosmesis using the same scoring guidelines that can then be compared to physician reported outcomes.

Grant Support: FUJIFILM Medical Systems/RSNA Research Resident, NCI U10CA180868, U10CA180822

#2765

Psychosocial predictors of life satisfaction among married women with breast cancer.

Chioma Asuzu, Ngozi Efosa-Urhoghide. _University of Ibadan, Ibadan, Nigeria_.

Introduction: Breast cancer presents a challenge to the totality of a woman's life. Correlates of life satisfaction among married women with breast cancer have not been very well explored. Purpose: This study seeks to determine the relationship that exists between life satisfaction with depression, cognitive emotional regulation, sexuality, self-concept, dejection, interpersonal conflict and social support among married women with breast cancer in Southwest Nigeria. It also seeks to ascertain which of these variables predict life satisfaction among the respondents.

Method: The variables in the study were assessed using modified standardized instruments which were revalidated for cultural suitability after ethical approvals were obtained in each of the 5 centres involved in the study. The instruments were given to breast cancer patients after informed consent to participate in the study have been obtained.

Result: The larger proportion {148 or 40.5%)} of the participants were in the age range of 46-55 years and 112 (30.7%) had stage III disease. Analysis of data showed that significant linear relationship exists between life satisfaction of married women with breast cancer and depression (r = .200**; p < .001), self-concept (r = .306**; p < .001), cognitive emotional regulation (r = .398**; p < .001), dejection (r = .226**; p < .001), interpersonal conflict (r = .352**; p < .001), anxiety (r =.316**; p < .001), sexuality (r =.305*; p < .001) and social support (r = .534; p < .001). There were also significant correlations among the independent variables.

Conclusion: Psychosocial factors are potent predictors of life satisfaction among married women with breast cancer. Based on the findings, it was recommended that psycho-social intervention based on the factors identified as causative agents of life satisfaction should be developed and implemented for married individuals.

#2766

Esophageal stenting in resource-limited settings.

Michael M. Mwachiro,1 Robert K. Parker,1 Stephen Burgert,1 Justus Lando,1 Sinkeet Rankeeti,1 Robert Chepkwony,1 Emmanuel Kiniga,1 Sanford Dawsey,2 Mark Topazian,3 Russell E. White1. 1 _Tenwek Hospital, Bomet, Kenya;_ 2 _National Cancer Institute, Bethesda, WA;_ 3 _Mayo Clinic, Rochester, MI_.

Background: Esophageal cancer is the 6th leading cause of cancer death globally, with geographical high-risk areas in Asia, the Middle East, and eastern and southern Africa. Esophageal squamous cell carcinoma (ESCC) is the more common variant in Africa. In Kenya, its incidence is 2nd in men after prostate cancer and 3rd in women after breast and cervix-uteri cancers. Late presentation is a common occurrence in developing countries and is multifactorial due to challenges in access to health care, low socioeconomic status and delayed or missed diagnosis. A large percentage of these tumors are thus unresectable and are only eligible for palliative care via stenting. Our hospital is a 300-bed referral center in southwestern Kenya, which is a hotspot for ESCC, and we see over 400 cases of ESCC annually.

Methods: We have developed a technique for placement of esophageal self-expanding metallic stents (SEMS) without fluoroscopy that is safe and easily reproducible. This is an outpatient procedure, with the majority done under conscious sedation, and routine followup is not necessary. The tumor margins are noted at time of video endoscopy, a guidewire is placed, and dilation done with Savary dilators as required. The SEMS are then loaded on the stent delivery device and deployed into the proper position based on measurements, and placement is subsequently confirmed via endoscopic visualization.

Results: A total of 3000 SEMS have been placed to date at our hospital, without using fluoroscopy. The male: female ratio has been 1.5:1, and the average age has been 60.4 years. The distribution of tumor locations was 67% in the middle and distal third. The most common complications were tumor overgrowth with obstruction and stent migration. Procedure related mortality was 0.3%. Post-procedure improvement in dysphagia score was seen in over 80% , and patient satisfaction was high. Initial data puts our post stent survival time around 8 months/ 250 days

Conclusions: Placement of SEMS for ESCC, without fluoroscopy, is a safe and reproducible procedure which has a low rate of adverse events. This procedure results in effective palliation of a difficult disease and can easily be done in resource-limited settings which have endoscopy capabilities. Current efforts are ongoing to increase opportunities for training endoscopists in this procedure and for provision of affordable stents in Africa.

#2767

Consistency of standing orders for primary prophylactic CSF within a national network of community oncology practices: SWOG intergroup trial S1415CD.

Scott Ramsey,1 Dawn Hershman,2 Ari Bell-Brown,1 Kate Watabayashi,1 Karma Kreizenbeck,1 Sean Sullivan,3 Aasthaa Bansal,1 William Barlow,4 Kathryn Arnold,5 Gary Lyman1. 1 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 2 _Columbia University Medical Center, New York, NY;_ 3 _University of Washington, Seattle, WA;_ 4 _CRAB, Seattle, WA;_ 5 _SWOG Statistical Center, Seattle, WA_.

BACKGROUND

Among the 400,000 individuals undergoing chemotherapy for breast, colorectal and lung cancer in the US, many are at risk of febrile neutropenia (FN). Prophylactic colony stimulating factor (CSF) use reduces the risk of FN, yet studies show that 55%-95% of CSF prescribing is inconsistent with clinical practice guidelines. There is lack of evidence and ambiguous guidelines for CSF use in regimens with an intermediate risk of FN. To address these issues, we are conducting a pragmatic trial to assess CSF prescribing and to generate evidence about CSF efficacy with intermediate risk regimens. To inform the design and sample size needs, we conducted a survey of current CSF order system use within a large network of community oncology practices.

METHODS

The study setting are sites within the NCI Community Oncology Research Program (NCORP), a national network across the U.S. and Puerto Rico that conducts multi-site cancer clinical trials. Between January-September 2016, 58 NCORP practices were surveyed on their existing systems for prescribing CSF prophylaxis.

RESULTS

8 clinics (14%) reported that their sites do not use any standing orders for CSF prophylaxis prescribing. Standing order set characteristics for the remaining 50 practices are shown in Table 1.

Table 1 Summary of CSF standing order implementation in 50 surveyed NCORP clinics

---

|  | |  | |  | |

|

|

Breast Cancer Regimens | Non-Small Cell Lung Cancer Regimens | Colorectal Cancer Regimens

Order Set Characteristic | Dose Dense

N (%) | High Risk

N (%) | Intermediate Risk

N (%) | Low Risk

N (%) | Intermediate Risk

N (%) | Low Risk

N (%) | Intermediate Risk

N (%) | Low Risk

N (%)

Automatically Included in Order Set (Standing Orders) | 46 (82%) | 31

(62%) | 7

(14%) | 0 | 8

(16%) | 0 | 7

(14%) | 0

Automatically Excluded in Order Set  | 0 | 0 | 6

(12%) | 19 (38%) | 5

(10%) | 19

(38%) | 8

(16%) | 19 (38%)

No automatic ordering- up to physician discretion to add or exclude orders | 4

(8%) | 19

(38%) | 37

(74%) | 31

(62%) | 37

(74%) | 31 (62%) | 35

(670%) | 31 (62%)

CONCLUSIONS

We observed wide variation in the current application of standing orders. Intermediate risk regimens had the most inconsistent practices, with a near equal number of clinics choosing either actively including or excluding CSF order sets. The majority of clinics using standing orders included CSF for dose dense and high risk regimens but only 19 (38%) actively excluded CSF for low risk regimens. These results support the need for more evidence to inform clearer guidelines on CSF use in intermediate risk regimens and studies that evaluate the effects of existing CSF standing orders on guideline adherence and patient outcomes.

Funding:PCORI (PCS-1402-09988) and NCORP grant (5UG1CA189974)

#2768

Treosulfan induces differential gonadal toxicity profile compared with Busulfan.

Mattan Levi,1 Salomon Stemmer,1 Jerry Stein,2 Ruth Shalgi,1 Irit Ben-Aharon1. 1 _Institute of Oncology , Davidoff Center, Rabin Medical Center, Beilinson Campus, Petah-Tiqva, Israel;_ 2 _Schnieder Children Medical Center, Petah-Tiqva, Israel_.

Background: Treosulfan (L-treitol-1,4-bis-methanesulfonate), an alkylating agent with myeloablative and immunosuppressive traits, has been increasingly incorporated as a main conditioning protocol for hematopoietic stem cell transplantation (HSCT) in pediatric malignant and non-malignant diseases. Treosulfan has been shown to present lower toxicity profile compared with conventional myeloablative regimens such as busulfan. Yet, while busulfan is considered highly gonadotoxic, the gonadal toxicity profile of treosulfan remains to be elucidated. Our aim was to study the short and long term gonadal effect of treosulfan in comparison to busulfan.

Methods: Mature 2 months old male and 3 months old female mice ("pubertal cohort") were injected with treosulfan 2000 mg/kg or busulfan 40 mg/kg and were sacrificed one week, one month or six months post drug administration. Immature male and female mice ("prepubertal cohort") were injected with treosulfan 750 mg/kg or busulfan 15 mg/kg and were sacrificed one week or three months post drug administration. Testicular function was assessed by measurements of sperm count and motility, testes and epididymides weight, serum anti-Mullerian Hormone (AMH) and testicular ID4 or GFRA1 mRNA (markers for stem spermatogonia). Immunohistochemistry was performed to evaluate testicular proliferation (Ki-67), apoptosis (TUNEL) and meiotic-active spermatocytes (DAZL-PCNA). Ovarian function was assessed by measurements of ovary weight, serum AMH, follicles count and ovarian SOHLH2, NOBOX of FIGLA mRNA (markers for primordial follicles). Immunohistochemistry was performed to evaluate ovarian proliferation (Ki-67), apoptosis (TUNEL) and growing follicles (AMH-PCNA).

Results: Treosulfan testicular toxicity was milder compared to busulfan toxicity, in both mature and immature male mice, while stem spermatogonia were spared. However, ovarian toxicity of both treosulfan and busulfan was severe and permanent in both mature and immature female mice; possibly by a short term irreversible reduction of reserve primordial follicles in the ovaries.

Conclusion: Our results indicate that treosulfan exerts a differential gonadal toxicity profile compared with busulfan, manifested by mild testicular toxicity and severe ovarian toxicity. 

### Predictive Biomarkers 2

#2769

Project Survival: Prospective clinical study utilizing multiomics and artificial intelligence to discover novel molecular markers for detection, stratification, and outcome in pancreatic cancer.

Rangaprasad Sarangarajan,1 John Crowley,2 Amy Stoll-D'Astice,2 Valerie Bussberg,3 Cindy Nguyen,1 Leonardo O. Rodrigues,1 Emily Chen,3 Eric Michael Grund,1 Vivek K. Vishnudas,1 Michael Kiebish,1 Viatcheslav R. Akmaev,1 Manuel Hidalgo,4 Niven R. Narain,1 A. James Moser4. 1 _BERG, LLC, Framingham, MA;_ 2 _Cancer Research And Biostatistics, Seattle, WA;_ 3 _BERG Health, LLC, Framingham, MA;_ 4 _Beth Israel Deaconess Medical Center, Boston, MA_.

Pancreatic adenocarcinoma is the third leading cause of cancer death and has an extremely poor response to first line therapies. There is a critical unmet need to discover and implement effective diagnostic panels to stratify this disease and personalize treatment. Project Survival is a prospective study designed to discover biomarkers for patient diagnosis, stratification, and prognostics for pancreatic cancer. The multisite study is in year 2 of enrolling subjects within 6 categories: healthy volunteers with a relative with pancreatic cancer (N=50), pancreatitis (N=50), pancreatic cystic neoplasm (N=50), suspicious pancreatic masses with pathology other than pancreatic cancer (N=50), early stage (N=200) and metastatic pancreatic cancer (N=200). The study analyzes matched subject sera, plasma, buffy coat, saliva, urine, and tumor/adjacent normal tissues and integrates them with full clinical annotation. Multiple time points per subject per year are taken longitudinally over the course of the six year timeline enabling dynamic modeling. Samples are analyzed by Mass Spectrometry for the proteome, signaling lipidome, structural lipidome, and metabolome. The BERG Interrogative Biology® platform utilizes Artificial Intelligence to integrate multiomic profiles with medical annotation and clinical endpoints. Utilizing the power of the Bayesian Network learner, bAIcis™ (BERG Artificial Intelligence Clinical Information System), multiomic profiles were aligned to the longitudinal clinical information and subjected to the AI-algorithms that inferred probabilistic cause-and-effect relationships among molecular and clinical variables inferring markers of pancreatic cancer status and defining the interconnectivity of molecular features with clinical phenotype. Network features linking clinical endpoints and key network pressure points will be identified as molecular drivers. The drivers of clinical endpoints will be analyzed to rank potential biomarkers.

#2770

Bcl-2 overexpression is a predictive biomarker for the combination of ABT-199 with chemotherapy in diffuse large B-cell lymphoma cell lines.

Ian McConnell, Elyse Anderson, Victor Yazbeck. _VCU Massey Cancer Center, Richmond, VA_.

Diffuse Large B-Cell Lymphoma (DLBCL) is the most common subtype of Non-Hodgkin's Lymphoma. The anti-apoptotic Bcl-2 protein is expressed in certain DLBCL subtypes such as "Double-Hit" where it mediates resistance to chemotherapy. ABT-199 is a BH3-mimetic that specifically targets Bcl-2, and has shown excellent clinical activity in several hematological malignancies. The anti-apoptotic protein Mcl-1 mediates resistance to ABT-199, and can be inhibited with chemotherapy. However, despite variability in the Bcl-2 expression levels, current clinical trials involving combinations of ABT-199 and chemotherapy for the treatment of patients with DLBCL use a one-size-fits-all approach regardless of Bcl-2 expression levels. We hypothesized that high Bcl-2 expression levels correlates with sensitivity to the combination of ABT-199 and chemotherapy in DLBCL. We examined the activity of ABT-199 in four DLBCL cell lines with varying Bcl-2 levels (CARNAVAL, OCI-ly18, SU-DHL-4 and SU-DHL-8) as determined by cell viability (Cell Titer Glo Assay) and apoptosis (Annexin-V/PI staining and FACS) analyses. CARNAVAL and SU-DHL-8 have the highest and lowest Bcl-2 expression levels, respectively, as revealed by immunoblotting. Of the four cell lines, CARNAVAL displayed the highest sensitivity (IC50 = ~7 nM) while SU-DHL-8 was resistant (IC50 = >10 µM) with the other 2 cell lines exhibiting intermediate sensitivity (45.9 nM for OCI-ly18 and 2.6 µM for SU-DHL-4). We then selected CARNAVAL and SU-DHL-8 in order to further examine the effect of the combination of ABT-199 with classic chemotherapy agents (etoposide and doxorubicin) used in DLBCL treatment. In CARNAVAL cells, the combination of either chemotherapy with ABT-199 led to a significant decrease in cell viability at 48 hrs compared to each single agent alone. This was associated with apoptosis induction as measured by Annexin-V/PI, caspase-3 and PARP cleavage, an increase in γ-H2AX (associated with DNA damage), and decrease in Mcl-1. Unlike CARNAVAL cells, ABT-199 did not enhance the activity of chemotherapy in SU-DHL-8 cells that lack Bcl-2 expression. These preclinical results support the use of Bcl-2 overexpression as a predictive biomarker for selecting patients that are likely to benefit from the combination of ABT-199 with chemotherapy in DLBCL. Current efforts are investigating the mechanism of resistance to the combination.

#2771

Correlation of tumor mutation burden and chemotherapy outcomes in colorectal cancer.

Sachin G. Pai, Benedito Carneiro, Aparna Kalyan, Ricardo Costa, Irene Helenowski, Alfred Radmeaker, Hiral Shah, Daniel Olson, Young Chae, Francis Giles. _Northwestern University, Chicago, IL_.

Background: The Cancer Genome Atlas (TCGA) has identified 16% of colorectal cancers (CRC) to have defects in mechanisms that repair spontaneous DNA damage, and consequently have high tumor mutation burden (TMB). Although, there is accumulating evidence for activity of immunotherapy on tumors harboring high-TMB, its impact on response to chemotherapy is unknown.

Methods: In this retrospective cohort study, we analyzed progression free survival (PFS) of 74 patients with metastatic CRC (61 colon & 13 rectal cancer) treated at tertiary care oncology clinics and underwent next-generation sequencing (NGS) of their tumor sample using FoundationOne® (Foundation Medicine Inc., Cambridge, MA). Most recent available specimen was analyzed at the time of diagnosis of metastatic disease. TMB was calculated by counting all synonymous and nonsynonymous variants as well as indels across a 1.25 megabase coding region spanning 315 genes. Low TMB (TMB-L) and Intermediate/High TMB (TMB- I/H) were defined as ≤ 5 mutations per base (MB) or ≥ 6/MB respectively. Demographic and clinical information (including imaging results, chemotherapy treatment) were obtained by chart review. Treatment was captured as 'oxaliplatin-based' if the chemotherapy regimen contained oxaliplatin (i.e., FOLFOX, XELOX) or 'irinotecan-based' if the regimen contained irinotecan (i.e., FOLFIRI). Subsequent modifications of dose or omission of the drug due to toxicity were not captured. Continuous variables were reported as medians and inter-quartile ranges and compared between groups via the Wilcoxon rank-sum test. Categorical variables were reported as frequencies and percentages and compared between groups via Fisher's exact test. Survival estimates were compared between groups via the log-rank test.

Results: There was no statistically different PFS in TMB-L (n=39) compared to TMB- I/H (n=26). (10.0 vs. 5.9 months, P = 0.18). In the TMB-L cohort, irinotecan-based chemotherapy (n=25) treated patients had improved PFS compared to oxaliplatin-based chemotherapy (n=10) treated CRC patients (11.9 vs. 6.5 months, P= <0.001). No difference in PFS was observed between the two treatment cohorts in TMB-I/H group. In stage 2 and 3 colon cancer patients, there was no difference in time to recurrence in the TMB-L and TMB-I/H cohorts, when patients were treated with oxaliplatin-based therapy in peri-operative setting (detailed statistics including survival curves will accompany final presentation).

Conclusion: TMB status may be a predictive biomarker in a subset of patients treated with chemotherapy, specifically in TMB-L cohort. Confirming these findings in a larger repository of tissue from studies comparing irinotecan vs. oxaliplatin-based regimen is recommended.

#2772

BCL-2 family expression profiling may identify distinct molecular subtypes of multiple myeloma with increased susceptibility to single agent Venetoclax.

Jenny Wu,1 Caleb Stein,2 Jeremy A. Ross,3 Franklin Peale,1 John D. Shaughnessy,4 Ryan Van Laar,5 Gareth Morgan,2 Jeffrey M. Venstrom,1 Elizabeth A. Punnoose,1 Yanwen Yanwen Jiang1. 1 _Genentech, Inc., South San Francisco, CA;_ 2 _University of Arkansas Medical Sciences, Little Rock, AR;_ 3 _Abbvie, North Chicago, IL;_ 4 _Mt. Sinai School of Medicine, NY;_ 5 _Signal Genetics, Little Rock, AR_.

Venetoclax (VEN) is being evaluated in relapsed/refractory multiple myeloma (R/R MM) patients as a single agent (NCT01794520). Improved objective response rates were observed in t(11;14) patients (40% in t(11;14)+ vs 6% in t(11;14)-), which were shown to associate with a favorable BCL-2 family expression profile (high BCL2:BCL2L1 (BCL-XL). Forty percent of t(11;14)+ population exhibited VEN favorable biomarker profile with an 88% ORR compared to 20% in t(11;14)+ patients with unfavorable profile. Thus, a favorable BCL-2 family expression profile may identify certain MM subgroups with increased sensitivity to the anti-tumor activity of VEN as a single agent. To better understand and identify the patient populations that may benefit from VEN, we retrospectively analyzed the prevalence of a favorable BCL-2 family expression profile in t(11;14)+ and other MM molecular subtypes in two published cohorts (GSE4581 and GSE9782). Our results showed that BCL2 expression varied significantly across molecular and cytogenetic subgroups. The t(11;14)+ subgroup expressed high BCL2 and the lowest BCL2L1 and MCL1 in both newly diagnosed (NDMM) and R/R MM patients. Correspondingly, the t(11;14) MM was enriched for the highest ratios of BCL2/MCL1 and BCL2/BCL2L1, further supporting the single agent VEN activity observed in this patient population. Based on prevalence study in cohort GSE9782, we observed 40% of t(11;14)+ R/R MM patients exhibited a favorable BCL-2 family expression profile, using clinical defined cutoffs, thus highly consistent with the VEN single agent trial. Furthermore, this favorable profile existed in other molecular subtypes, especially the ones that harbor abnormal MAF (23%) and D3 (37.5%) translocations, as well as dysregulated expression of cyclinD1 (21.2%)/D1+D2 (26.7%). The molecular subgroup with overexpression of cyclin D2 had the lowest prevalence of the favorable profile (7.5%). In the NDMM cohort, overall 27% patients had favorable profile. More specifically, the t(11;14) patients had the highest (61%) and D2 patients had the lowest (13%) prevalence of a favorable BCL-2 family profile. Collectively, our data suggests that MM subgroups are associated with distinct BCL-2 family expression profiles, and that the t(11;14) subgroup is particularly suited for single agent VEN treatment as indicated by the high prevalence of a favorable BCL-2 family expression profile. In addition, we further identified patients in the non t(11;14) MM subgroups with a favorable BCL-2 family expression profile that may also potentially benefit from VEN monotherapy.

#2773

A prospective observational study evaluating c-MET expression and EGFR gene mutation correlation with erlotinib response in second line treatment for advanced/metastatic non-small cell lung cancer patients.

Cheol-Kyu Park,1 Yoo-Duk Choi,2 In-Jae Oh,1 Jung-Eun Lee,3 Shin Yup Lee,4 Tae-Won Jang,5 Jeong-Seon Ryu,6 Young-Chul Kim1. 1 _Chonnam National University Hwasun Hospital, Hwasun, Jeonnam, Republic of Korea;_ 2 _Chonnam National University Medical School, Gwangju, Republic of Korea;_ 3 _Chungnam National University Hospital, Daejun, Republic of Korea;_ 4 _Kyungpook National University Medical Center, Daegu, Republic of Korea;_ 5 _Kosin University Gospel Hospital, Busan, Republic of Korea;_ 6 _Inha University Hospital, Incheon, Republic of Korea_.

Background: The c-MET is associated with tumor progression and acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). The aim of this study was to evaluate the prevalence and predictive role of c-MET and EGFR mutation in non-small cell lung cancer (NSCLC).

Methods: This was a multicenter prospective observational study for patients with stage IV or recurrent NSCLC who had progressive disease after 1st line chemotherapy and was followed by erlotinib for 2nd line treatment from September 2011 to September 2014. The primary outcome was to measure the rates of c-MET expression/amplification and EGFR mutation in tumor tissue. MET expression was evaluated by immunohistochemistry (IHC) and gene copy number was assessed by silver in situ hybridization (SISH). EGFR mutations were analyzed by Cobas™ version 1.

Results: A total of 196 patients were enrolled and major histologic types were adenocarcinoma (67%). c-MET was overexpressed (MET IHC-positive) in 44.4% of the patients and dominant in female and non-squamous histology. c-MET amplification and high polysomy (MET SISH-positive) were observed in 1.5% and 8.7%, but there was no specific clinical characteristics. The detection rate of EGFR mutation was 10.2% (19 deletion: 11, L858R: 7, exon 20 insertion: 2 cases). The response rate of erlotinib was significantly higher in EGFR sensitive mutant group (55% vs. 4%, p<0.001) and the median progression-free survival (PFS) was significantly longer than wild type (173 vs 58 days, p<0.001). c-MET positivity (IHC- or SISH-positive) did not show significant correlation with response or PFS. Patients with SISH-positive for c-MET tended to have shorter overall survival (OS) than patients with SISH-negative (127 vs 303 days, p=0.051).

Conclusion: In this observational study with 2nd line erlotinib treatment, we reconfirmed EGFR mutation is strong predictive marker, and EGFR-TKI use in EGFR wild-type patients should be considered carefully and may be harmful. c-MET overexpression was related to some clinical characteristics such as female and non-squamous histology. Although c-MET positivity was not associated with response or PFS, c-MET SISH may be predictive marker for OS.

#2774

Correlative analysis of systemic markers from a randomized trial assessing local consolidative therapy for non-small cell lung cancer (NSCLC) patients with oligometastatic disease.

Chad Tang, John V. Heymach, Alexandre Reuben, Hai Tran, James Welsh, Jennifer Wargo, Zhongxing Liao, Anne Tsao, Eric Prado, Stephen Swisher, Jianjun Zhang, Daniel Gomez. _MD Anderson Cancer Center, Houston, TX_.

Introduction: We present serum correlative analysis from a phase II randomized trial to identify markers associated with progression free survival (PFS) in NSCLC.

Methods: NSCLC patients presenting with oligometastatic disease (≤3 metastases) were enrolled on a multi-institution phase II randomized trial (NCT01725165). Patients who did not progress after ≥3 months of first-line systemic therapy were randomized to upfront local consolidative therapy (LCT arm) to all sites of disease or at later progression (no-LCT arm). For correlative analyses, peripheral blood was collected from consenting patients after completion of initial systemic therapy but prior to randomization (baseline) and at each follow up. Serum cytokine and angiogenesis factors (CAFs) were analyzed via magnetic bead-based multiplex assay (Merck Millipore). Peripheral blood T cell receptor (TCR) repertoire was profiled by immunoSEQ (Adaptive Biotechnologies). Peripheral cell-free DNA (cfDNA) was subjected to next generation sequencing of a panel of cancer genes. Comparisons of PFS, the primary endpoint of this study, were conducted via log-rank test.

Results: After enrollment of 74 patients with a median follow up of 19 months, this study was closed early after the Data Safety Monitoring Committee review determined efficacy in the LCT arm. Of 49 randomized patients, correlative blood draws were conducted on 33 patients. Considering all patients, high baseline EGF (cutpoint 50 pg/ml; median PFS: 5.7 vs 19.8 mo), Eotaxin (cutpoint 110 pg/ml; median PFS: 5.7 vs 21 mo), TNF alpha (cutpoint 8 pg/ml; median PFS: 3.6 vs 17.9 mo), or low baseline GRO (cutpoint 600 pg/ml; median PFS: 17.9 vs 3.6 mo) were associated with longer PFS (all P<0.05). When considering individual treatment arms, LCT arm patients exhibiting low baseline MMP-2 (cutpoint 90 ug/ml; median PFS: 17.9 vs 5.3 mo) and no-LCT arm patients exhibiting low baseline GRO (cutpoint 600 pg/ml; median PFS: 21.0 vs 1.8 mo) or high baseline FGF-2 (cutpoint 58 pg/ml; median PFS: 2.2 vs 21.0 mo) exhibited longer PFS (all P<0.05). TCR repertoire and cfDNA profile analyses are underway.

Conclusions: In this exploratory analysis, we identified baseline CAFs associated with PFS. These finding are being validated in a larger cohort of patients. If validated, CAFs could be a biomarker to select patients with oligometastatic NSCLC for aggressive local treatment.

#2775

CPS1 as a therapeutic target and prognostic indicator in LKB1-inactivated lung adenocarcinoma.

Muge Celiktas, Ichidai Tanaka, Satyendra Chandra Tripathi, Johannes F. Fahrmann, Clemente Aguilar-Bonavides, Pamela Villalobos, Oliver Delgado, Dilsher Dhillon, Jennifer B. Dennison, Edwin J. Ostrin, Hong Wang, Carmen Behrens, Kim-Anh Do, Adi F. Gazdar, Samir M. Hanash, Ayumu Taguchi. _MD Anderson Cancer Center, Houston, TX_.

Liver Kinase B1 (LKB1), encoded by STK11, is a tumor suppressor and somatically mutated in approximately 20% of lung adenocarcinoma. Aside from the effects of LKB1 inactivation on tumor initiation, LKB1-mutant cancers are biologically distinct from cancer with functional LKB1, and the loss of LKB1 uniquely confers invasive and metastatic properties in genetically engineered mouse models of cancer. While various pathways, including energy metabolism, cell polarity, and cell growth, are regulated by LKB1 and can play a pleiotropic role in cancer initiation and progression, no therapies are currently available for clinical use that specifically target LKB1 inactivation. Therefore, elucidation of the functional mechanisms associated with LKB1 inactivation has translational relevance.

We analyzed proteomic profiles of 45 lung adenocarcinoma cell lines with and without LKB1 inactivation to identify molecular features associated with LKB1 inactivation. Carbamoyl phosphate synthase 1 (CPS1) was identified as a markedly overexpressed protein in LKB1-inactivated lung adenocarcinoma cell lines. CPS1 is the first rate-limiting mitochondrial enzyme in the urea cycle, and plays an intricate role in arginine and pyrimidine metabolism. CPS1 knockdown reduced cell growth, decreased levels of metabolites associated with nucleic acid biosynthesis pathway, and contributed an additive effect when combined with conventional chemotherapy agents including gemcitabine, pemetrexed, or CHK1 inhibitor AZD7762. Tissue microarray analysis using 305 lung adenocarcinoma tumors revealed that CPS1 was expressed in 65.7% of LKB1-negative and only 5.0% of LKB1-positive lung adenocarcinomas. In addition, CPS1 expression was significantly and independently associated with poor overall survival of lung adenocarcinoma patients (P < 0.0001, HR = 3.03, 95% CI 1.74-5.25). Concordant with the results of tissue microarray, analysis of the Cancer Genome Atlas (TCGA) dataset consisting 403 lung adenocarcinomas revealed that high CPS1 mRNA expression was significantly associated with worse overall survival and is an independent prognostic indicator of lung adenocarcinoma (P = 0.005, HR = 2.31, 95% CI 1.28-4.16). Our findings suggest functional relevance of CPS1 and its association with worse outcome in LKB1-inactivated lung adenocarcinoma. Therefore, CPS1 is a promising therapeutic target in combination with other chemotherapy agents, as well as a prognostic biomarker, enabling us to personalize the treatment of LKB1-inactivated lung adenocarcinoma.

#2776

Genetic correlations with clinical response to ASTX727 in patients with myelodysplastic syndromes (MDS).

Haley E. Ramsey,1 Shilin Zhao,1 Yaomin Xu,1 Olatoyosi Otoyosi,2 Philip Amrein,3 Amy Dezern,4 Laura Michaelis,5 David P. Steensma,6 Stefan Faderl,7 Guillermo Garcia-Manero,8 James Lowder,9 Pietro Taverna,9 Michael R. Savona1. 1 _Vanderbilt University Medical Center, Nashville, TN;_ 2 _The University of Chicago, Chicago, IL;_ 3 _Massachusetts General Hospital, Boston, MA;_ 4 _Johns Hopkins University School of Medicine, Baltimore, MD;_ 5 _Medical College of Wisconsin, Milwaukee, WI;_ 6 _Dana-Farber Cancer Institute, Boston, MA;_ 7 _Hackensack University Medical Center, Hackensack, NJ;_ 8 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 9 _Astex Pharmaceuticals, Pleasanton, CA_.

DNA methyltransferase inhibitors (DNMTis) have been shown to alter the natural history of MDS and reduce related complications; however, many patients do not respond to DNMTi therapy or progress with limited effective secondary options. Therefore, the ability to preselect which patients are most likely to respond to DNMTis would be useful in personalizing MDS therapy. Earlier reports noted a trend toward improved response to therapy in the presence of somatic TET2 mutations (Bejar et al, 2014), and lower response rates in patients with mutations in DNMT3A (Ley et al, 2010).

Treatment with the DNMTi decitabine (DAC) requires 5 daily parenteral doses every month administered in the clinic. An orally administered DNMTi provides convenience with the potential for improved compliance, and allows for exploration of different extended dosing schedules which could produce alternative methylation of the genome and improve clinical response. DAC is not readily orally bioavailable due to rapid clearance by cytidine deaminase (CDA) present in the gut and liver. E7727, a novel CDA inhibitor, is orally bioavailable with an excellent safety profile in preclinical models. ASTX727 is a combination of E7727 and DAC, and has completed phase 1 dose escalation of 44 MDS or CMML patients in which pharmacokinetic (PK) and pharmacodynamic (PD) parameters of DAC were replicated with the oral agent.

During the dose escalation portion of the Phase 1 trial of ASTX727, bone marrow aspirates were obtained pre- and post- treatment from 28 patients (13 clinical responders), and genomic DNA from marrow cells was isolated for next-generation sequencing (NGS) with a panel of 37 myeloid neoplasia-associated genes. Consistent with previous reports, DNMT3A mutations were associated with poor response to ASTX727 (all 4 carriers failed therapy, P = 0.028). Conversely, of the 10 patients with TET2 mutations, a majority responded to treatment (6/10, P=0.444). ASXL1 mutations showed a complex combinatorial effect, as all three ASXL1-mutated patients with BCOR mutations, regardless of International Prognostic Scoring System (IPSS) risk category, were non-responders while all 3 patients ASXL1 who also carried a TET2 mutation responded.

ASTX727 is an oral DNMTi that revealed responses in this dose escalation Phase I study exhibiting a similar pharmacokinetic profile and achieving AUC range of IV DAC. As previously observed with DAC, ASTX727 may lead to improved responses in patients with specific gene mutations. This work shows preliminary effects on mutational burden and allele frequency concurrent with activity of ASTX727. Further analyses of patients treated at the RP2D in the Phase 2 trial are ongoing.

#2777

TMPRSS2-ERG predictive value for taxanes resistance according to prior second-line hormonal manipulations in metastatic castration resistant prostate cancer.

Mercedes Marín-Aguilera,1 Òscar Reig,1 Natalia Jiménez,1 Iván Victoria,1 Lydia Gaba,1 Sandra López,1 Javier Prato,2 Maria Verónica Pereira,1 Teresa Vilella,1 Montserrat Domenech,3 Josep Badal,3 Albert Font,4 Juan José García-Mosquera,4 Olatz Etxaniz,4 Cristina Carrato,4 Cristina Suárez,5 Joan Carles,5 Fabriccio Racc,5 Pedro Luis Fernández,1 Aleix Prat,1 Begoña Mellado1. 1 _Hospital Clinic of Barcelona/Institut d'Investigacions Biomèdiques Agust PI i Sunyer/Fundació Clínic per a la Recerca Biomèdica, Barcelona, Spain;_ 2 _Hospital Universitario de La Coruña, La Coruña, Spain;_ 3 _Fundació Althaia, Barcelona, Spain;_ 4 _Institut Català d'Oncologia, Badalona, Spain;_ 5 _Hospital Universitario Vall d'Hebron, Barcelona, Spain_.

Background: TMPRSS2-ERG is a genetic alteration specific of prostate cancer, present in primary tumors and maintained under castration resistant prostate cancer (CRPC) progression. It results in androgen-driven overexpression of ERG, which is involved in resistance to taxanes in preclinical models. In prior work, we found that TMPRSS2-ERG expression in blood correlated with docetaxel resistance in metastatic CPRC. Here, we investigated if TMPRSS2-ERG expression in primary tumors predicts taxanes resistance in CPRC and the potential impact of prior second-line hormonal manipulations with abiraterone (A) or enzalutamide (E).

Methods: Patients with metastatic CRPC treated with taxanes were included. Formalin-fixed paraffin-embedded (FFPE) tumors and peripheral blood mononuclear cells (PBMCs) fraction were tested for TMPRSS2-ERG by RT-qPCR. FFPE from hormone-sensitive disease (primary diagnosis) were retrospectively obtained. PBMCs were prospectively collected prior taxane initiation. TMPRSS2-ERG expression was tested by RT-qPCR. TMPRSS2-ERG detection was correlated with taxane response and clinical outcome.

Results: A total of 84 tumor samples from 74 patients were included: 65 (87.3%) treated with docetaxel, 19 (25.7%) with cabazitaxel and 10 (13.5%) with both. Forty-six tumor samples (54.7%) were TMPRSS2-ERG+ and 38 (45.2%) TMPRSS2-ERG-. Overall, no correlation between tumor TMPRSS2-ERG expression and taxanes response or clinical outcome was observed. In 42 (50%) samples matched tumor and PBMC samples were available at the time of this analysis: 23 (54.7%) had detectable TMPRSS2-ERG on tissue and 11 (26.2%) on PBMCs fraction. In 27 patients, taxanes were administered as a first-line therapy and in 15 after A or E progression. TMPRSS2-ERG was detected in PBMC from 8 (29.6%) and 3 (20%) patients without or with prior A or E. In patients without prior A or E, TMPRSS2-ERG expression in primary tumors predicted a lower median PSA-PFS (5.5 vs 10.1 months for TMPRSS2-ERG+ vs -, respectively; p<0.05). Similarly, when analyzing PBMCs samples from patients without prior A or E, PSA response was observed in 0% of TMPRSS2-ERG+ vs 55.6% of TMPRSS2-ERG- patients (p = 0.009). Median PSA-PFS, Rx-PFS were 2.9 vs 8.1 months (p < 0.01) and 3.2 vs 7.3 (p < 0.05), for TMPRSS2-ERG+ and TMPRSS2-ERG- patients. However, no significant differences were found either in PBMCs or FFPE samples in patients that received A or E prior to taxanes, regarding to PSA response, PSA-PFS, Rx-PFS and OS parameters between TMPRSS2-ERG+ and TMPRSS2-ERG- samples.

Conclusions: The role of TMPRSS2-ERG in taxane resistance may be different according to prior exposure to second-line hormone-therapy in CRPC. Prior androgen receptor inhibition may result in TMPRSS2-ERG downregulation and/or activation of alternative mechanisms of resistance. Further data according to this hypothesis will be presented.

#2778

Combination of DNA mismatch repair status and MACC1 expression in patients with stage II colon cancer: The BIOGRID studies.

Ulrich-Peter Rohr,1 Pia Herrmann,2 Katharina Ilm,2 Hai Zhang,3 Sabine Lohmann,4 Astrid Reiser,4 Andrea Muranyi,5 Janice Smith,2 Susen Burock,6 Marc Osterland,2 Katherine Leith,5 Shalini Singh,5 Patrick Brunhoeber,5 Rebecca Bowermaster,5 Jeanne Tie,7 Michael Christie,7 Hui-Li Wong,7 Paul Waring,7 Kandavel Shanmugam,5 Peter Gibbs,7 Ulrike S. Stein2. 1 _Divisional Medical and Scientific Affairs, Roche, Basel, Switzerland;_ 2 _Charité Universitätsmedizin Berlin and Max Delbrück Ctr. for Molecular Medicine, Berlin, Germany;_ 3 _Divisional Biomarker Group, Roche, Basel, Switzerland;_ 4 _Roche Diagnostics GmbH, Penzberg, Germany;_ 5 _Ventana Medical Systems, Inc., Tuscon, AZ;_ 6 _Charité Comprehensive Cancer Center, Berlin, Germany;_ 7 _University of Melbourne, Melbourne, Australia_.

We have previously identified the gene Metastasis-Associated in Colon Cancer 1 (MACC1). MACC1 acts a prognostic biomarker for tumor progression, metastasis and patient survival for a broad variety of solid cancer types. Here we assessed if the MACC1 gene could separate stage II colon cancer patients with proficient mismatch repair (pMMR) into high- and low-risk groups who might benefit from or be spared adjuvant chemotherapy based on their prognosis. In the Charité 1 discovery cohort (n=61), MACC1 expression and MSI status were assayed by qRT-PCR in cryo-preserved tumors from CRC patients. MSS/MSI-low/MACC1-low tumors showed better survival vs. MSS/MSI-low/MACC1-high (P&lt;0.0001). Patients with MSS/MSI-low/MACC1-low tumors had a similar prognosis as patients with MSI-H tumors. The Charité 2 comparison cohort (n=40) was used to translate MACC1 qRT-PCR analyses to FFPE samples. MACC1 expression was significantly higher in metachronously metastasizing tumors linked to shorter relapse-free survival (RFS), independent of the tissue type analyzed (cryo-preserved or FFPE). Next we translated MACC1 mRNA levels from qRT-PCR to MACC1 protein levels from immunohistochemistry (IHC) by comparing them in consecutive FFPE tumor sections in the BIOGRID 1 training cohort (n=189) enriched for disease recurrence. Chemotherapy-naïve patients with unfavorable pMMR status separated into MACC1-high and -low groups. Better RFS was seen in the pMMR/MACC1-low vs. pMMR/MACC1-high group using MACC1 mRNA and protein expression. pMMR/MACC1-low expression was seen in 12% and 8% of patients by qRT-PCR and IHC; interestingly, they had the same favorable prognosis as the deficient MMR (dMMR) group. Prognostic and predictive findings from BIOGRID 1 were confirmed in the independent BIOGRID 2 validation cohort (n=306) unenriched for recurrence. Better RFS was again seen in chemotherapy-naïve patients in the pMMR/MACC1-low (6%) vs. pMMR/MACC1-high group. No patients with pMMR/MACC1-low phenotype had disease recurrence. Remarkably, pooling BIOGRID 1 and 2, 5-year RFS was 100% and thus significantly longer in the pMMR/MACC1-low vs. pMMR/MACC1-high group (P=0.037). Taken together, MACC1 expression, measured by qRT-PCR or IHC, differentiates patients with unfavorable pMMR status. Patients with stage II colon cancer and pMMR/MACC1-low tumor status have a similar favorable prognosis as those patients with dMMR status who might not benefit from adjuvant therapy.

#2779

Assessing germline and somatic alterations in DNA repair pathway in cancer.

Gargi D. Basu,1 Tracey White,1 Janine LoBello,1 Ahmet Kurdoglu,1 Jeffrey Trent,2 Sen Peng,2 Matthew Halbert,1 Thomas Royce1. 1 _Ashion, Phoenix, AZ;_ 2 _Translational Genomics Research Institute, Phoenix, AZ_.

Introduction: DNA repair genes are involved with repair of single strand breaks as well as double strand breaks which ultimately maintain the genome integrity. The genes involved with DNA repair are frequently deregulated in cancer and these defects can be exploited therapeutically. Our study aims to explore somatic and germline changes in DNA repair genes across multiple cancer types.

Methods: Genomic profiling was performed on 226 tumor samples from 24 cancer types with ages ranging 19-82 years. We performed targeted exome sequencing of 562 genes in tumor and paired normal DNA which included 112 genes associated with cancer predisposition syndromes. In addition to identifying genomic changes in 33 key DNA repair genes (ATM, ATR, ARID1A, BAP1, BARD1, BRCA1, BRCA2, BRIP1, CDK12, CHEK1, CHEK2, ERCC2, ERCC3, ERCC4, FANCA gene family, MMR genes, NBS1, PALB2, PTEN, RAD50, RAD51, TP53 and POLE), we also characterized germline mutations using public databases (ClinVar, Ensembl).

Results: Profiled specimens included breast cancer (17.5%), ovarian cancer (15%), CRC (11%), NSCLC (10%), uterine (6%), sarcoma (6%) and other cancers. The most commonly deleted or mutated (somatic) DNA repair genes included TP53 (55%), PTEN (16%), ARID1A (13%), FANCA (11%), ERCC2 (10%), and ATM, BAP1, CDK12, CHEK2 at 6% each and BRCA1/2 combined at (9%). Deleterious mutations in MMR genes were noted in 3.5% (8/226) which included 1 case of germline PMS2-S46I mutation. Interestingly, a 55 yr old male African American CRC patient harbored germline XPC-P334H mutation along with somatic MLH1-Y548fs. No mutations were found in FANCC, FANCF, FANCG and RAD51. Germline analysis revealed a total of 47 pathogenic and presumed pathogenic variants out of which 61.7%(29/47) were DNA repair genes. A total of 13% (30/226) of cases harbored germline variants in DNA repair genes. Germline events included 9 cases with BRCA1 and BRCA2 clinically significant mutations. Presence of loss of heterozygosity (LOH) at the BRCA locus in 5/9 cases and somatic mutations in 3/9 cases were noted in germline BRCA mutated samples. Germline and somatic BRCA1 and BRCA2 alterations were present in 10% (22/226) of total cohort and as expected, the majority were present in breast and epithelial ovarian cancer. Genetic deficiencies in DNA repair pathway genes are being exploited therapeutically with PARP inhibitors as well as DNA damaging chemotherapeutics.

Conclusions: Exome sequencing identified subsets of patients with loss of function events in DNA repair genes that may be associated with benefit from PARP inhibitors and platinum agents. Somatic and germline biomarker testing revealed occurrence of BRCA1/2 as well as other DNA repair gene alterations across multiple cancers. It is imperative to explore the DNA repair pathway beyond BRCA1 and BRCA2 in patient selection for PARP inhibitors and DNA damaging agents and further investigation of this pathway is warranted in ongoing clinical trials.

#2780

High nuclear FTH1 protein expression predicts early disease recurrence for BRCA1/2 mutation carriers.

Antoinette Hollestelle, Mieke Timmermans, Renée Broeren-Foekens, Anita Trapman - Jansen, Carolien H. van Deurzen, Arzu Umar, John W. Martens, Maartje Hooning. _Erasmus Medical Ctr., Rotterdam, Netherlands_.

FTH1 is a 21kD subunit of the ferritin complex, which is involved in intracellular iron storage. In a previous study we found that FTH1 proteins can be located either in the cytoplasm or nucleus and, depending on its cellular localization, is associated with good or poor prognosis in triple negative breast cancer (TNBC). Furthermore, cytoplasmic localization of cFTH1 was associated with an increased anti-tumor (CD8+) T-cell / tumor-promoting (CD4+) T-cell ratio suggesting an immune modulatory role for FTH1. In the current study we have assessed the prognostic value of FTH1 expression for BRCA1/2 mutation carriers. FTH1 proteins were visualized by immunohistochemistry on a tissue microarray containing primary breast cancer specimens from 222 BRCA1/2 mutation carriers that were diagnosed between 1982 and 2008. Primary breast cancers from BRCA1 carriers were predominantly of the TNBC subtype, whereas primary breast cancers from BRCA2 mutation carriers were mainly ER-positive. The association between FTH1 expression and clinical outcome was evaluated by a Cox proportional hazards regression model using left truncation to adjust for study inclusion bias. The median follow-up time of the study was 9.2 years. High nuclear FTH1 (nFTH1), but not cytoplasmic FTH1 (cFTH1), was associated with decreased metastasis-free and disease-free survival as compared with low nFTH1 (multivariable HR=3.54, 95% CI=1.45-8.66, P=0.0056 adjusted for tumor size and nodal status; multivariable HR=2.71, 95% CI=1.49-4.92, P=0.0011 adjusted for tumor size). Neither nFTH1 nor cFTH1 were associated with breast cancer-specific or overall survival. This suggests that high nFTH1 expression in primary breast cancer predicts earlier disease recurrence for BRCA1/2 mutation carriers. Since we had observed that CD4+:CD8+ T-cell ratios were increased among TNBCs with high nFTH1, we hypothesize that nFTH1 promotes recruitment of immune suppressive CD4+/FOXP3+ regulatory T-cells in TNBC and BRCA1/2-associated tumors, suppressing CD4+/FOXP3- and CD8+ T-cell anti-tumor immunity.

#2781

Exosomal Del-1 as a potent diagnostic marker for breast cancer: A prospective cohort study.

Soo Jung Lee,1 Jeeyeon Lee,1 Yee Soo Chae,1 Jin Hyang Jung,1 Ho Yong park,1 Moon-Chang Baek,2 Pyong-Gon Moon2. 1 _Kyungpook National University Medical Center, Daegu, Republic of Korea;_ 2 _Cell and Matrix Research Institute, School of Medicine, Kyungpook National University, Daegu, Republic of Korea_.

Background: We previously demonstrated a diagnostic role of exosomal del-1 with two separated groups of breast cancer patients. In the current study, therefore, we aimed to confirm the diagnostic role in a prospective study with breast cancer by measuring plasma exosomal del-1 before and after surgery.

Patients and methods: To identify the optimal time of sampling after surgery, serial blood at day 1, 3, 5, and 7 after surgery was collected from 22 patients with early breast cancer. Thereafter, One hundred fifteen patients with breast cancer who underwent curative surgery were enrolled in the prospective cohort study to compare difference in plasma exosomal del-1 measured by ELISA at the time of diagnosis and post-surgery.

Results: Among all 22 patients for optimal sampling time after surgery, exosomal del-1 was higher than 0.5 at the time of diagnosis and then normalized at POD1. Among 115 patients for the confirmatory set, 109 (94.8%) patients showed a normalization of del-1 lower than 0.5 after surgery and 10 patients showed del-1> 0.4 . Median f/u duration of 22 months, 9 patients experienced relapse (4 locoregional and 5 distant), where 3 out of 6 in high group (>0.5), and 2 out of 4 in borderline group (0.4-0.5), and 4 out of 105 in normalized group (<0.4). In particular, patients who relapsed in higher del-1 group showed relatively earlier relapse compared to lower del-1 group.

Conclusion: In a prospective cohort study, we confirmed that exosomal del-1 has a potent diagnostic role in breast cancer. Furthermore, del-1 was also identified to dramatically decrease after curative surgery. Our current findings suggest its potential prognostic role as well as diagnostic role in breast cancer patients.

Changes of Exosomal Del-1 level between baseline and after-surgery (n=115)

---

sampling time | baseline | after surgery | p-value(paired t-test)

Del-1 (mean±SD) | 1.237±0.453 | 0.188±0.124 | <0.001

|  | |

#2782

The potential of p4EBP1 expression as predictive biomarker of mRCC.

Sei Naito, Osamu Ichiyanagi, Hiromi Ito, Hidenori Kanno, Tomoyuki kato, Yuuta Kurota, Atsushi Yamagishi, Mayu Yagi, Toshihiko Sakurai, Hayato Nishida, Hisashi Kawazoe, Tomohiro Shibasaki, Akira Nagaoka, Norihiko Tsuchiya. _Yamagata University, Yamagata, Japan_.

Introduction & objectives

Activation of Akt/mTOR pathway induces 4EBP1 phosphorylation, and enhances cell proliferation, anti-apoptotic effect, and angiogenesis in many types of cancers including renal cell carcinoma (RCC). As mTOR and angiogenetic proteins are main targets in metastatic RCC (mRCC) treatment. We assessed the correlation with survivals and phosphorylated 4EBP1 (p4EBP1) expression.

Materials & methods

We enrolled 254 non-mRCC patients who underwent primary surgery in Yamagata University between 2003 and 2010, and 59 mRCC patients whose resected primary lesion was available. Immunohistochemistry for p4EBP1 was performed on their FFPE samples. We assessed correlations between p4EBP1 expression manners and clinical features (disease-free interval [DFI] for non-mRCC patients, cause-specific survival [CSS] and progression-free survival [PFS] for mRCC patients). The CSS was calculated from mRCC diagnosis to death or last follow-up date. The PFS was calculated based on the durations patients were medicated. Univariate analysis was calculated by log-rank test and multivariate analysis was calculated by Cox-regression analysis.

Results

Non-mRCC patients with highly p4EBP1 expression were shorter DFI than those without high expression (p = 0.036). Their 5-year disease-free rates were 83.4% and 93.4%, respectively. The independent poor DFI factors were high p4EBP1 expression (HR; 3.4, p = 0.0054), grade (p = 0.0055), and pT stage (p < 0.0001).

In contrast, mRCC patients with p4EBP1 expression was longer CSS than those without expression (median CSSs; 56.7 and 32.2 months, p = 0.0246). The independent poor CSS factors were no p4EBP1 expression (hazard ratio [HR]; 3.3, p = 0.0409), grade 4 (HR; 8.7, p = 0.0006), and poor prognostic group on MSKCC criteria (HR; 4.2, p = 0.02770). Expression of p4EBP1 showed statistically longer PFS in mRCC patients with axitinib (median PFS; 9.2 and 2.5 months, p = 0.0255). The similar trends were shown in patients with other TKIs and mTOR inhibitors.

Conclusion

Since non-mRCC patients with the highly p4EBP1 expression had shorter DFI, expression of p4EBP1 should indicate aggressive RCC in nature. Nevertheless, mRCC patients with p4EBP1 expression had longer survival. These results mean that expression of p4EBP1 might be a predictive biomarker for TKIs and mTOR inhibitors.

#2784

Identifying biomarkers to predict cardiotoxicity of doxorubicin in individual patients.

Liqun Zhao, Baolin Zhang. _FDA, Silver Spring, MD_.

Doxorubicin is a highly effective anticancer agent but causes cardiotoxicity in many patients. This study aims to identify biomarkers to predict potential cardiotoxicity before the onset of cardiac tissue damage and dysfunction in individual cancer patients. First, we established a human cellular system to model the cardiac toxicity of oncology drugs using human induced pluripotent stem cells-derived cardiomyocytes (iPS-CMs). We tested a panel of clinically used drugs including anthracyclines (doxorubicin, daunorubicin, epirubicin and idarubicin), 5-FU, Taxol, Trastuzumab, and tyrosine kinase inhibitors (sunitinb, imatinib, and sorafinib). These drugs displayed varying degrees of cytotoxicity in iPS-CMs which are generally consistent with their known cardiotoxicity patterns. Second, we found that doxorubicin selectively upregulated the expression of death receptors in iPS-CMs. As a result, the treated iPS-CMs underwent spontaneous apoptosis that was further enhanced in the presence of specific cytokines. Based on these findings, we hypothesize that the baseline levels of the predefined cytokines in blood could be predictive of cardiotoxicity associated with doxorubicin treatment in individual patients. We are currently investigating the relationships between the levels of the predefined cytokine signature in blood and cardiac events in patients who received doxorubicin or related anthracycline therapy.

#2785

A comprehensive RNA-based assay for treatment prediction in non-small cell lung cancer patients.

Klaas Kok, Jaicong Wei, Anna Rybczynska, Martijn Terpstra, Anthonie van der Wekken, Jeroen Hilterman, Ed Schuuring, Rolf Sijmons, Harry Groen, Anke van den Berg. _UMCG, Groningen, Netherlands_.

Purpose: In late-stage lung cancer an increasing number of genomic aberrations is used to predict either sensitivity to, or resistance against, targeted therapies. These include gene mutations, gene fusions, amplifications and in the near future different relevant splice variants. Currently, detection of each type of aberration is carried out with different tests. We have set out to develop a novel all-in-one assay to detect the various types of aberrations, e.g. mutations, gene fusions and overexpression. The latter is assumed to be the biological marker that is underlying gene amplification.

Methods: Our assay is based on the Single Primer Enrichment Technology (SPET), allowing amplification of a target region using only a single sequence-specific primer. Our first custom-designed panel targets a comprehensive list of hotspots with therapeutic and prognostic significance in lung cancer, including EGFR, ALK, MET, KRAS, NRAS, PIK3CA, ROS1, BRAF, FGFR1 and RET. Using total RNA as input, we will be able to efficiently detect mutations, gene fusions and aberrant expression levels of selected genes. This test may also detect tumor-specific aberrations in platelets.

Results: As a proof of principle we analyzed RNA derived from 8 lung cancer-derived cell lines, from two frozen tumor biopsies, and from 10 FFPE biopsies all with confirmed genomic alterations by clinically approved assays. In total, conventional methods detected 18 small mutations, five gene fusions and three gene amplifications, that are all covered by our assay. Of these, 14 mutations, and four gene fusions were readily detected with our assay. The detection of three mutations failed because of low coverage of the target region, probably due to a less optimal design of the specific primer. One small deletion, and one gene fusion were not detected with our RNA-based assay, despite a high read coverage. Quantification of the expression level of the amplified genes is still under investigation.

Conclusion: Our preliminary data indicate this novel RNA-based assay not only efficiently identifies all crucial mutations in lung cancer cell lines, but also in small frozen and FFPE tumor biopsies of lung cancer patients. Our next goal is to test this novel method on RNA derived from platelets from cancer patients, providing a minimal invasive procedure to monitor the state of disease in those patients.

#2786

EGFR expression and phosphorylation in HNSCC predict response to EGFR inhibition but cell lines are not representative for the clinical situation.

Oliver von Ahsen,1 Sami S. Khaznadar,1 Martin Khan2. 1 _Bayer Pharma AG, Berlin, Germany;_ 2 _Klinikum Dahme-Spreewald, Koenigswusterhausen, Germany_.

Introduction Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer. Survival rates have not been improved for decades and conventional therapy is effective in only 50% of the patients. Based on broad expression in up to 80-90% of the HNSCC cases, epidermal growth factor receptor emerged as drug target but clinical efficacy of EGFR inhibitors in HNSCC is very limited. We therefore reinvestigated the EGFR expression levels necessary for response in cell lines and clinical samples.

Methods Standard procedures were used for IHC. The antibody clone D38B1 was used in 1:900 dilution for 2h at RT. Stainings were performed using the DAKO Envision system. EGFR expression and phosphorylation of Tyrosine-1173 were analysed by MSD (Mesoscale Discovery) in lysates from fresh frozen tumor or exponentially growing cells. For proliferation assays, 2000 cells per well were grown for 24 h before addition of inhibitors. Cell culture was continued for 72 h before testing viability using the CellTiter-Glo® Assay.

Results The majority (11/13) of HNSCC cell lines responded to the EGFR inhibitor Erlotinib. EGFR was highly expressed and phosphorylated in the Erlotinib responsive cell lines. Resistant cell lines displayed low level EGFR expression and phosphorylation. However, EGFR expression and phosphorylation in treatment naive clinical samples were significantly below the levels found in responding cell lines. In clinical samples EGFR was not overexpressed on the cellular level. Based on these findings, a clinical response to Erlotinib in HNSCC would not be expected.

Conclusion The prognostic value of EGFR expression has been used to argue for EGFR as a relevant target in HNSCC. Although most reviews claim that EGFR is overexpressed in HNSCC, clear data supporting this position are missing. Early studies tested the RNA levels and found the EGFR expression in tumors higher compared to control tissues. Studies using IHC assessed the association of EGFR expression with disease progression, but no comparison to expression in normal mucosa was described. Overexpression was based on percentage of positive cells not on the intensity of expression. We show similar levels of EGFR expression in growing keratinocytes and tumor cells. The often described overexpression only originated from a larger number of EGFR positive cells, not on overexpression on the cellular level. The high expression and functional relevance of EGFR in cell lines proves that EGFR activity is required for survival in cell culture. Our findings lead to the conclusion that this is not representative of the clinical situation. Definition of a response threshold for EGFR expression and clinical verification of this expression level is mandatory for the successful use of a predictive biomarker.

#2787

Identifying predictive markers of endocrine response in high-grade serous ovarian cancer using RNA sequencing.

Cristina Mapagu,1 Sian Fereday,2 Australian Ovarian Cancer Study Group, David D. Bowtell,2 Paul R. Harnett,3 Anna deFazio1. 1 _Westmead Institute for Cancer Research, University of Sydney, Sydney, Australia;_ 2 _Peter MacCallum Cancer Centre, East Melbourne, Australia;_ 3 _Crown Princess Mary Cancer Centre at Westmead Hospital, Westmead, Australia_.

Epithelial ovarian cancer is a complex disease and patients vary considerably in response to treatment. A subset of patients responds well to endocrine therapy, however hormone receptor positivity does not predict response and there are no biomarkers in clinical use to help select patients that would benefit from endocrine agents. We aimed to identify markers of endocrine response in high-grade serous ovarian cancer (HGSOC) by analysis of genes that are differentially expressed in tumors from patients that are endocrine responders compared with non-responders, combined with analysis of estrogen (E2) regulated genes that differ between E2 sensitive and E2 insensitive HGSOC cell lines.

Methods: Among women recruited to the Australian Ovarian Cancer Study, we identified 10 HGSOC patients treated with endocrine therapy (5 responders and 5 non-responders, based on GCIG CA125 response criteria) and performed RNAseq on cryopreserved tumor tissue. We also utilized paired E2 receptor (ER) positive HGSOC cell lines, derived from the same patient, with differential sensitivity to the growth effect of E2, PEO1 (E2 insensitive) and PEO4 (E2 sensitive). RNA was extracted following treatment with 0.1 nM E2 (or vehicle) for 24 hrs and gene expression changes in response to E2 were determined using RNAseq. Expression of selected ER responsive genes was validated using PCR array. Differential gene expression was determined using EdgeR. Pathway and Gene Ontology enrichment analysis of differentially expressed genes were performed using Metacore.

Results: Between endocrine responders and non-responders, 27 genes were significantly differentially expressed (False Discovery Rate (FDR)<0.05, absolute log2 Fold-Change (FC)≥1). In the paired HGSOC cell lines, significantly more genes were regulated by E2 in E2-sensitive PEO4, compared with E2-insensitive PEO1 (896 genes compared to 56 genes, respectively, FDR<0.05, absolute log2FC≥1). In response to E2, up-regulation of NRP1 in PEO1, up-regulation of LTBP1, MYC, PDZK1 and down-regulation of S100A6 and CYP1A1 in PEO4 were validated by PCR array profiling of ER-related genes. Genes regulated by E2 in cell lines and also differentially expressed between endocrine responders and non-responders included anoctamin 1 (ANO1) and NOTCH-Regulated Ankyrin Repeat Protein (NRARP). Both were up-regulated in endocrine responders and also up-regulated by E2 in PEO4.

Conclusion: The use of RNAseq in both tumors and cell line models was able to identify plausible sets of genes with prior evidence of association with endocrine sensitivity. In breast cancer, ANO1 has been associated with good prognosis following tamoxifen treatment, while NOTCH signaling, implicated in endocrine resistance, is negatively regulated by NRARP. Further validation is required to test the utility of these genes as predictive biomarkers that may aid in identifying HGSOC patients likely to respond to endocrine treatment.

#2788

**High ALDH1, S phase fraction, p16** INK4A **in esophageal squamous cell carcinoma could predict response to neoadjuvant chemotherapy.**

Rajeev Kumar,1 R. Ravi Kannan,1 Akalesh Kumar Verma,1 Anuradha Talukdar,1 Monoj Kumar Deka,1 Ritesh Tapkire,1 Litika Vermani,1 Sankar Kumar Ghosh2. 1 _Cachar Cancer Hospital and Research Centre, Assam, India;_ 2 _Assam University, Assam, India_.

Background: The prevalence of locally advanced esophageal squamous cell carcinoma (ESCC) remains high despite technological advancements in its diagnosis. This leads to prolonged multimodality treatment often resulting in poor response eventually leading to poor prognosis. Currently, there are no biomarkers available to predict response to neoadjuvant chemotherpy. Aldehyde dehydrogenase 1 (ALDH1), human epidermal growth factor receptor-2 (HER2), p16INK4A, ploidy and S phase fraction are considered significant biomarkers in various solid malignancies. However, there is paucity of data on their clinical significance in ESCC. In the present study, we investigated their significance in predicting the response to neoadjuvant chemotherapy (NACT) in ESCC patients.

Methods: Immunohistochemisty of ALDH1, HER2, p16INK4A and propidium iodide based cell cycle analysis through flow cytometer was performed in pre treatment biopsy sample collected from 108 ESCC patients who were presented at a comprehensive cancer centre in northeast India and all of them subsequently received neo adjuvant chemotherapy. Results: ALDH 1, HER2 and p16INK4A were found positive in 65.7%, 7.4% and 22% of pre treatment ESCC specimens respectively. ALDH1 expression correlates with poor response to neo adjuvant chemotherapy (P<0.001). A significant proportion of poor responders were found to be smokers (P=0.004). Poor responders also tends to correlate with increased mortality (P=0.009). HER2 couldn't predict response to NACT. However, all HER2 positive cases were associated with high ALDH1 expression (P=0.034). 50% of those with pathologically complete response were p16 positive on the contrary only 15% non responders were positive for p16 (P<0.001). 84% of the ESCC patients reported aneuploid tumor status and responder's were found having high S phase fraction (P=0.041). Conclusion: High ALDH1 expression can predict poor response whereas high p16 and S phase fraction can indicate good response to neoadjuvant chemotherapy in ESCC patients. HER2 seems having no clinical significance in the response assessment of neo adjuvant chemotherapy in ESCC.

#2789

PID, a novel immunofluorescence nanotechnology, as a histopathology-based EGFR biomarker assay enhances predictive power for the Erbitux response in subsets of PDXs.

Henry Qixiang Li,1 Jia Xue,1 Hisatake Okada,2 Etsuko Futaya,2 Yoshikazu Kurihara,2 Likun Zhang,3 Xiaoyu An,3 Sheng Guo3. 1 _Crown Bioscience, Inc., Beijing, China;_ 2 _Konica Minolta, Beijing, Japan;_ 3 _Crown Bioscience, Inc., Santa Clara, CA_.

Immunohistochemistry (IHC) is the most common used assay for detecting/quantifying defined protein biomarkers in specific tissue location in clinical/preclinical settings as prognostic/drug-predictive biomarker assays. However, IHC suffers from several key drawbacks, limiting its applications, including semi-quantitative nature, inconsistence, low sensitivity, narrow dynamic detection range and inadequacy for multi-target analysis, etc. An alternative method is immunofluorescence (IF), but with even lower usage for its disadvantageous photo-instability. A sensitive, quantitative and robust assay is thus an urgent/unmet need. We have recently developed a novel fluorescence nanoparticle (PID, phosphor integrated dye) with highly compact phosphor inside. PID has several key advanced features: 1) enhanced fluorescence (30,000x brighter over the conventional fluorescence and significantly above tissue auto-fluorescence), 2) enhanced photo-stability (stained slide is stable for >2 years); 3) highly quantitative by broadened detection dynamic range through enumeration of the bright fluorescent dots (nanoparticles) instead of signal amplification as in IHC (digital vs. analog); 4) fine subcellular imaging; 5) potentials for multi-color and H&E/bright light imaging. PDX is a well-accepted experimental cancer model mimicking original patients in histo- & molecular pathology(1), as well as drug response. We previously conducted a series of mouse clinical trial (MCT) of Erbitux on PDX cohorts of NSCLC, CRC, gastric and esophageal cancers (2,3), showing specific subsets of high-EGRF-expressing PDXs responded to the drug, where EGFR expression was measured by either RNAseq (mRNA) or IHC (protein). To assess PID-EGFR as a new biomarker assay platform, we analysed and compared its quantification to RNAseq and IHC, particularly in terms of the correlation to the drug efficacy. Our data demonstrates a greatly enhanced correlation of PID scores to mRNA levels in all cancer types, and to the tumor growth inhibition of gastric (3) and esophageal (4) carcinoma, but not in CRC (2) and NSCLC (Li et al., unpublished) as anticipated. The results demonstrated that PID-EGFR is a significantly improved histopathology based EGFR protein quantification assay over IHC-EGFR assay, warranting further clinical confirmation.

References

1. Guo S, et al. Cancer research 2016;76(16):4619.

2. Chen D, et al. Oncotarget 2015;6(38):40815

3. Zhang L, et al. Sci Rep 2013;3:2992. 4. Zhu et al. 2016.

#2790

PRC2 mediated repression of SMARCA2 predicts for EZH2 inhibitor activity in tumors with SWI/SNF mutations.

Thomas Januario, Xiaofen Ye, Russell Bainer, Bruno Alicke, Margaret Solon, Benjamin Haley, Zora Modrusan, Stephen Gould, Hartmut Koeppen, Robert L. Yauch. _Genentech, Inc., South San Francisco, CA_.

A synthetic lethality caused by EZH2 inhibition in the context of SNF5 mutations is supported by both preclinical and recent clinical data, however the extent of the synthetic lethal relationship in the context of other SWI/SNF subunit mutations is not well understood. We determined that a subset of SMARCA4 mutant cancer models are sensitive to EZH2 inhibition. EZH2 inhibition resulted in a heterogenous phenotypic response characterized by senescence and/or apoptosis amongst models, and further lead to tumor growth inhibition in vivo. The differential sensitivity to EZH2 inhibition was not caused by a differential pharmacodynamic effect of the drug, nor differences in basal histone methylation or PRC2 subunit levels. However, expression of the SWI/SNF subunit, SMARCA2, delineated sensitivity amongst SMARCA4 mutant models tested. Expression of SMARCA2 further delineated sensitivity amongst other SWI/SNF mutant models tested, including SNF5 and ARID1A mutants. We determined that SMARCA2 is under PRC2 mediated suppression and the derepression of SMARCA2 was necessary for apoptosis, but not senescence, in response to EZH2 inhibition. SMARCA2 has been shown to be concurrently lost in a high percentage of SNF5 mutant malignant rhabdoid tumors and SMARCA4 mutant SCCOHT tumors, however we determined that ≈15% of SMARCA4 mutant NSCLCs concurrently lose SMARCA2. Our data supports monitoring SMARCA2 expression as a predictive biomarker for EZH2-targeted therapies that are currently being developed in the context of SWI/SNF mutant cancers.

#2791

CDC25A overexpression is correlated with tumor response to radiotherapy in locally advanced rectal cancer.

Ming-Yii Huang. _Kaohsiung Medical University, Kaohsiung, Taiwan_.

Background. Radiotherapy is increasingly used in preoperative approaches for locally advanced rectal cancer to reduce local recurrence. However, the principal limitation is large variability in response among different individuals due to tumor heterogeneity. In current study, we compared gene expression profiles between radiation responder and non-responder in cancerous tissues to identify radiation-related molecules that can be used to evaluate the tumor response of radiation.

Methods. We investigated five genes (CDC25A, VAV1, TP73, BRCA1 and ZAP70) which were significantly related to prognostic factors (tumor size, advanced stage, invasive depth, lymph node metastasis and differentiation) from 110 colorectal cancer tissues confirmed by membrane array and RT-PCR methods in our previous study. Therefore, we further investigate those protein overexpressions by immunohistochemical stain in pretherapeutic rectal cancerous tissues. The expression profiles were evaluated according to the frequency of positive staining in the cytoplasm and/or nucleus of cancer cells. Its expression was classified as positive in cases with more than 50% positive-stained cells, with other samples being classified as negative. Cancer tissues that expressed scores of + were regarded as the overexpression group, whereas those with scores of negative staining were regarded as the non-overexpression group.

Results. We focused on these 5 genes and validated data with pretherapeutic biopsies from 36 locally advanced rectal cancer patients, and found that overexpression of CDC25A, VAV1 and ZAP70 were correlated with tumor response (P=0.001, 0.033, and 0.016, respectively). The multivariate logistic regressions with adjustment of age, sex and stage, CDC25A overexpression more than 2-fold were significantly correlated with tumor response (OR = 2.667, 95%CI = 1.591-4.470, P=0.03). Positive findings of CDC25A immunohistochemistry stain were also present in the tumor specimens of responders.

Conclusion. We identified radiation-related genes in rectal cancer and demonstrated that CDC25A may play an important role in response to radiotherapy. These findings suggest that CDC25A may be a novel marker for predicting the effectiveness of radiotherapy in clinical patients.

#2792

The small molecule imipridone ONC201 is active in tumor types with dysregulation of the DRD2 pathway.

Neel Madhukar,1 Varun Vijay Prabhu,2 Etienne Dardenne,1 Faye Doherty,3 Alexander VanEngelenburg,3 Rohinton Tarapore,2 Mathew Garnett,4 Ultan McDermott,4 Cyril Benes,5 Wolfgang Oster,2 Wafik El-Deiry,6 Mark Stein,7 David Rickman,1 Joshua Allen,2 Olivier Elemento1. 1 _Weill Cornell Medicine, New York, NY;_ 2 _Oncoceutics, Inc, Philadelphia, PA;_ 3 _HistoTox Labs, Inc, Boulder, CO;_ 4 _Wellcome Trust Sanger Institute, Hinxton, United Kingdom;_ 5 _Massachusetts General Hospital, Boston, MA;_ 6 _Fox Chase Cancer Center, Philadelphia, PA;_ 7 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ_.

ONC201 is the lead small molecule of the imipridone class of anti-cancer compounds that is currently being evaluated in phase I/II advanced cancer clinical trials. ONC201 is a highly selective antagonist of the G protein-coupled receptor dopamine receptor D2 (DRD2) that has exhibited promising anti-cancer efficacy and an exceptional safety profile. In the current study, we evaluated the influence of the DRD2 pathway on the responsiveness of tumors to ONC201 in preclinical and clinical studies. In vitro and in vivo studies have previously demonstrated robust ONC201 efficacy in glioblastoma (Allen et al 2013) and lymphoma (Ishizawa et al 2016) models. ONC201 Phase I trials have also revealed evidence of clinical benefit in endometrial cancer (Stein et al 2016). In vitro efficacy profiling of ONC201 in the Genomic of Drug Sensitivity in Cancer (GDSC) collection of cell lines confirmed broad-spectrum anti-cancer efficacy with particularly high sensitivity in lymphoma, neuroblastoma, endometrial and brain cancer. DRD2 is overexpressed in many cancers and DRD2 antagonism kills cancer cells via the same signaling pathways that are altered in response to ONC201. Results from the Project Achilles screen indicate that anti-cancer effects of DRD2 knockdown in various tumor types correlated with overall ONC201 efficacy. In particular, we noted that lymphoma cells are highly sensitive to DRD2 knockdown- a tumor type where ONC201 performs well. Gene expression analysis of samples in the Cancer Genome Atlas (TCGA) revealed high DRD2 expression in ONC201-sensitive tumor types, such as lymphoma and glioblastoma, and that high expression of DRD2 in glioma was associated with a poor prognosis. High DRD2 expression was also observed in neuroendocrine prostate cancer relative to other prostate cancer subtypes. In immunohistochemistry analyses of patient-derived tumor tissue microarrays, DRD2 overexpression was particularly noted in endometrial cancer, neuroblastoma and pheochromocytoma relative to normal tissues. The anti-cancer activity of ONC201 in pheochromocytoma and neuroendocrine prostate cancer was confirmed in cell viability assays. In ONC201-treated patients, ELISA was used to quantitate serum prolactin levels, a clinical biomarker of DRD2 antagonism. A 2-fold mean induction of prolactin, was detected in the serum of ONC201-treated patients, in accordance with physiological DRD2 antagonism. Interestingly, expression of DRD5 (a D1-like dopamine receptor), which counteracts DRD2 signaling, was significantly negatively correlated with ONC201 in vitro potency in the NCI60 and GDSC dataset (P <.05). Furthermore, a missense DRD5 mutation was identified in cancer cells with acquired resistance to ONC201. In conclusion, the DRD2 pathway is expressed in ONC201-sensiive tumors and may provide biomarkers of response.

#2793

Evidence for WT1 as a potential target for immunotherapy of lethal ovarian cancer.

Julia H. Carter,1 James A. Deddens,2 Gretchen Mueller,1 Thomas G. Lewis,1 Mariah K. Dooley,1 Jackson O. Pemberton,3 Larry E. Douglass1. 1 _Wood Hudson Cancer Research Lab., Newport, KY;_ 2 _University of Cincinnati, Cincinnati, OH;_ 3 _St Elizabeth Healthcare, Edgewood, KY_.

Epithelial ovarian cancer is the leading cause of death from gynecologic cancers with 22,280 new cases anticipated in 2016 and 14,240 deaths. Most patients are diagnosed at late stages and have a poor prognosis. Radical surgery and chemotherapy are the primary treatment. When relapse occurs there are few treatment options. The overall survival rate of women with ovarian cancer is unchanged in 50 years. Approximately 70% of epithelial ovarian cancers are serous including low grade serous tumors, some of which may evolve from serous borderline tumors, and high grade serous carcinomas. The latter are thought to originate in the fallopian tube. There are eight other subtypes of invasive ovarian cancer. All invasive ovarian cancers have recently been classified into two subtypes, Type 1 (indolent) and Type 2 (aggressive). The Wilms' Tumor 1 (WT1) protein is expressed in some normal tissues including the fallopian tube and ovary as well as in tumors such as ovarian and breast cancers and in leukemia. WT1 is a highly immunogenic tumor associated antigen. Mutated p53 is found in many high grade ovarian cancers. Recent reports indicate that WT1 and p53 may interact physically and functionally. Both WT1 and mutated p53 have been suggested as prognostic markers in ovarian cancer. We sought to determine the role of co-expression of mutated p53 and WT1 in overall survival of patients with ovarian cancer. Histologic sections were prepared from archived FFPE surgical specimens donated by St. Elizabeth Healthcare (Northern KY) and fully annotated by hospital records. H&E stained sections were diagnosed and graded by a board certified pathologist (LED) and included histologic sections from 41 borderline tumors and 98 Type 1 and Type 2 cancers. Adjacent sections were stained immunohistochemically using monoclonal antibodies (DAKO) to WT1 and p53. For survival analysis p53 was considered mutated if 75-100% of the tumor nuclei were stained (missense mutations) or if all nuclei were negative (truncated protein) and WT1 was considered either non-expressed (no stain) or expressed (some or all nuclei stained). While either mutated p53 or WT1 were prognostic alone of patient survival with Type 1 and Type 2 tumors, WT1 was a better predictor as seen by log-rank Chi-square. The 20 year survival probability of patients with tumors negative for both mutated p53 and WT1 was 70% and was significantly better than other patients (p < 0.0001). The 20 year survival probability for tumors negative for WT1 but expressing mutated p53 was 41%, and was 9% for tumors expressing WT1 and non-mutated p53. Patients with tumors expressing both WT1 and mutated p53 had a 20 year survival probability of 6%. Given these data and the immunogenicity of the WT1 tumor associated antigen we conclude that IHC detection of mutated p53 and WT1 in invasive ovarian cancers would be useful in stratifying patients eligible for immunologic approaches targeting WT1 for therapy of lethal ovarian cancers.

#2794

Indotecan (LMP400), imidotecan (LMP776) and LMP744: A new class of non-camptothecin Top1 inhibitors selective for homologous recombination deficient (HRD) cells.

Laetitia Marzi, Keli Agama, Zoe Weaver Ohler, Ludmila Szabova, Shyam Sharan, Junko Murai, Muthana Al Abo, Yves Pommier. _NCI, Bethesda, MD_.

To relax DNA supercoiling during transcription and replication, topoisomerase I (Top1) induces transient DNA cleavage complexes, which are trapped by anticancer drugs, leading to DNA double-strand breaks (DSB) that need to be repaired by homologous recombination (HR). BRCA1, BRCA2 and PALB2, which are key components for HR, lead to "synthetic lethality" with PARP inhibitors. Topotecan and irinotecan (camptothecin derivatives) are the only FDA-approved Top1 inhibitors. In spite of their wide usage they are plagued by their chemical instability, being drug efflux substrates, having short half-life, and dose-limiting bone marrow and gastro intestinal toxicity. It is now possible to overcome those limitations with the non-camptothecin indenoisoquinolines (LMP400, LMP776 and LMP744), which are in clinical trials. To rationally select patients for phase 2 clinical trials based on cancer-specific genomic alterations, we have determined whether the LMPs present a "synthetic lethality" toward BRCA1, BRCA2 or PALB2 deficiency, and whether this selectivity could be enhanced by combining them with the recently approved PARP inhibitor, olaparib.Using isogenic DT40 cell lines, with BRCA1, BRCA2 or PALB2 deficiencies, we assessed the role of HR in the cellular responses to the LMPs. Survival and cell cycle modifications were tested after treatment with the LMPs as single agents, as well as in combination with olaparib. We found that BRCA1-, BRCA2- and PALB2-deficient cells are 3 to 5 times hypersensitive to the LMPs (IC50's for LMP400 and LMP744= 10 nM for HR-deficient cells vs. 45 nM for WT cells, and IC50 for LMP776 around 5 nM vs. 18 nM for WT cells). Cell cycle analyses confirmed the death of these HR-deficient cells. Moreover, combination treatments showed a significant synergy between each of the three LMPs and olaparib (Combination index<0.7). The HR-deficient cells being markedly more sensitive than WT cells (taken as surrogate for normal tissues) to both treatments, led to a better response to the combination in those cells.Our results show that the LMPs are active at nanomolar concentrations, selectively in HR-deficient cells, indicative of a "synthetic lethality" of the indenoisoquinolines (LMPs) with HRD genotypes. They also demonstrate that the LMPs synergize with olaparib. These findings provide a rationale for personalized treatment and Phase 2 clinical trials with the indenoisoquinolines in combination with PARP inhibitors in HR-deficient cancers.

#2795

Integrating multiomics discovery approaches to identify biomarkers of therapeutic resistance in metastatic colorectal cancer through analyses of multiple sequential tumor and liquid biopsies; Qcroc01: NCT00984048.

Karen Gambaro,1 Maud Marques,2 Ryan Morin,3 Claudia Kleinman,2 Michael Witcher,2 Simon Turcotte,4 Benoit Samson,5 Bernard Lespérance,6 Yoo-Joung Ko,7 Richard Dalfen,8 Eve St-Hilaire,9 Lucas Sideris,10 Felix Couture,11 Sabine Tejpar,12 Ronald Burkes,13 Mohammed Harb,14 Francine Aubin,4 Thierry Alcindor,15 Errol Camlioglu,2 Adriana Aguilar,2 Mathilde Couetoux du Tertre1,1 Suzan McNamara,1 Adrian Gologan,2 Petr Kavan,2 Gerald Batist2. 1 _Quebec- Clinical Research on Cancer (Q-CROC), Montréal, Quebec, Canada;_ 2 _Segal Cencer Centre-Jewish General Hospital, Montréal, Quebec, Canada;_ 3 _Simon Fraser University, Vancouver, British Columbia, Canada;_ 4 _Centre Hospitalier de l'Université de Montréal, Montréal, Quebec, Canada;_ 5 _Charles LeMoyne Hospital, Montréal, Quebec, Canada;_ 6 _Sacré-Coeur de Montréal, Montréal, Quebec, Canada;_ 7 _Sunnybrook Health Science Centre, Toronto, Ontario, Canada;_ 8 _St. Mary's Hospital, Montréal, Quebec, Canada;_ 9 _Georges Dumont Hospital, Moncton, New Brunswick, Canada;_ 10 _Hôpital Maisonneuve Rosemont, Montréal, Quebec, Canada;_ 11 _Hôtel-Dieu de Quebec, Quebec, Quebec, Canada;_ 12 _Katholieke Universiteit Leuven, Leuven, Belgium;_ 13 _Mount Sinai Hospital, Toronto, Ontario, Canada;_ 14 _Moncton Hospital, Moncton, New Brunswick, Canada;_ 15 _McGill University Health Centre, Montréal, Quebec, Canada_.

Colorectal cancer (CRC) is the 2nd leading cause of cancer related-death in Canada. Clinical responses of metastatic (m)CRC to first-line treatment range from 35 to 60%, but even responders inevitably develop therapeutic resistance. Studies aiming at understanding mechanisms of resistance have largely investigated primary tumors. However, selective pressures during therapy can lead to the expansion of resistant clones and tumor heterogeneity. This highlights the need to characterize the molecular changes of metastasis and plasma over time of treatment and response to decipher tumor evolution and therapeutic resistance mechanisms.

In this multicenter study, 52 tissue samples from liver metastasis were collected at baseline (pre-biopsies) and at the time of resistance (post-biopsies) in responder and non-responder mCRC patients (n=44) undergoing the same standard first-line treatments. Multiple post-biopsies also have been harvested in 4 patients, to allow the assessment of tumor heterogeneity and as well as the evolution of the genomic complexity after treatment exposure.

Analyses were carried out across multiple omic platforms to identify resistant signatures and characterize molecular changes during treatment. Biopsies were profiled using exome and transcriptome sequencing as well as high-density SNP array analysis to capture chromosomal anomalies, loss of heterozygosity (LOH) and copy number variations (CNV). Additionally, serial blood samples were collected for proteomic, ctDNA and cytokine analysis.

Our preliminary analysis of transcriptomes performed on serial biopsies from a set of 11 patients identified genes consistently overexpressed at resistance. Cytogenetics analysis showed similar genomic profiles of matched pre- and post-biopsies and allowed the establishment of LOH and CNV catalogues of liver metastasis, while exome sequencing revealed cumulative somatic mutations over time of treatment, which suggests subclonal and acquired "driver" mutations of resistance. Plasma-derived ctDNA analysis was performed to investigate the mutational status during treatment and whether they correlate with their relative levels in biopsies. Immune gene expression analysis of a test set of 27 metastases revealed strong clustering of 7 metastases due to overexpression of transcripts related to active immune response, allowing to define novel subgroups of patients based on immune response status. Our study, using a multi-omic strategy and integration of independent molecular platforms to profile liver metastasis samples of responder and non-responder mCRC patients, constitutes an innovative approach to identify clinical biomarkers and molecular signature of resistance, which may enhance individualization of cancer medicine and customized therapy.

#2796

Development of a RAD51-based assay for determining homologous recombination proficiency and PARP inhibitor sensitivity.

Bose S. Kochupurakkal,1 Kalindi Parmar,1 Jean-Bernard Lazaro,1 Christine Unitt,2 Qing Zeng,1 Hunter Reavis,1 Chirag Ganesa,1 Shan Zhou,1 Joyce Liu,1 Sangeetha Palakurthi,1 Kyle Strickland,2 Brooke Howitt,2 Panagiotis Konstantinopoulos,1 Paul Kirschmeier,1 Joseph Geradts,2 Ronny Drapkin,3 Ursula Matulonis,1 Alan D'Andrea,1 Geoffrey Shapiro1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Brigham and Women's Hospital, Boston, MA;_ 3 _University of Pennsylvania, Philadelphia, PA_.

Homologous recombination (HR) repair deficiency confers sensitivity to inhibitors of poly(ADP-ribose) polymerase (PARP). To date, the identification of tumors with impaired HR has relied on genomic features, including mutational signature, LOH-based HRD assays or gene expression analyses defining 'BRCAness'. These tests analyze history of the tumor rather than providing a functional assessment of HR status at the time of diagnosis. Therefore, development of a functional assay for HR status in tumors is essential to make accurate treatment decisions. Here, we describe a RAD51-based immunohistochemical (IHC) assay that identifies HR status. We first screened commercial anti-RAD51 antibodies and identified a monoclonal antibody that detects RAD51 foci in HR-competent normal fibroblasts and shows no evidence of foci in HR-deficient (BRCA2-/-) VU423 fibroblasts after γ-irradiation. Conditions for detecting RAD51 foci in FFPE samples were identified using HR-deficient and HR-proficient triple-negative breast cancer cell lines. HR-deficient, PARP inhibitor-sensitive cell lines exhibited high levels of nuclear RAD51 and no evidence of foci, whereas HR-proficient, PARP inhibitor-resistant cells had low levels of nuclear RAD51 and foci. This result was confirmed in a BRCA1-mutated, PARP inhibitor-sensitive PDX model, where there was no evidence of foci although RAD51 levels were high. We further evaluated the pattern of RAD51 staining in 13 high-grade serous ovarian cancer (HGSOC) PDX models, for which sensitivity to olaparib had been characterized. The only olaparib-sensitive model demonstrated complete absence of RAD51 staining. Among the other 12 olaparib-resistant models, RAD51 foci were detectable, both before and after irradiation. The presence or absence of RAD51 foci correlated with olaparib sensitivity and not with BRCA mutation status. Therefore, tumors that are HR-deficient and PARP inhibitor-sensitive are characterized by either high RAD51 nuclear staining without foci, or absence of RAD51 staining. To validate these findings, we analyzed RAD51 staining patterns in a cohort of 50 primary HGSOCs from patients subsequently treated with platinum-based chemotherapy. Among these 50 samples, 45 demonstrated either RAD51 nuclear staining without foci or an absence of RAD51 staining. Five samples demonstrated RAD51 staining with foci. The median survivals of these groups were 6.1 and 1.5 years, respectively. In conclusion, we have developed a robust IHC assay for determining the functional HR-status in tumor samples. Further work will be required to determine if the staining patterns observed predict PARP inhibitor sensitivity among primary patient samples. Funded by a Biomarker Supplement to UM1 CA186709, NIH Grant P50 CA168504, SU2C Ovarian Cancer Dream Team and BCRF grant.

#2797

MiR-137 and MiR-496 target Del-1 and affect triple negative breast cancer progression.

Jeeyeon Lee, Yee Soo Chae, Soo Jung Lee, Jin Hyang Jung, Ho Yong Park, Moon-Chang Baek. _Kyungpook National University Medical Center, Daegu, Republic of Korea_.

Background: Although Del-1 was recently proposed as a new biomarker for early breast cancer in our previous studies, the mechanisms of Del-1 expression are barely understood. In the current study, we selected two microRNAs (miR-137 and - 496), potentially affecting Del-1 expression in breast cancer and examined their impact on Del-1 expression in a variety of breast cancer cell lines to identify their potential role in Del-1 expression and thereby breast cancer development or progression.

Methods: Del-1 mRNA and miR-137/- 496 levels were measured by qRT-PCR among breast epithelial (MCF10A) and cancer cells (MDA-MB-231, MCF7, SK-BR3 and T-47D). The effects of miR-137/- 496 on cell proliferation and invasion were detected using MTT, wound healing and Transwell assays. Furthermore, luciferase reporter assay was used to identify the direct regulation of Del-1 by miR-137 or - 496 in MDA-MB-231 cells. Plus, we analyzed the expressions of miR-137 or - 496 and Del-1 mRNA from 20 patients with triple negative early breast cancer.

Results: miR-137 and - 496 levels were low in all breast cancer cell lines. As Del-1 mRNA expression was remarkably higher in MDA-MB-231 compared to the other breast cancer cell lines, further functional analyses were done with MDA-MB-231 representing triple negative breast cancer subtype. Both miR-137 and miR-496 were revealed to directly bind at the 3'-UTR of Del-1. Del-1 by Luciferase reporter assay and Del-1 expression was upregulated by inhibitors and reversed by both mimics of both miR-137 and miR-496. Furthermore, both miR-137 and miR-496 were also demonstrated to inhibit cell proliferation, migration and invasion of MDA-MB-231, suggesting that these miRNAs affect cancer progression via Del-1. MiR-137 and miR-496 were remarkably down-regulated in 7 out of 12 triple negative breast cancer tissues, in particular with high Ki67 and high histologic grade.

Conclusion: Although Del-1 was recently introduced as a new biomarker for triple negative breast cancer, the mechanisms of Del-1 expression were barely identified. The current study firstly demonstrated that microRNA 137 and 496 are involved in Del-1 regulation by binding at Del-1 gene, affecting cancer progression by altering Del-1 expression.

#2798

Identification of gamma secretase inhibitor (GSI)-responsive GBM patients: Involvement of NOTCH, p53, and PI3K/MAPK signaling.

Chen Zhang, Martinez-Ledesma Juan, Jie Ding, Feng Gao, Shaofang Wu, Xiaolong Li, Erik Philip Sulman, Dimpy Koul, WK Alfred Yung. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Glioblastoma (GBM) is the most common and lethal primary intracranial tumor. The recent standard-of-care treatment consisting of surgery, followed by radiotherapy and temozolomide, just prolongs the median survival period of GBM patients to 14.6 months. What impedes the survival is due to the extensive heterogeneity among GBM patients at both the cellular and the molecular levels. In our study, we focused on the precision medicine strategy targeting NOTCH signaling. According to recent Phase I/II clinical trial results, only a few GBM patients showed satisfactory response to GSIs. However, little is known regarding how to distinguish GSI-responsive patients. Here, we illustrated that it required (1) high NOTCH activity, (2) wild-type p53 and elevated P53 activity, (3) increased MAPK activity, and (4) decreased PI3K/AKT activity for GBM to respond to GSIs. In our study, we tested a series of patient-derived primary glioma stem cells (GSCs) to two GSIs (RO4929097 and BMS-708163). GSI-sensitive lines were identified and GSI compounds impaired cell viability and sphere formation ability in these cell lines. GSIs also induced cell death of the sensitive GSCs. To find out the signature of the GSI-sensitive GSCs, we did enrichment and correlation analyses against RNA sequencing, RPPA, and methylation data of the GSI-sensitive and resistant GBM lines. We found that GSI-sensitive GSCs harbored significantly elevated Notch activity (high NOTCH1 and NICD1 expression, and low Notch1 methylation). Interestingly, GSI-responsive cells also possessed increased P53 activity (increased expression of P53 targets-P21, BAX and TIGAR), high wild-type p53 frequencies, increased MAPK activity (elevated p-Erk1/2-Thr202/Tyr204 expression), and decreased PI3K/AKT activity (high PTEN expression and low p-AKT-Thr308, RAPTOR, p-4E-BP1-Ser65 expression) (P<0.05). In an attempt to strengthen GSI effect, we showed that MDM2 inhibitor Nutlin-3 (retards P53 degradation and increases P53 activity) combining RO4929097 exhibited synergistic effect on suppressing cell viability of GSI-sensitive cells with wild-type p53. In summary, we elucidated the genetic characteristics (high NOTCH, wild-type p53, high MAPK, and low PI3K/AKT) of GSI sensitive GSCs. Co-administration of Nutlin-3 enhanced GSI function in the subset of GBMs. Our findings indicated genetic markers could be used to identify patients responsive to GSIs, providing a new therapeutic strategy to treat this population of GBM patients.

## TUMOR BIOLOGY:

### Cell Culture and Animal Models of Cancer 3

#2800

Transforming growth factor-beta 2 (TGF-β2) antisense oligonucleotide (ASO) OT-101 synergizes with chemotherapy in preclinical tumor models.

Osmond D'Cruz,1 Cynthia Lee,1 Vuong Trieu,2 Larn Hwang2. 1 _Autotelic Inc, Costa Mesa, CA;_ 2 _Oncotelic Inc, Agoura Hills, CA_.

Background: Overexpression of TGF-β2 has been implicated in the malignant progression of tumors by inducing immunosuppression, proliferation, angiogenesis and metastasis. Clinical failures of TGF-β inhibitors in targeting the tumor promoting arm of TGF-β signaling is attributed to multiple isoforms and receptor functions. OT-101 (Trabedersen) is a phosphorothioate ASO designed to specifically target human TGF-β2 mRNA with superior clinical activity in patients with TGF-β2-overexpressing tumors. Herein, we report the synergizing effect of OT-101 with chemotherapy in multiple human tumor xenograft models for further exploration of clinical combinations.

Methods: The in vivo efficacy studies of intraperitoneal (IP, 16 or 50 mg/kg) or subcutanous (SC, 1-64 mg/kg) repeated administration (qdx3/wk or qdx21) of OT-101 as single agent and in combination with Gemcitabine (15 mg/kg, qdx2/wk, IP), Dacarbazine (1-10 mg/kg, qdx4/wk, IP) or Paclitaxel (10 mg/kg, qdx5, IV) were evaluated in nude mice (10/subgroup) bearing either (i) orthotopic human L3.6pl pancreatic cancer (PAC), (ii) human metastatic C8161 melanoma, (iii) subcutaneous glioblastoma (U87-MG) or (iv) subcutaneous ovarian (SK-OV-3) tumors. Mice were monitored thrice a week for adverse effects, body weight loss, tumor size and survival outcome. The incidence of lymph node and liver surface and micro-metastases as well as size and weight of the pancreatic tumors were determined. Tumor sections were stained with anti-5-bromo-2'-deoxy-uridine antibody to determine tumor cell proliferation and with anti-CD31/PECAM-1 antibody to determine vascularization.

Results: OT-101 significantly reduced tumor growth (p = 0.0084), lymph node metastasis (p = 0.023), and tumor angiogenesis (p <0.0001) in the orthotopic PAC model. Mean tumor vessel density was significantly reduced (p <0.0001) in all groups in comparison to untreated control. OT-101 demonstrated synergy in tumor growth inhibition and increased survival in human malignant melanoma (C8161, p = 0.038, vs. Dacarbazine alone), glioblastoma (U87-MG, p = 0.001 vs. Paclitaxel) and ovarian (SKOV-3, p <0.05 vs. Paclitaxel) cancer models when combined with either Dacarbazine (C8161) or Paclitaxel (U87-MG and SK-OV-3). No syngergy was observed with Gemcitabine (PAC). The combination regimen tested was effective and tolerable. Significant antitumor activity was achieved at human dose equivalent of 80 mg/m2/day which is well below the optimized clinical dose used for IV infusion of patients at 140 mg/m2/day.

Conclusions: The preclinical data lay the groundwork for further exploration of combination therapy in the clinic as well as demonstrating that TGF-β2 is a druggable target. Of interest is the preferential synergy between OT-101 and Paclitaxel or Dacarbazine, but not with Gemcitabine.

#2801

Identification of oncogenic/metastatic driver genes that cooperate with p53 or p53/Rb-loss to induce triple-negative breast cancer.

Ronak Ghanbari Azarnier, Agatha Zuchelkowski, Philip.E Chung, Zhe Jiang, Eldad Zacksenhaus. _University of Toronto, Toronto General Research Institute, Toronto, Ontario, Canada_.

Triple-negative breast cancer (TNBCs) is an aggressive subtype with poor prognosis; drug-resistant metastases are common and highly lethal for which identification of therapeutic targets is of major medical interest.

Previously we demonstrated that deletion of p53 and Rb in mouse mammary epithelium induced TNBC-like tumors but with limited capacity to metastasize. Therefore, we decided to employ sleeping beauty (SB) mutagenesis system that can identify oncogenic networks responsible to drive primary and metastatic tumors. In order to do this, we generated mice in which p53 (p53f/f) or p53 plus Rb (p53f/f:Rbf/f ) are deleted and the SB transposon is mobilized from chromosome 9 as well as controls in which both p53 and Rb are wild type.

Forty primary tumors and forty metastases from each cohort will be harvested to identify common integration sites followed by deep sequencing. Then, the top 5-10 candidate oncogenes/tumor suppressors will be validated to determine whether their loss/activation would accelerate tumorigenesis/metastasis.

H&E staining of mammary tumors collected so far from different genotypes revealed histologically diverse tumor types which includes but not limited to poorly differentiated tumors with areas of mesenchymal/spindle-like cells, acinar adenocarcinomas, and well differentiated tumors. All mice analyzed developed multiple mammary tumors in more than one mammary gland with average latency of 108, 158 and 170 days for p53/Rb deleted (N=7), p53 deleted (N=12), and wild type mice (N=2), respectively (p < 0.05). However, we did not always observe macro metastases in mice with primary mammary tumors and we decided to perform primary tumor survival surgery when tumors are oversized and let the mice live longer to develop metastases. With this technique, 3 out of 4 mice that underwent tumor removal surgery developed lung metastases when sacrificed. In total, lung metastases were observed in 5 out of 28 mice analyzed so far with/without surgery (18%) which histologically resemble their primary tumors.

Since removing tumors from multiple glands is challenging, we decided to take a transplantation approach of the primary mammary epithelial cells of mid-pregnancy (13-16 dpc) p53f/f:Rbf/f:SB\+ females and injecting them into the mammary glands of 3-4 week old NSG mice. So far, four NSG mice developed mammary tumors with average latency of 176 days. These tumors resemble spindle-shape histology.

SB mutagenesis can promote metastasis in mouse models with limited dissemination potential such as the model proposed herein. The proposed experiments will identify novel oncogenic events that cooperate with p53-loss or combined p53/Rb-loss to induce primary and metastatic TNBCs which may serve as therapeutic targets for this aggressive disease. I have generated 3 sets of cohort of SB mutagenesis to identify these oncogenic events which are currently being analyzed in details.

#2802

High fat diet increases development of hepatocellular carcinoma in glycine N-methyltransferase deficient mice.

Michael N. VanSaun,1 Alisha Mendonsa,2 Fanuel Messaggio,1 Nagaraj Nagathihalli,1 Lee Gorden3. 1 _Univ. of Miami, Miami, FL;_ 2 _Seattle Childrens' Research Institute, Seattle, WA;_ 3 _Vanderbilt University, Nashville, TN_.

Introduction: Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide. HCC typically arises in patients with chronic liver disease or cirrhosis, yet it is increasingly associated with non-alcoholic fatty liver disease (NAFLD), specifically nonalcoholic steatohepatitis (NASH) in the absence of cirrhosis. NAFLD is associated with obesity, metabolic syndrome, and/or patients with type II diabetes. Our previous studies have shown that high fat diet induced hepatic steatosis increases proliferation of hepatocytes and the growth of malignant tumors in a murine model. Glycine N-MethylTransferase (GNMT) expression is lost in over 95% of HCC, and mice deficient in GNMT develop spontaneous HCC by 6 months of age. We hypothesized that GNMT deficient mice would have an increased susceptibility for the development and growth of HCC when a fed high fat diet.

Methods: Wildtype and GNMT deficient mice were placed on lean diet (LD, 13% calories from fat) or high fat diet (HFD, 42% calories from fat) at eight weeks of age. An initial cohort of mice were sacrificed after 3 months on diet (6 months of age) to assess for early tumor burden. A second cohort of mice was analyzed by magnetic resonance imaging (MRI) after 6 months on diet (9 months of age) and then sacrificed to assess for late stage disease. All mice were assessed for body weight, liver weight, pancreatic weight, and proliferative index (Ki67).

Results: GNMT deficient mice failed to gain weight when placed on HFD, which remained at levels equivalent to wildtype LD mice. At three months of age, wildtype mice on HFD had significantly enlarged livers due to hepatic steatosis. HFD fed GNMT deficient mouse livers were nearly 50% the size of wildtype livers and contained only minimal fatty deposits. Further, livers from HFD and LD fed GNMT mice were equivalent after 3 months, yet they were larger than wildtype mice fed LD. After six months on diet, MRI analysis showed significantly larger livers in HFD fed GNMT mice compared to LD fed GNMT mice due to extensive tumor burden. All wildtype mice lacked any tumors after six months regardless of diet. Histological analysis revealed a heightened cellular proliferation via Ki67 staining in GNMT deficient livers compared to wildtype livers. In comparison, GNMT silencing also occurs in pancreatic cancer, yet none of the GNMT deficient mice developed pancreatic tumors. However, small focal areas of pancreatitis were detected regardless of diet. Additionally, pancreatic weight was significantly decreased in HFD fed GNMT deficient mice compared the LD GNMT deficient mice.

Conclusions: While high fat diet did not induce obesity in GNMT deficient mice, it significantly increased cellular proliferation and primary tumor growth in the liver. Understanding dietary factors that impact the microenvironment of the liver and contribute to HCC development and progression is vital to finding new therapeutics for this malignancy.

#2803

The NCI mouse repository: Cancer models and miRNA-ES cell resource.

Parirokh Awasthi, Fitzgerald Debra, Jones N. Stephen. _Leidos Biomedical Research, Inc., Frederick, MD_.

The NCI Mouse Repository, located at the Frederick National Laboratory for Cancer Research (FNLCR), Frederick, Maryland, is an NCI-funded resource of approximately 150 genetically-engineered mouse cancer models and associated strains, including mice bearing conditional and point-mutant alleles in cancer-related genes. In addition, the Repository houses a unique collection of over 1500 different mouse ES cell clones bearing conditionally-activated miRNA transgenes to facilitate in vivo exploration of miRNA functions. The NCI Mouse Repository makes strains and mESCs available to all members of the scientific community (academic, non-profit, and commercial). The mouse models and ES cell clones are cryo-archived and distributed as frozen germplasm or cells.

Requests may be placed through the NCI Mouse Repository website (https://ncifrederick.cancer.gov/Lasp/MouseRepository/Default.aspx). In addition to the request form, this website includes detailed description for each strain accepted into the Repository and the associated publications provided by the donating scientist. The miR-harboring ESCs, originally generated at the Cold Spring Harbor Laboratories for the NCI, are described in full detail and include validation data for each miRNA ES cell clone. These resources are available for nominal cost to NCI, NIH, and other US government-funded investigators, as well as to Investigators at non-profit organizations.

#2804

Identifying therapeutically relevant mouse and patient-derived xenograft (PDX) models of human cancer using the mouse tumor biology database (MTB) data resource.

Dale A. Begley, Debra M. Krupke, Steven B. Neuhauser, Joel E. Richardson, John P. Sundberg, Janan T. Eppig, Carol J. Bult. _The Jackson Laboratory, Bar Harbor, ME_.

The laboratory mouse is the foremost model organism for interrogating the genetic and molecular basis of human cancer and is a powerful platform for identifying therapeutically effective targets for prevention and treatment of cancer. Research using genetically engineered mouse models (GEMMs) have led to important advances in our understanding of the genetic basis of cancer susceptibility, the function of tumor suppressors and oncogenes, and therapy responses in preclinical and co-clinical studies. Patient Derived Xenograft (PDX) models are an increasingly important model system for in vivo studies of human cancer. These models are created by implanting patient tumors into immunodeficient or humanized mouse hosts and are a powerful translational research platform for preclinical and co-clinical studies. The number of GEMM and PDX mouse models increases significantly every year and the diverse cancer-related data about human cancer models tend to be distributed in ways that makes it difficult for researchers to integrate and interpret the information to find the most relevant model for their research. The Mouse Tumor Biology database (http://tumor.informatics.jax.org) is an expertly curated resource for information and data about genetically defined mouse strains and PDX models of human cancer. MTB provides query tools to enable integrated searches and visualization of these varied data, thus facilitating the assessment of novel mouse models of human cancer and potential preventative and therapeutic treatments. Enforcement of controlled vocabularies and standard gene, allele and strain nomenclature within MTB facilitates precise and comprehensive queries of MTB for pertinent mouse models. MTB contains data from spontaneous or endogenously induced tumors from genetically defined mice including tumor classification, incidence, Quantitative Trait Loci, pathology reports, images and genetic changes in the tumor (somatic) and background strain (germline) genomes. The PDX resource enables queries based on tumor type, cancer diagnosis and genomic properties of the engrafted tumors. Information in MTB is obtained from curation of peer-reviewed scientific publications and direct data submissions from individual investigators and large-scale programs. New features in MTB include the Faceted Tumor Search Form and a Reported Mouse Models table linking the most common fatal human cancers to reported equivalent mouse models. MTB contains over 77,000 Tumor Frequencies and over 2,200 Pathology Reports with over 6,600 images from over 4,200 references. MTB provides access to detailed clinical, pathological, expression and genomics data from over 400 PDX models. Information in MTB is integrated with cancer models data from other bioinformatics resources including PathBase, the Gene Expression Omnibus and ArrayExpress. MTB is supported by NCI grant CA089713.

#2805

A panel of orthotopic glioblastoma multiforme (GBM) patient derived xenograft (PDX) mouse models for efficacy evaluation of drugs.

Mette M. Jensen,1 Camilla S. Knudsen,1 Lotte K. Kristensen,1 Mette K. Nedergaard,2 Michael J. Wick,3 Kyriakos P. Papadopoulos,3 Anthony W. Tolcher,3 Andreas Kjaer,4 Carsten H. Nielsen1. 1 _Minerva Imaging, Copenhagen, Denmark;_ 2 _Rigshospitalet, Copenhagen, Denmark;_ 3 _South Texas Accelerated Research Therapeutics (START), San Antonio, TX;_ 4 _Department of Clinical Physiology, Nuclear Medicine & PET and Cluster for Molecular Imaging, Rigshospitalet and University of Copenhagen, Copenhagen, Denmark_.

Background:

Patients with glioblastoma multiforme (GBM) have a poor prognosis and few treatment options; hence new treatments are needed. Subcutaneous patient derived xenograft (PDX) models are increasingly used for efficacy studies in drug development. However, orthotopic implantation confers a translational advantage as the cancer develops in a microenvironment more closely mimicking that of the original patient tumor. Also the major impact of the blood brain barrier that must be taking into account when targeting brain tumors as GBM in terms of drug bioavailability is better represented in the orthotopic models.

The aim of this study was therefore to develop a panel of orthotopic GBM PDX models for pre-clinical efficacy studies of new drugs. The models were then used to study the efficacy of standard of care such as temozolomide (TMZ) and external radiation therapy (XRT).

Methods: Low passage subcutaneous tumors from six different PDX GBM models designated ST108, ST112, ST146, ST545, ST610 and ST2473 were digested and used for intracranial stereotactic injection in nude mice. Tumor take and growth was determined by T2-weighted magnetic resonance imaging (MRI). At confirmed tumor take mice were either treated with TMZ (100mg/kg/day for 5 days) or whole brain XRT (2 Gy/day for 5 days). Control groups receiving vehicle or sham XRT were included depending on treatment regiment. Final endpoint was survival by humane endpoints and tumors were fixed in formalin for histological evaluation.

Results: MRI confirmed tumor take in all models within 5 weeks of implantation. The take rate was > 80% across all models. TMZ showed efficacy in the orthotopic ST610 GBM PDX model evaluated by MRI on day 14 (16.2±2.9 mm3 vs. 76.8±13.1 mm3, p=0.016), whereas the ST146 model displayed resistance to TMZ on day 14 (12.7±5.6 mm3 vs. 26.5±11.9 mm3, p=0.26). The median survival was 60 days vs. 14 days in the ST610 model (TMZ vs. vehicle, p=0.0005) and 27 days vs. 13 days in the ST146 model (TMZ vs. vehicle, p=0.007). XRT showed efficacy in the orthotopic ST2473 model. Tumor volume was significantly smaller in treated vs. sham animals 11 days after inclusion (6.9±1.4 mm3 vs. 28.9±3.3 mm3, p=0.001). Also, a survival benefit was observed in XRT treated animals compared to sham. Histology confirmed the presence of orthotopic tumors and typical GBM pathology characteristics such as pseudopalisading tumor cells surrounding necrosis and micro vascular proliferation were identified.

Conclusion: Six different orthotopic GBM PDX models were established from low passage subcutaneous PDX models. Models sensitive and resistant to TMZ were identified and histological GBM characteristics were identified. Together, the established panel of orthotopic PDX models can be used as a relevant translational platform for testing of new drugs in a setting that more closely mimics the GBM tumor microenvironment and the impact of the blood brain barrier in patients.

#2806

Generation and characterization of mouse model of Pmepa1 conditional knockout in prostate epithelia.

Shashwat Sharad, Talai Barbiev, Yingjie Song, Denise Young, Taduru Sreenath, Albert Dobi, Shiv Srivastava. _Uniformed Services University, Rockville, MD_.

Introduction and Objectives: Prostate cancer (CaP) is the most common non-skin malignancy diagnosed in American men and desfunctions of androgen receptor (AR) plays an essential role in prostate tumorigenesis. PMEPA1 is an androgen and TGF-β -induced gene abundant in prostate, which was identified to degrade AR protein via NEDD4 E3 ligase mediated pathway. Reduced or loss of PMEPA1 expression, commonly detected in prostate tumors, led to increased AR protein and activated AR signaling. PMEPA1 inhibited TGF-β receptor 1 meditated signaling by a negative feedback loop. It was reported that loss of PMEPA1 facilitates bone metastasis of CaP through blocking TGF-β signaling. To further investigating the biological function of PMEP1 gene in CaP tumorigenesis, particularly via AR and TGF-β, we generated Pmepa1 prostate conditional knockout mouse model.

Methods: C57BL/6 mice were utilized for generation of Pmepa1 conditional knockout model. Pmepa1 gene was conditionally deleted in mouse prostate epithelium by ARR2PB-Cre-Lox system. Male mice of genotypes of wild-type, Pmepa1 flox/wild-type-ARR2PB-Cre, Pmepa1 flox/flox-ARR2PB-Cre were euthanized at the age of 3 months for analysis. The prostate tissue was collected for frozen and formalin fixation for histology analysis, and other major organs including heart, lung, liver, spleen, bladder and testis were also collected for control. The tissue were sectioned and stained with Hematoxylin & Erosin (H&E). Total RNA and protein were harvested by homogenizing tissue. The protein levels of Pmepa1, AR and Nkx3.1 were analyzed by immunohistochemistry (IHC) and immunoblotting, and the transcript levels of these genes were evaluated by in situ hybridization (ISH) and quantitative-PCR (Q-PCR).

Results: The expression of Pmepa1 protein was found to focus on lateral lobe, consistent with AR protein expression pattern. The data of Q-PCR and ISH showed that transcript level of Pmepa1 was dramatically suppressed in Pmepa1 flox/flox and flox/wild-type ARR2PB-Cre mice. Compared to wild-type ones, Pmepa1 protein was shown significantly decreased in heterozygous and homozygous knockout mice by immunoblotting and IHC staining. And the protein level of AR was stronger in Pmepa1 conditional deleted mouse. There was no dramatic morphology change in mouse prostate gland by H&E staining.

Conclusions: Conditional deletion of Pmepa1 gene in mouse prostate epithelium led to enhanced AR protein and activating AR signaling. The effects of Pmepa1 gene loss in mouse prostate gland on other signaling such as TGF- β and prostate tumorigenesis are needed to be further evaluated.

Funding: This study was supported by CPDR, USUHS, HU0001-10-2-0002 to DGM.

#2807

Developing a variety of primary mouse tumor allograft models for evaluating efficacy of PARP inhibitor.

Gavin Jiagui Qu, Davy Xuesong Ouyang, Li Chen, Likun Zhang, Annie Xiaoyu An, Jie Cai, Henry Q. X. Li. _Crown Bioscience, Inc., Taicang, Jiangsu, China_.

Recent success of multiple poly (adenosine diphosphate [ADP]) ribose polymerase inhibitors (PARPi) in treating BRCA mutant ovarian cancers has fueled the development of PARPi in several areas, including expanding its use in other BRCA mutant or wildtype cancers; exploring resistant mechanisms and overcoming resistance; seeking for its combination with chemotherapy, radiotherapy and emerging immunotherapy to achieve persistent efficacy. Especially considering the mechanism of action of PARPi, its use can increase the mutational loads in tumors with defects in homologous recombination and DNA double strand break repair, which potentially would enhance the immunogenicity of the tumors. Therefore it would be very interesting to test the effectiveness of combining PARPi with anti-PD1/PD-L1 or anti-CTLA4. However, relevant animal models are lacking for such studies.Syngeneic mouse tumors offer about 30 viable in vivo models for proof of concept studies. However, these models suffer from several limitations: 1) only a few available and responsive to the current checkpoint inhibitors -- for given strains of mouse, there are only very few choices of cells/disease types; 2) not mimicking patient tumors since they are in vitro immortalized cell lines; 3) not reflective of patient disease pathways thus unfit for common target agents, either in mono- or combination therapy. We have established allografts of spontaneous mouse tumors (MuPrime®) derived from genetically engineered mouse models (GEMMs) as a new type of immuno-oncology models with following advantages: 1) its primary nature of "stem cell diseases" and relevant tumor microenvironment; 2) its diverse cancer types/strains of mice; 3) engineered oncogenic drivers as seen in human diseases, deriving from a wide range of available GEMMs, suitable for targeted agents. Thus far, we have built a small library of allografts and are testing them to facilitate pharmacological investigation, particularly those of immuno-oncology.We have found a few models, including an APCmin/+ skin squamous cell carcinoma and a P53 null sarcoma responded well to PARPi Niraparib. Continuous treatment with Niraparib led to development of partial or complete resistance. We are now profiling the resistant models to find out their general mutation loads, as well as specific biomarkers render resistant mechanisms. We are also treating the tumors with mouse PD1 antibody to test whether presumably enhanced mutation loads would sensitize these tumors to immunotherapy. The data of these studies will be presented at the meeting.

#2809

Role of c-Met in the development and progression of Tpl2-related skin cancer.

Nicole F. Bonan,1 David Kowalski,1 Kaitlin Kudlac,1 Lauren G. Falkenberg,1 Erik Maradiaga,1 Kira Flaherty,1 Jonathan S. Wiest,2 Katie L. DeCicco-Skinner1. 1 _American Univ., Washington, DC;_ 2 _NCI, Rockville, MD_.

Cutaneous squamous cell carcinoma (cSCC), a form of non-melanoma skin cancer, is the second most common form of cancer in the United States, with over one million cases diagnosed annually. Tpl2 (MAP3K8) is a serine threonine protein kinase in the mitogen-activated protein kinase (MAPK) signal transduction cascade. Tpl2 was identified by our laboratory as having a tumor suppressive function in skin carcinogenesis, with Tpl2 knockout mice developing significantly more papillomas and squamous cell carcinomas than matched wildtype (WT) controls. Cellular mesenchymal to epithelial transition factor (c-Met) is a receptor heightened in malignant skin cancers and involved in Tpl2 signaling. When bound to its ligand, hepatocyte growth factor (HGF), c-Met can regulate cell proliferation, survival, angiogenesis and invasion. The aim of this study was to ascertain whether c-Met signaling contributes to the heightened skin tumorigenesis in Tpl2-/- mice. Forty-four WT and Tpl2 -/- mice were subjected to a two-stage chemical carcinogenesis protocol for one year to induce skin tumors. Starting at the time of promotion, half of the WT and Tpl2 -/- mice were placed on normal diet and half received diet plus 30mg/kg Capmatinib, a pharmacological inihibitor of c-Met. This diet continued for the duration of the study. No adverse health effects were found in Capmatinib-treated mice. Similar to what we previously reported, Tpl2 -/- mice developed tumors earlier than WT controls and had statistically higher tumor incidence and overall tumor burden than WT mice. The number of tumor bearing Tpl2 -/- and Tpl2 -/- \+ Capmatinib fed mice was similar, although Capmatinib fed mice had a 60% reduction in overall tumor burden. Studies are underway to determine if Capmatinib affected the frequency of malignant conversion in Tpl2 -/- mice. In summary, our studies demonstrate that heightened c-Met signaling contributes to the higher tumor formation in Tpl2-/- mice and should be evaluated further as a possible therapeutic target in Tpl2-related skin cancers.

#2810

Using CRISPR/Cas9 to generate primary soft tissue sarcoma in genetically engineered and wild-type mice.

Jianguo Huang,1 Mark Chen,1 Melodi J. Whitley,1 Hsuan-Cheng Kuo,1 Andrea Walens,1 Yvonne M. Mowery,1 David V. Mater,1 William Eward,1 Diana M. Cardona,1 Lixia Luo,1 Yan Ma,1 Christopher E. Nelson,1 Jacqueline N. Robinson-Hamm,1 Charles A. Gersbach,1 Rebecca D. Dodd,2 David G. Kirsch1. 1 _Duke University Medical Center, Durham, NC;_ 2 _University of Iowa, Iowa City, IA_.

Genetically engineered mouse models (GEMMs) that employ site-specific recombinase (SSR) technology are important tools for pre-clinical studies, but this approach is costly and time-consuming. Here, we show that the CRISPR/Cas9 system can be used to efficiently complement existing GEMMs of sarcoma and generate primary sarcomas in wild type mice. Mice with the genotype KrasLSL-G12D/+; Rosa26LSL-Cas9-EGFP/+ received intramuscular delivery of an adenovirus expressing Cre recombinase and a single guide RNA (sgRNA) targeting Trp53. Cre-mediated expression of oncogenic Kras and Cas9, in combination with CRISPR/Cas9-mediated knockout of Trp53, was sufficient to generate primary soft tissue sarcomas. These tumors arose with kinetics similar to those generated using the Cre-loxP system to activate oncogenic Kras and delete Trp53 alleles. Additionally, we injected an adenovirus containing Cas9 and sgRNAs targeting Nf1 and Trp53 into the sciatic nerve of wild type mice. These mice formed malignant peripheral nerve sheath tumors (MPNSTs) in the same timeframe as MPNSTs generated using the Cre-loxP system to delete Nf1 and Ink4a/Arf alleles in GEMMs. These data demonstrate that CRISPR/Cas9 can be used to generate soft tissue sarcomas in wild type mice. Moreover, these results suggest that this technology can complement existing GEMMs for rapid assessment of tumor-modifying genes. These tools should decrease the time and expense associated with employing autochthonous mouse models of sarcoma for preclinical research.

#2811

Comparative study of anti-PD1 and CSF1R inhibition on tumor-infiltrating lymphocytes and macrophage populations across a panel of syngeneic tumor models.

Yuki Kato Maves, Hooman Izadi, Elvira C. Talaoc, Deborah Yan, Charlene Echegaray, Andrew Calinisan, Krystal Moya, Heather Venant, Mitchell Garland, Radhika Iyer, Shounak Gosh, Stephanie Songco, Jayant Thatte, Tommy Broudy. _Crown Biosciences, San Diego, CA_.

Immunotherapy represents an extremely promising treatment approach for cancer patients. The success in this field has been signified by multiple recent approvals of checkpoint inhibitors across a range of cancer types. Subsequently, there is an increasing need to understand why certain patients and diseases benefit from these treatments while others do not, as well as how to maximize the benefits from these treatments.

To address some of these questions, we used an in vivo screening platform, MuScreen, which allows for evaluation of multiple syngeneic models and treatment modalities.We have characterized baseline and treated tumor immune infiltrates for a panel of syngeneic models using flow cytometry. The correlation between responsiveness and effects of checkpoint inhibitors (e.g., anti-PD-1), and macrophage targeted agents (e.g., CSF1R inhibition) on immune infiltrates including tumor-associated macrophages (TAMs) were investigated in 6 syngeneic models. Furthermore, M1/M2 polarization of macrophages was investigated to explore the potential role of these cells in establishing an immunosuppressed tumor microenvironment that may affect response to therapy.

Here, we show that the baseline immune cell population varies across models, and that immune checkpoint inhibitors and macrophage-targeted agents had variable efficacy across different tumor models. We also present the correlation of immune cell infiltration, including tumor infiltrating lymphocytes (TILs), TAMs, and responsiveness to immunotherapy intervention.

Having an in-depth understanding of the immune make up of a model in which an immunomodulatory compound is screened is paramount for successful translation into the clinic. A well-characterized and fully profiled panel of syngeneic models such as the MuScreen platform allows for finding the right model for screening test compounds to be used alone or in combination with immunotherapy.

#2812

**Development and characterization of the ultra immunodeficient B6;129-** Rag2tm1FwaIL2rgtm1Rsky **/DwlHsd (R2G2) mouse model.**

Sheryl J. Wildt, Jamie McClellan, Mandy Horn. _Envigo RMS, Indianapolis, IN_.

The "R2G2" (B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd) knockout mouse is the latest advancement to provide an alternative option in the highly immunodeficient mouse model category for the oncology, immunology and other biomedical research communities. This model was generated by backcrossing an IL2rg (common gamma) knockout model to a RAG2 (recombinase activating gene) knockout model. The resulting mouse lacks various cytokines including IL-2, IL-4, IL-7, IL-9 and IL-15. In addition, this model lacks B cells, T cells, NK cells and has a deficit in lymphocyte development. The R2G2 model is not only ultra immunodeficient, but provides a model that is less leaky and more tolerant to gamma radiation than traditional SCID models. This model is particularly useful for both tumor transplantation and humanization studies. Herein we describe the development and characterization of the R2G2 model, which includes breeding history, growth curve, CBC/Chemistry, flow cytometry and both huHSC and huPMBC humanization studies.

#2813

An interactome analysis for personalized chemotherapy using PDX/NOG models of non-small cell lung cancer.

Tsuyoshi Chijiwa,1 Daisuke Komura,2 Mizuha Haraguchi,3 Akira Noguchi,4 Hidemitsu Sato,5 Hiroaki Ito,5 Haruhiko Nakayama,5 Makoto Katayama,6 Naoki Miyao,3 Naruaki Matsui,3 Yuichi Tateishi,7 Hiroshi Suemizu,7 Yoshiyasu Nakamura,8 Daisuke Furukawa,9 Takayuki Isagawa,2 Hiroto Katoh,2 Shumpei Ishikawa,2 Masato Nakamura,9 Yohei Miyagi8. 1 _Saitama Medical Center, Jichi Medical University, Saitama, Japan;_ 2 _Medical Research Institute, Tokyo Medical and Dental University, Japan;_ 3 _Nihon Koukan Hospital, Saitama, Japan;_ 4 _St. Marianna University School of Medicine, Saitama, Japan;_ 5 _Kanagawa Cancer Center, Saitama, Japan;_ 6 _Kawasaki Municipal Hospital, Saitama, Japan;_ 7 _Central Institute for Experimental Animals, Japan;_ 8 _Research Institute, Kanagawa Cancer Center, Japan;_ 9 _Tokai University School of Medicine, Japan_.

Lung cancer is one of the most common malignant diseases in the world, and its prognosis is generally poor. It is crucial to elucidate the biological mechanisms underlying lung cancer and accelerate the development of new treatment strategies. Xenografts derived from engrafting fresh surgical specimens directly into immunodeficient mice have recently enabled the development of more relevant in vivo models of human cancers. These patient-derived xenograft (PDX) models, established by the direct transfer of tumor tissue, retain similar morphologies, heterogeneities, and molecular signatures as the original cancers, and, thus, may be used in promising personalized medicine for cancer. We previously reported the rapid and efficient establishment of PDXs using super immunodeficient NOG mice (PDX/NOG model). In the present study, we analyzed the gene expression and cancer-stroma interaction profiles of PDXs established from NSCLC patients. We also discussed the possibility of individual PDX/NOG model simulations for personalized cancer chemotherapy. Ten NSCLC lines of PDX/NOG (Age 43-78 years; 8 men and 2 women; 7 adenocarcinoma, 1 adenosquamous carcinoma, 1 squamous carcinoma, and 1 large cell carcinoma) were presented. In these cases, clinical information regarding chemotherapy for donor patients was retrieved where possible. Genome sequencing and comprehensive analyses of tumor-stroma interactions (CAncer-STromal INteractome analysis, CASTIN) were performed on mRNA. CASTIN showed tumor-stroma interactions in PDX/NOG comprehensively and quantitatively at the gene expression level by distinguishing gene arrangements in human tissue (Cancer) from mouse tissue (Stroma) as signal strengths (ligand dependency (%) / receptor dependency (%)). Interactions of EGF(cancer)-EGFR(stroma) were observed from 1.2 (15%/0%) to 11.5 (94%/80%) as widely-distributed. Interactions of VEGFA(cancer)-KDR(stroma) were observed from 49.1 (31%/51%) to 301.7 (83%/100%) as closely-distributed. These interactions of EGF-EGFR or VEGFA-KDR in PDX/NOG closely reflected the clinical effectiveness of an EGFR inhibitor (Cetuximab) or VEGF-A inhibitor (Bevacizumab). The CASTIN results of PDX/NOG appear to be reliable for clinical simulations of chemotherapy and will definitely assist in the selection of the most sensitive anti-cancer drug. The fast and efficient establishment of individual PDXs will contribute to personalized anti-cancer therapies.

#2814

Effect of kif14 overexpression on tumor formation in mice in a lifespan study.

Natalie Pitt, Kamakshi Sishtla, Mehdi Shadmand, Rania Sulaiman, Anthony L. Sinn, Keith Condon, George E. Sandusky, Timothy W. Corson. _Indiana University School of Medicine, Indianapolis, IN_.

KIF14 is a mitotic kinesin and microtubule-associated molecular motor that plays an essential role in the last stages of cytokinesis. In multiple cancer types, KIF14, overexpression in tumors correlates with stage, aggressiveness, and poor patient outcomes. There is considerable interest in KIF14 as a possible oncogene, since the KIF14 locus is in a common region of genomic gain in multiple cancers.

In this study, wild type BDF-1 mice and a strain constitutively overexpressing Kif14, known as Kif14-Tg mice, were evaluated at the end of their natural lifespan, specifically for the occurrence of tumors. There were approximately fifty mice per genotype, and eighty of these mice have died. These mice were necropsied and the following tissues were collected: kidney, liver, lung, spleen, seminal vesicles, ovary, mammary gland, bone marrow, pituitary gland, eye, and brain. Any gross nodules were also collected. These tissues were then fixed in 10% neutral buffered formalin, processed, sectioned, and stained with hematoxylin and eosin.

Although there was no significant difference in mouse weights through the lifespan or in the survival curve between the Kif14-Tg and their wild type littermates, a significant increase in follicular lymphoma and diffuse large B cell lymphoma was seen in Kif14-Tg mice compared to the wild type mice. Thirty-three mice developed either a follicular lymphoma or a diffuse large B cell lymphoma, and twenty-four of those mice were Kif14-Tg mice. These were the two most common tumors in Kif14-Tg mice. Other lesions and tumors that were seen in both strains included thymic lymphoma, sarcomas, myeloid dysplasia, and other carcinomas. Nontumorous lesions were seen in both strains of mice including hydronephrosis and telangiectasia in multiple organs. Ballooning degeneration of the lens of the eyes was observed in five mice from the strain Kif14-Tg.

Our finding of increased follicular lymphoma and diffuse large B cell lymphoma proves the first evidence that Kif14 can promote tumor formation in a wild-type background. This is the first evidence that Kif14 may have a role in lymphoma, but complements our earlier finding that Kif14 overexpression can accelerate tumor formation in a mouse model of retinoblastoma. Together, these outcomes further support Kif14's potential as an oncogene.

#2815

Patient derived orthotopic xenograft (PDOX) model developed from needle biopsy specimen replicated clinical outcomes in advanced pancreatic cancer.

A-Ra Jeon,1 Sun Il Choi,2 Sang-Jae Park,2 Sung-Sik Han,2 Eun Kyung Hong,2 Sun-Young Kong,2 Min Kyeong Kim,1 Kyong-Ah Yoon,3 Young-Hwan Koh,2 Ju Hee Lee,2 Woo Jin Lee,2 Young Hwa Kang,2 Sang Myung Woo,2 Yun-Hee Kim2. 1 _Graduate School of Cancer Science & Policy, National Cancer Center, Republic of Korea; _2 _National Cancer Center, Goyang-si, Republic of Korea;_ 3 _Konkuk University, Seoul, Republic of Korea_.

Introduction:

A patient-derived xenograft (PDX) as a preclinical model can reflect the pathological and molecular features and drug responsibility of a patient's tumor. PDXs have been generated by subcutaneously implanting surgically resected tumor fragments into immunodeficient mice. Unfortunately, 80 % of pancreatic cancer patients is unresectable and they show highly aggressive and metastatic tumors. In most cases, diagnoses are confirmed histologically and diagnosed through endoscopic ultrasonography-guided/fine-needle aspiration (EUS/FNA) or percutaneous liver biopsy. However, they are not suitable for subcutaneous implantation into mice due to their threadlike shape and very small size. Therefore, the lack of patient-derived model system in pancreatic cancer is in desperate need of new strategy to cover unresectable patients as well as resectable. Here, we pioneered the development of a unresectable patient-derived orthotopic xenograft (PDOX) model satisfied with short period and high success rate, and made the best use of them as the test model for drug response.

Methods and Results:

PDOX models were established with a technique of direct surgical orthotopic implantation into the pancreatic tail of Athymic nude mice. PDOX models using fine needle biopsy had a higher success rate of engraftment, nearly 40% (17 cases among 44 cases) in patients with a time to progression of <6 months. The histopathological characteristics of the PDOX tumors are similar with those of the patient's original tissues. Additionally, genetic alterations were retained between patient tissues (F0) and PDOX tumors (F1~F3) from the mutation analysis using a comprehensive cancer panel. For drug responsibility, we selected refractory or sensitive PDOX groups to Gemcitabine and Abraxane based on their clinical information. As results after treatment with Gemcitabine, Abraxane and combination, selected PDOX models showed the drug sensitivity significantly correlated with the drug responsibility of original patients. Tumor growth was monitored by animal MRI every 2 weeks during 2 months. Typically, xenograft models using immortalized pancreatic cancer cell line, CFPAC-1, showed tumor regression to all treatment set.

Conclusion:

Collectively, our PDOX models using needle biopsy specimens can reflect the characteristics of unresectable patients and give the expectation the drug responsibility. They furthermore might be valuable in a screening and development system for targeted therapeutics, which can be applied to a wide range of pancreatic cancer patients.

#2816

The Jackson Laboratory Repository: Mouse models for cancer research.

Deborah Boswell, Stephen Rockwood, Michael Sasner, The JAX Repository Team. _The Jackson Laboratory, Bar Harbor, ME_.

The mouse is a well-established model organism for research of the biology, prevention, diagnosis, and treatment of cancer. Among the advantages of this valuable biomedical research tool are a relatively short reproductive cycle, an accelerated lifespan, and the ease of genetic manipulation and modification. Serving as a resource to the scientific community, the JAX Repository collection encompasses over 10,000 mouse strains and acquires hundreds of new models each year. Some of these new models are highlighted in this poster. Many of these strains have applications for modeling human cancer, including genetically engineered mouse models for specific cancers and xenograft models. Research tool strains, such as cas9-expressing (CRISPR) lines, a suite of immunodeficient mice, and recombinase expressing strains, are available to investigators to generate customized mutant lines for specialized purposes. JAX offers supplementary research tools that include specialized strain platforms and other useful resources. The JAX patient-derived xenograft (PDX) cancer model resource leverages the immunodeficient NSG strain for tumor engraftment.

The Repository maintains a searchable online resource to find mice (www.jaxmice.jax.org) and an Oncology Therapeutic Area page (www.jax.org/jax-mice-and-services/solutions-by-therapeutic-area/oncology) as a launch pad to featured JAX strains and resources related to oncology research.

The Repository has a rigorous quality control program that inspects and confirms expected mutation identity and genetic background. JAX mouse lines are also screened for alleles (GFP, cre, lacZ, etc.) that may contaminate strains in mouse lines shared between labs—an underappreciated problem that may contribute to inconsistencies of reproducibility. In addition, the Reproductive Sciences group at JAX safeguards each strain by cryopreservation, and can create cohorts for studies rapidly using IVF technology. Researchers can submit their strains to be considered for inclusion in the Repository by using the on-line form available at The Jackson Laboratory website: www.jax.org/donate-a-mouse.

The Jackson Laboratory Repository is supported by the NIH, The Howard Hughes Medical Institute, and other private charitable foundations.

#2817

Enriched environment inhibits progression of acute myeloid leukemia.

Run Xiao, Stephen M. Bergin, Michael A. Caligiuri, Lei Cao. _The Ohio State University Comprehensive Cancer Center, Columbus, OH_.

Background: Environmental factors and lifestyle have profound effects in the initiation, promotion and progression of cancer. Our recent work on environmental enrichment (EE), a housing environment boosting mental health, has revealed a novel phenotype characterized by a robust reduction in adiposity, resistance to diet-induced obesity (DIO), enhanced insulin sensitivity, lower serum IGF-1 and leptin levels, higher serum adiponectin level, enhanced immune functions, and inhibition in melanoma and colon cancer growth. We have teased out one key mechanism underlying the anticancer effect of EE: the stimulation of brain-derived neurotrophic factor (BDNF) expression in the hypothalamus and the subsequent activation of a specific neuroendocrine axis, the hypothalamic-sympathoneural-adipocyte (HSA) axis. However, the EE's effects on hematologic malignancy are largely unknown. Here we generalized this intervention to acute myeloid leukemia (AML) using the MLL-partial tandem duplication (PTD) and Flt3-internal tandem duplication (ITD) double knock-in mouse (MllPTD/WT:Flt3ITD/WT) that recapitulates features of counterpart human AML.

Methods: For transplantation of AML cells, Ly5.2 spleen cells isolated from leukemic MllPTD/WT: Flt3ITD/WT mice were resuspended in PBS. Syngenic Ly5.1 recipient C57Bl/6 male mice (CD45.1+) were sublethally irradiated (400 cGy). Five to six hours later, leukemic cells (4×106 per mouse) were injected through the lateral tail vein. Those mice were randomly assigned to either standard laboratory cage (SE) or EE housing. To monitor AML progression, white blood cell (WBC) counts were measured from peripheral blood. Survival experiments were performed.

Results: EE mice had a median survival 14% longer than SE mice (SE = 36 ± 1.1 days, EE = 41 ± 1.2 days, p = 0.038) and improved hazard rate (log-rank test z = 1.99, p = 0.047). Based on WBC counts (>10.9 k/µL), 100% of SE mice and 80% of EE mice had apparent AML at three weeks post injection. At this time point, EE had a substantially lower WBC counts (SE = 76.9 k/µL, EE = 26.4 k/µL, p = 0.0006). WBCs from EE mice had lower neutrophil, monocytes, eosinophils and basophils counts, but had similar lymphocytes, resulting in a reduced neutrophil to lymphocyte ratio. Red blood cell (RBC) and platelet data were the same except for a slightly reduced mean corpuscular hemoglobin concentration (MCHC) in EE mice. At four weeks, EE mice were substantially more active and mobile than SE mice. At five weeks, at which point 3 SE and 5 EE mice remained alive, WBC counts remained lower in EE mice.

Conclusions: This preliminary work demonstrated EE substantially improved progression-free survival and conferred a significant survival benefit. We will elucidate the underlying mechanisms of EE's anti-AML effect by assessing metabolic modulation and immune regulation.

#2818

PDX model of the enriched pleural effusion from advanced lung cancer patient for testing on targeted drug sensitivity and resistance.

Feifei Zhang,1 Ke Wang,1 Wenhua Xu,1 Yang Yang,1 Kedong Ouyang,1 Shun Lu,2 Danyi Wen,1 Taiping Chen1. 1 _LIDE Biotech Ltd, Shanghai, China;_ 2 _Shanghai Chest Hospital, Shanghai, China_.

Advanced lung cancer of late stage patients is selectively sensitive to available targeted therapeutics and often developing resistance to the early sensitive drug treatment. Surgery tumor tissues are usually not available to establish patient derived xenograft (PDX) models which is critical to evaluate drug sensitivity and resistance. We applied CD45 magnet beads to enrich the tumor cell population from pleural effusion specimens of advanced lung cancer. Seven PDX models were successfully established in this way. Based on genetic profiling of these models, TKI drug related EGFR mutations and ALK fusion were confirmed (EGFR: L858R, T790M, Exon 19 insertion; ALK fusion). Further in vivo efficacy studies were designed to test on multiple drug sensitivity and resistance including Gefitinib, Erlotinib, Ceritinib, Alectinib, AZD9291. Tumor growth inhibition (TGI) were well matched with corresponding gene mutations among the models. In particularly, T790M model was resistant to Erlotinib and quite sensitive for AZD9291. In conclusion, the models established here are very useful to test on potency of many current investigational TKI drugs and provide base for drug resistant model establishment.

#2819

**Intestinal estrogen receptor beta attenuates colon cancer signaling pathways** in vivo **.**

Linnea Pettersson,1 Ashish Saxena,2 Trang Vu,2 Jan-Åke Gustafsson,2 Amena Archer,3 Cecilia Williams3. 1 _Karolinska Institute, Stockholm, Sweden;_ 2 _Center for Nuclear Receptors and Cell Signaling, Houston, TX;_ 3 _Scilifelab, Stockholm, Sweden_.

Colorectal cancer (CRC) is the third most common form of cancer and the second leading cause for cancer deaths. Several epidemiological, in vitro and in vivo studies suggest that Estrogen receptor β (ERβ) could be a possible target for CRC prevention and treatment, but the potential mechanism is not demonstrated. ERβ is expressed in intestinal epithelial cells, as well as in some intestinal immune cells and other tissues. Previous studies have seen increased CRC after full ERβ knockout, but it is not clear if this is mediated by intestinal ERβ. The specific aim of our study is to determine the role of intestinal ERβ during colon cancer development in vivo. We deleted ERβ specifically in the intestine epithelia of mice (iERβKO), and induced CRC using Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS) treatment. After 9 weeks treatment, all mice presented colitis and two iERβKO mice but none of the wild-type (WT) mice had developed tumors. qPCR showed that the expression of pro-inflammatory markers was higher in iERβKO treated mice than in untreated or WT mice, Immunohistochemistry (IHC) of proliferative markers showed that iERβKO exhibited higher cell proliferation in the top of the crypts compared to controls. After 16 weeks treatment, all mice developed tumors. qPCR showed that the expression of mesenchymal markers was higher in treated iERβKO mice compared to treated WT mice. Our results demonstrate that intestinal ERβ attenuates colon cancer-related pathways.

#2820

Use of a genetically engineered mouse model and RNA sequencing to identify genes that are aberrantly regulated by mutant p53 in prostate cells following irradiation.

Ruth L. Vinall,1 Qian Chen,2 George Talbott,3 Neil Hubbard,2 Clifford Tepper,2 Alexander Borowsky2. 1 _California Northstate Univ., Elk Grove, CA;_ 2 _University of California, Davis, Sacramento, CA;_ 3 _California Northstate University, Elk Grove, CA_.

Our group has previously demonstrated that the Trp53 R270H mutation can drive prostate cancer (CaP) initiation in a genetically engineered mouse model, and that the human equivalent, TP53 R273H, can promote development of castration resistant growth of LNCaP cells as well as resistance to commonly used therapeutic agents. The primary objective of the current study was to identify genes that may contribute to the development of these gain-of-function phenotypes.

Wildtype mice and mice that were heterozygous or homozygous for the Trp53 R270H mutation (referred to as Trp53 +/+, Trp53 +/ R270H, or Trp53 R270H/R270H, respectively) that were ~3 months old were exposed to 5 Gy radiation to activate and stabilize p53, consequently increasing its expression. Mouse prostates were harvested 6 hours post-irradiation and either processed for subsequent histological/immunohistochemistry (IHC) analysis or snap-frozen for subsequent RNA extraction and transcriptome profiling with RNA-Sequencing (RNA-Seq) analysis. P53 expression was determined by IHC. RNA-Seq data were processed to quantify transcript levels and to assess differential gene expression between the 3 groups.

PIN lesions were observed in 3-month-old Trp53 R270H/R270H mice prostates, but not in Trp53 +/+ or Trp53 +/ R270H mice prostates. IHC analysis demonstrated that p53 was stabilized in the majority of prostate cells from Trp53 +/+, Trp53 +/ R270H, or Trp53 R270H/R270H mice 6 hours post-irradiation. RNA-Seq analysis of RNA isolated from irradiated mice prostates identified 1,444 genes that were differentially expressed in Trp53 +/+ versus Trp53 R270H/R270H mice prostate cells, and 796 genes that were differentially expressed in Trp53 +/+ versus Trp53 +/ R270H mice. Statistically significant differences in gene expression between the 3 groups were observed for 1,378 genes, including a number of p53 target genes, such as Cdkn1a, Bax, Bcl2, Kras, Mdm2, and Id4.

Our data identify multiple genes that may contribute to prostate cancer initiation and/or progression through p53 gain-of-function and loss-of-function mechanisms. It is possible that further analysis of these genes may lead to the development of new therapies and/or biomarkers for prostate cancer patients as well as guide the usage of currently available therapies in men at risk of developing CaP and CaP patients who harbor TP53 mutations.

#2821

Impact of SCID mouse gender on tumorigenicity, xenograft growth and drug-responses in patient derived orthotopic xenograft models (PDOX) of malignant brain tumors.

Lin Qi, Mari Kogiso, Yuchen Du, Huiyuan Zhang, Frank Braun, Holly Lindsay, Sibo Zhao, Sarah Injac, Patricia Baxter, Zhigang Liu, Yujing Zhang, Jack Su, Adekunle Adesina, Andrew Walter, Jeffery Murray, Javad Nazarian, Will Parsons, Murali Chintagumpala, Xiao-nan Li. _Texas Children's Cancer Center, Houston, TX_.

Brain tumor is the leading cause of cancer related death in children. To develop new therapies and to understand tumor biology, many efforts are made to develop patient derived orthotopic xenograft mouse models (PDOX). There are, however, some concerns about the potential impact of animal genders on tumor take rate, xenograft growth and in vivo drug responses. To systematically analyze such impact, we retrospectively examined >1,000 mice of 59 PDOX models, including glioblastoma (GBM), medulloblastoma (MB), primitive neuroectodermal tumor (PENT) and ependymoma (EPN) and diffuse intrinsic pontine glioma (DIPG), in which there were age-matched (within 2 wks differences) with relatively even numbers of male and female animals. All the animals were injected with identical number of tumor cells (1x10^5) in the mouse brains matching that in human patients. Animals were monitored daily and euthanized when they were moribund. Differences between male and female mice were analyzed by t-test. In 25 GBM models (15 adult and 10 pediatric) involving 419 mice, there was an average of number of mice was 17.8±9.5 mice per model (male: female = 9.88±5.8:7.93±3.6) (P>0.05). In 26 MB models with total 488 mice, the average animal number was 17.24±8.8 mice per model (male:female= 9.54±5.34 :7.71±3.45) (P>0.05). The tumor take rate was near >95% in both male and female mice (P>0.05). In GBM models, the survival times was 94.1±21.7 days in male mice and 92.3±20.8 days in female (P>0.05); wherease in MB models, they were 134.95±39.86 day in male and 121.08±31.68 days in female mice (P>0.05). The impact of animal gender on drug responses in vivo is essential to the preclinical drug testing. A total of 468 mice from 11 GBM, 5 MB, 3 EPN, 3 DIPG and 2 PNET were treated a series of standard and investigational drugs/compounds, including radiation, oncolytic virus (SVV-001), ABT888, BMI1-inhibitor, MLN8237, TMZ, Flavopiridol, SYC-435, SYC-719, SYC-836, Olig 2 inhibitor, VPA, SAHA, Echinomycin, MCB613, and PARPi. The overall survival times were 104.55±23.63 day in male mice, and 98.64±25.66 in female mice (P>0.05). The only model that exhibited the increased responses in male mice was found in IC-2305GBM treated with TMZ and PTC-596 (P<0.05). In conclusion, our data demonstrated that the gender of SCID mice does not have major impact on animal development nor in drug responses, and SCID mice of both genders are appropriate for brain tumor PDOX model development.

#2822

Establishing the use of Vortex technology for investigating circulating tumor cells in mouse models of breast cancer.

Vishnu Ramani,1 Rakhi Gupta,1 Melanie Triboulet,1 Corinne Renier,2 Steve C. Crouse,2 Elodie Sollier-Christen,2 Stefanie S. Jeffrey1. 1 _Stanford University, School of Medicine, Stanford, CA;_ 2 _Vortex Biosciences, Menlo Park, CA_.

Background: Circulating tumor cells (CTCs) are precursors of metastatic disease and are an important indicator of the disease progression and outcome of many cancers. Though several recent studies have advanced our understanding of CTCs, many critical areas of CTC biology remain largely unexplored. Murine models of cancer offer a unique opportunity to address this issue. However, small sample volumes, low number of CTCs, difficulty to access ports for blood collection are challenges that diminish the utility of mouse models for studying CTCs. In our previous studies, we had validated the use of Vortex technology for the label-free capture of CTCs from blood samples of different cancers. In this current study, we now report our recent, unpublished data on the adaptation of Vortex platform for enriching and characterizing murine and human tumor cells from mouse blood.

Methods: By spiking tumor cells into small volumes of mouse blood (200-400 µl) from cardiac puncture, we tested the impact of i) different dilutions of mouse blood (10X, 20X, 40X); ii) varying numbers of tumor cells; iii) different cell types (MDA-MB-231, 4T1 and EMT6); and iv) sample recycling on capture efficiency and purity. Blood was processed through the Vortex plastic chip and, to accurately identify and enumerate the enriched tumor cells, we developed a staining protocol that labels human- or murine-specific cytokeratin, vimentin, and CD45.

Results: Our results reveal that over a range of dilutions tested, just after one cycle, 10X dilution of mouse blood was superior, yielding a capture efficiency of 38.83% and a purity of 33% for MDA-MB-231 cells, compared to 20X (34.16% efficiency, 31.1% purity), and 40X (31.83% efficiency, 31% purity) dilutions. We are currently testing whether recycling the blood flow-through would increase the recovery. Even when as many as ~7500 tumor cells were spiked in mouse blood, the Vortex platform was able to successfully enrich tumor cells with high consistency and purity. Importantly, we were now able to successfully isolate human and murine tumor cell lines with varying levels of epithelial cell adhesion molecule (EpCAM) expression (4T1 - high, MDA-MB-231 - moderate to low) spiked into mouse blood. Experiments confirming the viability and growth rate of cancer cells isolated from mouse blood using MTT assay will also be presented.

Conclusion: In summary, our results reveal the use of the Vortex technology for studying CTCs in murine models of cancer, with the ability to handle volumes of blood as low as 200uL while having a capacity to capture extremely large number of CTCs. While this study was focused on breast cancer, our workflow is easily adapted to other malignancies. Studies are underway to grow viable CTCs from tumor bearing animals and isolated by this platform for subsequent growth in 3D culture systems for future biologic and drug testing studies.

#2823

Immunodeficient mice differentially sensitize to estrogen and exhibit severe estrogen-related adverse effects in orthotopic breast cancer model.

Tiina E. Kähkönen,1 Mari I. Suominen,1 Jenni Mäki-Jouppila,1 Jussi Halleen,1 Azusa Tanaka,2 Michael P. Seiler,2 Jenni Bernoulli1. 1 _Pharmatest Services Ltd, Turku, Finland;_ 2 _Taconic Biosciences, Hudson, NY_.

Estrogen-induced growth induction and stimulation of hormone receptor positive breast cancer is commonly acknowledged and thus external estrogen stimulus is used in animal models to support and accelerate growth of the tumors. However, estrogen is known to have adverse effects in the animals and the experiments may have to be preliminary terminated due to animal welfare issues. The aim of the study was to characterize and to compare estrogen caused adverse effects in athymic nude and NOG mice in orthotopic breast cancer model.

Athymic nude (Hsd:Athymic Nude-Foxn 1nu) and CIEA NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac, provided by Taconic Biosciences) were implanted with estrogen (17β-estradiol, E2) releasing hormone rods (5 µg/day) prior to cancer cell inoculation. 5x106 BT-474 (ER, PR and HER+) human breast cancer cells were inoculated to left inguinal mammary fat pad of the mice and the tumor growth was monitored by measuring the tumor volumes twice a week by caliber. The clinical condition and weight development of the mice was carefully monitored with special attention to E2-caused adverse effects. If the mice met the termination criteria (i.e. significant weight loss or general worsening of the overall health) they were sacrificed individually before the intended end of the study.

Nude mice exhibited severe estrogen-caused adverse effects as no adverse effect were observed in NOG mice. Most common adverse effect was redness and skin lesions in the urogenital area observed in 63% of the animals. More severe effects were observed in the lower urinary track including urinary stones and bladder obstruction in 67% of the mice. Also internal effects were observed including appearance change in kidneys and spleen in size and color. Due to severity of these effects 50% of the mice had to preliminary sacrificed. Tumor take (100% vs 85%) and final tumor volumes in average (110 mm2 vs 120 mm2) were comparable between nude and NOG mice.

A clear mouse strain specific impact was observed in the study. Nude mice sensitize to estrogen and exhibit severe estrogen-related adverse effects as no effects were observed in NOG mice. In breast cancer studies where E2 supplementation is needed for sufficient tumor growth, the choice of mouse strain should be carefully considered. Special attention should be paid to possible adverse effects keeping in mind the animal welfare issues.

#2824

Validation of a panel of patient-derived xenograft models for prostate caner and cell line models for preclinical drug evaluation.

Nektaria Papadopoulou,1 Jane Wrigley,1 Andrew McKenzie,1 Jason King,1 Louise Wainwright,1 Kelly Jones,1 Anne Collins,2 Rajendra Kumari1. 1 _Crown Bioscience UK, Loughborough, United Kingdom;_ 2 _University of York, York, United Kingdom_.

Background: Prostate cancer is the second most common cancer worldwide for males, and the fourth most common cancer overall, with more than a million new cases diagnosed. However, progress toward understanding the biology of prostate cancer and the development of new therapies has been hampered by the lack of in vivo models that adequately represent the spectrum of benign, latent, aggressive, and metastatic forms of the human disease. Here we report the validation of a panel of patient-derived xenograft (PDX) models and their utilization in preclinical studies alongside cell line models of prostate cancer.

Methods: Prostate cancer samples obtained from patients undergoing surgery were collected with ethical consent, disaggregated and established subcutaneously in Rag2-/-γC-/- mice (The Jackson Laboratory) to generate PDX models. Tumor material was diagnosed on the basis of operative histology and immunohistochemistry (IHC for PSA, androgen receptor expression). Tumor growth in both Rag2-/-γC-/- and NSG mice was evaluated in comparison to cell line models such LNCaP and PC3M. Briefly tumours were measured 3 times a week and tumour volumes were estimated using the formula 0.5 (LxW2) by measuring the tumour in two dimensions using electronic callipers for the duration of the study. For orthotopic PC3M, tumour growth was checked once weekly by bioluminescent imaging (BLI). Briefly, the mice were injected (s.c.) with 150mg/kg D-Luciferin prior to imaging, anaesthetised following administration of D-Luciferin and placed into the imaging chamber (Spectrum CT) and imaged for luminescence (ventral view).

Results: We have established a bank of transplantable prostate cancer PDX and stocks are maintained in liquid nitrogen which can be resuscitated in in both Rag2-/-γC-/- and NSG mice. Two models were reported to be hormone sensitive and 2 models represented castrate resistant prostate cancer (CRPC) one of which has a TMPRSS-ETS fusion. In comparison cell-derived LNCaP requires testosterone to grow whereas the bioluminescent PC3M CRPC orthotopic model develops metastasis in the liver, lungs, lumbar lymph nodes, fore limbs and hind limbs representing the late stage of cancer.

Conclusions: We have characterised both cell and patient-derived prostate cancer xenograft models which will provide a clinically relevant platform spanning the different stages of the disease for preclinical drug evaluation.

#2825

Gene expression profile in genetically engineered mouse model of lung cancer correlates with that in human lung adenocarcinoma.

Imayavaramban Lakshmanan, Satyanarayana Rachagani, Moorthy Ponnusamy, Seema Chugh, Samikshan Dutta, Sakthivel Muniyan, Surinder Batra, Apar Kishore. _University of Nebraska Medical Center, Omaha, NE_.

Introduction: Despite recent advances in the understanding of the biology of lung cancer, there are no agents that can target Kras, the most common mutation seen in lung adenocarcinoma. Development of genetically engineered mouse (GEM) models with krasG12Dand p53R172H+ will help us to understand the biology of kras mutated lung cancer and develop specific targeted therapeutic agents.

Material and Methods: We have developed two GEM models of lung adenocarcinoma harboring the KrasG12D mutation [KrasG12D;AdCre (KA) and KrasG12D; p53R172H;AdCre (KPA)] by conditional activation in the lung. We performed RNA-seq analysis from KA and KPA tumor tissues and compared them to normal lungs from littermate controls. The results were compared with publicly available human data (TCGA, cBioportal). In addition, we have developed two novel syngeneic cell lines from KA and KPA tumors, which will be used to test the efficacy of candidate drugs.

Results: Hyaluronan binding protein (Habp2) was top most upregulated gene [LogFC 12.7 (KA) 10.4 (KPA)] in both KA and KPA tumor tissues compared to normal. The TCGA and cBioportal data suggest Habp2 is overexpressed in lung adenocarcinoma as opposed to other histologic subtypes. We then analyzed Habp2 associated gene signature and observed that fibrinogen alpha chain (FGA), fibrinogen gamma chain (FGG), hyaluronan synthase 1 (HES1), serpin peptidase inhibitor, clade E1(Serpine1) were also upregulated in both models. On comparison of KA and KPA tumors, we observed that St6galnac1(ST6 N Acetylgalactosaminide Alpha-2,6 Sialyltransferase 1) was upregulated only in the KPA model, suggesting that p53R175H mutation leads to St6galnac1 overexpression.

Conclusion: Hyaluronan binding protein (Habp2) was top most upregulated gene in the KA and KPA models, while St6galnac1expression appeared to be associated with p53R175H mutation. Gene expression data from our GEM models of lung cancer agree with the TCGA and cBioportal analyses. Thus these GEM models seem to replicate human lung cancer and can be used to test drugs for in vivo therapeutic efficacy, prior to use in humans.

#2826

A novel intraperitoneal metastatic xenograft mouse model for survival outcome assessment of esophageal adenocarcinoma.

Md Sazzad Hassan, Niranjan Awasthi, Roderich E. Schwarz, Margaret A. Schwarz, Urs von Holzen. _Indiana University School of Medicine-South Bend, South Bend, IN_.

Introduction: Esophageal adenocarcinoma (EAC) has become the dominant type of esophageal cancer in United States. EAC is the fastest growing cancer in the western world and the overall 5 year survival rate of EAC is below 20 percent. Most patients present with locally advanced or widespread metastatic disease, where current treatment is largely ineffective. Prognosis for EAC patients remains poor even with combination therapies due to high resistance to chemotherapy. Therefore, new therapeutic approaches are urgently needed. Improvement of esophageal adenocarcinoma outcome requires well-characterized animal models in which to evaluate novel therapeutics. In this study we aimed to establish a peritoneal dissemination xenograft mouse model of esophageal adenocarcinoma that would support survival outcome analyses.

Methods: To find the best candidate cell line from 7 esophageal adenocarcinoma cell lines of various origin, we injected them intraperitoneally and subcutaneously into severe combined immunodeficiency (SCID) mice and examined the tumor progression and survival outcomes. Human esophageal adenocarcinoma cell lines of Caucasian/Hispanic origin ESO26, OE33, ESO51, SK-GT-2, OE19, OACM5.1C and Flo-1 originating from gastroesophageal junction, distal esophagus and gastric cardia/fundus were injected intraperitoneally/subcutaneously into SCID mice. The peritoneal/xenograft tumor formation and mouse survival were compared among different groups.

Results: All cell lines injected subcutaneously formed tumors within 3 months at variable rates. All cell lines except OACM5.1C formed intraperitoneal tumors within 3 months at variable rates. Median animal survival with peritoneal dissemination was 108 days for ESO26 cells (5X106), 65 days for OE33 cells (5X106), 88 days for ESO51 cells (5X106), 76 days for SK-GT-2 cells (5X106), 55 days for OE19 cells (5X106), 45 days for OE19 cells (10X106) and 82 days for Flo-1 cells (5X106). Interestingly, only in the OE19 model all mice (7/7 for 5X106 and 5/5 for10X106) developed bloody ascites with liver metastasis after intraperitoneal injection. The median survival time of these animals was the shortest (45 days for 10X106 cells). In addition, median survival was significantly increased after paclitaxel treatment compared with the control group (57 days versus 45 days, p=0.0034) along with a significant decrease of the relative subcutaneous tumor volume (p=0.023).

Conclusion: Peritoneal esophageal adenocarcinoma xenografts were successfully established after intraperitoneal injection of OE19 cells. This animal model of peritoneal dissemination for survival outcome will provide a useful survival outcome assessment model for the preclinical evaluation of cancer therapeutics in esophageal adenocarcinoma.

#2827

Establishing a mouse model to investigate GRB7-mediated signaling in development and triple-negative breast cancer.

Kristopher A. Lofgren, David R. Meier, Steven E. Cash, Paraic A. Kenny. _Gundersen Medical Foundation, La Crosse, WI_.

Triple-negative breast cancer (TNBC) lacks expression of the estrogen and progesterone steroid receptors and HER2; thus TNBC patients cannot benefit from the recent successes of targeted therapies, and instead rely on cytotoxic chemotherapeutic combinations. As such, tumor recurrence is high and patient prognosis is poor. GRB7 is a cytoplasmic adaptor protein required for cell migration, invasion, and proliferation in TNBC. We have previously reported that a high GRB7 mRNA level was the strongest predictor of tumor recurrence in a cohort of 246 TNBC patients treated with doxorubicin. GRB7 was confirmed in a smaller, independent cohort as an indicator of poor prognosis. After the observation of both decreased doxorubicin sensitivity and elevated GRB7 expression levels in TNBC tumors, we hypothesized that GRB7 was mediating a signaling pathway critical for drug resistance and cell survival.

To determine the role of GRB7 in mouse development and tumorigenesis, the current study focuses on the Grb7tm1a(EUCOMM)Wtsi knockout-first transgenic mouse, in which the insertion of the 1L2_Bact_P cassette upstream of the critical exons of GRB7 renders an effective knockout of GRB7, and the inclusion of loxP sites flanking critical exons of GRB7 facilitates the creation of tissue-specific, conditional GRB7 deletions. Herein, we investigate GRB7 expression in the homozygous Grb7tm1a mouse and derivative tissue-specific deletions, and evaluate their suitability for modeling GRB7-mediated events in mammary gland development and tumorigenesis in a TNBC model. Primary cell lines from the Grb7tm1a mouse and breast cancer cell lines will then be used to delineate the effects of altered GRB7 levels on the mechanism of developmental and TNBC cell signaling, respectively.

### Gene Expression in Metastatic Progression

#2828

Role of NEFL in aggressive prostate cancer progression.

Tanya C. Burch,1 Ian O. Oduor,2 Dean A. Troyer,1 Julius O. Nyalwidhe1. 1 _Eastern Virginia Medical School, Norfolk, VA;_ 2 _Old Dominion University, Norfolk, VA_.

Introduction: Prostate cancer (PCa) is the most prevalent cancer amongst men and the second most common cause of cancer related-deaths in the US. Prostate cancer has a heterogeneous spectrum of clinical outcomes with phenotypes ranging from indolent asymptomatic cases to very aggressive metastatic and lethal forms. The development of aggressive castration-resistant prostate cancer and neuroendocrine prostate cancer, which defy currently available treatments, represent the most challenging aspect of this disease. Our long term objective is to identify the molecules and characterize the mechanisms that are involved in PCa disease transformation and progression to aggressive lethal disease. In this study, we show that the over-expression of NEFL, a putative tumor suppressor genes (TSG), correlates with aggressive phenotypes in a subset of prostate cancers with aggressive neuroendocrine-like features. Identifying and characterizing mechanisms by which NEFL modulates progression of PCa will potentially allow for better discrimination of aggressive neuroendocrine prostate cancers and provide targets for better treatment and clinical management opportunities of the disease.

Methods: We have compared the mRNA and protein expression profiles of NEFL prostate cancer cell lines and prostatectomy tissues with different malignancy phenotypes. Validation of differential NEFL expression has been done by Western blot, qRT-PCR, IHC and mass spectrometry. Functional assays and oncogenic properties have been assessed after siRNA mediated knockdown of NEFL gene aggressive prostate cancer cell lines.

Results: We demonstrate significant correlations between of NEFL expression and the acquisition of aggressive PCa with neuroendocrine-like phenotypes. This was validated by Western blot and qRT-PC. SiRNA knockdown of NEFL suppresses the invasive and migratory capacity of PCa cells and tumor colony formation in soft agar assays. The molecular interactome of NEFL was characterized by LC/MS/MS. To our knowledge, this study is the first to demonstrate that NEFL up-regulation correlates with a subset of aggressive prostate cancer disease.

Conclusions: We demonstrate that NEFL is differentially expressed in PCa cells lines and prostatectomy tissues with a significantly higher abundance in cells with clinically aggressive and lethal neuroendocrine-like malignancy phenotypes. These findings are currently being validated using disease stratified tumor micro arrays. Further studies are focusing on uncovering the mechanisms that are modulated by NEFL and are responsible for PCa disease progression. These could assist in the development of novel diagnostic and therapeutic strategies for the disease.

#2829

Identification of transcriptomic signatures of organotropism in pancreatic cancer metastasis.

Yi Zhong, Jun Fan, Jinlong Huang, Christine A. Iacobuzio-Donahue. _Memorial Sloan Kettering Cancer Center, New York, NY_.

Metastatic disease is the most common cause of death in pancreatic ductal adenocarcinoma (PDA) patients. The most common sites of metastatic spread of PDA are to the liver, lung, and peritoneum. The acquisition of advanced stage PDA tissue samples has greatly improved our descriptive understanding of pancreatic cancer metastasis; however, important aspects of PDA metastatic cell biology remain poorly understood. We have previously shown that distant metastases arise from genetically evolved subclones within the primary site of PDA. To understand the biologic basis of PDA metastatic subclone formation we established a transgenic mouse model of PDA based on heterozygous inactivation of Tgfbr2 in association with p48-Cre mediated activation of mutant Kras and Trp53 alleles (KPTC mice). This model revealed that metastatic efficiency and organotropism are related but independently controlled phenomena by mutant Trp53 gain of function in association with TGFb signaling. Pertubation of TGFb signaling reduced liver but not lung metastasis due to a profound extravasation deficiency characterized by sinusoidal growth and lack of desmoplastic stroma compared to KPC mice. Analogous findings were confirmed in liver samples from patients indicating their clinical relevance. Portal vein colonization as a direct mode of access to the liver was observed in both mice and humans. To better understand the cell- autonomous features that promote organ specific metastasis, we have established cell lines from primary and metastatic PDAs of KPC mice representing five different target organ sites (liver, lung, peritoneum, omentum, lymph node). RNAseq and WGCNA performed on extracted RNA from these established cell lines indicate gene expression modules that are unique to metastases. Among metastases, gene expression modules have also been identified that are unique to local versus distant metastases. Analyses are ongoing to functionally validate these modules in additional samples of mouse and human PDA. These findings establish a novel paradigm for understanding pancreatic cancer metastasis and the observed clinical latencies of liver versus lung metastases specifically. Identification of cell-autonomous and micro-environmental mechanisms that support PDA organotropism have the potential to reveal novel targets for therapeutic intervention and modulation of the lethal metastatic phenotype.

#2830

Secreted protein acidic and rich in cysteine antigen and autoantibodies in sera of prostate cancer patients: Potential use in diagnosis /prognosis.

Anshu Rastogi,1 Andy Martinez,2 Amina Ali,1 Shyh-Han Tan,1 Jennifer Cullen,1 Yongmei Chen,1 Gyorgy Petrovics,1 Albert Dobi,1 Lakshmi Ravindranath,1 Denise Young,1 Isabell Sesterhenn,3 Jacob Kagan,4 Sudhir Srivastava,4 David McLeod,1 Inger Rosner,5 Shiv Srivastava,6 Alagarsamy Srinivasan7. 1 _Center for Prostate Disease Research, Rockville, MD;_ 2 _WRNMMC/Center for Prostate Disease Research, Rockville, MD;_ 3 _Joint Pathology Center, Silver Spring, MD;_ 4 _National Cancer Institute, NIH, Bethesda, MD;_ 5 _Uniformed Services Univ. of the Health Sci/WRNMMC, Bethesda, MD;_ 6 _Uniformed Services Univ. of the Health Sci/Center for Prostate Disease Research, Rockville, MD;_ 7 _Uniformed Services Univ. of the Health Sci., Rockville, MD_.

Objectives: Improvements in blood based biomarkers for detecting clinically significant prostate cancer (CaP) or for distinguishing between indolent and aggressive CaP are critical in enhancing the management of this most common non-skin cancer of men in the US. These biomarkers include tumor associated antigens (TAAs) and autoantibodies (AAbs) against TAAs in patient sera. SPARC/Osteonectin is highly expressed in metastatic cancers (glioblastoma, melanoma, breast cancer and prostate cancer) and promotes bone metastasis and epithelial-mesemchymal transition. Our comparative transcriptome analyses of well/moderately differentiated CaP with poorly differentiated CaP, defined SPARC as central node in the network of genes with common regulatory elements (NKXH_NKXH_HOX) associating with poorly differentiated CaP. Further, we reported associations of high levels of SPARC mRNA or protein with Gleason 8-10 or poorly differentiated primary tumors and association with metastatic progression. The aims of this study are: i) Quantify SPARC antigen in the sera CaP patients; ii) Are AAbs against SPARC present in the serum of CaP patients?; iii) Is there a correlation between SPARC AAb level in patient sera and disease status? iii) Does SPARC AAb level vary according to distinct ethnic groups?

Methods: Sera from CaP patients and healthy controls were evaluated for SPARC antigen using a commercial enzyme-linked immunosorbent assay (ELISA) kit. AAbs against SPARC were determined by ELISA utilizing recombinant full-length human SPARC protein as a substrate. For evaluation we used Caucasian American (CA, n=117) and African American (AA, n=111) CaP patients comprising Gleason 6-10, and healthy controls (CA, n=52; AA, n=45).

Results: SPARC antigen levels in the sera showed a difference in the overall case versus control groups (p=0.0016) and this trend was observed only in AA (p=0.0015) in comparison to CA patients (p=0.1709). SPARC AAbs were detected in the sera and the values were significant in the overall case versus control (p< .0001) and also in CA (p<.0001) and AA (p<.0001) CaP patients with levels significantly lower in patients compared to controls. AAb reactivity for CaP patients was similar between CA and AA groups.

Conclusions: This study demonstrated the presence of AAbs against SPARC in CaP patient serum for the first time. Of note, highly significant differences were noted between CaP patient (low) and controls (high) sera, across different ethnic groups. These data suggest further evaluation of SPARC AAbs as a promising serum biomarker for CaP.

Source of Funding: This research was supported by the Center for Prostate Disease Research, Uniformed Services University Grant HU0001-10-2-0002.

#2831

Identification of cell specific expression signatures using NGS global gene expression profiles from LCM procured breast carcinoma and adjacent stromal cells.

Jeoffrey J. Schageman,1 Kristi Lea,1 Sarah A. Andres,2 Kelli S. Bramlett,1 James L. Wittliff2. 1 _Thermo Fisher Scientific, Austin, TX;_ 2 _Univ. of Louisville Health Sciences Ctr., Louisville, KY_.

Our goal is to determine relationships of gene expression profiles to patient-associated characteristics, breast carcinoma pathology and biomarker status with clinical outcome to improve assessment of prognosis and therapy selection.

Procedures: De-identified frozen tissue biopsies of human breast carcinomas which were collected and stored under stringently controlled conditions were employed. Patient-related features (e.g., age, race, menopausal status, nodal status, clinical treatment and response) and results from biochemical analyses of established tumor markers (e.g., estrogen and progestin receptors, EGF receptor, HER-2/neu oncoprotein) guided tissue selection. Structural integrity of a tissue was first evaluated by light microscopy of H&E stained sections. To decipher uniqueness and clinical utility of gene expression profiles from specific cell types, Laser Capture Microdissection (LCM) with a PixCell IIeTM (Arcturus®, Thermo Fisher Scientific) was used to non-destructively collect either carcinoma cells or adjacent stromal cells on CapSureTM LCM caps. A second intact serial tissue section was processed identically to that of the section used for LCM as a control. Each of the two independent cell captures from the same tissue section and the second intact tissue section were independently extracted for RNA. RNA was assessed for integrity, purified and subjected to novel analyses using the Ion AmpliSeqTM RNA Breast Cancer Research Panel containing 1174 genes. Sequencing workflow included templating on the Ion Chef TM System and multiplexed sequencing on the Ion S5TM XL sequencing system and Ion 540TM chip.

Results: Each sequencing library yielded approximately 4-5 million reads with a mapping rate of >98% that was achieved after alignment to the human transcriptome. Each sample was sequenced in triplicate with technical reproducibility near 99% with a mean of 1035 genes classified as detected. Cluster analyses of gene expression profiles revealed clear distinctions between stromal and carcinoma cells procured by LCM compared to intact tissue driven by strongly differentially expressed genes such as vimentin (VIM), MPRIP, stanniocalcin-2 (STC2) and estrogen receptor beta (ESR2).

Conclusions: Collectively, the results indicate that molecular signatures of breast carcinoma cells procured by LCM reveal unique gene expression profiles compared to those of the intact tissue section or the adjacent stromal cells. These novel findings are likely to discern molecular features unique to a breast carcinoma that may be related to clinical outcome to improve assessment of patient prognosis and therapy selection. Supported in part by a grant from the Phi Beta Psi Charity Trust (JLW & SAA) and a CTSP Award from the Commonwealth of Kentucky (JLW). For research use only.

#2832

Class I histone deacetylase inhibitors impair tumor growth and metastatic programs.

Oliver H. Krämer, Nicole Kiweler. _Institut für Toxikologie Universitätsmedizin der Johannes Gutenberg-Universität Mainz, Mainz, Germany_.

(a) introductory sentence indicating the purposes of the study

Metastasis formation is associated with poor patient prognosis. Epithelial-mesenchymal and mesenchymal-epithelial (EMT/MET) transition cycles are key for the formation, survival, and homing of metastatic cells. Inhibitors against epigenetic modulators of the histone deacetylase family (HDACi) are promising, clinically tested epigenetic drugs that may combat cancer proliferation and spread.

(b) brief description of pertinent experimental procedures

We chose a systems toxicology approach to analyze the relevance of HDACs and their inhibitors for metastatic cell growth. We combined in vivo tests with morphological, cellular, functional, and global proteomic analyses. This strategy follows the 3R principle by Russel and Burch. Of the four classes of HDACs (I-IV), the class I subgroup is most important for tumorigenesis. We therefore analyzed how the pharmacological inhibition of class I HDACs affects the growth and spreading of syngeneic kidney cancer cells into the lungs of BALB/c mice and how such drugs affect tumor cell growth, apoptosis, migration, and gene expression patterns controlling EMT/MET in transformed kidney and breast cells. Furthermore, we investigated how the genetic elimination of HDACs affects cancer cell growth and EMT/MET. We used flow cytometry, immunoblot, immunofluorescence, qPCR, proteomics on a global scale, RNAi against the class I HDACs HDAC1/HDAC2, and in vivo tumor analyses.

(c) summary of the new, unpublished data

Both the inhibition as well as the elimination of HDAC1/HDAC2 evokes growth arrest, morphological alterations, and apoptosis. HDACi block tumor cell migration, integrin-dependent cell adhesion, and EMT induced by the cytokine TGF-β. Global proteomics and qPCR analyses illustrate that HDACi disrupt EMT/MET cycles and metastatic spread. Moreover, these assays reveal pathways through which HDACi propel cancer cell apoptosis.

(d) statement of the conclusions

Class I HDACi can modulate the EMT/MET balance that is required for the detrimental process of metastasis. Hence, HDACi should be considered further as clinical treatment option.

#2833

**Epithelial cell adhesion molecule (EpCAM) is associated with prostate cancer progression and chemo-/radio-resistance** in **** vitro **and** in vivo **.**

Jie Ni,1 Paul Cozzi,1 Julia Beretov,1 Junli Deng,2 Joseph Bucci,1 Peter Graham,1 Yong Li3. 1 _St George Hospital, Kogarah, Australia;_ 2 _University of New South Wales, Kogarah, Australia;_ 3 _St George Hospital / Univ. of New South Wales, Kogarah, Australia_.

Aims: Prostate cancer (CaP) is the most common cancer in males in Australia which caused more than 3000 deaths in 2015. EpCAM is a transmembrane protein that is expressed at low levels in a variety of human epithelial tissues, but overexpressed in most solid tumors. Our previous study indicated that EpCAM was strongly expressed in metastatic CaP cell lines, primary human CaP tissues and lymph node metastasis and is a biomarker involved in CaP progression, and chemo-/radio-resistance. However, the role of EpCAM in CaP progression and therapeutic resistance is still uncertain. The aim of this study was to investigate the role of EpCAM in CaP progression and chemo-/radio-resistance as well as underlying mechanisms in vitro and in vivo.

Methods: EpCAM gene was knocked down (KD) in PC-3, DU145 and LNCaP CaP cell lines using shRNA. Proliferation assay, colony formation assay, docetaxel (DTX) and radiation dose-response assay were carried out to evaluate the effect of KD of EpCAM on proliferation and therapeutic response of CaP cells in vitro. Subcutaneous (s.c) and orthotopic CaP animal models were established using PC-3-EpCAM-KD and PC-3-EpCAM-scramble (scr) control cells in NOD/SCID mice, to assess the effect of EpCAM on CaP tumourigenecity, chemotherapy (DTX) and radiation response. Signal transduction proteins in PI3K/Akt/mTOR signaling pathway, as well as proliferation, apoptotic and radiation response markers were evaluated by immunohistochemistry in xenografts.

Results: KD of EpCAM reduced CaP proliferative potential and enhanced DTX and radiation sensitivity in three CaP cell lines. Both s.c and orthotopic EpCAM-KD xenografts displayed suppressed tumor growth and increased DTX and radiation responsiveness compared to EpCAM-scr control xenografts in NOD/SCID mice. Marked down-regulation of PI3K/Akt/mTOR pathway proteins (p-Akt and p-mTOR) and proliferation marker (Ki-67) and significant up-regulation of apoptotic (Caspase-3) and radiation (ɣ-H2AX) responses to chemo-/radio-therapies were found in EpCAM-KD xenografts, compared with control xenografts. In addition, Kaplan-Meier curve analysis demonstrated that KD of EpCAM improved median survival (MS) of tumor-bearing mice by 21.5 days compared with the control group (HR=26.94, CI95% 4.317-168.1, p=0.0004) and that KD of EpCAM improved MS of tumor-bearing mice which received docetaxel (50mg/kg, single dose, i.p.) by 11 days (HR=20.95, CI95% 3.599-121.9, p=0.0007), and radiotherapy (2Gy/day for 4 days) by 12 days (HR=11.00, CI95% 2.11-57.36, p=0.0044) compared with the control group, respectively.

Conclusions: EpCAM plays an important role in CaP progression and chemo-/radio-resistance via PI3K/Akt/mTOR signaling pathway in vitro and in vivo and it is a promising therapeutic target for the treatment of CaP. EpCAM targeted therapy combined with chemo-/radio-therapy could be a novel modality for treatment-resistant CaP.

#2834

Metastatic colorectal cancer: characterization of distinct histological growth patterns which demonstrate different response to current treatment regimes.

Vincent Palmieri,1 Anthoula Lazaris,2 Hussam Alamri,2 Abdellatif Amri,2 Nisreen Ibrahim,1 Pablo Zoroquiain,2 Peter Vermeulen,3 Peter Metrakos2. 1 _McGill University, Montreal, Quebec, Canada;_ 2 _McGill University Health Centre - Research Institute, Montreal, Quebec, Canada;_ 3 _Translational Cancer Research Unit (TCRU), GZA Hospitals, Antwerp, Belgium_.

Colorectal cancer (CRC) is the third most common cancer in North America, and the third leading cause of cancer-related death. About 50% of patients will be diagnosed with CRC liver metastasis (CRCLM) during the course of their disease. We have identified two major histological growth patterns (HGP) in CRCLM resected from patients. In the desmoplastic HGP (DHGP), the tumor cells are enclosed within a desmoplastic stromal ring, physically separating them from the normal liver tissue. In the replacement HGP (RHGP), the tumor cells replace normal hepatocytes without disrupting adjacent cells or structures within the liver parenchyma. We have recently shown that patients with RHGP lesions who received neoadjuvant bevacizumab plus chemotherapy had a worse pathological response and five-year overall survival. Our group has also shown that RHGP CRCLM is resistant to anti-angiogenic therapy due to it promoting vessel co-option as a means of tumor vascularization, rather than VEGF-dependent angiogenesis as seen in DHGP. However, the molecular mechanisms that drive the CRCLM HGPs remain unknown.

This project aims to shed light on the biological processes that underlie the diversity of HGPs, with an emphasis on the key genes and pathways that support the RHGP. To test this, we have: Aim 1) performed RNAseq on chemonaïve RHGP and DHGP CRCLM lesions to identify pathways that are significantly upregulated in the RHGP. We have validated these genes via real-time PCR (RT-PCR), and a subset by immunohistochemistry (IHC). Aim 2) To further refine the RHGP gene signature and to examine the effects of cell-cell interactions on hepatocyte and tumor cell gene expression, individual cells have been isolated from distinct regions within RHGP CRCLM tissues using laser capture microdissection (LCM), followed by single-cell transcriptomics analyses.

Our preliminary data have identified three major pathways with differentially expressed genes: i. cell motility, ii. cell junctions, and iii. ECM-receptor interactions, which are upregulated in RHGP relative to the DHGP and normal liver. A panel of RHGP-specific genes has been identified and validated via RT-PCR. We selected a subset of these genes and are performing IHC to further validate and locate their expression. We have also performed LCM and are in the process of performing transcriptomics analyses – the data will be presented.

We expect to gain some insight into the mechanisms responsible for driving and/or maintaining the RHGP lesions. In the longer term, this work will result in identification of targets in the HGPs that will help stratify patients in terms of treatment. It would appear that we have fewer treatment options for the RHGP patients and it would be important to utilize the data to develop new treatment strategies for those patients. These new therapies would lead to further clinical trials.

#2835

Potential opposing roles of AKT isoforms in indolent versus aggressive prostate cancer.

Karina A. Miller,1 Hyun-Kyung Ko,2 Irwin H. Gelman1. 1 _Roswell Park Cancer Inst., Buffalo, NY;_ 2 _Cleveland Clinic, Cleveland, OH_.

Prostate cancer (CaP) is the most commonly diagnosed non-cutaneous cancer and the second highest contributor to cancer-related deaths in U.S. men. Localized CaP has a 5-year survival rate of approximately 100%, but disseminated, metastatic disease (mCaP) dramatically reduces 5-year survival to <30%. A known driving force in the progression of CaP is activation of the PI3K/Akt signaling pathway through loss of PTEN, with widespread effects on cell proliferation and migration. Additional genomic changes, such as the loss of TP53 or RB1, or the upregulation of MYC, correlate with increased incidence and aggressiveness of mCaP. However, only a fraction of men with high levels of circulating and disseminated CaP cells progress to overt, macro-metastatic disease - the lethal phenotype in CaP. Importantly, little is known regarding the drivers of indolent vs. aggressive mCaP. We used two transgenic (Tg) mouse models of mCaP with clinically relevant genetic alterations which exhibit Akt activation and Rb1 loss, yet one produces indolent mCaP and the other, systemic aggressive mCaP. Specifically, mice lacking Rb1 and the metastasis-suppressor, SSeCKS/Akap12, develop high-grade prostatic intraepithelial neoplasia (HG-PIN) and disseminated disease in the lymph nodes whereas those lacking Pten and Rb1 develop CaP and aggressive mCaP. The metastasis-suppressing functions of Akap12 relates to its ability to scaffold Src and PKC, thereby attenuating their oncogenic signaling pathways. We will show data suggesting that the pathological differences between these two Tg models is based on the differential use of PI3K-p110 and Akt isoforms, with aggressiveness correlating with the downregulation of Smad4, a known metastasis-suppressor in Tg CaP models. Additionally, differences in Akt isoform usage likely controls the expression of neurogenesis genes that are especially upregulated in castration-recurrent CaP that fails second-line androgen receptor antagonist inhibitors such as enzalutamide. By comparing the differential transcriptomes and signaling networks in primary-site tumors, lymph node metastases, and CaP cell lines derived from these two Tg models, several new potential therapeutic targets have been identified that could be exploited to prevent or treat aggressive mCaP.

#2836

Mitochondrial genomic backgrounds differentially affect nuclear DNA methylation and gene expression.

Carolyn J. Vivian,1 Amanda E. Brinker,1 Stefan Graw,1 Devin C. Koestler,1 Christophe Legendre,2 Gerald C. Gooden,3 Bodour Salhia,3 Danny R. Welch1. 1 _Univ. of Kansas Cancer Ctr., Kansas City, KS;_ 2 _Translational Genomics Research Institute, Phoenix, AZ;_ 3 _University of Southern California, Los Angeles, CA_.

Accumulating data implicate mitochondrial genetics and metabolism as contributors to metastatic efficiency. With only 13 proteins encoded by the mtDNA genome, the mechanism(s) by which the coordination of multiple genes involved in metastasis could occur is unclear. We hypothesized that mitochondrial DNA (mtDNA) polymorphisms would differentially regulate nuclear DNA (nDNA) methylation and gene expression. To test the hypotheses, we created Mitochondrial Nuclear Exchange (MNX) mice, by transferring an oocyte nucleus from strainx into an enucleated oocyte from strainy. The mice show that mammary tumor formation and metastasis are regulated by inherited mitochondrial polymorphisms (PMID: 26471915). Age-matched, male mouse brains were collected and pooled from four litter mates representing each of the wild-type and MNX mice (8 groups). Genome-wide nDNA methylation (Mouse methyl-Seq) and gene expression (RNA-Seq) were measured and results validated in independent replicate samples. Significant, selective and reproducible differential DNA methylation and gene expression were observed and validated between strains having identical nDNA, but different mtDNA. While the signal(s) from mitochondria altering methylation is not fully defined, the findings support the hypothesis and demonstrate how mitochondrial genetic alterations could be one of several quantitative trait loci involved in complex phenotypes, such as metastasis. The results also suggest that mtDNA polymorphisms could serve as predictive biomarkers for cancer aggressiveness. Support: Susan G. Komen for the Cure (SAC11037), Natl Fndn Cancer Res, Biomedical Research Training Program, Kansas Bioscience Authority, CA134981, P30-CA168524.

#2837

Differential expression of Protocadherin-7 (PCDH7) in prostate cancer (PCa) cells: a direct association between PCDH7 and castrate resistant PCa.

Gauri Shishodia,1 Sweaty Koul,1 Qin Dong,1 Hari K. Koul2. 1 _LSU Health Sciences Center - Shreveport, Shreveport, LA;_ 2 _FWCC/LSU Health Sciences Center - Shreveport, Shreveport, LA_.

Introduction: PCa is the most commonly diagnosed disease in American men and the second leading cause of death among them. Most of PCa deaths are a result of distant metastasis and due to emergence of castrate resistant PCa. Protocadherin-7 (PCDH7), a Cadherin superfamily transmembrane protein that is known to function in cell-cell recognition and adhesion, is reported to be overexpressed in breast cancer and non-small cell lung cancer (NSCLC). PCDH7 has been shown to mediate brain metastasis of breast cancer. Overexpression of PCDH7 was reported to be associated with poor clinical outcome of NSCLC. However, there is no report on involvement of PCDH7 in PCa tumorigenesis and metastasis. In the present study we evaluated expression of PCDH7 in androgen dependent and androgen independent PCa cells and in TCGA and Trento/Cornell/Broad 2016 clinical data sets.

Materials and Methods: PCa cells- LNCaP, C4-2b, DU145, PC3, 22Rv1 and RWPE1 were used in this study. Protein expression was checked by Western Blotting. mRNA levels were checked by quantitative real time PCR.

Results: Our data reveal high PCDH7 protein expression in PC3 cells and low levels of PCDH7 in LNCaP and RWPE1 cells. qRT-PCR confirmed an increased transcript level of PCDH7 in PC3 and low levels in LNCaP cells. We also observed elevated transcript levels for PCDH7 in castrate resistant and Enzalutamide refractory PCa (22Rv1) cells. These data suggest a direct relationship between castrate resistance and PCDH7 expression. Our results also show an inverse relationship between PCDH7 and E-Cadherin and a direct relationship between PCDH7 and EMT markers, indicating expression of PCDH7 is associated with tumor progression. Interestingly, there was no correlation between PCDH7 with tumor grade or metastasis from TCGA data, but analysis of NEPC (Trento/Cornell/Broad 2016) data set revealed amplification of PCDH7 in 9% of NEPC tumors. Collectively, these data suggest PCDH7 expression may be associated with advanced PCa in general and NEPC phenotype PCa in particular.

Conclusion: Our results suggest direct association of PCDH7 expression and castrate resistance in PCa cell lines. Role of PCDH7 in castrate resistant PCa needs further evaluation.

#2838

CtBP1 expression depletion on primary tumor deregulates miRNA expression and impairs development of spontaneous metastases on a prostate cancer and metabolic syndrome model.

Guillermo N. Dalton, Georgina Scalise, Juliana Porretti, Cintia Massillo, Paula L. Farré, Paola De Luca, Adriana De Siervi. _Instituto de Biología y Medicina Experimental (IByME) - CONICET, C.A.B.A, Argentina_.

Prostate cancer (PCa) is the second cancer in incidence in men worldwide. Approximately 20 % of cases continue with advanced or metastatic disease, and at this stage, it turns out incurable due to the lack of effective therapies. Hence, the need to identify new actionable targets is crucial. Metabolic syndrome (MeS) is a physiopathological disorder that increases PCa risk and aggressiveness. C-terminal Binding Protein (CtBP1) is a transcriptional corepressor that is activated by NADH binding. Previously our group established a MeS and PCa mice model that identified to CtBP1 as a novel link associating both diseases. Moreover, cell adhesion molecules play a significant role in cancer progression and metastasis. Thus, we found that CtBP1 diminished the capability of PCa cell lines to adhere to a collagen matrix, directly modulating expression of several cell adhesion genes, including repression of the epithelial marker CDH1 and induction of the mesenchymal marker VIM. The aim of this work was to investigate MeS/CtBP1 impact over PCa progression from in situ prostate carcinoma to metastatic disease. RNA was isolated from xenografts generated on MeS mice from CtBP1 depleted PC3 cells (PC3.shCtBP1) or control (PC3.PGIPZ); and hybridized to a miRNA expression microarray (Affymetrix GeneChip® miRNA 4.0). After data normalization and analysis we identified and validated a list of 11 miRNAs regulated by CtBP1 relevant to cell adhesion and PCa progression. To investigate CtBP1 role in spontaneous PCa metastasis, NOD SCID gamma (NSG) mice were fed with control or high fat diets during 12 weeks to induce MeS. Then PC3.shCtBP1 or PC3.PGIPZ cells were injected s.c. on MeS and control animals. Body weight and tumor size were measured 1 and 3 times a week, respectively. Thirty days after cell inoculation, tumors were around 1 cm, with no significant differences between treatments; however mice showed around 20% weight loss. Mice were sacrificed and tumors, lungs and livers were collected for RNA isolation and histopathological analysis. Using human GAPDH specific primers and RT-qPCR from lungs, we found that CtBP1 depletion led to a significant decrease of lung metastases, especially in the MeS group. In addition, Hematoxylin & Eosin stains from lung sections detected the lowest number and size of metastatic foci in the CtBP1 depleted xenografts generated in MeS animals. Gene expression comparison between primary tumors and metastases showed that epithelial markers, such as E-cadherin, were induced in xenografts and almost undetected in metastasis. Accordingly, mesenchymal markers expression, such as Vimentin, was low in xenografts and triggered on metastases. Our study uncovers for the first time the role of CtBP1 in PCa progression and its molecular targets in MeS mice.

#2839

Systematic enrichment analysis of gene expression profiling studies identifies SPARC dependent consensus pathways implicated in bladder cancer.

Bincy Anu John, Neveen Said. _Wake Forest Health Sciences, Winston-Salem, NC_.

Bladder cancer (urothelial cancer of the bladder) is the most common malignancy affecting the urinary system with increasing incidence and mortality. Treatment of bladder cancer has not advanced in the past 30 years. Therefore, there is a crucial unmet need for novel therapies, especially for advanced disease. We have recently reported Secreted Protein Acidic and Rich in Cysteine (SPARC) as a tumor suppressor in urinary bladder cancer that inhibits the multistep cascades on tumor initiation, progression and metastasis. SPARC gene and protein expression was among candidates associated with the tumorigenicity of the isogenic T24/T24t cell lines. We reported that SPARC depletion in human bladder cancer cell lines including T24 cells increased their tumorigenicity and metastatic potential. In addition, SPARC protein and transcript expression are significantly downregulated in advanced urothelial cancer. To gain insight on the molecular mechanisms of the tumor suppressor effects of SPARC in urothelial cancer, we did whole transcriptome profiling of T24 cells depleted of SPARC to identify SPARC-signature associated with bladder cancer invasiveness, progression, and metastasis. Subsequently, we used gene signature enrichment analysis (GSEA) pathway enrichment tools to define functionally related genes that are consistently up- or down- regulated as a function of loss of SPARC. We found that loss of SPARC expression is associated with enrichment of multiple oncogenic signaling pathways that are enriched in muscle invasive urothelial cancer and have been associated with poor prognosis. These pathways include K-ras, Myc-oncogene, p53 pathways as well as DNA damage/repair. Consistent with our earlier reports, loss of SPARC is associated with genes and proteins involved in angiogenesis, inflammation and hypoxia. In addition, we have identified novel signatures involved in unfolded protein response, mTOR signaling, glycolysis and cholesterol metabolism. These comprehensive signatures not only reveal SPARC-dependent networks that cooperate to elicit its biological responses but enables coherent understanding of the etiology of complex bladder cancer disease. Importantly, these signatures identify novel therapeutic opportunities for bladder cancer.

#2840

Integrative approach to biomarker discovery by performing comparative analysis of two cancers, hepatocellular carcinoma and endometrioid endometrial carcinoma, using genetics and transcriptomics from RNA sequencing data.

Jean-Noel Billaud, Stuart Tugendreich, Debra Toburen. _QIAGEN Bioinformatics, Redwood City, CA_.

Hepatocellular Carcinoma (HCC) and Endometrioid Endometrial Carcinoma (EEC) are two lethal diseases of public health importance worldwide. Using QIAGEN's RNA sequencing solution, we were able to highlight key molecular and biological processes that indicate similarities in the tumor progression toward metastasis between one EEC patient and the three HCC patients. We have identified ITGB1-001 isoform as a potential common biomarker between these two complex cancers. We have also shown these two cancers share activated pathways such as actin cytoskeleton signaling, and RAF1 as an upstream regulator indicating similar transcriptional program between these patients. Furthermore, we have identified as a potential therapeutic target the master regulator CTGF that connects a network of 286 downstream targets to invasion of tumor cells. We have filtered a set of unique variants in the EEC patient that affect a network of upstream regulators common with HCC. This approach may be useful in the context of precision or personalized medicine. Examining the gene expression in tumors from groups of patients with each disease revealed that at a molecular level, early stages of EEC resemble established HBV-positive, HCV-negative, liver cirrhosis positive HCC.

#2841

Investigating drivers of disease progression in invasive lobular carcinoma.

Nilgun Tasdemir,1 Matthew Sikora,2 Zhu Li,1 Kevin Levine,1 Ahmed Basudan,1 Soumya Luthra,1 Esther Elishaev,1 Uma Chandran,1 George C. Tseng,1 Rachel Jankowitz,1 David J. Dabbs,1 Priscilla McAuliffe,1 Nancy E. Davidson,1 Steffi Oesterreich1. 1 _University of Pittsburgh, Pittsburgh, PA;_ 2 _University of Colorado, Denver, CO_.

Invasive lobular carcinoma (ILC) is the second most common type of breast cancer following invasive ductal carcinoma (IDC) and accounts for 10-15% of all cases. Unlike the masses or lumps formed by IDCs, ILCs grow as small, dis-cohesive cells in a single-file pattern within dense layers of extracellular matrix. Paradoxically, while patients with ILC display favorable prognostic and predictive factors (ER+, PR+, HER2-, low Ki67), their long-term response to endocrine therapy is worse than patients with IDC. Despite such histological and clinical differences, ILC has remained a gravely understudied subtype of breast cancer. Thus, there is urgent need to investigate molecular drivers of ILC progression in order to develop more effective therapeutic strategies and improve patient outcome. To this end, we used the Nanostring platform to measure the expression of 577 copy number variation-associated genes in 131 primary ILC tumors and identified two candidate drivers that exhibit higher expression in tumors from patients with recurrent versus non-current disease: CTTN (cortical actin binding protein) and QSOX1 (quiescin sulfhydryl oxidase). In follow-up studies, we observed high CTTN and QSOX1 expression in human ILC cell lines, tumors and patient-derived xenografts. In addition, QSOX1 expression was further increased in ILC ovarian metastases compared to their matched primary tumors. In functional in vitro studies, RNAi-mediated inhibition of CTTN led to decreased adhesion and haptotaxis to Collagen I, while QSOX1 knockdown diminished the growth of human ILC cell lines. We are currently assessing the in vivo therapeutic utility of CTTN and QSOX1 inhibition in tumor growth and metastasis using human ILC cell line and patient-derived xenograft mouse models. This study greatly advances our understanding of ILC disease mechanisms and identifies novel therapeutic targets towards improving the clinical outcome of patients with this understudied subtype of breast cancer.

#2842

Dissecting the transcriptional profiles of metastatic and primary disease across cancer types.

Laxmi Silwal-Pandit,1 Vigdis Nygaard,1 Hege Russnes,1 Vegar Johansen Dagenborg,1 Veronica Skarpeteig,1 Olga Østrup,2 Silje Nord,1 Vivi Ann Flørnes,3 Anne Hansen Ree,4 Kjersti Flatmark,1 Anne-Lise Børresen-Dale,5 Ole Christian Lingjærde,6 Gunhild Mari Mælandsmo1. 1 _Institute for Cancer Research, Oslo University Hospital, Oslo, Norway;_ 2 _Rigshospitalet - Copenhagen University Hospital, Oslo, Denmark;_ 3 _Oslo University Hospital, Oslo, Norway;_ 4 _Akershus University Hospital, Oslo, Norway;_ 5 _Institute for Clinical medicine, University of Oslo, Oslo, Norway;_ 6 _Institute for Informatics, University of Oslo, Oslo, Norway_.

Cancer metastasis is the principal cause of death in individuals with cancer; nevertheless, the molecular basis of metastases is poorly understood. Genetic and epigenetic changes in malignant cells and their interaction with the tumor microenvironment are presumably key events in the establishment of metastases. In this study, we pursue to provide insights into potential importance of various fundamental cancer-related pathways in the metastatic process. We have performed a pathway - based analysis on mRNA expression data (Agilent 8X60K array) from colorectal liver metastases (CLM; N = 38 patients, n = 44 metastases), breast lymph-node metastases (BCM; N = n = 43) and melanoma lymph-node metastases (MLM; N = n= 44). For each of the 186 pathways in the KEGG pathway database, each sample is represented as points in a 'n' dimensional space according to their gene expression values, where 'n' is the number of genes in the pathway. We first calculated centroids for each of the three metastases types (CLM, BCM and MLM) and then calculated the Euclidean distance between centroids to get a measure of how similar or different each cancer type is for that particular pathway. The findings were compared to the corresponding primary cancer types and normal tissue available from TCGA to uncover biological processes specific to metastases. The pathways were functionally classified into genetic information processing, cellular processes, environmental information processing and metabolic pathways as per KEGG database. Across metastases from different cancer types, pathways involved in genetic information processing (e.g. translation, transcription, protein folding and degradation, DNA replication and repair) and metabolic pathways were more similar than the pathways involved in cellular processes (e.g. cell-cycle, apoptosis, adherence junction), and signal transduction pathways (e.g. Notch, MTOR, JAK STAT) responsible for environmental information processing. The genetic information processing pathways were more similar across cancer types also in the primary setting than the pathways involved in cellular and environmental information processing, whereas the metabolic pathways were more similar across cancer types in the metastatic setting compared to the primary setting. Preliminary findings suggest that the basic molecular architecture of the primary tumors is maintained, except for the metabolic pathways, which become more similar in the metastatic setting; and hence may be a common denominator across cancer types during metastatic process.

#2843

DNMT3B mediated DNA methylation and epigenetic reprogramming of metastatic cancer cells.

Jae Young So,1 Nicolas Skrypek,1 Xiang Wang,1 Anand Merchant,1 Howard Yang,1 Wei-Dong Chen,1 Gangqing Hu,2 Bhagelu R Achyut,1 Meggie Cam,1 Keji Zhao,2 Maxwell Lee,1 Li Yang1. 1 _NCI/NIH, Bethesda, MD;_ 2 _NHLBI/NIH, Bethesda, MD_.

Approximately 90% of cancer patients die from metastasis. In many cases, current therapies are designed based on the primary tumor characterization and are ineffective against metastatic disease. It is not clear whether metastatic cancer cells acquire additional genetic and epigenetic alterations, if so, what are the underlying mechanisms. Using several tumor models, we demonstrated that DNMT3B, de nova DNA methyltransferase, was increased in metastatic nodules in comparison to that from the primary tumors. DNMT3B knockdown or overexpression significantly inhibited or increased metastatic colonization of cancer cells. To understand the underlying molecular mechanisms of DNMT3B regulation, we performed DNMT3B ChIP-seq, RNA-seq, and DNA methylation-seq comparing lung metastases with primary tumors and 4T1 cells. We identified genes that are differentially methylated and expressed. Ingenuity Pathway Analysis revealed DNMT3B targeting multiple oncogenic signaling pathways. Bioinformatics analysis of human databases demonstrated significantly higher expression levels of DNMT3B in metastases than primary tumors in breast, prostate and melanoma cancer patients. In summary, our study demonstrated that DNMT3B as a key epigenetic regulator which plays critical roles for tumor metastasis. Our work suggests DNMT3B as potential therapeutic targets for breast cancer patients with metastatic disease.

#2844

Identification of genes associated with pancreatic cancer metastasis by genome-wide CRISPR Cas9 screening.

Youjia Li,1 Huiyi Feng,1 Guangjie Liu,2 Chi Hin Wong,1 Chi Han Li,1 Yangchao Chen1. 1 _CUHK, Hong Kong, Hong Kong;_ 2 _GIBH, Guangzhou, China_.

Pancreatic cancer is an extremely lethal cancer with a 5-year survival rate of 7%. One major reason why pancreatic cancer is hard to treat is its metastasis nature. Genes that related to the tumorigenesis and progression of pancreatic cancer were identified after decades of efforts. Several researches had revealed that pancreatic cancer originated from the successive accumulation of gene mutations, especially KRAS2, CNKD2A, TP53 and SMAD4. However, little is known about genes that regulate pancreatic cancer metastasis. Here we used a genome wide CRISPR Cas9 screening to identify the gene(s) associated with the metastasis of pancreatic cancer. We transduced pancreatic cancer cell line with lentivirus-based genome wide CRISPR sgRNA library to establish mutated cell library, and orthotopically injected the cell library into nude mice and waited for primary tumor growth and metastasis, then collected primary and metastatic tumors after several months. By next generation sequencing and analysis, we identified some gene candidates which may play important roles on pancreatic cancer metastasis.

#2845

A Rb phosphorylation code associated with lung cancer metastasis.

Jaileene Perez-Morales,1 Darielys Mejias-Morales,2 Bryan Torres-Collazo,3 Harry Negron-Pagan,1 Pedro Santiago-Cardona1. 1 _Ponce Health Sciences University, Ponce, PR;_ 2 _Pontifical Catholic University, Ponce, PR;_ 3 _University of Puerto Rico, Ponce, PR_.

Lung cancer is characterized by its poor prognosis, aggressiveness, and proclivity for early metastasis. Non-small cell lung cancer (NSCLC) it's the most common type of lung cancer with a five-year survival rate of 18%. Most predictors of metastasis and recurrence of NSCLC rely on post-resection evaluation of tumor histology, which is a severe limitation since only 15% of the patients are diagnosed with resectable disease. Hence, there is a need to characterize biomarkers with metastasis-predicting value in pre-resection small biopsy specimens. In addition to the proclivity for metastasis, another feature of lung cancers is the inactivation of the retinoblastoma protein (Rb). The retinoblastoma protein (Rb) is a tumor suppressor inactivated due to hyper-phosphorylation in most human cancers, including NSCLC. Our preliminary data relates the Rb S249/T821 phosphorylation signature to an epithelial-to-mesenchymal transition (EMT), a trait strongly associated to metastasis. We found that cells that have hyperphosphorylated Rb in residues S249/T821 express EMT markers such as decreased expression of E-cadherin and integrin α5 as well as increased expression of N-cadherin, Vimentin, and p-FAK. We propose to focus on the cell adhesion and migration-related kinase Cdk5 with its activator p39 as the kinase responsible for engendering the Rb S249/T821 phosphorylation signature. Our hypothesis is that Rb phosphorylation in S249 and T821 due to CDK5 activity can have prognostic value by being associated with a metastatic phenotype and EMT. Studies performed in a tissue microarray in a population of NSCLC adenocarcinoma that had undergone EMT found an up-regulation in p39 and that its expression is correlated with metastasis. Knockdown of CDK5 induced a decrease in phosphorylation of Rb S249/T821 with a decrease in E-cadherin expression. These data suggest that CDK5-dependent phosphorylation of Rb can affect cell adhesion by regulating E-cadherin. Introducing p39 into Rb hypo-phosphorylated cell line induced an increase in phosphorylation of Rb residues. Furthermore, the CDK5-p39-Rb axis might point to the explanation and prediction of the metastatic phenotype in lung cancer.

#2846

Identify urinary biomarkers for colorectal carcinoma liver metastasis.

Meng Cai,1 Yulin Sun,1 Jiajia Gao,1 Lina Zhao,1 Fang Liu,1 Wei Sun,2 Zhixiang Zhou,1 Xiaohang Zhao1. 1 _National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China; _2 _Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China_.

Colorectal cancer (CRC) is the third leading prevalent cause of death from cancer in adults, and more than one third of CRC patients are accompanied by liver metastasis. The disease begins as a benign adenomatous polyp, and it subsequently develops into an advanced adenoma and gradually progresses to an invasive cancer. The driving factors behind CRC comprise a series of successive accumulated gene mutations that follow the order "APC-KRAS-TP53-DCC". Moreover, urine is an important display window for tumor-derived exosomes. Here, to explore the roles of TP53 in CRC and identify novel urinary biomarkers for colorectal cancer metastatic to the liver, a comprehensive proteomic analysis of exosomal proteins from HCT116 TP53-wild type (WT), TP53-knockout (KO) and constructed TP53 (R273H)-mutant (MT) cells, as well as urine samples from CRC patients with and without liver metastasis was performed using the isobaric tag for relative and absolute quantitation (iTRAQ)-2D-LC-MS/MS strategy. The exosomes from the MT and KO cells exhibited significantly reduced sizes compared with the WT cells. A total of 3437 protein groups with ≥2 matched peptides were identified. Specifically, hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) was consistently down-regulated in the exosomes from the MT and KO cells. Functional studies demonstrated that low HGS levels were responsible for the decreased exosome size. TP53 regulated HGS expression and thus HGS-dependent exosome formation. Furthermore, the HGS expression was gradually increased concomitant with CRC carcinogenesis and was an independent poor prognostic factor. In addition, HGS was one of the differentially expressed urine proteins between CRC liver metastasis and healthy individuals. In conclusion, HGS mediates TP53-regulated exosome formation. HGS may serve as a novel histological and urinary prognostic biomarker and a candidate target for therapeutic interventions in CRC.

#2847

**Loss of heterozygosity (LOH) within the hypoxia inducible factor 3A (** HIF3A **) locus in liver metastasis (LM) from primary colorectal cancer (CRC).**

Takahito Kitajima, Minoru Koi, Alexander Worix, John M. Carethers. _University of Michigan, Ann Arbor, MI_.

Background and Aims: HIF3α is a hypoxia-regulated transcription factor that represses the effects of HIF1α and HIF2α under hypoxia at transcriptional and posttranslational levels. We previously found that LOH at the HIF3A locus is frequent in LM (59%) and the expression of HIF3A protein is reduced in LM compared to primary CRC. At present, the smallest overlapping LOH (SOL-LOH) region has been mapped to the intron 8 of the HIF3A, spanning from 46,312,911 to 46,315,651 on chromosome 19 in LMs. This genomic region contains multiple FOXA1 binding sites embedded in the two DNase I hypersensitive sites (UCSC Genome Browser, https://genome.UCSC.edu). About 30% of the human population carries a deletion within our SOL-LOH region (1000 Genomes Consortium: esv3644527, Chr19: 46,314,109-46,315,478). In this study, we determined whether esv3644527 deletion affects on the expressions of HIF3A, HIF2A, HIF1A and their target genes under normoxic and hypoxic conditions.

Methods: For LOH mapping, 75 resected LMs derived from primary CRC were analyzed for LOH using 8 polymorphic markers. We obtained three EBV-transformed B cell lines with heterozygous esv3644527 deletion and the one with the homozygous deletion from the Coriell Institute. The expression levels of 23 genes related to HIF pathways were determined by qRT-PCR.

Results and Discussion: The expression levels of HIF3A mRNA were too low to compare among these B cell lines by qRT-PCR. However, the expression levels of HIF2A and its target gene, angiopoietin 2 (ANGPT2), are significantly elevated in B cells with a homozygous deletion compared to B cells with a heterozygous deletion under both normoxic and hypoxic conditions. These observations are compatible with the results obtained from a previous study where pulmonary endothelial cells from HIF3A-knockout mice expressed elevated levels of HIF2A and ANGPT2 (Kobayashi-S et al., Genes Cells, 2015, 20:224-241), suggesting that deletion of the FOXA1 sites present in this region may regulate the HIF3A expression.

Conclusions: Our results show that about 60% of LMs exhibit LOH in the 2.7 Kb region surrounding intron 8 of the HIF3A locus. Deletion within this region may lead to upregulation of HIF2A and ANGPT2 by negatively regulating HIF3A expression. To prove our hypothesis, colon cancer cell lines with deleted FOXA1 sites will be assessed.

#2848

Identifying and targeting competing endogenous RNA (ceRNAs) networks to inhibit lung metastasis in triple negative breast cancer.

Pelin Ersan,1 Unal Tokat,1 Erol Eyupoglu,1 Umar Raza,1 Yasser Riazalhosseini,2 Can Alkan,1 Denis Thieffry,3 Daniel Gautheret,4 Ozgur Sahin1. 1 _Bilkent University, Ankara, Turkey;_ 2 _McGill University, Montreal, Quebec, Canada;_ 3 _Ecole Normale Supérieure, Paris, France;_ 4 _Université Paris-Sud, Paris, France_.

Introduction: Triple negative breast cancer (TNBC), the most aggressive breast cancer subtype, has high incidence rate of lung metastasis. Not only protein coding transcripts, but also non-coding transcriptome, such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), have active roles in cancer progression and metastasis. Additionally, lncRNAs can act as sponges for miRNAs. Here, we aimed i) to construct the first mRNA-miRNA-lncRNA competing endogenous RNA (ceRNA) network controlling metastasis in TNBC, and ii) to prevent lung metastasis by targeting identified central candidate genes.

Material/Method: We established primary tumor and human-in-mouse (HIM) and mouse-in-mouse (MIM) lung metastasis models using TNBC cell lines in nude and Balb/c mice, respectively. We visualized both primary and metastatic tumors using in vivo imaging system, harvested tumors and performed both RNA and small RNA sequencing. We obtained differentially expressed miRNAs, mRNAs and lncRNAs between primary and metastatic tumors. Using several bioinformatics tools, we did enrichment analyses, miRNA target predictions, and network construction. Identified central lncRNAs were overexpressed or knocked down and will be tested in in vitro and vivo metastasis-related assays.

Results and Conclusions: We obtained 45 and 91 miRNAs which were differentially expressed between primary and metastatic tumors in HIM and MIM models, respectively. A miRNA family with an established role in metastasis as well as several other miRNAs was identified as highly differentially expressed in the same direction in both models. Moreover, 1127 and 3350 mRNAs, and 85 and 111 lncRNAs were differentially expressed in HIM and MIM models, respectively. Metastasis-related processes based on differentially expressed mRNAs were enriched in the data. We then integrated these three layers of data, functional enrichments, pathway maps and target predictions to construct the first ceRNA network controlling lung metastasis in TNBCs. Currently, we are testing the functional roles of candidate lncRNAs in in vitro and in vivo metastasis assays. Ultimately, our study will uncover novel lncRNAs that can be used as potential targets and/or biomarkers in breast-to-lung metastasis.

Funding: This study is supported by TUBITAK-CNRS Bilateral Grant with project number 214S364.

#2849

Identification of active enhancers as potential biomarkers of aggressive colorectal cancer.

Zhiquan Wang. _Mayo Clinic, Rochester, MN_.

Colorectal cancer is the third most common cancer and second leading cause of cancer-related deaths in the United States. Tumor metastasis is the primary cause of mortality. Even with aggressive surgical and therapeutic management approaches, more than one third of patients will develop metastatic disease. Understanding the molecular changes that drive tumor cells to the metastatic state is key to developing targeted therapies and identifying early-stage markers for progression. To identify changes that promote cell invasiveness, a hallmark of tumor progression, we exposed the pre-metastatic SW-480 colorectal cancer cell line to multiple rounds of selection for the capacity to invade through a synthetic extra-cellular matrix. Following repeated selection, invasiveness increased by more than 10-fold. Epithelial markers, such as E-cadherin, were repressed and markers commonly associated with tumor cells that have undergone epithelial-mesenchymal transition, such as N-cadherin and Vimentin, were upregulated. Likewise, invasive cells showed increased affinity for ECM components. Paired ChIP-seq and RNA-seq revealed likely roles for epigenetic alterations in driving gene expression changes. Furthermore, the epigenetic changes followed ordered patterns of activation and inactivation, suggesting that these epigenetic signatures may provide insight into early mechanisms associated with epithelial-mesenchymal transition. Collectively, our data suggest that early epigenetic markers may be better early predictors of colorectal cancer aggressiveness than gene expression patterns and support the use of this in vitro metastasis model to study the molecular mechanisms of cancer progression.

#2850

A systems genetics approach reveals Rnaseh2c-immune response axis that affects metastasis severity in breast cancer.

Sarah Deasy, Kent Hunter. _NIH, Bethesda, MD_.

Metastatic breast cancer is a devastating disease with a 5-year survival rate of only 26%. This is due to a lack of effective therapies against established metastases and an inability to identify high risk patients who would benefit from specific adjuvant therapies to prevent metastatic progression. We have shown in mouse models that spontaneously arising tumors metastasize with different severity based on the mouse genetic background. Using systems genetics approaches we have identified genes correlated with metastasis and survival in both mice and humans. Rnaseh2c was identified as a novel candidate metastasis susceptibility gene. This gene encodes a scaffolding subunit of the Ribonuclease H2 enzyme which removes ribonucleotides misincorporated into the DNA. Experimentally modulating Rnaseh2c expression in a murine mammary cancer cell line resulted in significant changes in pulmonary metastasis, confirming this gene as a metastasis modifier. Mutations in Rnaseh2c are known to cause Aicardi-Goutieres Syndrome, a neurological autoinflammatory disorder that overlaps clinically with congenital viral infections and the autoimmune disease Systemic Lupus Erythematosus. Given this, we hypothesized that altered expression of Rnaseh2c in breast cancer cells affects metastasis by engaging the immune system. To investigate immune system involvement, we analyzed metastasis in immunocompromised mice. T cell deficiency ablated the effect of reduced Rnaseh2c expression on metastasis, revealing for the first time an Rnaseh2c-immune response axis in metastasis. Gene ontology pathway analysis following mRNA-sequencing of Rnaseh2c knockdown and overexpression tumors revealed that 20% of the genes with altered expression are involved in immune system-related pathways, including T cell signaling and antigen presentation. Furthermore, genes with significant changes included Type I interferons, T cell markers, and immune regulators. These results confirm that Rnaseh2c is a novel metastasis modifier gene and validate our hypothesis that the immune system is mediating the effect of Rnaseh2c on metastasis. This mechanism highlights a potential new target for combination with immune modulatory therapies to combat this devastating disease and adds to a panel of genes we identified that together could determine patients with high risk for metastasis. 

### Imaging Cancer Metabolism, Therapeutic Targets, and Treatment Response

#2851

**Cellular glutamine pool size change in response to glutaminase inhibition detected by kinetic analysis of ** 18 **F4-fluoroglutamine PET.**

Austin R. Pantel, Hsiaoju Lee, Shihong Li, Robert K. Doot, Robert H. Mach, David A. Mankoff, Rong Zhou. _University of Pennsylvania, Philadelphia, PA_.

INTRODUCTION

Oncogene-dependent reliance on glutamine is a cancer vulnerability that can be exploited for therapeutic gain. Inhibitors of glutaminase, the enzyme that catalyzes the conversion of glutamine to glutamate, have been developed and have shown antitumor effects in several tumor models. The specific and potent glutaminase inhibitor CB-839 demonstrated marked antitumor efficacy in a triple-negative breast cancer (TNBC) cell line with inherently high glutaminase activity (HCC-1806), but not in an estrogen receptor-positive (ER+) cell line with inherently low glutaminase activity. An early phase 1 clinical trial of CB-839 in combination with paclitaxel has shown encouraging results in TNBC patients. Cell-line specific efficacy of glutaminase inhibitors supports the need for clinical biomarkers that can predict and evaluate therapeutic efficacy.

As a minimally metabolized glutamine analog with similar transport properties, 18F4-Fluoroglutamine ([18F]4F-Gln) is an ideal radiotracer to infer glutamine pool size through estimates of tracer distribution volume (DV). In this study, we demonstrate differences in DV of [18F]4F-Gln in two xenografts with different levels of glutaminolysis (TNBC vs. ER+) using dynamic PET imaging, as well as show the effect of anti-glutaminase therapy on [18F]4F-Gln DV.

METHODS

TNBC (HCC-1806) and ER+ (MCF-7) xenografts were established in athymic nu/nu mice. Mice were scanned in a dedicated animal PET scanner at baseline and after CB-839 administration. Dynamic imaging was obtained for one hour upon injection of [18F]4F-Gln (300-350 μCi) via the tail vein. Kinetic analysis was performed with PMOD.

RESULTS

[18F]4F-Gln uptake was largely reversible with Logan plots demonstrating late linearity and k3 in a two-compartment (trapping) model was low (less than 0.01/min in most cases). Strong correlation was seen in DV estimates by Logan plot and a single-compartmental model (R2>0.9), but not with estimates from a two-compartment model. MCF-7 xenografts demonstrated greater than 60% larger DV at baseline than TNBC xenografts indicative of increased cellular glutamine concentrations in MCF-7 xenografts. An increase in DV was detected in TNBC xenografts post-glutaminase inhibition (>30% mean change), but not in MCF-7 xenografts.

CONCLUSION

Estimates of [18F]4F-Gln DV offer the ability to non-invasively infer tumor glutaminolysis. Low [18F]4F-Gln uptake in highly glutaminolytic tumors and an increase in [18F]4F-Gln uptake with glutaminase inhibition is concordant with changes in cellular glutamine concentration as measured by MR spectroscopy of tumor extracts. Our studies indicate promise for the use of [18F]4F-Gln as a predictive and pharmacodynamic marker for glutaminase-targeted therapy.

Supported by: Komen SAC130060, DE-SE0012476, R21-CA198563

We thank Calithera Inc. for providing CB-839 and Dr. Susan Demo for discussions.

#2852

**Monitoring the impact on metabolic flux** in vivo **of a newly developed LDH inhibitor using hyperpolarized 13C magnetic resonance spectroscopic imaging.**

Nobu Oshima,1 Shun Kishimoto,1 Kristin Beebe,1 Keita Saito,1 Kazutoshi Yamamoto,1 Jeffery Brender,1 Anastasia Sowers,1 Ganesha Rai,2 Bryan T. Mott,2 David J. Maloney,2 James B. Mitchell,1 Murali K. Cherukuri,1 Leonard M. Neckers1. 1 _National Cancer Institute, NIH, Bethesda, MD;_ 2 _National Center for Advancing Translational Sciences, NIH, Rockville, MD_.

[aim] Increased lactate production is a feature of many neoplasms, and Lactate Dehydrogenase A (LDH-A) plays a key role in conversion of pyruvate to lactate. LDHA inhibition, therefore, is considered to be a promising approach toward developing a new therapeutic strategy for cancer treatment focused on targeting cancer metabolism. Non-invasive imaging approaches able to monitor metabolic fluxes in vivo will be useful for this purpose.

Hyperpolarized 13C Magnetic Resonance Imaging (MRI) has been well known as a valuable technology to investigate metabolic processes in tumor xenografts, allowing us to perform dynamic 13C-metabolic flux analysis in vivo. Use of [1-13C]pyruvate with this technology provides the ability to monitor LDHA activity in real time through dynamic observation of conversion of [1-13C]pyruvate to [1-13C]lactate.

This study aimed to monitor drug efficacy of a newly developed LDH inhibitor (LDHI, obtained from National Cancer Institute Experimental Therapeutics Program, NExT) in a xenograft tumor model using 13C MRI technology with hyperpolarized 13C-labeled pyruvate.

[Results] Hyperpolarized [1-13C]pyruvate MR studies were performed before and after LDHI administration to assess the impact on metabolic flux in vivo. Using hyperpolarized [1-13C]pyruvate MR Spectroscopy (MRS), we found that lactate production was significantly suppressed by LDHI administration in MiaPaca (a glycolytic pancreatic cancer cell line) tumors, as was the [1-13C]lactate to [1-13C]pyruvate ratio ([1-13C]-Lac/Pyr), which was calculated from the areas under the curves (AUC) using time-intensity data. This ratio decreased from 1.08 to 0.128 (88.1% decrease) 30 minutes after intravenous administration of the LDHI. In addition, hyperpolarized [1-13C]pyruvate MRS revealed that LDHI significantly suppressed lactate production in a dose dependent manner. Furthermore, Chemical Shift Imaging with 13C MRI demonstrated that the [1-13C]lactate signal in each voxel clearly decreased, compared to that before LDHI administration. The sum of [1-13C]lactate signals in the tumor region decreased after LDHI administration, resulting in a significant decrease in the tumor-specific [1-13C] Lac/Pyr ratio (1.463±0.31 before LDHI to 0.134±0.036 30 minutes after LDHI administration, a 90.67±2.56% decrease, n=3, p<0.01).

[Conclusions] These results indicate that hyperpolarized 13C-MRI is a useful method to evaluate on-target efficacy of novel LDH inhibitors in vivo, and this technique can be used to determine optimum dose and exposure time of the LDHI in the tumor region. The current method can be of great value in providing an in vivo pharmacodynamic biomarker for this novel anti-cancer therapeutic targeting deregulated tumor metabolism.

#2853

Imaging tumor metabolism to guide treatment of breast cancer with drugs targeted at PI3K alpha.

Susana Ros, Paula D'Santos, De-en Hu, Ankita S. Batra, Alan J. Wright, Elizabeth Mannion, Alejandra Bruna, Carlos Caldas, Kevin M. Brindle. _CRUK Cambridge Institute, University of Cambridge, Cambridge, United Kingdom_.

PI3KCA (which encodes the phosphoinositide-3 kinase alpha isoform) is the most frequently mutated oncogene in breast cancer. Recently, several alpha isoform-specific PI3K inhibitors have been developed and have entered into early-phase clinical trials. However, intrinsic and acquired resistance limits their utility. Non‐invasive imaging methods could be used to guide treatment by not only allowing selection of the most effective drug in individual patients, but also by detecting intrinsic and acquired resistance.

We have been evaluating the effectiveness of a selective PI3K alpha inhibitor, Taselisib (GDC-0032 Genentech S.A.), by monitoring the rate of [1-13C]lactate production after injection of hyperpolarized [1-13C]pyruvate in cell lines and patient-derived xenografts (PDX) models of breast cancer using 13C magnetic resonance spectroscopic imaging. Hyperpolarization of the 13C nucleus increases its sensitivity to detection in the MR experiment by 104 \- 105x, thus allowing real time imaging of tumor metabolism. Preliminary data suggest that labeled lactate production can distinguish sensitive and resistant breast cancer models to Taselisib a few days after treatment. We are currently broadening the panel of PDXs analyzed and also including genetically engineered mechanistic-models of drug resistance.

The present study highlights the effectiveness of hyperpolarized 13C spectroscopy/spectroscopic imaging for detecting early response or resistance to next generation PI3K inhibitors. This clinically applicable technique can provide insights into in vivo response in real-time and identify new strategies to increase the efficacy of therapy in individual patients.

#2854

The potential of hyperpolarized 13-C magnetic resonance spectroscopy to monitor the effect of combretastatin based vascular disrupting agents.

Ane B. Iversen,1 Morten Busk,1 Lotte B. Bertelsen,1 Christoffer Laustsen,1 Ole L. Munk,1 Thomas Nielsen,1 Thomas R. Wittenborn,1 Johan Bussink,2 Jasper Lok,2 Hans Stodkilde-Jorgensen,1 Michael R. Horsman1. 1 _Aarhus University Hospital, Aarhus, Denmark;_ 2 _Radboud University Medical Center, Nijmegen, Netherlands_.

Purpose: Targeting tumor vasculature using vascular disrupting agents (VDAs) is an attractive therapy. These agents induce rapid physiological changes long before any growth inhibitory effects become apparent. Detecting these early changes should allow us to predict anti-tumor effects. Hyperpolarized ¹³C magnetic resonance spectroscopy (HPMRS) allows dynamic measurements of the metabolism of 13C-labeled substrates and may be a new approach for reliably monitoring early effects of VDAs. The aim of this study was to investigate the potential of HPMRS to achieve this in a pre-clinical model.

Methods: Mice bearing 200 cubic mm foot implanted C3H mammary carcinomas were intraperitoneally injected with combretastatin-A4-phosphate (CA4P) or the A1 analogue OXi4503. Tumor response was assessed by determining necrosis development, measured histologically 24-hours after treatment, and tumor growth time (TGT; time to reach 5 times treatment volume). Estimates of tumor perfusion and metabolism were performed 3h (CA4P) and 6h (OXi4503) after treatment. Perfusion was measured from Hoechst 33342 uptake in histological sections and by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) at 7-Tesla. Metabolic changes were assessed following intravenous injection of hyperpolarized [1-¹³C]pyruvate and observing the conversion to lactate with HPMRS in a 9.4-Tesla scanner. Additional mice were positron emission tomography (PET) scanned using a Mediso nanoScan PET/MRI scanner following administration of fluorodeoxyglucose (FDG). Statistical comparisons were made using a one-way analysis of variance (ANOVA), with significance level of p<0.05.

Results: A dose response relationship was found between injected drug doses and both tumor necrosis induction and TGT, with OXi4503 having a larger effect than CA4P. Two equally effective doses were selected for further analysis (250 mg/kg for CA4P and 50 mg/kg for OXi4503). Both drugs significantly decreased perfusion relative to controls; the respective mean (with 1 SE) values for CA4P and OXi4503 were decreased to 57% (50-64) and 38% (28-48) using Hoechst 33342, and 61% (57-65) and 61% (54-68) using DCE-MRI. FDG uptake (mean tumor-to-brain ratio) also significantly reduced to 43% (33-53) and 33% (25-41) for CA4P and OXi4503, respectively. However, an unaltered pyruvate to lactate ratio was found; the values being 98% (89-107) for CA4P and 113% (102-124) for OXi4503.

Conclusion: No change in the HPMRS values was observed, despite changes in all other parameters, indicating that the metabolic fate of pyruvate remains unaltered despite reduced delivery of oxygen and nutrients. This could be because our data provides measures of whole-tumor mean responses that include a viable metabolically unaltered tumor rim and a tumor core with no blood flow (i.e., no pyruvate delivery) following VDA treatment.

#2855

**Monitoring tumor growth and oxygen distribution of prostate cancer xenografts** in vivo **using ultrasound and photoacoustic imaging.**

Qiuhua Hu,1 Ellca Ratther,1 Katrina Sweeney,1 Pamela J. Russell,1 Colleen C. Nelson,1 Brian W. Tse,2 Elizabeth D. Williams,1 Brett G. Hollier1. 1 _Australia Prostate Cancer Research Centre - Queensland, Chronic Disease & Aging, Cancer Program, Institute of Health & Biomedical Innovation, School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD, Australia; _2 _Preclinical Imaging Facility, Translational Research Institute, Brisbane, QLD, Australia_.

Introduction

Prostate cancer (PCa) is the second leading cause of cancer-related death in men. Initially, PCa growth and progression is primarily driven by androgen stimulation of the androgen receptor (AR). As such, the standard treatments for PCa target the androgen/AR axis and are collectively termed androgen targeted therapies (ATTs). In spite of initial tumor regression, tumor cells ultimately adapt and become resistant to ATTs and progress to castration resistant PCa (CRPC) or metastatic CRPC. The tumor microenvironment (TME) has long been recognized to modulate the response of tumors to therapies, however, the impact of ATTs on the TME and their contribution to the development of resistance has not been investigated. This project aims to evaluate the potential of non-invasive photoacoustic imaging combined with ultrasound imaging to assess the alterations of the TME in response to ATTs in vivo.

Methods

PCa cell lines, PC-3 (androgen-independent) and LNCaP (androgen-sensitive), were subcutaneously implanted into male severe combined immune deficiency mice. When the tumor size reached 200-300 mm3, mice were castrated and subsequently treated with enzalutamide once serum PSA had reached pre-castration levels. Ultrasound and photoacoustic (US-PA) scanning (Vevo LAZR) was performed at intervals during PCa progression to simultaneously assess changes in tumor growth, anatomical structure, vasculature, and intratumoral oxygen saturation.

Results

We provide evidence that US-PA imaging is a feasible approach to monitor TME and the response of subcutaneous prostate tumors to ATTs in vivo. PC-3 tumors (nPC-3=11) showed no response to castration in volume, but slight changes in total hemoglobin and oxygen saturation. Growth of LNCaP tumors and their subsequent responses to castration were variable (nLNCaP=10). We identified a strong correlation between ultrasound and caliper-derived tumor volumes in these models and in the majority of tumors the tumor center was more hypoxic than the peripheral tumor area, which is likely due to low oxygen and nutrient transmission and/or cell apoptosis. However, in a subset of tumors higher oxygen levels were observed in the center compared with regions nearer to the skin surface, a pattern associated with a higher rate of tumor growth compared to the rest of the cohort. Importantly, we were able to detect a relatively increase in oxygen levels between pre- and post-castration tumors in individual mice.

Conclusion

We have successfully used a non-invasive imaging method to simultaneously assess multiple biological parameters in PCa xenografts in vivo. The information gained in this study will provide a broader view of the prostate tumor response to ATTs, which has the potential to reveal novel mechanisms of therapy resistance.

#2856

Measuring tumor metabolism in cancer spheroids and biopsies.

Sui Seng Tee. _Memorial Sloan Kettering Cancer Center, New York, NY_.

Introductory Sentence

Real-time metabolic flux can be measured non-invasively using hyperpolarized magnetic resonance spectroscopy in cancer spheroids as well as tumor biopsies.

Experimental Procedures

LnCAP cells were resuspended in a 1:1 mixture of Matrigel:alginate (1x105 cells/ml) and grown in a porous hollow fiber (A/G Technology Corporation, NY). [1-13C] pyruvate was polarized in a SpinLab (General Electric, NY) before dissolving with appropriate buffers. NMR studies were performed on a 1 Tesla Magritek Spectrometer (Magritek, CA). HP spectra were acquired (10o degree flip angle with repetition time of 4s for 25 scans in total). Cell tracking used the manual tracking function in ImageJ. Xenograft biopsies were obtained from LnCAP tumors using a 2mm punch.

Data Summary

LnCAP cells seeded sparsely enable cancer spheroid formation remain viable as assessed by Live/DEAD fluorescent staining. Histological sectioning revealed spheroids of approximately 200 µm in diameter. Direct deposition of these threads into a 5mm NMR tube allows measurement of metabolic flux using hyperpolarized [1-13C] pyruvate resulting in the formation of [1-13C] lactate. To modulate metabolism, we used an allosteric AKT inhibitor, MK2206. This drug resulted in relocalization of AKT from the plasma membrane as well as a reduction in cell motility from 0.72 ± 0.04 to 0.46 ± 0.03 μm/s (n=5, p<0.05). Administration of hyperpolarized pyruvate resulted in approximately 30% of lactate production 24 hr after treatment. Histological staining of spheroids after treatment revealed sustained decrease of AKT phosphorylation after treatment. These measurements are in good accordance with measurements of lactate secretion in the same cells grown under standard conditions, with MK2206 resulting in loss of AKT Ser473 phosphorylation as early as 2 hr post-treatment, followed by a decrease in lactate secretion in the media from 5.16 ± 0.2 to 1.35 ± 0.05 mM/106 cells (n=3, p>0.05) 24 hr after treatment.

To ensure that these methods are applicable clinical settings, LnCAP xenograft biopsies (~60mg) were deposited directly into an NMR tube to measure hyperpolarized [1-13C] pyruvate metabolism. Line-width of the pyruvate peak was measured to be 1.72 ± 0.19 Hz (n=3) in the first spectra, demonstrating good homogeneity of the magnetic field and conversion to lactate was observed. Future experiments will focus on measuring the metabolism of patient-derived xenografts and effects of treatment response.

Conclusions

Metabolic measurements using hyperpolarized pyruvate can be performed on cancer spheroids as well as biopsies, allowing quantification of metabolism to complement on-going clinical trials utilizing hyperpolarized MR imaging.

#2858

**Immuno-PET with site-specific labeled** 89 **Zr-DFO-trastuzumab improves immuno-reactivity and tumor uptake in a subcutaneous HER2 positive xenograft mouse model of ovarian adenocarcinoma.**

Lotte K. Kristensen,1 Camilla Christensen,2 Camilla S. Knudsen,1 Mette M. Jensen,1 Brian J. Agnew,3 Andreas Kjaer,2 Carsten H. Nielsen1. 1 _Minerva Imaging, Copenhagen, Denmark;_ 2 _Department of Clinical Physiology, Nuclear Medicine & PET and Cluster for Molecular Imaging, Rigshospitalet and University of Copenhagen, Copenhagen, Denmark; _3 _Thermo Fisher Scientific, Eugene, OR_.

Introduction: The combination of PET with the specificity of monoclonal antibodies (mAbs), immuno-PET, is an attractive approach to improve tumor detection and mAb quantification. 89Zirconium (89Zr) is particularly well-matched for the long circulatory half-life of intact mAbs. Previously, the use of 89Zr has been limited by the lack of suitable methods for stable coupling to mAbs. In addition, the conventional labeling strategies with random introduction of a chelator potentially reduce antibody binding and affinity towards target. Here we demonstrate the application of an enzyme- and click chemistry-mediated methodology for site-specifically labeled 89Zr immuno-PET imaging probes and compare them to a random labeled probe.

Experimental procedures: Trastuzumab was conjugated to the p-SCN-Bn-Deferoxamine (p-SCN-Bn-DFO) chelator in three alternate ways: (1) randomly on lysine residues or site-specifically on enzymatically treated glycans using either (2) ß-galactosidase or (3) endoglycosidase S2. DFO-trastuzumab was radiolabeled with 89Zr (89Zr-DFO-trastuzumab) and injected into SK-OV-3 tumor-bearing NMRI nude mice. In addition, a dose-escalation study was performed with co-injection of either 100 µg or 500 µg of unlabeled trastuzumab. At 24, 70 and 110 hours post-injection, mice underwent small animal PET/CT imaging for longitudinal assessment. Mice were euthanized and organs resected for ex vivo biodistribution after the last imaging time-point. Additionally, the immuno-reactivity and tracer stability in buffer and plasma were assessed for all three tracers.

Results: All tracers were found to be stable for up to 5 days post-labeling. PET imaging at 24, 70 and 110 hours after injection of 89Zr-DFO-trastuzumab revealed a gradual increase in tumor uptake and image contrast over the time-course. At 70 hours post-injection mean tumor uptakes were 6.7 ± 1.7, 13.9 ± 3.3 and 15.3 ± 3.8 % injected dose per gram tissue (%ID/g), for random, ß-galactosidase or endoglycosidase S2 labeled probes, respectively. Site-specific labeling significantly increased tumor PET uptake (One-way ANOVA, p < 0.0001) at all time-points compared to random labeling. This was further supported by an immuno-reactivity of 93% for site-specific labeled probes compared to 80% for the random labeled trastuzumab. Titration with unlabeled trastuzumab did not affect tumor uptake considerably and the ex vivo biodistribution confirmed the data obtained by in vivo PET imaging.

Conclusions: 89Zr-DFO-trastuzumab is well suited for specific immuno-PET imaging of HER2 positive ovarian cancer and site-specific labeling of trastuzumab presents with markedly higher immuno-reactivity and tumor specificity. These findings support the further development of site-specific radiolabeled monoclonal antibodies for immuno-PET.

#2859

Sensitive, specific detection of Her-2 positive tumors in mice using superparamagnetic relaxometry (SPMR).

Erika C. Vreeland,1 Kayla E. Minser,1 Caroline L. Weldon,1 Andrew Gomez,1 Todor Karaulanov,1 Helen J. Hathaway,2 William H. Anderson,1 Christopher Nettles,1 Dale L. Huber,3 Giulio Paciotti1. 1 _Imagion Biosystems, Albuquerque, NM;_ 2 _University of New Mexico, Albuquerque, NM;_ 3 _Sandia National Laboratories, Albuquerque, NM_.

Superparamagnetic Relaxometry (SPMR) is a non-invasive technique that utilizes superconducting quantum interference device (SQUID) detectors to localize and quantify the magnetization of superparamagnetic iron oxide (Fe3O4) nanoparticles (NPs) specifically bound to cancerous tumors. In an SPMR measurement, polyethylene glycol (PEG) coated NPs are functionalized with a tumor-targeting monoclonal antibody and injected intravenously. NPs that reach and bind to the target tissue are measured by the MRX™ instrument, while unbound nanoparticles, such as those freely circulating in the bloodstream, are not detected.

Here, we demonstrate the use of SPMR for specific cancer detection using long-circulating anti-Her2 antibody conjugated PrecisionMRX® NPs in vitro and in vivo. The stability and biofunctionality of conjugated nanoparticles were measured by dynamic light scattering, gel electrophoresis, and ELISA. A cell competition assay was developed to measure specific binding of NPs to Her2 positive (BT474) and Her2 negative (MCF7) cells in vitro. Specificity was defined by the ability of the native antibody to competitively block the binding of the anti-Her2 conjugated NPs to the Her-2 antigen expressed on the cell surface. For in vivo studies, nude mice with xenograft BT474 tumors were intravenously injected with anti-Her2 NPs at a dose of 20 mg/kg of body mass, while control mice were injected with PEG only NPs. Mice were measured individually on the MRX™ instrument at successive time points over the course of 24 hours. At selected intervals during the 24-hour period, blood, tumor, and organs were harvested and analyzed for SPMR signals and anti-Her2 content.

In vitro, the anti-Her2 NPs exhibited specific binding to BT474 cells, with little to no binding in MCF7 cells. In vivo, MRX measurements of mice injected with anti-Her2 NPs showed a measurable magnetic signal in the tumor that reached a near maximum approximately four hours after injection. Conversely, mice injected with unconjugated nanoparticles had no measurable tumor uptake. Finally, 24 hours post-injection, 4 – 8% of NPs and anti-Her2 were measurable in the blood, indicating long-term stability of the NP construct in circulation. Together, these results suggest targeted delivery of conjugated NPs to cancerous tissue in vivo and the utility of SPMR for the sensitive and specific detection of cancer in vivo.

This work was performed, in part, at the Center for Integrated Nanotechnologies, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science. Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-AC04-94AL85000.

#2860

89Zr-trastuzumab immunoPET imaging to monitor src status after treatment in HER2 breast cancer.

Brooke N. McKnight,1 Nerissa T. Viola-Villegas2. 1 _Wayne State University, Detroit, MI;_ 2 _Karmanos Cancer Institute, Detroit, MI_.

Around 30% of patients with early stage breast cancer have recurrent disease due to resistance. One of the putative causes of acquired resistance can be attributed to the hyperactivation of Src kinases, which are particularly stabilized by aberrant HER2 signaling, which itself is overexpressed in nearly 30% of early stage breast cancers. HER2 downstream effectors include Src, thus, we aim to investigate the use of a HER2-specific monoclonal antibody positron emission tomography (PET) probe, 89Zr-trastuzumab, to monitor Src therapy via surrogate HER2 expression. We hypothesize that 89Zr-trastuzumab can be a surrogate imaging tool for monitoring Src treatment.

89Zr-trastuzumab was previously developed and has demonstrated excellent specificity for breast cancer. Dasatinib-treated HER2+ BT474 (IC50 ~ 1.3 μM), SK-BR-3 (IC50 ~ 5.5 µM), and JIMT-1 (IC50 ~ 8 μM) breast cancer cells lines have shown a negative correlation between treatment and HER2. We next conducted tumorigenic studies using athymic nu/nu mice bearing JIMT-1 or BT474 xenografts, which were treated for 7 or 14 days with dasatinib (75 mg/kg/day). At the end of each treatment, the tumors were subsequently imaged with 18F-FDG, and then followed by 89Zr-trastuzumab, 24 h later. Images were analyzed using AsiPro (show version here and decay-corrected to the time of injection. Tumor uptake values were measured by drawing volumes-of-interest, expressed as % injected dose per gram (%ID/g) of tissue on regions showing the most binding. 18F-FDG did not demonstrate a predictive response in both treated and placebo tumors, whereas, HER2 PET was able to show differential response in treated vs. control cohorts. In JIMT-1 mice imaged with 89Zr-trastuzumab, a two-fold difference (p < 0.01) was observed between untreated and both treated groups (8.0 ± 1.5 %ID/g, n= 7 vs. 7-day treated: 3.8 ± 1.5 %ID/g, n=4, and 14-day-treated: 4.5 ± 1.3 %ID/g, n= 4) mice imaged with 89Zr-trastuzumab. Confirmatory ex vivo western blots of JIMT-1 tumors have shown a decrease in HER2 as treatment time increases, with a decrease in p-Src (Y-416). In vivo studies with BT-474 xenografts are currently underway. Immunohistochemistry on excised tumors, as well as treatment on a metastatic patient-derived xenograft model is currently in progress.

We have shown that HER2 PET can potentially be a surrogate marker of Src treatment. The findings from this study potentially afford a powerful tool to non-invasively detect and monitor changes in Src-targeted therapy in man at the early phases of treatment.

#2861

Dose-dependent tissue distribution and tumor targeting of Notch3-ADC using fluorescence molecular tomography imaging.

Anand Giddabasappa,1 Parul Gupta,1 Mauricio Leal,2 Jonathan Golas,2 Fengping Li,2 Bing Yang,1 Antonio Esparza,1 Christopher Winkelmann,1 Kenneth Geles3. 1 _Pfizer Inc, San Diego, CA;_ 2 _Pfizer Inc, Pearl River, NY;_ 3 _Pfizer Inc, Pearl River, CA_.

Background: NOTCH3, a cell surface receptor involved in cell-cell communications, is over-expressed or amplified in certain human tumors. NOTCH3 is known to regulate proliferation, differentiation and survival of cancer cells or cancer stem cells and thus an important therapeutic target. NOTCH3 antibody drug conjugate (ADC) is comprised of humanized anti-NOTCH3 antibody conjugated to an auristatin based cytotoxic payload. NOTCH3-ADC has shown promising results in pre-clinical tumor models. In this study we evaluated the kinetics, dose-dependent tissue distribution, tumor accumulation and targeting specificity of the NOTCH3-ADC in OVCAR3 xenograft model using fluorescence molecular tomography (FMT) imaging. The NOTCH3-ADC is mouse cross-reactive thus providing an accurate assessment of biodistribution.

Methods: NOTCH3-ADC was conjugated to the near-infrared dye, AlexaFluor680 (AF680). The in vitro cellular binding was evaluated by cell-based ELISA. In vivo biodistribution was evaluated using OVCAR3 subcutaneous xenograft model. NOTCH3-ADC-AF680 (1mg/kg; 3mg/kg and 10mg/kg) was injected when the tumors were ~300mm3 and imaged 5 min, 24, 48, 96, 168 and 240 h post-injection. Ex vivo FMT imaging, pharmacokinetic analysis and immunohistochemistry (IHC) was performed at 48 and 240 h after whole-body perfusion. An in vivo receptor competition FMT study was performed by injecting excess of unconjugated anti-NOTCH3 antibody (Ab) or a non-targeted control Ab.

Results: In vitro cell binding studies showed that conjugation of AF680 to NOTCH3-ADC did not change its binding ability. A dose-dependent tumor regression was also observed after a single injection of NOTCH3-ADC-AF680. In vivo FMT imaging showed dose-dependent whole-body clearance kinetics of NOTCH3-ADC. Dose-dependent accumulation in the tumors was observed with peak accumulation at 24-48 h post-injection and a slow decline at later time-points. A maximum accumulation of ~10 %ID/g was observed which was independent of the dose of NOTCH3-ADC-AF680. Ex vivo FMT quantitation of tumor was consistent with the IHC for antibody and LC/MS analysis of released payload. Pharmacological competition with excess unlabeled control Ab did not block tumor accumulation of NOTCH3-ADC-AF680, whereas excess unlabeled NOTCH3-Ab blocked ~47% of NOTCH3-ADC-AF680 accumulation. There was no significant specific accumulation of NOTCH3-ADC in other organs as observed by FMT imaging or IHC.

Conclusions: These imaging studies provided understanding of the kinetics, tumor accumulation and biodistribution of NOTCH3-ADC. Further this work showcase the utility of non-invasive FMT imaging in better understanding of pharmacology and behavior of biologic drugs.

#2862

Tumor-targeting salmonella typhimurium a1-r inhibited angiogenesis of osteosarcoma cells visualized by color-coded imaging in the in vivo absorbable gelatin sponge assay.

Tasuku Kiyuna. _University of California, San Diego, CA_.

INTRODUCTION We already have reported a color-coded imaging model that showed the sponge agngiogenesis assay using absorbable gelatin sponge implanted in nestin-driven green fluorescent protein (ND-GFP) nude mice and that osteosarcoma cells promote angiogenesis in this sponge assay. We report here that Salmonella typhimurium A1-R (A1-R) inhibits angiogenesis of osteosarcoma cells in the sponge angiogenesis assay in ND-GFP mice.

METHODS Sponge was initially transplanted subcutaneously in the flank of transgenic ND-GFP nude mice. Seven days after transplantation of sponge, skin flaps were made and 143B-RFP human osteosarcoma cells expressing red fluorescent protein (RFP) were injected into the transplanted sponge. After establishment of tumors in the sponge, the control-group mice were treated with PBS via tail-vein injection and A1-R treated group with A1-R likewise. Skin flaps were made at days 14, 21, and 28 after transplantation of the sponge to allow imaging of vascularization in the sponge using a variable-magnification small animal imaging system and confocal fluorescence microscopy.

RESULTS: Vessel length in the sponge was measured after treatment with FV10-ASW Fluoview software. Nascent blood vessels grew in the sponge in a time-dependent manner. A random 3 fields were quantified in each group). The mean length of ND-GFP expressing blood vessels in the sponge in mice with A1-R treated group were 9.40, 12.40, and 10.12 mm/mm2, at days 14, 21, and 28, respectively. The mean length of ND-GFP expressing blood vessels in the sponge in mice of the control group were 8.86, 14.50, and 15.81 mm/mm2 on day 14, 21, and 28, respectively. ND-GFP expressing blood vessels of the mice in the osteosarcoma cells treated with A1-R had shorter vessels than the control group (on days 28, P < 0.05), suggested the extent of nascent blood vessel growth was significantly inhibited by A1-R treatment. Besides, we evaluated the RFP colored area of the sponge in both groups, which almost represent the viable tumor size. The mean RFP colored area of A1-R treated group in the sponge were 4.49, 7.53 and 8.15 mm2, at days 14, 21, and 28, respectively and that of control group were 3.78, 6.40, and 7.49 mm2 at days 14, 21, and 28, respectively. The size of possibly viable RFP colored tumor area between A1-R treated group and control group at days 28 did not show significant area difference, indicated A1-R treated osteosarcoma cells showed shorter nascent vessel length than control group, not due to the tumor size difference but due to the difference of angiogenesis inhibitory effect. As a result of both outcomes above, the extent of nascent blood vessel growth was significantly inhibited by A1-R treatment on days 28.

CONCLUSION: In the present study, we reported the effect of angiogenesis inhibition of A1-R in vivo sponge assay, and suggest A1-R has potential for anti-angiogenic target therapy for osteosarcoma.

#2863

Magnetic resonance imaging based analysis of tumor growth and vascular parameters in animal model of GBM following IV formulated of HET0016 treatments.

Ali S. Arbab,1 Meenu Jain,1 Bhagelu Achyut,1 Kartik Angara,1 Mohammad H. Rashid,1 Asm Iskander,1 Thaiz F. Borin,1 Wenbo Zhi,1 Roxan Ara,1 Irina Lebedyeva,1 Hassan Bagher-Ebadian2. 1 _Augusta University, Augusta, GA;_ 2 _Henry Ford Health System, Detroit, MI_.

Glioblastoma (GBM) is a hypervascular primary brain tumor with poor prognosis. HET0016 a selective CYP450 inhibitor has been shown to inhibit angiogenesis and tumor growth. Therefore, to explore novel treatments, we have generated an improved intravenous (IV) formulation of HET0016 with HPßCD and tested in animal models of human and syngeneic GBM. Tumor growth and vascular parameters (tumor blood volume, permeability and, extravascular and extracellular space volume) were determined by in vivo dynamic contrast enhanced (DCE) magnetic resonance imaging (MRI). The pharmacokinetics of HPßCD-HET0016 were evaluated in plasma and tumor tissues using IV and IP routes of administration. IV treatment with HPßCD-HET0016 decreased tumor growth, tumor blood volume, permeability and, extravascular and extracellular space volume, when compared with the vehicle group (p<0.05). Similar growth inhibition was also observed in syngeneic GL261 GBM (p<0.05). Survival studies using patient derived xenografts of GBM (PDX), showed prolonged survival to 25-27 weeks in animals treated combinedly with focal radiation, IV HET0016 and TMZ (p<0.05). Administration of a single dose resulted in 7-fold higher levels of HET0016 in the IV group in plasma and 3.6-fold higher levels in tumor tissue at 60 min compared to that of IP route. We observed reduced expression of markers of cell proliferation (Ki-67), decreased neovascularization (laminin and αSMA) and migration (MHC-1) in the treated group (p<0.05). Decreased tumor growth was associated with reduced expression of pro-angiogenic markers (IL-8, MCP-1, VEGF, HIF-1α, and VE-Cadherin), inflammation (p-NFκB), intermediates of MAPK pathway (p-AKT, p-ERK and p-STAT1, EGFR), arachidonic acid metabolism (COX-1, CYP4A11) and increased expression of anti-angiogenic markers (Ang2, Angiostatin and Tie-2). Our results indicate that the improved IV formulation of HPßCD-HET0016 is effective in inhibiting tumor growth through decreasing proliferation, migration, and neovascularization. Furthermore, HET0016 significantly enhanced sensitivity of TMZ and prolonged survival in PDX GBM.

#2864

**Tumor targeting and imaging using** 64 **Cu labeled cyclic RGD conjugated human serum albumin via click chemistry.**

Cho Rong Park, Myung Geun Song, Ji-Yong Park, Hyewon Youn, June-Key Chung, Jae Min Jeong, Yun-Sang Lee, Keon Wook Kang. _Seoul National University, Seoul, Republic of Korea_.

PURPOSE: The RGD specifically recognizes the integrin αvβ3 which is overexpressed on various malignant tumors. One major drawback of small peptide such as RGD, however, is short half-life in the blood, which greatly compromises their targeting efficacy. To improve blood circulation time and targeting efficacy, we developed cyclic RGDyK (cRGDyK) conjugated human serum albumin (HSA) using click chemistry reaction.

METHODS: HSA was conjugated with DBCO-NHS (dibenzyl cyclooctyne) under physiologically favorable reaction condition for the preparation of strain promoted azide-alkyne reaction. Using this reaction group, DBCO-HSA was conjugated with N3-c RGDyK (azido cRGDyK) for integrin αvβ3 targeting, N3-FNR648 for fluorescence labeling and 64Cu labeled N3-NOTA (3-azidopropyl-NOTA) for radiolabeling. Cell binding of cRGDyK-HSA were analyzed with FNR648 labeled probes. Cellular uptake of 64Cu-cRGDyK-HSA were tested for integrin αvβ3 specific binding at cell level. PET images were acquired after tail vein injection of 64Cu-HSA, 64Cu-cRGDyK-HSA in integrin αvβ3 positive tumor (SK-OV3, ovarian cancer cell line) bearing BALB/c nude mice. PET signals were quantitatively analyzed with PET image analysis program, AMIDE.

RESULTS: The number of cRGDyK on DBCO-HSA conjugates was confirmed using

MALDI-TOF MS. cRGDyK-HSA was successfully conjugated with 64Cu labeled N3-NOTA. Integrin αvβ3 mRNA and protein was highly expressed in SK-OV3 cell line. At fluorescence labeled probes were treated in SK-OV3, cRGDyK-HSA were highly bound to cell membrane and this pattern were decreased with pre-treatment of excess cRGDyK. When compared to cellular uptake level, 64Cu-cRGDyK-HSA were more accumulated at integrin αvβ3 positive cells (SK-OV3) than integrin αvβ3 negative cells (P < 0.05) and there were no difference in 64Cu-HSA. Serial PET images were acquired 0, 4, 24, 48 hours after tail vein injection of 64Cu-cRGDyK-HSA. Radioactivity of 64Cu-cRGDyK-HSA in SK-OV3 tumor was higher than that of 64Cu-HSA. 64Cu-labeled-cRGDyK-HSA can be observed at 48 hours after injection, which shows longer circulation time in mice.

CONCLUSION: We successfully conjugated cyclic RGDyK to HSA using click chemistry approach. We demonstrated that cRGDyK-HSA specifically bind to integrin αvβ3 in in vitro and in vivo model. And in animal model, 64Cu-labeled-cRGDyK-HSA can be observed at 48 hours after injection, which shows longer circulation time. Our results indicated that cRGDyK-HSA have a potential to diagnosis and therapy response monitoring of tumor expressing integrin αvβ3.

#2865

The peptide transporter K16ApoE increases drug delivery across the blood brain barrier in an experimental animal model of melanoma brain metastases.

Synnøve Nymark Aasen,1 Heidi Espedal,2 Olivier Keunen,3 Christopher Florian Holte,2 Habib Baghirov,4 Rolf Bjerkvig,2 Tine Veronica Karlsen,2 Olav Tenstad,2 Dag Erlend Olberg,5 Gobinda Sarkar,6 Robert B Jenkins,6 Frits Thorsen2. 1 _Haukeland University Hospital, Bergen, Norway;_ 2 _University of Bergen, Bergen, Norway;_ 3 _Luxembourg Institute of Health, Luxembourg, Luxembourg;_ 4 _Norwegian University of Science and Technology, Trondheim, Norway;_ 5 _Oslo University Hospital, Oslo, Norway;_ 6 _Mayo Clinic, Rochester, MN_.

Introduction: Patients with brain metastases await a dismal prognosis. Regardless of the continuous progress in drug development, a major problem is the delivery of drugs across the blood brain barrier (BBB) and into the metastatic neoplasms. The BBB excludes almost all compounds, in particular highly charged, hydrophilic or large compounds, and most of the current chemotherapeutic agents are thus unable to penetrate the BBB. Varying strategies to transiently open the BBB have been studied previously. Here, we describe a peptide transporter comprising 16 lysine residues and 20 amino acid residues corresponding to the low density lipoprotein receptor (LDLR) binding domain of apolipoprotein E (ApoE). We show that the peptide (K16ApoE) is able to transiently open the BBB for drug-delivery into experimental brain metastases.

Experimental procedures: A systemic study of the ability of the peptide to open the BBB was conducted by dynamic contrast enhanced magnetic resonance imaging (DCE MRI) in nonobese diabetic/severe combined (nod/scid) mice. The BBB permeability was studied after administering 200 μg of the peptide intravenously. Further, cellular effects after treatment with the peptide was investigated in vitro using confocal microscopy, flow cytometry and impedance experiments. The biodistribution of the peptide was studied in blood plasma and several organs using 125I labeled K16ApoE. Finally, a treatment study was initiated, treating the animals with the peptide in combination with the B-RAF inhibitor Dabrafenib, only Dabrafenib or vehicle.

Summary: After injecting the K16ApoE peptide into the mice, a transient opening of the BBB for up to 4 hours was clearly demonstrated by DCE-MRI. Microscopy showed that the peptide disrupted brain endothelial cell monolayers, reducing the barrier properties of the cells. The impedance experiments displayed that the permeability through endothelial cell barriers was increased after treatment with K16ApoE, and a dose-dependent cell death pattern was observed at higher concentrations of K16ApoE.The peptide did not affect endothelial cell tight junctions. The biodistribution study showed that the peptide was eliminated from blood plasma in less than five minutes through the kidneys. The treatment study displayed that the group of animals receiving K16ApoE followed by Dabrafenib had smaller tumor volumes than the other two animal groups.

Conclusions: We have shown that the peptide opens the BBB and facilitates a therapeutic window of 4 hours. The peptide did in combination with Dabrafenib decrease the number of experimental brain metastases in our studies. Thus, the current strategy could also have the potential to improve the treatment of patients with brain metastatic disease.

#2866

Volumetric optoacoustic imaging of tumor cell death using a targeted imaging agent.

Bangwen Xie,1 Michal Tomaszewski,1 André A. Neves,1 Stefanie R. Mullins,2 David Tice,3 Richard Sainson,2 Sarah Bohndiek,1 Robert W. Wilkinson,2 Kevin M. Brindle1. 1 _University of Cambridge, Cambridge, United Kingdom;_ 2 _MedImmune Limited, Cambridge, United Kingdom;_ 3 _MedImmune LLC, Gaithersburg, MD_.

Multispectral optoacoustic tomography (MSOT) generates high-resolution cross-sectional images in less than a second. MEDI3039, is a newly described agonist of the TNF-related apoptosis-inducing ligand receptor2 (TRAILR2), which has been shown in preclinical studies to preferentially induce cell death in cancer versus normal cells1. We show here that MSOT, when used with a near infra-red (NIR) fluorophore-labelled protein domain of Synaptotagmin-I (C2Am-750, ~15kDa) that binds to phosphatidylserine (PS) exposed by apoptotic and necrotic cells2, can be used to image MEDI3039-induced cell death within the entire tumor region. Non-specific probe retention was assessed using a site-directed mutant (iC2Am-680 or iC2Am-750).

PS-specific binding of C2Am-750 to MEDI3039-treated TRAIL-sensitive Colo205 and TRAIL-resistant HT-29 human colon adenocarcinoma cells in vitro was evaluated by flow cytometry and confocal microscopy. The capability of C2Am-750 to detect cell death in vivo was assessed in mice bearing Colo205 or HT-29 xenografts, following a single dose (0.4 mg/kg, i.v.) of MEDI3039. All mice then received an i.v. injection of a 1:1 mixture of 0.1 µmole/kg C2Am-750 and iC2Am-680 16 h post treatment, followed by fluorescence imaging (FLI) measurements using an IVIS 200 camera at 0 and 3 h post probe injection. FLI measurements in treated Colo205 xenografts showed significantly increased retention of C2Am-750 uptake versus size-matched untreated tumors (6.0-fold, P value <0.0001), but there was no significant difference between treated and untreated HT-29 xenografts. iC2Am-680 retention was negligible in all groups. Next, volumetric MSOT measurements of C2Am-750 and iC2Am-750 (0.2 µmole/kg) retention in MEDI3039-treated Colo205 tumors were made using an iTheraMedical inVision 256 system at different wavelengths (660-900nm) and time points (3-24 h). C2Am-750 signal was markedly increased in tumors at 3 h post probe injection. Maximal C2Am-750 signal appeared in the center of treated tumors, whereas there was negligible iC2Am-750 retention. Subsequent histological analyses showed that C2Am-750 signal was strongly correlated with both cleaved caspase-3 and TUNEL staining of dead cells.

We have shown that real-time volumetric MSOT measurements with a NIR fluorophore-labelled C2Am, can be used to detect early tumor cell death within the entire tumor region following TRAILR2 agonist treatment. This study shows that this imaging technique can be used to characterize tumor heterogeneity and to determine the most appropriate therapy at an early stage post drug administration.

1.

Swers, J.S. et al. Multivalent scaffold proteins as superagonists of TRAIL receptor 2-induced apoptosis. Molecular cancer therapeutics 12, 1235-1244 (2013).

2.

Alam, I.S. et al. Comparison of the C2A domain of synaptotagmin-I and annexin-V as probes for detecting cell death. Bioconjugate chemistry 21, 884-891 (2010).

#2867

FDG-PET imaging as a potential biomarker of mitochondrial targeting by CPI-613, a novel inhibitor of mitochondrial metabolism.

Kiran Solingapuram Sai,1 Zuzana Zachar,2 Frankis Almaguel,1 Shawn D. Stuart,2 Michael S. Dahan,2 Moises Guardado,2 Stephanie Rideout,1 Minghui Wang,1 Anirudh Sattiraju,1 Paul M. Bingham,2 Boris Pasche,1 Akiva Mintz1. 1 _Wake Forest University School of Medicine, Winston Salem, NC;_ 2 _Stony Brook University, Stony Brook, NY_.

CPI-613 is a lipoate analogue that has been shown to inhibit the pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase (KGDH) complexes selectively in tumor cells (reviewed in Exp.Rev.Clin.Pharma. 7, 837). These two enzymes control the vast majority of carbon flow into the tricarboxylic acid (TCA) cycle and play a central role in mitochondrial metabolism. PDH converts pyruvate into acetyl-CoA, which in turn can enter the TCA cycle for cellular respiration. Since pyruvate is the final product of glycolysis, PDH serves to link glycolysis to the TCA cycle. Early clinical trials with CPI-613 have demonstrated very promising clinical responses in pancreatic cancer, leukemia, and lymphoma when used in combination with standard chemotherapy. Moreover, apparently homeostatic responses of tumor cells to CPI-613 inhibition of mitochondrial metabolism include compensatory upregulation of glucose uptake. Thus, we hypothesized that we can use 18F-Fludeoxyglucose (FDG)-PET/CT, a translational imaging approach that measures cellular glucose uptake, as a biomarker for CPI-613 cellular delivery and its targeting of mitochondrial metabolism. Therefore, in this work we evaluated the in vitro and in vivo glucose uptake in cancer cells and tumor xenografts after treatment with CPI-613. To measure glucose uptake in vitro we pulsed BxPc3 pancreatic cancer cells with 3H 2-deoxyglucose for 15 or 60 minutes following 2 hours of treatment with CPI-613. We observed a significant upregulation (~100% increase) of cellular 2-deoxyglucose uptake, consistent with a compensatory increase in glucose uptake as a result of successful targeting of mitochondrial metabolism. We therefore examined whether this upregulation occurs in vivo using FDG-PET/CT. Mice bearing BxPc3 flank tumors were treated with 50mg/kg of CPI-613 and underwent FDG-PET/CT scans 4 hours and 24 hours post-CPI-613 treatment. Similar to the in vitro response, tumors treated with CPI-613 exhibited a 75% increase in 18F-FDG uptake compared to untreated controls at 4 hours post therapy. In the 24 hour post-therapy scans, tumor 18F-FDG was significantly decreased, indicating tumor cell killing, which corresponds to previously published data that demonstrated efficacy of CPI-613 against BxPc3 tumor xenografts. These results indicate the potential for using the initial FDG flare seen on PET imaging as a biomarker to detect mitochondrial targeting by CPI-613 immediately after CPI-613 treatment, a strategy that may be used, after further clinical validation, to stratify responders to this novel mitochondrial inhibitor.

#2868

18 **F-FDG PET/CT-based early treatment response evaluation of nanoparticle-assisted photothermal therapy.**

Jesper Tranekjaer Joergensen,1 Kamilla Norregaard,1 Marina Simón Martín,1 Fredrik Melander,2 Lotte K. Kristensen,1 Pól Martin Bendix,3 Thomas L. Andresen,2 Lene B. Oddershede,3 Andreas Kjaer1. 1 _University of Copenhagen and Rigshospitalet, Copenhagen, Denmark;_ 2 _Technical University of Denmark, Copenhagen, Denmark;_ 3 _University of Copenhagen, Copenhagen, Denmark_.

Aim: Nanoparticle-assisted photothermal therapy is a new promising therapeutic strategy that utilizes photo-absorbing nanoparticles irradiated with near infrared light to generate a local temperature increase in tumor tissue. Here we used small animal 18F-FDG PET/CT to evaluate the treatment response of silica-gold nanoshell (NS)-assisted photothermal therapy in human tumor xenografts in mice.

Method: NMRI nude mice had ~ 106 human neuroendocrine tumor cells (H727) inoculated into the left flank. After 4-5 weeks, animals were divided into three groups: one receiving NS and laser irradiation (NS group, n = 9), one receiving saline and laser irradiation (saline group, n = 9), and one receiving NS but no laser irradiation (sham group, n = 5). All animals were PET scanned with 18F-FDG one day before treatment (baseline), followed by iv administration of either NS or saline. Approximately 24 hours after injection, the animals were placed on a laser treatment platform and the tumors were irradiated with an 807 nm diode laser for 5 minutes using a laser intensity of 1.8 W/cm2. During irradiation, the temperature at the surface of the tumor was measured using thermographic imaging. The mice were 18F-FDG PET scanned again at day 1 after treatment and tumor growth was followed by caliper measurements with the humane endpoint defined as a tumor volume of 1,000 mm3. PET and CT images were co-registered, and regions of interest were manually drawn on whole tumor regions. 18F-FDG uptake was quantified as mean percentage of injected dose per grams of tissue (%ID/g) and the treatment response was evaluated based on the reduction in tumor uptake of 18F-FDG between baseline and day 1.

Results: Thermographic imaging showed that the tumor surface on average reached 49.2 ± 3.3 °C in the NS group. In comparison, the average temperatures reached 44.5 ± 1.9 °C in the saline group and 33 ± 1.0 °C in the sham group. This was consistent with an overall inhibited tumor growth, as well as improved survival in the NS group compared to the sham and saline groups. The tumor accumulation of 18F-FDG uptake was comparable between groups at baseline but PET imaging revealed a significant reduction in tumor uptake at day 1 in the NS group ( 84 ± 8 % (day 1/baseline)) compared to the saline (= 108 ± 21 %; p < 0.01) and sham (= 110 ± 11 %; p < 0.05) groups. Moreover, the change in 18F-FDG tumor uptake (day 1/baseline), was used to stratify animals into responders and non-responders, where the responding group matched inhibited tumor growth and improved survival.

Conclusion: In this study we showed that 18F-FDG PET could be used for early response monitoring of the therapeutic outcome of nanoparticle-assisted photothermal therapy in human tumor xenografts in mice. Based on this, we suggest that PET can also be used for optimization of therapy, both for guiding treatment planning and early identification of non-responders for which the treatment strategy should then be changed.

#2869

**Comparison of** 18 **F-FDG and** 18 **F-FLT PET imaging for early response monitoring of nanoparticle-assisted photothermal cancer therapy.**

Kamilla Norregaard,1 Jesper Tranekjaer Joergensen,1 Marina Simón Martín,1 Lene B. Oddershede,2 Andreas Kjaer1. 1 _Rigshospitalet and University of Copenhagen, København N, Denmark;_ 2 _University of Copenhagen, København N, Denmark_.

Aim: Nanoparticle-assisted photothermal therapy relies on photo-absorbing agents that accumulate in tumors and upon irradiation with near-infrared light, generate hyperthermal temperatures sufficient to ablate surrounding cancerous tissue. In this study, we used small animal PET imaging to compare the tracers 18F-FDG and 18F-FLT, that are glucose and thymidine analogues respectively, for the assessment of silica-gold nanoshell (NS)-assisted photothermal therapy in a colorectal cancer mouse model.

Method: Mouse colorectal tumors (CT26) were established subcutaneously in female Balb/c mice by inoculation of ~ 3x105 cells. When the tumors reached a volume of ~150 mm3, the animals were divided into treatment groups receiving either: NS and laser irradiation (NS group), saline and laser irradiation (saline group), or NS but no laser irradiation (sham group). 24 h after intravenous injection of either NS or saline, the tumors were irradiated for 5 minutes with an 807 nm diode laser with an intensity of 1.5 W/cm2 (day 0) meanwhile the temperature development was monitored using thermographic imaging. To evaluate the treatment response, all animals were PET/CT scanned with either 18F-FDG or 18F-FLT the day before treatment (baseline) and two days after treatment (day 2). In addition, the change in tumor volume was assessed by CT imaging on day 7. Throughout the study, tumor growth was monitored using caliper measurements and animals were euthanized when the tumor volume exceeded 1,000 mm3. Regions of interest were manually drawn on the entire tumor volume on co-registered PET and CT images and the PET tracer uptake quantified as the mean percentage of injected dose per grams of tissue (%ID/g).

Results: During laser irradiation, NS-laden tumors reached hyperthermal temperatures that led to inhibited tumor growth and improved survival compared to the saline and sham treated groups. In the NS group, PET imaging showed a reduction in tumor uptake of 18F-FDG of nearly 50% at day 2 compared to baseline. In the saline and sham groups the tumor uptake of 18F-FDG was at comparable levels at baseline and day 2. In addition, a positive correlation was found between the change in tumor uptake between baseline and day 2 and the change in tumor volume on day 7 in all animals scanned with 18F-FDG. Preliminary data from animals scanned with 18F-FLT indicates that the tumor uptake is also strongly reduced after NS-assisted photothermal therapy and that the change in 18F-FLT uptake correlates with inhibited tumor growth on day 7.

Conclusion: Both 18F-FDG and 18F-FLT PET imaging show great potential for early evaluation of the treatment response after nanoparticle-assisted photothermal therapy and preliminary data suggest that both tracers can be used to predict treatment outcome.

#2870

Optical imaging of bombesin and transferrin receptor expression are as effective as 18FDG in assessing drug efficacy.

Jen-Chieh Tseng, Jeffrey D. Peterson. _Perkin Elmer, Hopkinton, MA_.

Physical measurement of tumor volume reduction is the most commonly used method used to assess tumor progression and treatment efficacy in mouse tumor xenograft models, but the detection of tumor size changes can require repeated drug dosing and tumor measurements for several weeks. However, 18F-FDG PET imaging of altered glucose metabolism can be a more sensitive tool for early cancer detection/diagnosis as well as treatment assessment; cancer cells are known to have abnormally increased cellular metabolism that can be inhibited by drug treatment. To illustrate this, we used HCT-116 human colorectal tumor xenografts in nu/nu mice with sorafenib treatment, a clinically approved tyrosine protein kinase inhibitor (5 d treatment, 40 mg/kg). Treatment is known to inhibit PDGFR and VEGFR, as well as Raf kinases that regulate energy metabolism in tumors. Sofie G8 PET imaging of treated mice using 18F-FDG revealed a significant drop in tumor metabolism with as little as 2-3 days of treatment, a time in which there is typically little or no effect on tumor size. This approach was relatively low through-put, and required special procedures to accommodate use of radioactivity, but offered the option of daily imaging of tumor status. We also explored alternative optical imaging approaches that could offer higher through-put imaging as well as the potential for multiplex imaging. There is no fluorescent equivalent of 18F-FDG, so we focused on bombesin- and transferrin-receptors as potentially useful biomarkers for drug-induced inhibition in tumor metabolism. Bombesin receptors are upregulated in a variety of tumors and are important in energy metabolism and tumor growth. The rapid recycling kinetics also make this receptor highly sensitive to cellular metabolic changes. Transferrin receptors are also upregulated in most tumors and provide critical iron transport function vital for their increased enzymatic, proliferative, and metabolic requirements. We used targeted near infrared (NIR) fluorescent imaging probes, BombesinRSense™ 680 (BRS-680) and Transferrin-Vivo™ 750 (TfV-750), to monitor changes in receptor expression in HCT-116 tumor xenografts during the course of sorafenib treatment. Interestingly, both BR-680 and TfV-750 FLI on the IVIS® SpectrumCT yielded data quite similar to our results using 18F-FDG PET; reduction in these probes can be measured as early as 48-72 hours, in the absence of significant reduction of tumor size/viability. As expected, both PET and FLI were also highly effective at imaging sorafenib effects 7-8 days later (3-4 days following a 5-day sorafenib treatment regimen), with datasets in good agreement with physical measurement of changes in tumor size. These results suggest that BRS-680 and TfV-750 can serve as fluorescent surrogates for 18F-FDG PET both in measuring early metabolic changes and ultimate therapeutic outcomes following cancer treatment.

#2871

Non-invasive monitoring of the efficacy of anticancer therapeutic agent in lung orthotopic xenograft models of NSCLC using Bio-Luminescence Imaging (BLI).

Sanchareeka Dey, Sarada Preeta Kalainayakan, Poorva Ghosh, Li Zhang. _University of Texas at Dallas, Richardson, TX_.

Non-Small Cell Lung Cancer (NSCLC) patients have a poor rate of survival owing to diagnosis at a later stage. This necessitates development of novel therapeutic strategies that limit rapid progression of NSCLC and imaging techniques that monitor such changes in vivo. There has been a significant growth of bioluminescence imaging applications in small laboratory animal models in recent years, owing to its high sensitivity, efficiency, and non-invasive nature.

Our lab has previously demonstrated that a therapeutic agent that disrupts mitochondrial respiration effectively hampers proliferation of NSCLC cell lines in vitro. With an effort to investigate the potential of this therapeutic agent in clinical settings, we used a lung orthotopic xenograft mouse model along with bioluminescence imaging (BLI) to continuously monitor the efficacy of the drug in vivo. Briefly, we implanted NSCLC cell line transduced with lentiviral particles carrying luciferase sequence, in the left lung of 6-8 weeks old female NOD/SCID mice to generate lung orthotopic xenografts. The therapeutic agent was administered intravenously over several weeks. Tumor growth was then compared between the untreated versus the treated mice, non-invasively through bioluminescence imaging (Perkin Elmer's IVIS Lumina III Imager). On sacrificing the mice, the lungs were harvested, formalin fixed and paraffin embedded for Immunohistochemistry.

Our BLI data show that in the mice treated with the therapeutic agent, tumors are localized to the left lung (site of implantation) as opposed to the control mice where the tumors metastasized to the right lung. The BLI data indicates significant decrease in radiance (total flux in photons per second) in mice subjected to treatment when compared to control mice.

We show that the therapeutic agent significantly inhibits progression and metastasis of human NSCLC cell line orthotopically injected in the lungs of NOD/SCID mice. The results suggest that it might be potent anti-cancer drug as it is effective in a model that recapitulates the natural environment of lung tumorigenesis. Immunohistochemical studies are underway to discern the mechanistic details.

#2872

Quantitative mass spectrometry imaging of erlotinib in skin rashes of cancer patients receiving erlotinib.

Meiko Nishimura,1 Hiroaki Aikawa,2 Mitsuhiro Hayashi,2 Yu Mizutani,1 Kei Takenaka,1 Yoshinori Imamura,1 Naoko Chayahara,3 Masanori Toyoda,1 Naomi Kiyota,1 Toru Mukohara,4 Akinobu Hamada,2 Hironobu Minami1. 1 _Kobe University Graduate School of Medicine, Kobe, Japan;_ 2 _National Cancer Center, Japan;_ 3 _Kobe Minimally Invasive Cancer Center, Kobe, Japan;_ 4 _Kobe University Hospital, Kobe, Japan_.

Background

The development of rashes is the most common adverse event observed in cancer patients treated with epidermal growth factor receptor-targeted tyrosine kinase inhibitors (EGFR-TKI) such as erlotinib. A significant relationship exists between the severity of a rash and survival in various cancers. However, whether erlotinib becomes distributed to the skin and whether its concentration is higher in a rash than in normal skin of treated cancer patients remains unknown. Here, using quantitative mass spectrometry imaging (qMSI), we successfully visualized the distribution of erlotinib in rashes and normal skin of patients with advanced pancreatic cancer receiving this drug.

Methods

We studied five patients with advanced pancreatic cancer who developed rashes after treatment with gemcitabine (1000 mg/m² by intravenous infusion for 30 minutes on days 1, 8, and 15 every 4 weeks) and erlotinib (given orally at 100 mg/day). We biopsied both rashes and normal skin, and compared the distribution of erlotinib using matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). The plasma concentration of erlotinib was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Results

Erlotinib concentrations in five patients were 2.55 ± 1.46 ng/mm3 in normal skin and 3.18 ± 1.53 ng/mm3 (mean ± SD) in rashes (p=0.123). In four out of five patients, the erlotinib concentration in rashes was higher than that in normal skin. The epidermis showed the strongest expression of EGFR in skin as judged by immunohistological staining. Erlotinib showed a greater tendency for distribution to the epidermis than subcutaneous

tissue. There was no correlation between erlotinib plasma concentrations vs. concentrations in rashes or normal skin.

Conclusions

Using qMSI, we, for the first time, visualized the distribution of erlotinib in skin tissue of pancreatic cancer patients receiving erlotinib. We found a tendency for a higher concentration of erlotinib in rashes than in normal skin. A greater distribution of erlotinib to the epidermis than subcutaneous tissue suggests erlotinib may directly bind EGFR expressed in the epidermis.

#2873

Imaging tumor heterogeneity after multikinase inhibitor therapy in rat hepatocellular carcinoma.

Nina M. Muñoz,1 Adeeb A. Minhaj,1 Kiersten L. Maldonado,1 Charles Kingsley,1 Hideyuki Nishiofuku,2 Keith A. Michel,1 Andrea C. Cortes,1 James A. Bankson,1 Asif Rashid,1 Rony Avritscher1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Nara Medical University, Nara, Japan_.

Purpose To assess tumor tissue perfusion and vascular permeability through multiparametric functional imaging, and to evaluate their correspondence with histopathologic parameters of necrosis and hypoxia in a rat model of hepatocellular carcinoma treated with a multikinase inhibitor.

Materials and Methods Rat hepatoma McA-RH7777 cells were implanted in the left liver lobe of nineteen male Buffalo rats. Exactly 2 weeks after tumor inoculation, the animals were randomly assigned to remain untreated (n=10) or to receive a daily dose of 7.5 mg/kg sorafenib by oral gavage (n=9) for 2 additional weeks. T2-weighted spin-echo magnetic resonance imaging (MRI), dynamic contrast-enhanced (DCE) and contrast-enhanced ultrasound (CEUS) were performed weekly. All tumors were harvested 4 weeks post-implantation >90 minutes after injecting 60mg/kg pimonidazole. Tissue sections were stained for hematoxylin-eosin and pimonidazole for quantitative assessment of necrosis and hypoxia, respectively. Differences between treatment groups were assessed using the Mann-Whitney test, and the correlation of imaging and histopathology parameters was determined by Spearman correlation analysis.

Results In spite of the relatively low dose and short treatment duration, the response to sorafenib therapy was characterized by a significantly higher median tumor necrosis (60 vs 15%, P < 0.0001) and increased tumor tissue hypoxia (35 vs 18.5% pimonidazole binding, P < 0.0001). Similarly, the sorafenib regimen caused a substantial decrease in vascular permeability, measured by DCE-MRI Ktrans (P = 0.002) as well as reduction in tumor perfusion evaluated by CEUS peak enhancement (PE) (P =0.043). Our findings show a strong correlation between the two histopathologic parameters we assessed, tumor tissue necrosis and hypoxia (r = 0.645, P=0.002). Furthermore, our results demonstrate significant correlation of functional imaging parameters of vascular permeability, namely DCE-MRI Ktrans and DCE-MRI normalized initial area under the curve (NIAUC), with histopathologic tissue hypoxia (r=-0.663, P= 0.002, and r= -0.512, P=0.029, respectively). In contrast, histopathologic tumor necrosis appears more strongly correlated with a functional imaging parameter of tumor perfusion, specifically, CEUS PE (r = -0.547, P = 0.028).

Conclusion Tumors exhibited wide heterogeneity in vascular perfusion and hypoxia after treatment. Interestingly, functional MRI parameters appear to correlate more strongly with tissue oxygenation, whereas functional CEUS parameters correlate with tumor viability. Novel predictive imaging biomarkers of treatment response may be developed through further analyses of spatial tumor heterogeneity in our animal model.

#2874

**Inhibition of tumor angiogenesis by** Salmonella typhimurium **A1-R in the** in vivo **Gelfoam color-coded imaging assay.**

Tasuku Kiyuna,1 Yasunori Tome,2 Takashi Murakami,1 Ming Zhao,1 Fuminori Kanaya,2 Robert M. Hoffman1. 1 _AntiCancer, Inc, San Diego, CA;_ 2 _University of the Ryukyus, Okinawa, Japan_.

We previously demonstrated a color-coded angiogenesis imaging model using Gelfoam® implanted in nestin-driven green fluorescent protein (ND-GFP) nude mice in which nascent blood vessels express GFP. In this assay, we demonstrated osteosarcoma promotes angiogenesis. We report here that Salmonella typhimurium A1-R (S. typhimurium A1-R) inhibits angiogenesis of osteosarcoma cells in the Gelfoam® angiogenesis assay in ND-GFP mice. Gelfoam® was transplanted subcutaneously in the flank of transgenic ND-GFP nude mice. Seven days after transplantation, skin flaps were made and 143B human osteosarcoma cells, expressing red fluorescent protein (RFP), were injected in the transplanted Gelfoam®. After establishment of tumor in the Gelfoam®, the control group mice were treated with PBS by tail vein injection. The treated group was treated by S. typhimurium A1-R, also by tail vein injection. Skin flaps were made at days 14, 21, and 28 after transplantation of the Gelfoam® in order to allow imaging of vascularization. The OV100 variable magnification small animal imaging system and FV1000 confocal fluorescence microscope were used to visualize vascularization. Nascent blood vessels grew in the Gelfoam® in a time-dependent manner. A random 3 fields were quantified in each group. The mean length of ND-GFP-expressing blood vessels in the Gelfoam® in mice were treated with S. typhimurium A1-R was 9.40, 12.40, and 10.12 mm/mm2, at days 14, 21, and 28, respectively. The mean total length of ND-GFP-expressing blood vessels in the Gelfoam® in mice of the control group were 8.86, 14.50, and 15.81 mm/mm2 on day 14, 21, and 28, respectively. ND-GFP expressing blood vessels of the mice in the osteosarcoma cells treated with S. typhimurium A1-R had shorter vessels than the control group (on day-28, P<0.05). There was not a significant difference in tumor size between the 2 groups, thereby indicating a specific inhibition of angiogenesis by S. typhimurium A1-R. In the present study, we reported that S. typhimurium A1-R inhibited angiogenesis of human osteosarcoma cells in the in vivo Gelfoam® color-coded imaging assay.

### Signaling in Cancer Stem Cells

#2875

Knockout of ST8SIA1 inhibits tumorigenesis in triple negative breast cancer by inducing PTEN and ganglioside GM1 mediated tumor growth arrest.

Khoa Nguyen, Yuanqing Yan, Chandra Bartholomeusz, Naoto Ueno, Kim-Anh Do, Michael Andreeff, V. Lokesh Battula. _UT MD Anderson Cancer Ctr., Houston, TX_.

We found that ganglioside GD2 is a biomarker for breast cancer stem cell (BCSCs) in triple negative breast cancer (TNBC) and that GD2 biosynthesis is tightly regulated by enzyme ST8SIA1 (GD3 synthase) in these cells. We have reported that ST8SIA1 is highly expressed in TNBC and its expression is highly correlated with TP53 mutations in primary tumors (based on TCGA data set). Here we hypothesized that ST8SIA1 has a functional role in BCSC mediated tumorigenesis in TNBC. To test this hypothesis, we deleted ST8SIA1 in SUM159 cells using CRISPR-Cas9 technology. As expected, deletion of ST8SIA1 in SUM159 cells reduced the absolute number of GD2+ cells from 17 ± 1.5% to 0.3 ± 0.1%. Although there was no significant difference in 2D cell growth, anchorage-independent growth by soft-agar and the mammosphere formation assays revealed a complete loss of colony formation in ST8SIA1-KO cells. Moreover, transplantation of ST8SIA1-KO- or Cas9 control- SUM159 cells in mammary fat pad of NSG mice revealed that ST8SIA1-KO cells failed to produce tumor formation even after a 15-wk after implantation. In addition, most of the cas9 control cell injected mice died within 4 wk after cell implantation whereas no deaths were observed in the ST8SIA1-KO cells as long as 100 d after tumor implantation. To investigate the mechanism involved in tumor growth inhibition associated with the loss of ST8SIA1 expression, we analyzed mRNA expression in ST8SIA1-KO- and Cas9 control- SUM159 cells by RNA sequencing. At p < 0.05 and fold change >2, we found 1502 genes down-regulated and 842 genes up-regulated in the ST8SIA1-KO-cells compared to controls. Ingenuity pathway analysis revealed that several stem cell-associated signaling pathways, including wnt, stat3, NFκB, nanog, and IL8, were down-regulated. Conversely, the potent tumor suppressor PTEN was induced in the ST8SIA1-KO-cells compared to controls. In addition, gene ontology by gene set enrichment analysis (GSEA) revealed inhibition of oncogenic signaling pathways including KRAS, p53, AKT, and RB. We have also identified a significant down-regulation of breast cancer-associated genes including BCL11A, PDGFRb, VCAM1, CXCR4, and wnt5a. Interestingly DKK1, which acts as an antagonist for wnt-β-catenin signaling, was up-regulated in the ST8SIA1-KO cells. These findings were validated by qRT-PCR, flow cytometry and western blot analysis. Ganglioside GM1 has been reported to have major in anti-tumor effect by inhibiting ERK signaling. Flow cytometry analysis revealed that ST8SIA-KO cells expressed ~10-fold higher GM1 compared to Cas9 control cells. In addition, pERK was down-regulated in ST8SIA-KO cells, suggesting a loss of tumorigenic potential of these cells. In conclusion, our data suggests that inhibition of ST8SIA1 in TNBC cells depletes BCSCs and inhibits tumorigenesis by down-regulating oncogenic pathways and up-regulation of PTEN and GM1 mediated signaling.

#2876

Loss of the stem cell and basal lineage regulator LBH delays onset of basal-like triple negative breast cancer.

Kilan C. Ashad-Bishop, Karoline Briegel. _Univ. of Miami, Miami, FL_.

There is increasing evidence that basal-like triple negative breast cancer (TNBC) originates from luminal mammary epithelial cells. Specific gene signatures have already been experimentally proven to serve as genetic lineage switches that, when overexpressed, transform pre-malignant luminal cells to oncogenic cells with basal, stem-like characteristics. Conversely, depletion of these genes in oncogenic cells with basal, stem-like characteristics leads to luminal differentiation. These studies suggest that during the earliest steps of neoplastic transformation, specific gene sets can alter cell fate decisions and differentiation status in mammary epithelial cells, which ultimately contributes to the heterogeneity of breast tumors.

Our lab has identified a novel Wnt/β-Catenin target gene, Limb bud and heart (LBH) that is majorly overexpressed in TNBC. LBH is a regulator of the basal mammary stem cell lineage and repressor of luminal differentiation via induction of ΔNp63 and repression of estrogen receptor alpha. LBH is required for the self-renewal and maintenance of adult basal mammary stem cells, which tend to be enriched in TNBC. Knockout studies in mice have shown that genetic ablation of LBH does not impair embryogenesis or normal adult organ function, making it a possible therapeutic target.

Using crosses between MMTV-Wnt-1 transgenic mice and K14Cre LBHloxP knockout mice, we are studying the effect of LBH ablation in the basal cells of the mammary epithelium downstream of ectopic Wnt expression in the mammary gland. We postulated that LBH may be an effector of Wnt-driven TNBC, therefore its inhibition would lead to decreased Wnt-induced mammary gland hyperplasia and tumor formation. In our model, LBH ablation in the basal mammary epithelium of female Wnt transgenic mice reduces mammary gland hyperplasia and delays tumor onset. MMTV-Wnt-1 driven, LBH-null tumors also exhibit histopathological differences indicative of a luminal to basal conversion.

There is an urgent need to elucidate mechanisms underlying TNBC development and progression and find reliable avenues for treatment. Our data indicates a role for Lbh as a novel effector for Wnt-driven TNBC and further studies may prove antagonism of Lbh to be a novel method to control TNBC progression.

#2877

IKK-mediated signaling controls prostate tumor initiating cells.

Sara E. Conard, Aaron Ebbs, Albert S. Baldwin. _University of North Carolina at Chapel Hill, Chapel Hill, NC_.

The heterogeneous nature of prostate cancer tumors is thought to play an important role in the decreased effectiveness of existing therapies. Tumor initiating cells (TICs) are capable of self-renewal and comprise a subset of the tumor mass. These cells are proposed to drive the growth and metastasis of tumors, and are considered to be resistant to traditional cytotoxic and targeted therapies. Several studies have shown that NF-κB signaling is increased in recurrent prostate cancer and enriched in prostate TICs. We sought to determine the potential of an IKK/NF-κB-driven mechanism for inherent or acquired resistance by IKK-mediated control of a subset of prostate tumor initiating cells. These studies were performed by using established prostate cancer cell lines, murine prostate organoids, and a murine prostate cancer animal model. We have found that inhibition of IKKα and IKKβ, upstream regulators of noncanonical and canonical NF-κB signaling, block self-renewal of several PTEN-deficient prostate cancer cell lines. Furthermore, we have also found that IKK is important for tumorigenicity and stemness seen in prostate cancer cells as measured by colony formation and extreme limiting dilution assays. Loss of canonical NF-κB (p65/RelA) decreased stemness while loss of noncanonical did not alter tumorsphere formation. Interestingly, Pten-/- tumors with loss of Ikkα or Ikkβ displayed decreased levels of self-renewing cells as measured by CD49fhigh expression, a known prostate basal cell marker. Isolated murine Pten-/- cells were 2x more efficient than Pten-/-Ikkα-/- or Pten-/-Ikkβ-/- cells in forming prostate organoids suggesting that loss of Ikk decreased the number of self-renewing cells needed for formation. Taken together, we conclude that IKK-mediated signaling is important for maintenance of prostate tumor initiating cells and further studies will address whether IKK-mediated signaling provides a mechanism for evading current therapies.

#2878

Anti-cancer effects of retinoic acid in CRC occurs via decreased growth of ALDH+ cancer stem cells and increased differentiation of stem cells to neuroendocrine cells.

Shirin R. Modarai,1 Lynn M. Opdenaker,1 Anindita Gupta,1 Jeremy Z. Fields,2 Bruce M. Boman1. 1 _Helen F. Graham Cancer Ctr., Newark, DE;_ 2 _CaTx, PA_.

Our studies on tissues from colon cancer patients show that in the development of colorectal cancer (CRC), stem cell (SC) overpopulation underlies tumor initiation and progression. Because aldehyde dehydrogenase (ALDH) is a marker for SCs in several tissues and is a key enzyme in retinoid acid (RA) signaling, we investigated the role of RA signaling in normal and malignant colonic SCs. Our overall hypothesis is that RA signaling regulates growth and differentiation of ALDH+ colonic SCs and dysregulation of RA signaling contributes to SC overpopulation in CRC development. To begin to investigate underlying mechanisms, we analyzed CRC cell lines/tissues to see if retinoid receptors RXR and RAR are exclusively expressed in ALDH+ SCs and if the RA pathway components change during CRC development. We determined whether RA signaling influences SC proliferation, differentiation, self-renewal, and SC population size. RXR & RAR were selectively expressed in ALDH+ colonic SCs, but not in MCM2+ proliferative cells, suggesting that RA signaling mainly occurs in SCs. Western blotting/immunostaining of CRCs revealed that RA signaling components, including RAR and RXR, become overexpressed in parallel with ALDH overexpression, which coincides with the known overpopulation of ALDH+ SCs that occurs during CRC development. Treatment of cells with RA ligands (ATRA, 9-cis RA) inhibited proliferation, reduced sphere formation, decreased ALDH+ SC population size, and induced differentiation along the neuroendocrine cell (NEC) lineage. Taken together, our findings indicate that: (1) retinoid signaling, by regulating ALDH+ colonic SCs, reduces SC proliferation, self-renewal and SC population size, and promotes SC differentiation to NECs, and (2) dysregulation of RA signaling in colonic SCs likely contributes to overpopulation of ALDH+ SCs and CRC growth. Thus, RA signaling, by inducing increased differentiation of the SC population, suggests a novel therapeutic target.

#2879

Dj-1 confers maintenance of stemness and radioresistance by regulating EGFRvIII expression in glioma stem cells.

Myung-Jin Park,1 Jeong-Yub Kim,1 Hee-Jin Kim,1 Ji-Soo Kim,1 Jeong-Chul Kim,1 Dea-Hee Lee,2 Yoo-Jin Na2. 1 _Korea Institute of Radiological and Medical Sciences, Seoul, Republic of Korea;_ 2 _Korea University, Seoul, Republic of Korea_.

DJ-1 is involved in many key cellular processes including cell proliferation, transcriptional regulation, differentiation, oxidative stress protection, and mitochondrial function maintenance. Deregulation of DJ-1 has been implicated in the pathogenesis of many human diseases such as Parkinson's disease, amyotrophic lateral sclerosis, infertility, and cancer. DJ-1 was initially identified as an oncogene product involved in human tumorigenesis in numerous cancer types. Alteration in expression of DJ-1 is associated with tumorigenesis in several types of cancers. Herein, we aimed to clarify the effect of DJ-1 on stemness and radioresistance of glioma stem cells (GSCs). Firstly, we found that the reduction in expression of DJ-1 in brain tumor tissues when compared with tumor adjacent normal brain tissues, as indicated by reduced DJ-1 staining. Knock-down of DJ-1 inhibited self-renewal activity of GSCs in vitro as evidenced by neurosphere formation, limiting dilution, and soft agar clonogenic assays. DJ-1 knock-down suppressed maintenance of stemness by suppressing the expression of Notch intracellular domain, Sox2, and phosphorylated STAT3 proteins. Intriguingly, DJ-1 knock-down inhibited the expression of EGFRvIII in GSCs which might be the cause of suppression of stemness pathways. In addition, knockdown of DJ-1 induced apoptosis, reduced cell invasiveness, and sensitized GSCs to ionizing radiation. Based on these data, we propose that DJ-1 could be considered as a crucial diagnostics and therapeutics target for malignant glioma.

#2880

GANT61-mediated constraint of Fibulin-5/prorenin receptor/BMI1 signaling impacts nasopharyngeal carcinoma stemness and metastasis.

Hsin-Ting Tasi, Chang-Han Chen. _Institute for Translational Research in Biomedine, Kaohsiung, Taiwan_.

Background: Cancer stem cells (CSCs) of NPC exhibit higher tumorigenesis and metastatic potential. Novel therapies based on molecular targets of CSCs have become the focus to cure NPC. GANT61, a GLI1 inhibitor, has been reported to exhibit potent anticancer effect on many cancers; here, we evaluated whether GANT61 has inhibitory effects on NPC-CSCs and further explored the possible mechanism.

Methods: Condition medium was used to enrich NPC stem-like cells. The NPC stem-like cells characteristics were examined by Q-RT-PCR, Western blotting, MTT, BudU, colony formation, sphere formation, and subcutaneous and metastatic xenograft models. Genetic, pharmacological and biochemical approaches were applied in NPC stem-like cells to investigate molecular signals, to assess the efficacies under the inhibitors treatment, and to examine the correlation among the molecules in vitro, in vivo and NPC specimens.

Results: In the present study, we had demonstrated that NPC tumorsphere cells (NPC-TCs) possess CSC properties. The proliferation, colony formation, spheroid formation, migration, and invasion of NPC-TCs were dramatic prevented in the presence of GANT61 in vitro and in vivo. ChIP reveals that Gli1 preferentially binds to the Fibulin-5 promoter, and Fibulin-5 transcriptional/post-transcriptional levels in NPC-TCs were regulated upon GANT61 treatment. However, ectopic expression of Fibulin-5 in NPC-TCs not only enhanced self-renewal and ALDH1 expression, but also reduced the effects of GANT61-eilicted anti-tumor in vitro and in vivo. We further identified that a novel signaling, prorenin receptor/BMI1 pathway was modulated by Fibulin-5 and involved in Fibulin-5-raised self-renewal capacity of NPC-TCs in vitro and in vivo. Notably, GANT61 inhibited NPC-TCs phenotypes was associated with suppression of Fibulin-5/prorenin receptor/BMI1 signaling. Finally, a significant correlation was observed among GLI1, Fibulin-5, AT1R and BMI1 in the specimens of NPC.

Conclusions: Our data highlight that Fibulin-5/prorenin receptor/BMI1 signaling has critical role in maintaining stem-like properties of NPC-CSCs and can be efficiently targeted by GANT61 representing a preclinical therapeutic strategy to repress NPC-CSCs.

#2881

CXCL1 and CCL5, induced by ionizing radiation, reprogram nontumorigenic cancer cells into cancer stem cells in breast cancer.

Justine Bailleul-Dubois,1 Nadège Bidan,1 Raphaëlle Mouttet-Audouard,2 Mélanie Arcicasa,2 Karine Hannebicque,2 Yuki Takayama,3 Samuel Meignan,2 Xuefen Le Bourhis,1 Chann Lagadec1. 1 _INSERM U908, Villeneuve d'Ascq, France;_ 2 _Centre Oscar Lambret, Lille, France;_ 3 _LIMMS/CNRS-IIS UMI2080, SMMIL-E, Tokyo, Japan_.

Identification of cancer stem cells (CSC) in solid tumors - with self-renewal, multipotency, tumorigenesis, and therapy resistance capacities - has opened path to new targeting therapeutic approaches. However, CSC targeting alone might not be sufficient to eradicate a tumor. Indeed, recent studies showed that cancer cells are plastic, and conventional therapies, such as radiotherapy, can lead to cancer cells (non-CSC) reprogramming into iCSC (induced-CSC). The goal of our work is to identify the molecular mechanisms responsible for treatment-induced CSC emergence. First, we have shown that conditioned media from irradiated non-CSC is sufficient to induce iCSC reprogramming. These results suggest that cell plasticity might be actively regulated by diffusible factors secreted by irradiated cells. By using proteins arrays and ELISA, we demonstrated that the secretion of a specific cocktail of chemokines is induced by ionizing radiation, such as CXCL1 and CCL5. Interestingly, recombinant CXCL1 and CCL5 treatments increase the sphere forming capacity (SFC) of isolated non-CSC treated population. Concomitantly, treatment with neutralizing antibodies targeting CXCL1 and CCL5 leads to a decreased CSC number (ALDH+ cells). We also studied the expression of the corresponding chemokines receptors, by flow cytometry. First, we saw that reprogrammable ALDH- cells are enriched for CXCL1 and CCL5 receptors expressing cells compare to unsorted population or ALDH+ population (CSC). We analysed the reprogramming potential of isolated ALDH-/receptor-positive cells versus ALDH-/receptor-negative cells. The ALDH-/receptor-positive-derived cell population is more able to form spheres and overcomes the receptor-negative-derived population when the two populations are mixed and tested for their sphere forming capacity. The use of pharmacological inhibitors against the receptors induce a slight decrease of CSC. Taken together, our results indicate the involvement of chemokines, in particular CXCL1 and CCL5, in the reprogramming mechanism. To validate the implication of CXCL1 and CCL5 and in a pre-clinical perspective, we are currently beginning an in vivo experiment to study the effects of CXCL1 and CCL5 inhibition on tumor development and on CSC enrichment in vivo.

#2882

The miR-200b-ZEB1 circuit regulates diverse stemness of human hepatocellular carcinoma.

Sen-Yung Hsieh, Su-Chun Tsai. _Chang Gung Memorial Hospital, Taoyuan, Taiwan_.

Background & aims: Hepatocellular carcinoma (HCC) can be derived from cancer stem cells (CSCs), which contribute to tumor initiation, metastasis, chemoresistance, and post-treatment recurrence. A great variety of HCC CSCs resulting in diverse clinical manifestations have been reported. We aimed to elucidate how CSC diversity is regulated.

Methods: MicroRNAs deregulated in HCC were identified by using array-based microRNA profiling. Diversity of HCC CSCs was assayed by flow-cytometry, in vitro and in vivo assays for tumorigenicity.

Results: MiR-200b downregulation occurred in early HCC and associated with shorter disease-free and overall survival. Ectopic expression of miR-200b or silencing of ZEB1 led to a decrease in CD13+ and CD24+ HCC CSCs and an increase in EpCAM+ HCC CSCs. MiR-200b directly suppressed BMI1 and ZEB1 expressions. ZEB1 transcriptionally upregulated CD13 and CD24 expression and downregulated EpCAM expression via directly targeting their promoters. Neither miR-200b nor ZEB1 had obvious effects on CD133 or CD90 expression. Silencing CD13 or CD24 expression suppressed tumorigenicity of HCC cells. Ectopic expression of CD24 reversed the suppression of tumorigenicity when these HCC cells had been ectopically expressing miR-200b. Clinically, miR-200b downregulation was coupled with ZEB1 upregulation in approximately two-thirds of HCC patients. ZEB1 expression was positively correlated with CD13 and CD24 expressions, while EpCAM expression was positively correlated with miR-200b expression in HCCs.

Conclusions: The miR-200b-ZEB1 circuit is a master regulator of diverse stemness of HCC, which distincts HCCs into those containing CD13+/CD24+ CSCs from those containing EpCAM+ CSCs, and a thus potential target for anti-HCC therapy.

#2883

Do Wnt3A-mediated canonical Wnt/Beta-catenin signals regulate stem cell phenotypes in brain metastasis-specific TNBC.

Jennifer H. Carlson, Pradip De, Casey Williams, Nandini Dey, Brian Leyland-Jones. _Avera Cancer Institute, Sioux Falls, SD_.

Introduction: Triple negative (TN) and basal-like subtypes portend one of the worst prognoses in BC and have the most challenging diagnosis among patients due to the aggressive nature of the disease. BC stem cells (BCSCs) have a distinct role in breast oncogenesis and CD44(+)/CD24(-) BCSCs play an important role in the clinical behavior of TNBC. Unique growth abilities of BCSCs including self-renewal, differentiation potential and resistance to anti-cancer agents contribute to the development and overall aggressiveness of recurrent metastatic lesions. Patients with TNBC as compared to non-TNBC have significantly higher incidences of recurrence or distant metastasis and the poorer outcome has been associated with increased frequency of BCSCs phenotypes. Aim: We previously reported that Wnt pathway (WP) is associated with metastasis (Dey et al., 2013) and controls metastasis-associated (MA) tumor cell phenotypes in TNBC (De et al., 2016) and here we tested the role of WP in the regulation of BCSCs and MA tumor cell phenotypes using brain-metastasis specific TNBC model (MDA-MB231BR).

Methods: MDA-MB231BR cells were stimulated with WP specific ligand or LWnt3A conditioned media (CM) and MA tumor cell phenotypes were tested following WP inhibition. To model distant metastasis in vitro, we have used a long-term 3D-matrigel assay in the presence of LWnt3ACM. Four BCSCs markers, CD44, CD44V6, CD24 and CD133 were selected for the flow cytometric analyses of cells following LWnt3ACM stimulation and downregulation of WP by sulindac sulfide.

Results: Specific stimulation of WP by LWnt3ACM led to the clonogenic growth from a single cell. Inhibition of WP blocked micro-metastatic growth of these colonies. Although WP stimulation alone did not alter % of CD44(+)/CD24(-) population in 2D format, inhibition of WP following treatment with sulindac sulfide caused a significant decrease in the % of CD44(+)/CD24(-) population (66%) as compared to the control (79%). To further understand the mechanistic long-term role of BCSCs during the micro-metastatic 3D growth of colonies, we have developed a novel method to identify the % of BCSCs in the micro-metastatic 3D colonies. After 15 days of 3D colony growth, cells are recovered from matrigel and the live cell fraction is analyzed for CD24, CD44, CD44v6 and CD133 by flow cytometric analysis. We previously presented data demonstrating the CD44+/CD24- fraction is enriched in 3D colonies as compared to correspoding 2D cells. Here we've extended our analysis and examined 3D colonies following either stimulation or inhibition of WP.

Conclusion: Our data demonstrates that WP mediates the BCSCs phenotype(s) of TNBC in a context-dependent manner. Studies are on-going to delineate the mechanistic role of different BCSCs markers particularily CD44 and CD44v6 in signaling the formation of WP-mediated micro-metastatic 3D colonies, the results of which will be presented at the meeting.

#2884

Interferon Beta (IFN-B) re-activates canonical type I IFN signaling to differentiate breast cancer stem cells (CSCs) and suppress mesenchymal/CSC driven tumor recurrence.

Mary R. Doherty,1 Damian J. Junk,1 HyeonJoo Cheon,2 George R. Stark,2 Mark W. Jackson1. 1 _CWRU, Cleveland, OH;_ 2 _Lerner Research Institute, Cleveland Clinic, Cleveland, OH_.

Tumor recurrence remains a major therapeutic obstacle to curing breast cancer. Cytokines within the breast tumor microenvironment (TME) influence recurrence following chemotherapy by altering the balance between mesenchymal CSC (MES/CSC) and epithelial non-CSC (EP/non-CSC). Here, we find that transformed breast MES/CSC express elevated levels of Un-phosphorylated Interferon Stimulated Gene Factor 3 (U-ISGF3), which is composed of STAT1 and STAT2 lacking phosphorylation of their tyrosine residues, and IRF9. Elevated expression of the U-ISGF3 proteins in breast cancer has previously been linked with resistance to ionizing radiation, thus we hypothesized that the U-ISGF3 complex is important for maintaining MES/CSC properties. However, knockdown of IRF9, the critical DNA binding component of this complex, or STAT1 did not alter the MES/CSC phenotype. Moreover, ISGF3 transcriptional targets were actually repressed in MES/CSC relative to EP/non-CSC, suggesting that elevated U-ISGF3 may act as a repressor in MES/CSC. Interestingly, we found that an MES/CSC program independent of IFN-B, was responsible for mediating U-ISGF3 expression. We therefore hypothesized that stimulating MES/CSC with IFN-B would phosphorylate the ISGF3 proteins and engage canonical IFN-B mediated inhibition of MES/CSC properties. Acute exposure to IFN-B led to the phosphorylation of ISGF3 in MES/CSC and induced the transcription of IFN-B responsive genes. Continuous exposure to IFN-B maintained Phosphorylated ISGF3 (P-ISGF3) which partially reverted the MES/CSC phenotype, resulting in the repression of mesenchymal markers (Vimentin and Slug), re-expression of Ep/non-CSC markers (CD24) and functionally inhibited colony formation in soft agar and migratory capability. In line with these observations, data from triple-negative breast cancer (TNBC) patients confirmed that increased expression of IFN-B responsive genes correlates with decreased expression of a CSC gene signature, and correlates with improved recurrence-free survival. Taken together, our findings demonstrate that IFN-B mediated signaling promotes the differentiation of MES/CSC by phosphorylating U-ISGF3, resulting in the suppression of MES/CSC-driven tumor recurrence. Future studies will examine the mechanism by which IFN-B mediated P-ISGF3 suppresses MES/CSC properties and whether elevation of U-ISGF3 can predict which patients would be candidates for an IFN-B therapy to phosphorylate STAT1 and STAT2, thus suppressing MES/CSC tumor recurrence.

#2885

Activation of IL1α signaling by HER2 overexpression promotes breast cancer stem cells.

Shou Liu,1 Ji shin Lee,2 Hexin Chen1. 1 _University of South Carolina, Columbia, SC;_ 2 _Chonnam National University, Republic of Korea_.

Breast cancer is a heterogeneous disease and only a small proportion of cells known as cancer stem cells (CSCs) have ability to generate tumor. Recently, it has been reported that HER2 overexpression increase breast cancer stem cells (BCSCs). However, the underlying mechanism is not clearly known. In this study, we explored the molecular mechanism how HER2 increase BCSCs. Our data has demonstrated HER2 induces BCSCs by indirectly activating NF-kB and STAT3 signaling. To figure out the direct target of HER2, we did the microarray and found that HER2 upregulates the expressions of IL1a and IL6, two major cytokines which directly activates NFkB and STAT3, respectively. Interestingly, we found that IL1a also indirectly activates STAT3. IL1α induced STAT3 was blocked when IL6 was knock out from cells. We found that HER2 enhances IL1α transcription by activating PU.1 which is known transcription factor of IL1a. Finally, we studied effect of IL1a on tumorigenesis, and found that IL1α KO dramatically attenuates the tumor growth in NOD/SCID mice. Taken together, we found that HER2 induces BCSCs by upregulating IL1a and IL6 expression. We for first time demonstrating that IL1α plays a critical role in HER2-induced expansion of BCSC population and may be a potential therapeutic target for HER2 positive breast cancer.

#2886

CD44/SMURF1 signaling maintains cancer stem cell-like invasive cells in head and neck squamous cell carcinoma.

Ali Khammanivong,1 Raj Gopalakrishnan,2 Erin B. Dickerson3. 1 _University of Minnesota Masonic Cancer Research Center, Minneapolis, MN;_ 2 _University of Minnesota School of Dentistry, Minneapolis, MN;_ 3 _University of Minnesota College of Veterinary Medicine, Minneapolis, MN_.

Head and neck squamous cell carcinoma (HNSCC) is highly invasive and resistant to therapies, where treatments often result in high rates of failure and disease recurrence. A subpopulation of tumor-initiating cancer stem cells (CSC) is thought to be responsible for metastatic invasion and drug resistance in many types of cancer, including HNSCC. CD44 is a known CSC marker in HNSCC but its role in maintaining CSC populations is not well understood. We previously reported that SMAD specific E3 ubiquitin protein ligase 1 (SMURF1) inhibition of bone morphogenetic protein (BMP) signaling is essential for maintaining a CD44-high CSC-like population in HNSCC. In this study, we sought to determine how CD44, a receptor for hyaluronic acid (HA) that constitutes a major component of the tumor stroma, is involved in the regulation of SMURF1 and the maintenance of a HNSCC CSC phenotype. CSC-like cells were enriched from HNSCC cell lines as CD44-high cells. CD44 signaling was stimulated by exogenous HA treatment and inhibited by CD44 knockout (KO) using an inducible CRISPR/Cas9 system. We then assessed changes in SMURF1 protein level, BMP signaling, transwell migration, Matrigel colony formation, and invasion through three-dimensional organotypic culture (OTC) following CD44 modulation. CD44-high cells were found to have increased extracellular HA production. Treatment with exogenous HA reduced BMP signaling, as determined by a reduction in phospho-SMAD1/5/8 levels, and increased transwell migration of CD44-high cells. CD44-KO reduced SMURF1 protein expression and inhibited Matrigel colony formation of an invasive and recurrent HNSCC-derived CD44-high cells but only partially reduced colony formation of a less-invasive cell line derived from a primary HNSCC. Knockout of CD44 expression showed a slight reduction in transwell migration of invasive cells. CD44-high cells also recapitulated an invasive and CSC-like growth pattern in OTC assays. In contrast, CD44-KO inhibited OTC invasion and the epithelial-to-mesenchymal transition (EMT) phenotype, resulting in increased apical epithelial growth and differentiation. CD44 reconstitution appeared to restore the invasive and EMT phenotype. Based on our current findings, CD44 may be crucial for maintaining an anchorage-independent and invasive phenotype of HNSCC but plays a minor role in carcinoma cell migration.

#2887

Macrophage conditioned medium promotes colon cancer stem cell phenotype via the hedgehog signaling pathway.

Fan Fan, Rui Wang, Delphine R. Boulbes, Huiyuan Zhang, Stephanie S. Watowich, Rajat Bhattacharya, Ling Xia, Xiang-Cang Ye, Lee M. Ellis. _UT MD Anderson Cancer Ctr., Houston, TX_.

Background: Contradicting studies have shown that increased macrophages infiltration in colorectal cancer (CRC) could either result in anti-tumoral or in pro-tumoral effects. Because of these inconsistencies, we sought to determine the role of conditioned medium from macrophages, in particular classically activated macrophages, on the development of the CSC phenotype in CRC cells, which is believed to mediate tumor growth and chemoresistance.

Methods: Murine (CT26) and human (HCP-1) CRC cell lines were treated with conditioned media (CM) from lipopolysaccharide (LPS)-activated murine macrophages. Viability studies were performed using the MTT assay, annexin V-FITC assay, and Western blot analysis. The CSC population was assessed using the sphere-forming assay and aldehyde dehydrogenase assay.

Results: The results showed that 1) murine LPS-activated macrophage CM induced apoptosis in murine and human CRC cells, 2) murine and human LPS-activated macrophage CM secreted paracrine factors that promoted the CSC phenotype in murine and human CRC cells via the Sonic hedgehog (SHH)-Gli signaling pathway, which is known to drive self-renewal and 3) these effects were blocked by depletion of SHH in macrophage CM.

Conclusions: Our data show that LPS-activated macrophages play an active role in promoting the CSC phenotype through activation of the SHH-Gli signaling pathway.

#2888

Dopamine signaling and therapeutic resistance in GBM.

Seamus P. Caragher, Cheol H. Park, Fatemeh Atashi, Shivani Baisiwala, Atique U. Ahmed. _Northwestern University, Chicago, IL_.

Glioblastoma multiforme (GBM), the most common and aggressive primary brain tumor in adults, is characterized by aggressive recurrence after conventional treatment, which include surgery followed by radiation and chemotherapy. This recurrence reilies on GBM cells' ability to promote therapeutic resistance. The recently developed Cancer Stem Cell (CSC) hypothesis argues that GBMs are driven by a rare subset of cells called Glioma Stem Cells (GSCs). A significant corollary of the CSC hypothesis is that GSCs have the ability to survive intensive radio- and chemotherapy and give rise to recurrent disease. Recent studies from our laboratory and others have shown a high degree of plasticity in GBM cells that indicated normal GBM cells acquire a stem-like state during anti-glioma therapy. Termed conversion, this molecular process increases the frequency of therapy resistant GSCs and promotes disease recurrence. We hypothesize that a drug capable of blocking tumor cell plasticity would prevent the generation of therapy-resistant GSCs, thereby block GBM recurrence. Based on this concept, we have developed a plasticity-based high-throughput drug screening method for GSCs. Our initial screening has identified several novel FDA-approved compounds that act as antagonists of neurotransmitter receptors. Further investigation revealed that treatment of patient-derived xenograft (PDX) GBM lines with temozolomide (TMZ) led to the formation of a population of cells that express both CD133, a marker of CSCs, and dopamine receptors 2 and 3 (DRDs) (control mean CD133+/DRD2+: 19.7%, TMZ mean: 83.8%, p=.0001). PDX lines treated with a dopamine receptor agonist exhibited increased expression of SOX2, OCT4 and c-Myc, GSC-markers. However, this treatment did not increase the expression of Ki67 in the GSC compartment, a marker for cell proliferation (mean DMSO: 63.8%, mean TMZ: 57.4%). Thus, DRD signaling may influence the plasticity driven GSC niche. Utilizing a CD133-reporter cell line, we found that two anti-psychotics, chlorprothioxene and loxapine, prevented the induction of CD133 expression caused by treatment with TMZ. Finally, we treated PDX lines with chlorpromazine, a newer version chlorprothioxene, in conjunction with TMZ, which resulted in increased therapeutic effecacy (p<.0001 by MTT assay). Overall, these data suggest that dopamine receptor signaling and stress-induced cellular plasticity interact in GBM. We proposed that dopamine receptors represent a strategy by which specific molecular changes induces by canonical chemotherapeutic agents can be inhibited to prevent the induction of resistance in surviving cells.

#2889

V-ATPase control of EV signaling in glioma stem cells.

Irene Bertolini,1 Andrea Terrasi,1 Andrea Di Cristofori,1 Silvano Bosari,2 Valentina Vaira3. 1 _University of Milan, Milan, Italy;_ 2 _University of Milan & Fondazione Ca' Granda, Milan, Italy; _3 _University of Milan & Fondazione INGM, Milan, Italy_.

Background: Recent evidences highlighted that GBM secreted microvesicles (EVs), particularly exosomes (Exo) and large oncosomes (LO), play a major role in the cross-talk between tumor cell and non-neoplastic parenchyma. How GBMs manage to thrive in a highly unfavorable, acidic microenvironment is still unclear, but recent work from our group has identified the vacuolar pump H+-ATPase (V-ATPase) as an important effector of GBM growth and glioma stem cells (GSC) maintenance. Additionally, in ExoCarta database V-ATPase subunits have been described in Exo from different cancer cell types.

Taken together, these data identify V-ATPase as an important driver of gliomagenesis, and a novel, actionable therapeutic target for disease intervention. However, the role of V-ATPase in reprogramming the GBM microenvironment has not been previously investigated.

Methods: Exo and LO were isolated by an Invitrogen kit and serial centrifugation, respectively, from media of patients' derived GBM neurospheres, enriched in GSC (NS, n=12) or differentiated cultures (n=8). For EVs internalization studies, Exo or LO were stained using FM 1-43 FX dye and the process was followed live for 30' and at selected time points (30'-4h-24h), using a confocal microscopy or flow cytometry (FACS). Electron microscopy, FACS (of Exo stained with CellTrace and SytoRNA in combination with CD63 coated beads), Nanosight and immunoblotting (for CD63, CD9 and Clathrin) analyses were used to confirm EVs subtypes. Cultures from patients' derived brain tumor margins or primary GBM (differentiated and not) were used as EVs-recipient cells. miRNA profiling was performed using Taqman Low density arrays and analyzed by R packages. Gene Ontology analysis was performed by DAVID. The study was approved by the Institutional Ethical Committee.

Results: NS are able to produce different EVs, which are internalized by recipient cells after 4 and up to 24 hours of co-culture. Both Exo than LO from NS are able to significantly increase cell growth in recipient cells (brain tumor margins and primary GMB differentiated monolayers), and this effect is stronger with EVs produced by NS with higher V-ATPase expression (V-ATPaseHIGH NS). Primary GBM cells after co-culture with EVs are able to produce a higher number of NS and V-ATPase activity block by BafilomycinA1 in NS-producing EVs completely revert this effect. Finally, the co-culture of V-ATPaseLOW NS with EVs from V-ATPaseHIGH NS increases their motility in collagen matrixes.

At molecular level, profiling of Exo-derived miRNAs distinguishes differentiated cultures from NS and, among NS, V-ATPaseHIGH cultures. In silico analysis and annotation of miRNA target genes from V-ATPaseHIGH–derived Exo showed an enrichment of cancer, cell cycle and PI3K/Akt pathways.

Conclusions: Altogether, these data point toward the central role of different EV types in GBM communication and suggest a role of the V-ATPase proton pump in regulating EV's contents.

#2890

MicroRNA-mediated upregulation of the WNT signaling activities in human breast cancer stem cells.

Yohei Shimono,1 Taichi Isobe,2 Andrei Turtoi,3 Junko Mukohyama,1 Toru Mukohara,1 Akira Suzuki,1 Vincent Castronovo,3 Hironobu Minami1. 1 _Kobe University, Kobe, Japan;_ 2 _Stanford University, Stanford, CA;_ 3 _University of Liege, Liege, Belgium_.

The canonical WNT signaling plays a critical role in many adult stem cells, including those of the breast and intestine. The fact that the canonical WNT signaling is implicated in both stem cell self-renewal and cancer suggests that normal physiological regulator of stem cell functions might be "hijacked" in cancer. Adenomatous polyposis coli (APC) is a component of the destruction complex that destabilizes β-catenin and suppresses the activity of the canonical WNT signaling.

MicroRNAs (miRNAs) are important regulators of stem cell functions. We have previously reported a set of 37 miRNAs that are upregulated or downregulated in human breast cancer stem cells (BCSCs, a CD44+CD24-/lowlineage\- population of human breast cancer cells) as compared to non-tumorigenic breast cancer cells (NTCs). Among them, miR-200c targets BMI1 that is a critical regulator of the stem cell maintenance, and strongly impairs the functions of human BCSCs in vivo.

In this study, we compared the expression profiles of miRNAs, mRNAs and proteins between BCSCs and NTCs isolated from the patient specimens of human breast cancers and patient-derived tumor xenografts (PDXs) established by their transplantation. Luciferase assays were performed using the plasmid in which the 3'UTR region of candidate mRNA was cloned downstream of a luciferase minigene. The effect of miRNAs on the activity of WNT signaling was evaluated using a TCF reporter plasmid. Finally, the abilities to form organoids and to form tumors in immunodeficient mice were evaluated using the human BCSCs infected with the miRNA inhibitor expressing lentivirus.

We found that miR-142 was highly upregulated in BCSCs, but was hardly expressed in NTCs in the patient breast cancer specimens. We confirmed that miR-142 targeted the sequence within the 3'UTR of APC mRNA and suppressed APC protein expression. Accordingly, miR-142 activated the canonical WNT signaling pathway in an APC-suppression dependent manner. The results of mRNA and protein expression profiling of the BCSCs isolated from human breast cancer PDXs suggested that the canonical WNT signaling was activated in BCSCs. Finally, inhibition of miR-142 in the BCSCs suppressed the tumor growth in vivo.

These results suggest that the miR-142, a miRNA frequently upregulated in human BCSCs, could provide at least a part of the molecular mechanism for aberrant activation of the canonical WNT signaling in breast cancer in which APC mutations are much less frequent than colon cancer.

#2891

Retinoic acid directs breast cancer cell state changes through regulation of TET2-aPKC pathway .

Meng-Ju Wu, Chun-Ju Chang. _Purdue University, West Lafayette, IN_.

The key molecular mechanism governing the cancer cell state (stem cell-like state vs. differentiation state) to control the cancer stem cell (CSC) pool remains elusive. This study provides the first evidence showing that all-trans retinoic acid (ATRA) induces the interaction and chromatin recruitment of a novel RARB-TET2 complex to epigenetically activate a specific cohort of gene targets, including MiR-200c. TET2-activated miR-200c further targets and suppresses aPKC, a cell polarity protein that plays a pivotal role in directing asymmetric division of mammalian stem cells to sustain the stem cell pool. Our data reveals that pharmacological concentration of ATRA effectively down-regulates aPKC through activation of miR-200c, leading to a decrease of the stem cell-like populations from non-tumorigenic mammary epithelial cells and non-aggressive breast cancer cells. However, aggressive breast cancer cells that manifest TET2-miR200c dysregulation sustain a CSC pool highly resistant to ATRA, where inhibition of aPKC directs the resistant CSCs to the luminal cell-like state and sensitization to Tamoxifen, resulting in abrogation of mammary tumor growth and progression. Together, these findings elucidate a novel RARB-TET2-miR200c-aPKC signaling pathway that directs cancer cell state changes and also provide previously unidentified therapeutic implications for aPKC inhibitors in diminishment of breast CSCs to eradicate breast cancer.

#2892

Oncostatin M elicits cellular plasticity through cooperative STAT3-SMAD3 signaling.

Benjamin L. Bryson, Damian J. Junk, Jacob Smigiel, Neetha Parameswaran, Mary R. Doherty, Courtney A. Bartel, Mark Jackson. _Case Western Reserve University, Cleveland, OH_.

Increasing evidence suggests that tumor cell plasticity promotes metastasis and tumor recurrence, resulting in cancer patient mortality. While it is clear that the tumor microenvironment (TME) contributes to tumor cell plasticity, the specific TME factors that actively generate tumor cell plasticity are largely unknown. Here, we identify TME cytokines that promote epithelial-mesenchymal plasticity, and acquisition of cancer stem-cell (CSC) properties. A screen of 27 TME cytokines identified multiple Interleukin-6 family members as inducers of mesenchymal/CSC properties, with Oncostatin M (OSM) being the most potent. Importantly, OSM induced plasticity was mediated by STAT3, but also dependent on TGF-β signaling and downstream SMAD3. Inhibition of functional TGF-β/SMAD signaling by expressing a dominant-negative TGF-β receptor, treating with a TGF-β receptor inhibitor, or suppressing SMAD3 expression using a SMAD3-shRNA prevented the OSM-induced mesenchymal/CSC properties. OSM-activated STAT3 binds to SMAD3 and promotes SMAD3 nuclear localization and binding to the SNAIL promoter, increasing transcription of the SNAIL gene. The epithelial-mesenchymal transition induced by the STAT3/SMAD3 axis results in a highly invasive and metastatic phenotype and the emergence of CSC properties, including therapeutic resistance. Importantly, maintenance of the OSM-induced mesenchymal/CSC state requires sustained exposure to the cytokine, as removal of OSM results in a marked phenotypic reversion. A high-throughput screen for small molecule inhibitors of the OSM-induced mesenchymal/CSC phenotype has been performed and are currently being confirmed. We propose that, targeted blockade of the STAT3/SMAD3 axis in tumor cells may represent a novel therapeutic approach to prevent the plasticity associated with metastasis and tumor recurrence.

#2893

IGF2 is essential for tumor initiating cell activity in human colorectal cancer.

Taronish D. Dubash,1 Christine Siegl,1 Sebastian M. Dieter,1 Joachim Weischenfeldt,2 Alexandros P. Drainas,2 Laura Schwarzmueller,1 Malgorzata Oles,2 Balca Mardin,2 Mikolaj Slabicki,1 Huber Wolfgang,2 Martin Schneider,3 Jan Korbel,2 Hanno Glimm,1 Claudia R. Ball1. 1 _National Ctr. for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 2 _European Molecular Biology Laboratory (EMBL), Heidelberg, Germany;_ 3 _Heidelberg University Hospital, Heidelberg, Germany_.

A small fraction of all cells within individual tumours from colorectal cancer (CRC) patients drives long term tumor growth and metastases in immune-compromised mice. Targeting these tumor-initiating cells (TIC) may improve the long-term outcome in advanced CRC. To identify candidate genes which drive proliferation and survival of TIC, we have performed a large scale high throughput loss of function shRNA screen in three-dimensional TIC enriched patient spheroids. Spheroids were transduced with the barcoded Cellecta decipher library comprising 27,500 shRNAs targeting 5043 genes associated with cell signaling pathways (Module 1). Two weeks later, cells were harvested for DNA isolation, barcode amplification and high-throughput barcode sequencing. Amongst others, we found 5/6 shRNAs targeting Insulin growth factor 2 (IGF2) scoring within a 20% depletion threshold, presenting depletion levels very similar to positive control shRNAs. mRNA expression profiling and qPCR analyses demonstrated low to moderate expression of the IGF2 gene product in the majority of patient spheroid cultures analyzed (n=17). In contrast, two out of 15 patient derived spheroid cultures analyzed demonstrated very pronounced IGF2 overexpression (>250 fold). Integrative SCNA profiling, expression and TAD profiling using the CESAM algorithm, followed by 4C Seq, demonstrated a tandem duplication of the IGF2 locus in these two patients which interrupts a IGF2 adjacent TAD boundary and results in de novo contact domain formation between the IGF2 promoter and a normally hidden distant super-enhancer. A dual luciferase reporter assay revealed that the hijacked enhancer is functionally active in human CRC cells and thereby may drive unphysiological IGF2 expression following enhancer hijacking. To assess the functional relevance of IGF2 tandem duplications, spheroids were transduced with RFP expressing lentiviral vectors encoding for 3 shRNAs targeting IGF2 as well as control shRNAs targeting EIF3A or scrambled shRNA. Strikingly, IGF2 knockdown led to a marked reduction of RFP\+ cells in competitive proliferation assays and markedly reduced viability assessed by ATPlight assay. Moreover, IGF2 knockdown strongly reduced tumor formation following xenotransplantation into immune deficient NSG mice. These data demonstrate that IGF2 is required for survival, proliferation and tumor-initiation of primary human TIC enriched spheroids. Notably, oncogenic miRNA-483 is encoded within intron 8 of IGF2, however, its function in IGF2 locus tandem duplicated cells remains elusive. Understanding the mechanisms of IGF2 dependency and the role of miRNA-483 in this context will be essential for the future development of therapeutic approaches targeting IGF2 expression in this patient subset harbouring tandem-duplications of the IGF2 locus.

#2894

Epinephrine-induced gamma-secretase/Notch signaling in pancreatic cancer stem cells.

Xuemin Xu, Chen Hu, Hildegard M. Schuller. _Univ. of Tennessee, Knoxville, TN_.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer deaths with 5-year survivals below 10%. Cancer stem cells represent a small population of cells in PDAC and are thought to drive the development, progression, metastasis and drug resistance of this cancer. The Notch pathway regulates the self-renewal of pancreatic cancer stem cells as well as angiogenesis and drug resistance. Gamma-secretase cleaves the Notch-1 receptor, resulting in release of the Notch intracellular domain (NICD) that then translocates to the nucleus and activates the transcription of target genes. Aberrant Notch signaling activated by γ-secretase activity is associated with progression, angiogenesis and drug resistance of PDAC and is being explored as a target for PDAC therapy. Smoking and psychological stress are risk factors for PDAC. However, the potential modulation of γ-secretase/Notch by these risk factors has not been investigated to date. Nicotine and psychological stress both cause hyperactivity of the sympathetic nervous system and adrenal glands, resulting in increased blood and organ levels of the stress neurotransmitters norepinephrine (NOR) and epinephrine (EPI), which are the physiological agonists of beta-adrenergic receptors (β-ARs). In turn, β-ARs are coupled to the stimulatory G-protein Gαs that stimulates the formation of intracellular cAMP, which activates multiple signaling pathways in a cell type-specific manner. Using spheroid formation assays and Western blotting, our data show, -for the first time,- that the stress neurotransmitter Epi significantly increases the self-renewal of pancreatic cancer stem cells by increasing the expression of the activated forms (c-terminal) of the γ-secretase component presenilin1 and of Notch1 (NICD). These responses were inhibited by the β-AR antagonist (beta-blocker) propranolol and by the inhibitory neurotransmitter γ-aminobutyric acid (GABA) that inhibits cAMP formation via activation of Gαi-coupled GABAB receptors. These findings suggest that the γ-sexretasesecretase/Notch pathway in pancreatic cancer stem cells is under regulatory control by neurotransmitters, with Epi as stimulator and GABA as inhibitor. These data could open up new avenues for the prevention and therapy of PDAC. Additional experiments are currently underway to further investigate mechanistic aspects of this novel concept. Supported by RO1CA042829 with NIH.

#2895

Octamer-4/microRNA-1246 signaling axis drives Wnt/β-catenin activation in liver cancer stem cells.

Stella Chai,1 Kai-Yu Ng,1 Man Tong,1 Eunice Y. Lau,2 Terence K. Lee,2 Kwok Wah Chan,1 Yun Fei Yuan,3 Tan To Cheung,1 Siu Tim Cheung,4 Xiao Qi Wang,1 Nathalie Wong,4 Chung Mau Lo,1 Kwan Man,1 Xin Yuan Guan,1 Stephanie K. Ma1. 1 _The University of Hong Kong, Pokfulam, Hong Kong;_ 2 _The Hong Kong Polytechnic University, Hong Kong, Hong Kong;_ 3 _Sun Yat-Sen University Cancer Center, Guangzhou, China;_ 4 _The Chinese University of Hong Kong, Hong Kong, Hong Kong_.

Hepatocellular carcinoma (HCC), the main type of liver cancer in human, is one of the most prevalent and deadly malignancies in the world. Despite advances in therapy, prognosis remains dismal, largely attributed by our limited understanding on information related to the progressive development of the disease, particularly in their cancer-initiating and stem cell-like properties. Wnt/β-catenin signaling is activated in CD133 liver cancer stem cells (CSC), a subset of cells known to be a root of tumor recurrence and therapy resistance in HCC. However, the regulatory mechanism of this pathway in CSCs remains unclear. Here, we show that human miRNA, miR-1246, promotes cancer stemness including self-renewal, drug resistance, tumorigencity and metastasis by activation of Wnt/β-catenin pathway via suppressing the expression of AXIN2 and GSK3β, two key members of the β-catenin destruction complex. This observation was validated by both in vitro and in vivo functional / cell biological studies on HCC cell lines with or without miR-1246 expression modulated by lentiviral based knockdown and overexpression strategies as well as in miR-1246 repressed HCC cells with concomitant expression of wild-type or constitutively active β-catenin. Clinically, high endogenous and circulating miR-1246 was identified in HCC clinical samples and correlated with a worse prognosis. Further functional analysis identified Oct4 to be the direct upstream regulator of miR-1246, which cooperatively drive β-catenin activation in liver CSCs. In conclusion, our findings not only uncover the non-canonical regulation of Wnt/β-catenin in liver CSCs by Oct4/miR-1246 signaling axis, but also provide a novel diagnostic marker as well as therapeutic intervention for HCC.

#2896

STAT1 dependent interferon-related DNA damage resistance signature (IRDS) as a survival mechanism in castrate resistant prostate cancer (CRPC).

Supreet Agarwal, Kerry McGowen, Keith Jansson, Mike Beshiri, Fathi Elloumi, Maggie Cam, Kathy Kelly. _NCI, Bethesda, MD_.

Using the organoid culture system and aggressive Pten/Tp53-null mouse model (GEMM), we have previously shown that the prostate cancer cell population harbors two classes of self-renewing luminal progenitors which are resistant to in vivo castration and to androgen receptor (AR) inhibitors (enzalutamide) ex vivo. Understanding signaling pathways governing intrinsic survival/self-renewal ability of luminal progenitors in castrate conditions can highlight pathways that play a role in acquired drug resistance. To identify mechanisms of castration resistance in luminal progenitors, we performed RNAseq analysis of luminal progenitor organoids derived from wild-type(WT) and Pten/Tp53-null mice (intact and castrated (two weeks), n=5; each). Gene enrichment analysis identified key signaling pathways altered in luminal tumor organoids (AR signaling, lipid metabolism, protein secretion, inflammation etc.) that have also been described for FACS-purified human prostate luminal (CD49flo) fraction. Interestingly, we found no difference in transcriptional profiles of intact and castrated tumor organoids, suggesting intrinsic survival ability of luminal progenitors upon castration. Of note, we observed most significant enrichment of STAT1-dependent IRDS in luminal progenitor tumor organoids relative to wild type luminal organoids. IRDS comprises of a subset of STAT1-driven genes that have been previously associated with survival of cancer cells and with breast cancer therapy resistance. Our analysis of human prostate cancer datasets revealed IRDS as a prognostic marker for progression in the TCGA primary prostate cancer cohort (p<0.05). Further, high IRDS-expressing CRPC samples (SU2C dataset) were enriched for low AR signaling (r= -0.33, p< 0.05). CRPC patients in IRDS-hi cohort showed enrichment for cancer stem cell phenotype and for genes associated with drug resistance, Consistent with the bioinformatics analysis real time PCR, immunofluorescence and western blot analysis of ex-vivo organoid cultures of castration-resistant Pten/Tp53-null tumor organoids showed higher protein expression of STAT1 and IRDS genes in luminal tumor organoids relative to luminal WT organoids. In vivo, castrated prostate tumors showed higher STAT1 levels than the intact tumors. Treatment with enzalutamide of luminal tumor organoids resulted in time dependent increase in STAT1 expression. STAT1 KD in tumor organoids decreased number of progeny organoids in subsequent generations suggesting either a direct or indirect effect upon self-renewal. Overall, our initial findings suggest STAT1 dependent signaling as a potential mechanism of androgen-independent survival in prostate cancer.

#2897

Oncogenic Kras activation in gastric adenocarcinoma promotes cancer stem cell phenotypes including metastasis & chemotherapy resistance.

Changhwan Yoon,1 Kevin K. Chang,1 Jacob Till,2 Sandra W. Ryeom,2 Sam S. Yoon1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Abramson Family Cancer Research Institute, Philadelphia, PA_.

INTRODUCTION:

The Cancer Genome Atlas found that the receptor tyrosine kinase (RTK)-Ras signaling pathway is altered in half of gastric adenocarcinomas (GAs). We added oncogenic Kras to the Atp4b-Cre;Cdh1fl/fl;Trp53fl/fl mouse model of GA and found accelerated tumorigenesis and metastasis. We also found Kras activity to be higher in gastric cancer stem-like cells (CSCs). Thus we hypothesized that Kras activty is critical for maintenance of gastric CSCs and promotes CSC phenotypes.

METHODS:

Human and murine GA cell lines were examined. The effect of Kras pathway inhibition was examined in GA spheroid cells and monolayer cells in various in vitro assays. Kras activity and Kras pathway inhibition was examined in CSCs, primary tumors and metastases in our GA mouse model. The combination of chemotherapy and Kras pathway inhibition was tested in human GA xenografts.

RESULTS:

Kras inhibition with shRNA or MEK inhibitor PD0325901 decreased the ability of GA cells to form spheroids and deceased expression of the stem cell transcription factor, Sox2. CD44(+) gastric CSCs had 65.8-75.4% higher migration, 64.2-79.5% higher invasion, and 63.1-70.2% more anchorage-independent growth compared to unselected cells. These properties could all blocked by 71.1-82.7% with Kras shRNA or PD0325901. In our GA mouse model, PD0325901 starting at 6 weeks of life increased median survival from 76 days to 95 days. Primary tumors and metastases from these mice with and without treatment with MEK inhibition are being analyzed for level of CD44 CSCs, Kras activity, and extent of metastases. Gastric CSCs were resistant to 5-fluorouracil and cisplatin chemotherapy, and this chemotherapy resistance could be reversed in human GA xenografts transducing cells with Kras shRNA.

CONCLUSION:

Kras is more active in gastric CSCs than non-CSCs and promotes malignant phenotypes including metastasis and chemotherapy resistance. Kras pathway inhibition can block these CSC phenotypes, and thus may prove useful in various combination therapies.

#2898

**ST8SIA1 is up-regulated in triple negative breast cancer and its expression is positively correlated with** TP53 **mutations and a cancer stem cell gene signature.**

V. Lokesh Battula, Yuanqing Yan, Khoa Nguyen, Kim-Anh Do, Michael Andreeff. _UT MD Anderson Cancer Ctr., Houston, TX_.

We discovered ganglioside GD2 as a breast cancer stem cell (BCSCs) marker in triple negative breast cancer (TNBC). GD2 biosynthesis is tightly regulated by the enzyme ST8SIA1 (GD3 synthase). However, expression of ST8SIA and its association with different breast cancer sub-types is not known. Here we hypothesize that ST8SIA1 is up-regulated in TNBC or "basal-type" breast tumors and associated with stemness in primary breast tumors. To investigate ST8SIA1 expression in primary tumors, we analyzed RNAseq data from the cancer genome atlas (TCGA) data base, which includes data from 1105 primary and metastatic breast tumors as well as adjacent normal tissues. We found that ST8SIA1 expression varied widely among different breast tumors. Interestingly, "basal-type" tumors expressed the highest levels of ST8SIA1 compared to all other types of breast cancers including luminal-A or luminal-B or HER2-enriched tumors (p < 0.01). In addition, TNBC (n = 115) expressed 4.6-fold higher ST8SIA1 levels compared to hormone receptors positive tumors including ER+ or PR+ or HER2+ tumors (n = 852, p<0.001). Survival analysis by log-rank test indicated that patients with ST8SIA1high tumors survive shorter (median survival 2.6 years) compared to patients with ST8SIA1low tumors (median survival 4.3 years). Next, we investigates the association between ST8SIA1expression and the most commonly mutated genes in breast cancer. We chose the top 20 most frequently mutated genes in TCGA dataset and examined their correlation with ST8SIA1 mRNA expression. We found that, among the top 20 mutations, TP53 had a very strong positive correlation with ST8SIA1 expression (p < 0.00001). In fact, the expression of ST8SIA1 was > 2-fold higher in TP53-mutated compared to wild type tumors. The other positively correlated mutation was a nuclear envelop protein called the spectrin repeat containing protein (nuclear envelope 1 or SYNE1; p < 0.05). Mutations in GATA3 were the most negatively correlated (p < 0.001) with ST8SIA1 expression. Interestingly, GATA3 plays a role in epithelial cell differentiation in the mammary gland, supporting the notion that ST8SIA1 is a stem cell-associated gene. In addition, correlation of ST8SIA1 mRNA expression with other genes revealed that FOXA1, the protein which is co-expressed with GATA3 and serves as negative predictor of "basal-type" of breast cancer, was down regulated in ST8SIA1high tumors. In conclusion, ST8SIA1 is associated with "basal-type" TNBC tumors and it has a strong positive correlation with TP53 mutations and negative correlation with GATA3 mutations. Knowing that TP53 mutations have a major role in tumorigenesis and drug resistance, these data suggest that ST8SIA1 could be a potential therapeutic target in patients with TP53-mutant TNBC.

#2899

Hypoxia-inducible factor 1-α maintains sarcoma stem-like cells in hypoxic regions of tumors and promotes migration and invasion via upregulation of platelet-derived growth factor receptors.

Kevin K. Chang, Changhwan Yoon, Sam S. Yoon. _Memorial Sloan Kettering Cancer Center, New York, NY_.

Introduction: Sarcomas often thrive in hypoxic conditions to proliferate and metastasize. To survive in such an environment, sarcomas upregulate hypoxia inducible factor-1α (HIF-1α), which enhances the transcription of over 150 genes mediating tumor metabolism, angiogenesis, and metastasis. HIF-1α may be important for the maintenance of a subset of tumor cells referred to as "sarcoma stem-like cells" or SSCs. Platelet-derived growth factor receptor (PDGFR) is important in mesenchymal biology and may be a potential target of HIF-1α.

Methods: Three human sarcoma cell lines were examined: HT1080 (fibrosarcoma), SK-LMS-1 (leiomyosarcoma), and DDLS8817 (dedifferentiated liposarcoma). Cell lines were grown as spheroids on ultra-low attachment dishes to enrich for SSCs. The roles of HIF-1α and PDGFR-α/β in promoting SSC phenotypes, such as migratory/invasive capacity, were assessed via several in vitro assays under hypoxic conditions.

Results: Sarcoma cell lines grown as spheroids had increased levels of HIF-1α and increased phosphorylation of PDGFR-α/β as compared to when grown as monolayers; these effects were even more pronounced when sarcoma spheroids were grown under hypoxic conditions. Hypoxic conditions also increased migratory and invasive capacity in spheroid cells by 65-73% as compared to normoxic conditions. HIF-1α knockdown using shRNA in the three cell lines decreased expression of self-renewal proteins and decreased spheroid formation by 67-80%. HIF-1α knockdown decreased expression of epithelial-mesenchymal transition (EMT) transcription factors and reduced migratory and invasive capacity by 80-87% under normoxic conditions and 85-87% under hypoxic conditions. Phosphorylation of PDGFR-α/β was decreased in sarcoma cell lines following HIF-1α knockdown under normoxic and hypoxic conditions. PDGFR-α/β inhibition with imatinib reversed chemotherapy resistance in normally resistant SSCs, and treatment of HT1080 xenografts with doxorubicin chemotherapy and imatinib reduced tumor formation by 85% as compared to control and by 77% as compared to chemotherapy alone.

Conclusions: The HIF-1α/PDGFR axis supports the maintenance of SSCs, especially under hypoxic conditions, and promotes SSC phenotypes such as migratory/invasive capacity and chemotherapy resistance. Combining chemotherapy with PDGFR-α/β inhibition may be a promising strategy to reverse chemotherapy resistance and prevent metastasis in sarcomas.

#2900

Sigma-1 receptor control tumorigenic and stem cell-like phenotype in human cancers.

Gianluca Civenni,1 Martina Marchetti,1 Shusil Pandit,1 Celeste De Monte,1 Federica Sereni,1 Jessica Merulla,1 Sabrina Zadic,1 Marco Losa,1 Sara Allegrini,1 Domenico Albino,1 Sarah Mapelli,1 Erik Laurini,2 Bernhard Wunsch,3 Sabrina Pricl,2 Giuseppina M. Carbone,1 Carlo V. Catapano1. 1 _Institute of Oncology Research, Bellinzona, Switzerland;_ 2 _University of Trieste, Trieste, Italy;_ 3 _University of Münster, Munster, Germany_.

Tumor-initiating stem-like cancer cells drive tumor progression, metastasis and treatment failure. Understanding the pathways driving self-renewal and expansion of cancer stem-like cells (CSCs) in human cancers may provide actionable therapeutic targets for developing novel treatment strategies. Metabolic reprogramming and mitochondrial homeostasis are emerging as key features of CSCs. The sigma-1 receptor (Sig-1R) is a ligand-activated chaperone protein localized at the ER-mitochondria membrane and involved in inter-organelle signaling, mitochondrial homeostasis and stress response. Sig-1R is up-regulated in many human cancers, although its role in tumorigenesis is unclear. Here, we examined the role of Sig-1R on survival and expansion of cancer cells with stem-like properties using genetic knockdown and selective pharmacological antagonists. Transient and stable depletion of Sig-1R using small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs), respectively, affected clonogenic and tumor-sphere forming capability of prostate cancer cell lines. Cell proliferation and viability under standard culture conditions were minimally affected. Selective high affinity Sig-1R antagonists also suppressed clonogenicity and tumor-sphere formation, reproducing the effects of genetic depletion. Notably, stable Sig-1R knockdown with shRNAs drastically reduced development of tumor xenografts in mice, indicating a reversal of tumorigenic and stem-like properties upon depletion of Sig-1R. Sig-1R knockdown impaired mitochondrial function with reduced mitochondrial membrane potential (flow cytometry JC-1 assay) and respiratory capacity (Seahorse Mito Stress assay). These effects were more evident under metabolic stress induced by glucose starvation and were associated with drastic changes in morphology and intracellular distribution of mitochondria. Similar metabolic effects were seen with selective Sig-1R antagonists. These findings indicate that Sig-1R sustains tumorigenic properties by enhancing mitochondrial homeostasis and metabolic adaptability of stem-like cancer cells ensuring their long-term survival and self-renewal capability. Targeting the Sig-1R with selective antagonists could be an innovative approach to cancer treatment capable of preventing survival and expansion of tumor-initiating stem-like cancer cells in human cancers.

#2901

**Wnt/p53 regulate stemness of mucoepidermoid carcinoma cells** in vitro **.**

Christie Rodriguez-Ramirez, April Andrews, Shaomeng Wang, Jacques E. Nor. _University of Michigan-Ann Arbor, Ann Arbor, MI_.

Mucoepidermoid carcinoma (MEC) is the most common malignant salivary gland cancer, yet very little is known about the pathobiology of this disease. As a result, no mechanism-based therapies have been developed for the treatment of MEC. Little to no response has been observed with the use of conventional chemotherapies. Consequently, standard of care is limited to surgery and/or radiation therapy, resulting in high patient morbidity. Work done by our group showed that cancer stem cells (CSCs), identified by ALDHhighCD44high cells, are drivers of MEC tumorigenesis. MI-773 is an inhibitor of the MDM2/p53 protein-protein interaction that results in p53 accumulation and activation of p53-mediated signaling. Our group found that treatment of MEC cell lines (UM-HMC-1, UM-HMC-3A, UM-HMC-3B) with sub-lethal doses of MI-773 significantly decreases the CSC population in vitro and in xenograph tumors. Moreover, recent studies have implicated p53-regulated mi-RNAs in the transcriptional regulation of several components of the Wnt/beta-Catenin pathway. We hypothesize that the canonical Wnt and p53 pathways crosstalk to regulate MEC stem cell maintenance. To address this hypothesis, we isolated or measured the fraction of the CSCs via flow cytometry, conducted sphere assays that measure survival/self-renewal of CSCs, and used western blots for cell signaling studies. We found that Wnt1, active beta-Catenin, and p53 protein levels are higher in CSCs than the non-CSCs in MEC cell lines. Moreover, we found that MI-773 reduces the expression of key Wnt pathway proteins, such as Wnt1 while also decreasing the number of spheres formed by MEC cells. Opposite results are observed when p53 is silenced. Notably, inhibiting the beta-Catenin pathway with PNU74654 or JW67 also decreases the fraction of CSCs and inhibits sphere formation. Collectively, these results indicate that both the Wnt and p53 pathways are important for MEC stem cell maintenance. Revealing pathways important for MEC stem cell maintenance will help identify effective mechanism-based therapies to sensitize MEC to conventional chemotherapies. This work was funded by grants R01-DE21139 and R01-DE23220 from the NIH/NIDCR.

#2902

Epithelial-type CD133+ stem-like lung cancer cells emerge higher drug resistance through MDFIC-mediated Wnt/β-catenin signaling pathway.

Chao-Ju Chen,1 Chih-Jen Yang,1 Ming-Shyang Huang,1 Yu-Peng Liu2. 1 _Kaohsiung Medical University Hospital, Kaohsiung, Taiwan;_ 2 _Kaohsiung Medical University, Kaohsiung, Taiwan_.

Background: The epithelial-to-mesenchymal transition (EMT) has been described to promote drug resistance and cancer stem cell (CSC) properties. Many studies demonstrated that a major proportion of circulating tumor cell (CTC) exhibits EMT and CSC characteristics. A recent finding revealed the presence of epithelial type (EpCAM+) CTC was associated with poor outcome, whereas the mesenchymal type (EpCAM-) CTC were not. We therefore hypothesize that the epithelial-type CSC may exhibit higher drug resistant ability.

Methods: We isolated epithelial type (E+) and mesenchymal type (E-) CD133+ cells and CD133- cells from PC14 lung cancer cell line by fluorescence assisted cell sorting. We used western blot, QPCR, immunofluorescence, sphere formation assay and tumor xenograft assay to characterize the 4 subpopulations. The drug resistant ability was determined by cell viability assay and in vivo drug response assays. The drug resistant signature was identified by comparing the gene expression profile from four subpopulations and Gene Expression Omnibus (GEO) database. Knockdown and overexpression of MDFIC in PC14 subpopulations were established by using lentivirus vectors. Immunoprecipitation and subcellular fractionation were performed for drug resistant mechanism investigation.

Results: The epithelial type PC14 CD133+ cells (E+/CD133+ subpopulation) exhibited higher sphere formation ability and was more resistant to the treatment of chemotherapy agents compared to the mesenchymal type CD133+ cells (E-CD133+ subpopulation) in vitro and in vivo. Gene expression profiling showed 86 genes were bioinformatically predicted as drug resistant signature and were correlated with the disease free survival of the patients with lung cancer. Among these genes, the mRNA level of 20 genes were significantly related to the patient's prognosis in the GSE31210 dataset. Human I-mfa domain-containing protein (MDFIC) was highly expressed in E+/CD133+ subpopulation. Knockdown and overexpression of MDFIC modulates drug resistance ability in cancer cells. MDFIC increased the level of free β-catenin through binding and stabilizing the axin-GSK3-β-β-catenin destruction complex and increased the transcriptional activity of Wnt/β-catenin signaling.

Conclusion: The epithelial-type CD133+ stem-like lung cancer cells are more resistant to the chemotherapy through MDFIC-mediated Wnt/β-catenin signaling activation.

#2903

1-Methoxyphaseollidin: Novel gamma secretase inhibitor targeting notch-1 signaling in breast cancer stem cells.

Venkatesh Kolluru,1 Deeksha Pal,1 Becca Baby,1 Houda Alatassi,1 Arun Kumar Sharma,2 Murali Ankem,1 Chendil Damodaran1. 1 _University of Louisville, Louisville, KY;_ 2 _Pennsylvania State University, Hershey, PA_.

We recently showed that two different ALDH+ and CD44+/CD24-/low breast cancer stem cells (BSCSs) exhibited stem cell characteristics that include self-renewal, extensive proliferation, the ability to form non-adherent spherical clusters, chemotherapy resistance and high Notch1 expression. We have identified a compound compound: 6-(3-methylbut-2-enyl) coumestrol (Pso) and treatment with Pso resulted in growth inhibition and an EMT phenotype in both BCSCs and BC cells. Oral Pso administration at physiologically achievable doses (25 mg/kg/BW) suppressed the growth of BCSCs and BC xenografts without toxicity. In the current studies, we identified several novel Pso-derived analogs that may be more potent than the parent compound. One such compound, 1-methoxyphaseollidin (1MP), obtained via three main functional group changes: (i) translocation of the isoprenyl moiety from the phenyl ring fused to the pyran ring (as in Pso) to the phenyl ring adjacent to the furan ring, (ii) removal of the carbonyl group from the pyran ring, and (iii) introduction of a methoxy group at the 1-position, inhibited Notch1 activity and growth of both BSCS and BC cells at nM concentration (IC50: 300nM), which is 100 times more potently than Pso in cell culture models. Molecular studies suggest that 1MP inhibits Notch signaling pathways (Hes1, Hey1 and Presenilin) in both BCSC and BC cells. Further, downregulation of AKT signaling (pAKT (S473), p65 and BCl-2 were seen in 1MP treated cells. Docking studies suggest that 1MP binds outside of the catalytic unit of γ-secretase and induces a conformational change, resulting in Notch1 inhibition in both BCSCs and BC cells. More importantly, administration of 1MP significantly inhibited the growth of BCSC and BC tumors without causing gastrointestinal toxicity in tumor-bearing mice. H&E staining suggests that 1MP treated tumors show infiltrate to margins are less as compared to vehicle treated mice's. We believe targeting notch1 signaling and optimizing 1MP could be an effective therapeutic strategy for treating breast cancer.

#2904

Targeting cancer stem-like cells in triple negative breast cancer cells through non-canonical notch signaling.

Fokhrul Hossain,1 Claudia Sorrentino,1 Ayse Bilyeu,1 Judy Crabtree,1 Antonio Pannuti,1 Todd Golde,2 Barbara Osborne,3 Lucio Miele1. 1 _LSUHSC, New Orleans, LA;_ 2 _University of Florida, FL;_ 3 _UMass Amherst, MA_.

Triple negative breast cancer (TNBC) is a heterogeneous group of clinically aggressive diseases. TNBC patients have high risk of recurrence and metastasis, and current treatment options remain limited. Cancer stem-like cells (CSCs) have been linked to cancer initiation, progression and chemotherapy resistance. Therefore CSC-targeted therapies are keenly sought. There is strong evidence for the involvement of Notch signaling in TNBC. Notch1 is highly expressed in Basal-like 1 (BL1) and especially Mesenchymal-Stem-Like (MSL) TNBCs. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. Moreover, strong evidence supports key roles of different Notch paralogs in breast CSCs. Here, we demonstrate that Notch1 promotes cell survival in MDA-MB-231 cells, representative of MSL TNBC, in part by activating NF-κB. Notch activation by Jagged1-expressing stromal cells enhances transcription of the anti-apoptotic gene cIAP-2 (BIRC3), a known NF-κB target. This event is dependent on recruitment to the cIAP-2 promoter of NF-κB subunits, IKKα and Notch1. Short term exposure of MDA-MB-231 cells (MSL, PTEN wild-type), but not MDA-MB-468 cells (BL1, PTEN-null) to recombinant Jagged1 leads to AKT phosphorylation. This is suppressed by dual mTORC1/2 inhibitors, AKT inhibitors and IKKα inhibitors but not Everolimus (mTORC1-selective inhibitor). These observations support a model where canonical and non-canonical mechanisms downstream of Notch1 trigger AKT phosphorylation and NF-κB activation in PTEN wild type TNBC cells. Rapid AKT phosphorylation downstream of Notch1 requires mTORC2, PI3K and IKKα, and contributes to NF-κB activation. This suggests a bidirectional crosstalk between the IKKα and AKT arms of this Jagged1-activated pathway. We demonstrate that recombinant Jagged1 increases the cellular metabolism of TNBC cells and knockdown of Notch1 or IKKα by siRNA decreases mitochondrial respiration and glycolysis. We have found that CSCs derived from MDA-MB-231 cells have increased Notch1, p-AKT, and oxidative metabolism. AKT inhibition or IKKα inhibition decreases both mitochondrial respiration and glycolysis of TNBC derived CSCs. Pharmacological inhibition of Notch cleavage by gamma secretase inhibitor (PF-03084014) in combination with AKT inhibitor (MK-2206) or NF-κB inhibitor (Bay11-7082) blocks CD90hi or CD44+CD24low sorted secondary mammospheres formation. These data suggest that combination treatments affecting Notch, NF-kB and AKT pathways have potential therapeutic importance in targeting CSCs of TNBC cases with wild type PTEN and high Notch1 expression.

### Tumor Evolution and Heterogeneity 1

#2905

Clinical and genomic resistance to second generation androgen blockade in paired biopsies of metastatic castration-resistant prostate cancer.

G. Celine Han,1 Justin Hwang,1 Stephanie A. Mullane,1 Carrie Cibulskis,2 Zhenwei Zhang,1 Rana R. McKay,1 PCF-SU2C Dream Team, Scott L. Carter,1 William C. Hahn,1 Mary-Ellen Taplin,1 Eliezer M. Van Allen1. 1 _Dana-Farber Cancer Inst., Boston, MA;_ 2 _Broad Institute, Boston, MA_.

Background

Recent "next generation" androgen deprivation therapies (ADT), such as abiraterone and enzalutamide, have improved survival in patients with castrate-resistant prostate cancer (CRPC). Despite therapy, most patients develop resistance to these agents. We investigated the genetic basis of tumor evolution and clinical resistance to next generation ADT in CRPC by using whole exome sequencing (WES) on paired pretreatment and post-resistance biopsies from CRPC patients.

Methods

Matched "trios" of germline, pre-treatment and post-resistant tumor samples were obtained from 7 patients treated with abiraterone (n=4) and enzalutamide (n=3) and WES was performed. Clinical data, including PSA and radiographic measurements, was used to classify patients as intrinsically resistant or initially responsive to treatment. Quality control, mutation and indel calling, copy number variation identification were performed using analytical pipelines at the Broad Institute. Tumor purity and ploidy were inferred, and phylogenetic analysis was performed using ABSOLUTE and Phylogic, respectively to identify resistance associated alterations in the context of clinical phenotypes.

Results

We identified multiple putative mechanisms and genetic categories of resistance to next generation ADT in CRPC. One abiraterone patient acquired an AR mutation (L702H) in the post-treatment sample, previously reported to be associated with poor outcomes and to confer resistance to this therapy. Amplification of MYC was associated with resistance in two other abiraterone patients with pre-existing AR gain: one patient with initial response to therapy acquired a focal gain of MYC in the resistant sample, and the other harbored a pre-existing MYC amplification, but had no response to therapy. In three enzalutamide patients, all acquired alterations in cell cycle genes (CDKN2A mutation, focal amplifications in regions spanning CDK4/6, and CCND3 focal amplification) at the time of resistance that were absent in pre-treatment tumors. In parallel, we confirm CDK4/6 overexpression was sufficient to promote enzalutamide resistance in prostate cancer cell models. Additional candidate genomic events associated with CRPC were also implicated with this approach, such as RSPO3 amplification.

Conclusion

This study outlines an approach to identify clinical genetic resistance mechanisms of next generation ADT in CRPC through integration of genomic data from serial biopsies, clinical patient outcomes, and preclinical functional screening. These findings confirm previously known potential resistance mechanisms, such as AR and MYC activation, and suggest putative mechanisms

linked to therapeutic agents, which may inform novel targets for treating CRPC patients.

#2906

Characterization of genetic intratumor heterogeneity of colorectal cancer and matching organoids.

Sigrid S. Arnadottir,1 Maria Jeppesen,2 Philippe Lamy,1 Iver Nordentoft,1 Michael Knudsen,1 Søren Vang,1 Mogens R. Madsen,3 Jacob Thastrup,4 Ole Thastrup,4 Claus L. Andersen1. 1 _Dept. of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark;_ 2 _Digestive Disease Center, Bispebjerg Hospital, University of Copenhagen, Copenhagen, Denmark;_ 3 _Surgical Research Unit, Department of Surgery, Herning Regional Hospital, Herning, Denmark;_ 4 _2cureX, Birkerød, Denmark_.

Background: In the recent years it has become evident that intratumor heterogeneity of solid tumors, such as colorectal cancer (CRC), complicates development of efficient therapy strategies. A minor clone of the tumor might have the ability for treatment resistance and hence the ability for re-establishing the tumor. Organoid cultures derived from the patients' tumors can be used for ex vivo drug screening prior to treatment of the patient. However this approach is only efficient if the organoids represent the heterogeneity of the tumor. In this study we aim to characterize the genetic intratumor heterogeneity of CRC by performing whole exome sequencing (WES) on multiple biopsies per tumor, and to see how well the heterogeneity is reflected in matching organoid cultures.

Methods: From five CRC patients, three biopsies were collected from separate areas of each tumor. Each biopsy was divided in two: one half was fresh frozen for DNA purification, and the other half was grown in vitro as organoid culture. When available, lymph node metastases (LNMs) were included. WES was performed using SeqCap EZ Exome v3.0, and Illumina NextSeq500. Matched germline DNA was used as reference to identify somatic mutations and copy number variations (CNVs) using Mutect2 and FACETS, respectively.

Results: Each tumor contained multiple mutations that were present in all biopsies and in the organoids as well, representing a common ancestral branch. However all tumor biopsies and organoids also contained private mutations. These constituted on average 10% of the mutations (range 3-18%) indicating spatial genetic heterogeneity in all tumors. Each organoid had private mutations not seen in the tumor area of origin and vice versa, indicating that each area contained multiple clones and only a subset was represented in the organoids. The extent of mutational differences between organoids and their area of origin was similar to the mutational differences between tumor areas. Implying that organoids do not reflect their local origin better than a single biopsy reflects the whole tumor. In one patient with LNMs it was observed that the mutational profile of the metastases resembled only one of the examined tumor areas. Surprisingly, not all the ancestral mutations found in the tumor biopsies were observed in the LNMs, suggesting that at least two clones co-existed in the area of origin and just one of these formed the metastases.

Conclusion: In the five patients studied; spatial genetic heterogeneity was observed, meaning that multiple biopsies are needed to picture the whole tumor. Genetic heterogeneity was also observed between primary tumor and metastases and our data support that the metastases were formed from a single cancer clone that did not dominate the primary tumor. From a genetic point of view, organoids do not seem to fully reflect the tumor area of origin and less so the whole tumor, indicating that care should be taken when using organoids as models of the primary tumor.

#2907

**Extracellular matrix remodeling triggered by lysyl oxidase inhibition promote the lung adenocarcinoma to squamous cell carcinoma transition independent of** LKB1 **status.**

Shun Yao, Hongbin Ji. _Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sc, Shanghai, China_.

Introduction: Lung adeno-squamous cell carcinoma represents as the most malignant subtype of non-small cell lung cancer. We have previously shown that Lkb1-deficiency triggered the lung adenocarcinoma (ADC) to squamous cell carcinoma (SCC) transition (AST) in mice through extracellular matrix remodeling, in which lysyl oxidase (LOX) plays an important role. Considering the high frequency of P53 mutations in human lung cancer, we reason it's attractive to test if LOX inhibition could modulate the lung cancer plasticity and subtype transition independent of LKB1 status.Methods: KrasG12D/P53L/L (KP) mice were used to treat with BAPN or DPA,two LOX inhibitors, daily via intraperitoneal injection post 4 weeks of Adeno-Cre infection for 6-8 weeks and then harvested the lungs for pathological analyses. The histology and expression of related markers were analyzed by H&E, IHC staining, quantitative PCR and western blot. Results: BAPN/DPA treatment could result in a dramatic reduction of collagen deposition. We found no significant changes of total tumor number, but decreased average tumor size and tumor burden in treatment groups. Detailed pathological analyses showed that a few tumors from treatment group displayed typical squamous pathology, with the expression of squamous markers P63 and K14. In contrast, the tumors from control groups were adenomatous pathology and only expressing ADC markers TTF1 and SP-C. We further found that either BAPN or DPA treatment in KP mice promoted apoptosis and inhibited cell proliferation in ADC. Interestingly, we found the transited SCC showed almost no growth arrest and low apoptosis rate after BAPN or DPA treatment, indicative of the resistance to LOX inhibitors. ECM deprivation is a major cause of oxidative stress that contributes to the accumulation of reactive oxygen species (ROS) through the deregulation of certain metabolic pathways. We then checked the level of 8-hydroxydeoxyguanosine (8-oxo-dGuo), a marker of DNA oxidative modification by ROS. In treatment groups, 8-oxo-dGuo was significantly lower in SCC than ADC. In our previous work done in KL model, we found that the ROS level was significantly up-regulated by LKB1 inactivation, which functionally modulated the AST. However, in KP mouse model, either LKB1 or p-AMPK level showed no significant change with or without BAPN/DPA treatment.Conclusions: These data demonstrated that pharmacological LOX inhibition could trigger the ADC to SCC transition independent of LKB1 status, highlight a non-cancer-cell autonomous role in phenotypic transition. Moreover, this lineage transition confers lung cancer with strong survival capability under environmental stress as well as the acquisition of drug resistance.

#2908

Single-cell genetic analysis of ductal carcinoma in situ with and without synchronous invasive breast cancer by multiplex FISH delineates specific patterns of tumor clonality and heterogeneity.

Irianna Marie Torres,1 Leanora Hernandez,1 Jausheng Tzeng,2 Russell Schwartz,3 Alejandro Schaffer,4 Edward Gertz,4 Stephen Brower,2 Miguel Sanchez,2 Gert Auer,5 Kerstin Heselmeyer-Haddad,1 Ried Thomas1. 1 _National Cancer Institute, Rockville, MD;_ 2 _Englewood Hospital and Medical Center, Englewood, NJ;_ 3 _Carnegie Mellon University, Pittsburgh, PA;_ 4 _NCBI, Bethesda, MD;_ 5 _Karolinska Hospital, Stockholm, Sweden_.

We previously studied synchronous Ductal Carcinomas in Situ (DCIS) and Invasive Ductal Carcinomas (IDC) using a novel approach of multiplexing FISH probes that allows us to simultaneously assess the copy numbers of up to 20 loci within intact nuclei providing new insights into tumor clonality and heterogeneity. A high degree of chromosomal instability already in DCIS, and frequently, but not always, a direct clonal evolution from DCIS to IDC was detected. We now ask whether this degree of instability is also present in DCIS that did not progress to IDC and are therefore analyzing FFPE material from 20 patients with either low-grade and high-grade DCIS who did not present with invasive breast carcinoma during their follow-up (5-10 years), in addition to 10 patients who presented with synchronous IDC. The multiplex FISH assay used targets five oncogenes (COX2, MYC, CCND1, HER2, ZNF217) and three tumor suppressor genes (DBC2, CDH1, TP53) frequently altered in breast carcinomas. To date, we have analyzed two paired cases of synchronous DCIS and IDC, DCIS-IDC 3 and 4, and three cases of DCIS without IDC, OP-DCIS 1, 4 and 5. The paired cases exhibited very similar aberration patterns for synchronous DCIS and IDC indicating the invasive carcinoma is closely related to the DCIS lesion. Specifically, the major clones in DCIS 3 (low-grade) and IDC 3 showed a diploid tumor cell population with a gain of COX2, and losses of DBC2, MYC, TP53, and HER2. Of note, the only difference observed was a gain of ZNF217 in the DCIS which was not seen in the major clone of the invasive carcinoma. The other paired case, DCIS 4 (high-grade) and IDC 4, showed major clones of triploid tumor cell populations with gains of COX2, CCND1 and MYC and losses of DBC2, CDH1, TP53 and ZNF217. The three DCIS cases without synchronous or subsequent IDC exhibited varying degrees of aberration and complexity patterns in their clonal populations. Case OP-DCIS 1, a high-grade DCIS, revealed a tetraploid cell population which showed a major clone with an amplification of CCND1 and a MYC gain combined with losses of DBC2, CDH1, TP53 and HER2. A low-grade DCIS, OP-DCIS 4, exhibited a diploid cell population with a major clone showing loss of CDH1 as the only aberration. The third case OP-DCIS 5 (low-grade DCIS) exhibited a diploid cell population with one major clone showing losses of CDH1, MYC, DBC2, TP53, HER2 and CCND1. Our preliminary observations show a tendency of diploid lesions with a predominance of loss patterns for low-grade DCIS, while high-grade DCIS seem to reveal higher ploidy with more complex gain and loss patterns. However, also low-grade DCIS with mainly loss patterns progress to invasive cancers as seen in case DCIS-IDC3. We expect that the analysis of the remaining cases will further elucidate the dynamics of DCIS lesions which will hopefully help to assess and stratify progression risk in patients with DCIS.

#2909

High-precision quantification of clonal evolution during comparative treatments of triple negative breast cancer.

Hyunsoo Kim,1 Pooja Kumar,1 Francesca Menghi,1 Eliza Cerveira,1 Mallory Romanovitch,1 Guru Ananda,1 Joshy George,1 Henry Chen,2 Susan Mockus,1 Chengsheng Zhang,1 Yan Yang,2 James Keck,2 R. Krishna Murthy Karuturi,1 Carol Bult,3 Charles Lee,1 Edison Liu,3 Jeffrey H. Chuang1. 1 _The Jackson Laboratory for Genomic Medicine, Farmington, CT;_ 2 _The Jackson Laboratory Mice, Clinical, and Research Services, Sacramento, CA;_ 3 _The Jackson Laboratory for Mammalian Genetics, Bar Harbor, ME_.

Population heterogeneity within tumors is essential to the development of drug resistance. However, precise quantification of cellularity levels of subpopulations, and in particular how they evolve in response to treatment, has been challenging. Here we describe the high precision characterization of subclonal evolution within triple-negative breast cancer patient-derived xenografts (PDXs) generated from three patients. For each patient model, we established multiple PDXs and treated them in cohorts of 5-10 mice each for the therapies cyclophosphamide, doxorubicin, cisplatin, or docetaxel, with treatments lasting one month. In all three patient models, the average behavior across mice was a reduction in size in response to docetaxel, but growth under doxorubicin and cyclophosphamide. For cisplatin treatment, one of the three models showed tumor shrinkage while the other two models showed continued growth but at a rate lower than the doxorubicin or cyclophosphamide cohorts. To determine the evolutionary behaviors underlying these observations, we initially performed exome panel sequencing of 34 residual tumor samples from these and untreated xenografts. Computational mutation and copy number analysis indicated sample-specific differences in tumor populations both in response to treatment and due to genetic drift. However, they also revealed measurement uncertainties related to exome capture efficiency, locus-specific read counts, and computational copy number estimation that limited quantitative inference of evolutionary behaviors. To solve this problem we used droplet digital PCR (ddPCR) to measure variant allele frequencies and local copy number at selected loci from the prior residual tumors as well as additional samples from replicate cohort treatments and cultured conditionally reprogrammed progenitor cells. In total we performed 1665 ddPCR measurements across 150 cancer samples. These ddPCR measurements reduced sample-specific uncertainty in variant allele frequency to ~2% and copy number to ~0.2, allowing for precise identification of subclones and their cellularities in each sample. We observed several common modes of evolution within these tumors including selective sweeps, spatial diffusion, and stable coexistence between distinct subpopulations. In the samples from one patient model, we observed frequent symbiotic growth of two distinct subpopulations having differential cisplatin sensitivity, such that the degree of clonal selection during cisplatin treatment was proportional to the tumor volume change on a mouse-to-mouse basis. This study demonstrates how high precision genomic characterization across comparatively treated samples can reveal treatment-relevant subclonal ecology, as well as the mutations that distinguish populations with different behaviors.

#2910

Inter metastatic genetic heterogeneity is a characteristic feature of recurrent pancreatic cancer.

Hitomi Sakamoto, Alvin Makohon-Moore, Marc Attiyeh, Liguo Zhang, Christine A. Iacobuzio-Donahue. _Memorial Sloan Kettering Cancer Center, New York, NY_.

Pancreatic cancer (PDA) is a 4th leading cause of cancer death with an overall 5-year survival of 8%. Surgical resection remains the only option for cure but only 10-15% of newly diagnosed patients are eligible. While surgery clearly provides a survival benefit compared to those patients who do not undergo potentially curative resection, the vast majority of patients will recur in the pancreatic remnant, liver, lungs, and/or peritoneum. Thus, there is an unmet need to better understand the fundamental features of recurrent disease. To explore the fundamental genomic features of recurrent PDA, patients were selected from participating Gastrointestinal Cancer Rapid Medical Donation Program (GICRMDP). 10 patients who underwent surgical resection; 8 patients had a whipple procedure, 2 patients had a distal pancreatectomy and each patient received a different type of chemotherapy after surgery, was evaluated. We performed 250x whole exome sequencing (WES) of 82 unique samples from these 10 patients, using frozen samples from autopsy and FFPE tissues from matching surgical specimen. In addition to the treatment naïve primary tumor, one or more samples of the local recurrence and multiple metastases were studied. The mean depth of coverage for all samples was 317x. Copy number variations were assessed by FACETs. MSK-IMPACT was performed for orthogonal validation of a subset of variants. We found that compared to the treatment naïve resected tumor, recurrent disease contained novel mutations in genes associated with MAPK and PI3K signaling. The most common mutational signature was Signature 1 followed by Signature 3. All patients who received a platinum drug as a form of therapy had a greater proportion of Signature 3 and 9 mutations in the recurrent disease than the primary tumor. Phylogenetic studies revealed that recurrent disease is the result of three patterns: 1) dissemination of disease to distant organs before surgery was performed, 2) dissemination of disease after surgery from residual microscopic disease in the pancreatic remnant, or 3) development of a second primary tumor in the remnant pancreas. This third scenario was identified in one of six additional patients studied by IMPACT. We conclude that recurrent pancreatic cancer is multifactorial, and at least two of these scenarios could be eliminated by total pancreatectomy, thereby increasing the rate of cure by surgical resection. Our study serves as a window to an understanding of the clonal dynamics of recurrent pancreatic cancer.

#2911

Single cell mass cytometry analysis of human lung adenocarcinoma.

Deon B. Doxie, Jonathan M. Lehman, Yong Zou, Maria S. Ortega, Caroline E. Maier, Jonathan M. Irish, Pierre P. Massion. _Vanderbilt Univiersity, Nashville, TN_.

Introduction: Lung cancer is the leading cause of cancer-related mortality in the world. Lung adenocarcinoma is the most common subtype. Tumor heterogeneity among adenocarcinomas presents a challenge in the management of the disease. Understanding heterogeneity may have implications in understanding the biological processes driving progression. Single cell platforms like mass cytometry offer an opportunity to profile tumor heterogeneity and to identify populations of driven by the activation of signaling pathways.

Methods: Adenocarcinomas were collected at the time of surgery and dissociated into suspension and cryopreserved. Mass cytometry analysis of tumors and adenocarcinoma cell lines was performed with a 30 marker antibody panel and rhodium intercalator dye to identify dead cells. The antibody panel included markers to characterize identity, cell cycle status, and signaling events. Biaxial gating or unsupervised analysis approaches SPADE and viSNE were used to compare major populations of cells.

Results: Validation of mass cytometry panels was performed with adenocarcinoma cell lines and human tumors. Adenocarcinoma cell lines PC9 (lung), A549 (lung), H520 (squamous) and SW620 (colon) exhibited phenotypically distinct cells between samples by expression of cytokeratin, CK7, and EGFR. Furthermore, PD-L1 expression was expressed on subsets within cell lines. Disaggregation of tumors was optimized with dissociation that included collagenase and DNase to release viable cells within 1 hour. Detection of viable cells was optimized with rhodium intercalator viability dye and histone H3 to identify nucleated cells. Using the 30 marker panel and viSNE analysis four populations were identified within tumors as infiltrating leukocytes, endothelial cells, fibroblast, and epithelial tumor cells. Epithelial cells were found to exhibit cellular heterogeneity between patients based on the expression of cytokeratin, CK7, TTF1, EGFR, vimentin, CD44, and MET. Preliminary signaling data identified basal kinase activity active in infiltrating leukocytes and cancer cells. Cancer cells and infiltrating immune cells had basal p-STAT5 activation, and cancer cells had high basal p-AKT implicated in dysregulated cell growth.

Conclusions: Populations of infiltrating stromal cells and epithelial (cancer) cells were identified in lung adenocarcinoma. The single cell phenotyping from tumors was consistent with the profile found in two lung adenocarcinoma cell lines. Preliminary differences in basal signaling pathways responsible for growth were observed within adenocarcinoma cell populations. This work demonstrates the feasibility of mass cytometry to identify and characterize tumor heterogeneity. Work is underway to define phenotypes within the epithelial and leukocyte populations that could predict tumor behavior and immune response within the microenvironment. This work is supported by NCI CA196405 to PPM.

#2912

Size based enrichment and sorting of Ov90 cancer cells and clusters with a new multistage filtration cartridge reveals distinct phenotypes.

Anne Meunier,1 Sara kheireddine,1 J. Alejandro Hernández-Castro,1 Teodor Veres,2 David Juncker1. 1 _McGill University, Montreal, Quebec, Canada;_ 2 _National Research Council of Canada, Boucherville, Quebec, Canada_.

Background: Circulating tumor cells (CTCs) are released in blood from the primary tumor, but although very heterogeneous both in size and marker expression, are very rare and provide information not available from the primary tumor. The identification of CTC cells and clusters could advance our understanding of metastasis and help personalize therapy.1,2 Notably, CTC clusters were shown to have a higher metastatic potential than single cells3 but the process remains poorly understood.

We present a multistage filtration system with pore sizes from 20 down to 8 μm for the size-selective enrichment of Ov90 ovarian cancer cells and clusters from blood. Each captured cell population was released, cultured and characterized independently.

Methods: We developed a 3D printed multistage filtration cartridge and polymer filters with 20, 15, 12, 10 and 8 μm-diameter pores. Filters were stacked from 20 (top) to 8 μm (bottom) and used to enrich and sort Ov-90 cells spiked in 1:6 mL of blood:PBS. Captured cells were released by removing individual filters from the cartridge, reverse flowing OSE medium, and then cultured separately.

Results: Ov-90 clusters were found mostly on the top filter (20 μm) and interestingly, few small clusters (3-4 cells) were found on the 8 and 10 μm filters, suggesting alignment of cluster cells as they pass through the pores.4 Cell and nucleus diameters were measured, and a general correlation was found between filter pore size and cell and nucleus size. Interestingly, nucleus size was found to be the single most significant parameter in determining passage of single cells and small clusters through pores.

Following cell culture, two distinct phenotypes were observed: cell captured on small pore filters (8-12 μm) grew primarily in a monolayer. Cells captured on filters with larger pores (15-20 μm) first grew as monolayer, but rapidly formed cell aggregates that subsequently detached from the surface. Staining for E-Cadherin, a cell-cell adhesion protein, revealed a loss of expression of cells from filter with larger pores.

Conclusion: We developed a new multistage filtration method and selectively enriched and sorted cells based primarily on their nucleus size. We identified two Ov-90 populations with different growth behaviors with low E-cadherin expression on the cells forming clusters, which is known to correlate with metastasis. The application of multistage filters may also reveal different CTC populations based on nucleus and cell size.

References:

(1) Baccelli, I. et al. A. Nat. Biotech. 2013, 31, 539-544.

(2) Pecot, C. V. et al. Cancer discovery 2011, 1, 580-586.

(3) Cheung, K. J. et al. Proc. Natl. Acad. Sci. U.S.A. 2016, 113, E854-E863.

(4) Au, S. H. et al. Proc. Natl. Acad. Sci. U.S.A. 2016, 113, 4947-4952.

#2913

**Emergence of** RAS **or** EGFR **mutant clones affects duration of response to EGFR blockade in colorectal cancers.**

Sabrina Arena,1 Beth Van Emburgh,1 Giulia Siravegna,1 Luca Lazzari,1 Giovanni Crisafulli,1 Giorgio Corti,2 Benedetta Mussolin,2 Federica Baldi,3 Michela Buscarino,2 Alice Bartolini,2 Emanuele Valtorta,4 Joana Vidal,5 Beatriz Bellosillo,5 Giovanni Germano,2 Filippo Pietrantonio,6 Agostino Ponzetti,7 Joan Albanell,5 Salvatore Siena,4 Andrea Sartore-Bianchi,4 Federica Di Nicolantonio,1 Clara Montagut,5 Alberto Bardelli1. 1 _University of Torino, Candiolo, Italy;_ 2 _Candiolo Cancer Institute - FPO, Candiolo, Italy;_ 3 _University of Torino, Torino, Italy;_ 4 _Niguarda Cancer Center, Milano, Italy;_ 5 _Hospital del Mar, Barcelona, Spain;_ 6 _Istituto Nazionale dei Tumori, Milano, Italy;_ 7 _San Giovanni Battista Hospital, Torino, Italy_.

Cetuximab and panitumumab are monoclonal anti-EGFR antibodies (moAbs) currently used for the treatment of advanced RAS wild type colorectal cancers (CRC). Emergence of acquired resistance invariably limits the efficacy of these agents, and the dynamics of clonal evolution during anti-EGFR blockade are poorly understood. At progression, RAS mutations represent the most common genetic alterations, while EGFR extracellular domain (ECD) mutations are acquired by a smaller cohort of patients. We found that the mutation profile correlates with the clinical outcome of patients; in particular those who develop RAS mutations upon EGFR blockade achieve reduced tumor shrinkage and shorter duration of response respect to patients in which EGFR ECD mutations emerge during therapy. We investigated in preclinical models the potential role of RAS and EGFR ECD mutations during the emergence of acquired resistance, by tracking the evolution of clones in a genetically barcoded population of CRC cells chronically treated with cetuximab. We observed that therapeutic (target therapy, chemotherapy) and environmental (reduced nutrient condition) pressures differentially shape the clonal composition of CRC cell populations, leading to the emergence of clones with the highest fitness in presence of the external pressure. In conclusion, a multistep clonal evolution process characterizes the development of drug resistance and is associated with the clinical outcome of CRC patients treated with anti-EGFR antibodies.

#2914

Molecular characterization with single-cell resolution of CTCs and FFPE specimens from the same lung adenocarcinoma patients reveals the extent of intra-tumor heterogeneity.

Mario Terracciano,1 Francesco Gelsomino,2 Francesco Bacchi,1 Francesca Fontana,1 Claudio Forcato,1 Alberto Ferrarini,1 Michelangelo Fiorentino,3 Valentina Del Monaco,1 Giulio Bassi,1 Chiara Mangano,1 Chiara Bolognesi,1 Paola Tononi,1 Genny Buson,1 Gianni Medoro,1 Nicolò Manaresi,1 Michele Tognetto,2 Andrea Ardizzoni2. 1 _Menarini Silicon Biosystems SpA, Castel Maggiore, Bologna, Italy;_ 2 _Policlinico Sant'Orsola – Malpighi, Bologna, Italy;_ 3 _Policlinico Sant'Orsola-Malpighi, Bologna, Italy_.

Introduction

Intra-tumor heterogeneity can hide genomic and genetic features, which may be key driver of disease progression. Routinely, only one biological specimen per patient is generally analyzed, which may only partially represents the genetics of the tumor. Here we report a multi-approach analysis of Circulating Tumor Cells (CTCs) and formalin-fixed paraffin-embedded (FFPE) tumor tissue-derived cells (TCs) obtained from the same patients, to investigate the underlying genetic heterogeneity.

Methods

Peripheral blood (PB) and FFPE tumor tissue were collected from two advanced lung adenocarcinoma patients, treated with cisplatin-pemetrexed and carboplatin-pemetrexed respectively as first line therapy. The first patient was previously diagnosed an ALK-traslocation and treated with an ALK-inhibitor. PB was enriched with either an EpCAM-based or EpCAM-independent method: the cell output of the latter was stained with Cytokeratin-PE, CD45-APC and DAPI. Matched FFPE sections were obtained from pleural effusion cell blocks for the first patient or from primary tumor tissue for the second one; after dissociation, cells were stained with Vimentin-APC, Keratin-FITC and DAPI. The DEPArray™ platform was used to detect and collect pure single CTCs or TCs, along with WBCs or stromal cells as controls. Whole genome amplified DNA of single CTCs and TCs was used to profile genome-wide copy-number aberrations (CNAs) using the Ampli1™ LowPass kit; single nucleotide variants were analyzed on CTCs WGA products and on pools of TCs using Ampli1™ CHP custom panel and DEPArray™ OncoSeek panel respectively.

Results

No clinically significant variants were detected in CTCs and FFPE samples; however the copy-number profiles of single TCs and CTCs revealed an overabundance of gains and losses, confirming the aberrant nature of tumor cells. In the first patient, all single cells showed a pattern of shared alterations, with a common amplification of the genome region comprising MYC gene (also confirmed by depth-of-coverage in targeted panel). A hierarchical unsupervised clustering clearly separated WBCs, from the group of TCs and CTCs, characterized by some emerging clones and low inter-cell heterogeneity. The analysis of the copy-number profiles of cells from the second patient showed an opposite situation; unsupervised clustering of low-pass profiles highlighted an independent group formed by single TCs clearly distinct from the highly heterogeneous cluster formed by CTCs.

Conclusions

The precision granted by analysis of pure cells derived from multiple specimens from the same patient, together with the combination of low-pass whole-genome sequencing and targeted sequencing, reveals unexpected genetic similarities and diversities, and provides fundamental information to understand intra-tumor heterogeneity.

#2915

The timing of mutational burst events impact the growth of tumors in the colon.

Chelsie K. Sievers, Tien N. Vo, Perry J. Pickhardt, Bryan D. Pooler, Kristina A. Matkowskyj, Dawn M. Albrecht, Quincy Rosemarie, Michael A. Newton, Richard B. Halberg. _University of Wisconsin, Madison, WI_.

Basic and clinical scientists believe that benign polyps in the colon progress to cancers through the slow, stepwise accumulation of mutations. Interestingly, only a small percentage of all tumors progress, whereas a significant number remain static in size, regress, or resolve completely. The mechanisms underlying these differential fates are unknown, and mechanisms of tumor evolution during this premalignant phase are still under investigation and continued debate. We previously reported that sub-clonal diversity arises early in small human adenomas and contributes to the growth of tumors in the colon. An emerging hypothesis is that colorectal tumors form and progress via a process of punctuated equilibrium, where multiple copy number alterations and mutational events happen simultaneously in a burst-like fashion. In this study, we prospectively test this concept using a mouse model in which tumor induction is spatially and temporally controlled via a non-surgical delivery of adenovirus expressing CRE recombinase and a temporally controlled mutational burst via administration of the carcinogen Azoxymethane. Tumors are induced at a similar rate regardless the timing of the burst relative to tumor induction with a mean tumor incidence of 62% at three weeks post induction. However, tumor growth may be affected by the timing of the burst. Animals that had a mutational burst event prior to tumor induction or those that had a late mutational burst event had a lower average in vivo growth rate compared to controls and those with an early burst event, but the average in vivo tumor size was comparable across all groups. Taken together, these preliminary data provide evidence that the timing of a mutational burst event contributes to tumor growth. This prospective experiment is being extended through computer modeling and statistical inference to compare in silico mutational landscapes to a cohort of colon polyps removed from patients at normal screening. The findings will provide new insights into the earliest stages of tumorigenesis.

#2916

The clonal evolution of glioblastoma.

Jennifer Brooke Goldstein,1 Ravesanker Ezhilarasan,1 Mona Jaffari,1 Alessandro Carugo,1 Giulio Draetta,1 Roeland Verhaak,2 Sahil Seth,1 Erik Sulman,1 Phillip Andrew Futreal1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _The Jackson Laboratory, ME_.

Background: Glioblastoma (GBM) is an aggressive cancer that often recurs despite multimodal therapy. Median survival is 12-15 months. Genomic profiling studies have shown marked tumor heterogeneity with distinct mutations among treated and untreated samples. The Cancer Genome Atlas (TCGA), revealed that mutations in TP53, CDKN2A, PTEN, EGFR and NF1 are predominant in untreated tumors. Of 19 patients who progressed on therapy, seven samples were hypermutated with mutations in mismatch repair proteins, mainly MSH6. We proposed to use the Cellecta Lentiviral-based tagging library, a novel model system to assess the role of intratumor heterogeneity (ITH) in the presumptive tumor-initiating fraction of primary GBM and in the development of resistance to temozolomide (TMZ). From a pool of 30,000,000 barcodes, the system enables the genetic integration of a unique barcode sequence into each cell. Each barcode can be quantitatively tracked via next-generation sequencing, allowing for dynamic monitoring of the subclonal architecture.

Methods: GSC 272 and 627 (sensitive and resistant to TMZ) were derived from core biopsies of GBM patients. Cells were transduced with a lentiviral vector containing luciferase. Cells were expanded and infected with the Cellecta Lentiviral library. Multiplicity of infection was determined. Transduced cells were injected into B6.Cg-Foxn1nu/J mice brains using a guide screw system. Half of the mice were treated with TMZ via gavage at two weeks. Brain tumors were removed and total DNA was extracted. Barcode inserts were amplified and the Illumina Sequencing platform was used for barcode quantification. Additionally, whole exome sequencing was performed to assess mutation status.

Results: Confirmation of luciferase signal has been performed using a Luciferase Reporter Assay. Multiplicity of infection was found to be 0.15 and 1 for GSC 6-27 and 272 respectively. Initial methylation studies showed GSC 272 to have methylation of MGMT while GSC 627 did not. There was no difference in OS in the TMZ Resistant (GSC 627) untreated vs. treated cohort (with a median OS of 55 days in both groups (N= 23 vs 25 respectively, P= 0.563). TMZ Sensitive (GSC 272) untreated mice did not survive as long as the treated cohort (N= 24 vs. 25, Median OS 43 vs. 206 days, P= 4.61e-09). Barcode and whole exome sequencing results is currently being analyzed.

Conclusions: The Cellecta lentiviral tagging system is an innovative way to track ITH and clonal evolution in glioblastoma orthotopic models. We hope to discover novel insights into TMZ treatment response and resistance.

#2917

High complexity molecular indexing reveals that time-dependent cell plasticity underpins response to EGFR targeted therapy.

Aaron Boudreau, Richard Bourgon, Marie Evangelista. _Genentech, Inc., South San Francisco, CA_.

Non-small-cell lung cancer (NSCLC) patients harboring epidermal growth factor receptor (EGFR)-mutant tumors often receive erlotinib, a 1st generation EGFR inhibitor, as a frontline therapy. Although many patients initially experience positive response with erlotinib treatment, resistance invariably occurs in the majority of cases - however, it remains unclear the full extent by which resistance is pre-determined by genomic aberration, stochastic by potentially epigenetic mechanisms, or a combination thereof. Using a lentiviral DNA barcode library to molecularly index PC-9, an EGFR mutant (exon 19 del. E746-A750) cell line model, we used next-generation DNA sequencing to trace the fate of over 1.6 million individual cells following erlotinib treatment. Similar to previous reports, we show that multiple subpopulations are present in PC-9 cells - those that are exquisitely sensitive to erlotinib, and those that are inherently resistant, representing ~2% of the population. Surprisingly, in the absence of erlotinib treatment, we show that cells cultured over time transitioned between these two states while maintaining population equilibrium. Using mathematical modeling, we identified several parameters of state switching or flux that were required to maintain this dynamic equilibrium. Understanding the molecular basis by which cells maintain cell state plasticity may enable new strategies to limit intra-tumor heterogeneity and may ultimately improve recurrence-free survival in NSCLC following erlotinib therapy.

#2918

Analysis of matched pre and post cisplatin-treated muscle-invasive bladder cancer reveals a candidate cisplatin mutational signature.

David Liu,1 Daniel Keliher,2 Philip Abbosh,3 Kent Mouw,1 Diana Miao,1 Mariel Boyd,4 Jean Hoffman-Censits,5 Gopa Iyer,4 Sara Tolaney,1 Jaegil Kim,6 Gad Getz,6 Scott Carter,1 Joaquim Bellmunt,1 Elizabeth R. Plimack,3 Jonathan E. Rosenberg,4 Eliezer M. Van Allen1. 1 _Dana Farber Cancer Institute, Boston, MA;_ 2 _Brown University, Providence, RI;_ 3 _Fox Chase Cancer Center, Philadelphia, PA;_ 4 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 5 _Sidney Kimmel Cancer Center at Thomas Jefferson University, Philadelphia, PA;_ 6 _Broad Institute of Harvard and MIT, Cambridge, MA_.

Background: Mutational signatures of exposure to DNA damaging agents such as UV irradiation, tobacco, and temozolomide have been described; however a cisplatin mutational signature has not been identified. We analyzed mutational changes in matched pre- and post-cisplatin based neoadjuvant chemotherapy (NAC)-treated muscle-invasive bladder cancer (MIBC) samples to identify a cisplatin mutational signature.

Methods: Whole exome sequencing and mutation calling was performed on matched germline and pre- and post-cisplatin based NAC (MVAC and GC) tumor samples from 46 MIBC patients with gross residual disease (≥ pT2) at cystectomy. After quality control, samples from 30 patients were analyzed. For each tumor, we characterized single nucleotide mutations (C>A, C>T, C>G, T>A, T>C, T>G) within a tri-nucleotide context, and used an optimized non-negative matrix factorization (NMF) to discover signatures of mutational processes in pre-NAC tumors, post-NAC tumors, and among mutations unique to the post-NAC samples.

We compared our discovered signatures to previously described human mutational signatures and to a signature of cisplatin exposure in a DT40 (chicken lymphoblast) cell line (normalized for a human exome context). We also performed strand asymmetry analysis to search for evidence of transcription-coupled repair.

Results: In both pre and post-NAC tumors we identified mutational signatures matching those attributed to APOBEC activity and nucleotide excision repair pathway defects, consistent with past studies in MIBC. In post-NAC tumors we identified an additional novel mutational signature, with modest overall cosine similarity (0.61) to the DT40 cisplatin signature, but similar C>A and T>A motifs. There was a strong correlation in inferred mutational activity (Pearson R = 0.98) when we replaced the novel signature with the DT40 signature in the post-NAC tumors, which was highly unlikely to be due to chance (p < 0.001, empiric null distribution). Further, we found evidence of transcriptional strand bias in C>A (p = 0.00025) and T>A (p = 4.2e-06) motifs with depletion of coding strand mutations, consistent with transcription coupled repair of platinum crosslinks at GpG and ApG motifs in the non-coding strand. Finally, we were able to rediscover the novel signature when limiting analyses to mutations unique to post-NAC tumors, consistent with mutational activity during chemotherapy.

Conclusions: Analysis of matched pre- and post- cisplatin treated MIBC identified a novel signature in post-cisplatin treated samples that (1) has mutational activity similar to a preclinical cisplatin mutational signature; (2) has a transcription strand bias consistent with known repair characteristics of platinum-induced DNA damage; and (3) arises in tumors following cisplatin chemotherapy. This may represent a cisplatin-induced mutational signature in human tumors.

#2919

Quantitative mapping of epidermal growth factor receptor activation in single breast cancer cells treated with targeted therapies.

Phuong Le, Kristopher Kilian, Andrew Smith. _University of Illinois at Urbana Champaign, Urbana, IL_.

Twenty percent of breast cancers are triple-negative, lacking estrogen receptor, progesterone receptor, and HER2. There is currently no specific FDA-approved targeted therapy for triple-negative breast cancer patients. Approximately half of triple-negative breast cancers overexpress epidermal growth factor receptor (EGFR), however phase II trials have only shown a 5% response rate with targeted inhibition. A quantitative analysis of EGFR signaling pathway dynamics of single cells at the single molecule level has the potential to shed light on the mechanism of lack of response to targeted therapy, as well as the cell-to-cell heterogeneity of response. Here, we combine single-molecule 3D imaging of EGFR using fluorescent quantum dots (QDs) with cell geometry normalization by micro-patterning to quantitatively map EGFR endocytosis in single cancer cells. Triple negative MDA-MB-231 cells conformed to defined geometries (rectangular with different aspect ratios) were grown on soft polyacrylamide gels after micro-contact printing islands of fibronectin. Two color QD conjugates of EGFR and its ligand, EGF, are used to track ligand binding and receptor internalization upon activation in the absence and presence of different families and dosages of pharmaceutical EGFR inhibitors. We demonstrated that microcontact-printing normalizes cell geometry while maintaining EGFR localization so that EGFR signaling response can be quantitatively assessed on a single-cell basis and that QD conjugates can specifically label EGFR and EGF. Quantitative single-molecule imaging at single-cell level revealed high level of heterogeneity in the amount and spatial localization of EGF binding and EGFR endocytosis among the cell population in the presence and absence of EGFR inhibitor, gefitinib. Using this platform, we will investigate if spatial organization of EGFR clusters within individual cells contribute to single-cell heterogeneous response to EGFR inhibitors. We anticipate quantitative mapping of EGFR activity with drug treatment will help to elucidate why EGFR drug treatments fail and provide a means to develop combination therapy to address heterogeneity in triple negative-breast cancer.

#2920

**Characterizing human melanoma treatment response** in vivo **using single-cell mass cytometry analysis of longitudinal tumor biopsies.**

Deon B. Doxie,1 Allison R. Greenplate,1 Kirsten E. Diggins,1 Caroline E. Maier,1 Jeffrey A. Sosman,2 Mark C. Kelley,1 Jonathan M. Irish1. 1 _Vanderbilt Univiersity, Nashville, TN;_ 2 _Northwestern University, Chicago, IL_.

Introduction: Metastatic melanoma that harbor BRAFV600E mutations are treated with inhibitors that inhibit MAPK signaling downstream of BRAF. Although these treatments improve patient survival, the majority of tumors develop resistance. It is increasingly clear that the presence and emergence of cell subsets in the tumor microenvironment shapes treatment responses. Mass cytometry is a single cell platform capable of measuring >35 proteins on millions of tumor cells. Here, mass cytometry was used to characterize matched tumor samples obtained before, during, and after therapy in order to quantify the impact of therapy on tumor cell populations.

Methods: Melanoma tumors from adults with metastases were biopsied or surgically resected from 12 patients with consent. Patients received two weeks of BRAFV600E inhibitor dabrafenib followed by two weeks of dabrafenib and MEK inhibitor trametinib. Tumors were enzymatically digested and cryopreserved with protocols for mass cytometry (Leelatian and Doxie et al., Cytometry B 2016). Samples were stained with a 30+ antibody panel focused on melanoma identity and markers of lineage and trafficking for leukocytes, fibroblast, and endothelial cells. Viable nucleated cells were identified by total histone H3 and rhodium dye exclusion. Expert biaxial gating and viSNE analysis were used to identify, computationally map, and track populations and their subsets over time. Tissue microarrays were also analyzed by histology to confirm findings observed by mass cytometry.

Results: More than 99% of cells were characterized and fell into one of the four major populations. Melanoma phenotypes included loss of MHC class I and/or expression of one or more markers of identity including SOX10, SOX2, nestin, S100β, and MCAM. Significant decreases in nestin, MCAM, MHC I and S100β (p < 0.01, p < 0.05, p < 0.05, and p<0.05, respectively) were observed in cells after treatment. Decreases in nestin and S100β populations identified by mass cytometry were observed in histology analysis of TMAs from matched tumors. Analysis of pre- and post-therapy melanoma cells and BRAF mutated cell lines indicated pre-therapy tumor cells had phenotypes most similar to cell lines. Strikingly, 6hrs or 1 day of treatment of cell lines with dabrafenib and trametinib did not result in decrease in nestin expression. The nestin low post-therapy phenotype of melanoma tumor cells was present in all melanoma cell lines at a low abundance (average of 7.0% +/- 3.9% in cell lines, N = 7).

Conclusions: Single cell analysis and longitudinal monitoring of patient's revealed tumor ecosystems experience rapid changes in cellular diversity during targeted therapy. Lower nestin expression distinguished treated melanoma tumor cells. The in vitro results suggest that in vivo remodeling of the tumor microenvironment results from a shift in host-tumor cellular interactions and not a direct response to inhibitors.

#2921

Integrated exome- and single cell RNA-sequencing reveals a clone's genomic instability and multifaceted fitness.

Noemi Andor,1 Carlo C. Maley,2 Hanlee P. Ji1. 1 _Stanford Univ., Palo Alto, CA;_ 2 _Arizona State University, Tempe, AZ_.

DNA-damage therapy is the main line of defense against most cancers, especially at advanced stages of progression. DNA-damage therapy damages the cell's DNA. We have identified that a cell's cumulative extent of DNA damage - even when successfully repaired - is an indicator for future tolerance to new insults. This finding points to a quantitative measure of genomic instability as biomarker of sensitivity to DNA-damaging agents. However, quantifying genomic instability is difficult, especially because each individual tumor harbors clones with their own distinct genomic characteristics. Here we address the challenge of quantifying the complex phenotype of genomic instability across the clonal subpopulations that coexist within an individual tumor.

We identified coexisting clones from bulk-exome sequencing and calculated the copy number profile of each clone's genome. We integrated mutations detected in both, single-cell RNA-sequencing (scRNA-seq) and exome sequencing, to assign single cells to clones. Because cells in G2/M phase of the cell cycle (further referred to as G2/M clones) have twice the ploidy compared to cells in other cell cycle phases, their presence represents an opportunity for validation. Using 15 gastric cancer cell lines - each sorted by cell cycle phase - we validated: i) clone-specific copy number and ii) assignment of single cells to clones. Here, the predicted size of G2/M clones is an indicator of how correct we quantify clone-specific copy number. The degree to which expression of cell cycle genes is exclusive to G2/M clones is an indicator as to how correct we assign single cells to clones. In addition to the dominant quiescent clone and the G2/M clone, we identified other discrete subclones in a subset of cell lines, suggesting that genetic intra-tumor heterogeneity is sometimes maintained in-vitro. This approach identified clone-specific markers that can be used for cytometry separation of cells of specific clonal subpopulations.

Overall, we demonstrate the power of combining exome sequencing and scRNA-seq to reveal how many clones are present in a tumor sample, how genomically unstable each clone is and how fit is each clone. When combined, these two techniques synergize into a powerful strategy to investigate the relationship between genomic instability, clone fitness and DNA-damage sensitivity.

#2922

Targeting physical and stromal sources of tumor heterogeneity with photodynamic therapy-based combinations.

Imran Rizvi,1 Utkan Demirci,2 Tayyaba Hasan1. 1 _Massachusetts General Hospital, Boston, MA;_ 2 _Stanford University School of Medicine, Canary Center for Early Cancer Detection, Palo Alto, CA_.

Tumor heterogeneity and drug resistance to conventional therapies remain major causes of treatment failure, recurrence and dismal survival rates for patients with advanced stage cancers. A range of cellular, architectural, and physical cues in the tumor microenvironment influence the intrinsic and acquired resistance mechanisms that lead to treatment failure. These include communication with heterocellular stromal partners and physical forces such as hydrodynamic shear stress, which remain understudied as determinants of tumor heterogeneity and variability in treatment response. Strategies that leverage photodynamic therapy (PDT), a mechanistically-distinct modality, to regionally target and prime stubborn tumor populations may be essential to overcoming key barriers to durable cancer management while minimizing toxicity from traditional agents. A multi-faceted approach is needed to evaluate and optimize PDT-based combination therapies, including the development of bioengineered 3D models that integrate cues such as physical forces and heterotypic cellular communication. Here the impact of hydrodynamic stress and stromal biology is evaluated in the context of ovarian cancer (OvCa). Metastatic OvCa spreads predominantly via flushing of ascites along preferential fluidic pathways and communicates with the local microenvironment, including the extracellular matrix (ECM) and tumor endothelial cells (TECs), to form peritoneal implants. A microfluidic model that supports 3D tumor growth was developed to investigate the role of fluidic stress on the heterogeneity of metastatic OvCa. The motivation for this study was based on clinical observations that the most stubborn tumors are often found in regions such as the peritoneal gutter, which is subjected to fluidic stress from ascites and is a common site of resistance and recurrence. Tumor nodules cultured under flow showed increased epithelial-mesenchymal transition (EMT) compared to non-flow cultures. Molecular and morphological changes consistent with EMT included a transcriptionally-regulated significant decrease in E-cadherin, a significant increase in vimentin, and significant decrease in fractal dimension, a metric adapted to quantify spindle-like morphology. A concomitant significant post-translational upregulation of epidermal growth factor receptor (EGFR) expression and activation, and chemoresistance was seen. The impact of heterotypic communication between TECs and OvCa cells was investigated in a 3D model. Tumors grown in the presence of TECs were differentially susceptible to chemotherapy and benzoporphyrin derivative (BPD)-based PDT and showed increased heterogeneity in response to treatment. This heterogeneity was overcome by PDT priming of tumors prior to chemotherapy. The potential value of using bioengineered models to guide customized PDT-based combinations will be presented.

#2923

Label-free enrichment and integrated full-length mRNA transcriptome analysis of single live circulating tumor cells from breast cancer patients.

Naveen Ramalingam,1 Yi Fang Lee,2 Lukasz Szpankowski,1 Anne Leyrat,1 Brian Fowler,1 Jovina Tan,3 Chong Tracy Lu,1 Ninez Delos Angeles,3 Chad Sanada,1 Cassandra Greene,1 Kyle Hukari,1 Andrew Wu,2 Yoon-Sim Yap,4 Jay West,1 Ali Asgar Bhagat2. 1 _Fluidigm Corporation, South San Francisco, CA;_ 2 _Clearbridge BioMedics Pte Ltd, Singapore, Singapore;_ 3 _Fluidigm Singapore Pte Ltd, Singapore, Singapore;_ 4 _National Cancer Centre, Singapore, Singapore_.

Background Label-free methods for isolating circulating tumor cells (CTCs) are attractive because they provide an opportunity to analyze a larger set of CTCs that may otherwise be missed due to variable or no expression of protein (label) markers. Understanding genetic and functional heterogeneity in CTCs allows us to gain insight into the mechanisms underscoring metastasis, drug resistance, and tumor aggressiveness. Currently, a simple workflow for isolation and molecular characterization of single CTCs by mRNA sequencing is lacking. In order to address this challenge, we developed a label-free workflow to isolate CTCs from breast cancer patients for full-length mRNA sequencing analysis by integrating the ClearCell® FX System with the Polaris™ system. The ClearCell FX system processes blood samples from cancer patients and enriches for CTCs in a label-free antibody-independent manner. The low level of nonspecifically isolated white blood cells from ClearCell FX is further depleted on the Polaris system by negative enrichment of viable CTCs. This unique integration of systems will enable researchers to perturb single CTCs in a controlled environment, monitor and measure the response due to perturbation, and link these response measurements to downstream genomic and transcriptomic analysis.

Method and Results CTCs from 7.5 mL of peripheral blood sample from breast cancer patients were enriched using ClearCell FX. To differentiate larger blood cells from putative CTCs, we stained the enriched cells with Alexa Fluor® 647-conjugated CD45 and CD31 to identify leukocytes and endothelial cells, respectively. Calcein AM (live cell marker) and CellTracker™ Orange (universal cell marker) were added to identify live cells. Single CTCs were selected on Polaris (Fluidigm) system, lysed and reverse-transcribed, and cDNA were preamplified on the Polaris integrated fluidic circuit (IFC). Sequencing libraries were generated using the Nextera® kit and sequenced on Illumina® MiSeq™ and NextSeq™ systems. We successfully processed blood samples from four patients. Sequenced data showed high-quality metrics, with read depth of up to 2.5 million reads (MiSeq) or 60 million reads (NextSeq), with a low percentage of mapped reads to ribosomal RNA and mitochondrial RNA. Unsupervised hierarchical clustering of gene expression data showed clustering by patient, but considerable heterogeneity was also observed among the CTCs from the same patient. We will provide insights into full-length mRNA transcriptome of single CTCs from triple negative breast cancer patient.

Conclusion We present the feasibility of integrating two microfluidics platforms to capture single CTCs for transcriptome and functional study. Our data suggests that the heterogeneity of tumor sample and characterization of metastatic processes can be elucidated from single-cell mRNA sequencing of CTCs.

#2924

Circulating DNA demonstrates convergent evolution and common resistance mechanisms during treatment of colorectal cancer.

Alain R. Thierry,1 Brice Pastor,1 Zhi-Qin Camilla Jiang,2 anastasia Katsiampoura,2 Christine Parseghian,2 Jonathan Loree,2 Michael J. Overman,2 Cynthia Sanchez,1 Safia El Messaoudi,3 Marc Ychou,4 Scott Kopetz2. 1 _INSERM U896, Montpellier, France;_ 2 _MD Anderson Cancer Center, Houston, TX;_ 3 _DiaDx SAS, Montpellier, France;_ 4 _Institut du Cancer de Montpellier, Montpellier, France_.

Malignant tumors shed cell free DNA into the blood stream. Analyzing this new biological source may have important implications in a shift towards personalized medicine for diagnosing and/or monitoring malignancies. We recently showed the first clinical validation and clinical utility of circulating DNA (cfDNA) analysis in oncology by testing RAS/BRAF hotspot mutations in plasma from metastatic colorectal cancer patients (mCRC) (Nat Med. 2014 Apr;20(4):430-5). In addition, liquid biopsies allow the tracking of clonal dynamics and detection of mutations during treatment. We evaluated under blinded conditions the ability of cfDNA to detect RAS/BRAF mutations in the plasma of 42 mCRC patients treated on a phase Ib/II trial of FOLFOX and dasatnib, with or without cetuximab. Prior to treatment, sequencing of archival tissue detected mutations in 25/42 patients (60%), while the cfDNA assay detected mutations in 37/42 patients (88%). Our cfDNA assay was able to detect mutations with allele frequencies as low as 0.01%. After exposure to treatment, 41/42 patients (98%) had a cfDNA detected RAS/BRAF mutation. Of 22 patients followed with serial measurements who were RAS/BRAF mutant at baseline, 11 (50%) developed a second mutation following treatment and 3 (14%) no longer had detectable levels of another mutant allele. Of RAS/BRAF wild type tumors at baseline, 4/5 (80%) developed new mutations. Patients may harbor mutations at very low frequency down to 0.01% before initiation or during treatment revealing the need of a high sensitive technique to detect mutant subclones. cfDNA quantitative measurements from this study closely mirrored changes in CEA and CT scan results, highlighting the importance of obtaining quantitative data beyond the mere presence of a mutation. Combined qualitative and quantitative cfDNA analysis allows tracking acquired resistance by studying the real-time clonal evolution of the tumor and might help physicians to adjust patient treatment. Our findings demonstrate the development of new RAS/BRAF mutations in patients regardless of whether they had pre-existing mutations in the pathway, demonstrating a convergent evolutionary pattern.

#2925

Distribution of copy number alterations defines clonal populations involved in colorectal cancer evolution.

Isabel Quintanilla, Elena Asensio, Maria Pellisé, Antoni Castells, Miriam Cuatrecasas, Jordi Camps. _IDIBAPS, Barcelona, Spain_.

Colorectal cancer progresses in a multi-step manner with adenoma being the most well-known precursor lesion. Malignant polyp, an adenoma that contains a focus of adenocarcinoma, is a suitable model to study the colorectal tumor evolution. Although the mutations that lead adenoma to evolve into carcinoma have been previously described, the copy number changes involved in this malignant transformation have not been fully explored. To understand how these genomic alterations contribute to carcinogenesis, we used sequential fluorescence in situ hybridization using probes for the oncogenes EGFR, MYC, CDX2, and ZNF217 and the tumor suppressor genes SMAD7 and APC in order to analyze the copy number changes in 23 cases of malignant polyps and 10 cases of low grade dysplasia (LGD) adenomas based on single cell analyses. We found the levels of genomic heterogeneity increased from LGD adenomas to malignant polyps, with the adenoma component having a lower degree of chromosomal instability than the adenocarcinoma component. Despite the intercellular heterogeneity, we observed different patterns of evolution. The gain of ZNF217 was the earliest event as it was found to be in some cases of the LGD adenoma, and was also important in the neoplastic transformation of malignant polyps. Other notable genomic imbalances we observed during the malignant transformation were the gain of CDX2 and the loss of SMAD7. Interestingly, in a significant proportion of cases we observed the gain of all loci analyzed to be the decisive step in the transition from adenoma to carcinoma, suggesting a whole genome duplication event. Lastly, in half of the malignant polyps studied the main clone in the carcinoma component was already present in the adjacent adenoma component, although in some cases at a low frequency only detected by single cell analysis. Our data suggest that copy number changes are early events in colorectal carcinogenesis that can determine the evolution from adenoma to carcinoma.

#2926

Histologically defined intratumoral sequencing uncovers evolutionary cues into conserved molecular events driving gliomagenesis.

Antony Prabhu, Pravin Kesarwani, Shiva Kant, Stewart Graham, Prakash Chinnaiyan. _William H. Beaumont Hospital, Royal Oak, MI_.

Glioblastoma (GBM) represents an archetypal example of a heterogeneous malignancy, harboring regions of invasion, necrosis, and vascularization. To begin to understand the diverse molecular consequences of this complex tumor ecology, we analyzed RNA-seq data generated from commonly identified intratumoral structures in GBM that were isolated and enriched using laser capture micro-dissection. Structures included the infiltrative, leading edge, central tumor core, and cellular regions in areas of necrosis. From this RNA-seq database consisting of 119 structural regions from 37 individual tumors generated from ~12,000 hematoxylin and eosin histologic images, we validated significant intra-tumoral heterogeneity in GBM, with cells derived from the infiltrating leading edge almost exclusively harboring the proneural molecular subtype, while cells in regions of necrosis displaying the mesenchymal subtype. In addition, we made the striking observation that when evaluating the tumors transcriptional profiles in the context of their derived structural regions, there was a relative small amount of inter-tumoral heterogeneity in GBM, with significant clustering of transcriptional profiles of specific structural regions from different tumors. To begin to understand the specific molecular events driving regional growth and their contributory role in gliomagenesis, we analyzed the transcriptional programs in the context of evolutionary progression. This level of analysis identified numerous pathways associated with neuronal receptor signaling, autophagy, and fatty acid metabolism as early events in tumorigenesis. Traditional cancer related signaling pathways emerged as key mediators driving progression, with glioma and p53 signaling ranking highest on the list. As the tumor progressed to harbor regions of necrosis, in addition to expected changes in VEGF and tumor signaling, we uncovered global metabolic changes associated with progression, with metabolic pathways comprising 10 of the top 20 pathways unique to this region. Collectively, these findings suggest that the observed intra-tumoral heterogeneity in GBM may be a conserved, predictable consequence to its complex microenvironment and therapeutic strategies rationally designed to target these unique, unequivocally present tumor cell biomes may lead to therapeutic gains in this otherwise fatal malignancy.

#2927

Proteogenomic heterogeneity in metastatic lung adenocarcinoma revealed from rapid/warm autopsy.

Nitin Roper, Tapan K. Maity, James Gao, Abhilash Venugopalan, Xu Zhang, Romi Biswas, Constance Cultraro, David Kleiner, Stephen Hewitt, Javed Khan, Guha Udayan. _National Cancer Institute, Bethesda, MD_.

Introduction: Intratumor heterogeneity has been characterized among multiple cancer types. However, in lung adenocarcinoma, recent work has been limited to early stage primary tumors and intertumor heterogeneity has not been well-studied. Most importantly, an integrated tumor heterogeneity analysis at the level of somatic variants, copy number, transcript and protein expression, and the phosphoproteome is outstanding.

Methods: In order to characterize both intra-tumor and inter-tumor heterogeneity of metastatic lung adenocarcinoma, we applied whole exome sequencing, RNA-seq, CNV-seq and mass spectrometry-based proteomic analyses on 33 tumor regions from metastatic sites including lung, liver and kidney, obtained by rapid/warm autopsy from 4 patients with Stage IV lung adenocarcinoma. The autopsy procedure was initiated between 2-4 hours of death.

Results: We found considerable intertumor heterogeneity with organ-specific, branched evolution that was consistent across DNA, RNA and protein analyses. Intratumor heterogeneity differed depending on oncogene-status: oncogene-negative tumors (without RTK/RAS/RAF mutations or known fusion gene) had significantly higher genomic intratumor heterogeneity than oncogene positive tumors. The degree of heterogeneity at the genomic and proteomic level was patient-specific. The proteomic analysis complemented genomic variants-based clonal evolution analysis. High-confidence driver mutations (KRAS, EGFR, TP53, CTNNB1) uniformly occurred early in the evolution of metastatic lung adenocarcinoma consistent with the concept of these mutations as likely 'founders'. In contrast, other known driver mutations occurred more often in later stages among specific organ branches. Notably, oncogene-negative tumors carried significantly more driver mutations than oncogene-positive tumors suggesting that genomic alterations may have a larger role in the metastatic process versus the early evolution of lung adenocarcinoma in oncogene-negative vs. oncogene-positive tumors. Among all patients, lung cancer specific focal copy number alterations frequently occurred within metastatic branch points. Single metastatic site biopsies would therefore be unlikely to capture all significant driver mutations and copy number alterations, particularly among oncogene-negative tumors.

Conclusion: Metastatic lung adenocarcinoma evolves through a branched, organ-specific, process with acquisition of significant driver mutations and copy number changes, particularly among oncogene-negative tumors. The branched evolution is ultimately influenced by proteomic and phosphoproteomic alterations affecting key signaling pathways that may not be always a result of genomic changes.

#2928

Pervasive associations between mutational processes and driver mutations across cancer types.

Daniel P. Temko,1 Benjamin Schuster-Boeckler,2 Simone Severini,1 Ian Tomlinson,2 Trevor Graham3. 1 _University College London, London, United Kingdom;_ 2 _University of Oxford, Oxford, United Kingdom;_ 3 _Barts Cancer Institute, Queen Mary University of London, London, United Kingdom_.

More than 30 distinct mutational signatures, each the result of an underlying mutational process, have now been described in human cancers -- we have sought to address the extent to which these mutational processes shape the spectrum of driver mutations across cancer types.

Using publicly available genomic data from 10,188 cancers across 22 common cancer types, we have statistically assessed the association between mutational process activity and the presence of individual pathogenic driver mutations. Specifically, we determined the sample-specific exposures for each mutational signature. For each recurrent driver mutation in each cancer type, we tested for a difference in the exposure to each mutational signature in samples with the driver mutation compared to those without. We used an independent analysis based on the causal SNV(s) for each driver mutation to test our hypothesis that many of the associations between driver mutations and mutational signatures are causative in nature.

Our method is novel in linking specific pathogenic amino-acid changes to mutational process activity. This allows us to investigate mutational causality in carcinogenesis, and permits us to delineate the inter-related effects of mutation and clonal selection.

Our data indicate that both extrinsic (mutagen driven) and intrinsic (DNA replication and repair) associated mutational processes have pan-cancer associations with multiple distinct driver mutations, many of which are causative in nature. Of particular note is the identification of a pan-cancer driving role for both the APOBEC and microsatellite instability (MSI) associated mutational processes, an early causative role for defective DNA proof reading in colorectal and endometrial cancers, and the identification of a general 'mutational sensitivity' to oncogenic PIK3CA mutations in many cancer types. In addition, our analysis also provides quantitative understanding of the relative effects of the distinct evolutionary forces of mutation and selection, via a detailed analysis of the mutually exclusive mutations to the IDH1 and IDH2, and KRAS and BRAF, genes respectively. Taking into account mutational signatures, our data suggest preferential selection for IDH1 R132H compared to other common IDH1 and IDH2 mutations and preferential selection for KRAS G12D compared to other common KRAS and BRAF mutations, in a subset of cancer types.

These data demonstrate the functional importance of mutational processes in shaping the landscape of driver mutations across cancer types, informing our understanding of the relative effects of mutation and selection in carcinogenesis. Our study has ramifications for both cancer prevention, by providing knowledge of how removing mutagen exposure could impact carcinogenesis, and for treatment choice by indicating the relationship of mutational processes and the likely presence of actionable or resistance mutations.

#2929

Exploratory evolved strategies that limit cancer growth for possible new therapeutic strategies.

Dominique Abrahams,1 Arig Ibrahim Hashim,1 Kim Luddy,1 Robert Gillies,1 Robert Gatenby,1 Joel Brown2. 1 _Moffitt Cancer Center, Tampa, FL;_ 2 _University of Illinois, Chicago, IL_.

Strong selection can promote mammalian evolution of a remarkably diverse phenotypic range over a short period of time. With appropriate selection forces, we tested the hypothesis that laboratory animals can evolve phenotypes that are resistant to the growth of implanted tumors. We propose that strong selection can produce evolution of different strategies to generate resistance to the growth of inoculated tumors,1. Changes in the supportive cell layer that decrease limited growth of cancer cell populations. 2. Increase of immunologic response to tumor antigens.

Hence, we examined the evolution of resistance in immuno-competent and immuno-deficient mice. Fixed number of cells from luciferase tagged LL/2 (Lewis lung carcinoma) cancer cells were implanted in groups of 10 male C57BL/6 and SCID mice. All tumor cells were obtained from frozen samples of a single large tumor population to eliminate any contribution from tumor cell evolution. Tumor growth in each animal was measured by calipers and luciferase imaging. The two animals that exhibited the slowest tumor growth in each cohort were bred with females from the same litter. Over 12 generations, the selection pressures resulted in emergence of SCID mice in which tumors grew at approximately 1/10th the rate compared their initial generation. The immunocompetent C57BL/6 mice evolved significant changes in immune-mediated parental tumor cell killing but this has yet resulted in significantly delayed tumor growth because the LL/2 cells rapidly evolve resistance strategies. Injection of the same tumor cells into unevolved wild type strains of both SCID and C57BL/6 mice produced rapid tumor growth identical to that seen in the first generation.

Using immunohistochemistry we observed decrease in blood supply among generations, proliferation, and apoptosis while no differences were observed in Glut-1 and CA-9 expression. To investigate this observation, first we examined changes in the molecular characteristics of the tumor cells during in-vivo growth by microarray on tumor cells isolated from "final" adapted cell population(s) in the animals that have been selected to decrease tumor growth compared to the wild type as well as normal cells in parallel. Our results showed that there are 158 genes different between tumors growing in the evolved and selected mice, among them are genes involved in extracellular matrix organization, hence we used second-harmonic generation (SHG) microscopy to image and quantify collagen,

our results showed significant increase in collagen at the edge as well as the core of the tumor of the evolved mice compared to wild type mice.

In conclusion our evolutionary study has reduced tumor growth in SCID mice but has had limited success in the C57BL/6. The SCID mice adaptation to tumor was likely through alteration in function of the supportive cell layer (Collagen), thus generating biomechanical forces and protective cellular events during tumor progression at early stages. 

## TUMOR BIOLOGY:

### Tumor Microenvironment 3

#2930

Niche microenvironments essential for tumor heterogeneity of primary cancer cells.

Shiki Fujino,1 Norikatsu Miyoshi,2 Masayuki Ohue,2 Yusuke Takayashi,2 Masayoshi Yasui,2 Hidekazu Takahashi,1 Naoyuki Haraguchi,1 Jhunichi Nishimura,1 Taishi Hata,1 Tsunekazu Mizushima,1 Yuichiro Doki,1 Masaki Mori1. 1 _Osaka University, Osaka, Japan;_ 2 _Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan_.

Primary culture of cancer cells derived from each patient's tumor can provide important information of the "individual tumor." It is general to use cell lines in basic research filed. However, cell lines are quite different from clinical cancers. Clinical cancer tissues are composed of not only cancer cells but also tumor microenvironments such as stromal cells and tumor vessels. The primary culture method of clinical cancer with tumor niche microenvironments has not been optimized. We have developed a simple 2D-culture method for primary colorectal cancer (CRC). We obtained 30 samples from surgically resected tumor. They were mechanically and enzymatically digested and fibrotic tissue and bacteria were excluded using customized two size filters. And we cultured the obtained cells on a matrigel-coated plate with embryonic stem (ES) cells culture medium. We named these cultured cancer cells, "isolated-tumor derived Cancer Cells (iCCs)." All iCCs grew and about 80 % of iCCs were successfully passaged. Twenty-three iCCs were transplanted into the subcutaneous layer of NOD-SCID mice, and the tumor growth and histology of iCCs were examined. The morphology was similar to each parental clinical tumor. And microarray analysis showed that RNA expression of iCCs was similar to each parental tumor. Furthermore, we examined the culture medium; our modified ES culture medium (ES-cultured iCCs) and 10% FBS medium (serum-cultured iCCs). The expression of surface markers regarding cancer stem cells such as CD44 and CD24 were different between ES-cultured iCCs and serum-cultured iCCs, and drug sensitivity of iCCs were also different. FACS analysis and immunocytochemistry revealed that iCCs contained PDGFR-positive cells. The results of multi-drug sensitivity assay were different between iCCs and cell lines especially in PDGFR inhibitor. Strong correlations were observed between the results of multi-drug sensitivity assay of iCCs and clinical outcomes of chemotherapy. We report an innovative primary culture method and the in vivo and in vitro analyses of iCCs, leading to the future application.

#2931

Targeting membrane-bound carbonic anhydrases in breast cancer to intervene in the metastatic phenotype.

Mam Y. Mboge,1 Zhijuan Chen,1 Brian P. Mahon,2 Chingkkuang Tu,1 Alyssa S. Wolff,1 John V. Mathias,1 Fabrizio Carta,3 Claudiu T. Supuran,3 Rob McKenna,1 Susan C. Frost1. 1 _Univ. of Florida, Gainesville, FL;_ 2 _National Institutes of Health, Bethesda, MD;_ 3 _Universita degli Studi di Firenze, Italy_.

Breast cancer is the second leading cause of cancer related deaths among women in the United States. Despite the tremendous progress that has been made towards treating localized tumors, nearly 40,000 women die each year, predominantly from metastatic drug resistance. The tumor microenvironment plays a pivotal role in determining tumor growth, invasion, metastasis, and therapeutic success or failure. Therefore, a therapy targeting the tumor microenvironment is needed to sufficiently preserve the quality of life of cancer patients, by inhibiting metastasis. Elevated levels of Carbonic anhydrase IX (CAIX) expression in primary breast cancers is a marker for highly aggressive and metastatic tumors, especially of the triple negative subtype (TNBC). It is also associated with hypoxia, extracellular acidification, and poor prognosis. Low pH (values of ~6.5-6.8) is toxic to normal cells in the tumor microenvironment while enhancing cancer cell proliferation and tumor growth. Our goal was to compare the structure of a CAIX-mimic bound to ureidosulfonamide inhibitors with the biological activity of these inhibitors in triple negative and estrogen receptor positive (ER+) breast cancer cell lines. CAIX is a reversible enzyme and at low pH (high proton concentration), the enzyme will consume protons, raising pH. Our hypothesis is that CAIX inhibition, in the context of an acidic microenvironment, will dysregulate its ability to maintain the acidic pH preferred by cancer cells which favors their growth and migration. In this study, we have shown the interaction of sulfonamide-based inhibitors with a CAIX-mimic using X-ray crystallography. These structures show that the inhibitors make multiple contacts within the active site cavity. This is consistent with the inhibitor-induced decrease in CAIX activity and to some extent expression. We have also investigated the effect of CA inhibition on breast cancer cell growth, proliferation, activation of cell death pathways and migration. This reveals that, although CA inhibition with the sulfonamide-based compounds inhibits cell growth and migration, it does not activate apoptotic pathways. In total, these observations indicate that CAIX is a viable small molecular drug target for the treatment of metastatic breast cancer.

#2932

**Immuno-inhibitory pathway, TIM-3/Galectin-9, in lung adenocarcinoma: clinical and** in vitro **analysis.**

Mikio Oka, Yoshihiro Ohue, Koji Kurose, Yumi Nishio, Midori Isobe, Eiichi Nakayama. _Kawasaki Medical School, Kurashiki, Japan_.

Introduction and Purpose:

Recently, immune checkpoint therapy has been incorporated into treatment of solid cancers including lung cancer, resulted in prolonged survival benefit. Checkpoint inhibitors target on PD-1/PD-L1 or CTLA-4 immuno-inhibitory molecules in immune or cancer cells. However, checkpoint therapy in lung adenocarcinoma (LAD) showed less than 20% response rate, suggesting existence of other immuno-inhibitory pathways.

Therefore, we focused on T cell immunoglobulin and mucin domain 3 (TIM-3) on the CD4 and CD8 T-cell surface and its ligand Galectin-9 in tumor cells, and analyzed the expression and function in LAD cells and tissues.

Materials and Methods:

TILs and PBMCs were obtained from 11 patients with LAD, and 194 LAD tissues resected surgically were analyzed. Expression of immuno-inhibitory molecules (PD-1, TIM-3, BTLA, LAG-3) was examined by flow cytometry using FACS on TILs and PBMCs, and PD-L1 and Galectin-9 expressions in LAD tissues was analyzed by immunohistochemistry using tissue microarray.

To investigate soluble Galectin-9 released from LAD cells, EGFR-mutated PC-9 and EGFR-wild-type OU-LC-SK cells were treated with EGFR-TKI afatinib at a concentration of 10 nM for 3 days. Then, the amount of Galectin-9 in culture supernatant was measured by ELISA. To investigate T-cell apoptosis induction by Galectin-9, established XAGE1-specifiic CD8 cloned T-cells were incubated with Galectin-9 protein for 8 hrs.

Results:

Increased expression in TILs compared to PBMCs was observed on PD-1 and TIM-3, but not on BTLA or LAG-3 in CD4 and CD8 T-cells. In LAD tissues, the frequencies of high PD-L1 and Galectin-9 expression in the tumor cell membrane or cytoplasm were 49% and 31%, respectively (in squamous cell carcinoma, the frequencies were 32% and 16%, respectively). Furthermore, correlated expression of PD-L1, Galectin-9 and CD3 (T-cell infiltration) was observed at the periphery of the tumor nest. Those findings suggest the relevance of the PD-1/PD-L1 and TIM-3/Galectin-9 pathways in the tumor microenvironment of LAD.

Next, soluble Galectin-9 released from the LAD cells was measured. Galectin-9 was detected only in the medium of EGFR-mutated LAD cells following treatment with afatinib. Moreover, apoptosis was induced in TIM-3-positive CD8 T-cell clones following interaction with Galectin-9 protein and this was inhibited by the addition of anti-Galectin-9 or an anti-TIM-3 antibody. The findings suggested that a significant amount of Galectin-9 could be released from LAD cells and induced T-cell apoptosis in tumor microenvironment.

Conclusions:

Our results suggested the relevance of the PD-1/PD-L1 and TIM-3/Galectin-9 immuno-inhibitory pathways in the tumor microenvironment of LAD, and that release of soluble Galectin-9 from LAD cells could negatively regulate T-cell function. For successful immune checkpoint therapy in LAD, simultaneous inhibition of TIM-3/Galectin-9 pathway may be needed.

#2933

Involvement of CXCL12-CXCR7 axis in adipocyte-induced TNBC metastasis.

Hyeongjwa Choi,1 Rosa Mistica C. Ignacio,1 Carla R. Gibbs,1 Eunsook Lee,2 Samuel E. Adunyah,1 Deok-Soo Son1. 1 _Meharry Medical College, Nashville, TN;_ 2 _Florida A &M University, Tallahassee, FL_.

Breast cancer (BC) is the most frequent tumor in women worldwide. Although its mortality has significantly decreased owing to advanced therapies, triple negative breast cancer (TNBC) is still difficult to treat because of lack of estrogen receptor, progesterone receptor and HER-2. TNBC accounts for 12-24% of total BC and contributes to the aggressiveness and poorer outcomes. The risk of BC increases significantly in obese women. Obesity is also associated with the worse clinical prognosis of BC. But the underlying mechanisms between obesity and BC, particularly TNBC, remain unclear. Therefore, we compare the molecular mechanisms by which human adipocyte conditioned media (CM) affect TNBC cells (BT549) and non-TNBC cells (MCF7A). Adipocyte CM had no effect on the proliferation of both TNBC and non-TNBC cells. However, adipocyte CM enhanced significantly migration in TNBC cells compared to non-TNBC cells. Next, we examined the signaling pathway by which adipocyte CM promote migration of TNBC cell. AKT and ERK were activated in both TNBC cells and non-TNBC cells. However, phospho-STAT3 was significantly increased in TNBC cells. Also, N-cadherin, a marker for epithelial to mesenchymal transition (EMT), was increased at the late time point in TNBC cells. Furthermore, CXCR7 was specifically increased in TNBC cells after treatment with adipocyte CM using a PCR array. CXCR7 is chemoattractive to CXCL12 which is highly expressed in the lung, the bone marrow, and the liver, common sites of BC metastasis. Taken together, the results indicate that adipocyte CM may promote metastasis of TNBC cells through the CXCL12-CXCR7 axis by activating STAT3.

#2934

Neoadjuvant chemotherapy influence changes of the immune response in non-small cell lung carcinomas immune response in non-small cell lung carcinomas.

Edwin Roger Cuentas,1 Carmen Behrens,2 Jaime Rodriguez-Canales,2 Mei Jiang,3 Apar Pataer,3 Arelene Correa,3 Stephen Swisher,3 Boris Sepesi,3 Annikka Weissferdt,3 Neda Kalhor,3 Jiexin Zhang,3 Jack Lee,3 John Heymach,2 Cesar Moran,3 Jianjun Zhang,3 Don Gibbons,3 Ignacio Wistuba3. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _UT MD Anderson Cancer Center., Houston, TX;_ 3 _UT MD Anderson Cancer Center, Houston, TX_.

Background: The clinical efficacy observed with PD-1/PD-L1 inhibitors in non-small cell lung carcinoma (NSCLC) has prompted to characterize the immune response in lung tumors treated with chemotherapy. The aim of this study was to determine the changes of the immune microenvironment in surgically resected NSCLCs from patients who received and did not receive neo-adjuvant chemotherapy.

Methods: We studied formalin-fixed and paraffin embedded (FFPE) tumor tissues from 112 stage II/III resected NSCLC, including 61 chemotherapy-naïve (adenocarcinoma, ADC=33; squamous cell carcinoma, SCC=28) and 51 chemotherapy-treated (ADC=31; SCC=20) tumors. mIF was performed using the Opal 7-color fIHC Kit™, scanning in the Vectra™ multispectral microscope and analyzed using the inForm™ software (Perkin Elmer, Waltham, MA). The markers studied were grouped in two 6-antibody panels: Panel 1, AE1/AE3 pancytokeratins, PD-L1 (clone E1L3N), CD3, CD4, CD8 and CD68; and Panel 2, AE1/AE3, PD1, Granzyme B, FOXP3, CD45RO and CD57.

Results: Positive PD-L1 expression (>5%) in malignant cells (MCs) was detected in 48% (n=53/112) of NSCLCs. Overall, chemotherapy-treated tumors showed significantly higher percentages of MCs expressing PD-L1 (median, 19.52%) than chemotherapy-naïve cases (median, 1.54%; P=0.008). Higher densities of lymphocytes expressing CD3+ (P=0.021), CD4+ (P=0.05), CD57 (P<0.001), CD45RO+ (P=0.019), and PD-1 (P=0.016) were detected in chemotherapy-treated NSCLCs compared with chemo-naïve tumors. In contrast, lower densities of FOXP3+ regulatory T cells and CD68+ macrophages but not statistical significant were detected in chemotherapy-naïve tumors when compared with chemotherapy-treated cases. Following chemotherapy ADCs exhibited significantly higher levels of CD57+ (P=0.008) and high density of PD-1 expressing by CD45RO+ cells (P=0.016) than chemotherapy-naïve tumors. Chemotherapy-treated SCCs demonstrated higher density of TAMs CD68+, CD57+,CD45RO+ and PD-1 cells than chemotherapy-naïve tumors (P<0.05). In chemotherapy-treated cancers, lower levels of CD4+ helper T cells was associated with worse overall survival (OS; P=0.04) in univariate analysis. In chemotherapy-treated ADC patients, lower levels of CD68-positive (P=0.010) and higher levels of FOXP3-positive cells correlated with worse OS (P=0.044).

Conclusions: Neo-adjuvant chemotherapy-treated NSCLCs exhibited higher levels of PD-L1 expression and T cell subsets regulation compared to chemotherapy-naïve tumors, suggesting that chemotherapy activates specific immune response mechanisms in lung cancer. These results provide an inclination towards the design of clinical trials combining neo-adjuvant chemotherapy and immunotherapy prior to surgical resection of locally advanced NSCLC. (Supported by CPRIT MIRA and UT Lung SPORE grants, and MD Anderson Moon Shot Program).

#2935

Sex disparity in the response of the immune microenvironment to colon cancer liver metastases.

Simon Milette,1 Ni Wang,2 Stéphanie Perrino,2 Pnina Brodt1. 1 _McGill University, Montreal, Quebec, Canada;_ 2 _Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada_.

Background Metastasis remains the most life-threatening aspect of malignant disease and a major clinical challenge. Myeloid-derived suppressor cells (MDSC) and regulatory T lymphocytes (Tregs), which are frequently found in the tumor microenvironment, are known to impair immune surveillance and promote a state of immune tolerance that leads to metastatic expansion. Previously, we have shown that the incidence of colon carcinoma liver metastases was reduced in TNF receptor 2 (TNFR2) deficient female mice, as a consequence of reduced recruitment of MDSCs and Tregs into the liver. Intriguingly however, no reduction in liver metastasis or MDSC/Treg accumulation was observed in TNFR2 deficient male mice, suggesting that their recruitment and survival were distinctly regulated. The objective of this study was, therefore, to elucidate the mechanisms underlying the sex disparity observed in the response of the immune microenvironment to liver metastases, in particular the role of sex hormones in immune response regulation in this context.

Methods Hormone-ablated female mice were generated using surgical ovariectomy (OVX). Estrogen-deprived mice, sham-operated controls and hormone reconstituted OVX mice were injected with syngeneic murine colon carcinoma MC-38 cells via the intrasplenic/portal route and experimental liver metastases enumerated 2 weeks later. MDSCs and Tregs recruited into the livers of tumor-bearing mice were analyzed 7-10 days post-tumor injection using flow cytometry, as well as IHC performed on liver cryostat sections.

Results We found that the number of experimental liver metastases was markedly reduced in ovariectomized female mice, as compared to sham-operated controls but was restored in estradiol supplemented OVX mice. Reduced incidence of hepatic metastases in OVX mice correlated with reduced accumulations of CD11b+Gr-1+ myeloid cells (MDSCs) and CD4+Foxp3+ cells (Tregs) at metastatic sites. IHC also revealed an increased CD8\+ : CD4+Foxp3+ (i.e. cytotoxic T cell : Treg) ratio around hepatic metastases in OVX mice as compared to sham-operated controls. Estradiol reconstitution in OVX mice restored MDSC and Treg cell accumulation to the levels observed in control mice.

Conclusion Together, our data identify female sex hormones as major players in the regulation of the tumor immune microenvironment and a potential contributing factor in the patients' response to immune-based cancer therapy.

Supported by the Canadian Institute for Health Research, MDEIE and FRSQ.

#2936

Molecular characterization of the immune subclass of hepatocellualr carcinoma.

Daniela Sia,1 Yang Jiao,1 Iris Martinez,1 Olga Kuchuk,1 Carlos Villacorta Martin,1 Manuel Castro de Moura,2 Juan Putra,1 Genis Camprecios,1 Swan Thung,1 Samuel Waxman,1 Vincenzo Mazzaferro,3 Manel Esteller,2 Augusto Villanueva,1 Josep Maria Llovet1. 1 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 2 _IDIBELL, Hospital Universitari Bellvitge, Barcelona, Spain;_ 3 _Fondazione IRCCS, Istituto Nazionale dei Tumori, Milan, Italy_.

Background: Immune checkpoint inhibitors have emerged as a promising therapeutic approach in different solid tumors, including hepatocellular carcinoma (HCC). Nonetheless, little is known about the immune-component of HCC or potential biomarkers of response to these therapies.

Aims: To perform comprehensive characterization of the HCC immunological profile and to identify biomarkers to select immunotherapy candidates.

Methods: We performed gene expression array deconvolution through non-negative matrix factorization in 228 resected HCCs. Characterization of the transcriptional landscape was conducted using >1,000 signatures representing distinct immune cells by gene set enrichment and nearest template prediction analyses. Presence of immune infiltration, tertiary lymphoid structure (TLS), PD-1 and PD-L1 immunostainings was investigated using immunohistochemistry. DNA methylation profile of 450K CpG sites was analyzed to identify those with significant differences for each group. Extensive validation of the immune classifier was performed in 728 independent HCC samples.

Results: Overall, an immune-related subclass of HCC was identified in ~27% of patients. The immune subclass was characterized by gene signatures identifying immune cells (i.e. T cells, TLS, cytotox, p<0.001), signatures of response to immune checkpoint therapy (p<0.001), presence of high immune infiltration (p=0.01), TLS (≥5 foci, 19/51 vs 33/168, p=0.01) and PD-1 and PD-L1 protein expression (p<0.05). The methylation levels of 363 CpG sites in 192 immune response gene promoters were able to capture the Immune class (ANOVA, p<0.05, Δβ>0.2 Tukey test). Integration with HCC molecular classifications revealed significant enrichment of the Immune subclass with IFN and S1 (p<0.001) and exclusion of the CTNNB1 and S2 (p<0.001) subclasses. The immune class contains two distinct microenvironment-based types: A) Exhausted immune response type (~35%) characterized by stromal activation, T cell exhaustion signatures, and presence of immunosuppressive components such as TGFB, LGALS1, M2 macrophages and pathways able to recruit myeloid-derived-suppressor cells (FDR<0.01); and B) Active immune response type (~65%) characterized by overexpression of adaptive immune response genes and IFN signaling (p<0.001). Tumors within the active immune response type showed a trend towards better survival vs rest (p=0.07).

Conclusions: Around 27% of HCC patients belong to the Immune class, characterized by activation of immune cells and signatures of response to immunotherapies. Within this subclass, two distinct types have been characterized by presenting active or exhausted immune responses, a feature that provides the rationale for precision medicine-based therapies.

#2937

Utilizing multiplex chromogenic IHC and digital image analysis to evaluate immune cell content and spatial distribution within NSCLC tumor tissue.

Lorcan Sherry,1 Mark Anderson,1 Marianne Cowan,1 Lee Dawson,2 Richard Bystry,2 Sarah-Jane Green2. 1 _OracleBio, Biocity Scotland, United Kingdom;_ 2 _Asterand Bioscience, Royston, United Kingdom_.

Chromogenic-based immunohistochemistry (IHC) is an established technique utilized within oncology R&D, allowing cells to be visualized within the context of their tumor microenvironment. However, when quantifying chromogenic stained sections via digital image analysis, the number of targets is usually limited due to constraints in deconvolving more than 3 chromogens present on the same tissue section image. The purpose of this study was to apply a customized algorithm, including a 5-colour deconvolution process to triplex chromogen IHC plus hematoxylin stained Non-Small Cell Lung Carcinoma (NSCLC) tumor tissues in order to quantify immune cell content and spatial distribution within both tumor and stroma regions of interest (ROI).

A multiplex IHC assay was developed and applied to FFPE sections of NSCLC tumor tissue to stain epithelial tumor cells (Pan CK, yellow), CD8+ cells (purple) and FoxP3+ nuclei (brown), counterstained with hematoxylin (blue). Immunostained whole slide images were generated for image analysis. A customized algorithm, which included a 5-chromogen color deconvolution process, was utilized within the Indica Labs HALO platform to separate the four IHC chromogens (including counterstain) and a fifth color (black) representing carbon deposit artefacts. Nuclear objects were formed by applying weighted optical density values for the brown, purple and blue colors, which were positive for either CD8 or FoxP3 and counterstain. A classifier was developed to automatically segment tumor from stroma. Each positive cell type was identified, using defined size and shape parameters, and quantified within each ROI.

IHC multiplex staining highlighted CD8+ cells and FoxP3+ nuclei to be present in both tumor and stroma compartments. Analysis data demonstrated the number of tumor and stroma CD8+ cells was 120 and 554 cells per mm2, while the number of tumor and stroma FoxP3+ nuclei was 20 and 19 nuclei per mm2. This gave a CD8+:FoxP3+ ratio of 6 within the tumor and 29 within the stroma. The average distance of CD8+ cells in the tumor or stroma to their nearest FoxP3+ nuclei was 92µm and 174µm respectively. The average distance of CD8+ cells to their nearest tumor cell was 39µm while the average distance of FoxP3+ nuclei to their nearest tumor cell was 29µm.

This study demonstrated multiplex chromogenic IHC as a valuable approach for quantifying multiple cell types within the context of their tumor microenvironment. Furthermore, up to five chromogen colors can be separated utilizing a custom 5-plex analysis algorithm, leading to a more in-depth evaluation of immune cell types present and their spatial distribution. This propounds a greater potential for interpretation of therapeutic mechanistic responses within the tumor microenvironment.

#2938

Tumour-associated macrophage polarisation and re-education with oncolytic viruses.

Munitta Muthana,1 Emer Murphy,1 Amy Kwan,1 Natalie Winder,1 Joe Conner2. 1 _Univ. of Sheffield Medical School, Sheffield, United Kingdom;_ 2 _Virttu Biologics, United Kingdom_.

Tumor-associated macrophages (TAMs) promote key processes in tumour progression through their plasticity and capacity to become polarised by tumours to have an immunosuppressive phenotype. However, the plasticity of these cells provides an opportunity for therapeutic 'reprogramming' towards a classically activated inflammatory phenotype thus preventing tumour-associated immunosuppression and inducing anti-tumour immunity. Oncolytic viruses (OV) represent a new class of anti-cancer therapeutics that promote anti-tumour responses through selective tumour cell killing and the induction of systemic anti-tumour immunity. We propose that TAMs can be targeted using an OV, leading to altered macrophage polarization and a reduction in tumour growth and metastasis. Experimental Design: In-vitro, cytotoxicity, viral replication and cytokine expression was assessed in a panel of primary human and murine macrophages following exposure to Sephrevir, an ICP34.5-deleted oncolytic herpes-simplex virus currently in Phase 1/2a trials. In-vivo, murine TS1-PyMT cells were implanted into the mammary fat pads of female FVB mice to establish a syngeneic immunocompetent mouse model of breast cancer. Tumour growth and overall survival were assessed following administration of Sephrevir given either by intratumoural (I.T) or intravenous injection (I.V). Immune cell enumeration and protective immunity were assessed post-mortem by immunofluorescence and flow cytometry. Results: Both primary human and mouse macrophages served as a host for oncolytic viral replication. Sephrevir was also found to attenuate TAM phenotypes as evidenced by increased "M1" receptor signatures (e.g. CD40, CD80, CD86, MHC II), increased pro-inflammatory gene expression (e.g. IL-1, IL-6, IL-8, CXCL10, TNF-a) and increased nitric oxide production. I.T and I.V administration of Sephrevir led to marked tumour shrinkage as well as the recruitment of cytotoxic T cells. Moreover, TAM polarisation was evident based on a change in signature with TAMs becoming skewed to IL-12hi/IL-10lo and a change in the iNOS/arginase1 ratio. Conclusions: Our results demonstrate that Sephrevir is associated with in vivo anti-tumour effects and importantly can be used for manipulating the phenotype of the abundant macrophage population located within the tumour microenvironment.

#2939

Tumor associated myeloid cell transcriptome signatures in an inducible Kras-positive lung adenocarcinoma murine model.

Clifton L. Dalgard,1 Mouna Lagraoui,1 Gauthaman Sukumar,1 Celeste Huaman,1 Corey A. Carter,2 Brian C. Schaefer1. 1 _Uniformed Services University, Bethesda, MD;_ 2 _John Murtha Cancer Center, Bethesda, MD_.

Tumorigenesis is modified by dynamic activities in the tumor microenvironment and mechanisms driving these alterations during tumor progression may be tumor- and patient-specific. Of well-established functions for tumor-associated stromal cells (e.g. angiogenesis, chemotaxis) immune system regulation by the tumor microenvironment is less well understood in their mechanism and effects. However, diminished activity of antigen-specific T cell lymphocytes tumor cytotoxicity is tumor promoting and recent cancer immunotherapy have targeted immune inhibitory checkpoints (e.g. anti-PD-L1) to restore T cell activity. Recent studies suggest that tumor-associated myeloid cells play a role in immunosuppression in several human cancers, including lung cancer. We hypothesize that early tumors of the lung promote changes in the phenotype of tumor-proximal myeloid cells, establishing an immunosuppressive microenvironment. As a component to observe this immunosuppressive role for myeloid cells, we performed specific transcriptome profiling of myeloid cells by immunomagnetic selection of CD11b-positive cells from lung tumors of a murine inducible Kras-positive, p53-negative cancer model. RNA sequencing of isolated myeloid cells as a function of tumorigenesis duration (3-18 weeks) establishes the kinetics of gene expression changes associated with tumor-supporting and immune-suppressing functions. Genome-wide expression analysis of myeloid cells between tumor-inducing adenovirus-Cre injected and normal control subjects resulted in identifying 1,883 genes with differential expression. Gene set enrichment analysis resulted in the stratification of samples by well-established gene signatures and tumor-promoting activity for angiogenesis (32 genes), lung tissue remodeling (47 genes) and cell survival (11 genes). Interestingly, a significant downregulated gene signature was observed in tumor-associated myeloid cells for immune cell chemotaxis (12 genes) and positive regulation of immune system activation (12 genes). These non-canonical gene signatures may identify a myeloid phenotype for immunosuppressive function with altered expression of genes such as L-selectin (Sell), C-C chemokine receptor type 7 (Ccr7), C-C motif chemokine (Ccl20) and CMRF35-like molecule 7 (CD300lb). The results shown are first steps in our tasks to define the phenotypes and mechanisms linking myeloid cells to tumor-associated immunosuppression in lung cancer.

#2940

**T** H **17: mediators of metastatic growth acceleration after surgery.**

Vikas Sud, Samer Tohme, Dirk J. van der Windt, Hai Huang, Allan Tsung. _University of Pittsburgh, Pittsburgh, PA_.

Surgery is known to accelerate the growth and progression of metastases in patients undergoing oncological resections with 50-60% patients having recurrent metastasis after resection making it a major reason for treatment failure. This impedes the efficacy of adjuvant chemotherapy and overall prognosis of the patient. Studies have proved that inflammatory cascade initiated by surgery that increases metastatic growth and progression is mediated through a specific mechanism of neutrophil cell death called Neutrophil Extracellular Traps (NETs). We investigated the mechanism of how NET formation after surgical stress increases metastatic growth by influencing a pro-tumor microenvironment in order to specifically identify targets for perioperative therapy to keep metastases dormant and improve the effect of chemotherapy and overall prognosis. 12 week old male C57BL/6J mice (WT) and mice in which NET formation was inhibited, ie, WT mice injected with DNAse and global homozygous knockout of peptidyl arginine deaminase 4 (PAD4), an essential nuclear protein for NET formation, underwent splenic injection of 100,000 cells of the murine colorectal cancer cell line, MC-38, followed with either 70% hepatic ischemia-reperfusion (IR) for 60 minutes after 7 days or no IR. Livers were harvested at multiple time points and assessed for changes in both innate and adaptive immune cell populations by flow cytometry. On gross examination, the livers of the NET deficient mice had fewer tumors as compared to the WT mice. Mice, which underwent IR after tumor cell injection in both groups showed an increase in nodules formation versus those with just tumor cell injection, however were still more in WT than NET deficient mice. Flow cytometry revealed a decrease in both TH17 (0.36% of T cells in no IR mice vs 0.0036% in IR mice) and CD8 cells (71% of T cells in no IR mice vs 34.8% in IR mice) in mice subjected to IR versus those with only tumor injection. We found that increased tumors after surgical stress, which were mediated by NET formation, were caused due to a decrease in anti-tumor activity of CD8 cells regulated via TH17 cells. If controlled in the perioperative period, it may reduce acceleration in metastatic foci and improve the overall prognosis. We are further investigating the mechanism of NETs influencing the tumor immune environment.

#2941

Inhibition of hypoxia-induced ectonucleoside triphosphate diphosphohydrolase 2 (ENTPD2) restrains myeloid-derived suppressor cell (MDSC) accumulation and sensitizes tumors to immune checkpoint inhibition.

David Kung-Chun Chiu, Aki Pui-Wah Tse, Iris Ming-Jing Xu, Robin Kit-Ho Lai, Hui-yu Koh, Felice Ho-Ching Tsang, Larry Lai Wei, Chun-Ming Wong, Irene Oi-Lin Ng, Carmen Chak-Lui Wong. _Univ. of Hong Kong, Hong Kong, Hong Kong_.

Background and Objective: Rapidly expanding knowledge on cancer immunology has introduced promising anti-cancer therapeutic approaches which involve the activation of T cells to combat cancer cells. Accumulating studies have indicated that the efficacy of immunotherapies is critically determined by the stromal cell components in tumors. Myeloid-derived suppressor cells (MDSCs), are regarded as one of the major immune cell types that possess immunosuppressive activities against T cells which allow cancers to escape immune surveillance and become non-responsive to immune checkpoints blockade. To increase the efficacy of immunotherapy, novel strategies to target MDSC in tumors are warranted. Hypoxia, oxygen (O2) shortage, frequently occurs in tumors due to abnormal vasculature. Using hepatocellular carcinoma (HCC) as a model, we have previously observed MDSC preferentially accumulates in hypoxic regions of human HCC tissues. Here, we aim to identify hypoxia-induced therapeutic targets that are critical for MDSC accumulation in tumors.

Experimental Procedures: Transcriptome sequencing in multiple HCC cell lines exposed to hypoxia and normoxia and HCC clinical specimens was performed to identify potential hypoxia-induced genes relevant to HCC development. MDSCs were isolated from HCC-bearing mice by magnetic bead sorting for different functional assays. LC-MS was performed to evaluate the level of extracellular metabolites. Flow cytometry was used to detect the frequencies of tumor-infiltrating MDSCs in orthotopic and subcutaneous HCC mouse models.

Results: We showed that hypoxia, through stabilization of hypoxia-inducible factor-1 (HIF-1), induced ectoenzyme, ectonucleoside triphosphate diphosphohydrolase 2 (ENTPD2/ CD39L1), in cancer cells, causing its over-expression in HCC clinical specimens. Over-expression of ENTPD2 was found as a poor prognostic indicator for HCC patients. Mechanistically, we demonstrated that ENTPD2 converted extracellular ATP to 5'-AMP which prevents the differentiation of monocytic MDSCs to dendritic cells, therefore promoting the maintenance of MDSCs in vitro and in vivo. Therapeutically, we found that ENTPD2 inhibitor POM-1 restrained MDSC accumulation and tumor growth, substantially enhancing the efficiency and efficacy of immune checkpoints inhibitors.

Conclusion: Our study reveals a novel mechanism whereby hypoxia/HIF-1 in cancer cells governs tumor-infiltrating MDSCs. Our data suggest that ENTPD2 may be a good prognostic marker and therapeutic target for cancer patients especially those receiving immune therapy.

#2942

Immunophenotypes in young-onset colorectal cancer.

M E. van Herk, M Lee, M Rytterdahl, M E. Kop, A F. Ramalheiro, R van der Breggen, T van Wezel, H Morreau, E S. Jordanova, N F. de Miranda. _Leiden university medical centre, Leiden, Netherlands_.

Colorectal cancer accounts for ten percent of new cancer cases and is the fourth most frequent cause of cancer-related deaths worldwide. In recent years, mainly due to screening programs, more patients are diagnosed in early stages of tumor progression, leading to a higher survival rate. However, the incidence of young (<50) patients diagnosed with colorectal cancer has been on the rise. These patients often have a poor prognosis due to the fact that tumors are diagnosed at advanced stages of tumor progression.

To our knowledge, no study has yet characterized immunophenotypes and immune evasive mechanisms specifically in young-onset colorectal cancers. To that end we have investigated the expression of HLA class I and PD-L1 in over 200 colorectal cancers derived from young-onset (< 50 years-old) patients. Furthermore, we applied a novel multispectral immunofluorescence technology to perform multiparameter immunophenotyping with CD3, CD8, PD-1, PD-L1, CD163, and Ki-67 in the same cohort.

We describe that HLA class I expression is maintained in the large majority of tumors allowing thus the development of neo-antigen targeted therapies. Interestingly, reduced expression of HLA class I but not total loss was associated with liver metastases, which suggests a specific selective pressure at this organ that might warrant tailored immune therapeutic interventions. As previous studies on colorectal cancer demonstrated, PD-L1 expression is limited and often restricted to immune cell compartments. The presence of immune cell infiltrates was related to the mutation background of tumors but also to their HLA class I phenotype: tumors with altered HLA class I expression were more likely to present traces of lymphocyte-mediated anti-tumor immunity.

Retained HLA class I expression in the majority of colorectal cancers associated with low infiltration by effector immune cells suggests the therapeutic induction of anti-tumor immune responses in young-onset colorectal cancers, for instance, by means of neo-antigen-targeted therapies. We are currently assessing the frequency of natural recognition of neo-antigens in an autologous setting in young-onset, late-stage colorectal cancers.

This work was supported by the Fight Colorectal Cancer-Michael's Mission-AACR Fellowship in Young-Onset, Late-Stage Colorectal Cancer Research awarded to N.F.C.C. de Miranda (15-40-1645-DEMI)

#2943

MYC functions as a master switch for natural killer cell-mediated immune surveillance of lymphoid malignancies.

Srividya Swaminathan, Adriane Mosley, Crista Horton, Daniel F. Liefwalker, Anja Deutzmann, Renumathy Dhanasekaran, Arvin Gouw, Andrew Gentles, Martin Eilers, Holden T. Maecker, Dean W. Felsher. _Stanford University, Stanford, CA_.

The MYC oncogene drives the pathogenesis of many hematopoietic malignancies, including Burkitt's lymphoma (BL) and Acute Lymphoblastic Leukemia (ALL). These malignancies are often "oncogene-addicted" to MYC. Using mass cytometry (CyTOF), we demonstrate that MYC-addicted T-ALL excludes specific immune subsets from the tumor microenvironment implicated in immune surveillance, including natural killer (NK) cells. MYC inhibition clears malignant lymphocytes from the spleen and restores the normal splenic NK composition. Concordantly, peripheral blood of T-ALL patients have reduced percentages of activated NK cells as compared to healthy individuals. We show that MYC excludes activated NK cells from sites of lymphomagenesis by suppressing ERK1/2-STAT1/2-Type I IFN signaling, in both murine and human lymphomas. Furthermore, MYC-associated BL patients with higher than median expression of STAT1/2, and cytotoxic NK cell genes, PRF1 and Granzymes, have favorable clinical outcomes. Hence, oncogenic MYC appears to causally and reversibly suppress NK-mediated immune surveillance during lymphomagenesis.

#2944

miR-130a and -145 reprogram myeloid derived suppressor cells and enhance anti-tumor immunity.

Hiroki Ishii,1 Suman K. Vodnala,1 Bhagelu R. Achyut,2 Christine M. Hollander,1 Ashish Lal,1 Li Yang1. 1 _National Cancer Institute, Rockville, MD;_ 2 _Georgia Regents University, Augusta, GA_.

Myeloid derived suppressor cells (MDSCs) are increased in tumor bearing condition, and elicit immunosuppression via type 2 polarization. We have previously reported that TGF-β signaling in MDSCs is essential for tumor metastasis. TGFβR2, a receptor essential for TGF-β signaling, is elevated in MDSCs. Deletion of Tgfbr2 in MDSCs, the gene encoding TGFβR2, significantly decreased tumor metastasis. However, it is unclear how TGFβR2 is regulated in MDSCs. We identify microRNA-130a and -145, which directly target Tgfbr2 in MDSCs and inhibit the expression of TGF-β receptor II (TβRII). Both miRs levels were lower in MDSCs from advanced tumor bearing mice correlating to increased TβRII level. Overexpression of miR-130a or -145 in MDSCs shift their immunological phenotype from type 2 to type 1 polarization. Moreover, tumor metastasis was significantly suppressed in myeloid specific miR-130 transgenic mice. In pre-clinical mouse models, the number of lung metastasis was significantly decreased when mice were treated with combination therapy of miR-130a or -145 with paclitaxel when compared with miR-130a, -145, or paclitaxel alone. We proposed that reprogramming of MDSCs by miR-130a and -145 could be a novel strategy in therapeutic treatment for breast cancer metastasis.

#2945

Impact of intratumoral mature dendritic cells on prognosis of the patients with esophageal squamous cell carcinoma.

Junya Nishimura, Hiroaki Tanaka, Yuichiro Miki, Tatsuro Tamura, Tatsunari Fukuoka, Go Ohira, Masatsune Shibutani, Sadaaki Yamazoe, Kenjiro Kimura, Takahiro Toyokawa, Hisashi Nagahara, Ryosuke Amano, Kazuya Muguruma, Kiyoshi Maeda, Kosei Hirakawa, Masaichi Ohira. _Osaka City University Graduate School of Medicine, Osaka, Japan_.

[Background] Promising efficacy of the immunotherapy with anti-PD-1 antibody for esophageal squamous cell carcinoma (ESCC) is expected. Tumor-infiltrating lymphocytes (TILs)have been found to be associated with favorable outcome of patients in many cancers including ESCC. However, the contributing factor of TILs in the primary tumor remains unclear. Dendritic cells (DCs) are the most potent antigen-presenting cells to induce cytotoxic T lymphocytes. [Purpose] The aim of this study was to examine the correlation of mature dendritic cells with CD8+ cells in the tumor in order to clarify the impact of DCs on locally infiltrated lymphocytes in ESCC. [Object and Methods] Formalin-fixed, paraffin-embedded blocks of primary lesions were collected from 80 patients with ESCC who underwent surgical treatment at Osaka City University Hospital. Immunohistochemical analysis using anti-LAMP-3 antibody and CD8 antibody was used to evaluate intratumoral DCs and TILs. The number of LAMP-3⁺DCs and TILs were counted at ×400 magnification. We divided the 80 cases into two groups according to the median number of intratumoral DCs and TILs to examine the clinicopathological features. [Results] LAMP-3⁺DCs were predominantly observed in peritumoral area and sparsely found in intratumoral area, whereas CD8⁺T cells distributed between both peritumoral and intratumoral areas. The number of DCs was significantly associated with increasing TNM stage. For example, the median number of DCs was 59.9 in pathological T1, 34.2 in T2/3, 62.4 in pN1, 34.1 in pN1/2/3. In addition, many patients with high infiltaraion of DCs had positive lymphatic invasion (Ly). The number of intratumoral DCs was significantly correlated with the number of TILs which were observed in intratumoral regions. Patients in the high infiltration of DCs group showed a significant better prognosis than patients in the low infiltration group. Double staining showed that LAMP3⁺DC-CD8⁺T cell clusters were seen in peritumoral area.

#2946

Prognostic effect of tumor-infiltrating immune cell composition in non-small cell lung cancer by histology.

Menno Tamminga, T. Jeroen Hiltermann, Ed Schuuring, Rudolf S. Fehrmann, Harry J. Groen. _University Medical Centre Groningen, Netherlands_.

Introduction: The immune system plays an important role in tumor progression and treatment response, for example with the new immune modulating therapies. In lung cancer the effect of the individual immune cell types is unclear. In our study, we used publicly available expression data to find evidence whether there are differences in the composition of immune cell fractions within the microenvironment between squamous cell carcinoma and adenocarcinoma of the lung.

Methods: We searched the Gene Expression Omnibus (GEO) for human NSCLC samples. Analysis was confined to samples hybridized to the Affymetrix HG-U133 Plus 2.0 (GEO accession number GPL570) platforms. Expression profiles were downloaded and curated for duplicates and corrupt files. Different immune cell fractions were estimated using CIBERSORT and the LM22 leukocyte signature matrix. We combined this information with available clinical data. We used Cox regression analyses to evaluate the prognostic impact of the different immune fractions and Mann-Whitney U tests to test significant differences.

Results: Comparing the immune cell composition of adenocarcinoma (n=587) and squamous cell carcinoma (n=254), we observed in adenocarcinoma increased percentages of naïve and memory B-cells, resting and active CD4+ T-cells, regulatory T-cells, active NK cells, monocytes, M2 macrophages, resting dendritic cells and resting mast cells, and decreased percentages of plasma cells , CD8+ T-cells, follicular helper cells, resting NK cells, gamma delta T-cells, M0 and M1 macrophages and active mast cells. In adenocarcinoma, a higher percentage of memory B-cells in the immune infiltrate was associated with increased survival (HR 0.97, 95% CI 0.95-0.99, p=0.02), while higher percentages of neutrophils (HR of 1.07, 95% CI 1.01-1.13, p=0.02), M0 macrophages (1.02, 95% CI 1.01-1.04, p=0.01) and follicular helper cells (1.07, 95% CI 1.02-1.13, p=0.01) were associated with a worse survival. In both histologies, the resting mast cell immune fraction showed a positive association with survival (adenocarcinoma HR 0.96, 95% CI 0.93-0.99, squamous cell carcinoma HR 0.93, 95% CI 0.89-0.98, p<0.01 in both histologies).In squamous cell carcinoma, a borderline association with a worse prognosis was observed with the naïve CD4+ T-cell fraction (HR of 1.1, 95% CI 1.00-1.22, p=0.053) and the regulatory T-cell fraction (HR of 1.11, 95% CI 0.99-1.24, p=0.067).

Conclusion: In NSCLC significant differences in intratumoral immune cell composition were observed between adenocarcinoma and squamous cell carcinoma.

#2947

Intratumoral injection of the toll-like receptor 4 agonist G100 induces a T-cell response in the soft tissue sarcoma microenvironment.

Yongwoo D. Seo,1 Edward Y. Kim,1 Ernest U. Conrad,2 Ryan B. O'Malley,1 Sara Cooper,3 Bailey Donahue,3 Lee D. Cranmer,4 Hailing Lu,5 Frank Hsu,5 Elizabeth T. Loggers,4 Taylor Hain,3 Darin J. Davidson,1 Lynn Bonham,4 Venu G. Pillarisetty,1 Gabrielle M. Kane,1 Stanley R. Riddell,3 Robin L. Jones,4 Seth M. Pollack3. 1 _University of Washington, Seattle, WA;_ 2 _Seattle Childrens Hospital, Seattle, WA;_ 3 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 4 _Seattle Cancer Care Alliance, Seattle, WA;_ 5 _Immune Design, Seattle, WA_.

Introduction: Soft tissue sarcomas (STS) are heterogeneous mesenchymal tumors which are both morbid and lethal. G100 is a stable oil-in-water emulsion of glucopyranosyl lipid adjuvant, a highly potent toll-like receptor 4 (TLR4) agonist, which has been utilized for intratumoral (IT) injections and as vaccine adjuvants without significant toxicity. We hypothesized that IT G100 would induce a robust local and potentially systemic anti-tumor immune response in the STS microenvironment, leading to improved outcomes.

Methods: 15 metastatic STS patients who had a superficial injectable lesion were treated with weekly IT G100 for 8-12 weeks; 12 patients received concurrent radiation for 2 weeks at the start, while 3 got IT G100 alone for 6 weeks prior to radiation. Biopsies and blood were collected pre and post treatment, and flow cytometry was performed on fresh tumor samples. T-cell receptor (TCR) deep sequencing of tumor-infiltrating lymphocytes (TIL) and peripheral blood mononuclear cells (PBMC) was performed on 7 patients. RECIST v1.1 and the Common Terminology Criteria for Adverse Events were used to monitor clinical outcomes.

Results: Patients had a median of 3 (0~5) prior lines of therapy and mean tumor size of 5.6cm (1~20cm). No grade 3 or higher treatment-related toxicity was observed, and local tumor control was achieved in 93% (14/15). 6 (40%) had stable disease after treatment, and 1 (P06) had complete regression of injected tumor. This tumor had a high percentage of infiltrating pre-treatment immune cells (12% CD45+ on flow cytometry versus 2.7% for all other tumors). TCR sequencing showed that the increase in clonality of PBMC after treatment was greater in P06 (389%) compared to 6 other patients (mean 34%). There was also higher overlap in TCR sequence between TIL versus PBMC after treatment (13% versus 22%), suggesting systemic expansion of tumor-specific T-cells. In 7 patients evaluable for tumor-associated macrophages (tumors with >1000 CD45+CD11b+ cells), 71% had a shift from an M2 to M1 phenotype. In all patients who received G100 alone, there was an increase in T-cell infiltration into tumor after treatment. In one patient (P14), the proportion of CD3+ live cells in tumor went from <1% to 62%; of these, 51% were CD4+ and 44% were CD8+. TIL from P14 had a 251% increase in clonality after treatment; at the same time, percentage of live tumor cells that were PD-L1\+ increased from 0.02% to 1.3%.

Conclusion: IT G100 provides a potentially viable agent for local control of metastatic STS. With or without radiation, G100 appears to shift the tumor microenvironment into a more inflammatory state with significant infiltration of T cells. The increase in clonality in PBMC and TIL, as well as increased overlap of tumor-associated versus peripheral TCR sequences, suggest induction of a tumor-specific response. Combination of G100 with other immunomodulators may further enhance the adaptive anti-tumor response.

#2948

**A distinct CD8** \+ **tumor infiltrating lymphocyte subset is associated with high TIL density, enhanced cytotoxicity and improved survival in patients with lung cancer.**

Anusha Preethi Ganesan,1 Oliver Wood,2 Eva Garrido-Martin,3 Serena Chee,2 Toby Mellows,3 James Clarke,3 Daniela Samaniego-Castruita,1 Divya Singh,1 Gregory Seumois,1 Aiman Altezani,4 Edwin Woo,4 Peter Friedman,3 Gareth Thomas,3 Emma King,3 Tilman Sanchez-Elsner,3 Pandurangan Vijayanand,1 Christian H. Ottensmeier2. 1 _La Jolla Inst. for Allergy & Immunology, San Diego, CA; _2 _Cancer Sciences Unit, University of Southampton, Southampton, United Kingdom;_ 3 _University of Southampton, Southampton, United Kingdom;_ 4 _Southampton Univeristy Hospital NHS Trust, Southampton, United Kingdom_.

High tumor infiltrating lymphocyte (TIL) density predicts for good prognosis in several cancers and therapies that boost the anti-tumor responses of cytotoxic lymphocytes (CTLs) have shown promise in the clinic. However, clinical responses to currently available immunotherapeutic agents vary considerably, the molecular basis of which is unclear. We performed global transcriptional profiling of CTLs in tumors and adjacent non-tumor tissue from 36 patients with early stage lung cancer to define the molecular features associated with robustness and heterogeneity of anti-tumor immune responses. We observed major differences in the transcriptional program of tumor-infiltrating CTLs (CD8+ TILs) that is shared across tumor subtypes. Pathway analysis revealed enrichment of genes in cell cycle, TCR activation and co-stimulation pathways, indicating tumor-driven expansion of presumed tumor antigen-specific CTLs. We also observed marked heterogeneity in the expression of molecules associated with TCR activation and immune checkpoints, and their expression was positively correlated with the density of tumor-infiltrating CTLs. Interestingly, TILhigh tumors were also enriched for a distinct CD8\+ tumor infiltrating lymphocyte subset that appeared to have enhanced cytotoxicity and independently predicted improved survival in patients with lung cancer. In summary, we define the molecular fingerprint of tumor-infiltrating CTLs and identify a number of novel targets that may be important in modulating the magnitude and specificity of anti-tumor immune responses in lung cancer.

#2949

TGF-β is a key mediator of NK cell dysfunction in gliolastoma.

Hila Shaim, Abdullah Alsuliman, Konrad Gabrusiewicz, Jun Wei, John Yu, Rafet Basar, May Daher, Lucila Kerbauy, Mayela Mendt, Muharrem Muftuoglu, Li Li, Enli Liu, Nobuhiko Imahashi, Sonny Ang, Young Gi, Pinaki Banerjee, David Marin, Richard Champlin, Elizabeth Shpall, Amy Heimberger, Katayoun Rezvani. _MD Anderson Cancer Center, Houston, TX_.

Background: NK cells play a crucial role in the antitumor immune response and are involved in controlling tumor formation, progression and metastases. Glioblastoma (GBM) is the most devastating brain tumor, associated with very poor prognosis. While immunotherapy emerged as a promising approach for anti cancer therapy, GBM appears immune resistant. Here, we studied the role of NK cells in targeting glioblastoma and possible mechanisms of NK immune evasion.

Methods: NK cells were isolated from freshly resected GBM tissue, matching peripheral blood and the blood of healthy controls. Flow cytometry was used to characterize the cells and their ability to produce cytokines, and chromium release assay was perfumed to assess their cytotoxicity. In vitro assays were performed by co-culturing GBM stem cells (GSCs) with healthy donor NK cells.

Results: We found that whereas NK cells were abundant in primary GBM tissue and could efficiently target GBM stem cells (GSCs), GBM infiltrating NK cells (TiNKs) displayed an abnormal phenotype with downregulation of many activating receptors including CD16, NKG2D, DNAM, NKp30, NKp46, 2B4 and NKG2C and upregulation of inhibitory proteins such as PD-1. This inhibitory phenotype was associated with impaired NK cell function when compared with NK cells isolated from the peripheral blood of patients and healthy donors. GSC-NK cell-cell contact resulted in release of TGF-β by GCSs, which in turn led to NK dysfunction through constitutive activating of the p-Smad pathway. TGF-β activation, in turn, is partially mediated by the matrix metalloproteases MMP-2 and MMP-9, secreted by GSCs upon contact with NK cells and enhanced upon TGF-β exposure. We demonstrated that inhibition of the TGF-β axis, in particular by the small molecule inhibitor, galunisertib, can prevent GSC-induced NK cell dysfunction but is unable to inactivate the p-Smad pathway, thus, cannot reverse existing dysfunction.

Conclusions: Our results indicate that NK-GBM cross-talk plays an important role in tumor escape and highlight the importance of developing future adoptive transfer therapies with the intent of limiting tumor escape from antitumor immunity.

#2950

Biomarkers of immune infiltration for multiplex immunofluorescence in breast cancer.

April Swoboda, Galina Khramtsova, Lise Sveen, Andrey Khramtsov, Rita Nanda, Olufunmilayo Olopade. _University of Chicago Medicine, Chicago, IL_.

Introduction: Only a subset of patients have a clinical response to cancer immunotherapy. The molecular mechanisms that mediate the immunological response or tolerance to treatment are just beginning to be understood. Several tumor signaling pathways are involved in those mechanisms: Wnt/β-catenin, STAT3, NFkB, PI3K/PTEN/AKT, and TP53. To characterize the tumor, its immune infiltrate, and its microenvironment, we examined expression of several biomarkers and determined the relationships among them in various breast cancer subtypes.

Methods: 148 breast cancer (BCa) cases from the University of Chicago Breast Cancer SPORE tissue bank under IRB approved protocols were classified into four biological subtypes based on immunohistochemical (IHC) staining: luminal A, luminal B, Her2 and basal-like. IHC staining for PD-L1, CD8, FoxP3, CD68, CD163, and β-catenin was performed on tissue microarrays. CD8+ and FoxP3+ cells were counted manually and recalculated for 1 mm2. Macrophage phenotype was determined using single and double staining with CD68 for total population and CD68/CD163 for M2. CD68+ and CD163+ cells were counted manually, confirmed by Image Analysis, and recalculated for 1 mm2. PD-L1 and β-catenin (membrane-associated, cytosolic and nuclear) were scored as negative, weak positive, moderate, and strong.

Results: The study cohort subtypes were 20.9% basal-like, 10.4% Her2, 58.3% luminal A, and 10.4% luminal B. The ratio of M2 to M1 cells increased with disease progression (p<0.001). Her2 and basal-like subtypes had a significantly higher percentage of M2 cells as compared to the luminal A subtype (p<0.001). The cases with M2 macrophage phenotype had a higher ratio of FoxP3 regulatory T cells (Tregs) to cytotoxic CD8+ cells. PD-L1 expression was present in 23% of specimens. However, the intensity of expression was markedly different across the BCa subtypes. PD-L1 high expressing tumors had, on average, greater FoxP3+ cell infiltrate and low or absent CD8+ T cells. The ratio of FoxP3 Tregs to CD8+ cells in basal-like tumors were 1.7, 2.5, and 4 -fold higher than in Her2, luminal A, and luminal B tumors, respectively (p<0.05 for all correlations). There was a statistically significant decrease in membrane-associated β-catenin in tumor compared to normal tissue (p<0.0001). The proportion of tumors positive for cytosolic or nuclear β-catenin in basal-like tumors was statistically higher than all other tumor subtypes. High β-catenin nuclear protein expression was predominantly observed in tumors that lacked CD8+ T cells. Ongoing analysis will evaluate the association of these biomarkers with clinical outcomes.

Conclusions and Future Directions: Our data suggests that IHC using a panel of antibodies may be a robust and suitable method for evaluating level of immune infiltration. Future studies will evaluate multiplex immunofluorescence of multiple biomarkers in breast tissue.

#2951

The tumor microenvironment in cholangiocarcinoma is dominated by an immunosuppressive infiltrate.

Nathania M. Figueroa, Brian Belt, Ankit Patel, Booyeon Han, Margaret Hill, William Alexander, Michael O'Dell, Aditi Murthy, Kelli Connolly, Scott A. Gerber, Aram Hezel, David Linehan. _University of Rochester Medical Center, Rochester, NY_.

Purpose: To characterize the dynamics of the immune response to cholangiocarcinoma (CCA) in a genetically engineered mouse model of CCA in order to identify immune pathways susceptible to targeted immunotherapies.

Experimental Design: Genetically modified mice with targeted Kras activation and loss of p53 (Alb-Cre/LSL-KRASG12D /p53Lox/+) in the liver spontaneously develop CCA recapitulating the histopathologic features of human disease. To evaluate the in vivo immune response to CCA, tumors from Kras-p53 mice and normal livers were excised and processed for histologic examination, flow cytometry, and gene expression analysis. Immunohistochemistry (IHC) and flow cytometry were performed on myeloid and T cell markers to distinguish immune cell subsets. Cell lines established from isolated CCA tumors were used to assess the functional impact between the immune system and tumor cells in co-culture in-vitro studies.

Results: Kras-p53 murine hepatic tumors were highly desmoplastic with a heavy fibrotic stromal compartment. IHC analysis demonstrated a prominent inflammatory leukocyte infiltrate compared to normal liver. Flow cytometry of single cell tumor suspensions showed the immune reaction was dominated by CD11b+ monocytic (Ly6C+) and granulocytic (Ly6G+) myeloid cells, and Foxp3+ regulatory T cells (T reg). In vitro studies revealed tumor educated myeloid cells expressed higher levels of genes associated with an immunosuppressive phenotype, including Arginase 1 (p=<0.007). Invasion assays analyzing the migration potential of CCA cell lines co-cultured with CD11b+ cells showed increased invasive properties in these tumor cells when compared to naive controls (p=<0.0001). Additionally, increased tumor-initiating properties were seen by qRT-PCR in tumor cell lines having undergone similar co-culture experiments. These data suggest the immune response to CCA is predominantly immunosuppressive and tumor supportive.

Conclusion: CCA tumors from Alb-Cre/LSL-KRASG12D /p53Lox/+ mice have a prominent immunosuppressive infiltrate recapitulating features of the immune reaction in human disease. Thus, Kras-p53 mice provide an ideal model to test targeted immunotherapy for the treatment of CCA.

#2952

The novel cathepsin L/K inhibitors KGP94 and KGP207 prevent M0 to M2 macrophage differentiation and macrophage-mediated pro-tumor functions.

Samantha S. Dykes, Dietmar W. Siemann. _University of Florida, Gainesville, FL_.

Tumor-associated macrophages play many important roles in tumor progression, including facilitating tumor cell invasion and angiogenesis. Due to their numerous pro-tumor functions, tumor-associated macrophages represent an important cell population in the design of targeted anti-cancer therapies. Interleukin-4-mediated activation of macrophages resulted in an M2-like, or pro-tumor macrophage phenotype, often associated with increased expression of proteases including the lysosomal cathepsin L. Cathepsin L is important in both macrophage and tumor cell invasion, whereby secreted cathepsin L degrades the extracellular matrix, allowing for cell infiltration. The present study examined the role of cathepsin L in M0 to M2 differentiation and macrophage-mediated tumor cell invasion using the novel cathepsin L/K inhibitors KGP94 and KGP207 [Dr. Kevin Pinney, Baylor University]. KGP94 and KGP207 prevented M2 macrophage motility and invasion in in vitro migration and invasion assays. Moreover, KGP94 and KGP207 reduced macrophage-stimulated invasion of 4T1 murine breast cancer cells. Together, these data suggested that cathepsin L was necessary for macrophage invasion and motility and for macrophage-mediated invasion of breast cancer cells. KGP94 and KGP207 treatment partially prevented IL-4-stimulated M0 to M2 differentiation of macrophages as determined by a decrease in the IL-4-induced expression of the M2 marker Arginase-1 upon drug treatment. Additionally, exogenous recombinant cathepsin L partially stimulated the expression of Arginase-1 in M0 macrophages. Together, these data suggest that cathepsin L plays a role in macrophage M0 to M2 differentiation. In conclusion, the novel cathepsin L/K inhibitors KGP94 and KGP207 prevented M0 to M2 differentiation, macrophage invasion, and macrophage-stimulated invasion of breast cancer cells. These data highlight the importance of cathepsin L in macrophage functions and suggest that cathepsin L inhibition is a viable approach for the treatment of tumors associated with high macrophage infiltration.

#2953

Tumor-associated macrophages infiltration highly associated with PD-L1 expression in gastric cancer.

Kazuto Harada,1 Jeannelyn S Estrella,1 Arlene M Correa,1 Lang Ma,1 Xiaoquan Dong,1 Yan Xu,1 Wayne L Hofstette,1 Kazuki Sudo,1 Hisashi Onodera,2 Koyu Suzuki,2 Akihiro Suzuki,2 Randy L Johnson,1 Zhenning Zhenning,3 Shumei Song,1 Jaffer A Ajani,1 Hisashi Onodera2. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _St. Lukes University, Tokyo, Japan;_ 3 _First Hospital of China Medical University, Houston, China_.

Objectives: programmed death ligand 1 (PD-L1) is key protein for tumor to acquire the evacuation form T cell immune response and PD-L1 inhibitors are useful for an immune checkpoint blockade therapy. Tumor-associated macrophages (TAM) facilitate not only tumor cell progression, but also immunosuppression, which prevent tumor cell attack by T cell. Therefore, TAM is considered to be associated with PD-L1 expression, however it remain unclear in gastric cancer (GC).

Methods: We performed immunohistochemical staining of PD-L1, CD68 (pan-macrophage) and CD163 (M2 macrophage) in 217 GC sample tissue microarray. The area of CD68 and CD163 positive cells were evaluated by using by the Cytoplasmic V2.0 algorithm on the Aperio ImageScope software.

Results: 31 samples (14.3%) were positive for PD-L1 expression. The mean of rate for CD68 and CD163 positive area were 6.8±0.38% and 6.2±0.30%, respectively. CD163 positive macrophage infiltration is significantly more in diffuse type tumor than in intestinal type tumor (diffuse (n=111): 6.9%, intestinal (n=91): 5.3%, p=0.0056), but CD68 positive macrophage infiltration is similar in both type (p=0.38). Interestingly, PD-L1 expression tumor have significantly more CD68 and CD163 positive macrophage infiltration (CD68: PD-L1 negative 6.3%, positive 10.4%, p=0.0002, CD163: PD-L1 negative 5.7%, positive 9.2%, p<0.0001). PD-L1 expression and CD68 and CD163 positive area had no relationship between any clinicopathological features.

Conclusions: Macrophages infiltration in GC is highly associated with PD-L1 expression of tumor cells. M2 macrophage infiltration is significantly associated with diffused type of GAC. This result suggests that TAM

infiltration is associated with the immunosuppression through upregulating PD-L1 expression in GC cells. Thus, macrophages infiltration can serve as a potential therapeutic target.

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#2954

The role of hedgehog signaling in breast cancer progression through macrophage polarization.

Ann Hanna, Lalita A. Shevde. _University of Alabama at Birmingham, Birmingham, AL_.

The tumor microenvironment is comprised of stromal cells that constantly crosstalk with the cancer cells. Stromal cells, specifically tumor infiltrating immune cells, secrete cytokines and chemokines that regulate host responses to cancer through tumor-suppressing or tumor-promoting mechanisms. Macrophages within the tumor microenvironment are very plastic and can modulate different functions that promote tumor growth or tumor suppression, depending on their phenotype. Classically activated macrophages (M1) suppress tumor growth by mounting a pro-inflammatory immune response; while alternatively activated macrophages (M2) promote tumor progression by inducing an anti-inflammatory response which suppresses immune system function.

Hedgehog (Hh) signaling is essential for normal mammalian embryonic development by modulating vital functions such as cell proliferation, differentiation, and angiogenesis. Although the Hh signaling pathway is tightly controlled, it often is deregulated, thus promoting tumorigenesis and tumor progression. Aberrant Hh signaling has particularly been implicated in breast cancer progression and metastasis.

In this project, we investigate the role of Hh signaling in polarizing cancer associated macrophages toward the tumor-promoting M2 phenotype in a breast cancer model. We have discovered that using small molecule inhibitors to inhibit Hh signaling attenuates the cytokine profile associated with M2 macrophages, while adding recombinant Hh ligand potentiates it. Our data thus far supports a role for Hh signaling in alternatively polarizing macrophages. We hypothesize that Hh inhibitors will reduce the abundance of M2 macrophages and prevent metastasis.

#2955

Gr1-MDSCs and Tregs modulate the prostate cancer progression.

Sanjay Kumar,1 James Stokes,1 Udai Singh,2 Karyn Scissum Gunn,1 Upender Manne,3 Selvarangan Ponnazhagan,3 Manoj K. Mishra1. 1 _Alabama State Univ., Montgomery, AL;_ 2 _University of South Carolina, SC;_ 3 _University of Alabama Birmingham, AL_.

Myeloid-derived suppressor cells (MDSCs) and Regulatory T cells (Tregs) are the major components of immunosuppressive network that play a critical role in tumor immune evasion. The mechanisms by which tumors induce expansion of suppressive cells and the crosstalk between Gr1-MDSCs (Gr1+, CD11b+, F4/80+) and Tregs relative to TGF-β secretion remain incompletely defined. Prior studies have suggested that MDSCs may contribute to Treg recruitment in cancer. Herein, the goal of this investigation is to examine the role of TGF-β mediated generation of Tregs and Gr1-MDSCs during prostate cancer progression and clearance. To achieve this goal, matrigel system was used along with transgenic adenocarcinoma of mouse prostate (TRAMP-C1, C2 and C3 cell) in C57BL/6 mice. Interestingly, TRAMP-C3 cells are characterized as non-tumorigenic; however, TRAMP-C1 and TRAMP-C2 cells do form tumor. Mice were administered with serial log concentration of TRAMP (C1, C2 and C3) cells along with the matrigel. After three weeks, matrigels along with or without tumor were excised from tumor bearing mice, single cell suspensions was prepared and cells were flow analyzed for GR1-MDSCs and Tregs. Additionally, the TGF-β level was also estimated (colorimetric) in culture supernatant in context to MDSCs and Treg expansion. Our initial findings suggest that the expression of Gr1-MDSCs (Gr1+, CD11b+, F4/80+) and Foxp3\+ Tregs increased in TRAMP-C1 and C2 as compared to TRAMP-C3 bearing C57BL/6 mice. These findings indicate that the modulation of Gr1-MDSCs and Tregs could help in tumor suppression.

#2956

Chemical library screen identifies compounds that target S100A8/S100A9 complex and MDSC accumulation.

Eunmi Lee,1 Maria Ouzounova,1 Raziye Piranlioglu,1 Abdeljabar El Andaloussi,1 Sena Arbag,2 Gang Zhou,1 Hasan Korkaya1. 1 _Augusta Univ. Cancer Ctr., Augusta, GA;_ 2 _Hacettepe University, Ankara, Turkey_.

We previously demonstrated that MDSC subsets accumulated in primary tumor and distant organs regulate tumor plasticity. The mouse transcriptome analysis of in vitro co-cultures and samples from syngeneic mouse model revealed that granulocytic subset of myeloid-derived suppressor cells (gMDSCs) from metastatic 4T1 tumor bearing mice regulate several hundred genes in tumor cells upon co-culture. The top genes are S100A8, S100A9, MMP8, FPR1, CCL3, and TGFβ2 which also predicted poor survival in human solid tumors including breast cancer. Therefore, we called these 6 genes as "metastatic gene signature". It has been reported that S100A8/S100A9 heterotetramer, called calprotectin, play a key role in inflammation-associated cancer progression. To investigate the role of calprotectin (S100A8/A9) in tumor-mediated immunosuppression and metastasis, we first utilized Tasquinimod, a small molecule targeting S100A9 only. However, Tasquinimod treatment of 4T1 tumor-bearing mice had a moderate anti-tumor activity which may be due to a limited activity on granulocytic MDSC accumulation. In addition, we determined that there was a significant upregulation of S100A8 in MDSCs from Tasquinimod treated mice. This data suggested that inhibiting only S100A9 leads to activation of S100A8 and thus may be ineffective targeting of MDSCs. We therefore run a computational screen of the NCI chemical library against the crystal structure of calprotectin (S100A8/S100A9) and identified 40 lead compounds. We then performed in vitro screening assay to identify compounds that inhibit MDSC induction. We identified 3 compounds that significantly suppressed gMDSC differentiation. We are currently performing in vivo studies with these 3 candidate drugs in our murine breast cancer model and will present our findings at the AACR meeting. We believe that this study will provide a novel drug targeting S100A8/S100A9 heterotetramer, a key molecule in MDSC induction and its regulatory functions during tumor progression.

#2956A

Cancer invasion of mammary epithelial cells in 3D culture shows YAP-independent mechanotransduction.

Joanna Y. Lee, Jessica Chang, Sungmin Nam, Ovijit Chaudhuri. _Stanford University, Stanford, CA_.

83% of non-invasive breast cancers are diagnosed as ductal carcinoma in situ (DCIS). While some DCIS tumors remain confined in the mammary duct, others progress into invasive ductal carcinoma (IDC). The mechanisms underlying invasion are not well understood. Current diagnostic methods cannot accurately predict which DCIS cases will progress to IDC, and unnecessary treatment affects long-term health and quality of life, with radiation potentially promoting malignancy-inducing mutations. One possible regulator of invasion may be extracellular matrix (ECM) stiffness. Increased ECM stiffness has been correlated with invasion and 3D culture models of normal mammary epithelium show that enhanced stiffness induces an invasive phenotype. However, the mechanisms underlying stiffness-induced invasion remain unclear. Studies have converged upon the finding that YAP, a transcriptional regulator that is deregulated in diverse cancers, is the transducer of ECM stiffness. However, these studies were primarily performed in 2D culture and involved col-1, a ligand that activates distinct signaling pathways and is not normally found in the BM. Here, we examined the gene expression profiles of 3D cultured MCF10A cells during stiffness-induced invasion with and without col-1. We generated interpenetrating networks (IPNs) of reconstituted basement membrane (rBM) and alginate, which allow stiffness to be tuned in the absence of col-1 and independently of cell adhesion ligand concentration and matrix pore size. Traditionally used hydrogels composed of rBM and col-1, with stiffness tuned by increasing concentrations of col-1, were also produced for 3D culture. Our results show that enhanced 3D stiffness increases MCF10A cell invasion and proliferation in both the presence and absence of col-1. However, in contrast to results from 2D culture, invasive phenotypes in 3D cultured cells did not correlate with nuclear localization of YAP, indicating lack of YAP activity. The dispensible nature of YAP in 3D stiffness sensing was supported by RNA-seq analysis, which showed a lack of increased gene expression of YAP downstream targets in conditions of enhanced stiffness. RNA-seq identified 389 differentially expressed genes in response to enhanced 3D culture stiffness. By relating these genes to transcription factors using ChIP-Seq data provided by ENCODE, we identified p300, FOS, STAT3, NELFE, and TAF1 as potential mechanotransducers of stiffness-induced invasion. Furthermore, highly expressed stiffness-induced genes were validated by immunofluorescence to identify potential indicators for invasion. Our RNA-seq results may lead to the development of a PCR-based prognostic that allows accurate determination of cancer invasion risk in breast cancer patients, informing course of treatment.

#169

Preclinical efficacy and sensitivity determinants of evofosfamide in molecularly defined models of head and neck squamous cell carcinoma.

Francis W. Hunter,1 Avik Shome,1 Dan Li,1 Way W. Wong,1 Peter Tsai,1 Nooriyah Poonawala,1 Purvi M. Kakadiya,1 Troy M. Ketelä,2 Maria K. Kondratyev,2 Courtney R. Lynch,1 Tet-Woo Lee,1 Khanh B. Tran,1 Jules B. Devaux,1 Rachel Zussman,1 Cho R. Hong,1 Dennis Kee,3 Andrew M. Macann,4 Anthony J. Hickey,1 Stefan K. Bohlander,1 Cristin G. Print,1 William R. Wilson,1 Bradly G. Wouters,2 Stephen M. Jamieson1. 1 _University of Auckland, Auckland, New Zealand;_ 2 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada;_ 3 _LabPLUS, Auckland, New Zealand;_ 4 _Auckland City Hospital, Auckland, New Zealand_.

Tumor hypoxia is prevalent in head and neck squamous cell carcinoma (HNSCC), where it limits radiotherapy outcomes. Hypoxia-activated prodrugs (HAPs) have been developed to target hypoxic regions of tumors. These agents undergo oxygen-sensitive reductive activation, thereby delivering cytotoxic species within hypoxic cells. This study investigated the efficacy and sensitivity determinants of the clinical-stage HAP evofosfamide (TH-302) using molecularly-characterized models of HNSCC. We deployed a collection of 27 HPV-negative HNSCC cell lines derived from lesions of varying TNM stages and primary, nodal or recurrent sites. The collection was characterized for gene expression by RNA-seq, from which somatic variants were also called. Their transcriptomic features were investigated in the context of pan-cancer TCGA data by hierarchical clustering. The potency and hypoxic selectivity of 3 HAPs - evofosfamide, PR-104A and SN30000 - were assessed by antiproliferative assay in 22 lines and compared to bromo-isophosphoramide mustard (Br-IPM), cisplatin and 5-FU. The antitumor activity of evofosfamide (50 mg/kg qdx5 for 2-3 cycles with or without a single 10 Gy dose of radiation on day 5 of cycle 1) was evaluated in HNSCC xenografts in addition to a PDX isolated from an SCC of the glottic larynx. The hypoxic fraction at baseline and after 5 days of treatment was quantified by pimonidazole staining. Genetic modifiers of sensitivity to evofosfamide and its cytotoxic metabolite Br-IPM were explored through whole-genome CRISPR-Cas9 screens using the GeCKO v2 library. High-throughput screens with a custom shRNA pool were performed in one HNSCC and two pancreatic ductal adenocarcinoma cell lines to identify reductases responsible for the activation of evofosfamide in hypoxic cells. Evofosfamide was more potent and more selective for hypoxic HNSCC cells in vitro than PR-104A or SN30000. Cell line sensitivity to evofosfamide was correlated with Br-IPM and cisplatin but not with PR-104A, SN30000 or 5-FU, indicating distinct sensitivity determinants. Evidence of antitumor activity with evofosfamide was observed in vivo. CRISPR screens identified potential evofosfamide sensitivity genes that were reproducibly enriched following drug exposure. Reductase-focused RNA interference screens defined a cluster of sensitivity genes that mapped to mitochondrial electron transport, whereas shRNA's targeted against presumed activating enzymes such as POR were not enriched. Concentration-dependent oxidation of cytochrome a and decreased respiration was observed in cells exposed to evofosfamide, suggesting reduction by mitochondrial complexes. This study provides a rationale for the clinical evaluation of evofosfamide with radiotherapy in genetically defined subsets of HNSCC patients.

### Tumor Microenvironment 4

#2957

T-cell receptor immunosequencing reveals novel insights into the immune response to human pancreatic cancer.

Yongwoo D. Seo,1 Florencia G. Jalikis,1 Xiuyun Jiang,1 Marissa Vignali,2 Harlan Robins,3 Venu G. Pillarisetty1. 1 _University of Washington, Seattle, WA;_ 2 _Adaptive Biotechnologies, Seattle, WA;_ 3 _Fred Hutchinson Cancer Research Center, Seattle, WA_.

Introduction Despite advancements in therapy, pancreatic ductal adenocarcinoma (PDA) remains an aggressive cancer with high mortality. It is characterized by dense inflammation, including many T cells; however, it is unclear whether these T cells signify a true antitumor response. In the setting of disappointing early results of immunotherapy in PDA, we sought to gain a deeper understanding of the tumor-associated T-cell response.

Methods With IRB approval, we obtained archival resected PDA tumors in paraffin-embedded blocks from 54 patients. We performed histopathology to identify blocks containing lymph nodes (LN) and performed T-cell receptor (TCR) immunosequencing on DNA extracted from immediately adjacent tissue. To address possible intratumoral heterogeneity, we analyzed duplicate samples from separate blocks for 10 patients. Productive clonality was defined as 1-Pielou's evenness for TCR rearrangements encoding a functional protein; TCR fraction was calculated from number of observed templates and the amount of input DNA. Two-tailed t-tests were used to compare subgroups. TCR sequence overlap between each of the 10 pairs of duplicate samples were calculated at the amino acid level.

Results Among samples that did not contain LN, mean TCR fraction was 0.27, and mean clonality was 0.15 (typical peripheral blood mononuclear cell clonality is 0.08). TCR fraction was positively correlated with clonality (R2=0.23, p=0.007), though this correlation was only significant in patients who received neoadjuvant chemotherapy (R2=0.19, p=0.03). TCR fraction was higher in patients with positive nodal status (0.32 vs. 0.23, p=0.02), but lower in patients who received neoadjuvant therapy (0.22 vs. 0.33, p=0.02). There was no survival difference between high or low TCR fraction or clonality. Samples containing LN (not included in previous analyses) had higher TCR fraction (0.49 vs. 0.27, p<0.0001) but lower clonality (0.09 vs. 0.15, p=0.0002); this relationship was maintained regardless of nodal status. Among 10 pairs of duplicate samples, there was 53% mean overlap of TCR amino acid sequences, compared to 0.1% for pairs of unmatched samples (p<0.0001).

Conclusion Here we demonstrate evidence of clonal expansion of T cells in the human PDA microenvironment. Extensive overlap of TCR sequences between two distinct samples for each of 10 pairs analyzed further suggests that the clonal expansion may be specific to tumor antigens. Neoadjuvant therapy may also influence clonal expansion, as only treated patients had a positive correlation between TCR fraction and clonality. Finally, the presence of a lymph node appears to skew TCR sequencing data toward a less clonal but more numerous population. This suggests that the most rigorous analysis of TCRs in a solid tumor microenvironment needs to exclude blocks that contain lymph nodes in order to characterize the true intratumoral population of T cells.

#2958

**Transcriptome analysis of astrocytoma** versus **non-neoplastic human microglia.**

Thais Fernanda Galatro,1 Antonio M. Lerario,2 Sueli M. Oba-Shinjo,1 Bart J. Eggen,3 Suely K. Marie1. 1 _University of São Paulo, São Paulo, Brazil;_ 2 _University of Michigan, Ann Arbor, MI;_ 3 _University of Groningen, Groningen, Netherlands_.

Diffuse gliomas are primary brain tumors characterized by infiltrative growth and high heterogeneity, rendering the disease mostly incurable. Advances in genetic analysis revealed that molecular alterations predict patients' overall survival and clinical outcome. However, glioma tumorigenicity is not exclusively caused by its genetic alterations. The crosstalk between tumor cells and the surrounding microenvironment plays a crucial role in modulating glioma growth and aggressiveness. The most abundant non-neoplastic cells in this microenvironment belong to the myeloid lineage, comprising CNS-resident microglia and infiltrating monocytes/macrophages (iTAMs). Understanding the dynamics between tumor and myeloid cells and the changes leading to pro-tumorigenic activation of innate immunity cells would elucidate potential treatment alternatives.

Microglia were isolated from the parietal cortex of 16 autopsy samples of cognitively intact humans (non-neoplastic, NN) and from 6 glioma samples (three GBMs, one anaplastic astrocytoma and two low grade astrocytoma, TU-glia), and their mRNA expression profile was determined by deep sequencing. The comparison of these transcriptome datasets provided an incomparable landscape of the crosstalk between immune and tumor cells.

Our preliminary analysis shows that differences between NN microglia and TU-uglia go beyond inflammation specific genes. While there is overexpression of anti-inflammatory markers (CD163, IL2RA, CXCL2 and TGFB1), implying pro-tumorigenic characteristics, there is also high expression of genes indicative of an acute, pro-inflammatory response, such as IL1B, CCL5, CCR7 and TNFA. Further analysis showed that TU-uglia expressed high levels of genes related to extracellular matrix (ECM) remodeling, such as fibronectin (FN1) and tenascin-C (TNC), as well as their ligands (ITGA4 and ITGB1), indicating that microglia, and not tumor cells, might be the main source for those proteins in the tumor microenvironment. We were also able to collect one sample of iTAMs from a GBM case. Despite not providing a significant number for statistical analysis, we can already see major differences in gene expression between this sample and our TU-uglia cohort. We identified surface receptors differentially expressed between both cell types (i.e.: ROBO1 and PDGFRA for TU-uglia, MARCO and MET for iTAMs), as well as transcription factors associated with stemness and proliferation, such as KLF4, SOX2 and OCT4 (the last two exclusive for TU-uglia). These results demonstrate that different activation signaling pathways occur within the same tumor sample. Microglia and iTAMs are subject to a number of signals from different clones of tumor cells, driving different responses along the progression of the disease, increasing its tumorigenicity. Better knowledge of this dynamics will help improve our understanding of gliomas and alternative treatment options.

#2959

Changes in the tumor stroma after MFP treatment in a MPA-induced murine mammary carcinoma model.

Gonzalo Ricardo Sequeira,1 Ana Sahores,1 Tomas Dalotto-Moreno,1 Silvia Vanzulli,2 Laura Polo,1 Virginia Novaro,1 Caroline Lamb,1 Mariana Salatino,1 Claudia Lanari1. 1 _Inst. de Biología y Medicina Experimental, Buenos Aires, Argentina;_ 2 _Academia Nacional de Medicina, Buenos Aires, Argentina_.

The role of the immune system in the regression of mammary carcinomas using endocrine therapies has been poorly investigated. The main goals of this study were: a) to evaluate differences in antiprogestin-induced regression of hormone receptor positive mammary carcinomas growing in immunocompetent or immunodeficient mice; b) to characterize the tumor infiltrating immune cells in mifepristone (MFP)-treated murine mammary carcinomas and c) to evaluate whether MFP treatment elicits immune protection in tumor re-challenge assays. Bone marrow (BM) cells from BALB/c-GFP mice were intravenously inoculated into NSG mice to establish the NSG/BM-GFP mouse model. GFP cells were detected in spleen, bone marrow, and blood from NSG/BM-GFP mice two months after BM-GFP cell inoculation. The murine mammary carcinoma 59-2-HI, originally induced by the administration of medroxyprogesterone acetate to BALB/c mice, regresses completely after MFP treatment. Tumors were orthotopically inoculated into NSG or NSG/BM-GFP female mice two months after BM grafting. When the tumors reached a size of approximately 50 mm2, low dose MFP pellets (0.2 mg) were administered in order to inhibit tumor growth. To characterize the infiltrating immune cells, tumors were excised after drug administration, disaggregated, and isolated cells were analyzed by FACs. For the re-challenge assay, tumors were excised 6 days after MFP treatment and animals were orthotopically re-inoculated with the same tumor in the opposite flank. Sham operated animals were used as controls. MFP inhibited the growth of tumors transplanted in either NSG or NSG/BM-GFP+ and no differences were observed between both groups, suggesting that the immune system is not a key factor mediating antiprogestin-induced tumor regression. Tissue remodeling was associated with an increase in lymphocytes (CD8+; T cells) and macrophages (CD11b+ F480+; p&lt0.01 at day 3 and 6), and a decrease in the Treg subpopulation (CD4+ CD25+ Foxp3+; p&lt0.05). These results led us to hypothesize that antiprogestin treatment could elicit a protective antitumor immune memory response by delaying tumor recurrence. To test this hypothesis we performed re-challenge assays using animals in which primary tumors have been previously excised. All tumors of control mice reached the ethical limit size (200 mm2) before day 47, while only half of the tumors in the MFP-treated group reached this size by this time (hazard ratio: 1.98). We conclude that antiprogestins induce tumor regression regardless of the presence of an impaired immune system and that the reduced intra-tumor Treg/CD8+ ratio observed in regressing tumors might be used as a predictive marker of treatment response. The re-challenge experiments agree with the hypothesis that regressing tumors expose intracellular antigens that generate a protective immune memory response, which in turn, could be associated with the long free relapse survival induced by endocrine therapy.

#2960

Adipocytes sequester and metabolize daunorubicin.

Xia Sheng,1 Jean-Hugues Parmentier,1 Jonathan Tucci,1 Hua Pei,2 Omar Cortez-Toledo,1 Christina Dieli-Conwright,3 Matthew Oberley,1 Michael Neely,1 Etan Orgel,1 Stan Louie,2 Steven D. Mittelman1. 1 _USC/Children's Hospital Los Angeles, Los Angeles, CA;_ 2 _USC School of Pharmacy, Los Angeles, CA;_ 3 _USC School of Dentistry, Los Angeles, CA_.

Obesity is associated with poorer outcome from many cancers, including childhood acute lymphoblastic leukemia (ALL). We have previously shown that adipocytes protect ALL cells from the anthracycline, daunorubicin (DNR). We therefore investigated whether adipocytes sequester and/or metabolize DNR in the ALL microenvironment. Using fluorescence and LC/MS measures, we demonstrated that adipocytes absorb DNR, reducing the intracellular DNR concentration in co-cultured BV173 ALL cells (after 48 hours, median fluorescent intensity of ALL cultured with adipocytes was 1.7±1.0 vs. 5.0±1.7 of those cultured alone, p<0.01). Mouse adipocytes convert DNR to the less active metabolite, daunorubicinol (DNR-ol); over 48 hours, media DNR decreased to 0.1±0.2 (vs. 24.8±8.76 ng/mL in control wells). At the same time, DNR-ol increased to 15.0±1.9 (vs. 0.7±0.6 in control wells; both p<0.05). Similar conversion of DNR to DNR-ol was observed in both mouse adipose explants and human adipose tissue biopsy samples ex vivo. qPCR confirmed human subcutaneous adipose tissue expresses several enzymes capable of metabolizing DNR, including AKR1A1, 1B1, 1C1, 1C2, 1C3, 7A2, and CBR1 and 3 (expression ranged between 20 and 195% β-actin). Using immunohistochemistry, we confirmed expression of AKR1C1, 1C2, and 1C3 in bone marrow adipocytes of children during the first month of treatment for ALL. Finally, two hours after an intravenous dose of DNR in mice, we found that the DNR-ol to DNR ratio was higher in subcutaneous (0.60±0.26) and omental (0.55±0.21) adipose than in white blood cells (0.16±0.11), bone marrow (undetectable DNR-ol), and spleen (undetectable DNR-ol). Together, these data demonstrate that adipocytes sequester and inactivate DNR, likely due to their expression of AKR and CBR enzymes. These findings uncover a novel and important mechanism which could promote local anthracycline resistance by cancer cells in microenvironment rich in adipocytes, such as bone marrow, omentum, and breast.

#2961

Targeting stromal-derived Dickkopf-3 (DKK3) for the treatment of pancreatic ductal adenocarcinoma (PDAC).

Liran Zhou,1 Hongmei Husted,1 Todd Moore,1 Mason Lu,1 Defeng Deng,1 Yan Liu,1 Vijaya Ramachandran,1 Thiruvengadam Arumugam,1 Baoan Ji,2 Huamin Wang,1 Jeffrey E. Lee,1 Craig D. Logsdon,1 Rosa F. Hwang1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Mayo Clinic, Rochester, MN_.

Introduction: We and others have shown that pancreatic stellate cells (PSCs) in the tumor-associated stroma of PDAC promote tumor progression and resistance to therapy but the precise mechanisms are unclear. We investigated the role of PSC-derived DKK3, a member of the Dickkopf family of glycoproteins, in PDAC progression, metastasis and response to chemotherapy.

Methods: We evaluated expression of DKK3 in human PDAC tissue and cell lines, human PSCs and in a genetically engineered mouse model (GEMM) of PDAC. The paracrine and autocrine effects of DKK3 on PDAC and PSCs were examined by treatment with exogenous DKK3 and gain- and loss of function assays for proliferation, migration, invasion, and gemcitabine-induced apoptosis. The effects of DKK3 on PDAC progression and metastasis were determined by shRNA neutralization and genetic ablation in orthotopic xenograft models and the KPC autochthonous model of PDAC. We developed novel monoclonal antibodies (mAbs) against DKK3 and tested their ability to neutralize DKK3 and prolong survival in mouse models of PDAC.

Results: DKK3 was expressed at 4.5 times higher levels in human PDAC by Affymetrix profiling compared to normal pancreas and was present in 99% (118/119) of samples on a tissue microarray with moderate to high expression in 59%. In a GEMM of PDAC, DKK3 appeared early with preneoplastic PanIN lesions with increased expression in invasive carcinoma. DKK3 was strongly expressed by PSCs with minimal to no expression in PDAC cells and knockdown by shRNA reduced PSC proliferation and migration by 60% and 84% compared to controls (p<0.001). Treatment of Panc1 and BxPC3 cells with DKK3 stimulated migration and invasion by 100-300% (p<0.001) and proliferation of DKK3-silenced Panc1 cells was decreased by 80% (p<0.001). Overexpression of DKK3 in L3.6pl cells increased colony formation in gemcitabine by >90% (p<0.001) with 65% reduction in apoptosis (p<0.01), indicating that DKK3 contributes to PDAC resistance to chemotherapy. When we ablated DKK3 in KPC mice by breeding with DKK3-knockout mice, tumor growth was inhibited and survival increased by 45% (p=0.0002). In addition, fewer PanIN lesions developed in DKK3-null mice suggesting that DKK3 may contribute to the early developmental stages of PDAC. DKK3 neutralizing mAbs abrogated DKK3-mediated induction of PDAC cell migration, invasion and resistance to gemcitabine in vitro. Furthermore, treatment with DKK3 mAb significantly inhibited primary tumor growth, reduced peritoneal metastases and prolonged survival in an orthotopic model of PDAC by 43% compared to control mAb (p=0.005; HR 0.24, 95% CI 0.01-0.30).

Conclusions: These data are the first report, to our knowledge, of a tumor-promoting function of DKK3 and our results suggest that neutralization of DKK3 may be an effective approach as a primary treatment for PDAC and to enhance responsiveness to chemotherapy.

#2962

Mechanobiology of epithelia on native basement membrane and relevance for cancer invasion.

Marija Plodinec,1 Philipp Oertle,1 Daphne Assgeirson,1 Willi Halfter,2 Serenella Eppenberger Castori,3 Ellen C. Obermann,1 Alexandre Glentis,4 Roderick Y. LIM1. 1 _Univ. of Basel, Basel, Switzerland;_ 2 _Univ. of Pittsburgh, Pittsburgh, PA;_ 3 _Univ. Hospital Basel, Basel, Switzerland;_ 4 _Institute Curie, Paris, France_.

The onset of metastasis occurs when cancer cells invade and breach the basement membrane (BM) that provides mechanical support to epithelial tissues. Yet, it remains unclear what triggers cancer cells to breach the BM, and how 'triggered' cells in fact breach the BM. We have established an in vitro assay using native BM interface for culturing epithelial cells. Using atomic force microscopy (AFM) with other high-resolution microscopies and TER (trans-epithelial resistance), we have correlated the mechano-cellular attributes of the BM/epithelium interface to its biochemical and structural properties. We demonstrated that the internal limiting membrane (ILM) isolated from human retinas acts as a native substrate for culturing epithelial cells in terms of BM composition, architecture and stiffness. These are required to act jointly in order to achieve apico-basal polarity, tissue barrier formation and stiffness properties of the epithelial layer similarly to secretory epithelia in vivo. The native BM serves several advantageous over reconstituted Matrigel, an ECM extracts that originates from mouse tumor ascites. Besides variations in thickness and biochemical composition, we find that Matrigel is mechanically 100-fold more compliant (i.e., softer) than native BMs. When tumorigenic variants of cells are used, we demonstrated that cancer cell invasion is associated with a decrease in cellular stiffness correlated to changes in cell and BM morphology. In addition, we showed that activation of ß1 integrin by the stiffness and architecture of the native alpha - 5 laminin chain has a key role, not only as previously thought for maintenance of cell polarity but also for the establishment of a physiological mechanophenotype. On the other hand it is well know that during cancer progression in vivo, cancer cells can perforate the BM using proteolysis. Whether stromal cells also play a role and what kind of role in this process is unclear. Therefore, we asked if carcinoma‐associated fibroblasts (CAFs) isolated from cancer patients promote cancer cell invasion through a BM. In the presence of CAFs, moderately invasive cancer cells invade in a matrix metalloproteinase‐independent manner. Using live imaging and atomic force microscopy, we showed that CAFs actively pull, stretch and soften the BM, forming gaps through which cancer cells can migrate. By exerting contractile forces, CAFs alter the organization and the physical properties of the BM, making it permissive for cancer cell invasion. Finally, we propose that, in addition to proteolysis, mechanical interactions between CAFs and BM represent an alternative mechanism of BM breaching. Given their mechano-biological relevance, native BMs allow us to further understand how mechanosignaling occurs between the epithelia and the surrounding stromal layers at the BM interface during cancer invasion and progression.

#2963

Inhibition of stromal p38MAPK abrogates breast cancer metastasis.

Sheila A. Stewart,1 Bhavna Murali,1 Qiaho Ren,1 Xinming Su,1 Kevin Flannagan,1 Xianmin Luo,1 Jasmine Sponagel,1 Yujie Fu,1 Elise Alspach,1 Kathlees Leahy,1 Roberta Faccio,1 Kathleen Weilbaecher,1 Joseph Monahan2. 1 _Washington University, St. Louis, MO;_ 2 _Confluence Life Sciences, St. Louis, MO_.

Bone metastasis is a devastating and fatal complication of breast cancer for which we lack effective therapies. Thus, identifying therapies that effectively limit metastases will significantly reduce comorbidities and improve long-term survival. Recently we demonstrated that the p38MAPK pathway sustains the pro-tumorigenic senescence-associated secretory phenotype (SASP) and targeting this pathway limits the tumor-promoting capabilities of senescent cells and cancer-associated fibroblasts (CAFs). Because we found that a significant percentage of p38MAPK-dependent SASP factors are expressed in the stroma associated with breast cancer lesions, we asked whether targeting p38MAPK could limit primary and metastatic breast cancer growth. While p38MAPK inhibition modestly limited primary tumor growth, we found that its inhibition significantly reduced breast cancer bone metastases by specifically targeting the stromal compartment. Further, p38MAPK inhibition was as effective as paclitaxel at limiting tumor growth in the bone but in contrast to paclitaxel, which failed to protect from cancer-induced bone loss, p38MAPK inhibition also protected against devastating bone loss. This contrasts our p38MAPK approach from zoledronic acid, which limits bone loss but fails to slow tumor growth in already engrafted tumors. Analysis of the mechanism(s) responsible for this reduced metastasis suggests that p38MAPK inhibition targets reactive and/or senescent osteoblasts within the bones of animals harboring metastatic lesions. Because we find that senescent osteoblasts are present in human bone, we postulate that they promote metastatic outgrowth and thus p38MAPK inhibition limits the pro-metastatic activities of these cells. Finally, we will present recent data from our preclinical model that demonstrates that inhibition of the p38MAPK pathway can drastically reduce metastasis from the primary site. We propose that p38MAPK is an important stromal-specific therapy for breast cancer metastasis to the bone.

#2964

Stromal Hedgehog pathway activation suppresses growth and metastases of lung adenocarcinoma.

Sahba Kasiri, Baozhi Chen, Alexandra Wilson, Umaru Barrie, Ummay Marriam, Zhiqun Zeng, Luc Girard, James Kim. _UT Southwestern, Dallas, TX_.

Aberrant activation of the Hedgehog (Hh) signaling pathway, a crucial developmental pathway, drives the tumor growth of Gorlin-type cancers. However, recent data suggest that paracrine activation of the pathway is tumor suppressive rather than oncogenic in sporadic epithelial cancers. The role of the pathway in non-small lung cancer is poorly understood. Thus, we explored the role of stromal Hh pathway activation in growth of lung tumor epithelia. Human lung adenocarcinoma cell lines were used to probe SHH mRNA and protein expression. Co-culture of high SHH expressing cell lines with murine embryonic and lung fibroblasts were used to confirm and probe the role of paracrine SHH expression on the growth of lung cancer cells. The in vivo role of paracrine SHH was tested using autochthonous lung cancer models with conditional KRASG12Dactivation, p53 loss,, and SHH loss compared to wild-type SHH. In human lung adenocarcinoma patients, higher expression of SHH mRNA in lung adenocarcinoma correlated with poor overall and progression free survival. A scan of 35 human lung adenocarcinoma cell lines revealed heterogeneous expression of SHH and IHH with high expression found predominantly in mutant K-Ras lines. Co-culture of high SHH expressing tumor epithelial cells and Shh-Light2 reporter cell lines demonstrated that SHH activated the fibroblast reporter in a paracrine manner, rather than an autocrine effect on cancer cells. Treatment with the SMO inhibitor, KAAD-cyclopamine, also inhibited the growth of tumor epithelial cells in co-culture with NIH-3T3 fibroblast cells but the effect was decreased when co-cultured with lung fibroblasts. Genetic loss of SHH in an autochthonous mouse model, LSL-KrasG12D/+;Trp53fl/fl; Shhfl/fl (KPS) did not affect overall survival compared to LSL-KrasG12D/+;Trp53fl/fl (KP) mice However, early inhibition of the Hh pathway by anti-SHH/IHH antibody, 5E1, on KP mice resulted in significantly worse survival rates with increased metastatic burden compared to IgG treatment. Analysis of KP tumors revealed unexpected high levels of IHH mRNA by in situ hybridization that may account for the survival differences seen between genetic ablation and pharmaceutical inhibition of the Hh ligands. In conclusion, the Hh signaling pathway acts upon lung stromal cells in a paracrine fashion. Inhibition of Hh activity in vivo worsened mortality rate due to increase in tumor growth and metastases. Furthermore, mutant Kras lung cancers express high levels of IHH that dominates the tumor suppressive effect of our mouse models.

#2965

**Myeloid depletion reverses established T-ALL growth** in vivo **.**

Todd A. Triplett, Aram Lyu, Wesley Godfrey, Lauren I.E. Ehrlich. _University of Texas at Austin, Austin, TX_.

T cell acute lymphoblastic leukemia (T-ALL) is a hematological cancer that arises from developing T-cells. Most T-ALL research has focused on elucidating cell-intrinsic genetic permutations that promote tumor proliferation and survival, which has resulted in a detailed description and understanding of the genetic mutations associated with T-ALL in patient cells. However, to date these studies have not resulted in effective targeted therapies, which may potentially be due to the heterogeneity of genetic lesions within and between patients or the acquisition of new mutations. Thus, we need to broaden our understanding of other factors promoting T-ALL growth to identify alternative therapeutic targets that are effective across mutational landscapes.

To this end, our studies seek to identify extrinsic signals in the tumor microenvironment that enable T-ALL growth. Our previous studies used a systematic and unbiased evaluation of endogenous stromal subsets from the thymic TME to reveal that myeloid cells, primarily dendritic cells (DC), were necessary and sufficient for ex vivo survival of T-ALL cells. Furthermore, transcriptional profiling revealed elevated expression of Igf1r and Igf1 by T-ALL and DC from the TME, respectively. Using multiple molecular approaches, inhibition of IGF1R revealed that IGF1R signaling was necessary for DC-mediated survival of T-ALL from multiple tumor locations. These findings have relevance to human T-ALL as pervasive TEC-free regions containing extensive DC networks were evident in T-ALL patient samples, and previous studies have verified that IGF1R, a direct target of NOTCH1, is expressed at high levels in T-ALL patients.

In these studies, we sought to determine whether myeloid cells were required for tumor survival in vivo. After transplantation, tumors became established systemically in lymphoid and non-lymphoid tissues of the recipients. Immunofluorescence analysis revealed extensive myeloid networks around and between T-ALL tumor cells in multiple tissues. As for primary tumors, transplanted T-ALL cells also required the presence of DC from the TME for survival ex vivo. Importantly, myeloid cell depletion in transplanted hosts with established T-ALL resulted in substantial reduction in tumor cell burden in all tissues examined, thus demonstrating a fundamental role for these cells in supporting T-ALL in vivo. To identify key survival pathways activated in T-ALL cells by myeloid cells, we have compared comprehensive trancscriptomic and phosphoproteomic profiling from tumor cells in the presence or absence of myeloid cells in transplant models. Preliminary analysis of T-ALL versus WT thymocytes has confirmed hyper-activated IGF1R signaling, as indicated by hyper-phosphorylation of downstream targets, such as IRS1. Collectively, these studies provide the first evidence that myeloid cells directly support T-ALL in vivo, suggesting that these cells and their associated signals may be auspicious therapeutic targets.

#2966

Stromal platelet derived growth factor receptor-beta (PDGFRbeta) promotes breast cancer progression.

Gina M. Sizemore, Anisha M. Hammer, Katie A. Thies, Steven T. Sizemore, Anthony J. Trimboli, B. Eason Hildreth, Raleigh D. Kladney, Arnab Chakravarti, Gustavo Leone, Michael C. Ostrowski. _Ohio State University, Columbus, OH_.

Over the past decade it has become evident that the tumor microenvironment (TME) actively participates in carcinogenesis. Tumor-associated fibroblasts, for example, modulate neighboring tumor epithelium through growth factor secretion to initiate and promote tumor growth. The platelet derived growth factor receptors (PDGFRs), PDGFRalpha and PDGFRbeta, are receptor tyrosine kinases activated by PDGF that may be critical and actionable mediators of breast tumor-stromal communication. PDGFRs are predominately expressed in breast tumor stroma while their cognate ligands are specifically expressed in tumor epithelium and associated endothelium. In some cancers, tumor-derived PDGFs act on the TME to recruit tumor associated fibroblasts; however, this role has not been described in breast cancer. To begin to evaluate a role for PDGFRbeta, we utilized publicly available gene expression data to confirm upregulation in tumor stroma compared to tumor epithelium. Importantly, PDGFRB is increased in tumor stroma compared to normal stroma. To directly test whether stromal PDGFR activation promotes tumor growth, we co-injected murine mammary tumor cells with or without PDGFR-expressing mouse mammary fibroblasts (MMFs) orthotopically in FVB/N mice. MMF inclusion increased tumor cell proliferation as well as associated angiogenesis while systemic treatment with imatinib mesylate, a small molecule inhibitor for PDGFR, restored both proliferation and angiogenesis back to baseline. These findings indicated the importance of PDGFR signaling in tumor initiation leading us to develop a mouse model of stromal-specific PDGFRbeta activation using the Fsp-cre transgene previously published by our group (henceforth referred to as "PDGFRbeta mutant"). PDGFRbeta mutant mammary glands exhibit increased tertiary side-branching and epithelial proliferation confirming a stromal-specific PDGFRbeta effect on neighboring epithelium during development. Further, MMFs isolated from the PDGFRbeta mutant mice exhibit increased motility towards PDGF-B expressing tumor cells in vitro, which implies increased response and recruitment of the mutant MMFs towards an expanding tumor. To test whether PDGFRbeta mutant mice harbor a mammary TME supportive of increased tumor growth, we injected murine mammary tumor cells orthotopically into either control or PDGFRbeta mutant mice finding that the time required to meet early removal criteria (tumor &gt1.2cm3) was shorter in the mutant mice compared to controls. Ongoing studies are evaluating whether systemic PDGFR inhibition will abrogate this observed increase in tumorigenesis. In summary, our data suggest that stromal PDGFRbeta signaling is pro-tumorigenic in breast cancer and that inhibition using well-described PDGFR inhibitors could be a valid therapeutic approach for women whose tumors express increased PDGF-to-PDGFR tumor-stromal signaling.

#2967

Pancreatic cancer cell growth requires lipids released by tumor-induced stroma autophagy.

Petrus R. De Jong,1 Sean-Luc Shanahan,2 Morgan A. Brand,1 Alejandro D. Campos,1 Anagha Srirangam,1 Nikolas Marino,1 Claudia P. Miller,1 Olga Zagnitko,1 Adam D. Richardson,1 David A. Scott,1 Brian P. James,1 Andrew P. Hodges,2 Ally Perlina,2 Alexey M. Eroshin,2 Randall French,3 Malene Hansen,2 Sally A. Litherland,4 Andrew M. Lowy,3 J. Pablo Arnoletti,5 Garth Powis1. 1 _SBP NCI-Designated Cancer Center, La Jolla, CA;_ 2 _SBP Medical Discovery Institute, La Jolla, CA;_ 3 _University of California, San Diego, La Jolla, CA;_ 4 _Florida Hospital Cancer Institute, Orlando, FL;_ 5 _Institute for Surgical Advancement/ Florida Hospital Center for Specialized Surgery, Orlando, FL_.

Pancreatic ductal adenocarcinoma (PDAC) is non-resectable in the majority of patients and highly resistant to chemotherapy, resulting in a poor survival. The tumor microenvironment and hypoxia are important modifiers of cancer progression in PDAC. Understanding the metabolic vulnerabilities of PDAC in the harsh tumor microenvironment may lead to novel therapeutic approaches with improved clinical efficacy. First, we found that PDAC cells showed beneficial effects of co-cultured stroma cells, but only under lipid-free serum conditions. To study the metabolic crosstalk between cancer cells and stroma in more detail, we performed an untargeted metabolomic screen of PDAC cells and fibroblasts co-cultured in normoxia and hypoxia, and performed RNA-seq profiling in parallel. We found that stromal cells are metabolically more responsive to co-culture than cancer cells. PDAC cells induce catabolic carbohydrate and protein metabolism in stromal cells, particularly in hypoxia. In contrast, 13C-based metabolic flux assays demonstrated that stromal cells display enhanced anabolic lipid metabolism in co-culture with PDAC cells. Furthermore, de novo synthesized 13C-labeled fatty acids in stromal cells were taken up by PDAC cells. In particular, PDAC cells showed extensive scavenging of lysophospholipids (lyso-PLs) from the culture medium, which was increased in co-culture under hypoxic conditions. These data were confirmed by analyzing portal vein plasma samples isolated from pancreatic cancer patients before and after surgery. In addition, we found metabolites and expression levels of metabolic enzymes from the glycerophospholipid pathway to be enriched in PDAC cells in co-culture and hypoxia. By using fibroblasts, human pancreatic stellate cells and patient-derived cancer-associated fibroblasts (CAFs), we demonstrate direct transfer of lyso-PLs from stromal to PDAC cells via lipid droplets. The transfer of lyso-PLs was abrogated by pharmacological inhibitors of autophagy, or by siRNA-mediated knockdown of autophagy genes in stromal and tumor cells. These data suggest that PDAC cells cause stroma cells to undergo autophagy, and reprogram stroma metabolism to obtain complex lipid species for their metabolic needs in the lipid-starved tumor microenvironment.

#2968

Examining the role of breast cancer Neuregulin1 and macrophage ErbB3 in intravasation.

Ramon Cabrera, Serena Chiang, Jeffrey Segall. _Albert Einstein College of Medicine, Bronx, NY_.

The purpose of our studies is to investigate the role of Neuregulin1 (NRG1) and ErbB3 signaling between breast cancer cells and macrophages in facilitating tumor cell intravasation. The interaction of breast cancer cells with other cell types within the tumor microenvironment plays an important role in metastasis. These interactions are thought to influence tumor cell invasion and intravasation, two important steps in the process of metastasis. Our studies are specifically interested in examining the signaling occurring between tumor cells and macrophages. Previous studies have established the presence of paracrine signaling between breast cancer cells and macrophages, where colony stimulating factor 1 (CSF-1) produced by the tumor cells stimulates the production of epidermal growth factor (EGF) by macrophages, leading to chemotactic invasion of the tumor cells. In addition to this paracrine loop signaling between tumor cells and macrophages, it has been seen that macrophage expression of ErbB3, a member of the EGFR family of receptor tyrosine kinases, may play a role in facilitating tumor cell invasion. In order to examine the effects of signaling between tumor cells and macrophages in intravasation, we utilize an in vitro transendothelial migration (iTEM) assay. This assay uses transwells coated with matrigel and endothelial cells in order to mimic the entry of tumor cells into blood vessels. We show that using an ErbB3 blocking antibody results in a significant reduction of macrophage-induced transendothelial migration of breast cancer cells. Additionally, reduction of expression of the ErbB3 receptor ligand Neuregulin1 in tumor cells yields a similar result. Stimulation of macrophages with NRG1 leads to increased expression of Jagged1 (JAG1), a ligand of the Notch receptor. Activation of the Notch receptor pathway has been shown to be involved in tumor cell invasion. Overall our studies look to further examine the interaction between tumor cells and macrophages, and these observations indicate that ErbB3, NRG1, and JAG1 could all serve as novel targets in metastasis and the tumor microenvironment.

#2969

Paracrine effect of the endothelium on prostate cancer cells.

Verónica Torres-Estay, Patricia Fuenzalida, Catalina Ascencio, Carla Cembrano, Daniela Carreño, Néstor Corro, Viviana Montecinos, Gareth Owen, Xavier Figueroa, Julio Amigo, Juan Carlos Saéz, Alejandro Godoy. _Pontificia Universidad Católica de Chile, Santiago, Chile_.

Prostate cancer (CaP) is the most commonly diagnosed cancer and the second leading cause of cancer deaths among males in the United States. Androgen deprivation therapy (ADT) is the standard treatment for advanced or metastatic CaP. However, during ADT, CaP progresses from an androgen-sensitive (AS-CaP) to a more aggressive, and eventually lethal, castration-resistant (CRPC) phenotype. There is evidence to suggest that the prostate tumor mass is under tight control of endothelial microvasculature due to an increase in angiogenesis by tumor cells. Nevertheless, now there is evidence to support that this influence is not one-directional and that the endothelial cells secrete a large number of active substances (angiocrine factors), which may directly or indirectly influence tumor growth and progression. However, the direct impacts of the endothelium on prostate tumor progression or the molecular mechanisms that are involved in this communication remain unclear. Here we investigated the potential influence of endothelium-derived paracrine factors on prostate cancer biology and the role of connexins in these interactions, since connexins play a major role in cell-cell communication and form a bidirectional signaling pathway to assemble gap junctions and alter cell behaviors. We measured the effect of conditioned media (CM) obtained from a primary culture of human endothelial cells isolated from umbilical vein (HUVEC) on viability, proliferation, migration and invasion of CaP cell lines (LNCaP, LNCaP-C4-2 and PC3) and in the metastatic potential by in vivo assays using co-injection of CaP cell with HUVEC or injection of CaP cells pre-incubated with CM from HUVEC in a zebrafish embryo model. Finally, we studied the expression and the role of connexins on this stimulation using pharmacological (GJIC inhibitors) approaches. All together, our results showed that CM from endothelial cell induces an increases in the viability and proliferation in all CaP cell lines (LNCaP, LNCaP-C4-2 and PC3) but only increases migration of the CRPC cell lines (LNCaP-C4-2 and PC3). We also observed in our in vivo model that endothelial cells either through cell-cell interaction or by paracrine communication increases the metastatic ability of the CaP cells. Moreover, the increase in viability and migration of CaP cells observed with CM from endothelial cells was blocked using inhibitors of gap junctions. Real-time PCR analyses detected an up-regulation of Cx43 mRNA after exposition to CM from endothelial cells. Our data suggest that angiocrine communication between endothelial cells and CaP cells increases proliferation and migration of more aggressive CaP cells which could be important for the acquisition of the aggressive phenotype of the disease, and this interaction could be mediated by Cx43. Delineation of such critical players may culminate in identifying therapeutic targets or biomarkers to counteract CaP, especially advanced CaP.

#2970

Mitigating tumor-stroma metabolic symbiosis for cancer therapy.

Dhruv Kumar, Jacob New, Vikalp Vishwakarma, Hemant Chavan, Partha Kasturi, Sufi M. Thomas. _Kansas University Medical Center, Kansas City, KS_.

Head and neck squamous cell carcinoma (HNSCC) affects 40,000 patients annually and is associated with <50% 5-year survival. There is an urgent need to better understand the biology of the disease in order to develop more effective therapeutic approaches. HNSCC tumors are dysplastic with up to 80% fibroblasts. We recently reported that tumor-associated fibroblasts (TAFs) make HNSCC more aggressive. Furthermore, we reported that TAFs produce hepatocyte growth factor (HGF), which binds to the c-Met receptor expressed on HNSCC to drive aggressiveness. Although we did not detect HGF secretion from HNSCC cell lines, we reported that paracrine activation of c-Met by TAF-secreted HGF facilitates HNSCC progression. Reciprocal signaling between the tumor and stroma has been reported in several cancers to facilitate tumor growth, invasion and resistance to therapy. Recent studies have shown that c-Met activation promotes glycolysis. Although highly glycolytic, the mechanisms regulating HNSCC glycolysis remain unknown. We show that TAF-secreted HGF through c-Met activation on HNSCC (a) induces aerobic glycolysis accompanied by lactate production, and b) regulates expression of basic fibroblast growth factor (bFGF). Studies have shown that HNSCC tumors have high lactate levels resulting from increased glycolysis, and this correlates with reduced survival. Our data demonstrate that TAF-secreted HGF increases key glycolytic enzymes including hexokinase II. Furthermore, HGF increases glycolysis and lactate production from HNSCC. We demonstrate that HGF also increases levels of the bidirectional lactate transporter, monocarboxylate transporter 1 (MCT1). We demonstrate that MCT1 levels are increased in both HNSCC and TAFs under co-culture conditions. In addition, HGF stimulation increases levels of MCT1 in HNSCC indicating a possible mechanism whereby HNSCC remove the excess lactate generated during glycolysis. The mechanisms whereby HNSCC tumors survive highly acidic conditions remain unknown. Since MCT1 levels are increased in TAFs as well, we sought to determine if TAFs utilize the lactate as a carbon source to generate energy. Indeed, we found that bFGF secreted by HNSCC, binds to its cognate FGF receptor (FGFR) on TAFs to facilitate latate utilization through mitochondrial oxidative phosphorylation (OXPHOS). Thus there exists a metabolic symbiosis between HNSCC and TAFs that contribute to tumor growth. Through these studies, we delineate the mechanisms of glycolysis regulation in HNSCC and demonstrate that inhibition of the cross-talk between HNSCC and TAFs can be used as a novel therapeutic approach.

#2971

The prognostic relevance of inflammatory cytokines and growth factors elaborated from adipose-derived stem cells in breast cancer.

Nicole Werwie,1 Kelsey Sadlek,1 Eric Lundstrom,1 Daniel Berrebi,1 Gerald Hobbs,1 Linda Vona-Davis2. 1 _West Virginia University, Morgantown, WV;_ 2 _West Virginia University Cancer Institute, Morgantown, WV_.

Introduction: Given the high rates of obesity and aggressive breast cancers, it is paramount to understand how adipose tissue supports the tumor microenvironment. We previously found that adipose-derived stem cells cultured with triple-negative tumor cells enhanced the expression of numerous pro-inflammatory cytokines. This finding is significant as it could explain, in part, how obesity contributes to worse outcomes in breast cancer. To further expand this study, we probed the Cancer Genome Atlas (TCGA) breast invasive carcinoma dataset for the expression profiles of the inflammatory cytokines we found to be highly up-regulated in vitro. We predicted TCGA data would demonstrate a correlation between cytokine overexpression and worse clinical scenarios.

Methods: Cytokines and growth factors were measured in the media of adipose stromal cells and MDA-MB-231 tumor cells via membrane-based antibody array. To validate clinical significance, we compared each cytokine's mRNA expression profile, available from primary tumor samples of TCGA breast invasive carcinoma dataset (n = 1,005), to indices of tumor progression and survival status. For survival analysis, samples were dichotomized as high or low expression by individual cytokines (z score ≥ 1.5). Cytokine and growth factor expression was further stratified according to molecular subtype of breast cancer (luminal A, luminal B, HER2, triple negative) and receptor status. Ingenuity pathway analysis (IPA) was performed to identify candidate markers related to obesity or inflammation.

Results: High levels of FGF7 and CCL5 protein were found in the conditioned media of tumor and adipose stem cell co-cultures and together they showed a significant impact on breast cancer survival within TCGA data. FGF7, CCL5, together with CCL2, IL6, and IL6R were further analyzed for expression across molecular subtypes of breast cancer. IL6R, CCL5, and CCL2 expression varied significantly between breast cancer subtypes, with mean expression highest in triple-negative tumors. After dichotomizing the samples for estrogen receptor status, IL6R, CCL5, and CCL2 showed significantly higher mean expression in ER negative tumors. Greater expression of these cytokines in tumors was correlated with reduced patient survival in triple-negative tumors and ER negative subtypes. IPA analysis indicated IL6, IL6R, CCL5, and CCL2, but not FGF7, cooperate through inflammatory pathways to promote tumor progression.

Conclusions: Genomic data analysis supports in vitro findings that IL6R, CCL5, and CCL2 overexpression, either independently or synergistically, predict a worse prognosis in breast cancer. For adipose-driven, hormone receptor negative breast tumors, these cytokines and growth factors could serve as novel therapeutic targets. (Supported by NIH P20GM103434 and NIGMS U54GM104942)

#2972

SPINK1, a soluble factor released by the therapy-damaged tumor microenvironment, promotes prostate cancer resistance.

Fei Chen,1 Da Fu,2 Eric Lam,3 Yu Sun1. 1 _Institute of Health Sciences, Shanghai Institutes for Biological Sciences, CAS, Shanghai, China;_ 2 _Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai, China;_ 3 _Department of Surgery and Cancer, Imperial College London, London, United Kingdom_.

Cancer evolution is driven by not only the genetic and/or epigenetic alterations of cancer cells, but also diverse factors that are derived from the surrounding tumor microenvironment (TME). Upon chemotherapy or radiation, stromal cells in the TME become senescent and develop a senescence-associated secretory phenotype (SASP) that is characterized by secretion of a large number of cytokines, chemokines, growth factors, and proteases. In most cases, the SASP is functionally regulated by the DNA damage secretory program (DDSP) and pathophysiologically responsible in vivo for disease exacerbation. We recently disclosed significant upregulation of the serine protease inhibitor Kazal type I (SPINK1) in primary normal human prostate fibroblasts after exposure to DNA damaging agents. SPINK1 plays critical roles in cancer cell proliferation, survival and motility in multiple human malignancies, but its influence as a soluble TME-associated factor on cancer progression remains unknown. In this study, we performed a series of molecular and cellular studies to define the biological roles of SPINK1 in an activated TME. Promoter analysis indicated that SPINK1 expression in the fibroblasts is regulated by multiple transcriptional factors including the NF-κB complex in response to genotoxic stress. Fibroblast-derived SPINK1 can significantly enhance the aggressiveness of prostate cancer cells including accelerated proliferation, increased migration, elevated invasion and more importantly, enhanced chemoresistance. SPINK1 triggers a typical epithelial-mesenchymal transition (EMT) in cancer cells, a process mediated by EGFR/PI3K/Akt and MAPK/Erk signaling pathways. Consistent with the in vitro data, our in vivo studies suggested that SPINK1 expression in the prostate TME substantially promoted cancer survival and disease progression. Clinical investigation revealed increased expression of SPINK1 in the stroma of multiple organ types including the prostate, lung and breast of cancer patients after chemotherapy, implying the systemic induction of SPINK1 by anticancer agents. Overall, our study demonstrates that SPINK1 is a soluble biomarker of therapeutically damaged TME and represents a potentially exploitable molecular target in human prostate cancer clinics.

#2974

Obesity-induced modifications in the mammary gland microenvironment promote TNBC progression even after weight loss.

Alyssa J. Cozzo, Ottavia Zattra, Alex J. Freemerman, Liza Makowski. _UNC-Chapel Hill, Chapel Hill, NC_.

Obesity contributes to 20% of US female cancer deaths and is linked to increased risk for triple negative breast cancer (TNBC), as well as increased risk of vascular invasion, metastasis, and mortality in breast cancer patients. We previously showed that obesity-induced elevation of hepatocyte growth factor (HGF) signaling through its receptor, cMET, accelerated development of tumors in the C3(1)-TAg GEMM of TNBC compared to low fat diet (LFD)-fed lean controls. Weight loss significantly reduced HGF/cMET signaling in normal mammary glands and cMET in tumors, and reversed high fat diet (HFD)-driven tumor progression, while crizotinib-mediated cMET inhibition limited tumor development and microvascular density in both lean and obese C3(1)-TAg mice. To differentiate tumor-intrinsic versus microenvironment-associated mechanisms, we employed an orthotopic transplant model of a C3(1)-TAg TNBC cell line to investigate tumor growth dynamics in lean, obese, or formerly obese (FOb) microenvironments. We hypothesized that TNBC tumors derived from lean C3(1)-TAg mice and transplanted into obese mice would grow more rapidly, contain more macrophages (MFs) and exhibit greater blood vessel density than tumors in lean or FOb mice due to obesity-induced MF and/or endothelial secretion of HGF. At 8 weeks of age, female FVB/NJ mice were randomized to diet groups (N=20/diet): 1) lean (LFD, 10% kcal from fat), 2) obese (HFD, 60% kcal from fat) 3) or FOb (HFD, 5 weeks; LFD, 5 weeks). Following 10 weeks on diets mice were injected with 2x105 luciferase-tagged C3(1)-TAg cells. Tumor volume was measured by calipers. At 21 days post-injection mice were sacrificed for tissue collection. Normal mammary gland, tumors, and tumor-adjacent adipose + tumor-draining lymph node were collected for histological examination and gene expression analysis. Mammary fat pad mass in obese FVB/NJ mice (0.27 g) was significantly greater than lean (0.14 g) or FOb animals (0.11 g) (P<0.0001). Tumor volume at sacrifice was significantly greater in obese vs lean animals (190 mm3 vs 89.96 mm3, respectively; P<0.05). However, mean final tumor volume in FOb animals (148.0 mm3) did not differ significantly from either lean or obese mice. Our findings show that obesity accelerated tumor progression relative to lean mice. C3(1)-TAg cells transplanted into FOb mice resulted in an intermediate rate of tumor growth, suggesting that obesity-mediated changes to the microenvironment were not fully corrected by weight loss despite normalization of body weight and adiposity. To elucidate how the FOb mammary microenvironment persists in driving TNBC tumor promotion, planned analyses include histological examination of candidate mediators, including MF infiltration and regional lymph node metastasis by diet group, as well as quantification of pro-growth and pro-angiogenic signaling molecules (e.g., HGF, VEGF, Ang2) in lysates of tumors and tumor-adjacent adipose.

#2975

Collagen type XI alpha 1 confers cisplatin chemoresistance in ovarian cancer through inhibitor of apoptosis proteins (IAPs).

Miran Rada,1 Jennifer Cha,2 Jessica Sage,1 Sandra Orsulic,3 Dong-Joo Cheon1. 1 _Albany Medical College, Albany, NY;_ 2 _Rensselaer Polytechnic Institute, Troy, NY;_ 3 _Cedars-Sinai Medical Center, Los Angeles, CA_.

Ovarian cancer is the most lethal gynecological cancer, which affects one in 70 females in the United States during their lifetime. While cisplatin is the frontline treatment for ovarian cancer, chemoresistance severely limits the treatment success. The goal of this study is to identify novel therapeutic targets to overcome cisplatin resistance. We recently identified Collagen type XI alpha 1 (COL11A1) as a novel biomarker associated with cisplatin resistance in ovarian cancer. COL11A1 is a component of the tumor stroma and its expression is restricted to cancer-associated fibroblasts (CAFs) adjacent to tumor cells. We further showed that COL11A1 protects ovarian cancer cells from cisplatin-induced apoptosis, yet the underlying mechanism is largely unknown. To understand the molecular mechanism by which COL11A1 protects cancer cells from cisplatin-induced apoptosis, we overexpressed COL11A1 in ovarian cancer cells and performed RNA-Seq. Our RNA-Seq data revealed that COL11A1 did not change the levels of genes involved in cisplatin transport, inactivation, and DNA repair pathways in cancer cells, which are well-established mechanisms to confer cisplatin resistance. Rather, COL11A1 enhances the expression of the Inhibitors of Apoptosis Proteins (IAPs), including XIAP, BIRC2 (c-IAP1) and BIRC3 (c-IAP2). Using a full-length COL11A1 protein, genetic manipulation, and co-culture method, we confirmed that COL11A1 significantly increases the expression of IAPs upon cisplatin treatment. The inhibition of XIAP, BIRC2 and BIRC3 with an IAP antagonist BV6 attenuated the ability of COL11A1 to inhibit cisplatin-mediated apoptosis. Our data indicates that COL11A1 activates Akt, and Akt is known to phosphorylate XIAP at serine-87, which protects it from ubiquitination and degradation in response to cisplatin. Collectively, our data identify IAP as a mediator of COL11A1's action in blocking cisplatin-induced apoptosis.

#2976

Critical role of spatial location of hypoxia and its association with astrocytes in the resistance of brain tumor to therapy.

Chiu-Min Lin, Chi-Shiun Chiang. _National Tsing-Hua University, Hsinchiu, Taiwan_.

Many reports have shown that hypoxia and gliosis could promote tumor cells by shielding from the immune response and relapsing, but their roles on brain tumor therapies are still unclear. Using an invasive murine astrocytoma tumor model, ALTS1C1, we found that tumor invasion front is associated with a specific hypoxia region, naming tumor peripheral hypoxia, contributes to brain tumor therapy resistant and may be responsible for tumor recurrence after the therapy. Using this tumor model, we found that the overgrowing tumor caused the shortage of vessels and resulted in the hypoxia in the early stage of tumor progression, which is conventionally called chronic hypoxia or avascular hypoxia. When tumor continues to grow, a rim of hypoxia area was formed at tumor edge and subsequently activated nearby astrocytes. At the same time, the outward reactive astrocytes enhanced tumor invasion into normal parenchyma tissues. As a result, a specific hypoxia was produced in tumor peripheral, which has relative high microvascular density (MVD) and is associated with highly reactive astrocytosis. We explored the role of this region on the response to therapies. By quantitative counting the apoptotic tumor cells across the tumor cross-section following radiation- or chemo-therapy, we found that cells at the active astrocytic region (the peripheral region) are more resistant than cells in tumor core. These results indicate that the peripheral hypoxia, not the chronic hypoxia, is the main cause of the failure of brain tumor therapy. Most importantly, we found that the formation of tumor peripheral hypoxia might be associated with the activation of astrocytes. Therefore, tactics that can suppress astrocytosis and prevent the formation of peripheral hypoxia may be alternative approaches for complementing the shortage of traditional brain tumor therapy.

#2977

Secretory autophagy in tumor associated fibroblasts promotes head and neck squamous cell carcinoma progression and emerges as a novel therapeutic target.

Jacob New, Levi Arnold, Megha Ananth, Sameer Alvi, Wen-Xing Ding, Sufi M. Thomas. _University of Kansas Medical Center, Kansas City, KS_.

As the fifth most common cancer worldwide, head and neck squamous cell carcinoma (HNSCC) is a leading global health burden. Despite therapeutic advancements, the survival rate has remained relatively unchanged for the last fifty years. To improve treatment and survival, there is a need to better understand the underlying biology of this disease. The tumor microenvironment's role in promoting cancer progression and resistance to therapy has gathered great attention. In HNSCC, the predominant microenvironment cell type is tumor associated fibroblasts (TAFs). Studies in our lab and others have demonstrated the significant tumor promoting role of TAFs; however, little is understood of the underlying biology of TAFs. In this study, we identify that TAFs have upregulated an unconventional pathway of secretion: secretory autophagy. Although paradigmatically a degradation pathway, there has been a growing appreciation for a novel role of autophagy in cellular secretion. We hypothesized HNSCC induces secretory autophagy in the TAFs, modulating secreted factors responsible for tumor progression. We assessed the role of autophagy inhibition in alleviating TAF-facilitated HNSCC progression, and uncovered a significant reduction in proliferation, migration, and invasion of the cancer cells. This was achieved by both siRNA knockdown of Beclin-1, and therapeutic inhibition of the lysosome through chloroquine. We discovered that NFs placed in co-culture with HNSCC had a significantly upregulated level of autophagy marker LC3-II. By characterizing the role of known HNSCC secreted factors in inducing TAF autophagy, we identified basic fibroblast growth factor is responsible for the LC3-II accumulation. To understand which TAF factors are secreted by an autophagy dependent mechanism, we assessed TAF-conditioned media in the presence of autophagy knockdown. Key cytokines, such as IL-6 and IL-8, were identified as autophagy dependent. Rescue of these cytokines in autophagy inhibited TAF-conditioned media restored HNSCC migration. Although autophagy has been identified as upregulated in a variety of cancer types, no one has previously characterized the role of autophagy in HNSCC, and both early pre-clinical and clinical studies of autophagy inhibition as a cancer therapy have been limited by the lack of a specifically targeted inhibitor. We demonstrate a significant decrease in HNSCC progression in preclinical models by using a new and highly specific small molecule inhibitor of autophagy, SAR405, which inhibits Vps34, an upstream autophagy pathway kinase. In summary, we uncover a novel role for secretory autophagy in the tumor microenvironment which promotes tumor progression, and can be uniquely targeted for therapy.

#2978

Discrete super-enhancer networks in pancreatic cancer cells and cancer associated fibroblasts are targeted and modulated by triptolide.

Pawan Noel, Serina Ng, Ruben Muñoz, Daniel Von Hoff, Haiyong Han. _Translational Genomics Research Institute (TGen), Phoenix, AZ_.

Background: Pancreatic cancer (PC) is the 3rd leading cause of adult cancer death in the USA. Pancreatic ductal adenocarcinoma (PDAC) accounts for >90% of all PC cases and has a dismal 5-year survival rate of < 6%, mostly due to lack of reliable methods for early detection, and limited treatment options. A unique, highly fibrotic and hypo-vascularized tumor stroma or "desmoplastic reaction" (DR) forms the major barrier to currently available therapeutics. Activated cancer associated fibroblasts (CAFs) play an important role in driving and maintaining the DR in PDAC. Thus therapeutic regimens that reprogram pancreatic CAFs can potentially enhance sensitivity to anti-tumor agents and may improve patient outcomes. Recent studies implicate the role of super-enhancers (SE) in maintaining cell specific gene expression networks in both normal and diseased cells. Here we aim to define SE networks in pancreatic CAFs and epithelial tumor cells and explore the role of triptolide in modulating SE signatures.

Methods: Using genomic techniques on control vs triptolide treated pancreatic tumor and CAF cells, we measured occupancy of BRD4 and acetylated histone 3 at lysine27 (H3K27ac), marks of active SEs, using ChIP-seq. RNA-seq revealed differentially regulated genes via SE modulation by triptolide. Immunoblotting was performed to validate the effect of triptolide on the protein signature associated with dysregulated SEs.

Results: Top ranked SEs between BRD4 and H3K27ac marks showed extensive overlap, but were highly distinct between cancer cells and CAFs. The SEs in tumor cells associated with genes related to transcription (e.g.POLR2E, PARK7, MYC), whereas those in CAFs associated with genes involved in desmoplasia/fibrosis (e.g. COL1A1, COL1A2, TGFBI). Triptolide significantly altered SE activation at diverse and discrete genomic loci in both cell types. Specifically, SE regions associated with COL1A2 and TGFBI showed reduced H3K27ac binding in CAFs exposed to triptolide but not in tumor cells, which showed reduced histone signal at RNA Pol II region. Transcriptional deregulation by triptolide exceeded in CAFs vs tumor cells (1282 vs 23 genes upregulated, 1496 vs 321 genes downregulated). Finally, triptolide resulted in reduced expression of BRD4 and RNA Pol II in both tumor and CAFs, while c-MYC and Collagen I were specifically and significantly downregulated in tumor and CAFs, respectively.

Conclusions: Our results suggest that pancreatic CAFs and tumor cells harbor distinct SE networks that are characteristic of their different cell types and states. Triptolide might exert its antitumor activity by potentially targeting distinct SEs in different cells. Epigenetic reprogramming of transcription by exploiting SE modulating compounds like triptolide alone or in combination with current standard of care may provide more effective treatment options for PC patients.

#2979

Inhibition of palladin in cancer associated fibroblast impedes pancreatic ductal adenocarcinoma associated desmoplasia.

Jennifer I. Alexander,1 Edna Cukierman2. 1 _Drexel University College of Medicine, Philadelphia, PA;_ 2 _Fox Chase Cancer Center, Philadelphia, PA_.

Pancreatic Ductal Adenocarcinoma (PDAC) claims 97% of patient lives in less than 5 years after diagnosis. Treatment of PDAC is challenged by the collagen-rich, highly dense, fibrous-like extracellular matrix (ECM) known as desmoplasia. Desmoplasia is initiated by activated cancer associated fibroblasts (CAFs) and disseminates throughout the stromal compartment in a vicious cycle involving alterations to the physical/structural and biochemical pathways in neighboring naïve fibroblasts that ultimately enriches the CAF population. This desmoplastic-ECM (D-ECM) encapsulates the tumor mass, thereby limiting therapeutic perfusion. Paradoxically, ablation of D-ECM induces a more aggressive PDAC progression.

Although the underlying biology remains unclear, the D-ECM is a consequence of cytoskeletal rearrangements occurring amid the CAFs. The goal of this study is to delineate the cytoskeleton dynamics engaged as a potential mechanism to restore the innate tumor-suppressive properties of a normal ECM. As such, this study focuses on the actin cross-linker palladin which has been identified as an independent prognostic marker in PDAC progression. We postulate palladin has an isoform-specific role in PDAC desmoplasia as well as cancer progression.

Using an in vivo-mimetic 3D fibroblast-derived ECM model, we explore the isoform-specific role of palladin during D-ECM onset as well as during D-ECM-induced CAF activation in PDAC patient derived CAFs in which the major palladin isoforms (isoforms 3 and 4) were knocked out via CRISPR/Cas9 gene editing. Using these cells, we examined the activation status of the CAFs while characterizing the ECM produced via quantitative immunoblots, real-time polymerase chain reaction, and detailed microscopy analyses. We then validated the effects of palladin knockout CAFs activity in influencing kras mutated cancer cells' metabolic activity, invasion and proliferation.

Our data indicates palladin isoforms 3 and 4 expression are necessary to drive CAF activation as well as perpetuate the D-ECM phenotype whereby knockdown of either isoform prevents CAF activation while limiting cancer cell behavior. Additionally, the ECMs produced by the palladin knockout CAFs were no longer capable of activating naïve fibroblast in addition to decreasing the inherent aggressive nature of kras-mutant cancer cells. These results are similar to previous findings regarding the restrictive nature of a normal ECM on CAF activation and cancer cell behavior.

These findings suggest that palladin isoforms 3 and 4 are required for desmoplastic onset and D-ECM-induced fibroblastic activation and cancer cell progression. Together, these results propose manipulation of specific palladin isoforms could signify a strategy to restore a normal tumor-suppressive stroma in PDAC.

#2980

**Cystathionine-beta-synthase (CBS)-derived hydrogen sulfide (H** 2 **S) supports proliferation, migration and bioenergetics in colon cancer-associated fibroblasts (CAFs).**

Katalin Modis, Manjit Maskey, Paul Johnson, Csaba Szabo, Mark R. Hellmich, Celia Chao. _Univ. of Texas Medical Branch, Galveston, TX_.

Introduction: Our group has recently demonstrated that CBS, one of the H2S-producing enzymes, is abundantly overexpressed in human colorectal cancer (CRC) tissue specimens resulting in an increased H2S production. In order to extend our observations, we have now compared, using immunohistochemistry, CBS expression in carcinoma-associated fibroblasts (CAFs) compared to normal colonic fibroblasts (NCFs) in human colon adenocarcinoma samples. The overall goal of our studies is to characterize how the CBS/H2S axis contributes to the malignant phenotype of the CAFs in the tumor microenvironment.

Methods: Human CAFs and NCFs were collected from CRC under an IRB approved protocol and cultured in vitro. Western blot analysis of both NCF and CAF was performed to determine CBS protein expression levels. SiRNA/shRNA-mediated silencing of CBS in CAFs was performed. A pharmacological inhibitor of CBS, aminooxyacetic acid (AOAA), in both CAF and NCF cultures was also utilized. Hydrogen sulfide (H2S) levels were measured using the fluorogenic probe 7-Azido-4-methylcoumarin. We also performed proliferation assays using a coulter counter in CAFs, NCFs, and CAFs subjected to siRNA/shRNA-mediated CBS silencing or AOAA. Control cells were transduced with shNT vectors. Cells were also treated with the slow-releasing H2S donor GYY4137. We also conducted cell migration assays in transwell chambers (migration of CAFs toward conditioned media (CM) collected either from HCT116 cells or normal colon mucosal cells (NCM356). Finally, the XF24 Extracellular Flux Analyzer (Seahorse) was used to measure bioenergetic function in CAFs subjected to siRNA/shRNA-mediated silencing of CBS or treated with AOAA.

Results: CBS protein expression and H2S production in CAFs was increased compared to NCFs. The H2S donor, GYY 4137, increased the proliferation rate of CAFs but not of NCFs. CBS silencing or AOAA treatment reduced CAF migration toward conditioned media from HCT116 cells. Genetic or pharmacological inhibition of CBS in CAF cells suppressed key bioenergetic parameters including basal respiration, ATP production, maximal respiratory capacity, and spare respiratory capacity compared to NCF cells or CAF shNT cells.

Conclusions: Thus, CBS/H2S axis serves as a mechanism to support CAFs' function. In these cells, H2S contributes to maintaining their bioenergetic functions, which, in turn, promotes their proliferation and migration toward colon cancer cells. We hypothesize that these processes may contribute CRC progression.

#2981

Identification of circulating stroma-related biomarkers for pancreatic ductal adenocarcinoma.

Andrea Resovi,1 Luca Porcu,1 Alessia Anastasia,1 Paola Allavena,2 Paola Cappello,3 Anna Falanga,4 Giulia Taraboletti,1 Mariarosa Bani,1 Dorina Belotti,1 Raffaella Giavazzi1. 1 _Istituto di ricerche farmacologiche Mario Negri, Milano e Bergamo, Italy;_ 2 _Humanitas Clinical and Research Center, Rozzano, Italy;_ 3 _CeRMS, University of Torino, Torino, Italy;_ 4 _Department of Immunohaematology and Transfusion Medicine, Bergamo, Italy_.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive epithelial malignancies characterized by a relevant amount of tumor stroma. It is usually diagnosed late and has limited treatment options. Plasmatic markers capable of detecting the disease in its early stages and monitoring treatment effectiveness might change the fate of PDAC patients. This study investigates the potential value of circulating stroma-related molecules as PDAC biomarkers capable to detect the disease and to monitor treatment response. Thirty-eight stroma-related candidate biomarkers were selected from published proteomic studies on PDAC. Levels of these molecules were tested by ELISA and Multiplex Luminex assays in a first cohort of plasma samples from PDAC patients and healthy subjects. Thirteen molecules -extracellular matrix proteins and proteolytic fragments, matrix-degrading enzymes and their inhibitors, growth factors and adhesion molecules-, were found to be differentially expressed in PDAC plasma compared to healthy subjects. A second analysis in an independent cohort of PDAC patients, chronic pancreatitis patients and healthy controls confirmed the previous findings and allowed restricting the number of biomarkers up-regulated in the plasma of PDAC patients. The plasma analysis of two genetically engineered mouse models of pancreatic cancer [Pdx-1-Cre;LSL-KrasG12D and Pdx-1-Cre;LSL-KrasG12D/+;LSL-Trp53R172H/+] revealed an up-regulation of four markers in association with PanIN development, indicating their potential role in the early diagnosis of pancreatic cancer. These markers were also elevated in the plasma of mice bearing patient derived orthotopic PDAC xenografts (PDAC-PDX), but not in a mouse model of caerulein-induced chronic pancreatitis. Their level was associated with PDAC-PDX tumor growth and response to gemcitabine combined with nab-paclitaxel. In conclusion this analysis has identified a panel of stroma-related potential biomarkers associated with tumor progression and drug response of PDAC.

Supported by Associazione Italiana per la Ricerca sul Cancro (AIRC 5 per mille grant n. 12182) and Fondazione "Eugenio Morandi" ONLUS per lo studio e la cura dei tumori del pancreas.

#2982

Metastatic and non-metastatic colorectal cancer cells differentially regulate fibroblast cell cycle via extracellular vesicles.

Rahul Bhome, Louise M. House, Tilman Sanchez-Elsner, Stephen M. Thirdborough, Emre Sayan, Alex H. Mirnezami. _University of Southampton, Southampton, United Kingdom_.

Cancer-associated fibroblasts are critical to tumor progression. There exists a dynamic crosstalk between cancer and stromal compartments, which maintains a permissive tumor microenvironment. Extracellular vesicles (EVs) play a significant role in this intercellular communication. Colorectal cancer (CRC) cells can be categorized according to epithelial-mesenchymal transition (EMT) status, and therefore metastatic capacity. We aimed to investigate the effect of EMT on EV-mediated cancer-fibroblast signaling.

CRC cell lines (DLD-1, HCT116, SW620 and SW480) were characterized by western blotting to determine EMT status. EVs were isolated from conditioned media by serial centrifugation and validated by transmission electron microscopy, western blotting and nanoparticle tracking analysis. Fluorescently labeled EVs and cells were detected and evaluated by flow cytometry and fluorescence microscopy. Increasing concentrations of EVs from CRC cells were co-cultured with fibroblasts for 24h. Activation/inhibition of signaling pathways was examined by western blotting. EV microRNA (miRNA) profiles were obtained, validated by qPCR and submitted for target and pathway analysis.

DLD-1, HCT116 and SW620 cells express E-cadherin and are considered epithelial, whereas SW480 lacks E-cadherin, expresses ZEB-1, and is considered mesenchymal. EVs were spherical, enriched in ALIX, TSG101, CD63 and had a mean diameter of 90nm. EVs from CRC cells were shown to transfer directly to primary ex vivo patient-derived fibroblasts and fibroblast cell lines. Transfer of EVs from epithelial CRC cells abrogated ERK activity in fibroblasts, even at the lowest concentration, and was associated with reduced fibroblast proliferation, whereas EVs from mesenchymal cells had no effect. MiRNA profiling of EVs from epithelial and mesenchymal CRC cells showed a 10-fold upregulation of miR-143-3p in epithelial compared to mesenchymal EVs. MiRNA target analysis and experimental validation show that miR-143-3p directly targets KRAS and HRAS, providing a potential miRNA-orchestrated mechanism of action for the downregulation of fibroblast ERK activity in the tumor microenvironment.

Importantly, CRC cellular ERK activity is not reflected in fibroblasts treated with CRC EVs, suggesting that EVs do not directly transmit ERK protein or mRNA. However, miRNAs are the most stable EV cargo, and we show that epithelial but not mesenchymal CRC EVs contain upregulated miRNAs, which target critical components of the ERK pathway. Downregulation of ERK activity has been shown to induce fibroblast senescence, a phenotype linked to cancer progression. We hypothesize that differential regulation occurs because epithelial CRC cells are juxtaposed with fibroblasts in the tumor core, where senescent cancer associated fibroblasts are frequently observed, whereas mesenchymal CRC cells are at the invasive front or in the circulation.

#2983

Tumor induced stromal STAT1 deregulates mammary tissue homeostasis and accelerates breast cancer.

Victoria R. Zellmer, Patricia M. Schnepp, Sarah L. Fracci, Xuejuan Tan, Erin N. Howe, Siyuan Zhang. _University of Notre Dame, Notre Dame, IN_.

The tumor microenvironment (TME) – the dynamic tissue space in which the tumor exists – plays a significant role in tumor initiation and is a key contributor in cancer progression. Little is known about the tumor-induced changes in the adjacent tissue stroma. Herein, we sought to explore tumor-induced changes in the TME at the morphological and molecular level to further characterize cancer progression. We show that tumor-adjacent mammary glands (TAGs) display altered branch morphology, increased expression of αSMA particularly in myofibroblasts, and an increased capacity to form mammospheres in 2D suspension culture. FACS analysis showed that TAGs contain an increased number of Lin-CD24+/CD49+ enriched mammary gland stem cell (MaSCs) population, suggesting deregulated tissue homeostasis in TAGs. We conducted comparative transcriptomics on TAGs and contralateral control glands. Meta-analysis on differentially expressed genes from our RNA-seq dataset plus two breast cancer stromal patient microarray datasets identified shared upregulation of STAT1, which we verified in tumor-adjacent tissues. Knockdown of STAT1 in caveolin-deficient mouse embryonic fibroblasts (CAFs) cocultured with human breast cancer cells altered cancer cell proliferation, further suggesting the role of STAT1 as a stromal contributor of tumorigenesis. Furthermore, in our proof-of-concept in vivo experiment, co-treatment with fludarabine, a FDA-approved STAT1 activation inhibitor and DNA synthesis inhibitor, in combination with doxorubicin, showed enhanced therapeutic efficacy in treating mouse mammary gland tumors. Our results demonstrate that stromal STAT1 expression could promote tumor progression and is a potential therapeutic target for breast cancer.

#2984

The chemokine CCL2/CCR2 signaling mediated fibroblasts-cancer cells crosstalk promotes basal like breast cancer progression.

Min Yao, Wei Bin Fang, Fang Fan, Nehemiah Alvarez, Patrick E. Fields, Nikki Cheng. _Univerisity of Kansas Medical Center, Kansas City, KS_.

Cancer associated fibroblasts are the most abundant stromal cells in breast cancer, but their function in cancer progression has not been fully understood. We previously identified that the chemokine CCL2 was highly expressed in breast cancer associated fibroblasts, and high stromal CCl2 expression predicted poor outcome in basal like breast cancer. CCL2 is known to recruit monocyte/macrophage and promotes cancer progression. We previously found that recombinant CCL2 can directly signal to breast cancer cells and promote cell survival and invasion in vitro. In this study, we aimed to determine the functional importance of CCL2 signaling mediated fibroblast-cancer cell interactions in breast cancer progression. We used a fibroblast and cancer cell co-graft mouse model as the main functional assay. We generated primary fibroblasts from mouse mammary tumor and human breast cancer, and confirmed most of they expressed high level of CCL2. When co-grafted with the human basal breast cancer cell line MCF10A-CA1D into nude mice, fibroblasts enhanced xenograft growth. Stable knockdown of CCL2 expression from fibroblasts significantly reduced its ability in tumor growth promotion, while knockdown CCL2 from cancer cells did not. Decreased CCL2 production from fibroblasts resulted in increased apoptosis and autophagy in tumor samples. To determine the importance of direct CCL2 signaling to cancer cell, we generated the CCL2 receptor CCR2 mutant CA1D cancer cell lines by CRISPR-Cas9 targeting technology. Mutation of CCR2 in cancer cell significantly reduced tumor growth when co-grafted with CCL2 secreting fibroblasts. Lastly, we tested continuous delivery of CCL2 neutralizing antibody in the co-graft tumor model, but observed minimal therapeutic effect. Further examination of blood CCL2 level revealed an increased production of CCL2 from fibroblasts and mouse host after antibody treatment, which may contribute to the lack of therapeutic effect. In summary, our studies demonstrated the importance of CCL2/CCR2 signaling mediated fibroblasts-cancer cell interaction in basal like breast cancer progression. The CCL2 signaling pathway can be potentially served as therapeutic target, but requires development of efficient targeting strategy.

#2985

Interaction between the malignant melanoma cell of the primary lesion and the metastatic lesion.

Takaharu Hatano,1 HIsashi Motomura,1 Heishiro Fujikawa,1 Masakazu Yashiro,2 Kishu Kitayama3. 1 _Department of plastic and reconstructive surgery, Osaka City University Graduate School of Medicine, Osaka, Japan;_ 2 _Department of Surgical Oncology, Osaka City University Graduate School of Medicine, Oncology Institute of Geriatrics and Medical Science, Osaka City University Graduate School of Medicine, Osaka, Japan;_ 3 _Department of Surgical Oncology, Osaka City University Graduate School of Medicine, Osaka, Japan_.

Introduction: The prognosis of patients with malignant melanoma is extremely poor because of the frequent metastasis. We have occasionally experienced the clinical cases that malignant melanoma patients rapidly developed distant metastases after the surgical resection of primary cancer lesion. These cases suggested that a primary tumor may control a metastatic tumor. We hypothesized that primary melanoma cells might produce a factor(s) which regulate the progression of metastatic melanoma cells. Then we investigated the growth and invasion interaction between primary malignant melanoma cells and high metastatic melanoma cells. Materials and methods: We used two malignant melanoma cell lines, B16 as a parent primary melanoma cell line and a selected daughter cell line B16/BL6 with high metastatic ability. We investigated the invasive interaction between the parent B16 cells and daughter B16/BL6 cells in vitro. The conditioned medium from B16 cells and B16/BL6 cells was added to B16 cells or B16/BL6 cells. Then we examined the effect of these conditioned medium on the motility and proliferation of melanoma cells by MTT assay or invasion assay at 24 hours and 72 hours after the addiction of the conditioned medium. Results:The conditioned medium from B16 cells significantly (p=0.02) suppressed the invasion ability of B16/BL6 cells at 24 hours after the addiction of the conditioned medium. In contrast, the invasion ability of B16 cells were not affected by the conditioned medium from B16/BL6 cells at 24 hours. Each conditioned medium did not affect on the proliferation of both melanoma cell lines. On the other hand, at 72 hours after the addiction of the conditioned medium from B16 cells significantly (p=0.000003) suppressed the growth of B16/BL6 cells. Conclusion: Primary melanoma cells might down-regulate the progression of metastatic melanoma cells by a soluble factor(s).

## CLINICAL RESEARCH:

### Clinical Biomarkers

#2986

Atezolizumab in metastatic TNBC (mTNBC): Long-term clinical outcomes and biomarker analyses.

Peter Schmid,1 Cristina Cruz,2 Fadi S. Braiteh,3 Joseph Paul Eder,4 Sara Tolaney,5 Irene Kuter,6 Rita Nanda,7 Cathie Chung,8 Philippe Cassier,9 Jean-Pierre Delord,10 Michael Gordon,11 Yijin Li,12 Bo Liu,12 Carol O'Hear,12 Marcella Fasso,12 Luciana Molinero,12 Leisha A. Emens13. 1 _Barts Cancer Institute, London, United Kingdom;_ 2 _Vall d'Hebron University Hospital, Barcelona, Spain;_ 3 _Comprehensive Cancer Centers of Nevada, Las Vegas, NV;_ 4 _Yale School of Medicine, New Haven, CT;_ 5 _Dana Farber Cancer Institute, Boston, MA;_ 6 _Massachusetts General Hospital, Boston, MA;_ 7 _University of Chicago Medical Center, Chicago, IL;_ 8 _The Angeles Clinic and Research Institute, Los Angeles, CA;_ 9 _Centre Leon Berard, Lyon, France;_ 10 _Institut Claudius Regaud, Toulouse, France;_ 11 _Pinnacle Oncology Hematology, Scottsdale, AZ;_ 12 _Genentech, Inc., South San Francisco, CA;_ 13 _Johns Hopkins University School of Medicine, Baltimore, MD_.

This abstract has been withheld from publication due to its inclusion in the AACR Annual Meeting 2017 Official Press Program. It will be posted online following its presentation.

#2987

Validation and clinical feasibility of a comprehensive genomic profiling assay to identify likely immunotherapy responders through tumor mutational burden (TMB).

Daniel S. Lieber,1 Mark R. Kennedy,1 Douglas B. Johnson,2 Jonathan E. Rosenberg,3 Marcin Kowanetz,4 Joel R. Greenbowe,1 Garrett M. Frampton,1 Caitlin F. Connelly,1 Alexa B. Schrock,1 Jeffrey S. Ross,1 Philip J. Stephens,1 Siraj M. Ali,1 Vincent A. Miller,1 David A. Fabrizio1. 1 _Foundation Medicine, Inc., Cambridge, MA;_ 2 _Vanderbilt University, Nashville, TN;_ 3 _Memorial Sloan Kettering Cancer Center, New York, MA;_ 4 _Genentech, Inc., South San Francisco, CA_.

Background

Patients across a range of disease types have demonstrated robust and durable responses using checkpoint inhibitor therapies (CPITs). Given the limitations of immuno-histochemical based testing, identifying a unified, quantitative metric to determine potential response to CPITs remains an urgent need. Tumor mutational burden (TMB) measures the number of somatic protein coding mutations per target sequence in a tumor specimen. This measure has been associated with response and survival for multiple CPITs across an array of indications. In this study we describe Foundation Medicine's (FMI) work to develop and validate a TMB result as part of our comprehensive genomic profiling assays and summarize clinical feasibility in NSCLC, melanoma and bladder cancer.

Methods

We developed an analysis method to determine TMB based on data from our comprehensive genomic profiling assays. TMB is calculated by counting all synonymous and non-synonymous somatic variants across 315 or 405 genes, excluding germline alterations and known or likely driver alterations. The mutation count is normalized by the coding target territory to achieve a mutation density of mutations per megabase (mut/Mb). To determine accuracy, we compared TMB values from our comprehensive genomic profiling assay against a CLIA-validated whole-exome sequencing (WES) method on 29 patients. Precision was assessed over 10 clinical samples replicated 4-6 times. Lower limit of sample tumor purity was determined through dilutions of tumor/normal pairs from 80% to 5% tumor. Clinical feasibility was assessed by analyzing TMB versus immunotherapy-based survival in a cohort of 65 metastatic melanoma patients, 150 urothelial carcinoma patients and 463 NSCLC patients. Additionally, we examined the relationship between TMB and microsatellite instability status (MSI), an independent biomarker associated with response to CPITs.

Results

Foundation Medicine's TMB measure provides accurate and precise results across a range of tumor mutational burden values on samples with as little as 20% tumor purity. Using cohort specific thresholds, TMB was significantly associated with improved survival to CPITs in NSCLC, melanoma and bladder cancer. Using data from over 40,000 patient samples, we also show significant overlap between high TMB and high MSI samples and show that MSI-High specimens represent a subset of TMB-High specimens.

Conclusions

We have developed and validated the tumor mutational burden (TMB) biomarker as part of our comprehensive cancer genomic profiling assays. Initial clinical feasibility results demonstrate that TMB can be used to predict the likely response to anti-PD-1/PD-L1 CPITs across a growing number of indications including NSCLC, melanoma and bladder cancer.

#2988

Immunogenomic analyses of tumor cells and microenvironment in patients with advanced melanoma before and after treatment with nivolumab.

Timothy A. Chan,1 Nadeem Riaz,1 Jonathan J. Havel,1 Vladimir Makarov,1 Alexis Desrichard,1 Jennifer S. Sims,1 F. Stephen Hodi,2 Salvador Martín-Algarra,3 William H. Sharfman,4 Shailender Bhatia,5 Wen-Jen Hwu,6 Thomas F. Gajewski,7 Craig L. Slingluff,8 Sviatoslav M. Kendall,1 Han Chang,9 John-William Sidhom,10 Jonathan P. Schneck,11 Nils Weinhold,1 Christine E. Horak,9 Walter J. Urba12. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _University of Navarra, Pamplona, Spain;_ 4 _The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD;_ 5 _University of Washington, Seattle, WA;_ 6 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 7 _University of Chicago Gordon Center for Integrative Science, Chicago, IL;_ 8 _University of Virginia, Charlottesville, VA;_ 9 _Bristol-Myers Squibb, Princeton, NJ;_ 10 _Johns Hopkins University, Baltimore, MD;_ 11 _Johns Hopkins School of Medicine, Baltimore, MD;_ 12 _Earle A. Chiles Research Institute, Portland, OR_.

Background: Response to checkpoint blockade may be dependent on tumor mutational load and the presence of antigen-specific effector T cells in the tumor microenvironment; however, how blockade modulates these features during therapy is unclear. We assessed genomic changes in tumors from patients (pts) with advanced melanoma receiving nivolumab (nivo) who progressed on ipilimumab (ipi-P) or were ipi-naive (ipi-N).

Methods: Tumor biopsies were collected pretreatment and 4 weeks post first nivo dose from ipi-N or ipi-P pts treated with nivo 3 mg/kg Q2W in the phase 1 open-label CA209-038 study (NCT01621490). Biopsies from 68 pts were analyzed by whole exome, transcriptome, and/or TCR sequencing (paired biopsies from 41, 42, and 34 pts, respectively).

Results: Objective response rate (ORR) in the overall cohort (n=85) was 27% with similar ORR in ipi-N and ipi-P cohorts. In the genomic cohort (n=68), ORR was 23% with a similar number of complete or partial responses (CR/PR) in ipi-N and ipi-P pts (n=7 and n=8, respectively). Prior to treatment, mutational and neoantigen load were comparable, regardless of previous treatment. Following nivo treatment, both mutational and neoantigen load were reduced 5-fold in pts who responded (CR/PR; n=9) and 1.2-fold in pts with stable disease (SD, n=13) compared with a 1.1-fold increase in pts with progressive disease (PD, n=19). Intratumoral heterogeneity analysis before and after nivo demonstrated that CR/PR pts generally lost tumor mutation clones/subclones. Novel tumor mutation clones were observed in on-treatment samples from 2 CR/PR pts and all pts who progressed on nivo. Transcriptome analyses revealed significant increases in distinct tumor immune cell subsets (CD8+ T cells and NK cells) and immune checkpoint gene expression (LAG3, CTLA4, PCDC1, and CD274 [PD-L1]) following nivo, which were more pronounced in pts with CR/PR vs PD (log2 fold-changes of 1.24, 1.07, 1.71, and 0.74, respectively). Consistent with the transcriptome analyses, tumor-infiltrating lymphocytes, as assessed by immunohistochemistry, generally increased following nivo in pts who responded: 2.8 vs 1.9-fold change in CR/PR/SD vs PD in the ipi-P cohort; 4.8 vs 1.8-fold change in CR/PR/SD vs PD in the ipi-N cohort. Differences in treatment-related TCR repertoire diversity changes were apparent between pts who responded within the ipi-N and ipi-P cohorts: a decrease in the evenness of T-cell clonotype distribution was observed among pts with CR/PR/SD relative to pts with PD in the ipi-N cohort (P=0.036), but not in the ipi-P cohort.

Conclusion: Nivo and ipi modulate T-cell repertoire and tumor mutational heterogeneity in pts with advanced melanoma, presenting potential mechanisms of action underlying successful nivo therapy. These data also show that prior ipi treatment may influence biological response to nivo, but further investigation is warranted.

#2989

Expression of immune checkpoints proteins and their prognostic relevance in prostate cancer.

Claudia Hube-Magg, Cornelia Schroeder, Ronald Simon, Martina Kluth, Till Krech, Franziska Büscheck, Frank Jacobsen, Doris Höflmayer, Lars Budäus, Hartwig Huland, Markus Graefen, Guido Sauter, Thorsten Schlomm. _Univ. Medical Ctr. Hamburg-Eppendorf, Hamburg, Germany_.

Immune checkpoint proteins including programmed death ligand 1, 2 (PD-L1, PD-L2) and B7-H3 play important roles for immune evasion in many cancer types. PD-L1 is an established therapeutic target in lung cancer and in a growing number of other indications. The clinical success of PD-L1 inhibitors has accelerated the development of drugs also targeting PD-L2 and B7-H3. In prostate cancer, studies on the prevalence and clinical relevance of these proteins have been inconclusive. Here, we performed immunohistochemical analysis of PD-L1, PD-L2 and B7-H3 in our large prostate cancer prognosis tissue microarray (TMA) containing tissue samples from more than 17,000 patients. Normal prostate glands were negative for PD-L1 and B7-H3, but positive for PD-L2. We found that 47% of 12,808 interpretable cancers showed B7-H3 expression, and 92% of 9,463 cancers showed PD-L2 expression, while expression of PD-L1 was virtually absent (0.2% of 17,392 tumors) in epithelial cells. Stroma cells and immune cells were entirely negative for PD-L1 and B7-H3, while some faint staining of lymphocytes was occasionally observed for PD-L2. In cancers, PD-L2 and B7-H3 staining had different prognostic impact, which depended on the ERG status: B7-H3 expression was linked to young patient age, advanced stage, high Gleason grade, lymph node metastasis (p<0.0001 each), presence of PTEN deletion (p<0.0001), and poor prognosis in ERG negative cancers (p<0.0001), while PD-L2 expression was linked to beneficial tumor features and good prognosis exclusively in tumors with ERG fusion (p<0.0001). In a multivariate analysis including pT stage, Gleason grade, nodal stage, resection margin and preoperative PSA level, B7-H3 expression was an independent prognostic factor (p=0.0004). In summary, the results of our study identify a subset of young patients with ERG negative aggressive prostate cancers that might benefit from anti-B7-H3 therapy in the future. PD-L2 expression appears to protect tumors from progression and does not seem to be suitable as a potential target in prostate cancer. PD-L1 expression is extremely rare in this disease, suggesting that only few prostate cancer patients might benefit from PD-L1 inhibitors.

#2990

Polyfunctional anti-CD19 CAR T cells determined by single-cell multiplex proteomics associated with clinical activity in patients with advanced non-Hodgkin's lymphoma.

John Rossi,1 Patrick Paczkowski,2 Yueh-wei Shen,1 Kevin Morse,2 Brianna Flynn,2 Alaina Kaiser,2 Colin Ng,2 Kyle Gallatin,2 Tom Cain,2 Rong Fan,3 Sean Mackay,2 James Heath,4 Steven A. Rosenberg,5 James N. Kochenderfer,6 Jing Zhou,2 Adrian Bot1. 1 _Kite Pharma, Santa Monica, CA;_ 2 _IsoPlexis, Branford, CT;_ 3 _Yale School of Engineering and Applied Science, Yale University, New Haven, CT;_ 4 _Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA;_ 5 _Surgery Branch, National Institutes of Health, Bethesda, MD;_ 6 _Experimental Transplantation and Immunology Branch, National Institutes of Health, Bethesda, MD_.

Introduction: Autologous anti-CD19 CAR T cells have shown promising clinical efficacy in B cell malignancies, with T cell expansion and blood levels for IL-15, IL-10 and Granzyme B as correlates of objective response and toxicity (Kochenderfer et al. J Clin Oncol 2016; 34:LBA3010). It is unclear, however, which key immune programs in CAR T cells impact their in vivo expansion and clinical outcome. We evaluated in detail the functionality of anti-CD19 CAR T cells by using single-cell proteomics analysis (Lu et al. PNAS 2015;113:607-615). We explored how the polyfunctionality of pre-infusion CAR T cell products, post-stimulation with the CD19 antigen in vitro, associated with CAR T cell expansion in vivo and objective response.

Methods: Product T cells were separated into CD4+ or CD8+ T cell subsets using microbeads. CD4+ or CD8+ fractions were then co-cultured with CD19-K562 targets or NGFR-K562 control cells, at a 1:2 ratio for 20 hrs. Single cells were then analyzed using a 32-plex panel of secreted cytokines, chemokines, and cytotoxic molecules. Specifically, T cells were loaded onto a single-cell barcode chip capable of assaying 32 secreted proteins/cell. The polyfunctional profile and strength (pSI) of each sample was determined (Ma et al. Cancer Discov 2013;3:418-429) and analyzed relative to in vivo expansion of the CAR T cells and patient response to the CAR T cell therapy. CAR T cell expansion in blood was measured by quantitative PCR.

Results: Single-cell pSI of patient CAR T cells showed a statistically significant association (p = 0.011) with objective response (complete or partial response) to the therapy. While product pSI showed variability across patients, the median pSI was 2+ times higher for responders versus non-responders. The polyfunctional profiles for both CD4+ and CD8+ cells were dominated by effector molecules, stimulatory cytokines and chemokines. Polyfunctional CD4+ and CD8+ subsets with IFN-γ, IL-8 and/or MIP-1α correlated best with patient outcome, with CD8+ T cells showing co-expression of Granzyme B, and CD4+ T cells also comprising IL-17A+IL8+ and IL5+IL8+ subsets. While CAR expansion in vivo also correlated with objective response (p = 0.032), the association between product pSI and CAR cell expansion in vivo did not reach statistical significance (p = 0.079), suggesting that they bring independent contributions to predicting objective response. In support of that, a composite index integrating pSI and CAR T cell expansion in vivo associated best with clinical response (p = 0.005).

Conclusion: Polyfunctionality of CAR T cells, in conjunction with their expansion in vivo, correlates with clinical outcome in an anti-CD19 CAR T cell clinical trial. Single-cell multiplexed proteomics measurements may provide powerful insight into the clinical performance of CAR T cell products.

[J.R. and P.P. contributed equally to this study.]

#2991

Integrative analysis of molecular and drug response data from clinical samples and PDTXs to identify pharmacogenomic associations in breast cancer.

Maurizio Callari, Rajbir N. Batra, Ankita Sati Batra, Wendy Greenwood, Suet-Feung Chin, Alejandra Bruna, Oscar M. Rueda, Carlos Caldas. _University of Cambridge, Cambridge, United Kingdom_.

Breast cancer is the most common cancer among women showing high clinical and molecular heterogeneity. Current clinical management causes patients overtreatment with implications on both patients' quality of life and healthcare costs. Moreover, intrinsic or acquired tumor resistance to treatment leads to incurable metastatic progression in a significant proportion of patients. Consequently, there is an urgent need for better predictive biomarkers and a better understanding of the mechanisms driving response to treatment. As part of the METABRIC initiative, we fully molecularly characterized 2000 breast primary tumors, measuring gene expression, copy number aberration, somatic mutations and methylation. In addition, a biobank of breast cancer patient-derived tumor xenograft (PDTX) models (n=92) has been generated in our lab and a comprehensive molecular characterization was also obtained. We recently demonstrated that breast cancer PDTXs maintain originating cancers intra-tumor heterogeneity, hence representing a more relevant preclinical model than cell lines. An ex-vivo drug screening was performed in these models generating response data (IC50 and AUC) for 100 different drugs, including "best in class" PI3K, PARP and CDK4/6 inhibitors, novel biological and chemical inhibitors of HER2, ER, IGF1R and HER3, as well as standard of care agents. Here we derived signatures of pathway activation/disruption by integrating different data types in the METABRIC cohort. Their association with previous breast cancer classifications, as well as their prognostic significance was studied. The predictive power of these signatures was investigated in the PDTX cohort to identify novel pharmacogenomics associations. They were tested independently as well as in combination, to derive molecular predictors of response to treatment. We found known and novel associations between genomic/transcriptomic features and drug response. For example, our results confirmed the known association between estrogen receptor-related genes and response to tamoxifen treatment. We also identified markers of response to inhibitors of the PI3K/AKT/mTOR pathway. Selected findings were validated in clinical cohorts as well as in independent PDTX models. In conclusion, by integrating molecular data from large cohorts of clinical samples and PDTX we have generated a computational framework for the systematic identification of pharmacogenomics associations in breast cancer and to generate hypothesis for rational drug-drug combinations.

#2992

**Identification of** NOTCH1 **inactivating mutation as a therapeutic vulnerability to PI3K/mTOR pathway inhibition in head and neck squamous cell carcinoma (HNSCC).**

Vaishnavi Sambandam, Li Shen, Pan Tong, Tuhina Mazumdar, Curtis Pickering, Jeffrey N. Myers, Jing Wang, Mitchell Frederick, Faye M. Johnson. _MD Anderson Cancer Center, Houston, TX_.

Background: Genomic alterations in the PI3K/mTOR pathway occur in 54% of HNSCC patients. However, clinical trials of PI3K/mTOR pathway inhibitors had limited success even in those tumors with pathway alterations, including PIK3CA mutations. To target genomic alterations in HNSCC, we tested the efficacy of 7 PI3K/mTOR pathway inhibitors in 59 HNSCC cell lines and determined the association between drug sensitivity and molecular characteristics in order to identify biomarkers of response.

Methods: We systematically analyzed the association between drug sensitivity and genomic alterations in 59 HNSCC lines.

Results: NOTCH1mut lines are significantly sensitive to PI3K/mTOR pathway inhibitors: GSK2126458 (13/16), BYL719 (6/16), PQR309 (13/16), BKM120 (14/16), BEZ235 (12/16), BAY806942 (14/16) and GDC0980 (13/16 lines). In contrast to PIK3CAmut cell lines, all 7 NOTCH1mut lines tested underwent apoptosis (14.3 fld; P<0.005). After PI3K/mTOR inhibition, NOTCH1mut lines showed significantly reduced clonogenic growth in vitro (0.4/ 0.9 fold in HN31/ PCI15B; P<0.05) and significant tumor growth inhibition in vivo using orthotopic oral xenograft mouse models (1.7 and 2 fold in UMSCC22A and HN31; P<0.01). To determine if NOTCH1 mediates resistance, we conditionally expressed cleaved NOTCH1 by Dox-inducible system in a NOTCH1mut line (UMSCC22A). This rescued PI3K/mTOR inhibitor-induced apoptosis (0.5 fold; P<0.05) and reduced colony formation in vitro. As no canonical pathways account for the underlying mechanism of sensitivity, we measured the level of 301 proteins by reverse phase protein array (RPPA) in 3 NOTCH1mut and 3 NOTCH1WT lines after GSK2126458 treatment. Glutaminase and Glutamate Dehydrogenase were differentially expressed in NOTCH1mut lines. Thus, we hypothesized that PI3K/mTOR inhibition in NOTCH1mut lines induced reactive oxygen species (ROS)-mediated apoptosis via metabolic alterations. Consistent with this hypothesis, NOTCH1mut lines exhibited increased ROS production; Metabolic pathway inhibitors targeting Glycolysis, Pentose Phosphate pathway and Glutaminolysis, in combination with GSK2126458 decreased cell viability in NOTCHWT lines.

Conclusion: In contrast to PIK3CAmut cells, NOTCH1mut HNSCC cells underwent apoptosis after PI3K/mTOR pathway inhibition in vitro and decreased tumor size in vivo. The ectopic activation of NOTCH1 rescued NOTCH1mut HNSCC cells from PI3K/mTOR inhibitor-mediated apoptosis. The underlying mechanism may involve differential effects on tumor metabolism and ROS production. This work is significant because inactivating NOTCH1 mutations, which occur in 18% of HNSCC patients and SCCs of the lung, esophagus, and other sites, may serve as a biomarker for response. Our future work may uncover previously unknown crosstalk between the PI3K/mTOR and NOTCH pathways in SCCs.

### Innate Immune Mechanisms in Cancer Treatment

#2993

STING signaling in breast tumor microenvironment modulates immune checkpoint blockade efficacy in the neu-N mouse model of breast cancer.

Jeremy B. Foote,1 Marlene Kok,1 James M. Leatherman,1 Todd D. Armstrong,1 Bridget Marcinkowski,1 David B. Kanne,2 Elizabeth M. Jaffee,1 Thomas W. Dubensky,2 Leisha A. Emens1. 1 _Johns Hopkins University, School of Medicine, Baltimore, MD;_ 2 _Aduro BioTech, Berkley, CA_.

Background: The presence of tumor infiltrating T cells (TILs) is associated with improved clinical outcomes in multiple tumor types and is also necessary for response to immune checkpoint blockade. While T cell responses to some tumors occur spontaneously, the majority of cancers are not naturally recognized by the immune system. The lack of response is attributed primarily to insufficient T cell infiltration into the tumor microenvironment (TME). Activating the STING pathway represents one strategy for increasing infiltration of T cells into the TME. This induces interferon-β (IFN-β) production, leading to dendritic cell (DC) activation and priming of tumor antigen specific CD8+ T cells that mediate tumor regression. The intratumoral injection (IT) of ML-RR-S2-CDA (ADU-S100), a synthetic cyclic dinucleotide STING agonist, has antitumor efficacy in several aggressive mouse tumor models, including B16 melanoma, CT26 colon carcinoma, Panc02 pancreatic carcinoma, and 4T1 triple negative breast cancer. However, the impact of antigen-specific tolerance on tumor regression associated with STING-activation remains poorly understood. Therefore, we evaluated the efficacy of IT ADU-S100 in both non-tolerant parental FVB/N and the immune tolerant neu/N transgenic mice bearing established HER-2+ breast tumors.

Methods: First we first evaluated the impact of IT ADU-S100 on tumor regression, survival, innate sensing, and priming of HER-2 specific CD8+ T cells in both tumor-bearing non-tolerant FVB/N control and tolerant neu/N mice. Then we determined whether modulating the most highly expressed immune checkpoints on tumor infiltrating CD8+ T cells enhanced intratumoral STING activation with ADU-S100 in the tolerant neu/N mouse model of HER-2+ breast cancer.

Results: ADU S-100 induced HER-2-specific CD8+ T cell priming and durable tumor clearance in 100% of non-tolerant, FVB/N mice. In contrast, ADU S-100 failed to sufficiently prime HER-2-specific CD8+ T cells in tolerant neu/N mice, delaying tumor growth and clearing tumors in only 10% of mice. No differences in IFN-β production, DC priming, or HER-2-specific CD8+ T cell trafficking were detected between FVB/N and neu/N mice. However, activation and expansion of HER-2-specific CD8+ T cells was defective in neu/N mice. Immune cell infiltrates of untreated tumor-bearing neu/N mice expressed high levels of PD1 and OX40 on CD8+ T cells, and high levels of PD-L1 on both myeloid and tumor cells. Modulating PD-L1 and OX40 signaling combined with IT ADU S-100 enhanced HER-2-specific CD8+ T cell activity, clearing tumors from 40% of neu/N mice.

Conclusions: Intratumoral STING activation synergizes with PD1 pathway-blockade and OX40 receptor stimulation to overcome immune tolerance and prime tumor antigen-specific CD8+ T cell responses that mediate effective tumor regression.

#2994

Engineered STING-targeting immunotherapy delays B16 melanoma tumor growth and significantly improves animal survival as compared to an immune-checkpoint inhibitor.

Andrea Amalfitano, Fadel S. Alyaqoub, Yasser A. Aldhamen, Abdulraouf Abbas, Cristiane Pereira-Hicks, Sarah Godbehere, Christopher M. Waters. _Michigan State University, East Lansing, MI_.

Background: The stimulator of interferon genes (STING) is a cytosolic innate sensor activated by cyclic di-nucleotides (CDNs) including the bacterially derived bis-(3′-5′)-cyclic-dimeric-guanosine monophosphate (c-di-GMP), leading to the induction of type I interferons. We recently published that an engineered adenovirus (Ad) vector delivering a potent, bacterially derived diguanylate cyclase (AdVCA0848) in vivo can generate sustained c-di-GMP levels in vivo and induce beneficial immune responses, including induction of IFN-β, activation of NK cells and DCs, as well as enhance antigen-specific T cell responses. Here, we determined if in vivo use of AdVCA0848 can also induce potent anti-tumor responses. Methods: Experiments to be presented include those that subcutaneously placed 150,000 B16F10 melanoma cells into C57BL/6 mice. On day 7 post tumor implant (PTI), tumor-bearing mice were randomly divided into control groups (receiving intra-tumoral (i.t.) vehicle treatments or Ad controls, ± i.p administration of non-specific antibodies) and experimental groups, (single i.t. administrations with 2x1010 vps of AdVCA0848 alone, or in combination with repeated i.p. administrations of 200 μg of an anti-PD1 antibody (RMP-1-14) on days 7,11,15 and 19 PTI. Results: As compared to appropriate controls, we have repeatedly found that a single i.t. injection of AdVCA0848 significantly slowed B16 tumor growth (p<0.01), a result that correlated with high levels of animal survival (p<0.001). These levels of efficacy significantly exceeded those achieved with lone use of a PD-1 inhibitor. Under these same conditions, combined use of the AdVCA0848 therapy with anti-PD1 treatments trended to further improve animal survival (50% survival at day 27 PTI), as compared to lone use of either therapy (36.4% and 26.7% survival at day 27 PTI for AdVCA0848 and PD-1 treatments, respectively). Multiple innate and adaptive immune response readouts and immunohistological evaluations will be presented. Conclusions: The growth of established B16 melanoma tumors was rapidly inhibited after a single i.t. treatment with the engineered STING activator AdVCA0848, and this efficacy resulted in significant improvements in animal survival, at rates that were also significantly better than those achieved using a PD-1 antagonist. Furthermore, the i.t. administration of AdVCA0848 trended to show further improved efficacy when combined with a PD-1 antagonist, suggesting synergy. The AdVCA0848 platform is a novel, off the shelf immune-therapeutic that can now be justified for use to improve anti-tumor responses in melanoma, augment currently approved FDA immune therapies for melanoma (such as PD-1 targeted therapies), as well may show efficacy in other aggressive cancers.

#2995

Development of a novel highly potent TLR8 selective agonist for cancer immunotherapy.

Yuxun Wang, Heping Yang, Longsheng Wang, Guangliang Fu, Fang Bao, Fang Liu, Shuwen Ren, Huanping Li, Haixia Ji, Yajun Yu, Zhiheng Wu, Panhu Zhu, Hui Xu, Yaqiao Gao, Pei Wang, Shoujun Chen, Daxin Gao. _Denovo Pharmatech, Shanghai, China_.

The cancer drug discovery field is experiencing unprecedented revolution accompanied by growing excitement from researchers, drug developers, patients and investors, partly due to recent clinical success of cancer immunotherapy. Human immune defense system comprises both innate and adaptive immune pathways. All the targets drugged by the recently approved cancer immunotherapeutic agents including the immune checkpoint proteins PD-1, PD-L1 and CTLA-4 function in adaptive immune pathways. In contrast, targets involved in the innate immune pathway have not matured to regulatory approval for systemic use though several candidates are now in preclinical and clinical development. Drugs targeting innate immunity represent great opportunity for more rapid and broader spectrum anti-cancer effect than adaptive immunity. Furthermore, combinations of drugs targeting innate and adaptive immunity are expected to produce strong synergistic efficacy owing to their complementary nature as body's immune defense. Toll-like receptors (TLRs) are a family of proteins that recognize pathogen associated molecular patterns (PAMPs). Their primary function is to activate innate immune responses while also involved in facilitating adaptive immune responses. Different TLRs exert distinct functions by activating varied immune cascades. TLR8 is more broadly expressed in immune cells than other TLRs such as TLR7 and TLR9. One of the major causes of cancer immunotherapy failure is potent suppression of immune response by Treg cells. TLR8 is the only TLR that has been shown to be necessary and sufficient to reverse the suppressive function of Treg cells leading to strong tumor inhibition. Therefore, agonists targeting TLR8 are expected to be effective cancer therapy. However, there is no approved TLR8 selective agonist at present. There is only one TLR8 selective agonist in clinical development. Through structure-based drug design, we discovered a novel, highly potent and selective small molecule TLR8 agonist, DN-A1. DN-A1 exhibited strong in vitro cellular activity with EC50 at 4.23 nM, about 30-fold more potent than the only drug candidate in clinical trials. The activity was highly selective for TLR8 over other TLRs. DN-A1 displayed superior in vitro ADMET and in vivo PK profiles. DN-A1 showed clean CYP profile with IC50 over 10 μM for all major CYP isoenzymes tested including 3A4, 1A2, 2C9, 2C19 and 2D6. DN-A1 had favorable hERG parameter with IC50 over 30 μM whereas the reference compound's hERG IC50 was 3.84 μM suggesting potential cardiac toxicity. DN-A1 significantly impeded tumor growth as a single agent and was well-tolerated in mouse tumor models. Taken together, DN-A1 warrants further development as a potential best-in-class preclinical drug candidate for TLR8-mediated cancer immunotherapy.

#2996

Immune checkpoint protein VISTA suppresses Toll-like receptor signaling and the production of inflammatory cytokines.

Wenwen Xu,1 Ying Yuan,2 Na Li,1 Yongwei Zheng,1 Kamal Rajasekaran,1 Halli Miller,1 Michael Olson,1 Demin Wang,1 Subramaniam Malarkannan,1 Li Wang1. 1 _Medical College of Wisconsin, Milwaukee, WI;_ 2 _Shanghai University of Traditional Chinese Medicine, Shanghai, China_.

Introduction: V-domain Ig suppressor of T-cell activation (VISTA, gene Vsir) is an inhibitory immune-checkpoint molecule that suppresses CD4+ and CD8+ T cell activation. Previous studies have shown that Vsir-/- mice developed chronic inflammatory phenotypes, and Vsir-/- CD4+ and CD8+ T cells were hyper-responsive towards self- and foreign antigens. Whether VISTA regulates innate immunity is still unknown.

Methods: Peritoneal macrophages from WT or Vsir-/- mice were isolated and stimulated with TLR agonists such as CpG (TLR9), R848 (TLR7), LPS (TLR4), Pam3csk4 (TLR2), and poly (I:C) (TLR3). Alternatively, human monocyte THP-1 cells overexpressing VISTA were stimulated by TLR2 agonist Pam3CSK4. The activation of TLR signaling pathways and the production of inflammatory cytokines were examined by Western Blotting, gel shift assay, or ELISA. The ubiquitination status of key signaling molecules such as TRAF6, IRAK1/4 and MyD88 was examined by immunoprecipitation and Western Blotting. To examine the role of VISTA in regulating TLR-mediated inflammatory responses in the context of cancer vaccine, tumor-bearing mice were treated with VISTA-specific monoclonal antibody (mAb) and a peptide vaccine containing TLR agonists. The production of inflammatory cytokines and chemokines within the tumor microenvironment (TME) was examined via quantitative RT-PCR.

Results: Vsir-/- macrophages were hyper-responsive towards TLR2/4/7/9 agonists, but not TLR3 agonist, resulting in increased production of inflammatory cytokines IL-6, IL-12, and TNFa. Analysis of signaling cascade revealed that VISTA inhibited the activation of MyD88-dependent TLR signaling, via suppressing the activation of MAPKs, and the activation of transcription factors AP-1 and NF-kB. Consistent with the role of VISTA in regulating TLR-mediated innate immunity, treatment with VISTA-blocking mAb augmented levels of inflammatory cytokines and chemokines within the TME, and synergized with TLR/peptide vaccine, resulting in an optimal therapeutic outcome.

Conclusions: Our study establishes that VISTA critically regulates the inflammatory responses of myeloid cells mediated by TLR signaling. In the context of cancer vaccine therapy, VISTA-blocking mAb treatment enhanced levels of inflammatory cytokine and chemokines within the TME, which is critical for the development of optimal tumor-specific T cell responses and the tumor-controlling therapeutic outcome.

#2997

The tetravalent bispecific antibody AFM13 engages and primes innate immune cells for anti-cancer immunity.

Jens Pahl,1 Joachim Koch,2 Uwe Reusch,2 Thorsten Gantke,2 Adelheid Cerwenka,1 Martin Treder2. 1 _German Cancer Research Center, Heidelberg, Germany;_ 2 _Affimed GmbH, Heidelberg, Germany_.

AFM13 is a tetravalent bispecific antibody with bivalent binding to both CD30 and CD16A. It has been shown to engage NK-cells through CD16A with high affinity and specificity, resulting in strong NK-cell cytotoxicity, and is currently being tested in Phase 2 monotherapy and in combination with pembrolizumab in Phase 1b clinical trials. We have previously shown that AFM13-dependent activation of NK-cell cytotoxicity towards CD30+ tumor cells is more pronounced than that of anti-CD30 mAbs. In addition, AFM13 enhances NK-cell sensitivity to low doses of IL-2 and IL-15, leading to an increased NK-cell proliferative potential. Here, we have extended the panel of phenotypic markers on NK-cells that are modulated after exposure to CD30+ tumor cells in the presence of AFM13. Targeting some of these markers may enable the development of novel combination therapies. Moreover, we have analyzed the kinetics of NK-cell responses to AFM13 exposure. Even though short-term exposure to AFM13 significantly enhanced NK-cell cytotoxicity, long-term exposure led to a partial, transient functionally exhausted phenotype in vitro, which could be fully restored by cytokine stimulation for several days in the absence of AFM13. Importantly, these recovered cells displayed high cytotoxicity towards CD30+ target cells in the presence of AFM13. Interestingly, the transient NK-cell exhaustion was not related to the expression of typical exhaustion markers or insufficient levels of perforin and granzyme. These data may warrant the development of novel metronomic application regimens of AFM13. Further studies imply that immune cells other than NK-cells are able to inhibit growth of CD30+ tumor cells in an AFM13-dependent manner. This appears to be strictly dependent on CD16A and a specific cytokine milieu. Taken together, AFM13 specifically enhances the cytotoxic, proliferative and cytokine-producing potential of NK-cells, parameters that can be utilized to monitor NK-cell responses during AFM13 therapy. Moreover, based on our data, engagement of CD16A+ cells to the tumor site might enable several innate immune effector functions within the tumor microenvironment for synergistic anti-tumor activity.

#2998

Elotuzumab can costimulate NK cell responses by engaging SLAMF7.

Tatiana Pazina,1 Ashley Mentlik James,1 Alexander W. MacFarlane,1 Natalie A. Bezman,2 Robert F. Graziano,2 Michael D. Robbins,2 Adam D. Cohen,3 Kerry S. Campbell1. 1 _Fox Chase Cancer Ctr., Philadelphia, PA;_ 2 _Bristol-Myers Squibb, Princeton, NJ;_ 3 _University of Pennsylvania, Philadelphia, PA_.

Elotuzumab (Elo) is an IgG1 monoclonal antibody targeting SLAMF7 (CS1, CRACC, CD319) protein, which is highly and uniformly expressed on multiple myeloma cells. Improved survival has been observed in multiple myeloma patients treated with Elo in combination with immunomodulatory drugs such as dexamethasone and lenalidomide. Previous work showed that the main mechanism of Elo action in vitro is CD16-mediated antibody dependent cellular cytotoxicity (ADCC) via Fc interaction with CD16 (FcγRIIIa) receptor on NK cells. However, SLAMF7 is also expressed on NK cells where it transmits an activating signal. We hypothesized that Elo can directly activate NK cells via SLAMF7 ligation. Utilizing non-fucosylated Elo that has higher affinity to CD16 and Elo mutant that has an Fc region mutation that abrogates binding to CD16, we confirmed that Elo promotes NK cell activation and degranulation in a CD16-dependent manner. Specifically, non-fucosylated Elo had higher potency, whereas Elo mutant did not stimulate degranulation or CD69 expression.

To test for co-stimulatory effects of Elo and Elo mutant we measured calcium signaling responses triggered by those antibodies in combination with antibodies to engage the activating receptors NKp46 and NKG2D. Elo or Elo mut alone had no effect on calcium signaling, but when added in combination with multimeric engagement of NKp46 and NKG2D both antibodies co-stimulated calcium signaling responses.

We conclude that Elo stimulates NK cells via interaction with CD16, but direct engagement with SLAMF7 can also transduce co-activating signals to potentiate calcium signals generated through NKp46 and NKG2D in a CD16-independent manner. Our results provide evidence that direct engagement with SLAMF7 by Elo has the potential to reduce the threshold of NK cell activation triggered through other activating receptors.

#2999

**NKG2A checkpoint receptor expression on tumor-infiltrating CD8** + **T cells restrains efficacy of immunotherapy.**

Thorbald van Hall,1 Nadine van Montfoort,1 Sytse Piersma,1 Marjolein Sluijter,1 Marij J. Welters,1 Pascale André,2 Nicolai Wagtmann,2 Sjoerd H. van der Burg1. 1 _Leiden University Medical Center, Leiden, Netherlands;_ 2 _Innate Pharma, Marseille, France_.

The inhibitory receptor CD94-NKG2A is expressed on subsets of NK cells and cytotoxic CD8+ T cells, but not CD4+ T helper cells. It's ligand is the non-classical MHC class I molecule HLA-E, commonly over-expressed in human cancers. We previously demonstrated that high HLA-E expression in ovarian carcinoma and NSCLC may neutralize the survival benefit of T cell infiltration, suggesting that NKG2A could represent an immune checkpoint that tumors may exploit for immune evasion. We thus examined NKG2A expression on human and mouse Tumor Infiltrating Lymphocyte (TIL) populations and tested blockade of this receptor in mouse tumor models. Multicolor flow cytometry analysis showed higher frequency of NKG2A+CD8+ T cells in TIL samples obtained from biopsies of human head- and neck- carcinomas (n=17) compared to peripheral blood CD8+ T cells. Frequency of NKG2A+ TIL was higher in patients with measurable immune reactivity to HPV16-viral antigens than in patients without tumor immunity (14.8% vs 4.7%, respectively). Interestingly, only subsets of the NKG2A+ T cells co-expressed other inhibitory receptors, including PD-1 and TIM3, suggesting the existence of a 'dedicated' T cell subset for NKG2A. In our mouse tumor models we observed similar findings and, moreover, demonstrated that therapeutic vaccines comprising tumor antigens increased the frequency of NKG2A+-positive CD8+ TIL up to 80%. Similarly, expression of Qa-1, the mouse homologue of HLA-E, on tumor cells also increased after therapeutic vaccination. As a proof of concept that the NKG2A - Qa-1 axis limits the efficacy of immunotherapy, NKG2A blocking antibodies were applied in the TC-1 model together with therapeutic peptide vaccination. Combination treatment with vaccine and NKG2A blockade significantly improved the therapeutic efficacy compared to the vaccine alone, both in terms of survival (P<0.05, median survival 44 vs 31 days) as well as overall response rate, defined as decreased tumor size of more than 30% (93.8% vs 43.8%, respectively). Similar results were obtained in Qa-1-deficient mice, suggesting that Qa-1 on tumor cells is of importance. In conclusion, inhibitory receptor NKG2A is enriched on CD8+ TIL and functions as an immune checkpoint that restrains therapeutic efficacy of a cancer vaccine.

### Predictors and Drivers of Pediatric Tumors

#3000

Defining the subclonal landscape of high-risk neuroblastoma.

Jo Lynne Harenza, Maura A. Diamond, Derek A. Oldridge, Olivia Padovan-Merher, Pichai Raman, Yael P. Mosse, John M. Maris. _Children's Hospital of Philadelphia, Philadelphia, PA_.

We recently showed that relapsed neuroblastoma (NB) tumors harbor an increased somatic mutational burden, enriched for ALK or RAS-MAPK activating lesions, nominating targeted therapeutic strategies for a condition that is largely incurable. We also showed that these mutations can be present in subclonal tumor cell populations at diagnosis, suggesting that they may contribute to acquired chemoradiotherapy resistance. We hypothesize that subclonal mutations in NB oncogenes are common at diagnosis, are biomarkers for treatment failure, and can be targeted in conjunction with standard chemotherapy to improve patient survival. Here, we focus on the first aspect of this broad hypothesis by presenting the subclonal landscape of diagnostic, high-risk neuroblastomas. A meta-analysis of our previously published primary/relapse (N=23), whole exome (N=232) and whole genome (N=159) diagnostic data, and recent sequencing study of 78 diagnostic and 67 relapse cases were used to inform design of a custom amplicon panel for ultra-deep sequencing. The 28-gene panel covers 57 mutations in frequently mutated genes. Here, 250 high-risk primary tumors were sequenced to an average depth of 50,000X. More subclonal mutations were called (mean=36) compared to clonal alterations (mean=3), demonstrating the complex subclonal architecture in these tumors. We validated five clonal ALK mutations we originally discovered and identified 15 additional pathogenic ALK mutations with mutant allele frequencies (MAFs) ranging from 0.03-23%. Known ALK activating mutations were found at codons 1170, 1174, 1196, 1245, 1275 in 15 of 38 diagnostic primary tumors. This 39.5% frequency is much higher than the 14% frequency defined by us using Sanger or other sequencing methods. Improvement of subclonal mutation detection of actionable, relapse-specific driver mutations demonstrates the clinical utility of utilizing this sequencing methodology at diagnosis to enable timely improvement of outcomes for children with high-risk refractory NB.

#3001

Germline mutations in cancer predisposition genes and risk for subsequent neoplasms among long-term survivors of childhood cancer in the St. Jude Lifetime Cohort.

Zhaoming Wang,1 Carmen L. Wilson,1 John Easton,1 Dale Hedges,1 Qi Liu,2 Gang Wu,1 Michael Rusch,1 Michael Edmonson,1 Shawn Levy,3 Jennifer Q. Lanctot,1 Eric Caron,1 Kyla Shelton,1 Kelsey Currie,1 Matthew Lear,1 Heather L. Mulder,1 Donald Yergeau,1 Celeste Rosencrance,1 Bhavin Vadodaria,1 Yadav Sapkota,1 Russell J. Brooke,1 Wonjong Moon,1 Evadnie Rampersaud,1 Xiaotu Ma,1 Shuoguo Wang,1 Ti-Cheng Chang,1 Stephen Rice,1 Andrew Thrasher,1 Aman Patel,1 Cynthia Pepper,1 Xin Zhou,1 Xiang Chen,1 Wenan Chen,1 Angela Jones,3 Braden Boone,3 Deo Kumar Srivastava,1 Chimene A. Kesserwan,1 Kim E. Nichols,1 James R. Downing,1 Melissa M. Hudson,1 Yutaka Yasui,1 Leslie L. Robison,1 Jinghui Zhang1. 1 _St. Jude Children's Research Hospital, Memphis, TN;_ 2 _University of Alberta, Edmonton, Alberta, Canada;_ 3 _HudsonAlpha, Huntsville, AL_.

This abstract has been withheld from publication due to its inclusion in the AACR Annual Meeting 2017 Official Press Program. It will be posted online following its presentation.

#3002

Therapeutic targeting of PGBD5-induced DNA repair dependence in pediatric solid tumors.

Anton Henssen,1 Casie Reed,1 Eileen Jiang,1 Wong Wai,1 Elisa de Stanchina,1 Yasumichi Kuwahara,2 Hajime Hosoi,2 Johannes H. Schulte,3 Neil Ganem,4 John Petrini,5 Alex Kentsis1. 1 _Mem. Sloan Kettering Cancer Ctr., New York, NY;_ 2 _Kyoto Prefectural University of Medicine, Tokyo, Japan;_ 3 _Charité- Universitätsmedizin Berlin, Berlin, Germany;_ 4 _Boston University, Boston, MA;_ 5 _Molecular Biology Program, New York, NY_.

Despite intense efforts, the cure rates of childhood solid tumors are not satisfactory. Resistance to intensive chemotherapy is common, and targets for molecular therapies are largely undefined, particularly for tumors caused by aberrant developmental and epigenetic factors. We have now found that the majority of childhood solid tumors, rhabdoid tumors, neuroblastoma, medulloblastoma and Ewing sarcoma, express an active DNA transposase PGBD5. Using functional genetic approaches, we observed that mouse and human cells deficient in non-homologous end joining (NHEJ) DNA repair cannot tolerate the expression of PGBD5. In a chemical screen of DNA repair inhibitors, we identified the ATR/ATM inhibitor AZD6738 as a specific sensitizer of PGBD5-dependent DNA damage and apoptosis. Ectopic expression of PGBD5 was sufficient to induce hypersensitivity to AZD6738, and endogenous PGBD5 was necessary for its anti-tumor effects. We found that PGBD5-expressing tumor cells accumulate unrepaired DNA damage in response to AZD6738 treatment, and undergo apoptosis in both dividing and G1 phase cells in the absence of measureable DNA replication stress. Accordingly, AZD6738 exhibits nanomolar potency against the majority of neuroblastoma, medulloblastoma, Ewing sarcoma and rhabdoid tumor cells tested, while sparing non-transformed human and mouse embryonic fibroblasts in vitro. Finally, AZD6738 exhibits excellent single-agent activity against high-risk neuroblastoma and medulloblastoma xenografts, with induction of apoptosis and tumor regression in vivo. These findings delineate a therapeutically actionable synthetic lethality for the majority of childhood solid tumors.

#3003

Exome analysis of known hereditary cancer genes in 122 children with rhabdomyosarcoma.

Talia Wegman-Ostrosky,1 Rajesh Patidar,1 Sivasish Sindiri,1 Jack Shern,1 Douglas S. Hawkins,2 Daniel Catchpoole,3 Jun S. Wei,1 Stephen Skapek,4 Javed Khan,1 Douglas R. Stewart1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Seattle Children's, Seattle, WA;_ 3 _University Of Sydney, Sydney, Australia;_ 4 _UT Southwestern Department of Pediatrics, Dallas, TX_.

Introduction. Rhabdomyosarcoma (RMS) accounts for 5% of all pediatric cancer and is the most prevalent soft tissue tumor in childhood and adolescents. RMS is thought to arise from primitive mesenchymal stem cells directed towards myogenesis. Between 7-33% of RMS cases arise from a hereditary cancer syndrome, like LFS or NF1. We analyzed germline genetic variants in hereditary cancer genes in 122 children with RMS.

Methodology. In 122 children with RMS and 1001 cancer-free adults, we examined germline exome data to determine the frequency of genetic variants in 51 cancer genes known to underlie syndromes associated with RMS. DNA was extracted from blood or buccal cells using standard methods. Exome enrichment was performed with NimbleGen SeqCap EZ Human Exome Library v3.0+UTR, on an Illumina HiSeq. Annotation of each exome variant was performed using a custom software pipeline. We evaluated all variants that passed quality controls with a population minor allele frequency (MAF) <0.1%. The cataloging of the variants was based on the ACMG classification as pathogenic (P), likely pathogenic (LP), or variable of unknown significance.

Results We compared the age, gender, histologic type and localization of the primary RMS of the patients with and without P/LP variants in the 51 genes. In the patients without P/LP variants, the mean age of diagnosis was 5 years and the most frequent site of diagnosis was head and neck. In the group with P/LP variants, the mean age of diagnosis was 10 years, and the most frequent site was pelvis.

In the 51 genes that were analyzed we found 9 P and 12 LP variants in 15 genes: TP53, ATM, MSH6, PMS, DICER1, FANCA, RECQL4, PTEN, WRN, RB1, BUB1B, RET, APC, FANCM and TSC2; genes with 2 variants include WRN, PTEN, BUB1B, FANCA and RET. Most of the variations were stopgain, follow by missense, frameshift insertion and splicing genetic variations. Ten of this genes are associated with an autosomal dominant pattern of inheritance. In one 15-year-old female patient with an alveolar RMS in the paraspinal area we found P/LP variants in PTEN and PMS2. The frequency of P/LP variants in cases was 16% and 3% in controls.

Conclusions. To our knowledge, this is the first study where multiple germline variation where analyses in children with RMS. We found P/LP variation in 16% of all the cases (pending orthogonal confirmation); the mean age of diagnosis was 10 years old and their primary tumor site was in the pelvis (50%). Identification of P/LP variation in genes underlying RMS-associated syndromes has implications for follow-up, screening and management of these patients and their families. We acknowledge the Children's Oncology Group in helping to assemble the RMS cohort.

#3004

Comparison of somatic alterations in the genome and transcriptome of 1,705 pediatric leukemia and solid tumors: a report from the Children's Oncology Group (COG) - NCI TARGET Project.

Xiaotu Ma,1 Yu Liu,1 Yanling Liu,1 Michael Edmonson,1 Charles Gawad,1 Xin Zhou,1 Yongjin Li,1 Michael Rusch,1 John Easton,1 Mark Wilkinson,1 Leandro C. Hermida,2 Sean Davis,2 Malcolm Smith,2 Jaime Guidry Auvil,2 Paul Meltzer,2 Ching C. Lau,3 Elizabeth Perlman,4 John M. Maris,5 Soheil Meshinchi,6 Stephen P. Hunger,5 Daniela S. Gerhard,2 Jinghui Zhang1. 1 _St Jude Children's Research Hospital, Memphis, TN;_ 2 _National Cancer Institute, NIH, Bethesda, MD;_ 3 _Texas Children's Cancer and Hematology Centers, Houston, TX;_ 4 _Ann and Robert H. Lurie Children's Hospital of Chicago, Chicago, IL;_ 5 _Children's Hospital of Philadelphia, Philadelphia, PA;_ 6 _Fred Hutchinson Cancer Research Center, Seattle, WA_.

To discover common and sub-type specific somatic alterations affecting key biological processes in pediatric cancers, we analyzed point mutations, copy number alterations, gene fusions and structural alterations detected from paired tumor-normal whole genome sequencing (n=655), whole exome sequencing (n=1,108), and RNA-seq data (n=913) of 1,705 leukemia and solid tumors. Our cohort consists of 693 B-lineage Acute Lymphoblastic Leukemia (B-ALL), 264 T-ALL, 211 Acute Myeloid Leukemia (AML), 318 Neuroblastoma (NBL), 128 Wilms Tumor (WT), and 91 Osteosarcoma (OS) with a median mutation rate of 0.28-0.58 per Mb. We identified 130 potential driver genes based on significance of variant recurrence and pathogenicity within each cancer type and across all cancer types. Seventy-two (55%) driver genes were significant in one cancer type, thirty eight were significant in > 1 leukemia subtype, thirteen (NRAS, WT1, MYCN, PTEN, TP53, KRAS, RB1, ATRX, PTPN11, MLLT1, BCOR, SETD2, NF1) were significant in both leukemia and solid tumor while the remaining seven (MGA, SF3B1, ASXL1, BCORL1, STAG2, ACTB, NIPBL) were significant only in pan-cancer analysis. The number of mutated driver genes per sample ranged from 0.8 in WT to 5.8 in T-ALL, lower when considering only point mutations (from 0.3 in NBL to 3.1 in T-ALL). The most frequently mutated biological processes affecting both leukemia and solid tumor were transcription factors (56% of samples), cell cycle (41%), epigenetic regulators (36%), Ras signaling (21%), PI-3K (11%), and the MYC complex (7%). By contrast, the JAK signaling pathway was mutated only in leukemia (16%) while mutations in the NOTCH signaling pathway were exclusive to T-ALL (77%).

Aberrant transcription may also affect the normal function of a driver gene. For example, the RAS signaling pathway was mutated in B-ALL (35%), T-ALL (15%), AML (37%) and NBL (4.3%). Aside from the known KRAS 4a isoform found in all cancer types, we discovered two novel KRAS isoforms present in 71.1% of B-ALL, 67.9% of T-ALL, 71.3% of AML and 3.0% of NBL but not in WT or OS. Allele-specific expression (ASE) was detected in 205 (6.8%) of 3,016 expressed somatic mutations, and 97% (32 out of 33) of truncation mutations on autosomes exhibit reduced expression of the mutant allele likely due to nonsense mediated decay. Two ASE mutations, WT1 D447N in a cytogenetically normal AML and JAK2 D873N in a B-ALL, were selected for single-cell sequencing and successfully validated.

Only 44% of our driver genes match those identified in adult cancer. This, coupled with our finding that point mutations only accounted for 48% of the driver alterations, may provide new insight into the design of precision treatment for pediatric cancer. Our presented data will be made public at NCI's Genome Data Commons (gdc.cancer.gov) and can be explored on our ProteinPaint data portal (pecan.stjude.org).

#3005

Whole-genome sequencing identified novel non-coding mutations causal of oncogene activation in T-cell acute lymphoblastic leukemia.

Maoxiang Qian,1 Shaoyan Hu,2 Hui Zhang,1 Yu Guo,3 Jin Yang,2 Xujie Zhao,1 Lin Wan,2 Jun Lu,2 Jian Pan,2 Meimei Chang,3 Shirley K. Kham,4 Yong Cheng,1 Chunliang Li,1 Allen E. Yeoh,4 Anders Skanderup,3 Jun J. Yang1. 1 _St. Jude Children's Research Hospital, Memphis, TN;_ 2 _Children's Hospital of Soochow University, Suzhou, China;_ 3 _Genome Institute of Singapore, Singapore;_ 4 _National University Children's Medical Institute, Singapore_.

There is growing evidence that non-coding sequences in human genome often function as transcriptional regulatory elements of protein-coding genes. In fact, germline polymorphisms and somatically acquired mutations within regulatory DNA can profoundly alter chromatin structure and modify gene transcription, directly contributing to tumorigenesis. However, there is a paucity of unbiased genome-wide characterization of somatic non-coding mutations in cancer. Using T-cell acute lymphoblastic leukemia (T-ALL) as a model disease, we herein report a systematic interrogation of driver non-coding genomic alterations by paired whole-genome and transcriptome sequencing of 31 children with T-ALL. To identify non-coding mutations with potential regulatory impact in a genome-wide fashion, our analytical pipeline consisted of 3 approaches: 1) the "hotspot analysis" for recurring mutations at the nearby positions, 2) the "regional recurrence analysis" for predefined regulatory regions with significant enrichment of non-coding mutations, 3) the "transcriptional factor analysis" for mutations that potentially result in gain/loss of transcription factor binding sites and alter expression of adjacent genes. Remarkably, T-ALL oncogenes LMO1 and TAL1 emerged as loci with the most significant recurring non-coding mutations. At the LMO1 locus, 3 patients (9.7%) showed an identical single-nucleotide mutation proximal to the transcription start site of the long isoform of LMO1. This recurring mutation resulted in the gain of a canonical Myb binding site (AACGG) and ~120-fold increase in LMO1 transcription compared to patients with wildtype genotype. TAL1 overexpression was observed in 15 patients, of whom 11 had intrachromosomal rearrangement (STIL-TAL1 fusion). The remaining 4 patients had somatic insertion that created a MYB-mediated super enhancer, consistent with recent reports. LMO1 enhancer mutation was further confirmed in an independent validation cohort (N=26), in which we additionally identified a novel intrachromosomal rearrangement between MED17 and LMO1 resulting in transcriptional activation of the latter. In a panel of T-ALL cell lines, LMO1 enhancer mutation was again associated with dramatic overexpression of LMO1, an active enhancer histone mark (H3K27ac), Dnase hypersensitivity, and allele-specific binding of MYB. Interestingly, we also observed robust binding of TAL1, CREBBP, RUNX1, ETS1, ELF1 and RNA Polymerase II at this site. Reporter gene assay confirmed the MYB-mediated transcription activation effects of this LMO1 enhancer mutation in vitro. In this genome-wide investigation of non-coding mutations in T-ALL, we identified novel enhancer mutations with drastic effects on oncogene activation. Our findings expand the understanding of how genomic alterations in regulatory DNA contribute to cancer pathogenesis.

#3006

Oncogenic activity of H3.3 K27M in a spontaneous DIPG mouse model.

Jon D. Larson, Lawryn H. Kasper, Gang Wu, Yiping Fan, David Finkelstein, Hongjian Jin, Barbara S. Paugh, Andre B. Silveira, Beisi Xu, Xiaoyan Zhu, Junyuan Zhang, Helen R. Russell, Peter J. McKinnon, David W. Ellison, Jinghui Zhang, Suzanne J. Baker. _St. Jude Children's Research Hospital, Memphis, TN_.

Pediatric diffuse high-grade gliomas (HGG) cause a devastatingly poor outcome. Diffuse intrinsic pontine gliomas (DIPG) are brainstem HGGs found almost exclusively in children and represent approximately 50 percent of all pediatric HGGs. Recurrent, clonal somatic mutations in histone H3 are a molecular hallmark that distinguish the genetic mechanisms underlying pediatric and adult HGG, and indicate a strong link between epigenetic dysfunction and pediatric brain tumorigenesis. H3 K27M mutations found in nearly 80 percent of DIPGs and over half of non-brainstem HGGs occurring in thalamus or other midline structures, induce a dominant loss of genome-wide H3K27me3. In contrast, mutually exclusive H3.3 G34R/V mutations occur in approximately 14 percent of cortical HGG tumors in older children through young adulthood suggesting a distinct developmental origin compared to K27M mutant tumors. DNA methylation and gene expression patterns distinguish G34R/V and K27M tumors, but the oncogenic mechanisms and reasons for brain region selectivity of histone mutations remain uncertain. To study these mutations in the developing mammalian brain and investigate how they contribute to oncogenesis, we generated conditionally activated, epitope-tagged knock-in mice to express K27M, G34R or non-mutated H3.3 proteins from the endogenous H3f3a locus. Untransformed embryonic neural precursor or astrocyte cells expressing H3.3 K27M demonstrated H3K27me3 loss and a growth advantage in vitro with brain region-specific, stage-dependent expression and epigenetic signatures. DIPGs frequently harbor genetic alterations in TP53 and PDGFRα in addition to H3.3 K27M, and mice with induced brain-specific Trp53 deletion plus mutated PDGFRαV544ins expression developed spontaneous HGG in vivo. Induction of H3.3 K27M significantly accelerated PDGFRαV544ins;Trp53 knockout HGG formation involving the brainstem. Importantly, the non-mutated H3.3 epitope-tagged protein did not affect tumor burden or latency. The H3.3 K27M tumors recapitulate characteristics observed in human DIPG including histopathological features, strong nuclear K27M expression and robust loss of H3K27me3 throughout the tumors. We relate the genetic, epigenetic and transcriptional landscapes of these models to primary pediatric DIPG to help identify their developmental origins and oncogenic mechanisms downstream of histone H3.3 K27M. 

## EPIDEMIOLOGY:

### Cancer Epidemiology and Prevention

#3007

Tobacco smoking, alcohol use and risk of hepatocellular carcinoma and intrahepatic cholangiocarcinoma: The Liver Cancer Pooling Project.

Jessica Leigh Petrick,1 Peter T. Campbell,2 Jill Koshiol,1 Jake E. Thistle,1 Gabriella Andreotti,1 Laura E. Beane-Freeman,1 Julie E. Buring,3 Andrew T. Chan,3 Dawn Q. Chong,4 Michele M. Doody,1 Susan M. Gapstur,2 John Michael Gaziano,3 Edward Giovannucci,5 Barry I. Graubard,1 I-Min Lee,3 Linda M. Liao,1 Martha S. Linet,1 Julie R. Palmer,6 Jenny N. Poynter,7 Mark P. Purdue,1 Kim Robien,8 Lynn Rosenberg,6 Catherine Schairer,1 Howard D. Sesso,3 Rashmi Sinha,1 Meir J. Stampfer,3 Marcia Stefanick,9 Jean Wactawski-Wende,10 Xuehong Zhang,3 Anne Zeleniuch-Jacquotte,11 Neal D. Freedman,1 Katherine A. McGlynn1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _American Cancer Society, Atlanta, GA;_ 3 _Brigham and Women's Hospital, Boston, MA;_ 4 _Massachusetts General Hospital and Harvard Medical School, Boston, MA;_ 5 _Harvard School of Public Health, Boston, MA;_ 6 _Boston University, Boston, MA;_ 7 _University of Minnesota, Minneapolis, MN;_ 8 _George Washington University, Washington, DC;_ 9 _Stanford University School of Medicine, Stanford, CA;_ 10 _University at Buffalo, Buffalo, NY;_ 11 _New York University School of Medicine, New York, NY_.

Background: Since 1980, liver cancer has been among the most rapidly increasing cancer types in the United States (US), with 5-year survival rates of approximately 17%. While tobacco and alcohol are known to be associated with primary liver cancer, it is unclear whether they only increase the risk of hepatocellular carcinoma (HCC), the most common type of liver cancer, or whether they also increase risk of intrahepatic cholangiocarcinoma (ICC), second most common histologic type. Additionally, it is unclear what amount of alcohol consumption is associated with an increased risk of liver cancer. As liver cancer is a rare cancer type, we conducted a study of pooled data from the National Cancer Institute Cohort Consortium to examine the associations between smoking and alcohol use and liver cancer, stratified by histologic subtype.

Methods: In the Liver Cancer Pooling Project, a consortium of US-based cohort studies, we pooled data from 1,518,741 individuals (HCC n=1,423, ICC n=410) in 14 cohorts. Multivariable-adjusted hazard ratios (HRs) and 95% confidence intervals (CI) were estimated using proportional hazards regression. Cubic splines were used to model the association between alcohol and liver cancer risk.

Results: Compared to never smokers, both former and current smokers at study baseline had an increased risk of ICC (HR=1.32, 95% CI: 1.03-1.68 and HR=1.47, 95% CI: 1.07-2.02, respectively) and HCC (HR=1.24, 95% CI: 1.08-1.43 and HR=1.86, 95% CI: 1.57-2.20, respectively). This finding was consistent for heavier smoking intensity, longer duration of smoking, and more pack-years of smoking. Heavy alcohol consumption was associated with an 87% increased risk of HCC (HR≥7 drinks/day v. non-drinker=1.87, 95% CI: 1.41-2.47) and a non-significant 68% increased risk of ICC (HR≥5 drinks/day v. non-drinker=1.68, 95% CI: 0.99-2.86). Risk of HCC significantly increased at 4.5 alcoholic drinks per day, while risk of ICC was non-significantly increased with any amount of consumption.

Conclusions: These findings suggest that, in a US population, cigarette smoking is associated with an increased risk of both histologic subtypes of primary liver cancer - HCC and ICC. In contrast, alcohol consumption was primarily associated with an increased risk of HCC. These results suggest that smoking cessation and alcohol reduction programs could be important intervention opportunities for these lethal cancer types.

#3008

Obesity, physical activity, and breast cancer survival among older breast cancer survivors in the CPS-II Nutrition Cohort.

Maret L. Maliniak, Alpa V. Patel, Marjorie L. McCullough, Peter T. Campbell, Corinne R. Leach, Susan M. Gapstur, Mia M. Gaudet. _American Cancer Society, Atlanta, GA_.

Background: Body mass index (BMI) and physical activity and their relation to breast cancer-specific survival have not been well-studied in older survivors (≥65 years of age at diagnosis), despite their comprising nearly half of today's 3.5 million breast cancer survivors. Additional data are needed to determine if specific guidelines for older survivors are warranted.

Objective: To examine the associations of pre- and post-diagnosis BMI and recreational physical activity with breast cancer-specific and all-cause mortality among women with non-metastatic breast cancer, overall and stratified by age 65 years at diagnosis.

Methods: In the Cancer Prevention Study-II Nutrition Cohort, 4,927 women diagnosed with breast cancer between 1992 and 2011 completed biennial surveys and were followed for cause-specific mortality. Pre-diagnosis exposures were assessed at the questionnaire completed ≥6 months before diagnosis, and post-diagnosis exposures were assessed at the first questionnaire completed ≥2 years after diagnosis to avoid capturing short-term changes due to treatment. BMI was computed as weight (kg) divided by height squared (m2). Physical activity was based on the summary MET-hours/week of recreational activity. Multivariable Cox proportional hazards models were used to calculate hazard ratios (HR) and 95% confidence intervals (CI).

Results: Over a mean follow-up time of 9.8 years after diagnosis, 1,394 deaths, including 452 from breast cancer, occurred. The median age at diagnosis was 70 years (77% ≥65 years). Compared with normal weight (BMI 18.5-<25 kg/m2), class II-III obesity (BMI ≥35 kg/m2) was associated with higher risk of breast cancer-specific mortality in pre-diagnosis (HR: 2.2; 95% CI: 1.5-3.2) and post-diagnosis analyses (HR: 1.6; 95% CI: 1.0, 2.5), although the association was limited to women ≥65 years of age at diagnosis. Pre-diagnosis but not post-diagnosis BMI was positively associated with all-cause mortality among both age groups (P-trend <0.0001). Pre-diagnosis physical activity was not associated with breast cancer-specific mortality overall or in either age group. There was a significant inverse trend for post-diagnosis physical activity and breast cancer-specific mortality overall (P-trend=0.03) and among women <65 years of age at diagnosis (P-trend=0.01) but not among women ≥65 years of age at diagnosis (P-trend=0.21). Pre- and post-diagnosis physical activity were significantly associated with reduced all-cause mortality in both age strata.

Conclusion: Our results suggest that pre- and post-diagnostic morbid obesity is associated with increased risk of death from breast cancer in older, but not younger, breast cancer survivors. Conversely, physical activity may reduce risk of breast cancer death in survivors <65 years of age at diagnosis. However, both were associated with overall longevity, regardless of age.

#3009

Lipid-lowering drug use and risk of fatal prostate cancer in the Atherosclerosis Risk in Communities (ARIC) Study.

Alison M. Mondul,1 Corinne E. Joshu,2 John Barber,2 Anna Prizment,3 Nrupen Bahvsar,4 Josef Coresh,2 Elizabeth Selvin,2 Aaron Folsom,3 Elizabeth A. Platz2. 1 _Univ. of Michigan, Ann Arbor, MI;_ 2 _Johns Hopkins Bloomberg School of Public Health, Baltimore, MD;_ 3 _University of Minnesota, Minneapolis, MN;_ 4 _Duke University, Durham, NC_.

Background: Studies show that lipid-lowering medications, particularly statins, may be protective for more aggressive prostate cancer. Lethal prostate cancer is often considered an optimal outcome in epidemiologic studies when the aim is to identify modifiable risk factors with the potential to reduce morbidity and mortality from this disease, yet this outcome remains understudied. Further, lipid drug use and prostate cancer risk is understudied in black men, whose risk of fatal prostate cancer is 2-3 times higher than that of white men. Thus, we studied lipid medication use and fatal prostate cancer risk in the ARIC Study, which includes ~25% black participants.

Methods: The ARIC Study is a prospective cohort that included 15,792 participants (7,082 men) recruited from four US communities. We conducted a prospective cohort analysis of 6,547 men who attended visit 2 (1990-1992, the start of the statin era) and were followed for cancer death through 12/31/2012. Death from prostate cancer (n=90) as the underlying cause was obtained from death certificates supplemented with medical records. Updated information on medication use was collected throughout the study follow-up during study visits and annual telephone calls. Lipid medication use was modeled as a time-dependent variable in two ways: 1) current use (yes/no), and 2) duration of use (<10, ≥10 years). Cox proportional hazards regression was used to estimate the hazard ratio (HR) and 95% confidence interval (CI) of prostate cancer death overall and by race. All models were adjusted for age (years), race, and study center. Additional models were further adjusted for height, BMI, smoking, diabetes, and education.

Results: The prevalence of lipid medication use was 17% by visit 4, partway into the statin drug era, and 76% of those medications were statins. After adjustment for age, race, and study center, men who used lipid medications were statistically significantly less likely to die from prostate cancer than men who did not use lipid medications (HR=0.56, 95% CI=0.33-0.95). This finding was slightly attenuated after further mutlivariable adjustment (HR=0.62, 0.36-1.08). The statistically significant inverse association appeared to be restricted to men who used lipid medications for ≥10 years (vs. never use: HR=0.39, 95% CI=0.19-0.82, p-trend=0.02). Lipid medication use was inversely associated with a reduced risk of prostate cancer death in both white and black men. Conclusions: Use of lipid medications was associated with a lower risk of fatal prostate cancer in both black and white men. Whether the fact that black men are less likely to receive/take lipid medications could partly explain the black-white disparity in prostate cancer in the US requires further study.

Support: NHLBI contracts, NCI grant, NPCR

#3010

Broad reduction in cancer incidence in patients treated with warfarin: a prospective cohort study.

Gry S. Haaland,1 Ragnhild S. Falk,2 Oddbjørn Straume,1 James B. Lorens1. 1 _University of Bergen, Bergen, Norway;_ 2 _University of Oslo, Oslo, Norway_.

BACKGROUND

Evidence shows that the vitamin K-antagonist warfarin, a popular anti-coagulant in clinical use for decades, has anti-tumor activity. This is recently attributed to disruption of post-translational modification of Gas6, the common ligand of the Axl receptor tyrosine kinase family. Gas6-Axl signaling is associated with malignancy and is required for tumoriogenesis and progression in several preclinical cancer models. Warfarin is shown to inhibit Axl signaling-dependent malignant traits and enhance anti-tumor immune responses at doses that do not achieve anticoagulation. The objective of this study is to investigate the association between warfarin use and cancer incidence in a large unselected Norwegian cohort. Our results reveal a remarkable reduction in cancer incidence associated with warfarin use across a wide range of tumor types.

METHODS

A cohort selected from the Norwegian population registry included all persons born between 1924-1954, living in Norway 2006-2012 (n=1 256 725). We cross-referenced this cohort using the unique Norwegian National ID Number to: 1) the Cancer Registry of Norway and retrieved information on all cancer cases 2006-2012; 2) information on filled warfarin prescriptions (ATC: B01AA03) from the Norwegian Prescriptions Database (2004-2012). Warfarin-use was defined > 6 months and minimum 2 years between first warfarin prescription and cancer diagnosis. We also performed a subgroup analysis on persons prescribed warfarin for atrial fibrillation/flutter (n=33 313) compared to non-users. Mantel-Haenzel method was used to calculate the incidence rate ratio (IRR), adjusting for sex and age.

RESULTS

In this cohort, 92 942 persons were classified as warfarin users, and we observed 132 687 cancer cases in the 7-year study period. We observed a significantly lower sex and age-adjusted risk for cancer development across all malignancy types in the warfarin user group compared to the non-user group (IRR: 0.842, 95% CI, 0.824-0.861). The association was similar among many cancers including major types (prostate IRR: 0.687,95% CI 0.653-0.722; lung IRR: 0.801, 95% CI 0.749-0.856; breast IRR: 0.903, 95% CI 0.817-0.998). Given the expected confounding effect of thrombotic disease on cancer incidence, we conducted a subgroup analysis among patients prescribed warfarin for atrial fibrillation/flutter (AF-group), a subgroup with reduced comorbidity. Warfarin users in the AF-group showed a stronger overall cancer risk reduction (IRR: 0.619, 95%CI 0.592-0.646), including all major cancer types, particular lung cancer. (Prostate, IRR: 0.604, 95%CI 0.552-0.662; Lung IRR: 0.391, 95%CI 0.332-0.460; Breast IRR: 0.720, 95%CI 0.594-0.871)

CONCLUSION

We show that warfarin use is associated with a broad cancer protective effect in a large unselected patient cohort. Subgroup analysis of arrhythmia patients to reduce confounders reinforced the notion that warfarin exerts important anti-tumor effects.

#3011

Dietary fat, fatty acids, and ovarian cancer risk: Preliminary findings from the Shanghai Women's Health Study.

Eftitan Y. Akam,1 Harvey J. Murff,2 Yong-Bing Xiang,3 Nikhil K. Khankari,1 Hui Cai,1 Xiao O. Shu,2 Wei Zheng,2 Alicia Beeghly-Fadiel2. 1 _Division of Epidemiology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN;_ 2 _Division of Epidemiology, Department of Medicine and Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN;_ 3 _Department of Epidemiology, Shanghai Cancer Institute, Shanghai, China_.

Introduction: Dietary fat is an essential nutrient for human growth and development, but epidemiological studies have linked excess fat to the development of multiple malignancies. The few existing studies on ovarian cancer risk are inconsistent, possibly due to differences in sources and subtypes of fat. For example, omega-3 (n-3) polyunsaturated fatty acids (PUFAs) are thought to be antineoplastic, while omega-6 (n-6) PUFAs may promote carcinogenesis.

Approach: We evaluated dietary fat in relation to ovarian cancer risk in the prospective Shanghai Women's Health Study (SWHS). Intake levels were determined from food frequency questionnaires (FFQ) administered at baseline and during follow-up; specific nutrient components were based on the sum of specific foods multiplied by nutrient content from the Chinese Food Composition Table. Cox proportional hazards regression was used to estimate relative risks (RR) and 95% confidence intervals (CI) for dietary fat and fatty acid components categorized into quintiles. Minimal models included age and residual adjustment for total energy intake; fully adjusted models also included education, years of menstruation, years of oral contraceptive use, parity, years of breastfeeding, family history of ovarian cancer, smoking, drinking, NSAID use, BMI, and physical activity.

Results: Among 73,144 SWHS participants, we identified a total of 219 ovarian cancer cases in 1,114,828 person-years of follow-up. We found significant dose-response relationships between increasing total PUFA (P-trend=0.032) and n-6 PUFA (P-trend=0.022) intakes and decreased ovarian cancer risk. In both minimally and fully adjusted models, women with the highest quintile of total dietary PUFAs were approximately 35% less likely to develop ovarian cancer (RR=0.65, 95% CI: 0.43-1.00), and women with the highest n-6 PUFA intake had a nearly 40% lower risk (RR=0.61, 95% CI: 0.40-0.93). Total fat, monounsaturated fat, and n-3 PUFA intake were not significantly related to ovarian cancer risk.

Conclusions: Findings from our preliminary analysis of a large prospective cohort of Chinese women suggest that total PUFA intake may be protective of ovarian cancer, and that this association may be driven by n-6 rather than n-3 PUFAs. Further evaluation, including stratification, sensitivity analysis, and evaluation of source of dietary fatty acids, is currently underway.

#3012

Long-term aspirin use and total and cancer-specific mortality.

Yin Cao,1 Meir Stampfer,2 Walter Willett,2 Donna Spiegelman,2 JoAnn Manson,3 Eric Rimm,2 Molin Wang,2 Shuji Ogino,3 Charles Fuchs,4 Edward Giovannucci,2 Andrew Chan1. 1 _Massachusetts General Hospital and Harvard Medical School, Boston, MA;_ 2 _Harvard T.H. Chan School of Public Health, Boston, MA;_ 3 _Brigham and Women's Hospital and Harvard Medical School, Boston, MA;_ 4 _Dana Farber Cancer Institute, Boston, MA_.

This abstract has been withheld from publication due to its inclusion in the AACR Annual Meeting 2017 Official Press Program. It will be posted online following its presentation.

#3013

Duration of oral contraceptive use and the prevention of gynecologic cancers: Modification by modifiable factors.

Kara A. Michels, Louise A. Brinton, Ruth M. Pfeiffer, Britton Trabert. _Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Bethesda, MD_.

Background: Although oral contraceptive (OC) use is ubiquitous in the United States, the influence of OCs on the development of cancers is not fully understood. In 2013, an Agency for Healthcare Research and Quality Evidence Report identified several data gaps with regard to OC use and its associations with cancer, including a need to understand if associations are consistent across subpopulations of users, including smokers and obese women. Methods: To address this data gap, we used Cox proportional hazards models to examine duration of OC use (never/less than 1 year [reference], 1 to 4, 5 to 9, 10+ years) and subsequent development of incident ovarian or endometrial cancer among participants in the NIH-AARP Diet and Health Study. We further examined effect modification by a variety of modifiable risk factors, including body mass index (BMI), cigarette smoking, alcohol consumption, and physical activity, via interaction terms and a likelihood ratio test. Models were adjusted for age, race, age at menarche and the modifiers of interest. Results: For ovarian cancer (n=1,241 cases, 149,502 non-cases), a reduction in risk was conferred by OC use that linearly decreased with duration of use (hazard ratios [HR] ranging from 0.83 to 0.60 across categories of duration). We also identified linear, inverse associations between increasing duration of OC use and ovarian cancer risk across most potential modifiers. Some of the most pronounced reductions were observed among long-term OC users (10+ years) who were current smokers (HR compared to non-users of 0.44, 95% confidence interval [CI] 0.20, 0.97) or who engaged in moderate physical activity (0.44, CI 0.23, 0.85). For endometrial cancer (n=2,337 cases, n=112,132 non-cases), we observed pronounced reductions in risk associated with long-term OC use among women with obese BMIs (0.36, CI 0.25, 0.52) and those who exercised rarely (0.40, CI 0.29, 0.56) or moderately (0.50, CI 0.33, 0.75). However, long-term use was not associated with risk reductions among women with BMIs of less than 25 or among women who exercised at least three times per week. Conclusions: OC use is consistently associated with reduced risks of ovarian cancer across most strata of modifiable risk factors. For endometrial cancer risk, OC use seems to offer the greatest risk reduction among women in previously identified high-risk categories (i.e., overweight/obese BMI, physically inactive), findings which merit further discussion as these women may be more likely to have comorbid conditions that would contraindicate OC use (e.g., hypertension, diabetes).

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

### Identification of Molecular Targets

#3014

**BCL3 over-expression contributes an** in vivo **growth advantage in a B-cell lymphoma xenograft model and is a risk factor for ibrutinib relapse in CLL.**

Timothy L. Chen, Bonnie K. Harrington, Larry Beaver, Amy S. Ruppert, Nyla A. Heerema, Xiaoli Zhang, Hatice Gulcin Ozer, Amy J. Johnson, Jennifer A. Woyach, Erin Hertlein, John C. Byrd. _The Ohio State University, Columbus, OH_.

Chronic lymphocytic leukemia (CLL) relies on chronic B-cell receptor (BCR) signaling, and as such is effectively treated with Bruton's tyrosine kinase (BTK) inhibitors. However patients who relapse on BTK inhibitors such as ibrutinib have an extremely poor prognosis without additional intervention. Therefore, identifying and characterizing risk factors that predict relapse to BTK inhibitors is important. A series of 308 CLL patients have been enrolled on various ibrutinib clinical trials at the Ohio State University. Seven of these patients were identified to carry the translocation t(14;19), which leads to over-expression of B-cell leukemia 3 (BCL3), Of these 7, 6 (85%) have relapsed on ibrutinib while only 27% of patients (83/308) overall have progressed. BCL3 is known to regulate NF-κB transcription and influence B-cell function, and we found that overall BCL3 expression is increased in CLL compared to normal B-cells, therefore hypothesized that BCL3 may provide a competitive advantage to promote ibrutinib resistance. In order to test the role of BCL3 in ibrutinib resistance using an in vitro system, we overexpressed BCL3 or an empty vector (EV) control in ibrutinib responsive B-cell lines (BCL3 was at least 2-fold over-expressed relative to endogenous BCL3). Cells were treated with vehicle or 1uM ibrutinib for 1 hour followed by a washout, and proliferation and viability were evaluated at various time points. Ibrutinib inhibited cell proliferation and induced apoptosis, however BCL3 expression did not abrogate these effects. However, while examining BCR signaling proteins we found that BCL3 expression enhanced phosphorylation of BTK, suggesting that BCL3 stabilizes activated BTK to circumvent ibrutinib treatment. We next engrafted these EV or BCL3 over-expressing cells into the left or right flank, respectively, of immune-compromised (NSG) mice. Mice were sacrificed when either tumor reached a volume of 2000cm3, and the tumors were isolated and weighed. Tumors which developed from the BCL3 over-expressing cells at sacrifice were larger than those which developed from the EV cells by an average of 1332g. We also evaluated a disseminated tumor model where HBL-1 EV or BCL3 over-expressing cells were injected via the tail vein. Disease was monitored by weekly peripheral blood flow cytometry for human CD19. We saw that 6 of 11 mice intravenously engrafted with HBL-1 BCL3 present with peripheral disease when the mice meet removal criteria, whereas only 1 of 11 mice engrafted with HBL-1 EV cells developed peripheral disease. We conclude that the presence of t(14;19) indicates a higher risk of relapse in patients undergoing ibrutinib therapy, and these patients should be closely monitored for evidence of progression. Our studies also suggest that BCL3 promotes more aggressive disease in a lymphoma xenograft model, and may be involved in tumor migration in vivo.

#3015

Selective inhibition of mutant KRAS cell and tumor growth by PHT-7.3, an inhibitor of the KRas signaling nanocluster protein Cnk1.

Roisin Delaney,1 Marco Maruggi,1 Martin Indarte,2 Robert Lemos,1 Geoff Grandjean,3 Lynn Kirkpatrick,2 Garth Powis1. 1 _Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA;_ 2 _PHusis Therapeutics Inc., La Jolla, CA;_ 3 _Sanford Burnham Prebys Medical Discoavery Institute, La Jolla, CA_.

Activating mutations of KRas is the most common proto-oncogenic event in human cancer but there remains no effective therapy for patients harboring mutated KRas (mut-KRas). Despite intense efforts, tight nucleotide binding, few defined pockets, and redundant localization signals have impeded the development of compounds that bind or inhibit KRas. We have identified connector enhancer of kinase suppressor of Ras 1 (Cnk1) as a critical mediator for growth driven by mut-KRas in human cancer cells. Cnk1 co-localizes with mutant KRas at the membrane and deletion of Cnk1 abrogates KRas activation and the activation of the Ras effectors Ral and Rho. Cnk1 deletion caused cells with mutant KRas to accumulate at the G1 checkpoint similar to selective deletion of mutant KRas itself. Following a screen and initial structural optimization a small molecule probe compound PHT-7.3 was identified and shown to bind selectively to the pleckstrin homology PH domain of Cnk1 preventing Cnk1 and mut-KRas co-localization. PHT-7.3 inhibited mut-KRas but not wt-KRas non small cell lung cancer (nsclc) cell growth, and selectively blocks mut-KRas downstream signaling in cells. PHT-7.3 exhibited cytostatic antitumor activity in the mut-KRas(G12S) A549 and mut-KRas(G12V) H441 nsclc xenografts, but not in the wt-KRas H1975 nsclc xenograft. Mut-KRas downstream signaling was inhibited by PHT-7.3 in the xenografts with downregulation of activated Rho and Ral signaling. PHT-7.3 showed further increased antitumor activity in A549 xenografts in combination with erlotinib or trametinib. Thus, the work identifies the PH domain of Cnk1 as a druggable target whose inhibition selectively blocks mutant-KRas activation, and PHT-7.3 as a lead agent in the development of therapies for KRas tumors.

#3016

Identification of protein arginine methyltransferase 1 as novel epigenetic vulnerability in KRAS/p53 mutant PDAC primary patient models.

Virginia Giuliani, Bhavatarini Vangamudi, Erika Suzuki, Meredith Miller, Chiu-Yi Liu, Alessandro Carugo, Christopher Bristow, Guang Gao, Jing Han, Yuting Sun, Ningping Feng, Edward Chang, Joseph Marszalek, Jeffrey Kovacs, Maria Emilia Di Francesco, Carlo Toniatti, Timothy Heffernan, Philip Jones, Giulio Draetta. _UT MD Anderson Cancer Ctr., Houston, TX_.

Pancreatic ductal adenocarcinoma (PDAC) is a rapidly progressing disease associated with less than 10% 5-year survival rate. Various treatment regimens failed to improve survival of PDAC patients, thus a critical need exists to identify druggable targets essential for tumor maintenance. We developed a powerful in vivo platform that enables the identification of new molecular drivers in the PDAC context where activating mutation of KRAS gene and loss of p53 dominate the genetic landscape. Through an in vivo loss of function screen performed in KRAS/p53 mutant PDAC primary patient models, we identify protein arginine methyltransferase 1 (PRMT1) as top scoring hit. This novel dependency in PDAC was subsequently validated in multiple PDAC models using both shRNA mediated as well as CRISPR base genetic inhibition and we demonstrated that PRMT1 knockdown induces a significant growth inhibition in vitro. Methylation of arginine 3 on histone H4 (H4R3me2a) as well as global arginine methylation was also evaluated and showed a dramatic reduction upon PRMT1 knockdown, correlating observed phenotype with target engagement. To further confirm a role for PRMT1 in tumor maintenance, we developed inducible PRMT1 knockdown in a primary patient model and showed a dramatic tumor growth inhibition (TGI) in vivo upon PRMT1 knockdown. PRMT1 is the primary enzyme responsible for arginine asymmetric demethylation, however other members of the Type I family are also involved in this process and we evaluated the role of protein arginine methyltransferase 4 (PRMT4) and 6 (PRMT6) in our workhorse model. Surprisingly, no significant phenotypic response was observed upon genetic inhibition of PRMT4 or PRMT6 suggesting no redundancy between different PRMT type I and a unique dependency on PRMT1. To strengthen and complement the genetic validation, we leveraged a PRMT Type I inhibitor and confirmed in vitro results as well as in vivo efficacy at tolerated doses (xenograft vs allograft). Key models have been prioritized in order to inform on PRMT1 dependency and to refine responder population. Our research has identified and validated for the first time an arginine methyltransferase as a novel genetic vulnerability in PDAC and strongly suggest PRMT1 as a new therapeutic opportunity in PDAC cancers.

#3017

Inhibition of YAP/TAZ-dependent transforming properties in LATS1/2 deleted RPE1 cells by MAPKAPK5 targeting.

Min Hwan Kim, Joon Kim. _Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea_.

Purpose: Recent evidence suggests the Hippo pathway effectors YAP/TAZ play important roles in tumor pathogenesis, including tumorigenesis, metastasis, and drug resistance. Loss of LATS1/2, the negative regulators of YAP/TAZ, has been reported in various human tumors, and suggested to activate YAP/TAZ in tumorigenic process. However, the molecular consequence of LATS1/2 genetic loss in benign human cells has not been addressed, and therapeutic targets for tumors with LATS1/2 loss have not been explored. We aimed to investigate the transforming potential of LATS1/2 loss in human RPE1 cells, and to find targetable kinases that support YAP/TAZ activation in tumors with LATS1/2 loss

Method: To examine the effect of LATS1/2 loss, both LATS1 and LATS2 alleles were deleted by CRISPR-Cas9 genome editing in RPE1-hTERT cells. YAP/TAZ activity of LATS1/2-null RPE1 cells was measured by immunofluorescence (IF) staining and qRT-PCR. Transforming properties of LATS1/2 loss were monitored on both 2-dimensional (2D) and 3D culture, and the centrosome number and DNA contents of cells were determined by IF and propidium iodide staining. LATS1/2-null RPE1 cells were subjected to an image-based kinome-wide siRNA library screening for identifying YAP/TAZ downregulating hits.

Results: The LATS1/2-null RPE1 cells showed persistent nuclear localization and transcriptional activation of YAP/TAZ. In contrast to wild-type RPE1 cells, the LATS1/2-null RPE1 cells showed high proliferation rate, loss of contact inhibition, and sphere formation capacity in matrigel 3D culture, demonstrating transforming activity of LATS1/2 loss. Moreover, LATS1/2 knockout caused genomic instability and centrosome overduplication in RPE1 cells that might be related to defects in mitotic checkpoint regulation. Next, we performed an image-based siRNA library screening to find potential therapeutic targets for tumors with LATS1/2 loss. We found that the inhibition of a p38 MAPK pathway component, MAPKAPK5, suppresses YAP/TAZ nuclear localization and activation in LATS1/2-null RPE1 cells. MAPKAPK5 physically interacted with YAP, and RNAi-mediated MAPKAPK5 inhibition decreased YAP/TAZ protein level and transcriptional activity. MAPKAPK5 depletion resulted in suppression of LATS1/2-null RPE1 cell proliferation on 2D culture as well as sphere formation capacity on 3D culture. Conversely, constitutively active MAPKAPK5 mutant expression in RPE1 cells induced YAP/TAZ nuclear enrichment.

Conclusion: These results suggest that LATS1/2 genetic loss drives transforming properties in human RPE1 cells, and MAPKAPK5 inhibition suppresses YAP/TAZ activation induced by LATS1/2 loss. We suggest that MAPKAPK5 is a novel positive regulator of YAP/TAZ and may serve as a therapeutic target for YAP/TAZ-driven tumors.

#3018

RET rearrangements as promising therapeutic targets in breast cancer.

Bhavna S. Paratala,1 Jeffrey S. Ross,2 Casey B. Williams,3 Whitney Petrosky,1 Kirstin A. Williams,3 Jon Chung,2 Sonia C. Dolfi,1 Shridar Ganesan,1 Siraj Ali,2 Brian Leyland-Jones,3 Kim M. Hirshfield1. 1 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ;_ 2 _Foundation Medicine, Cambridge, MA;_ 3 _Avera Cancer Institute Center for Precision Oncology, Sioux Falls, SD_.

Background: Receptor tyrosine kinase alterations have played a significant role in therapeutic decisions for cancer due to their oncogenic nature and response to targeted small molecule kinase inhibitors. Increased genomic profiling of tumors using hybrid-capture based next-generation sequencing approaches now reveal the presence of previously unknown fusions and alterations involving kinases in a diverse set of cancers. Here we report the presence and therapeutic significance of recurrent and novel fusions involving RET, a known oncogenic tyrosine kinase receptor, in breast cancer.

Methods: Comprehensive genomic profiling on formalin-fixed, paraffin embedded patient tumor tissues was performed using FoundationOne platform that covers the entire coding region for 315 cancer-related genes and introns of 28 genes involved in rearrangements at a depth of 500-1000X (Foundation Medicine, MA). Out of 23 rearrangements, two representative RET fusion expression vectors were synthesized and expressed in non-tumorigenic cell lines (breast MCF10A and mouse 3T3 fibroblasts) and were evaluated for RET kinase signaling, drug response, and tumorigenicity.

Results: RET gene fusions, the canonical NCOA4-RET and a novel, noncanonical RASGEF1A-RET fusion, were identified in two separate breast cancers and both include exons required to retain the intact kinase domain of Ret. The novel RASGEF1A-RET fusion includes the non-coding region of RASGEF1A potentially resulting in a truncated RET protein using an alternate internal start site in exon 11 of RET. In vitro characterization of both fusions expressed in mouse 3T3 and human MCF10a cell lines revealed constitutive kinase activation and subsequent downstream signaling as evidenced by phosphorylation of Ret, Erk and Akt. This is the first reported noncanonical RET rearrangement resulting in a 5' truncated but functional RET kinase. Non-tumorigenic cell lines with stable expression of either rearrangement showed transformed phenotypes assessed by changes in morphology, enhanced growth rate, colony forming ability, and tumor formation in mice. RET fusion-transformed cells were exquisitely sensitive to treatment with RET inhibitors when evaluated in both short-term and long-term functional assays. NCOA4-RET was found by CGP in an index case of metastatic ER+/HER2+ breast cancer that had radiographic evidence of disease progression while on trastuzumab, pertuzumab, and anastrazole. Subsequent treatment with cabozantinib plus anastrazole led to a rapid clinical and radiographic response.

Conclusion: CGP techniques involving hybrid-capture based approaches can identify previously unreported but recurrent RET gene fusions in breast cancer. Here, we show that RET fusions including both canonical and non-canonical complex rearrangements are functional and may represent promising therapeutic targets in selected breast cancer patients.

#3019

The role of TACC3 in the progression from ductal carcinoma in situ to invasive breast cancer.

Loredana Campo, Maya Mathew, Eun-Kyoung breuer, William Small. _Loyola University Medical Center, Maywood, IL_.

Ductal carcinoma in situ (DCIS) is a non-invasive breast cancer with the potential to become invasive/metastatic. The development of DCIS to invasive breast cancer is an important aspect of breast tumor aggressiveness, however, little is known about the genetic and molecular alterations involved in this progression. Also, analysis of molecular markers and genetic signatures of DCIS and invasive breast cancer have failed to identify transition-specific biomarkers or pathways. Hence, identifying the molecular mechanisms by which DCIS progresses to invasive breast cancer is critical for the success of diagnosis, prognosis and therapy of breast cancer. Members of the transforming acidic coiled-coil (TACC) protein family are key players in the maintenance of centrosome integrity, the regulation of microtubule assembly, and spindle stability during mitosis. TACC3 has been shown to be involved in the regulation of centrosome-microtubule dynamic networks, cell growth and differentiation, and transcription/gene expression through interactions with other molecules. Mounting evidence indicates that high levels of TACC3 may contribute to breast cancer development. Here, we investigated the significance of TACC3 in the transition of DCIS to invasive breast cancer. First, we examined the expression of TACC3 in non-tumorigenic human mammary epithelial MCF10A cells and MCF10DCIS.COM cells, which are known to form DCIS-like lesions in mice and to progress to invasive cancer, and found that the expression of TACC3 is elevated in MCF10DCIS.COM cells compared to MCF10A cells. By analyzing tissue microarray data, we found that expression of TACC3 gradually increased from normal breast to DCIS, and further increased in invasive breast cancer. In addition, ectopic expression of TACC3 in MCF10DCIS.COM cells promoted cell proliferation, migratory and invasive capabilities as well as the epithelial-mesenchymal transition (EMT)-like phenotype. Moreover, MCF10DCIS.COM cells stably expressing TACC3 exhibited increased expression of stemness markers. Interestingly, a small molecule inhibitor of TACC3, KHS101 treatment in MCF10DCIS.COM cells significantly suppressed cell proliferation and the PI3K/AKT signaling pathway while having little effect on MCF10A cells. Our findings, therefore, suggest that TACC3 may play an important role in the progression from DCIS to invasive breast cancer and that targeting TACC3 could be a promising new therapeutic strategy for DCIS patients.

#3020

Novel therapeutic targets in head and neck cancer.

Maria Kondratyev,1 Aleksandra Pesic,1 Troy Ketela,1 Azin Sayad,1 Stephano Marastoni,1 Carl Virtanen,1 Laurie Ailles,1 Soroush Samadian,1 Mikhail Bashkurov,2 Marianne Koritzinsky,3 Brad Wouters3. 1 _UHN, Oakville, Ontario, Canada;_ 2 _Lunanfeld, Toronto, Ontario, Canada;_ 3 _UHN, Toronto, Ontario, Canada_.

HNSCC is 6th most common malignancy in the world. Despite advances in diagnosis and treatment, the survival rates remain low due to frequent recurrences the biology of which remains unclear. Using functional genomic technologies, we identified new therapeutic targets for metastatic disease in HNSCC. Whole genome shRNA screens were conducted in matched sets of cell lines derived from primary tumors and respective metastatic sites, identifying genes essential for cell survival only following metastasis. To test if knockdown of selected targets inhibits metastasis in a therapeutic setting, we established orthotropic model of HNSCC that metastasize to lymph nodes in the mouse. Components of Notch pathway were identified as essential for survival of cells derived from metastatic sites. Whole exome sequencing identified a novel mutation in one of the EGF domains of Notch3 that was acquired only in the metastatic line. Utilizing CRISPR methodology, we established that "fixing" the mutation results in reversal of metastatic phenotype of the cells, making them Notch independent similar to their primary tumor counterparts. Mutations in EGF domains have been reported to influence interaction with specific ligands, dictating which ligand can activate Notch signaling. Our data indicate that a distinct set of target genes is induced upon interaction between Notch3 and Jag2 ligand. Furthermore, our results indicate that suppression of Notch3 improves survival in mice bearing orthotropic tumors derived from the metastatic HNSCC lines. Overall, our data demonstrate that metastatic cells from head and neck tumors acquire dependency on Notch3 signaling. Novel treatments targeting components of this pathway may prove effective in targeting metastatic cells alone or in combination with conventional therapies.

## IMMUNOLOGY:

### Adaptive Immunity in the Tumor Microenvironment

#3021

CCR5 inhibition: macrophage repolarization therapy for colorectal cancer.

Niels Halama,1 Inka Zoernig,1 Anna Berthel,1 Christoph Kahlert,2 Fee Klupp,3 Meggy Suarez-Carmona,1 Karsten Brand,3 Juergen Krauss,1 Felix Lasitschka,3 Alexis Ulrich,3 Juergen Weitz,2 Martin Schneider,3 Markus Buechler,3 Laurence Zitvogel,4 Thomas Herrmann,5 Axel Benner,6 Christina Kunz,6 Stephan Luecke,6 Christoph Springfeld,1 Christine S. Falk,7 Dirk Jaeger1. 1 _National Ctr. for Tumor Diseases, Heidelberg, Germany;_ 2 _University Hospital Dresden, Dresden, Germany;_ 3 _University Hospital Heidelberg, Heidelberg, Germany;_ 4 _Institute Gustave Roussy, Paris, France;_ 5 _Medizinische Klinik Idar-Oberstein, Idar-Oberstein, Germany;_ 6 _German Cancer Research Center, Heidelberg, Germany;_ 7 _Hannover Medical School, Hannover, Germany_.

In patients with metastatic colorectal cancer (CRC), the local immune response influences the clinical course. An in-depth analysis of the invasive margin of human CRC liver metastases revealed a distinct immunological microenvironment. Within this microenvironment, two distinct subsets of myeloid cells induce an influx of T cells into the invasive margin via CXCL9/CXCL10. CCL5 is produced by these T cells and stimulates pro-tumoral effects via CCR5, creating an exploitive loop. CCR5 was found on macrophages, lymphocytes and on the vast majority of tumor cells. Inhibition of CCR5 in patient-derived functional in vitro organotypic culture models showed a promising macrophage repolarization with anti-tumoral effects. These effects are mediated by activation of an antiviral program in macrophages, leading to interferon and reactive oxygen species production and subsequent selective tumor cell death. These anti-tumoral effects were confirmed in a phase I trial with a CCR5 antagonist in 14 patients with liver metastases of advanced refractory CRC. Treatment with the oral CCR5 Inhibitor was very well tolerated and objective responses were seen, especially in combination with previously ineffective chemotherapy. Biopsies revealed mitigation of tumor-promoting inflammation within the tumor tissue, confirming the validity of the explant model and highlighting the feasbility of this approach. It furthermore shows the proof-of-concept for macrophage repolarization in cancer patients.

#3022

Novel hydrocarbon stapled peptide inhibitors suppresses β-catenin dependent cancer growth and immunomodulates colorectal tumors.

David Zhu,1 Joy Jin2. 1 _WntRx/Lab Central, Boston, MA;_ 2 _Harvard University, Boston, MA_.

Mutations in several components of the canonical Wnt pathway function as drivers in a range of common solid tumor cancers, including about 90% of sporadic colorectal cancers (CRCs), the third most common cancer in the US. In particular, activation of β-catenin (β-cat), a key Wnt signaling component, can initiate tumorigenesis in the intestine and promote cancer stem cell metastasis6,7. As such, targeting the Wnt pathway is an attractive approach to the development of cancer therapeutics. However, Wnt inhibitors tested to date, including those that disrupt β-cat activity, have either had limited efficacy or unacceptable side effect profiles1. Here, we developed a potent and robust, selective lead compound targeting oncogenic Wnt activity by disrupting the highly specific interaction of β-cat with its co-activators BCL9/B9L, which has been shown to abrogate transactivation. By using hydrocarbon stapled peptide technology and an acutely sensitive high throughput biochemical screening assay, we have identified several promising peptides that possess a strong pharmacokinetic profile. These peptides (i) exhibit robust activity in mouse models; (ii) inhibit cancer cell proliferation, metastasis, and stem cell progression; and (iii) modulate the immune microenvironment, demonstrating the feasibility of such peptide development and selection. Synergy is demonstrated between the stapled peptides and anti-PD1 antibodies, which has significant implications for combination therapy with commercial immune checkpoint blockers that have become standard of care for second-line melanoma treatment. These peptides represent a promising new potential therapeutic class for patients with CRC, for which traditional chemotherapy and targeted inhibitors have had limited therapeutic success.

#3023

The antitumor activity of immunotoxins is enhanced by tofacitinib.

Nathan Simon,1 Antonella Antignani,1 Stephen Hewitt,1 Massimo Gadina,2 Masanori Onda,1 Christine Alewine,1 David Joseph Fitzgerald1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _National Institute of Arthritis and Musculoskeletal and Skin Disease, Bethesda, MD_.

Treatment outcomes of certain aggressive cancers have not improved significantly in the last decade, necessitating development of new therapies. Recombinant immunotoxins (IT) are fusion proteins consisting of the enzymatic domain of a cytotoxin linked to an antibody fragment targeting a cellular surface protein. While the combination of selective targeting and potent cell-killing make ITs well suited for targeted cancer therapy, clinical experience has indicated that patients can generate anti-toxin neutralizing antibodies after one cycle of treatment. Mice treated with the FDA-approved JAK inhibitor, tofacitinib, produced significantly lower anti-toxin antibody titers, suggesting that tofacitinib could be used in combination with immunotoxins to improve therapeutic efficacy. Accordingly, the combination of tofacitinib and IT was tested both in vitro and in vivo using two models of aggressive human cancers: pancreatic ductal adenocarcinoma and triple-negative breast cancer. In vitro combination studies examined the effects on oncogenic signaling pathways and looked for enhancement of cytotoxic potency. Results demonstrated some tofacitinib-mediated inhibition of key signaling pathways but no enhancement of immunotoxin killing. By contrast, when xenograft tumor models were challenged, immunotoxin activity was enhanced greatly by co-treating with tofacitinib. Tofacinib treatments alone did not demonstrate any antitumor activity. The combination of tofacitinib and immunotoxin decreased tumor growth and extended survival compared to either treatment alone. Examination of post-treatment tumor samples and sera from mice indicated that JAK inhibition resulted in alterations of the tumor microenvironment, including a decrease in cancer-promoting cytokine levels and reductions in recruitment of suppressive immune cells to tumor tissue. This study demonstrates that the inhibition of the JAK-STAT pathway can significantly enhance the ability of cytotoxin-based therapeutic agents to elicit anti-tumor effects through alteration of the tumor microenvironment.

#3024

**CD4** + **outperform CD8** + **central memory-derived CAR T cells, mediating persistent antitumor responses and long-term eradication of glioblastoma.**

Dongrui Wang, Renate Starr, Brenda Aguilar, Alfonso Brito, Brenda Chang, Aniee Sarkissian, Behnam Badie, Michael E. Barish, Stephen J. Forman, Christine E. Brown. _City of Hope, Duarte, CA_.

Purpose: To compare antitumor effector function mediated by CD4+ and CD8+ chimeric antigen receptor (CAR)-T cells targeting IL13 receptor α2 (IL13Rα2) for treatment of glioblastoma (GBM).

Experimental Design: CD4+ and CD8+ IL13Rα2-specific CAR-T cells, derived from enriched central memory T cells (Tcm), were evaluated for their antitumor potential both in vitro by recursive challenge with GBM cells and in vivo in established orthotopic GBM xenograft models. Further, GBM-stimulated CD4+ and CD8+ CAR-T cells were examined for their exhaustion- and memory-associated phenotypes at transcriptional and protein levels, which would account for their differential antitumor activity.

Results: While CD8+ CAR-T cells mediated robust short-term cytolytic effector function, they became rapidly exhausted and lost antitumor potential after repeated stimulation with tumor cells. CD4+ CAR-T cells, by contrast, persisted and retained effector potency even after repetitive tumor challenge. CD4-mediated cytotoxicity acted through the granzyme B/perforin pathway and was independent of the CD8+ cells. Activated CD4+ CAR-T cells augmented the proliferation of CD8+ cells, but failed to protect the CD8+ cells against stimulation-induced exhaustion. In NSG mice bearing GBM xenografts, CD4+ CAR-T cell treatment led to long-term tumor-free survival, while CD8+ CAR-T cell treated mice showed short-term tumor regression followed by antigen-positive relapse and associated T cell exhaustion. Upon GBM stimulation, CD4+ cells expressed less co-inhibitory receptors and more memory-associated markers than the CD8+ T cell subset. Further, a comprehensive transcriptional analysis revealed that when activated, CD4+ cells retained expression of genes involved in survival- and self-renewal-related pathways, indicating a less exhausted status and a better functional persistence in comparison to the CD8+ cells.

Conclusion: We observed a superior anti-GBM activity mediated by CD4+ CAR-T cells over the CD8+ subset, featured by their ability to maintain long-term immune response and reduce stimulation-induced exhaustion. It is thus inferred that the frequency of CD4+ cells could be an important criteria for evaluating the efficacy of CAR-T cells as immunotherapeutic products.

#3025

Preclinical characterization of a novel monoclonal antibody targeting a neo-antigen expressed in ovarian and GI malignancies.

Kristen Zeligs,1 Philip M. Arlen,2 Kwong Tsang,2 Lidia Hernandez,3 Massimo Fantini,2 Christina M. Annunziata3. 1 _Walter Reed National Military Medical Center, Bethesda, MD;_ 2 _Precision Biologics, Inc., Rockville, MD;_ 3 _National Cancer Institute, Bethesda, MD_.

Objectives: We have developed a novel humanized IgG1 monocolonal antibody, NEO-201, which targets a neoantigen expressed on ovarian and GI malignancies. This antigen has homology to CEACAM; however, it has specificity to tumor tissues and not normal tissues. Our aims were as follows: (1) to evaluate the expression of this antigen on tumor cell lines derived from various ovarian and GI cancers; and (2) to determine the functionality of NEO-201.

Methods: Expression of the antigen was identified by western blot analysis and flow cytometry. Functionality was determined by an antibody dependent cell-mediated cytotoxicity (ADCC) assay using the antigen-expressing cell lines identified by western blot analysis. Cell lines not expressing the antigen were used as negative controls. The effector cells used in the ADCC assay were natural killer (NK) cells isolated from PBMCs of normal donors and high-affinity activated NK (haNK) cells. haNK cells are NK-92 cells genetically engineered to express the high-affinity variant of human CD16 (FcgRIIIA-V158) and the human IL-2 gene. haNK cells have little or no inhibitory expression of killer cell immunoglobulin-like receptors, a unique feature that may optimize their highly cytotoxic activity against a broad range of malignancies.

Results: Using western blot analysis, 2 of 5 ovarian cancer cell lines (OV90 and PE01) and 1 of 2 colorectal cancer cell lines (LS174T) stained positively for NEO-201. Flow cytometry analysis on ovarian, colorectal, and pancreatic cancer cell lines confirmed the expression of the NEO-201 neoantigen on 1 of 2 ovarian cancer cell lines (OV90), 1 of 2 colorectal cancer cell lines (LS174T), and 2 of 2 pancreatic cancer cell lines (ASPC-1 and CFPAC-1). ADCC assay was performed using OV90 and ASPC-1 tumor cell lines. Cell lines not expressing the neoantigen were used as negative controls. Killing of tumor cells expressing the neoantigen was also observed with NEO-201 alone; however, the lysis was significantly augmented when NK cells from the PBMCs of normal donors or haNK cells were included in the ADCC assay.

Conclusions: These finding demonstrate that the NEO-201 neoantigen is expressed on several tumor cell lines representing ovarian and GI malignancies. NEO-201 can mediate ADCC activity in the presence of NK cells and NK cell lines. In addition, NEO-201 can induce apoptosis in tumor cell lines expressing the NEO-201 neoantigen. Furthermore, preliminary studies demonstrated that this neoantigen is expressed in numerous cancer types (not shown). In vivo studies as well as IND-related studies are planned.

#3026

Targeting GPX4 in tumor-associated stromal cells increases inflammatory-cell infiltration.

Shrikanta Chattopadhyay,1 Cherrie Huang,1 Ninib Baryawno,1 Nicolas Severe,1 Vasanthi Viswanathan,2 Carlotta Costa,1 David Scadden,1 Stuart Schreiber2. 1 _Massachusetts General Hospital, Boston, MA;_ 2 _Broad Institute, Cambridge, MA_.

The lack of a T-cell inflamed microenvironment in tumors limits responsiveness to many immunotherapies. T-cell exclusion is often mediated by a dense infiltration of fibroblast-like stromal cells. Up to 55% of triple-negative breast cancers have 'stroma-rich' tumors with markedly lower T-cell inflammation. Here we report a therapeutic strategy that can potentially convert stroma-rich tumors into T-cell inflamed tumors by forcing stromal cells to secrete 5-lipoxygenase products which are powerful chemo-attractants for T cells.

We were initially interested in identifying selective inhibitors of stromal-cell function. To achieve this, we used phenotype-based small-molecule screening in which the phenotype of stroma-induced cancer cell migration in vitro was a surrogate for stroma-induced metastasis in vivo. We identified a compound, RSL3 that inhibited this migration. RSL3 was selectively cytotoxic to stromal cells over cancer cells, in comparisons of immortalized cell lines as well as comparisons of patient-derived primary breast cancer cells to cancer-associated fibroblasts (CAFs). We therefore undertook studies to identify its mechanism of action.

RSL3 was recently reported to target the redox enzyme glutathione peroxidase 4 (GPX4). GPX4 metabolizes lipid peroxides so we performed metabolomic profiling of RSL3-treated stroma-cancer co-cultures and found elevated arachidonic acid products of lipoxygenase enzymes. Stromal cells were found to contain >10-fold higher levels of lipoxygenase products than carcinoma cells. Blocking either 5-lipoxygenase (5-LO) or 15-lipoxygenase (15-LO) with selective inhibitors abrogated RSL3's cytotoxicity to stromal cells. Thus, high lipoxygenase activity in stromal cells increases their susceptibility to GPX4 inhibition.

Because 5-LO products like leukotriene B4 are powerful chemo-attractants for myeloid cells and T cells, we studied the impact of GPX4 knockdown in vivo using xenografts of cancer cells co-injected with stromal cells. GPX4 knockdown resulted in a large increase in myeloid-cell infiltration into tumors but, surprisingly, T-cell infiltration was suppressed. We reasoned that 15-LO products are immunosuppressive based on recent findings that 15-LO gene amplifications are inversely correlated with T-cell infiltration in breast cancers in The Cancer Genome Atlas. Consistent with this, GPX4 inhibition of stroma-cancer co-cultures suppressed T-cell chemotaxis but combined inhibition of GPX4 and 15-LO significantly enhanced T-cell chemotaxis over untreated controls in vitro. We are undertaking in vivo testing of this combination.

In summary, our unbiased chemical biology approach has revealed a therapeutic strategy to promote T-cell inflammation. We envision this to be a priming strategy for stroma-rich cancers to become responsive to a variety of different immunotherapies thereby unleashing their full curative potential.

#3027

Dissecting mechanisms of anti-PD-1 therapy with massively parallel single-cell RNA-sequencing.

Brian C. Miller,1 Marc H. Wadsworth,2 Kevin Bi,1 Travis K. Hughes,2 Robert Manguso,1 Arlene H. Sharpe,3 Alex K. Shalek,2 Nicholas Haining1. 1 _Dana-Farber Cancer Inst., Boston, MA;_ 2 _Massachusetts Institute of Technology, Cambridge, MA;_ 3 _Harvard Medical School, Boston, MA_.

Anti-PD-1 therapy is an important new treatment option for many different types of malignancies, but overall response rates are less than 40%. Limited understanding of how anti-PD-1 treatment changes the tumor immune microenvironment is a barrier to identifying rational combination therapies and understanding mechanisms of immunotherapy resistance. To overcome this barrier, we set out to understand the mechanisms by which anti-PD-1 therapy augments the anti-tumor immune response using single-cell genomics. We have developed a massively parallel single-cell RNA-sequencing platform ("Seq-Well") that uses a fabricated chip with nearly 100,000 nanowells into which barcoded beads and individual cells are distributed prior to lysis and RNA capture. We used this platform to comprehensively define the global expression profile of all major immune lineages in the tumor microenvironment in a mouse tumor model of immunotherapy. Mice were implanted with the B16F10 melanoma transplantable model of cancer and treated with anti-PD-1 or control antibodies. Tumors were harvested and CD45+ tumor-infiltrating leukocytes isolated by FACS. In a single experiment we were able to sequence the transcriptomes of over 600 individual cells, allowing us to clearly distinguish different immune lineages within the tumor microenvironment. We detect two transcriptionally distinct populations of CD8+ T cells, one that is highly proliferative (as marked by Ki-67), and one that has higher expression of cytotoxic markers (i.e. perforin). The Ki-67hi population is enriched for a gene expression signature from terminally exhausted CD8+ T cells in chronic viral infection, suggesting that this is a more exhausted subset. Treatment with anti-PD-1 globally alters the tumor microenvironment, including enriching for CD8+ T cells in the Prfhi subpopulation compared with the Ki-67hi more terminally exhausted population. Studies to understand changes in the immune infiltrate of immunotherapy resistant tumors are currently ongoing. In conclusion, massively parallel single-cell RNA-sequencing allows us to dissect the mechanisms by which checkpoint blockade controls tumor growth, revealing shifts in the differentiation state of exhausted CD8+ T cells induced by checkpoint blockade.

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

### Breakthroughs in the Control, Function, and Drivers of Oncogenesis

#3028

Reactive oxygen species induced nuclear translocation of c-MET via INTERNET mechanism.

Mei-Kuang Chen,1 Yi Du,2 Yu-Yi Chu,3 Mien-Chie Hung2. 1 _The University of Texas Health Science Center at Houston, Graduate School of Biomedical Science, Houston, TX;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _National Cheng Kung University, Tainan, Taiwan_.

c-MET is one of the receptor tyrosine kinases (RTKs) that are overexpressed in malignant cancer types including breast cancer. c-MET is traditionally known for its role in signaling transduction from the cell surface, our recent publication provided evidence that c-MET can be translocated into the nucleus and phosphorylate PARP1 in response to reactive oxygen species (ROS) stimulation (Du et al. Nat Med 2016). Oxidative stress is a common phenomenon in cancer cells due to alteration of metabolism, and constitutive oxidative stress related to ROS has been observed in breast cancer cells. In this study, we demonstrated that hepatocyte growth factor (HGF) as well as hydrogen peroxide (H2O2) can induce retrograde translocation of c-MET holoreceptor via a membrane-bound vesicle transport mechanism and translocate into the nucleus through INTERNET (integral trafficking from the ER to the nuclear envelope transport pathway) (Wang et al. J Biol Chem 2012) in breast cancer cells. While c-MET nuclear transportation increases dramatically under H2O2 treatment, our findings provide a putative mechanism by which breast cancer cells adapt to oxidative stress and suggest that INTERNET might be a general nuclear translocation mechanism shared among RTKs.

#3029

An in vivo role for Argonaute 2 in KRAS driven pancreatic cancer.

Sunita Shankar, Jean Tien, Vijaya L. Dommeti, Sylvia Zelenka-Wang, Ingrid A. Apel, Jiaqi Shi, Chandan Kumar-Sinha, Arul M. Chinnaiyan. _University of Michigan, Ann Arbor, MI_.

Oncogenic mutations in RAS provide a compelling yet intractable therapeutic target. Using co-immunoprecipitation mass spectrometry, we recently identified an interaction between RAS and Argonaute 2 (AGO2), the core component of the RNA silencing machinery exerting translational control of mRNA transcripts. RAS and AGO2 co-sediment and co-localize in intracellular endomembrane bound organelles. The AGO2 N-terminal domain directly binds the KRAS Switch II region, irrespective of GDP/GTP bound to RAS. Functionally, we observed that AGO2 was required for maximal oncogenic KRAS levels and AGO2 knock-down attenuates cell proliferation in mutant KRAS-dependent cells. Intriguingly, AGO2 mediated microRNA processing (RISC activity) was attenuated in cancer cells expressing mutant KRAS compared to those expressing wild type RAS. Yet, these initial investigations using cell line models do not reveal a clear role for AGO2 in KRAS driven oncogenesis. Therefore, we probed the role of AGO2 in the well-established pancreatic ductal adenocarcinoma (PDAC) mouse model driven by oncogenic KRAS.

Towards this end, we generated LSL-KrasG12D; Pft1a Cre; AGO2flox/flox (KCA) mice. Preliminary analysis (8-10 weeks of age) suggests that compared to LSL-KRASG12D (KC) mice, homozygous loss of AGO2 results in increased KRAS driven Acinar to Ductal Metaplasia (ADM) phenotype considered as precursors of PanIN (pancreatic intraepithelial neoplasia) and PDAC. This mirrors the phenotype of loss of Dicer in the same model further reinforcing a role for microRNAs in restraining KRAS oncogenic programs. Yet unlike Dicer, loss of AGO2 alone did not cause any gross or histological changes in the development of the mouse pancreata. In an effort to understand the underlying mechanisms that are regulated by AGO2, we also studied the activated levels of a variety of signaling molecules in pancreata obtained from different genotypes. Primary analysis indicates that wild type RAS levels are elevated during AGO2 loss and has a bearing on PDAC initiation. Using both Western blot and Immunohistochemical analyses, we demonstrate a previously unknown and critical role for AGO2 in KRAS-MAPK signaling pathway.

#3030

Mutant p53-driven metastasis explored utilizing a traceable, conditional osteosarcoma model.

Rasoul Pourebrahimabadi, Yun Zhang, Mark J. McArthur, Guillermina Lozano. _MD Anderson Cancer Center, Houston, TX_.

TP53 is the most frequently mutated gene in cancer and missense mutations in particular account for the majority of mutations. Mutant p53 proteins exert oncogenic Gain-of-Function (GOF) properties that contribute to increased migration and invasion in culture and metastasis in vivo. To elucidate how mutant p53 drives metastasis, we developed a traceable somatic osteosarcoma model that initiates with a single p53 mutation (p53R172H or p53R245W), or p53 loss specifically in osteoblasts using Osx-cre. To mark tumor cells, we utilized the mTmG allele which expresses membrane-targeted Tomato (mT) prior to Cre-mediated excision and membrane-targeted green fluorescent protein (mG) after excision so the tumor cells become GFP+ and the stromal cells remain RFP+. In this high penetrant, short latency mouse model, tumor micro-metastasis can be detected by GFP expression in the context of a normal stroma and immune system. The cohort has developed osteosarcoma with complete penetrance and an average survival of 304± 65 day. We observed metastasis in 55% of p53 mutant osteosarcomas which was significantly higher than 26% metastasis observed in p53 null tumors (p<0.01). In p53 mutant osteosarcomas, the metastasis rate was found significantly associated with low p53 copy number. Spectral karyotyping (SKY) on early passage cells derived from somatic p53 mutant tumors exhibited marked aneuploidy with modal chromosome numbers near tetraploid. To characterize the transcriptional profile associated with mutant p53 GOF, we analyzed and compared the RNA-Seq transcriptomes of eight tumors derived from p53R172H mutant mice with eight p53 null tumors. Comparative analysis of gene expression identified a signature of snRNA genes to be strongly associated with mutant p53 gain of function. Functional annotation of upregulated genes in mutant tumors was also highly enriched for chromatin modifying enzymes. These findings contribute to our understanding of the role of mutant p53 GOF in metastasis. Our long term goal is to study how tumor-stromal interactions affect tumor development and metastasis. This understanding will have broad translational significance in diagnosis and treatment of tumors with mutant p53.

#3031

PHIP is a therapeutic target for triple negative solid tumors.

David De Semir,1 Vladimir Bezrookove,1 Mehdi Nosrati,1 Altaf A. Dar,1 Nathan Salomonis,2 Richard W. Sagebiel,1 Pierre Desprez,1 Robert J. Debs,1 Dirk Schadendorf,3 Mohammed Kashani-Sabet1. 1 _California Pacific Medical Center Research Institute, San Francisco, CA;_ 2 _Cincinnati Children's Hospital Medical Center, Cincinnati, OH;_ 3 _University Hospital Essen, Essen, Germany_.

Targeted therapy relies on the classification of tumors according to the major molecular drivers of the malignant phenotype, which can then help decide the therapeutic treatment. However, a substantial subset of solid tumors does not express these markers, exemplified by the triple-negative subtype of breast cancer. Shared molecular factors that promote the progression of these tumors, and that represent a target for their therapy, are missing. We previously described a role for PHIP (Pleckstrin Homology domain-Interacting Protein) in the progression of melanoma, and demonstrated PHIP activation in triple-negative melanomas. Analysis of the TCGA profiling efforts in melanoma, breast and lung cancer revealed PHIP expression to be enriched in triple-negative breast cancer and in the bronchiad subtype of triple-negative lung cancer. Here we show the broad-based role of PHIP in the progression of triple-negative subtypes of three solid tumors (breast and lung cancer, and melanoma) defined by different mutational drivers and targeted therapies. By using a shRNA-based targeting of PHIP in in vitro and in vivo models, we have suppressed the malignant phenotype of triple-negative MDA-MB-231 and MDA-MB-436 human breast carcinoma cells. In addition, PHIP knockdown resulted in significant anti tumor effects in H1703 and Calu3 human lung cancer cells lacking mutations in EGFR, KRAS, and ALK, and in two short-term triple wild type melanoma cultures lacking mutations in BRAF, NRAS, and NF1. Suppression of PHIP expression resulted in inhibition of both tumor cell proliferation by cell survival and colony formation assays and invasion into matrigel in each of the tumor models examined, and was accompanied by suppression of pAKT, CCND1, TLN1, and ITGβ1 expression when assessed by western-blot or quantitative immunofluorescence. The PHIP protein contains two bromodomains that can be therapeutically targeted by small molecules. However, the functional activity of the PHIP bromodomains has been poorly characterized. We show that PHIP co-localizes with and binds to the acetylated histone modification H4K91ac, and that both are coordinately regulated upon growth factor stimulation suggesting a new function for PHIP as a chromatin remodeler. To conclude, our results identify a novel role for PHIP in the progression of solid tumors lacking the major molecular drivers, and suggest PHIP as a druggable target for the therapy of these malignancies.

#3032

Direct regulation of alternative splicing by Smad3 through PCBP1 is essential to the tumor-promoting role of transforming growth factor beta1.

Veenu Tripathi, Katherine M. Sixt, Xuan Xu, Ying E. Zhang. _National Cancer Institute, NIH, Bethesda, MD_.

In advanced stages of cancers, TGF- β promotes tumor progression in conjunction with inputs from receptor tyrosine kinase pathways. However, the mechanisms that underpin the signaling cooperation and convert TGF- β from a potent growth inhibitor to a tumor promoter are not fully understood. In this work we report that TGF- β directly regulates alternative splicing of cancer stem cell marker CD44 through a phosphorylated T179 of SMAD3-mediated interaction with RNA-binding protein PCBP1. We show that TGF- β and EGF respectively induce SMAD3 and PCBP1 to colocalize in SC35 positive nuclear speckles, and the two proteins interact in the variable exon region of CD44 pre-mRNA to inhibit spliceosome assembly in favor of expressing the mesenchymal isoform CD44s over the epithelial isoform CD44E. We further show that the SMAD3-mediated alternative splicing is essential to the tumor-promoting role of TGF- β and has a global influence on protein products of genes instrumental to epithelial to mesenchymal transition and metastasis.

#3033

The PRL family of tyrosine phosphatases as a novel drug targets in acute lymphoblastic leukemia.

Jessica S. Blackburn. _University of Kentucky, Lexington, KY_.

The aggressive and unpredictable behavior of acute lymphoblastic leukemia (ALL) presents a major clinical challenge in both the pediatric and adult setting. The development of new and better chemotherapies for this disease requires a detailed understanding of the genes and pathways that drive ALL malignancy. Phosphatases are emerging as new drug targets with important roles in leukemia progression. To determine the extent to which phosphatases play a role in ALL malignancy, we used CRISPR/Cas9 to knock out expression of 255 individual phosphatases in human T-cell acute lymphoblastic leukemia cell lines and screened for deleterious effects on cell viability. Top hits from this screen included the protein tyrosine phosphatase type IVa family, also known as the PRLs. PRL3 is genomically amplified in a subset of human T-ALL, and more than 50% of human ALL patients have significantly higher expression of PRL2 and/or PRL3, compared to normal lymphocytes, suggesting that these PRLs may play an important role in ALL malignancy. Transgenic Myc-induced ALL models in zebrafish showed that overexpression of PRL2 and PRL3 significantly shortened latency of primary and relapse ALL, enhanced leukemia stem cell self-renewal (PRL2) and prevented apoptosis after standard chemotherapy treatment (PRL3). Finally, a specific PRL inhibitor strongly induced apoptosis of PRL-expressing ALL cells in a dose-dependent manner, in both zebrafish models in vivo and human cell lines in vitro. We have identified several FDA-approved, general phosphatase inhibitors that have potent anti-PRL activity and are capable of killing ALL cells in vitro. Current work is focused on moving these inhibitors into pre-clinical testing using patient-derived xenografts. Pull-down approaches are also being used to identify the substrates of PRL phosphatase activity that are critical to ALL survival and may represent new, tractable drug targets for the treatment of ALL. <!--EndFragment-->

#3034

A programmed ribosomal frameshifting defect potentiates the transforming activity of the JAK2-V617F mutation.

Yousuf A. Khan,1 Sergey O. Sulima,2 Joe Kendra,1 Vivek M. Advani,1 John E. Jones,1 Joseph Briggs,1 Kim de Keersmaecker,2 Jonathan D. Dinman1. 1 _University of Maryland College Park, Potomac, MD;_ 2 _KU Leuven Department of Oncology, Leuven, Belgium_.

The JAK-STAT pathway is one of the major mechanisms through which cellular proliferation, differentiation, migration, survival and apoptosis are regulated in response to external stimuli. Promiscuous activation of this pathway is a major driver in the pathogenesis of myeloproliferative neoplasms, often characterized by clonal expansion of mature myeloid cells. The JAK2-V617F allele is the most common and well characterized mutation linked to this class of leukemias. A change of G to T at nucleotide 1849 of the coding sequence (CDS) results in substitution of valine to phenylalanine at codon 617. JAK2-V617F mutant cells have strongly increased activation of JAK-STAT signaling, which can partially be explained by its ability abolish inhibition of kinase activity and increase the Km of the enzyme. It is however unclear if these effects are sufficient to explain the strong activation of the JAK-STAT pathway induced by the V617F mutation. Here, we demonstrate that this mutation destabilizes a programmed -1 ribosomal frameshift (-1 PRF) signal in the JAK2 mRNA, leading to decreased rates of -1 PRF promoted by this element. There is an inverse relationship between -1 PRF and mRNA stability. Thus, decreased -1 PRF results in increased abundance of the JAK2 mRNA and increased JAK2 protein expression. Prior studies established that elevated expression levels of JAK proteins are sufficient to transform cells in vitro, suggesting that -1 PRF may normally help to limit JAK2 expression. Silent protein coding changes in the -1 PRF signal slippery sequence ablating JAK2 frameshifting also increased JAK2 expression and phenocopied the transforming activity of JAK2-V617F in cell culture. Further, the combination of the V617F and slippery site mutations conferred an additive effect on cellular transformation. The presence at least three additional -1 PRF signals in the JAK2 mRNA suggests that -1 PRF plays a significant role in controlling JAK2 expression.

### High Throughput Genomics Dissecting Cancer Development and Vulnerabilities

#3035

Decomposing oncogenic transcriptional signatures to generate maps of divergent cellular states.

William J. Kim,1 William C. Hahn,1 Jill Mesirov,2 Pablo Tamayo2. 1 _Dana-Farber Cancer Institute, Cambridge, MA;_ 2 _University of California San Diego, San Diego, CA_.

The systematic sequencing of the cancer genome has provided a powerful framework for identifying genetic alterations in cancer. However, a deeper understanding of the functional consequences of these alterations is necessary to guide appropriate therapeutic strategies. Here, we describe Onco-GPS (OncoGenic Positioning System), a data-driven analysis framework, and associated experimental and computational methodology, to organize individual tumor samples with shared oncogenic alterations onto a reference map defined by their underlying cellular states. Using this approach, we generated series of RAS/MAPK sub-signatures that reflect known and novel downstream pathways. These sub-signatures were then used to classify KRAS and BRAF mutant cancers into sub groups or cell states, as represented in the novel visual paradigm "OncoGPS map". OncoGPS approach was first applied to cancers with BRAF mutations. The approach not only accurately classified three groups of BRAF mutant cancers that aligned with sensitivity to BRAF inhibitors, but also identified resistance mechanisms associated with each state, validating the overall approach. OncoGPS also identified four major cell states associated with KRAS mutant cancers. Further characterization of the four cell states showed that multiple molecular features representing epithelial-EMT states and activation of NF-κB pathway delineated two major "axes" of KRAS mutant cancers. In addition to the pathways and cell states defined in each group, the OncoGPS approach is able to identify effective drug combinations which was experimentally tested and confirmed. These results show that the Onco-GPS is an effective analytic framework to explore the complex landscape of oncogenic cellular states across cancers, and a powerful framework to summarize knowledge, establish relationships, and generate more effective disease models for research or as part of individualized precision medicine paradigms.

#3036

Real-time genomic characterization of metastatic pancreatic cancer to enable precision medicine.

Andrew J. Aguirre,1 Scott Carter,1 Nicholas Camarda,1 Arezou Ghazani,1 Jonathan Nowak,1 Annacarolina Da Silva,1 Lauren Brais,1 Dorisanne Ragon,1 Devin McCabe,1 Lori Marini,1 Kristin Anderka,2 Karla Helvie,1 Nelly Oliver,1 Ana Babic,1 Paul Shyn,3 Douglas Rubinson,1 Anuj Patel,1 James Cleary,1 Nadine McCleary,1 Matthew Kulke,1 Thomas Clancy,1 Leona Doyle,1 Jason Hornick,1 Christine Ardito-Abraham,4 Ruth Yu,5 Michael Downes,5 Ronald Evans,5 Richard A. Moffitt,6 Jen Jen Yeh,6 William C. Hahn,1 Charles Fuchs,1 Robert Mayer,1 Nikhil Wagle,1 David Tuveson,7 Levi A. Garraway,1 Brian M. Wolpin1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Broad Institute, Cambridge, MA;_ 3 _Brigham and Women's Hospital, Boston, MA;_ 4 _The Lustgarten Foundation, Bethpage, NY;_ 5 _Salk Institute, La Jolla, CA;_ 6 _University of North Carolina, Chapel Hill, NC;_ 7 _Cold Spring Harbor Laboratory and The Lustgarten Foundation, Cold Spring Harbor, NY_.

Pancreatic ductal adenocarcinoma (PDAC) is currently the fourth-leading cause of cancer-related death in the United States and is projected to become the second leading cause by 2030. Most patients present with advanced disease and die within 12 months of diagnosis. Recent genomic studies of primary pancreatic cancer resection specimens have identified several molecular alterations and genomic subtypes of the disease that may guide precision medicine approaches to clinical management. However, the molecular landscape of metastatic PDAC has been less well characterized. Moreover, biopsy-driven studies in metastatic PDAC have been historically very challenging due to the aggressive course of this disease as well as the low-volume and heterogeneous nature of biopsies that makes deep molecular characterization difficult. Insufficient genomic analysis of a patient's tumor early in their disease course is a major barrier to enrollment on clinical trials of targeted therapies. To address these limitations, we have implemented a multi-disciplinary clinical and research biopsy protocol to enable real time comprehensive molecular characterization of metastatic PDAC biopsy specimens. We have performed core needle biopsies of metastatic lesions in the liver or peritoneal cavity in 42 patients at the time of initial presentation. A low rate of complications was observed, with only a single patient having a self-limited hemorrhagic complication after liver biopsy. On average, 4-6 separate biopsy specimens were collected from each patient for histopathology and genomic analysis. Whole exome sequencing (WES) was performed in a CLIA-certified laboratory and a comprehensive molecular report of somatic alterations and selected pathogenic germline variants was returned to the referring clinician with a typical turn-around time of 3-5 weeks. We observed a striking incidence of recurrent germline and somatic alterations in DNA-damage repair genes, such as BRCA2, ATM and CHEK2. We also observed alterations in genes with known therapeutic implications, such as BRAF, RNF43, STK11 and ROS, and in select cases, these results guided choice of second or third line therapy. In parallel to WES, we performed RNA sequencing on bulk tumor tissue and readily identified expression signatures defining multiple subtypes of tumor and stroma that may have prognostic or therapeutic implications for tumor- or stroma-directed therapies. Collectively, these results demonstrate the feasibility and value of real-time genomic characterization of metastatic PDAC and provide a path forward for improved stratification and enrollment of PDAC patients on molecularly defined clinical trials.

#3037

Dissecting treatment resistance in patients with ovarian cancer and PDX-models using single-cell RNA-sequencing.

Benjamin Izar,1 Itay Tirosh,2 Elizabeth Stover,1 Asaf Rotem,1 Parin Shah,1 Mike Cuoco,2 Chris Rodman,2 Joyce Liu,1 Ursula Matulonis,1 Orit Rozenblatt-Rosen,2 Levi Garraway,1 Aviv Regev2. 1 _Dana-Farber Cancer Institute, Cambridge, MA;_ 2 _Broad Institute, Cambridge, MA_.

Background: Ovarian cancer (OvCa) is frequently associated with malignant effusions, which are complex ecosystems with heterogeneous populations of malignant cells and non-malignant cells. Bulk RNA-seq or whole-exome sequencing (WES) only reflect average cellular behavior and thereby mask intrinsic cell diversity with potential relevance for treatment resistance.

Approach: To overcome some of these barriers, we applied single-cell RNA-sequencing (scRNA-seq) to malignant and non-malignant cells isolated from patients with platinum treatment resistant disease. Furthermore, we used patient-derived xenograft (PDX) cohorts, in which we isolated cells for scRNA-seq from vehicle tumors (VEH), treated the other models with carboplatin, and harvested cells at the time of minimal residual disease (MRD) or disease progression (PROG).

Results: To date, we have profiled ~12000 single cells from 12 patients with treatment naïve (n=3) or platinum-resistant disease (n=9), including sequential sampling in 3 patients with resistant disease. We observed significant inter- and intra-individual transcriptional heterogeneity in malignant cells. A recurrent pattern across resistant patients was the differential expression of inflammatory pathways in a subset of cells. In a patient with three consecutive specimens, we observed increasing accumulation of cells expressing a cell state characterized by tumor necrosis factor alpha (TNF-a) signaling, Importantly, these cells were genetically identical to the entire population, supporting the hypothesis that non-encoded mechanisms conferred treatment resistance. In a BRCA-mutant patient, unbiased analysis identified a stemness program in a subpopulation of cells, which was genetically identical to other cells, indicating phenotypic conversion. To systemically interrogate mechanisms of resistance to platinum therapy, sequenced single cells isolated from PDX models at three time points (VEH, MRD and PROG). In a BRCA-WT PDX model, resistant cells isolated at MRD and PROG shared a transcriptional program that was dominated by expression of a STAT3 program. Ex vivo cultures from platinum-resistant patients were exquisitely sensitivity to JAK/STAT3-inhibitor. Live cell imaging revealed that STAT3-inhibition prevented spheroid formation, attachment and clearance through a mesothelial monolayer in vitro.

Conclusion: Our results indicate that non-encoded mechanisms play an important role in the development of treatment resistance in ovarian cancer. Our initial studies indicate an important role of inflammatory pathways in treatment resistance, in particular STAT3 signaling, which can be overcome with specific inhibitors at nanomolar concentrations. These data suggests that single-cell profiling can be performed on clinical ovarian cancer specimens and may yield novel therapeutic avenues for patients with treatment-resistant ovarian cancer.

#3038

Bridging the gap between NGS data and its usability: cancer gene discovery through massive-scale transcriptomic analyses and development of a powerful web-tool for dissemination of these findings.

Yashar Niknafs, Nicholas Molen, Balaji Pandian, Matthew Iyer, Arul Chinnaiyan. _Univ. of Michigan, Ann Arbor, MI_.

Background: In a prior transcriptomic analysis focused on lncRNA interrogation using some of these data, we discovered tens of thousands of novel lncRNAs (Iyer and Niknafs et al, Nature Genetics, 2015). Building on this analysis, we recently developed a markedly improved bioinformatics tool for novel gene/isoform discovery from massive RNA-seq datasets named TACO (Niknafs et al, Nature Methods, In press, tacorna.github.io). TACO produces high-fidelity transcript structure predictions from large RNA-seq datasets. We now set out to comprehensively leverage both TACO and the publicly available RNA-seq data for discovery of novel transcriptional cancer associations. Additionally, in order to widely disseminate these findings in an accessible manner, we have built a web-tool that provides the scientific community access to these data and analyses.

Methods: We have downloaded, curated, and processed 23,623 RNA-seq samples largely from the TCGA, ICGC, GTEx, and CCLE, comprising 37 tissue types and over 35 cancer types. RNA-seq data processing was performed using STAR, Cufflinks, Kallisto, and TACO. The web tool for visualization and access to these data and analyses was built using a JavaScript-based server infrastructure (Noje.js) and a relational PostgreSQL database.

Results: Generation of a consensus transcriptome from this large-scale RNA-seq dataset via TACO resulted in the discovery of tens of thousands of novel transcriptional elements, including intergenic non-coding RNAs and novel splice isoforms of known genes. Such an expansive RNA-seq cohort that includes many normal tissue samples enabled statistically powerful cancer association expression analyses that revealed a myriad of novel cancer genes, especially in tissues for which there was previously little-to-no normal tissue RNA-seq data (e.g., brain and pancreas). Many of these novel transcriptional elements discovered using TACO were also found to be cancer associated. We have built a web-tool to facilitate further analysis and discovery using these data and analyses by the scientific community. The web-tool provides a powerful and intuitive interface for researchers with little-to-no bioinformatics expertise to leverage large-scale RNA-seq datasets.

Conclusion: Here we present the largest reported compendium of RNA-seq data, and reveal many novel cancer gene associations. Using a new, powerful gene discovery tool, TACO, we identify a multitude of novel transcriptional elements that are also cancer associated. Despite the abundance of publicly available RNA-seq data, necessary computing resources, data storage, and bioinformatics expertise are barriers to usability of these data by scientific community. Our RNA-seq expression web-tool bridges this gap, and enables users to powerfully interrogate cancer expression across dozens of tumor and tissue types.

#3039

Combinatorial CRISPR-Cas9 gene knockout to enable genetic interaction mapping in human cells.

John Paul Shen, Dongxin Zhao, Roman Sasik, Jens Luebeck, Ana Bojorquez-Gomez, Katherine Licon, Trey Ideker, Prashant Mali. _UCSD, La Jolla, CA_.

Genetic interactions, in particular negative or 'synthetic-lethal' interactions for which simultaneous disruption of two genes causes cell killing, have implications for therapeutic development. The feasibility of this approach has been demonstrated with the recent approval of the drug olaparib, an inhibitor of PARP1, specifically for tumors with loss-of-function mutations in BRCA1/2. However, beyond olaparib, further applications of synthetic-lethal cancer therapy have been limited by poor understanding of the important genetic interactions in a cancer cell, and how these vary from one cancer type to another or from patient to patient. To enable systematic mapping of these genetic interaction networks, we developed a CRISPR-Cas9 screening methodology for knocking out single and pairs of genes in high-throughput. Here, we combined multiplex targeting with array-based oligonucleotide synthesis to create dual-gRNA libraries covering up to 10^5 defined gene pairs. In these libraries, each construct bears two gRNAs, with each gRNA designed to target either a gene or a scrambled non-targeting sequence absent from the genome. We conducted genetic interaction screens by transducing the dual-gRNA lentiviral library into a population of cells stably expressing Cas9, maintaining these cells in exponential growth over the course of four weeks, then sampling the relative changes in gRNAs at days 3, 14, 21 and 28 post-transduction. To robustly quantify gene fitness and genetic interactions, we developed a computational analysis framework that integrates all samples across the multiple days of the experiment. Using this method we evaluated all pairwise gene knockout combinations among a panel of 73 genes divided between tumor-suppressor genes (TSG) and cancer-relevant drug targets (DT); including negative controls this amounted to a total of 23,652 combinations. Experiments were performed in three cancer cell lines: HeLa, A549 and 293T. In total 162 therapeutically relevant interactions were identified, of which 146 (90.1%) were private to one cell line. None of the interactions were observed in all three cell lines. These patterns replicated in low throughput assays with combinatorial drugs at 80% precision. In summary, we have introduced a combinatorial CRISPR-Cas9 genetic interaction mapping technology that successfully identifies many therapeutically-relevant genetic interactions in cancer and shows the great importance of cellular context on the architecture of the genetic interaction network. Recognizing that there will be great diversity in genetic interaction between different tumors it will be important to perform future studies across a large number of samples, which is enabled by the high-throughput method we have developed.

#3040

Functional genomic screening identifies USP11 as a novel regulator of ERα transcription in breast cancer.

Lisa Dwane,1 Aisling E. O'Connor,2 Laoighse Mulrane,2 Annette M. Dirac,3 Karin Jirstrom,4 John P. Crown,5 Rene Bernards,3 William M. Gallagher,2 Tríona Ní Chonghaile,1 Darran P. O'Connor1. 1 _Royal College of Surgeons Ireland, Dublin, Ireland;_ 2 _Conway Institute, University College Dublin, Dublin, Ireland;_ 3 _Netherlands Cancer Institute, Dublin, Netherlands;_ 4 _Malmö University Hospital, Dublin, Sweden;_ 5 _St Vincent's University Hospital, Dublin, Ireland_.

Approximately 70% of breast cancers overexpress the estrogen receptor α (ERα) and depend on this key transcriptional regulator for growth and differentiation. The discovery of novel mechanisms controlling ERα function represent major advances in our understanding of breast cancer progression and potentially offer attractive new therapeutic opportunities. Here, we investigated the role of deubiquitinating enzymes (DUBs), which act to remove ubiquitin moieties from proteins, in regulating transcriptional activity of ERα in breast cancer.

To identify DUBs involved in the regulation of ERα transcriptional activity, we performed an RNAi loss-of-function screen using a library of shRNA vectors targeting all human DUB genes. The DUB library consisted of pools of four non-overlapping shRNAs targeting all 108 known or putative DUBs (432 shRNAs in total). We found that suppression of a number of DUBs markedly repressed or enhanced the activity of an estrogen-response-element (ERE) luciferase reporter following estradiol (E2) stimulation. Of particular interest, suppression of the BRCA2-associated DUB, USP11, was found to down-regulate ERα transcriptional activity.

Subsequent validation using two individual siRNAs targeted to USP11 revealed a notable reduction in expression of endogenous ERα target genes in the ZR-75-1 cell line, as quantified using qRT-PCR. Immunoprecipitation of ERα revealed no physical interaction with USP11, however E2 stimulation resulted in translocation of USP11 to the nucleus, suggesting a potential role in E2-induced transcription. Furthermore, USP11 expression was found to be upregulated in the estrogen-independent cell line LCC1 when compared to their parental MCF7 cells. Knockdown of USP11 in LCC1 cells resulted in decreased mRNA expression of a panel of ERα target genes, suggesting a role for USP11 in an estrogen independent setting.

To support the prognostic relevance of USP11, immunohistochemical staining of a breast cancer tissue microarray (n=144) was performed. Kaplan-Meier analysis of this cohort revealed a highly significant association between poor overall survival (OS) (p=0.030) and breast cancer-specific survival (BCSS) (p=0.041). In silico analysis of publically available breast cancer gene expression datasets further supported an association between high USP11 mRNA levels and poor prognosis. We observed a significant correlation between high expression of USP11 mRNA in ER-positive patients and poor distant metastasis-free survival (DMFS) (HR 2, CI 1.37-2.91, p=0.00023). This correlation was also significant in ER-positive patients who had received endocrine therapy only (HR 2.9, CI 1.63-5.15, p=0.00015).

These results suggest a role for USP11 in driving cellular growth and identify USP11 as novel therapeutic target in breast cancer.

#3041

Duplex Sequencing detects cancer-associated mutations arising during normal aging: Clonal evolution over a century of human lifetime.

Jesse J. Salk,1 Elisabeth Maritschnegg,2 Paul Speiser,2 Robert Zeillinger,2 Rosana Risques,1 Lawrence A. Loeb1. 1 _University of Washington, Seattle, WA;_ 2 _Medical University of Vienna, Vienna, Austria_.

High-throughput DNA sequencing has fundamentally changed practice standards in clinical oncology. We can now systematically examine the dysfunctional genes and pathways of individual tumors and can often pair these with targeted therapies. Moreover, NGS has improved our ability to resolve molecular heterogeneity, such as results from evolving subclones within a tumor or admixtures of cancer-derived and normal DNA in liquid biopsies. However, this remains imperfect. With standard NGS, sequencing errors create a background that obscures variants present at less than 1-2%. Methods of tag-based error correction improve accuracy, but most rely on hemimolecular tagging and are thus still limited by certain types of errors caused by DNA damage. Our group pioneered Duplex Sequencing, an error correction technology that independently genotypes and compares both single-stranded halves of individual DNA duplexes. This approach improves sequencing accuracy by >100,000-fold and allows detection of a single mutation among ten million unmutated nucleotides. We carried out Duplex Sequencing on uterine lavages from women with and without high-grade serous ovarian cancer to determine the sensitivity for identifying tumor-derived TP53 mutations as a screening test. Under blinded conditions we detected tumor-derived mutations in 80% of cancer cases. However, in addition, we found that nearly all women, including those without cancer, carried very low frequency TP53 mutations that did not originate from a tumor. These could not be explained by technical errors, as they exhibited a strong signature of positive selection, indicated by higher-than-random non-synonymous fraction, substantial clustering in cancer-associated hotspots and a spectrum, exonic distribution and predicted pathogenicity closely mirroring TP53 mutations in cancer databases. Furthermore, abundance of these "biological background" mutations increased with age. To more broadly investigate the extent of clonal evolution in normal aging, we used Duplex Sequencing to examine tissue specimens from two individuals without cancer, differing in age by 101 years. We assessed relative mutation load in 22 cancer-associated genes and one non-cancer associated gene in 19 different tissues. Initial results indicate that the extent of age-associated mutation accumulation varies by organ and the pattern of apparent clonal selection is influenced by both tissue type and genetic context. Our findings illustrate the near-universal presence of cancer-like mutations arising from age-associated processes in multiple non-neoplastic tissues. Somatic clonal evolution appears to be a ubiquitous part of human life. Beyond many biological implications, this highlights an important specificity challenge that must be accounted for when using ultra-accurate sequencing technologies for cancer screening and other sensitive diagnostics.

### The Riddle of the Noncoders: New Functional Roles for miRNAs, Long Noncoding RNAs, and mRNAs

#3042

MIR142 **loss-of-function mutations promote leukemogenesis through derepression of** ASH1L **resulting in increased** HOX **gene expression.**

Juo-Chin Yao,1 Terrence N. Wong,1 Maria Trissal,1 Rahul Ramaswamy,1 Yaping Sun,2 Pavan R. Reddy,2 Daniel C. Link1. 1 _Washington University in St. Louis, St. Louis, MO;_ 2 _University of Michigan, Ann Arbor, MI_.

MIR142 mutations have been identified in acute myeloid leukemia (AML) and non-Hodgkins lymphoma. In AML, all MIR142 mutations localize to the miR-142-3p seed sequence. We show that mutated MIR142 is unable to suppress several well-known targets of miR-142-3p. Interestingly, the mutations of miR-142-3p result in their preferential loading into the RNA-induced silencing complex, leading to the degradation of miR-142-5p. Accordingly, miR-142-5p expression is decreased in MIR142 mutated AML. Hence, MIR142 mutations in AML disrupt both miR-142-3p/5p functions. Thus, we modeled the effect of MIR142 mutations on hematopoiesis using Mir142-/- mice. We show that loss of miR142 results in significant increases in myeloid hematopoietic stem/progenitor cells (HSPCs), including granulocyte-macrophage progenitors, myeloid-biased multipotent progenitors (CD150- CD48+ Flk2- Kit+ Sca+ lineage-) and CD229\- myeloid-biased HSCs (CD150+ CD48\- Kit+ Sca+ lineage-). In contrast, there are significant decreases of megakaryocyte-erythroid progenitors and erythroid precursors. Although the number of HSCs is normal in Mir142-/- mice, HSC transplantation suggest that they are myeloid-biased. In AML, MIR142 mutations are commonly found in conjunction with mutations of IDH1/2. To assess the importance of this association, we transduced wildtype or Mir142-/- HSPCs with retrovirus expressing IDH2 R172K and then transplanted into lethally irradiated recipients. Expression of IDH2 R172K alone was sufficient to induce a lethal myeloproliferative neoplasm (MPN). In contrast, Mir142-/- alone did not result in MPN. However, loss of Mir142 cooperates with IDH2 R172K to produce a more severe MPN, with increased CD34+ blasts and more severe anemia. Moreover, secondary transplantation shows that Mir142-/- x IDH2 R172K cells but not IDH2 R172K cells efficiently engraft and induce MPN, suggesting that loss of miR142 increases leukemia-initiating activity. We identify the histone methyltransferase ASH1L as a target gene of miR142 that contributes to altered hematopoiesis in Mir142-/- mice. The 3'-untranslated region of ASH1L has four miR-142-3p binding sites, and luciferase reporter assay shows that miR142 suppresses its translation by 80%. Consistent with this observation, Ashl1 protein expression is 3-fold higher in Mir142-/- bone marrow. ASH1L is a key positive regulator of HOX gene expression. Accordingly, we observed markedly (5-10 fold) increased HoxA9/A10 expression in myeloid progenitors in Mir142-/- mice. Likewise, HoxA9/A10 expression is increased in CD34+ blasts from Mir142-/- x IDH2 R172K transplanted mice. Of note, increased HoxA9 expression has been shown to cooperate with mutant IDH1 to induce AML in mice. Together, these findings support a model in which loss-of-function mutations of MIR142 contribute to hematopoietic malignancies by derepressing ASH1L and inducing HOXA9/10 gene expression.

#3043

**A novel** Cpa3-Cre; miR-9fl/fl **mouse reveals a functional role for miR-9 in promoting mast cell invasion via up-regulation of CMA1.**

Joelle M. Fenger,1 Feng Xu,1 Xaioli Zhang,1 Peter Y. Yu,1 Misty D. Bear,2 Shanice L. Reynolds,1 Michael L. Pennell,1 Vincenzo Coppola,1 William C. Kisseberth,1 Stephen J. Galli,3 Cheryl A. London1. 1 _The Ohio State University, Columbus, OH;_ 2 _Kansas State University, Manhattan, KS;_ 3 _Stanford University, Columbus, CA_.

Introduction: Our laboratory previously demonstrated that miR-9 promotes the invasive properties of normal and malignant mast cells and that overexpression of miR-9 is associated with spontaneous metastasis in canine mast cell tumors (MCTs), a well-established large animal model of malignant mast cell disease. The purpose of this study was to further investigate the biologic effect and mechanisms by which mir-9 enhances normal mast cell invasion.

Methods: Bone marrow-derived mast cells (BMMCs) were generated from Cpa3-Cre; miR-9Δfl/Δfl, and Cpa3-Cre; miR-9Δfl/Δfl mice and Taqman miRNA assays were used to quantify mature miR-9 expression. BMMCs were evaluated for differences in proliferative capacity, the ability to migrate through Matrigel, sensitivity to degranulation, and the expression and release of growth factor expression and release following chemical stimulation. RNA sequencing was performed by the OSUCCC Genomics Resource and changes in protein and mRNA expression were validated with Western blotting and quantitative PCR.

Results and Conclusions: We generated a transgenic mouse carrying a floxed STOP-miR-9 transgene (miR-9Δfl/Δfl) in which expression of miR-9 occurs only after Cre-mediated recombination and crossed these mice with Cpa3-Cre mice which express Cre recombinase under the control of the carboxypeptidase A3 (Cpa3) promoter, restricting expression of miR-9 to mast cells and basophils. Bone marrow-derived mast cells (BMMCs) derived from Cpa3-Cre;miR-9Δfl/Δfl mice significantly enhanced the invasive phenotype of mast cells. Moreover, the enforced expression of miR-9 in BMMCs markedly changed the production and release of cytokine and growth factors in response to chemical stimulation, including substantial up-regulation of IL-4, IL-13, and GM-CSF transcript and enhanced TGF-β1 release. RNA sequencing revealed a unique transcriptional profile associated with miR-9 over-expression in the Cpa3-Cre;miR-9Δfl/Δfl derived BMMCs and identified increased expression of several mast cell-restricted proteases involved in extracellular matrix and tissue remodeling including CMA1 and MCP-6. Furthermore, transduction of Cpa3-Cre;miR-9Δfl/Δfl derived BMMCs with CMA1 shRNA abrogated miR-9-dependent mast cell invasion. Studies are underway to investigate the regulatory pathway mediated by miR-9 that contributes to CMA1 up-regulation and promotes the invasive phenotype. In summary, these data provide evidence supporting a critical role for miR-9 in mast cell invasion and suggest that dysregulation of miR-9 may contribute to pathologic conditions involving mast cell-mediated tissue remodeling.

#3044

Rapid decay of engulfed extracellular miRNA by XRN1 exonuclease promotes transient epithelial-mesenchymal transition.

Joséphine Zangari,1 Marius Ilie,1 Florian Rouaud,2 Laurie Signetti,1 Mickaël Ohanna,2 Robin Didier,2 Barnabé Roméo,1 Dana Goldoni,1 Nicolas Nottet,3 Cathy Staedel,4 Jocelyn Gal,5 Bernard Mari,3 Baharia Mograbi,1 Paul Hofman,1 Patrick Brest1. 1 _Université Côte d'Azur, FHU Oncoage, Nice, France;_ 2 _Université Côte d'Azur, Nice, France;_ 3 _Université Côte d'Azur, Valbonne, France;_ 4 _Université de Bordeaux, Bordeaux, France;_ 5 _Université Côte d'Azur, Antoine Lacassagne Cancer Center, Nice, France_.

Background: Extracellular vesicles (EVs) have been shown to play an important role in intercellular communication as carriers of DNA, RNA and proteins. While the intercellular transfer of miRNA thorough EVs has been widely studied, the stability of extracellular miRNA (ex-miRNA) once engulfed by recipient cell remains to be determined.

Materials and Methods: Lung cancer cells (A549 cell line) were incubated with neutrophils or their supernatant or isolated EVs from these supernatant. Kinetics of expression of miR-223-3p were followed by RT-qPCR. FOXO1 expression and EMT expression markers were used as endpoints to follow effective effect or miR-223-3p in recipient cells.

Results: We identified the ex-miRNA-directed phenotype to be transient due to the rapid decay of the ex-miRNA. We demonstrated that the ex-miR-223-3p transferred from neutrophils to cancer cells was functional as demonstrated by the decreased expression of its target, FOXO1, and the occurrence of epithelial-mesenchymal transition reprogramming. We showed that the engulfed ex-miRNA, unlike endogenous miRNA, was unstable, enabling dynamic regulation and a return to a non-invasive phenotype within less than 8 hours. This transient phenotype could be modulated by targeting XRN1/PACMAN exonuclease. Indeed, its silencing was associated with slower decay of ex-miR-223-3p and subsequently prolonged the invasive properties.

Conclusion: We showed that the "steady step" level of engulfed miRNA (ex-miR-223-3p) and its subsequent activity was dependent on the presence of a donor cell in the surroundings to constantly fuel the recipient cell with ex-miRNAs and of XRN1 exonuclease, which is involved in the decay of these imported miRNAs.

#3045

Engineering Mesenchymal Stem Cells (MSCs) to support tumor-targeted delivery of exosome-encapsulated microRNA-379.

Killian P. O'Brien, Katie Gilligan, Sonja Khan, Brian Moloney, Kerry Thompson, Pierce Lalor, Peter Dockery, Helen Ingoldsby, Michael J. Kerin, Roisin M. Dwyer. _National University of Ireland Galway, Galway, Ireland_.

Mesenchymal Stem Cells (MSCs) are multipotent stromal cells known to migrate specifically to the sites of tumors and metastases, raising potential as tumor-targeted delivery vehicles for therapeutic agents. MSCs secrete exosomes containing genetic material including microRNAs (miRs), that are effectively taken up by cells. Previous work has shown miR-379 to be significantly reduced in breast tumor tissue, highlighting a potential tumor suppressor role. This study aimed to establish a mechanism of action for miR-379, and to engineer MSCs to secrete exosomes enriched with the miR for tumor-targeted delivery. Methods: RNA was isolated from breast tumors and matching lymph node metastases from the same patients, and miR-379 expression quantified by RQ-PCR. HCC1954 breast cancer cells were transduced with lentivirus to express elevated miR-379 (HCC-379) or a control sequence (HCC-NTC). Cells were implanted into the mammary fat pad (MFP) of mice and tumor progression monitored. Subsequently, MSCs were transduced to generate MSC-379 and MSC-NTC. MSC-secreted exosomes were isolated by differential centrifugation, microfiltration and ultracentrifugation. The morphology, size and number of isolated exosomes was characterized using Transmission Electron Microscopy, Western Blot and Nanoparticle Tracking Analysis respectively. Exosomal miRs were analysed by RQ-PCR. MFP tumors were established using HCC1954-luciferase cells, followed by IV injection of MSC-379 or MSC-NTC cells, or sequential doses of exosomes derived from either cell population. Results: miR-379 expression was significantly reduced in lymph node metastases compared to primary tumors from the same patients (p=0.009), supporting a tumor suppressor role. Analysis of HCC-379 and HCC-NTC tumor growth In Vivo showed no difference in tumor size, however an increase in tumour necrosis (5-50%) and decrease in lymph node invasion was observed in HCC-379 tumors. Investigation of a potential role for miR-379 in regulating COX-2, revealed an inverse relationship (r = -0.48, p=0.02) at a mRNA level, further confirmed at a protein level. MSC-379 cells showed no significant change in migratory or proliferative capacity In Vitro. MSC-secreted exosomes were confirmed to be the correct morphology and size (30-120nm), and to express the exosome-associated protein CD63. An increase in miR-379 (>5 fold) was observed in exosomes secreted by MSC-379 compared to MSC-NTC cells. Administration of MSC-379 or MSC-NTC cells, or exosomes derived from either cell population, was well tolerated In Vivo with no adverse effects observed. Monitoring tumor response to therapy using IVIS is ongoing. Conclusion: The data presented supports miR-379 as a potent tumor suppressor in breast cancer, mediated in part through regulation of COX-2. Engineering tumor-targeted MSCs to secrete exosomes enriched with miR-379 holds exciting potential as a novel therapy for breast cancer.

#3046

Identification of lncRNAs involved in regulation of androgen receptor splice variants in prostate cancer through CRISPR-Cas9-based screen.

Tsui-Ting Ho, Pratirodh Koirala, Nanjiang Zhou, Yin-Yuan Mo. _Univ. of Mississippi Medical Ctr. Cancer Inst., Jackson, MS_.

Prostate cancer is the second leading cause of death in men in the USA. Despite the high initial response rates to androgen deprivation therapy (ADT), these tumors ultimately develop the resistance, i.e., castration-resistant prostate cancer (CRPC). One of the contributing factors is constitutive expression of androgen receptor (AR) splice variants such as AR3, a major splice variant identified in clinical specimens. However, little is known about long non-coding RNAs (lncRNAs) on regulation of AR splicing. In this study, we generate CRISPR/Cas9-based libraries against a focus group of lncRNAs and an AR3 mini-gene reporter to identify lncRNAs involved in regulation of AR splicing. The principles of this screening system is that only sgRNAs capable of promoting AR3 expression will be enriched in SAM (Synergistic Activation Mediator) library, whereas these clones are lost from the knockout gRNA library after selection. This screen identifies several candidate lncRNA sgRNAs and dual gRNAs (targeting Lnc-DC/ Har1B/ LINC00568/ DHRS4-AS1/ SNHG5). Further characterization confirms the regulation of their corresponding endogenous genes and AR3 expression. Importantly, overexpression of Lnc-DC increases AR3 level and enhances tumor cell growth in response to androgen deprivation. Moreover, while ectopic expression of DHRS4-AS1 increases, DHRS4-AS1 knockout suppresses AR3. Finally, rescue experiments restore the ability of DHRS4-AS1 to induce AR3, further supporting a role for Lnc-DC and DHRS4-AS1 in AR3 regulation. Together, we demonstrate that the CRISPR/Cas9-based screening system provides a powerful tool for lncRNA research. As a result, these lncRNAs may serve as biomarkers to predict the response to ADT or therapeutic targets for treatment of CRPC.

#3047

**PRC2-mediated silencing of circRNA** CDR1as **drives** miR-7 **-independent melanoma metastasis.**

Doug Hanniford,1 Rana Moubarak,1 Jochen Imig,1 Alejandro Ulloa,1 Beatriz Sanchez Sendra,1 Alcida Karz,2 Iman Osman,1 Ioannis Aifantis,1 Eva Hernando1. 1 _New York University School of Medicine, New York, NY;_ 2 _Duke University, Durham, NC_.

Circular RNAs are a novel class of non-coding RNAs with functions that remain poorly characterized in normal and pathological conditions. CDR1as is a non-canonical circRNA observed to act as a sponge for miR-7 in brain tissues. Analysis of RNA-seq data of melanocytes and melanoma cell lines and short-term cultures revealed loss of CDR1as expression as a hallmark of melanoma cells. We confirmed silencing of CDR1as in melanoma cells and tissues by RT-qPCR using divergent primers. Clinically, we observed CDR1as loss associated with metastatic progression and poor patient outcomes in a cohort of fresh-frozen melanoma tissue samples. Depletion of CDR1as in melanoma cell lines enhanced invasion in vitro and lung metastasis in vivo, demonstrating functional significance of CDR1as silencing. Surprisingly, CDR1as depletion had no clear effect on miR-7 activity in melanoma cells, and miR-7 inhibition was insufficient to rescue CDR1as silencing-induced invasion. Moreover, GSEA analyses of proteomic profiling of melanoma cells depleted of CDR1as revealed reductions of proteins involved in oxidative phosphorylation (OXPHOS) and mitochondrial function, suggesting CDR1as loss may alter metabolism of melanoma cells. Mining of CLIP-Seq data sets and subsequent RIP-PCR revealed direct interactions of CDR1as with the IGF2BP family of proteins and TARDBP, each of which are involved in regulation of mitochondrial function. These data suggest that CDR1as could regulate the subcellular localization and/or function of proteins involved in OXPHOS and mitochondrial respiration.

To understand the upstream mechanism driving CDR1as loss in melanoma, we sought to examine the primary transcript from which CDR1as arises. Interestingly, examination of the CDR1as locus revealed an upstream long non-coding RNA, LINC00632, as a plausible primary transcript of CDR1as. CDR1as and LINC00632 expression is strikingly correlated across human tissues and in melanoma cell lines (r>.65). Moreover, depletion of LINC00632 using GapmeRs reduced expression of CDR1as, suggesting they arise from the same precursor transcript. Intriguingly, from data mining of ChIP-Seq datasets, we observed that the PRC2-dependent repressive chromatin mark H3K27me3, is abundant in the genomic locus of LINC00632 in melanoma cells. We validated the presence of H3K27me3 at LINC00632 by ChIP-PCR in melanoma cells lacking CDR1as expression. Strikingly, treatment of such melanoma cell lines with the EZH2 inhibitor, GSK126, induced robust re-expression of both CDR1as and LINC00632, and removal of H3K27me3 at LINC00632.

Our data document that CDR1as is epigenetically silenced in melanoma, and its loss promotes melanoma invasion and metastasis. Moreover, CDR1as loss could contribute to metabolic adaptations during metastasis through misregulation of RNA binding proteins.

#3048

A noncoding function of TYRP1 mRNA promotes melanoma growth.

Marie-Dominique Galibert,1 David Gilot,1 Mélodie Migault,1 Laura Bachelot,1 Fabrice Journé,2 Aljosja Rogiers,3 Emmanuelle Donnou-Fournet,1 Ariane Mogha,1 Nicolas Mouchet,1 Marie-Laure Pinel-Marie,4 Bernard Mari,5 Tristan Montier,6 Sébastien Corre,1 Arthur Gautron,1 Florian Rambow,3 Petra El Hajj,2 Rania Ben Jouira,7 Sophie Tartare-Deckert,7 Jean-Christophe Marine,3 Brice Felden,4 Ghanem Ghanem2. 1 _CNRS UMR6290 - IGDR / University of Rennes, Rennes, France;_ 2 _Institut Jules Bordet, Université Libre de Bruxelles, Brussels, Belgium;_ 3 _VIB Center for Cancer Biology, Leuven, Belgium;_ 4 _INSERM U835, Université de Rennes I, Rennes, France;_ 5 _CNRS UMR 7275, Sophia Antipolis, France;_ 6 _INSERM U1078, Université de Bretagne Occidentale, Université Européenne de Bretagne, Brest, France;_ 7 _INSERM U1065, C3M, Nice, France_.

RNAs competition to bind miRNA has been proposed to influence biological systems whether these RNAs are implicated in diseases onset is unclear. Here, we report that TYRP1 mRNA, in addition to encoding tyrosinase-related protein 1 (TYRP1), indirectly governs cell proliferation by sequestering miR-16 on non-canonical miRNA response elements (MREs). Consequently, sequestered miR-16 is no longer able to repress its mRNA targets such as RAB17, which we show is involved in melanoma cell proliferation and invasion. Restoration of miR-16 tumor suppressor function can be achieved in vitro by silencing TYRP1 or increasing miR-16 expression levels. Importantly, TYRP1-dependent miR-16 sequestration can also be overcome in vivo using small oligonucleotides that mask miR-16 binding sites on TYRP1 mRNA. Together, our findings assign a pathogenic noncoding function to TYRP1 mRNA and underscore miRNA displacement as a new antimelanoma targeted therapeutic approach.

## TUMOR BIOLOGY:

### Early Stages of Metastatic Dissemination

#3049

Tracing the origin of disseminated tumor cells in breast cancer using single-cell sequencing.

Jonas Demeulemeester,1 Parveen Kumar,2 Elen K. Møller,3 Silje Nord,3 David C. Wedge,4 April Peterson,5 Randi R. Mathiesen,3 Renathe Fjelldal,3 Masoud Z. Esteki,2 Koen Theunis,2 Elia F. Gallardo,2 Jason Grundstad,5 Elin Borgen,3 Lars O. Baumbusch,3 Anne-Lise Børresen-Dale,3 Kevin P. White,5 Vessela N. Kristensen,3 Peter Van Loo,1 Thierry Voet,2 Bjørn Naume3. 1 _The Francis Crick Institute, London, United Kingdom;_ 2 _KU Leuven – University of Leuven, Leuven, Belgium;_ 3 _Oslo University Hospital, Oslo, Norway;_ 4 _Wellcome Trust Sanger Institute, Cambridge, United Kingdom;_ 5 _University of Chicago, Chicago, IL_.

Background. Single-cell micro-metastases of solid tumors often occur in the bone marrow. These disseminated tumor cells (DTCs) may resist therapy and lay dormant or progress to cause overt bone and visceral metastases. The molecular nature of DTCs remains elusive, as well as when and from where in the tumor they originate. Here, we apply single-cell sequencing to identify and trace the origin of DTCs in breast cancer.

Results. We sequence the genomes of 63 single cells isolated from six non-metastatic breast cancer patients. By comparing the cells' DNA copy number aberration (CNA) landscapes with those of the primary tumors and lymph node metastasis, we establish that 53% of the single cells morphologically classified as tumor cells are DTCs disseminating from the observed tumor. The remaining cells represent either non-aberrant 'normal' cells or 'aberrant cells of unknown origin' that have CNA landscapes discordant from the tumor. Further analyses suggest that the prevalence of aberrant cells of unknown origin is age-dependent, and that at least a subset is hematopoietic in origin. Evolutionary reconstruction analysis of bulk tumor and DTC genomes enables ordering of CNA events in molecular pseudo-time and traced the origin of the DTCs to either the main tumor clone, primary tumor subclones, or subclones in an axillary lymph node metastasis.

Conclusions. Single-cell sequencing of bone marrow epithelial-like cells, in parallel with intra-tumor genetic heterogeneity profiling from bulk DNA, is a powerful approach to identify and study DTCs, yielding insight into metastatic processes. A heterogeneous population of CNA-positive cells is present in the bone marrow of non-metastatic breast cancer patients, only part of which are derived from the observed tumor lineages.

#3050

Polymorphisms in the arntl2 promoter affect breast cancer metastasis susceptibility.

Ngoc-Han Ha, Kent Hunter. _National Cancer Institute, Bethesda, MD_.

Breast cancer mortality is primarily due to metastatic lesions rather than primary tumors, yet relatively little is known regarding the mechanisms of metastatic breast cancer, making it difficult to identify patients who are at risk for metastatic disease. Our hypothesis suggests that inherited germline mutations contribute to metastatic disease and that these single nucleotide polymorphisms (SNPs) could be used to predict outcome in breast cancer patients. To investigate the effect of inherited SNPs on metastasis, we used a mouse genetics approach comparing strains with high (FVB) and low (MOLF) metastatic phenotypes and identified Arntl2, a circadian rhythm transcription factor, as a gene whose differential expression predicted outcome in breast cancer patients. To identify SNP differences in Arntl2 between MOLF and FVB, we performed whole genome sequencing of MOLF and compared it to the FVB genome. Overlapping the data with DNase hypersensitivity sites revealed 10 SNPs in the predicted promoter of Arntl2. To test the causative role of the SNPs on Arntl2 expression in vivo, metastatic cell lines were engineered using the CRISPR-Cas9 approach to specifically replace the FVB Arntl2 promoter with that of MOLF. In agreement with our hypothesis, substitution of the MOLF promoter reduced Arntl2 transcript levels and subsequently decreased lung metastases in orthotopic implantation assays. In vitro pulldown experiments with strain-specific promoter probes revealed potential differential binding of chromatin modifier proteins, demonstrating the significance of the SNPs in regulating Arntl2 transcription. Finally, analysis of SNPs associated with Arntl2 expression in a cohort of Chinese breast cancer patients revealed significant correlation of Arntl2 expression with overall survival, validating this gene as a marker in humans. Since Arntl2 is a transcription factor, current studies are focused on identifying Arntl2-regulated genes to investigate downstream pathways involved in metastasis. This study has important implications regarding the role of circadian rhythm in cancer progression and provides a potential mechanism to explain the increased risk of breast cancers in nightshift workers. Furthermore, this provides the first evidence that transcriptional control elements can be engineered using CRISPR-Cas9 to establish the causative role of SNPs in inherited susceptibility to cancer metastasis.

#3051

Mechanism of early dissemination and metastasis in Her2+ mammary cancer.

Kathryn Harper,1 Maria Soledad Sosa,1 David Entenberg,2 Hedayatollah Hosseini,3 Julie Cheung,1 Rita Nobre,1 Alvaro Avivar-Valderas,1 Chandandaneep Nagi,1 Nomeda Girnius,4 Roger Davis,4 Eduardo Farias,1 John Condeelis,2 Christoph A. Klein,3 Julio A. Aguirre-Ghiso1. 1 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 2 _Albert Einstein College of Medicine, New York, NY;_ 3 _University of Regensburg, Regensburg, Germany;_ 4 _University of Massachusetts Medical School, Worcester, MA_.

Metastasis is the leading cause of cancer related deaths and these lesions develop from disseminated cancer cells (DCC) that can remain dormant. Metastasis initiating cells are thought to originate from a subpopulation present in progressed invasive tumors. However, DCCs detected in patients before the manifestation of breast cancer metastasis contain fewer genetic abnormalities than primary tumors or than DCCs from patients with metastases. These findings and those in pancreatic cancer and melanoma models argued that dissemination might occur during early stages of tumor evolution. Yet, the mechanisms that might allow early-disseminated cancer cells (eDCC) to complete all steps of metastasis were unknown. Here we show that in early lesions (EL), before any overt primary tumor (PT) masses are detected, there is a sub-population of Her2+/P-p38lo/P-ATF2lo/TWISThi/E-cadherinlo early cancer cells that are invasive and disseminate to target organs. Intra-vital imaging and organoid studies of early lesions revealed that Her2+ eDCC precursors locally invaded, intravasated and lodged in target organs. Her2+ eDCCs activated a Wnt-dependent EMT-like dissemination program but without complete loss of epithelial phenotype that was reversed by Her2 or Wnt inhibition. Surprisingly, while the majority of eDCCs are TWISThi/E-cadherinlo and dormant, they eventually initiate metastasis. Our work identifies a mechanism for early dissemination whereby Her2 aberrantly activates a program similar to mammary ductal branching that spawns eDCCs capable of forming metastasis after a dormancy phase.

#3052

**Defining the role of EMT in breast cancer dormancy and metastasis** in vivo **.**

Kay T. Yeung, Jing Yang. _UC San Diego, San Diego, CA_.

Breast cancer patients can develop metastases from dormant tumor cells years after primary tumor resection and treatment. Despite the clinical significance of metastatic latency, the exact cellular and molecular events regulating tumor dormancy remains largely unknown due to challenges in detecting single dormant tumor cells in vivo and characterizing the dormant tumor microenvironment. A developmental program termed Epithelial Mesenchymal Transition (EMT) has been shown to play an important role in tumor cell dissemination, tumor dormancy, and chemoresistance. During EMT, stationary epithelial cells lose their epithelial characteristics, including adherent junctions and apical-basal polarity, and acquire mesenchymal shape and properties. These cells gain the ability to migrate into the circulation, lodge into a distant organ, and form dormant tumor niches. We have previously shown that reversal of EMT is a key driving force in dormant tumor cell proliferation and macrometastasis formation in a skin carcinoma mouse model. Here, we present a novel metastatic breast cancer mouse model in which HER2-driven primary breast tumor cells are labelled with red fluorescent protein with inducible EMT transcription factor TWIST1. We established this mouse model by crossing the TetON-TWIST1 mice with the MMTV-rtTA mice to generate the MMTV-rtTA/TetON-TWIST1 double transgenic mice. To generate HER2-driven breast tumors in these mice, we administered concentrated lentivirus that expresses both the HER2 gene and the tdTomato fluorescent marker under one promoter via intraductal injection. This allows for rapid and synchronized development of primary tumors which recapitulate the formation of sporadic human breast cancer which arises in single cells in otherwise normally developed breast tissue. TWIST1 expression in the luminal layer of the mammary gland was then induced via oral doxycycline. Upon expression of TWIST1, Her2-driven breast tumor cells were found in circulation and in the lungs as micrometastases. We determined that disseminated breast tumor cells mostly remained in dormancy until TWIST expression was switched off by doxycycline withdrawal. We showed that tumor dormancy is partly due to the inability to reverse EMT and regain proliferative capacity. Lastly, using our mouse model to track disseminated breast cancer cells, we will characterize the associated dormant residing niche to further our understanding of tumor dormancy in connection with EMT and immune escape.

#3053

Stability and stemness of the hybrid epithelial-mesenchymal phenotype.

Mohit Kumar Jolly,1 Dongya Jia,1 Satyendra C. Tripathi,2 Steve Mooney,3 Muge Celiktas,2 Samir M. Hanash,2 Sendurai A. Mani,2 Kenneth J. Pienta,4 Eshel Ben-Jacob,1 Herbert Levine1. 1 _Rice University, Houston, TX;_ 2 _UT MD Anderson Cancer Center, Houston, TX;_ 3 _University of Waterloo, Waterloo, Ontario, Canada;_ 4 _Johns Hopkins University, Baltimore, MD_.

Transitions between epithelial and mesenchymal phenotypes – EMT and MET – are hallmarks of cellular plasticity during embryonic development and cancer metastasis. During these transitions, cells can also adopt a hybrid epithelial/mesenchymal (hybrid E/M) phenotype enabling them to migrate collectively as observed during gastrulation, wound healing, and clusters of Circulating Tumor Cells (CTCs). The hybrid E/M phenotype has largely been tacitly assumed to be 'metastable', i.e. transient state. Here, we integrate mathematical modeling with in vitro experiments to identify certain 'phenotypic stability factors' (PSFs) - GRHL2, OVOL2 and ΔNP63α that can stabilize a hybrid E/M phenotype. We show that H1975 (NSCLC cell line) cells can display a hybrid E/M phenotype stably and migrate collectively, a behavior that is impaired by knockdown of GRHL2 or OVOL2. Further, our computational model predicts that these PSFs can also associate hybrid E/M phenotype with high tumor-initiating potential, a prediction strengthened by the observation that the higher levels of one or more of these PSFs may predict poor patient outcome. Overall, our results suggest that a hybrid E/M phenotype need not be 'metastable', and bolster the notion that a hybrid E/M phenotype, but not necessarily full EMT, associates with aggressive tumor progression.

#3054

Tracing EMT with fluorescent biosensors (chromobodies) in living cancer cells.

Julia Maier, Theresa Plaga, Stefanie Egetemaier, Bjoern Traenkle, Ulrich Rothbauer. _University of Tuebingen, Reutlingen, Germany_.

Epithelial-mesenchymal transition (EMT) is a complex cellular program, involved in the progression of epithelial cancers to a metastatic stage. During this process characteristic epithelial marker proteins are repressed in favor of mesenchymal biomarkers, such as the intermediate filament protein vimentin. There is an ongoing need for reliable cell-based EMT model systems, which can be adapted to screening campaigns for novel anti-metastatic therapies. Imaging-based screening systems are restricted to endpoint immunofluorescence studies that provide only little information about the dynamics of the process, or to ectopic expression of EMT markers, fused to fluorescent proteins. However, overexpression of EMT marker proteins, particularly vimentin, have been shown to influence the EMT phenotype of the cells.

Here we introduce chromobodies as an innovative technology to monitor EMT in living non-small lung cancer cells. Chromobodies are fluorescently-labeled single-domain-antibodies that are functional within in living cells and directly target endogenous proteins. We generated chromobodies against the EMT biomarkers vimentin, actin and β-catenin and stably introduced them into the genome of A549 cells. Upon induction of EMT with transforming growth factor β (TGF- β), we could visualize the dynamic reorganization of endogenous vimentin, actin and β-catenin in the established EMT chromobody models in 2D as well as 3D cell culture. By means of these chromobody cell lines we further developed a 2D high content analysis system to visualize and quantify spatiotemporal changes of the endogenous biomarkers vimentin and actin during the EMT process. In a proof-of-principle screen, using known EMT-affecting reagents (e.g. TGF receptor I inhibitors LY364947 and SB505124) as well as EMT-unrelated agents, we were able to show that this high content analysis system can be applied to select compounds that inhibit TGF-β-induced EMT.

In summary, live cell imaging of EMT markers with chromobodies provides a novel versatile approach to study EMT in real time and allows the identification of EMT inhibitors as potential candidates for anti-metastatic therapies by high content screening.

#3055

A permanent lung imaging window reveals, for the first time, the steps of extravasation, seeding and growth of early metastatic dissemination.

Sonia E. Voiculescu, Yarong Wang, Maja Oktay, John Condeelis, David Entenberg. _Albert Einstein College of Medicine, Bronx, NY_.

Metastatic disease is the major cause of cancer mortality and is responsible for over 1/2 million deaths each year in the U.S. alone. Of the three most common sites of metastasis observed clinically (bone, lung and liver), the most difficult to study using intravital microscopy is the lung; a delicate organ in perpetual motion. We have recently developed and validated an implantable, permanent, lung imaging window which allows high-resolution multiphoton imaging of the intact, breathing (without ventilation), murine lung over days to weeks of repeated imaging. This window does not use vacuum to immobilize the lung tissue thereby avoiding well know artifacts associated with vacuum lung windows. Using our new window, we have been able to visualize, with single cell resolution, the steps of metastasis in a clinically relevant, spontaneously-metastasizing murine breast cancer model. We present the first direct visualization of tumor cell arrival, extravasation, and progression to micro-metastases. Further, we have observed, for the first time, the activity of the tripartite structure called Tumor MicroEnvironment of Metastasis (TMEM) in metastatic lung lesions directly demonstrating the mechanism of dissemination from metastatic lung tumors. Using this approach to imaging live lung tissue serially, within a single animal, we are investigating the mechanisms underlying the fate of tumor cells arriving in the lung with the ultimate goal of directly identifying signals behind seeding and survival of metastatic tumor cells and their responses to interventions in real time.

### Tumor and Stromal Cell Interactions

#3056

Genetic deletion of insulin-like growth factor binding protein-7 (IGFBP7) promotes hepatocellular carcinoma (HCC): a novel role of IGFBP7 in regulating antitumor immune surveillance.

Maaged A. Akiel, Chunqing Guo, Xia Li, Devaraja Rajasekaran, Rachel G. Mendoza, Nidhi H. Jariwala, Chadia L. Robertson, Mark A. Subler, Jolene J. Windle, Paul B. Fisher, Devanand Sarkar. _Virginia Commonwealth University, Richmond, VA_.

In the US, the incidence and mortality rates of hepatocellular carcinoma (HCC) are alarmingly increasing since no effective therapy is available for the advanced disease. Activation of IGF signaling is a major oncogenic event in diverse cancers, including HCC. Insulin-like growth factor binding protein-7 (IGFBP7) inhibits IGF signaling by binding to IGF-1 receptor (IGF-1R) and functions as a potential tumor suppressor for hepatocellular carcinoma (HCC). IGFBP7 abrogates tumors by inducing cancer-specific senescence and apoptosis and inhibiting angiogenesis. We now document that Igfbp7 knockout (Igfbp7-/-) mouse shows constitutive activation of IGF signaling, presents with pro-inflammatory and immunosuppressive microenvironment, and develops spontaneous tumors in lungs and liver and markedly accelerated carcinogen-induced HCC. Loss of Igfbp7 resulted in increased proliferation and decreased senescence in hepatocytes and mouse embryonic fibroblasts that could be blocked by an IGF-1 receptor inhibitor. A significant inhibition of genes regulating immune surveillance was observed in Igfbp7-/- livers which was associated with marked inhibition in antigen cross-presentation by Igfbp7-/- dendritic cells. IGFBP7 overexpression inhibited growth of HCC cells in syngeneic immunocompetent mice which could be abolished by depletion of CD4+ or CD8+ T lymphocytes. Our studies unravel modulation of immune response as a novel component of pleiotropic mechanisms by which IGFBP7 suppresses HCC. Even though HCC has an immunosuppressive milieu, immune targeted therapies are beginning to demonstrate significant objective responses in clinical trials. IGFBP7 might be an effective anti-HCC therapeutic by directly inhibiting cancer cells and stimulating an anti-tumor immune response.

#3057

Gut microbiome controls liver metastasis.

Chi Ma, Qiong Fu, Tim Greten. _NCI/NIH, Bethesda, MD_.

Aim: The gut microbiome can modify tumor immunity and has been suggested to be involved in the development of liver cancer. However, it remains unknown how the gut microbiome controls hepatic immune responses. This study was designed to exam the effect of the gut microbiome on liver antitumor immunity, and to study potential mechanism.

Experimental procedure: Antibiotic cocktail in drinking water was given to remove mouse gut microbiota. Control mice were kept on regular water. EL4 thymoma cells were injected s.c. to induce spontaneous liver metastasis. B16 melanoma and CT26 colon cancer cells were injected intrasplenically to form liver metastasis. Lung metastasis was induced by tail injection of tumor cells.

Results: Antibiotic cocktail efficiently depleted gut bacteria. Removing gut commensal bacteria did not affect the growth of primary s.c. tumors, but impaired formation of liver metastasis in different models. The inhibitory effect on liver metastasis by removing gut microbiome was found after intrasplenic injection of tumor cells to form liver metastasis using both B16 melanoma and CT26 colon cancer tumor cells. Interestingly, formation of lung metastasis caused by tail vein injection of B16 cells was not impaired by antibiotics treatment, suggesting a liver specific effect. The inhibition of liver metastasis by antibiotic treatment was absent in Rag1 knockout mice, suggesting that the mechanism is mediated by the adaptive immune system.

Conclusion: Our results suggest that the gut microbiome affects the liver immune microenvironment and modulates antitumor immunity.

#3058

Syndecan-1 mediates breast cancer metastasis to the brain through IL-8 and PECAM-1 signaling.

Sierra Mosticone Wangensteen,1 Megan Sayyad,1 Madhavi Puchalapalli,1 Megan Sullivan,2 Jamie Singh,1 Briana Ratchford,1 Jayda Abrams,1 Majid Jahromi,1 Bin Hu,1 Michael Idowu,1 Jennifer Koblinski1. 1 _Virginia Commonwealth University, Richmond, VA;_ 2 _NorthShore University Health System, Evanston, IL_.

Brain metastasis is a devastating, late-stage event affecting approximately 10-30% of breast cancer patients. However, it is not well understood how breast cancer cells migrate across the blood-brain barrier (BBB) and invade into the brain. Syndecans (Sdcs) are cell surface heparan sulfate proteoglycans (HSPGs) that have previously been linked to breast cancer progression and metastasis. Preliminary findings from our lab indicate that Sdc1 may be involved in breast cancer metastasis to the brain. In clinical samples from breast cancer patients with brain metastases, 67% of the brain metastasis samples stained positive for Sdc1 expression. In an experimental mouse model of metastasis, we found that silencing expression of Sdc1 in MDA-231 breast cancer cells greatly reduced metastasis specifically to the brain, with no difference in lung or bone metastases. Additionally, silencing expression of Sdc1 in MDA-231 cells resulted in a reduction in cancer cell migration across an in vitro BBB transwell model system. These findings prompted us to investigate the mechanism through which Sdc1 facilitates breast cancer cell migration across the BBB. Since BBB endothelial cell junctions have been reported to be disrupted in metastasis to the brain, immunofluorescence was performed to examine localization of junction proteins in our BBB model following addition of conditioned medium (CM) from MDA-231 non-silenced (NS1) control and Sdc1KD cells. We observed disruption in PECAM-1 localization at BBB endothelial cell junctions upon addition of CM from MDA-231 NS1 cells with markedly less disruption occurring upon treatment with CM from Sdc1KD cells. These results suggest that paracrine factors secreted from MDA-231 cells may facilitate breast cancer cell migration across the BBB by affecting PECAM-1 localization on BBB endothelial cells. By performing a cytokine array using CM from MDA-231 cells, we determined that Sdc1KD CM contained lower levels of IL-8 than MDA-231 NS1 cell CM. These findings were confirmed by ELISA, qRT-PCR, and multiplex analysis. We then went on to treat endothelial cells in the xCELLigence in vitro BBB model system with IL-8 and observed a sufficient decrease in cell index readings, suggesting that IL-8 affects BBB barrier permeability. Taken together, our results suggest that Sdc1 supports breast cancer cell migration across the BBB through a signaling mechanism involving IL-8 and PECAM-1. Elucidating this mechanism will allow for the development of therapeutic strategies to combat breast cancer cell metastasis to the brain.

[S.M.W. and M.S. contributed equally to this work.]

#3059

UHRF1 deficiency exerts anti-tumor immune responses in non-small cell lung cancer.

Handan Xiang, Junying Chen, Xiao-Fan Wang. _Duke University, Durham, NC_.

Immune surveillance is essential for effective tumor clearance. However, the tumor microenvironment is typically impermissive for proper stimulation of both innate and adaptive immune populations. Epigenetic inhibitors have recently been shown to function as immune sensitizing reagents in the suppressive microenvironment. In our search for novel epigenetic regulators with immunomodulatory functions, we found ubiquitin-like with PHD and ring finger domain-containing protein 1 (UHRF1) being crucial for the suppression of immune activation. Loss of UHRF1 in cancer cells led to significant reduction of tumor masses in a syngeneic lung cancer model. The tumor shrinkage was due to increased infiltration of T cells with the enhanced ability to proliferate and produce IFN-gamma. Mechanistically, lung cancer cells deficient of UHRF1 had elevated expression of endogenous retrovirus genes due to DNA demethylation, which associated with enhanced type I interferon responses. Type I interferons are potent antitumor effector molecules by promoting immune cell infiltration into the tumor microenvironment, yet they can also dampen T cell activation by upregulating PD-L1 expression in tumor cells. Surprisingly, UHRF1 deficient cancer cells failed to upregulate PD-L1 even in the presence of excessive type I interferons, indicative of additional regulatory pathways for PD-L1. We further found that UHRF1 directly interacted with PD-L1 promoter regions and that UHRF1 promoted PD-L1 transcription. Therefore, the inhibition of UHRF1 augmented type I interferon responses while diminished PD-L1 expression, which synergistically contributed to the immune activation phenotype. Moreover, in non-small cell lung cancer (NSCLC) patients, UHRF1 expression was positively correlated with PD-L1 expression as well as poor prognosis. Together, our data suggested that UHRF1 represents a novel immuno-oncology target for NSCLC.

#3060

Role of curcumin in targeting cancer-associated fibroblasts and modulation of tumor microenvironment in dendritic cell-based immunotherapy.

Sheefa Mirza,1 Nayan Jain,1 Rakesh Rawal2. 1 _Gujarat University, Ahmedabad, India;_ 2 _The Gujarat Cancer and Research Institute, Ahmedabad, India_.

INTRODUCTION: Cancer associated fibroblasts (CAFs), main component of tumor microenvironment (TME), modulate the recruitment and functions of tumor-associated immune cells by secreting various growth hormones, miRNAs and cytokines; thus having an important role in generation of immunosuppressive TME which is yet to be elucidated. Curcumin is known to have various properties including capability to modulate numerous target proteins including transcription factors, receptors, kinases, cytokines, enzymes and growth factors. Thus, aim of the study was to evaluate the effect of miRNAs and cytokines released by lung cancer patients' derived CAFs and to assess immunomodulatory potential of curcumin on DC maturation by targeting these CAFs through modulating their TME.

METHODOLOGY: CAFs were cultured from lung cancer patient derived tumor tissue biopsy and characterised using CAF-specific markers. Further, immature DCs (imDCs) were differentiated in presence of rGM-CSF and rIL-4 for 5-6 days. These imDCs were cultured in the presence of conditioned media derived from CAFs (CAFs-CM) as well as NFs (Normal Fibroblasts, NFs-CM) at day 6 for 48 hours to evaluate the effect of CAFs on DC maturation. Mature DCs (mDCs) were characterized by the presence of maturation markers CD80, CD83, CD86 and CTLA4 using qRT-PCR. Moreover, expression of miR-221, miR-222, miR-155, miR-142-3p and miR-146a was assessed to evaluate the role of epigenetic regulators on DC maturation. Cytokine profiling of CAFs-CM as well as CAFs-CM treated with curcumin was conducted.

RESULTS: α-SMA+Vimentin+ cells were considered as CAFs. A significant upregulation of CD80, CD83 and CD86 was observed when cultured in the presence of NFs-CM while a remarkable downregulation of these markers was found when cultured in CAFs-CM. CTLA-4 was down regulated in NFs-CM as compared to CAFs-CM, suggesting the role of CAFs in generation of regulatory DCs. Amongst all miRNAs, miR-146a was shown to be up regulated dramatically in CAF-DCs (DCs cultured in CAFs-CM) as well as in CAFs-CM, suggesting the immunosuppressive role of miR-146a. Further, an increased expression of miR-146a was positively correlated with increased expression of anti-inflammatory cytokines like IL-6, IL-10, TGF-β and decreased expression of TNF-α (pro-inflammatory) in CM derived from CAF-DCs. Moreover, curcumin had the potential to convert regulatory DCs facilitated by CAFs into mDCs, which were characterized by high expression of co-stimulatory molecules, low expression of CTLA4, lower levels of immune suppressive cytokines production, lower levels of miR-146a.

CONCLUSION: These findings provide insight into understanding the immunomodulatory role of curcumin in targeting CAFs and modulating the tumor microenvironment, thus enhancing antitumor immune response in DC based therapy.

#3061

Tumor-derived PAI-1 promotes macrophage M2 polarization by stimulating an autocrine IL-6 /STAT3 pathway.

Marta H. Kubala, Veronica R. Placencio, Yves A. DeClerck. _Children's Hospital Los Angeles, Los Angeles, CA_.

Macrophages can exhibit a spectrum of activation states ranging from a pro-inflammatory and antitumorigenic M1 (CD80+; IL-12high; iNOShigh) to a pro-tumorigenic M2 [CD163+; IL-10high; CD206+; Arg+ (mouse)] phenotype. Plasminogen activator inhibitor-1 (PAI-1) is overexpressed in many cancers and although it acts as a protease inhibitor it is paradoxically correlated with poor outcome. PAI-1 exerts its pro-tumorigenic role via pro-angiogenic and anti-apoptotic functions. Increasing evidence points towards the importance of PAI-1 in inflammation and tumorigenesis, but its influence on the immune component of the tumor microenvironment has not yet been investigated. Here we report a novel observation that PAI-1 is a regulator of macrophage polarization. We observe that in PAI-1 KO mice xenotransplanted with 3 human cancer cell lines (HT1080, A549, HCT116) in which PAI-1 was knocked down (KD), tumors were less infiltrated with macrophages with lower Arg expression compared to WT mice implanted with PAI-1 pos. tumors. We demonstrate that recombinant PAI-1 through its uPA interactive domain increased the expression of M2 polarization markers (CD163, IL-10) and decreased or did not affect the expression of M1 polarization markers (iNOS, CD80) in human peripheral blood monocytes. Further investigation demonstrated that treatment of monocytes with PAI-1 induced a rapid (2 h.) increase in IL-6 mRNA and secretion (4 h.) of the protein that was followed by phosphorylation (8-24 h.) of signal transduction and activation of transcription 3 (STAT3) in monocytes. Further linking STAT3 activation to PAI-1, we observed a decreased pSTAT3 in monocytes co-cultured with HT1080 cells upon PAI-1 downregulation. We found that STAT3 activation was downstream of IL-6 and responsible for M2 polarization, as the blockage of the IL-6 receptor with a function blocking antibody (tocilizumab) or inhibition of STAT3 activation by a JAK2/STAT3 inhibitor (ruxolitinib) prevented M2 polarization of monocytes by PAI-1. The contribution of PAI-1 to M2 polarization of macrophages was tested in PAI-1 KO mice implanted with human tumors in which PAI-1 expression was controlled by doxycycline. These experiments demonstrated that induction of PAI-1 in established tumors increased the presence of tumor associated macrophages (TAM) (F4/80+) and decreased the presence of M1 (iNOS+) TAM. Further supporting the presence of an autocrine PAI-1/IL-6 pathway in macrophage polarization in human cancers, a meta-analysis of gene expression array data indicated strong correlations between PAI-1 and IL-6 expression in breast (P=<0.05) and colon (P<0.01) cancers and between PAI-1 and CD163 in colon cancer (P<0.0001). The data thus identify a new pro-tumorigenic function for PAI-1 in the communication between tumor cells and macrophages where tumor-derived PAI-1 activates an autocrine IL-6/STAT3 pathway that promotes M2 polarization.

#3062

Metformin inhibits TGFβ-induced stromal ECM remodeling to impede invasion in ovarian cancer.

Peter C. Hart, Shermeen Sheikh, Ernst Lengyel, Iris Romero. _University of Chicago, Chicago, IL_.

Recently, our group has elucidated that metformin, the commonly used medication for type 2 diabetes, has anti-ovarian cancer (OvCa) effects in preclinical models and use of the drug is associated with improved OvCa survival in retrospective epidemiological studies. While the cytotoxic effects of metformin in ovarian cancer cell lines is well demonstrated, the impact of metformin on tumor-stromal interactions in the tumor microenvironment is not well defined. Currently, our work has focused on the impact of metformin on the tumor microenvironment and cancer cell behavior in physiologic models of tumor metastasis. Using a 3D organotypic model, we demonstrate that OvCa cell invasion is inhibited by metformin. In evaluating the molecular mechanism mediating this effect we show that OvCa cells co-cultured with stromal cells or stimulated with TGFβ1 upregulate the production of the extracellular matrix (ECM) proteins fibronectin and collagen 1 and that this effect is attenuated by metformin. Interestingly, metformin's inhibition of ECM production was not dependent on AMP-activated kinase (AMPK), a common metformin target. To expand on this finding we performed label-free shotgun proteomics and identified collagens, fibronectin and TGFBR2 to be among the proteins most highly repressed in response to metformin exposure in OvCa cells. Finally, we identified that this repression of ECM remodeling by metformin was associated with an overall reduction in TGFBR2 expression and consequent SMAD/STAT3 activity. Taken together, our data indicate that metformin may have novel targets in the tumor microenvironment where it targets pro-tumorigenic effects of the stromal compartment by repressing TGFβ-dependent stromal cell ECM secretion, thereby inhibiting ovarian cancer invasion and metastasis.

### Advancing Clinical Trial Design in Regulatory Science and Policy

#3594

Adaptively randomized seamless-phase multiarm platform trial: Glioblastoma Multiforme Adaptive Global Innovative Learning Environment (GBM AGILE).

Donald A. Berry,1 Todd Graves,2 Jason Connor,2 Brian Alexander,3 Timothy Cloughesy,4 Anna Barker,5 Scott M. Berry,2 for the GBM AGILE Global Alliance. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Berry Consultants, LLC, Austin, TX;_ 3 _Dana Farber Cancer Institute, Boston, MA;_ 4 _The Ronald Reagan UCLA Medical Center, Boston, CA;_ 5 _Arizona State University, Phoenix, AZ_.

Traditional phase 3 clinical trials compare an experimental arm with control. They inefficiently use patients, time, and finances. Dramatic and rapid changes in biology makes such trials untenable. We describe an alternative drug development strategy that we are using in a particular setting, the trial GBM AGILE (Glioblastoma Multiforme Adaptive Global Innovative Learning Environment).

The trial's design employs many innovations. Some aspects are similar to those of I-SPY 2 (see 4 articles in July 7, 2016 NEJM) but GBM AGILE extends I-SPY 2 in many ways. (1) It is a Bayesian platform trial that simultaneously evaluates many treatment arms (including combinations) from many companies. (2) Arms are added to the trial at any time and leave when they have been evaluated, whether positively or negatively. (3) An arm's sample size is adaptive and based on frequent analyses of the trial results. (4) Every arm has an initial stage in which it is randomized adaptively: arms performing better in disease subtypes are assigned with higher probability to such patients. (5) An arm that performs sufficiently well in a disease subset moves seamlessly into a small (50-patient) confirmatory, registration stage in the same subset, with equal randomization against control. (6) All experimental arms are compared against a common control arm that is assigned to 20% of patients in every subtype; a bridging model takes advantage of having many arms in the trial and many comparisons among arms, and enables indirect randomization comparisons of all arms with all controls. (7) Patient subtypes are defined by line of therapy, MGMT methylation status for newly diagnosed patients, and biomarkers associated with targeted therapies, although adaptive randomization enables us to draw conclusions about off-target effects.

The many possible subtypes means that there are many possible drug indications. So there are many possible "error types" and no single definition of statistical power. For example, the trial may conclude that a drug's indication is "recurrent, biomarker-positive" disease when in truth it is "all recurrent" disease. We show how the design addresses this issue and we define "pure type I error."

GBM AGILE's primary endpoint is overall survival (OS). To make the design more efficient we incorporate evaluations of patients' statuses over time using a longitudinal model based on periodic MRI assessments and performance status. The longitudinal model and its components are not end points but rather provide auxiliary information that enables multiply imputing OS for surviving patients.

We represent the trial's coordinating committees that are made up of more than 150 enthusiastic and devoted disease experts and advocates from around the globe, including from Australia and China. The U.S. FDA has been enormously helpful in designing GBM AGILE, especially as regards its potential for drug and biomarker registration.

Our approach provides a model for other diseases, including those outside of cancer.

#3595

Under-reporting of research biopsies in clinical trials in oncology.

Christine M. Parseghian, Kanwal Raghav, James Yao, Lee M. Ellis, Alda Tam, Michael J. Overman. _U.T. MD Anderson Cancer Center, Houston, TX_.

Purpose: Research biopsies are frequently incorporated into clinical trials in oncology, and are often a mandatory requirement for trial enrollment. However, limited information is available regarding the extent and completeness of research biopsy reporting.

Methods: We identified a cohort of therapeutic clinical trials wherein at least one image-guided non-diagnostic research biopsy was performed between 1/2005 and 10/2010 from a large interventional radiology database at MD Anderson Cancer Center. Study protocols were compared with the highest level of corresponding publication as a manuscript, registry report, or abstract.

Results: A total of 866 research biopsies were performed across 46 clinical trials (median 19 biopsies/trial). After a median follow-up time of 5.8yrs from study completion, 35 trials (76%) had published manuscripts (18 reported biopsy results), 8 (17%) had either a registry or abstract report (1 reported biopsy results), and 3 (7%) were not reported. In total, 19 trials (41%) reported biopsy results. Of these 19 trials, 6 (32%) trials under-reported the number of biopsies performed. Only 14 of 32 (42%) trials with mandatory research biopsies reported on these biopsies. Comments regarding biopsy quality/adequacy were reported in only 5 trials. Complications from these biopsies occurred in 39 pts (grade 3 in 6 pts) in 8 trials, but only 1 (13%) trial reported biopsy complications. Factors associated with better reporting of research biopsies are shown in the Table. When limited to only published manuscripts, results were unchanged. Academic sponsorship and a larger number of biopsies, especially serial biopsies, were associated with increased reporting of research biopsies.

Conclusion: Despite ethical obligations to report research biopsies, 59% of trials do not report on these biopsies. Although complications occur, they are rarely reported. Clinical trial design efforts to ensure that an adequate number of research biopsies are obtained may lead to improved reporting of research biopsy biomarker results.

Variable | N | Trials reporting biopsies (%) | P-value

---|---|---|---

Mandatory or optional biopsy | |  | 1

Mandatory (+/- optional) | 33 | 14 (42)

|

Optional | 13 | 5 (38)

|

Total number of biopsies per trial | |  | 0.002

<10 | 19 | 3 (16)

|

10-30 | 22 | 11 (50)

|

>30 | 5 | 5 (100)

|

Trials with serial biopsies (No. of pts.) | |  | 0.0001

1-5 | 11 | 0 (0)

|

>=5 | 18 | 13 (72)

|

Study sponsorship | |  | 0.03

Industry | 24 | 6 (25)

|

Academic | 22 | 13 (59)

|

Clinical trial | |  | 0.77

Multicenter | 21 | 8 (38)

|

Single center | 25 | 11 (44)

|

Phase of clinical trial | |  | 0.51

I or I/II | 33 | 15 (45)

|

II or III | 13 | 4 (31)

|

#3596

How many tumor indications should be initially studied in clinical development of next-generation immunotherapies.

Cong Chen,1 Qiqi Deng,2 Linchen He,3 Devan Mehrotra,1 Eric H. Rubin,1 Robert A. Beckman4. 1 _Merck & Co., Inc., Collegeville, PA; _2 _Boehringer Ingelheim Pharmaceuticals Inc, Ridgefield, CT;_ 3 _New York University, NY;_ 4 _Georgetown University Medical Center, DC_.

An experimental oncology immunotherapy may have the potential to be effective in a large number of tumor indications. Once a recommended Phase II dose (RP2D) is determined, under resource constraint, a natural strategy is to conduct Phase II proof-of-concept (POC) trials in two waves. A cohort of potential tumor indications is selected for the first wave investigation and the second wave investigation in a different cohort of tumor indications is initiated only after the drug has been demonstrated to be effective in the first wave. Immunotherapy development is a dynamic environment with rapidly evolving mechanistic understanding, constant flow of new data and frequent changes in the competitive landscape. How many tumor indications should be investigated in the first wave given the uncertainties? We attempt to answer this question by maximizing a benefit-cost ratio, defined to be the expected number of effective tumor indications correctly identified in the two waves divided by the expected total sample size for the POC trials in the two waves and the total sample size for the Phase III trials triggered by those with a positive outcome in the first wave. It is found that the optimal number of the first wave POC trials is in a range of approximately three to six, which may vary with the resource constraint but is otherwise robust to the key factors we have considered. A recommendation is made on how much resource should be invested in the first wave.

#3597

Experiences and lessons from innovative dose escalation designs in early-phase oncology.

Jiawen Zhu,1 Ulrich Beyer,2 Somnath Sarkar,1 Gwen Nichols,1 William Pao,2 Daniel Sabanés Bové2. 1 _F. Hoffmann-La Roche Ltd, New York, NY;_ 2 _F. Hoffmann-La Roche Ltd, Basel, Switzerland_.

Introduction: Since 2010, Roche-sponsored early development trials in oncology have successfully implemented innovative dose escalation (D/E) strategies including modified continual reassessment methods (CRM) and other Bayesian adaptive designs. Compared to standard 3+3 designs, these methods allow for flexibility to address a variety of clinical questions and to estimate more accurately a molecule's maximum tolerated dose (MTD). However, CRM designs are sometimes considered as complex and difficult to implement and are not easily understood by clinicians (Le Tourneau et al., 2009, Iasonos et al. 2014). Here, we share our experiences and learnings from the initial exploration, introduction, and wide-spread implementation of CRM in early oncology clinical trials.

Methods: We conducted a thorough internal review of the current statistical methods and clinical strategies used in CRM designs. In addition, we systematically collected feedback on the internal experience through surveys and interviews of participating biostatisticians, pharmacologists, clinicians and operations managers.

Results: CRMs using two parameter logistic regression models and escalation with overdose control (Neuenschwander et al., 2008) are the most commonly used CRM designs in Roche-sponsored oncology D/E trials. Compared to trials in which 3+3 designs are used, we experienced significant advantages with CRM designs including: flexibility in cohort sizes; formal integration of relevant prior pre-clinical/clinical knowledge of the dose-toxicity relationship; and contribution of all dose limiting toxicities (DLTs) to an MTD determination. On the other hand, we learned about some limitations. The CRM designs modeled binary DLT events where the grade of an event was not considered. Thus, in a cohort where severe DLT event(s) were observed, clinical judgment typically superseded the CRM recommendation towards a lower dose; this decision rule was a priori defined in the protocol. In certain situations, inconsistent prior-information in the dose-toxicity relationship was observed. Therefore, prior assumptions had to be carefully assessed through simulations using multiple dose-toxicity scenarios.

Conclusions: Overall, utilization of CRM designs was considered beneficial to Roche early development trials. Based on our experience, CRM designs are flexible and can be tailored to address a variety of clinical research questions. Trial simulation analyses were critical for us to understand the performance of CRM designs, including the accuracy of estimated MTD, trial duration/sample size, and sensitivity to prior assumptions. Furthermore, the introduction and implementation of CRM designs required and promoted strong multidisciplinary collaborations, especially during the design and study protocol set-up phases and the D/E recommendation phase. 
